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

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

CD4陽性T細胞に発現するA20(tnfaip3) はTh2型アレルギー性気道炎症を抑制する

研究科: 千葉大学大学院医学薬学府（先端医学薬学専攻）学位記番号: 千大院医薬博甲第医1412号博士（医学）千葉大学 = Chiba Universit

Table 1 - Fueling Open Innovation for Malaria Transmission-Blocking Drugs: Hundreds of Molecules Targeting Early Parasite Mosquito Stages

Despite recent successes at controlling malaria, progress has stalled with an estimated 219 million cases and 435,000 deaths in 2017 alone. Combined with emerging resistance to front line antimalarial therapies in Southeast Asia, there is an urgent need for new treatment options and novel approaches to halt the spread of malaria. Plasmodium, the parasite responsible for malaria propagates through mosquito transmission. This imposes an acute bottleneck on the parasite population and transmission-blocking interventions exploiting this vulnerability are recognized as vital for malaria elimination

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

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

Lead optimization of a pyrrole-based dihydroorotate dehydrogenase inhibitor series for the treatment of malaria

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

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

The requirement for next-generation antimalarials to be both curative and transmission-blocking necessitates the identification of previously undiscovered druggable molecular pathways. We identified a selective inhibitor of the; Plasmodium falciparum; protein kinase; Pf; CLK3, which we used in combination with chemogenetics to validate; Pf; CLK3 as a drug target acting at multiple parasite life stages. Consistent with a role for; Pf; CLK3 in RNA splicing, inhibition resulted in the down-regulation of more than 400 essential parasite genes. Inhibition of; Pf; CLK3 mediated rapid killing of asexual liver- and blood-stage; P. falciparum; and blockade of gametocyte development, thereby preventing transmission, and also showed parasiticidal activity against; P. berghei; and; P. knowlesi; Hence, our data establish; Pf; CLK3 as a target for drugs, with the potential to offer a cure-to be prophylactic and transmission blocking in malaria

Biochemical Screening of Five Protein Kinases from <i>Plasmodium falciparum</i> against 14,000 Cell-Active Compounds

<div><p>In 2010 the identities of thousands of anti-<i>Plasmodium</i> compounds were released publicly to facilitate malaria drug development. Understanding these compounds’ mechanisms of action—i.e., the specific molecular targets by which they kill the parasite—would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children’s Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of <i>Plasmodium falciparum</i> calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC₅₀ < 1 μM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple <i>Plasmodium</i> kinase targets without harming human cells is challenging but feasible.</p></div

Assessment of compound promiscuity with human kinases.

<p>Kinobeads were incubated with K562 cell extract either in the presence of vehicle (DMSO) or TCAMS compound, respectively (20 μM-0.03 μM). Protein kinases captured by the beads (140–150 kinases per experiment) were quantified following tryptic digestion, isobaric peptide tagging, and LC-MS/MS analysis. Kinases were identified as potential targets by virtue of their reduced capture in the presence of excess TCAMS compounds. Apparent dissociation constants (K_d’s) were calculated from the extent to which capture of each kinase was reduced at each compound concentration. K_d values from duplicate experiments generally agreed with each other quite well (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149996#pone.0149996.s002" target="_blank">S2 Fig</a>). Colored bands indicate kinase-ligand complexes with apparent pK_d’s of ≥6, with darker shades denoting higher pK_d’s. Kinases that did not have an apparent pK_d of ≥6 for any of the compounds are not represented; only names of every other targeted kinase are shown due to space limitations. These results are summarized numerically in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149996#pone.0149996.t003" target="_blank">Table 3</a>.</p

A comparison of different CDPK inhibitors’ cytotoxicity to human cells.

<p>Inhibition of HepG2 cell growth at compound concentrations of 10 μM is shown for CDPK4 inhibitors in scaffolds D and G (top) and for CDPK1 inhibitors in scaffolds F and H (bottom).</p