Lead Optimization Native Kinase Profiling and Inhibitor Selectivity Screening

KiNativ™ is a kinase screening platform that utilizes chemical probes to label, selectively purify and detect native kinases in samples ranging from cell lysates to animal tissues. Off-target profiling against full-length kinases, with endogenous post-translation modifications and protein:protein interactions still intact, ensures that selectivity data are truly representative of a compound's in vivo activity.

• Targeted LC-MS² permits evaluation of up to 180 native kinases per run (>200 kinases/proteome)
• Automated sample preparation and targeted data collection provide quantitative, reproducible data
• Better correlation with in vivo results compared to recombinant kinase assays

Selectivity Profiling Results Using KiNativ™

Six reference kinase inhibitors were profiled against two cell lines, HL60's and PC3's. A total of 209 kinases were evaluated and K_d's were determined. Only kinases inhibited by one of the five clinical compounds are illustrated here.

 

KiNativ™ Accurately Predicts In Vivo Inhibitor Potency

KiNativ™ consistently demonstrates higher correlation with in vivo and cell-based assays compared to corresponding substrate-based assays with recombinant kinases. Several examples illustrated here highlight how KiNativ™ can identify the most efficacious inhibitors at the earliest stages of drug discovery.

Evaluation of TAK1 inihbitors

Three structural classes of TAK1 inhibitors were evaluated using a recombinant kinase assay, KiNativ™, and a cellular phosphorylation assay. The recombinant assay shows potent activity for all three inhibitors, whereas KiNativ™ accurately identifies compound #1 as the only active inhibitor.

B-Raf inhibition and cellular efficacy

Several reference B-Raf inhibitors were evaluated by a recombinant V600E-B-Raf substrate-based assay, KiNativ™ (A375 cells), an A375 cell proliferation assay, and a cellular Erk phosphorylation assay (at 2μM in A375 cells). GW5074 appears to be the most potent V600E-B-Raf inhibitor by the substrate-based assay but is a poor B-Raf inhibitor in KiNativ™ and does not inhibit A375 (V600E B-Raf) proliferation or Erk phosphorylation. Overall the KiNativ™ data correlates well with cellular data for all compounds tested.

Accurate prediction of in vivo potency

Data for gleevec and dasatinib were compared relative to the reported in vivo potency. While both Ambit and KiNativ™ correlate with relative response, the absolute potency is more accurate withKiNativ™.