The impact of whole genome sequence data on drug discovery - A malaria case study

Background: identification and validation of a drug discovery target is a p rominent step in drug development. In the post-genomic era it is possible t o reevaluate the association of a gene with a specific biological function to see if a homologous gene can subsume this role. This concept has special relevance to drug discovery in human infectious diseases, like malaria. A trophozoite cysteine protease (falcipain-1) from the papain family, thought to be responsible for the degradation of erythrocyte hemoglobin, has been considered a promising target for drug discovery efforts owing to the antim alarial activity of peptide based covalent cysteine protease inhibitors. Th is led to the development of non-peptidic non-covalent inhibitors of falcip ain-1 and their characterization as antimalarials. it is now clear from seq uencing efforts that the malaria genome contains more than one cysteine pro tease and that falcipain-1 is not the most important contributor to hemoglo bin degradation. Rather, falcipain-2 and falcipain-3 appear to account for the majority of cysteine hemoglobinase activity in the plasmodium trophozoi te. Materials and Methods: We have modeled the falcipain-2 cysteine protease fr om one of the major human malaria species. Plasmodium falciparum and compar ed it to our original work on falcipain-1. As with falcipain-1, computation al screening of the falcipain-2 active site was conducted using DOCK. Using structural superpositions within the protease family and evolutionary anal ysis of substrate specificity sites, we focused on the commonalities and th e protein specific features to direct our drug discovery effort. Results: Since 1993, the size of the Available Chemicals Directory had incr eased from 55313 to 195419 unique chemical structures. For falcipain-2, eig ht inhibitors were identified with IC50's against the enzyme between I and 7 muM. Application of three of these inhibitors to infected erythrocytes cu red malaria in culture, but parasite death did not correlate with food vacu ole abnormalities associated with the activity of mechanistic inhibitors of cysteine proteases like the epoxide E64. Conclusions: Using plasmodial falcipain proteases, we show how a protein fa mily perspective can influence target discovery and inhibitor design. We su spect that parallel drug discovery programs where a family of targets is co nsidered, rather than serial programs built on a single-therapeutic focus, will become the dominant industrial paradigm. Economies of scale in assay d evelopment and in compound synthesis are expected owing to the functional a nd structural features of individual family members. One of the remaining c hallenges in post-genomic drug discovery is that inhibitors of one target a re likely to show some activity against other family members. This lack of specificity may lead to difficulties in functional assignments and target v alidation as well as a complex side effect profile.