Active Small-molecule Inhibitor Research Articles

Discovery of an orally active small-molecule irreversible inhibitor of protein disulfide isomerase for ovarian cancer treatment

Protein disulfide isomerase (PDI), an endoplasmic reticulum chaperone protein, catalyzes disulfide bond breakage, formation, and rearrangement. The effect of PDI inhibition on ovarian cancer progression is not yet clear, and there is a need for potent, selective, and safe small-molecule inhibitors of PDI. Here, we report a class of propynoic acid carbamoyl methyl amides (PACMAs) that are active against a panel of human ovarian cancer cell lines. Using fluorescent derivatives, 2D gel electrophoresis, and MS, we established that PACMA 31, one of the most active analogs, acts as an irreversible small-molecule inhibitor of PDI, forming a covalent bond with the active site cysteines of PDI. We also showed that PDI activity is essential for the survival and proliferation of human ovarian cancer cells. In vivo, PACMA 31 showed tumor targeting ability and significantly suppressed ovarian tumor growth without causing toxicity to normal tissues. These irreversible small-molecule PDI inhibitors represent an important approach for the development of targeted anticancer agents for ovarian cancer therapy, and they can also serve as useful probes for investigating the biology of PDI-implicated pathways.

Clinical Pharmacokinetics and Pharmacodynamics of Linagliptin

Linagliptin is an orally active small-molecule inhibitor of dipeptidyl peptidase (DPP)-4, which was first licensed in the US, Europe, Japan and other territories in 2011 to improve glycaemic control in adults with type 2 diabetes mellitus. Linagliptin is the first and thus far the only DPP-4 inhibitor, and oral antihyperglycaemic drug in general, to be approved as a single-strength once-daily dose (5 mg). Compared with other available DPP-4 inhibitors, linagliptin has a unique pharmacokinetic/pharmacodynamic profile that is characterized by target-mediated nonlinear pharmacokinetics, concentration-dependent protein binding, minimal renal clearance and no requirements for dose adjustment for any intrinsic or extrinsic factor. After single or multiple oral doses of 1-10 mg, linagliptin displays less than dose-proportional increases in maximum plasma concentration (C(max)) and area under the plasma concentration-time curve (AUC). Linagliptin is rapidly absorbed after oral administration, with C(max) occurring after approximately 90 minutes, and reaches steady-state concentrations within 4 days. With the therapeutic dose, steady-state C(max) (11-12 nmol/L) and AUC (∼150 nmol · h/L) are approximately 1.3-fold greater than after a single dose, indicating little drug accumulation with repeat dosing. Linagliptin exhibits concentration-dependent protein binding in human plasma in vitro (99% at 1 nmol/L to 75-89% at >30 nmol/L) and has a large apparent volume of distribution, demonstrating extensive distribution into tissues. The nonlinear pharmacokinetics of linagliptin are best described by a two-compartmental model that incorporates target-mediated drug disposition resulting from high-affinity, saturable binding to DPP-4. The oral bioavailability of linagliptin estimated with this model is approximately 30%. Linagliptin has a long terminal half-life (>100 hours); however, its accumulation half-life is much shorter (approximately 10 hours), accounting for the rapid attainment of steady state. The majority of linagliptin is eliminated as parent compound, demonstrating that metabolism plays a minor role in the overall pharmacokinetics in humans. The main, pharmacologically inactive S-3-hydroxypiperidinyl metabolite accounted for approximately 17% of the total drug-related compounds in plasma. Linagliptin is eliminated primarily in faeces, with only around 5% of the oral therapeutic dose excreted in the urine at steady state. Linagliptin potently inhibits DPP-4 (inhibition constant 1 nmol/L), and trough drug concentrations achieved with therapeutic dosing inhibit >80% of plasma DPP-4 activity, the threshold associated with maximal antihyperglycaemic effects in animal models. There are no clinically relevant alterations in linagliptin pharmacokinetics resulting from renal impairment, hepatic impairment, coadministration with food, race, body weight, sex or age. In vitro, linagliptin is a weak substrate and weak inhibitor of cytochrome P450 (CYP) 3A4 and permeability glycoprotein (P-gp) but not of other CYP isozymes or ATP-binding cassette transporters. Clinical studies have revealed no relevant drug interactions when coadministered with other drugs commonly prescribed to patients with type 2 diabetes, including the narrow therapeutic index drugs warfarin and digoxin. Linagliptin plasma exposure is reduced by potent inducers of CYP3A4 or P-gp. Linagliptin has demonstrated a large safety window (>100-fold the recommended daily dose) and clinically relevant antihyperglycaemic effects in patients with type 2 diabetes.

