International Journal of Drug Design and Discovery Volume Issue April June

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1 International Journal of Drug Design and Discovery Volume 4 Issue 2 April June Fragment Based Drug Discovery - A Tool for Drug Discovery M. P. Toraskar*, N. S. Singasane, J. B. Pichake and Vilasrao Kadam Department Of Pharmaceutical Chemistry,BharatiVidyapeeth s College of Pharmacy, Sector 8, C.B.D, Belapur, Navi Mumbai , Maharashtra, India. ABSTRACT: Sinc ce the early 1990s, several technological and scientific advances such as combinatorial chemistry, high-throughput screening and the sequencing of the human genome have been developed for discovery of new drug entity. However, the return on investment in terms of marketed products has not met expectations. Fragment-based drug design is another tool for drug discovery that has emerged in the past decade. The goal is to build drug leads in pieces, by identifying small molecular fragments and then either linking them or expanding them. Fragment-based drug design is a new approach that has been successfully applied to challenging targets, such as protein-protein interactions. Fragment-based drug design uses x-ray crystallography, NMR or other physical techniques to screen fragment libraries for specific binding to a target protein. Knowledge of exactly how the fragments bind to the protein target allows the hits to be optimized by growing the fragments or by combining and linking different fragments. KEYWORDS: Fra agment based drug discovery, High throughput screening, Tethering, Non Tethering, Nuclear Magnetic Resonance, X-Ray Crystallography. Introduction Over the past few years a novel method for designing new leads in drug discovery has been developed as a fragment- of new based drug discovery (FBDD) approach. The advent technologies, such as high-throughpu ut screening (HTS) and combinatorial chemistry, has produced new tools for the discovery of biologically active molecules. The basic method of fragment based drug discovery is shown in Figure 1. The definitionn of a fragment varies, but usually refers to molecules weighing less than Da, with fewer than heavy atoms. In the past decade, fragment-baserational and focused approach thatt concentrates on the drug discovery has emerged as a more quality, rather than the quantity of hits and leads. Small- of molecule drug discovery has always been a struggle attrition, but in the past few years pressures have mounted to increase efficiency at all stages of the process. A potential solution for lead identification and optimization, fragment- The based lead discovery is becoming increasingly popular. goal is to build drug leads in pieces, by identifying small molecular fragments and then either linking them or expanding them to generate a lead (Drug) molecule. Fragment-based drug discovery (shown in fig. 2) is a new approach that has been successfully applied to * For correspondence: Dr. MrunmayeeToraskar Tel: ; Mob: Fax: , rupalitoraskar@yahoo.com, mrunmayeetoraskar@gmail.com, namrata.singasane@gmail.com challenging targets, such as protein-protein interactions. While 3-D protein structures have been used in drug discovery for many years now, fragment-based drug discovery usess x-ray crystallography, NMR, MS or other physical techniques to screen fragment libraries for specific binding to a target protein. Knowledge of exactly how the fragments bind to the protein target allows the hits to be optimized by growing the fragments or by combining and linking different fragments to generate a hit, whichh is subsequently optimized to produce a lead, and (in the best cases) a drug 1,2. Fig. 1 Fragment Based Drug Discovery. Finding novel compounds as starting points for optimization is one of the major challenges in drug discovery research. Fragment-based methods have emerged in the past 10 years as an effective way to sample chemical diversity with a limited number of low molecular weight compounds. In FBDD about low molecular 1083

2 1084 International Journal of Drug Design and Discovery Volume 4 Issue 2 April June 2013 weight fragments are typically screened using biophysical techniques. Ideally, the experimental binding mode will be established by X-ray crystallography y or NMR. Fragments are then grown to form new interactions. Although fragment hits have low potency due to their small size, they form high quality interactions and can readily be optimized into potent lead molecules and ultimately clinicall candidates. Limitations of HTS High-throughput screening (HTS) (Shown in fig. 3) is the traditional approach for the discovery of most medicinal chemistry leads. Despitee its many successes, the method has its drawbacks. It is a complex and expensive method. The HTS approach is also limited in terms of the number of complex fully-formed compounds that can practically be made and stored in a collection. Even very large libraries represent only a small fraction of the vast universe of potential compounds that could be made. The quality of the leads emanating from HTS has also fallen below expectations, with many requiring extensive optimization and even then failing at the last hurdle. Thus, after a decadee of HTS, there is a need for a more efficient approach to lead discovery. Also there is a increasee in a molecular weight while lead optimization 3. Fig. 2 Fragment Based Drug Design 4. Fig. 3 Conventional (HTS) and Fragment Based Drug Design 5 5.

