br Fragment based drug discovery FBDD is
Fragment-based drug discovery (FBDD) is a powerful method to discover drug leads and has been widely adopted in both academia and industry. FBDD can be used to explore chemical AZD 0530 space with libraries which are smaller in size, producing drug leads with high ligand-binding efficiency. It also provides a rational path to high-affinity lead compounds that possess drug–like properties. The fragment-based approach is particularly well-suited for target-based drug discovery because the structural requirements for inhibition or inactivation can be used to guide the choice of pharmacophore and other structural motifs during drug design. The human African trypanosomiasis (sleeping sickness), a disease caused by the kinetoplastid protozoans and , is one of the neglected tropical diseases (NTDs). The disease is fatal if untreated, and current treatment options available for the disease are ineffective and have well-documented adverse effects. It is important to note that the incidence of sleeping sickness has decreased in the last decade, although the need to develop new and effective drugs remains a key objective in controlling and eradicating the disease. A promising drug candidate in clinical trials for human African trypanosomiasis is the nitroimidazole fexinidazole. Fexinidazole is also being developed as a potential treatment for Chagas Disease., The major cathepsin L-like cysteine protease in rhodesiense, rhodesain, is a validated drug target. The cysteine protease is essential for the survival and infectivity of the parasite. Its important role in the ability of the parasite to proliferate has been investigated by several groups., , Steverding et al. have also shown that pharmacological inhibition of rhodesain is lethal to A number of potent inhibitors of rhodesain that also have antitrypanosomal activity have been reported. Among them are peptide-based covalent inhibitors with Michael acceptors such as 1 and 2 and thiosemicarbazone based inhibitors for example 3 and 4 (). Furthermore, a peptide derived covalent inhibitor of its homologue in , K777, was recently a promising pre-clinical drug candidate for the treatment of Chagas disease (American trypanosomiasis), which highlights the importance of rhodesain as a drug target., This provides the rationale that covalent or non-covalent inhibitors of rhodesain may be advanced into the drug development pipeline against African sleeping sickness. To discover rhodesain inhibitors, we employed the irreversible tethering method to discover covalent inhibitors of cysteine proteases. In this method, a mixture of cysteine reactive electrophilic fragments were incubated with the cysteine protease, allowing the best binding fragments to covalently and irreversibly modify the catalytic cysteine of the protease, and the covalent cysteine protease–inhibitor complexes were subsequently detected using mass spectrometry methods. The fragments can subsequently be elaborated into drug leads while retaining the original Michael acceptor electrophile. The originally developed method requires mass spectrometry to identify fragment hits, and this requirement limits the widespread use of this technology. We thought to expand the method and asked if the electrophilic fragments can be screened individually in enzymatic assays to identify weak and irreversible fragment inhibitors. Notably, such an approach would contradict current practices in academia and industry, in which reactive compounds are removed from compound screening collections. We envisioned that if successful, this strategy would substantially expand the use of irreversible tethering in laboratory settings in which mass spectrometry services are not available. Thus, a previously made library of 200 electrophilic fragments was screened for inhibitor activity against rhodesain in in vitro enzymatic assays., The active hits were then investigated for their antitrypanosomal activity and cytotoxicity to human Hep G2 cells. The initial screen of the fragment library (10μM) against rhodesain in 384-well assay plates led to the identification of seven positive hits (). These seven compounds caused ⩾85% inhibition of rhodesain. A follow-up assay using 200μL reaction mixtures in 96-well plates to confirm the activity of the active compounds led to the identification of compounds and as hits, the other five compounds caused <40% inhibition of rhodesain at 10μM (, ). Fragments and were recently reported as inhibitors of papain, a cysteine protease structurally and biochemically related to rhodesain. Fragments and seem to be better inhibitors of rhodesain than papain judging by the / values, however, it is important to note that the optimal assay conditions for the two cysteine proteases are different, and the assay conditions may influence the observed inactivation constants. Since K777 has a vinyl sulfone electrophile as a Michael acceptor, we investigated if vinyl sulfone analogues of and were capable of inhibiting rhodesain. We have found that compound , which is the vinyl sulfone analogue of , was also active in our assays, but it was less reactive towards the cysteine protease than the acrylate () (). However, compound , the vinyl sulfone analog of , was inactive in the enzymatic assay, suggesting that unique structural features of drive its activity against rhodesain.