Neuropeptide 26RFa (QRFP) is a key regulator of glucose homeostasis and its activity is markedly altered in obese/hyperglycemic mice.

Recent studies have shown that the hypothalamic neuropeptide 26RFa regulates glucose homeostasis by acting as an incretin and increasing insulin sensitivity. In this study, we further characterized the role of the 26RFa/GPR103 peptidergic system in the global regulation of glucose homeostasis using a 26RFa receptor antagonist and also assessed whether a dysfunction of the 26RFa/GPR103 system occurs in obese hyperglycemic mice. First, we demonstrate that administration of the GPR103 antagonist reduces the global glucose-induced incretin effect and insulin sensitivity whereas, conversely, administration of exogenous 26RFa attenuates glucose-induced hyperglycemia. Using a mouse model of high-fat diet-induced obesity and hyperglycemia, we found a loss of the antihyperglcemic effect and insulinotropic activity of 26RFa, accompanied with a marked reduction of its insulin-sensitive effect. Interestingly, this resistance to 26RFa is associated with a downregulation of the 26RFa receptor in the pancreatic islets, and insulin target tissues. Finally, we observed that the production and release kinetics of 26RFa after an oral glucose challenge is profoundly altered in the high-fat mice. Altogether, the present findings support the view that 26RFa is a key regulator of glucose homeostasis whose activity is markedly altered under obese/hyperglycemic conditions.

Predicting the relative binding affinity of mineralocorticoid receptor antagonists by density functional methods.

In drug discovery, prediction of binding affinity ahead of synthesis to aid compound prioritization is still hampered by the low throughput of the more accurate methods and the lack of general pertinence of one method that fits all systems. Here we show the applicability of a method based on density functional theory using core fragments and a protein model with only the first shell residues surrounding the core, to predict relative binding affinity of a matched series of mineralocorticoid receptor (MR) antagonists. Antagonists of MR are used for treatment of chronic heart failure and hypertension. Marketed MR antagonists, spironolactone and eplerenone, are also believed to be highly efficacious in treatment of chronic kidney disease in diabetes patients, but is contra-indicated due to the increased risk for hyperkalemia. These findings and a significant unmet medical need among patients with chronic kidney disease continues to stimulate efforts in the discovery of new MR antagonist with maintained efficacy but low or no risk for hyperkalemia. Applied on a matched series of MR antagonists the quantum mechanical based method gave an R(2) = 0.76 for the experimental lipophilic ligand efficiency versus relative predicted binding affinity calculated with the M06-2X functional in gas phase and an R(2) = 0.64 for experimental binding affinity versus relative predicted binding affinity calculated with the M06-2X functional including an implicit solvation model. The quantum mechanical approach using core fragments was compared to free energy perturbation calculations using the full sized compound structures.

Synthesis of functionalized cinnamaldehyde derivatives by an oxidative Heck reaction and their use as starting materials for preparation of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase inhibitors.

Cinnamaldehyde derivatives were synthesized in good to excellent yields in one step by a mild and selective, base-free palladium(II)-catalyzed oxidative Heck reaction starting from acrolein and various arylboronic acids. Prepared α,ß-unsaturated aldehydes were used for synthesis of novel α-aryl substituted fosmidomycin analogues, which were evaluated for their inhibition of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate reductoisomerase. IC(50) values between 0.8 and 27.3 µM were measured. The best compound showed activity comparable to that of the most potent previously reported α-aryl substituted fosmidomycin-class inhibitor.

Discovery of retinoic acid receptor agonists as proliferators of cardiac progenitor cells through a phenotypic screening approach.

Identification of small molecules with the potential to selectively proliferate cardiac progenitor cells (CPCs) will aid our understanding of the signaling pathways and mechanisms involved and could ultimately provide tools for regenerative therapies for the treatment of post-MI cardiac dysfunction. We have used an in vitro human induced pluripotent stem cell-derived CPC model to screen a 10,000-compound library containing molecules representing different target classes and compounds reported to modulate the phenotype of stem or primary cells. The primary readout of this phenotypic screen was proliferation as measured by nuclear count. We identified retinoic acid receptor (RAR) agonists as potent proliferators of CPCs. The CPCs retained their progenitor phenotype following proliferation and the identified RAR agonists did not proliferate human cardiac fibroblasts, the major cell type in the heart. In addition, the RAR agonists were able to proliferate an independent source of CPCs, HuES6. The RAR agonists had a time-of-differentiation-dependent effect on the HuES6-derived CPCs. At 4 days of differentiation, treatment with retinoic acid induced differentiation of the CPCs to atrial cells. However, after 5 days of differentiation treatment with RAR agonists led to an inhibition of terminal differentiation to cardiomyocytes and enhanced the proliferation of the cells. RAR agonists, at least transiently, enhance the proliferation of human CPCs, at the expense of terminal cardiac differentiation. How this mechanism translates in vivo to activate endogenous CPCs and whether enhancing proliferation of these rare progenitor cells is sufficient to enhance cardiac repair remains to be investigated.

