Case McNamara - Academia.edu (original) (raw)

Papers by Case McNamara

Scientific Reports, 2016

The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are re... more The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4. We show here that S. cerevisiae also acquires mutations in a gene encoding a P-type ATPase (ScPMA1) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace ScPma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits ScPma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a ScPma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae. This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity.

Science, 2008

Antigenically variable M proteins are major virulence factors and immunogens of the human pathoge... more Antigenically variable M proteins are major virulence factors and immunogens of the human pathogen group A Streptococcus (GAS). Here, we report the ∼3 angstrom resolution structure of a GAS M1 fragment containing the regions responsible for eliciting type-specific, protective immunity and for binding fibrinogen, which promotes M1 proinflammatory and antiphagocytic functions. The structure revealed substantial irregularities and instabilities throughout the coiled coil of the M1 fragment. Similar structural irregularities occur in myosin and tropomyosin, explaining the patterns of cross-reactivity seen in autoimmune sequelae of GAS infection. Sequence idealization of a large segment of the M1 coiled coil enhanced stability but diminished fibrinogen binding, proinflammatory effects, and antibody cross-reactivity, whereas it left protective immunogenicity undiminished. Idealized M proteins appear to have promise as vaccine immunogens.

ACS Infectious Diseases, 2016

In order to identify the most attractive starting points for drugs that can be used to prevent ma... more 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.

Journal of medicinal chemistry, Jan 18, 2015

Several of the enzymes related to the folate cycle are well-known for their role as clinically va... more Several of the enzymes related to the folate cycle are well-known for their role as clinically validated antimalarial targets. Nevertheless for serine hydroxymethyltransferase (SHMT), one of the key enzymes of this cycle, efficient inhibitors have not been described so far. On the basis of plant SHMT inhibitors from an herbicide optimization program, highly potent inhibitors of Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) SHMT with a pyrazolopyran core structure were identified. Cocrystal structures of potent inhibitors with PvSHMT were solved at 2.6 Å resolution. These ligands showed activity (IC50/EC50 values) in the nanomolar range against purified PfSHMT, blood-stage Pf, and liver-stage P. berghei (Pb) cells and a high selectivity when assayed against mammalian cell lines. Pharmacokinetic limitations are the most plausible explanation for lack of significant activity of the inhibitors in the in vivo Pb mouse malaria model.

ACS Chemical Biology, 2014

Aminopyrazoles are a new class of antimalarial compounds identified in a cellular antiparasitic s... more Aminopyrazoles are a new class of antimalarial compounds identified in a cellular antiparasitic screen with potent activity against Plasmodium falciparum asexual and sexual stage parasites. To investigate their unknown mechanism of action and thus identify their target, we cultured parasites in the presence of a representative member of the aminopyrazole series, GNF-Pf4492, to select for resistance. Whole genome sequencing of three resistant lines showed that each had acquired independent mutations in a P-type cation-transporter ATPase, PfATP4 (PF3D7_1211900), a protein implicated as the novel Plasmodium spp. target of another, structurally unrelated, class of antimalarials called the spiroindolones and characterized as an important sodium transporter of the cell. Similarly to the spiroindolones, GNF-Pf4492 blocks parasite transmission to mosquitoes and disrupts intracellular sodium homeostasis. Our data demonstrate that PfATP4 plays a critical role in cellular processes, can be inhibited by two distinct antimalarial pharmacophores, and supports the recent observations that PfATP4 is a critical antimalarial target.

ACS Medicinal Chemistry Letters, 2014

Imidazopyridine 1 was identified from a phenotypic screen against P. falciparum (Pf) blood stages... more Imidazopyridine 1 was identified from a phenotypic screen against P. falciparum (Pf) blood stages and subsequently optimized for activity on liver-stage schizonts of the rodent parasite P. yoelii (Py) as well as hypnozoites of the simian parasite P. cynomolgi (Pc). We applied these various assays to the cell-based lead optimization of the imidazopyrazines, exemplified by 3 (KAI407), and show that optimized compounds within the series with improved pharmacokinetic properties achieve causal prophylactic activity in vivo and may have the potential to target the dormant stages of P. vivax malaria.

Science, 2011

Most malaria drug development focuses on parasite stages detected in red blood cells, even though... more 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.

