Pharmacophore-Based Virtual Screening to Discover New Active Compounds for Human Choline Kinase α1.

Choline kinase (CK) catalyses the transfer of the ATP γ-phosphate to choline to generate phosphocholine and ADP in the presence of magnesium leading to the synthesis of phosphatidylcholine. Of the three isoforms of CK described in humans, only the α isoforms (HsCKα) are strongly associated with cancer and have been validated as drug targets to treat this disease. Over the years, a large number of Hemicholinium-3 (HC-3)-based HsCKα biscationic inhibitors have been developed though the relevant common features important for the biological function have not been defined. Here, selecting a large number of previous HC-3-based inhibitors, we discover through computational studies a pharmacophore model formed by five moieties that are included in the 1-benzyl-4-(N-methylaniline)pyridinium fragment. Using a pharmacophore-guided virtual screening, we then identified 6 molecules that showed binding affinities in the low µM range to HsCKα1. Finally, protein crystallization studies suggested that one of these molecules is bound to the choline and ATP-binding sites. In conclusion, we have developed a pharmacophore model that not only allowed us to dissect the structural important features of the previous HC-3 derivatives, but also enabled the identification of novel chemical tools with good ligand efficiencies to investigate the biological functions of HsCKα1.

Ligand binding and functional selectivity of L-tryptophan metabolites at the mouse aryl hydrocarbon receptor (mAhR).

The aryl hydrocarbon receptor (AhR) is a nuclear receptor regulating a wide range of biological and toxicological effects. Metabolites of L-tryptophan are able to bind and activate AhR, providing a link between tryptophan catabolism and a novel mechanism of protective tolerance, referred to as "disease tolerance". The notion that pharmacologic modulation of genes associated with endotoxin tolerance would be beneficial in clinical settings dominated by acute hyperinflammatory responses to infection thrusts AhR into the limelight as an interesting druggable target. Combining homology modeling, docking studies, and molecular dynamic simulations with mutagenesis experiments and gene profiling, in this work we report that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and two different L-tryptophan metabolites, namely L-Kynurenine and FICZ (6-formylindolo[3,2-b]carbazole), are able to bind to mAhR, exploiting different key interactions with distinct set of fingerprint residues. As a result, they stabilize different conformations of mAhR that, in turn, selectively regulate downstream signaling and transcription of specific target genes. Collectively, these results open new avenues for the design and development of selective AhR modulators that, by targeting specific receptor conformations associated with specific AhR functions, may offer novel therapeutic opportunities in infectious diseases and other morbidity that may be associated with the receptor.

Synthesis and quantitative structure-property relationships of side chain-modified hyodeoxycholic acid derivatives.

Bile acids have emerged as versatile signalling compounds of a complex network of nuclear and membrane receptors regulating various endocrine and paracrine functions. The elucidation of the interconnection between the biological pathways under the bile acid control and manifestations of hepatic and metabolic diseases have extended the scope of this class of steroids for in vivo investigations. In this framework, the design and synthesis of novel biliary derivatives able to modulate a specific receptor requires a deep understanding of both structure-activity and structure-property relationships of bile acids. In this paper, we report the preparation and the critical micellization concentration evaluation of a series of hyodeoxycholic acid derivatives characterized by a diverse side chain length and by the presence of a methyl group at the alpha position with respect to the terminal carboxylic acid moiety. The data collected are instrumental to extend on a quantitative basis, the knowledge of the current structure-property relationships of bile acids and will be fruitful, in combination with models of receptor activity, to design and prioritize the synthesis of novel pharmacokinetically suitable ligands useful in the validation of bile acid-responsive receptors as therapeutic targets.

Exploring the effect of PARP-1 flexibility in docking studies.

Poly(ADP-ribose)polymerase-1 (PARP-1) is an enzyme belonging to the ADP-ribosyltransferase family. A large body of works has validated PARP-1 as an attractive drug target for different therapeutic areas, including cancers and ischemia. Accordingly, sampling the conformational space of the enzyme is pivotal to understand its functions and improve structure-based drug discovery approaches. In the first part of this study we apply replica exchange molecular dynamic (REMD) simulations to sample the conformational space of the catalytic domain of PARP-1 in the ligand-bound and unbound forms. In the second part, we assess how and to what extend the emerging enzyme flexibility affects the performance of docking experiments of a library of PARP-1 inhibitors. This study pinpoints a putative key role of conformational shifts of Leu324, Tyr325 and Lys242 in opening an additional binding site pocket that affects the binding of ligands to the catalytic cleft of PARP-1. Furthermore, it highlights the improvement of the enrichment factor of active ligands obtained in docking experiments when using conformations generated with REMD simulations of ligand-bound PARP-1.

Probing the Binding Site of Bile Acids in TGR5.

