Gordon Alton

Cytoreductive antitumor activity of PF-2341066, a novel inhibitor of anaplastic lymphoma kinase and c-Met, in experimental models of anaplastic large-cell lymphoma

A t(2;5) chromosomal translocation resulting in expression of an oncogenic kinase fusion protein known as nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) has been implicated in the pathogenesis of anaplastic large-cell lymphoma (ALCL). PF-2341066 was recently identified as a p.o. bioavailable, small-molecule inhibitor of the catalytic activity of c-Met kinase and the NPM-ALK fusion protein. PF-2341066 also potently inhibited NPM-ALK phosphorylation in Karpas299 or SU-DHL-1 ALCL cells (mean IC50 value, 24 nmol/L). In biochemical and cellular screens, PF-2341066 was shown to be selective for c-Met and ALK at pharmacologically relevant concentrations across a panel of >120 diverse kinases. PF-2341066 potently inhibited cell proliferation, which was associated with G1-S–phase cell cycle arrest and induction of apoptosis in ALK-positive ALCL cells (IC50 values, ∼30 nmol/L) but not ALK-negative lymphoma cells. The induction of apoptosis was confirmed using terminal deoxyribonucleotide transferase–mediated nick-end labeling and Annexin V staining (IC50 values, 25–50 nmol/L). P.o. administration of PF-2341066 to severe combined immunodeficient-Beige mice bearing Karpas299 ALCL tumor xenografts resulted in dose-dependent antitumor efficacy with complete regression of all tumors at the 100 mg/kg/d dose within 15 days of initial compound administration. A strong correlation was observed between antitumor response and inhibition of NPM-ALK phosphorylation and induction of apoptosis in tumor tissue. In addition, inhibition of key NPM-ALK signaling mediators, including phospholipase C-γ, signal transducers and activators of transcription 3, extracellular signal-regulated kinases, and Akt by PF-2341066 were observed at concentrations or dose levels, which correlated with inhibition of NPM-ALK phosphorylation and function. Collectively, these data illustrate the potential clinical utility of inhibitors of NPM-ALK in treatment of patients with ALK-positive ALCL. [Mol Cancer Ther 2007;6(12):3314–22]

Characterization of PF-4708671, a novel and highly specific inhibitor of p70 ribosomal S6 kinase (S6K1).

S6K1 (p70 ribosomal S6 kinase 1) is activated by insulin and growth factors via the PI3K (phosphoinositide 3-kinase) and mTOR (mammalian target of rapamycin) signalling pathways. S6K1 regulates numerous processes, such as protein synthesis, growth, proliferation and longevity, and its inhibition has been proposed as a strategy for the treatment of cancer and insulin resistance. In the present paper we describe a novel cell-permeable inhibitor of S6K1, PF-4708671, which specifically inhibits the S6K1 isoform with a Ki of 20 nM and IC50 of 160 nM. PF-4708671 prevents the S6K1-mediated phosphorylation of S6 protein in response to IGF-1 (insulin-like growth factor 1), while having no effect upon the PMA-induced phosphorylation of substrates of the highly related RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated kinase) kinases. PF-4708671 was also found to induce phosphorylation of the T-loop and hydrophobic motif of S6K1, an effect that is dependent upon mTORC1 (mTOR complex 1). PF-4708671 is the first S6K1-specific inhibitor to be reported and will be a useful tool for delineating S6K1-specific roles downstream of mTOR.

Effects of p38 MAPK Inhibition on Early Stages of Diabetic Retinopathy and Sensory Nerve Function

Purpose. p38 mitogen-activated protein kinase (MAPK) is known to play a regulatory role in inflammatory processes in disease. Inflammation has been linked also to the development of diabetic retinopathy in rodents. This study was conducted to evaluate the effect of a p38 MAPK inhibitor on the development of early stages of diabetic retinopathy in rats. Methods. Streptozotocin-diabetic rats were assigned to two groups-treated with the p38 MAPK inhibitor PHA666859 (Pfizer, New York, NY) and untreated-and compared with age-matched nondiabetic control animals. Results. At 2 months of diabetes, insulin-deficient diabetic control rats exhibited significant increases in retinal superoxide, nitric oxide (NO), cyclooxygenase (COX)-2, and leukostasis within retinal microvessels. All these abnormalities were significantly inhibited by the p38 MAPK inhibitor (25 mg/kgBW/d). At 10 months of diabetes, significant increases in the number of degenerate (acellular) capillaries and pericyte ghosts were measured in control diabetic rats versus those in nondiabetic control animals, and pharmacologic inhibition of p38 MAPK significantly inhibited all these abnormalities (all P < 0.05). This therapy also had beneficial effects outside the eye in diabetes, as evidenced by the inhibition of a diabetes-induced hypersensitivity of peripheral nerves to light touch (tactile allodynia). Conclusions. p38 MAPK plays an important role in diabetes-induced inflammation in the retina, and inhibition of p38 MAPK offers a novel therapeutic approach to inhibiting the development of early stages of diabetic retinopathy and other complications of diabetes.

