Structure-Activity Relationship Studies of Tetrahydroquinolone Free Fatty Acid Receptor 3 Modulators.

Free fatty acid receptor 3 (FFA3, previously GPR41) is activated by short-chain fatty acids, mediates health effects of the gut microbiota, and is a therapeutic target for metabolic and inflammatory diseases. The shortage of well-characterized tool compounds has however impeded progress. Herein, we report structure-activity relationship of an allosteric modulator series and characterization of physicochemical and pharmacokinetic properties of selected compounds, including previous and new tools. Two representatives, 57 (TUG-1907) and 63 (TUG-2015), showed improved solubility and preserved potency. Of these, 57, with EC50 = 145 nM and a solubility of 33 µM, showed high clearance in vivo but is a preferred tool in vitro. In contrast, 63, with EC50 = 162 nM and a solubility of 9 µM, showed lower clearance and seems better suited for in vivo studies. Using 57, we demonstrate for the first time that FFA3 activation leads to calcium mobilization in murine dorsal root ganglia.

Selective Allosteric Modulation of N-Terminally Cleaved, but Not Full Length CCL3 in CCR1.

Chemokines undergo post-translational modification such as N-terminal truncations. Here, we describe how N-terminal truncation of full length CCL3(1-70) affects its activity at CCR1. Truncated CCL3(5-70) has 10-fold higher potency and enhanced efficacy in ß-arrestin recruitment, but less than 2-fold increased potencies in G protein signaling determined by calcium release, cAMP and IP3 formation. Small positive ago-allosteric ligands modulate the two CCL3 variants differently as the metal ion chelator bipyridine in complex with zinc (ZnBip) enhances the binding of truncated, but not full length CCL3, while a size-increase of the chelator to a chloro-substituted terpyridine (ZnClTerp), eliminates its allosteric, but not agonistic action. By employing a series of receptor mutants and in silico modeling we describe residues of importance for chemokine and small molecule binding. Notably, the chemokine receptor-conserved Glu2877.39 interacts with the N-terminal amine of truncated CCL3(5-70) and with Zn2+ of ZnBip, thereby bridging their binding sites and enabling the positive allosteric effect. Our study emphasizes that small allosteric molecules may act differently toward chemokine variants and thus selectively modulate interactions of specific chemokine subsets with their cognate receptors.

Structure-Activity Investigations and Optimisations of Non-metabolite Agonists for the Succinate Receptor 1.

The succinate receptor 1 (SUCNR1) is a receptor for the metabolite succinate, which functions as a metabolic stress signal in the liver, kidney, adipose tissue and the retina. However, potent non-metabolite tool compounds are needed to reveal the physiological role and pharmacological potential of SUCNR1. Recently, we published the discovery of a computationally receptor-structure derived non-metabolite SUCNR1 agonist series with high target selectivity. We here report our structure-activity exploration and optimisation that has resulted in the development of agonists with nanomolar potency and excellent solubility and stability properties in a number of in vitro assays. Ligand-guided receptor models with high discriminative power between binding of active and inactive compounds were developed for design of novel chemotypes.

Receptor structure-based discovery of non-metabolite agonists for the succinate receptor GPR91.

OBJECTIVE: Besides functioning as an intracellular metabolite, succinate acts as a stress-induced extracellular signal through activation of GPR91 (SUCNR1) for which we lack suitable pharmacological tools. METHODS AND RESULTS: Here we first determined that the cis conformation of the succinate backbone is preferred and that certain backbone modifications are allowed for GPR91 activation. Through receptor modeling over the X-ray structure of the closely related P2Y1 receptor, we discovered that the binding pocket is partly occupied by a segment of an extracellular loop and that succinate therefore binds in a very different mode than generally believed. Importantly, an empty side-pocket is identified next to the succinate binding site. All this information formed the basis for a substructure-based search query, which, combined with molecular docking, was used in virtual screening of the ZINC database to pick two serial mini-libraries of a total of only 245 compounds from which sub-micromolar, selective GPR91 agonists of unique structures were identified. The best compounds were backbone-modified succinate analogs in which an amide-linked hydrophobic moiety docked into the side-pocket next to succinate as shown by both loss- and gain-of-function mutagenesis. These compounds displayed GPR91-dependent activity in altering cytokine expression in human M2 macrophages similar to succinate, and importantly were devoid of any effect on the major intracellular target, succinate dehydrogenase. CONCLUSIONS: These novel, synthetic non-metabolite GPR91 agonists will be valuable both as pharmacological tools to delineate the GPR91-mediated functions of succinate and as leads for the development of GPR91-targeted drugs to potentially treat low grade metabolic inflammation and diabetic complications such as retinopathy and nephropathy.

