Influence of DNA template choice on transcription and inhibition of Escherichia coli RNA polymerase.

In recent decades, quantitative transcription assays using bacterial RNA polymerase (RNAP) have been performed under widely diverse experimental conditions. We demonstrate that the template choice can influence the inhibitory potency of RNAP inhibitors. Furthermore, we illustrate that the sigma factor (σ(70)) surprisingly increases the transcription efficiency of templates with nonphysiological nonprokaryotic promoters. Our results might be a useful guideline in the early stages of using RNAP for drug discovery.

Computational investigation of the binding mode of bis(hydroxylphenyl)arenes in 17ß-HSD1: molecular dynamics simulations, MM-PBSA free energy calculations, and molecular electrostatic potential maps.

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) catalyzes the last step of the estrogen biosynthesis, namely the reduction of estrone to the biologically potent estradiol. As such it is a potentially attractive drug target for the treatment of estrogen-dependent diseases like breast cancer and endometriosis. 17ß-HSD1 belongs to the bisubstrate enzymes and exists as an ensemble of conformations. These principally differ in the region of the ßFαG'-loop, suggesting a prominent role in substrate and inhibitor binding. Although several classes of potent non-steroidal 17ß-HSD1 inhibitors currently exist, their binding mode is still unclear. We aimed to elucidate the binding mode of bis(hydroxyphenyl)arenes, a highly potent class of 17ß-HSD1 inhibitors, and to rank these compounds correctly with respect to their inhibitory potency, two essential aspects in drug design. Ensemble docking experiments resulted in a steroidal binding mode for the closed enzyme conformations and in an alternative mode for the opened and occluded conformers with the inhibitors placed below the NADPH interacting with it synergically via π-π stacking and H-bond formation. Both binding modes were investigated by MD simulations and MM-PBSA binding free energy estimations using as representative member for this class compound 1 (50 nM). Notably, only the alternative binding mode proved stable and was energetically more favorable, while when simulated in the steroidal binding mode compound 1 was displaced from the active site. In parallel, ab initio studies of small NADPH-inhibitor complexes were performed, which supported the importance of the synergistic interaction between inhibitors and cofactor.

Homology modeling meets site-directed mutagenesis: An ideal combination to elucidate the topology of 17ß-HSD2.

17ß-Hydroxysteroid dehydrogenase type 2 (17ß-HSD2) catalyzes the conversion of highly active estrogens and androgens into their less active forms using NAD+ as cofactor. Substrate and cofactor specificities of 17ß-HSD2 have been reported and potent 17ß-HSD2 inhibitors have been discovered in a ligand-based approach. However, the molecular basis and the amino acids involved in the enzymatic functionality are poorly understood, as no crystal structure of the membrane-associated 17ß-HSD2 exists. The functional properties of only few amino acids are known. The lack of topological information impedes structure-based drug design studies and limits the design of biochemical experiments. The aim of this work was the determination of the 17ß-HSD2 topology. For this, the first homology model of 17ß-HSD2 in complex with NAD+ and 17ß-estradiol was built, using a multi-fragment "patchwork" approach. To confirm the quality of the model, fifteen selected amino acids were exchanged one by one using site directed mutagenesis. The mutants' functional behavior demonstrated that the generated model was of very good quality and allowed the identification of several key amino acids involved in either ligand or internal structure stabilization. The final model is an optimal basis for further experiments like, for example, lead optimization.

Novel estrone mimetics with high 17beta-HSD1 inhibitory activity.

17Beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) catalyzes the reduction of estrone into estradiol, which is the most potent estrogen in humans. Lowering intracellular estradiol concentration by inhibition of this enzyme is a promising new option for the treatment of estrogen-dependent diseases like breast cancer and endometriosis. Combination of ligand- and structure-based design resulted in heterocyclic substituted biphenylols and their aza-analogs as new 17beta-HSD1 inhibitors. The design was based on mimicking estrone, especially focusing on the imitation of the D-ring keto group with (substituted) heterocycles. Molecular docking provided insights into plausible protein-ligand interactions for this class of compounds. The most promising compound 12 showed an inhibitory activity in the high nanomolar range and very low affinity for the estrogen receptors alpha and beta. Thus, compound 12 is a novel tool for the elucidation of the pharmacological relevance of 17beta-HSD1 and might be a lead for the treatment of estrogen-dependent diseases.

