A Novel ACK1/TNK2 Small Molecule Inhibitor, (R)-9bMS

Need for ACK1 inhibitor

ACK1 integrates signals from various receptor tyrosine kinases (RTKs), e.g. EGFR, PDGFR, Insulin receptor (IR), HER-2 and FGFR. Thus, ACK1 inhibitor could effectively block signal from multiple RTKs and would have significant anti-proliferative effects in prostate, breast, ovarian, pancreatic and lung cancer patients. However, ACK1-specific inhibitor is currently not available for clinical trial.

 

Identification of the novel ACK1 inhibitor, (R)-9b

We generated compound libraries which were screened for ACK1 inhibition using a novel ELISA (Enzyme-Linked Immunosorbent Assay) that was developed in our lab. Subsequently, IC50 for those compounds that inhibited ACK1 activity by >90% were determined in vitro. Further, various cancer cells were treated with these compounds to identify an inhibitor that is active in vivo.  

The most potent compound was identified to be (R)-9b with in vitro IC50 48 nM. The mesylate salt of this compound is (R)-9bMS. It suppressed proliferation of various prostate cancer cell lines, and was especially active against castration resistant prostate cancer (CRPC) cell line C4-2b (IC50 ~350 nM). 

 

Specificity of the novel ACK1 inhibitor, (R)-9b

(R)-9bMS was subjected to extensive kinase profiling (n=369 kinases) to determine its selectivity. Apart from ACK1, only 11 other kinases were inhibited in vitro to >90% (Table S2 and Figure S3C in Cancer Cell 2017; Pubmed ID:  28609657). The Human Protein Atlas reveals that 8 of these 11 kinases, TNK1, LRRK2, JAK1–3, TYK2, and NUAK1 and NUAK2, are either not expressed or expressed at very low levels in prostate tissues and cancers. The remaining three kinases, FLT3, PKCmu, and SIK2, have not yet been shown to be critical for CRPCs. Thus, while in vitro studies indicate other potential targets, at least in CRPCs, ACK1 appears to be the predominant target of (R)-9b.

 

Prostate tumor (CRPC) suppression by ACK1 inhibitor, (R)-9bMS

(R)-9bMS was highly soluble in aqueous phase which allowed to perform xenograft studies. It suppressed prostate tumor growth in castrated mice and thus was highly effective in suppressing CRPC growth.

 

Structure of (R)-9b mesylate salt

Compound (R)-9b (yellow) docked to ACK1 (pdb 4EWH), with ACK1 shown in ribbon representation with helices shown in red, β-strands shown as yellow arrows and loops shown in green.

Compound (R)-9b (yellow) docked to ACK1 (pdb 4EWH), with ACK1 shown in ribbon representation with helices shown in red, β-strands shown as yellow arrows and loops shown in green.

Expanded stick model of (R)-9b (yellow) docked to the ATP binding site of ACK1, showing the amino acid residues of the kinase hinge in stick representation.