Abstract
We have recently reported the implementation of a structural biology-based drug design platform for the identification of protein kinase inhibitors which utilize hydrophobic clusters to ...stabilize the inactive state of a kinase. These clusters are not formed in active kinases and result in the conversion of the ATP binding cleft into a non-polar environment which is sterically and electrostatically incompatible with ATP binding. We have industrialized this approach, designated the ArQule Kinase Inhibitor Platform (AKIPTM), and synthesized a library of more than 15,000 small molecules through the application of an in silico guided process. This has allowed us to rapidly generate leads to a variety of kinases, including receptor tyrosine kinases, non-receptor tyrosine kinases, and serine-threonine kinases. By virtue of their unique binding modes, many of these inhibitors would not be identified in standard assays using highly activated kinases. We have therefore deliberately re-engineered standard biochemical kinase assays using unphosphorylated inactive kinases to aid in the optimization of these inhibitors. In addition to these biochemical assays, we have also implemented a variety of well-established technologies not commonly used early in the hit generation process, including a thermal shift assay (TSA), affinity mass spectrometry, endogenous tryptophan fluorescence detection, an ATP-exclusion assay using a non-hydrolyzable ATP analogue, classical kinetic analysis to assess ATP-dependence and mechanism-of-inhibition, X-ray crystallography, and finally, cross-competition experiments with known inhibitors. Using the cumulative knowledge gained from these technologies throughout the hit generation, hit-to-lead, and lead optimization stages has enabled us to make informed decisions and resulted in identification of many potent ATP-independent inhibitors. The AKIP technology to date has produced at least one clinical candidate, ARQ 092, which potently inhibits AKT with a high degree of selectivity amongst the human kinome. Further examples of the application of these various technologies will be provided for a diverse range of kinases, including c-Met, FGFR, Ack, and TNIK.
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 2914. doi:1538-7445.AM2012-2914
Abstract
ARQ 197 is a small molecule that inhibits c-MET kinase and is being evaluated clinically both as a monotherapy and in combination with other anti-cancer agents, including gemcitabine. ...Prompted by clinical observations, in vitro studies were conducted to explore the combination of ARQ 197 and gemcitabine with the goal of determining the most effective exposure regimen and to further refine the strategy of an ongoing phase 1b combination clinical trial. Gemcitabine and ARQ 197 both induce cell cycle arrest in cancer cells, but in different phases. Gemcitabine induces a G1/S-phase arrest, while ARQ 197 arrests cells in the G2/M phase. We hypothesized that the efficacy of combining these two agents might be enhanced by deliberate pulsatile treatment with ample time allowed for gemcitabine-treated cells to re-enter the cell cycle and to therefore potentially increase their vulnerability to ARQ 197-induced cell cycle arrest and apoptosis. A 7-day MTS cytotoxicity assay was employed in which the cells were seeded on Day 0, treated with gemcitabine for 24 h on Day 2, drug free medium added on Day 3, and ARQ 197 added on Day 5 for 24 h and then replaced with drug free medium. Cell viability was assayed with MTS reagent at the end of Day 7. To determine whether this pulsatile schedule was more advantageous then a regimen with no intervening “drug holiday”, control cultures were not replenished with drug free medium on Day 3 and ARQ 197 was added on Day 5. When the pulsatile schedule was employed, the combinatorial cytotoxicity of these two agents was significantly enhanced in several cancer cell lines (including NSCLC, bladder, pancreatic, breast, ovarian and uterine carcinomas). Mechanistic studies confirmed the G1/S-phase arrest of gemcitabine-treated cells, and G2/M arrest was observed following exposure to ARQ 197 in this regimen. Furthermore, this potentiation was also observed if ARQ 197 was exposed to the cancer cells as the first agent on Day 2 and gemcitabine added as the second agent on Day 5, as long as the pulsatile regimen was employed. These data suggest that modifications to dosing schedules of ARQ 197 and gemcitabine may improve the potential for synergistic anti-tumor activity in the clinic.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5376. doi:10.1158/1538-7445.AM2011-5376
A series of N-hydroxy-3-phenyl-2-propenamides were prepared as novel inhibitors of human histone deacetylase (HDAC). These compounds were potent enzyme inhibitors, having IC50s < 400 nM in a ...partially purified enzyme assay. However, potency in cell growth inhibition assays ranged over 2 orders of magnitude in two human carcinoma cell lines. Selected compounds having cellular IC50 < 750 nM were tested for maximum tolerated dose (MTD) and for efficacy in the HCT116 human colon tumor xenograft assay. Four compounds having an MTD ≥ 100 mg/kg were selected for dose−response studies in the HCT116 xenograft model. One compound, 9 (NVP-LAQ824), had significant dose-related activity in the HCT116 colon and A549 lung tumor models, high MTD, and low gross toxicity. On the basis, in part, of these properties, 9 has entered human clinical trials in 2002.
