Search

In cystic fibrosis (CF), excessive furin activity plays a critical role in the activation of the epithelial sodium channel (ENaC), dysregulation of which contributes to airways dehydration, ineffective mucociliary clearance (MCC) and mucus obstruction. Here, we report a highly selective, cell-permeable furin inhibitor, BOS-318 that derives selectivity by eliciting the formation of a new, unexpected binding pocket independent of the active site catalytic triad. Using human ex vivo models, BOS-318 showed significant suppression of ENaC which led to enhanced airway hydration and an ~30-fold increase in MCC rate. Furin inhibition also protected ENaC from subsequent activation by neutrophil elastase, a soluble protease dominant in CF airways. Additional therapeutic benefits include protection against Pseudomonas aeruginosa exotoxin A-induced epithelial cell death. Our findings demonstrate the utility of selective furin inhibition as a mutation-agnostic approach that can correct features of CF airways pathophysiology in a manner expected to deliver therapeutic value.

GlaxoSmithKline and Astex Pharmaceuticals recently disclosed the discovery of the potent H-PGDS inhibitor GSK2894631A 1a (IC50 = 9.9 nM) as part of a fragment-based drug discovery collaboration with Astex Pharmaceuticals. This molecule exhibited good murine pharmacokinetics, allowing it to be utilized to explore H-PGDS pharmacology in vivo. Yet, with prolonged dosing at higher concentrations, 1a induced CNS toxicity. Looking to attenuate brain penetration in this series, aza-quinolines, were prepared with the intent of increasing polar surface area. Nitrogen substitutions at the 6- and 8-positions of the quinoline were discovered to be tolerated by the enzyme. Subsequent structure activity studies in these aza-quinoline scaffolds led to the identification of 1,8-naphthyridine 1y (IC50 = 9.4 nM) as a potent peripherally restricted H-PGDS inhibitor. Compound 1y is efficacious in four in vivo inflammatory models and exhibits no CNS toxicity.

Funding Acknowledgements Nothing to declare OnBehalf None Background Peri-coronary fat attenuation index (pFAI) has emerged as a clinical marker of coronary inflammation, which is measurable from standard coronary CT angiography (CCTA). It compares well with gold-standard methods for the assessment of coronary inflammation and can predict future cardiovascular events. pFAI could prove invaluable to differentiate an inflammatory from noninflammatory coronary artery status, helping unravel the mechanisms subtending an event classified as myocardial infarction with nonobstructive coronary arteries (MINOCA) or Tako-Tsubo syndrome (TTs). Methods and Results Patients admitted with MINOCA and TTs diagnosis between 2011 and 2018, who had both CCTA and CMR performed during or shortly after the acute phase, were selected and pFAI measured in their index CCTA. pFAI was also measured in a control subjects who had CCTA for atypical chest pain work-up, no obstructive coronary artery disease found in their CCTA and no cardiac events at a minimum 2-year follow-up. In the n = 106 MINOCA/TTs patients selected, mean pFAI averaged for the 3 coronary arteries was -68.37 ± 8.29 vs -78.03 ± 6.20 in the n = 106 controls (p confirmed also when comparing mean pFAI in each single coronary artery between MINOCA/TTs and controls (p particular, were also more frequently found (p controls. Conclusions In MINOCA and TTs patients, CCTA is not only able to detect otherwise angiographically invisible atherosclerotic plaques, but its diagnostic yield can be further expanded using the simple off-line measurement of pFAI for the characterization of peri-coronary fat tissue. In MINOCA/TTs mean pFAI clearly demonstrates higher values in comparison with controls, a finding which has been previously associated with coronary artery inflammation. We speculate that this newly-available diagnostic tool in the future may help select patients for new therapies, for example therapies targeting coronary inflammation. I was able to build a table. Abstract 1176 Figure.

[Image: see text] Bone Morphogenetic Protein 1 (BMP1) inhibition is a potential method for treating fibrosis because BMP1, a member of the zinc metalloprotease family, is required to convert pro-collagen to collagen. A novel class of reverse hydroxamate BMP1 inhibitors was discovered, and cocrystal structures with BMP1 were obtained. The observed binding mode is unique in that the small molecule occupies the nonprime side of the metalloprotease pocket providing an opportunity to build in metalloprotease selectivity. Structure-guided modification of the initial hit led to the identification of an oral in vivo tool compound with selectivity over other metalloproteases. Due to irreversible inhibition of cytochrome P450 3A4 for this chemical class, the risk of potential drug–drug interactions was managed by optimizing the series for subcutaneous injection.

RIP1 regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP1 kinase that are suitable for advancement into the clinic have yet to be described. Herein, we report our lead optimization of a benzoxazepinone hit from a DNA-encoded library and the discovery and profile of clinical candidate GSK2982772 (compound 5), currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis. Compound 5 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking many TNF-dependent cellular responses. Highlighting its potential as a novel anti-inflammatory agent, the inhibitor was also able to reduce spontaneous production of cytokines from human ulcerative colitis explants. The highly favorable physicochemical and ADMET properties of 5, combined with high potency, led to a predicted low oral dose in humans.

RIP1 regulates cell death and inflammation and is believed to play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases and cancer. While small-molecule inhibitors of RIP1 kinase have been advanced to the clinic for inflammatory diseases and CNS indications, RIP1 inhibitors for oncology indications have yet to be described. Herein we report on the discovery and profile of GSK3145095 (compound 6). Compound 6 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking RIP1 kinase-dependent cellular responses. Highlighting its potential as a novel cancer therapy, the inhibitor was also able to promote a tumor suppressive T cell phenotype in pancreatic adenocarcinoma organ cultures. Compound 6 is currently in phase 1 clinical studies for pancreatic adenocarcinoma and other selected solid tumors.

