WO2001068137A2 - Compositions for regulating memory consolidation - Google Patents

Abstract

The present invention makes available methods and reagents for facilitating LTP, e.g., to increase memory function such as long-term memory and recall ability.

Description

NMGN-pWO-009 - 1 -

Methods and Compositions for Regulating Memory Consolidation

Government Support

This invention was partially funded by grants from certain government agencies; the government has certain rights to the invention.

Background of the Invention

Behavioral research has found that the human mind consolidates memory at certain key time intervals. The initial phase of memory consolidation occurs in the first few minutes after we are exposed to a new idea or learning experience. The next phase occurs during our sleep that night. If a learning experience has on-going meaning to us, the next week or so serves as a further period of memory consolidation. In effect, in this phase, the material moves from short-term memory to long-term memory for storage.

It has been known for several decades that the formation of long-term memory requires gene expression. The prevailing hypothesis for the formation of long-term memory (LTM) is that introduction of a memory item alters the pattern of existing neuronal connectivity to form a neuronal network that will subserve the information for long-term storage. Modulation of synaptic efficacy is induced by changes in synaptic transmission within selected synapses or alteration in synaptic contacts. These changes are in turn supported by molecules that underlie transmission or synaptic remodeling. It is suggested that modulation of gene expression is needed for LTM formation to overcome the relative short lifetime of proteins in neurons (as compared with enduring memory).

In animal models of learning and memory, the requirement for de novo protein synthesis around the time of training has long been a definitive property of long-term memory that separates it from other types of memory retention. Thus, the storage of long-term memory is associated with cellular program(s) of gene expression, altered protein synthesis, and the growth of new synaptic connections. For instance, recent work suggests that this property of memory formation may have a specific molecular underpinning that involves cAMP -responsive transcription and that is mediated through the cAMP response element binding protein (CREB) family of transcription factors. NMGN-pWO-009 - 2 -

CREB is a nuclear protein that modulates the transcription of genes with cAMP response (CREs) elements in their promoters. Increases in the concentration of cAMP or other signals can trigger the phosphorylation and activation of CREB. Activation of CREB triggers transcription of genes containing CREs. Some of the newly synthesized proteins are additional transcription factors that ultimately give rise to the activation of late response genes, whose products are responsible for the modification of synaptic efficacy leading to LTM.

CREB subserves the formation of memories of various types of tasks that utilize different brain structures. Evidence is available suggesting that CREB regulates the transcription of genes that subserve LTM. In Nplysia, for example, CREB activation has been interfered with by microinjection of CRE containing oligonucleotides into cultured neurons. In Drosophila, CREB function has been disrupted using a reverse genetic approach. Thus, LTM has been specifically blocked by the induced expression of a CREB repressor isoform, and enhanced by the induced expression of an activator isoform. In mouse, the role of CREB has been confirmed by behavioral analyses of a knock-out line with a targeted mutation in the CREB gene. In these mutants, learning and short-term memory are normal, whereas long-term memory is disrupted. On the whole, the data suggest that encoding of long-term memories involves highly conserved molecular mechanisms. Animals with lesions of the medial temporal lobes and related thalamic structures show a profound disruption of memory consolidation. We have previously demonstrated that fornix-dependent lesion-induced amnesia is associated with abnormal regulation of gene expression in specific subregions of the hippocampus. See, for example, Taubenfeld et al. (1999) Nat Neurosci 2:309-10. In normal animals, inhibitory avoidance training produces a rapid and persistent increase in the phosphorylation of CREB, which is a necessary step in the regulation of CRE-mediated gene expression required for memory consolidation. The change in CREB phosphorylation is largely confined to hippocampal fields CAl and dentate gyrus, and lasts at least 6 hours after training. Animals with fornix lesions learn the inhibitory avoidance and display memory at control levels for up to 6 hours, however, by 24 hours they exhibit amnesia. The amnesic animals also fail to exhibit any increase in hippocampal CREB NMGN-pWO-009 - 3 -

phosphorylation after training. Our results suggest that hippocampal inputs passing through the fornix regulate consolidation of this form of memory via regulation of CREB-mediated gene expression in hippocampal neurons.

Initial learning is likely to result from changes in the transmission of synapses conveying information about where the animal is in space. Whether or not these changes are made permanent depends on the timely occurrence of new gene expression. Signals impinging on hippocampal neurons via the fornix contribute to memory consolidation by modulating the gene expression required for the establishment of long- term memory. Identification of the critical chemical signals and their transduction pathways can suggest treatments for amnesia associated with damage to the temporal lobe memory system.

Summary of the Invention

The present invention relates to methods, compounds, and pharmaceutical preparations for increasing long-term potentiation and/or improving long-term memory in animals, such as humans, by administering one or more agents which, for example, alone or in combination, modulate PKA and PKC dependent pathways, thereby increasing MAPK/MEK activity and resulting in improved memory functions.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Nols. 154 and 155 (Wu et al. eds.), NMGN-pWO-009 - 4-

Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I- IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Brief Description of the Drawings

Figure 1 illustrates the synergistic effect on long-term memory of conjoint treatment with a cholinergic/glutaminergic agent and an adrenergic/dopaminergic agent. Each position on the chart indicates the averaged result for a sample of six rats injected immediately after training with a single injection as indicated for each column. Figure 2 depicts results of memory testing on fornix-lesioned rats treated with a series of memory-enhancing compositions (A = oxotremorine, B = clenbuterol, C = oxotremorine + clenbuterol).

Detailed Description of the Invention

I. Overview The present invention relates to the discovery of a synergistic effect of two signal transduction pathways in long-term memory consolidation and memory, and demonstrates that treatment with agents that activate both pathways is sufficient to produce significant enhancement of long-term memory. In general, the invention provides a method for potentiating long-term memory by treatment, e.g., by jointly administering agents that mimic adrenergic-dependent activation of cAMP and cholinergic-dependent or glutamergic-dependent activation of protein kinase (PKC).

While not wishing to be bound by any particular theory, experimental evidence suggests that PKA is positively coupled to Rap-1 and B-Raf, which activate MAPK kinase (MEK), leading to MAPK phosphorylation and activation of CREB. Similarly, experimental evidence suggests that PKC interacts with Ras and/or Raf-1, thereby activating MEK. As the alternate name "cAMP-dependent protein kinase" suggests, PKA is agonized by cAMP, suggesting that this pathway may be stimulated by adenylate cyclase activators (including agents which stimulate dopamine receptor D1/D5 (DAR) and/or β-adrenergic receptor (βAR), cAMP analogs, and cAMP pliosphodiesterase inhibitors). PKC is activated by diacylglycerol, and thus can be NMGN-pWO-009 - 5 -

activated by phospholipase C and agents which stimulate metabotropic glutamate receptors (mGLURs) and/or muscarinic acetylcholine receptor (lriAChR). Activation of

PKC also results in MAPK activation and activation of CREB. It is therefore specifically contemplated that agents which act intracellularly to stimulate cAMP and PKC can be administered according to the method of the present invention. In preferred embodiments, the subject activators are organic molecules having a molecular weight less than 2500 amu, more preferably less than 1500 amu, and even more preferably less

^■ than 750 amu, and are capable of regulating at least some of the biological activities cAMP and PKC, and upstream or downstream regulators thereof. Thus, the methods of the present invention include the use of MAPK/MEK activators, such as small molecules, which agonize cAMP and PKC pathways to improve long-term memory

(LTM) in animal subjects.

In another aspect, the present invention provides pharmaceutical preparations comprising, as an active ingredient, one or more compounds which (alone or collectively) stimulate activators of cAMP and PKC. The subject compounds are formulated in an amount sufficient to improve LTP in an animal. The subject preparations and methods can be treatments using MAPK/MEK activators effective for human and/or animal subjects. In addition to humans, other animal subjects to which the invention is applicable extend to both domestic animals and livestock, raised either as pets or for commercial purposes. Examples are dogs, cats, cattle, horses, sheep, hogs, and goats.

Still another aspect of the invention relates to the use of PKC agonists and cAMP agonists, in combination, for lessening the severity or prophylacticly preventing the occurrence of learning and/or memory defects in an animal, and thus, altering the learning ability and/or memory capacity of the animal. As a result, the compounds of the present invention may be useful for treating or preventing memory impairment, e.g., due to toxicant exposure, brain injury, epilepsy, mental retardation in children, and dementia resulting from a disease, such as Parkinson's disease, Alzheimer's disease, AIDS, head trauma, Huntington's disease, Pick's disease, Creutzfeldt- Jakob disease, and stroke. The invention also relates to the use of a compound which stimulates both PKC NMGN-pWO-009 - 6-

and PKA pathways, thereby obviating the need for administration of two separate pharmaceutically active agents.

II. Definitions

For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

The term "adrenergic" refers to neurons which secrete neurotransmitters chemically related to adrenaline (epinephrine). Examples are dopamine, norepineplirme, epinephrine. Such agents are also referred to as catecholamines, which are derived from the amino acid tyrosine. The term "biogenic amines" refers to a class of neurotransmitters which include catecholamines (e.g., dopamine, norepinephrine, and epinephrine) and serotonin.

The term "catecholamines" refers to neurotransmitters that have a catechol ring (e.g., a 3,4-dihydroxylated benzene ring). Examples are dopamine, norepinephrine, and epinephrine. The term "cholmergic" refers to neurons which secrete neurotransmitter synthesized from choline.

The term "dopamine" refers to an adrenergic neurotransmitter.

The terms "MAPK activator" and "MAPK/MEK activator" refer to an agent or combination of agents which induce or increase MAPK induction, e.g., by activation of cholinergic, glutammergic, dopaminergic, and/or adrenergic signal pathways and/or increasing the level and/or activity of PKC and/or cAMP in a cell. Exemplary MAPK activators include ligands of cholinergic, glutammergic, and adrenergic receptors, as well as cAMP agonists and PKC agonists. The subject method can be deployed as a conjoint therapy, e.g., by using a cholinergic/glutaminergic agonist molecularly distinct from a dopaminergic/adrenergic agonist administered conjointly therewith. In other embodiments, both functionalities can be combined in one molecule.

A "cAMP agonist", as the term is used herein, refers to any agent which increases the level or activity of cAMP or mimics cAMP in a cell, including agents which activate adrenergic receptors, adenylate cyclase, or PKA, as well as agents which NMGN-pWO-009 - 7 -

mimic or increase the level of cAMP, and agents which inhibit cAMP phosphodiesterase(s).

A "PKC agonist", as the term is used herein, refers to any agent which increases the level or activity of PKC in a cell, including agents which activate cholmergic receptors and/or glutaminergic receptors, phospholipase C, Ras/Rat-1 kinase or PKC, as well as agents which mimic or increase the level of diacyl glycerol, etc.

The term "ED50" means the dose of a drug which produces 50% of its maximum response or effect.