3 M. P. Toraskar et al : Fragment Based Drug Discovery A Tool for Drug Discovery 1085 Steps in Fragment Based Drug Discovery Fragment Library Design In contrast to HTS, fragment-based lead discovery involves the identification of low molecular weight chemical fragments (also known as scaffolds or templates) from very much smaller compound libraries which are tested for binding affinity against the target of interest. These fragments are then combined or optimized to generate lead compounds. While designing the library, Lipinski s Rule of Five and Rule of Three is applied to evaluate drug likeness or determine if a chemical compound with a certain pharmacological or biological activity has properties that would make it a likely orally active drug in humans. The rule describes molecular properties important for a drug's pharmacokinetics in the human body, including their absorption, distribution, metabolism, and excretion ("ADME"). However, the rule does not predict if a compound is pharmacologically active 6.To promote lead like properties fragment usually comply with LIPINSKI S RULE OF 5 and LIPINSKI S RULE OF 3.The requirements are as below: LIPINSKI S RULE OF 5: i) ClogP equal to 5; (ii) Not more than 5 hydrogen bond donors (OH and NH groups); (iii) Not more the 10 hydrogen bond acceptors (notably N and O); and (iv) A molecular weight under LIPINSKI S RULE OF 3: (i) ClogP equal to 3; (ii) Not more than 3 hydrogen bond donors and three acceptor; and (iii) A molecular weight under In FBDD, as fragment become smaller their statistical likelihood of showing binding to features within any site becomes higher although their binding to features within any site becomes higher although their binding efficiency may diminish. Fragment-based lead discovery finds that the resulting molecules are likely to have a higher ligand efficiency than molecules discovered through conventional methods. An investigation of roughly 150 ligands, many of them drugs, reveals that the free energy of binding increases roughly linearly with increasing ligand size up to about 15 atoms, beyond which there is very little increase; the maximum free-energy contribution per heavy atom is roughly 1.5 kcal/mol 9. That study, as well as an earlier study, inspired the concept of ligand efficiency, defined as the ratio of the free energy of ligand binding to the number of non-hydrogen atoms. Hopkins et al. have defined ligand efficiency (LE) simply as: LE= ΔG/ HAC -RTln(IC 50 )/ HAC Where ΔG is the free energy of binding of the ligand for a specific protein, HAC is number of heavy atoms in the ligand and the IC 50 represents the measured potency of the ligand for the protein 9.This has been extended to other properties such as polar surface area and molecular weight. Because fragments should have higher ligand efficiencies than typical high-throughput screening (HTS) hits, they should produce ultimately more efficient drugs, which should have better pharmaceutical properties (lower molecular weight, better pharmacokinetics, lower toxicity, etc.). Fragments are also less likely to have interfering functionality that would prevent them from binding to a target protein 9,10,11. Fragment Screening Techniques A small, less complex molecule is as a rule a weaker binder, which means that fragments are more difficult to detect using conventional HTS techniques. One approach is to screen compounds at high concentrations ( μM compared with the 10-30μM concentrations typically used in HTS) 12. Instead of conventional bioassays, various biophysical screening methods are used including Nuclear magnetic resonance (NMR), X-ray crystallography and mass spectrometry (MS). NMR and X-ray crystallography are particularly suitable as they can provide significant structural understanding of the ligand-protein binding event; this is critical in prioritizing fragment hits and optimizing them into leads. Method employed to identify low molecular weight fragment are: i)tethering; and ii)non-tethering. There are two methods under nontethering method: i) NMR; and ii)x-ray crystallography. Tethering The basic method of Tethering is shown in Fig To facilitate the drug discovery process, many researchers are turning to fragment-based approaches to find lead molecules more efficiently which allows for the identification of small-molecule fragments that bind to specific regions of a protein target. These fragments can then be elaborated, combined with other molecules, or combined with one another to provide high-affinity drug leads. To be useful for drug discovery, fragments should be small (molecular weight preferably lower than 250 Da), heavily functionalized, and contain no toxicophores 14,15. Fragments for Tethering must also contain a thiol or disulfide bond. Tethering relies on reversible covalent bond formation between the fragment and the protein of interest. Tethering requires relatively little protein mg to screen more than 10,000 fragments.