Targeting the unactivated conformations of protein kinases for small molecule drug discovery

Background: The number of drugs in active clinical development or on the market that target the unactivated conformational states of protein kinases is growing and represents a significant portion of kinase research at biopharmaceutical companies. These non-classical kinase inhibitors have a mode of action which may overcome some of the liabilities of classical ATP-site inhibitors that substantially overlap the space that ATP occupies in the activated kinase. Objective: This review will discuss state-of-the-art methods of inhibiting protein kinases by targeting the unactivated conformations of the enzyme with small molecules directed to the ATP binding region. Methods: Biochemical and structural biology publications and public domain crystal structures were evaluated to identify key concepts in drug discovery for unactivated protein kinase inhibitors that target the ATP binding region. Conclusion: The potential for enhanced selectivity, potency and duration of pharmacological action may allow non-classical kinase therapeutics to be used for chronic dosing in non-life-threatening indications. Moreover, by targeting additional conformational space on the kinase protein it is possible that new chemical matter will be discovered such that current intellectual property limitations on traditional ATP-site chemical scaffolds may be circumvented.

Study of the PDK1/AKT signaling pathway using selective PDK1 inhibitors, HCS, and enhanced biochemical assays.

The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP(3)) through interaction with their pleckstrin-homology domains. A biochemical method was developed to measure the kinase activity of PDK1 and AKT1/2, utilizing nickel-chelating coated lipid vesicles as a way to mimic the membrane environment. The presence of these vesicles in the reaction buffer enhanced the specific activity of the His-tagged PDK1 (full-length, and the truncated kinase domain) and the activity of the full-length His-tagged AKT1 and AKT2 when assayed in a cascade-type reaction. This enhanced biochemical assay is also suitable for measuring the inhibition of PDK1 by several selective compounds from the carbonyl-4-amino-pyrrolopyrimidine (CAP) series. One of these inhibitors, PF-5168899, was further evaluated using a high content cell-based assay in the presence of CHO cells engineered with GFP-PDK1.

Identification of novel pyrrolopyrazoles as protein kinase C β II inhibitors

A novel series of pyrrolopyrazole-based protein kinase C β II inhibitors has been identified from high-throughput screening. Herein, we report our initial structure-activity relationship studies with a focus on optimizing compound ligand efficiency and physicochemical properties, which has led to potent inhibitors with good cell permeability.

Therapeutic modulation of inflammatory gene transcription by kinase inhibitors

Altered gene expression contributes to the aetiology of inflammatory disease by modulation of the concentration of disease-related proteins. The expression of inflammatory genes is controlled through the concerted actions of specific transcription factors. Signal transduction networks positively or negatively regulate the activity of these transcription factors. Key components of these networks are protein kinases, which phosphorylate substrates on tyrosine, threonine or serine residues. During the disease process, pro-inflammatory signalling at the cell surface leads to a cascade of kinase activation, which ultimately culminates in modulation of the activity of transcription factors. Thus, pharmacological inhibition of protein kinases is a potential therapeutic strategy to treat inflammation. There are approximately 500 protein kinases in the human genome. Targeted small molecule inhibitors of these kinases should allow for tissue- and disease-specific therapies of unprecedented selectivity. Heralding this new era in molecular medicine is imatinib (Gleevec™, Norvartis) a recently marketed tyrosine kinase inhibitor. This review focuses on kinase inhibitors that are currently in development for inflammatory diseases and the transcription factors that are involved.

Potent and selective thiophene urea-templated inhibitors of S6K.

S6K1 (p70 S6 kinase-1) is thought to play a critical role in the development of obesity and insulin resistance, thus making it an attractive target in developing medicines for the treatment of these disorders. We describe a novel thiophene urea class of S6K inhibitors. The lead matter for the development of these inhibitors came from mining the literature for reports of weak off-target S6K activity. These optimized inhibitors exhibit good potency and excellent selectivity for S6K over a panel of 43 kinases.