The "CREB" family of proteins (sometimes referred to as the ATF family) is best known for the three members that can mediate cAMP -responsive transcription:

CREB itself, CREM and ATF-1 (DeGroot et al. (1993) Mol Endocrinol 7:145-153).

These basic-region, leucine-zipper proteins bind to DNA sequences, called cAMP response element (CRE) sites, which are often found in the upstream regulatory regions of genes whose synthesis is cAMP-responsive. Molecular analysis has shown that CRE sites, and their interaction with CREB family members, are necessary for cAMP responsiveness. After the catalytic subunit of PKA translocates to the nucleus, it can directly phosphorylate the serine residue at position 133 on CREB, thus activating the protein and directly linking the cAMP transduction pathway to the induction of new gene expression (Backsai et al. (1993) Science 260: 222-226; and Hagiwara et al. (1993) Mol Cell Biol 13 :4852-4859).

The term "interact" as used herein is meant to include detectable interactions between molecules, such as can be detected using, for example, a two-hybrid assay. The term "interact" is also meant to include "binding" interactions between molecules. Interactions may be protein-protein or protein-nucleic acid in nature. The term "modulation" as used herein refers to both upregulation, i.e., stimulation, and downregulation, i.e., suppression, of a response.

An "effective amount" of, e.g., a MAPK/MEK activator, with respect to the subject method of treatment, refers to an amount of the activator in a preparation which, when applied as part of a desired dosage regimen brings about, for example, a change in NMGN-pWO-009 - 8 -

phosphorylation of CREB and enhanced LTM according to clinically acceptable standards.

The term "LD₅₀" means the dose of a drug which is lethal in 50% of test subjects. A "patient" or "subject" to be treated by the subject method can mean either a human or non-human animal.

The term "prodrug" is intended to encompass compounds which, under physiological conditions, are converted into the therapeutically active agents of the present invention. A common method for making a prodrug is to include selected moieties which are hydrolyzed under physiological conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by an enzymatic activity of the host animal.

The term "therapeutic index" refers to the therapeutic index of a drug defined as LD₅₀/ED₅₀.

The term "acylamino" is art-recognized and refers to a moiety that can be represented by the general formula:

wherein R₉ is as defined above, and R'^ represents a hydrogen, an alkyl, an alkenyl or - (CH2)_m- 8₅ where m and Rg are as defined above.

Herein, the term "aliphatic group" refers to a straight-chain, branched-chain, or cyclic aliphatic hydrocarbon group and includes saturated and unsaturated aliphatic groups, such as an alkyl group, an alkenyl group, and an alkynyl group.

The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively. NMGN-pWO-009 - 9-

The terms "alkoxyl" or "alkoxy" as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, -O-(CH2)_m-R8, where m and Rg are described above.

The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3-C30 for branched chains), and more preferably 20 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.

Moreover, the term "alkyl" (or "lower alkyl") as used throughout the specification, examples, and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the NMGN-pWO-009 - 10 -

like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carboiiyl-substituted alkyls, -CF3, -CN, and the like.

Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Throughout the application, preferred alkyl groups are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl. The term "alkylthio" refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In preferred embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-alkynyl, and -S-(CH2)_m-R8_> wherein m and Rg are defined above. Representative alkylthio groups include methylthio, ethylthio, and the like. The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the

general formula: wherein R9, RJQ ^an(l R-'lO ^eacrι independently represent a hydrogen, an alkyl, an alkenyl, -(CH2)_m-R8, or R9 and RJQ taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; Rg represents an aryl, a cycloalkyl, a cycloalkenyl. a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In preferred embodiments, only one of R9 or

R10 can be a carbonyl, e.g., R9, RJQ and the nitrogen together do not form an imide. In even more preferred embodiments, R9 and RJQ (^anι^ optionally R'10) each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH2)_m-R8- Thus, the term "alkylamine" as used herein means an amine group, as defined above, having a NMGN-pWO-009 - 11 -

substituted or unsubstituted alkyl attached thereto, i.e., at least one of R9 and R^Q is an alkyl group.

The term "amido" is art-recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula:

wherein R9, RJO ^{are s} defined above. Preferred embodiments of the amide will not include imides which may be unstable.

The term "aralkyl", as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).

The term "aryl" as used herein includes 5-, 6-, and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics." The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN, or the like. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The term "carbocycle", as used herein, refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon. NMGN-pWO-009 - 12 -

The term "carbonyl" is art-recognized and includes such moieties as can be represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R represents a hydrogen, an alkyl, an alkenyl, -(CH2)_m-R8 or a pharmaceutically acceptable salt, R'ι j represents a hydrogen, an alkyl, an alkenyl or -(CH2)_m-R8, where m and Rg are as defined above.

Where X is an oxygen and R \ or R'u is not hydrogen, the formula represents an

"ester". Where X is an oxygen, and Rj j is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when \ \ is a hydrogen, the formula represents a "carboxylic acid". Where X is an oxygen, and R'u is hydrogen, the formula represents a "formate". In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiocarbonyl" group. Where X is a sulfur and R ι or R'n is not hydrogen, the formula represents a "thioester." Where X is a sulfur and R \ is hydrogen, the formula represents a "thiocarboxylic acid." Where X is a sulfur and \ \' is hydrogen, the formula represents a "thiolformate." On the other hand, where X is a bond, and Rj j is not hydrogen, the above formula represents a

"ketone" group. Where X is a bond, and Rj \ is hydrogen, the above formula represents an "aldehyde" group.

The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium.

The terms "heterocyclyl" or "heterocyclic group" refer to 3- to 10-membered ring structures, more preferably 3- to 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can also be polycycles. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, NMGN-pWO-009 - 13 -

isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.

As used herein, the term "nitro" means -NO2; the term "halogen" designates -F, - Cl, -Br or -I; the term "sulfhydryl" means -SH; the term "hydroxyl" means -OH; and the term "sulfonyl" means -SO2-.

A "phosphonamidite" can be represented in the general formula:

wherein R9 and R\ Q are as defined above, Q2 represents O, S or N, and R4 represents a lower alkyl or an aryl, Q2 represents O, S or N.

A "phosphoramidite" can be represented in the general formula:

wherein R9 and R1 Q are as defined above, and Q2 represents O, S or N.

A "phosphoryl" can in general be represented by the formula:

NMGN-pWO-009 - 14-

wherein Q represented S or O, and R45 represents hydrogen, a lower alkyl or an aryl.

When used to substitute, for example, an alkyl, the phosphoryl group of the phosphorylalkyl can be represented by the general formula:

Q-i Q

-Q2 or -Q OR 4. 6

OR 4, 6 OR 4, 6 wherein Q represented S or O, and each R46 independently represents hydrogen, a lower alkyl or an aryl, Q2 represents O, S or N. When Q\ is an S, the phosphoryl moiety is a "phosphorothioate".

The terms "polycyclyl" or "polycyclic group" refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings. Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.

The phrase "protecting group" as used herein means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. The field of protecting group chemistry has been reviewed (Greene, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 2" ed.; Wiley: New York, 1991).

A "selenoalkyl" refers to an alkyl group having a substituted seleno group attached thereto. Exemplary "selenoethers" which may be substituted on the alkyl are selected from one of -Se-alkyl, -Se-alkenyl, -Se-alkynyl, and -Se-(CH2)_m-R-8_> m and Rs being defined above. NMGN-pWO-009 - 15 -

As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

The term "sulfamoyl" is art-recognized and includes a moiety that can be represented by the general formula:

in which R9 and RJQ are as defined above.

The term "sulfate" is art recognized and includes a moiety that can be represented by the general formula:

in which R41 is as defined above. NMGN-pWO-009 - 16 -

The term "sulfonamido" is art recognized and includes a moiety that can be represented by the general formula:

0₂

in which R9 and '\ \ are as defined above.

The term "sulfonate" is art-recognized and includes a moiety that can be represented by the general formula: :

in which R41 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms "sulfoxido" or "sulfinyl", as used herein, refers to a moiety that can be represented by the general formula:

in which R44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

The term "sulfonyl", as used herein, refers to a moiety that can be represented by the general formula:

R ₄ in which R44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

Analogous substitutions can be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, NMGN-pWO-009 - 17-

iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g., alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, j?-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively. The terms triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, p- toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ms, Ts represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, methanesulfonyl, and p- toluenesulfonyl respectively. A more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations. The abbreviations contained in said list, and all abbreviations utilized by organic chemists of ordinary skill in the art are hereby incorporated by reference. Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)- isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, NMGN-pWO-009 - 18 -

such as carboxyl, diastereomeric salts may be formed with an appropriate optically active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers. Contemplated equivalents of the compounds described above include compounds which otherwise correspond thereto, and which have the same general properties thereof (e.g., the ability to inhibit hedgehog signaling), wherein one or more simple variations of substituents are made which do not adversely affect the efficacy of the compound. In general, the compounds of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here.

For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Also for purposes of this invention, the term "hydrocarbon" is contemplated to include all permissible compounds having at least one hydrogen and one carbon atom. In a broad aspect, the permissible hydrocarbons include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic organic compounds which can be substituted or unsubstituted.

III. Exemplary Compounds of the Invention.

As described in further detail below, it is contemplated that the subject methods can be carried out using a variety of different small molecules which can be readily identified, for example, by such drug screening assays as described herein.

A. PKC Pathway Activators

A number of different targets exist for increasing the activity of PKC. PKC agonists may include phorbol esters, such as phorbol 12-myristate 13 -acetate (PMA), phorbol 12,13-dibutyrate (PDBu), and phorbol 12,13-didecanoate (PDD), tetradecanoyl phorbol- 13 -acetate (TPA), etc., diacyl glycerols, such as 1,2-didecanoylglycerol, 1,2- NMGN-pWO-009 - 19 -

dioctanoyl-sn-glycerol, 1 -oleoyl-2-acetyl-sn-glycerol, 1 -stearoyl-2-arachidonoyl-sn- glycerol, 1,2-dioleoylglycerol, l-stearoyl-2-linoleoyl-sn-glycerol, l-O-hexadecyl-2-O- arachidonoyl-sn-glycerol, etc., compounds provided in U.S. Patents No. 5,750,091 and 5,962,504, farnesyl thiotriazole, ingenol 3,20-dibenzoate and other ingenol esters, (-)-7- octylindolactam V and other indolactams, mezerin, N-heptyl-5-chloronaphthalene-l- sulfonamide, thymeleatoxin, and others.

Compounds which activate PLC include phenylephrine.