Design, Synthesis, and Evaluation of Novel Tyrosine-Based DNA Gyrase B Inhibitors.

The discovery and synthesis of new tyrosine-based inhibitors of DNA gyrase B (GyrB), which target its ATPase subunit, is reported. Twenty-four compounds were synthesized and evaluated for activity against DNA gyrase and DNA topoisomerase IV. The antibacterial properties of selected GyrB inhibitors were demonstrated by their activity against Staphylococcus aureus and Enterococcus faecalis in the low micromolar range. The most promising compounds, 8a and 13e, inhibited Escherichia coli and S. aureus GyrB with IC50 values of 40 and 30 µM. The same compound also inhibited the growth of S. aureus and E. faecalis with minimal inhibitory concentrations (MIC90 ) of 14 and 28 µg/mL, respectively.

Discovery of substituted oxadiazoles as a novel scaffold for DNA gyrase inhibitors.

DNA gyrase and topoisomerase IV are type IIa topoisomerases that are essential bacterial enzymes required to oversee the topological state of DNA during transcription and replication processes. Their ATPase domains, GyrB and ParE, respectively, are recognized as viable targets for small molecule inhibitors, however, no synthetic or natural product GyrB/ParE inhibitors have so far reached the clinic for use as novel antibacterial agents, except for novobiocin which was withdrawn from the market. In the present study, a series of substituted oxadiazoles have been designed and synthesized as potential DNA gyrase inhibitors. Structure-based optimization resulted in the identification of compound 35, displaying an IC50 of 1.2 µM for Escherichia coli DNA gyrase, while also exhibiting a balanced low micromolar inhibition of E. coli topoisomerase IV and of the respective Staphylococcus aureus homologues. The most promising inhibitors identified from each series were ultimately evaluated against selected Gram-positive and Gram-negative bacterial strains, of which compound 35 inhibited Enterococcus faecalis with a MIC90 of 75 µM. Our study thus provides further insight into the structural requirements of substituted oxadiazoles for dual inhibition of DNA gyrase and topoisomerase IV.

Linker-switch approach towards new ATP binding site inhibitors of DNA gyrase B.

Due to increasing emergence of bacterial resistance, compounds with new mechanisms of action are of paramount importance. One of modestly researched therapeutic targets in the field of antibacterial discovery is DNA gyrase B. In the present work we synthesized a focused library of potential DNA gyrase B inhibitors composed of two key pharmacophoric moieties linked by three types of sp3-rich linkers to obtain three structural classes of compounds. Using molecular docking, molecular dynamics and analysis of conserved waters in the binding site, we identified a favourable binding mode for piperidin-4-yl and 4-cyclohexyl pyrrole-2-carboxamides while predicting unfavourable interactions with the active site for piperazine pyrrole-2-carboxamides. Biological evaluation of prepared compounds on isolated enzyme DNA gyrase B confirmed our predictions and afforded multiple moderately potent inhibitors of DNA gyrase B. Namely trans-4-(4,5-dibromo-1H-pyrrole-2-carboxamide)cyclohexyl)glycine and 4-(4-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)piperidin-1-yl)-4-oxobutanoic acid with an IC50 value of 16 and 0.5 µM respectively.

Molecular Mechanism of Action for Allosteric Modulators and Agonists in CC-chemokine Receptor 5 (CCR5).