With the goal of discovering more selective anti-inflammatory drugs, than COX inhibitors, to attenuate prostaglandin signaling, a fragment-based screen of hematopoietic prostaglandin D synthase was performed. The 76 crystallographic hits were sorted into similar groups, with the 3-cyano-quinoline 1a (FP IC50 = 220,000 nM, LE = 0.43) being a potent member of the 6,6-fused heterocyclic cluster. Employing SAR insights gained from structural comparisons of other H-PGDS fragment binding mode clusters, the initial hit 1a was converted into the 70-fold more potent quinoline 1d (IC50 = 3,100 nM, LE = 0.49). A systematic substitution of the amine moiety of 1d, utilizing structural information and array chemistry, with modifications to improve inhibitor stability, resulted in the identification of the 300-fold more active H-PGDS inhibitor tool compound 1bv (IC50 = 9.9 nM, LE = 0.42). This selective inhibitor exhibited good murine pharmacokinetics, dose-dependently attenuated PGD2 production in a mast cell degranulation assay and should be suitable to further explore H-PGDS biology.

Inhibitors of the tissue factor (TF)/factor VIIa complex (TF-FVIIa) are promising novel anticoagulants which show excellent efficacy and minimal bleeding in preclinical models. Starting with an aminoisoquinoline P1-based macrocyclic inhibitor, optimization of the P' groups led to a series of highly potent and selective TF-FVIIa inhibitors which displayed poor permeability. Fluorination of the aminoisoquinoline reduced the basicity of the P1 group and significantly improved permeability. The resulting lead compound was highly potent, selective, and achieved good pharmacokinetics in dogs with oral dosing. Moreover, it demonstrated robust antithrombotic activity in a rabbit model of arterial thrombosis.

A novel series of potent and selective hexokinase 2 (HK2) inhibitors, 2,6-disubstituted glucosamines, has been identified based on HTS hits, exemplified by compound 1. Inhibitor-bound crystal structures revealed that the HK2 enzyme could adopt an "induced-fit" conformation. The SAR study led to the identification of potent HK2 inhibitors, such as compound 34 with greater than 100-fold selectivity over HK1. Compound 25 inhibits in situ glycolysis in a UM-UC-3 bladder tumor cell line via (13)CNMR measurement of [3-(13)C]lactate produced from [1,6-(13)C2]glucose added to the cell culture.

Indoleamine-2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that catalyzes the rate-limiting step in the kynurenine pathway of tryptophan (TRP) metabolism. As an inflammation-induced immunoregulatory enzyme, pharmacological inhibition of IDO1 activity is currently being pursued as a potential therapeutic tool for the treatment of cancer and other disease states. As such, a detailed understanding of the mechanism of action of established and novel IDO1 inhibitors remains of great interest. Comparison of a newly-developed IDO1 inhibitor (GSK5628) to the existing best-in-class compound, epacadostat (Incyte), allows us to report on a unique inhibition mechanism for IDO1. Here, we demonstrate that GSK5628 inhibits IDO1 by competing with heme for binding to a heme-free conformation of the enzyme (apo-IDO1) while epacadostat coordinates its binding with the iron atom of the IDO1 heme cofactor. Comparison of these two compounds in cellular systems reveals a long-lasting inhibitory effect of GSK5628, undescribed for other known IDO1 inhibitors. Detailed characterization of this apo-binding mechanism for IDO1 inhibition may help design superior inhibitors or may confer a unique competitive advantage over other IDO1 inhibitorsvis-à-visspecificity and pharmacokinetic parameters.

On the basis of a crystal structure of a phenylpyrrolidine lead and subsequent molecular modeling results, we designed and synthesized a novel series of macrocyclic FVIIa inhibitors. The optimal 16-membered macrocycle was 60-fold more potent than an acyclic analog. Further potency optimization by incorporation of P1' alkyl sulfone and P2 methyl groups provided a macrocycle with TF/FVIIa Ki = 1.6 nM, excellent selectivity against a panel of seven serine proteases, and FVII-deficient prothrombin time EC2x = 1.2 μM. Discovery of this potent, selective macrocyclic scaffold opens new possibilities for the development of orally bioavailable FVIIa inhibitors.

Compound 2 was previously identified as a potent inhibitor of factor XIa lacking oral bioavailability. A structure-based approach was used to design analogs of 2 with novel P1 moieties with good selectivity profiles and oral bioavailability. Further optimization of the P1 group led to the identification of a 4-chlorophenyltetrazole P1 analog, which when combined with further modifications to the linker and P2' group provided compound 32 with FXIa Ki=6.7 nM and modest oral exposure in dogs.

The structure-activity relationships (SAR) of six-membered ring replacements for the imidazole ring scaffold is described. This work led to the discovery of the potent and selective pyridine (S)-23 and pyridinone (±)-24 factor XIa inhibitors. SAR and X-ray crystal structure data highlight the key differences between imidazole and six-membered ring analogs.

Human fatty acid synthase (hFAS) is a complex, multifunctional enzyme that is solely responsible for the de novo synthesis of long chain fatty acids. hFAS is highly expressed in a number of cancers, with low expression observed in most normal tissues. Although normal tissues tend to obtain fatty acids from the diet, tumor tissues rely on de novo fatty acid synthesis, making hFAS an attractive metabolic target for the treatment of cancer. We describe here the identification of GSK2194069, a potent and specific inhibitor of the β-ketoacyl reductase (KR) activity of hFAS; the characterization of its enzymatic and cellular mechanism of action; and its inhibition of human tumor cell growth. We also present the design of a new protein construct suitable for crystallography, which resulted in what is to our knowledge the first co-crystal structure of the human KR domain and includes a bound inhibitor.

Antithrombotic agents that are inhibitors of factor XIa (FXIa) have the potential to demonstrate robust efficacy with a low bleeding risk profile. Herein, we describe a series of tetrahydroquinoline (THQ) derivatives as FXIa inhibitors. Compound 1 was identified as a potent and selective tool compound for proof of concept studies. It exhibited excellent antithrombotic efficacy in rabbit thrombosis models and did not prolong bleeding times. This demonstrates proof of concept for the FXIa mechanism in animal models with a reversible, small molecule inhibitor.

