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MCSS has proved to be a valuable tool in the arsenal ofmethods used for drug discovery. In this chapter, we have focused on the application of MCSS to proteins, but it should be noted that the method has been applied to both DNA and RNA and fruitful results have been obtained 42, 43 . As MCSS finds energetically favorable positions of chemical fragments in a binding site of interest, additional methods are needed to construct ligands from MCSS data. A variety of different approaches have been...

Thymidylate Synthase Proof of Principle

We first applied Tethering to thymidylate synthase (TS). This enzyme converts deoxyuridine monophosphate (dUMP) to thymidine monophosphate (dTMP), an activity essential for DNA synthesis. The cancer drug 5-fluorouracil irreversibly inhibits TS, and a selective inhibitor of a non-human form of the enzyme could yield a new antibiotic or antifungal drug 11 . In addition to its biological interest, TS was ideally suited for developing Tethering 12 . It is well characterized both structurally and...

Kdvidity EE KdN1

An example with a bivalent ligand and receptor (N 2) is shown in Fig. 2.2b. Kitov and Bundle 22 proposed an alternative definition of avidity (K vidity,KB), given in Eq. (2) K .ra + 1 Kd,N-1 + + 1 Kd.O-1 (2) where Kd N , Kd,N-i, and Kd,i are the dissociation constants of receptor-ligand species with N receptor-ligand interactions (i N), N-1 interactions (i N-1), and one interaction (i 1), respectively, to the completely dissociated multivalent receptor and multivalent ligand (i 0 Fig. 2.2)....

Ligands Bound to Structured RNA

The Tar RNA stem-loop of HIV is an attractive target for drug discovery. During the viral life cycle, the Tar RNA binds tat protein and facilitates assembly of a competent transcriptional complex. Mei and co-workers screened > 100 000 compounds for inhibitors of the Tar-tat interaction. They identified four ligands that bound the RNA and studied them using ESI-MS 26 . The compounds were shown to make contacts with the three-base internal UCC bulge, through chemical replacement ofthe upper...

Spleen Tyrosine Kinase Case Study

Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in hematopoietic cells. Syk is an inflammatory disease target that controls degranulation of mast cells in asthma 48, 49 . The crystal structure of Syk was obtained by SGX at 2.5 A resolution 50 in the context of our human protein kinase pipeline. While initial crystal forms of Syk were undergoing optimization for crystallographic fragment screening, it was noted that the active site of...

Characterization of Protein Ligand Binding Sites

The MSCS method was not possible before the age of structural biology. Rather, the characterization of protein-ligand binding sites depended on complex biochemical investigations. Primarily through the use of kinetic experiments, these investigations sought to discover several types of information the identities of the residues that constitute the binding site, their arrangement on the protein surface and the affinity of a ligand to that binding site. The identity of residues involved in the...

ESIMS for Linking Lowaffinity Ligands

The low hit rates for RNA targets in traditional HTS assay formats can be traced to difficulties in detecting and accurately measuring low-affinity interactions between small molecules and the RNA. We have developed a high-throughput MS-based assay that directly measures ligand affinities of 0.01-1000.0 M for RNA targets. In contrast to traditional HTS assays, the MS-based assay accurately quantifies binding affinity, stoichiometry, and specificity over a wide range of ligand KD values. This...

Bivalency in the Immune System

Immunology Drugs

IgG and IgE antibodies, prime components of the immune system, are bivalent proteins containing two identical receptors (Fab sites Fig. 2.12) 21 . When binding bivalently to a surface (Fig. 2.12a) or to a soluble bivalent ligand (Fig. 2.12b), we postulate that the enhancement (P) for a given antibody is inversely proportional to the monovalent dissociation constant (K fflnlty) and directly proportional to the effective concentration (Ceff) of ligand near an available receptor (Fig. 2.12). If we...

Discovery of Highly Potent AChE by In Situ Click Chemistry

The target-guided click chemistry approach was first tested with acetylcholine esterase (AChE) (Fig. 15.4). The enzyme plays a key role in neurotransmitter hydrolysis in the central and peripheral nervous system 55, 56 . It has two separate binding sites on either end of a narrow gorge 57 . For fragment assembly by the 326 15 Click Chemistry for Drug Discovery Step 1. Identification of Anchor Molecule 326 15 Click Chemistry for Drug Discovery Step 1. Identification of Anchor Molecule enzyme,...

Dynamic Combinatorial Chemistry The Principle

Cem Combinatorial Chemistry

Dynamic combinatorial chemistry DCC is a recent and rapidly developing approach for ligand or receptor identification, based on the implementation of dynamic assembly and recognition processes 10-20 . It offers a possible alternative to the static approaches of traditional combinatorial chemistry. The method is based on the generation of a dynamic combinatorial library DCL of interchangeable constituents. Such a DCL consists of continually interchanging library members generated by reversible...

SAR by NMR

SAR by NMR was reported in 1996 by Shuker, Hajduk, Meadows, and Fesik 1 as a fragment assembly approach to inhibitor design, using NMR as a structural guide. It is essentially a five-step method 75 that involves screening for weak- binding fragments in two binding sites. In step 1, NMR is used to screen for a weak binding ligand in a first site. In step 2, the ligand is optimized for higher affinity, using NMR structural assays to ensure that binding mode is retained. In step 3, the protein is...

Introduction

Traditional drug discovery usually begins with a search for small molecule hits that demonstrate modest IC50 lt 10 M in vitro activity against the molecular target of interest. Such hits are subsequently optimized into preclinical drug candidates using iterative, trial-and-error methods and or structure-directed design. The most commonly used approaches for finding hits include high-throughput screening HTS of large compound libraries typically 100 000-2 000 000 compounds or modification of...

Acknowledgments

The Authors gratefully acknowledge the support of many people at Astex Therapeutics who have contributed to aspects of the work presented in this chapter. We also thank Chris Murray, Miles Congreve, Ian Tickle, and Tom Blundell for helpful discussions. T.G.D. and R.L.M.v.M. contributed equally to this work. 1 Campbell, S. F. 2000, Science, art and drug discovery a personal perpsective, Clin.Sci. 99, 255-260. 2 Oprea,T. I., Davis, A. M.,Teague, S. J., Leeson, P. D. 2001, Is there a difference...

NMR Screening Using WaterLOGSY

We screened our Drug Fragment Library, Focused Kinase Library and Privileged Fragment Library against a variety of targets including different kinases and a serine protease, using the Water-LOGSY method. Where possible, we set up the experiments in a competition format in which an initial screen for fragment binding was followed by a displacement step with a known tight binder to discriminate fragments binding at the protein active site from nonspecific binders. In order to identify these...