ABSTRACT
Most phenotypic screens aiming to discover new antimalarial chemotypes begin with low cost, high-throughput tests against the asexual blood stage (ABS) of the malaria parasite life cycle. Compounds active against the ABS are then sequentially tested in more difficult assays that predict whether a compound has other beneficial attributes. Although applying this strategy to new chemical libraries may yield new leads, repeated iterations may lead to diminishing returns and the rediscovery of chemotypes hitting well-known targets. Here, we adopted a different strategy to find starting points, testing â¼70,000 open source small molecules from the Global Health Chemical Diversity Library for activity against the liver stage, mature sexual stage, and asexual blood stage malaria parasites in parallel. In addition, instead of using an asexual assay that measures accumulated parasite DNA in the presence of compound (SYBR green), a real time luciferase-dependent parasite viability assay was used that distinguishes slow-acting (delayed death) from fast-acting compounds. Among 382 scaffolds with the activity confirmed by dose response (<10 µM), we discovered 68 novel delayed-death, 84 liver stage, and 68 stage V gametocyte inhibitors as well. Although 89% of the evaluated compounds had activity in only a single life cycle stage, we discovered six potent (half-maximal inhibitory concentration of <1 µM) multistage scaffolds, including a novel cytochrome bc1 chemotype. Our data further show the luciferase-based assays have higher sensitivity. Chemoinformatic analysis of positive and negative compounds identified scaffold families with a strong enrichment for activity against specific or multiple stages.
Subject(s)
Antimalarials/isolation & purification , Drug Discovery , Life Cycle Stages/drug effects , Plasmodium falciparum/drug effects , Small Molecule Libraries/pharmacology , Antimalarials/chemistry , Antimalarials/pharmacology , Cheminformatics/methods , Drug Evaluation, Preclinical , High-Throughput Screening Assays , Plasmodium falciparum/genetics , Small Molecule Libraries/chemistryABSTRACT
Current therapeutics for chronic infection with hepatitis B virus (HBV) rarely induce functional cure due to the immunotolerant status of patients. Small molecule agonists targeting toll-like receptor 7 (TLR7) have been shown to elicit a functional cure in animal models of HBV but sometimes with poor tolerability due to immune-related toxicities. In an effort to increase the therapeutic window of TLR7 agonists to treat chronic hepatitis B (CHB), we developed an oral TLR7 agonist, APR002, designed to act locally in the gastrointestinal tract and liver, thus minimizing systemic exposure and improving tolerability. Here, we describe the pharmacokinetic/pharmacodynamic (PK/PD) profile of APR002 in mice and uninfected woodchucks as well as the safety and antiviral efficacy in combination with entecavir (ETV) in woodchucks with CHB. Treatment of woodchucks chronically infected with woodchuck hepatitis virus (WHV) with weekly oral doses of APR002 was well-tolerated. While APR002 and ETV single agents did not elicit sustained viral control, combination therapy resulted in durable immune-mediated suppression of the chronic infection. These woodchucks also had detectable antibodies to viral antigens, enhanced interferon-stimulated gene expression, and loss of WHV covalently closed circular DNA. Conclusion: APR002 is a novel TLR7 agonist exhibiting a distinct PK/PD profile that in combination with ETV can safely attain a functional cure in woodchucks with chronic WHV infection. Our results support further investigation of liver-targeted TLR7 agonists in human CHB.
ABSTRACT
A series of novel 8-aminoquinolines (8-AQs) with an aminoxyalkyl side chain were synthesized and evaluated for in vitro antiplasmodial properties against asexual blood stages, liver stages, and sexual stages of Plasmodium falciparum. 8-AQs bearing 2-alkoxy and 5-phenoxy substituents on the quinoline ring system were found to be the most promising compounds under study, exhibiting potent blood schizontocidal and moderate tissue schizontocidal in vitro activity.