Compounds which activate muscarinic acetylcholine receptors include aceclidine, demecarium bromide, dexpanthenol, echothiophate iodide, isoflurophate, neostigmine bromide, neostigmine methylsulfate, physostigmine, physostigmine salicylate, physostigmine sulfate, pyridostigmine bromide, acetylcholine chloride, arecolme HBr, bethanechol chloride, carbachol, cis-dioxolane, (4-hydroxy-2-butynyl)-l- trimethylammonium m-chlorocarbonilate chloride, methacholine chloride, metoclopramide HC1, muscarine chloride, nicotine tartrate, cis-2-methyl-5- trimethylammonium methyl- 1,3 -oxathiolane iodide, oxotremorine, oxotremorine sesquifumarate, or pilocarpine HC1; agents which may enhance acetylcholine synthesis, storage or release, such as phosphatidylcholine, 4-aminopyridine, bifemelane, 3,4- diaminopyridine, choline, vesamicol, secoverine, bifemelane, tetraphenylurea, and nicotinamide; postsynaptic receptor agonists such as ethyl nipecotate and levacecarnine; and compounds such as those provided by U.S. Patent Nos. RE36,375, 5,888,999, 5,668,144, 5,403,845, etc. Methods for increasing cholinergic neurotransmission include the administration of precursors to ACh (e.g., choline, lecithin) to increase endogenous ACh stores available for release, the administration of acetylcholinesterase inhibitors (e.g., physostigmine) to prevent the synaptic degradation of ACh, the administration of cholinergic receptor agonists (i.e., arecoline, oxotremorine) to stimulate postsynaptic receptors directly and the administration of transmitter releasing agents (i.e., 4-aminopyridine) to augment the release of neuronal ACh (Davidson et al. Acta Psychiatr Scand, Suppl 366:47-57, 1991).

Agonists for metabotropic glutamate receptors include (2S,lR,2R,3R)-2- (2S,rR,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV), (2S,1'S,2'S)-

(carboxycyclopropyl)glycine, (+)-2-aminobicyclo[3.1.0]hexane-2-6-dicarboxylic acid NMGN-pWO-009 - 20 -

(LY354740), LY379268, amino-4-phosphonobutyric acid, lS,3R-aminocyclopentane- 2,3-dicarboxylic acid, trans-azetidine-2,4-dicarboxylic acid, S-4C3HPG, L-AP4, (RS)- 3,5-dihydroxyphenylglycine (DHPG), (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), (S)-(+)-2-(3'-carboxybicyclo[l.l.l]pentyl)-glycine (UPF 596), ibotenate, quisqualate, trans-l-aminocyclopentane-l,3-dicarboxylic acid, (lS,3R)-l-aminocyclopentane-l,3- dicarboxylic acid, (S)-4-carboxyphenylglycine, (lR,3S)-l-aminocyclopentane-l,3- dicarboxylic acid, L-CCG-I, and L-cystein-sulfinic acid, and compounds provided by U.S. Patent 5,981,195.

B. cAMP Pathway Activators A number of different classes of compounds increase the level or activity of cAMP, thereby activating PKA. For example, compounds which may activate adenylate cyclase include forskolin (FK), cholera toxin (CT), pertussis toxin (PT), prostaglandins (e.g., PGE-1 and PGE-2), auranofin, thyrotropin, and colforsin. β-Adrenergic receptor agonists (sometimes referred to herein as "β-adrenergic agonists"), which thereby activate adenylate cyclase, include albuterol, bambuterol, bitolterol, BRL-47672, butopamine, carbuterol, cimaterol, clenbuterol, clorprenaline, denopamine, dioxethedrine, dobutamine, dopamine, dopexamine, ephedrine, epinephrine, etafedrine, ethylnorepinephrine, fenoterol, flerobuterol, formoterol, hexoprenaline, ibopamine, isoetharine, isoprenaline (isoproterenol), mabuterol, metaproterenol, methoxyphenamine, MJ-9184-1, norepinephrine, orciprenaline, oxyfedrine, pirbuterol, prenalterol, procaterol, protokylol, quinterenol, QH-25, R-804, ractopamine, reproterol, rimiterol, ritodrine, salbutamol, salmefamol, salmeterol, soterenol, sulfonterol, tazolol, terbutaline, tetrahydropapaveroline, tretoquinol, trimetoquinol, tulobuterol, xamoterol, zinterol, erythro-DL-l-(7-methylindan-4-yloxy)- 3-isopropylaminobutan-2-ol, (R*,R*)-4-[2-({2-[(3-chlorophenyl)-2- hydroxyethyl]amino}propyl)phenyl]phenoxy-acetic acid, {2-hydroxy-5-[2-({2-hydroxy- 3-[4-(l-methyl-4-trifluoromethyl)-lH-imidazol-2-yl]phenoxy}propyl)amino]- ethoxy}benzamide monomethanesulfonate, disodium (R,R)-5-[2-[[2-(3-chlorophenyl)-2- hydroxyethyl]amino]-propyl]-l,3-benzodioxol-2,2-dicarboxylate, and erythro-DL-l-(7- methylindan-4-yloxy)-3 -isopropylaminobutan-2-ol. NMGN-pWO-009 - 21 -

Dopamine receptor agonists include SKF-38393, SKF-81297, ABT-431, C-APB, SCH-23390, SCH-39166, SKF-77434, 6-chloro-7,8-dihydroxy-3-allyl-l-phenyl-2,3,4,5- tetrahydro-lH-3-benzazepine hydrobromide (SKF-82958), SKF 82957, (+/-)-6-chloro- PB hydrobromide, SKF 82958, CY204-283, dihydrexidine (DHX), SKF-75670, A77636, A68930, SKF 82526 (fenoldopam), and compounds provided in U.S. Patent No. 5,597,832.

Compounds which may inhibit a cAMP phosphodiesterase include amrinone, milrinone, xanthine, methylxanthine, anagrelide, cilostamide, medorinone, indolidan, rolipram, 3 -isobutyl-1 -methylxanthine (IBMX), c elerytlirine, cilostazol, glucocorticoids, 8-methoxymethyl-3-isobutyl-l-methylxanthine, KW-6, 8-methylamino- 3 -isobutyl-1 -methylxanthine (MIMAX), dibenzoquinazoline diones, trequinsin, lixazinone, Y-590, imazodan, quazinone, ICI 153,110, bemorandan, siguazodan, adibendan, enoximone, simobendan, saterinone, sulmazole, RO 20-1724 (imidazoiidinone), denbufylline, zaprinast, dipyridamole,griseolic acid, aminophylline, trequensin, nothiazines, vinpocetine, HA-558,dipyridamole, etazolate, caffeine, indomethacin, theophylline, 2-o-propoxyphenyl-8-azapurin-6-one, papverine, methyl isobutylxanthine (MIX), and fenoxamine.

Analogs of cAMP which may be useful in the present method include dibutyryl- cAMP (db-cAMP), (8-(4)-chlorophenylthio)-cAMP (cpt-cAMP), 8-[(4-bromo-2,3- dioxobutyl)thio]-cAMP, 2-[(4-bromo-2,3-dioxobutyl)thio]-cAMP, 8-bromo-cAMP, dioctanoyl-cAMP, Sp-adenosine 3':5'-cyclic phosphorothioate, 8-piperidino-cAMP, N⁶- phenyl-cAMP, 8-methylamino-cAMP, 8-(6-aminohexyl)amino-cAMP, 2'-deoxy-cAMP, N⁶,2'-O-dibutryl-cAMP, Sp-adenosine-3',5'-cyclic monophosphorothioate (Sp-cAMPS), N ,2'-O-disuccinyl-cAMP, Sp-5,6-dichloro-l -b-D-ribofuranosylbenzimidazole-3',5'- monophosphorothioate, N⁶-monobutyryl-cAMP, 2'-O-monobutyryl-cAMP, 2'-O- monobutyryl-8-bromo-cAMP, N⁶-monobutyryl-2'-deoxy-cAMP, and 2'-O- monosuccinyl-cAMP.

Above-listed compounds useful in the subject methods may be modified to increase the bioavailability, activity, or other pharmacologically relevant property of the compound. For example, forskolin has the formula: NMGN-pWO-009 - 22 -

Forskolin

Modifications of forskolin which have been found to increase the hydrophilic character of forskolin without severely attenuating the desired biological activity include acylation of the hydroxyls at C6 and/or C7 (after removal of the acetyl group) with hydrophilic acyl groups. In compounds wherein C6 is acylated with a hydrophilic acyl group, C7 may optionally be deacetylated. Suitable hydrophilic acyl groups include groups having the structure -(CO)(CH₂)_nX, wherein X is OH or NR₂; R is hydrogen, a C_rC alkyl group, or two Rs taken together form a ring comprising 3-8 atoms, preferably 5-7 atoms, which may include heteroatoms (e.g., piperazine or morpholine rings); and n is an integer from 1-6, preferably from 1-4, even more preferably from 1-2. Other suitable hydrophilic acyl groups include hydrophilic amino acids or derivatives thereof, such as aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, tyrosine, etc., including amino acids having a heterocyclic side chain. Forskolin, or other compounds listed above, modified by other possible hydrophilic acyl side chains known to those of skill in the art may be readily synthesized and tested for activity in the present method.

In addition to the specific agents mentioned above, other agents that may be modulate cAMP activity include, but are not limited to: pituitary adenylyl cyclase- activating polypeptide (PACAP), gastric inhibitory peptide (GIP), peptide-YY (PYY), glucagon-like peptide (GLP-1), secretin, vasoactive intestinal peptide (NIP), parathyroid hormone-related peptide (PTHrP), corticotropin-releasing hormone/corticotropin- releasing factor (CRH/CRF), and calcitonin gene-related peptide (CGRP); the neurotransmitters serotonin, epinephrine, histamine, and vasopressin (or surrogate NMGN-pWO-009 - 23 -

agonists of the receptors for these agents that elevate intracellular cAMP). Additionally, compounds set forth in U.S. Patent No. 5,385,915 may be useful in the disclosed methods.

Similarly, variants, prodrug forms, or derivatives of any of the above-listed compounds may be effective as MAPK/MEK activators in the subject method. Those skilled in the art will readily be able to synthesize and test such derivatives for suitable activity.

In certain embodiments, the subject MAPK/MEK activators can be chosen on the basis of their selectivity for MAPK activation. In certain embodiments, it may be advantageous to administer two or more of the above cAMP agonists, preferably of different types. For example, use of an adenylate cyclase agonist in conjunction with a cAMP phosphodiesterase antagonist may have an advantageous or synergistic effect.

In certain preferred embodiments, the subject agents modulate MAPK activity with an ED50 of 1 mM or less, more preferably of 1 μM or less, and even more preferably of 1 nM or less.

C. Generation of Animal Models to Test Agents

Applicants have previously described an animal model for studying fornix- mediated memory consolidation. See, for example, Taubenfield et al., Supra. The fornix-lesioned animals can be used for drug screening, e.g., to identify appropriate combinations and dosages of the subject compositions which enhance memory consolidation. The lesioned mammal can have a lesion of the fornix or a related brain structure that disrupts memory consolidation (e.g., perirhinal cortex, amygdala, medial septal nucleus, locus coeruleus, hippocampus, mammallary bodies). Lesions in the mammal can be produced by mechanical or chemical disruption. For example, the fornix lesion can be caused by surgical ablation, electrolytic, neurotoxic and other chemical ablation techniques, or reversible inactivation such as by injection of an anesthetic, e.g., tetrodotoxin or lidocaine, to temporarily arrest activity in the fornix.