4 1086 International Journal of Drug Design and Discovery Volume 4 Issue 2 April June 2013 Fig. 4 Tethering. The target protein contains a cysteine residue within or near the targeted site, it can be used directly. Otherwise, site-directed mutagenesis can introduce a cysteinee residue. The cysteine residue should generally be within 5 to 10 Ǻ of the site of interestt and should be relatively surface exposed to facilitate thiol-disulfide exchange. Once the cysteine is in place, the protein is reacted with a library of disulfide-containinconditions. In theory, the cysteine should form a disulfide fragments under partially reducing with each of the fragments, and the exchange should occur rapidly. Assuming that the reactivity of the disulfide in each fragment is equivalent and assuming no noncovalent interaction between the fragments and the protein, the mixture at equilibrium should consist of the protein disulfide-bonded to each fragment in equal proportion. However, if one of the fragments has inherent affinity for the protein of interest, and if it binds to the protein near the introduce cysteine, then the thiol-disulfide equilibrium will be shifted in favor of the disulfide for this fragment, and this protein-fragment complex will predominate. In other words, the fragment will be selected by the protein 13,16. Non-Tethering method NMR Screening New methodologies for NMR screening (a) Reporter screening By this method, (shown in fig. 5) not the binding to the protein target of the test compounds is directly observed, but the ability of a test compound to displace a known ligand that is added to the mixture of protein and test compounds as a reporter ligand or spy molecule, which binds to the protein with medium affinity. Bound reporter ligands can be readily distinguished from unbound ligands by their resonance signals 17,18. Fig. 5 In this experiment, the resonance signals of a reporter ligand (triangles) are detected in the presencee of the target protein and test compounds in 1D proton spectra. (a) Without protein target, the signals of the reporter ligand are sharp, as commonly observed for small, unbound molecules. (b) In the presence of the target protein but in the absence of test compounds, the reporter ligand is bound to the target protein with moderate affinity, and its relaxation rate is increased, as evidenced by the severe line broadening of the reporter ligand resonances in the NMR spectrum. (c) After adding test compounds that bind to the same site as the reporter ligand with higher affinity, the reporter ligand is displaced from the binding site, so that it becomes unbound. Unbound reporter ligand can be readily distinguished from bound reporter ligand by its sharp resonances in the NMR spectrum. If testt compounds are added that do not bind to the protein target, bind more weakly than the reporter ligand, or bind to a different, noncompetitivee binding site, the test compounds would not displace the reporter ligand from its binding site, and therefore it would remainbound to the target. This would be manifested by broad lines in the NMR spectrum of the reporter ligand, as seen heree 17,18.

