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WO2008134069A2 - Agents comportant un ligand ancré - Google Patents

Agents comportant un ligand ancré Download PDF

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Publication number
WO2008134069A2
WO2008134069A2 PCT/US2008/005528 US2008005528W WO2008134069A2 WO 2008134069 A2 WO2008134069 A2 WO 2008134069A2 US 2008005528 W US2008005528 W US 2008005528W WO 2008134069 A2 WO2008134069 A2 WO 2008134069A2
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Prior art keywords
binding
thrombin
agent
ligand
nucleic acid
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PCT/US2008/005528
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English (en)
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WO2008134069A3 (fr
Inventor
Weihong Tan
Zehui Cao
Youngmi Kim
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University Of Florida Research Foundation
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Publication of WO2008134069A3 publication Critical patent/WO2008134069A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity

Definitions

  • the invention relates generally to agents including therapeutic agents and drugs that act as ligands that specifically bind to molecular targets such proteins. Binding of the agent to the target mediates a biological activity of the target molecule.
  • the invention relates to anticoagulant agents that act as ligands that selectively bind to proteins of the blood coagulation cascade, to inhibit their blood clotting activities.
  • a vast number of biological reactions are controlled by the interaction of an effector molecule, or "ligand” with a "binding site,” or “active site” in or on a target molecule.
  • ligand-receptor interactions are well known among such interactions.
  • antigen-antibody interactions are interactions between nucleic acid ligands such as aptamers and protein-based target molecules.
  • the mechanism of action of many modern drugs is to either stimulate or inhibit a target molecule (e.g., a receptor on a cell surface, or a circulating molecule such as blood coagulation factor) by specifically binding to the target molecule.
  • a target molecule e.g., a receptor on a cell surface, or a circulating molecule such as blood coagulation factor
  • the potency of the drug will depend in part on the efficiency with which the drug is able to bind to its target molecule.
  • the invention provides a new class of molecular agents having greatly increased binding efficiency for a wide range of protein-based molecular targets, such as many known receptors and other proteins that serve as targets for a wide variety of drugs in current use.
  • One aspect of the invention is an anchored ligand agent for efficient binding to a target protein.
  • the agent comprises: a nucleic acid molecule comprising an anchoring portion for binding to a first site in a target protein or peptide; a nucleic acid molecule comprising a binding portion for binding to a second site in said target protein or peptide; and a molecule comprising a linker portion that joins said anchoring nucleic acid to said binding nucleic acid.
  • the anchoring portion of the agent (“molecular anchor, MA”) can bind to the first site on the target protein with a binding affinity (Kd) of about 0.7 nM.
  • the second site in the target protein or peptide is associated with a specific biological activity, upon binding with a ligand.
  • An anchored ligand in accordance with the invention is designed to specifically bind to such a biologically active site in the target molecule, inorder to effect a desired biological response.
  • at least one of the nucleic acid molecules is DNA-based ligand such as an aptamer.
  • the invention provides bivalent molecules, e.g., comprising DNA- based ligands such as an aptamers, optionally connected by a linker.
  • exemplary bivalent molecules are set forth in Example 2.
  • the linker can be a molecule of sufficient length to permit simultaneous binding of the anchoring portion to the first site on the target molecule, and binding of the binding portion to the second site on the target protein, wherein said binding results in a biological response.
  • Anchored ligand agents in accordance with the invention can comprise hydrophilic linker molecules (typically monomers) of variable lengths, such as a polyethylene glycol (PEG), a poly vinyl alcohol (PVA), a polyglycolide, a vinyl ether, or a phosphoramidite.
  • an anticoagulant agent comprising: a nucleic acid molecule comprising an anchoring portion for binding to a first site in a protein associated with blood coagulation; a nucleic acid molecule comprising a binding portion for binding to a second site in said blood coagulation protein; and a linker portion joining said anchoring nucleic acid to said binding nucleic acid.
  • At least one of the nucleic acid molecules is an aptamer that specifically binds to the blood coagulation protein thrombin.
  • Anticoagulant agents in accordance with the invention exhibit increased thrombin-inhibiting ability, relative to that of a thrombin-inhibiting aptamer that is not linked to an anchoring portion. Other aspects of the invention are discussed below.
  • FIG. IA is a schematic diagram illustrating the equilibrium between binding to a target molecule 105 and dissociation from the target molecule 105 by a free ligand 115 and a competing substrate molecule 120.
  • FIG. IB is a schematic diagram illustrating the equilibrium between binding and dissociation from a target molecule 105 by an anchored ligand 140 which comprises one portion of an anchored ligand agent 150 in accordance with an embodiment of the invention, and a competing substrate molecule 120.