Three new halogenated monoterpenes, 2, 3, and 4, along with the known compounds halomon (1) and two analogues, 5 and 6, were isolated from the Madagascar red marine alga Portieria hornemannii. The ...structures of all three new compounds were determined by NMR spectroscopy in combination with mass spectrometric data analysis. Two of these monoterpenes (1 and 2) were low micromolar inhibitors of DNA methyl transferase-1.
Protein kinase inhibitors with enhanced selectivity can be designed by optimizing binding interactions with less conserved inactive conformations because such inhibitors will be less likely to ...compete with ATP for binding and therefore may be less impacted by high intracellular concentrations of ATP. Analysis of the ATP-binding cleft in a number of inactive protein kinases, particularly in the autoinhibited conformation, led to the identification of a previously undisclosed non-polar region in this cleft. This ATP-incompatible hydrophobic region is distinct from the previously characterized hydrophobic allosteric back pocket, as well as the main pocket. Generalized hypothetical models of inactive kinases were constructed and, for the work described here, we selected the fibroblast growth factor receptor (FGFR) tyrosine kinase family as a case study. Initial optimization of a FGFR2 inhibitor identified from a library of commercial compounds was guided using structural information from the model. We describe the inhibitory characteristics of this compound in biophysical, biochemical, and cell-based assays, and have characterized the binding mode using x-ray crystallographic studies. The results demonstrate, as expected, that these inhibitors prevent activation of the autoinhibited conformation, retain full inhibitory potency in the presence of physiological concentrations of ATP, and have favorable inhibitory activity in cancer cells. Given the widespread regulation of kinases by autoinhibitory mechanisms, the approach described herein provides a new paradigm for the discovery of inhibitors by targeting inactive conformations of protein kinases.
Abstract
The AKT family of serine-threonine kinases is a critical signal transduction node serving a variety of cellular functions including survival, proliferation, protein synthesis, and glucose ...metabolism. Small molecule inhibitors of AKT are emerging as potential targeted agents to treat cancers that exhibit aberrant AKT pathway signaling. A screening strategy was employed to identify inhibitors which utilize the intrinsic negative regulatory function of hydrophobic clusters in the ATP-binding cleft to inhibit the kinase activity of AKT1. ARQ 092 was identified through this screening paradigm following in vitro and in vivo optimization and was selected for further development. Crystallographic studies provided evidence that this mechanism of inhibition of AKT had been achieved. ARQ 092 binds to inactive, unphosphorylated AKT1 with sub-nanomolar affinity (Kd = 0.28 nM measured by surface plasma resonance) and biochemically inhibits all three isoforms (IC50 values of 3 nM, 4.5 nM, and 5.5 nM respectively for AKT1, AKT2, and AKT3). ARQ 092 displayed inhibition kinetics against AKT1 which were not affected by ATP concentrations up to 1000 M. When screened against a panel of over 300 kinases, ARQ 092 inhibited only three kinases within 100-fold of its IC50 against AKT1. ARQ 092 potently inhibited AKT phosphorylation (Ser473 & Thr308) in AN3CA human endometrial carcinoma cells (EC50 values of 39 nM and 61 nM, respectively for p-Ser473 & p-Thr308) and demonstrated concentration-dependent inhibition of phosphorylation of the downstream AKT substrate PRAS40. ARQ 092 inhibited growth of AN3CA cells (GI50 = 555 nM), A2780 cells (GI50 = 400 nM), and IGROV-1 cells (GI50 = 66 nM), in addition to LNCaP, ZR-75–1, and BT-474 human cancer cell lines (GI50 values ranging from 1 to 4 μM). Following a single oral dose of ARQ 092, inhibition of AKT and PRAS40 phosphorylation was documented in vivo in both AN3CA human endometrial tumor and BT474 human breast tumor xenograft models. The growth of AN3CA tumor xenografts was markedly suppressed after daily oral administration of ARQ 092 for 10 days. Finally, ARQ 092 was shown to have good pharmaceutical properties and was advanced into preclinical development.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A230.
