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PDF - 60K, Linear correlation observed between tumor growth inhibition (% T/C) or tumor regression and the fraction of time over the in vivo S473P-Akt IC80 in different cancer cell line-derived tumor xenografts implanted in nude mice (represented as dots) and nude rats (represented as triangles) following NVP-BYL719 treatment (R2=0.77, p

Somatic PIK3CA mutations are frequently found in solid tumors, raising the hypothesis that selective inhibition of PI3Kα may have robust efficacy in PIK3CA-mutant cancers while sparing patients the side-effects associated with broader inhibition of the class I phosphoinositide 3-kinase (PI3K) family. Here, we report the biologic properties of the 2-aminothiazole derivative NVP-BYL719, a selective inhibitor of PI3Kα and its most common oncogenic mutant forms. The compound selectivity combined with excellent drug-like properties translates to dose- and time-dependent inhibition of PI3Kα signaling in vivo, resulting in robust therapeutic efficacy and tolerability in PIK3CA-dependent tumors. Novel targeted therapeutics such as NVP-BYL719, designed to modulate aberrant functions elicited by cancer-specific genetic alterations upon which the disease depends, require well-defined patient stratification strategies in order to maximize their therapeutic impact and benefit for the patients. Here, we also describe the application of the Cancer Cell Line Encyclopedia as a preclinical platform to refine the patient stratification strategy for NVP-BYL719 and found that PIK3CA mutation was the foremost positive predictor of sensitivity while revealing additional positive and negative associations such as PIK3CA amplification and PTEN mutation, respectively. These patient selection determinants are being assayed in the ongoing NVP-BYL719 clinical trials. Mol Cancer Ther; 13(5); 1117–29. ©2014 AACR.

PDF - 129K, Legends for Supplementary Figures 1 through 4 and Supplementary Tables 1 through 7. Supplementary Table 1. Determination of NVP-BYL719 in vitro effects on human protein kinases. Supplementary Table 2. Activity profile of NVP-BYL719 in the Invitrogen Kinase panel. Supplementary Table 3. Activity profile of NVP-BYL719 in the Ambit Kinase panel. Supplementary Table 4. NVP-BYL719 Kd (nmol/L) determination in the Ambit Kinase panel for the hits found to be inhibited >90%. Supplementary Table 5. NVP-BYL719 anti-tumor effects in diverse cancer cell line-derived xenograft models. Supplementary Table 6. CCLE cell lines responsive to NVP-BYL719. Supplementary Table 7 Anti-tumor effect of NVP-BYL719 in patient-derived xenograft models carrying PIK3CA genetic alterations.

PDF - 71K, Rat1-myr-p110alpha (blue), beta (green) or delta (red) cells were treated with increasing concentrations of NVP-BYL719 for 30 minutes. Levels of S473P-Akt in cell extracts were quantified by Reverse Phase Protein Array as described in (18) and plotted as percentage of untreated control cells. The graph illustrates n=3 independent experiments. IC50s (plus/minus) SD and IC80s (plus/minus) SD of n=3 independent experiments are reported..

PDF - 92K, NVP-BYL719 does not inhibit mTOR and PIKKs involved in DNA damage-repair processes. A. TSC1 -/- MEFs cells were grown in a 96-well format and treated for 1 h with increased concentrations of RAD001 or NVP-BYL719 (from 0.5 nmol/L to 10 ?mol/L in 1 third dilution steps) and immediately fixed. S235/236P-RPS6 levels were measured and IC50 determined with the Excel module XLfit. Background (no primary Ab incubated); BL, Baseline. B: TSC1 -/- MEFs cells were treated with increasing concentrations of NVP-BYL719 as indicated or RAD001 at 500 nmol/L or an equivalent DMSO concentration for 30 minutes. Levels of S235/236P-RPS6 and total RPS6 in protein- normalized lysates were detected by Western blot analyzis using an activation-state specific antibody, followed by incubation with species- specific HRP-labeled secondary antibody and signal development by ECL. C: 24 h post seeding, A549 cells were treated at the same time with Actinomycin D (Act D) at a concentration of 5 ?mol/L (an agent used to induce DNA damage), and with increasing concentrations of NVP-BYL719 as indicated or with the vehicle control (DMSO) for 1 h. Levels of S15P-p53 and tubulin in protein-normalized lysates were detected by Western blot analysis using an activation-state specific antibody, followed by incubation with species- specific HRP-labeled secondary antibody and signal development by ECL. D: 24 h post seeding, U2OS cells were pre-treated for 1 h with increased concentrations of NVP-BYL719 or KU55933 a specific small molecular mass inhibitor of ATM (Supplementary reference 1) at a concentration of 10 ?mol/L or with the vehicle control (DMSO). The cells were then irradiated with 15 Gy and re-incubated at 37 degrees C for 1 h and then lysed. Levels of S1981P-ATM in protein- normalized lysates were detected by Western blot analysis using an activation-state specific antibody, followed by incubation with species- specific HRP-labeled secondary antibody and signal development by ECL.

PDF - 37K, NVP-BYL719 was tested at 10 different concentrations (from 0.5 nmol/L to 10 μmol/L in 1 third dilution steps) against CLK2 (A) and LRRK2 (B). SelectScreenTM Kinase Profiling Service uses XLfit from IDBS to determine the IC50 values (IC50 CLK2=621 nmol/L; IC50 LRRK2=3'220 nmol/L). The dose response curve is curve fit to model number 205 (sigmoidal dose-response model).