To further illustrate, fimbrio-fornix (rodents) and fornix (primates) lesions can be created by stereotatic ablation. In particular, neurons of the fornix structure are NMGN-pWO-009 - 24-

axotomized, e.g., by transection or aspiration (suction) ablation. A complete transection of the fornix disrupts adrenergic, cholinergic and GABAergic function and electrical activity, and induces morphological reorganization in the hippocampal fomiation. In general, the fornix transection utilized in the subject method will not disconnect the parahippocampal region from the neocortex. In those embodiments, the fornix transection will not disrupt functions that can be carried out by the parahippocampal region independent of processing by the hippocampal formation, and hence would not be expected to produce the full-blown amnesia seen following more complete hippocampal system damage. In one embodiment, the animal can be a rat. Briefly, the animals are anesthetized, e.g., with intraperitoneal injections of a ketamine-xylazine mixture and positioned in a Kopf stereotoxic instrument. A sagittal incision is made in the scalp and a craniotomy is performed extending 2.0 mm posterior and 3.0 mm lateral from Bregma. An aspirative device, e.g., with a 20 gauge tip, is mounted to a stereotaxic frame (Kopf Instruments) and fimbria-fornix is aspirated by placing the suction tip at the correct sterotaxic location in the animals brain. Unilateral aspirative lesions are made by suction through the cingulate cortex, completely transecting the fimbria fornix unilaterally, and (optionally) removing the dorsal tip of the hippocampus as well as the overlying cingulate cortex to inflict a partial denervation on the hippocampus target. See also, Gage et al, (1983) Brain Res. 268:27 and Gage et al. (1986) Neuroscience 19:241.

In another exemplary embodiment, the animal can be a monkey. The animal can be anesthetized, e.g., with isoflurane (1.5-2.0%). Following pretreatment with mannitol (0.25 g/kg, iv), unilateral transections of the left fornix an be performed, such as described by Kordower et al. (1990) J. Comp. NeuroL, 298:443. Briefly, a surgical drill is used to create a parasagittal bone flap which exposes the frontal superior sagittal sinus. The dura is retracted and a self-retaining retractor is used to expose the interhemispheric fissure. The corpus callosum is longitudinally incised. At the level of the foramen of Monro, the fornix is easily visualized as a discrete 2-3 mm wide white fiber bundle. The fornix can be initially transected using a ball dissector. The cut ends of the fornix can then be suctioned to ensure completeness of the lesion. NMGN-pWO-009 - 25 -

In still other illustrative embodiments, the fornix lesion can be created by excitotoxically, or by other chemical means, inhibiting or ablating fornix neurons, or the cells of the hippocampus which are innervated by fornix neurons. In certain preferred embodiments, the fornix lesion is generated by selective disruption of particular neuronal types, such as fornix cholinergic and adrenergic neurons.

For instance, the afferant fornix signals to the hippocampus due to cholmergic neurons can be ablated by atropine blockade. Another means for ablation of the cholinergic neurons is the use of 192IgG-saporin (192IgG-sap), e.g., intraventricularly injection into the fornix and hippocampus. The agents such as 6-OHDA and ibotenic acid can be used to selectively destroy fornix dopamine neurons as part of the ablative regimen.

In preferred embodiments, the animal is a non-human mammal, such as a dog, cat, horse, cow, pig, sheep, goat, chicken, monkey, ape, rat, rabbit, etc. In certain prefen^*ed embodiments, the animal is a non-human primate. In other preferred embodiment, the animal is a rodent.

There are a variety of test for cognitive function, especially learning and memory testing, which can be carried our using the lesioned and normal animals. Learning and/or memory tests include, for example, inhibitory avoidance, contextual fear conditioning, visual delay non-match to sample, spatial delay non-match to sample, visual discrimination, Barnes circular maze, Morris water maze, and radial arm maze tests.

An exemplary passive avoidance test utilizes an apparatus that consists of a lit chamber that can be separated from a dark chamber by a sliding door. At training, the animal is placed in the lit chamber for some period of time, and the door is opened. The animal moves to the dark chamber after a short delay - the latency - that is recorded. Upon entry into the dark chamber, the door is shut closed and a foot shock is delivered. Retention of the experience is determined after various time intervals, e.g., 24 or 48 hours, by repeating the test and recording the latency. The protocol is one of many variants of the passive avoidance procedures (for review, see Rush (1988) Behav Neural Biol 50:255). NMGN-pWO-009 - 26 -

An exemplary maze testing embodiment is the water maze working memory test. In general, the method utilizes an apparatus which consists of a circular water tank. The water in the tank is made cloudy by the addition of milk powder. A clear plexiglass platform, supported by a movable stand rest on the bottom of the tank, is submerged just below the water surface. Normally, a swimming rat cannot perceive the location of the platform but it may recall it from a previous experience and training, unless it suffers from some memory impairment. The time taken to locate the platform is measured and referred to as the latency. During the experiment, all orientational cues such as ceiling lights, etc., remain unchanged. Longer latencies are generally observed with rats with some impairment to their memory.

Another memory test includes the eyeblink conditioning test, which involves the administration of white noise or steady tone that precedes a mild air puff which stimulates the subject's eyeblink.

Still another memory test which can be used is fear conditioning, e.g., either "cued" and "contextual" fear conditioning. In one embodiment, a freeze monitor administers a sequence of stimuli (sounds, shock) and then records a series of latencies measuring the recovery from shock induced freezing of the animal.

Another memory test for the lesioned animals is a holeboard test, which utilizes a rotating holeboard apparatus containing (four) open holes arranged in a 4-corner configuration in the floor of the test enclosure. A mouse is trained to poke its head into a hole and retrieve a food reward from a "baited" hole which contains a reward on every trial. There is a food reward (e.g., a Froot Loop) in every exposed hole which is made inaccessible by being placed under a screen. The screen allows the odor of the reward to emanate from the hole, but does not allow access to the reinforcer. When an individual hole is baited, a reward is placed on top of the screen, where it is accessible. The entire apparatus rests on a turntable so that it may be rotated easily to eliminate reliance on proximal (e.g., olfactory) cues. A start tube is placed in the center of the apparatus. The subject is released from the tube and allowed to explore for the baited ("correct") hole.

As set out above, one use for the fornix-lesioned animals is for identifying combinations of MAPK/MEK activators, e.g., from amongst various test agents, which are able to enhance or inhibit memory consolidation. In general, the subject method NMGN-pWO-009 - 27 -

utilizes an animal which has been manipulated to create at least partial disruption of fornix-mediated signalling to the hippocampus, the disruption affecting memory consolidation and learned behavior in the animal. The animal is conditioned with a learning or memory regimen which results in learned behavior in the mammal in the absence of the fornix lesion. Combinations of potential MAPK/MEK activators are administered to the animal in order to assess their effects on memory consolidation. An increase in learned behavior, relative to the absence of the test agents, indicates that the administered combination enhances memory consolidation.

In the methods of the present invention, retention of the learned behavior can be determined, for example, after at least about 12-24 hours, 14-22 hours, 16-20 hours and or 18-19 hours after completion of the learning phase to determine whether the agents promote memory consolidation. In a particular embodiment, retention of the learned behavior can be determined 24 hours after completion of the learning phase.

As used herein, a "control mammal" can be an untreated lesion mammal (i.e., a lesion animal receiving no agents or not the same combinations to be assessed), a trained control mammal (i.e., a mammal that undergoes training to demonstrate a learned behavior without any lesion) and/or an untrained control mammal (i.e., a mammal with or without a lesion, that receives no training to demonstrate a learned behavior).

D. Pharmaceutical Preparations of MAPK/MEK Activators In another aspect, the present invention provides pharmaceutical preparations comprising the subject MAPK/MEK activators. The MAPK MEK activators for use in the subject method may be conveniently formulated for administration with a biologically acceptable, non-pyrogenic, and/or sterile medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof. The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists. As used herein, "biologically acceptable medium" includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation. The use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the activity of NMGN-pWO-009 - 28 -

the MAPK MEK activators, its use in the pharmaceutical preparation of the invention is contemplated. Suitable vehicles and their formulation inclusive of other proteins are described, for example, in the book Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable "deposit formulations".

Pharmaceutical formulations of the present invention can also include veterinary compositions, e.g., pharmaceutical preparations of the MAPK/MEK activators suitable for veterinary uses, e.g., for the treatment of live stock or domestic animals, e.g., dogs.

Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinacious biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a MAPK/MEK activator at a particular target site. The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, controlled release patch, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral and topical administrations are preferred.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous NMGN-pWO-009 - 29-

system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms such as described below or by other conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular MAPK/MEK activators employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. NMGN-pWO-009 - 30 -

In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous, intracerebroventricular and subcutaneous doses of the compounds of this invention for a patient will range from about 0.0001 to about 100 mg per kilogram of body weight per day.

If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. The term "treatment" is intended to encompass also prophylaxis, therapy and cure.

The patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general. The compound of the invention can be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with other antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Conjunctive therapy, thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered.

While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation

(composition). The MAPK/MEK activators according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine.

Thus, another aspect of the present invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more of the compounds described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially NMGN-pWO-009 - 31 -

formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; or (4) intravaginally or intrarectally, for example, as a pessary, cream or foam. However, in certain embodiments the subject compounds may be simply dissolved or suspended in sterile water. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject regulators from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as socium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

As set out above, certain embodiments of the present MAPK/MEK activators may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts" in this respect, refers to the NMGN-pWO-009 - 32 -

relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J Pharm. Set 66:1-19)

The pharmaceutically acceptable salts of the subject compounds include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like. In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include NMGN-pWO-009 - 33 -

ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, for example, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.

Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. NMGN-pWO-009 - 34-

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste. In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be NMGN-pWO-009 - 35 -

made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. NMGN-pWO-009 - 36-

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof. It is known that sterols, such as cholesterol, will form complexes with cyclodextrins. Thus, in preferred embodiments, where the inhibitor is a steroidal alkaloid, it may be formulated with cyclodextrins, such as α-, β- and γ-cyclodextrin, dimethyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin.

Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active MAPK/MEK activator.

Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain NMGN-pWO-009 - 37-

customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the MAPK/MEK activators in the proper medium. Absorption enhancers can also be used to increase the flux of the MAPK/MEK activators across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable NMGN-pWO-009 - 38 -

pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drag form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.

When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

The addition of the active compound of the invention to animal feed is preferably accomplished by preparing an appropriate feed premix containing the active compound in an effective amount and incorporating the premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containing the active ingredient can be blended into the feed. The way in which such feed premixes and complete rations can be prepared and administered are described in reference books

(such as "Applied Animal Nutrition", W.H. Freedman and CO., San Francisco, U.S.A.,

1969 or "Livestock Feeds and Feeding" O and B books, Corvallis, Ore., U.S.A., 1977).

