Allan Wissner

Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase

HER-2 belongs to the ErbB family of receptor tyrosine kinases, which has been implicated in a variety of cancers. Overexpression of HER-2 is seen in 25–30% of breast cancer patients and predicts a poor outcome in patients with primary disease. Trastuzumab (Herceptin), a monoclonal antibody to HER-2, is specifically approved for HER-2-positive breast cancer but is active only in a subset of these tumors. Blocking HER-2 function by a small molecule kinase inhibitor, therefore, represents an attractive alternate strategy to inhibit the growth of HER-2-positive tumors. HKI-272 is a potent inhibitor of HER-2 and is highly active against HER-2-overexpressing human breast cancer cell lines in vitro. It also inhibits the epidermal growth factor receptor (EGFR) kinase and the proliferation of EGFR-dependent cells. HKI-272 reduces HER-2 receptor autophosphorylation in cells at doses consistent with inhibition of cell proliferation and functions as an irreversible binding inhibitor, most likely by targeting a cysteine residue in the ATP-binding pocket of the receptor. In agreement with the predicted effects of HER-2 inactivation, HKI-272 treatment of cells results in inhibition of downstream signal transduction events and cell cycle regulatory pathways. This leads to arrest at the G1-S (Gap 1/DNA synthesis)-phase transition of the cell division cycle, ultimately resulting in decreased cell proliferation. In vivo, HKI-272 is active in HER-2- and EGFR-dependent tumor xenograft models when dosed orally on a once daily schedule. On the basis of its favorable preclinical pharmacological profile, HKI-272 has been selected as a candidate for additional development as an antitumor agent in breast and other HER-2-dependent cancers.

Kinase Domain Mutations in Cancer: Implications for Small Molecule Drug Design Strategies

Kinases have emerged as ubiquitous but highly challenging targets for drug discovery. The human genome has 518 kinases and many of these play critical roles in cell growth and apoptosis, making them interesting drug targets for oncology. Kinase targets including epidermal growth factor receptor (EGFR), Raf, Src, and breakpoint cluster regionsAbelson’s kinase (bcr-Abl) emerged early in the study of oncogenic proteins. Despite years of effort involving a compelling breadth of accumulated preclinical design experience (reviewed extensively by Liao and Andrews) and clinical experience (reviewed by LaRusso and Eder ), relatively few kinase inhibitors have been approved (Table 1). As an additional complication, clinical use of these inhibitors has led to the emergence of drug resistant tumors. In many patients, response to small molecule kinase inhibitors has been followed by tumor resurgence, which rendered these inhibitors less effective than expected. This resistance has been linked to a number of mechanisms that include the amplification of the oncogenic kinase gene and alternative signaling pathways or plasticity in signaling. However, in many instances, resistance has been traced to individual or groups of mutations in the drug targets that make the tumors unresponsive in the clinic. These mutants alter the binding properties of the drugs as shown by in vitro studies. When viewed across multiple cancer targets, the location of these mutations forms a compelling pattern with a number of common mechanisms elucidated with reference to this pattern. Significantly, recent characterization of mutations in the EGFR kinase has included structural and kinetic studies that have challenged assumptions about how individual drug resistance mutants are understood and to what extent mechanisms can be generalized across kinases by homology alone. This review will provide a brief overview of kinase structure and function as it pertains to drug discovery, describe the location and importance of clinical mutations, and review the emerging understanding of their impact based on sequence homology, protein crystal complexes, and biochemical/biophysical information. Underlying this discussion is our appreciation that the current clinical arsenal of small molecule kinase inhibitors only contains the first weapons to be deployed in a long war against drug resistance mutations occurring in multiple kinases that target multiple cancers. Table 1 provides a list of kinase inhibitors approved to date for various cancer indications within the U.S. Since 2001, eight inhibitors targeted to the kinase catalytic domain have been approved for clinical use led by imatinib mesylate for chronic myeloid leukemia (CML). CML has been traced to the effect of a characteristic mutation in which part of the breakpoint cluster region (BCR) gene was spliced into the Abelson kinase (Abl) gene creating a hybrid BCR-Abl gene. Known as the “Philadelphia chromosome”, the splice occurs upstream of the kinase domain. Nevertheless, pathway inhibition, by imatinib treatment (Figure 1), provides a significant benefit to patients until the emergence of resistant tumor strains. Gene sequencing efforts on the resulting resistant tumors identified a number of mutations, many in the kinase domain, which reduced the * To whom correspondence should be addressed. Telephone: 845 602 8819. Facsimile: 845 602 5682. E-mail: bikkerj@wyeth.com. a Abbreviations: Abl, Abelson kinase; AML, Acute Myeloid Leukemia; AMP-PNP, 5′-adenylyl,γ-imidodiphosphate; ATP, adenosine triphosphate; Bcr-Abl, breakpoint cluster regionsAbelson kinase; EGF(R), epidermal growth factor (receptor); CML, chronic myeloid leukemia; CMML, chronic myelomonocytic leukemia; CMPD, chronic myeloproliferative disorders; DFSP, dermatofibrossarcoma protruberans; Erb-B1/2/3/4, erythroblastic leukemia viral oncogene homologue 1/2/3/4; Erb-B1 is synonymous with EGFR and HER-1; GIST, gastroInstestinal stromal tumor; HER-1/2/3/4, human EGF receptor-1/2/3/4; HES, hypereosinophilic syndrome; NSCLC, non-small-cell lung cancer; PDGF(R), platelet derived growth factor (receptor); PKA, protein kinase A; SMCD, systemic mast cell disease; VEGF(R), vascular endothelial growth factor (receptor); WT, wild type.  Copyright 2009 by the American Chemical Society