Science, 2008

Antigenically variable M proteins are major virulence factors and immunogens of the human pathoge... more Antigenically variable M proteins are major virulence factors and immunogens of the human pathogen group A Streptococcus (GAS). Here, we report the ∼3 angstrom resolution structure of a GAS M1 fragment containing the regions responsible for eliciting type-specific, protective immunity and for binding fibrinogen, which promotes M1 proinflammatory and antiphagocytic functions. The structure revealed substantial irregularities and instabilities throughout the coiled coil of the M1 fragment. Similar structural irregularities occur in myosin and tropomyosin, explaining the patterns of cross-reactivity seen in autoimmune sequelae of GAS infection. Sequence idealization of a large segment of the M1 coiled coil enhanced stability but diminished fibrinogen binding, proinflammatory effects, and antibody cross-reactivity, whereas it left protective immunogenicity undiminished. Idealized M proteins appear to have promise as vaccine immunogens.

Science, 2010

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Proteins: Structure, Function, and Bioinformatics, 2011

Establishing a quantitative understanding of the determinants of affinity in protein-protein inte... more Establishing a quantitative understanding of the determinants of affinity in protein-protein interactions remains challenging. For example, TEM-1/BLIP and SHV-1/BLIP are homologous βlactamase/β-lactamase inhibitor protein complexes with disparate K i values (3 nM and 2 μM, respectively), and a single substitution, D104E in SHV-1, results in a 1000-fold enhancement in binding affinity. In TEM-1, E104 participates in a salt bridge with BLIP K74, while the corresponding SHV-1 D104 does not in the wild type SHV-1/BLIP co-structure. Here, we present a 1.6 Å crystal structure of the SHV-1 D104E/BLIP complex that demonstrates that this point mutation restores this salt bridge. Additionally, mutation of a neighboring residue, BLIP E73M, results in salt bridge formation between SHV-1 D104 and BLIP K74 and a 400-fold increase in binding affinity. To understand how this salt bridge contributes to complex affinity, the cooperativity between the E/K or D/K salt bridge pair and a neighboring hot spot residue (BLIP F142) was investigated using double mutant cycle analyses in the background of the E73M mutation. We find that BLIP F142 cooperatively stabilizes both interactions, illustrating how a single mutation at a hot spot position can drive large perturbations in interface stability and specificity through a cooperative interaction network.

Proceedings of the National Academy of Sciences, 2008

The growing resistance to current first-line antimalarial drugs represents a major health challen... more 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 highthroughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of Ϸ1.7 million compounds, we identified a diverse collection of Ϸ6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (<1.25 M). 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.

PLoS Genetics, 2013

Malaria parasites elude eradication attempts both within the human host and across nations. At th... more 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 (9.5610 26 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 (1.0-9.7610 29 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.

Nature Chemical Biology, 2008

Calcium-dependent protein kinases play a crucial role in intracellular calcium signaling in plant... more 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.

Journal of Innate Immunity, 2009

disease; M1 isolates were uniformly resistant. We conclude increased resistance to host cathelici... more disease; M1 isolates were uniformly resistant. We conclude increased resistance to host cathelicidin and killing within phagocyte extracellular traps contribute to the propensity of M1 GAS strains to produce invasive infections.

Cell Host & Microbe, 2012

With renewed calls for malaria eradication, nextgeneration antimalarials need be active against d... more With renewed calls for malaria eradication, nextgeneration 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.

BMC Bioinformatics, 2014

Whole-genome sequencing represents a powerful experimental tool for pathogen research. We present... more Whole-genome sequencing represents a powerful experimental tool for pathogen research. We present methods for the analysis of small eukaryotic genomes, including a streamlined system (called Platypus) for finding single nucleotide and copy number variants as well as recombination events. We have validated our pipeline using four sets of Plasmodium falciparum drug resistant data containing 26 clones from 3D7 and Dd2 background strains, identifying an average of 11 single nucleotide variants per clone. We also identify 8 copy number variants with contributions to resistance, and report for the first time that all analyzed amplification events are in tandem. The Platypus pipeline provides malaria researchers with a powerful tool to analyze short read sequencing data. It provides an accurate way to detect SNVs using known software packages, and a novel methodology for detection of CNVs, though it does not currently support detection of small indels. We have validated that the pipeline detects known SNVs in a variety of samples while filtering out spurious data. We bundle the methods into a freely available package.

Antimicrobial Agents and Chemotherapy, 2014

Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-am... more 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 M 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 [IC 50 ] for hypnozoites, KAI407, 0.69 M, and PQ, 0.84 M; for developing liver stages, KAI407, 0.64 M, and PQ, 0.37 M). 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.

ACS Chemical Biology, 2011

Decoquinate has single-digit nanomolar activity against in vitro blood stage Plasmodium falciparu... more 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 À1 . 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.