Factor VIIa (FVIIa) inhibitors have shown strong antithrombotic efficacy in preclinical thrombosis models with limited bleeding liabilities. Discovery of potent, orally active FVIIa inhibitors has been largely unsuccessful due to the requirement of a basic P1 group to interact with Asp189 in the S1 binding pocket, limiting their membrane permeability. We have combined recently reported neutral P1 binding substituents with a highly optimized macrocyclic chemotype to produce FVIIa inhibitors with low nanomolar potency and enhanced permeability.

This manuscript describes the discovery of a series of macrocyclic inhibitors of FXIa with oral bioavailability. Assisted by structure based drug design and ligand bound X-ray crystal structures, the group linking the P1 moiety to the macrocyclic core was modified with the goal of reducing H-bond donors to improve pharmacokinetic performance versus 9. This effort resulted in the discovery of several cyclic P1 linkers, exemplified by 10, that are constrained mimics of the bioactive conformation displayed by the acrylamide linker of 9. These cyclic P1 linkers demonstrated enhanced bioavailability and improved potency.

Inhibitors of the Tissue Factor/Factor VIIa (TF-FVIIa) complex are promising novel anticoagulants that show excellent efficacy and minimal bleeding in preclinical models. On the basis of a zwitterionic phenylglycine acylsulfonamide 1, a phenylglycine benzylamide 2 was shown to possess improved permeability and oral bioavailability. Optimization of the benzylamide, guided by X-ray crystallography, led to a potent TF-FVIIa inhibitor 18i with promising oral bioavailability, but promiscuous activity in an in vitro safety panel of receptors and enzymes. Introducing an acid on the pyrrolidine ring, guided by molecular modeling, resulted in highly potent, selective, and efficacious TF-FVIIa inhibitors with clean in vitro safety profile. The pyrrolidine acid 20 showed a moderate clearance, low volume of distribution, and a short t 1/2 in dog PK studies.

Aminoisoquinoline and isoquinoline groups have successfully replaced the more basic P1 benzamidine group of an acylsulfonamide factor VIIa inhibitor. Inhibitory activity was optimized by the identification of additional hydrophobic and hydrophilic P′ binding interactions. The molecular details of these interactions were elucidated by X-ray crystallography and molecular modeling. We also show that decreasing the basicity of the P1 group results in improved oral bioavailability in this chemotype.

The human, cytosolic enzyme isocitrate dehydrogenase 1 (IDH1) reversibly converts isocitrate to α-ketoglutarate (αKG). Cancer-associated somatic mutations in IDH1 result in a loss of this normal function but a gain in a new or neomorphic ability to convert αKG to the oncometabolite 2-hydroxyglutarate (2HG). To improve our understanding of the basis for this phenomenon, we have conducted a detailed kinetic study of wild-type IDH1 as well as the known 2HG-producing clinical R132H and G97D mutants and mechanistic Y139D and (newly described) G97N mutants. In the reductive direction of the normal reaction (αKG to isocitrate), dead-end inhibition studies suggest that wild-type IDH1 goes through a random sequential mechanism, similar to previous reports on related mammalian IDH enzymes. However, analogous experiments studying the reductive neomorphic reaction (αKG to 2HG) with the mutant forms of IDH1 are more consistent with an ordered sequential mechanism, with NADPH binding before αKG. This result was further confirmed by primary kinetic isotope effects for which saturating with αKG greatly reduced the observed isotope effect on (D)(V/K)NADPH. For the mutant IDH1 enzyme, the change in mechanism was consistently associated with reduced efficiencies in the use of αKG as a substrate and enhanced efficiencies using NADPH as a substrate. We propose that the sum of these kinetic changes allows the mutant IDH1 enzymes to reductively trap αKG directly into 2HG, rather than allowing it to react with carbon dioxide and form isocitrate, as occurs in the wild-type enzyme.

Two novel series of meta-linked phenylglycine-based macrocyclic FVIIa inhibitors have been designed to improve the rodent metabolic stability and PK observed with the precursor para-linked phenylglycine macrocycles. Through iterative structure-based design and optimization, the TF/FVIIa Ki was improved to subnanomolar levels with good clotting activity, metabolic stability, and permeability.

Inhibitors of Factor VIIa (FVIIa), a serine protease in the clotting cascade, have shown strong antithrombotic efficacy in preclinical thrombosis models with minimal bleeding liabilities. Discovery of potent, orally active FVIIa inhibitors has been largely unsuccessful because known chemotypes have required a highly basic group in the S1 binding pocket for high affinity. A recently reported fragment screening effort resulted in the discovery of a neutral heterocycle, 7-chloro-3,4-dihydroisoquinolin-1(2H)-one, that binds in the S1 pocket of FVIIa and can be incorporated into a phenylglycine FVIIa inhibitor. Optimization of this P1 binding group led to the first series of neutral, permeable FVIIa inhibitors with low nanomolar potency.

The recent discovery of the role of receptor interacting protein 1 (RIP1) kinase in tumor necrosis factor (TNF)-mediated inflammation has led to its emergence as a highly promising target for the treatment of multiple inflammatory diseases. We screened RIP1 against GSK's DNA-encoded small-molecule libraries and identified a novel highly potent benzoxazepinone inhibitor series. We demonstrate that this template possesses complete monokinase selectivity for RIP1 plus unique species selectivity for primate versus nonprimate RIP1. We elucidate the conformation of RIP1 bound to this benzoxazepinone inhibitor driving its high kinase selectivity and design specific mutations in murine RIP1 to restore potency to levels similar to primate RIP1. This series differentiates itself from known RIP1 inhibitors in combining high potency and kinase selectivity with good pharmacokinetic profiles in rodents. The favorable developability profile of this benzoxazepinone template, as exemplified by compound 14 (GSK'481), makes it an excellent starting point for further optimization into a RIP1 clinical candidate.

Pyridine-based Factor XIa (FXIa) inhibitor (S)-2 was optimized by modifying the P2 prime, P1, and scaffold regions. This work resulted in the discovery of the methyl N-phenyl carbamate P2 prime group which maintained FXIa activity, reduced the number of H-bond donors, and improved the physicochemical properties compared to the amino indazole P2 prime moiety. Compound (S)-17 was identified as a potent and selective FXIa inhibitor that was orally bioavailable. Replacement of the basic cyclohexyl methyl amine P1 in (S)-17 with the neutral p-chlorophenyltetrazole P1 resulted in the discovery of (S)-24 which showed a significant improvement in oral bioavailability compared to the previously reported imidazole (S)-23. Additional improvements in FXIa binding affinity, while maintaining oral bioavailability, was achieved by replacing the pyridine scaffold with either a regioisomeric pyridine or pyrimidine ring system.

