Peter R. Young

Discovery of Selective Irreversible Inhibitors for Bruton’s Tyrosine Kinase

The importance of B cells in rheumatoid arthritis (RA) pathogenesis has been recently demonstrated in several clinical studies using the anti-CD20 antibody rituximab, which selectively depletes B cells. A recent phase III clinical trial led to the FDA approval of rituximab for a subset of RA patients. Bruton’s tyrosine kinase (Btk), a member of Tec family kinases, is a key component in the B-cell receptor signal pathway (BCR). Upon activation by upstream kinases (for example, Lyn and Syk), Btk phosphorylates and thereby activates phospholipase-Cg (PLCg), leading to several important downstream events including calcium ion transportation, NF-kB activation, and (auto)antibody generation. Previous biological studies (genetic loss of function and siRNA knockdown) strongly suggest that Btk is also a mediator of proinflammatory signals. Taken together, these studies indicate Btk may be a potential target for the treatment of RA. However, despite the previous discovery of LFM-A13 as a selective Btk inhibitor, there is no published study that has demonstrated that inhibition of Btk activity leads to in vivo efficacy in an animal model of rheumatoid arthritis. As ATP binding sites in kinases are highly conserved, it is a formidable task to develop selective ATP competitive kinase inhibitors. Among several approaches, the use of electrophilic inhibitors has been shown as a viable method to achieve selectivity. Considering the relative scarcity of knowledge on “chemical knockdown” of Btk activity, it is crucial to discover a potent and selective tool compound for this kinase. Herein, we describe the discovery of a selective, irreversible Btk inhibitor and its efficacy in a mouse RA model. An initial campaign to scan for scaffolds capable of inhibiting Btk’s kinase activity identified compound 1 as having

CRA-024781: a novel synthetic inhibitor of histone deacetylase enzymes with antitumor activity in vitro and in vivo

CRA-024781 is a novel, broad spectrum hydroxamic acid–based inhibitor of histone deacetylase (HDAC) that shows antitumor activity in vitro and in vivo preclinically and is under evaluation in phase I clinical trials for cancer. CRA-024781 inhibited pure recombinant HDAC1 with a Ki of 0.007 μmol/L, and also inhibited the other HDAC isozymes HDAC2, HDAC3/SMRT, HDAC6, HDAC8, and HDAC10 in the nanomolar range. Treatment of cultured tumor cell lines grown in vitro with CRA-024781 resulted in the accumulation of acetylated histone and acetylated tubulin, resulting in an inhibition of tumor cell growth and the induction of apoptosis. CRA-024781 parenterally administered to mice harboring HCT116 or DLD-1 colon tumor xenografts resulted in a statistically significant reduction in tumor growth at doses that were well tolerated as measured by body weight. Inhibition of tumor growth was accompanied by an increase in the acetylation of α-tubulin in peripheral blood mononuclear cells, and an alteration in the expression of many genes in the tumors, including several involved in apoptosis and cell growth. These results reveal CRA-024781 to be a novel HDAC inhibitor with potent antitumor activity. [Mol Cancer Ther 2006;5(5):1309–17]

The glycotope-specific RAV12 monoclonal antibody induces oncosis in vitro and has antitumor activity against gastrointestinal adenocarcinoma tumor xenografts in vivo

RAV12 is a chimeric antibody that recognizes an N-linked carbohydrate antigen (RAAG12) strongly expressed on multiple solid organ cancers. More than 90% of tumors of colorectal, gastric, and pancreatic origin express RAAG12, and a majority of these tumors exhibit uniform RAAG12 expression. RAV12 exhibits potent cytotoxic activity in vitro against COLO 205 colon tumor cells via an oncotic cell death mechanism. RAV12-treated COLO 205 cells undergo morphologic changes consistent with oncosis, including cytoskeletal rearrangement, rapid plasma membrane swelling, and cell lysis. RAV12 inhibited the growth of RAAG12-expressing gastrointestinal tumor xenografts in athymic mice. In the case of SNU-16 tumor cells, twice weekly treatment of established s.c. tumors with 10 mg/kg RAV12 caused a ∼70% suppression of tumor growth at the end of the study. This preclinical data has led to the initiation of a phase I/IIA clinical study of RAV12 in patients with metastatic or recurrent adenocarcinoma. [Mol Cancer Ther 2007;6(3):856–65]

CRA-026440: a potent, broad-spectrum, hydroxamic histone deacetylase inhibitor with antiproliferative and antiangiogenic activity in vitro and in vivo

CRA-026440 is a novel, broad-spectrum, hydroxamic acid–based inhibitor of histone deacetylase (HDAC) that shows antitumor and antiangiogenic activities in vitro and in vivo preclinically. CRA-026440 inhibited pure recombinant isozymes HDAC1, HDAC2, HDAC3/SMRT, HDAC6, HDAC8, and HDAC10 in the nanomolar range. Treatment of cultured tumor cell lines grown in vitro with CRA-026440 resulted in the accumulation of acetylated histone and acetylated tubulin, leading to an inhibition of tumor cell growth and the induction of apoptosis. CRA-026440 inhibited ex vivo angiogenesis in a dose-dependent manner. CRA-026440 parenterally given to mice harboring HCT116 or U937 human tumor xenografts resulted in a statistically significant reduction in tumor growth. CRA-026440, when used in combination with Avastin, achieved greater preclinical efficacy in HCT 116 colorectal tumor model. Inhibition of tumor growth was accompanied by an increase in the acetylation of α-tubulin in peripheral blood mononuclear cells and an alteration in the expression of many genes in the tumors, including several involved in angiogenesis, apoptosis, and cell growth. These results reveal CRA-026440 to be a novel HDAC inhibitor with potent antitumor activity. [Mol Cancer Ther 2006;5(7):1693–701]

Discovery of a new chemical series of BRD4(1) inhibitors using protein-ligand docking and structure-guided design.

Bromodomains are key transcriptional regulators that are thought to be druggable epigenetic targets for cancer, inflammation, diabetes and cardiovascular therapeutics. Of particular importance is the first of two bromodomains in bromodomain containing 4 protein (BRD4(1)). Protein-ligand docking in BRD4(1) was used to purchase a small, focused screening set of compounds possessing a large variety of core structures. Within this set, a small number of weak hits each contained a dihydroquinoxalinone ring system. We purchased other analogs with this ring system and further validated the new hit series and obtained improvement in binding inhibition. Limited exploration by new analog synthesis showed that the binding inhibition in a FRET assay could be improved to the low μM level making this new core a potential hit-to-lead series. Additionally, the predicted geometries of the initial hit and an improved analog were confirmed by X-ray co-crystallography with BRD4(1).