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Reversible, Competitive Inhibitors 8.1

Challenges

Initially, the pharmaceutical industry was reluctant to enter the PTP inhibitor field, and in particular to developing active site-directed inhibitors. The highly conserved structure of the catalytic domains of even distantly related PTPs with little primary sequence identity in combination with the expectation of a large protein family (Charbonneau and Tonks 1992; Pot and Dixon 1992a; Denu and Dixon 1998) seemed to represent insurmountable obstacles for the development of highly specific, low molecular weight inhibitors for therapeutic use (reviewed in Tonks 2003). This was also the situation in the early days in the kinase field (Cohen 2002). In the latter case, the development of a relatively specific and clinically extremely efficient kinase inhibitor, Gleevec, for the treatment of chronic myelogenous leukemia and other malignant diseases has changed the perception completely in the industry (see chapters by Cohen and Wakeling, this volume).

In our opinion, the major challenge in developing PTP inhibitors is not related to the conserved structure of PTPs as such, but rather to the very nature of the PTP catalytic machinery (Sect. 2). Indeed, we and others have demonstrated that highly selective and potent PTP inhibitors can be made (see Sect. 9). However, while the above-described low pKa value of the catalytic cysteine in PTPs is essential for its function as a nucleophile both in the first step of catalysis and for the susceptibility to redox regulation, this same property is one of the major challenges in the identification and development of PTP inhibitors. Thus, it is a common experience among laboratories that perform high-throughput screenings to get high hit rates when searching for PTP inhibitors, in many cases caused by oxidation or alkyla-

tion of the active site cysteine (Bleasdale et al. 2001; van Montfort et al. 2003) (see below). It should be noted, however, that redox-induced conformational changes of PTPs as discussed above (Sect. 6) may represent a novel opportunity for the design of inhibitors that only influence already activated signaling pathways (Sect. 10).

Another challenge relates to the nature of the pocket of the active site of PTPs. In contrast to kinases, for which inhibitors have been developed that bind to a relatively hydrophobic pocket in the ATP binding site, the active site in PTPs is a water-molecule-filled, fairly shallow, cavity that first turns into a pocket upon closure of the WPD loop (Sect. 2). A prerequisite for this loop closure seems to be binding of the phosphate moiety of pTyr to the P-loop. Thus, many structure-based approaches are based on development of inhibitors that mimic the binding of phosphate to the P-loop. Since pTyr at neutral pH is doubly negatively charged, most competitive PTP inhibitors developed to date are similarly charged molecules. As a consequence, problems with cellular uptake, pharmacokinetic properties, and oral availability may be expected.

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