In vitro activity: ML-281, a quinoxalinone derivative, is a novel, potent and selective STK33 inhibitor with IC50 value of 14 nM. ML281 was identified from a high-throughput screen using compounds in the Molecular Libraries Small Molecule Repository (MLSMR). It showed low nanomolar inhibition of purified recombinant STK33 and a distinct selectivity profile as compared to other STK33 inhibitors. 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.

Kinase Assay: ML281 was then profiled against a panel of 83 kinases chosen for diversity and toxicity. ML281 was found to be extremely selective and inhibits only two kinases other than STK33 in the panel tested at a level of 25% or more: FLT3 (a proto-oncogene) and KDR (VEGF R2 associated with vascularization). Full data for the 83 kinases tested are provided in the Supporting Information.

Cell Assay: ML281 was finally profiled in cellular assays to test the hypothesis of STK33 synthetic lethality in KRAS-dependent cell lines. Figure Figure44 shows the relative viability of two KRAS-dependent (NOMO-1 and SKM-1) and two KRAS-independent (THP-1 and U937) AML-derived cell lines. ML281 does not appear to significantly alter viability of any of the tested cell lines at concentrations of up to 10 μM. This experiment has been repeated with more than 20 KRAS-dependent and KRAS-independent cell lines, and no significant correlation between KRAS dependency and cell viability was found (see the Supporting Information). Whether ML281 is not inhibiting STK33 in cells (due to off-target effects or high plasma protein binding) or whether STK33 inhibition is not synthetic lethal to KRAS-dependent cell line remains to be determined. In this context, biomarkers of STK33 activity should greatly help the cancer research community to answer that question.