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  Nmr-solve method for rapid identification of bi-ligand drug candidatesUSPTO Application #: 20070298439Title: Nmr-solve method for rapid identification of bi-ligand drug candidates Abstract: Methods for rapidly identifying drug candidates that can bind to an enzyme at both a common ligand site and a specificity ligand site, resulting in high affinity binding. The bi-ligand drug candidates are screened from a focused combinatorial library where the specific points of variation on a core structure are optimized. The optimal points of variation are identified by which atoms of a ligand bound to the common ligand site are identified to be proximal to the specificity ligand site. As a result, the atoms proximal to the specificity ligand site can then be used as a point for variation to generate a focused combinatorial library of high affinity drug candidates that can bind to both the common ligand site and the specificity ligand site. Different candidates in the library can then have high affinity for many related enzymes sharing a similar common ligand site. (end of abstract) Agent: Mcdermott, Will & Emery - San Diego, CA, US Inventors: Daniel S. Sem, Maurizio Pellecchia, Anna Tempczyk-Russell USPTO Applicaton #: 20070298439 - Class: 435007800 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay, Involving Nonmembrane Bound Receptor Binding Or Protein Binding Other Than Antigen-antibody Binding The Patent Description & Claims data below is from USPTO Patent Application 20070298439. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application is a continuation-in-part of application Ser. No. 09/326,435, filed Jun. 4, 1999, the content of which is hereby incorporated by reference. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates to drug discovery methods, more specifically to NMR methods for identifying atoms of interest in enzyme ligands for generating and screening combinatorial libraries of bi-ligand drug candidates. [0004] 2. Background Information [0005] Widespread and sometimes indiscriminate use of antibiotics has allowed certain strains of disease-causing bacteria to become resistant to commonly available antibiotics. As a result, the need for more effective antimicrobial drugs is becoming more pressing. One approach to developing such drugs is to find compounds that bind to essential enzymes in bacteria. When such enzymes have two adjacent binding sites, it is especially useful to find "bi-ligand" drugs that can bind at both sites simultaneously. Such drugs are likely to bind extremely tightly, inactivating the enzyme and ultimately killing the bacteria. [0006] The rapid discovery and development of bi-ligand drugs has been difficult. Bi-ligand drug candidates have been identified using rational drug design, but previous methods are time-consuming and require a precise knowledge of structural features. When searching for a drug that binds to an enzyme at two binding sites, it would be particularly useful to understand how a ligand binds to the enzyme. Specifically, which atoms in the ligand interact with which portions of the enzyme's binding sites? [0007] Recent advances in nuclear magnetic spectroscopy (NMR) have allowed the determination of the three-dimensional interactions between a ligand and an enzyme in a few instances. However, these efforts have been limited by the size of the enzyme and can take years to map and analyze the complete structure of the complexes of enzyme and ligand. [0008] Thus, there is a need to more rapidly identify which atoms in the ligand interact with which portions of the enzyme binding sites so that focused combinatorial libraries can be generated and screened for more effective drugs. The present invention satisfies this need and provides related advantages as well. SUMMARY OF THE INVENTION [0009] The present invention provides a method for rapidly identifying drug candidates that can bind to an enzyme having at least two binding sites. The first site on the enzyme is the "common ligand site" where a known ligand can bind to the enzyme, as well as to other related enzymes. The second site is a "specificity ligand site" adjacent to the common ligand site. Thus, the method identifies bi-ligand drug candidates that can bind at both the common ligand site and the specificity ligand site. As a result, the candidates can bind with high affinity to the enzyme. As a further result, the candidates can be used to bind to related enzymes sharing a similar common ligand site. [0010] The bi-ligand drug candidates are screened from a combinatorial library. Like other combinatorial libraries, a number of diverse compounds can be generated off of a core structure. In the case of a bi-ligand library, this core structure can be a mimic of the common ligand. The mimic can then be derivatized with varying groups at a selected point to generate the diversity of drug candidates in the library. The library is "focused" by optimizing the specific points on the mimic where variation occurs. [0011] The optimal points of variation on the ligand are identified by determining which atoms are proximal to the specificity ligand site when the mimic is bound to the common ligand site. These atoms are identified by first determining which amino acids of the enzyme are proximal to the specificity ligand site, and then identifying which atoms on the bound common ligand mimic are proximal to these amino acids. NMR methods using the nuclear Overhauser effect (NOE) are particularly useful for identifying proximal atoms. Accordingly, this technique has been named Nuclear Magnetic Resonance-Structure Oriented Library Valency Engineering or NMR-SOLVE.sup.SM. As a result of NMR-SOLVE.sup.SM, the identified proximal atoms can then be used as a point for variation to generate a focused combinatorial library of high affinity drug candidates that can bind to both the common ligand site and the specificity ligand site