Protease-activated receptor-2 ligands reveal orthosteric and allosteric mechanisms of receptor inhibition.

Protease-activated receptor-2 (PAR2) has been implicated in multiple pathophysiologies but drug discovery is challenging due to low small molecule tractability and a complex activation mechanism. Here we report the pharmacological profiling of a potent new agonist, suggested by molecular modelling to bind in the putative orthosteric site, and two novel PAR2 antagonists with distinctly different mechanisms of inhibition. We identify coupling between different PAR2 binding sites. One antagonist is a competitive inhibitor that binds to the orthosteric site, while a second antagonist is a negative allosteric modulator that binds at a remote site. The allosteric modulator shows probe dependence, more effectively inhibiting peptide than protease activation of PAR2 signalling. Importantly, both antagonists are active in vivo, inhibiting PAR2 agonist-induced acute paw inflammation in rats and preventing activation of mast cells and neutrophils. These results highlight two distinct mechanisms of inhibition that potentially could be targeted for future development of drugs that modulate PAR2.

Functionalized 3-amino-imidazo[1,2-a]pyridines: a novel class of drug-like Mycobacterium tuberculosis glutamine synthetase inhibitors.

3-Amino-imidazo[1,2-a]pyridines have been identified as a novel class of Mycobacterium tuberculosis glutamine synthetase inhibitors. Moreover, these compounds represent the first drug-like inhibitors of this enzyme. A series of compounds exploring structural diversity in the pyridine and phenyl rings have been synthesized and biologically evaluated. Compound 4n was found to be the most potent inhibitor (IC(50)=0.38+/-0.02 microM). This compound was significantly more potent than the known inhibitors, l-methionine-SR-sulfoximine and phosphinothricin.

Mineralocorticoid Receptor Antagonists.

Two mineralocorticoid receptor antagonists, spironolactone and eplerenone, are currently approved by the FDA. Several non-steroid based ligands are in clinical trials for indications including heart failure, hypertension and diabetic kidney disease, and even more structurally distinct chemical series are reported in the literature with preclinical data from animal models. Design of new ligands that are both selective over the other oxosteroid receptors (GR, PR and AR) and possess properties compatible with oral dosing, despite the overall lipophilic binding pocket of MR, remains a challenge. High-throughput screening has been successfully used to identify novel starting points in several drug discovery programs, and these were optimized using property based drug design, often aided by protein-ligand X-ray complex structures.

Identification of Mineralocorticoid Receptor Modulators with Low Impact on Electrolyte Homeostasis but Maintained Organ Protection.

The mechanism-based risk for hyperkalemia has limited the use of mineralocorticoid receptor antagonists (MRAs) like eplerenone in cardio-renal diseases. Here, we describe the structure and property-driven lead generation and optimization, which resulted in identification of MR modulators ( S)-1 and ( S)-33. Both compounds were partial MRAs but still demonstrated equally efficacious organ protection as eplerenone after 4 weeks of treatment in uni-nephrectomized rats on high-salt diet and aldosterone infusion. Importantly, and in sharp contrast to eplerenone, this was achieved without substantial changes to the urine Na+/K+ ratio after acute treatment in rat, which predicts a reduced risk for hyperkalemia. This work led to selection of ( S)-1 (AZD9977) as the clinical candidate for treating MR-mediated cardio-renal diseases, including chronic kidney disease and heart failure. On the basis of our findings, we propose an empirical model for prediction of compounds with low risk of affecting the urinary Na+/K+ ratio in vivo.

Evaluation of the amino acid binding site of Mycobacterium tuberculosis glutamine synthetase for drug discovery.

A combination of a literature survey, structure-based virtual screening and synthesis of a small library was performed to identify hits to the potential antimycobacterial drug target, glutamine synthetase. The best inhibitor identified from the literature survey was (2S,5R)-2,6-diamino-5-hydroxyhexanoic acid (4, IC(50) of 610+/-15microM). In the virtual screening 46,400 compounds were docked and subjected to a pharmacophore search. Of these compounds, 29 were purchased and tested in a biological assay, allowing three novel inhibitors containing an aromatic scaffold to be identified. Based on one of the hits from the virtual screening a small library of 15 analogues was synthesized producing four compounds that inhibited glutamine synthetase.