Subject(s)
Aminoquinolines/chemistry , Antimalarials/chemistry , Plasmodium falciparum/growth & development , Aminoquinolines/chemical synthesis , Aminoquinolines/pharmacology , Antimalarials/chemical synthesis , Antimalarials/pharmacology , Cell Survival/drug effects , Hep G2 Cells , Humans , Life Cycle Stages/drug effects , Plasmodium falciparum/drug effects , Structure-Activity RelationshipABSTRACT
To discover leads for next-generation chemoprotective antimalarial drugs, we tested more than 500,000 compounds for their ability to inhibit liver-stage development of luciferase-expressing Plasmodium spp. parasites (681 compounds showed a half-maximal inhibitory concentration of less than 1 micromolar). Cluster analysis identified potent and previously unreported scaffold families as well as other series previously associated with chemoprophylaxis. Further testing through multiple phenotypic assays that predict stage-specific and multispecies antimalarial activity distinguished compound classes that are likely to provide symptomatic relief by reducing asexual blood-stage parasitemia from those which are likely to only prevent malaria. Target identification by using functional assays, in vitro evolution, or metabolic profiling revealed 58 mitochondrial inhibitors but also many chemotypes possibly with previously unidentified mechanisms of action.
Subject(s)
Antimalarials/pharmacology , Chemoprevention , Drug Discovery , Malaria/prevention & control , Plasmodium/drug effects , Antimalarials/chemistry , Antimalarials/isolation & purification , Antimalarials/therapeutic use , Drug Evaluation, Preclinical , Humans , Mitochondria/drug effects , Plasmodium/growth & developmentABSTRACT
UNLABELLED: Mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) are associated with parasite resistance to the imidazolopiperazines, a potent class of novel antimalarial compounds that display both prophylactic and transmission-blocking activity, in addition to activity against blood-stage parasites. Here, we show that pfcarl encodes a protein, with a predicted molecular weight of 153 kDa, that localizes to the cis-Golgi apparatus of the parasite in both asexual and sexual blood stages. Utilizing clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene introduction of 5 variants (L830V, S1076N/I, V1103L, and I1139K), we demonstrate that mutations in pfcarl are sufficient to generate resistance against the imidazolopiperazines in both asexual and sexual blood-stage parasites. We further determined that the mutant PfCARL protein confers resistance to several structurally unrelated compounds. These data suggest that PfCARL modulates the levels of small-molecule inhibitors that affect Golgi-related processes, such as protein sorting or membrane trafficking, and is therefore an important mechanism of resistance in malaria parasites. IMPORTANCE: Several previous in vitro evolution studies have implicated the Plasmodium falciparum cyclic amine resistance locus (PfCARL) as a potential target of imidazolopiperazines, potent antimalarial compounds with broad activity against different parasite life cycle stages. Given that the imidazolopiperazines are currently being tested in clinical trials, understanding their mechanism of resistance and the cellular processes involved will allow more effective clinical usage.
Subject(s)
Antimalarials/pharmacology , Drug Resistance, Multiple , Genetic Loci , Mutation , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , Recombination, GeneticABSTRACT
In order to identify the most attractive starting points for drugs that can be used to prevent malaria, a diverse chemical space comprising tens of thousands to millions of small molecules may need to be examined. Achieving this throughput necessitates the development of efficient ultra-high-throughput screening methods. Here, we report the development and evaluation of a luciferase-based phenotypic screen of malaria exoerythrocytic-stage parasites optimized for a 1536-well format. This assay uses the exoerythrocytic stage of the rodent malaria parasite, Plasmodium berghei, and a human hepatoma cell line. We use this assay to evaluate several biased and unbiased compound libraries, including two small sets of molecules (400 and 89 compounds, respectively) with known activity against malaria erythrocytic-stage parasites and a set of 9886 diversity-oriented synthesis (DOS)-derived compounds. Of the compounds screened, we obtain hit rates of 12-13 and 0.6% in preselected and naïve libraries, respectively, and identify 52 compounds with exoerythrocytic-stage activity less than 1 µM and having minimal host cell toxicity. Our data demonstrate the ability of this method to identify compounds known to have causal prophylactic activity in both human and animal models of malaria, as well as novel compounds, including some exclusively active against parasite exoerythrocytic stages.
