Melanie Valenti

The identification of 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) as a potent, selective, orally bioavailable inhibitor of class I PI3 kinase for the treatment of cancer

Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis, biological activity, and further profiling of these compounds are described. This work resulted in the discovery of 17, GDC-0941, which is a potent, selective, orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer.

NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

We describe the biological properties of NVP-AUY922, a novel resorcinylic isoxazole amide heat shock protein 90 (HSP90) inhibitor. NVP-AUY922 potently inhibits HSP90 (K(d) = 1.7 nmol/L) and proliferation of human tumor cells with GI(50) values of approximately 2 to 40 nmol/L, inducing G(1)-G(2) arrest and apoptosis. Activity is independent of NQO1/DT-diaphorase, maintained in drug-resistant cells and under hypoxic conditions. The molecular signature of HSP90 inhibition, comprising induced HSP72 and depleted client proteins, was readily demonstrable. NVP-AUY922 was glucuronidated less than previously described isoxazoles, yielding higher drug levels in human cancer cells and xenografts. Daily dosing of NVP-AUY922 (50 mg/kg i.p. or i.v.) to athymic mice generated peak tumor levels at least 100-fold above cellular GI(50). This produced statistically significant growth inhibition and/or regressions in human tumor xenografts with diverse oncogenic profiles: BT474 breast tumor treated/control, 21%; A2780 ovarian, 11%; U87MG glioblastoma, 7%; PC3 prostate, 37%; and WM266.4 melanoma, 31%. Therapeutic effects were concordant with changes in pharmacodynamic markers, including induction of HSP72 and depletion of ERBB2, CRAF, cyclin-dependent kinase 4, phospho-AKT/total AKT, and hypoxia-inducible factor-1alpha, determined by Western blot, electrochemiluminescent immunoassay, or immunohistochemistry. NVP-AUY922 also significantly inhibited tumor cell chemotaxis/invasion in vitro, WM266.4 melanoma lung metastases, and lymphatic metastases from orthotopically implanted PC3LN3 prostate carcinoma. NVP-AUY922 inhibited proliferation, chemomigration, and tubular differentiation of human endothelial cells and antiangiogenic activity was reflected in reduced microvessel density in tumor xenografts. Collectively, the data show that NVP-AUY922 is a potent, novel inhibitor of HSP90, acting via several processes (cytostasis, apoptosis, invasion, and angiogenesis) to inhibit tumor growth and metastasis. NVP-AUY922 has entered phase I clinical trials.

Pharmacologic Characterization of a Potent Inhibitor of Class I Phosphatidylinositide 3-Kinases

Extensive evidence implicates activation of the lipid phosphatidylinositide 3-kinase (PI3K) pathway in the genesis and progression of various human cancers. PI3K inhibitors thus have considerable potential as molecular cancer therapeutics. Here, we detail the pharmacologic properties of a prototype of a new series of inhibitors of class I PI3K. PI103 is a potent inhibitor with low IC50 values against recombinant PI3K isoforms p110alpha (2 nmol/L), p110beta (3 nmol/L), p110delta (3 nmol/L), and p110gamma (15 nmol/L). PI103 also inhibited TORC1 by 83.9% at 0.5 micromol/L and exhibited an IC50 of 14 nmol/L against DNA-PK. A high degree of selectivity for the PI3K family was shown by the lack of activity of PI103 in a panel of 70 protein kinases. PI103 potently inhibited proliferation and invasion of a wide variety of human cancer cells in vitro and showed biomarker modulation consistent with inhibition of PI3K signaling. PI103 was extensively metabolized, but distributed rapidly to tissues and tumors. This resulted in tumor growth delay in eight different human cancer xenograft models with various PI3K pathway abnormalities. Decreased phosphorylation of AKT was observed in U87MG gliomas, consistent with drug levels achieved. We also showed inhibition of invasion in orthotopic breast and ovarian cancer xenograft models and obtained evidence that PI103 has antiangiogenic potential. Despite its rapid in vivo metabolism, PI103 is a valuable tool compound for exploring the biological function of class I PI3K and importantly represents a lead for further optimization of this novel class of targeted molecular cancer therapeutic.