TGR5 is a G-protein-coupled receptor (GPCR) mediating cellular responses to bile acids (BAs). Although some efforts have been devoted to generate homology models of TGR5 and draw structure-activity relationships of BAs, none of these studies has hitherto described how BAs bind to TGR5. Here, we present an integrated computational, chemical, and biological approach that has been instrumental to determine the binding mode of BAs to TGR5. As a result, key residues have been identified that are involved in mediating the binding of BAs to the receptor. Collectively, these results provide new hints to design potent and selective TGR5 agonists.

Patented TGR5 modulators: a review (2006 - present).

INTRODUCTION: The G protein-coupled receptor TGR5 is a key player of the bile acid signaling network, and its activation has been proved to increase the glycemic control, to enhance energy expenditure and to exert anti-inflammatory actions. Accordingly, TGR5 ligands have emerged in drug discovery and preclinical appraisals as promising agents for the treatment of liver diseases, metabolic syndrome and related disorders. AREAS COVERED: Recent advances in the field of TGR5 modulators are reviewed, with a particular attention on patent applications and peer-reviewed publications in the past 6 years. EXPERT OPINION: Activation of TGR5 showed to protect mice from diabesity and insulin resistance, to improve liver functions, as well as to attenuate the development of atherosclerosis. However, although the efficacy of TGR5 agonists in mice is encouraging, further studies are needed to determine their potential in humans and to evaluate carefully the balance between the therapeutic benefits and adverse effects associated with the target. The development of new TGR5 selective ligands to support studies in animal models will surely facilitate the understanding of the complexity of TGR5 signaling network.

Fitting the complexity of GPCRs modulation into simple hypotheses of ligand design.

G-protein coupled receptors (GPCR(s)) are a large family of membrane-bound receptors that mediate a wide range of physiologic responses to hormones, neurotransmitters and dietary lipids, which represent an important class of drug targets. Significant chemical space regions have been explored both in the academia and by pharmaceutical companies, in the quest for new GPCR modulators as potential therapeutic agents. This accumulated body of evidence provides new opportunities to evaluate potential features of GPCR agonists and antagonists, and how to distinguish them. In this study, the chemical space covered within the WOMBAT database by GPCRs modulators was investigated with the aim of identifying specific molecular determinants that distinguish GPCR agonists from antagonists. While instrumental to get insights into the design strategies of GPCRs modulators, the results of this study provide novel clues on the molecular mechanisms that underlie the complexity of GPCR modulation.

Avicholic Acid: A Lead Compound from Birds on the Route to Potent TGR5 Modulators.

Grounding on our former 3D QSAR studies, a knowledge-based screen of natural bile acids from diverse animal species has led to the identification of avicholic acid as a selective but weak TGR5 agonist. Chemical modifications of this compound resulted in the disclosure of 6α-ethyl-16-epi-avicholic acid that shows enhanced potency at TGR5 and FXR receptors. The synthesis, biological appraisals, and structure-activity relationships of this series of compounds are herein described. Moreover, a thorough physicochemical characterization of 6α-ethyl-16-epi-avicholic acid as compared to naturally occurring bile acids is reported and discussed.

Novel method for generating structure-based pharmacophores using energetic analysis.

We describe a novel method to develop energetically optimized, structure-based pharmacophores for use in rapid in silico screening. The method combines pharmacophore perception and database screening with protein-ligand energetic terms computed by the Glide XP scoring function to rank the importance of pharmacophore features. We derive energy-optimized pharmacophore hypotheses for 30 pharmaceutically relevant crystal structures and screen a database to assess the enrichment of active compounds. The method is compared to three other approaches: (1) pharmacophore hypotheses derived from a systematic assessment of receptor-ligand contacts, (2) Glide SP docking, and (3) 2D ligand fingerprint similarity. The method developed here shows better enrichments than the other three methods and yields a greater diversity of actives than the contact-based pharmacophores or the 2D ligand similarity. Docking produces the most cases (28/30) with enrichments greater than 10.0 in the top 1% of the database and on average produces the greatest diversity of active molecules. The combination of energy terms from a structure-based analysis with the speed of a ligand-based pharmacophore search results in a method that leverages the strengths of both approaches to produce high enrichments with a good diversity of active molecules.

Charting the chemical space of target sites: insights into the binding modes of amine and amidine groups.

Nowadays there is growing awareness that the translation of the increasing number of lead compounds into clinical candidates is still a slow and often inefficient process. In order to facilitate the lead optimization procedure, due consideration must be given to the use of the right bioisosteric replacements. Very recently, we reported that exploring a chemical space of binding sites is a more effective strategy for studying the bioisosteric relationships existing among functional groups. As a continuation of our work in this field, we report herein the construction of a chemical space covered by binding sites of small molecules containing diverse amine and amidine groups. The analysis of the differences in some properties of the binding sites of these functional groups allow for gaining insights into the binding modes of positively charged groups. In addition, this study pinpoints that different types of interactions and bioisosteric relationships exist among primary, secondary, tertiary, quaternary amine, and amidine moieties.

Highlights at the gate of tryptophan catabolism: a review on the mechanisms of activation and regulation of indoleamine 2,3-dioxygenase (IDO), a novel target in cancer disease.