Irreversible inhibition of epidermal growth factor receptor tyrosine kinase with in vivo activity by N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (CL-387,785).

It has been shown previously that 4-anilino quinazolines compete with the ability of ATP to bind the epidermal growth factor receptor (EGF-R), inhibit EGF-stimulated autophosphorylation of tyrosine residues in EGF-R, and block EGF-mediated growth. Since millimolar concentrations of ATP in cells could reduce the efficacy of 4-anilino quinazolines in cells and the activity of these compounds would not be sustained once they were removed from the body, we reasoned that irreversible inhibitors of EGF-R might improve the activity of this series of compounds in animals. Molecular modeling of the EGF-R kinase domain was used to design irreversible inhibitors. We herein describe one such inhibitor: N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]2-butynamide, known as CL-387,785. This compound covalently bound to EGF-R. It also specifically inhibited kinase activity of the protein (IC50 = 370+/-120 pM), blocked EGF-stimulated autophosphorylation of the receptor in cells (ic50 approximately 5 nM), inhibited cell proliferation (IC50 = 31-125 nM) primarily in a cytostatic manner in cell lines that overexpress EGF-R or c-erbB-2, and profoundly blocked the growth of a tumor that overexpresses EGF-R in nude mice (when given orally at 80 mg/kg/day for 10 days, daily). We conclude that CL-387,785 is useful for studying the interaction of small molecules with EGF-R and may have clinical utility.

Inhibitors of Src tyrosine kinase: the preparation and structure–activity relationship of 4-anilino-3-cyanoquinolines and 4-anilinoquinazolines

Src is a nonreceptor tyrosine kinase involved in signaling pathways that control proliferation, migration, and angiogenesis. Increased Src expression and activity are associated with an increase in tumor malignancy and poor prognosis. Several quinolines and quinazolines were identified as potent and selective inhibitors of Src kinase activity.

