The Plasmodium proteasome is an emerging antimalarial target due to its essential role in all the major life cycle stages of the parasite and its contribution to the establishment of resistance to artemisinin (ART)-based therapies. However, because of a similarly essential role for the host proteasome, the key property of any antiproteasome therapeutic is selectivity. Several parasite-specific proteasome inhibitors have recently been reported, however, their selectivity must be improved to enable clinical development. Here we describe screening of diverse libraries of non-natural synthetic fluorogenic substrates to identify determinants at multiple positions on the substrate that produce enhanced selectivity. We find that selection of an optimal electrophilic "warhead" is essential to enable high selectivity that is driven by the peptide binding elements on the inhibitor. We also find that host cell toxicity is dictated by the extent of coinhibition of the human β2 and β5 subunits. Using this information, we identify compounds with over 3 orders of magnitude selectivity for the parasite enzyme. Optimization of the pharmacological properties resulted in molecules that retained high potency and selectivity, were soluble, sufficiently metabolically stable and orally bioavailable. These molecules are highly synergistic with ART and can clear parasites in a mouse model of infection, making them promising leads as antimalarial drugs.

High-throughput screening of a library of small polar molecules against Mycobacterium tuberculosis led to the identification of a phthalimide-containing ester hit compound (1), which was optimized for metabolic stability by replacing the ester moiety with a methyl oxadiazole bioisostere. A route utilizing polymer-supported reagents was designed and executed to explore structure-activity relationships with respect to the N-benzyl substituent, leading to compounds with nanomolar activity. The frontrunner compound (5h) from these studies was well tolerated in mice. A M. tuberculosis cytochrome bd oxidase deletion mutant (ΔcydKO) was hyper-susceptible to compounds from this series, and a strain carrying a single point mutation in qcrB, the gene encoding a subunit of the menaquinol cytochrome c oxidoreductase, was resistant to compounds in this series. In combination, these observations indicate that this novel class of antimycobacterial compounds inhibits the cytochrome bc1 complex, a validated drug target in M. tuberculosis.

Introduction of water-solubilizing groups on the 5-phenyl ring of a 2-aminopyrazine series led to the identification of highly potent compounds against the blood life-cycle stage of the human malaria parasite Plasmodium falciparum. Several compounds displayed high in vivo efficacy in two different mouse models for malaria, P. berghei-infected mice and P. falciparum-infected NOD-scid IL-2Rγ mice. One of the frontrunners, compound 3, was identified to also have good pharmacokinetics and additionally very potent activity against the liver and gametocyte parasite life-cycle stages.

The 2-aminopyridine MMV048 was the first drug candidate inhibiting phosphatidylinositol 4-kinase (PI4K), a novel drug target for malaria, to enter clinical development. In an effort to identify the next generation of PI4K inhibitors, the series was optimized to improve properties such as solubility and antiplasmodial potency across the parasite life cycle, leading to the 2-aminopyrazine UCT943. The compound displayed higher asexual blood stage, transmission-blocking, and liver stage activities than MMV048 and was more potent against resistant and clinical isolates. Excellent antiplasmodial activity translated into high efficacy in and humanized NOD-γ mouse models. The high passive permeability and high aqueous solubility of UCT943, combined with low to moderate intrinsic clearance, resulted in sustained exposure and high bioavailability in preclinical species. In addition, the predicted human dose for a curative single administration using monkey and dog pharmacokinetics was low, ranging from 50 to 80 mg. As a next-generation PI4K inhibitor, UCT943, based on the combined preclinical data, has the potential to form part of a single-exposure radical cure and prophylaxis (SERCaP) to treat, prevent, and block the transmission of malaria.

A drug repositioning approach was leveraged to derivatize astemizole (AST), an antihistamine drug whose antimalarial activity was previously identified in a high-throughput screen. The multistage activity potential against the Plasmodium parasite's life cycle of the subsequent analogues was examined by evaluating against the parasite asexual blood, liver, and sexual gametocytic stages. In addition, the previously reported contribution of heme detoxification to the compound's mode of action was interrogated. Ten of the 17 derivatives showed half-maximal inhibitory concentrations (IC 50 s) of <0.1 μM against the chloroquine (CQ)-sensitive Plasmodium falciparum NF54 (Pf NF54) strain while maintaining submicromolar potency against the multidrugresistant strain, Pf K1, with most showing low likelihood of cross-resistance with CQ. Selected analogues (Pf NF54-IC 50 < 0.1 μM) were tested for cytotoxicity on Chinese hamster ovarian (CHO) cells and found to be highly selective (selectivity index > 100). Screening of AST and its analogues against gametocytes revealed their moderate activity (IC 50 : 1−5 μM) against late stage P. falciparum gametocytes, while the evaluation of activity against P. berghei liver stages identified one compound (3) with 3-fold greater activity than the parent AST compound. Mechanistic studies showed a strong correlation between in vitro inhibition of β-hematin formation by the AST derivatives and their antiplasmodium IC 50 s. Analyses of intracellular inhibition of hemozoin formation within the parasite further yielded signatures attributable to a possible perturbation of the heme detoxification machinery.

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.

A novel 2,8-disubstituted-1,5-naphthyridine hit compound stemming from the open access Medicines for Malaria Venture Pathogen Box formed a basis for a hit-to-lead medicinal chemistry program. Structure-activity relationship investigations resulted in compounds with potent antiplasmodial activity against both chloroquine sensitive (NF54) and multidrug resistant (K1) strains of the human malaria parasite Plasmodium falciparum. In the humanized P. falciparum mouse efficacy model, one of the frontrunner compounds showed in vivo efficacy at an oral dose of 4 × 50 mg·kg. In vitro mode-of-action studies revealed Plasmodium falciparum phosphatidylinositol-4-kinase as the target.

On the basis of our recent results on a novel series of imidazopyridazine-based antimalarials, we focused on identifying compounds with improved aqueous solubility and hERG profile while maintaining metabolic stability and in vitro potency. Toward this objective, 41 compounds were synthesized and evaluated for antiplasmodial activity against NF54 (sensitive) and K1 (multidrug resistant) strains of the malaria parasite Plasmodium falciparum and evaluated for both aqueous solubility and metabolic stability. Selected compounds were tested for in vitro hERG activity and in vivo efficacy in the P. berghei mouse model. Several compounds were identified with significantly improved aqueous solubility, good metabolic stability, and a clean hERG profile relative to a previous frontrunner lead compound. A sulfoxide-based imidazopyridazine analog 45, arising from a prodrug-like strategy, was completely curative in the Plasmodium berghei mouse model at 4 × 50 mg/kg po.