The role of citrate as a physiological modulator of mammalian acetyl-CoA carboxylases (ACCs) has been well studied; however, the mechanism has not been clearly defined. In the current study, we found that citrate activated recombinant human ACC2 by more than approximately 1000-fold, but activated recombinant human ACC1 only by approximately 4-fold. The data fit best to a model which accounts for cooperative binding of two citrate molecules. Citrate activates ACCs at lower concentrations and inhibits at higher concentrations with apparent K(d) values of 0.8+/-0.3 and 3.4+/-0.6 mM, and apparent K(i) values of 20+/-8 and 38 +/-8 mM for ACC1 and ACC2, respectively. In the absence of added citrate, both ACC1 and ACC2 were inactivated by avidin rapidly and completely. Addition of 10 mM citrate protected ACC2 from avidin inactivation; however, protection by citrate was less pronounced for ACC1. In response to citrate treatment, different aggregation patterns for the two isoforms were also observed by dynamic light scattering. Although formation of aggregates by both isoforms was sensitive to citrate, with Mg2+ and Mg-citrate addition only formation of the ACC2 aggregates showed a dependence on citrate concentration. Mass spectrometry data indicated phosphorylation of Ser79 of ACC1 (a serine known to regulate activity), and the corresponding Ser221 of ACC2. Taken together, these data suggest that recombinant human ACC1 and ACC2 are differentially activated by citrate, most likely through conformational changes leading to aggregation, with ACC2 being more sensitive to this activator.

Introduction of the phenyl piperidinone and phenyl pyridinone P4 moieties in the anthranilamide scaffold led to potent, selective, and orally bioavailable inhibitors of factor Xa. Anthranilamide 28 displayed comparable efficacy to apixaban in the rabbit arteriovenous-shunt (AV) thrombosis model.

The synthesis, structural activity relationships (SAR), and selectivity profile of a potent series of phenylalanine diamide FXIa inhibitors will be discussed. Exploration of P1 prime and P2 prime groups led to the discovery of compounds with high FXIa affinity, good potency in our clotting assay (aPPT), and high selectivity against a panel of relevant serine proteases as exemplified by compound 21. Compound 21 demonstrated good in vivo efficacy (EC50=2.8μM) in the rabbit electrically induced carotid arterial thrombosis model (ECAT).

BMS-654457 ((+) 3′-(6-carbamimidoyl-4-methyl-4-phenyl-1,2,3,4-tetrahydro-quinolin-2-yl)-4-carbamoyl-5′-(3-methyl-butyrylamino)-biphenyl-2-carboxylic acid) is a small-molecule factor XIa (FXIa) inhibitor. We evaluated the in vitro properties of BMS-654457 and its in vivo activities in rabbit models of electrolytic-induced carotid arterial thrombosis and cuticle bleeding time (BT). Kinetic studies conducted in vitro with a chromogenic substrate demonstrated that BMS-654457 is a reversible and competitive inhibitor for FXIa. BMS-654457 increased activated partial thromboplastin time (aPTT) without changing prothrombin time. It was equipotent in prolonging the plasma aPTT in human and rabbit, and less potent in rat and dog. It did not alter platelet aggregation to ADP, arachidonic acid and collagen. In vivo, BMS-654457 or vehicle was given IV prior to initiation of thrombosis or cuticle transection. Preservation of integrated carotid blood flow over 90 min (iCBF, % control) was used as a marker of antithrombotic efficacy. BMS-654457 at 0.37 mg/kg + 0.27 mg/kg/h produced almost 90 % preservation of iCBF compared to its vehicle (87 ± 10 and 16 ± 3 %, respectively, n = 6 per group) and increased BT by 1.2 ± 0.04-fold (P

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.

A multidisciplinary, fragment-based screening approach involving protein ensemble docking and biochemical and NMR assays is described. This approach led to the discovery of several structurally diverse, neutral surrogates for cationic factor VIIa P1 groups, which are generally associated with poor pharmacokinetic (PK) properties. Among the novel factor VIIa inhibitory fragments identified were aryl halides, lactams, and heterocycles. Crystallographic structures for several bound fragments were obtained, leading to the successful design of a potent factor VIIa inhibitor with a neutral lactam P1 and improved permeability.

In an effort to identify a potential back-up to apixaban (Eliquis®), we explored a series of diversified P4 moieties. Several analogs with substituted gem-dimethyl moieties replacing the terminal lactam of apixaban were identified which demonstrated potent FXa binding affinity (FXa Ki), good human plasma anticoagulant activity (PT EC2x), cell permeability, and oral bioavailability.

Dethiobiotin synthetase (DTBS; E.C. 6.6.6.6), the penultimate enzyme in the biosynthesis of the essential vitamin biotin, is a new potential target for novel herbicides. Inhibitors were designed based on mechanistic and structural information. The in-vitro activities of these potential inhibitors versus the bacterial enzyme are reported here. Mimics of 7,8-diaminopelargonic acid (DAPA) or the DAPA carbamate reaction intermediate were substrates or partial substrates for the enzyme. Synergistic binding with ATP was noted with compounds which contained an amino functionality. NMR studies and X-ray structures confirmed that the inhibitors could be phosphorylated by the enzyme. Several series of potential inhibitors were designed to take advantage of this partial substrate activity by generating potentially more tightly bound phosphorylated inhibitors in situ. Structure-activity relationships for these series based on both substrate and inhibitory activity are described herein. An X-ray structure for one of these inhibitors is also discussed. Although considerable potential for inhibitors of this type was demonstrated, none of the compounds reported showed sufficient herbicidal activity to be a commercial proposition.