Table S1. Specific mutations of genes listed in Table 1. Table S2. Foundation Medicine sequencing calls of cancer-specific gene panel. Table S3. Gene lists corresponding to microarray analysis described in Figures 1A and 1B: (A) binimetinib and (B) ribociclib. Significant genes (in bold type) are defined as having an adjusted p-value of 0.25 or less.

This document contains Supplementary information, including materials and methods as well as Figure Legends for the Supplementary Figures.

Purpose: To test second-line personalized medicine combination therapies, based on genomic and proteomic data, in patient-derived xenograft (PDX) models.Experimental Design: We established 12 PDXs from BRAF inhibitor–progressed melanoma patients. Following expansion, PDXs were analyzed using targeted sequencing and reverse-phase protein arrays. By using multi-arm preclinical trial designs, we identified efficacious precision medicine approaches.Results: We identified alterations previously described as drivers of resistance: NRAS mutations in 3 PDXs, MAP2K1 (MEK1) mutations in 2, BRAF amplification in 4, and aberrant PTEN in 7. At the protein level, re-activation of phospho-MAPK predominated, with parallel activation of PI3K in a subset. Second-line efficacy of the pan-PI3K inhibitor BKM120 with either BRAF (encorafenib)/MEK (binimetinib) inhibitor combination or the ERK inhibitor VX-11e was confirmed in vivo. Amplification of MET was observed in 3 PDX models, a higher frequency than expected and a possible novel mechanism of resistance. Importantly, MET amplification alone did not predict sensitivity to the MET inhibitor capmatinib. In contrast, capmatinib as single agent resulted in significant but transient tumor regression in a PDX with resistance to BRAF/MEK combination therapy and high pMET. The triple combination capmatinib/encorafenib/binimetinib resulted in complete and sustained tumor regression in all animals.Conclusions: Genomic and proteomic data integration identifies dual-core pathway inhibition as well as MET as combinatorial targets. These studies provide evidence for biomarker development to appropriately select personalized therapies of patients and avoid treatment failures. Clin Cancer Res; 22(7); 1592–602. ©2015 AACR.See related commentary by Hartsough and Aplin, p. 1550

A. PCA of protein expression profiles of WM3965 control and progression tumor grafts. B. unsupervised hierarchical clustering of proteins identified in the PCA.

Purpose: Recently, several studies reported a strong functional link between histone deacetylases (HDAC) and the development of tumors of the large intestine. However, despite the importance of these molecules, comparably little is known on expression patterns and functions of specific HDAC isoforms in colorectal cancer.Experimental Design: We characterized class I HDAC isoform expression patterns in a cohort of 140 colorectal carcinomas by immunohistochemistry. In addition, effects of HDAC inhibition by valproic acid and suberoylanilide hydroxamic acid, and specific HDAC isoform knockdown by short interfering RNA, were investigated in a cell culture model.Results: We found class I HDACs highly expressed in a subset of colorectal carcinomas with positivity for HDAC1 in 36.4%, HDAC2 in 57.9%, and HDAC3 in 72.9% of cases. Expression was significantly enhanced in strongly proliferating (P = 0.002), dedifferentiated (P = 0.022) tumors. High HDAC expression levels implicated significantly reduced patient survival (P = 0.001), with HDAC2 expression being an independent survival prognosticator (hazard ratio, 2.6; P = 0.03). Short interfering RNA–based inhibition of HDAC1 and HDAC2 but not HDAC3 suppressed growth of colon cancer cells in vitro, although to a lesser extent than chemical HDAC inhibitors did.Conclusions: The strong prognostic impact of HDAC isoforms in colorectal cancer, the interactions of HDACs with tumor cell proliferation and differentiation in vivo, and our finding that HDACs are differentially expressed in colorectal tumors suggest that the evaluation of HDAC expression in clinical trials for HDAC inhibitors might help to identify a patient subgroup who will exceptionally profit from such a treatment.

Supplementary Figure S1. Neuroblastoma cell lines are sensitive to binimetinib. Supplementary Figure S2. Gene expression profiling of human neuroblastoma cell lines with regards to relative sensitivity to MEK1/2 and CDK4/6 inhibition Supplementary Figure S3. Combined MEK and CDK4/6 inhibition does not induce significant levels of apoptosis or senescence. Supplementary Figure S4. G1 arrest induced by MEK1/2 and CDK4/6 inhibition is reversible. Supplementary Figure S5. Gene expression profiling of human neuroblastoma cell lines with regards to the synergy observed from combined binimetinib-ribociclib treatment. Figure S6. Neuroblastoma xenografts are sensitive to combined binimetinib-ribociclib treatment.