Abstract 4779: K-983, an orally available small molecule inhibitor of FGFR, demonstrated prolonged survival in a mouse orthotopic gastric cancer model

Abstract The receptor tyrosine kinase FGFR and its ligand fibroblast growth factor (FGF) play important roles in cell growth, migration, and survival. Dysregulation of the FGFR/FGF signaling pathway has been found in many human malignancies. K-983 was identified as a novel, potent, and orally active small molecule inhibitor of FGFR. The anti-tumor activity of K-983 was evaluated in a rat xenograft models. K-983 displayed dose dependent anti-tumor efficacy. K-983 demonstrated dose dependent inhibition of FGFR phosphorylation and prolonged survival in a mouse peritoneal dissemination model of gastric cancer. Furthermore, K-983 was examined using an orthotopic model of human scirrhous gastric cancer in nude mouse. K-983 (80 mg/kg) was orally administered once daily to the mice for 28 days. Histopathological examination revealed that K-983 inhibited tumor growth at inoculated site, invasion in gastric wall and metastasis to other organs. K-983 also significantly prolonged the survival of mice in this orthotopic tumor model. In summary, these results demonstrated the promising therapeutic potential of K-983 in anticancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4779. doi:1538-7445.AM2012-4779

Abstract 1716: FGFR1 gene amplification predicts the antitumor response of FGFR inhibitor LY2874455 in human lung cancer cells

Abstract Lung cancer is the leading cause of cancer-related death for both men and women in the world. FGFR receptor tyrosine kinase signaling pathway has been implicated in tumor cell growth, survival, angiogenesis, and metastasis. Activation of the FGFR pathway via FGFR over-expression, and chromosomal amplification and translocation as well as genetic mutation contributes to the tumor progression and the resistance to anti-EGFR and -VEGF treatment in lung cancer. Targeting FGFR pathway is therefore an attractive therapeutic strategy for lung cancer. We have discovered and developed a novel, ATP competitive and orally active small molecule inhibitor of FGFR, LY2874455, currently in clinic development. LY2874455 is a potent inhibitor against four FGFRs, and demonstrates a broad-spectrum of anti-tumor activity in a number of tumor xenograft models. In the current study, we evaluated the anti-tumor activity of LY2874455 in lung tumor cells with or without FGFR1 gene amplification. FGFR1 is known to be amplified in human lung cancer cells NCI-H1581, NCI-H520 and DMS-114, but not in NCI-H460, A549 and NCI-H1299. The mRNA expression levels of FGFR1 are shown to be higher in the FGFR1 amplified cells than those in the FGFR1 non-amplified cells. Consistent with this, the FGFR1 amplified cells exhibited the much more robust b-FGF-induced phosphorylation of FRS2 and ERK, which are also more sensitive to the inhibition by LY2874455 than that in the FGFR1 non-amplified cells. Furthermore, the FGFR1 amplified cells exhibit the better responses to the growth inhibition and apoptosis induction by LY2874455 than those in the FGFR1 non-amplified cells. Therefore, these findings suggest that FGFR1 plays an important role in lung cancer cell growth and survival, and that LY2874455 may provide a promising treatment option for some lung cancer patients whose tumor harbors FGFR1 gene amplification. The in vivo anti-tumor response of LY2874455 is being further investigated in the tumor xenograft models derived from these FGFR1 amplified or non-amplified human lung cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1716. doi:1538-7445.AM2012-1716

