Establishment of a Structure–Activity Relationship of 1<i>H</i>‑Imidazo[4,5‑<i>c</i>]quinoline-Based Kinase Inhibitor NVP-BEZ235 as a Lead for African Sleeping Sickness

Compound NVP-BEZ235 (<b>1</b>) is a potent inhibitor of human phospoinositide-3-kinases and mammalian target of rapamycin (mTOR) that also showed high inhibitory potency against Trypanosoma brucei cultures. With an eye toward using <b>1</b> as a starting point for anti-trypanosomal drug discovery, we report efforts to reduce host cell toxicity, to improve the physicochemical properties, and to improve the selectivity profile over human kinases. In this work, we have developed structure–activity relationships for analogues of <b>1</b> and have prepared analogues of <b>1</b> with improved solubility properties and good predicted central nervous system exposure. In this way, we have identified <b>4e</b>, <b>9</b>, <b>16e</b>, and <b>16g</b> as the most promising leads to date. We also report cell phenotype and phospholipidomic studies that suggest that these compounds exert their anti-trypanosomal effects, at least in part, by inhibition of lipid kinases

Identification and Characterization of Hundreds of Potent and Selective Inhibitors of <i>Trypanosoma brucei</i> Growth from a Kinase-Targeted Library Screening Campaign

<div><p>In the interest of identification of new kinase-targeting chemotypes for target and pathway analysis and drug discovery in <i>Trypanosomal brucei,</i> a high-throughput screen of 42,444 focused inhibitors from the GlaxoSmithKline screening collection was performed against parasite cell cultures and counter-screened against human hepatocarcinoma (HepG2) cells. In this way, we have identified 797 sub-micromolar inhibitors of <i>T. brucei</i> growth that are at least 100-fold selective over HepG2 cells. Importantly, 242 of these hit compounds acted rapidly in inhibiting cellular growth, 137 showed rapid cidality. A variety of <i>in silico</i> and <i>in vitro</i> physicochemical and drug metabolism properties were assessed, and human kinase selectivity data were obtained, and, based on these data, we prioritized three compounds for pharmacokinetic assessment and demonstrated parasitological cure of a murine bloodstream infection of <i>T. brucei rhodesiense</i> with one of these compounds (NEU-1053). This work represents a successful implementation of a unique industrial-academic collaboration model aimed at identification of high quality inhibitors that will provide the parasitology community with chemical matter that can be utilized to develop kinase-targeting tool compounds. Furthermore these results are expected to provide rich starting points for discovery of kinase-targeting tool compounds for <i>T. brucei,</i> and new HAT therapeutics discovery programs.</p></div

Summary of average properties of the overall screening set and specific compounds.

<p>Summary of average properties of the overall screening set and specific compounds.</p

The project assay cascade.

<p>The project assay cascade.</p

Compound composite scoring schema.

a<p>“Fast” is defined as pEC₅₀≥6 at 18 h.</p>b<p>“Cidal” is defined as pEC₉₉≥6 in the reversibility experiments. Only compounds that were rapidly acting and/or had an MPO score ≥4 were tested in the cidality assay; compounds not tested in the cidal assay are assigned a score of 0.</p><p>Compound composite scoring schema.</p

Scatter plot showing the average pEC₅₀ at the 18 hour time point for the 242 compounds tested in the cidality assay.

<p>Compounds are colored on the basis of reversibility behavior (cidal = red; static = green).</p

(A) Peripheral blood levels of NEU-1200 (blue), NEU-1207 (red) and NEU-1053 (green) after intraperitoneal administration of 5 mg/kg single dose to NMRI mice (n = 3).

<p>The average and standard deviation of the mean for each time point are represented in the plot. Blood levels of NEU-1053 after IP administration was observed till 24h post-dose. Y-axis is represented in log scale. (B) Animal survival plot showing the effects of dosing of NEU-1053 (ip 20/mg/kg/day; orange) versus DMSO control (black line) in <i>T. b. brucei</i> or (C) <i>T. b. rhodesiense</i> infection. Parasitemia was checked on days indicated by circles.</p

Representation of 797 hit compounds.

<p>(a) Plot of LLE vs LE, color coded based on MPO score (min = 1.1, Red; max = 5.75, Green). (b) plot of molecular weight vs cLogP. Compounds with MPO score ≥4 are colored green.</p

Highest-ranking cluster representatives for clusters 1-24.

<p>Each compound's composite score is shown in parentheses.</p

(A) Ź trend over single shot campaign duration.

<p>(B) Distribution of %inhibition response in single shot assay. Plates assayed were run on 3 different days, identified by different colors in plots A and B. (C) Correlation of 4,574 compounds in dose response assay (n = 2).</p