ABSTRACT
Preventing transmission is an important element of malaria control. However, most of the current available methods to assay for malaria transmission blocking are relatively low throughput and cannot be applied to large chemical libraries. We have developed a high-throughput and cost-effective assay, the Saponin-lysis Sexual Stage Assay (SaLSSA), for identifying small molecules with transmission-blocking capacity. SaLSSA analysis of 13,983 unique compounds uncovered that >90% of well-characterized antimalarials, including endoperoxides and 4-aminoquinolines, as well as compounds active against asexual blood stages, lost most of their killing activity when parasites developed into metabolically quiescent stage V gametocytes. On the other hand, we identified compounds with consistent low nanomolar transmission-blocking activity, some of which showed cross-reactivity against asexual blood and liver stages. The data clearly emphasize substantial physiological differences between sexual and asexual parasites and provide a tool and starting points for the discovery and development of transmission-blocking drugs.
Subject(s)
Antimalarials/pharmacology , Drug Evaluation, Preclinical/methods , High-Throughput Screening Assays/methods , Malaria/parasitology , Plasmodium falciparum/drug effects , Humans , Malaria/transmission , Plasmodium falciparum/physiologyABSTRACT
Renewed global efforts toward malaria eradication have highlighted the need for novel antimalarial agents with activity against multiple stages of the parasite life cycle. We have previously reported the discovery of a novel class of antimalarial compounds in the imidazolopiperazine series that have activity in the prevention and treatment of blood stage infection in a mouse model of malaria. Consistent with the previously reported activity profile of this series, the clinical candidate KAF156 shows blood schizonticidal activity with 50% inhibitory concentrations of 6 to 17.4 nM against P. falciparum drug-sensitive and drug-resistant strains, as well as potent therapeutic activity in a mouse models of malaria with 50, 90, and 99% effective doses of 0.6, 0.9, and 1.4 mg/kg, respectively. When administered prophylactically in a sporozoite challenge mouse model, KAF156 is completely protective as a single oral dose of 10 mg/kg. Finally, KAF156 displays potent Plasmodium transmission blocking activities both in vitro and in vivo. Collectively, our data suggest that KAF156, currently under evaluation in clinical trials, has the potential to treat, prevent, and block the transmission of malaria.
Subject(s)
Antimalarials/pharmacology , Imidazoles/pharmacology , Malaria, Falciparum/drug therapy , Malaria, Falciparum/transmission , Piperazines/pharmacology , Animals , Inhibitory Concentration 50 , Mice , Mice, Inbred ICR , Plasmodium falciparum/drug effects , Sporozoites/drug effectsABSTRACT
Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 µM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 µM, and PQ, 0.84 µM; for developing liver stages, KAI407, 0.64 µM, and PQ, 0.37 µM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class for P. vivax malaria prophylaxis and potentially a radical cure.
Subject(s)
Antimalarials/pharmacology , Imidazoles/pharmacology , Malaria/drug therapy , Plasmodium cynomolgi/drug effects , Pyrazines/pharmacology , Animals , Antimalarials/therapeutic use , Drug Evaluation, Preclinical/methods , Female , Hepatocytes/parasitology , Imidazoles/therapeutic use , In Vitro Techniques , Liver/parasitology , Macaca mulatta/parasitology , Malaria/parasitology , Malaria/prevention & control , Mice , Mice, Inbred ICR , Pyrazines/therapeutic use , Sporozoites/drug effectsABSTRACT
Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.