Supplementary Table S1: Summary of melanoma patients with clinical follow-up. Figure Legends

Purpose: Neuroblastoma is treated with aggressive multimodal therapy, yet more than 50% of patients experience relapse. We recently showed that relapsed neuroblastomas frequently harbor mutations leading to hyperactivated ERK signaling and sensitivity to MEK inhibition therapy. Here we sought to define a synergistic therapeutic partner to potentiate MEK inhibition.Experimental Design: We first surveyed 22 genetically annotated human neuroblastoma-derived cell lines (from 20 unique patients) for sensitivity to the MEK inhibitor binimetinib. After noting an inverse correlation with sensitivity to ribociclib (CDK4/6 inhibitor), we studied the combinatorial effect of these two agents using proliferation assays, cell-cycle analysis, Ki67 immunostaining, time-lapse microscopy, and xenograft studies.Results: Sensitivity to binimetinib and ribociclib was inversely related (r = −0.58, P = 0.009). MYCN amplification status and expression were associated with ribociclib sensitivity and binimetinib resistance, whereas increased MAPK signaling was the main determinant of binimetinib sensitivity and ribociclib resistance. Treatment with both compounds resulted in synergistic or additive cellular growth inhibition in all lines tested and significant inhibition of tumor growth in three of four xenograft models of neuroblastoma. The augmented growth inhibition was attributed to diminished cell-cycle progression that was reversible upon removal of drugs.Conclusions: Here we demonstrate that combined binimetinib and ribociclib treatment shows therapeutic synergy across a broad panel of high-risk neuroblastoma preclinical models. These data support testing this combination therapy in relapsed high-risk neuroblastoma patients, with focus on cases with hyperactivated RAS–MAPK signaling. Clin Cancer Res; 23(7); 1785–96. ©2016 AACR.

Discovery efforts leading to the identification of ervogastat (PF-06865571), a systemically acting diacylglycerol acyltransferase (DGAT2) inhibitor that has advanced into clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) with liver fibrosis, are described herein. Ervogastat is a first-in-class DGAT2 inhibitor that addressed potential development risks of the prototype liver-targeted DGAT2 inhibitor PF-06427878. Key design elements that culminated in the discovery of ervogastat are (1) replacement of the metabolically labile motif with a 3,5-disubstituted pyridine system, which addressed potential safety risks arising from a cytochrome P450-mediated

[Image: see text] The atypical chemokine receptor CXCR7 has been studied in various disease settings including immunological diseases and heart disease. Efforts to elucidate the role of CXCR7 have been limited by the lack of suitable chemical tools with a range of pharmacological profiles. A high-throughput screen was conducted to discover novel chemical matter with the potential to modulate CXCR7 receptor activity. This led to the identification of a series of diphenylacetamides confirmed in a CXCL12 competition assay indicating receptor binding. Further evaluation of this series revealed a lack of activity in the functional assay measuring β-arrestin recruitment. The most potent representative, compound 10 (K(i) = 597 nM), was determined to be an antagonist in the β-arrestin assay (IC(50) = 622 nM). To our knowledge, this is the first reported small molecule β-arrestin antagonist for CXCR7, useful as an in vitro chemical tool to elucidate the effects of CXCL12 displacement with β-arrestin antagonism in models for diseases such as cardiac injury and suitable as starting point for hit optimization directed toward an in vivo tool compound for studying CXCR7 receptor pharmacology.

[Image: see text] CD33/Siglec 3 is a myeloid lineage cell surface receptor that is known to regulate microglia activity. Multiple genome-wide association studies (GWAS) have identified genetic variants in the CD33 gene that convey protection from late-onset Alzheimer’s disease. Furthermore, mechanistic studies into GWAS-linked variants suggest that disease protection is attributed to the alternative splicing of exon 2 of the CD33 pre-mRNA. Using a phenomimetic screen, a series of compounds were found to enhance the exclusion of CD33 exon 2, acting as a chemomimetic of the GWAS-linked gene variants. Additional studies confirmed that meyloid lineage cells treated with several of these compounds have a reduced full-length V-domain containing CD33 protein, while targeted RNA-seq concordantly demonstrated that compound 1 increases exon 2 skipping in cellular mRNA pools. These studies demonstrate how pharmacological interventions can be used to manipulate disease-relevant pre-mRNA splicing and provide a starting point for future efforts to identify small molecules that alter neuroimmune function that is rooted in the human biology of neurodegenerative disease.

The atypical chemokine receptor C-X-C chemokine receptor type 7 (CXCR7) is an attractive therapeutic target for a variety of cardiac and immunological diseases. As a strategy to mitigate known risks associated with the development of higher molecular weight, basic compounds, a series of pyrrolidinyl-azolopyrazines were identified as promising small-molecule CXCR7 modulators. Using a highly enabled parallel medicinal chemistry strategy, structure-activity relationship studies geared towards a reduction in lipophilicity and incorporation of saturated heterocycles led to the identification of representative tool compound 20. Notably, compound 20 maintained good potency against CXCR7 with a suitable balance of physicochemical properties to support in vivo pharmacokinetic studies.

Research on digital platform ecosystems is growing rapidly. While the relevance of third-party applications is commonly known, scholars have made only minor attempts to analyze knowledge sharing between platform owners and third-party developers. We find that third-party application development is a knowledge intensive task that requires knowledge to cross organizational boundaries. In this paper, we use computational analytic methods to analyze knowledge sharing in a digital platform ecosystem. We collected trace data about a third-party developer ecosystem with frequent knowledge exchange between the platform owner and third-party developers. We developed a web scraper and retrieved all 4866 pages of SAP’s developer community that were tagged ‘SAP Cloud Platform’. Next, we used text mining to render a topic model. Based on the latent dirichlet allocation algorithm, we extracted 25 topics that were frequently discussed in the community. We clustered the topics into the following six meta-topics: User Accounts and Authentication, Connectivity, Cloud Database, Specific Technologies, SAP Resources, and Installation. Platform owners can use our approach to (1) identify frequently discussed topics, (2) generate meta-knowledge in these topics and (3) use the meta-knowledge to improve their platform core and its boundary resources.

