Development of a Highly Selective Plasmodium falciparum Proteasome Inhibitor with Anti-malaria Activity in Humanized Mice.

Plasmodium falciparum proteasome (Pf20S) inhibitors are active against Plasmodium at multiple stages-erythrocytic, gametocyte, liver, and gamete activation stages-indicating that selective Pf20S inhibitors possess the potential to be therapeutic, prophylactic, and transmission-blocking antimalarials. Starting from a reported compound, we developed a noncovalent, macrocyclic peptide inhibitor of the malarial proteasome with high species selectivity and improved pharmacokinetic properties. The compound demonstrates specific, time-dependent inhibition of the ß5 subunit of the Pf20S, kills artemisinin-sensitive and artemisinin-resistant P. falciparum isolates in vitro and reduces parasitemia in humanized, P. falciparum-infected mice.

Harnessing evolutionary fitness in Plasmodium falciparum for drug discovery and suppressing resistance.

Drug resistance emerges in an ecological context where fitness costs restrict the diversity of escape pathways. These pathways are targets for drug discovery, and here we demonstrate that we can identify small-molecule inhibitors that differentially target resistant parasites. Combining wild-type and mutant-type inhibitors may prevent the emergence of competitively viable resistance. We tested this hypothesis with a clinically derived chloroquine-resistant (CQ(r)) malaria parasite and with parasites derived by in vitro selection with Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors. We screened a chemical library against CQ(s) and CQ(r) lines and discovered a drug-like compound (IDI-3783) that was potent only in the CQ(r) line. Surprisingly, in vitro selection of Plasmodium falciparum resistant to IDI-3783 restored CQ sensitivity, thereby indicating that CQ might once again be useful as a malaria therapy. In parallel experiments, we selected P. falciparum lines resistant to structurally unrelated PfDHODH inhibitors (Genz-666136 and DSM74). Both selections yielded resistant lines with the same point mutation in PfDHODH:E182D. We discovered a compound (IDI-6273) more potent against E182D than wild-type parasites. Selection of the E182D mutant with IDI-6273 yielded a reversion to the wild-type protein sequence and phenotype although the nucleotide sequence was different. Importantly, selection with a combination of Genz-669178, a wild-type PfDHODH inhibitor, and IDI-6273, a mutant-selective PfDHODH inhibitor, did not yield resistant parasites. These two examples demonstrate that the compromise between resistance and evolutionary fitness can be exploited to design therapies that prevent the emergence and spread of resistant organisms.

In vitro resistance selections for Plasmodium falciparum dihydroorotate dehydrogenase inhibitors give mutants with multiple point mutations in the drug-binding site and altered growth.

Malaria is a preventable and treatable disease; yet half of the world's population lives at risk of infection, and an estimated 660,000 people die of malaria-related causes every year. Rising drug resistance threatens to make malaria untreatable, necessitating both the discovery of new antimalarial agents and the development of strategies to identify and suppress the emergence and spread of drug resistance. We focused on in-development dihydroorotate dehydrogenase (DHODH) inhibitors. Characterizing resistance pathways for antimalarial agents not yet in clinical use will increase our understanding of the potential for resistance. We identified resistance mechanisms of Plasmodium falciparum (Pf) DHODH inhibitors via in vitro resistance selections. We found 11 point mutations in the PfDHODH target. Target gene amplification and unknown mechanisms also contributed to resistance, albeit to a lesser extent. These mutant parasites were often hypersensitive to other PfDHODH inhibitors, which immediately suggested a novel combination therapy approach to preventing resistance. Indeed, a combination of wild-type and mutant-type selective inhibitors led to resistance far less often than either drug alone. The effects of point mutations in PfDHODH were corroborated with purified recombinant wild-type and mutant-type PfDHODH proteins, which showed the same trends in drug response as the cognate cell lines. Comparative growth assays demonstrated that two mutant parasites grew less robustly than their wild-type parent, and the purified protein of those mutants showed a decrease in catalytic efficiency, thereby suggesting a reason for the diminished growth rate. Co-crystallography of PfDHODH with three inhibitors suggested that hydrophobic interactions are important for drug binding and selectivity.