The small molecule metal ion chelators bipyridine and terpyridine complexed with Zn2+ (ZnBip and ZnTerp) act as CCR5 agonists and strong positive allosteric modulators of CCL3 binding to CCR5, weak modulators of CCL4 binding, and competitors for CCL5 binding. Here we describe their binding site using computational modeling, binding, and functional studies on WT and mutated CCR5. The metal ion Zn2+ is anchored to the chemokine receptor-conserved Glu-283VII:06/7.39 Both chelators interact with aromatic residues in the transmembrane receptor domain. The additional pyridine ring of ZnTerp binds deeply in the major binding pocket and, in contrast to ZnBip, interacts directly with the Trp-248VI:13/6.48 microswitch, contributing to its 8-fold higher potency. The impact of Trp-248 was further confirmed by ZnClTerp, a chloro-substituted version of ZnTerp that showed no inherent agonism but maintained positive allosteric modulation of CCL3 binding. Despite a similar overall binding mode of all three metal ion chelator complexes, the pyridine ring of ZnClTerp blocks the conformational switch of Trp-248 required for receptor activation, thereby explaining its lack of activity. Importantly, ZnClTerp becomes agonist to the same extent as ZnTerp upon Ala mutation of Ile-116III:16/3.40, a residue that constrains the Trp-248 microswitch in its inactive conformation. Binding studies with 125I-CCL3 revealed an allosteric interface between the chemokine and the small molecule binding site, including residues Tyr-37I:07/1.39, Trp-86II:20/2.60, and Phe-109III:09/3.33 The small molecules and CCL3 approach this interface from opposite directions, with some residues being mutually exploited. This study provides new insight into the molecular mechanism of CCR5 activation and paves the way for future allosteric drugs for chemokine receptors.

New N-phenyl-4,5-dibromopyrrolamides and N-Phenylindolamides as ATPase inhibitors of DNA gyrase.

Following the withdrawal of novobiocin, the introduction of a new ATPase inhibitor of DNA gyrase to the clinic would add the first representative of this mechanistic class to the antibacterial pipeline. This would be of great importance because of the well-known problems associated with antibacterial resistance. Using structure-based design and starting from the recently determined crystal structure of the N-phenyl-4,5-dibromopyrrolamide inhibitor-DNA gyrase B complex, we have prepared 28 new N-phenyl-4,5-dibromopyrrolamides and N-phenylindolamides and evaluated them against DNA gyrase from Escherichia coli. The most potent compound was 2-((4-(4,5-dibromo-1H-pyrrole-2-carboxamido)phenyl)amino)-2-oxoacetic acid (9a), with an IC50 of 0.18 µM against E. coli gyrase. A selected set of compounds was evaluated against DNA gyrase from Staphylococcus aureus and against topoisomerase IV from E. coli and S. aureus, but the activities were weaker. The binding affinity of 2-((4-(4,5-dibromo-1H-pyrrole-2-carboxamido)phenyl)amino)-2-oxoacetic acid (9a) to E. coli gyrase was studied using surface plasmon resonance. In the design of the present series, the focus was on the optimisation of biological activities of compounds - especially by varying their size, the position and orientation of key functional groups, and their acid-base properties. The structure-activity relationship (SAR) was examined and the results were rationalised with molecular docking.

N-Phenyl-4,5-dibromopyrrolamides and N-Phenylindolamides as ATP Competitive DNA Gyrase B Inhibitors: Design, Synthesis, and Evaluation.