[Image: see text] Sodium-phosphate cotransporter 2a, or NaPi2a (SLC34A1), is a solute-carrier (SLC) transporter located in the kidney proximal tubule that reabsorbs glomerular-filtered phosphate. Inhibition of NaPi2a may enhance urinary phosphate excretion and correct maladaptive mineral and hormonal derangements associated with increased cardiovascular risk in chronic kidney disease–mineral and bone disorder (CKD-MBD). To date, only nonselective NaPi inhibitors have been described. Herein, we detail the discovery of the first series of selective NaPi2a inhibitors, resulting from optimization of a high-throughput screening hit. The oral PK profile of inhibitor PF-06869206 (6f) in rodents allows for the exploration of the pharmacology of selective NaPi2a inhibition.

Diabetes is a metabolic disease characterized by elevated levels of plasma glucose. When untreated, diabetes increases the risk of developing co-morbidities such as cardiovascular disease. Several drugs, often used as part of combination therapies, have been approved to treat the disease, but these drugs will eventually fail to effectively control blood glucose levels, at which point insulin replacement therapy is required. A medical need exists for new antidiabetic drugs that exhibit good efficacy with improved safety/toleration profiles and can be added on top of existing therapies, or that can provide additional benefits beyond glucose lowering such as pancreatic beta (β)-cell protection.This review analyzes drug targets and applicants of patents that published between 2011-2016 claiming novel small or large molecules for the treatment of diabetes, and compares the results to the 2008-2010 time period.A majority of patent activity around the discovery of new antidiabetic drugs in 2011-2016 was directed against 15 targets, most of which were also the focus of drug discovery efforts in the 2008-2010 time period. The top targets by total patent counts were DPP4, GLP1R, INSR, GPR119, and SGLT2 (SLC5A2). With the exception of GPR119, these are the pharmacological targets of some of the best-selling antidiabetic drugs currently on the market. The top targets of patent families with the largest size counts, a metric useful in assessing patent value and applicant interest, were AMPK, CALCR, DPP4, and GLP1R. The patent analysis identified several emerging targets with greater patent activity in 2011-2016 compared to 2008-2010, including FFAR1, FFAR4, and FGFR1. Most of the patent activity in 2011-2016 was directed at established and precedented diabetes targets, the modulation of which may lead to improvements in glucose control and a delay in the progression of the disease. Few targets were identified that promote pancreatic β-cell regeneration and β-cell health, areas where future opportunities may exist for developing transformative drug therapies that may potentially lead to cures for diabetes.

Allostery describes the functional coupling between sites in biomolecules. Recently, the role of changes in protein dynamics for allosteric communication has been highlighted. A quantitative and predictive description of allostery is fundamental for understanding biological processes. Here, we integrate an ensemble-based perturbation approach with the analysis of biomolecular rigidity and flexibility to construct a model of dynamic allostery. Our model, by definition, excludes the possibility of conformational changes, evaluates static, not dynamic, properties of molecular systems, and describes allosteric effects due to ligand binding in terms of a novel free-energy measure. We validated our model on three distinct biomolecular systems: eglin c, protein tyrosine phosphatase 1B, and the lymphocyte function-associated antigen 1 domain. In all cases, it successfully identified key residues for signal transmission in very good agreement with the experiment. It correctly and quantitatively discriminated between positively or negatively cooperative effects for one of the systems. Our model should be a promising tool for the rational discovery of novel allosteric drugs.

Purpose: Neuroblastoma is treated with aggressive multimodal therapy, yet more than 50% of patients experience relapse. We recently showed that relapsed neuroblastomas frequently harbor mutations leading to hyperactivated ERK signaling and sensitivity to MEK inhibition therapy. Here we sought to define a synergistic therapeutic partner to potentiate MEK inhibition. Experimental Design: We first surveyed 22 genetically annotated human neuroblastoma-derived cell lines (from 20 unique patients) for sensitivity to the MEK inhibitor binimetinib. After noting an inverse correlation with sensitivity to ribociclib (CDK4/6 inhibitor), we studied the combinatorial effect of these two agents using proliferation assays, cell-cycle analysis, Ki67 immunostaining, time-lapse microscopy, and xenograft studies. Results: Sensitivity to binimetinib and ribociclib was inversely related (r = −0.58, P = 0.009). MYCN amplification status and expression were associated with ribociclib sensitivity and binimetinib resistance, whereas increased MAPK signaling was the main determinant of binimetinib sensitivity and ribociclib resistance. Treatment with both compounds resulted in synergistic or additive cellular growth inhibition in all lines tested and significant inhibition of tumor growth in three of four xenograft models of neuroblastoma. The augmented growth inhibition was attributed to diminished cell-cycle progression that was reversible upon removal of drugs. Conclusions: Here we demonstrate that combined binimetinib and ribociclib treatment shows therapeutic synergy across a broad panel of high-risk neuroblastoma preclinical models. These data support testing this combination therapy in relapsed high-risk neuroblastoma patients, with focus on cases with hyperactivated RAS–MAPK signaling. Clin Cancer Res; 23(7); 1785–96. ©2016 AACR.