Posttranslational modifications of histones play important roles in chromatin structure and genomic stability. Distinct lysine residues in histones are targets for covalent binding of biotin, catalyzed by holocarboxylase synthetase (HCS) and biotinidase (BTD). Histone biotinylation has been implicated in heterochromatin structures, DNA repair, and mitotic chromosome condensation. To test whether HCS and BTD deficiency alters histone biotinylation, and to characterize phenotypes associated with HCS and BTD deficiency, HCS- and BTD-deficient flies were generated by RNA interference (RNAi). Expression of HCS and BTD decreased by 65–90% in RNAi-treated flies, as judged by mRNA abundance, BTD activity, and abundance of HCS protein. Decreased expression of HCS and BTD caused decreased biotinylation of K9 and K18 in histone H3. This was associated with altered expression of 201 genes in HCS-deficient flies. Lifespan of HCS- and BTD-deficient flies decreased by up to 32% compared to wild-type controls. Heat tolerance decreased by up to 55% in HCSdeficient flies compared to controls, as judged by survival times; effects of BTD deficiency were minor. Consistent with this observation, HCS deficiency was associated with altered expression of 285 heat-responsive genes. HCS and BTD deficiency did not affect cold tolerance, suggesting stressspecific effects of chromatin remodeling by histone biotinylation. This is the first study to provide evidence that HCS-dependent histone biotinylation affects gene function and phenotype, suggesting that the complex phenotypes of HCS- and BTD-deficiency disorders may reflect chromatin structure changes. 1This

Five active site residues, Thr11, Glu12, Lys15, Lys37, and Ser41, implicated by the protein crystal structure studies of Escherichia coli DTBS, were mutated to determine their function in catalysis and substrate binding. Nine mutant enzymes, T11V, E12A, E12D, K15Q, K37L, K37Q, K37R, S41A, and S41C, were overproduced in an E. coli strain lacking a functional endogenous DTBS gene and purified to homogeneity. Replacement of Thr11 with valine resulted in a 24,000-fold increase in the Km(ATP) with little or no change in the Kd(ATP), KM(DAPA) and DTBS k(cat), suggesting an essential role for this residue in the steady-state affinity for ATP. The two Glu12 mutants showed essentially wild-type DTBS activity (slightly elevated k(cat)'s). Unlike wild-type DTBS, E12A had the same apparent KM(DAPA) at subsaturating and saturating ATP concentrations, indicating a possible role for Glu12 in the binding synergy between DAPA and ATP. The mutations in Lys15 and Lys37 resulted in loss of catalytic activity (0.01% and0.9% of wild-type DTBS k(cat) for K15Q and the Lys37 mutant enzymes, respectively) and higher KM's for both DAPA (40-fold and100-fold higher than wild-type for the K15Q and Lys37 mutant enzymes, respectively) and ATP (1800-fold and10-fold higher than wild-type for K15Q and the K37 mutant enzymes, respectively). These results strongly suggest that Lys15 and Lys37 are crucial to both catalysis and substrate binding. S41A and S41C had essentially the same k(cat) as wild-type and had moderate increases in the DAPA and ATP KM and Kd (ATP) values. Replacement of Ser41 with cysteine resulted in larger effects than replacement with alanine. These data suggest that the H-bond between N7 of DAPA and the Ser41 side chain is not very important for catalysis. The catalytic behavior of these mutant enzymes was also studied by pulse-chase experiments which produced results consistent with the steady-state kinetic analyses. X-ray crystallographic studies of four mutant enzymes, S41A, S41C, K37Q, and K37L, showed that the crystals were essentially isomorphous to that of the wild-type DTBS. The models of these mutant enzymes were well refined (1.9 -2.6 A) and showed good similarity to the wild-type enzyme (rmsd of C alpha atoms: 0.16-0.24 A). The crystal structure of S41C complexed with DAPA, Mn2+/Mg2+, and AMPPCP revealed a localized conformational change (rotations of side chains of Cys41 and Thr11) which can account for the changes in the kinetic parameters observed for S41C. The crystal structures of the Lys37 mutant enzymes showed that the positive charge of the side chain of Lys37 is indispensable. Mutations of Lys37 to either glutamine or leucine resulted in a shift of the metal ion (up to 0.5 A) together with side chains of other active site residues which could disrupt the subtle balance between the positive and negative charges in the active site. The conformational change of the phosphate binding loop (Gly8-X-X-X-X-X-Gly14-Lys15-Thr16) upon nucleotide binding observed previously [Huang, W., Jia, J., Gibson, K. J., Taylor, W. S., Rendina, A. R., Schneider, G.,Lindqvist, Y. (1995) Biochemistry 34, 10985] appears to be important to attain the proper active site scaffold.

Graves' orbitopathy (GO) is the most common and important extrathyroidal manifestation of Flajani-Basedow-Graves' disease, with autoimmune etiology. In most cases they are mild forms, in 3-5% they are severe and progressive. For therapeutic purposes, it is classified according to the severity (mild, moderate-severe or sight threatening), to the activity (active if clinical activity score is=3), and to the impact on quality of life. The choice of medical or surgical therapy depends on the activity of the disease. Therapy for mild GO consists of abolition of risk factors, local treatments, oral administration of selenium. Therapy for moderate-severe and active GO consists of administration of intravenous, oral, topic and local (retrobulbar, peribulbar and subconjunctival) glucocorticoids (GC). The therapy of choice, after careful selection of patients, is pulse therapy with intravenous GC, with 79% of response. Orbital radiotherapy is effective in 60% of cases; diabetes mellitus and hypertension are absolute contraindications. Contemporary administration of oral GC and orbital radiotherapy are more effective than single therapies. Marginal and not validated therapies are cyclosporine, somatostatin analogues, TNF-a inhibitors and rituximab. The treatment for dysthyroid optic neuropathy (DON) consists of combination of steroids, orbital radiotherapy and, if necessary, orbital decompression surgery. The surgical therapies are orbital decompression and rehabilitative surgery.

