Topographical Mapping of Isoform-Selectivity Determinants for J-Channel-Binding Inhibitors of Sphingosine Kinases 1 and 2.

Sphingosine kinase enzymes (SK1 and SK2) catalyze the conversion of sphingosine into sphingosine 1-phosphate and play a key role in lipid signaling and cellular responses. Mapping of isoform amino acid sequence differences for SK2 onto the recently available crystal structures of SK1 suggests that subtle structural differences exist in the foot of the lipid-binding "J-channel" in SK2, the structure of which has yet to be defined by structural biology techniques. We have probed these isoform differences with a ligand series derived from the potent SK1-selective inhibitor, PF-543. Here we show how it is possible, even with relatively conservative changes in compound structure, to systematically tune the activity profile of a ligand from ca. 100-fold SK1-selective inhibition, through equipotent SK1/SK2 inhibition, to reversed 100-fold SK2 selectivity, with retention of nanomolar potency.

Inhibitory Kappa B Kinase α (IKKα) Inhibitors That Recapitulate Their Selectivity in Cells against Isoform-Related Biomarkers.

IKKß plays a central role in the canonical NF-kB pathway, which has been extensively characterized. The role of IKKα in the noncanonical NF-kB pathway, and indeed in the canonical pathway as a complex with IKKß, is less well understood. One major reason for this is the absence of chemical tools designed as selective inhibitors for IKKα over IKKß. Herein, we report for the first time a series of novel, potent, and selective inhibitors of IKKα. We demonstrate effective target engagement and selectivity with IKKα in U2OS cells through inhibition of IKKα-driven p100 phosphorylation in the noncanonical NF-kB pathway without affecting IKKß-dependent IKappa-Bα loss in the canonical pathway. These compounds represent the first chemical tools that can be used to further characterize the role of IKKα in cellular signaling, to dissect this from IKKß and to validate it in its own right as a target in inflammatory diseases.

Doing the methylene shuffle--further insights into the inhibition of mitotic kinesin Eg5 with S-trityl L-cysteine.

S-Trityl L-cysteine (STLC) is an inhibitor of the mitotic kinesin Eg5 with potential as an antimitotic chemotherapeutic agent. We previously reported the crystal structure of the ligand-protein complex, and now for the first time, have quantified the interactions using a molecular dynamics based approach. Based on these data, we have explored the SAR of the trityl head group using the methylene shuffle strategy to expand the occupation of one of the hydrophobic pockets. The most potent compounds exhibit strong (<100 nM) inhibition of Eg5 in the basal ATPase assay and inhibit growth in a variety of tumour-derived cell lines.