SummaryAchieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here, we identify a lipid kinase, phosphatidylinositol 4-kinase (PI4K), as the target of imidazopyrazines, a novel 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 PI4K, altering the intracellular distribution of phosphatidylinositol 4-phosphate. Collectively, our data define PI4K 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.

SummaryWith 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.

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

Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crystal structure of dengue virus MTase with a bound SAH derivative revealed that its N6-substituent bound in this cavity and induced conformation changes in residues lining the pocket. These findings demonstrate that one of the major hurdles for the development of methyltransferase-based therapeutics, namely selectivity for disease-related methyltransferases, can be overcome.Methyltransferases (MTases) 3 play key roles in normal physiology and human diseases through methylating DNA, RNA, and proteins. Almost all MTases use S-adenosyl-L-methionine (SAM) as a methyl donor and generate S-adenosyl-Lhomocysteine (SAH) as a by-product. Pharmacological modulation of MTases by small molecules represents a novel approach to therapeutic intervention in cancer and other diseases (1). However, because the core domains of various MTases are conserved, designing inhibitors that specifically block the disease-related MTase without affecting other MTases, has been challenging. The ability to rationally design and generate selective inhibitors would have profound implications for development of new medicines for many methyltransferase-mediated diseases.Dengue virus (DENV), from genus Flavivirus in the family Flaviviridae, is the most prevalent mosquito-borne viral pathogen that infects humans. The four serotypes of DENV (DENV-1 to -4) pose a public health threat to 2.5 billion people worldwide, and cause 50 -100 million human infections each year. Neither vaccine nor antiviral therapy is currently available for DENV. The flavivirus MTase methylates the guanine N7 and ribose 2Ј-O positions of the viral RNA cap in a sequential manner (i.e. GpppA-RNA 3 m7GpppA-RNA 3 m7GpppAm-RNA) (2, 3). Recent studies have shown that flavivirus MTase is critical for viral replication and, therefore, represents a valid target for antiviral therapeutics (4 -6). We therefore examined the feasibility to design inhibitors that specifically modulate flavivirus MTase. EXPERIMENTAL PROCEDURESPreparation of DENV-3 MTases-The DNA fragment representing the MTase domain of DENV-3 was cloned into expression vector pGEX4T1 (Amersham Biosciences). Ala-substitution mutant MTases were prepared using a standard overlapping PCR procedure. Recombinant MTases, containing an N-terminal GST, were expressed in Escherichia coli. BL21 cells and purified through a GSTPrep TM FF 16/10 column (GE Healthcare). The GST tag was then cleaved by thrombin and removed from the MTases using the GST column. The MTases were further purified through gel filtration to ensure protein purity was Ͼ95%. The p...

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic cells by a unique mechanism that consists in a calcium-dependent, direct translocation of the CyaA catalytic domain across the plasma membrane of the target cells. CyaA possesses a series of a glycine-and aspartate-rich nonapeptide repeats (residues 1006 -1613) of the prototype GGXG(N/D)DX(L/I/F)X (where X represents any amino acid) that are characteristic of the RTX (repeat in toxin) family of bacterial cytolysins. These repeats are arranged in a tandem fashion and may fold into a characteristic parallel ␤-helix or ␤-roll motif that constitutes a novel type of calcium binding structure, as revealed by the three-dimensional structure of the Pseudomonas aeruginosa alkaline protease. Here we have characterized the structure-function relationships of various fragments from the CyaA RTX subdomain. Our results indicate that the RTX functional unit includes both the tandem repeated nonapeptide motifs and the adjacent polypeptide segments, which are essential for the folding and calcium responsiveness of the RTX module. Upon calcium binding to the RTX repeats, a conformational rearrangement of the adjacent non-RTX sequences may act as a critical molecular switch to trigger the CyaA entry into target cells.The adenylate cyclase toxin (CyaA) 2 is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough (1-3). The 1706 residue-long CyaA is a bi-functional protein endowed with both catalytic (adenylate cyclase) and hemolytic activities (2, 4, 5). Synthesized as an inactive precursor, it is converted to the active toxin by a post translational palmitoylation of two internal lysine residues (Lys 860 and Lys 983 ) (6, 7). This active CyaA toxin is then able to deliver its catalytic domain directly across the plasma membrane of a variety of eukaryotic cells and disrupts their physiological functions by uncontrolled synthesis of cAMP (5, 8 -11), leading to the cell death by apoptosis (12)(13)(14). CyaA is constructed in a modular fashion; the calmodulinactivated catalytic domain is located in the 400-amino-proximal residues, whereas the C-terminal moiety (residues 400 -1706) is endowed with hemolytic activity (4, 5, 15, 16), which results from its ability to form cation-selective channels in membranes (17,18). It also mediates the binding and internalization of the toxin into eukaryotic cells (5,11,19). The hemolytic and the RTX domains display structural characteristics that link CyaA to the RTX (repeat in toxin) family of bacterial toxins (20, 21). Indeed, it contains a pore-forming domain (from residues 500 -700) with four hydrophobic segments (17,18,22,23), the target site for the post-translational palmitoylation (7, 24), 30 -40 copies of a characteristic glycine-and aspartate-rich nonapeptide repeats (residues 1006 -1613) of the prototype GGXG(N/ D)DX(U)X (X represents any amino acid, and U represents any large ...