Discovery of Dual-Stage Malaria Inhibitors with New Targets.

Malaria remains a major global health problem, with more than half of the world population at risk of contracting the disease and nearly a million deaths each year. Here, we report the discovery of inhibitors that target multiple stages of malaria parasite growth. To identify these inhibitors, we took advantage of the Tres Cantos Antimalarial Compound Set (TCAMS) small-molecule library, which is comprised of diverse and potent chemical scaffolds with activities against the blood stage of the malaria parasite, and investigated their effects against the elusive liver stage of the malaria parasite using a forward chemical screen. From a screen of nearly 14,000 compounds, we identified and confirmed 103 compounds as dual-stage malaria inhibitors. Interestingly, these compounds show preferential inhibition of parasite growth in liver- versus blood-stage malaria parasite assays, highlighting the drug susceptibility of this parasite form. Mode-of-action studies were completed using genetically modified and drug-resistant Plasmodium parasite strains. While we identified some compound targets as classical antimalarial pathways, such as the mitochondrial electron transport chain through cytochrome bc1 complex inhibition or the folate biosynthesis pathway, most compounds induced parasite death through as yet unknown mechanisms of action. Importantly, the identification of new chemotypes with different modes of action in killing Plasmodium parasites represents a promising opportunity for probing essential and novel molecular processes that remain to be discovered. The chemical scaffolds identified with activity against drug-resistant Plasmodium parasites represent starting points for dual-stage antimalarial development to surmount the threat of malaria parasite drug resistance.

Drug screen targeted at Plasmodium liver stages identifies a potent multistage antimalarial drug.

Plasmodium parasites undergo a clinically silent and obligatory developmental phase in the host's liver cells before they are able to infect erythrocytes and cause malaria symptoms. To overcome the scarcity of compounds targeting the liver stage of malaria, we screened a library of 1037 existing drugs for their ability to inhibit Plasmodium hepatic development. Decoquinate emerged as the strongest inhibitor of Plasmodium liver stages, both in vitro and in vivo. Furthermore, decoquinate kills the parasite's replicative blood stages and is active against developing gametocytes, the forms responsible for transmission. The drug acts by selectively and specifically inhibiting the parasite's mitochondrial bc(1) complex, with little cross-resistance with the antimalarial drug atovaquone. Oral administration of a single dose of decoquinate effectively prevents the appearance of disease, warranting its exploitation as a potent antimalarial compound.

Diverse evolutionary pathways challenge the use of collateral sensitivity as a strategy to suppress resistance.

Drug resistance remains a major obstacle to malaria control and eradication efforts, necessitating the development of novel therapeutic strategies to treat this disease. Drug combinations based on collateral sensitivity, wherein resistance to one drug causes increased sensitivity to the partner drug, have been proposed as an evolutionary strategy to suppress the emergence of resistance in pathogen populations. In this study, we explore collateral sensitivity between compounds targeting the Plasmodium dihydroorotate dehydrogenase (DHODH). We profiled the cross-resistance and collateral sensitivity phenotypes of several DHODH mutant lines to a diverse panel of DHODH inhibitors. We focus on one compound, TCMDC-125334, which was active against all mutant lines tested, including the DHODH C276Y line, which arose in selections with the clinical candidate DSM265. In six selections with TCMDC-125334, the most common mechanism of resistance to this compound was copy number variation of the dhodh locus, although we did identify one mutation, DHODH I263S, which conferred resistance to TCMDC-125334 but not DSM265. We found that selection of the DHODH C276Y mutant with TCMDC-125334 yielded additional genetic changes in the dhodh locus. These double mutant parasites exhibited decreased sensitivity to TCMDC-125334 and were highly resistant to DSM265. Finally, we tested whether collateral sensitivity could be exploited to suppress the emergence of resistance in the context of combination treatment by exposing wildtype parasites to both DSM265 and TCMDC-125334 simultaneously. This selected for parasites with a DHODH V532A mutation which were cross-resistant to both compounds and were as fit as the wildtype parent in vitro. The emergence of these cross-resistant, evolutionarily fit parasites highlights the mutational flexibility of the DHODH enzyme.