Grand Challenge in Inflammation

Acute inflammation is a normal response to infection and is beneficial for the host to survive. However, most human diseases are due to sterile inflammation. When allowed to continue unchecked, sterile inflammation can result in autoinflammatory disorders, neurodegenerative disease, or cancer. A variety of safe and effective anti-inflammatory agents are available for short term control of acute inflammation including aspirin and non-steroidal anti-inflammatory drugs (NSAIDs). For the most part, even short term glucocorticoids are safe and effective. But attacking the upstream causes of inflammation is required for long-term treatment. There is new era of anti-inflammatory agents, which includes “biologicals” such as anti-cytokine therapies, kinase inhibitors, statins, histone deacetylase inhibitors, and PPAR agonists. Reducing pain, inflammation, and fever with salicylate-containing plant extracts can be traced throughout written human history. One hundred fifty years ago, Felix Hoffman acetylated salicylic acid and created aspirin. Aspirin inhibits the cyclooxygenase (COX) enzymes COX-1 and COX-2, which synthesize the inflammatory mediators prostaglandins and thromboxanes. The ability to block production of prostaglandins and thromboxanes accounts for aspirin being the world's most used therapeutic agent. Second to aspirin are NSAIDs, which target COX-2 and hence the synthesis of prostaglandins, particularly PGE2. Synthetic forms of natural cortisol (termed glucocorticoids) are widely used to treat many inflammatory diseases and despite their serious side effects, glucocorticoids remain a mainstay of inflammation reduction. Yet, it is still the challenge of the pharmaceutical chemist to develop more effective and less toxic agents to treat the signs and symptoms of acute inflammation as well as the long-term consequences of chronic inflammatory diseases. Inflammation is a dynamic process with pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1 playing central roles. A number of “biologicals” have been developed to treat inflammation by reducing the activity of specific cytokines or their receptors (anti-cytokine therapies), block lymphocyte trafficking into tissues, prevent the binding of monocyte–lymphocyte co-stimulatory molecules, or deplete B lymphocytes. Current anti-cytokine therapies have found a place in the treatment of autoimmune diseases, such as rheumatoid arthritis, inflammatory bowel disease, psoriasis, and others. Without question, neutralization of specific pro-inflammatory cytokines has demonstrated their causative role in inflammation and has changed the lives of millions of patients with these diseases. One drawback of anti-cytokine therapies is decreased host immune defense against infection and possibly cancer. Nevertheless, the benefits of anti-cytokine therapies outweigh the risks and the risks can be reduced. Compared to the consequences of long-term glucocorticoid treatment to stem inflammation, anti-cytokine therapies are a major improvement. Indeed, organ toxicities are rarely, if ever, observed with anti-cytokine therapies as they operate almost exclusively in extracellular rather than intracellular compartments. Kinases that act downstream of cytokine receptors have become new targets to tame inflammation, and orally active small molecule inhibitors of intracellular signaling kinases will likely be the new frontier of anti-inflammatory agents. However, because many intracellular signaling molecules are involved in normal cellular functions, the effective concentration that does not elicit organ toxicity will need to be carefully determined. Statins, a safe class of drugs used for lowering serum cholesterol, also have anti-inflammatory properties. Orally active inhibitors of histone deacetylases, which are currently in clinical use, are effective anti-inflammatory drugs. Naturally occurring resolvins are also being developed as anti-inflammatory agents. Thus the challenge is to develop anti-inflammatory agents that are orally active, safe, and effective for treating acute or chronic inflammation.

Clinical Development of AVL-292; A Potent, Selective Covalent Btk Inhibitor for the Treatment of B Cell Malignancies,

Abstract 3485Targeted therapies that suppress B cell receptor (BCR) signaling have emerged as promising agents in the treatment of several B cell malignancies. Bruton’s tyrosine kinase (Btk) plays a key role in promoting B cell proliferation and survival through participation in the BCR signaling pathway and represents a promising new drug target. AVL-292 is a covalent, highly selective, orally active small molecule inhibitor of Btk currently being evaluated in a Phase 1b clinical trial in relapsed, refractory B cell malignancies including Chronic Lymphocytic Leukemia (CLL) and non-Hodgkin lymphomas. AVL-292 forms a covalent bond with Cys481 in Btk and potently inhibits Btk in biochemical (IC50 < 0.5nM) and cellular assays (EC50 1–10 nM) including anti-IgM stimulation of BCR signaling, B cell proliferation and activation. A quantitative pharmacodynamic assay to determine the level of AVL-292 bonded to Btk in vitro or in vivo was developed and this drug-target engagement by AVL-292 was shown to correlate directly with inhibition of Btk enzyme activity and substrate phosphorylation. To rationally determine the dose and dose frequency of AVL-292 most likely to benefit patients and to reduce the potential for sub-therapeutic dosing in initial oncology patient cohorts, AVL-292 was administered to healthy adult subjects in a double-blind, placebo controlled, single ascending dose study. This study assessed safety, pharmacokinetics, and quantitatively measured Btk protein levels and AVL-292-Btk engagement in freshly isolated peripheral B lymphocytes. In healthy human subjects, AVL-292 was found to be safe and well tolerated following oral administration at dose levels ranging from 0.5–7.0 mg/kg. AVL-292 plasma levels and pharmacodynamic measurement of Btk engagement was dose-proportional across cohorts. All subjects that received 1.0 mg/kg AVL-292 achieved >80% Btk engagement and mean peak plasma levels (Cmax 365 ng/mL) of AVL-292 were rapidly achieved (Tmax median 40 min). Subjects receiving 2.0 mg/kg AVL-292 had a mean peak plasma concentration of 542 ng/mL. All subjects demonstrated >84% Btk engagement at this dose, with 5 of 6 subjects achieving >98% drug-target engagement. Although AVL-292 plasma levels declined substantially by 8 hours, Btk engagement persisted throughout 24 hours, demonstrating that covalent inhibition of Btk with AVL-292 enables prolonged duration of activity without high levels of circulating drug. These results suggest that a once daily dosing schedule is sufficient for sustained Btk inhibition. Furthermore, the Btk protein level in circulating B lymphocytes from all study subjects was evaluated and the mean level was found to be 417.7 pg Btk/mg total protein. Interestingly, this finding in normal B cells correlates well with preclinical ex vivo analysis of Btk protein in primary CLL cells where comparable Btk protein levels were found. This suggests that the AVL-292 dose range and schedule identified for complete Btk engagement in this healthy volunteer trial is likely to inform appropriate dose selection in the subsequent phase 1b oncology study, allowing more rapid identification of a safe and clinically effective dose. Disclosures:Evans:Avila Therapeutics: Employment, Equity Ownership. Tester:Avila Therapeutics: Employment, Equity Ownership. Aslanian:Avila Therapeutics: Employment, Equity Ownership. Chaturvedi:Avila Therapeutics: Employment, Equity Ownership. Mazdiyasni:Avila Therapeutics: Employment, Equity Ownership. Sheets:Avila Therapeutics: Employment, Equity Ownership. Nacht:Avila Therapeutics: Employment, Equity Ownership. Stiede:Avila Therapeutics: Employment, Equity Ownership. Witowski:Avila Therapeutics: Employment, Equity Ownership. Lounsbury:Avila Therapeutics: Employment, Equity Ownership. Petter:Avila Therapeutics: Employment, Equity Ownership. Singh:Avila Therapeutics: Employment, Equity Ownership. Westlin:Avila Therapeutics: Employment, Equity Ownership.

