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RAS proteins are central in the proliferation of many types of cancer, but a general approach toward the identification of pan-mutant RAS inhibitors has remained unresolved. In this work, we describe the application of a binding pharmacophore identified from analysis of known RAS binding peptides to the design of novel peptides. Using a chemically divergent approach, we generated a library of small stapled peptides from which we identified compounds with weak binding activity. Exploration of structure-activity relationships (SARs) and optimization of these early compounds led to low-micromolar binders of KRAS that block nucleotide exchange.

A high-throughput screen (HTS) of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) resulted in several series of compounds with the potential for further optimization. Informatics was used to identify active chemotypes with lead-like profiles and remove compounds that commonly occurred as actives in other HTS screens. The activities were confirmed with IC50 measurements from two orthogonal assay technologies, and further analysis of the Hill slopes and comparison of the ratio of IC50 values at 10 times the enzyme concentration were used to identify artifact compounds. Several series of compounds were rejected as they had both high slopes and poor ratios. A small number of compounds representing the different leading series were assessed using isothermal titration calorimetry, and the X-ray crystal structure of the complex with PFKFB3 was solved. The orthogonal assay technology and isothermal calorimetry were demonstrated to be unreliable in identifying false-positive compounds in this case. Presented here is the discovery of the dihydropyrrolopyrimidinone series of compounds as active and novel inhibitors of PFKFB3, shown by X-ray crystallography to bind to the adenosine triphosphate site. The crystal structures of this series also reveal it is possible to flip the binding mode of the compounds, and the alternative orientation can be driven by a sigma-hole interaction between an aromatic chlorine atom and a backbone carbonyl oxygen. These novel inhibitors will enable studies to explore the role of PFKFB3 in driving the glycolytic phenotype of tumors.

The design of compounds that selectively inhibit a single kinase is a significant challenge, particularly for compounds that bind to the ATP site. We describe here how protein-ligand crystal structure information was able both to rationalize observed selectivity and to guide the design of more selective compounds. Inhibition data from enzyme and cellular screens and the crystal structures of a range of ligands tested during the process of identifying selective inhibitors of FGFR provide a step-by-step illustration of the process. Steric effects were exploited by increasing the size of ligands in specific regions in such a way as to be tolerated in the primary target and not in other related kinases. Kinases are an excellent target class to exploit such approaches because of the conserved fold and small side chain mobility of the active form.

Deregulation of the cell cycle has long been recognized as an essential driver of tumorigenesis, and agents that selectively target key cell cycle components continue to hold promise as potential therapeutics. We have developed AZD5438, a 4-(1-isopropyl-2-methylimidazol-5-yl)-2-(4-methylsulphonylanilino) pyrimidine, as a potent inhibitor of cyclin-dependent kinase (cdk) 1, 2, and 9 (IC50, 16, 6, and 20 nmol/L, respectively). In vitro, AZD5438 showed significant antiproliferative activity in human tumor cell lines (IC50 range, 0.2–1.7 μmol/L), causing inhibition of the phosphorylation of cdk substrates pRb, nucleolin, protein phosphatase 1a, and RNA polymerase II COOH-terminal domain and blocking cell cycling at G2-M, S, and G1 phases. In vivo, when orally administered at either 50 mg/kg twice daily or 75 mg/kg once daily, AZD5438 inhibited human tumor xenograft growth (maximum percentage tumor growth inhibition, range, 38–153; P < 0.05). In vivo, AZD5438 reduced the proportion of actively cycling cells. Further pharmacodynamic analysis of AZD5438-treated SW620 xenografts showed that efficacious doses of AZD5438 (>40% tumor growth inhibition) maintained suppression of biomarkers, such as phospho-pRbSer249/Thr252, for up to 16 hours following a single oral dose. A comparison of different schedules indicated that chronic daily oral dosing provided optimal cover to ensure antitumor efficacy. These data indicate that broad cdk inhibition may provide an effective method to impair the dysregulated cell cycle that drives tumorigenesis and AZD5438 has the pharmacologic profile that provides an ideal probe to test this premise. [Mol Cancer Ther 2009;8(7):1856–66]

Modification of imidazo[1,2-a]pyridine CDK inhibitors lead to identification of less lipophilic imidazo[1,2-b]pyridazine series of CDK inhibitors. Although several equivalent compounds from these two series have similar structure and show similar CDK activity, the SAR of the two series differs significantly. Protein inhibitor structure determination has confirmed differences in binding mode and given some understanding of these differences in SAR. Potent and selective imidazo[1,2-b]pyridazine inhibitors of CDK2 have been identified, which show >1 microM plasma levels following a 2mg/kg oral dose to mice.