  • the anchored ligand is molecularly anchored close to the target molecule it is not free to diffuse away from the target following dissociation. This proximity to the target greatly increases the probability of re-binding to the target molecule.
  • the binding efficiency of the anchored ligand agent is greatly increased, as compared with a free ligand having the same chemical structure.
  • FIG. 2 is a schematic diagram illustrating the design and mechanism of action of an anchored ligand agent effective as an anticoagulant agent with thrombin inhibitory activity, as compared with an unanchored ligand, in accordance with the invention.
  • FIG. 3 is a graph showing comparison of the normalized clotting times of thrombin bound to different aptamer inhibitors. Clotting time of thrombin alone was defined as 1.
  • the inset is a graph showing comparison of clotting times for anchored ligand agents in accordance with the invention having the indicated numbers of linker molecules (spacers).
  • FIG. 4 is a graph depicting real time monitoring of light scattering intensity generated by the coagulation process in the presence of different aptamers or linked aptamers agents in accordance with the invention. Fibrinogen was added at 0 second.
  • FIG. 5 is a graph showing effect of the complementary sequence (cDNA of 15Apt) on anticoagulation of A-8-MA, a thrombin inhibitor in accordance with the present invention. Scattering intensity of the thrombin, A-8-MA and fibrinogen reaction mixture was monitored. Excess cDNA of 15 Apt was added at around 500 th second.
  • FIG. 6A-B are schematics of the exemplary molecules of the invention, (a) 15Apt, monovalent ligand, has constant ON and OFF and diffuses into bulk solution immediately after dissociation from thrombin, resulting in low inhibitory function, (b) In contrast, when linked to
  • 27Apt to form a bivalent ligand 15 Apt can rapidly return to the binding site after dissociation due to confined molecular diffusion by 27Apt that is still in the bound state to thrombin.
  • FIG 7 depicts a comparison of the normalized clotting times of thrombin bound to different NA inhibitors.
  • Clotting time of thrombin alone was defined as 1, and the relative values based on it are plotted.
  • 15Apt alone showed a threefold increase of the clotting time, but any delay was observed from the 27Apt-treated sample.
  • Bi-xS bivalent NA candidates
  • FIG. 8 depicts real-time monitoring of scattering light generated by the coagulation process in the presence of different monovalent or bivalent NA ligands (Bi-xSs).
  • Bi-xSs monovalent or bivalent NA ligands
  • FIG 7A-C depict a comparison of binding kinetics,
  • Thrombin-bound 15 Apt MBA (blue) showed slower hybridization kinetics compared to the free form.
  • thrombin-bound Bi-8S MBA red showed a 51.7 times slower dissociation rate.
  • the k a ' of 15Apt domain of Bi-8S is about 62 times stronger than free 15Apt.
  • FIG. 8 depicts reversible inhibitory function.
  • Red T- 15 Apt was added at around 200 seconds to the incubation of Bi-8S, thrombin and fibrinogen.
  • Black fibrinogen was added to thrombin at 0 seconds in the absence of any inhibitors.
  • Blue Bi-8S incubated with thrombin and fibrinogen (no T-15Apt).
  • FIG. 9A-B depict comparison of anticoagulant potency of Bi-8S and 15Apt using human plasma and aPTT and PT measurements, (a) shows dosage-dependent aPTT plotted for each NA inhibitor, and the maximal aPTT is shown inside the figure, (b) shows dosage-dependent PT, and the maximal PT recorded appears inside the figure.
  • FIG. 10A-B depict an investigation of concentration effect of T-15Apt in binding comparison, (a) 15 Apt/thrombin complex was treated with different amounts of T- 15 Apt. (b) Ma MBl /thrombin complex was treated with different amounts of T- 15 Apt. As shown in the figure, there was no noticeable kinetics change.
  • FIG. 11 depicts an investigation of the dissociation of T'- 15 Apt. The preincubated mixture of F-T'-15Apt and short 15Apt-Q for 30 mins was treated with 15Apt. The increased fluorescence signal of the sample was obtained from the dissociation of T'-15Apt, which is very rapid reaction. Since this dissociation between 15Apt and T'-15Apt is much faster than the association of 15Apt to thrombin, it does not interfere the measurement of k' on . DETAILED DESCRIPTION OF THE INVENTION
  • the stimulus for, or inhibition of, a vast number of biological reactions is based on the interaction of an effector molecule, or "ligand” with a "binding site,” or “active site” in or on a target molecule.
  • ligand effector molecule
  • binding site or “active site” in or on a target molecule.