Malaria affects around 240 million people around the world every year. The microscopic parasite responsible for the disease are carried by certain mosquitoes and gets transmitted to humans through bites. These parasites are increasingly acquiring genetic mutations that make anti-malaria medication less effective, creating an urgent need for alternative treatment approaches. Several new malaria drugs being explored in preclinical research work by binding to an enzyme known as DHODH and preventing it from performing its usual role in the parasite. Previous work found that, in some cases, malaria parasites that evolved resistance to one type of DHODH inhibitor (by acquiring mutations in their DHODH enzyme) then became more vulnerable to another kind. It may be possible to leverage this 'collateral sensitivity' by designing treatments which combine two DHODH inhibitors and therefore make it harder for the parasites to evolve resistance. To investigate this possibility, Mandt et al. first tested several DHODH inhibitors to find the one that was most potent against drug-resistant parasites. In subsequent experiments, they combined TCMDC-125334, the best candidate that emerged from these tests, with a DHODH inhibitor that works well against vulnerable parasites. However, the parasites still rapidly evolved resistance. Further work identified a new DHODH mutation that allowed the parasites to evade both drugs simultaneously. Together, these findings suggest that the DHODH enzyme may not be the best target for new malaria drugs because many it can acquire many possible mutations that confer resistance. Such results may inform other studies that aim to harness collateral sensitivity to fight against a range of harmful agents.

Development of an ectopic huLiver model for Plasmodium liver stage infection.

Early Plasmodium falciparum and P. vivax infection requires parasite replication within host hepatocytes, referred to as liver stage (LS). However, limited understanding of infection dynamics in human LS exists due to species-specificity challenges. Reported here is a reproducible, easy-to-manipulate, and moderate-cost in vivo model to study human Plasmodium LS in mice; the ectopic huLiver model. Ectopic huLiver tumors were generated through subcutaneous injection of the HC-04 cell line and shown to be infectible by both freshly dissected sporozoites and through the bite of infected mosquitoes. Evidence for complete LS development was supported by the transition to blood-stage infection in mice engrafted with human erythrocytes. Additionally, this model was successfully evaluated for its utility in testing antimalarial therapeutics, as supported by primaquine acting as a causal prophylactic against P. falciparum. Presented here is a new platform for the study of human Plasmodium infection with the potential to aid in drug discovery.

The anticancer human mTOR inhibitor sapanisertib potently inhibits multiple Plasmodium kinases and life cycle stages.

Compounds acting on multiple targets are critical to combating antimalarial drug resistance. Here, we report that the human "mammalian target of rapamycin" (mTOR) inhibitor sapanisertib has potent prophylactic liver stage activity, in vitro and in vivo asexual blood stage (ABS) activity, and transmission-blocking activity against the protozoan parasite Plasmodium spp. Chemoproteomics studies revealed multiple potential Plasmodium kinase targets, and potent inhibition of Plasmodium phosphatidylinositol 4-kinase type III beta (PI4Kß) and cyclic guanosine monophosphate-dependent protein kinase (PKG) was confirmed in vitro. Conditional knockdown of PI4Kß in ABS cultures modulated parasite sensitivity to sapanisertib, and laboratory-generated P. falciparum sapanisertib resistance was mediated by mutations in PI4Kß. Parasite metabolomic perturbation profiles associated with sapanisertib and other known PI4Kß and/or PKG inhibitors revealed similarities and differences between chemotypes, potentially caused by sapanisertib targeting multiple parasite kinases. The multistage activity of sapanisertib and its in vivo antimalarial efficacy, coupled with potent inhibition of at least two promising drug targets, provides an opportunity to reposition this pyrazolopyrimidine for malaria.