Subject(s)
1-Phosphatidylinositol 4-Kinase/antagonists & inhibitors , Malaria/drug therapy , Malaria/parasitology , Plasmodium/drug effects , Plasmodium/enzymology , 1-Phosphatidylinositol 4-Kinase/chemistry , 1-Phosphatidylinositol 4-Kinase/genetics , 1-Phosphatidylinositol 4-Kinase/metabolism , Adenosine Triphosphate/metabolism , Animals , Binding Sites , Cytokinesis/drug effects , Drug Resistance/drug effects , Drug Resistance/genetics , Fatty Acids/metabolism , Female , Hepatocytes/parasitology , Humans , Imidazoles/metabolism , Imidazoles/pharmacology , Life Cycle Stages/drug effects , Macaca mulatta , Male , Models, Biological , Models, Molecular , Phosphatidylinositol Phosphates/metabolism , Plasmodium/classification , Plasmodium/growth & development , Pyrazoles/metabolism , Pyrazoles/pharmacology , Quinoxalines/metabolism , Quinoxalines/pharmacology , Reproducibility of Results , Schizonts/cytology , Schizonts/drug effects , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolismABSTRACT
Malaria parasites elude eradication attempts both within the human host and across nations. At the individual level, parasites evade the host immune responses through antigenic variation. At the global level, parasites escape drug pressure through single nucleotide variants and gene copy amplification events conferring drug resistance. Despite their importance to global health, the rates at which these genomic alterations emerge have not been determined. We studied the complete genomes of different Plasmodium falciparum clones that had been propagated asexually over one year in the presence and absence of drug pressure. A combination of whole-genome microarray analysis and next-generation deep resequencing (totaling 14 terabases) revealed a stable core genome with only 38 novel single nucleotide variants appearing in seventeen evolved clones (avg. 5.4 per clone). In clones exposed to atovaquone, we found cytochrome b mutations as well as an amplification event encompassing the P. falciparum multidrug resistance associated protein (mrp1) on chromosome 1. We observed 18 large-scale (>1 kb on average) deletions of telomere-proximal regions encoding multigene families, involved in immune evasion (9.5×10(-6) structural variants per base pair per generation). Six of these deletions were associated with chromosomal crossovers generated during mitosis. We found only minor differences in rates between genetically distinct strains and between parasites cultured in the presence or absence of drug. Using these derived mutation rates for P. falciparum (1.0-9.7×10(-9) mutations per base pair per generation), we can now model the frequency at which drug or immune resistance alleles will emerge under a well-defined set of assumptions. Further, the detection of mitotic recombination events in var gene families illustrates how multigene families can arise and change over time in P. falciparum. These results will help improve our understanding of how P. falciparum evolves to evade control efforts within both the individual hosts and large populations.
Subject(s)
Antigens , Atovaquone/administration & dosage , Drug Resistance, Multiple , Host-Parasite Interactions , Plasmodium falciparum , Antigenic Variation/drug effects , Antigenic Variation/genetics , Antigens/drug effects , Antigens/genetics , Cytochromes b/genetics , Drug Resistance, Multiple/drug effects , Drug Resistance, Multiple/genetics , Evolution, Molecular , Genome, Protozoan/drug effects , High-Throughput Nucleotide Sequencing , Host-Parasite Interactions/genetics , Host-Parasite Interactions/immunology , Humans , Malaria, Falciparum/genetics , Malaria, Falciparum/immunology , Mitosis/genetics , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/immunology , Multidrug Resistance-Associated Proteins/metabolism , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Plasmodium falciparum/immunologyABSTRACT
With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited.
Subject(s)
Antimalarials/pharmacology , Enzyme Inhibitors/pharmacology , Fungi/chemistry , Isocoumarins/pharmacology , Lysine-tRNA Ligase/antagonists & inhibitors , Plasmodium falciparum/enzymology , Antimalarials/isolation & purification , Cell Line , Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/isolation & purification , Humans , Inhibitory Concentration 50 , Isocoumarins/isolation & purification , Parasitic Sensitivity Tests , Plasmodium falciparum/drug effects , Protein Biosynthesis/drug effects , Protozoan Proteins/antagonists & inhibitorsABSTRACT
On the basis of the initial success of optimization of a novel series of imidazolopiperazines, a second generation of compounds involving changes in the core piperazine ring was synthesized to improve antimalarial properties. These changes were carried out to further improve the potency and metabolic stability of the compounds by leveraging the outcome of a set of in vitro metabolic identification studies. The optimized 8,8-dimethyl imidazolopiperazine analogues exhibited improved potency, in vitro metabolic stability profile and, as a result, enhanced oral exposure in vivo in mice. The optimized compounds were found to be more efficacious than the current antimalarials in a malaria mouse model. They exhibit moderate oral exposure in rat pharmacokinetic studies to achieve sufficient multiples of the oral exposure at the efficacious dose in toxicology studies.
