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...

Info

The initial publications on lead-like properties suggested that small, simple molecules would have higher hit rates and lower affinities than the typical larger, more complex molecules used in classic high-throughput screening (HTS) discovery programs. This concept has now been demonstrated by SGX Pharmaceuticals 49 , who reported typical fragment screening hit rates of 15 for several different target classes. Such high hit rates suggest that it is not necessary to rely on previously known...

Bivalency in the Immune System

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...

Metalmediated

Carbonic anhydrases are active drug targets for a variety of diseases from glaucoma to cancer, and these zinc-containing metalloenzymes also contain a deep active site that can bind a variety of metal chelators. Simple molecules such as ben-zenesulfonamide bind to human carbonic anhydrase I (hCA-I) with low-micromo-lar affinity 48 . Researchers at North Dakota State University in Fargo found that they could enhance this affinity by two orders of magnitude by making two-prong inhibitors 49...

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...

Pyramid Evolution Integration of Crystallography and NMR

The examples described above show that the Pyramid approach, based on crystal-lographic screening, is well suited for identifying low affinity fragments and has been highly successful in generating hits against a number of different targets. To further expand the potential of our fragment-based hit generation process, we have now broadened our crystallographic screening platform by complementing it with NMR spectroscopy, which is the primary alternative technique able to effec tively detect...

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...

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...

Ic50

Chem. 43 6 - EC 1.1.1.21 aldo keto 20001062-1070 reductase aldose reductase WAY-121365 7.136 IC50 26 184.2 J. Med. Chem. 39 20 - EC 1.13.11.34 lipoxygenase arachidonate 5-1996 3951 -3970 d oxygen ase I ipoxyg enase 244.3 J. Med. Chem. 41 12 - EC 1.4.3.4 flavin 232.3 J. Med. Chem. 41 12 - EC 1.4.3.4 flavin