Nature, 2013

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential ... more 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.

Scientific Reports, 2016

The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are re... more The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4. We show here that S. cerevisiae also acquires mutations in a gene encoding a P-type ATPase (ScPMA1) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace ScPma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits ScPma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a ScPma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae. This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity.

Science, 2008

Antigenically variable M proteins are major virulence factors and immunogens of the human pathoge... more Antigenically variable M proteins are major virulence factors and immunogens of the human pathogen group A Streptococcus (GAS). Here, we report the ∼3 angstrom resolution structure of a GAS M1 fragment containing the regions responsible for eliciting type-specific, protective immunity and for binding fibrinogen, which promotes M1 proinflammatory and antiphagocytic functions. The structure revealed substantial irregularities and instabilities throughout the coiled coil of the M1 fragment. Similar structural irregularities occur in myosin and tropomyosin, explaining the patterns of cross-reactivity seen in autoimmune sequelae of GAS infection. Sequence idealization of a large segment of the M1 coiled coil enhanced stability but diminished fibrinogen binding, proinflammatory effects, and antibody cross-reactivity, whereas it left protective immunogenicity undiminished. Idealized M proteins appear to have promise as vaccine immunogens.

ACS Infectious Diseases, 2016

In order to identify the most attractive starting points for drugs that can be used to prevent ma... more 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.

Journal of medicinal chemistry, Jan 18, 2015

Several of the enzymes related to the folate cycle are well-known for their role as clinically va... more Several of the enzymes related to the folate cycle are well-known for their role as clinically validated antimalarial targets. Nevertheless for serine hydroxymethyltransferase (SHMT), one of the key enzymes of this cycle, efficient inhibitors have not been described so far. On the basis of plant SHMT inhibitors from an herbicide optimization program, highly potent inhibitors of Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) SHMT with a pyrazolopyran core structure were identified. Cocrystal structures of potent inhibitors with PvSHMT were solved at 2.6 Å resolution. These ligands showed activity (IC50/EC50 values) in the nanomolar range against purified PfSHMT, blood-stage Pf, and liver-stage P. berghei (Pb) cells and a high selectivity when assayed against mammalian cell lines. Pharmacokinetic limitations are the most plausible explanation for lack of significant activity of the inhibitors in the in vivo Pb mouse malaria model.

ACS Chemical Biology, 2014

Aminopyrazoles are a new class of antimalarial compounds identified in a cellular antiparasitic s... more Aminopyrazoles are a new class of antimalarial compounds identified in a cellular antiparasitic screen with potent activity against Plasmodium falciparum asexual and sexual stage parasites. To investigate their unknown mechanism of action and thus identify their target, we cultured parasites in the presence of a representative member of the aminopyrazole series, GNF-Pf4492, to select for resistance. Whole genome sequencing of three resistant lines showed that each had acquired independent mutations in a P-type cation-transporter ATPase, PfATP4 (PF3D7_1211900), a protein implicated as the novel Plasmodium spp. target of another, structurally unrelated, class of antimalarials called the spiroindolones and characterized as an important sodium transporter of the cell. Similarly to the spiroindolones, GNF-Pf4492 blocks parasite transmission to mosquitoes and disrupts intracellular sodium homeostasis. Our data demonstrate that PfATP4 plays a critical role in cellular processes, can be inhibited by two distinct antimalarial pharmacophores, and supports the recent observations that PfATP4 is a critical antimalarial target.

ACS Medicinal Chemistry Letters, 2014

Imidazopyridine 1 was identified from a phenotypic screen against P. falciparum (Pf) blood stages... more Imidazopyridine 1 was identified from a phenotypic screen against P. falciparum (Pf) blood stages and subsequently optimized for activity on liver-stage schizonts of the rodent parasite P. yoelii (Py) as well as hypnozoites of the simian parasite P. cynomolgi (Pc). We applied these various assays to the cell-based lead optimization of the imidazopyrazines, exemplified by 3 (KAI407), and show that optimized compounds within the series with improved pharmacokinetic properties achieve causal prophylactic activity in vivo and may have the potential to target the dormant stages of P. vivax malaria.

Science, 2011

Most malaria drug development focuses on parasite stages detected in red blood cells, even though... more 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 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 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.

