Publications by authors named "Florence I Raynaud"

81 Publications

Triplet Therapy with Palbociclib, Taselisib, and Fulvestrant in -Mutant Breast Cancer and Doublet Palbociclib and Taselisib in Pathway-Mutant Solid Cancers.

Cancer Discov 2020 Sep 21. Epub 2020 Sep 21.

Breast Unit, The Royal Marsden Hospital NHS Foundation Trust, London, United Kingdom.

Cyclin-dependent kinase 4/6 (CDK4/6) and PI3K inhibitors synergize in -mutant ER-positive HER2-negative breast cancer models. We conducted a phase Ib trial investigating the safety and efficacy of doublet CDK4/6 inhibitor palbociclib plus selective PI3K inhibitor taselisib in advanced solid tumors, and triplet palbociclib plus taselisib plus fulvestrant in 25 patients with -mutant, ER-positive HER2-negative advanced breast cancer. The triplet therapy response rate in mutant, ER-positive HER2-negative cancer was 37.5% [95% confidence interval (CI), 18.8-59.4]. Durable disease control was observed in -mutant ER-negative breast cancer and other solid tumors with doublet therapy. Both combinations were well tolerated at pharmacodynamically active doses. In the triplet group, high baseline cyclin E1 expression associated with shorter progression-free survival (PFS; HR = 4.2; 95% CI, 1.3-13.1; = 0.02). Early circulating tumor DNA (ctDNA) dynamics demonstrated high on-treatment ctDNA association with shorter PFS (HR = 5.2; 95% CI, 1.4-19.4; = 0.04). Longitudinal plasma ctDNA sequencing provided genomic evolution evidence during triplet therapy. SIGNIFICANCE: The triplet of palbociclib, taselisib, and fulvestrant has promising efficacy in patients with heavily pretreated -mutant ER-positive HER2-negative advanced breast cancer. A subset of patients with -mutant triple-negative breast cancer derived clinical benefit from palbociclib and taselisib doublet, suggesting a potential nonchemotherapy targeted approach for this population.
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http://dx.doi.org/10.1158/2159-8290.CD-20-0553DOI Listing
September 2020

First-in-Human Study of AT13148, a Dual ROCK-AKT Inhibitor in Patients with Solid Tumors.

Clin Cancer Res 2020 Sep 2;26(18):4777-4784. Epub 2020 Jul 2.

The Royal Marsden NHS Foundation Trust, London, United Kingdom.

Purpose: AT13148 is an oral AGC kinase inhibitor, which potently inhibits ROCK and AKT kinases. In preclinical models, AT13148 has been shown to have antimetastatic and antiproliferative activity.

Patients And Methods: The trial followed a rolling six design during dose escalation. An intrapatient dose escalation arm to evaluate tolerability and a biopsy cohort to study pharmacodynamic effects were later added. AT13148 was administered orally three days a week (Mon-Wed-Fri) in 28-day cycles. Pharmacokinetic profiles were assessed using mass spectrometry and pharmacodynamic studies included quantifying p-GSK3β levels in platelet-rich plasma (PRP) and p-cofilin and p-MLC2 levels in tumor biopsies.

Results: Fifty-one patients were treated on study. The safety of 5-300 mg of AT13148 was studied. Further, the doses of 120-180-240 mg were studied in an intrapatient dose escalation cohort. The dose-limiting toxicities included hypotension (300 mg), pneumonitis, and elevated liver enzymes (240 mg), and skin rash (180 mg). The most common side effects were fatigue, nausea, headaches, and hypotension. On the basis of tolerability, 180 mg was considered the maximally tolerated dose. At 180 mg, mean and AUC were 400 nmol/L and 13,000 nmol/L/hour, respectively. At 180 mg, ≥50% reduction of p-cofilin was observed in 3 of 8 posttreatment biopsies.

Conclusions: AT13148 was the first dual potent ROCK-AKT inhibitor to be investigated for the treatment of solid tumors. The narrow therapeutic index and the pharmacokinetic profile led to recommend not developing this compound further. There are significant lessons learned in designing and testing agents that simultaneously inhibit multiple kinases including AGC kinases in cancer.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-0700DOI Listing
September 2020

Phase I Trial of the PARP Inhibitor Olaparib and AKT Inhibitor Capivasertib in Patients with - and Non--Mutant Cancers.

Cancer Discov 2020 Oct 12;10(10):1528-1543. Epub 2020 Jun 12.

Royal Marsden Hospital, London, United Kingdom.

Preclinical studies have demonstrated synergy between PARP and PI3K/AKT pathway inhibitors in and (-deficient and -proficient tumors. We conducted an investigator-initiated phase I trial utilizing a prospective intrapatient dose- escalation design to assess two schedules of capivasertib (AKT inhibitor) with olaparib (PARP inhibitor) in 64 patients with advanced solid tumors. Dose expansions enrolled germline -mutant tumors, or wild-type cancers harboring somatic DNA damage response (DDR) or PI3K-AKT pathway alterations. The combination was well tolerated. Recommended phase II doses for the two schedules were: olaparib 300 mg twice a day with either capivasertib 400 mg twice a day 4 days on, 3 days off, or capivasertib 640 mg twice a day 2 days on, 5 days off. Pharmacokinetics were dose proportional. Pharmacodynamic studies confirmed phosphorylated (p) GSK3β suppression, increased pERK, and decreased BRCA1 expression. Twenty-five (44.6%) of 56 evaluable patients achieved clinical benefit (RECIST complete response/partial response or stable disease ≥ 4 months), including patients with tumors harboring germline mutations and wild-type cancers with or without DDR and PI3K-AKT pathway alterations. SIGNIFICANCE: In the first trial to combine PARP and AKT inhibitors, a prospective intrapatient dose- escalation design demonstrated safety, tolerability, and pharmacokinetic-pharmacodynamic activity and assessed predictive biomarkers of response/resistance. Antitumor activity was observed in patients harboring tumors with germline mutations and wild-type cancers with or without somatic DDR and/or PI3K-AKT pathway alterations..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0163DOI Listing
October 2020

Metabolomic changes of the multi (-AGC-) kinase inhibitor AT13148 in cells, mice and patients are associated with NOS regulation.