A systems biology approach reveals the role of a novel methyltransferase in response to chemical stress and lipid homeostasis.

Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors.

Abstract P6-15-17: Synergy of Ixabepilone and Dasatinib in Triple Negative Breast Cancer Cell Lines

Abstract Background: Treatment options are very limited for breast cancers that do not express HER2 or estrogen and progesterone receptors (Triple Negative Breast Cancer; TNBC). Dasatinib is an orally active small molecule inhibitor of the src kinase. It has been proposed that dasatinib inhibits growth of breast cancer cells by modulating epidermal growth factor receptor (EGFR) signaling. Approximately 70% of breast cancers over express EGFR family members as well as c-Src. Src is co-expressed with at least one of the EGFRs. Both src and EGFR1 expression have been associated with TNBC. Dasatinib has previously shown activity as a single agent in TNBC cell lines. However, dasatinib has limited single agent activity in a cohort of chemotherapy refractory TNBC patients according to a phase I/II study. We believe that dasatinib may sensitize breast cancer cells to anti-mitotic agents. Ixabepilone is a potent anti-mitotic agent that has shown significant response in TNBC patients, both in the neoadjuvant setting and in the metastatic setting, including those cancers that are taxane refractory. Therefore, combining these two drugs may have utility in this setting and may lead to inhibition of tumor cell proliferation, invasion, metastasis, angiogenesis, and tumor cell survival. Objectives: To study the activity of dasatinib combined with Ixabepilone in breast cancer cell lines. Methods: A panel of 8 breast cancer cell lines was used in these experiments. These included MCF-7(ER+), T-47D (ER +), SKBR3 (HER2+), and five triple negative cell lines: BT-549, HS578T, MDA-MB-157, MDA-MB-231, MDA-MB-436. First, these cell lines were evaluated for src expression by Western blot as well as sensitivity to dasatinib alone, ixabepilone alone and then the combination of these two drugs using a high-throughput, 384- well CellTiter-Blue cell viability assay. Percent viability was calculated from four replicate cell wells after 72-hour drug incubation and IC50s were determined. Experiments were repeated a minimum of three times. Synergistic, additive, or antagonistic effects were determined using the combination index (CI) method developed by Chou and Talalay. Results: TNBC cell lines expressed higher levels of src protein than cell lines with positive expression of HER2, ER and/or PR by Western blot analysis. Viability assays suggested a correlation between src expression and dasatinib IC50. Combinations of dasatinib and ixabepilone were determined to have combinational indexes &amp;lt;1 in all cell lines tested, suggestive of synergistic activity. Conclusion: This study demonstrates that in a range of breast cell lines with varying levels of src expression, dasatinib combined with ixabepilone is a synergistic combination and may prove to be beneficial to patients with very limited treatment options. Figure available in online version. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-17.