VI. Synthetic Schemes and Identification of Active Regulators NMGN-pWO-009 - 39 -

The subject MAPK/MEK activators, and derivatives thereof, can be prepared readily by employing known synthetic methodology. As is well known in the art, these coupling reactions are carried out under relatively mild conditions and tolerate a wide range of "spectator" functionality. Additional compounds may be synthesized and tested in a combinatorial fashion, to facilitate the identification of additional MAPK/MEK activators which may be employed in the subject method. a. Combinatorial Libraries

The compounds of the present invention, particularly libraries of variants having various representative classes of substituents, are amenable to combinatorial chemistry and other parallel synthesis schemes (see, for example, PCT WO 94/08051). The result is that large libraries of related compounds, e.g. a variegated library of compounds represented above, can be screened rapidly in high throughput assays in order to identify potential MAPK/MEK activator lead compounds, as well as to refine the specificity, toxicity, and/or cytotoxic-kinetic profile of a lead compound. For instance, PKA, PKC, cAMP, and other bioactivity assays, such as may be developed using cells e.g., transfected with a suitable reporter construct, as described below, can be used to screen a library of the subject MAPK/MEK activators for those having agonist activity toward PKC, PKA, or downstream or upstream regulators or effectors thereof.

Simply for illustration, a combinatorial library for the purposes of the present invention is a mixture of chemically related compounds which may be screened together for a desired property. The preparation of many related compounds in a single reaction greatly reduces and simplifies the number of screening processes which need to be carried out. Screening for the appropriate physical properties can be done by conventional methods. Diversity in the library can be created at a variety of different levels. For instance, the substrate aryl groups used in the combinatorial reactions can be diverse in terms of the core aryl moiety, e.g., a variegation in terms of the ring structure, and/or can be varied with respect to the other substituents.

A variety of techniques are available in the art for generating combinatorial libraries of small organic molecules such as the subject MAPK/MEK activators. See, for example, Blondelle et al. (1995) Trends Anal. Chem. 14:83; the Affymax U.S. NMGN-pWO-009 - 40-

Patents 5,359,115 and 5,362,899: the Ellman U.S. Patent 5,288,514: the Still et al. PCT publication WO 94/08051; the ArQule U.S. Patents 5,736,412 and 5,712,171; Chen et al. (1994) JACS 116:2661: Kerr et al. (1993) JACS 115:252; PCT publications WO92/10092, WO93/09668 and WO91/07087; and the Lerner et al. PCT publication WO93/20242). Accordingly, a variety of libraries on the order of about 100 to 1 ,000,000 or more diversomers of the subject MAPK/MEK activators can be synthesized and screened for particular activity or property.

In an exemplary embodiment, a library of candidate MAPK/MEK activator diversomers can be synthesized utilizing a scheme adapted to the techniques described in the Still et al. PCT publication WO 94/08051 , e.g., being linked to a polymer bead by a hydrolyzable or photolyzable group, optionally located at one of the positions of the candidate regulators or a substituent of a synthetic intermediate. According to the Still et al. technique, the library is synthesized on a set of beads, each bead including a set of tags identifying the particular diversomer on that bead. The bead library can then be "plated" with cells for which a MAPK/MEK activator is sought. The diversomers can be released from the bead, e.g., by hydrolysis.

Many variations on the above and related pathways permit the synthesis of widely diverse libraries of compounds which may be tested as MAPK/MEK activators and regulators of CREB phosphorylation. b. Screening Assays

Another aspect of the present invention relates to method for identifying agents which, by modulating the function of certain genes and/or gene products, can be used to modify long-term memory consolidation in animals. As described in further detail below, test agents can be assessed in a cell-based or cell-free assay for ability to inhibit or potentiate MAPK activity, or the activity of any component of a MAPK-activating pathway. As is well known in the art, suitable genes can range from cell surface receptors and secreted proteins to transcription factors. Accordingly, the invention contemplates such drug-screening formats which detect compounds that, for example, modulate an enzymatic activity, a half-life, or a cellular localization of a component of a cAMP or PKC pathway, or the interaction of such a component with other proteins, nucleic acids, carbohydrates or other biological molecules, etc. Any of a variety of NMGN-pWO-009 - 41 -

assay formats may be employed and, in light of the present disclosure, will be comprehended by a skilled artisan.

. Monitoring the influence of compounds on cells may be applied not only in basic drug screening, but also in clinical trials. In such clinical trials, the expression of a panel of genes may be used as a "read-out" of a particular drug's therapeutic effect.

(i) Cell-Free Assays

In many drug-screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays performed in cell-free systems, such as may be derived with purified or semi-purified proteins, are often preferred as "primary" screens in that they permit rapid development and relatively easy detection of an alteration in a molecular target mediated by a test compound. Moreover, effects of cellular toxicity and/or bioavailability of the test compound can generally be ignored in an in vitro assay, which is focused primarily on the effect of the drug on the molecular target as may be indicated by an alteration of binding affinity with upstream or downstream elements or with intrinsic enzymatic activity. Many of the proteins, enzymes, and receptors identified in cAMP and PKC signal pathways will be amenable to some form of cell-free assay formats. Soluble proteins, be they cytoplasmic or extracellular, can be recombinantly expressed and at least partially purified, or provided as lysates, for use in cell-free assays. Membrane-associated proteins can, in certain instances, be purified in detergent or liposomes, or isolated as part of a cell membrane fraction or organelle preparation.

Accordingly, in an exemplary screening assay of the present invention, a reaction mixture is generated including a MAPK-regulator and one or more proteins (or nucleic acids) which interact with the MAPK-regulator, such molecules being referred to herein as 'interactors'. Examples of interactors include proteins that function upstream (including both activators and repressors of the MAPK-regulator), and proteins or nucleic acids which function downstream of the MAPK-regulator, whether they are positively or negatively regulated by it. The reaction mixture also includes one or more test compounds. Detection and quantification of complexes of the MAPK-regulator with upstream or downstream interactor provide a means for determining a compound's NMGN-pWO-009 - 42-

efficacy at inhibiting or potentiating complex formation between the MAPK-regulator and the interactors. The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. In one control assay, isolated and purified MAPK-regulator is added to a composition containing the interactor, and the formation of a complex is quantitated in the absence of the test compound.

Complex formation between the MAPK-regulator and a binding partner may be detected by a variety of techniques. Modulation of the formation of complexes can be quantitated using, for example: detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled proteins; by immunoassay; or by chromatographic detection.

Typically, it will be desirable to immobilize either the MAPK-regulator or its interacting partner to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of the MAPK-regulator to an upstream or downstream element, in the presence and absence of a candidate agent, can be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase/ MAPK- regulator (GST/MR) fusion proteins can be adsorbed onto glutathio'ne sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione-derivatized microtitre plates, which are then combined with a cell lysate or other preparation including the interactor and the test compound. The mixture may then be incubated under conditions conducive to complex formation (in the absence of the test compound), e.g., at physiological conditions for salt and pH, though slightly more stringent conditions may be desired. Following incubation, the beads are washed to remove any unbound interactor, and the matrix immobilized and the amount of interactor in the matrix determined, or in the supernatant after the complexes are subsequently dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of interactor found in the bead fraction quantitated from the gel using standard electrophoretic techniques. NMGN-pWO-009 - 43 -

Other techniques for immobilizing proteins or nucleic acids on matrices are also available for use in the subject assay. For instance, either the MAPK-regulator or its cognate binding partner can be immobilized utilizing conjugation of biotin and streptavidin. For instance, biotinylated MAPK-regulators can be prepared from biotin- NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96-well plates (Pierce Chemical). Alternatively, antibodies reactive with the MAPK-regulator, but which do not interfere with binding of upstream or downstream binding partners, can be derivatized to the wells of the plate, and the MAPK-regulator trapped in the wells by antibody conjugation. As above, preparations of an interactor and a test compound are incubated in the MAPK-regulator-presenting wells of the plate, and the amount of complex trapped in the well can be quantitated. Exemplary methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the MAPK-regulator binding partner, or which are reactive with the MAPK-regulator and compete with the binding partner; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the binding partner, either intrinsic or extrinsic activity. In the instance of the latter, the enzyme can be chemically conjugated or provided as a fusion protein with an interactor. To illustrate, the interactor can be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of polypeptide trapped in the complex can be assessed with a chromogenic substrate of the enzyme, e.g., 3,3'-diamino-benzadine tetrahydrochloride or 4-chloro-l-naphthol. Likewise, a fusion protein comprising the polypeptide and glutathione-S-transferase can be provided, and complex formation quantitated by detecting the GST activity using l-chloro-2,4-dinitrobenzene (Habig et al (1974) J. Biol. Chem. 249:7130).

For processes which rely on immunodetection for quantitating one of the proteins trapped in the complex, antibodies against the protein, such as anti-MAPK- regulator antibodies, can be used. Alternatively, the protein to be detected in the complex can be "epitope tagged" in the form of a fusion protein which includes a second polypeptide sequence for which antibodies are readily available (e.g., from commercial sources). For instance, the GST fusion proteins described above can also be used for NMGN-pWO-009 - 44-

quantification of binding using antibodies against the GST moiety. Other useful epitope tags include mycepitopes (e.g., see Ellison et al. (1991) J. Biol. Chem. 266:21150- 21157) which includes a 10-residue sequence from c-myc, as well as the pFLAG system (International Biotechnologies, Inc.) or the pEZZ-protein A system (Pharmacia, N.J.). Other cell-free embodiments include assays which detect an intrinsic activity of a

MAPK-regulator or a complex including a MAPK-regulator, and identify compounds that increase or inhibit that activity. For instance, the reaction mixture can be generated to include the MAPK-regulator, a substrate for an enzymatic activity of the MAPK- regulator, and the test agent. The rate of conversion of the substrate to product is determined, and can be compared to such control samples as the MAPK-regulators and substrate admixed alone. Test agents which are inhibitors of the MAPK-regulator activity will decrease the rate of conversion of the substrate to product, whereas test agents that increase that rate are likely to be agonists of the MAPK-regulator activity.

In preferred embodiments, the substrate is readily detectable, e.g., the conversion of substrate to product produces a colorimetric or fluorometric change in the reaction mixture which is detectable by spectroscopic means, or creates or destroys an epitope which is detectable by immunoassay.

(ii) Cell-Based Assays

In addition to cell-free assays, such as described above, the known MAPK- regulators may also be used in cell-based assays for identifying small molecule agonists/antagonists and the like. The ability of a test agent to alter the activity of a MAPK-regulator in the cell may include directly detecting the formation of complexes including the MAPK-regulator, detecting an intrinsic enzymatic activity of the MAPK- regulator, directly detecting a change in cellular localization of the MAPK-regulator, detecting a post-translational modification to the MAPK-regulator or a change in the stability of the MAPK-regulator, or detecting the downstream consequence of any one of such events.