Science, 2008

Antigenically variable M proteins are major virulence factors and immunogens of the human pathoge... more Antigenically variable M proteins are major virulence factors and immunogens of the human pathogen group A Streptococcus (GAS). Here, we report the ∼3 angstrom resolution structure of a GAS M1 fragment containing the regions responsible for eliciting type-specific, protective immunity and for binding fibrinogen, which promotes M1 proinflammatory and antiphagocytic functions. The structure revealed substantial irregularities and instabilities throughout the coiled coil of the M1 fragment. Similar structural irregularities occur in myosin and tropomyosin, explaining the patterns of cross-reactivity seen in autoimmune sequelae of GAS infection. Sequence idealization of a large segment of the M1 coiled coil enhanced stability but diminished fibrinogen binding, proinflammatory effects, and antibody cross-reactivity, whereas it left protective immunogenicity undiminished. Idealized M proteins appear to have promise as vaccine immunogens.

Science, 2010

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Proteins: Structure, Function, and Bioinformatics, 2011

Establishing a quantitative understanding of the determinants of affinity in protein-protein inte... more Establishing a quantitative understanding of the determinants of affinity in protein-protein interactions remains challenging. For example, TEM-1/BLIP and SHV-1/BLIP are homologous βlactamase/β-lactamase inhibitor protein complexes with disparate K i values (3 nM and 2 μM, respectively), and a single substitution, D104E in SHV-1, results in a 1000-fold enhancement in binding affinity. In TEM-1, E104 participates in a salt bridge with BLIP K74, while the corresponding SHV-1 D104 does not in the wild type SHV-1/BLIP co-structure. Here, we present a 1.6 Å crystal structure of the SHV-1 D104E/BLIP complex that demonstrates that this point mutation restores this salt bridge. Additionally, mutation of a neighboring residue, BLIP E73M, results in salt bridge formation between SHV-1 D104 and BLIP K74 and a 400-fold increase in binding affinity. To understand how this salt bridge contributes to complex affinity, the cooperativity between the E/K or D/K salt bridge pair and a neighboring hot spot residue (BLIP F142) was investigated using double mutant cycle analyses in the background of the E73M mutation. We find that BLIP F142 cooperatively stabilizes both interactions, illustrating how a single mutation at a hot spot position can drive large perturbations in interface stability and specificity through a cooperative interaction network.

Proceedings of the National Academy of Sciences, 2008

The growing resistance to current first-line antimalarial drugs represents a major health challen... more 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 highthroughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of Ϸ1.7 million compounds, we identified a diverse collection of Ϸ6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (<1.25 M). 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.

PLoS Genetics, 2013

Malaria parasites elude eradication attempts both within the human host and across nations. At th... more 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 (9.5610 26 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 (1.0-9.7610 29 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.

Nature Chemical Biology, 2008

Calcium-dependent protein kinases play a crucial role in intracellular calcium signaling in plant... more 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.

Journal of Innate Immunity, 2009

disease; M1 isolates were uniformly resistant. We conclude increased resistance to host cathelici... more disease; M1 isolates were uniformly resistant. We conclude increased resistance to host cathelicidin and killing within phagocyte extracellular traps contribute to the propensity of M1 GAS strains to produce invasive infections.

Cell Host & Microbe, 2012

With renewed calls for malaria eradication, nextgeneration antimalarials need be active against d... more With renewed calls for malaria eradication, nextgeneration 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.

BMC Bioinformatics, 2014

Whole-genome sequencing represents a powerful experimental tool for pathogen research. We present... more Whole-genome sequencing represents a powerful experimental tool for pathogen research. We present methods for the analysis of small eukaryotic genomes, including a streamlined system (called Platypus) for finding single nucleotide and copy number variants as well as recombination events. We have validated our pipeline using four sets of Plasmodium falciparum drug resistant data containing 26 clones from 3D7 and Dd2 background strains, identifying an average of 11 single nucleotide variants per clone. We also identify 8 copy number variants with contributions to resistance, and report for the first time that all analyzed amplification events are in tandem. The Platypus pipeline provides malaria researchers with a powerful tool to analyze short read sequencing data. It provides an accurate way to detect SNVs using known software packages, and a novel methodology for detection of CNVs, though it does not currently support detection of small indels. We have validated that the pipeline detects known SNVs in a variety of samples while filtering out spurious data. We bundle the methods into a freely available package.

Antimicrobial Agents and Chemotherapy, 2014

Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-am... more 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 M 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 [IC 50 ] for hypnozoites, KAI407, 0.69 M, and PQ, 0.84 M; for developing liver stages, KAI407, 0.64 M, and PQ, 0.37 M). 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.

ACS Chemical Biology, 2011

Decoquinate has single-digit nanomolar activity against in vitro blood stage Plasmodium falciparu... more 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 À1 . 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.

Nature, 2013

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential ... more 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.