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)=110nM. 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 pK(a)=5.0 and pK(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.
D000818:Animals; D001667:Binding, Competitive; D018819:Dipeptidyl Peptidase 4 / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D006801:Humans; D007532:Isoleucine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008956:Models, Chemical; D011480:Protease Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D012371:Robotics / Q000379:methods*; D013844:Thiazoles / Q000378:metabolism / Q000494:pharmacology
D011480:Protease Inhibitors; D013844:Thiazoles; C114061:isoleucyl-thiazolidide; D007532:Isoleucine; D018819:Dipeptidyl Peptidase 4