Metabolomics 2020 04 13;16(4):50. Epub 2020 Apr 13.

Division of Cancer Therapeutics, The Institute of Cancer Research, London, SW7 3RP, UK.

Introduction: To generate biomarkers of target engagement or predictive response for multi-target drugs is challenging. One such compound is the multi-AGC kinase inhibitor AT13148. Metabolic signatures of selective signal transduction inhibitors identified in preclinical models have previously been confirmed in early clinical studies. This study explores whether metabolic signatures could be used as biomarkers for the multi-AGC kinase inhibitor AT13148.

Objectives: To identify metabolomic changes of biomarkers of multi-AGC kinase inhibitor AT13148 in cells, xenograft / mouse models and in patients in a Phase I clinical study.

Methods: HILIC LC-MS/MS methods and Biocrates AbsoluteIDQ™ p180 kit were used for targeted metabolomics; followed by multivariate data analysis in SIMCA and statistical analysis in Graphpad. Metaboanalyst and String were used for network analysis.

Results: BT474 and PC3 cells treated with AT13148 affected metabolites which are in a gene protein metabolite network associated with Nitric oxide synthases (NOS). In mice bearing the human tumour xenografts BT474 and PC3, AT13148 treatment did not produce a common robust tumour specific metabolite change. However, AT13148 treatment of non-tumour bearing mice revealed 45 metabolites that were different from non-treated mice. These changes were also observed in patients at doses where biomarker modulation was observed. Further network analysis of these metabolites indicated enrichment for genes associated with the NOS pathway. The impact of AT13148 on the metabolite changes and the involvement of NOS-AT13148- Asymmetric dimethylarginine (ADMA) interaction were consistent with hypotension observed in patients in higher dose cohorts (160-300 mg).

Conclusion: AT13148 affects metabolites associated with NOS in cells, mice and patients which is consistent with the clinical dose-limiting hypotension.
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http://dx.doi.org/10.1007/s11306-020-01676-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7154022PMC
April 2020

Quizartinib-resistant FLT3-ITD acute myeloid leukemia cells are sensitive to the FLT3-Aurora kinase inhibitor CCT241736.

Blood Adv 2020 04;4(7):1478-1491

Cancer Research UK Cancer Therapeutics Unit, Division of Cancer Therapeutics, The Institute of Cancer Research, Sutton, United Kingdom.

Internal tandem duplication of FLT3 (FLT3-ITD) is one of the most common somatic mutations in acute myeloid leukemia (AML); it causes constitutive activation of FLT3 kinase and is associated with high relapse rates and poor survival. Small-molecule inhibition of FLT3 represents an attractive therapeutic strategy for this subtype of AML, although resistance from secondary FLT3 tyrosine kinase domain (FLT3-TKD) mutations is an emerging clinical problem. CCT241736 is an orally bioavailable, selective, and potent dual inhibitor of FLT3 and Aurora kinases. FLT3-ITD+ cells with secondary FLT3-TKD mutations have high in vitro relative resistance to the FLT3 inhibitors quizartinib and sorafenib, but not to CCT241736. The mechanism of action of CCT241736 results in significant in vivo efficacy, with inhibition of tumor growth observed in efficacy studies in FLT3-ITD and FLT3-ITD-TKD human tumor xenograft models. The efficacy of CCT241736 was also confirmed in primary samples from AML patients, including those with quizartinib-resistant disease, which induces apoptosis through inhibition of both FLT3 and Aurora kinases. The unique combination of CCT241736 properties based on robust potency, dual selectivity, and significant in vivo activity indicate that CCT241736 is a bona fide clinical drug candidate for FLT3-ITD and TKD AML patients with resistance to current drugs.
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http://dx.doi.org/10.1182/bloodadvances.2019000986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160287PMC
April 2020

Achieving Target Depletion through the Discovery and Optimization of Benzimidazolone BCL6 Degraders.

J Med Chem 2020 04 10;63(8):4047-4068. Epub 2020 Apr 10.

Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein-protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3,5)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2-benzo[]imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing.
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http://dx.doi.org/10.1021/acs.jmedchem.9b02076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184563PMC
April 2020

International Ring Trial of a High Resolution Targeted Metabolomics and Lipidomics Platform for Serum and Plasma Analysis.

Anal Chem 2019 11 8;91(22):14407-14416. Epub 2019 Nov 8.

Duke Proteomics and Metabolomics Shared Resource , Duke School of Medicine , 701 W. Main Street , Durham , North Carolina 27701 , United States.