Synthesis, biological evaluation and molecular modelling studies of novel ACD- and ABD-ring steroidomimetics as inhibitors of CYP17.

Two novel classes of non-steroidal substrate mimetics were synthesised and examined for their potency as inhibitors of human CYP17. Selected compounds were tested for inhibition of hepatic CYP enzymes 3A4, 1A2, 2C9 and 2C19. The most promising compound 15 showed a good inhibition of the target enzyme (31% and 66% at 0.2 and 2 microM, respectively), and little inhibition of the most important hepatic enzyme CYP3A4 (6% and 19% inhibition at 0.2 and 2 microM, respectively) and the key enzyme of glucocorticoid biosynthesis CYP11B1 (3% and 23% inhibition at 0.2 and 2 microM, respectively). Docking studies revealed that this compound does not assume the same binding mode as steroidal ligands.

Synthesis, biological evaluation, and molecular modeling studies of methylene imidazole substituted biaryls as inhibitors of human 17alpha-hydroxylase-17,20-lyase (CYP17)--part II: Core rigidification and influence of substituents at the methylene bridge.

Thirty-five novel substituted imidazolyl methylene biphenyls have been synthesized as CYP17 inhibitors for the potential treatment of prostate cancer. Their activities have been tested with recombinant human CYP17 expressed in Escherichia coli. Promising compounds were tested for selectivity against CYP11B1, CYP11B2, and hepatic CYP enzymes 3A4, 1A2, 2B6 and 2D6. The core rigidified compounds (30-35) were the most active ones, being much more potent than Ketoconazole and reaching the activity of Abiraterone. However, they were not very selective. Another rather potent and more selective inhibitor (compound 23, IC(50)=345 nM) was further examined in rats regarding plasma testosterone levels and pharmacokinetic properties. Compared to the reference Abiraterone, 23 was more active in vivo, showed a longer plasma half-life (10h) and a higher bioavailability. Using our CYP17 homology protein model, docking studies with selected compounds were performed to study possible interactions between inhibitors and amino acid residues of the active site.

Synthesis, biological evaluation and molecular modelling studies of methyleneimidazole substituted biaryls as inhibitors of human 17alpha-hydroxylase-17,20-lyase (CYP17). Part I: Heterocyclic modifications of the core structure.

Novel chemical entities were prepared via Suzuki and S(N) reaction as AC-ring substrate mimetics of CYP17. The synthesised compounds 1-31 were tested for activity using human CYP17 expressed in Escherichia coli. Promising compounds were tested for selectivity against hepatic CYP enzymes (3A4, 2D6, 1A2, 2C9, 2C19, 2B6). Two potent inhibitors (27, IC50 = 373 nM/28, IC50 = 953 nM) were further examined in rats regarding their effects on plasma testosterone levels and their pharmacokinetic properties. Compound 28 was similarly active as abiraterone and showed better pharmacokinetic properties (higher bioavailability, t(1/2) 9.5 h vs 1.6 h). Docking studies revealed two new binding modes different from the one of the substrates and steroidal inhibitors.

CYP17 inhibitors. Annulations of additional rings in methylene imidazole substituted biphenyls: synthesis, biological evaluation and molecular modelling.

Twenty-one novel compounds originating from two classes of annulated biphenyls were synthesized as mimetics of the steroidal A- and C-rings and examined for their potency as inhibitors of human CYP17. Selected compounds were tested for inhibition of the hepatic CYP enzyme 3A4. Potent CYP17 inhibitors were found for each class, compound 9 (17 and 71% at 0.2 and 2 microM, respectively) and 21 (591 nM). Compound 21 showed only weak inhibition of CYP3A4 (32 and 64% at 2 and 10 microM, respectively). Both compounds, however, exhibited moderate to strong inhibition of the glucocorticoid-forming enzyme CYP11B1. The most interesting compounds were docked into our protein model. They bound into one of the modes which we have previously published. New interaction regions were identified.

Imidazolylmethylbenzophenones as highly potent aromatase inhibitors.

Suppression of tumor and plasma estrogen levels by inhibition of aromatase is one of the most effective treatments for postmenopausal breast cancer patients. Starting from an easy, synthetically accessible, benzophenone scaffold, a new class of potent aromatase inhibitors was synthesized, endowed with high selectivity with respect to 17 alpha-hydroxylase/17,20-lyase (CYP17). Compounds 1b and 1d proved to be among the most potent inhibitors described so far.

Composing compound libraries for hit discovery--rationality-driven preselection or random choice by structural diversity?

