Discovery and evaluation of non-basic small molecule modulators of the atypical chemokine receptor CXCR7.

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.

Population pharmacokinetics and pharmacodynamics of BYL719, a phosphoinositide 3-kinase antagonist, in adult patients with advanced solid malignancies.

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 week(1) 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.

Discovery of 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamides as potent agonists of TGR5 via sequential combinatorial libraries.

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.

Discovery of the Potent and Selective MC4R Antagonist PF-07258669 for the Potential Treatment of Appetite Loss.

The melanocortin-4 receptor (MC4R) is a centrally expressed, class A GPCR that plays a key role in the regulation of appetite and food intake. Deficiencies in MC4R signaling result in hyperphagia and increased body mass in humans. Antagonism of MC4R signaling has the potential to mitigate decreased appetite and body weight loss in the setting of anorexia or cachexia due to underlying disease. Herein, we report on the identification of a series of orally bioavailable, small-molecule MC4R antagonists using a focused hit identification effort and the optimization of these antagonists to provide clinical candidate 23. Introduction of a spirocyclic conformational constraint allowed for simultaneous optimization of MC4R potency and ADME attributes while avoiding the production of hERG active metabolites observed in early series leads. Compound 23 is a potent and selective MC4R antagonist with robust efficacy in an aged rat model of cachexia and has progressed into clinical trials.

Conservation and diversification of dileucine signal recognition by adaptor protein (AP) complex variants.

The clathrin-associated, heterotetrameric adaptor protein (AP) complexes, AP-1, AP-2, and AP-3, recognize signals in the cytosolic domains of transmembrane proteins, leading to their sorting to endosomes, lysosomes, lysosome-related organelles, and/or the basolateral membrane of polarized epithelial cells. One type of signal, referred to as "dileucine-based," fits the consensus motif (D/E)XXXL(L/I). Previous biochemical analyses showed that (D/E)XXXL(L/I) signals bind to a combination of two subunits of each AP complex, namely the AP-1 γ-σ1, AP-2 α-σ2, and AP-3 δ-σ3 hemicomplexes, and structural studies revealed that an imperfect variant of this motif lacking the (D/E) residue binds to a site straddling the interface of α and σ2. Herein, we report mutational and binding analyses showing that canonical (D/E)XXXL(L/I) signals bind to this same site on AP-2, and to similar sites on AP-1 and AP-3. The strength and amino acid requirements of different interactions depend on the specific signals and AP complexes involved. We also demonstrate the occurrence of diverse AP-1 heterotetramers by combinatorial assembly of various γ and σ1 subunit isoforms encoded by different genes. These AP-1 variants bind (D/E)XXXL(L/I) signals with marked preferences for certain sequences, implying that they are not functionally equivalent. Our results thus demonstrate that different AP complexes share a conserved binding site for (D/E)XXXL(L/I) signals. However, the characteristics of the binding site on each complex vary, providing for the specific recognition of a diverse repertoire of (D/E)XXXL(L/I) signals.

Identification of novel series of pyrazole and indole-urea based DFG-out PYK2 inhibitors.

Previous drug discovery efforts identified classical PYK2 kinase inhibitors such as 2 and 3 that possess selectivity for PYK2 over its intra-family isoform FAK. Efforts to identify more kinome-selective chemical matter that stabilize a DFG-out conformation of the enzyme are described herein. Two sub-series of PYK2 inhibitors, an indole carboxamide-urea and a pyrazole-urea have been identified and found to have different binding interactions with the hinge region of PYK2. These leads proved to be more selective than the original classical inhibitors.

Shaping a screening file for maximal lead discovery efficiency and effectiveness: elimination of molecular redundancy.

High Throughput Screening (HTS) is a successful strategy for finding hits and leads that have the opportunity to be converted into drugs. In this paper we highlight novel computational methods used to select compounds to build a new screening file at Pfizer and the analytical methods we used to assess their quality. We also introduce the novel concept of molecular redundancy to help decide on the density of compounds required in any region of chemical space in order to be confident of running successful HTS campaigns.

Critical Assessment of State-of-the-Art Ligand-Based Virtual Screening Methods.

The availability of large chemical libraries containing hundreds of millions to billions of diverse drug-like molecules combined with an almost unlimited amount of compute power to achieve scientific calculations has led investors and researchers to have a renewed interest in virtual screening (VS) methods to identify biologically active compounds. The number of in silico screening tools and software which employ the knowledge of the protein target or known bioactive ligands is increasing at a rapid pace, creating a crowded computational landscape where it has become difficult to assess the real advantages and disadvantages in terms of accuracy and efficiency of each individual VS technology. In the current work, we evaluate the performance of several state-of-the-art commercial software for 3D ligand-based VS against well-known 2D methods using an internally curated benchmarking data set. Our results show that the best individual methods can differ significantly based on the data set, and that combining them using data fusion techniques results in improved enrichment in the top 1 % of retrieved hits. Although 2D methods alone can already provide a significant enrichment in the number of predicted active compounds, the combination of data-fused 2D results with just one out of the best 3D methods (ROCS, FLAP or Blaze) further improves early enrichment and the likelihood of identifying additional chemotypes.

Discovery of Small-Molecule CD33 Pre-mRNA Splicing Modulators.

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.

Discovery of Ervogastat (PF-06865571): A Potent and Selective Inhibitor of Diacylglycerol Acyltransferase 2 for the Treatment of Non-alcoholic Steatohepatitis.