Identification, Characterization and Initial Hit-to-Lead Optimization of a Series of 4-Arylamino-3-Pyridinecarbonitrile as Protein Kinase C theta (PKCθ) Inhibitors

The protein kinase C (PKC) family of serine/threonine kinases is implicated in a wide variety of cellular processes. The PKC theta (PKCtheta) isoform is involved in TCR signal transduction and T cell activation and regulates T cell mediated diseases, including lung inflammation and airway hyperresponsiveness. Thus inhibition of PKCtheta enzyme activity by a small molecule represents an attractive strategy for the treatment of asthma. A PKCtheta high-throughput screening (HTS) campaign led to the identification of 4-(3-bromophenylamino)-5-(3,4-dimethoxyphenyl)-3-pyridinecarbonitrile 4a, a low microM ATP competitive PKCtheta inhibitor. Structure based hit-to-lead optimization led to the identification of 5-(3,4-dimethoxyphenyl)-4-(1H-indol-5-ylamino)-3-pyridinecarbonitrile 4p, a 70 nM PKCtheta inhibitor. Compound 4p was selective for inhibition of novel PKC isoforms over a panel of 21 serine/threonine, tyrosine, and phosphoinositol kinases, in addition to the conventional and atypical PKCs, PKCbeta, and PKCzeta, respectively. Compound 4p also inhibited IL-2 production in antiCD3/anti-CD28 activated T cells enriched from splenocytes.

Syntheses and EGFR and HER-2 kinase inhibitory activities of 4-anilinoquinoline-3-carbonitriles: analogues of three important 4-anilinoquinazolines currently undergoing clinical evaluation as therapeutic antitumor agents

The syntheses and biological evaluations of 4-anilinoquinoline-3-carbonitrile analogues of the three clinical lead 4-anilinoquinazolines Iressa, Tarceva, and CI-1033 are described. The EGFR and HER-2 kinase inhibitory activities and the cell growth inhibition of the two series are compared with each other and with the clinical lead EKB-569. Similar activities are observed between these two series.

Synthesis and Structure–Activity Relationships of 3-Cyano-4-(phenoxyanilino)quinolines as MEK (MAPKK) Inhibitors

A series of 3-cyano-4-(phenoxyanilino)cyanoquinolines has been prepared as MEK (MAP kinase kinase) inhibitors. The best activity is seen with alkoxy groups at both the 6- and 7-positions. The lead compounds show low nanomolar IC50s against MAP kinase kinase, and have potent inhibitory activity in tumor cells.

Substituted 4-anilino-7-phenyl-3-quinolinecarbonitriles as Src kinase inhibitors

A series of substituted 4-anilino-7-phenyl-3-quinolinecarbonitriles has been prepared as Src kinase inhibitors. Optimal activity is observed with compounds that have basic amines attached via the para position of the 7-phenyl ring, and a hydrogen atom at the C-6 position. The best compounds are low nanomolar inhibitors of Src kinase, and have potent activity against Src-transformed fibroblast cells.

MEK (MAPKK) inhibitors. Part 2: structure-activity relationships of 4-anilino-3-cyano-6,7-dialkoxyquinolines.

A series of 4-anilino-3-cyano-6,7-dialkoxyquinolines with different substituents attached to the 4-anilino group has been prepared that are potent MEK (MAP kinase kinase) inhibitors. The best activity is obtained when a phenyl or a thienyl group is attached to the para-position of the aniline through a hydrophobic linker, such as an oxygen, a sulfur, or a methylene group. The most active compounds show low nanomolar IC(50)s against MEK (MAP kinase kinase), and have potent growth inhibitory activity in LoVo cells (human colon tumor line).

Synthesis and evaluation of 4-Anilino-6,7-dialkoxy-3-quinolinecarbonitriles as inhibitors of kinases of the Ras-MAPK signaling cascade

4-[3-Chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)]anilino-6,7-diethoxy-3-quinolinecarbonitrile (3) was identified as a MEK1 kinase inhibitor with exceptional activity against LoVo cells. The structure-activity relationships of the C-4 aniline substituents were explored, and water-solubilizing groups were added at the C-7 position to improve physical properties. Secondary cellular assays revealed that a compound possessing the appropriate aniline substituents inhibited MEK1 as well as MAPK phosphorylation, thereby acting as a dual inhibitor of the Ras-MAPK signaling cascade.