HAT Trick: p300, Small Molecule, Inhibitor

p300 is an acetyltransferase that targets histone and nonhistone proteins for lysine acetylation. Based on a p300 structure, Bowers et al. (2010) have conducted an in silico screen for active site small molecule inhibitors of p300. We describe this important discovery and its potential therapeutic applications.

Abstract 4511: LY2801653 treatment increases survival of mice bearing H441 orthotopic lung tumors

Abstract Lung cancer is the leading cause of cancer-related death for both men and women in the world. c-Met receptor tyrosine kinase (RTK) signaling pathway has been implicated in tumor growth, angiogenesis, and metastasis. Sustained activation, over-expression, or mutation of c-Met is associated with poor prognosis in several tumors, including NSCLC. Therefore, c-Met is emerging as a novel target of therapeutic potential for lung cancer. We have discovered and developed a novel, ATP competitive, and orally active small molecule inhibitor of c-Met, LY 2801653. It is a potent inhibitor of c-Met phosphorylation in vivo, and demonstrated anti-tumor activity in several xenograft tumor models. In the current study, we evaluated the efficacy of LY2801653 in both c-Met dependant and c-Met independent NSCLC cells in vitro and in vivo orthotopic lung models that recapitulated the local and regional growth pattern seen in lung cancer patients. In c-Met dependant H441 model, LY2801653 demonstrated the significant inhibition of both primary tumor (90% inhibition) and metastasis (65% inhibition of lymph node and 70% inhibition of chest wall); whereas in c-Met independent H1299 model, LY281653 only inhibited primary tumor growth (70% inhibition). The benefit of LY2801653 treatment was further demonstrated in the survival of mice bearing H441 lung tumors. Treatment with LY2801653 significantly increased the survival of mice to 192% compared to that in the vehicle treated mice. The anti-tumor and/or anti-metastasis mechanism of LY2801653 was also investigated. These results demonstrated therapeutic potential of LY2801653 in the treatment of lung cancer. 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 4511.

Abstract 3611: LY2801653, an orally available small molecule inhibitor of c-Met, demonstrated broad antitumor efficacy in patient derived xenograft models

Abstract The receptor tyrosine kinase c-Met and its ligand hepatocyte growth factor (HGF) play important roles in cell growth, migration, and survival. Dysregulation of the c-Met/HGF signaling pathway has been found in many human malignancies. LY2801653 was identified and developed as a novel, potent, and orally active small molecule inhibitor of human c-Met. It demonstrated dose dependent inhibition of c-Met phosphorylation in xenograft tumors with a long lasting PD effect. In the current study, the anti-tumor activity of LY2801653 was evaluated in a panel of human patient derived xenograft models. LY2801653 displayed potent anti-tumor efficacy in a number of non small cell lung, renal, pancreatic, and breast tumor models. Examination of c-Met expression in these tumors by immunohistochemistry (IHC) revealed a good correlation between response and c-Met expression in the tumor tissue. Interestingly, the two non small cell lung adenocarcinoma models LXFA526 and LXFA1647 that were resistant to EGFR inhibitors Cetuximab and Erlotinib, displayed high sensitivity towards LY2801653. High level of genomic amplification of c-Met locus was found in these models, consistent with the hypothesis that c-Met activation represents one of the resistant mechanisms to EGFR inhibition. LY2801653 treatment led to increase in functional vessel areas, and decrease in tumor hypoxia. Enhanced anti-tumor efficacy was achieved when Erlotinib was combined with LY2801653. In summary, these results demonstrated the promising therapeutic potential of LY2801653 in mono and combination therapy of cancer. 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 3611.

Hydroxyamidine Inhibitors of Indoleamine-2,3-dioxygenase Potently Suppress Systemic Tryptophan Catabolism and the Growth of IDO-Expressing Tumors