Subject(s)
Antimalarials/pharmacology , Imidazoles/pharmacology , Malaria, Falciparum/drug therapy , Piperazines/pharmacology , Plasmodium falciparum/drug effects , Animals , Antimalarials/chemical synthesis , Antimalarials/chemistry , Antimalarials/pharmacokinetics , Biological Availability , Caco-2 Cells , Humans , Imidazoles/chemical synthesis , Imidazoles/chemistry , Imidazoles/pharmacokinetics , Malaria, Falciparum/parasitology , Mice , Mice, Inbred BALB C , Piperazines/chemical synthesis , Piperazines/chemistry , Piperazines/pharmacokinetics , Plasmodium falciparum/metabolism , Rats , Rats, Wistar , Structure-Activity RelationshipABSTRACT
BACKGROUND: Malaria remains a disease of devastating global impact, killing more than 800,000 people every year-the vast majority being children under the age of 5. While effective therapies are available, if malaria is to be eradicated a broader range of small molecule therapeutics that are able to target the liver and the transmissible sexual stages are required. These new medicines are needed both to meet the challenge of malaria eradication and to circumvent resistance. METHODS AND FINDINGS: Little is known about the wider stage-specific activities of current antimalarials that were primarily designed to alleviate symptoms of malaria in the blood stage. To overcome this critical gap, we developed assays to measure activity of antimalarials against all life stages of malaria parasites, using a diverse set of human and nonhuman parasite species, including male gamete production (exflagellation) in Plasmodium falciparum, ookinete development in P. berghei, oocyst development in P. berghei and P. falciparum, and the liver stage of P. yoelii. We then compared 50 current and experimental antimalarials in these assays. We show that endoperoxides such as OZ439, a stable synthetic molecule currently in clinical phase IIa trials, are strong inhibitors of gametocyte maturation/gamete formation and impact sporogony; lumefantrine impairs development in the vector; and NPC-1161B, a new 8-aminoquinoline, inhibits sporogony. CONCLUSIONS: These data enable objective comparisons of the strengths and weaknesses of each chemical class at targeting each stage of the lifecycle. Noting that the activities of many compounds lie within achievable blood concentrations, these results offer an invaluable guide to decisions regarding which drugs to combine in the next-generation of antimalarial drugs. This study might reveal the potential of life-cycle-wide analyses of drugs for other pathogens with complex life cycles.
Subject(s)
Antimalarials/pharmacology , Malaria/drug therapy , Plasmodium berghei/drug effects , Plasmodium falciparum/drug effects , Plasmodium yoelii/drug effects , Animals , Antimalarials/chemistry , Antimalarials/classification , Culicidae/parasitology , Drug Resistance, Multiple , Humans , Liver/parasitology , Malaria/parasitology , Malaria/transmission , Malaria, Falciparum/drug therapy , Malaria, Falciparum/parasitology , Malaria, Falciparum/transmission , Mice/parasitology , Plasmodium berghei/growth & development , Plasmodium falciparum/growth & development , Plasmodium yoelii/growth & development , Species SpecificityABSTRACT
Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.