  • well known among such interactions are receptor-ligand interactions, antigen- antibody interactions, and interactions between nucleic acid ligands such as aptamers with protein-based target molecules.
  • the potency of the drug will depend in great part on the efficiency with which the drug is able to bind to its target molecule.
  • Many potential therapeutic molecules are limited in their usefulness due to low binding efficiency to their target molecules. Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
  • the invention addresses an aspect of this deficiency by providing a novel molecular agent useful as a drug and for many other applications.
  • the agent comprises a nucleic acid ligand molecule useful for effecting a biological activity, linked to a molecular anchor.
  • Agents in accordance with the invention can be used to selectively and efficiently bind to a target molecule such as a protein (for example in order to stimulate or inhibit the biological function mediated by the target molecule).
  • Agents in accorance with the invention are based on a novel molecular engineering strategy that provides for the anchoring of the attachment portion of the agent to the target molecule by means of a "molecular anchor” (MA) that binds with high affinity to the target molecule.
  • the MA is an aptamer.
  • the anchored portion of the agent is tethered to the binding portion of the agent by the linker portion, which is the equivalent of a "molecular rope.”
  • the binding portion of the agent upon selective binding to its binding site on the target molecule, either stimulates or inibits a biological response that is mediated by the target molecule.
  • the active binding portion of the agent is restricted by in its ability to diffuse very far from the vicinity of the binding site on the molecular target.
  • the binding portion of the agent (or "ligand portion") is available to interact repeatedly with the binding site on the molecular target.
  • the ligand portion of an agent of the invention is able to bind to the target molecule with much higher efficiency than is possible for the corresponding unanchored ligand.
  • agents of the invention are those comprising ligands that selectively bind to known drug targets with high affinity, effectively enhancing the potency of many known drugs.
  • An agent in accordance with the invention comprises an attachment portion, a binding portion (also termed a "ligand portion,") and a linker portion.
  • Particularly preferred agents comprise nucleic acid ligands in the form of aptamers that specifically bind to selected target molecules with very high affinity.
  • One preferred agent in accordance with the invention comprises nucleic acid probe linked to a molecular anchor (MA).
  • the molecular anchor (MA) comprises a binding portion that binds with high affinity to a first binding site in the target molecule.
  • a linker molecule is attached on one end to the MA and on the other end to a nucleic acid probe.
  • the nucleic acid probe is selected to bind specifically to an "active" binding site in or on the target molecule that is associated with biological activity.
  • the binding portion of the MA that effects the biological effect is a second aptamer.
  • the linker between the two aptamers can be any suitable hydrophilic molecule that exhibits minimal nonspecific interactions with biomolecules.
  • Suitable hydrophilic polymers can include, e.g., a poly vinyl alcohol (PVA), a polyglycolide, a polyethylene glycol and a vinyl ether. Not all suitable polymers may be generally available as DNA synthesizer reagents.
  • Commercially available linkers that permits optimal control of the length of the linker include, e.g., spacer phosphoramidites such as Spacer Phosphoramidite 18, Spacer Phosphoramidite 9, and Spacer Phosphoramidite C3 (the listed spacers having different lengths) from Glen Research (Sterling, VA).
  • Figures IA and IB schematically illustrate various features of the design and mechanism of action of an anchored ligand agent in accordance with the invention.
  • Both figures illustrate a schematic target molecule 105 having two binding sites, suspended in a solution.
  • One of the binding sites on target molecule 105 is a ligand binding site 110, which is shown unoccupied in the drawing on the left in FIG. IB.
  • the other binding site is a molecular anchor (MA) binding site 125, further discussed infra.
  • Figure IA schematically illustrates the typical phenomenon of competitive binding of a free ligand 115 and a competing substrate molecule 120 for a single ligand binding site 110 on a target molecule 105. In the drawing on the left, the ligand binding site 110 is shown occupied by a free ligand molecule 115, whereas in the drawing on the right, site 110 is occupied by a competing substrate molecule 120 that is also present in the solution.
  • the selective binding of a ligand that is free in a solution to a corresponding binding site on a target molecule represents an equilibrium in which the ligand is either associated with (bound to) the binding site on the target molecule, or it is dissociated from the binding site, and is free to diffuse away from the target molecule into the solution at large.
  • the rate of dissociation of a particular ligand from a target molecule is governed by the strength of the molecular interaction between the ligand and the target (known as the "binding affinity"). Binding affinity is generally expressed in terms of a dissociation constant (Kd).
  • a dissociation constant in the range of 200-500 nM is considered to reflect a relatively low binding affinity (resulting in a tendancy for the ligand to dissociate from the target molecule and diffuse away from the target into the solution).