A challenge facing metabolomics in the analysis of large human cohorts is the cross-laboratory comparability of quantitative metabolomics measurements. In this study, 14 laboratories analyzed various blood specimens using a common experimental protocol provided with the Biocrates AbsoluteIDQ p400HR kit, to quantify up to 408 metabolites. The specimens included human plasma and serum from male and female donors, mouse and rat plasma, as well as NIST SRM 1950 reference plasma. The metabolite classes covered range from polar (e.g., amino acids and biogenic amines) to nonpolar (e.g., diacyl- and triacyl-glycerols), and they span 11 common metabolite classes. The manuscript describes a strict system suitability testing (SST) criteria used to evaluate each laboratory's readiness to perform the assay, and provides the SST Skyline documents for public dissemination. The study found approximately 250 metabolites were routinely quantified in the sample types tested, using Orbitrap instruments. Interlaboratory variance for the NIST SRM-1950 has a median of 10% for amino acids, 24% for biogenic amines, 38% for acylcarnitines, 25% for glycerolipids, 23% for glycerophospholipids, 16% for cholesteryl esters, 15% for sphingolipids, and 9% for hexoses. Comparing to consensus values for NIST SRM-1950, nearly 80% of comparable analytes demonstrated bias of <50% from the reference value. The findings of this study result in recommendations of best practices for system suitability, quality control, and calibration. We demonstrate that with appropriate controls, high-resolution metabolomics can provide accurate results with good precision across laboratories, and the p400HR therefore is a reliable approach for generating consistent and comparable metabolomics data.
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http://dx.doi.org/10.1021/acs.analchem.9b02908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310668PMC
November 2019

High Proliferation Rate and a Compromised Spindle Assembly Checkpoint Confers Sensitivity to the MPS1 Inhibitor BOS172722 in Triple-Negative Breast Cancers.

Mol Cancer Ther 2019 10;18(10):1696-1707

Cancer Research UK Cancer Therapeutics Unit, Division of Cancer Therapeutics, The Institute of Cancer Research, London, United Kingdom.

BOS172722 (CCT289346) is a highly potent, selective, and orally bioavailable inhibitor of spindle assembly checkpoint kinase MPS1. BOS172722 treatment alone induces significant sensitization to death, particularly in highly proliferative triple-negative breast cancer (TNBC) cell lines with compromised spindle assembly checkpoint activity. BOS172722 synergizes with paclitaxel to induce gross chromosomal segregation defects caused by MPS1 inhibitor-mediated abrogation of the mitotic delay induced by paclitaxel treatment. In pharmacodynamic experiments, BOS172722 potently inhibits the spindle assembly checkpoint induced by paclitaxel in human tumor xenograft models of TNBC, as measured by inhibition of the phosphorylation of histone H3 and the phosphorylation of the MPS1 substrate, KNL1. This mechanistic synergy results in significant efficacy, with robust tumor regressions observed for the combination of BOS172722 and paclitaxel versus either agent alone in long-term efficacy studies in multiple human tumor xenograft TNBC models, including a patient-derived xenograft and a systemic metastasis model. The current target indication for BOS172722 is TNBC, based on their high sensitivity to MPS1 inhibition, the well-defined clinical patient population with high unmet need, and the synergy observed with paclitaxel.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-1203DOI Listing
October 2019

phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy.

Autophagy 2020 06 13;16(6):1044-1060. Epub 2019 Sep 13.

Cancer Research UK Cancer Imaging Centre, Division of Radiotherapy and Imaging, The Institute of Cancer Research London and Royal Marsden Hospital , London, UK.

Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using C-labeled choline and C-magnetic resonance spectroscopy and western blotting, we show increased choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues.

Abbreviations: AKT: AKT serine/threonine kinase; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; ChoPL: choline phospholipid; CHKA: choline kinase alpha; CHPT1: choline phosphotransferase 1; CTCF: corrected total cell fluorescence; CTP: cytidine-5'-triphosphate; DCA: dichloroacetate; DMEM: dulbeccos modified Eagles medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; ER: endoplasmic reticulum; GDPD5: glycerophosphodiester phosphodiesterase domain containing 5; GFP: green fluorescent protein; GPC: glycerophosphorylcholine; HBSS: hanks balances salt solution; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPCAT1: lysophosphatidylcholine acyltransferase 1; LysoPtdCho: lysophosphatidylcholine; MRS: magnetic resonance spectroscopy; MTORC1: mechanistic target of rapamycin kinase complex 1; PCho: phosphocholine; PCYT: choline phosphate cytidylyltransferase; PLA2: phospholipase A2; PLB: phospholipase B; PLC: phospholipase C; PLD: phospholipase D; PCYT1A: phosphate cytidylyltransferase 1, choline, alpha; PI3K: phosphoinositide-3-kinase; pMAFs: pancreatic mouse adult fibroblasts; PNPLA6: patatin like phospholipase domain containing 6; Pro-Cho: propargylcholine; Pro-ChoPLs: propargylcholine phospholipids; PtdCho: phosphatidylcholine; PtdEth: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RPS6: ribosomal protein S6; SCD: stearoyl-CoA desaturase; SEM: standard error of the mean; SM: sphingomyelin; SMPD1/SMase: sphingomyelin phosphodiesterase 1, acid lysosomal; SGMS: sphingomyelin synthase; WT: wild-type.
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http://dx.doi.org/10.1080/15548627.2019.1659608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7469489PMC
June 2020

Differences in Signaling Patterns on PI3K Inhibition Reveal Context Specificity in -Mutant Cancers.

Mol Cancer Ther 2019 08 1;18(8):1396-1404. Epub 2019 Jul 1.

Division of Cancer Therapeutics, The Institute of Cancer Research, London, United Kingdom.