Subject(s)
Antimalarials/pharmacology , Drug Discovery , Imidazoles/pharmacology , Liver/parasitology , Malaria/drug therapy , Piperazines/pharmacology , Plasmodium/drug effects , Animals , Antimalarials/chemistry , Antimalarials/pharmacokinetics , Antimalarials/therapeutic use , Cell Line, Tumor , Drug Evaluation, Preclinical , Drug Resistance , Erythrocytes/parasitology , Humans , Imidazoles/chemistry , Imidazoles/pharmacokinetics , Imidazoles/therapeutic use , Malaria/parasitology , Malaria/prevention & control , Mice , Mice, Inbred BALB C , Molecular Structure , Piperazines/chemistry , Piperazines/pharmacokinetics , Piperazines/therapeutic use , Plasmodium/cytology , Plasmodium/growth & development , Plasmodium/physiology , Plasmodium berghei/cytology , Plasmodium berghei/drug effects , Plasmodium berghei/growth & development , Plasmodium berghei/physiology , Plasmodium falciparum/cytology , Plasmodium falciparum/drug effects , Plasmodium falciparum/growth & development , Plasmodium falciparum/physiology , Plasmodium yoelii/cytology , Plasmodium yoelii/drug effects , Plasmodium yoelii/growth & development , Plasmodium yoelii/physiology , Polymorphism, Single Nucleotide , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Random Allocation , Small Molecule Libraries , Sporozoites/drug effects , Sporozoites/growth & developmentABSTRACT
Decoquinate has single-digit nanomolar activity against in vitro blood stage Plasmodium falciparum parasites, the causative agent of human malaria. In vitro evolution of decoquinate-resistant parasites and subsequent comparative genomic analysis to the drug-sensitive parental strain revealed resistance was conferred by two nonsynonymous single nucleotide polymorphisms in the gene encoding cytochrome b. The resultant amino acid mutations, A122T and Y126C, reside within helix C in the ubiquinol-binding pocket of cytochrome b, an essential subunit of the cytochrome bc(1) complex. As with other cytochrome bc(1) inhibitors, such as atovaquone, decoquinate has low nanomolar activity against in vitro liver stage P. yoelii and provides partial prophylaxis protection when administered to infected mice at 50 mg kg(-1). In addition, transgenic parasites expressing yeast dihydroorotate dehydrogenase are >200-fold less sensitive to decoquinate, which provides additional evidence that this drug inhibits the parasite's mitochondrial electron transport chain. Importantly, decoquinate exhibits limited cross-resistance to a panel of atovaquone-resistant parasites evolved to harbor various mutations in cytochrome b. The basis for this difference was revealed by molecular docking studies, in which both of these inhibitors were shown to have distinctly different modes of binding within the ubiquinol-binding site of cytochrome b.
Subject(s)
Antimalarials/pharmacology , Cytochromes b/antagonists & inhibitors , Decoquinate/pharmacology , Plasmodium falciparum/drug effects , Animals , Antimalarials/administration & dosage , Antimalarials/chemistry , Crystallography, X-Ray , Cytochromes b/genetics , Cytochromes b/metabolism , Decoquinate/administration & dosage , Decoquinate/chemistry , Drug Discovery , Drug Resistance, Fungal/drug effects , Drug Resistance, Fungal/genetics , Female , Humans , Mice , Mice, Inbred ICR , Models, Molecular , Molecular Structure , Parasitic Sensitivity Tests , Plasmodium falciparum/genetics , Structure-Activity RelationshipABSTRACT
Starting from a hit series from a GNF compound library collection and based on a cell-based proliferation assay of Plasmodium falciparum, a novel imidazolopiperazine scaffold was optimized. SAR for this series of compounds is discussed, focusing on optimization of cellular potency against wild-type and drug resistant parasites and improvement of physiochemical and pharmacokinetic properties. The lead compounds in this series showed good potencies in vitro and decent oral exposure levels in vivo. In a Plasmodium berghei mouse infection model, one lead compound lowered the parasitemia level by 99.4% after administration of 100 mg/kg single oral dose and prolonged mice survival by an average of 17.0 days. The lead compounds were also well-tolerated in the preliminary in vitro toxicity studies and represents an interesting lead for drug development.
