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1.
Nature ; 504(7479): 248-253, 2013 Dec 12.
Article in English | MEDLINE | ID: mdl-24284631

ABSTRACT

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/metabolism
2.
PLoS Genet ; 9(2): e1003293, 2013.
Article in English | MEDLINE | ID: mdl-23408914

ABSTRACT

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/immunology
3.
Antimicrob Agents Chemother ; 58(3): 1586-95, 2014.
Article in English | MEDLINE | ID: mdl-24366744

ABSTRACT

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 effects
4.
Antimicrob Agents Chemother ; 58(9): 5060-7, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24913172

ABSTRACT

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 effects
5.
Genome Res ; 20(11): 1534-44, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20829224

ABSTRACT

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.


Subject(s)
Chromosomal Instability , Chromosome Mapping , Clindamycin , Drug Resistance/genetics , Malaria, Falciparum/parasitology , Plasmodium falciparum/genetics , Antimalarials/therapeutic use , Base Sequence , Chromosomal Instability/genetics , Chromosome Mapping/methods , Clindamycin/therapeutic use , DNA Copy Number Variations , Female , Gene Frequency , Genome, Protozoan , Genotype , Humans , Infant , Malaria, Falciparum/diagnosis , Malaria, Falciparum/drug therapy , Malaria, Falciparum/genetics , Male , Models, Biological , Models, Molecular , Molecular Sequence Data , Pedigree , Peru , Pregnancy , Telomere/genetics
6.
PLoS Med ; 9(2): e1001169, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22363211

ABSTRACT

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 Specificity
7.
Proc Natl Acad Sci U S A ; 105(26): 9059-64, 2008 Jul 01.
Article in English | MEDLINE | ID: mdl-18579783

ABSTRACT

The growing resistance to current first-line antimalarial drugs represents a major health challenge. To facilitate the discovery of new antimalarials, we have implemented an efficient and robust high-throughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of approximately 1.7 million compounds, we identified a diverse collection of approximately 6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (<1.25 microM). Most known antimalarials were identified in this screen, thus validating our approach. In addition, we identified many novel chemical scaffolds, which likely act through both known and novel pathways. We further show that in some cases the mechanism of action of these antimalarials can be determined by in silico compound activity profiling. This method uses large datasets from unrelated cellular and biochemical screens and the guilt-by-association principle to predict which cellular pathway and/or protein target is being inhibited by select compounds. In addition, the screening method has the potential to provide the malaria community with many new starting points for the development of biological probes and drugs with novel antiparasitic activities.


Subject(s)
Antimalarials/analysis , Antimalarials/pharmacology , Computational Biology , Animals , Antimalarials/chemistry , Antimalarials/therapeutic use , Cluster Analysis , Drug Evaluation, Preclinical , Drug Resistance/drug effects , Folic Acid Antagonists/analysis , Folic Acid Antagonists/chemistry , Folic Acid Antagonists/pharmacology , Malaria/drug therapy , Models, Molecular , Parasites/drug effects , Plasmodium falciparum/drug effects , Plasmodium falciparum/enzymology , Reproducibility of Results , Structure-Activity Relationship , Tetrahydrofolate Dehydrogenase/chemistry
8.
Nat Chem Biol ; 4(6): 347-56, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18454143

ABSTRACT

Calcium-dependent protein kinases play a crucial role in intracellular calcium signaling in plants, some algae and protozoa. In Plasmodium falciparum, calcium-dependent protein kinase 1 (PfCDPK1) is expressed during schizogony in the erythrocytic stage as well as in the sporozoite stage. It is coexpressed with genes that encode the parasite motor complex, a cellular component required for parasite invasion of host cells, parasite motility and potentially cytokinesis. A targeted gene-disruption approach demonstrated that pfcdpk1 seems to be essential for parasite viability. An in vitro biochemical screen using recombinant PfCDPK1 against a library of 20,000 compounds resulted in the identification of a series of structurally related 2,6,9-trisubstituted purines. Compound treatment caused sudden developmental arrest at the late schizont stage in P. falciparum and a large reduction in intracellular parasites in Toxoplasma gondii, which suggests a possible role for PfCDPK1 in regulation of parasite motility during egress and invasion.


Subject(s)
Adenine/analogs & derivatives , Antimalarials/pharmacology , Cyclohexylamines/pharmacology , Gene Expression Regulation, Enzymologic/genetics , Malaria/parasitology , Plasmodium falciparum/enzymology , Protein Kinases/drug effects , Protein Kinases/genetics , Protozoan Proteins/antagonists & inhibitors , Adenine/chemistry , Adenine/pharmacology , Adenine/therapeutic use , Animals , Antimalarials/chemistry , Antimalarials/therapeutic use , CHO Cells , Cell Line , Cell Proliferation/drug effects , Cricetinae , Cricetulus , Cyclohexylamines/chemistry , Cyclohexylamines/therapeutic use , Drug Evaluation, Preclinical , Enzyme Activation/drug effects , Gene Expression Regulation, Enzymologic/drug effects , HeLa Cells , Humans , Life Cycle Stages/drug effects , Malaria/drug therapy , Malaria/immunology , Male , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Structure , Molecular Weight , Movement/drug effects , Oligonucleotide Array Sequence Analysis/methods , Parasitic Sensitivity Tests , Plasmodium falciparum/growth & development , Protein Kinases/physiology , Protozoan Proteins/genetics , Protozoan Proteins/physiology , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Small Molecule Libraries , Stereoisomerism , Structure-Activity Relationship , Tissue Distribution
9.
Bioorg Med Chem Lett ; 20(14): 4027-31, 2010 Jul 15.
Article in English | MEDLINE | ID: mdl-20610151

ABSTRACT

A novel family of 1H-imidazol-2-yl-pyrimidine-4,6-diamines has been identified with potent activity against the erythrocyte-stage of Plasmodium falciparum (Pf), the most common causative agent of malaria. A systematic SAR study resulted in the identification of compound 40 which exhibits good potency against both wild-type and drug resistant parasites and exhibits good in vivo pharmacokinetic properties.


Subject(s)
Antimalarials/chemistry , Plasmodium falciparum/drug effects , Pyrimidines/chemistry , Animals , Antimalarials/pharmacokinetics , Antimalarials/pharmacology , Drug Discovery , Pyrimidines/pharmacokinetics , Pyrimidines/pharmacology , Structure-Activity Relationship
10.
ACS Infect Dis ; 6(4): 613-628, 2020 04 10.
Article in English | MEDLINE | ID: mdl-32078764

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 ∼70,000 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/chemistry
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