5 M. P. Toraskar et al : Fragment Based Drug Discovery A Tool for Drug Discovery 1087 Fig. 6 Principle of the SLAPSTIC experiment. Small, unbound organic compounds in solution have typically sharp resonances (spectrum at the top). When they bind to a paramagnetic spin-labeled protein target, their signal intensity is drastically reduced or completely quenched by paramagnetic relaxation enhancement (spectrum at the bottom) 19,20. (b) Spin labels for NMR screening Spin labels can be used to identify and characterize intermolecular interactions. This method (shown in Fig. 6) utilizes a spin labeled compound as a first-site ligand. Screening this complex with a library of compounds allows identification of compounds that bind simultaneously with the first; spin labeled ligand, in a neighboring binding site (second site). Second-site ligands can then be identified from quenching of their NMR signals by the spin-labeled first ligand 19,20. (c) NMR screening based on methyl group chemical shifts As an alternative to NMR screening by observation of protein target resonances in 2D [ 15 N, 1 H]-HSQC spectra, Fesik and coworkers suggested to monitor 13 C/ 1 H chemical shift changes of methyl group resonances in 2D [ 13 C, 1 H]- HSQC spectra. Selective methyl group labeling on a perpetuated background is advantageous for screening high molecular weight protein targets (MW > 50 kda). Application of this method demands labeling of the methyl groups with 13 C. In NMR binding studies that monitor methyl group chemical shift changes, the sensitivity is increased about threefold compared with the corresponding experiments based on 15 N/ 1 H chemical shift observation 21. (d) 3-FABS 3-FABS (three fluorine atoms for biochemical screening) shown in Fig. 7 allows rapid and reliable functional screening of compound libraries, performed at protein and substrate concentrations comparable to the ones utilized by standard HTS techniques. 3-FABS monitor 19 F signal intensitiess rather than 1H signals. The experiment is only applicable to enzymes and allows measurement of accurate IC 50 values.3-fabs require labeling of the substrate with a CF 3 moiety. During the assay, the enzymatic reaction is performed with the CF 3 -labeled substrate and quenched after an established delay that depends on the enzyme and their action conditions. Fluorine NMR spectroscopy is then used to monitor the substrate and the enzymatically modified reaction product 22,23. Fig. 7 Schematic diagram showing the principle of the 3- FABS method. First, fluorine-containing moiety, like CF 3 3, is introduced in the substrate as a sensor or reporter group. The chemical modification of the substrate by the enzyme (here represented as addition of a chemical fragment denoted A ) induces changes in the electronic cloud of the CF 3 moiety. This results in distinct chemical shifts for the 19 product and the substrate F signals, and therefore chemical shift changes in the 19 F NMR spectra [22, 23].

6 1088 International Journal of Drug Design and Discovery Volume 4 Issue 2 April June 2013 (e) Affinity tags Protein protein inter actions can be detected by a novel NMR reporter system based on affinity tags. In this approach, one of the binding partners is fused to a ligand- weight reporter ligand is bound. Protein protein interactions are then monitored via changes in the NMR relaxation of binding domain, wheree a medium-affinity, low-molecular the reporter ligand because the parameters of the reporter ligand spectra dependd on the molecular weight of the protein ligand complex (among other factors, e.g. affinity constant, protein and ligand concentration), changes of these spectral parameters can be used to probe changes in the molecular composition of the ternary protein protein ligand complex. One of the principal advantages of this technique is the relatively low consumption of unlabeled proteins 24. (f) SAR by NMR Using Chemical Shift Mapping SAR by NMR is carried out in five main steps (shown in Fig. 8): (i) Identification of ligands with highh binding affinity from a library of compounds by using 2D 1H-15N HSQC; (ii) Optimization of ligands by chemical modification; (iii) Identification of ligands binding in the presence of saturating amounts of optimized ligands from step 2 by using 2D 1H- 15N HSQC; (iv) Optimization of ligands for the second site; and