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 Kd's were determined. Only kinases inhibited by one of the five clinical compounds are illustrated here.

Selectivity Profiling Results Using KiNativ™

 

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.

Evaluation of TAK1 inhibitors

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.

B-Raf inhibition and cellular efficacy

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™.

Accurate prediction of in vivo potency

* Manley, PW; et. al., Biochim. Biophys. Acta, 2005 Dec 30; 1754(1-2):3-13
** Du, J; et. al., Nat. Biotechnol. 2009 Jan; 27(1):77-83

Contact KiNativ™Contact KiNativ™

For more information or a consultation on your specific profiling needs please contact us.

KiNativ™ Customer Service
ActivX Biosciences, Inc.
Telephone: (858) 526-2515
Fax: (858) 587-4878