Data are crucial for tailoring health apps to personal needs. Nonetheless, a users' privacy and security need to be preserved, particularly since health technologies are able to gather a broad range of data over a long time period. In order to guarantee an appropriate level of security and privacy, the perceptions of end users need to be evaluated to enhance adoption of the technologies. In this paper, we conduct a Delphi study to gain insights into the perception of certain data elements used in popular health apps. As health apps are now commonly used both privately and at the workplace, both are compared in this study. As input for the Delphi study, an exploratory market analysis has been utilized. Our work reveals a mismatch between the perception of sensitivity of data and the usage rate of common health apps. Additionally, variations have been identified between the private and corporate health context, providing implications for both practice and theory.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that downregulates low-density lipoprotein (LDL) receptor (LDL-R) levels on the surface of hepatocytes, resulting in decreased clearance of LDL-cholesterol (LDL-C). Phenotypic screening of a small-molecule compound collection was used to identify an inhibitor of PCSK9 secretion, (R)-N-(isoquinolin-1-yl)-3-(4-methoxyphenyl)-N-(piperidin-3-yl)propanamide (R-IMPP), which was shown to stimulate uptake of LDL-C in hepatoma cells by increasing LDL-R levels, without altering levels of secreted transferrin. Systematic investigation of the mode of action revealed that R-IMPP did not decrease PCSK9 transcription or increase PCSK9 degradation, but instead caused transcript-dependent inhibition of PCSK9 translation. In support of this surprising mechanism of action, we found that R-IMPP was able to selectively bind to human, but not E. coli, ribosomes. This study opens a new avenue for the development of drugs that modulate the activity of target proteins by mechanisms involving inhibition of eukaryotic translation.

Adenosine monophosphate-activated protein kinase (AMPK) is a protein kinase involved in maintaining energy homeostasis within cells. On the basis of human genetic association data, AMPK activators were pursued for the treatment of diabetic nephropathy. Identification of an indazole amide high throughput screening (HTS) hit followed by truncation to its minimal pharmacophore provided an indazole acid lead compound. Optimization of the core and aryl appendage improved oral absorption and culminated in the identification of indole acid, PF-06409577 (7). Compound 7 was advanced to first-in-human trials for the treatment of diabetic nephropathy.

Purpose: To test second-line personalized medicine combination therapies, based on genomic and proteomic data, in patient-derived xenograft (PDX) models. Experimental Design: We established 12 PDXs from BRAF inhibitor–progressed melanoma patients. Following expansion, PDXs were analyzed using targeted sequencing and reverse-phase protein arrays. By using multi-arm preclinical trial designs, we identified efficacious precision medicine approaches. Results: We identified alterations previously described as drivers of resistance: NRAS mutations in 3 PDXs, MAP2K1 (MEK1) mutations in 2, BRAF amplification in 4, and aberrant PTEN in 7. At the protein level, re-activation of phospho-MAPK predominated, with parallel activation of PI3K in a subset. Second-line efficacy of the pan-PI3K inhibitor BKM120 with either BRAF (encorafenib)/MEK (binimetinib) inhibitor combination or the ERK inhibitor VX-11e was confirmed in vivo. Amplification of MET was observed in 3 PDX models, a higher frequency than expected and a possible novel mechanism of resistance. Importantly, MET amplification alone did not predict sensitivity to the MET inhibitor capmatinib. In contrast, capmatinib as single agent resulted in significant but transient tumor regression in a PDX with resistance to BRAF/MEK combination therapy and high pMET. The triple combination capmatinib/encorafenib/binimetinib resulted in complete and sustained tumor regression in all animals. Conclusions: Genomic and proteomic data integration identifies dual-core pathway inhibition as well as MET as combinatorial targets. These studies provide evidence for biomarker development to appropriately select personalized therapies of patients and avoid treatment failures. Clin Cancer Res; 22(7); 1592–602. ©2015 AACR. See related commentary by Hartsough and Aplin, p. 1550

Modulating protein activity with small molecules binding to cryptic pockets offers great opportunities to overcome hurdles in drug design. Cryptic sites are atypical binding sites in proteins that are closed in the absence of a stabilizing ligand and are thus inherently difficult to identify. Many studies have proposed methods to predict cryptic sites. However, a general approach to prospectively sample open conformations of these sites and to identify cryptic pockets in an unbiased manner suitable for structure-based drug design remains elusive. Here, we describe an all-atom, explicit cosolvent, molecular dynamics (MD) simulations-based workflow to sample the open states of cryptic sites and identify opened pockets, in a manner that does not require a priori knowledge about these sites. Furthermore, the workflow relies on a target-independent parameterization that only distinguishes between binding pockets for peptides or small-molecules. We validated our approach on a diverse test set of seven proteins with crystallographically determined cryptic sites. The known cryptic sites were found among the three highest-ranked predicted cryptic sites, and an open site conformation was sampled and selected for most of the systems. Crystallographic ligand poses were well reproduced by docking into these identified open conformations for five of the systems. When the fully open state could not be reproduced, we were still able to predict the location of the cryptic site, or identify other cryptic sites that could be retrospectively validated with knowledge of the protein target. These characteristics render our approach valuable for investigating novel protein targets without any prior information.