Disruption of the Plasmodium falciparum Life Cycle through Transcriptional Reprogramming by Inhibitors of Jumonji Demethylases.

Little is known about the role of the three Jumonji C (JmjC) enzymes in Plasmodium falciparum (Pf). Here, we show that JIB-04 and other established inhibitors of mammalian JmjC histone demethylases kill asexual blood stage parasites and are even more potent at blocking gametocyte development and gamete formation. In late stage parasites, JIB-04 increased levels of trimethylated lysine residues on histones, suggesting the inhibition of P. falciparum Jumonji demethylase activity. These epigenetic defects coincide with deregulation of invasion, cell motor, and sexual development gene programs, including gene targets coregulated by the PfAP2-I transcription factor and chromatin-binding factor, PfBDP1. Mechanistically, we demonstrate that PfJmj3 converts 2-oxoglutarate to succinate in an iron-dependent manner consistent with mammalian Jumonji enzymes, and this catalytic activity is inhibited by JIB-04 and other Jumonji inhibitors. Our pharmacological studies of Jumonji activity in the malaria parasite provide evidence that inhibition of these enzymatic activities is detrimental to the parasite.

Lead Optimization of a Pyrrole-Based Dihydroorotate Dehydrogenase Inhibitor Series for the Treatment of Malaria.

Malaria puts at risk nearly half the world's population and causes high mortality in sub-Saharan Africa, while drug resistance threatens current therapies. The pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is a validated target for malaria treatment based on our finding that triazolopyrimidine DSM265 (1) showed efficacy in clinical studies. Herein, we describe optimization of a pyrrole-based series identified using a target-based DHODH screen. Compounds with nanomolar potency versus Plasmodium DHODH and Plasmodium parasites were identified with good pharmacological properties. X-ray studies showed that the pyrroles bind an alternative enzyme conformation from 1 leading to improved species selectivity versus mammalian enzymes and equivalent activity on Plasmodium falciparum and Plasmodium vivax DHODH. The best lead DSM502 (37) showed in vivo efficacy at similar levels of blood exposure to 1, although metabolic stability was reduced. Overall, the pyrrole-based DHODH inhibitors provide an attractive alternative scaffold for the development of new antimalarial compounds.

Setting Our Sights on Infectious Diseases.

In May 2019, the Wellcome Centre for Anti-Infectives Research (WCAIR) at the University of Dundee, UK, held an international conference with the aim of discussing some key questions around discovering new medicines for infectious diseases and a particular focus on diseases affecting Low and Middle Income Countries. There is an urgent need for new drugs to treat most infectious diseases. We were keen to see if there were lessons that we could learn across different disease areas and between the preclinical and clinical phases with the aim of exploring how we can improve and speed up the drug discovery, translational, and clinical development processes. We started with an introductory session on the current situation and then worked backward from clinical development to combination therapy, pharmacokinetic/pharmacodynamic (PK/PD) studies, drug discovery pathways, and new starting points and targets. This Viewpoint aims to capture some of the learnings.

In vitro selection predicts malaria parasite resistance to dihydroorotate dehydrogenase inhibitors in a mouse infection model.

Resistance has developed in Plasmodium malaria parasites to every antimalarial drug in clinical use, prompting the need to characterize the pathways mediating resistance. Here, we report a framework for assessing development of resistance of Plasmodium falciparum to new antimalarial therapeutics. We investigated development of resistance by P. falciparum to the dihydroorotate dehydrogenase (DHODH) inhibitors DSM265 and DSM267 in tissue culture and in a mouse model of P. falciparum infection. We found that resistance to these drugs arose rapidly both in vitro and in vivo. We identified 13 point mutations mediating resistance in the parasite DHODH in vitro that overlapped with the DHODH mutations that arose in the mouse infection model. Mutations in DHODH conferred increased resistance (ranging from 2- to ~400-fold) to DHODH inhibitors in P. falciparum in vitro and in vivo. We further demonstrated that the drug-resistant parasites carrying the C276Y mutation had mitochondrial energetics comparable to the wild-type parasite and also retained their fitness in competitive growth experiments. Our data suggest that in vitro selection of drug-resistant P. falciparum can predict development of resistance in a mouse model of malaria infection.

