Structure-Activity Relationship and Mode-Of-Action Studies Highlight 1-(4-Biphenylylmethyl)-1H-imidazole-Derived Small Molecules as Potent CYP121 Inhibitors.

CYP121 of Mycobacterium tuberculosis (Mtb) is an essential target for the development of novel potent drugs against tuberculosis (TB). Besides known antifungal azoles, further compounds of the azole class were recently identified as CYP121 inhibitors with antimycobacterial activity. Herein, we report the screening of a similarity-oriented library based on the former hit compound, the evaluation of affinity toward CYP121, and activity against M. bovis BCG. The results enabled a comprehensive SAR study, which was extended through the synthesis of promising compounds and led to the identification of favorable features for affinity and/or activity and hit compounds with 2.7-fold improved potency. Mode of action studies show that the hit compounds inhibit substrate conversion and highlighted CYP121 as the main antimycobacterial target of our compounds. Exemplified complex crystal structures of CYP121 with three inhibitors reveal a common binding site. Engaging in both hydrophobic interactions as well as hydrogen bonding to the sixth iron ligand, our compounds block a solvent channel leading to the active site heme. Additionally, we report the first CYP inhibitors that are able to reduce the intracellular replication of M. bovis BCG in macrophages, emphasizing their potential as future drug candidates against TB.

Biophysical Screening of a Focused Library for the Discovery of CYP121 Inhibitors as Novel Antimycobacterials.

The development of novel antimycobacterial agents against Mycobacterium tuberculosis (Mtb) is urgently required due to the appearance of multidrug resistance (MDR) combined with complicated long-term treatment. CYP121 was shown to be a promising novel target for inhibition of mycobacterial growth. In this study, we describe the rational discovery of new CYP121 inhibitors by a systematic screening based on biophysical and microbiological methods. The best hits originating from only one structural class gave initial information about molecular motifs required for binding and activity. The initial screening procedure was followed by mode-of-action studies and further biological characterizations. The results demonstrate superior antimycobacterial efficacy and a decreased toxicity profile of our frontrunner compound relative to the reference compound econazole. Due to its low molecular weight, promising biological profile, and physicochemical properties, this compound is an excellent starting point for further rational optimization.

Structure-Activity Relationships of 2-Sufonylpyrimidines as Quorum-Sensing Inhibitors to Tackle Biofilm Formation and eDNA Release of Pseudomonas aeruginosa.

Drug-resistant Pseudomonas aeruginosa (PA) strains are on the rise, making treatment with current antibiotics ineffective. Hence, circumventing resistance or restoring the activity of antibiotics by novel approaches is of high demand. Targeting the Pseudomonas quinolone signal quorum sensing (PQS-QS) system is an intriguing strategy to abolish PA pathogenicity without affecting the viability of the pathogen. Herein we report the structure-activity relationships of 2-sulfonylpyrimidines, which were previously identified as dual-target inhibitors of the PQS receptor PqsR and the PQS synthase PqsD. The SAR elucidation was guided by a combined approach using ligand efficiency and ligand lipophilicity efficiency to select the most promising compounds. In addition, the most effective inhibitors were rationally modified by the guidance of QSAR using Hansch analyses. Finally, these inhibitors showed the capacity to decrease biofilm mass and extracellular DNA, which are important determinants for antibiotic resistance.

Discovery and Biophysical Evaluation of First Low Nanomolar Hits Targeting CYP125 of M.†tuberculosis.

Tuberculosis, which is predominantly caused by Mycobacterium tuberculosis (Mtb), is still the most lethal bacterial infection with 1.5 million casualties in 2014. Moreover, the fact that the appearance of resistant mutants and long-term treatment are coupled with economic problems in developing countries hampers an efficient therapy. Interference with the essential cholesterol metabolism of Mtb could be a promising novel strategy to fight Mtb infections. CYP125, a P450 enzyme in Mtb, has been shown to play an important role in this metabolic pathway. For this reason, we used a combined screening approach involving surface plasmon resonance spectroscopy and a heme coordination assay to identify new CYP125 binders by employing a focused P450-inhibitor library. We identified the first hits with high affinity and favorable ligand efficiencies. Furthermore, frontrunner compounds also showed selectivity toward CYP121 specific to Mtb and required for its survival. To date, these are the first compounds targeting CYP125 with low nanomolar affinity.

Application of Dual Inhibition Concept within Looped Autoregulatory Systems toward Antivirulence Agents against Pseudomonas aeruginosa Infections.