It is increasingly appreciated that drug response to different cancers driven by the same oncogene is different and may relate to differences in rewiring of signal transduction. We aimed to study differences in dynamic signaling changes within mutant (), non-small cell lung cancer (NSCLC), colorectal cancer, and pancreatic ductal adenocarcinoma (PDAC) cells. We used an antibody-based phosphoproteomic platform to study changes in 50 phosphoproteins caused by seven targeted anticancer drugs in a panel of 30 cell lines and cancer cells isolated from 10 patients with cancers. We report for the first time significant differences in dynamic signaling between colorectal cancer and NSCLC cell lines exposed to clinically relevant equimolar concentrations of the pan-PI3K inhibitor pictilisib including a lack of reduction of p-AKTser473 in colorectal cancer cell lines ( = 0.037) and lack of compensatory increase in p-MEK in NSCLC cell lines ( = 0.036). Differences in rewiring of signal transduction between tumor types driven by cancers exist and influence response to combination therapy using targeted agents.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679718PMC
August 2019

C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-ones: Studies towards the identification of potent, cell penetrant Jumonji C domain containing histone lysine demethylase 4 subfamily (KDM4) inhibitors, compound profiling in cell-based target engagement assays.

Eur J Med Chem 2019 Sep 17;177:316-337. Epub 2019 May 17.

Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London, UK. Electronic address:

Residues in the histone substrate binding sites that differ between the KDM4 and KDM5 subfamilies were identified. Subsequently, a C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one series was designed to rationally exploit these residue differences between the histone substrate binding sites in order to improve affinity for the KDM4-subfamily over KDM5-subfamily enzymes. In particular, residues E169 and V313 (KDM4A numbering) were targeted. Additionally, conformational restriction of the flexible pyridopyrimidinone C8-substituent was investigated. These approaches yielded potent and cell-penetrant dual KDM4/5-subfamily inhibitors including 19a (KDM4A and KDM5B Ki = 0.004 and 0.007 μM, respectively). Compound cellular profiling in two orthogonal target engagement assays revealed a significant reduction from biochemical to cell-based activity across multiple analogues; this decrease was shown to be consistent with 2OG competition, and suggests that sub-nanomolar biochemical potency will be required with C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one compounds to achieve sub-micromolar target inhibition in cells.
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http://dx.doi.org/10.1016/j.ejmech.2019.05.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580095PMC
September 2019

Metabolism of the dual FLT-3/Aurora kinase inhibitor CCT241736 in preclinical and human in vitro models: Implication for the choice of toxicology species.

Eur J Pharm Sci 2019 Nov 3;139:104899. Epub 2019 Apr 3.

Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London, United Kingdom. Electronic address:

CCT241736 is a dual fms-like tyrosine kinase 3 (FLT3)/Aurora kinase inhibitor in development for the treatment of acute myeloid leukaemia. The successful development of any new drug relies on adequate safety testing including preclinical toxicology studies. Selection of an appropriate preclinical species requires a thorough understanding of the compound's metabolic clearance and pathways, as well as other pharmacokinetic and pharmacodynamic considerations. In addition, elucidation of the metabolising enzymes in human facilitates improved clinical prediction based on population pharmacokinetics and can inform drug-drug interaction studies. Intrinsic clearance (CL) determination and metabolite profiling of CCT241736 in human and four preclinical species (dog, minipig, rat and mouse) was undertaken in cryopreserved hepatocytes and liver microsomes. Recombinant human cytochrome P450 bactosomes (rCYP) were utilised to provide reaction phenotyping data and support prediction of metabolic pathways. CCT241736 exhibited low CL in both hepatocytes and liver microsomes of human, dog, minipig and rat, but considerably higher CL in mouse. CYP3A4 and CYP3A5 were identified as the major enzymes responsible for biotransformation of CCT241736 in human, exclusively forming five out of seven metabolites. Minipig showed greatest similarity to human with regard to both overall metabolic profile and abundance of specific metabolites relative to parent compound, and is therefore proposed as the most appropriate toxicological species. The greatest disparity was observed between human and dog. Based on metabolic profile, either mouse or rat is a suitable rodent species for toxicology studies.
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http://dx.doi.org/10.1016/j.ejps.2019.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892276PMC
November 2019

Effect of acute total sleep deprivation on plasma melatonin, cortisol and metabolite rhythms in females.

Eur J Neurosci 2020 01 2;51(1):366-378. Epub 2019 May 2.

Chronobiology, Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK.

Disruption to sleep and circadian rhythms can impact on metabolism. The study aimed to investigate the effect of acute sleep deprivation on plasma melatonin, cortisol and metabolites, to increase understanding of the metabolic pathways involved in sleep/wake regulation processes. Twelve healthy young female participants remained in controlled laboratory conditions for ~92 hr with respect to posture, meals and environmental light (18:00-23:00 hr and 07:00-09:00 hr <8 lux; 23:00-07:00 hr 0 lux (sleep opportunity) or <8 lux (continuous wakefulness); 09:00-18:00 hr ~90 lux). Regular blood samples were collected for 70 hr for plasma melatonin and cortisol, and targeted liquid chromatography-mass spectrometry metabolomics. Timepoints between 00:00 and 06:00 hr for day 1 (baseline sleep), day 2 (sleep deprivation) and day 3 (recovery sleep) were analysed. Cosinor analysis and MetaCycle analysis were performed for detection of rhythmicity. Night-time melatonin levels were significantly increased during sleep deprivation and returned to baseline levels during recovery sleep. No significant differences were observed in cortisol levels. Of 130 plasma metabolites quantified, 41 metabolites were significantly altered across the study nights, with the majority decreasing during sleep deprivation, most notably phosphatidylcholines. In cosinor analysis, 58 metabolites maintained their rhythmicity across the study days, with the majority showing a phase advance during acute sleep deprivation. This observation differs to that previously reported for males. Our study is the first of metabolic profiling in females during sleep deprivation and recovery sleep, and offers a novel view of human sleep/wake regulation and sex differences.
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http://dx.doi.org/10.1111/ejn.14411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7027445PMC
January 2020