Structural Characterization of Agonist Binding to Protease-Activated Receptor 2 through Mutagenesis and Computational Modeling.

Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor that is activated by proteolytic cleavage of its N-terminus. The unmasked N-terminal peptide then binds to the transmembrane bundle, leading to activation of intracellular signaling pathways associated with inflammation and cancer. Recently determined crystal structures have revealed binding sites of PAR2 antagonists, but the binding mode of the peptide agonist remains unknown. In order to generate a model of PAR2 in complex with peptide SLIGKV, corresponding to the trypsin-exposed tethered ligand, the orthosteric binding site was probed by iterative combinations of receptor mutagenesis, agonist ligand modifications, and data-driven structural modeling. Flexible-receptor docking identified a conserved binding mode for agonists related to the endogenous ligand that was consistent with the experimental data and allowed synthesis of a novel peptide (1-benzyl-1H[1,2,3]triazole-4-yl-LIGKV) with functional potency higher than that of SLIGKV. The final model may be used to understand the structural basis of PAR2 activation and in virtual screens to identify novel agonists and competitive antagonists. The combined experimental and computational approach to characterize agonist binding to PAR2 can be extended to study the many other G protein-coupled receptors that recognize peptides or proteins.

Microwave-enhanced alpha-arylation of a protected glycine in water: evaluation of 3-phenylglycine derivatives as inhibitors of the tuberculosis enzyme, glutamine synthetase.

A microwave-enhanced, palladium-catalyzed protocol for the alpha-arylation of a protected glycine in neat water is described. This reaction proceeds rapidly, under non-inert conditions, to afford a range of phenylglycine derivatives in moderate to good yields. Based on this alpha-arylation, a number of aryl L-methionine-SR-sulfoximine (MSO) analogues were prepared and evaluated for their Mycobacterium tuberculosis glutamine synthetase (TB-GS) inhibitory activity.

Virtual screening and bioassay study of novel inhibitors for dengue virus mRNA cap (nucleoside-2'O)-methyltransferase.

We report high-throughput structure-based virtual screening of putative Flavivirus 2'-O-methyltransferase inhibitors together with results from subsequent bioassay tests of selected compounds. Potential inhibitors for the S-adenosylmethionine binding site were explored using 2D similarity searching, pharmacophore filtering and docking. The inhibitory activities of 15 top-ranking compounds from the docking calculations were tested on a recombinant methyltransferase with the RNA substrate (7Me)GpppAC(5). Local and global docking simulations were combined to estimate the ligand selectivity for the target site. The results of the combined computational and experimental screening identified a novel inhibitor, with a previously unknown scaffold, that has an IC(50) value of 60 microM.

Quantitative structure-activity relationships of pine weevil antifeedants, a multivariate approach.

Antifeedant activity of mainly phenylpropanoic, cinnamic, and benzoic acids esters was tested on the pine weevil, Hylobius abietis (L.). Of 105 compounds screened for activity, 9 phenylpropanoates, 3 cinnamates, and 4 benzoates were found to be highly active antifeedants. To understand the structure-activity relationships of these compounds, a multivariate analysis study was performed. A number of molecular and substituent descriptors were calculated and correlated to results from two-choice feeding tests with H. abietis. Three local models were developed that had good internal predictive ability. External test sets showed moderate predictivity. In general, low polarity, small size, and high lipophilicity were characteristics for compounds having good antifeedant activity.

Phenotypic Screen for Cardiac Regeneration Identifies Molecules with Differential Activity in Human Epicardium-Derived Cells versus Cardiac Fibroblasts.

Activation and proliferation of resident cardiac progenitor cells has therapeutic potential to repair the heart after injury. However, research has been impeded by a lack of well-defined and characterized cell sources and difficulties in translation to screening platforms. Here, we describe the development, validation, and use of a 384-well phenotypic assay in primary human epicardium-derived cells (EPDCs) to identify compounds that induce proliferation while maintaining the progenitor phenotype. Using this assay, we screened 7400 structurally diverse compounds where greater than 90% are biologically annotated and known to modulate a broad range of biological targets. From the primary screen, we identified and validated hits and expanded upon the lead molecules of interest. A counterscreen was developed in human cardiac fibroblasts to filter out compounds with a general proliferative effect, after which the activity of selected molecules was confirmed across multiple EPDC donors. To further examine the mechanism of action of compounds with annotated targets, we performed knockdown experiments to understand whether a single known target was responsible for the proliferative effect, confirming results with protein expression and activity assays. Here, we were able to show that the annotated targets of compounds of interest were not responsible for the proliferative effect, which highlights potential differences in cell types and signaling pathways and possible polypharmacology. These studies demonstrate the feasibility of using relevant human primary cells in a phenotypic screen to identify compounds as novel biological tools and starting points for drug discovery projects, and we disclose the first small molecules to proliferate human primary EPDCs.

