Funding SourcesGlaxoSmithKline acknowledges financial support from Medicines for Malaria Venture (MMV).
' ACKNOWLEDGMENTGlaxoSmithKline acknowledges the perceptive and helpful advice from Jeremy Burrows and Mike Witty.
In the interest of identification of new kinase-targeting chemotypes for target and pathway analysis and drug discovery in Trypanosomal brucei, 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 T. brucei 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 in silico and in vitro 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 T. brucei rhodesiense 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 T. brucei, and new HAT therapeutics discovery programs.
A kinase-targeting cell-based high-throughput
screen (HTS) against Trypanosoma brucei was recently reported, and this screening set included the Published
Kinase Inhibitor Set (PKIS). From the PKIS was identified 53 compounds
with pEC50 ≥ 6. Utilizing the published data available
for the PKIS, a statistical analysis of these active antiparasitic
compounds was performed, allowing identification of a set of human
kinases having inhibitors that show a high likelihood for blocking T. brucei cellular proliferation in vitro. This observation
was confirmed by testing other established inhibitors of these human
kinases and by mining past screening campaigns at GlaxoSmithKline.
Overall, although the parasite targets of action are not known, inhibitors
of this set of human kinases displayed an enhanced hit rate relative
to a random kinase-targeting HTS campaign, suggesting that repurposing
efforts should focus primarily on inhibitors of these specific human
kinases. We therefore term this statistical analysis-driven approach “preferred lead repurposing”.
Compound
NVP-BEZ235 (1) 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 1 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 1 and have prepared analogues of 1 with improved solubility properties and good predicted central
nervous system exposure. In this way, we have identified 4e, 9, 16e, and 16g 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.
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