AIMS: In order to identify new scaffolds for drug discovery, surface plasmon resonance is frequently used to screen structurally diverse libraries. Usually, hit rates are low and identification processes are time consuming. Hence, approaches which improve hit rates and, thus, reduce the library size are required. METHODS: In this work, we studied three often used strategies for their applicability to identify inhibitors of PqsD. In two of them, target-specific aspects like inhibition of a homologous protein or predicted binding determined by virtual screening were used for compound preselection. Finally, a fragment library, covering a large chemical space, was screened and served as comparison. RESULTS & CONCLUSION: Indeed, higher hit rates were observed for methods employing preselected libraries indicating that target-oriented compound selection provides a time-effective alternative.

Mechanistic details for anthraniloyl transfer in PqsD: the initial step in HHQ biosynthesis.

PqsD mediates the conversion of anthraniloyl-coenzyme A (ACoA) to 2-heptyl-4-hydroxyquinoline (HHQ), a precursor of the Pseudomonas quinolone signal (PQS) molecule. Due to the role of the quinolone signaling pathway of Pseudomonas aeruginosa in the expression of several virulence factors and biofilm formation, PqsD is a potential target for controlling this nosocomial pathogen, which exhibits a low susceptibility to standard antibiotics. PqsD belongs to the ß-ketoacyl-ACP synthase family and is similar in structure to homologous FabH enzymes in E. coli and Mycobacterium tuberculosis. Here, we used molecular dynamics simulations to obtain the structural position of the substrate ACoA in the binding pocket of PqsD, and semiempirical molecular orbital calculations to study the reaction mechanism for the catalytic cleavage of ACoA. Our findings suggest a nucleophilic attack of the deprotonated sulfur of Cys112 at the carbonyl carbon of ACoA and a switch in the protonation pattern of His257 whereby Nδ is protonated and the proton of Nε is shifted to the sulfur of CoA during the reaction. This is in agreement with the experimentally determined decreased catalytic activity of the Cys112Ser mutant, whereas the Cys112Ala, His257Phe, and Asn287Ala mutants are all inactive. ESI mass-spectrometric measurements of the Asn287Ala mutant show that anthraniloyl remains covalently bound to Cys112, thus further supporting the inference from our computed mechanism that Asn287 does not take part in the cleavage of ACoA. Since this mutant is inactive, we suggest instead that Asn287 must play an essential role in the subsequent formation of HHQ in vitro.

Peptide-based investigation of the Escherichia coli RNA polymerase σ(70):core interface as target site.

The number of bacterial strains that are resistant against antibiotics increased dramatically during the past decades. This fact stresses the urgent need for the development of new antibacterial agents with novel modes of action targeting essential enzymes such as RNA polymerase (RNAP). Bacterial RNAP is a large multi-subunit complex consisting of a core enzyme (subunits: α(2)ßß'ω) and a dissociable sigma factor (σ(70); holo enzyme: α(2)ßß'ωσ(70)) that is responsible for promoter recognition and transcription initiation. The interface between core RNAP and σ(70) represents a promising binding site. Nevertheless, detailed studies investigating its druggability are rare. Compounds binding to this region could inhibit this protein-protein interaction and thus holo enzyme formation, resulting in inhibition of transcription initiation. Sixteen peptides covering different regions of the Escherichia coli σ(70):core interface were designed; some of them-all derived from σ(70) 2.2 region-led to a strong RNAP inhibition. Indeed, an ELISA-based experiment confirmed the most active peptide P07 to inhibit the σ(70):core interaction. Furthermore, an abortive transcription assay revealed that P07 impedes transcription initiation. In order to study the mechanism of action of P07 in more detail, molecular dynamics simulations and a rational amino acid replacement study were performed, leading to the conclusion that P07 binds to the coiled-coil region in ß' and that its flexible N-terminus inhibits the enzyme by interaction with the ß' lid-rudder-system (LRS). This work revisits the ß' coiled-coil as a hot spot for the protein-protein interaction inhibition and expands it by introduction of the LRS as target site.

Discovery of novel bacterial RNA polymerase inhibitors: pharmacophore-based virtual screening and hit optimization.

