[Show abstract][Hide abstract] ABSTRACT: 1. 4-[2((1R,2R)-2-Hydroxycyclohexylamino)-benzothiazol-6-yloxyl]-pyridine-2-carboxylic acid methylamide (BLZ945) is a small molecule inhibitor of CSF-1R kinase activity within osteoclasts designed to prevent skeletal related events in metastatic disease. Key metabolites were enzymatically and structurally characterized to understand the metabolic fate of BLZ945 and pharmacological implications. The relative intrinsic clearances for metabolites were derived from in vitro studies using human hepatocytes, microsomes and phenotyped with recombinant P450 enzymes. 2. Formation of a pharmacologically active metabolite (M9) was observed in human hepatocytes. The M9 metabolite is a structural isomer (diastereomer) of BLZ945 and is about 4-fold less potent. This isomer was enzymatically formed via P450 oxidation of the BLZ945 hydroxyl group, followed by aldo–keto reduction to the alcohol (M9). 3. Two reaction phenotyping approaches based on fractional clearances were applied to BLZ945 using hepatocytes and liver microsomes. The fraction metabolized (fm) or contribution ratio was determined for each metabolic reaction type (oxidation, glucuronidation or isomerization) as well as for each metabolite. The results quantitatively illustrate contribution ratios of the involved enzymes and pathways, e.g. the isomerization to metabolite M9 accounted for 24% intrinsic clearance in human hepatocytes. In summary, contribution ratios for the Phase I and Phase II pathways can be determined in hepatocytes.
[Show abstract][Hide abstract] ABSTRACT: Activation of the phosphoinositide 3-kinase (PI3K) pathway occurs frequently in breast cancer. However, clinical results of single-agent PI3K inhibitors have been modest to date. A combinatorial drug screen on multiple PIK3CA mutant cancers with decreased sensitivity to PI3K inhibitors revealed that combined CDK 4/6-PI3K inhibition synergistically reduces cell viability. Laboratory studies revealed that sensitive cancers suppress RB phosphorylation upon treatment with single-agent PI3K inhibitors but cancers with reduced sensitivity fail to do so. Similarly, patients' tumors that responded to the PI3K inhibitor BYL719 demonstrated suppression of pRB, while nonresponding tumors showed sustained or increased levels of pRB. Importantly, the combination of PI3K and CDK 4/6 inhibitors overcomes intrinsic and adaptive resistance leading to tumor regressions in PIK3CA mutant xenografts.
[Show abstract][Hide abstract] ABSTRACT: Somatic PIK3CA mutations are frequently found in solid tumors raising the hypothesis that selective inhibition of PI3Kalpha may have robust efficacy in PIK3CA-mutant cancers while sparing patients the side-effects associated with broader inhibition of the class I PI3K family. Here we report the biological properties of the 2-aminothiazole derivative NVP-BYL719, a selective inhibitor of PI3Kalpha and its most common oncogenic mutant forms. The compound selectivity combined with excellent drug-like properties translates to dose- and time-dependent inhibition of PI3Kalpha signaling in vivo, resulting in robust therapeutic efficacy and tolerability in PIK3CA-dependent tumors. Novel targeted therapeutics such as NVP-BYL719, designed to modulate aberrant functions elicited by cancer specific genetic alterations upon which the disease depends, require well-defined patient stratification strategies in order to maximize their therapeutic impact and benefit for the patients. Here we also describe the application of the Cancer Cell Line Encyclopedia as a preclinical platform to refine the patient stratification strategy for NVP-BYL719 and found that PIK3CA mutation was the foremost positive predictor of sensitivity while revealing additional positive and negative associations such as PIK3CA amplification and PTEN mutation, respectively. These patient selection determinants are being assayed in the ongoing NVP-BYL719 clinical trials.
Molecular Cancer Therapeutics 03/2014; · 5.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PI3 kinases are a family of lipid kinases mediating numerous cell processes such as proliferation, migration and differentiation. The PI3 Kinase pathway is often de-regulated in cancer through PI3Kα overexpression, gene amplification, mutations and PTEN phosphatase deletion. PI3K inhibitors represent therefore an attractive therapeutic modality for cancer treatment. Herein we describe how the potency of a benzothiazole fragment hit was quickly improved based on structural information and how this early chemotype was further optimized through scaffold hopping. This effort led to the identification of a series of 2-acetamido-5-heteroaryl imidazopyridines showing potent in vitro activity against all class I PI3Ks and attractive pharmacokinetic properties.