Pseudomonas aeruginosa quorum-sensing (QS) is a sophisticated network of genome-wide regulation triggered in response to population density. A major component is the self-inducing pseudomonas quinolone signal (PQS) QS system that regulates the production of several nonvital virulence- and biofilm-related determinants. Hence, QS circuitry is an attractive target for antivirulence agents with lowered resistance development potential and a good model to study the concept of polypharmacology in autoloop-regulated systems per se. Based on the finding that a combination of PqsR antagonist and PqsD inhibitor synergistically lowers pyocyanin, we have developed a dual-inhibitor compound of low molecular weight and high solubility that targets PQS transcriptional regulator (PqsR) and PqsD, a key enzyme in the biosynthesis of PQS-QS signal molecules (HHQ and PQS). In vitro, this compound markedly reduced virulence factor production and biofilm formation accompanied by a diminished content of extracellular DNA (eDNA). Additionally, coadministration with ciprofloxacin increased susceptibility of PA14 to antibiotic treatment under biofilm conditions. Finally, disruption of pathogenicity mechanisms was also assessed in vivo, with significantly increased survival of challenged larvae in a Galleria mellonella infection model. Favorable physicochemical properties and effects on virulence/biofilm establish a promising starting point for further optimization. In particular, the ability to address two targets of the PQS autoinduction cycle at the same time with a single compound holds great promise in achieving enhanced synergistic cellular effects while potentially lowering rates of resistance development.

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.

Combining in silico and biophysical methods for the development of Pseudomonas aeruginosa quorum sensing inhibitors: an alternative approach for structure-based drug design.

The present work deals with the optimization of an inhibitor of PqsD, an enzyme essential for Pseudomonas aeruginosa quorum sensing apparatus. Molecular docking studies, supported by biophysical methods (surface plasmon resonance, isothermal titration calorimetry, saturation transfer difference NMR), were used to illuminate the binding mode of the 5-aryl-ureidothiophene-2-carboxylic acids. Enabled to make profound predictions, structure-based optimization led to increased inhibitory potency. Finally a covalent inhibitor was obtained. Binding to the active site was confirmed by LC-ESI-MS and MALDI-TOF-MS experiments. Following this rational approach, potent PqsD inhibitors were efficiently developed within a short period of time. This example shows that a combination and careful application of in silico and biophysical methods represents a powerful complement to cocrystallography.

Biochemical and biophysical analysis of a chiral PqsD inhibitor revealing tight-binding behavior and enantiomers with contrary thermodynamic signatures.

Antivirulence strategies addressing bacterial pathogenicity without exhibiting growth inhibition effects represent a novel approach to overcome today's crisis in antibiotic development. In recent studies, we examined various inhibitors of PqsD, an enzyme involved in formation of Pseudomonas aeruginosa cell-to-cell signaling molecules, and observed desired cellular effects for 2-nitrophenyl derivatives. Herein, we investigated the binding characteristics of this interesting compound class using several biochemical and biophysical methods. The inhibitors showed time-dependent activity, tight-binding behavior, and interactions with the catalytic center. Furthermore, isothermal titration calorimetry (ITC) experiments with separated enantiomers revealed contrary thermodynamic signatures showing either enthalpy- or entropy-driven affinity. A combination of site-directed mutagenesis and thermodynamic profiling was used to identify key residues involved in inhibitor binding. This information allowed the proposal of experimentally confirmed docking poses. Although originally designed as transition state analogs, our results suggest an altered position for both enantiomers. Interestingly, the main difference between stereoisomers was found in the orientation of the hydroxyl group at the stereogenic center. The predicted binding modes are in accordance with experimental data and, thus, allow future structure-guided optimization.

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.

Validation of PqsD as an anti-biofilm target in Pseudomonas aeruginosa by development of small-molecule inhibitors.

2-Heptyl-4-hydroxyquinoline (HHQ) and Pseudomonas quinolone signal (PQS) are involved in the regulation of virulence factor production and biofilm formation in Pseudomonas aeruginosa. PqsD is a key enzyme in the biosynthesis of these signal molecules. Using a ligand-based approach, we have identified the first class of PqsD inhibitors. Simplification and rigidization led to fragments with high ligand efficiencies. These small molecules repress HHQ and PQS production and biofilm formation in P. aeruginosa. This validates PqsD as a target for the development of anti-infectives.