  • a Kd in the subnanomolar range would be considered to be a relatively high binding affinity (resulting in more persistent and frequent binding of the ligand to the target).
  • FIG. 1 illustrates a competitive binding situation in which free ligand molecules 115 compete with competing substrate molecules 120 for binding site 110.
  • the drawing illustrates the equilibrium that exists between binding of the ligand 115 (left drawing) and binding of the competing substrate molecule 120 (right drawing). It is readily apparent from FIG.
  • the present invention provides a novel and innovative solution to the problem of low binding efficiency of a ligand and resultant diffusion of the ligand away from its ligand-binding site on a target molecule by providing agents ("anchored ligands") that are molecularly engineered to greatly increase their binding efficiency to their particular target molecules, as compared to the corresponding "unanchored" ligand free in solution. As illustrated in FIG.
  • an anchored ligand agent 150 in accordance with the invention comprises three components: a molecular anchor (MA) (or “anchor portion”) 130; a binding portion (or “anchored ligand”) 140 capable of selectively binding to the ligand binding site 110 on the target molecule 105; and a linker portion 135.
  • MA molecular anchor
  • binding portion or “anchored ligand”
  • linker portion 135. Referring to the right-hand side of FIG. IB, there is illustrated the specific interaction of an anchored ligand agent 150 of the invention with its target molecule 105.
  • the interaction occurs at two sites: the molecular anchor portion 130 of the anchored ligand agent 150 specifically binds to the molecular anchor binding site 125 on the target molecule 105, whereas the anchored ligand portion 140 of the agent 150 specifically binds to the ligand binding site 110 on the target molecule 105.
  • the length of the linker portion 135 of the agent 150 is proportioned to allow flexibility and appropriate positioning of both the anchored ligand portion 140 and the MA portion 130 in their respective binding pockets in or on the target molecule 105.
  • FIG. IB illustrates the situation in which the anchored ligand 140 has dissociated from the binding site 110 in the target molecule 105. If free, the ligand 140 would be able to diffuse away from the target molecule 105 without constraint, as described above and illustrated in FIG. IA. However, due to its tethering to the molecular anchor 130 by means of the linker molecule 135, the anchored ligand 140 of the agent 150 is limited in the distance it can diffuse from the target molecule 105 by the length of the linker molecule 135.
  • the invention provides novel agents in accordance with the invention that can act as highly efficient anticoagulants.
  • the corresponding antidotes to these anticoagulants are also provided, as further discussed infra.
  • Anticoagulant drugs act by regulating proteins of the blood coagulation cascade. Although some anticoagulants have been in clinical use for decades, there is recognition that improved anticoagulants are greatly needed for safer and more effective treatments, due to the low efficiency and poor stability of presently available drugs [2]. Histrorically, thrombin has been one of the preferred molecular targets for anticoagulant therapeutics.
  • a widely used anticoagulant is heparin, which is known to inhibit the blood coagulation protein thrombin. Heparin inhibits thrombin in an indirect manner, by enhancing the activity of the natural antithrombin [3]. Unfortunately, heparin preparations exhibit diversity in molecular size, and demonstrate nonspecific binding to plasma proteins [4].
  • Fractionated low-molecular-weight heparins are known have a more predictable anticoagulant effect; however, these products are antithrombin-dependent and are not effective to inactivate thrombin that is bound to blood clots, fibrin, or fibrin derivatives [5, 6].
  • thrombin inhibitors are preferable for many clinical applications.
  • a promising development in the field of anticoagulation therapeutics is a DNA-based anticoagulant that is an aptamer, isolated by a systematic selection process [7]. Consisting of merely 15 bases in a single strand of DNA, this aptamer (abbreviated as "15Apt”) has been shown to bind to exosite 1 of the thrombin molecule and to inhibit its function in the coagulation cascade. The 15Apt aptamer has high selectivity for thrombin, and demonstrates very low to no cytotoxicity, due to its DNA nature.
  • a further unique advantage of a DNA-based anticoagulant therapy based on aptamers is that antidotes of aptamer drugs are readily available, based on their complementary DNAs (cDNAs). If necessary, the antidotes can be administered to effectively reverse the anticoagulation activity of the aptamers by virtue of the strong and rapid DNA-DNA hybridization that occurs between the aptamers and the antidotes [8, 9]. This is especially advantageous in anticoagulation therapy where, as discussed above, an over dosage of an anticoagulant can lead to serious bleeding complications.
  • cDNAs complementary DNAs
  • the invention provides in one aspect improved aptamer-based anchored ligand agents that are useful as anticoagulants.
  • improved aptamer-based anchored ligand agents that are useful as anticoagulants.