C–X–C chemokine receptor type 7 (CXCR7) is involved in cardiac and immune pathophysiology. We report the discovery of a novel 1,4-diazepine CXCR7 modulator, demonstrating for the first time the role of pharmacological CXCR7 intervention in cardiac repair. Structure–activity-relationship (SAR) studies demonstrated that a net reduction in lipophilicity (log D) and an incorporation of saturated ring systems yielded compounds with good CXCR7 potencies and improvements in oxidative metabolic stability in human-liver microsomes (HLM). Tethering an ethylene amide further improved the selectivity profile (e.g., for compound 18, CXCR7 Ki = 13 nM, adrenergic α 1a Kb > 10 000 nM, and adrenergic β 2 Kb > 10 000 nM). The subcutaneous administration of 18 in mice led to a statistically significant increase in circulating concentrations of plasma stromal-cell-derived factor 1α (SDF-1α) of approximately 2-fold. Chronic dosing of compound 18 in a mouse model of isoproterenol-induced cardiac injury further resulted in a st...

The medicinal chemistry and preclinical biology of imidazopyridine-based inhibitors of diacylglycerol acyltransferase 2 (DGAT2) is described. A screening hit 1 with low lipophilic efficiency (LipE) was optimized through two key structural modifications: (1) identification of the pyrrolidine amide group for a significant LipE improvement, and (2) insertion of a sp(3)-hybridized carbon center in the core of the molecule for simultaneous improvement of N-glucuronidation metabolic liability and off-target pharmacology. The preclinical candidate 9 (PF-06424439) demonstrated excellent ADMET properties and decreased circulating and hepatic lipids when orally administered to dyslipidemic rodent models.

Inhibition of triacylglycerol (TAG) biosynthetic enzymes has been suggested as a promising strategy to treat insulin resistance, diabetes, dyslipidemia, and hepatic steatosis. Monoacylglycerol acyltransferase 3 (MGAT3) is an integral membrane enzyme that catalyzes the acylation of both monoacylglycerol (MAG) and diacylglycerol (DAG) to generate DAG and TAG, respectively. Herein, we report the discovery and characterization of the first selective small molecule inhibitors of MGAT3. Isoindoline-5-sulfonamide (6f, PF-06471553) selectively inhibits MGAT3 with high in vitro potency and cell efficacy. Because the gene encoding MGAT3 (MOGAT3) is found only in higher mammals and humans, but not in rodents, a transgenic mouse model expressing the complete human MOGAT3 was used to characterize the effects of 6f in vivo. In the presence of a combination of diacylglycerol acyltransferases 1 and 2 (DGAT1 and DGAT2) inhibitors, an oral administration of 6f exhibited inhibition of the incorporation of deuterium-labeled glycerol into TAG in this mouse model. The availability of a potent and selective chemical tool and a humanized mouse model described in this report should facilitate further dissection of the physiological function of MGAT3 and its role in lipid homeostasis.

The chemokine receptor CXCR7 is an attractive target for a variety of diseases. While several small-molecule modulators of CXCR7 have been reported, peptidic macrocycles may provide advantages in terms of potency, selectivity, and reduced off-target activity. We produced a series of peptidic macrocycles that incorporate an N-linked peptoid functionality where the peptoid group enabled us to explore side-chain diversity well beyond that of natural amino acids. At the same time, theoretical calculations and experimental assays were used to track and reduce the polarity while closely monitoring the physicochemical properties. This strategy led to the discovery of macrocyclic peptide–peptoid hybrids with high CXCR7 binding affinities (Ki 5 × 10–6 cm/s). Moreover, bioactive peptide 25 (Ki = 9 nM) achieved oral bioavailability of 18% in rats, which was commensurate with the observed plasma clearance values upon intravenous administration.

Aims The aim was to characterize the population pharmacokinetics of BYL719 in cancer patients and assess the time course of tumour response in relation to drug exposure and dosing schedule. Methods Plasma samples and longitudinal tumour size measurements were collected from 60 patients with advanced solid malignancies who received oral BYL719 once daily (30–450 mg) or twice daily at 120 mg or 200 mg. Non-linear mixed effect modelling was employed to develop the population pharmacokinetic and pharmacodynamic model. Results The pharmacokinetics were best described by a one compartment disposition model and transit compartments accounting for the lag time in absorption. The typical population oral clearance and volume of distribution estimates with their between-subject variability (BSV) were 10 l h−1 (BSV 26%) and 108 l (BSV 28%), respectively. The estimated optimal number of transit compartments was 8.1, with a mean transit time to the absorption compartment of 1.28 h (BSV 32%). The between-occasion variability in the rate and extent of absorption was 46% and 26%, respectively. Tumour growth was modelled using a turnover model characterized by a zero order growth rate of 0.581 cm week1 and a first order death rate of 0.0123 week−1. BYL719 inhibited tumour growth with an IC50 of 100 ng ml−1 (BSV 154%). Model-based predictions showed potential for additional anti-tumour activity of twice daily dosing at total daily dose below 400 mg, but a loss of efficacy if administered less frequently than once daily. Conclusions The proposed model provides a valuable approach for planning future clinical studies and for designing optimized dosing regimens with BYL719.