Abstract 753: Novel, potent and selective small molecule inhibitors of 3-phosphoinositide-dependent kinase (PDK1)

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a crucial role in cell growth, proliferation and survival. Genomic aberrations in the PI3K pathway, such as mutational activation of PI3Kα or loss-of-function of the tumor suppressor PTEN, have been closely linked to the development and progression of a wide range of cancers. Inhibition of the key targets in the pathway, PI3K, AKT, mTOR & PDK1, may provide an effective treatment of cancer. In an effort to discover compounds that inhibit PDK1, we have developed a series of 3-Carbonyl-4-Amino-Pyrrolopyrimidine (CAP) compounds that are selective and potent PDK1 inhibitors. Early screening led to a viable starting point, PF-03772304, (4-amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-(6-methylamino-pyrazin-2-yl)-methanone, which has an IC50 of 94 nM for PDK1 and a ligand efficiency of 0.42. While potent, this lead was not selective against PI3K. Using structure-based drug design, this lead was modified to expand into the selectivity pocket of PDK1 (under the G-Loop), leading to the identification of a potent and pathway-selective compound, PF-05017255 ((4-Amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-{6-[(3S,4R)-4-(4-fluoro-phenyl)-tetrahydro-furan-3-ylamino]-pyrazin-2-yl}-methanone). PF-05017255 has a Ki of 0.6 nM for PDK1 and is more than 400-fold selective against other PI3K pathway kinases: PI3Kα, AKT, S6K and mTOR. For even greater kinase selectivity, we sought to lower the clogP of our lead (clogP for PF-05017255 is 3.0) to reduce the contribution from the hydrophobic effect. These efforts led to PF-05168899 (1-{(2R,3R)-3-[6-(4-Amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonyl)-pyrazin-2-ylamino]-2-phenyl-pyrrolidin-1-yl}-ethanone) with a Ki of 0.4 nM for PDK1, a clogP of 2.1, and greater than 1000-fold selectivity against PI3Kα, AKT, S6K, mTOR, CDK2, CHK1 and PAK4. PF-05168899 also showed little inhibitory effect (<50% at 1 uM) against 33 of 35 kinases in a broader panel, demonstrating significant inhibition only against CHK2 (94%) and AuroraB (54%). In addition, the most potent analogs (e.g. PF-05168889) inhibited the phosphorylation of AKT at the residue threonine 308 (IC50 40-200 nM) in a variety of cancer cell lines (e.g. H460, A549). The design, synthesis and SAR of this chemical series will be described. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 753.

Abstract 4482: Novel and selective small molecule inhibitors of 3-phosphoinositide-dependent kinase-1 inhibit the PDK-1/AKT signaling pathway and cell proliferation

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Receptor tyrosine kinases (RTKs), PTEN (phosphatase and tensin homolog deleted on chromosome 10), and PIK3CA (encodes the p110α subunit of phosphatidylinositol 3-kinase [PI3K]) frequently contribute to tumor progression through their ability to regulate the intracellular level of phosphatidylinositol-3,4,5-triphosphate (PIP3). 3-phosphoinositide-dependent kinase-1 (PDK-1), a serine/threonine kinase, activates the catalytic domain of numerous kinases by phosphorylating their T-loop sites. PDK-1 activity is required for activation of AKT, p70S6K, and RSK which lead to cell proliferation and transformation. The interaction of the pleckstrin homology (PH) domain of AKT with the membrane bound PIP3 confers a conformational change in AKT, allowing PDK-1 to phosphorylate AKT at the residue threonine-308 (T308). This T-loop activation at T308, along with the phosphorylation of the serine-473 residue by mTOR, fully activates the AKT pathway. Although the roles of many PDK-1 substrates have yet to be characterized, the oncogenic activity of aberrant PI3K pathway signaling through PDK-1 has been extensively validated. We have developed a series of 3-Carbonyl-4-Amino-Pyrrolopyrimidne (CAP) compounds that are potent inhibitors of human PDK-1 (full length and kinase domain) which demonstrate more than 100-fold selectivity against P70S6K, PI3K, AKT, and mTOR. In this study, representative compounds from the CAP series were used to perform a variety of anti-tumor assays. We demonstrate PDK-1 compounds inhibit the phosphorylation of T308 on AKT as well as downstream molecules of the PI3K pathway, such as S6 ribosomal protein (S6RP) in breast, lung and colon cancer cell lines harboring a PI3KCA mutation. Additionally, blockade of AKT and molecules in the PI3K pathway leads to the inhibition of cell proliferation and cell transformation in cancer cells. Our data suggest that the inhibition of PDK-1 activity is sufficient to induce anti-tumor activity in cancer cells through the PI3K-PDK1-AKT axis, and that a potent and specific PDK-1 inhibitor could potentially be developed as a therapeutic agent against several cancer types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4482.