[Show abstract][Hide abstract] ABSTRACT: Following the discovery of NVP-BEZ235, our first dual pan-PI3K/mTOR clinical compound, we sought to identify additional phosphoinositide 3-kinase (PI3K) inhibitors from different chemical classes with a different selectivity profile. The key to achieve these objectives was to couple a structure-based design approach with intensive pharmacologic evaluation of selected compounds during the medicinal chemistry optimization process. Here, we report on the biologic characterization of the 2-morpholino pyrimidine derivative pan-PI3K inhibitor NVP-BKM120. This compound inhibits all four class I PI3K isoforms in biochemical assays with at least 50-fold selectivity against other protein kinases. The compound is also active against the most common somatic PI3Kα mutations but does not significantly inhibit the related class III (Vps34) and class IV (mTOR, DNA-PK) PI3K kinases. Consistent with its mechanism of action, NVP-BKM120 decreases the cellular levels of p-Akt in mechanistic models and relevant tumor cell lines, as well as downstream effectors in a concentration-dependent and pathway-specific manner. Tested in a panel of 353 cell lines, NVP-BKM120 exhibited preferential inhibition of tumor cells bearing PIK3CA mutations, in contrast to either KRAS or PTEN mutant models. NVP-BKM120 shows dose-dependent in vivo pharmacodynamic activity as measured by significant inhibition of p-Akt and tumor growth inhibition in mechanistic xenograft models. NVP-BKM120 behaves synergistically when combined with either targeted agents such as MEK or HER2 inhibitors or with cytotoxic agents such as docetaxel or temozolomide. The pharmacological, biologic, and preclinical safety profile of NVP-BKM120 supports its clinical development and the compound is undergoing phase II clinical trials in patients with cancer.
Molecular Cancer Therapeutics 12/2011; 11(2):317-28. · 5.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phosphoinositide-3-kinases (PI3Ks) are important oncology targets due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein we describe the structure guided optimization of a series of 2-morpholino, 4-substituted, 6-heterocyclic pyrimidines where the pharmacokinetic properties were improved by modulating the electronics of the 6-position heterocycle, and the overall druglike properties were fine-tuned further by modification of the 4-position substituent. The resulting 2,4-bismorpholino 6-heterocyclic pyrimidines are potent class I PI3K inhibitors showing mechanism modulation in PI3K dependent cell lines and in vivo efficacy in tumor xenograft models with PI3K pathway deregulation (A2780 ovarian and U87MG glioma). These efforts culminated in the discovery of 15 (NVP-BKM120), currently in Phase II clinical trials for the treatment of cancer.Keywords: NVP-BKM120; phosphoinositide-3-kinase; PI3K/AKT3 pathway
[Show abstract][Hide abstract] ABSTRACT: This article addresses the issue of miscorrelation between hepatic injury biomarkers and histopathological findings in the drug development context. Our studies indicate that the use of toxicogenomics can aid in the drug development decision-making process associated with such miscorrelated data. BLZ945 was developed as a Colony-Stimulating Factor 1 Receptor (CSF-1R) inhibitor. Treatment of BLZ945 in rats and monkeys increased serum alanine aminotransferase (ALT) and aspartate transaminase (AST). However, liver hypertrophy was the only histopathological liver finding in rats, and there was no change in the livers of monkeys. Longer treatment of BLZ945 in rats for 6 weeks caused up to 6-fold elevation of ALT, yet hepatocyte necrosis was not detected microscopically. Toxicogenomic profiling of liver samples demonstrated that the genes associated with early response to liver injury, apoptosis/necrosis, inflammation, oxidative stress, and metabolic enzymes were upregulated. Studies are ongoing to evaluate the mechanisms underlying BL945-induced ALT and AST elevations.
International Journal of Toxicology 06/2011; 30(3):300-12. · 1.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although orphan drug applications required by the EMEA must include assessments of similarity to pre-existing products, these can be difficult to quantify. Here we illustrate a paradigm in comparing nilotinib to the prototype kinase inhibitor imatinib, and equate the degree of structural similarity to differences in properties. Nilotinib was discovered following re-engineering of imatinib, employing structural biology and medicinal chemistry strategies to optimise cellular potency and selectivity towards BCR-ABL1. Through evolving only to conserve these properties, this resulted in significant structural differences between nilotinib and imatinib, quantified by a Daylight-fingerprint-Tanimoto similarity coefficient of 0.6, with the meaning of this absolute measure being supported by an analysis of similarity distributions of similar drug-like molecules. This dissimilarity is reflected in the drugs having substantially different preclinical pharmacology and a lack of cross-intolerance in CML patients, which translates into nilotinib being an efficacious treatment for CML, with a favourable side-effect profile.