Introduction of a Methyl Group Curbs Metabolism of Pyrido[3,4- d]pyrimidine Monopolar Spindle 1 (MPS1) Inhibitors and Enables the Discovery of the Phase 1 Clinical Candidate N-(2-Ethoxy-4-(4-methyl-4 H-1,2,4-triazol-3-yl)phenyl)-6-methyl- N-neopentylpyrido[3,4- d]pyrimidine-2,8-diamine (BOS172722).

J Med Chem 2018 09 10;61(18):8226-8240. Epub 2018 Sep 10.

Cancer Research UK Cancer Therapeutics Unit at The Institute of Cancer Research , London SM2 5NG , United Kingdom.

Monopolar spindle 1 (MPS1) occupies a central role in mitosis and is one of the main components of the spindle assembly checkpoint. The MPS1 kinase is an attractive cancer target, and herein, we report the discovery of the clinical candidate BOS172722. The starting point for our work was a series of pyrido[3,4- d]pyrimidine inhibitors that demonstrated excellent potency and kinase selectivity but suffered from rapid turnover in human liver microsomes (HLM). Optimizing HLM stability proved challenging since it was not possible to identify a consistent site of metabolism and lowering lipophilicity proved unsuccessful. Key to overcoming this problem was the finding that introduction of a methyl group at the 6-position of the pyrido[3,4- d]pyrimidine core significantly improved HLM stability. Met ID studies suggested that the methyl group suppressed metabolism at the distant aniline portion of the molecule, likely by blocking the preferred pharmacophore through which P450 recognized the compound. This work ultimately led to the discovery of BOS172722 as a Phase 1 clinical candidate.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166229PMC
September 2018

Development and validation of a LC-MS/MS method for the quantification of the checkpoint kinase 1 inhibitor SRA737 in human plasma.

Bioanalysis 2017 Jul 10;9(13):1001-1010. Epub 2017 Jul 10.

Newcastle Cancer Centre Pharmacology Group, Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, NE2 4HH2, UK.

Aim: SRA737 is an orally active small-molecule inhibitor of checkpoint kinase 1 being investigated in an oncology setting. A HPLC-MS/MS method for quantifying plasma concentrations of SRA737 was validated.

Methods & Results: Sample preparation involved protein precipitation with acetonitrile following addition of CN-deuterated SRA737 as internal standard. A rapid and selective method was fully validated across a range of 5-20,000 ng/ml, exhibiting good sensitivity, overall precision (expressed as coefficient of variation) ≤8.0% and accuracy 96-102%. Consistently high recovery was observed, with no matrix effect and a lower limit of quantitation of 5 ng/ml.

Conclusion: A novel method for analyzing SRA737 in human plasma has been validated and is now being utilized for quantification of SRA737 in a Phase I trial.
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http://dx.doi.org/10.4155/bio-2017-0043DOI Listing
July 2017

Modulation of Plasma Metabolite Biomarkers of the MAPK Pathway with MEK Inhibitor RO4987655: Pharmacodynamic and Predictive Potential in Metastatic Melanoma.

Mol Cancer Ther 2017 10 21;16(10):2315-2323. Epub 2017 Jun 21.

Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London, United Kingdom.

MAPK pathway activation is frequently observed in human malignancies, including melanoma, and is associated with sensitivity to MEK inhibition and changes in cellular metabolism. Using quantitative mass spectrometry-based metabolomics, we identified in preclinical models 21 plasma metabolites including amino acids, propionylcarnitine, phosphatidylcholines, and sphingomyelins that were significantly altered in two B-RAF-mutant melanoma xenografts and that were reversed following a single dose of the potent and selective MEK inhibitor RO4987655. Treatment of non-tumor-bearing animals and mice bearing the PTEN-null U87MG human glioblastoma xenograft elicited plasma changes only in amino acids and propionylcarnitine. In patients with advanced melanoma treated with RO4987655, on-treatment changes of amino acids were observed in patients with disease progression and not in responders. In contrast, changes in phosphatidylcholines and sphingomyelins were observed in responders. Furthermore, pretreatment levels of seven lipids identified in the preclinical screen were statistically significantly able to predict objective responses to RO4987655. The RO4987655 treatment-related changes were greater than baseline physiological variability in nontreated individuals. This study provides evidence of a translational exo-metabolomic plasma readout predictive of clinical efficacy together with pharmacodynamic utility following treatment with a signal transduction inhibitor. .
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http://dx.doi.org/10.1158/1535-7163.MCT-16-0881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112418PMC
October 2017

Characterisation of CCT271850, a selective, oral and potent MPS1 inhibitor, used to directly measure in vivo MPS1 inhibition vs therapeutic efficacy.

Br J Cancer 2017 Apr 23;116(9):1166-1176. Epub 2017 Mar 23.

Cancer Research UK Cancer Therapeutics Unit, Division of Cancer Therapeutics, The Institute of Cancer Research, London, UK.