Validation of the protein kinase PfCLK3 as a multistage cross-species malarial drug target.

The requirement for next-generation antimalarials to be both curative and transmission-blocking necessitates the identification of previously undiscovered druggable molecular pathways. We identified a selective inhibitor of the Plasmodium falciparum protein kinase PfCLK3, which we used in combination with chemogenetics to validate PfCLK3 as a drug target acting at multiple parasite life stages. Consistent with a role for PfCLK3 in RNA splicing, inhibition resulted in the down-regulation of more than 400 essential parasite genes. Inhibition of PfCLK3 mediated rapid killing of asexual liver- and blood-stage P. falciparum and blockade of gametocyte development, thereby preventing transmission, and also showed parasiticidal activity against P. berghei and P. knowlesi Hence, our data establish PfCLK3 as a target for drugs, with the potential to offer a cure-to be prophylactic and transmission blocking in malaria.

Identification of Collateral Sensitivity to Dihydroorotate Dehydrogenase Inhibitors in Plasmodium falciparum.

Drug resistance has been reported for every antimalarial in use highlighting the need for new strategies to protect the efficacy of therapeutics in development. We have previously shown that resistance can be suppressed with a population biology trap: by identifying situations where resistance to one compound confers hypersensitivity to another (collateral sensitivity), we can design combination therapies that not only kill the parasite but also guide its evolution away from resistance. We applied this concept to the Plasmodium falciparum dihydroorotate dehydrogenase ( PfDHODH) enzyme, a well validated antimalarial target with inhibitors in the development pipeline. Here, we report a high-throughput screen to identify compounds specifically active against PfDHODH resistant mutants. We additionally perform extensive cross-resistance profiling allowing us to identify compound pairs demonstrating the potential for mutually incompatible resistance. These combinations represent promising starting points for exploiting collateral sensitivity to extend the useful lifespan of new antimalarial therapeutics.

Isoxazolopyrimidine-Based Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase with Antimalarial Activity.

Malaria kills nearly 0.5 million people yearly and impacts the lives of those living in over 90 countries where it is endemic. The current treatment programs are threatened by increasing drug resistance. Dihydroorotate dehydrogenase (DHODH) is now clinically validated as a target for antimalarial drug discovery as a triazolopyrimidine class inhibitor (DSM265) is currently undergoing clinical development. We discovered a related isoxazolopyrimidine series in a phenotypic screen, later determining that it targeted DHODH. To determine if the isoxazolopyrimidines could yield a drug candidate, we initiated hit-to-lead medicinal chemistry. Several potent analogues were identified, including a compound that showed in vivo antimalarial activity. The isoxazolopyrimidines were more rapidly metabolized than their triazolopyrimidine counterparts, and the pharmacokinetic data were not consistent with the goal of a single-dose treatment for malaria.

Identification of Fast-Acting 2,6-Disubstituted Imidazopyridines That Are Efficacious in the in Vivo Humanized Plasmodium falciparum NODscidIL2Rγ null Mouse Model of Malaria.

Optimization of a chemical series originating from whole-cell phenotypic screening against the human malaria parasite, Plasmodium falciparum, led to the identification of two promising 2,6-disubstituted imidazopyridine compounds, 43 and 74. These compounds exhibited potent activity against asexual blood stage parasites that, together with their in vitro absorption, distribution, metabolism, and excretion (ADME) properties, translated to in vivo efficacy with clearance of parasites in the PfSCID mouse model for malaria within 48 h of treatment.

A potent antimalarial benzoxaborole targets a Plasmodium falciparum cleavage and polyadenylation specificity factor homologue.