Bacterial DNA gyrase is a well-known and validated target in the design of antibacterial drugs. However, inhibitors of its ATP binding subunit, DNA gyrase B (GyrB), have so far not reached clinical use. In the present study, three different series of N-phenyl-4,5-dibromopyrrolamides and N-phenylindolamides were designed and prepared as potential DNA gyrase B inhibitors. The IC50 values of compounds on DNA gyrase from Escherichia coli were in the low micromolar range, with the best compound, (4-(4,5-dibromo-1H-pyrrole-2-carboxamido)benzoyl)glycine (18a), displaying an IC50 of 450 nM. For this compound, a high-resolution crystal structure in complex with E. coli DNA gyrase B was obtained, revealing details of its binding mode within the active site. The binding affinities of three compounds with GyrB were additionally evaluated by surface plasmon resonance, and the results were in good agreement with the determined enzymatic activities. For the most promising compounds, the inhibitory activities against DNA gyrase from Staphylococcus aureus and topoisomerases IV from E. coli and S. aureus were determined. Antibacterial activities of the most potent compounds of each series were evaluated against two Gram-positive and two Gram-negative bacterial strains. The results obtained in this study provide valuable information on the binding mode and structure-activity relationship of N-phenyl-4,5-dibromopyrrolamides and N-phenylindolamides as promising classes of ATP competitive GyrB inhibitors.

Discovery of 4,5,6,7-Tetrahydrobenzo[1,2-d]thiazoles as Novel DNA Gyrase Inhibitors Targeting the ATP-Binding Site.

Bacterial DNA gyrase and topoisomerase IV are essential enzymes that control the topological state of DNA during replication and validated antibacterial drug targets. Starting from a library of marine alkaloid oroidin analogues, we identified low micromolar inhibitors of Escherichia coli DNA gyrase based on the 5,6,7,8-tetrahydroquinazoline and 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole scaffolds. Structure-based optimization of the initial hits resulted in low nanomolar E. coli DNA gyrase inhibitors, some of which exhibited micromolar inhibition of E. coli topoisomerase IV and of Staphylococcus aureus homologues. Some of the compounds possessed modest antibacterial activity against Gram positive bacterial strains, while their evaluation against wild-type, impA and ΔtolC E. coli strains suggests that they are efflux pump substrates and/or do not possess the physicochemical properties necessary for cell wall penetration. Our study provides a rationale for optimization of this class of compounds toward balanced dual DNA gyrase and topoisomerase IV inhibitors with antibacterial activity.

Discovery of mono- and disubstituted 1H-pyrazolo[3,4]pyrimidines and 9H-purines as catalytic inhibitors of human DNA topoisomerase†IIα.

Human DNA topoisomerase IIα (htIIα) is a validated target for the development of anticancer agents. Based on structural data regarding the binding mode of AMP-PNP (5'-adenylyl-ß,γ-imidodiphosphate) to htIIα, we designed a two-stage virtual screening campaign that combines structure-based pharmacophores and molecular docking. In the first stage, we identified several monosubstituted 9H-purine compounds and a novel class of 1H-pyrazolo[3,4]pyrimidines as inhibitors of htIIα. In the second stage, disubstituted analogues with improved cellular activities were discovered. Compounds from both classes were shown to inhibit htIIα-mediated DNA decatenation, and surface plasmon resonance (SPR) experiments confirmed binding of these two compounds on the htIIα ATPase domain. Proposed complexes and interaction patterns between both compounds and htIIα were further analyzed in molecular dynamics simulations. Two compounds identified in the second stage showed promising anticancer activities in hepatocellular carcinoma (HepG2) and breast cancer (MCF-7) cell lines. The discovered compounds are suitable starting points for further hit-to-lead development in anticancer drug discovery.

Monocyclic 4-amino-6-(phenylamino)-1,3,5-triazines as inhibitors of human DNA topoisomerase IIα.

Human DNA topoisomerase IIα (htIIα) is a validated target for the development of anticancer agents. Starting from the available information about the binding of the purine-based htIIα inhibitors in the ATP binding site we designed a virtual screening campaign combining structure-based and ligand-based pharmacophores with a molecular docking calculation searching for compounds that would contain a monocycle mimetic of the purine moiety. We discovered novel 4-amino-6-(phenylamino)-1,3,5-triazines 6, 7 and 11 as monocyclic htIIα inhibitors targeting the ATP binding site. Compound 6 from the 1,3,5-triazine series also displayed cytotoxicity properties in hepatocellular carcinoma (HepG2) cell lines and selectivity against human umbilical vein endothelial (HUVEC) cell lines.