The crystal structures of six complexes of homodimeric Escherichia coli dethiobiotin synthetase with a variety of substrates, substrate analogs, and products have been determined to high resolution. These include (1) the binary complex of dethiobiotin synthetase and the N7-carbamate of 7,8-diaminononanoic acid, (2) the binary complex of enzyme and the alternate substrate, 3-(1-aminoethyl)-nonanedioic acid, (3) the binary complex of enzyme with the product ADP, (4) the quaternary complex of enzyme, ADP, the N7-carbamate of 7,8-diaminononanoic acid, and Ca2+, (5) the ternary complex of enzyme, the ATP analog adenylyl (beta, gamma-methylene)diphosphonate, and the N7-carbamate of 7,8-diaminononanoic acid, and (6) the quaternary complex of enzyme, the ATP analog adenylyl (beta, gamma-methylene)diphosphonate, 7,8-diaminononanoic acid, and Mn2+. One molecule of each substrate binds to one monomer of the enzyme. ADP and the ATP analogue bind to the classical mononucleotide binding fold with the phosphate groups close to the phosphate binding loop Gly8--Thr16 between beta-strand beta 1 and the N-terminus of alpha-helix alpha 1. The adenine ring is bound in a pocket between beta-strands beta 6 and beta 7. In the quaternary complex with Mn2+, the metal binding site is found in the vicinity of the beta- and gamma-phosphate groups. Two oxygen atoms from the phosphates and oxygen atoms from the side chains of Asp54, Thr16, and Glu115 are ligands to the Mn2+ ion in the quaternary complex. In the complex with ADP and the N7-carbamate of 7,8-diaminononanoic acid prepared in the presence of Ca2+ ions, a different metal binding site is found. The Ca2+ ion is coordinated to an oxygen atom of the alpha-phosphate group of the nucleotide, the side chain of Asp54, and solvent molecules. The 7,8-diaminononanoic acid substrate molecule interacts with residues from both subunits, making the dimer the minimal functional unit. The diamino group binds between the loops after beta 2 and beta 4, and the terminal carboxyl group at the hydrophobic tail of the substrate interacts with the amino terminus of helix alpha 5 and with the side chain of Tyr187 in helix alpha 6 of the second subunit at the monomer-monomer interface. Strong additional electron density close to the N7 nitrogen atom of the 7,8-diaminononanoic acid substrate in some complexes indicates that, even in the absence of added bicarbonate in the crystallization mixture, the carbamylated intermediate is formed in the crystal.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are known driver mutations in acute myeloid leukemia (AML) and other cancer types. Patient outcomes in AML have remained poor, especially for patients above 60 years of age who typically do not tolerate high dose chemotherapy and stem cell transplantation, leading to cure rates below 20%. The development of novel targeted therapies for defined AML subtypes is urgently desired. Inhibitors of mutants of the closely related IDH2 gene as well as IDH1 have recently been described and show promising pre-clinical and early phase clinical activity. However, the specific molecular and functional effects of IDH1 inhibitors in AML, including in primary patients' cells, have not been reported yet. Here, we report the development of novel allosteric inhibitors of mutant IDH1 for differentiation therapy of acute myeloid leukemia. A high-throughput biochemical screen targeting an IDH1 heterodimer composed of R132H and WT IDH1 led to the identification of a tetrahydropyrazolopyridine series of inhibitors. Structural and biochemical analyses revealed that these novel compounds bind to an allosteric site that does not contact any of the mutant residues in the enzymes active site and inhibit enzymatic turnover. The enzyme complex locked in the catalytically inactive conformation inhibits the production of the oncometabolite 2-hydroxyglutarate (2-HG). In biochemical studies, we observed potent inhibition of several different clinically relevant R132 mutants in the presence or absence of the cofactor NADPH, accompanied by significant decrease in H3K9me2 levels. Allosteric inhibitor treatment of primary AML patients' cells with different clinically relevant R132 mutants of IDH1 ex vivo uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block, increased cell death and induction of differentiation both at the level of leukemic blasts and immature stem-like cells. Allosteric inhibition of IDH1 also led to a decrease in blasts in an in vivo xenotransplantation model. At the molecular level, enhanced reduced representation bisulfite sequencing showed that treatment with allosteric IDH1 inhibitors led to a significant reversal of the DNA cytosine hypermethylation pattern induced by mutant IDH1, accompanied by gene expression changes of key sets of genes and pathways, including "Cell Cycle", "G1/S transition", "Cellular growth and proliferation", and "Cell death and survival". Taken together, our findings provide novel insight into the cellular and molecular effects of inhibition of mutant IDH1 in primary AML patients' cells. Furthermore, our study provides proof-of-concept for the molecular and biological activity of novel allosteric inhibitors for targeting of different mutant forms of IDH1 in leukemia, and opens new avenues for future investigations with these and other allosteric inhibitors for targeting mutant IDH1 in leukemia and other cancers. Disclosures Gao: GlaxoSmithKline: Employment. Pietrak:GlaxoSmithKline: Employment. Rendina:GlaxoSmithKline: Employment. Rominger:GlaxoSmithKline: Employment. Quinn:GlaxoSmithKline: Employment. Smallwood:GlaxoSmithKline: Employment. Wiggall:GlaxoSmithKline: Employment. Reif:GlaxoSmithKline: Employment. Schmidt:GlaxoSmithKline: Employment. Qi:GlaxoSmithKline: Employment. Zhao:GlaxoSmithKline: Employment. Joberty:GlaxoSmithKline: Employment. Faelth-Savitski:GlaxoSmithKline: Employment. Bantscheff:GlaxoSmithKline: Employment. Drewes:GlaxoSmithKline: Employment. Duraiswami:GlaxoSmithKline: Employment. Brady:GlaxoSmithKline: Employment. Concha:GlaxoSmithKline: Employment. Adams:GlaxoSmithKline: Employment. Schwartz:GlaxoSmithKline: Employment. McCabe:GlaxoSmithKline: Employment.