Malignant tumors arise, in part, because the immune system does not adequately recognize and destroy them. Expression of indoleamine-2,3-dioxygenase (IDO; IDO1), a rate-limiting enzyme in the catabolism of tryptophan into kynurenine, contributes to this immune evasion. Here we describe the effects of systemic IDO inhibition using orally active hydroxyamidine small molecule inhibitors. A single dose of INCB023843 or INCB024360 results in efficient and durable suppression of Ido1 activity in the plasma of treated mice and dogs, the former to levels seen in Ido1-deficient mice. Hydroxyamidines potently suppress tryptophan metabolism in vitro in CT26 colon carcinoma and PAN02 pancreatic carcinoma cells and in vivo in tumors and their draining lymph nodes. Repeated administration of these IDO1 inhibitors impedes tumor growth in a dose- and lymphocyte-dependent fashion and is well tolerated in efficacy and preclinical toxicology studies. Substantiating the fundamental role of tumor cell-derived IDO expression, hydroxyamidines control the growth of IDO-expressing tumors in Ido1-deficient mice. These activities can be attributed, at least partially, to the increased immunoreactivity of lymphocytes found in tumors and their draining lymph nodes and to the reduction in tumor-associated regulatory T cells. INCB024360, a potent IDO1 inhibitor with desirable pharmaceutical properties, is poised to start clinical trials in cancer patients.

Novel therapeutic approaches to squamous cell carcinoma of the head and neck using biologically targeted agents

Despite significant improvements in the treatment and outcomes of patients with squamous cell carcinoma of the head and neck (SCCHN) that have resulted from technological advances in radiation delivery and the use of cytotoxic chemotherapy, there is still a pressing need for novel therapies. In the last two decades, our understanding of the molecular biological basis of cancer has provided us with a new framework for developing specific targeted therapies. It is likely that the next wave of developments will include active small molecule inhibitors of epidermal growth factor receptor (EGFR) (and other members of the c-erbB family of receptors), antiangiogenic agents, and drugs that can increase proapoptotic signaling in cancer cells. As with cetuximab, it is most likely that these new agents will first find a niche in the context of combination regimens with standard anticancer therapeutics.

Abstract B138: SB2312, a novel and potent dual inhibitor of mTOR and PI3K with high target inhibition and antitumor efficacy

Abstract Background: Dysregulation of the PI3K signaling pathway, either through activating mutations, amplification of PI3K or deletion of PTEN has been linked to a wide range of hyperproliferative diseases. The development of small molecule modulators of key proteins in this signaling cascade has focused mainly on mTOR or PI3K. Recent investigations have shown a significant overlap in the activity of small molecule inhibitors against these two proteins. Inhibitors that are currently progressing through preclinical and early clinical development have been shown to affect both mTOR and PI3K to some extent. However, most reported inhibitors do not inhibit both of these kinases in a close dose range. Our drug discovery program has focused on equipotent inhibition of both targets. SB2312 is a novel compound with very favorable drug-like properties, giving rise to equivalent targeting of both mTOR and PI3K in cells and tumor tissue and resulting in a more efficient inhibition of this pathway compared to reference compounds currently under evaluation in clinical trials. Methods: In vitro-IC50 for mTOR and PI3K were determined using recombinant enzymes. Inhibitory activity of SB2312 on the mTOR/PI3K signaling pathway was determined by Western-blot analysis, Surefire assays, and proliferation assays in various cancer cell lines. Efficacious doses of SB2312 or reference compounds were selected after pharmacokinetic and biomarker analysis of PC3 tumor-bearing mice, treated with single oral doses. Anti-tumor efficacy of SB2312 and reference compounds was evaluated in mouse xenograft models after continuous daily oral dosing. Results: SB2312 is a potent and selective dual mTOR and PI3K inhibitor (IC50: 55 nM and 18 nM, respectively) and has no significant activity on a large panel of protein kinases or enzymes and receptors. Efficient and equivalent inhibition of mTOR and PI3K in human cell lines was demonstrated by blockade of the phosphorylation of S6KT389 (IC50= 70 nM) and AKTS473 (IC50= 21 nM). SB2312 inhibits the proliferation of a broad range of human tumor cell lines independently of their PI3K pathway mutation status (IC50= 112–882 nM) and is also effective against rapamycin-resistant cell lines. SB2312 has very favorable physicochemical and pharmacokinetic properties, with high oral bioavailability and tumor exposure. Efficient and sustained inhibition of PI3K and mTOR was demonstrated in tumor tissues from a PC3 tumor xenograft model, along with dose-dependent anti-tumor activity at very well tolerated doses. Tumor growth inhibition was 53%, 105%, and 117% at 11, 33, and 80 mg/kg SB2312, respectively, without any body weight loss at any of these doses. Summary: Herein, we report SB2312, a novel, potent and selective dual mTOR/PI3K inhibitor with excellent drug-like properties. Our studies show superior and sustained blockade of the mTOR/PI3K pathway and high antitumor efficacy at well tolerated doses. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B138.