The synthesis of analogues of tetrahydroquinoline angiotensin II antagonist, ZENECA ZD6888, bearing substituents on the biphenyl ring system is reported. Several of these compounds show comparable or superior activity to ZD6888 in an in vitro beinding assay and in inhibition of the AII-induced pressor response in normotensive rats.

Novel chiral sulfides, derived from pinene, have been prepared, converted to sulfonium salts and treated with base and aldehydes to generate non-racemic epoxides. Low to moderate enantioselectivities were obtained in the product epoxides and this possibly occurred as a result of using diastereomeric mixtures of sulfonium salts. Dibenzyl substituted sulfonium salts were also prepared to avoid formation of diastereomers but upon treatment with base and an aldehyde this gave the Stevens rearrangement product instead of epoxide.

IntroductionNon-small cell lung cancer (NSCLC) is characterized by a multitude of genetic aberrations with unknown clinical impact. In this study, we aimed to identify gene copy number changes that correlate with clinical outcome in NSCLC. To maximize the chance to identify clinically relevant events, we applied a strategy involving two prognostically extreme patient groups.MethodsShort-term (58 month; n = 47) were selected from a clinically well-characterized NSCLC patient cohort with available fresh frozen tumor specimens. The samples were analyzed using high-resolution single-nucleotide polymorphism array technology to assess gene copy number variations and array-based gene expression profiling. The molecular data were combined with information on clinical parameters.ResultsGenetic aberrations were strongly associated with tumor histology. In adenocarcinoma (n = 50), gene copy number gains on chromosome 8q21-q24.3 (177 genes) were more frequent in long-term than in short-term survivors. In squamous cell carcinoma (n = 28), gains on chromosome 14q23.1-24.3 (133 genes) were associated with shorter survival, whereas losses in a neighboring region, 14q31.1-32.33 (110 genes), correlated with favorable outcome. In accordance with copy number gains and losses, messenger RNA expression levels of corresponding genes were increased or decreased, respectively.ConclusionComprehensive tumor profiling permits the integration of genomic, histologic, and clinical data. We identified gene copy number gains and losses, with corresponding changes in messenger RNA levels that were associated with prognosis in adenocarcinoma and squamous cell carcinoma of the lung.

Novel chiral sulfides, derived from pinene, have been prepared, converted to sulfonium salts and treated with base and aldehydes to generate non-racemic epoxides. Low to moderate enantioselectivities were obtained in the product epoxides and this possibly occurred as a result of using diastereomeric mixtures of sulfonium salts. Dibenzyl substituted sulfonium salts were also prepared to avoid formation of diastereomers but upon treatment with base and an aldehyde this gave the Stevens rearrangement product instead of epoxide.

trans-1,3-Dithiane-1,3-dioxide reacts with high diastereoselectivity with aromatic aldehydes and the 1,3-dithiane-1,3-dioxide moiety can be easily converted to a thiolester without racemisation by carrying out a Pummerer reaction; the thiolester is a group that can be readily transformed into acids, esters, amides, ketones and aldehydes.

The relative configurations of 1,8-stereogenic centres can be controlled by coupling the tin(IV) chloride promoted reactions of aldehydes with 4-alkoxypent-2-enylstannanes, which proceed with excellent 1,5-induction, with a 2,3-Wittig rearrangement: this approach has been used to complete a diastereoselective synthesis of (±)-epipatulolide C 16 .

On the basis of an extension of the literature lead 1, a series of benzimidazoles have been synthesized and shown to be angiotensin II (AII) receptor antagonists. The structure-activity relationships of these new antagonists have been explored and the key binding interactions defined. Molecular mechanics calculations were carried out on analogues of imidazole AII antagonists and conformationally restricted analogues were synthesized. The benzimidazole antagonists displaced AII in binding studies in vitro with IC50 values in the range 10(-5)-10(-7) M and antagonized the hypertensive effects of AII in vivo (rats) following intravenous administration with ED50 values in the range of 5-20 mg/kg.