  • MA molecular anchor
  • Table 1 provides DNA sequence information pertaining to several preferred embodiments of anticoagulant agents in accordance with the invention. Table 1. DNA sequences
  • 15Apt is covalently linked to the MA.
  • the 15Apt is thus held closely around thrombin even after dissociation, resulting in a much higher probability of re- binding to thrombin.
  • the MA has similar or better affinity for thrombin than the 15Apt but binds to a different site. Because 15 Apt and MA are unlikely to be dissociated from the two binding sites on thrombin at the same time, and also because the MA is a stronger binder, the molecular assembly improves each other's binding strength by greatly increasing Ic 0n of the binding reaction while having roughly unchanged k of r-
  • 27Apt thrombin-binding DNA aptamer
  • SEQ ID NO:3 a 27-mer thrombin-binding DNA aptamer
  • This aptamer is an ideal choice as a MA in combination with an anchored ligand based on 15Apt (SEQ ID NO:2) for several reasons.
  • 27Apt has a strong affinity for thrombin, with an estimated Kd of 0.7 nM.
  • 27Apt is known to bind to a different site on the thrombin molecule than 15Apt, (i.e., exosite 2).
  • simultaneous binding on exosite 1 and 2 of thrombin is known to occur [15, 16].
  • hydrophilic polymers suitable for use as spacers for linking the two aptamers can include, e.g., polyethylene glycol (PEG), poly vinyl alcohol (PVA), polyglycolide, polyethylene glycol, vinyl ether.
  • PEG polyethylene glycol
  • PVA poly vinyl alcohol
  • polyglycolide polyethylene glycol
  • vinyl ether e.g., polyethylene glycol (PEG), poly vinyl alcohol (PVA), polyglycolide, polyethylene glycol, vinyl ether.
  • One particularly preferred spacer for a thrombin-based anticoagulant in accordance with the invention comprises 18 atoms.
  • an anchored anticoagulant agent of the invention comprises SEQ ID NO:2 and SEQ ID NO:3 linked by an 18-atom polyethylene glycol (PEG) chain (-2.1 nm).
  • SEQ ID NO:2 and SEQ ID NO:3 are linked by Spacer Phosphoramidite 18 from Glen Research.
  • This Example describes the design and testing of a DNA anticoagulant agent in accordance with the invention that comprises a molecule functioning as a molecular anchor (MA) coupled via a linker molecule to a nucleic acid ligand in the form of an anti-thrombin aptamer.
  • MA molecular anchor
  • This novel assembly demonstrates significantly improved anticoagulation efficiency, as compared to the free aptamer. Accordingly, this agent and those of similar design are believed to hold great potential for treating various diseases related to blood clotting disorders.
  • reagents All reagents for buffer preparation including DNA grade water and for HPLC purification were from Fisher Scientific Company L.L.C. (Pittsburgh, PA). A buffer resembling physiological conditions was used for all coagulation tests.
  • the buffer contained 25 mM Tris- HCl at pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , ImM CaCl 2 , and 5% (V/V) glycerol.
  • Human ⁇ -thrombin was purchased from Haematologic Technologies, Inc. (Essex Junction, VT). Fibrinogen was obtained from Sigma-Aldrich, Inc. (St. Louis, MO).
  • the HPLC was performed on a ProStar HPLC Station (Varian, Inc., Palo Alto, CA) equipped with fluorescence and a photodiode array detector.
  • a C-18 reverse phase column (Alltech Associates Inc., Flemington, NJ, C-18, 5 ⁇ M, 250x4.6 mm) was used.
  • Clotting time tests Two hundred ⁇ L of physiological buffer was added to a disposable transparent plastic cuvette (Fisher Scientific Company L.L.C., Pittsburgh, PA). Then 1 ⁇ L of 10 ⁇ M thrombin and 1 ⁇ L of 100 ⁇ M aptamer or linked aptamers (in the case of non-linked 15 Apt and 27Apt mixture, 1 ⁇ L of 100 ⁇ M of each was added) were added and incubated for 15 minutes. Subsequently, 4 ⁇ L of 20 mg/mL fibrinogen was mixed with the solution. Samples in the cuvette were examined continuously for the formation of a gel-like substance. The time when the sample became non-fluidic was recorded.
  • a typical clotting test was used to evaluate linker length effect on thrombin inhibition.
  • Ten fold free aptamer or A-L-MA was incubated with thrombin for 15 min before excess thrombin substrate, fibrinogen, was introduced. Cleaved by thrombin, fibrinogen generates fibrin monomers that rapidly crosslink with each other to form a polymer network.
  • the reaction solution turns from a fluidic form to a non-fluidic one, accompanied by a gel-like appearance.