In this paper, the design, fabrication and characterization of an amorphous silicon germanium carbide (a-SiGeC:H) photo sensitive bipolar junction transistor (PS-BJT) with three terminals are presented. Whereas the current gain of similar transistor devices presented in the past (Wu et al., 1984; Hwang et al., 1993; Nascetti and Caputo, 2002; Chang et al., 1985a,b; Wu et al, 1985; Hong et al., 1990) can only be controlled with photo induced charge generation, the n–i–δp–i–n structure developed features a contacted base to provide the opportunity to adjust the current gain optically and electrically, too. Electron microscope-, current-/voltage- and spectral measurements were performed to study the PS-BJT behavior and calculate the electrical and optical current gain. The spectral response maximum of the base–collector diode has a value of 170 mA/W applying a base–collector voltage of − 1 V and is located at 620 nm. The base–emitter diode reaches a sensitivity of 25.7 mA/W at 530 nm with a base-emitter voltage of − 3 V. The good a-Si:H transport properties are validated in a μτ-product of 4.6 × 10 − 6 cm 2 V s, which is sufficient to reach a continuous base- and photo-tunable current gain of up to − 126 at a base current of I B = + 10 nA and a collector–emitter voltage of V CE = − 3 V. The transistor obtains a maximum collector current of − 65.5 μA (V CE = − 3 V) and + 56.2 μA (V CE = + 3 V) at 10,000 lx 5300 K white-light illumination. At 3300 lx, the electrical current gain reaches a value of + 100 (V CE = + 2 V) at I B = 10 nA. With a negative base current of I B = − 10 nA the electrical gain can be adjusted between 87 (V CE = + 2 V) and − 106 (V CE = -3 V), respectively. When no base charge is applied, the transistor is “off” for V CE > − 3 V. Reducing the base current increases the electrical current gain. Operating with a voltage V CE of just ± 2 V, the device presented in this paper obtains no optical gain with an incident light power of 0.31 μW and 578 nm wavelength. The PS-BJT exhibits promising characteristics with a maximum current density greater than 65 μA μm − 1 and a tunable high current gain β. Possible applications exist in fields of control and drive circuits, in display and image devices, in photo interrupters, in photo sensitive matrices or in optoelectronic switches.

This paper presents an inventive unipolar amorphous silicon (a-Si:H) two terminal two color photo sensor, featuring two functional modes of operation. The sensor structure is green sensitive at − 8.5 V and reaches a sensitivity maximum of 130 mA/W. At lower absolute bias voltage values the spectral response at 550 nm is suppressed by an internal optical filter. The filter structure consists of an a-Si:H/a-SiGeC:H absorber interface, generating an interior trapping area with high defect densities. The combination of an internal band gap barrier and an area with high defect states causes localized charge recombination. The interface deteriorates transport properties locally to suppress green sensitivity while the detectors blue and red efficiencies exceed. The maximum suppression of the 530 nm response is 32.5 mA/W at − 5.5 V bias voltage. Additionally, we observe a shift of both two color sensitivity maxima. The peak at lower wavelength shifts from 350 nm to 480 nm. In consequence, the device is even able to detect signals in the near ultraviolet spectrum. The second peak shifts from 590 nm to 640 nm. At − 8.5 V the filter is deactivated and the maximum detector response is located at 530 nm. In reverse biased conditions the device's dynamic range remains constantly high and reaches values of 90 dB at 1000 lx white-light illumination. The interior 85 nm a-Si:H layer reduces the dark current density to 10 − 8 A/cm 2 at − 8 V. Possible sensor applications may exist in fields of medical diagnostics, fluorescence and spectrophotometrical measurements, chemical analysis or in colorimetric and hyperspectral imagery.

Background: Phosphatidylinositol-3-kinase (PI3K) pathway activation has been associated with resistance to anti-estrogen receptor (ER) therapy and PIK3CA mutated ER+ breast cancer (BC) cells are sensitive to inhibition of PI3K under estrogen deprivation. In an in vitro study, the combination of BYL719 and fulvestrant was synergistic in PIK3CA mutated BC cell lines. In the BYL719 single agent part of this study out of 21 ER+ BC patients (pts), 3 partial responses were observed and 5 pts remained on the study with stable disease for over 26 weeks. Median PFS in pts with ER+ PIK3CA mutated BC was 5.5 months (Gonzalez-Angulo, 2013). Methods: This phase I study was amended to determine the Maximum Tolerated Dose (MTD) of BYL719 in combination with the ER antagonist fulvestrant in PIK3CA mutated ER+ breast cancer pts. Dose escalation of BYL719 in combination with fixed dose fulvestrant used an adaptive Bayesian logistic regression model with overdose control. The starting dose of BYL719 was 300 mg once daily, 25% below the MTD of single agent BYL719. All pts received the standard dose of 500 mg fulvestrant every 4 weeks. No dose escalation of fulvestrant was permitted. Following the MTD declaration, a dose expansion cohort will open. Results: At the data cut-off (27 March 2013), 4 pts were enrolled in the initial cohort of 300 mg/q.d. BYL719 plus 500 mg/qmonth (with an additional dose at week 2) fulvestrant, followed by 8 pts into the subsequent 400 mg/q.d. BYL719 plus fulvestrant dose combination. No pts experienced dose limiting toxicity (DLT) in the 300 mg cohort, and 1 pt experienced a DLT of CTCAE grade 3 diarrhea, vomiting, fatigue, anorexia and bloating in the 400 mg cohort. The MTD of BYL719 in combination with fulvestrant has been declared as 400 mg/q.d. The most frequent BYL719 related toxicities (>15% pts), regardless of grade and treatment group, are diarrhea (42%), hyperglycemia (33%), decreased appetite (25%), nausea (25%), and fatigue (17%). Systemic drug exposure of oral BYL719 when given in combination with fulvestrant was found to be similar to the single agent data. The patients were typically pre-treated with numerous chemotherapy, hormonal or targeted therapies. One pt in the 300 mg/q.d. cohort had a confirmed partial response, and 2 pts in the 400 mg/q.d. cohort had a partial response (one confirmed and one only occurring after the data cut-off). From the 12 breast cancer pts treated, 2 (17%) discontinued treatment due to progressive disease, and the median exposure of all pts at the time of data cut-off was 9 weeks. Conclusions: This combination displays a favorable safety and PK profile, both comparable to the single agent BYL719 experience. BYL719 in combination with fulvestrant shows encouraging preliminary anti-tumor activity, which supports further investigation of this combination. Recruitment within the dose expansion is continuing to obtain a sample size of 20 patients carrying the PIK3CA mutation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-16-14.