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 O-dearylation of PF-06427878 to a reactive quinone metabolite precursor, and (2) modifications of the amide group to a 3-THF group, guided by metabolite identification studies coupled with property-based drug design.

Discovery of Diphenylacetamides as CXCR7 Inhibitors with Novel ß-Arrestin Antagonist Activity.

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 ß-arrestin recruitment. The most potent representative, compound 10 (K i = 597 nM), was determined to be an antagonist in the ß-arrestin assay (IC50 = 622 nM). To our knowledge, this is the first reported small molecule ß-arrestin antagonist for CXCR7, useful as an in vitro chemical tool to elucidate the effects of CXCL12 displacement with ß-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.

An ear-core interaction regulates the recruitment of the AP-3 complex to membranes.

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.

Specific regulation of the adaptor protein complex AP-3 by the Arf GAP AGAP1.

Arf1 regulates membrane trafficking at several membrane sites by interacting with at least seven different vesicle coat proteins. Here, we test the hypothesis that Arf1-dependent coats are independently regulated by specific interaction with Arf GAPs. We find that the Arf GAP AGAP1 directly associates with and colocalizes with AP-3, a coat protein complex involved in trafficking in the endosomal-lysosomal system. Binding is mediated by the PH domain of AGAP1 and the delta and sigma3 subunits of AP-3. Overexpression of AGAP1 changes the cellular distribution of AP-3, and reduced expression of AGAP1 renders AP-3 resistant to brefeldin A. AGAP1 overexpression does not affect the distribution of other coat proteins, and AP-3 distribution is not affected by overexpression of other Arf GAPs. Cells overexpressing AGAP1 also exhibit increased LAMP1 trafficking via the plasma membrane. Taken together, these results support the hypothesis that AGAP1 directly and specifically regulates AP-3-dependent trafficking.

Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 gamma-sigma1 and AP-3 delta-sigma3 hemicomplexes.

The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the gamma and sigma1 subunits of AP-1 and the delta and sigma3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the gamma/delta and sigma subunits of AP-1 and AP-3.

Class I histone deacetylase expression has independent prognostic impact in human colorectal cancer: specific role of class I histone deacetylases in vitro and in vivo.

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.

Cosolvent-Enhanced Sampling and Unbiased Identification of Cryptic Pockets Suitable for Structure-Based Drug Design.

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 parametrization 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.

Similarity searching and scaffold hopping in synthetically accessible combinatorial chemistry spaces.

Large collections of combinatorial libraries are an integral element in today's pharmaceutical industry. It is of great interest to perform similarity searches against all virtual compounds that are synthetically accessible by any such library. Here we describe the successful application of a new software tool CoLibri on 358 combinatorial libraries based on validated reaction protocols to create a single chemistry space containing over 10 (12) possible products. Similarity searching with FTrees-FS allows the systematic exploration of this space without the need to enumerate all product structures. The search result is a set of virtual hits which are synthetically accessible by one or more of the existing reaction protocols. Grouping these virtual hits by their synthetic protocols allows the rapid design and synthesis of multiple follow-up libraries. Such library ideas support hit-to-lead design efforts for tasks like follow-up from high-throughput screening hits or scaffold hopping from one hit to another attractive series.

Histone deacetylase inhibitors suppress the inducibility of nuclear factor-kappaB by tumor necrosis factor-alpha receptor-1 down-regulation.

Recently, the inhibition of histone deacetylase (HDAC) enzymes has attracted attention in the oncologic community as a new therapeutic opportunity for hematologic and solid tumors including non-small cell lung cancer (NSCLC). In hematologic malignancies, such as diffuse large B-cell lymphoma, the HDAC inhibitor (HDI), suberoylanilide hydroxamic acid (SAHA), has recently entered phase II and III clinical trials. To further advance our understanding of their action on tumor cells, we investigated the possible effect of HDI treatment on the functionality of the nuclear factor-kappaB (NF-kappaB) pathway in NSCLC. We found that in the NSCLC cell lines, A549 and NCI-H460, the NF-kappaB pathway was strongly inducible, for example, by stimulation with tumor necrosis factor-alpha (TNF-alpha). Incubation of several NSCLC cell lines with HDIs resulted in greatly reduced gene expression of TNF-alpha receptor-1. HDI-treated A549 and NCI-H460 cells down-regulated TNF-alpha receptor-1 mRNA and protein levels as well as surface exposure, and consequently responded to TNF-alpha treatment with reduced IKK phosphorylation and activation, delayed IkappaB-alpha phosphorylation, and attenuated NF-kappaB nuclear translocation and DNA binding. Accordingly, stimulation of NF-kappaB target gene expression by TNF-alpha was strongly decreased. In addition, we observed that SAHA displayed antitumor efficacy in vivo against A549 xenografts grown on nude mice. HDIs, therefore, might beneficially contribute to tumor treatment, possibly by reducing the responsiveness of tumor cells to the TNF-alpha-mediated activation of the NF-kappaB pathway. These findings also hint at a possible use of HDIs in inflammatory diseases, which are associated with the overproduction of TNF-alpha, such as rheumatoid arthritis or Crohn's disease.

Diabetes area patent participation analysis - part II: years 2011-2016.

INTRODUCTION: 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 (ß)-cell protection. AREAS COVERED: 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. EXPERT OPINION: 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 ß-cell regeneration and ß-cell health, areas where future opportunities may exist for developing transformative drug therapies that may potentially lead to cures for diabetes.

Discovery of Orally Bioavailable Selective Inhibitors of the Sodium-Phosphate Cotransporter NaPi2a (SLC34A1).

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.