Discovery of Antimalarial Azetidine-2-carbonitriles That Inhibit <i>P. falciparum</i> Dihydroorotate Dehydrogenase

Dihydroorotate dehydrogenase (DHODH) is an enzyme necessary for pyrimidine biosynthesis in protozoan parasites of the genus <i>Plasmodium</i>, the causative agents of malaria. We recently reported the identification of novel compounds derived from diversity-oriented synthesis with activity in multiple stages of the malaria parasite life cycle. Here, we report the optimization of a potent series of antimalarial inhibitors consisting of azetidine-2-carbonitriles, which we had previously shown to target <i>P. falciparum</i> DHODH in a biochemical assay. Optimized compound BRD9185 (<b>27</b>) has <i>in vitro</i> activity against multidrug-resistant blood-stage parasites (EC₅₀ = 0.016 μM) and is curative after just three doses in a <i>P. berghei</i> mouse model. BRD9185 has a long half-life (15 h) and low clearance in mice and represents a new structural class of DHODH inhibitors with potential as antimalarial drugs

Screening cascade for DOS screen (A) and potency comparison of hits from the DOS library between the novel axenic and intramacrophage assay (B).

<p>A: Screening cascade. Numbers of compounds that passed and failed the hit selection criteria are shown respectively in green and red. Numbers marked with an asterisk indicate that the selectivity window is unknown and may be ≥ 10 (no effect seen in HepG2 assay, activity in the cidal axenic assay pEC50<5.3). The cascade starts with screening 9,907 compounds in the novel axenic assay at a single concentration (nov. axenic SP). Identified hits are processed in potency format in both the novel axenic assay (axenic pot., for activity confirmation) and HepG2 assay (HepG2 pot., toxicity information against the human cell line HepG2). Active compounds that provide a ≥ 10-fold toxicity window are processed in the intramacrophage potency assay (intramac. pot.) for hit confirmation, resulting in 24 hits. B: Comparison of novel axenic and intramacrophage mean pEC₅₀ values for 141 compounds. The size of the markers is inversely related to the toxicity against the THP-1 cells (i.e. high toxicity–small symbol). Colour is by series (green: Ortho Azetidine Nitrile, blue: Azetidine Nitrile, orange: Povarov, red: SnAr 8-ortho, yellow: other). Data set represents three biological replicates for the novel axenic assay (with the exception of one compound with two replicates only) and at least two replicates for the intramacrophage assay.</p

Screening cascade.

<p>Novel axenic assay is primary, single concentration (SP) entry to the cascade, followed by confirmation, assessment of potency and selectivity using the novel axenic assay and a mammalian counterscreen assay (HepG2) in potency mode (pot.). Potent and selective hits are then profiled in the intramacrophage assay.</p

Library screen with novel axenic assay, comparison with intracellular results.

<p>15,667 compounds were screened in the novel axenic assay at 15μM. Comparison with the intracellular assay shows that 67 of the hits are also active in the intracellular assay whereas 71 of the hits are not. 280 novel axenic assay inactive compounds were hits in the intracellular assay, 170 of these compounds showed toxicity in the intracellular assay (in single point mode, at 50μM, or less than 3-fold selectivity window in potency assay) and are therefore considered false positives in the intracellular assay. Potencies were determined for 110 non-toxic compounds in the novel axenic and intracellular assays. 13 compounds were inactive in the intracellular assay and are added to the false positive count (13 + 170 = 183). 97 compounds had confirmed activity in the intracellular assay; 42 of these compounds showed activity in the novel axenic assay, while 54 did not. (INMAC = intracellular assay). Hit criteria as described in Table A in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004094#pntd.0004094.s001" target="_blank">S1 Text</a>.</p

Suitability of the <i>Leishmania donovani</i> assays for diversity screening.

<p>Suitability of the <i>Leishmania donovani</i> assays for diversity screening.</p

Limit-of-detection of historic and novel axenic assay and assay models.