Subject(s)
Antimalarials/chemical synthesis , Imidazoles/chemical synthesis , Piperazines/chemical synthesis , Amino Acids/chemical synthesis , Amino Acids/chemistry , Amino Acids/pharmacology , Aniline Compounds/chemical synthesis , Aniline Compounds/chemistry , Aniline Compounds/pharmacology , Animals , Antimalarials/chemistry , Antimalarials/pharmacology , Benzene Derivatives/chemical synthesis , Benzene Derivatives/chemistry , Benzene Derivatives/pharmacology , Cell Line , Drug Resistance , Female , Humans , Imidazoles/chemistry , Imidazoles/pharmacology , Inhibitory Concentration 50 , Malaria/drug therapy , Mice , Mice, Inbred BALB C , Piperazines/chemistry , Piperazines/pharmacology , Plasmodium berghei , Plasmodium falciparum/drug effects , Rats , Structure-Activity RelationshipABSTRACT
Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine-imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics.
Subject(s)
Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , Adenosine Triphosphate/metabolism , Antitubercular Agents/pharmacology , Glycerophosphates/metabolism , Imidazoles/pharmacology , Models, BiologicalABSTRACT
Recent reports of increased tolerance to artemisinin derivatives--the most recently adopted class of antimalarials--have prompted a need for new treatments. The spirotetrahydro-beta-carbolines, or spiroindolones, are potent drugs that kill the blood stages of Plasmodium falciparum and Plasmodium vivax clinical isolates at low nanomolar concentration. Spiroindolones rapidly inhibit protein synthesis in P. falciparum, an effect that is ablated in parasites bearing nonsynonymous mutations in the gene encoding the P-type cation-transporter ATPase4 (PfATP4). The optimized spiroindolone NITD609 shows pharmacokinetic properties compatible with once-daily oral dosing and has single-dose efficacy in a rodent malaria model.
Subject(s)
Antimalarials/pharmacology , Indoles/pharmacology , Malaria/drug therapy , Plasmodium berghei/drug effects , Plasmodium falciparum/drug effects , Plasmodium vivax/drug effects , Spiro Compounds/pharmacology , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Animals , Antimalarials/administration & dosage , Antimalarials/chemistry , Antimalarials/pharmacokinetics , Cell Line , Drug Discovery , Drug Resistance , Erythrocytes/parasitology , Female , Genes, Protozoan , Humans , Indoles/administration & dosage , Indoles/chemistry , Indoles/pharmacokinetics , Malaria/parasitology , Male , Mice , Models, Molecular , Mutant Proteins/antagonists & inhibitors , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation , Parasitic Sensitivity Tests , Plasmodium falciparum/genetics , Plasmodium falciparum/growth & development , Plasmodium vivax/growth & development , Protein Synthesis Inhibitors/administration & dosage , Protein Synthesis Inhibitors/chemistry , Protein Synthesis Inhibitors/pharmacokinetics , Protein Synthesis Inhibitors/pharmacology , Protozoan Proteins/biosynthesis , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Rats , Rats, Wistar , Spiro Compounds/administration & dosage , Spiro Compounds/chemistry , Spiro Compounds/pharmacokineticsABSTRACT
Here, we fully characterize the genomes of 14 Plasmodium falciparum patient isolates taken recently from the Iquitos region using genome scanning, a microarray-based technique that delineates the majority of single-base changes, indels, and copy number variants distinguishing the coding regions of two clones. We show that the parasite population in the Peruvian Amazon bears a limited number of genotypes and low recombination frequencies. Despite the essentially clonal nature of some isolates, we see high frequencies of mutations in subtelomeric highly variable genes and internal var genes, indicating mutations arising during self-mating or mitotic replication. The data also reveal that one or two meioses separate different isolates, showing that P. falciparum clones isolated from different individuals in defined geographical regions could be useful in linkage analyses or quantitative trait locus studies. Through pairwise comparisons of different isolates we discovered point mutations in the apicoplast genome that are close to known mutations that confer clindamycin resistance in other species, but which were hitherto unknown in malaria parasites. Subsequent drug sensitivity testing revealed over 100-fold increase of clindamycin EC(50) in strains harboring one of these mutations. This evidence of clindamycin-resistant parasites in the Amazon suggests that a shift should be made in health policy away from quinine + clindamycin therapy for malaria in pregnant women and infants, and that the development of new lincosamide antibiotics for malaria should be reconsidered.