Background: The main role of the cell cycle is to enable error-free DNA replication, chromosome segregation and cytokinesis. One of the best characterised checkpoint pathways is the spindle assembly checkpoint, which prevents anaphase onset until the appropriate attachment and tension across kinetochores is achieved. MPS1 kinase activity is essential for the activation of the spindle assembly checkpoint and has been shown to be deregulated in human tumours with chromosomal instability and aneuploidy. Therefore, MPS1 inhibition represents an attractive strategy to target cancers.

Methods: To evaluate CCT271850 cellular potency, two specific antibodies that recognise the activation sites of MPS1 were used and its antiproliferative activity was determined in 91 human cancer cell lines. DLD1 cells with induced GFP-MPS1 and HCT116 cells were used in in vivo studies to directly measure MPS1 inhibition and efficacy of CCT271850 treatment.

Results: CCT271850 selectively and potently inhibits MPS1 kinase activity in biochemical and cellular assays and in in vivo models. Mechanistically, tumour cells treated with CCT271850 acquire aberrant numbers of chromosomes and the majority of cells divide their chromosomes without proper alignment because of abrogation of the mitotic checkpoint, leading to cell death. We demonstrated a moderate level of efficacy of CCT271850 as a single agent in a human colorectal carcinoma xenograft model.

Conclusions: CCT271850 is a potent, selective and orally bioavailable MPS1 kinase inhibitor. On the basis of in vivo pharmacodynamic vs efficacy relationships, we predict that more than 80% inhibition of MPS1 activity for at least 24 h is required to achieve tumour stasis or regression by CCT271850.
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http://dx.doi.org/10.1038/bjc.2017.75DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418449PMC
April 2017

Pyrido[3,4-d]pyrimidin-4(3H)-one metabolism mediated by aldehyde oxidase is blocked by C2-substitution.

Xenobiotica 2017 Sep 26;47(9):771-777. Epub 2016 Oct 26.

a Cancer Research UK Cancer Therapeutics Unit , The Institute of Cancer Research , London , UK.

1. We have previously described C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one derivatives as cell permeable inhibitors of the KDM4 and KDM5 subfamilies of JmjC histone lysine demethylases. 2. Although exemplar compound 1 exhibited moderate clearance in mouse liver microsomes, it was highly cleared in vivo due to metabolism by aldehyde oxidase (AO). Similar human and mouse AO-mediated metabolism was observed with the pyrido[3,4-d]pyrimidin-4(3H)-one scaffold and other C8-substituted derivatives. 3. We identified the C2-position as the oxidation site by LC-MS and H-NMR and showed that C2-substituted derivatives are no longer AO substrates. 4. In addition to the experimental data, these observations are supported by molecular modelling studies in the human AO protein crystal structure.
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http://dx.doi.org/10.1080/00498254.2016.1230245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526139PMC
September 2017

Structure-Based Optimization of Potent, Selective, and Orally Bioavailable CDK8 Inhibitors Discovered by High-Throughput Screening.

J Med Chem 2016 Oct 7;59(20):9337-9349. Epub 2016 Oct 7.

Merck KGaA , Frankfurter Strasse 250, Darmstadt, 64293, Germany.

The mediator complex-associated cyclin dependent kinase CDK8 regulates β-catenin-dependent transcription following activation of WNT signaling. Multiple lines of evidence suggest CDK8 may act as an oncogene in the development of colorectal cancer. Here we describe the successful optimization of an imidazo-thiadiazole series of CDK8 inhibitors that was identified in a high-throughput screening campaign and further progressed by structure-based design. In several optimization cycles, we improved the microsomal stability, potency, and kinase selectivity. The initial imidazo-thiadiazole scaffold was replaced by a 3-methyl-1H-pyrazolo[3,4-b]-pyridine which resulted in compound 25 (MSC2530818) that displayed excellent kinase selectivity, biochemical and cellular potency, microsomal stability, and is orally bioavailable. Furthermore, we demonstrated modulation of phospho-STAT1, a pharmacodynamic biomarker of CDK8 activity, and tumor growth inhibition in an APC mutant SW620 human colorectal carcinoma xenograft model after oral administration. Compound 25 demonstrated suitable potency and selectivity to progress into preclinical in vivo efficacy and safety studies.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00597DOI Listing
October 2016

2,8-Disubstituted-1,6-Naphthyridines and 4,6-Disubstituted-Isoquinolines with Potent, Selective Affinity for CDK8/19.

ACS Med Chem Lett 2016 Jun 28;7(6):573-8. Epub 2016 Mar 28.

Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research , London SW7 3RP, UK.

We demonstrate a designed scaffold-hop approach to the discovery of 2,8-disubstituted-1,6-naphthyridine- and 4,6-disubstituted-isoquinoline-based dual CDK8/19 ligands. Optimized compounds in both series exhibited rapid aldehyde oxidase-mediated metabolism, which could be abrogated by introduction of an amino substituent at C5 of the 1,6-naphthyridine scaffold or at C1 of the isoquinoline scaffold. Compounds 51 and 59 were progressed to in vivo pharmacokinetic studies, and 51 also demonstrated sustained inhibition of STAT1(SER727) phosphorylation, a biomarker of CDK8 inhibition, in an SW620 colorectal carcinoma human tumor xenograft model following oral dosing.
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http://dx.doi.org/10.1021/acsmedchemlett.6b00022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4904262PMC
June 2016

p53 Loss in MYC-Driven Neuroblastoma Leads to Metabolic Adaptations Supporting Radioresistance.