The bacterial RNA polymerase (RNAP) is a validated target for broad spectrum antibiotics. However, the efficiency of drugs is reduced by resistance. To discover novel RNAP inhibitors, a pharmacophore based on the alignment of described inhibitors was used for virtual screening. In an optimization process of hit compounds, novel derivatives with improved in vitro potency were discovered. Investigations concerning the molecular mechanism of RNAP inhibition reveal that they prevent the protein-protein interaction (PPI) between σ(70) and the RNAP core enzyme. Besides of reducing RNA formation, the inhibitors were shown to interfere with bacterial lipid biosynthesis. The compounds were active against Gram-positive pathogens and revealed significantly lower resistance frequencies compared to clinically used rifampicin.

Novel small molecule inhibitors targeting the "switch region" of bacterial RNAP: structure-based optimization of a virtual screening hit.

Rising resistance against current antibiotics necessitates the development of antibacterial agents with alternative targets. The "switch region" of RNA polymerase (RNAP), addressed by the myxopyronins, could be such a novel target site. Based on a hit candidate discovered by virtual screening, a small library of 5-phenyl-3-ureidothiophene-2-carboxylic acids was synthesized resulting in compounds with increased RNAP inhibition. Hansch analysis revealed π (lipophilicity constant) and σ (Hammet substituent constant) of the substituents at the 5-phenyl moiety to be crucial for activity. The binding mode was proven by the targeted introduction of a moiety mimicking the enecarbamate side chain of myxopyronin into the hit compound, accompanied by enhanced RNAP inhibitory potency. The new compounds displayed good antibacterial activities against Gram positive bacteria and Gram negative Escherichia coli TolC and a reduced resistance frequency compared to the established antibiotic rifampicin.

Modulation of cytochromes P450 with xanthone-based molecules: from aromatase to aldosterone synthase and steroid 11ß-hydroxylase inhibition.

Imidazolylmethylflavones previously reported by us as aromatase inhibitors proved to be able to interact with aldosterone synthase (CYP11B2), a cytochrome P450 enzyme involved in the biosynthesis of the mineralcorticoid hormone aldosterone, and were used to obtain a pharmacophore model for this enzyme. Here, in the search for potential ligands for CYP11B2 and the related CYP11B1, a virtual screening of a small compounds library of our earlier synthesized aromatase inhibitors was performed and, according to the results and the corresponding biological data, led to the design and synthesis of a series of xanthones derivatives carrying an imidazolylmethyl substituent in position 1 and different substituents in position 4. Some very potent inhibitors were obtained; in particular, the 4-chlorine derivative was active in the low nanomolar or subnanomolar range on CYP11B2 and CYP11B1, respectively, proving that xanthone can be considered as an excellent scaffold, whose activity can be directed to different targets when appropriately functionalized.

Structural optimization of 2,5-thiophene amides as highly potent and selective 17ß-hydroxysteroid dehydrogenase type 2 inhibitors for the treatment of osteoporosis.

Inhibition of 17ß-HSD2 is an attractive mechanism for the treatment of osteoporosis. We report here the optimization of human 17ß-HSD2 inhibitors in the 2,5-thiophene amide class by varying the size of the linker (n equals 0 and 2) between the amide moiety and the phenyl group. While none of the phenethylamides (n = 2) were active, most of the anilides (n = 0) turned out to moderately or strongly inhibit 17ß-HSD2. The four most active compounds showed an IC50 of around 60 nM and a very good selectivity toward 17ß-HSD1, 17ß-HSD4, 17ß-HSD5, 11ß-HSD1, 11ß-HSD2 and the estrogen receptors α and ß. The investigated compounds inhibited monkey 17ß-HSD2 moderately, and one of them showed good inhibitory activity on mouse 17ß-HSD2. SAR studies allowed a first characterization of the human 17ß-HSD2 active site, which is predicted to be considerably larger than that of 17ß-HSD1.

Molecular basis of HHQ biosynthesis: molecular dynamics simulations, enzyme kinetic and surface plasmon resonance studies.