Biological properties of potent inhibitors of class I phosphatidylinositide 3-kinases: from PI-103 through PI-540, PI-620 to the oral agent GDC-0941

The phosphatidylinositide 3-kinase pathway is frequently deregulated in human cancers and inhibitors offer considerable therapeutic potential. We previously described the promising tricyclic pyridofuropyrimidine lead and chemical tool compound PI-103. We now report the properties of the pharmaceutically optimized bicyclic thienopyrimidine derivatives PI-540 and PI-620 and the resulting clinical development candidate GDC-0941. All four compounds inhibited phosphatidylinositide 3-kinase p110α with IC50 ≤ 10 nmol/L. Despite some differences in isoform selectivity, these agents exhibited similar in vitro antiproliferative properties to PI-103 in a panel of human cancer cell lines, with submicromolar potency in PTEN-negative U87MG human glioblastoma cells and comparable phosphatidylinositide 3-kinase pathway modulation. PI-540 and PI-620 exhibited improvements in solubility and metabolism with high tissue distribution in mice. Both compounds gave improved antitumor efficacy over PI-103, following i.p. dosing in U87MG glioblastoma tumor xenografts in athymic mice, with treated/control values of 34% (66% inhibition) and 27% (73% inhibition) for PI-540 (50 mg/kg b.i.d.) and PI-620 (25 mg/kg b.i.d.), respectively. GDC-0941 showed comparable in vitro antitumor activity to PI-103, PI-540, and PI-620 and exhibited 78% oral bioavailability in mice, with tumor exposure above 50% antiproliferative concentrations for >8 hours following 150 mg/kg p.o. and sustained phosphatidylinositide 3-kinase pathway inhibition. These properties led to excellent dose-dependent oral antitumor activity, with daily p.o. dosing at 150 mg/kg achieving 98% and 80% growth inhibition of U87MG glioblastoma and IGROV-1 ovarian cancer xenografts, respectively. Together, these data support the development of GDC-0941 as a potent, orally bioavailable inhibitor of phosphatidylinositide 3-kinase. GDC-0941 has recently entered phase I clinical trials. [Mol Cancer Ther 2009;8(7):1725–38] [Mol Cancer Ther 2009;8(7):1725–38]

Combining Hit Identification Strategies: Fragment- Based and in Silico Approaches to Orally Active 2-Aminothieno[2,3-D]Pyrimidine Inhibitors of the Hsp90 Molecular Chaperone.

Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential molecular therapeutic agents for the treatment of cancer. Here we describe novel 2-aminothieno[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors, which were designed by combining structural elements of distinct low affinity hits generated from fragment-based and in silico screening exercises in concert with structural information from X-ray protein crystallography. Examples from this series have high affinity (IC50 = 50-100 nM) for Hsp90 as measured in a fluorescence polarization (FP) competitive binding assay and are active in human cancer cell lines where they inhibit cell proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Several examples (34a, 34d and 34i) caused tumor growth regression at well tolerated doses when administered orally in a human BT474 human breast cancer xenograft model.

In vitro and In vivo Pharmacokinetic-Pharmacodynamic Relationships for the Trisubstituted Aminopurine Cyclin-Dependent Kinase Inhibitors Olomoucine, Bohemine and CYC202