Structure-Based Drug Design of Mineralocorticoid Receptor Antagonists to Explore Oxosteroid Receptor Selectivity.

The mineralocorticoid receptor (MR) is a nuclear hormone receptor involved in the regulation of body fluid and electrolyte homeostasis. In this study we explore selectivity triggers for a series of nonsteroidal MR antagonists to improve selectivity over other members of the oxosteroid receptor family. A biaryl sulfonamide compound was identified in a high-throughput screening (HTS) campaign. The compound bound to MR with pKi =6.6, but displayed poor selectivity over the glucocorticoid receptor (GR) and the progesterone receptor (PR). Following X-ray crystallography of MR in complex with the HTS hit, a compound library was designed that explored an induced-fit hypothesis that required movement of the Met852 side chain. An improvement in MR selectivity of 11- to 79-fold over PR and 23- to 234-fold over GR was obtained. Given the U-shaped binding conformation, macrocyclizations were explored, yielding a macrocycle that bound to MR with pKi =7.3. Two protein-ligand X-ray structures were determined, confirming the hypothesized binding mode for the designed compounds.

New Hits as Antagonists of GPR103 Identified by HTS.

Preclinical data indicate that GPR103 receptor and its endogenous neuropeptides QRFP26 and QRFP43 are involved in appetite regulation. A high throughput screening (HTS) for small molecule GPR103 antagonists was performed with the clinical goal to target weight management by modulation of appetite. A high hit rate from the HTS and initial low confirmation with respect to functional versus affinity data challenged us to revise the established screening cascade. To secure high quality data while increasing throughput, the binding assay was optimized on quality to run at single concentration. This strategy enabled evaluation of a larger fraction of chemical clusters and singletons delivering 17 new compound classes for GPR103 antagonism. Representative compounds from three clusters are presented. One of the identified clusters was further investigated, and an initial structure-activity relationship study is reported. The most potent compound identified had a pIC50 of 7.9 with an improved ligand lipophilic efficiency.

GPR103 antagonists demonstrating anorexigenic activity in vivo: design and development of pyrrolo[2,3-c]pyridines that mimic the C-terminal Arg-Phe motif of QRFP26.

GPR103, a G-protein coupled receptor, has been reported to have orexigenic properties through activation by the endogenous neuropeptide ligands QRFP26 and QRFP43. Recognizing that central administration of QRFP26 and QRFP43 increases high fat food intake in rats, we decided to investigate if antagonists of GPR103 could play a role in managing feeding behaviors. Here we present the development of a new series of pyrrolo[2,3-c]pyridines as GPR103 small molecule antagonists with GPR103 affinity, drug metabolism and pharmacokinetics and safety parameters suitable for drug development. In a preclinical obesity model measuring food intake, the anorexigenic effect of a pyrrolo[2,3-c]pyridine GPR103 antagonist was demonstrated. In addition, the dynamic 3D solution structure of the C-terminal heptapeptide of the endogenous agonist QRFP26(20-26) was determined using NMR. The synthetic pyrrolo[2,3-c]pyridine antagonists were compared to this experimental structure, which displayed a possible overlay of pharmacophore features supportive for further design of GPR103 antagonists.

Links between bacterial production, amino-acid utilization and community composition in productive lakes.

Influence of distribution and abundance of bacterial taxa on ecosystem function are poorly understood for natural microbial communities. We related 16S rRNA-based terminal restriction fragment length polymorphism to bacterial production and arginine uptake kinetics to test if functional features of bacterioplankton in four lakes could be predicted from community composition. Maximum arginine uptake rate (arginine V(max)) ranged from 10% to 100% of bacterial production. Owing to high growth efficiencies on arginine (63-77%), the bacterial community could potentially saturate its carbon demand using this single organic substrate, for example, during sudden surges of free amino acids. However, due to low in situ concentrations of arginine in these lakes (<0.9 microg l(-1)), actual uptake rates at ambient concentrations rarely exceeded 10% of V(max). Bacterial production and arginine V(max) could be predicted from a subset of bacterial ribotypes, tentatively affiliated with several bacterial divisions (Cyanobacteria, Actinobacteria, Bacteroidetes and Proteobacteria). Multivariate statistical analysis indicates that there were both highly important and less important ribotypes for the prediction of bacterial production and arginine V(max). These populations were either negatively or positively related to the respective functional feature, indicating contrasting ecological roles. Our study provides a statistically robust demonstration that, apart from environmental conditions, patterns in bacterial community composition can also be used to predict lake ecosystem function.