Optimization of a high-throughput screening hit led to the discovery of a new series of 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamides as highly potent agonists of TGR5. This novel chemotype was rapidly developed through iterative combinatorial library synthesis. It was determined that in vitro agonist potency correlated with functional activity data from human peripheral blood monocytes.

Lysophospholipase-like 1 (LYPLAL1) is an uncharacterized metabolic serine hydrolase. Human genome-wide association studies link variants of the gene encoding this enzyme to fat distribution, waist-to-hip ratio, and nonalcoholic fatty liver disease. We describe the discovery of potent and selective covalent small-molecule inhibitors of LYPLAL1 and their use to investigate its role in hepatic metabolism. In hepatocytes, selective inhibition of LYPLAL1 increased glucose production supporting the inference that LYPLAL1 is a significant actor in hepatic metabolism. The results provide an example of how a selective chemical tool can contribute to evaluating a hypothetical target for therapeutic intervention, even in the absence of complete biochemical characterization.

An innovative family of hydrogenated amorphous silicon (a-Si:H) multicolor p–i–n photo sensors, sensitive in the VIS and the near UV spectrum, is presented. Typical values of the quantum efficiency at 350 nm and 580 nm are 5.4% and 54.7%, respectively, with − 0.4 V and − 12 V bias. Electro-optical studies were performed to explore the effect of combining linearly graded a-SiGe:H/μc-SiGeC:H layers with linearly graded a‐SiC:H-layers. The devices presented additionally contain a buried a-Si:H region. Low-reflective aluminum doped zinc oxide (ZnO:Al) back contacts improve the spectral color separation. μτ-products and absorption coefficients of graded absorbers were determined. Discrete absorbers were substituted by a linear graded a-SiC:H absorption zone in the top structure, an interior a-Si:H region and a graded a-SiGe:H/a-SiC:H alloy combination. In this paper we demonstrate a reduction of interference fringes and operation at low bias voltages, combined with a highly precise adjustment of the spectral sensitivity, even in the near UV-spectrum. The device dynamic range exceeds 50 dB at 1000 lx white-light illumination. As the deposited upper layers adopt the roughness of μc-SiGeC:H clusters in the rear absorber, we present an in-situ structured front contact without etching ZnO:Al.

This work presents the development of three-terminal photodiodes which have their origin in bipolar pinipip-diode-structures [1,2]. The idea is to develop an unipolar diode and to contact a buried p-layer by an interior TCO-anode (transparent conductive oxide) instead of nip-stacking. The simulation of the optical material properties shows promising results. At first, both parts of the diode-structure were produced separately. The manufactured bottom (Cr/nip/TCO) and top (TCO/pip/TCO) parts were measured optically and electrically. These measurements are required to simulate the SR of the total-diode. Finally, a Cr/nip-a-Si:H/TCO/pip-a-Si:H/TCO multi-layer stack was deposited. The measured SR of the integrated diode validates the simulated data. The SR maximum shift amounts to 100nm, from 540nm by contacting the interior anode, to 640nm at the top anode. Furthermore, the curves are clearly split and do not enclose each other. The presented approach, with additional bandgap engineering, promises good prospects to improve color separation compared to currently existing detectors and should lead to a tunable multi-spectral photodiodes for high quality color recognition. Such a diode can be used in photonic devices, e.g. for safety and security applications.

AP-3 is a heterotetrameric adaptor involved in the biogenesis of lysosome-related organelles. The function of AP-3 as an adaptor relies on its ability to bind to membranes in an Arf-dependent fashion and to recognize sorting signals in the cytosolic tails of the transmembrane cargo. Here, we report an interdomain interaction involving the ear domain of the delta subunit and the sigma3 subunit of AP-3. This interaction interferes with the binding of AP-3 to Arf but not to dileucine-based sorting signals. As a consequence, the delta-ear inhibits the recruitment of AP-3 to membranes both in vitro and in vivo and impairs the sorting of lysosomal membrane proteins. These observations suggest a new regulatory mechanism for the recruitment of AP-3 to membranes involving delta-ear-sigma3 interactions.

We recently described two novel aryl binding sites of farnesyltransferase. The arylacetyl residue was designed as an appropriate substituent for our benzo-phenone-based AAX-peptidomimetic compound capable of occupying the near aryl binding site which is located next to the catalytic zinc ion. Non-thiol farnesyl-transferase inhibitors with micromolar to submicromolar activity were obtained.