v) Tethering the two ligands from step 2 and step 4 in various positions and checking again for binding 25. such as 0 ppm to -1 ppm) as shown in Fig. 9. If ligand binds, saturation will propagate from the selected receptor protons to other protons of receptor via spin diffusion and then the saturation is transferred to binding compounds by cross relaxation at the ligand-receptor interface. A control experiment is done by saturating a region of the spectrum thatt does not contain signal. The resulting difference spectrum yields only those resonances that have experienced saturation, namely those of the receptor and those of the compound that binds to the receptor. The receptor is typically present at very small concentrations so its resonance will not be visible 26. Fig. 8 SAR by NMR. (g) STD NMR Saturation Transfer Differencee STD involves selectively saturating a resonance that belongs to thereceptor (must find a region of the spectrum that contains only resonances from receptor Fig. 9 STD NMR 26. (h) WaterLOGSY (water-ligand observation by gradient spectroscopy) A variant of STD where saturation transfer involves bound water instead of protein i.e. saturate water resonancesnmr experiment that utilizes the large bulk water magnetization to transfer magnetization via the protein ligand complex to the free ligand molecules. Magnetizationn transfer from bulk water to ligand occurs via labile receptor protons within and remote from the ligand-bindingg site as well as from long-lived water molecules within the binding pocket. In this experiment, the resonances of the binding compounds appear with opposite sign than those of the nonbinding compounds and can thus be used as an NMR binding assay 27. X-ray crystallography Fragment-based drug discovery using x-ray crystallography as shown in fig.10 requires thatt a protein target that can readily form crystals with or without ligand and its three dimensional structure is available. It uses X-ray crystallography to screen fragment libraries for a target protein. A protein crystal is

7 M. P. Toraskar et al : Fragment Based Drug Discovery A Tool for Drug Discovery 1089 soaked in a solution containing about 10 compound fragments, which range in size from Daltons, for 3 to 24 hours.if a fragment binds to the protein in the crystal, the complex causes a change in the x-ray diffraction pattern as compared to the native, or unbound, diffraction pattern. Differencee maps between the complex and the native data show where the fragment is bound on the protein structure. The fragment must bind specifically to the protein molecules in the crystal to be detected. The exact binding constant cannot be determined by this method as in NMR, but subsequent refinement determines the fragments 3-D structure in the protein binding pocket. X-ray has an advantage because it provides knowledge of how the fragments bind in the active site. For protein crystallography, ten to a hundred milligrams of purified protein are needed to set up and grow large numbers of protein crystals, at least 100 or so. Each crystal is soaked in a sample of five to 10 compound fragments, mounted, and cryo-cooled for X- ray data collection. High-speed data collection can be accomplished at synchrotron facilities. Using beam line facility we can typically screen our entire library, about 1,000 fragments, in about 24 to 48 hours. X-ray crystallography has the advantage of defining the ligand-binding sites with more certainty than NMR and the binding orientations of the molecular fragments play a critical role in guiding efficient lead optimization programs. The concentrationn of the molecular fragment is typically greater than 20 mm, reflecting the low-affinity that is expected. Fragment libraries can be screened as singlets or in cocktails using X-ray crystallography. As the output from an X-ray experiment is a visual description of the bound compound (its electron density) it is possible to screen cocktails of compounds without the need to deconvolute. An optimum cocktail size is typically between 4-8 and is defined by the tolerance of the protein crystals to organic solvents and the concentration at which you wish to screen each fragment. For example, if the maximum tolerated solvent concentration is 240 mm then you can screen 8 compounds each at a concentration of 30 mm. Fig. 10 X-Ray Crystallography. The