  • the recorded the time of this transition for each sample normalized the data and compared them among the tested inhibitors. As shown in FIG. 3, 15Apt alone delayed the coagulation time by nearly 3 fold. If the same amount of each of the non-linked 15 Apt and the 27Apt was mixed and used, the coagulation time was only slightly increased (FIG. 3).
  • Figure 3 further shows that free 27 Apt indeed did not inhibit thrombin or noticeably affect the function of 15 Apt.
  • A-L-MA with a linker of 8 spacers led to a 9 fold increase of transition time over 15Apt (27 times total over thrombin alone).
  • 27Apt worked as an MA to tightly hold 15 Apt close to thrombin to ensure that 15 Apt competed favorably for thrombin over fibrinogen.
  • 15 Apt was linked to a scrambled DNA by 8 spacers, the resulting A-8-random DNA showed very limited thrombin inhibition.
  • linker length is shown in the inset of FIG.3.
  • A-L-MA with 4 spacers displayed much weaker anticoagulation than other A-L-Mas, and even free 15 Apt.
  • molecular anchors The consequence of a linker with insufficient length is that the MA will have limited flexibility for binding and thus will prevent the anchored ligand portion of the agent from reaching the binding pocket of thrombin, leading to a greatly reduced anticoagulation capability.
  • Six spacers seemed to be closer to the optimum length and 8 spacers gave the best inhibition of thrombin.
  • FIG. 5 shows the effect of the cDNA of 15Apt on anticoagulation of A-8-MA. Scattering light of the thrombin, A-8-MA and fibrinogen reaction mixture was monitored.
  • Human ⁇ -thrombin was purchased from Haematologic Technologies, Inc. (Essex Junction, VT). Fibrinogen and the sulfated hirudin fragment were obtained from Sigma-Aldrich, Inc. (St. Louis, MO). Universal Coagulation Reference Plasma (UCRP) and thromboplastin-DL for human sample testing were purchased from Pacific Hemostasis (Cape Town, South Africa). The aPTT assay reagent was from Trinity Biotech USA (Berkeley Heights, NJ). The bivalirudin was obtained from The Medicines Company (Parsippany, NJ).
  • the HPLC was performed on a ProStar HPLC Station (Varian Medical Systems, Palo Alto, CA) equipped with a fluorescence detector and a photodiode array detector.
  • a C-18 reverse phase column (Alltech, Cl 8, 5 ⁇ M, 250x4.6mm) was used.
  • S means one unit of spacer phosphoramidite. Dabcyl is a quencher and FAM is a fluorophore.
  • Clotting time tests To evaluate the inhibitory potency of each NA ligand, we measured the clotting timeof each sample containing only thrombin, each nucleic acid ligand, and fibrinogen substrate in physiological buffer. The theory behind the experiment is that the mixture of sample becomes non-fluidic when the fibrinogen is digested by thrombin. As a result, the different time points of this transition can be used as an indicator. Briefly, l ⁇ L of 10 ⁇ M thrombin and 1 ⁇ L of 100 ⁇ M monovalent or bivalent nucleic acid ligand were added to a disposable transparent plastic cuvette (Fisher Scientific Company L.L.C., Pittsburgh, PA) containing 200 ⁇ L physiological buffer and then incubated for 15 minutes.
  • a disposable transparent plastic cuvette Fisher Scientific Company L.L.C., Pittsburgh, PA
  • the excitation and emission wavelengths were both set at 580 nm, and the emission was detected at the right angle relative to the light excitation so that the excitation light did not interfere with the light scattering signal.
  • the initial rate of scattering increase represented the relative thrombin-inhibition strength of the tested sample. Initial rates were calculated from the linear range of the early slopes of the scattering profiles.
  • Reversible binding reaction using target DNAs To test the reversible binding of Bi-8S, we treated the sample mixture with target DNAs of 27Apt or 15 Apt. The mixture of sample, including fibrinogen, was prepared in the same way as indicated for the clotting time. About 500 seconds after fibrinogen was added to the reaction mixture, each target DNA of either 15 Apt or
  • aPTT and PT for each ligand using human plasma samples. Procedures applied were those recommended by the manufacturer. For aPTT determination, 50 ⁇ L of UCRP was pre-incubated at 37°C with a different amount of each ligand for 2 minutes; then 50 ⁇ L of aPPT-L was added and incubated for another 200 seconds. Next, 50 ⁇ L of pre-warmed CaC12 was added to initiate the intrinsic clotting cascade. Finally, the scattering signal was monitored until the signal was saturated.
  • Thrombin is a multifunctional protease involved in the regulation of homeostasis.
