PDF file, 63KB, Kinase inhibitory profile of SAR-020106 at 10 μMa.

ABSTRACT The use of bacterial transposon mutant libraries in phenotypic screens is a well-established technique for determining which genes are essential or advantageous for growth in conditions of interest. Standard, inactivating, transposon libraries cannot give direct information about genes whose over-expression gives a selective advantage. We report the development of a system wherein outward-oriented promoters are included in mini-transposons, generation of transposon mutant libraries in Escherichia coli and Pseudomonas aeruginosa and their use to probe genes important for growth under selection with the antimicrobial fosfomycin, and a recently-developed leucyl-tRNA synthase inhibitor. In addition to the identification of known mechanisms of action and resistance, we identify the carbon–phosphorous lyase complex as a potential resistance liability for fosfomycin in E. coli and P. aeruginosa. The use of this technology can facilitate the development of novel mechanism-of-action antimicrobials that are urgently required to combat the increasing threat worldwide from antimicrobial-resistant pathogenic bacteria.

Abstract The DNA damage response network ensures the fidelity of DNA replication and controls the repair of damage arising during cellular replication or from exogenous agents such as genotoxic drugs. Checkpoint Kinase 1 (CHK1) is a serine/threonine kinase occupying a central position in this complex network of cell regulatory and DNA repair mechanisms. G1/S, S or G2/M cell cycle checkpoints are activated in response to genotoxic antitumor drugs to provide an opportunity for repair of damaged DNA or to activate apoptotic pathways. Unlike normal cells, human cancer cells frequently have functional defects in the tumor suppressor p53 with consequent loss of G1/S checkpoint control and greater reliance on S and G2/M checkpoints. Thus CHK1 inhibitors which abrogate the S and G2/M checkpoints will selectively sensitize p53 deficient cancer cells to DNA damaging agents. CHK1 inhibition by siRNA and several small molecule inhibitors have confirmed this in preclinical studies. The challenges of improving the CHK1 potency and selectivity of our initial, fragment derived pyrazolopyridine inhibitors, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps. Initial hit compounds were optimised into potent inhibitors of CHK1 using iterative cycles of design, synthesis, assay and crystallography, progressing through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and isoquinolines. The potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, and potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts when dosed i.p. in nude mice. Further lead optimisation led to orally bioavailable analogues with good in vitro ADME and in vivo pharmacokinetic properties, exemplified by CCT244747. CCT244747 has demonstrated both in vivo pharmacodynamic modulation of signaling through CHK1 and potentiation of cytotoxic drugs in human tumor xenografts. In summary, we show how a fragment derived compound with weak, micromolar activity against CHK1 evolved through a scaffold hopping strategy to give the selective CHK1 isoquinoline inhibitor SAR-020106, from which optimisation of pharmacokinetic properties led to potent, selective and orally bioavailable CHK1 inhibitors such as CCT244747. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A235.

Iminosugars comprise the most attractive class of carbohydrate mimetics reported to date and are ideally positioned to take advantage of our increasing understanding of glycobiology in the search for new medicines. First-generation iminosugar drugs suffered from lack of adequate selectivity, resulting in considerable side-effects in the clinic. Current efforts directed towards second-generation compounds, encompassing a much greater range of structures and addressing a wider selection of biochemical targets, are enabling the identification and development of suitable candidates that benefit from improved activity and selectivity. Furthermore, second-generation compounds can address a variety of established targets that have previously proved refractory to other compound classes. This review focuses on the breadth of opportunities provided by second-generation leads from iminosugars (Seglins™).

Pyrazolopyridine inhibitors with low micromolar potency for CHK1 and good selectivity against CHK2 were previously identified by fragment-based screening. The optimization of the pyrazolopyridines to a series of potent and CHK1-selective isoquinolines demonstrates how fragment-growing and scaffold morphing strategies arising from a structure-based understanding of CHK1 inhibitor binding can be combined to successfully progress fragment-derived hit matter to compounds with activity in vivo. The challenges of improving CHK1 potency and selectivity, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps, which progressed through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and ultimately to imidazo[4,5-c]pyridines and isoquinolines. A potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, which potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts in nude mice.