fragment must bind specifically to the protein molecules in the crystal to be detected. Nonspecific binding, often a major problem in other high-throughput screening, is invisible in the x-ray data because nonspecific binders bind randomly and essentially do not affect the x-ray diffraction signal. Fragments identified using this technology bind too weakly to be considered hits their binding affinity is usually in the high µm to mm range. There are two basic strategiess to create higher affinity compounds from the fragments: extension and linking. High-affinity compounds can also be created by linking low-affinity fragments together. This requires multiple fragments that bind in close proximity, knowledge of their binding modes, and orientations, and finally, a suitable linker that can maintain the binding interactions of the individual fragments. NMR or mass spectroscopy is used more commonly in conjunction with fragment linking. Variations on this include fragment self-assembly in the presence of a binding site that brings two fragments into close proximity. where reactivee fragments can self-assemble Extension or growing consists of adding functionality to an initial fragment that explores the adjacent areas of the binding pocket to find favorable interactions that increase the binding affinity. This has been used successfully to develop inhibitors to a number of targets, including p38 MAP kinase inhibitor (Astex), DNA gyrase (Hoffman-La and urokinase (Abbott). Roche), Erm methyl transferase (Abbott), Knowledge of where specific chemical fragments bind to a protein can be used in a broad range of computational techniques. The information can be used in a variety of computational techniques, such as pharmacophore searches of available chemical libraries. The empirical data reduces the combinatorial complexity of the problem dramatically. These searches allow researchers to find or synthesize compounds that hopefully validate the binding hypothesis and extend the compound fragment into other parts of the pocket 28,29. Fragments to Lead Development A variety of strategies are available to do this: fragment evolution, fragment linking, fragment self- assembly and targeted libraries. Various substitutions or expansions are made to the initial hit (or fragment, in this case) in order to improve affinity and other properties. Fragment merging and linking generally involve combining elements from a fragment with elements from a known substrate, inhibitor, or another fragment to create a hybrid molecule. This approach can improve molecules potency as well as physicochemical or ADME (absorption, distribution, metabolism, and excretion) properties.

8 1090 International Journal of Drug Design and Discovery Volume 4 Issue 2 April June 2013 Fragment Evolution Initial fragments identified by direct binding techniques are built up into larger, more complex molecules that target additional interactions in the active site of the protein. This evolution leads to more tightly binding molecules which can be furtherr developed ncluding optimization of their drug- and like properties, for example, selectivity, oral activity efficacy. Fig. 11 Fragment Evolution (a) Fragment 1 binds to the receptor at one site. (b) The lead molecule is evolved by building away from the starting fragment and making good contact with the upper surface and then by growing into a second pocket. Identification of initial fragments using a direct binding technique is most useful if it is supported by some information about the binding mode of the fragment. With this type of informationn it is possible to develop hypotheses about how to build up larger and more complex molecules that target additional interactions in the active site of the protein. This evolution leads to tighter-binding molecules, which can then be further optimized. Fragment Linking Two fragments are identified that bind in separate sites but are close enough together to be chemically linked resulting in a larger, higher-affinity molecule. Here two fragments have been identified that bind in separate binding sites that are close enough to each other to be chemically linked. experimental energetics associated with optimally linked fragments has suggested thatt the rigid body entropy losss on protein binding constitutes a barrier of around three orders of magnitude