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are known driver mutations in acute myeloid leukemia (AML) and other cancer types. AML is hallmarked by a differentiation block and patient outcomes remain poor, especially for patients above 60 years of age who typically do not tolerate high dose chemotherapy and stem cell transplantation, leading to cure rates below 20%. Hence the development of novel targeted therapies for treatment of AML subtypes are required. Of note, inhibitors of mutants of the closely related IDH2 gene as well as IDH1 have recently been described and show promising pre-clinical and early phase clinical activity. However, the specific molecular and functional effects of IDH1 inhibitors in AML, including in primary patients' cells, have not been reported yet. Here, we report the development of novel allosteric inhibitors of mutant IDH1 for differentiation therapy of acute myeloid leukemia. A high-throughput biochemical screen targeting an IDH1 heterodimer composed of R132H and WT IDH1 led to the identification of a tetrahydropyrazolopyridine series of inhibitors. Structural and biochemical analyses revealed that these novel compounds bind to an allosteric site that does not contact any of the mutant residues in the enzymes active site and inhibit enzymatic turnover. The enzyme complex locked in the catalytically inactive conformation inhibits the production of the oncometabolite 2-hydroxyglutarate (2-HG). In biochemical studies, we observed potent inhibition of several different clinically relevant R132 mutants in the presence or absence of the cofactor NADPH, accompanied by significant decrease in H3K9me2 levels. Treatment of primary IDH1 mutant AML patients' cells ex vivo uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block, increased cell death and induction of differentiation both at the level of leukemic blasts and immature stem-like cells. Allosteric inhibition of IDH1 also led to a decrease in leukemic blasts in an in vivo xenotransplantation model. At the molecular level, enhanced reduced representation bisulfite sequencing showed that treatment with allosteric IDH1 inhibitors led to a significant reversal of the DNA cytosine hypermethylation pattern induced by mutant IDH1, accompanied by gene expression changes of key sets of genes and pathways, including “Cell Cycle”, “G1/S transition”, “Cellular growth and proliferation”, and “Cell death and survival”. Taken together, our findings provide novel insight into the effects of inhibition of mutant IDH1 in primary AML patients' cells and open avenues for future investigations with these and other novel allosteric inhibitors for targeting IDH1 mutants in leukemia and possibly in other cancers. Citation Format: Ujunwa C. Okoye-Okafor, Boris Bartholdy, Jessy Cartier, Enoch Gao, Beth Pietrak, Alan R. Rendina, Cynthia Rominger, Chad Quinn, Angela Smallwood, Ken Wiggall, Alexander Reif, Stan Schmidt, Hongwei Qi, Huizhen Zhao, Gerard Joberty, Maria Faelth-Savitski, Marcus Bantscheff, Gerard Drewes, Chaya Duraiswami, Pat Brady, Swathi-Rao Narayanagari, Ileana Antony-Debre, Kelly Mitchell, Heng Rui Wang, Yun-Ruei Kao, Maximilian Christopeit, Luis Carvajal, Laura Barreyro, Elisabeth Paietta, Britta Will, Nestor Concha, Nicholas D. Adams, Benjamin Schwartz, Michael T. McCabe, Jaroslav Maciejewski, Amit Verma, Ulrich Steidl. Novel allosteric IDH1 mutant Inhibitors for differentiation therapy of acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C38.

Apixaban is a potent, direct, selective, and orally active inhibitor of coagulation factor Xa. Rate constants for apixaban binding to free and prothrombinase-bound factor Xa were measured using multiple techniques. The inhibition mechanism was determined in purified systems and in a plasma prothrombin clotting time assay. Apixaban inhibits factor Xa with a K(i) of 0.25 nM at 37°C, an association rate constant of approximately 20 μM(-1) s(-1), and a dissociation half-life of 1-2 min. Under physiological conditions apixaban exhibits mixed-type inhibition and maintains high factor Xa affinity with a K(i) of 0.62 nM and association rate constant of 12 μM(-1) s(-1) for prothrombinase, and a K(i) of 1.7 nM and association rate constant of 4 μM(-1) s(-1) for the prothrombinase:prothrombin complex. Experiments in prothrombin depleted human plasma showed that the mechanism and kinetics of inhibition are maintained in plasma. The mechanistic detail derived from these experiments can be used to understand and interpret the pharmacodynamic action of apixaban.

SummaryFactor (F) VIIa in association with tissue factor (TF) is the primary in vivo initiator of blood coagulation and activates FX and FIX to generate thrombin, which plays a key role in the pathogenesis of thrombosis. We evaluated the enzyme kinetics, antithrombotic and antihaemostatic properties of BMS-593214, an active-site, direct FVIIa inhibitor. Studies were conducted in enzymatic assays, and in anesthetised rabbit models of electrically-induced carotid arterial thrombosis (AT), thread-induced vena cava venous thrombosis (VT) and cuticle bleeding time (BT). Antithrombotic efficacy of BMS-593214 given intravenously was evaluated for both the prevention and treatment of AT and VT. BMS-593214 displayed direct, competitive inhibition of human FVIIa in the hydrolysis of a tripeptide substrate with Ki of 5 nM. However, it acted as a noncompetitive inhibitor of the activation of the physiological substrate FX by TF/VIIa with Ki of 9.3 nM. BMS-593214 showed selectivity for FVIIa and exhibited species differences in TF-FVIIa-dependent anticoagulation with similar potency in human and rabbit plasma. BMS-593214 was efficacious in the prevention and treatment models of AT and VT with ED50 values of 1.1 to 3.1 mg/kg. Furthermore, BMS-593214 exhibited a wide therapeutic window with respect to BT. These results suggest that inhibition of FVIIa with small-molecule active-site inhibitors represents a promising antithrombotic approach for the development of new therapies for the prevention and treatment of AT and VT.

We previously disclosed a series of highly potent FXa inhibitors bearing alpha-substituted (CH(2)NR(1)R(2)) phenylcyclopropyl P4 moieties in the pyrazolodihydropyridone core system. Herein, we describe our continuous SAR efforts in this series. Effects of the C-3 substitution of the pyrazolodihydropyridone core and the alpha-substitution (R group) of the cyclopropyl ring on FXa binding affinity (FXa K(i)), human plasma anticoagulant activity (PT EC(2x)) and permeability are discussed. A set of compounds obtained from optimization of the R group and the C-3 substituent were orally bioavailable in dogs. Furthermore, representative compounds were highly efficacious in the rabbit arterio-venous shunt thrombosis model (EC(50)s=29-81nM).