Background Technologies based on DNA microarrays have the potential to provide detailed information on genomic aberrations in tumor cells. In practice a major obstacle for quantitative detection of aberrations is the heterogeneity of clinical tumor tissue. Since tumor tissue invariably contains genetically normal stromal cells, this may lead to a failure to detect aberrations in the tumor cells. Principal Finding Using SNP array data from 44 non-small cell lung cancer samples we have developed a bioinformatic algorithm that accurately models the fractions of normal and tumor cells in clinical tumor samples. The proportion of normal cells in combination with SNP array data can be used to detect and quantify copy number neutral loss-of-heterozygosity (CNNLOH) in the tumor cells both in crude tumor tissue and in samples enriched for tumor cells by laser capture microdissection. Conclusion Genome-wide quantitative analysis of CNNLOH using the CNNLOH Quantifier method can help to identify recurrent aberrations contributing to tumor development in clinical tumor samples. In addition, SNP-array based analysis of CNNLOH may become important for detection of aberrations that can be used for diagnostic and prognostic purposes.

An imidazole series of cyclin-dependent kinase (CDK) inhibitors has been developed. Protein inhibitor structure determination has provided an understanding of the emerging structure activity trends for the imidazole series. The introduction of a methyl sulfone at the aniline terminus led to a more orally bioavailable CDK inhibitor that was progressed into clinical development.

Because the majority of cancers exhibit direct or indirect deregulation of cyclin-dependent kinase (CDK) function, members of the CDK family are attractive targets for the development of anticancer agents. As part of an ongoing program, novel imidazopyridines were identified and developed as potent and selective CDK inhibitors. Here, we describe data on the in vitro biological activities of one of these compounds, AZ703. The selectivity profile of AZ703 was investigated in kinase assays against a range of CDK enzymes as well as a panel of protein kinases in vitro. IC50s were assessed against different tumor cell lines in vitro. The mechanism of action of AZ703 was determined by observing changes in phosphorylation of CDK substrates and cell cycle effects on tumor and normal cells. In vitro studies revealed that AZ703 is a selective inhibitor of CDK1 and CDK2 and displays a mode of action consistent with the induction of G1-, S-, and G2-M-phase arrest. AZ703 also showed potent antiproliferative activity across a wide range of tumor cell lines in vitro. Moreover, AZ703 induced reversible blockade of normal cells while causing tumor cells to undergo apoptosis. We have identified AZ703 as a novel selective imidazo[1,2-a]pyridine CDK inhibitor that shows promising antitumor properties in vitro. [Mol Cancer Ther 2006;5(3):655–64]

Exploration of SAR and optimisation of the imidazo[1,2-a]pyridine CDK inhibitors has lead to the discovery of novel, potent and selective inhibitors of the cyclin-dependent kinase CDK2. Understanding of SAR has identified positions of substitution, which allow modification of physical properties and offer the potential for in vivo optimisation.

High-throughput screening identified the imidazo[1,2-a]pyridine and bisanilinopyrimidine series as inhibitors of the cyclin-dependent kinase CDK4. Comparison of their experimentally-determined binding modes and emerging structure-activity trends led to the development of potent and selective imidazo[1,2-a]pyridine inhibitors for CDK4 and in particular CDK2.

Progesterone is an important hormone in breast cancer; the paracrine mediator of the progesterone receptor, RANKL plays a critical role in cancer stem cells, metastasis and tumorigenesis. Steroidal inhibitors of PR have shown modest clinical activity but have been limited by toxicity. AZ’4425 is a novel, potent non-steroidal progesterone receptor (PR) antagonist which selectively inhibits PR compared to other steroid hormone receptors (∼ 1000-fold ER, MR, GR, AR). AZ’4425 competitively inhibits progesterone binding, thus preventing progesterone-induced phosphorylation of PR, nuclear translocation and PR-induced transcription with an IC50 of 26nM. Anchorage independent growth of T47D cells is similarly inhibited with an IC50 of 25nM. Using cancer stem cell (CSC) assays (mammosphere formation and ALDH positivity) we have shown broad cellular activity in both ER/PR positive tumor cell lines and early and late stage clinical samples. Furthermore, AZ’4425 prevents the increase in CSCs, and miRNA changes (miR221/2 and miR200c) induced by anti-estrogen therapies, suggesting a potential role in prevention of acquired resistance. In vivo, the PR antagonist inhibits transcription of progesterone-induced genes including RANKL and SGK1. Notably, AZ’4425 results in significant inhibition of new tumor formation and results in stasis and regression of DMBA-induced tumors, both as monotherapy and in combination with letrozole. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C135. Citation Format: Christine M. Chresta, Denis Alferez, Georgia Cerillo, Emma Still, Adina Hughes, Graeme Walker, Jane Kendrew, Claire Sadler, Jayne Harris, Iain Simpson, Andrew P. Thomas, Al Rabow, Robert Clarke, Sacha Howell, Graham Richmond. AZ’4425 is a potent, selective, and orally bio-available progesterone receptor antagonist that has shown anti-tumor activity and inhibition of cancer stem cell proliferation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C135.