to the binding affinity, and that this barrier is essentially independent of molecular mass. The analysis implies that there should be a super-additives effect when two fragments are linked in an optimal fashion 30. Fragment Self-Assembly Separate fragments with complementary functional groups can thus be assembled in the presence of the target protein to form a larger, more potent molecule. In effect, the protein catalyzes the synthesis of its own inhibitor by selecting fragments that can cross-link to each other when brought close together. Fig. 13 Fragment Self-Assembly (a) Fragment 1 and 2 bind to the receptor sites simultaneously with reacting groups positioned within conformational reach of each other, ncreasing the effective molarity of reacting groups. (b) Lead molecule formed in the active site. Fig. 14 Lead progression via fragment optimization (a) Existing lead molecule discovered by fragment-based approach. (b) Lead molecule re-engineered to address optimization of a particular property (for example, selectivity, cell-based activity, oral activity or efficacy). The use of reactive fragments that are capable of selfas a protein) is a large and growing field. Here two separate fragments are linked together to form a larger and more assembly in the presence of a template molecule (such active inhibitor in the presence of the protein target itself 31. Lead Progression by Fragment Optimization Here we optimize or modify properties of lead moleculee for a particular property such as selectivity, oral activity or efficacy other than just binding potency of a lead. Fig. 12 Fragment Linking (a) Fragment 1 binds to the receptor at one site. (b) Fragment 2 binds to the receptor at an adjacent site. (c) Fragments joined together by linking group that allows the lead molecule to span both sites. For this to be an efficient lead-identification approach, one needs to both identify the initial fragments and also have a process that allows the appropriate linking to be achieved in an efficient manner. Such additives require that the contribution from the linker is negligible and that the loss in rigid-body entropy on binding of all components to the enzyme is very small. Recently, an analysiss of the

9 M. P. Toraskar et al : Fragment Based Drug Discovery A Tool for Drug Discovery 1091 Conclusion Fragment based drug discovery has been established asa powerful alternative to traditional high throughput screening techniques for identifying drug lead. The process continues to expand and evolve as understanding of protein ligand interaction and molecular recognition increases.the idea of fragment based drug discovery is that small molecules with an inherently lower binding affinity would make better starting points for a chemistry program. Starting from a low molecular weight compound with optimum binding to the protein, chemical modifications that increase molecular weight can more easily be applied to optimize the drug profile of a compound. The results show that FBDD is a powerful alternative approach for lead finding. Studies have showed the potential of using small weak binders, an approach they term Small is Beautiful, to facilitate the drug discovery cycle. It is also identified that the need for a commitment to screen at high concentrations and the development of NMR screening libraries as two areas of development for successful fragment based drug design with a shorter iteration cycle. The appropriate use of this technique improves ability to critically assess the identification and optimization of lead compound that have potential for generating new therapeutic agent. Abbreviations NMRNuclear magnetic resonance HTS High throughput screening FBDD Fragment based drug discovery MS Mass spectrometry HSQCHeteronuclear Single Quantum Coherence FABS Fluorine atoms for biochemical screening STD Saturation Transfer Difference References [1] Mestres, J.; and Veeneman, G.H. Identification of latent hits in compound screening Collections. J. Med. Chem. (2003), 46, [2] Jan W. F. Wasley, Book Review of Fragment Based Drug Discovery: A Practical approach, J. Med. Chem., (2009), [3] Gribbon, P.; Andreas, S. Highthroughput drug discovery: what can we expect from HTS Drug Discov. Today. (2005), 10, [4] Maly, D.J.;Choong,D.J.;Ellman, J.A. Proc.Natl. Acad.Sci. USA. (2000),97, [5] Erlanson,D.A.;Hansen, K.S.curr