Such assays can be simple binding assays. For instance, where the MAPK- regulator is a receptor, the assay can be used to identify compounds which bind to the receptor or effect the ability of the receptor to bind its ligand. In other embodiments, cells which are phenotypically sensitive to the presence or activity of the MAPK- NMGN-pWO-(J09 - 45 -

regulator, e.g., it produces a morphological change in the cell, over- or under-expresses a recombinant MAPK-regulator in the presence and absence of a test agent of interest, with the assay scoring for modulation in MAPK-regulator responses by the target cell which mediated by the test agent. As with the cell-free assays, agents which produce a statistically significant change in MAPK-regulator-dependent responses (either inhibition or potentiation) can be identified. For example, the level of expression of genes or gene products which are up- or down-regulated in response to the presence or activity of an MAPK-regulator can be detected. In preferred embodiments, the regulatory regions of such genes, e.g., the 5' flanking promoter and enhancer regions, are operably linked to a detectable marker (such as luciferase) which encodes a gene product that can be readily detected.

In the event that the MAPK-regulator themselves, or in complexes with other proteins, are capable of binding DNA and modifying transcription of a gene, a transcriptional based assay could be used, for example, in which an MAPK-regulator- responsive regulatory sequence is operably linked to a detectable marker gene.

In yet another aspect of the invention, the subject drug screening assays can utilized the MAPK-regulator to generate a "two-hybrid" assay (see, for example, U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and PCT Publication No. WO 94/10300). Briefly, the two-hybrid assay relies on reconstituting in vivo a functional transcriptional activator protein from two separate fusion proteins. In particular, the method makes use of chimeric genes which express hybrid proteins. To illustrate, a first chimeric gene can be generated with the coding sequence for a DNA-binding domain of a transcriptional activator fused in frame to the coding sequence for a MAPK-regulator. The second hybrid protein encodes a transcriptional activation domain fused in frame to another polypeptide, e.g., an interactor, which binds to the MAPK-regulator. If the two fusion proteins are able to interact, e.g., form a MAPK-regulator-dependent complex, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription of a reporter gene which is operably linked to a transcriptional regulatory site which is bound by the DNA-binding domain of the NMGN-pWO-009 - 46 -

first fusion proteins, and expression of the reporter gene can be detected and used to score for the interaction of the MAPK-regulator and sample proteins. a. Host Cells

Suitable host cells for generating the subject assay include prokaryotes, yeast, or higher eukaryotic cells, especially mammalian cells. Prokaryotes include gram-negative or gram-positive organisms. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman (1981) Cell 23:175) CV-1 cells (ATCC CCL 70), L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines. It will be understood that to achieve selection or screening, the host cell must have an appropriate phenotype.

If yeast cells are used, the yeast may be of any species which are cultivable and in which an exogenous receptor can be made to engage the appropriate signal transduction machinery of the host cell. Suitable species include Schizosaccharomyces pombe, Kluyverei lactis, and Ustilaqo maydis; Saccharomyces cerevisiae is preferred. Other yeast which can be used in practicing the present invention are Neurospora crassa, Aspergillus niger, Aspergillus nidulans, Pichia pastoris, Candida tropicalis, and Hansenula polymorpha. The term "yeast", as used herein, includes not only yeast in a strictly taxonomic sense, i.e., unicellular organisms, but also yeast-like multicellular fungi or filamentous fungi. The choice of appropriate host cell will also be influenced by the choice of detection signal. For instance, reporter constructs, as described below, can provide a selectable or screenable trait upon transcriptional activation (or inactivation) in response to a signal transduction pathway coupled to LTM protein of interest. The reporter gene may be an unmodified gene already in the host cell pathway, such as the genes responsible for growth arrest in yeast. It may be a host cell gene that has been operably linked to a "receptor-responsive" promoter. Alternatively, it may be a heterologous gene (e.g., a "reporter gene construct") that has been so linked. Suitable genes and promoters are discussed below. In other embodiments, second messenger generation can be measured directly in the detection step, such as mobilization of intracellular calcium or phospholipid metabolism are quantitated. In yet other embodiments indicator genes can be used to detect receptor-mediated signaling. NMGN-pWO-009 - 47 -

b. Screening and Selection Assays: Second Messenger Generation

When screening for bioactivity of test compounds, intracellular second messenger generation can be measured directly. A variety of intracellular effectors have been identified as being receptor- or ion channel-regulated, including adenylyl cyclase, cyclic AMP, phosphodiesterases, and phospholipase C, as well as a variety of ions.

When receptors that modulate cAMP are tested, it will be possible to use standard techniques for cAMP detection, such as competitive assays which quantitate

[3H]cAMP in the presence of unlabelled cAMP.

Certain receptors and ion channels stimulate the activity of phospholipase C which stimulates the breakdown of phosphatidylinositol 4,5-bisphosphate to 1,4,5-IP3 (which mobilizes intracellular Ca++) and diacylglycerol (DAG) (which activates protein kinase C). Inositol lipids can be extracted and analyzed using standard lipid extraction techniques. DAG can also be measured using thin-layer chromatography. Water- soluble derivatives of all three inositol lipids (IP1, IP2, IP3) can also be quantitated using radiolabelling techniques or HPLC.

The other product of PIP2 breakdown, DAG can also be produced from phosphatidyl choline. The breakdown of this phospholipid in response to receptor- mediated signaling can also be measured using a variety of radiolabelling techniques.

In the case of certain receptors and ion channels, it may be desirable to screen for changes in cellular phosphorylation. Such assay formats may be useful when the receptor of interest is a receptor kinase or phosphatase. For example, immunoblotting (Lyons and Nelson (1984) Proc. Natl. Acad. Sci. USA 81:7426-7430) using anti- phosphotyrosine, anti-phosphoserine or anti-phosphothreonine antibodies. In addition, tests for phosphorylation could be also useful when the receptor itself may not be a kinase, but activates protein kinases or phosphatase that function downstream in the signal transduction pathway.

The MAP kinase pathway is such a pathway, and that appears to mediate both mitogenic, differentiation and stress responses in different cell types. Stimulation of growth factor receptors results in Ras activation followed by the sequential activation of c-Raf, MEK, and p44 and p42 MAP kinases (ERKl and ERK2). Activated MAP kinase

then phosphorylates many key regulatory proteins, including p90RSK and Elk-1 that are phosphorylated when MAP kinase translocates to the nucleus. Homologous pathways exist in mammalian and yeast cells. For instance, an essential part of the S. cerevisiae pheromone signaling pathway is comprised of a protein kinase cascade composed of the products of the STEl l, STE7, and FUS3/KSS1 genes (the latter pair are distinct and functionally redundant). Accordingly, phosphorylation and/or activation of members of this kinase cascade can be detected and used to quantitate receptor engagement. Phosphotyrosine specific antibodies are available to measure increases in tyrosine phosphorylation and phospho-specific antibodies are commercially available (New England Biolabs, Beverly, MA).

In yet another embodiment, the signal transduction pathway of the targeted receptor or ion channel upregulates expression or otherwise activates an enzyme which is capable of cleaving a substrate which can be added to the cell. The signal can be detected by using a detectable substrate, in which case loss of the substrate signal is monitored, or alternatively, by using a substrate which produces a detectable product. In preferred embodiments, the conversion of the substrate to product by the activated enzyme produces a detectable change in optical characteristics of the test cell, e.g., the substrate and/or product is chromogenically or fluorogenically active. In an illustrative embodiment the signal transduction pathway causes a change in the activity of a proteolytic enzyme, altering the rate at which it cleaves a substrate peptide (or simply activates the enzyme towards the substrate). The peptide includes a fluorogenic donor radical, e.g., a fluorescence emitting radical, and an acceptor radical, e.g., an aromatic radical which absorbs the fluorescence energy of the fluorogenic donor radical when the acceptor radical and the fluorogenic donor radical are covalently held in close proximity. See, for example, USSN 5,527,681, 5,506,115, 5,429,766, 5,424,186, and 5,316,691; and Capobianco et al. (1992) Anal Biochem 204:96-102. For example, the substrate peptide has a fluorescence donor group such as 1-aminobenzoic acid (anthranilic acid or ABZ) or aminomethylcoumarin (AMC) located at one position on the peptide and a fluorescence quencher group, such as lucifer yellow, methyl red or nitrobenzo-2-oxo- 1,3-diazole (NBD), at a different position near the distal end of the peptide. A cleavage site for the activated enzyme will be disposed between each of the sites for the donor and acceptor groups. The intramolecular resonance energy transfer from the NMGN-pWO-0^~09 - 49 -

fluorescence donor molecule to the quencher will quench the fluorescence of the donor molecule when the two are sufficiently proximate in space, e.g., when the peptide is intact. Upon cleavage of the peptide, however, the quencher is separated from the donor group, leaving behind a fluorescent fragment. Thus, activation of the enzyme results in cleavage of the detection peptide, and dequenching of the fluorescent group.

In still other embodiments, the detectable signal can be produced by use of enzymes or chromogenic/fluorescent probes whose activities are dependent on the concentration of a second messenger, e.g., calcium, hydrolysis products of inositol phosphate, cAMP, etc. For example, the mobilization of intracellular calcium or the influx of calcium from outside the cell can be measured using standard techniques. The choice of the appropriate calcium indicator, fluorescent, bioluminescent, metallochromic, or Ca++-sensitive microelectrodes depends on the cell type and the magnitude and time constant of the event under study (Borle (1990) Environ Health Perspect 84:45-56). As an exemplary method of Ca++ detection, cells could be loaded with the Ca++sensitive fluorescent dye fura-2 or indo-1, using standard methods, and any change in Ca++ measured using a fluorometer.

As certain embodiments described above suggest, in addition to directly measuring second messenger production, the signal transduction activity of a receptor or ion channel pathway can be measured by detection of a transcription product, e.g., by detecting receptor/channel-mediated transcriptional activation (or repression) of a gene(s). Detection of the transcription product includes detecting the gene transcript, detecting the product directly (e.g., by immunoassay) or detecting an activity of the protein (e.g., such as an enzymatic activity or chromogenic/fluorogenic activity); each of which is generally referred to herein as a means for detecting expression of the indicator gene. The indicator gene may be an unmodified endogenous gene of the host cell, a modified endogenous gene, or a part of a completely heterologous construct, e.g., as part of a reporter gene construct.

In one embodiment, the indicator gene is an unmodified endogenous gene. In certain instances, it may be desirable to increase the level of transcriptional activation of the endogenous indicator gene by the signal pathway in order to, for example, improve the signal-to-noise ratio of the test system, or to adjust the level of response to a level NMGN-pWO-009 - 50-

suitable for a particular detection technique. In one embodiment, the transcriptional activation ability of the signal pathway can be amplified by the overexpression of one or more of the proteins involved in the intracellular signal cascade, particularly enzymes involved in the pathway. For example, increased expression of Jun kinases (JNKs) can potentiate the level of transcriptional activation by a signal in an MEK/MEKK pathway. This approach can, of course, also be used to potentiate the level of transcription of a heterologous reporter gene as well.