Purpose: To investigate pharmacokinetic-pharmacodynamic relationships for the trisubstituted aminopurine cyclin-dependent kinase inhibitors olomoucine, bohemine, and CYC202 (R-roscovitine; seliciclib) in the HCT116 human colon carcinoma model. Experimental Design: The in vitro activity of the agents was determined in a human tumor panel using the sulforhodamine B assay. The concentration and time dependence was established in HCT116 cells. Molecular biomarkers, including RB phosphorylation and cyclin expression, were assessed by Western blotting. Pharmacokinetic properties were characterized in mice following analysis by liquid chromatography-tandem mass spectrometry. Based on these studies, a dosing regimen was developed for CYC202 that allowed therapeutic exposures in the HCT116 tumor xenograft. Results: The antitumor potency of the agents in vitro was in the order olomoucine (IC50, 56 μmol/L) < bohemine (IC50, 27 μmol/L) < CYC202 (IC50, 15 μmol/L), corresponding to their activities as cyclin-dependent kinase inhibitors. Antitumor activity increased with exposure time up to 16 hours. The agents caused inhibition of RB and RNA polymerase II phosphorylation and depletion of cyclins. They exhibited relatively rapid clearance following administration to mice. CYC202 displayed the slowest clearance from plasma and the highest tumor uptake, with oral bioavailability of 86%. Oral dosing of CYC202 gave active concentrations in the tumor, modulation of pharmacodynamic markers, and inhibition of tumor growth. Conclusions: CYC202 showed therapeutic activity on human cancer cell lines in vitro and on xenografts. Pharmacodynamic markers are altered in vitro and in vivo, consistent with the inhibition of cyclin-dependent kinases. Such markers may be potentially useful in the clinical development of CYC202 and other cyclin-dependent kinase inhibitors.

Benzoquinone ansamycin heat shock protein 90 inhibitors modulate multiple functions required for tumor angiogenesis

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in maintaining the correct conformation and stability of its client proteins. This study investigated the effects of Hsp90 inhibitors on client protein expression and key cellular functions required for tumor angiogenesis. The benzoquinone ansamycin Hsp90 inhibitors geldanamycin and/or its derivatives 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin inhibited production of vascular endothelial growth factor (VEGF)-A by tumor cells and blocked proliferative responses of human endothelial cells at nanomolar concentrations. 17-AAG also significantly reduced endothelial cell migration, tubular differentiation, invasion through Matrigel, and secretion of urokinase-type plasminogen activator at concentrations at or below those that inhibited proliferation. 17-AAG significantly reduced expression of VEGF receptor (VEGFR)-2 and established Hsp90 client proteins in human endothelial cells in vitro as well as in mouse vena cava, mesenteric vessels, and blood vessels within human tumor xenografts in vivo; this was associated with decreased tumor microvessel density. Finally, we showed for the first time that Hsp90 inhibitors also reduce expression of VEGFR-1 on human vascular endothelial cells, VEGFR-3 on lymphatic endothelial cells in vitro, and all three VEGFRs on mouse vasculature in vivo. Thus, we identify Hsp90 inhibitors as important regulators of many aspects of tumor angiogenesis (and potentially lymphangiogenesis) and suggest that they may provide therapeutic benefit not only via direct effects on tumor cells but also indirectly by inhibiting the production of angiogenic cytokines and responses of activated endothelial cells that contribute to tumor progression and metastasis.[Mol Cancer Ther 2006;5(3):522–32]

The Bcl-2/Bcl-XL family inhibitor ABT-737 sensitizes ovarian cancer cells to carboplatin.

Purpose: The effective treatment of ovarian cancer is hampered by the development of drug resistance, which may be mediated by members of the Bcl-2 family of apoptosis regulators. ABT-737 is a recently described inhibitor of members of this family. We investigated whether this compound could sensitize ovarian cancer cells to chemotherapeutic agents. Experimental Design: The sensitivity of ovarian cancer cell lines to ABT-737 in combination with either carboplatin or paclitaxel was tested either in vitro by assessing cell growth/survival and apoptosis or in xenograft studies. Results: As a single agent, ABT-737 inhibited the growth of eight ovarian cancer cell lines, although with relatively poor potency. However, ABT-737, but not a less active enantiomer, increased the sensitivity of several cell lines to carboplatin. The increased sensitivity to carboplatin was accompanied by a decrease in time at which apoptosis was observed when assessed according to the number of attached cells, PARP cleavage, and nucleosome formation. ABT-737 was more effective at sensitizing IGROV-1 cells when ABT-737 was administered after carboplatin. In addition, ABT-737 significantly enhanced the activity of carboplatin in one of three primary cultures derived directly from ascitic tumor cells in patients recently treated with chemotherapy. Small interfering RNA directed to Bcl-XL also increased the sensitivity of ovarian cancer cell lines to carboplatin. ABT-737 was also able to augment the inhibition of IGROV-1 tumor xenograft growth beyond that obtained with carboplatin alone. Conclusions: These data suggest that ABT-737, in combination with carboplatin, may find utility in the treatment of patients with ovarian cancer.

