Subsite screening

Another contribution of NMR to the gyrase project was based on an approach termed subsite screening by NMR . For gyrase, the situation corresponded to case (1) as defined in section 19.2. Fig. 19.5 (see p. 428) provides a space-filling model of the active site of GyrB in the complex with a phenylnitroindazole as determined by X-ray crystallography... 

The integration of combinatorial chemistry, structure-based library design and virtual screening [268, 269] also resulted in successful applications [270, 271]. It ultimately should result in broader SAR information about directionality and physicochemical requirements of acceptable building blocks. This concept is based on feasible scaffolds for exploring protein subsites using parallel or combinatorial synthesis. 

Other type III peptidomimetic inhibitors of thrombin have been developed from screening leads (166, 167) such as inhibitor (94) (Fig. 15.40). SAR led to the design of (95)Inhibitor (96) was derived from docking studies with the 5-amidino indole nucleus, followed by addition of a lipophilic side-chain to interact with the important Sg subsite of thrombin. The crystal structures of both (95) and (96) in the active site of thrombin shows that the aromatic core, binds in the site as expected, but... 

Two libraries of a-ketoamide derivatives were generated on pins to map the S and S subsites of human heart chymase [234]. The libraries were screened directly on the pins by a method that measured enzyme absorption onto the pins and by an enzyme depletion assay. From the first library which was based on the sequence Z-Ile-Aa2-Aai-Phe-CO-Gly-GIy it was found that Pro-Gly provided the P3-P2 combination that gave rise to potent inhibition and maximum selectivity over chymotrypsin (library 17 Kj 1.0 nM). From the second library of peptidyl-3-fluorobenzylpyruvamides, (F)-Phe-CO-Glu-Asp-Arg-OMe was identified as an inhibitor of the chymase (library 18 K 1 /iM) but it was less active than the corresponding ketoamide. However it exhibited 100-fold selectivity over chymotrypsin [234]. 

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