Discovery of the first inhibitors of bacterial enzyme D-aspartate ligase from Enterococcus faecium (Aslfm).

The D-aspartate ligase of Enterococcus faecium (Aslfm) is an attractive target for the development of narrow-spectrum antibacterial agents that are active against multidrug-resistant E. faecium. Although there is currently little available information regarding the structural characteristics of Aslfm, we exploited the knowledge that this enzyme belongs to the ATP-grasp superfamily to target its ATP binding site. In the first design stage, we synthesized and screened a small library of known ATP-competitive inhibitors of ATP-grasp enzymes. A series of amino-oxazoles derived from bacterial biotin carboxylase inhibitors showed low micromolar activity. The most potent inhibitor compound 12, inhibits Aslfm with a Ki value of 2.9 µM. In the second design stage, a validated ligand-based pharmacophore modeling approach was used, taking the newly available inhibition data of an initial series of compounds into account. Experimental evaluation of the virtual screening hits identified two novel structural types of Aslfm inhibitors with 7-amino-9H-purine (18) and 7-amino-1H-pyrazolo[3,4-d]pyrimidine (30 and 34) scaffolds, and also with Ki values in the low micromolar range. Investigation the inhibitors modes of action confirmed that these compounds are competitive with respect to the ATP molecule. The binding of inhibitors to the target enzyme was also studied using isothermal titration calorimetry (ITC). Compounds 6, 12, 18, 30 and 34 represent the first inhibitors of Aslfm reported to date, and are an important step forward in combating infections due to E. faecium.

Structure-based discovery of substituted 4,5'-bithiazoles as novel DNA gyrase inhibitors.

Bacterial DNA gyrase is a well-established and validated target for the development of novel antibacterials. Starting from the available structural information about the binding of the natural product inhibitor, clorobiocin, we identified a novel series of 4'-methyl-N(2)-phenyl-[4,5'-bithiazole]-2,2'-diamine inhibitors of gyrase B with a low micromolar inhibitory activity by implementing a two-step structure-based design procedure. This novel class of DNA gyrase inhibitors was extensively investigated by various techniques (differential scanning fluorimetry, surface plasmon resonance, and microscale thermophoresis). The binding mode of the potent inhibitor 18 was revealed by X-ray crystallography, confirming our initial in silico binding model. Furthermore, the high resolution of the complex structure allowed for the placement of the Gly97-Ser108 flexible loop, thus revealing its role in binding of this class of compounds. The crystal structure of the complex protein G24 and inhibitor 18 provides valuable information for further optimization of this novel class of DNA gyrase B inhibitors.

In silico discovery and biophysical evaluation of novel 5-(2-hydroxybenzylidene) rhodanine inhibitors of DNA gyrase B.

Bacterial DNA gyrase is an established and validated target for the development of novel antibacterials. In our previous work, we identified a novel series of bacterial gyrase inhibitors from the class of 4-(2,4-dihydroxyphenyl) thiazoles. Our ongoing effort was designated to search for synthetically more available compounds with possibility of hit to lead development. By using the virtual screening approach, new potential inhibitors were carefully selected from the focused chemical library and tested for biological activity. Herein we report on a novel class of 5-(2-hydroxybenzylidene) rhodanines as gyrase B inhibitors with activity in low micromolar range and moderate antibacterial activity. The binding of the two most active compounds to the enzyme target was further characterised using surface plasmon resonance (SPR) and differential scanning fluorimetry methods (DSF).

In silico discovery of 2-amino-4-(2,4-dihydroxyphenyl)thiazoles as novel inhibitors of DNA gyrase B.

Cyclothialidines are a class of bacterial DNA gyrase B (GyrB) subunit inhibitors, targeting its ATP-binding site. Starting from the available structural information on cyclothialidine GR122222X (2), an in silico virtual screening campaign was designed combining molecular docking calculations with three-dimensional structure-based pharmacophore information. A novel class of 2-amino-4-(2,4-dihydroxyphenyl)thiazole based inhibitors (5-9) with low micromolar antigyrase activity was discovered.