  • thrombin hydrolyzes fibrinogen, and activates platelets and some blood coagulation factors that have procoagulant activity.
  • Disorders in blood clotting are tightly linked to many serious health issues including heart attack and stroke. Therefore, thrombin is typically the target in anticoagulation therapy for these diseases.
  • anticoagulant drugs currently on the market often suffer from indirect inhibition and sub-optimum selectivity, which could lead to side effects including bleeding (17-18).
  • thrombin NA aptamers There are two known thrombin NA aptamers. One is 15 base long ( 15 Apt) and binds to exosite 1, while the other, called 27Apt, is 27 base long and interacts with exosite 2 (19, 20) as shown in Figure 6.
  • the dissociation constant K d of 15Apt tends to be very high (up to 450 nM), depending on measurement methods(21-23), and K d of 27Apt is approximately 0.7 nM (20)
  • K d of 15Apt tends to be very high (up to 450 nM), depending on measurement methods(21-23), and K d of 27Apt is approximately 0.7 nM (20)
  • As a potential anticoagulant only 15 Apt should have the enzymatic inhibitory functions required for thrombin-mediated coagulation since it occupies the fibrinogen-binding exosite 1.
  • efforts to explore the anticoagulant effect of this aptamer have shown only limited progress due to
  • NA ligand of thrombin NA ligand of thrombin.
  • this type of construct is likely to be even more unpredictable, as its unique conformational structure can be disrupted, resulting in the loss of its binding property (24). Therefore, instead of modifying the aptamer sequence itself, we have linearly assembled two existing NA aptamers of thrombin to form a molecular assembly of thrombin aptamers. This assembly specifically improves the inhibitory function of thrombin due to the multivalent interaction mentioned above.
  • bivalent NA inhibitor We hypothesized that linear molecular assembly of two monovalent NA aptamers would result in a superior functional NA inhibitor of enzymatic reactions with multivalent binding properties. However, first we need to find out whether we could achieve enhanced inhibition even in the absence of multivalent interactions by simply mixing these two aptamers without covalent linkage. The second important question would be whether these two aptamers could interact with each other and subsequently cause the loss of inhibition after assembly. To address these issues, we performed typical clotting test using no aptamer, 15Apt alone, 27Apt alone, and a mixture of 15Apt and 27Apt. As shown in Figure 7, 15Apt alone delayed the coagulation time by nearly threefold, while 27Apt had no significant observed inhibition.
  • bivalent NA ligands were designed and evaluated with the purpose of optimizing the distance between the two different NA aptamers. This step is particularly critical in designing bivalent ligands, and we initially assumed that a shorter distance between the two aptamers would result in disruption of their simultaneous binding and, hence, less effective binding and inhibition. Therefore, we designed several potential bivalent NA ligands with linkers of different lengths composed of 4, 6, 8, or 10 spacer phosphoramidites and designated to Bi-xSs, as shown in Table 2. Considering that one spacer is about 2.1 nm long and that the inner diameter of thrombin is several nanometers, this represents a sufficiently ample range of lengths; i.e., from 8.4 to 21.0 nm.
  • Bi-4S produced an initial rate of 2903 cps/sec calculated from the early slope of the scattering profile, 2.8 times faster than that of 15Apt alone, 1049cps/sec, which means that 27Apt interferes with 15Apt in the binding process.
  • the initial reaction rates of Bi-8S and Bi-6S were much slower than the others, at 63 and 97 cps/sec, respectively. Since Bi-8S demonstrates an initial rate close to 16.6 times slower than free 15 Apt, it also represents a considerable improvement of antithrombin efficacy.
  • the anticoagulation trend among the tested inhibitors correlates very well with the results from the clotting tests.
  • Bi-IOS did not function as well as Bi-8S, resulting in an increased initial reaction rate.
  • Bi'-8S whose sequence of 27Apt domain is replaced by the scrambled one also showed no improvement in inhibiting the clotting process. Therefore, based on the evidence gathered from both clotting test and turbidity measurement using scattering light, we conclude that Bi-8S is the best design for improved thrombin inhibition.
  • binding kinetics studies Because bivalent interaction of the aptamer assembly with thrombin increases overall binding affinity, it is proposed as the mechanism for the enhanced inhibition. Since the binding affinity is directly related to kinetic parameters, such as k on and & O fr, of the thrombin/inhibitor interaction, we carried out experiments to investigate the impact of the molecular assembling on k on and & o rr of the reaction in order to reveal what actually causes the improved inhibition.
  • One important feature of aptamers is their binding to the target is often accompanied by changes in tertiary structures. This allows researchers to build various signal transduction mechanisms, such as FRET, into aptamers for sensitive target detection. In fact,
  • 15Apt was among the first aptamers to be built into molecular beacon aptamers (MBAs) for protein detection based on FRET.
