Inhibitors from Progressive Design

A very successful progressive design approach for thrombin is to start from a small building block, known or expected to bind non-covalently in a precise position in the active site, and expand this by progressive addition of substituents. We have called such building blocks needles and have described the discovery of a thrombin-specific needle and its evolution to a full-blown thrombin inhibitor with the use of sequential X-ray structural analyses (see [28]). The details will not be repeated here. The inhibitor, napsagatran, 5 is shown in Fig. 7.5. 

Napsagatran is, with Kj for thrombin of 270 pM and for trypsin 1.9 pM, one of the most potent and selective thrombin inhibitors known and will be used here to illustrate a number of principles. 

the needle itself, amidino-piperidine, has Kj for thrombin of 150 pM and for trypsin 360 pM and thus is 2.4 x selective for thrombin. This contrasts with the classical needle benzamidine, which has fQ for thrombin of 300 pM and 31 pM for trypsin and thus is 10 X selective for trypsin. This is perhaps unexpected, as it could be argued that benzamidine, being planar, is a much better analogue of the substrate arginine guanidinium group. The width of the recognition pocket (measured from 

1) Note added in proof This concept has been published by D.A. Erlanson, A.C. Braisted, D.R. Raphael, M. Randal, R.M. Stroud, E.M. Gor- 

An important principle, much neglected, is that the conformation of a potential inhibitor in solution is also relevant. The observed inhibition constant is a measure of fhe free energy change in fhe whole system on mixing enzyme and inhibitor. It is very unfavorable if fhe inhibitor prefers a conformation in solution of substantially lower energy fhan when bound to fhe enzyme. 

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A research program in progress at Raltech Scientific Services is designed to find inhibitors which will prevent nitrosamine formation in cosmetic products, A review of the literature (4) indicated that the oil phase of emulsions may play an important role in nitrosation chemistry. Thus, results from studies in water alone could be misleading when reduced to practice. 

It is easy to argue from the current eCB literature that pharmacological manipulations of eCB inactivation will be important for human health. It is also important that selective inhibitors of each of the inactivation processes be designed so that mechanistically interpretable studies of these processes can be accomplished. Although significant progress has been made in the development of these agents, it is clear that more selective inhibitors are needed. 

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