Abstract C194: In vivo characterization of AMG 900, an orally active small molecule inhibitor of aurora kinases in phase 1 clinical trials

Abstract Background: The aurora family of serine-threonine kinases (Aurora-A, -B, -C) regulate cell-cycle progression in mammalian cells. Aurora kinases A and B are essential for proper chromosome congression, segregation, and cytokinesis during mitosis, whereas aurora kinase C function appears restricted to male meiosis. Aurora kinase B is responsible for the direct phosphorylation of histone H3 on serine-10 (p-histone H3). Aurora kinase A and B expression is elevated in a variety of human cancers and is associated with advanced clinical staging and poor prognosis. The emergence of aurora kinases as key mitotic regulators and their potential role in tumorigenesis has focused substantial interest in developing selective small molecule inhibitors for the treatment of human cancers. AMG 900 is a novel ATP competitive small molecule inhibitor that is highly potent and selective for aurora kinases A, B, and C. Objective: The aim of this study was to characterize the in vivo effects of AMG 900 by establishing a pharmacokinetic-pharmacodynamic relationship and profiling its antitumor activity across multiple human xenografts models using a variety of dosing schedules. Results: In nude mice, oral administration of AMG 900 inhibited phosphorylation of histone H3 in tumors and bone marrow in a time- and dose-dependent manner. The degree of p-histone H3 suppression in cells correlated with plasma drug concentrations. The effect of AMG 900 was tested in a panel of human tumor xenograft models. Oral administration of AMG 900 at 15 mg/kg BID for two consecutive days per week or 3 mg/kg BID every day inhibited tumor growth (50–97% inhibition; p ≤ 0.0044) in all nine of the xenograft models tested compared with the vehicle control group. Importantly, three of these xenograft models are resistant to paclitaxel, docetaxel, and doxorubicin. Mice treated with efficacious doses of AMG 900 showed a transient loss of body weight (0–10%) and bone marrow cellularity, consistent with its on-mechanism effects on normal proliferating cells. Since aurora kinase inhibitors are known to cause neutropenia, studies were performed to investigate the effects of prophylactic treatment of PEGylated-G-CSF (SD/02). In mice, administration of SD/02 shortened the duration of AMG 900-induced neutropenia. Conclusion: Based on these preclinical activities, AMG 900 has the potential to treat advanced cancers, including tumors resistant to standard-of-care chemotherapeutic agents such as paclitaxel and docetaxel. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C194.

Abstract A261: In vitro characterization of AMG 900, an orally active small molecule inhibitor of aurora kinases in phase 1 clinical trials

Abstract Background: The aurora family of serine-threonine kinases (Aurora-A, -B, -C) regulate cell-cycle progression in mammalian cells. Aurora kinases A and B are essential for proper chromosome congression, segregation, and cytokinesis during mitosis, whereas aurora kinase C function appears restricted to male meiosis. Aurora kinase B is responsible for the direct phosphorylation of histone H3 on serine-10 (p-histone H3). Aurora kinase A and B expression is elevated in a variety of human cancers and is associated with advanced clinical staging and poor prognosis. The emergence of aurora kinases as key mitotic regulators and their potential role in tumorigenesis has focused substantial interest in developing selective small molecule inhibitors for the treatment of human cancers. Objective: The aim of this study was to characterize the in vitro effects of AMG 900 in tumor cell lines, including cell lines resistant to paclitaxel and three well-characterized aurora kinase inhibitors. Results: AMG 900 is a novel ATP competitive small molecule inhibitor that is highly potent and selective for aurora kinases A, B, and C. In cells, AMG 900 inhibits autophosphorylation of aurora kinases A and B as well as the phosphorylation of histone H3. The predominant cellular response of tumor cells to AMG 900 treatment is aborted cell division without a mitotic arrest, which leads to endoreduplication and cell death. The effect of AMG 900 was tested in a panel of 26 tumor cell lines to evaluate its potential to inhibit cell proliferation across multiple tumor types. AMG 900 inhibited the proliferation of tumor cell lines at low nanomolar concentrations (EC50 values 1–6 nM). Importantly, AMG 900 was equally potent against multidrugresistant (MDR) cell lines expressing ATP-binding cassette transporters (P-gp and BCRP1), whereas paclitaxel and three aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358) showed a loss of potency in these MDR-positive cell lines compared with matched parental cell lines. To further investigate potential mechanisms of resistance, an HCT116 cell line was adapted to grow in the presence of AZD1152, a selective inhibitor of aurora kinase B. The resulting AZD1152-resistant HCT116 subline harbors a mutation in one allele of the aurora kinase B gene (W221L substitution) and was negative for P-gp and BCRP1 expression. AMG 900 induced an accumulation of cells with ≥4N DNA content (EC50 value 5 nM) and inhibited colony formation in this AZD1152-resistant HCT116 subline. Conclusion: To our knowledge, AMG 900 is the first described inhibitor of aurora kinases A, B, and C that has uniform in vitro potency in MDR-positive tumor cell lines. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A261.

