Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H+-ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.

Advances in the genetics, function, and stage-specificity of Plasmodium kinases has driven robust efforts to identify targets for the design of antimalarial therapies. Reverse genomics following phenotypic screening against Plasmodia or related parasites has uncovered vulnerable kinase targets including PI4K, PKG, and GSK-3, an approach bolstered by access to human disease-directed kinase libraries. Alternatively, screening compound libraries against Plasmodium kinases has successfully led to inhibitors with antiplasmodial activity. As with other therapeutic areas, optimizing compound ADMET and PK properties in parallel with target inhibitory potency and whole cell activity becomes paramount toward advancing compounds as clinical candidates. These and other considerations will be discussed in the context of progress achieved toward deriving important, novel mode-of-action kinase-inhibiting antimalarial medicines.

A novel diazaspiro [3.4]octane series was identified from a Plasmodium falciparum whole-cell high-throughput screening campaign. Hits displayed activity against multiple stages of the parasite lifecycle, which together with a novel sp 3 -rich scaffold provided an attractive starting point for a hit-to-lead medicinal chemistry optimization and biological profiling program. Structure−activity-relationship studies led to the identification of compounds that showed low nanomolar asexual blood-stage activity (<50 nM) together with strong gametocyte sterilizing properties that translated to transmission-blocking activity in the standard membrane feeding assay. Mechanistic studies through resistance selection with one of the analogues followed by wholegenome sequencing implicated the P. falciparum cyclic amine resistance locus in the mode of resistance.

39New chemical matter is needed to target the divergent biology associated with the different 40 life cycle stages of Plasmodium. Here, we report the parallel screening of the Medicines for 41 Malaria Venture Pandemic Response Box to identify multistage-active and stage-specific 42 compounds against various life cycle stages of Plasmodium parasites (asexual parasites, 43 stage IV/V gametocytes, gametes, oocysts and liver stages) and for endectocidal activity. Hits 44 displayed unique chemotypes and included two multistage-active compounds, 16 asexual-45 targeted, six with prophylactic potential and ten gametocyte-targeted compounds. Notably, 46 four structurally diverse gametocyte-targeted compounds with potent transmission-blocking 47 activity were identified: the JmjC inhibitor ML324, two azole antifungals including 48 eberconazole, and the antitubercular clinical candidate SQ109. Besides ML324, none of these 49 have previously attributed antiplasmodial activity, emphasizing the success of de novo parallel 50 screening against different Plasmodium stages to deliver leads with novel modes-of-action. 51Importantly, the discovery of such transmission-blocking targeted compounds covers a 52 previously unexplored base for delivery of compounds required for malaria elimination 53 strategies. 55 56Malaria treatment solely relies on drugs that target the parasite but current treatment options 57 have a finite lifespan due to resistance development. Moreover, whilst current antimalarials 58 are curative of asexual blood stage parasitemia and associated malaria symptoms, they 59 cannot all be used prophylactically and typically do not effectively block transmission. This 60 limits their utility in malaria elimination strategies, where the latter dictates that chemotypes 61 should block human-to-mosquito (gametocyte and gametes) and mosquito-to-human 62 (sporozoites and liver schizonts) transmission. 63The transmission stages of malaria parasites are seen as parasite population 64 bottlenecks, 1 with as few as 100 sporozoites able to initiate an infection after migrating to the 65 liver where exoerythrocytic schizogony occurs. The subsequent release of thousands of 66 daughter cells, which in turn infect erythrocytes, initiates the extensive population expansion 67 that occurs during asexual replication. A minor proportion (~1%) 2 of the proliferating asexual 68 parasites will undergo sexual differentiation to form mature stage V gametocytes, a 10-14 day 69 process in the most virulent parasite Plasmodium falciparum. Only ~10 3 of these falciform-70 shaped mature gametocytes are taken up by the next feeding mosquito to transform into male 71 and female gametes in the mosquito's midgut. 3 Fertilization results in zygote development, 72 and a motile ookinete that passes through the midgut wall forms an oocyst from which 73 sporozoites develop, making the mosquito infectious.74 The sporozoite and gametocyte population bottlenecks have been the basis of enticing 75 arguments towards the development of chemotypes able to targe...

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We dissected halogen-aryl π interactions experimentally using a bicyclic N-arylimide based molecular torsion balances system, which is based on the influence of the non-bonded interaction on the equilibria between folded and unfolded states. Through comparison of balances modulated by higher halogens with fluorine balances, we determined the magnitude of the halogen-aryl π interactions in our unimolecular systems to be larger than -5.0 kJ mol , which is comparable with the magnitude estimated in the biomolecular systems. Our study provides direct experimental evidence of halogen-aryl π interactions in solution, which until now have only been revealed in the solid state and evaluated theoretically by quantum-mechanical calculations.

The application of dynamic ligation screening (DLS), a methodology for fragment-based drug discovery (FBDD), to the aspartic protease β-secretase (BACE-1) is reported. For this purpose, three new fluorescence resonance energy transfer (FRET) substrates were designed and synthesized. Their kinetic parameters (Vmax , KM , and kcat ) were determined and compared with a commercial substrate. Secondly, a peptide aldehyde was designed as a chemically reactive inhibitor (CRI) based on the Swedish mutation substrate sequence. Incubation of this CRI with the protease, a FRET substrate, and one amine per well taken from an amine library, which was assembled by a maximum common substructure (MCS) approach, revealed the fragment 3-(3-aminophenyl)-2H-chromen-2-one (1) to be a competitive BACE-1 inhibitor that enhanced the activity of the CRI. Irreversibly formed fragment combination products of 1 with the initial peptide sequence were active and confirmed the targeting of the active site through the ethane-1,2-diamine isostere. Finally, structure-assisted combination of fragment 1 with secondary fragments that target the S1 site in hit optimization yielded novel, entirely fragment-based BACE-1 inhibitors with up to 30-fold improved binding affinity. Interactions with the protein were explained by molecular modeling studies, which indicate that the new fragment combinations interact with the catalytic aspartic acid dyad, as well as with the adjacent binding sites required for potency.

A series of 2,4-disubstituted imidazopyridines, originating from a SoftFocus Kinase library, was identified from a high throughput phenotypic screen against the human malaria parasite Plasmodium falciparum. Hit compounds showed moderate asexual blood stage activity. During lead optimization, several issues were flagged such as cross-resistance against the multi-drug resistant K1 strain, in vitro cytotoxicity and cardiotoxicity, and were addressed through structureactivity and structure-property relationship studies. Pharmacokinetic properties were assessed in mouse for compounds showing desirable in vitro activity, selectivity window over cytotoxicity and microsomal metabolic stability. Frontrunner compound 37 showed good exposure in mice combined with good in vitro activity against the malaria parasite which translated into in vivo efficacy in the P. falciparum NOD-scid IL-2Rγ null (NSG) mouse model. Preliminary mechanistic studies suggest inhibition of hemozoin formation as a contributing mode of action.