[Show abstract][Hide abstract] ABSTRACT: PI3 Kinases are a family of lipid kinases mediating numerous cell processes such as proliferation, migration, and differentiation. The PI3 kinase pathway is often de-regulated in cancer through PI3Kα overexpression, gene amplification, mutations, and PTEN phosphatase deletion. PI3K inhibitors represent therefore an attractive therapeutic modality for cancer treatment. Herein we describe a novel series of PI3K inhibitors sharing a pyrimidine core and showing significant potency against class I PI3 kinases in the biochemical assay and in cells. The discovery, synthesis and SAR of this chemotype are described.
[Show abstract][Hide abstract] ABSTRACT: The inhibition of key receptor tyrosine kinases (RTKs) that are implicated in tumor vasculature formation and maintenance, as well as tumor progression and metastasis, has been a major focus in oncology research over the last several years. Many potent small molecule inhibitors of vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) kinases have been evaluated. More recently, compounds that act through the complex inhibition of multiple kinase targets have been reported and may exhibit improved clinical efficacy. We report herein a series of potent, orally efficacious 4-amino-3-benzimidazol-2-ylhydroquinolin-2-one analogues as inhibitors of VEGF, PDGF, and fibroblast growth factor (FGF) receptor tyrosine kinases. Compounds in this class, such as 5 (TKI258), are reversible ATP-competitive inhibitors of VEGFR-2, FGFR-1, and PDGFRbeta with IC(50) values <0.1 microM. On the basis of its favorable in vitro and in vivo properties, compound 5 was selected for clinical evaluation and is currently in phase I clinical trials.
Journal of Medicinal Chemistry 12/2008; 52(2):278-92. · 5.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Herein are described a series of novel heterocyclic analogs of the 4-amino-3-benzimidazol-2-ylhydroquinolin-2-one scaffold. These compounds are potent inhibitors of receptor tyrosine kinases and exhibit favorable pharmacokinetic profiles. The synthesis and SAR of these compounds are described.
[Show abstract][Hide abstract] ABSTRACT: Fms-like tyrosine kinase 3 (FLT3) encodes a receptor tyrosine kinase (RTK) for which activating mutations have been identified in a proportion of acute myelogenous leukemia (AML) patients and associated with poor clinical prognosis. Given the relevance of FLT3 mutations in AML, we investigated the activity of CHIR-258, an orally active, multitargeted small molecule, with potent activity against FLT3 kinase and class III, IV, and V RTKs involved in endothelial and tumor cell proliferation in AML models.
CHIR-258 was tested on two human leukemic cell lines in vitro and in vivo with differing FLT3 mutational status [MV4;11 cells express FLT3 internal tandem duplications (ITD) versus RS4;11 cells with wild-type (WT) FLT3].
Antiproliferative activity of CHIR-258 against MV4;11 was approximately 24-fold greater compared with RS4;11, indicating more potent inhibition against cells with constitutively activated FLT3 ITD. Dose-dependent down modulation of receptor phosphorylation and downstream signaling [signal transducer and activator of transcription 5 (STAT5) and extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase] in MV4;11 cells with CHIR-258 confirmed the molecular mechanism of action. Target modulation of phospho-FLT3, phospho-STAT5, and phospho-ERK in MV4;11 tumors was achieved at biologically active doses of CHIR-258. Tumor regressions and eradication of AML cells from the bone marrow were shown in s.c. and bone marrow engraftment leukemic xenograft models. Tumor responses were characterized by decreased cellular proliferation and positive immunohistochemical staining for active caspase-3 and cleaved poly(ADP-ribose) polymerase, suggesting cell death was mediated in part via apoptosis.
Our data indicate that CHIR-258 may be an effective therapy in FLT3-associated AML and warrants clinical trials.