Potent and Orally Active Small-Molecule Inhibitors of the MDM2−p53 Interaction

We report herein the design of potent and orally active small-molecule inhibitors of the MDM2-p53 interaction. Compound 5 binds to MDM2 with a K(i) of 0.6 nM, activates p53 at concentrations as low as 40 nM, and potently and selectively inhibits cell growth in tumor cells with wild-type p53 over tumor cells with mutated/deleted p53. Compound 5 has a good oral bioavailability and effectively inhibits tumor growth in the SJSA-1 xenograft model.

Pharmacogenetics of telatinib, a VEGFR-2 and VEGFR-3 tyrosine kinase inhibitor, used in patients with solid tumors

Summary Purpose Telatinib is an orally active small-molecule tyrosine kinase inhibitor of kinase insert domain receptor (KDR; VEGFR-2) and fms-related tyrosine kinase 4 (FLT4; VEGFR-3). This study aims at the identification of relationships between single nucleotide polymorphisms (SNPs) in genes encoding for transporter proteins and pharmacokinetic parameters in order to clarify the significant interpatient variability in drug exposure. In addition, the potential relationship between target receptor polymorphisms and toxicity of telatinib is explored. Methods Blood samples from 33 patients enrolled in a phase I dose-escalation study of telatinib were analyzed. For correlation with dose normalized AUC(0–12), ATP-binding cassette (ABC) B1 (ABCB1), ABCC1, and ABCG2 were the genes selected. For correlation with telatinib toxicity, selected genes were the drug target genes KDR and FLT4. Results No association between dose normalized AUC(0–12) and drug transporter protein polymorphisms was observed. In addition, no association between toxicity and KDR or FLT4 genotype or haplotype was seen. Conclusions Our pharmacogenetic analysis could not reveal a correlation between relevant gene polymorphisms and clinical and pharmacokinetic observations of telatinib.

Virtual High Throughput Screening Using Combined Random Forest and Flexible Docking

We present here the random forest supervised machine learning algorithm applied to flexible docking results from five typical virtual high throughput screening (HTS) studies. Our approach is aimed at: i) reducing the number of compounds to be tested experimentally against the given protein target and ii) extending results of flexible docking experiments performed only on a subset of a chemical library in order to select promising inhibitors from the whole dataset. The random forest (RF) method is applied and tested here on compounds from the MDL drug data report (MDDR). The recall values for selected five diverse protein targets are over 90% and the performance reaches 100%. This machine learning method combined with flexible docking is capable to find 60% of the active compounds for most protein targets by docking only 10% of screened ligands. Therefore our in silico approach is able to scan very large databases rapidly in order to predict biological activity of small molecule inhibitors and provides an effective alternative for more computationally demanding methods in virtual HTS.

Small-Molecule Antagonists for CXCR2 and CXCR1 Inhibit Human Melanoma Growth by Decreasing Tumor Cell Proliferation, Survival, and Angiogenesis

Melanoma, the most aggressive form of skin cancer, accounts for 75% of all skin cancer-related deaths and current therapeutic strategies are not effective in advanced disease. In the current study, we have investigated the efficacy of orally active small-molecule antagonist targeting CXCR2/CXCR1. Human A375SM melanoma cells were treated with SCH-479833 or SCH-527123, and their effect on proliferation, motility, and invasion was evaluated in vitro. We examined the downstream signaling events in the cells following treatment with antagonists. For in vivo studies, A375SM cells were implanted subcutaneously into athymic nude mice followed by administration of SCH-479833, SCH-527123, or hydroxypropyl-beta-cyclodextrin (20%) orally for 21 days and their effect on tumor growth and angiogenesis was evaluated. Our data show that SCH-479833 or SCH-527123 inhibited the melanoma cell proliferation, chemotaxis, and invasive potential in vitro. Treatment of melanoma cells with SCH-479833 or SCH-527123 also inhibited tumor growth. Histologic and histochemical analyses showed significant (P < 0.05) decreases in tumor cell proliferation and microvessel density in tumors. Moreover, we observed a significant increase in melanoma cell apoptosis in SCH-479833- or SCH-527123-treated animals compared with controls. Together, these studies show that selectively targeting CXCR2/CXCR1 with orally active small-molecule inhibitors is a promising therapeutic approach for inhibiting melanoma growth and angiogenesis.