In other embodiments, the sensitivity of an endogenous indicator gene can be enhanced by manipulating the promoter sequence at the natural locus for the indicator gene. Such manipulation may range from point mutations to the endogenous regulatory elements to gross replacement of all or substantial portions of the regulatory elements. In general, manipulation of the genomic sequence for the indicator gene can be carried out using techniques known in the art, including homologous recombination.

In another exemplary embodiment, the promoter (or other transcriptional regulatory sequences) of the endogenous gene can be "switched out" with a heterologous promoter sequence, e.g., to form a chimeric gene at the indicator gene locus. Again, using such techniques as homologous recombination, the regulatory sequence can be so altered at the genomic locus of the indicator gene.

In still another embodiment, a heterologous reporter gene construct can be used to provide the function of an indicator gene. Reporter gene constructs are prepared by operatively linking a reporter gene with at least one transcriptional regulatory element. If only one transcriptional regulatory element is included it must be a regulatable promoter, At least one the selected transcriptional regulatory elements must be indirectly or directly regulated by the activity of the selected cell-surface receptor whereby activity of the receptor can be monitored via transcription of the reporter genes.

Many reporter genes and transcriptional regulatory elements are known to those of skill in the art and others may be identified or synthesized by methods known to those of skill in the art. h. Exemplary Screening and Selection Assays: Reporter Genes Examples of reporter genes include, but are not limited to CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase, and NMGN-pWO-009 - 51 -

other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwin et al. (1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J. Mol. Appl. Gen. 2: 101), human placental secreted alkaline phosphatase (Cullen and Malim (1992) Methods in Enzymol. 216:362-368).

Transcriptional control elements for use in the reporter gene constructs, or for modifying the genomic locus of an indicator gene include, but are not limited to, promoters, enhancers, and repressor and activator binding sites. Suitable transcriptional regulatory elements may be derived from the transcriptional regulatory regions of genes whose expression is rapidly induced, generally within minutes, of contact between the cell surface protein and the effector protein that modulates the activity of the cell surface protein. Examples of such genes include, but are not limited to, the immediate early genes (see, Sheng et al. (1990) Neuron 4: 477-485), such as c-fos. Immediate early genes are genes that are rapidly induced upon binding of a ligand to a cell surface protein. The transcriptional control elements that are preferred for use in the gene constructs include transcriptional control elements from immediate early genes, elements derived from other genes that exhibit some or all of the characteristics of the immediate early genes, or synthetic elements that are constructed such that genes in operative linkage therewith exhibit such characteristics. The characteristics of preferred genes from which the transcriptional control elements are derived include, but are not limited to, low or undetectable expression in quiescent cells, rapid induction at the transcriptional level within minutes of extracellular simulation, induction that is transient and independent of new protein synthesis, subsequent shut-off of transcription requires new protein synthesis, and mRNAs transcribed from these genes have a short half-life. It is not necessary for all of these properties to be present.

Other promoters and transcriptional control elements, in addition to those described above, include the vasoactive intestinal peptide (VIP) gene promoter (cAMP responsive; Fink et al. (1988), Proc. Natl. Acad. Sci. 85:6662-6666); the somatostatin gene promoter (cAMP responsive; Montminy et al. (1986), Proc. Natl. Acad. Sci. 8.3:6682-6686); the proenkephalin promoter (responsive to cAMP, nicotinic agonists, and phorbol esters; Comb et al. (1986), Nature 323:353-356); the phosphoenolpyruvate NMGN-pWO-009 - 52 -

carboxy-kinase gene promoter (cAMP responsive; Short et al. (1986), J. Biol. Chem. 261:9721-9726); the NGFI-A gene promoter (responsive to NGF, cAMP, and serum; Changelian et al. (1989). Proc. Natl. Acad. Sci. 86:377-381); and others that may be known to or prepared by those of skill in the art. In the case of receptors which modulate cyclic AMP, a transcriptional based readout can be constructed using the cAMP response element binding protein, CREB, which is a transcription factor whose activity is regulated by phosphorylation at a particular serine (SI 33). When this serine residue is phosphorylated, CREB binds to a recognition sequence Icnown as a CRE (cAMP Responsive Element) found to the 5' of promotors Icnown to be responsive to elevated cAMP levels. Upon binding of phosphorylated CREB to a CRE, transcription from this promoter is increased.

Phosphorylation of CREB is seen in response to both increased cAMP levels and increased intracellular Ca levels. Increased cAMP levels result in activation of PKA, which in turn phosphorylates CREB and leads to binding to CRE and transcriptional activation. Increased intracellular calcium levels results in activation of calcium/calmodulin responsive kinase IV (CaM kinase IV). Phosphorylation of CREB by CaM kinase IV is effectively the same as phosphorylation of CREB by PKA, and results in transcriptional activation of CRE containing promotors.

Therefore, a transcriptional-based readout can be constructed in cells containing a reporter gene whose expression is driven by a basal promoter containing one or more CRE. Changes in the intracellular concentration of Ca⁺⁺ (a result of alterations in the activity of the receptor upon engagement with a ligand) will result in changes in the level of expression of the reporter gene if: a) CREB is also co-expressed in the cell, and b) either the endogenous yeast CaM kinase will phosphorylate CREB in response to increases in calcium or if an exogenously expressed CaM kinase IV is present in the same cell. In other words, stimulation of PLC activity will result in phosphorylation of CREB and increased transcription from the CRE-construct, while inhibition of PLC activity will result in decreased transcription from the CRE-responsive construct.

In preferred embodiments, the reporter gene is a gene whose expression causes a phenotypic change which is screenable or selectable. If the change is selectable, the phenotypic change creates a difference in the growth or survival rate between cells NMGN-pWO-009 - 53 -

which express the reporter gene and those which do not. If the change is screenable, the phenotype change creates a difference in some detectable characteristic of the cells, by which the cells which express the marker may be distinguished from those which do not. Selection is preferable to screening in that it can provide a means for amplifying from the cell culture those cells which express a test polypeptide which is a receptor effector.

The marker gene is coupled to the receptor signaling pathway so that expression of the marker gene is dependent on activation of the receptor. This coupling may be achieved by operably linking the marker gene to a receptor-responsive promoter. The term "receptor-responsive promoter" indicates a promoter which is regulated by some product of the target receptor's signal transduction pathway.

Alternatively, the promoter may be one which is repressed by the receptor pathway, thereby preventing expression of a product which is deleterious to the cell. With a receptor repressed promoter, one screens for agonists by linking the promoter to a deleterious gene, and for antagonists, by linking it to a beneficial gene. Repression may be achieved by operably linking a receptor- induced promoter to a gene encoding mRNA which is antisense to at least a portion of the mRNA encoded by the marker gene (whether in the coding or flanking regions), so as to inhibit translation of that mRNA. Repression may also be obtained by linking a receptor-induced promoter to a gene encoding a DNA binding repressor protein, and incorporating a suitable operator site into the promoter or other suitable region of the marker gene.

The following exemplary assays are intended to be instructive as to the types of assays which may be employed for identifying MAPK-regulators for use in the therapeutic methods disclosed herein. The following assays, as well as variants and modifications thereof, e.g., for assaying the activity of another receptor or kinase, may be employed for the purposes of identifying such compounds.

Exemplary Assays

PKC

As described in U.S. Patent No. 5,962,504, one can measure the ability of a given compound, such as a phorbol ester or analog thereof, to stimulate the protein kinase C-mediated incorporation of radioactive phosphate from P-adenosine triphosphate into a standard acceptor substrate such as histone HI. Tests of this nature NMGN-pWO-009 - 54 -

reveal a difference in potency between given agonists of as much as 10,000,000-fold or more [Dunn and Blumberg, Cancer Res. 43 4632-4637 (1983), Table 1].

As described in U.S. Patent No. 5,776,685, cells which are engineered to express a PKC acquire a tumor promoter-sensitive, PKC-related phenotype. The tumor promoter-sensitive phenotype, and in particular, a tumor promoter-induced alteration in the growth rate of the cells which express PKC, allow the cells to be used to in a relatively rapid, convenient, and inexpensive assay for evaluating the tumor promoter agonist or antagonist activity, or the PKC activator or inhibitor activity, of a compound e.g., a cosmetic or pharmaceutical product. An assay as described in U.S. Patent No. 5,759,787 may be used for PKC or any of the other kinases involved in MAPK-regulation. That patent provides efficient and sensitive methods and compositions for detecting, identifying and/or characterizing kinase activity and specific modulators of kinase activity, preferably protein kinase activity. The methods use a bifunctional kinase reaction product to 1) specifically capture and immobilize phosphorylated product as opposed to unreacted substrate and to 2) specifically detect the immobilized product as opposed to other immobilized molecules. One functionality of the product is provided by the phosphorylation reaction itself: the reaction introduces a novel molecular structural feature, or epitope, within the substrate, for which feature a specifically binding receptor is available. Conveniently, this feature comprises the phosphate group itself; for example, a phosphorylated serine or tyrosine residue of a peptide substrate. Alternatively, phosphorylation (or dephosphorylation, as the reaction can generally be run in reverse) of the substrate may induce a specifically-detectable conformational change which does not necessarily comprise the phosphate group. The assay may use any phosphorylation-dependent feature for which a specifically binding receptor can be obtained. Specific immune receptor, such as an antibody provide convenient such receptors.

PKA

For measuring effects of test compounds on PKA activity, one of the above assays may be suitable modified, or an assay such as one of those described in U.S. Patent No. 5,759,787; Casnellie, J. E. (1991) in Protein Phosphorylation, Part A.

Methods in Enzymology vol. 200 (Hunter, T., Sefton, B. M., eds) pp. 115-120, NMGN-pWO-009 - 55 -

Academic Press, San Diego; Kemp, B. E., and Pearson, R. B. (1991) in Protein Phosphorylation, Part A. Methods in Enzymology vol. 200 (Hunter, T., Sefton, B. M., eds) pp. 121-134, Academic Press, San Diego; Isbell, J. C, Christian, S. T., Mashburn, N. A., and Bell, P. D. (1995) Life Sci 57, 1701-1707; Erdbragger, W., Strohm, P., and Michel, M. C. (1995) Cell Signal 7, 635-642; Lutz, M. P., Pinon, D. I, and Miller, L. J. (1994) Anal. Biochem. 220, 268-274; Mcllroy, B. K., Walters, J. D., and Johnson, J. D. (1991) Anal. Biochem. 195, 148-152; Wang, Z. X., Cheng, Q., and Kililea, S. D. (1995) Anal. Biochem. 230, 55-61; or Zhao, Z. H., et al. (1991) Biochem. Biophys. Res. Comm. 176, 1454-1461 may be used to assay the kinase activity of any component of a MAPK-regulating pathway.