Benzoxaboroles are effective against bacterial, fungal and protozoan pathogens. We report potent activity of the benzoxaborole AN3661 against Plasmodium falciparum laboratory-adapted strains (mean IC50 32 nM), Ugandan field isolates (mean ex vivo IC50 64 nM), and murine P. berghei and P. falciparum infections (day 4 ED90 0.34 and 0.57 mg kg-1, respectively). Multiple P. falciparum lines selected in vitro for resistance to AN3661 harboured point mutations in pfcpsf3, which encodes a homologue of mammalian cleavage and polyadenylation specificity factor subunit 3 (CPSF-73 or CPSF3). CRISPR-Cas9-mediated introduction of pfcpsf3 mutations into parental lines recapitulated AN3661 resistance. PfCPSF3 homology models placed these mutations in the active site, where AN3661 is predicted to bind. Transcripts for three trophozoite-expressed genes were lost in AN3661-treated trophozoites, which was not observed in parasites selected or engineered for AN3661 resistance. Our results identify the pre-mRNA processing factor PfCPSF3 as a promising antimalarial drug target.

The Discovery of Novel Antimalarial Aminoxadiazoles as a Promising Nonendoperoxide Scaffold.

Since the appearance of resistance to the current front-line antimalarial treatments, ACTs (artemisinin combination therapies), the discovery of novel chemical entities to treat the disease is recognized as a major global health priority. From the GSK antimalarial set, we identified an aminoxadiazole with an antiparasitic profile comparable with artemisinin (1), with no cross-resistance in a resistant strains panel and a potential new mode of action. A medicinal chemistry program allowed delivery of compounds such as 19 with high solubility in aqueous media, an acceptable toxicological profile, and oral efficacy. Further evaluation of the lead compounds showed that in vivo genotoxic degradants might be generated. The compounds generated during this medicinal chemistry program and others from the GSK collection were used to build a pharmacophore model which could be used in the virtual screening of compound collections and potentially identify new chemotypes that could deliver the same antiparasitic profile.

Novel Antitubercular 6-Dialkylaminopyrimidine Carboxamides from Phenotypic Whole-Cell High Throughput Screening of a SoftFocus Library: Structure-Activity Relationship and Target Identification Studies.

A BioFocus DPI SoftFocus library of ∼35 000 compounds was screened against Mycobacterium tuberculosis (Mtb) in order to identify novel hits with antitubercular activity. The hits were evaluated in biology triage assays to exclude compounds suggested to function via frequently encountered promiscuous mechanisms of action including inhibition of the QcrB subunit of the cytochrome bc1 complex, disruption of cell-wall homeostasis, and DNA damage. Among the hits that passed this screening cascade, a 6-dialkylaminopyrimidine carboxamide series was prioritized for hit to lead optimization. Compounds from this series were active against clinical Mtb strains, while no cross-resistance to conventional antituberculosis drugs was observed. This suggested a novel mechanism of action, which was confirmed by chemoproteomic analysis leading to the identification of BCG_3193 and BCG_3827 as putative targets of the series with unknown function. Initial structure-activity relationship studies have resulted in compounds with moderate to potent antitubercular activity and improved physicochemical properties.

Antimalarial efficacy of MMV390048, an inhibitor of Plasmodium phosphatidylinositol 4-kinase.

As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all Plasmodium life cycle stages, apart from late hypnozoites in the liver. Efficacy was shown in the humanized Plasmodium falciparum mouse model, and modest reductions in mouse-to-mouse transmission were achieved in the Plasmodium berghei mouse model. Experiments in monkeys revealed the ability of MMV390048 to be used for full chemoprotection. Although MMV390048 was not able to eliminate liver hypnozoites, it delayed relapse in a Plasmodium cynomolgi monkey model. Both genomic and chemoproteomic studies identified a kinase of the Plasmodium parasite, phosphatidylinositol 4-kinase, as the molecular target of MMV390048. The ability of MMV390048 to block all life cycle stages of the malaria parasite suggests that this compound should be further developed and may contribute to malaria control and eradication as part of a single-dose combination treatment.