Abstract
Fibroblast growth factor receptor (FGFR) tyrosine kinase family members have gained increasing attention as potential therapeutic targets. Gene amplification, or gain-of-function ...translocations and mutations of these receptors, have been implicated in a variety of cancers including breast, gastric, bladder, ovarian, endometrial, lymphoma and myeloma. We have previously described the biological profiling of ARQ 087, a potent small-molecule pan-FGFR kinase inhibitor that shows anti-tumor activity in FGFR dysregulated tumor cells in vitro and in vivo. A number of cell lines with genetically-altered FGF receptors were identified that were sensitive in anti-proliferative assays to ARQ 087, including FGFR2 amplified gastric carcinoma lines KATO III and SNU-16, and endometrial lines AN3CA, MFE-296 and MFE-280 harboring FGFR2 mutants, as well as a breast cancer cell line with increased FGFR1 gene copy (MDA MB-157). Western blot pharmacodynamic analysis demonstrates that ARQ 087 inhibits FGFR phosphorylation and downstream pathway markers in these cell lines in a concentration-dependent manner. Using antibody arrays specific for known cytokines as well as regulatory proteins involved in angiogenesis and apoptosis, we have identified a number of protein biomarkers in cell lysates and in conditioned media of sensitive cell lines that changed in response to ARQ 087 exposure. Three proteins (cytochrome c, clusterin, and HTRA2) decreased in SNU-16 (ARQ 087-sensitive, FGFR amplified) but not DLD1 (ARQ 087-resistant, non FGFR expressing) cells. VEGF decreased and IL-1rα increased in SNU-16 but not in DLD1 conditioned media in response to ARQ 087. Furthermore, analysis of mouse plasma from animals bearing AN3CA tumors treated with ARQ 087 revealed a decrease in human VEGF, CXCL16, MMP-9, Serpin E1 and MIF and an increase in RANTES, IL-1β, IP-10, SDF-1, IL-1rα. TNFα, GM-CSF, IL-2, sTREM-1, sICAM-1, MIP-1β, IL-8. In summary, we have identified a number of cell lines from a broad cell line screening campaign that exhibited dysregulated FGFR pathway function and are sensitive to the anti-proliferative effects of ARQ 087. These cell lines, in turn, have been used as models in which to identify candidate biomarkers that may greatly facilitate subsequent clinical evaluation of this molecularly targeted anti-tumor agent.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3571. doi:10.1158/1538-7445.AM2011-3571
Abstract
Herein we describe the implementation of a biochemical and biophysical screening strategy to discover small molecules that inhibit Akt through a mechanism distinct from ATP-competitive ...inhibitors. A series of novel derivatives of the core scaffold 3H-imidazo4,5-bpyridine were identified and optimized. These Akt inhibitors demonstrated potent inhibition of intracellular Akt and downstream targets including PRAS40 activation in vitro. Pharmacodynamic and pharmacokinetic studies in vivo demonstrated the effectiveness of the series at inhibiting the activation of Akt and an additional downstream effector (p70S6) following oral dosing in mice. Co-crystallization studies with un-phosphorylated Akt1 revealed that as a consequence of binding these novel, potent and selective, ATP-independent inhibitors the ATP binding cleft is occupied by non-polar residues which are associated as tight clusters. The cleft is closed with a ‘hydrophobic lock’ which may function to sterically exclude the binding of both ATP and ATP-competitive inhibitors.