BACKGROUND: PQS (PseudomonasQuinolone Signal) and its precursor HHQ are signal molecules of the P. aeruginosa quorum sensing system. They explicate their role in mammalian pathogenicity by binding to the receptor PqsR that induces virulence factor production and biofilm formation. The enzyme PqsD catalyses the biosynthesis of HHQ. RESULTS: Enzyme kinetic analysis and surface plasmon resonance (SPR) biosensor experiments were used to determine mechanism and substrate order of the biosynthesis. Comparative analysis led to the identification of domains involved in functionality of PqsD. A kinetic cycle was set up and molecular dynamics (MD) simulations were used to study the molecular bases of the kinetics of PqsD. Trajectory analysis, pocket volume measurements, binding energy estimations and decompositions ensured insights into the binding mode of the substrates anthraniloyl-CoA and ß-ketodecanoic acid. CONCLUSIONS: Enzyme kinetics and SPR experiments hint at a ping-pong mechanism for PqsD with ACoA as first substrate. Trajectory analysis of different PqsD complexes evidenced ligand-dependent induced-fit motions affecting the modified ACoA funnel access to the exposure of a secondary channel. A tunnel-network is formed in which Ser317 plays an important role by binding to both substrates. Mutagenesis experiments resulting in the inactive S317F mutant confirmed the importance of this residue. Two binding modes for ß-ketodecanoic acid were identified with distinct catalytic mechanism preferences.

Structure optimization of 2-benzamidobenzoic acids as PqsD inhibitors for Pseudomonas aeruginosa infections and elucidation of binding mode by SPR, STD NMR, and molecular docking.

Pseudomonas aeruginosa employs a characteristic pqs quorum sensing (QS) system that functions via the signal molecules PQS and its precursor HHQ. They control the production of a number of virulence factors and biofilm formation. Recently, we have shown that sulfonamide substituted 2-benzamidobenzoic acids, which are known FabH inhibitors, are also able to inhibit PqsD, the enzyme catalyzing the last and key step in the biosynthesis of HHQ. Here, we describe the further optimization and characterization of this class of compounds as PqsD inhibitors. Structural modifications showed that both the carboxylic acid ortho to the amide and 3'-sulfonamide are essential for binding. Introduction of substituents in the anthranilic part of the molecule resulted in compounds with IC50 values in the low micromolar range. Binding mode investigations by SPR with wild-type and mutated PqsD revealed that this compound class does not bind into the active center of PqsD but in the ACoA channel, preventing the substrate from accessing the active site. This binding mode was further confirmed by docking studies and STD NMR.

Hydroxybenzothiazoles as new nonsteroidal inhibitors of 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1).

17ß-estradiol (E2), the most potent estrogen in humans, known to be involved in the development and progession of estrogen-dependent diseases (EDD) like breast cancer and endometriosis. 17ß-HSD1, which catalyses the reduction of the weak estrogen estrone (E1) to E2, is often overexpressed in breast cancer and endometriotic tissues. An inhibition of 17ß-HSD1 could selectively reduce the local E2-level thus allowing for a novel, targeted approach in the treatment of EDD. Continuing our search for new nonsteroidal 17ß-HSD1 inhibitors, a novel pharmacophore model was derived from crystallographic data and used for the virtual screening of a small library of compounds. Subsequent experimental verification of the virtual hits led to the identification of the moderately active compound 5. Rigidification and further structure modifications resulted in the discovery of a novel class of 17ß-HSD1 inhibitors bearing a benzothiazole-scaffold linked to a phenyl ring via keto- or amide-bridge. Their putative binding modes were investigated by correlating their biological data with features of the pharmacophore model. The most active keto-derivative 6 shows IC50-values in the nanomolar range for the transformation of E1 to E2 by 17ß-HSD1, reasonable selectivity against 17ß-HSD2 but pronounced affinity to the estrogen receptors (ERs). On the other hand, the best amide-derivative 21 shows only medium 17ß-HSD1 inhibitory activity at the target enzyme as well as fair selectivity against 17ß-HSD2 and ERs. The compounds 6 and 21 can be regarded as first benzothiazole-type 17ß-HSD1 inhibitors for the development of potential therapeutics.

Exploring the PDE5 H-pocket by ensemble docking and structure-based design and synthesis of novel ß-carboline derivatives.

By studying the co-crystal information of interactions between PDE5 and its inhibitors, forty new tetrahydro-ß-carbolines based-analogues were synthesized, and tested for their PDE5 inhibition. Some compounds were as active as tadalafil in inhibiting PDE5 and of better selectivity profile particularly versus PDE11A, the nature of the terminal ring and its nitrogen substituent are the main determinants of selectivity. Ensemble docking confirmed the role of H-loop closed conformer in activity versus its occluded and open forms. Conformational studies showed the effect of bulkiness of the terminal ring N-alkyl substituent on the formation of stable enzyme ligands conformers. The difference in potencies of hydantoin and piperazinedione analogues, together with the necessity of C-5/C-6 R-absolute configuration has been revealed through molecular docking.