Cancer Res 2016 05 29;76(10):3025-35. Epub 2016 Mar 29.

Division of Clinical Studies, The Institute of Cancer Research, London, United Kingdom.

Neuroblastoma is the most common childhood extracranial solid tumor. In high-risk cases, many of which are characterized by amplification of MYCN, outcome remains poor. Mutations in the p53 (TP53) tumor suppressor are rare at diagnosis, but evidence suggests that p53 function is often impaired in relapsed, treatment-resistant disease. To address the role of p53 loss of function in the development and pathogenesis of high-risk neuroblastoma, we generated a MYCN-driven genetically engineered mouse model in which the tamoxifen-inducible p53ER(TAM) fusion protein was expressed from a knock-in allele (Th-MYCN/Trp53(KI)). We observed no significant differences in tumor-free survival between Th-MYCN mice heterozygous for Trp53(KI) (n = 188) and Th-MYCN mice with wild-type p53 (n = 101). Conversely, the survival of Th-MYCN/Trp53(KI/KI) mice lacking functional p53 (n = 60) was greatly reduced. We found that Th-MYCN/Trp53(KI/KI) tumors were resistant to ionizing radiation (IR), as expected. However, restoration of functional p53ER(TAM) reinstated sensitivity to IR in only 50% of Th-MYCN/Trp53(KI/KI) tumors, indicating the acquisition of additional resistance mechanisms. Gene expression and metabolic analyses indicated that the principal acquired mechanism of resistance to IR in the absence of functional p53 was metabolic adaptation in response to chronic oxidative stress. Tumors exhibited increased antioxidant metabolites and upregulation of glutathione S-transferase pathway genes, including Gstp1 and Gstz1, which are associated with poor outcome in human neuroblastoma. Accordingly, glutathione depletion by buthionine sulfoximine together with restoration of p53 activity resensitized tumors to IR. Our findings highlight the complex pathways operating in relapsed neuroblastomas and the need for combination therapies that target the diverse resistance mechanisms at play. Cancer Res; 76(10); 3025-35. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-1939DOI Listing
May 2016

Multiparameter Lead Optimization to Give an Oral Checkpoint Kinase 1 (CHK1) Inhibitor Clinical Candidate: (R)-5-((4-((Morpholin-2-ylmethyl)amino)-5-(trifluoromethyl)pyridin-2-yl)amino)pyrazine-2-carbonitrile (CCT245737).

J Med Chem 2016 06 23;59(11):5221-37. Epub 2016 May 23.

Sareum Ltd. , Cambridge CB22 3FX, U.K.

Multiparameter optimization of a series of 5-((4-aminopyridin-2-yl)amino)pyrazine-2-carbonitriles resulted in the identification of a potent and selective oral CHK1 preclinical development candidate with in vivo efficacy as a potentiator of deoxyribonucleic acid (DNA) damaging chemotherapy and as a single agent. Cellular mechanism of action assays were used to give an integrated assessment of compound selectivity during optimization resulting in a highly CHK1 selective adenosine triphosphate (ATP) competitive inhibitor. A single substituent vector directed away from the CHK1 kinase active site was unexpectedly found to drive the selective cellular efficacy of the compounds. Both CHK1 potency and off-target human ether-a-go-go-related gene (hERG) ion channel inhibition were dependent on lipophilicity and basicity in this series. Optimization of CHK1 cellular potency and in vivo pharmacokinetic-pharmacodynamic (PK-PD) properties gave a compound with low predicted doses and exposures in humans which mitigated the residual weak in vitro hERG inhibition.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01938DOI Listing
June 2016

Rapid Discovery of Pyrido[3,4-d]pyrimidine Inhibitors of Monopolar Spindle Kinase 1 (MPS1) Using a Structure-Based Hybridization Approach.

J Med Chem 2016 04 7;59(8):3671-88. Epub 2016 Apr 7.

Cancer Research UK Cancer Therapeutics Unit, Division of Cancer Therapeutics, The Institute of Cancer Research , 15 Cotswold Road, Sutton, London, SM2 5NG, United Kingdom.

Monopolar spindle 1 (MPS1) plays a central role in the transition of cells from metaphase to anaphase and is one of the main components of the spindle assembly checkpoint. Chromosomally unstable cancer cells rely heavily on MPS1 to cope with the stress arising from abnormal numbers of chromosomes and centrosomes and are thus more sensitive to MPS1 inhibition than normal cells. We report the discovery and optimization of a series of new pyrido[3,4-d]pyrimidine based inhibitors via a structure-based hybridization approach from our previously reported inhibitor CCT251455 and a modestly potent screening hit. Compounds in this novel series display excellent potency and selectivity for MPS1, which translates into biomarker modulation in an in vivo human tumor xenograft model.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01811DOI Listing
April 2016

Plasma Metabolomic Changes following PI3K Inhibition as Pharmacodynamic Biomarkers: Preclinical Discovery to Phase I Trial Evaluation.

Mol Cancer Ther 2016 06 5;15(6):1412-24. Epub 2016 Apr 5.

Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London, United Kingdom. Drug Development Unit, The Royal Marsden NHS Foundation Trust, Sutton, United Kingdom.