Ortho-substituted biphenyl moieties are widely used in drug design. We herein report a successful use of the perpendicular conformation of the alpha-substituted phenylcyclopropyl groups to mimic the aplanar, biologically active conformation of the ortho-substituted biphenyl moieties to achieve structural diversity. This is exemplified by the design and synthesis of a series of highly potent pyrazole bicyclic-based Factor Xa (FXa) inhibitors bearing alpha-substituted phenylcyclopropyl P4 moieties. The designed perpendicular conformation was confirmed by the X-ray structure of FXa-bound compound 2r. The potential structural basis for the high FXa potency in the phenylcyclopropyl P4 analogs and their improved FXa inhibitory activities compared with the biphenyl P4 counterparts are discussed.

The selective grass herbicides diclofop, haloxyfop, and trifop (((aryloxy)phenoxy)propionic acids) and alloxydim, sethoxydim, and clethodim (cyclohexanediones) are potent, reversible inhibitors of acetyl-coenzyme A carboxylase (ACC) partially purified from barley, corn, and wheat. Although inhibition of the wheat enzyme by clethodim and diclofop is noncompetitive versus each of the substrates adenosine triphosphate (ATP), HCO{sub 3}{sup {minus}}, and acetyl-coenzyme A (acetyl-CoA), diclofop and clethodim are nearly competitive versus acetyl-CoA since the level of inhibition is most sensitive to the concentration of acetyl-CoA (K{sub is} < K{sub ii}). To conclusively show whether the herbicides interact at the biotin carboxylation site or the carboxyl transfer site, the inhibition of isotope exchange and partial reactions catalyzed at each site was studied with the wheat enzyme. Only the ({sup 14}C)acetyl-CoA-malonyl-CoA exchange and decarboxylation of ({sup 14}C)malonyl-CoA reactions are strongly inhibited by clethodim and diclofop, suggesting that the herbicides interfere with the carboxyl transfer site rather than the biotin carboxylation site of the enzyme. Double-inhibition studies with diclofop and clethodim suggest that the ((aryloxy)phenoxy)propionic acid and cyclohexanedione herbicides may bind to the same region of the enzyme.

Efforts to identify a suitable follow-on compound to razaxaban (compound 4) focused on modification of the carboxamido linker to eliminate potential in vivo hydrolysis to a primary aniline. Cyclization of the carboxamido linker to the novel bicyclic tetrahydropyrazolopyridinone scaffold retained the potent fXa binding activity. Exceptional potency of the series prompted an investigation of the neutral P1 moieties that resulted in the identification of the p-methoxyphenyl P1, which retained factor Xa binding affinity and good oral bioavailability. Further optimization of the C-3 pyrazole position and replacement of the terminal P4 ring with a neutral heterocycle culminated in the discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban, compound 40). Compound 40 exhibits a high degree of fXa potency, selectivity, and efficacy and has an improved pharmacokinetic profile relative to 4.

We previously reported a series of enantiopure cis-(1R,2S)-cyclopentyldiamine derivatives as potent and selective inhibitors of Factor Xa (FXa). Herein, we describe our approach to improve the metabolic stability of this series via core modifications. Multiple resulting series of compounds demonstrated similarly high FXa potency and improved metabolic stability in human liver microsomes compared with the cyclopentyldiamide 1. (3R,4S)-Pyrrolidinyldiamide 31 was the best overall compound with human FXa Ki of 0.50 nM, PT EC2x of 2.1 μM in human plasma, bioavailability of 25% and t1/2of 2.7 h in dogs. Further biochemical characterization of compound 31 is also presented.

In the search of Factor Xa (FXa) inhibitors structurally different from the pyrazole-based series, we identified a viable series of enantiopure cis-(1R,2S)-cycloalkyldiamine derivatives as potent and selective inhibitors of FXa. Among them, cyclohexyldiamide 7 and cyclopentyldiamide 9 were the most potent neutral compounds, and had good anticoagulant activity comparable to the pyrazole-based analogs. Crystal structures of 7-FXa and 9-FXa illustrate binding similarities and differences between the five- and the six-membered core systems, and provide rationales for the observed SAR of P1 and linker moieties.

The isocitrate dehydrogenase 1 (IDH1) and IDH2 genes are mutated in acute myelogenous leukemia, low-grade glioma, intrahepatic cholangiocarcinoma, and chondrosarcomas. IDH1 and IDH2 normally function to convert isocitrate into alpha-ketoglutarate. However, when these enzymes are mutated at select residues the mutant enzymes now convert α-KG into 2-hydroxyglutarate (2-HG). In normal cells, 2-HG levels are typically extremely low, but IDH1/2 mutant cells can accumulate up to 10 mM 2-HG. In an effort to counteract the neomorphic activity of mutant IDH enzymes, we identified and developed potent inhibitors of IDH1. The compounds inhibit IDH1 catalytic activity in biochemical assays and reduce 2-HG production in IDH1-mutant cell lines. Consistent with the fact that 2-HG inhibits α-KG dependent enzymes including histone demethylases and Tet family hydroxylases, these IDH1 inhibitors induce a decrease in several histone methylation marks and also DNA methylation. These data demonstrate that small molecule inhibitors can reverse many of the epigenetic effects of mutant IDH1. Note: This abstract was not presented at the meeting. Citation Format: Cynthia Rominger, Chad Quinn, Enoch Gao, Beth Pietrak, Alan Rendina, Angela Smallwood, Arthur Groy, Susan Korenchuk, Charles McHugh, Ken Wiggall, Alexander Reif, Stanley Schmidt, Hongwei Qi, Huizhen Zhao, Nestor Concha, Christopher Carpenter, Juan Luengo, Ryan Kruger, Benjamin Schwartz, Nicholas Adams, Michael T. McCabe. A novel inhibitor of IDH1 abrogates 2-HG production and reverses aberrant epigenetic alterations in IDH1 mutant cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3514. doi:10.1158/1538-7445.AM2015-3514