Initiate lead optimization

Fig. 7 Recommendations: Identification of reversible, competitive chemical leads for structure-based design of selective PTP inhibitors competitive type of inhibition in both buffer systems (i.e., no influence of Vmaxapp with increasing inhibitor concentration), and they should not be time dependent. In our case, we further require that lead compounds are general PTP inhibitors and consequently test a small battery of different PTPs. In the next round, we analyze for sensitivity to catalase (which would be an exclusion criteria) and include a specificity test by testing for activity against non-PTP enzymes with a catalytic cysteine such as papain (Fig. 8).

Fig. 8 Specificity check of putative PTP inhibitors in papain. OBA showing no inhibition and a non-specific PTP inhibitor demonstrating time-dependent inhibition of papain

Recently, we have as a final test included isothermal titration calorimetry to verify that the compounds indeed bind to the PTPs.

The power of our approach is probably best illustrated by the fact that OBA was selected as a potential lead structure exclusively on the basis of its behavior as a classical, textbook example of a reversible, competitive inhibitor. Indeed, the label on the vial containing the original HTS hit indicated a different structure, and it was not until after structural analysis that the true nature/structure of OBA was revealed.

In addition to the above PTP-specific problems, it appears that there is a general problem related to screening of compound libraries. Thus, further analysis of screening hits often reveals peculiar inhibition properties showing non-competitive binding with poor specificity and little relationship between structure and activity. In a careful analysis of 'general screening hits' it was found that the inhibition of such compounds could largely be eliminated by increasing the concentration of the enzymes by 10-fold despite a 1,000-fold excess of inhibitor (McGovern et al. 2002). Further analysis revealed that such compounds formed aggregates, and it was suggested this may account for the activity of many promiscuous screening hits (McGovern et al. 2002; McGovern and Shoichet 2003). It is advisable to add this simple procedure to our scheme depicted in Fig. 7.

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