PI3K plays a key role in cellular metabolism and cancer. Using a mass spectrometry-based metabolomics platform, we discovered that plasma concentrations of 26 metabolites, including amino acids, acylcarnitines, and phosphatidylcholines, were decreased in mice bearing PTEN-deficient tumors compared with non-tumor-bearing controls and in addition were increased following dosing with class I PI3K inhibitor pictilisib (GDC-0941). These candidate metabolomics biomarkers were evaluated in a phase I dose-escalation clinical trial of pictilisib. Time- and dose-dependent effects were observed in patients for 22 plasma metabolites. The changes exceeded baseline variability, resolved after drug washout, and were recapitulated on continuous dosing. Our study provides a link between modulation of the PI3K pathway and changes in the plasma metabolome and demonstrates that plasma metabolomics is a feasible and promising strategy for biomarker evaluation. Also, our findings provide additional support for an association between insulin resistance, branched-chain amino acids, and related metabolites following PI3K inhibition. Mol Cancer Ther; 15(6); 1412-24. ©2016 AACR.
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http://dx.doi.org/10.1158/1535-7163.MCT-15-0815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5321508PMC
June 2016

Synthesis and Evaluation of a 2,11-Cembranoid-Inspired Library.

Chemistry 2016 Apr 1;22(16):5657-64. Epub 2016 Mar 1.

Division of Cancer Therapeutics, The Institute of Cancer Research, London, SM2 5NG, UK.

The 2,11-cembranoid family of natural products has been used as inspiration for the synthesis of a structurally simplified, functionally diverse library of octahydroisobenzofuran-based compounds designed to augment a typical medicinal chemistry library screen. Ring-closing metathesis, lactonisation and SmI2 -mediated methods were exemplified and applied to the installation of a third ring to mimic the nine-membered ring of the 2,11-cembranoids. The library was assessed for aqueous solubility and permeability, with a chemical-space analysis performed for comparison to the family of cembranoid natural products and a sample set of a screening library. Preliminary investigations in cancer cells showed that the simpler scaffolds could recapitulate the reported anti-migratory activity of the natural products.
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http://dx.doi.org/10.1002/chem.201505093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869678PMC
April 2016

Discovery of potent and selective CDK8 inhibitors from an HSP90 pharmacophore.

Bioorg Med Chem Lett 2016 Mar 22;26(5):1443-51. Epub 2016 Jan 22.

Cancer Research UK Cancer Therapeutics Unit at The Institute of Cancer Research, London SW7 3RP, UK.

Here we describe the discovery and optimization of 3-benzylindazoles as potent and selective inhibitors of CDK8, also modulating CDK19, discovered from a high-throughput screening (HTS) campaign sampling the Merck compound collection. The primary hits with strong HSP90 affinity were subsequently optimized to potent and selective CDK8 inhibitors which demonstrate inhibition of WNT pathway activity in cell-based assays. X-ray crystallographic data demonstrated that 3-benzylindazoles occupy the ATP binding site of CDK8 and adopt a Type I binding mode. Medicinal chemistry optimization successfully led to improved potency, physicochemical properties and oral pharmacokinetics. Modulation of phospho-STAT1, a pharmacodynamic biomarker of CDK8, was demonstrated in an APC-mutant SW620 human colorectal carcinoma xenograft model following oral administration.
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http://dx.doi.org/10.1016/j.bmcl.2016.01.062DOI Listing
March 2016

Discovery of Potent, Selective, and Orally Bioavailable Small-Molecule Modulators of the Mediator Complex-Associated Kinases CDK8 and CDK19.

J Med Chem 2016 Feb 21;59(3):1078-101. Epub 2016 Jan 21.

Cancer Research UK Cancer Therapeutics Unit at The Institute of Cancer Research, London, SW7 3RP, U.K.

The Mediator complex-associated cyclin-dependent kinase CDK8 has been implicated in human disease, particularly in colorectal cancer where it has been reported as a putative oncogene. Here we report the discovery of 109 (CCT251921), a potent, selective, and orally bioavailable inhibitor of CDK8 with equipotent affinity for CDK19. We describe a structure-based design approach leading to the discovery of a 3,4,5-trisubstituted-2-aminopyridine series and present the application of physicochemical property analyses to successfully reduce in vivo metabolic clearance, minimize transporter-mediated biliary elimination while maintaining acceptable aqueous solubility. Compound 109 affords the optimal compromise of in vitro biochemical, pharmacokinetic, and physicochemical properties and is suitable for progression to animal models of cancer.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5362750PMC
February 2016

8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors.

J Med Chem 2016 Feb 7;59(4):1388-409. Epub 2016 Jan 7.

Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research , 15 Cotswold Road, London SM2 5NG, U.K.

We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770324PMC
February 2016

7-(Pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-based derivatives for kinase inhibition: Co-crystallisation studies with Aurora-A reveal distinct differences in the orientation of the pyrazole N1-substituent.

Bioorg Med Chem Lett 2015 Oct 6;25(19):4203-9. Epub 2015 Aug 6.

Cancer Research UK Cancer Therapeutics Unit at The Institute of Cancer Research, London, United Kingdom. Electronic address:

Introduction of a 1-benzyl-1H-pyrazol-4-yl moiety at C7 of the imidazo[4,5-b]pyridine scaffold provided 7a which inhibited a range of kinases including Aurora-A. Modification of the benzyl group in 7a, and subsequent co-crystallisation of the resulting analogues with Aurora-A indicated distinct differences in binding mode dependent upon the pyrazole N-substituent. Compounds 7a and 14d interact with the P-loop whereas 14a and 14b engage with Thr217 in the post-hinge region. These crystallographic insights provide options for the design of compounds interacting with the DFG motif or with Thr217.
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http://dx.doi.org/10.1016/j.bmcl.2015.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4577729PMC
October 2015