p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo–electron microscopy (cryo-EM) structures for adenosine diphosphate (ADP)–bound, full-length, hexameric wild-type p97 in the presence and absence of an allosteric inhibitor at resolutions of 2.3 and 2.4 angstroms, respectively. We also report cryo-EM structures (at resolutions of ~3.3, 3.2, and 3.3 angstroms, respectively) for three distinct, coexisting functional states of p97 with occupancies of zero, one, or two molecules of adenosine 5′-O-(3-thiotriphosphate) (ATPγS) per protomer. A large corkscrew-like change in molecular architecture, coupled with upward displacement of the N-terminal domain, is observed only when ATPγS is bound to both the D1 and D2 domains of the protomer. These cryo-EM structures establish the sequence of nucleotide-driven structural changes in p97 at atomic resolution. They also enable elucidation of the binding mode of an allosteric small-molecule inhibitor to p97 and illustrate how inhibitor binding at the interface between the D1 and D2 domains prevents propagation of the conformational changes necessary for p97 function.

SUMMARY Genetically hard-wired neural mechanisms must enforce behavioral reproductive isolation because interspecies courtship is rare even in sexually naïve animals of most species. We find that the chemoreceptor Gr32a inhibits male D. melanogaster from courting diverse fruit fly species. Gr32a recognizes non-volatile aversive cues present on these reproductively dead-end targets, and activity of Gr32a neurons is necessary and sufficient to inhibit interspecies courtship. Male-specific Fruitless (FruM), a master regulator of courtship, also inhibits interspecies courtship. Gr32a and FruM are not co-expressed, but FruM neurons contact Gr32a neurons, suggesting that these genes influence a shared neural circuit that inhibits inter-species courtship. Gr32a and FruM also suppress within-species intermale courtship, but we show that distinct mechanisms preclude sexual displays toward conspecific males and other species. Although this chemosensory pathway does not inhibit interspecies mating in D. melanogaster females, similar mechanisms appear to inhibit this behavior in many other male drosophilids.

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 appear to be required for hemoglobin hydrolysis by intraerythrocytic malaria parasites. Previous studies showed that peptidyl vinyl sulfone inhibitors of falcipain-2 blocked the development of P. falciparum in culture and exerted antimalarial effects in vivo. We now report the structure-activity relationships for inhibition of falcipain-2, falcipain-3, and parasite development by 39 new vinyl sulfone, vinyl sulfonate ester, and vinyl sulfonamide cysteine protease inhibitors. Levels of inhibition of falcipain-2 and falcipain-3 were generally similar, and many potent compounds were identified. Optimal antimalarial compounds, which inhibited P. falciparum development at low nanomolar concentrations, were phenyl vinyl sulfones, vinyl sulfonate esters, and vinyl sulfonamides with P 2 leucine moieties. Our results identify independent structural correlates of falcipain inhibition and antiparasitic activity and suggest that peptidyl vinyl sulfones have promise as antimalarial agents.Malaria is one of the most important infectious diseases in the world. Plasmodium falciparum, the most virulent human malaria parasite, is estimated to cause over 300 million new cases and 1 million deaths annually (33). Further complicating this grim scenario is the emergence of the widespread resistance of P. falciparum to available antimalarial drugs (25). New drugs to combat malaria are urgently needed.Among potential new targets for antimalarial chemotherapy are enzymes that mediate hemoglobin hydrolysis. Intraerythrocytic P. falciparum trophozoites derive amino acids for protein synthesis from the hydrolysis of host cell hemoglobin in an acidic food vacuole (12,20,27). Proteases that hydrolyze hemoglobin in the food vacuole include members of the aspartic protease (1), cysteine protease (38, 39), and metalloprotease (9) families. Cysteine protease inhibitors arrested the erythrocytic life cycle of P. falciparum (26). Examination of inhibitortreated parasites revealed abnormally swollen food vacuoles filled with undigested hemoglobin, indicating that the block in parasite development was due to the inhibition of hemoglobin hydrolysis (26).P. falciparum contains three fairly typical papain family cysteine proteases, known as falcipains (28,38,39). Falcipain-2 and falcipain-3 appear to be the principal cysteine protease hemoglobinases (38, 39). Both of these proteases localize to vacuolar parasite fractions and readily hydrolyze hemoglobin under physiological reducing conditions at acidic pHs (37). Falcipain-2 is considerably more active against small peptide substrates, but the specificities of the two proteases are similar; both enzymes display a strong preference for leucine at the P 2 position (38, 39). The role of falcipain-1 in hemoglobin hydrolysis is unknown.In earlier studies, peptidyl vinyl sulfones inhibited falcipain-2 activity and parasite development at nanomolar concentrations and were active in vivo against murine malaria (22,29,30). We have now init...

BackgroundSchistosoma flatworm parasites cause schistosomiasis, a chronic and debilitating disease of poverty in developing countries. Praziquantel is employed for treatment and disease control. However, its efficacy spectrum is incomplete (less active or inactive against immature stages of the parasite) and there is a concern of drug resistance. Thus, there is a need to identify new drugs and drug targets.Methodology/Principal FindingsWe show that RNA interference (RNAi) of the Schistosoma mansoni ortholog of human polo-like kinase (huPLK)1 elicits a deleterious phenotypic alteration in post-infective larvae (schistosomula or somules). Phenotypic screening and analysis of schistosomula and adult S. mansoni with small molecule inhibitors of huPLK1 identified a number of potent anti-schistosomals. Among these was a GlaxoSmithKline (GSK) benzimidazole thiophene inhibitor that has completed Phase I clinical trials for treatment of solid tumor malignancies. We then obtained GSKs Published Kinase Inhibitor Sets (PKIS) 1 and 2, and phenotypically screened an expanded series of 38 benzimidazole thiophene PLK1 inhibitors. Computational analysis of controls and PLK1 inhibitor-treated populations of somules demonstrated a distinctive phenotype distribution. Using principal component analysis (PCA), the phenotypes exhibited by these populations were mapped, visualized and analyzed through projection to a low-dimensional space. The phenotype distribution was found to have a distinct shape and topology, which could be elicited using cluster analysis. A structure-activity relationship (SAR) was identified for the benzimidazole thiophenes that held for both somules and adult parasites. The most potent inhibitors produced marked phenotypic alterations at 1–2 μM within 1 h. Among these were compounds previously characterized as potent inhibitors of huPLK1 in cell assays.Conclusions/SignificanceThe reverse genetic and chemical SAR data support a continued investigation of SmPLK1 as a possible drug target and/or the prosecution of the benzimidazole thiophene chemotype as a source of novel anti-schistosomals.

Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.