In addition we investigated if the previously reported papai
In addition, we investigated if the previously reported papain inhibitor can inhibit rhodesain, and found that it was not active at inhibiting rhodesain. The vinyl sulfone analogue of () was then synthesized and tested, and it was also inactive towards rhodesain. We then determined the time dependent inhibition constant / of compounds , and , which were 18.32, 13.22 and 3.47Ms, respectively (, ). Taken together, our preliminary SAR analysis indicates that a combination of specific structural features of the fragment and the electrophile make inhibitors of rhodesain. Crystal structure analysis of the fragment-rhodesain complexes is underway, and it should provide a structural ratonale for extensive SAR studies. Although the identified covalent inhibitors of rhodesain are fragments, and therefore need to be optimized further to increase their potency and selectivity, we asked if any of those compounds have antitrypanosomal activity. We rationalized that those fragments that display antitrypanosomal activity, yet are not toxic to human hepatocellular carcinoma (Hep G2), could be further optimized into inhibitors that display selective toxicity to but not to human cells. Therefore, compounds – were tested for growth inhibitory activity on as well as their cytotoxicity on Hep G2 cells. Compounds and have emerged as promising lead structures, since both compounds were potent at inhibiting rhodesain in vitro (/ values 18.32 and 13.22, respectively), and displayed selective toxicity to without being toxic to Hep G2 solution concentration calculator (). Importantly, compound was inactive in our in vitro assays, despite the fact that it was active in the antitrypanosomal assay. This indicates that may be reactive towards one or more other catalytic cysteines in , although the weak selectivity index of makes it a less desired lead compound. In conclusion, an electrophilic fragment library was evaluated for inhibitory activity against the cathepsin-L like cysteine protease rhodesain. The unique feature of this approach is that reactive compounds were screened in an enzymatic assay in a 384 well plate format to identify specific hits, which stands in sharp contrast to the currently accepted dogma in the pharmaceutical industry that reactive compounds must be excluded from all HTS screens, because reactive compounds can display promiscuous reactivity toward their protein targets. Our results show that in fact it is possible to screen a library of cysteine reactive fragments in enzymatic assays in a 384 well plate format if the library of the cysteine reactive fragments is properly designed. Furthermore, the non-peptidic nature of the identified inhibitors of rhodesain could result in better pharmacokinetic properties of the covalent rhodesain inhibitor drug leads. Furthermore, current known covalent inhibitors of rhodesain have two electron withdrawing groups present at the Michael acceptor site, which can increase the number of off-target effects for such inhibitors. In contrast, our fragment libraries have only one electron-withdrawing group at the Michael acceptor site, which should reduce the electrophilicity and non-specific reactivity of these fragments (). We envision that fragments that contain other electrophiles can be assembled and tested against other cysteine proteases either using mass spectrometry or enzymatic assays in the 96 or 384 well plate format, which will significantly expand the use of the irreversible tethering technology. Further optimization of the identified rhodesain inhibitor fragments into potent and selective lead compounds will be reported in the near future. Although compounds and were also previously identified as papain hits, we believe that we can achieve reasonable selectivity for rhodesain amongst other papain-family cysteine proteases upon growth of the fragment into a drug lead, similar to how selectivity amongst ATP competitive kinase inhibitors is achieved. Acknowledgments This work was supported in part by the US National Institutes of Health (SC2GM109782 to I.V.O., T32GM105538 to S.K), American Cancer Society Institutional Research Grant (93-037-18 to A.S.), Chemistry of Life Processes Institute Lambert Fellowship (Z.X.), the ACS Medicinal Chemistry Fellowship (S.K.) and Northwestern University (A.S.) A.S. is a Pew Scholar in the Biomedical Sciences, supported by the Pew Charitable Trusts. We thank Rama Mishra and the Center for Molecular Innovation and Drug Discovery for assisting with the initial design of the library of electrophilic fragments.