Genotoxic antitumor agents continue to be the mainstay of current cancer chemotherapy. These drugs cause DNA damage and activate numerous cell cycle checkpoints facilitating DNA repair and the maintenance of genomic integrity. Most human tumors lack functional p53 and consequently have compromized G1/S checkpoint control. This has led to the hypothesis that S and G2/M checkpoint abrogation may selectively enhance genotoxic cell killing in a p53 deficient background, as normal cells would be rescued at the G1/S checkpoint. CHK1 is a serine/threonine kinase associated with DNA damage linked S and G2/M checkpoint control. SAR-020106 (the structure will be shown on the poster) is an ATP competitive, potent and selective CHK1 inhibitor with an IC50 of 13.3nM on the isolated human enzyme. This compound abrogates an etoposide-induced G2 arrest with an IC50 of 55nM in HT29 cells, and significantly enhances the cell killing of gemcitabine and SN38 by 3.0 to 29-fold in a number of colon tumor lines in vitro and in a p53 dependent fashion. Biomarker studies have shown that SAR-020106 inhibits cytotoxic drug induced autophosphorylation of CHK1 at S296 and blocks phosphorylation of CDK1 at Y15 in a dose-dependent fashion both in vitro and in vivo. Cytotoxic drug combinations were associated with increased γH2AX and PARP cleavage consistent with SAR-020106 enhanced DNA damage and tumor cell death. Irinotecan and gemcitabine antitumor activity was enhanced by SAR-020106 in vivo with minimal toxicity. Orally bioavailable analogues of SAR-02106 are currently under development. In conclusion SAR-020106 represents a novel class of CHK1 inhibitors which can enhance antitumor activity with selected anticancer drugs in vivo and may therefore have clinical utility. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3518.

The Aurora kinases regulate multiple aspects of mitotic progression, and their overexpression in diverse tumor types makes them appealing oncology targets. An intensive research effort over the past decade has led to the discovery of chemically distinct families of small molecule Aurora kinase inhibitors, many of which have demonstrated therapeutic potential in model systems. These agents are also important tools to help dissect signaling pathways that are orchestrated by Aurora kinases, and the antiproliferative target of pan-Aurora inhibitors such as VX-680 has been validated using chemical genetic techniques. In many cases the nonspecific nature of Aurora inhibitors toward unrelated kinases is well established, potentially broadening the spectrum of cancers to which these compounds might be applied. However, unambiguously demonstrating the molecular target(s) for clinical kinase inhibitors is an important challenge, one that is absolutely critical for deciphering the molecular basis of compound specificity, resistance, and efficacy. In this paper, we have investigated amino acid requirements for Aurora A sensitivity to the benzazepine-based Aurora inhibitor MLN8054 and the close analogue MLN8237, a second-generation compound that is in phase II clinical trials. A crystallographic analysis facilitated the design and biochemical investigation of a panel of resistant Aurora A mutants, a subset of which were then selected as candidate drug-resistance targets for further evaluation. Using inducible human cell lines, we show that cells expressing near-physiological levels of a functional but partially drug-resistant Aurora A T217D mutant survive in the presence of MLN8054 or MLN8237, authenticating Aurora A as a critical antiproliferative target of these compounds.

Abstract Genotoxic antitumor agents continue to be the mainstay of current cancer chemotherapy. These drugs cause DNA damage and activate numerous cell cycle checkpoints facilitating DNA repair and the maintenance of genomic integrity. Most human tumors lack functional p53 and consequently have compromised G1-S checkpoint control. This has led to the hypothesis that S and G2-M checkpoint abrogation may selectively enhance genotoxic cell killing in a p53-deficient background, as normal cells would be rescued at the G1-S checkpoint. CHK1 is a serine/threonine kinase associated with DNA damage–linked S and G2-M checkpoint control. SAR-020106 is an ATP-competitive, potent, and selective CHK1 inhibitor with an IC50 of 13.3 nmol/L on the isolated human enzyme. This compound abrogates an etoposide-induced G2 arrest with an IC50 of 55 nmol/L in HT29 cells, and significantly enhances the cell killing of gemcitabine and SN38 by 3.0- to 29-fold in several colon tumor lines in vitro and in a p53-dependent fashion. Biomarker studies have shown that SAR-020106 inhibits cytotoxic drug–induced autophosphorylation of CHK1 at S296 and blocks the phosphorylation of CDK1 at Y15 in a dose-dependent fashion both in vitro and in vivo. Cytotoxic drug combinations were associated with increased γH2AX and poly ADP ribose polymerase cleavage consistent with the SAR-020106–enhanced DNA damage and tumor cell death. Irinotecan and gemcitabine antitumor activity was enhanced by SAR-020106 in vivo with minimal toxicity. SAR-020106 represents a novel class of CHK1 inhibitors that can enhance antitumor activity with selected anticancer drugs in vivo and may therefore have clinical utility. Mol Cancer Ther; 9(1); 89–100