  • MAAs molecular beacon aptamers
  • 15Apt and the 15 Apt domain of Bi-8S with a fluorophore and quencher pair to forml5Apt MBA and Bi-8S MBA 1 shown in Table 2.
  • the compact structure of the 15 Apt bound to thrombin was expected to differ considerably from the random coil structure in solution, thus giving different fluorescence intensity.
  • each MBA was incubated with T'- 15 Apt, and then thrombin was added while the fluorescence signal was monitored. Due to the weak binding affinity between T'-15Apt and 15Apt, thrombin would compete with T'-15Apt for binding to 15Apt or the 15Apt domain of the bivalent ligands. This study was based on three assumptions: 1) that T'-15Apt only interacted with 15Apt, 2) that the binding affinity of the T'- 15Apt is identical for both MBAs, and 3) that binding of 15Apt to thrombin is more favorable than to T'-15Apt.
  • the antidote effect of the binding aptamer One of the unique properties of NA aptamers is that the binding can be readily regulated using the complementary sequences. Strong binding affinity of a target is critical, but reversibility of binding is equally, if not more, important. Reversibility of binding directly impacts the pharmacology of drug treatments in the sense that the side effects of drugs could then be quenched by antidotes. Based on their binding affinity as measured by the Watson-Crick base paring of complementary sequences, NA ligands and their complementary NAs are shown to be the most effective drug/antidote pairs. To demonstrate this, the antidote effects of two aptamers' target sequences, T- 15 Apt and T-27Apt, were investigated.
  • the clotting mixture including thrombin and fibrinogen with Bi-8S, was treated with excess T- 15 Apt and T-27Apt separately while the scattering was monitored (Figure 10).
  • T- 15 Apt immediate scattering increase was seen, and the extent of final scattering intensity was comparable to that observed without any thrombin inhibitors ( Figure 10), indicating that the activity of thrombin is readily recovered by inactivation of 15 Apt, and the response is rapid.
  • the clotting mixture treated with target DNA of 27Apt showed slower change in the scattering signal (data not shown), suggesting that its effectiveness in reversing the inhibition of thrombin is limited.
  • Antithrombin potency ofBi-8S Recent studies related to its biological functions find that thrombin is critical in both blood clotting disorders and influencing tumor angiogenesis(24). Thus, after we demonstrated that Bi-8S is the best inhibitor of thrombin in buffer system, we tested Bi-8S in human plasma samples to further demonstrate the potency of the anticoagulant. Standard activated partial thromboplastin time (aPTT) (25) and Prothrombin Time (PT) (26) tests were utilized as described in Materials and Methods. The aPTT and PT are performance indicators measuring the efficacy of the contact activation pathway and extrinsic pathway of coagulation, respectively, as well as the common coagulation pathways.
  • aPTT Standard activated partial thromboplastin time
  • PT Prothrombin Time

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Abstract

L'invention concerne de nouveaux agents thérapeutiques comprenant des molécules de ligand telles que des aptamères, liées à un élément d'ancrage moléculaire (MA). En l'absence du MA, les ligands se diffusent rapidement dans une solution principale après s'être dissociés d'une molécule cible. Grâce au MA lié étroitement à la molécule cible, les molécules à ligand ancré de l'invention restent à proximité étroite du site actif de la molécule cible, même après s'être dissociées de la cible, ce qui permet au ligand de se lier à nouveau rapidement à sa cible. La réversibilité de la liaison au site actif peut être obtenue efficacement et rapidement au moyen d'antidotes comprenant les séquences d'ADN complémentaires. Dans une forme de réalisation préférée, l'invention concerne un agent anticoagulant très efficace dont l'efficacité de liaison à la thrombine est multipliée par rapport à un ligand correspondant à base d'aptamère non ancré.
PCT/US2008/005528 2007-04-26 2008-04-28 Agents comportant un ligand ancré WO2008134069A2 (fr)

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US5756291A (en) * 1992-08-21 1998-05-26 Gilead Sciences, Inc. Aptamers specific for biomolecules and methods of making
US20020037506A1 (en) * 2000-04-18 2002-03-28 Yun Lin Aptamer based two-site binding assay
CA2425208C (fr) * 2000-09-26 2013-04-09 Duke University Aptameres d'arn et procede d'identification desdits aptameres
US20050123936A1 (en) * 2003-01-16 2005-06-09 Ansari Aseem Z. Method, composition, and kit to design, evaluate, and/or test compounds that modulate regulatory factor binding to nucleic acids
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