In conclusion an electrophilic fragment library was

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 lipid metabolism 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.
, an anaerobic gram-positive, spore-forming bacterium that can induce fatal intestinal inflammatory disease, is the most prevalent cause of antibiotic-associated diarrhea and pseudomembranous colitis in nosocomial settings. Two exotoxins, toxin A (TcdA) and toxin B (TcdB), secreted by the bacterium, are primarily responsible for the induction of disease because strains lacking both toxins are avirulent. TcdA and TcdB are large homologous single-chain proteins that contain at least 4 distinct domains: an N-terminal glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a putative translocation domain, and a C-terminal receptor binding domain (RBD). Although the exact method of toxin entry into target cells remains elusive, a model of toxin action is emerging: after receptor-mediated endocytosis, which may involve regions in both the translocation domain and RBD,, the CPD and GTD eventually are translocated into the cytosol where cysteine protease self-cleaves and releases GTD from the rest of the toxin. GTD inactivates Rho guanosine triphosphatases, leading to the intoxication of host cells. Thus, CPD plays a key role in the cytosolic delivery of GTD effectors. Autoprocessing-deficient TcdA or TcdB mutants are still cytotoxic to host cells inducing cell rounding, Rho guanosine triphosphatase glucosylation, and apoptosis, but such activity is reduced compared with their wild-type toxins.,