Overexpression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers that correlate with poor prognosis. Antiphosphotyrosine and anti-EGFr affinity chromatography, isotope-coded LC-MS/MS, and immunoblot methods were combined to describe and measure signaling networks associated with EGF receptor activation and pharmacological inhibition. The squamous carcinoma cell line HN5, which overexpresses EGF receptor and displays sustained receptor kinase activation, was used as a model system, where pharmacological inhibition of EGF receptor kinase by erlotinib markedly reduced auto and substrate phosphorylation, Src family phosphorylation at EGFR Y845, while increasing total EGF receptor protein. Diverse sets of known and poorly described functional protein classes were unequivocally identified by affinity selection, comprising either proteins tyrosine phosphorylated or complexed therewith, predominantly through EGF receptor and Src family kinases, principally 1) immediate EGF receptor signaling complexes (18%); 2) complexes involved in adhesion and cell-cell contacts (34%); and 3) receptor internalization and degradation signals. Novel and known phosphorylation sites could be located despite the complexity of the peptide mixtures. In

OSI-930, a potent thiophene inhibitor of the Kit, KDR, and platelet-derived growth factor receptor tyrosine kinases, was used to selectively inhibit tyrosine phosphorylation downstream of juxtamembrane mutant Kit in the mast cell leukemia line HMC-1. Inhibition of Kit kinase activity resulted in a rapid dephosphorylation of Kit and inhibition of the downstream signaling pathways. Attenuation of Ras-Raf-Erk (phospho-Erk, phospho-p38), phosphatidyl inositol-3V kinase (phospho-p85, phospho-Akt, phospho-S6), and signal transducers and activators of transcription signaling pathways (phospho-STAT3/5/6) were measured by affinity liquid chromatography tandem mass spectrometry, by immunoblot, and by tissue microarrays of fixed cell pellets. To more globally define additional components of Kit signaling temporally altered by kinase inhibition, a novel multiplex quantitative isobaric peptide labeling approach was used. This approach allowed clustering of proteins by temporal expression patterns. Kit kinase, which dephosphorylates rapidly upon kinase inhibition, was shown to regulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2. Interactions with SH2 domain adapters [growth factor receptor binding protein 2 (Grb2), Cbl, Slp-76] and SH3 domain adapters (HS1, cortactin, CD2BP3) were attenuated by inhibition of Kit kinase activity. Functional crosstalk between Kit and the non -receptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk was observed.Inhibition of Kit modulated phosphorylation-dependent interactions with pathways controlling focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, Wasp, Fhl-3, G25K, Ack-1, Nap1, SH3P12/ponsin) and septin-actin complexes (NEDD5, cdc11, actin). The combined use of isobaric protein quantitation and expression clustering, immunoblot, and tissue microarray strategies allowed temporal measurement signaling pathways modulated by mutant Kit inhibition in a model of mast cell leukemia. [Mol Cancer Ther 2005;4(8):1186 -97]

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Activated Cdc42-associated kinase (ACK1) is a non-receptor tyrosine kinase originally identified by virtue of its binding to GTP-bound small GTPase Cdc42. Considerable attention has been paid to ACK1’s involvement in cancer in recent years. For example, gene amplification and over-expression of ACK1 were found in multiple cancers including lung, ovarian and prostate cancers and were associated with poor prognosis and metastatic phenotypes. Activated ACK1 has been shown to phosphorylate and activate androgen receptor function and to promote the progression of prostate cancer. More recently, activated ACK1 was found to phosphorylate and promote the activation of Akt, a protein kinase that plays a central role in growth, proliferation and cell survival in various cancers. Taken together, these literature data suggest that ACK1 is a potential target for cancer treatment. Several series of ACK1 inhibitors have been previously disclosed in literature. Unfortunately, compounds from these series suffer from poor oral pharmacokinetic (PK) properties which have prevented them from being utilized further for in vivo studies. Therefore, there is a clear need for potent, selective and orally bioavailable small molecule ACK1 inhibitors to further probe its role in cancer, both in the in vitro and in vivo setting. This report describes the medicinal chemistry effort towards a series of novel imidazo[1,5-a]pyrazine derived inhibitors of ACK1. Virtual screening led to the discovery of the initial hit, and subsequent exploration of structure-activity relationships and optimization of drug metabolism and pharmacokinetic properties led to the identification of potent, selective and orally bioavailable ACK1 inhibitors. Citation Format: Meizhong Jin, Jing Wang, Andrew Kleinberg, Mridula Kadalbajoo, Kam W. Siu, Andrew Cooke, Mark Bittner, Yan Yao, April Thelemann, Qunsheng Ji, Shripad Bhagwat, Kristen M. Mulvihill, Josef A. Rechka, Jonathan A. Pachter, Andrew P. Crew, David Epstein, Mark J. Mulvihill. Discovery of imidazo[1,5-a]pyrazine derived potent, selective and orally bioavailable ACK1 inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2463. doi:10.1158/1538-7445.AM2013-2463

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