There is increasing evidence that FGFR signaling plays an important role within human cancer, with members of FGFR family acting as driving oncogenes in a significant number of human tumors. Deregulation of FGFR-signaling has been documented within clinical samples of breast multiple myeloma, bladder, endometrial, gastric, squamous NSCLC and prostate cancers. This dysregulation most frequently occurs through gene amplification, or through genetically altered forms of FGFR proteins. This increasing body of evidence implicating FGFR signaling in cancer has provided rationale for the identification and testing of selective inhibitors of FGFR signaling in the clinic. In this presentation, we describe the progress of our FGFR tyrosine kinase inhibitor programme and report the discovery of N-(5-pyrazolyl)benzamide FGFR inhibitors. Early compounds in this series suffered from poor in vivo pharmacokinetic (PK) properties. The key site of metabolism was identified to be at a basic N-methyl group. This group was shown to be located in the solvent channel of the ATP binding site on binding to FGFR1, and modification could be made without causing major changes to intrinsic binding affinity. However, the first compounds identified with low metabolic clearance also showed a significant reduction in oral bioavailability, due to apparent low permeability and increased efflux potential. The characterization of these PK issues and the discovery of compounds which overcame them, through modulation of pKa, lipophilicity and masking of the polar groups, will be described. Leading compounds showed significant anti-tumor activity in xenograft tumors grown in mice. Detailed characterization of these compounds led to the identification of AZD4547, a potent and selective FGFR tyrosine kinase inhibitor currently in Phase I clinical studies. 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 3912. doi:1538-7445.AM2012-3912

The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signalling axis plays an important role in normal organ, vascular and skeletal development. Deregulation of FGFR signalling through genetic modification or over-expression of the receptors (or their ligands) has been observed in numerous tumour settings, whilst the FGF/FGFR axis also plays a key role in driving tumor angiogenesis. A growing body of data demonstrates that inhibition of FGFR signalling can result in anti-proliferative and/or pro-apoptotic effects, both in vitro and in vivo, thus confirming the validity of FGFR as a therapeutic target. AZD4547 is a small molecule inhibitor which competes with ATP for binding to FGF receptors 1, 2 and 3, thus inhibiting autophosphorylation and downstream signalling. Using in vitro kinase assays, we demonstrate that AZD4547 is a potent inhibitor of FGFR's 1,2 and 3 with IC50 values of 0.2, 2.5 and 1.8nM respectively. AZD4547 is also highly selective vs a panel of over 70 other kinases (no activity at 10μM) and importantly vs Kinase Insert Domain Receptor (KDR) (>120-fold window in cellular assays vs. FGFR2). In vitro treatment of a large panel of cell lines (>100) with AZD4547 resulted in varying anti-proliferative responses, the most potent of which were almost exclusively confined to those cell-lines in which FGFR signalling was de-regulated. Treatment of these sensitive cell lines with AZD4547 for 1hr, demonstrated potent inhibition of phosphorylation of FGF receptors and downstream substrates including p44/42 MAPK. The anti-proliferative effects of AZD4547 appear to be cell line dependent and vary between potent G1-phase cell cycle arrest and apoptotic induction. Chronic in vivo dosing of AZD4547 up to 12.5mg/kg qd was well tolerated and resulted in dose-dependent growth inhibition in a KMS11 tumour xenograft model. This in vivo anti-tumor activity is correlated with exposure to AZD4547 and associated pharmacodynamic modulation of phospho-FGFR. These data confirm the novelty of AZD4547 as a potent and selective agent for the therapeutic treatment of tumors with deregulated FGF/FGFR signalling. 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 3568. doi:10.1158/1538-7445.AM2011-3568

The relative configurations of 1,8-stereogenic centres can be controlled by coupling the tin(IV) chloride promoted reactions of aldehydes with 4-alkoxypent-2-enylstannanes, which proceed with excellent 1,5-induction, with a 2,3-Wittig rearrangement: this approach has been used to complete a diastereoselective synthesis of (±)-epipatulolide C 16 .

trans-1,3-Dithiane-1,3-dioxide reacts with high diastereoselectivity with aromatic aldehydes and the 1,3-dithiane-1,3-dioxide moiety can be easily converted to a thiolester without racemisation by carrying out a Pummerer reaction; the thiolester is a group that can be readily transformed into acids, esters, amides, ketones and aldehydes.