Indoleamine 2,3-dioxygenase (IDO) catalyzes the first and rate-limiting step of Kynurenine pathway along the major route of Tryptophan catabolism. The scientific interest in the enzyme has been growing since the observations of the involvement of IDO in the mechanisms of immune tolerance and in the mechanisms of tumor immuno-editing process. In view of this latter observation, in particular, preclinical studies of small molecule inhibitors of the enzyme have indicated the feasibility to thwart the immuno-editing process and to enhance the efficacy of current chemotherapeutic agents, supporting the notion that IDO is a novel target in cancer disease.This review covers the structural and conformational aspects of substrate recognition by IDO, including the catalytic mechanism and the so-far puzzling mechanisms of enzyme activation. Furthermore, we discuss the recent advances of medicinal chemistry in the field of IDO inhibitors.

Adamantyl-substituted retinoid-derived molecules that interact with the orphan nuclear receptor small heterodimer partner: effects of replacing the 1-adamantyl or hydroxyl group on inhibition of cancer cell growth, induction of cancer cell apoptosis, and inhibition of SRC homology 2 domain-containing protein tyrosine phosphatase-2 activity.

(E)-4-[3-(1-Adamantyl)-4'-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC) induces the cell-cycle arrest and apoptosis of leukemia and cancer cells. Studies demonstrated that 3-Cl-AHPC bound to the atypical orphan nuclear receptor small heterodimer partner (SHP). Although missing a DNA-binding domain, SHP heterodimerizes with the ligand-binding domains of other nuclear receptors to repress their abilities to induce or inhibit gene expression. 3-Cl-AHPC analogues having the 1-adamantyl and phenolic hydroxyl pharmacophoric elements replaced with isosteric groups were designed, synthesized, and evaluated for their inhibition of proliferation and induction of human cancer cell apoptosis. Structure-anticancer activity relationship studies indicated the importance of both groups to apoptotic activity. Docking of 3-Cl-AHPC and its analogues to an SHP computational model that was based on the crystal structure of ultraspiracle complexed with 1-stearoyl-2-palmitoylglycero-3-phosphoethanolamine suggested why these 3-Cl-AHPC groups could influence SHP activity. Inhibitory activity against Src homology 2 domain-containing protein tyrosine phosphatase 2 (Shp-2) was also assessed. The most active Shp-2 inhibitor was found to be the 3'-(3,3-dimethylbutynyl) analogue of 3-Cl-AHPC.

Molecular field analysis and 3D-quantitative structure-activity relationship study (MFA 3D-QSAR) unveil novel features of bile acid recognition at TGR5.

Bile acids regulate nongenomic actions through the activation of TGR5, a membrane receptor that is G protein-coupled to the induction of adenylate cyclase. In this work, a training set of 43 bile acid derivatives is used to develop a molecular interaction field analysis (MFA) and a 3D-quantitative structure-activity relationship study (3D-QSAR) of TGR5 agonists. The predictive ability of the resulting model is evaluated using an external set of compounds with known TGR5 activity, and six bile acid derivatives whose unknown TGR5 activity is herein assessed with in vitro luciferase assay of cAMP formation. The results show a good predictive model and indicate a statistically relevant degree of correlation between the TGR5 activity and the molecular interaction fields produced by discrete positions of the bile acid scaffold. This information is instrumental to extend on a quantitative basis the current structure-activity relationships of bile acids as TGR5 modulators and will be fruitful to design new potent and selective agonists of the receptor.

Molecular docking and spatial coarse graining simulations as tools to investigate substrate recognition, enhancer binding and conformational transitions in indoleamine-2,3-dioxygenase (IDO).

Indoleamine 2,3-dioxygenase (IDO) is an heme-containing enzyme involved in the regulation of important immunological responses and neurological processes. The enzyme catalyzes the oxidative cleavage of the pyrrole ring of the indole nucleus of tryptophan (Trp) to yield N-formylkynurenine, that is the initial and rate limiting step of the kynurenine pathway. Some indole derivatives have been reported to act as effectors of the enzyme by enhancing its catalytic activity. On the basis of the recent availability of the crystal structure of IDO, in this work we investigate substrate recognition and enhancer binding to IDO using molecular docking experiments. In addition, conformational transitions of IDO in response to substrate and enhancer binding are studied using coarse graining simulations with the program FIRST. The results enable us to identify (i) the binding site of enhancer modulators; (ii) the motion of an electrostatic gate that regulates the access of the substrate to the catalytic site of the enzyme; (iii) the movement of the anchoring region of a hairpin loop that may assist the shuttle of substrates/products to/from the catalytic site of IDO. These data, combined with available site-directed mutagenesis experiments, reveal that conformational transitions of IDO in response to substrate and enhancer binding are controlled by distinct combination of two conformational states (open and close) of the above structural motifs. On this basis, a molecular mechanism regarding substrate recognition and activity enhancement by indole derivatives is proposed.