Dopamine Receptors

U.S. Patent No. 5,882,855 describes an assay for measuring activation of a human dopamine Dl receptor. This assay may be modified by for any desired dopamine receptor by methods well known to those of skill in the art, e.g., by using cells transfected with a nucleic acid encoding a dopamine receptor of interest. mAChR

Standard binding assays, e.g., immunoprecipitations and yeast two-hybrid assays as described herein, can be performed to determine the ability of an mAChR polypeptide or a biologically active portion thereof to interact with (e.g., bind to) a binding partner, such as a G protein or phospholipase C. To determine whether an mAChR polypeptide or a biologically active portion thereof can modulate an acetylcholine response in an acetylcholine responsive cell, such cells can be transfected with a construct driving the overexpression of an mAChR polypeptide or a biologically active portion thereof. Methods for the preparation of acetylcholine responsive cells, e.g., intact smooth muscle cells or extracts from such cells are known in the art and described in Glukhova et al. (1987) Tissue Cell 19 (5):657-63, Childs et al. (1992) J. Biol. Chem. 267(32):22853-9, and White et al. (1996) J. Biol. Chem. 271 (25):15008-17. The cells can be subsequently treated with acetylcholine, and a biological effect of acetylcholine on the cells, such as phosphatidylinositol turnover or cytosolic calcium concentration can be measured using methods known in the art (see Hartzell H. C. et al. (1988) Prog. Biophys. Mol. Biol. 52:165-247). Alternatively, transgenic animals, e.g., mice

overexpressing a mAChR polypeptide or a biologically active portion thereof, can be used. Tissues from such animals can be obtained and treated with acetylcholine. For example, methods for preparing detergent-skinned muscle fiber bundles are known in the art (Strauss et al. (1992) Am. J. Physiol. 262:1437-45). The contractility of these tissues in response to acetylcholine can be determined using, for example, isometric force measurements as described in Strauss et al., supra. Similarly, to determine whether an mAChR polypeptide or a biologically active portion thereof can modulate an acetylcholine response in an acetylcholine responsive cell such as a gland cell, gland cells, e.g., parotid gland cells grown in tissue culture, can be transfected with a construct driving the overexpression of an mACHR polypeptide or a biologically active portion thereof. The cells can be subsequently treated with acetylcholine, and the effect of the acetylcholine on amylase secretion from such cells can be determined using, for example an enzymatic assay with a labeled substrate. The preferred assays used for mAChR activity will be based on phosphatidylinositol turnover such as those developed for the Ml, M3 and M5 classes of receptors (see E. Watson et al. The G Protein Linked Receptor: FactsBook (Academic Press, Boston, Mass., 1994)).

Exemplification

The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

In a first experiment, sessions were conducted in a two-compartment inhibitory: avoidance box (Coulbourn Instruments). The "light" compartment was made of white: Plexiglas and was illuminated by a 50-watt incandescent light bulb fastened to the cageL lid. The "dark" compartment was made of black Plexiglas and was not illuminated. Both , compartments had a floor made of 4 mm stainless steel rods spaced 1.2 cm apart.: Footshocks were delivered to the grid floor of the dark chamber via a constant currenl ; scrambler circuit. The two compartments were separated by a solenoid-operated sliding , door, and an electronic counter, activated by the opening or closing of the sliding door. I recorded acquisition and retention latencies. The inhibitory avoidance box was enclosed in a sound attenuated isolation box. NMGN-pWO-009 57 -

Inhibitory avoidance training began by placing a rat into the light compartmenl- with its head facing away from the door. After a period of ten seconds, the sliding dooi ^■ was automatically opened, allowing the rat access to the dark chamber. Two seconds aftei : the rat had fully entered the dark chamber, the door closed and an inescapable footshocl; : (0.8 mA for 2 seconds) was delivered to the grid floor. The rat was then removed from the : apparatus and immediately injected with either oxotremorine, clenbuterol, or a cocktail oi both these compounds. The rat is then returned to its home cage.

Retention testing was conducted 48 hours later, and involved each rat being placed in the light compartment of the apparatus, with its head facing away from the door. The door was opened 10 seconds later, and the latency to enter the dark chamber was recorded. No footshock was delivered on the retention test. The retention test was terminated at 900 seconds.

Drug treatments for enhancing memory consolidation were made at various intervals after training. The object of the treatment was to stimulate or enhance the gene response over the timecourse of memory consolidation. Results of this experiment are presented in Figure 1. ι

In a second experiment, forty-eight male, Long-Evans rats, weighing between 180 ^■ and 220 grams served as subjects in a second experiment. Subjects were individually housed in plastic cages with contact bedding, and maintained on a 12 hour on 12 hour off light dark cycle. Food and water was freely available. Prior to the experiment, the rats ι were allocated to one of eight different experimental conditions as outlined in the table below.

All animals were anesthetized with sodium pentobarbital (50 mg/kg, I. P) and placed in a stereotaxic apparatus, where a midline incision was made and the scalp retracted to expose the skull. Lesions of the fornix were made by drilling holes through the skull at the following coordinates: 0.3 mm and 0.8

mm posterior to Bregma, and 0.4 mm and 0.6 mm lateral to the midline. Monopolar electrodes (teflon coated wire, 125 μm in diameter) oriented laterally at 10 degrees from the vertical, were lowered at each site to a depth of 4.0 mm measured from the surface of the skull. DC current at 1 mA was passed through the electrodes for a duration of 12 seconds. The electrodes were then removed, and the wound sutured. Control animals received sham operations in which holes were drilled in the skull overlying the fornix. Postoperatively, the animals were kept warm and monitored closely until spontaneous movement occurred. Once stabilized, they were returned to their home cages and left to recover for seven days prior to behavioral testing.

Experimental sessions were conducted in a two-compartment inhibitory avoidance ^" box (Stoelting Physiology Research Instruments). The^' "light" compartment was made of white Plexiglas and was illuminated by a 50-watt incandescent light bulb fastened to the cage lid. The "dark" compartment was made of black Plexiglas and was not illuminated. Both compartments had a floor made of 4 mm stainless steel rods spaced 1.2 cm apart.. Footshocks could be delivered to the grid floor of the dark chamber via a constant current scrambler circuit. The two compartments were separated by a solenoid-operated sliding door, and an electronic counter, activated by the opening or closing of the sliding door, . recorded acquisition and retention latencies. The inhibitory avoidance box was located in a sound attenuated, non-illuminated room.

Inhibitory avoidance training began by placing the rat into the light compartment with its head facing away from the door. After a period of ten seconds, the sliding door ^■ was automatically opened, allowing the rat access to the dark chamber. One second after the rat had fully entered the dark chamber, the door was closed and an inescapable. footshock (1 mA for 2 seconds) was delivered to the grid floor. The rat was then; removed from the apparatus and immediately injected with either saline or drug depending on experimental condition. The rat was then returned to its home cage.

Retention testing was conducted 48 hours later, and involved each rat being placed into the light chamber with its head facing away from the door. The door was opened 10. seconds later, and the latency to enter the dark chamber was recorded. Footshock was not delivered on the retention test. The retention test was terminated at 940 seconds. Results of this test are presented in Figure 2. NMGN-pWO-009 - 59-

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

All patents, publications, and other references cited above are hereby incorporated by reference in their entirety.

Claims

NMGN-pWO-009 - 60-

We claim:

1. A method for enhancing memory consolidation in an animal, comprising administering to the animal an adrenergic neurotransmitter or agonist thereof, and a cholinergic or glutaminergic neuiOtransmitter or agonist thereof, in an amount sufficient to enhance long-term memory in the animal.

2. The method of claim 1, wherein the adrenergic neurotransmitter or agonist thereof potentiates and/or activates cAMP signal transduction.

3. The method of claim 1, wherein the adrenergic neurotransmitter or agonist thereof is a ligand of an adrenergic receptor.

4. The method of claim 1, wherein the adrenergic neurotransmitter or agonist thereof is acts intracellularly to potentiate and/or activate cAMP-dependent signal transduction.

5. The method of any of claims 1-4, wherein the adrenergic neurotransmitter or agonist thereof is a small organic molecule.

6. The method of claim 1, wherein the cholinergic or glutaminergic neurotransmitter or agonists thereof potentiates and/or activates protein kinase C (PKC) signal transduction.

7. The method of claim 1, wherein the cholinergic or glutaminergic neurotransmitter or agonist thereof is a ligand of a cholinergic receptor.

8. The method of claim 1, wherein the cholinergic or glutaminergic neurotransmitter or agonist thereof is a ligand of a glutaminergic receptor.

The method of claim 1, wherein the adrenergic neurotransmitter or agonist thereof is acts intracellularly to potentiate and/or activate PKC signal NMGN-pWO-009 - 61 -

transduction.

10. The method of any of claims 1 or 6-9, wherein the adrenergic neurotransmitter or agonist thereof is a small organic molecule.

11. The method of any of claims 1-10, wherein the adrenergic neurotransmitter or agonist thereof, and the cholinergic or glutaminergic neurotransmitter or agonists thereof are two separate molecules.

12. The method of any of claims 1-10, wherein the adrenergic neurotransmitter or agonist thereof, and the cholinergic or glutaminergic neurotransmitter or agonists thereof are provided in the same molecule.

13. A method for enhancing memory consolidation in an animal, comprising administering to the animal a cAMP agonist and a PKC agonist in an amount sufficient to enhance long-term memory in the animal.

14. A pharmaceutical preparation comprising an adrenergic neurotransmitter or agonist thereof, and a cholinergic or glutaminergic neurotransmitter or agonists thereof, in an amount sufficient to enhance long term memory in the animal.

15. The method of claim 2, wherein the cAMP agonist binds to a dopamine receptor.

16. The method of claim 2, wherein the cAMP agonist binds to a β-adrenergic receptor.

17. The method of claim 2 wherein the cAMP agonist is a cAMP analog.

18. The method of claim 2, wherein the cAMP agonist is a cAMP phosphodiesterase inhibitor. NMGN-pWO-009 - 62-

19. The method of claim 2, wherein the cAMP agonist agonizes adenylate cyclase activity.

20. The method of claim 2, wherein the PKC agonist binds to a metabotropic glutamate receptor.

21. The method of claim 2, wherein the PKC agonist binds to a muscarinic acetylcholine receptor.

22. The method of claim 2, wherein the PKC agonist agonizes phospholipase C (PLC).

23. The method of claim 2, wherein the PKC agonist is a phorbol ester or a diacyl glycerol.

24. The method of claim 2, wherein combination of the PKC agonist and cAMP agonist promotes CREB phosphorylation with an ED50 of 1 mM or less.

25. The method of claim 24, wherein the combination promotes CREB phosphorylation with an ED50 of 1 μM or less.