opin chem.biol. (2004), 8, [6] Teague, S.J.;Davis, A.M.; Leeson, P.D.; Oprea,T. The Design of Leadlike Combinatorial Libraries. Angew. Chem. Int. Ed. Engl. (1999), 38, [7] Lipinski, C. A., Lombardo, F., Dominy, B. W. & Feeney, P. J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. (2001),46, [8] Congreve, M.; Carr, R.; Murray, C.; Jhoti, H. A rule of three for fragment-based lead discovery? Drug Discov.Today(2003),8, [9] Kuntz, I.D.; Chen, K.; Sharp, K.A.; Kollman P.A. The maximal affinity of ligands. Proc. Natl. Acad. Sci. U.S.A. (1999), 96, [10] Kenneth G. Holden. Review of Lead Generation Approaches in Drug Discovery, J. Med. Chem., (2010), [11] Erlanson D.A.;McDowell, R.S and Tom O Brien. Fragment based Drug Discovery, Journal of Medicinal Chemistry, (2004), 47, [12] Maly,D.J.; Choong, I.C.; Ellman, J.A. Combinatorial target-guided ligand ligand assembly: Identification of potent subtype-selective c-src inhibitors. Proc Natl Acad Sci USA.(2000), 97, [13] Erlanson D.A.; Braisted A.C.; Raphael D.R.;Randal,M.; Stroud, RM.;, Gordon E M.; Wells J. A.Sitedirected ligand discovery. Proc. Natl.Acad. Sci. USA(2000), 97, [14] Fejzo,J.; Lepre, CA.; Peng, JW.; Bemis, GW.; Ajay,Murcko, M.A., and Moore, J.M. The SHAPES strategy: an NMR-based approach for lead generation in drug discovery. Chem. Biol.(1999),6, [15] Jacoby,E.; Davies, J.; Blommers M.J.;. Design of small molecule libraries for NMR screening and other applications in drug discovery. Curr. Top. Med. Chem. (2003),3, [16] Erlanson, D.A.; Wells, J.A.; Braisted, A.C. TETHERING: Fragment-Based Drug Discovery, (2004), [17] Dalvit, C.;Flocco, M.; Veronesi, M.; Stockman, B.J. Fluorine-NMR competition binding experiments for highthroughput screening of large compound mixtures. Comb. Chem. High Throughput Screen. (2002), 5, [18] Dalvit, C.;Fagerness, P.E.; Hadden, D.T.; Sarver, R.W.; Stockman, B.J. Fluorine-NMR experiments for highthroughput screening: theoretical aspects, practical considerations, and range of applicability. J. Am. Chem. Soc. (2003), 125, [19] Jahnke, W.; Spin labels as a tool to identify and characterize protein ligand interactions by NMR spectroscopy. Chembiochem(2002), 3,

10 1092 International Journal of Drug Design and Discovery Volume 4 Issue 2 April June 2013 [20] Jahnke, W.;Perez, L.B.; Paris, C.G.; Strauss, A.; Fendrich, G.; Nalin, C.M. Second-site NMR screening with a spinlabeledfirst ligand. J. Am. Chem. Soc. (2000), 122, [21] Hajduk, P.J., D. J. Augeri.; Mack,J.; Mendoza, R.; Yang, J.; Betz, S.F.; S. W. Fesik. NMR-based screening of proteins containing 13C labeledmethyl groups. J. Am. Chem. Soc.(2000),122, [22] Dalvit, C.,Ardini,E., Flocco, M., Fogliatto, G.P.,Mongelli, N., and Veronesi,M. A general NMR method for rapid, efficient,and reliable biochemical screening. J. Am. Chem. Soc.(2003), 125, [23] Dalvit, C.;Ardini E, Fogliatto,G.P.;Mongelli, N.; Veronesi, M.; Reliable high-throughput functional screening with3- FABS. Drug Discov. Today. (2004), 9, [24] Ludwiczek, M.L.;Baminger, B.; Konrat, R.;NMR probing of protein protein interactionsusing reporter ligands and affinity tags. J. Am. Chem. Soc. (2004),126, [25] Shuker, S. B., P. J. Hajduk. Meadows,R. P.; S. W. Fesik, S.W."Discovering high-affinity ligands for proteins: SAR by NMR." (1996), Science 274(5292), [26] Mayer, M.; Meyer, B."Characterization of ligand binding by saturation transfer difference NMR spectroscopy." (1999), Angewandte Chemie-International Edition 38(12), [27] Dalvit, C.; Pevarello,P.; Tato,M.; Veronesi,M.; Vulpetti, A.;Sundstrom, M. "Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water." Journal of Biomolecular Nmr.(2000), 18(1), [28] Nienaber, V. L.; Richardson, P. L.; Klighofer, V.; Bouska, J. J.; Giranda, V. L.; Greer, J. Discovering novel ligands for macromolecules using X-ray crystallographic screening. Nat. Biotechnol. (2000), 18, [29] Congreve, M. S.; Davis, D. J.; Devine, L.; Granata, C.; O Reilly, M.; Wyatt, P. G.; Jhoti, H. Detection of ligands from a dynamic combinatorial library by X-ray crystallography. Angew. Chem.,Int. Ed.(2003), 42, [30] Murray, C. W.; Verdonk, M. L. The consequences of translational and rotational entropy lost by small molecules on binding to proteins. J. Comput. Aided Mol. Des. (2002), 16, [31] Ramstrom, O. & Lehn, J. M. Drug discovery by dynamic combinatorial libraries. Nature Rev. Drug Discov. (2002), 1,