Application of robotics to steady state enzyme kinetics: analysis of tight-binding inhibitors of dipeptidyl peptidase IV

Using available commercial robotics and instrumentation, we developed a fully automated and rigorous steady state enzyme kinetic assay for dipeptidyl peptidase IV (DPP IV; E.C. 3.4.14.5). The automated assay was validated with isoleucyl thiazolidide, a potent inhibitor of DPP IV with K is=110 nM. Signal window analysis indicated that the assay had a 98% probability of detecting an inhibitor yielding 15% inhibition, with a predicted false positive rate of 0.13%. A mechanistic inhibition version of the automated assay was validated with isoleucyl 4-cyanothiazolidide, a very potent inhibitor of DPP IV. Isoleucyl 4-cyanothiazolidide was a competitive inhibitor of purified porcine DPP IV with K is=1 nM. Similar K is values were obtained for purified rat DPP IV and for DPP IV activity in human plasma from normal and diabetic donors. The pH dependence of K is for isoleucyl 4-cyanothiazolidide yielded a bell-shaped profile, with p K a=5.0 and p K b=7.6. To date, over 100,000 data points have been generated in profiling targeted compound libraries and in the analysis of tight-binding inhibitors of DPP IV. The data also show that robotic analysis is capable of producing full mechanistic inhibition analysis in a timely fashion to support drug discovery.