The dual-specificity protein kinase monopolar spindle 1 (Mps1) is a central component of the mitotic spindle assembly checkpoint (SAC), a sensing mechanism that prevents anaphase until all chromosomes are bioriented on the metaphase plate. Partial depletion of Mps1 protein levels sensitizes transformed, but not untransformed, human cells to therapeutic doses of the anticancer agent Taxol, making it an attractive novel therapeutic cancer target. We have previously determined the X-ray structure of the catalytic domain of human Mps1 in complex with the anthrapyrazolone kinase inhibitor SP600125. In order to validate distinct inhibitors that target this enzyme and improve our understanding of nucleotide binding site architecture, we now report a biophysical and structural evaluation of the Mps1 catalytic domain in the presence of ATP and the aspecific model kinase inhibitor staurosporine. Collective in silico, enzymatic, and fluorescent screens also identified several new lead quinazoline Mps1 inhibitors, including a low-affinity compound termed Compound 4 (Cpd 4), whose interaction with the Mps1 kinase domain was further characterized by X-ray crystallography. A novel biophysical analysis demonstrated that the intrinsic fluorescence of SP600125 changed markedly upon Mps1 binding, allowing spectrophotometric displacement analysis and determination of dissociation constants for ATP-competitive Mps1 inhibitors. By illuminating the structure of the Mps1 ATP-binding site our results provide novel biophysical insights into Mps1-ligand interactions that will be useful for the development of specific Mps1 inhibitors, including those employing a therapeutically validated quinazoline template.

Checkpoint kinase 1 (CHK1) is an oncology target of significant current interest. Inhibition of CHK1 abrogates DNA damage-induced cell cycle checkpoints and sensitizes p53 deficient cancer cells to genotoxic therapies. Using template screening, a fragment-based approach to small molecule hit generation, we have identified multiple CHK1 inhibitor scaffolds suitable for further optimization. The sequential combination of in silico low molecular weight template selection, a high concentration biochemical assay and hit validation through protein-ligand X-ray crystallography provided 13 template hits from an initial in silico screening library of ca. 15000 compounds. The use of appropriate counter-screening to rule out nonspecific aggregation by test compounds was essential for optimum performance of the high concentration bioassay. One low molecular weight, weakly active purine template hit was progressed by iterative structure-based design to give submicromolar pyrazolopyridines with good ligand efficiency and appropriate CHK1-mediated cellular activity in HT29 colon cancer cells.

The Aurora and Polo-like kinases are central components of mitotic signaling pathways, and recent evidence suggests that substantial cross-talk exists between Aurora A and Plk1. In addition to their validation as novel anticancer agents, small molecule kinase inhibitors are increasingly important tools to help dissect clinically relevant protein phosphorylation networks. However, one major problem associated with kinase inhibitors is their promiscuity toward "off-target" members of the kinome, which makes interpretation of data obtained from complex cellular systems challenging. Additionally, the emergence of inhibitor resistance in patients makes it clear that an understanding of resistance mechanisms is essential to inform drug design. In this study, we exploited structural knowledge of the binding modes of VX-680, an Aurora kinase inhibitor, and BI 2536, a Polo-like kinase inhibitor, to design and evaluate drug-resistant kinase mutants. Using inducible stable human cell lines, we authenticated mitotic targets for both compounds and demonstrated that Aurora A mutants exhibit differential cellular sensitivity toward the inhibitors VX-680 and MLN8054. In addition, we validated Aurora B as an important anti-proliferative target for VX-680 in model human cancer cells. Finally, this chemical genetic approach allowed us to prove that Aurora A activation loop phosphorylation is controlled by a Plk1-mediated pathway in human cells.

Authors' Therapeuti Sutton, Su Kingdom Note: Sup Cancer The Correspon Cancer Th Laboratorie Kingdom. P Mike.Walto

This paper presents results from a simulation study which investigated the use of Continuous Descent Arrival (CDA) procedures for conducting a descent through a busy terminal area, using conventional transport-category automation. This research was part of the Low Noise Flight Procedures (LNFP) element within the Quiet Aircraft Technology (QAT) Project, that addressed development of flight guidance, and supporting pilot and Air Traffic Control (ATC) procedures for low noise operations. The procedures and chart were designed to be easy to understand, and to make it easy for the crew to make changes via the Flight Management Computer Control-Display Unit (FMC-CDU) to accommodate changes from ATC. The test runs were intended to represent situations typical of what exists in many of today's terminal areas, including interruptions to the descent in the form of clearances issued by ATC.

The discovery and selection of suitable compounds to start a medicinal chemistry program is a fundamental stage in the process of drug discovery, where decisions are made to ultimately affect the probability of success for that project. Hit identification is a crucially important stage gate in the drug discovery process. As the development candidate that emerges from pre-clinical research invariably resembles the initial hit, it is important to make as well-informed a choice as possible when selecting which hits to progress and which to shelve. Decisions made during the hit identification process determine the chemistry direction in which a project proceeds, usually taking into account the potency of the hit, any structure–activity relationship generated during the hit-finding exercise, the potential scope for generating novel and patentable chemical matter, and the synthetic tractability of the series.