Clinical Cancer Research 08/2005; 11(14):5281-91. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The t(4;14) translocation that occurs uniquely in a subset (15%) of patients with multiple myeloma (MM) results in the ectopic expression of the receptor tyrosine kinase (RTK), fibroblast growth factor receptor 3 (FGFR3). Inhibition of activated FGFR3 in MM cells induces apoptosis, validating FGFR3 as a therapeutic target in t(4;14) MM and encouraging the clinical development of FGFR3 inhibitors for the treatment of these patients, who have a poor prognosis. We describe here the characterization of a novel, small-molecule inhibitor of class III, IV, and V RTKs, CHIR-258, as an inhibitor of FGFR3. CHIR-258 potently inhibits FGFR3 with an inhibitory concentration of 50% (IC50) of 5 nM in in vitro kinase assays and selectively inhibited the growth of B9 cells and human myeloma cell lines expressing wild-type (WT) or activated mutant FGFR3. In responsive cell lines, CHIR-258 induced cytostatic and cytotoxic effects. Importantly, addition of interleukin 6 (IL-6) or insulin growth factor 1 (IGF-1) or coculture on stroma did not confer resistance to CHIR-258. In primary myeloma cells from t(4;14) patients, CHIR-258 inhibited downstream extracellular signal-regulated kinase (ERK) 1/2 phosphorylation with an associated cytotoxic response. Finally, therapeutic efficacy of CHIR-258 was demonstrated in a xenograft mouse model of FGFR3 MM. These studies support the clinical evaluation of CHIR-258 in MM.
[Show abstract][Hide abstract] ABSTRACT: To evaluate whether beta-catenin signaling has a role in the regulation of angiogenesis in colon cancer, a series of angiogenesis-related gene promoters was analyzed for beta-catenin/TCF binding sites. Strikingly, the gene promoter of human vascular endothelial growth factor (VEGF, or VEGF-A) contains seven consensus binding sites for beta-catenin/TCF. Analysis of laser capture microdissected human colon cancer tissue indicated a direct correlation between up-regulation of VEGF-A expression and adenomatous polyposis coli (APC) mutational status (activation of beta-catenin signaling) in primary tumors. In metastases, this correlation was not observed. Analysis by immunohistochemistry of intestinal polyps in mice heterozygous for the multiple intestinal neoplasia gene (Min/+) at 5 months revealed an increase and redistribution of VEGF-A in proximity to those cells expressing nuclear beta-catenin with a corresponding increase in vessel density. Transfection of normal colon epithelial cells with activated beta-catenin up-regulated levels of VEGF-A mRNA and protein by 250-300%. When colon cancer cells with elevated beta-catenin levels were treated with beta-catenin antisense oligodeoxynucleotides, VEGF-A expression was reduced by more than 50%. Taken together, our observations indicate a close link between beta-catenin signaling and the regulation of VEGF-A expression in colon cancer.
Cancer Research 07/2003; 63(12):3145-53. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genetic studies have identified mutations in key regulators of the
Wnt/β-catenin pathway in a variety of cancers, most
frequently in colon cancers. However, whether the pathway is activated
in clinical cancer samples is not easily determined, and therefore it
is useful to find markers that could be surrogates to show activation
of the Wnt/β-catenin pathway. Gene expression profiles were
analyzed in SW620, a colon cancer cell line in which β-catenin levels
are stabilized as a consequence of truncated adenomatous polyposis coli
and were compared with profiles of the same cells transfected with
antisense oligodeoxynucleotides. Treatment of cells with β-catenin
antisense oligodeoxynucleotides resulted in a decrease in the levels of
axin2 and human naked cuticle (hnkd)
mRNAs. Interestingly, the proteins encoded by both of these mRNAs are
known inhibitors of the β-catenin pathway. In 30 human cell lines
derived from different origins, axin2 and
hnkd were expressed only in human colon cancer cell
lines that are known to have activating mutations in the
Wnt/β-catenin pathway. Further, levels of both
axin2 and hnkd mRNA were also found to be
elevated in about 65% of laser microdissected cells from human colon
tumors compared with laser microdissected cells of normal morphology
from the same patient samples. The increased expression of
axin2 and hnkd correlated with
truncations in adenomatous polyposis coli in the same patient samples.
These results reveal that it is possible to detect activation of a
carcinogenic pathway in human cancer samples with specific markers.
Proceedings of the National Academy of Sciences 12/2001; 98(26):14973-14978. · 9.81 Impact Factor