Figure 4.

Selectivity Profiling of JZ128. (A) CITe-Id profiling compliments KiNativ profiling (Left) Replicate CITe-Id results for JZ128 and JZ128-DTB in PC3 lysates with JZ128-DTB-labeled cysteine sites rank ordered for competitive dose–response to JZ128. Dashed box indicates sites with a competitive dose response threshold two standard deviations relative to the mean value of the null. See Figure S6A for individual treatment condition data and Table S2 for complete CITe-Id results. (Right) KiNativ results rank-ordered by kinase inhibition. Dashed box indicates kinases with >50% inhibition by JZ128 at 1 μM. See Table S3 for complete data set. (Middle) Venn diagram overlaying the kinase results for CITe-Id and KiNativ profiling. *SRC was competively bound by JZ128-DTB (CITe-Id) but not enzymatically inhibited (KiNativ). **JZ128 labeling of PIP4K2C C313 was identified using nontryptic protease digestion of the purified protein. All cysteine labeling sites were determined with CITe-Id except where noted for PIP4K2C. (B) CITe-Id data for kinase cysteines modified by JZ128-DTB with competitive dose responses (>3 std. dev. relative to the mean of the null). Bars represent the mean of the ratio of a condition from each biological replicate with standard deviation error bars and individual data points as gray circles. (C) Live PC3 cells expressing FLAG-HA-PKN WT were treated with JZ128. After lysis samples were treated with JZ128-DTB and FLAG-HA-PKN3 was enriched by tandem affinity purification. Bar plot shows competitive dose–response for the JZ128-DTB modified C840-containing PKN3 target peptide across two biological replicate experiments. (D) Dendrograms of the kinome depicting the covalent targets of JZ128 and THZ1 as identified by CITe-Id. Image generated using TREEspot Software Tool and reprinted with permission from KINOMEscan, a division of DiscoveRx Corporation.