of an enzyme of interest, as well as related enzymes sharing a similar common ligand site. BRIEF DESCRIPTION OF THE DRAWINGS [0012] FIG. 1a shows a schematic enzyme 1 having a specificity ligand site (SL site) 1a and a common ligand site (CL site) 1b. For purposes of illustration, an interface region 1c is shown between the SL site 1a and the CL site 1b. Enzyme atom 1d is within the interface region 1c and enzyme atom 1e is outside the interface region 1c. FIG. 1b shows a specificity ligand (SL) 2 bound to the SL site 1a and a common ligand (CL) 3 bound to the CL site 1b. The CL 3 has a reactive atom 3a within a reactive region 3b. A nonbound common ligand mimic (CL mimic) 4 is also shown, having individual atoms 4a, 4b. [0013] FIG. 2 illustrates the first stage of identifying an atom 1d of the interface region 1c that is proximal to the reactive atom 3a of the CL 3. In FIG. 2a, the reactive atom 3a of the CL 3 is perturbed. In turn, a nearby atom 1d in the interface region 1c of the enzyme 1 becomes perturbed, as shown in FIG. 2b. Atom 1e, which is outside of the interface region 1c , is more distant from the reactive atom 3a and is not detectably perturbed. Thus, detection of the perturbation of nearby atom 1d allows its identification as an atom proximal to the reactive atom 3a of the CL 3. [0014] FIG. 3 illustrates the second stage of identifying which CL mimics are proximal to the interface region 1c and identifying an atom 4a of the CL mimic 4 that is proximal to the part of the interface region 1c identified as 1d. In FIG. 3a, the atom 1d previously identified in the interface region 1c is perturbed. An atom 4a in the CL mimic 4 then becomes perturbed, as shown in FIG. 3b, but not atom 4b, which is more distant from interface atom 1d. Consequently, an atom 4a of the CL mimic 4 is identified that is proximal to the part of the interface region 1c identified as 1d. As a further benefit, FIG. 3c illustrates that the second stage can determine whether a particular CL mimic 5 binds to the CL site 1b, but does not bind proximally to the interface region 1c. Thus, if interface atom 1d is perturbed, it will be too distant from atoms of the distally binding CL mimic 5 so that atom 5a does not become perturbed [0015] Once a proximal atom 4a has been identified in the CL mimic 4, FIG. 4a illustrates a focused combinatorial library 6 of bi-ligand drug candidates 6a, 6b, 6c and 6d, having varying substituent groups attached to the identified proximal atom 4a. These are in contrast to drug candidates 7a to 7h from an unfocused combinatorial library 7 based on substitutions at other atoms such as 4b of the CL mimic 4 or substitutions to a distally binding CL mimic 5. Upon screening the focused library 6, a particular drug candidate 6c is selected for high binding affinity to the enzyme. As shown in FIG. 4b, the drug candidate 6c consists of the CL mimic 4 attached to a SL-binding moiety 8 through a linker 9. The drug candidate 6c can then bind the enzyme 1 tightly at both the SL site 1a and the CL site 1b [0016] FIG. 5 illustrates a variant method for identifying a proximal atom 4a. In FIG. 5a, a CL 3 is bound to the CL site 1b in the presence of nonbound CL mimic 4. An atom 3a of the CL 3 is perturbed with radiofrequency irradiation, transferring energy to a nearby atom 1d of the interface region 1c in an NOE experiment. The CL 3 then unbinds, as in FIG. 5b. In FIG. 5c, a CL mimic 4 then binds to the CL site 1b, so that the energy is then transferred from the atom 1d of the interface region 1c to a nearby atom 4a of the CL mimic 4, as shown in FIG. 5d. As a result, the variant method allows identification of an atom 4a of the CL mimic 4 that is proximal to the interface region 1c. [0017] FIG. 6 illustrates the identification of amino acids at the interface region. FIG. 6a shows the results of a 2D-HSQC NMR experiment with TROSY for an NADH-bound dehydrogenase. FIG. 6b shows the results of a comparable experiment with an NADD-bound dehydrogenase, where the NADD has a deuterium-for-hydrogen substitution on the 4-carbon position of the nicotinamide ring. As is well known, the 4-carbon of NADH is close to the interface region of dehydrogenases. In both figures, the x-axis represents the .sup.1H chemical shift and the y-axis represents the .sup.15N chemical shift. As a result of chemically perturbing the NADH to NADD, changes in the chemical shifts--represented by the two arrows--permit identification of amino acids at the interface region of the enzyme. [0018] FIG. 7 illustrates the identification of the proximal atom in the CL mimic using 3D-HSQC NOESY with TROSY. In FIG. 7a, the horizontal axis represents the .sup.1H chemical shift of protein, the vertical axis represents the .sup.15N chemical shift of protein and the oblique axis represents all .sup.1H chemical shifts. The broken arrows in FIG. 7a represent NOEs 10 resulting from radio frequency irradiation of the sample. These NOEs 10 are to ligand proton with chemical shift 11 The NOEs 10 allow identification of a proximal atom 4a of a CL mimic, shown in FIG. 7b. [0019] FIGS. 8 to 13 illustrate the results discussed in Example II. [0020] FIG. 8a shows the 2-D structure of the CL mimic designated TTE0001.002.D2. The positions of protons H1, H2, H3, H4, G1, G2 and G3 on the CL mimic are indicated. FIG. 8b shows a Lineweaver-Burk plot for the compound in competition with NADH for lactate dehydrogenase. [0021] FIG. 9 shows the 2-D structures of the SL mimic 2,6-pyridinedicarboxylate (2,6-PDC) (FIG. 9a), the common ligand NADH (FIG. 9b), and CL analog ACNADH (FIG. 9c), which is used for perturbation by chemical modification ACNADH differs from NADH by a substitution from amine in NADH to methyl in ACNADH, shown at the far right of FIGS. 9b and 9c. Furthermore, individual protons H.sub.1'A, H.sub.8A and H.sub.2N on ACNADH and corresponding atoms on NADH are indicated. Continue reading... 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