Dual blockade of the PI3K/AKT/mTOR (AZD8055) and RAS/MEK/ERK (AZD6244) pathways synergistically inhibits rhabdomyosarcoma cell growth in vitro and in vivo.

Purpose: To provide rationale for using phosphoinositide 3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathway inhibitors to treat rhabdomyosarcomas, a major cause of pediatric and adolescent cancer deaths. Experimental Design: The prevalence of PI3K/MAPK pathway activation in rhabdomyosarcoma clinical samples was assessed using immunohistochemistry. Compensatory signaling and cross-talk between PI3K/MAPK pathways was determined in rhabdomyosarcoma cell lines following p110α short hairpin RNA–mediated depletion. Pharmacologic inhibition of reprogrammed signaling in stable p110α knockdown lines was used to determine the target-inhibition profile inducing maximal growth inhibition. The in vitro and in vivo efficacy of inhibitors of TORC1/2 (AZD8055), MEK (AZD6244), and P13K/mTOR (NVP-BEZ235) was evaluated alone and in pairwise combinations. Results: PI3K pathway activation was seen in 82.5% rhabdomyosarcomas with coactivated MAPK in 36% and 46% of alveolar and embryonal subtypes, respectively. p110α knockdown in cell lines over the short and long term was associated with compensatory expression of other p110 isoforms, activation of the MAPK pathway, and cross-talk to reactivate the PI3K pathway. Combinations of PI3K pathway and MAP–ERK kinase (MEK) inhibitors synergistically inhibited cell growth in vitro. Treatment of RD cells with AZD8055 plus AZD6244 blocked reciprocal pathway activation, as evidenced by reduced AKT/ERK/S6 phosphorylation. In vivo, the synergistic effect on growth and changes in pharmacodynamic biomarkers was recapitulated using the AZD8055/AZD6244 combination but not NVP-BEZ235/AZD6244. Pharmacokinetic analysis provided evidence of drug–drug interaction with both combinations. Conclusions: Dual PI3K/MAPK pathway activation and compensatory signaling in both rhabdomyosarcoma subtypes predict a lack of clinical efficacy for single agents targeting either pathway, supporting a therapeutic strategy combining a TORC1/2 with a MEK inhibitor. Clin Cancer Res; 19(21); 5940–51. ©2013 AACR.

Small-Molecule Activation of p53 Blocks Hypoxia-Inducible Factor 1 alpha and Vascular Endothelial Growth Factor Expression In Vivo and Leads to Tumor Cell Apoptosis in Normoxia and Hypoxia

ABSTRACT The p53 tumor suppressor protein negatively regulates hypoxia-inducible factor 1α (HIF-1α). Here, we show that induction of p53 by the small-molecule RITA (reactivation of p53 and induction of tumor cell apoptosis) [2,5-bis(5-hydroxymethyl-2-thienyl) furan] (NSC-652287) inhibits HIF-1α and vascular endothelial growth factor expression in vivo and induces significant tumor cell apoptosis in normoxia and hypoxia in p53-positive cells. RITA has been proposed to stabilize p53 by inhibiting the p53-HDM2 interaction. However, induction of p53 alone was insufficient to block HIF-1α induced in hypoxia and has previously been shown to require additional stimuli, such as DNA damage. Here, we identify a new mechanism of action for RITA: RITA activates a DNA damage response, resulting in phosphorylation of p53 and γH2AX in vivo. Unlike other DNA damage response-inducing agents, RITA treatment of cells induced a p53-dependent increase in phosphorylation of the α subunit of eukaryotic initiation factor 2, requiring PKR-like endoplasmic reticulum kinase activity, and led to the subsequent downregulation of HIF-1α and p53 target proteins, including HDM2 and p21. Through the identification of a new mechanism of action for RITA, our study uncovers a novel link between the DNA damage response-p53 pathway and the protein translational machinery.