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

Identification of Fast-Acting 2,6-Disubstituted Imidazopyridines That Are Efficacious in the in Vivo Humanized <i>Plasmodium falciparum</i> NODscidIL2Rγ^<i>null</i> Mouse Model of Malaria

Optimization of a chemical series originating from whole-cell phenotypic screening against the human malaria parasite, <i>Plasmodium falciparum</i>, led to the identification of two promising 2,6-disubstituted imidazopyridine compounds, <b>43</b> and <b>74</b>. These compounds exhibited potent activity against asexual blood stage parasites that, together with their in vitro absorption, distribution, metabolism, and excretion (ADME) properties, translated to in vivo efficacy with clearance of parasites in the <i>Pf</i>SCID mouse model for malaria within 48 h of treatment

Identification of Fast-Acting 2,6-Disubstituted Imidazopyridines That Are Efficacious in the in Vivo Humanized <i>Plasmodium falciparum</i> NODscidIL2Rγ^<i>null</i> Mouse Model of Malaria

Optimization of a chemical series originating from whole-cell phenotypic screening against the human malaria parasite, <i>Plasmodium falciparum</i>, led to the identification of two promising 2,6-disubstituted imidazopyridine compounds, <b>43</b> and <b>74</b>. These compounds exhibited potent activity against asexual blood stage parasites that, together with their in vitro absorption, distribution, metabolism, and excretion (ADME) properties, translated to in vivo efficacy with clearance of parasites in the <i>Pf</i>SCID mouse model for malaria within 48 h of treatment

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

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

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

Summary of kinobead competition assays (results reflect two independent experiments).

<p>Summary of kinobead competition assays (results reflect two independent experiments).</p

Human cytotoxicity of inhibitors of 1, 2, or 3 of the <i>P</i>. <i>falciparum</i> kinases studied.

<p>Inhibition of HepG2 cell growth at compound concentrations of 10 μM were previously reported by Gamo et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149996#pone.0149996.ref003" target="_blank">3</a>].</p

Clustering of <i>P</i>. <i>falciparum</i> protein kinase hits into chemical scaffolds.

<p>Inhibition of HepG2 cell growth at compound concentrations of 10 μM were previously reported by Gamo et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149996#pone.0149996.ref003" target="_blank">3</a>]. For some scaffolds, target counts exceed the number of hits because some compounds hit more than one target.</p

Venn diagrams showing overlapping and non-overlapping targets of hit compounds.

<p>225 compounds had IC₅₀’s below 1 μM against at least one kinase (left); a subset of 72 compounds had IC₅₀’s below 100 nM against at least one kinase (right).</p