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 LB-1. doi:1538-7445.AM2012-LB-1
Abstract
Utilization of hydrophobic motifs present in auto-inhibited protein kinases has resulted in the identification of a series of 5,6-dihydrobenzo hquinazolin-2-amines with activity as ...fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors. Herein we describe the combination of a proprietary in silico design process, a new screening paradigm using an array of biochemical and biophysical technologies in conjunction with an established parallel chemistry process for the identification and optimization of a series of novel FGFR inhibitors. These potent FGFR inhibitors exhibit a preference for the inactive form of the kinase, are non-ATP competitive, and exhibit robust cellular pharmacodynamic inhibition as well as in vitro anti-proliferative effects in cells dependent on FGFR and significant anti-tumor activity in appropriate xenograft models in vivo. The design strategy, synthesis, structure activity relationships and in vitro and in vivo biology of selected inhibitors will be presented.
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 3905. doi:1538-7445.AM2012-3905
Abstract
Background: ARQ 621 is an allosteric, potent and selective inhibitor of Eg5, a microtubule-based ATPase motor protein involved in cell division. Eg5 inhibition is recognized as a potential ...therapeutic strategy in cancer supported by the observation that over-expression of Eg5 causes genomic instability and tumor formation in mice. In this clinical trial we sought to evaluate the safety and pharmacokinetics of ARQ 621 in patients with solid tumors.
Methods: Patients (pts) are being enrolled into this multi-cohort phase 1 trial at the initial i.v. dose of 10 mg/m2/week. Drug is administered weekly intravenously over 1-2 hours. Cohorts of 3 or 6 patients are based on a 3+3 dose escalation schedule and dose is increased according to a modified Fibonacci scheme. Treatment continues until disease progression or unacceptable toxicity.
Results: Recently expanded preclinical data with ARQ 621 shows anti-tumor activity with potencies in the low nanomolar range across a much wider range of human cancer cell types than previously reported, including colon, NSCLC, gastric, and hematologic cancer cell lines. Furthermore, no evidence of bone marrow toxicity and DNA damage is evident with ARQ 621 as we have seen with a leading Eg5 inhibitor. As of November 19, 2009, 18 pts (60% male; median age 60 yrs, ECOG PS 0 (N=3), PS 1 (N=13), PS 2 (N=2) were enrolled. The most common tumors treated with ARQ 621 were colorectal (N=6) and breast (N=2). Treatment-emergent adverse events (TEAEs) were reported in 13 pts. The most common (> 10%) include fatigue (27%), redness at infusion site 3 (16%), and anemia 3 (16%). Three pts experienced serious AEs (1 each of anemia, sepsis, pneumonia) reported as not drug related. Three pts (cholangiosarcoma, liposarcoma and colorectal carcinoma) remained stable for > 4 months.
Conclusions: Preclinical studies continue to elucidate the potential indications for Eg5 inhibition as well as the combinability of ARQ 621 with other molecularly targeted therapeutics. To date, therapy with ARQ 621 appears well tolerated, with no dose-limiting toxicity observed at doses and frequencies much higher than those achieved with the leading Eg5 inhibitors of comparable potencies. Dose-limiting toxicity has not been reached, and recruitment continues to identify the maximum tolerated dose and a recommended Phase 2 dose. Additional safety, pharmacokinetic, and preliminary efficacy data will be available for presentation.
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 2750.
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