<p>A: Detection limit of the historic axenic assay. Black line shows the linear regression (R² = 0.983, p < 0.0001). Dashed line shows average value of blanks. Blue line shows detection limit (3x standard deviation above the blanks value) for this assay. Vertical blue arrow indicates the number of cells at the limit of detection in this single experiment. Data is from a representative experiment of 4 with a minimum of 24 technical replicates. B: Detection limit of the novel axenic assay. Black line shows the linear regression (R² = 0.998, p < 0.0001). Dashed line shows average value of blanks. Red line shows detection limit for this assay (3x standard deviation above the blanks value). Red vertical arrow indicates the number of cells at the limit of detection in this single experiment. Data is from a representative experiment of 4 with a minimum of 24 technical replicates. Inset shows data from a similar range of cell densities as used in 1A. C and D: The coloured areas represent cell densities that can be detected with the respective assay formats (C: historic axenic assay, D: novel axenic assay). Red lines represent different cell-growth inhibition scenarios during the course of this assay: compounds that do not inhibit cell growth (marked “no Inhibition”), compounds that arrest cell growth, without killing cells (marked “static Inhibition”), and compounds that kill cells (marked with “cidal Inhibition”). The black double arrows represent the analysis window. Blue (historic assay) and red (novel assay) arrows show the fold difference between the starting density and detection limit.</p

Potency comparison of a series of structurally distinct compounds between the novel axenic and intramacrophage assay.

<p>In this scatterplot the mean pEC₅₀ values for control compounds representing a range of pharmacophores are compared between the novel and intramacrophage assay. Data set represents three or more replicates and the standard deviation is shown with error bars for each assay. Black line shows equipotency between both assays, blue line shows linear regression for compounds that are active in both assay (with outliers miltefosine and VL-2098 excluded, R² = 0.81).</p

A Potent and Selective Quinoxalinone-Based STK33 Inhibitor Does Not Show Synthetic Lethality in KRAS-Dependent Cells

The KRAS oncogene is found in up to 30% of all human tumors. In 2009, RNAi experiments revealed that lowering mRNA levels of a transcript encoding the serine/threonine kinase STK33 was selectively toxic to KRAS-dependent cancer cell lines, suggesting that small-molecule inhibitors of STK33 might selectively target KRAS-dependent cancers. To test this hypothesis, we initiated a high-throughput screen using compounds in the Molecular Libraries Small Molecule Repository (MLSMR). Several hits were identified, and one of these, a quinoxalinone derivative, was optimized. Extensive SAR studies were performed and led to the chemical probe ML281 that showed low nanomolar inhibition of purified recombinant STK33 and a distinct selectivity profile as compared to other STK33 inhibitors that were reported in the course of these studies. Even at the highest concentration tested (10 μM), ML281 had no effect on the viability of KRAS-dependent cancer cells. These results are consistent with other recent reports using small-molecule STK33 inhibitors. Small molecules having different chemical structures and kinase-selectivity profiles are needed to fully understand the role of STK33 in KRAS-dependent cancers. In this regard, ML281 is a valuable addition to small-molecule probes of STK33

Identification of Highly Specific Diversity-Oriented Synthesis-Derived Inhibitors of Clostridium difficile

In 2013, the Centers for Disease Control highlighted Clostridium difficile as an urgent threat for antibiotic-resistant infections, in part due to the emergence of highly virulent fluoroquinolone-resistant strains. Limited therapeutic options currently exist, many of which result in disease relapse. We sought to identify molecules specifically targeting <i>C. difficile</i> in high-throughput screens of our diversity-oriented synthesis compound collection. We identified two scaffolds with apparently novel mechanisms of action that selectively target <i>C. difficile</i> while having little to no activity against other intestinal anaerobes; preliminary evidence suggests that compounds from one of these scaffolds target the glutamate racemase. In vivo efficacy data suggest that both compound series may provide lead optimization candidates