Synthesis, molecular docking study and biological evaluation of new pyrrole scaffolds as potential antitubercular agents for dual targeting of enoyl ACP reductase and dihydrofolate reductase.

In this study, new series of N'-(2-(substitutedphenoxy)acetyl)-4-(1H-pyrrol-1-yl)benzohydrazides (3a-j) 4-(2,5-dimethyl-1H-pyrrol-1-yl)-N'-(2-(substitutedphenoxy)acetyl)benzohydrazides (5a-j) were synthesized, characterized and assessed as inhibitors of enoyl ACP reductase and DHFR. Most of the compounds exhibited dual inhibition against the enzymes enoyl ACP reductase and DHFR. Several synthesized substances also demonstrated significant antibacterial and antitubercular properties. A molecular docking analysis was conducted in order to determine the potential mechanism of action of the synthesized compounds. The results indicated that there were binding interactions seen with the active sites of dihydrofolate reductase and enoyl ACP reductase. Additionally, important structural details were identified that play a critical role in sustaining the dual inhibitory activity. These findings were useful for the development of future dual inhibitors. Therefore, this study provided strong evidence that several synthesized molecules could exert their antitubercular properties at the cellular level through multi-target inhibition. By shedding light on the mechanisms through which these compounds exert their inhibitory effects, this research opens up promising avenues for the future development of dual inhibitors with enhanced antibacterial and antitubercular properties. The study's findings underscore the importance of multi-target approaches in drug design, providing a strong foundation for the design and optimization of novel compounds that can effectively target bacterial infections at the cellular level.

Evaluation of Fusobacterium nucleatum Enoyl-ACP Reductase (FabK) as a Narrow-Spectrum Drug Target.

Fusobacterium nucleatum, a pathobiont inhabiting the oral cavity, contributes to opportunistic diseases, such as periodontal diseases and gastrointestinal cancers, which involve microbiota imbalance. Broad-spectrum antimicrobial agents, while effective against F. nucleatum infections, can exacerbate dysbiosis. This necessitates the discovery of more targeted narrow-spectrum antimicrobial agents. We therefore investigated the potential for the fusobacterial enoyl-ACP reductase II (ENR II) isoenzyme FnFabK (C4N14_ 04250) as a narrow-spectrum drug target. ENRs catalyze the rate-limiting step in the bacterial fatty acid synthesis pathway. Bioinformatics revealed that of the four distinct bacterial ENR isoforms, F. nucleatum specifically encodes FnFabK. Genetic studies revealed that fabK was indispensable for F. nucleatum growth, as the gene could not be deleted, and silencing of its mRNA inhibited growth under the test conditions. Remarkably, exogenous fatty acids failed to rescue growth inhibition caused by the silencing of fabK. Screening of synthetic phenylimidazole analogues of a known FabK inhibitor identified an inhibitor (i.e., 681) of FnFabK enzymatic activity and F. nucleatum growth, with an IC50 of 2.1 µM (1.0 µg/mL) and a MIC of 0.4 µg/mL, respectively. Exogenous fatty acids did not attenuate the activity of 681 against F. nucleatum. Furthermore, FnFabK was confirmed as the intracellular target of 681 based on the overexpression of FnFabK shifting MICs and 681-resistant mutants having amino acid substitutions in FnFabK or mutations in other genetic loci affecting fatty acid biosynthesis. 681 had minimal activity against a range of commensal flora, and it was less active against streptococci in physiologic fatty acids. Taken together, FnFabK is an essential enzyme that is amenable to drug targeting for the discovery and development of narrow-spectrum antimicrobial agents.

Novel FabI inhibitor disrupts the biofilm formation of MRSA through down-regulating the expression of quorum-sensing regulatory genes.

OBJECTIVES: The aim of this study was to explore the antibiofilm and antivirulence efficacy of benzylaniline 4k against MRSA. METHODS: The clinical MRSA strains were identified and used to evaluate their potential to form biofilm using crystal violet assay. The minimal inhibitory concentration (MIC) was determined using broth microdilution method. The expression of genes was detected using quantitative real-time PCR (qRT-PCR). Rabbit blood hemolytic assay was used to observe the inhibitory ability of alpha-hemolysin (Hla). RESULTS: Compound 4k showed potent antibacterial activity against 16 clinical MRSA with an MIC50 of 1.25 mg/L and MIC90 of 2.25 mg/L. The value of minimum biofilm eradication concentration (MBEC) against MRSA2858 biofilm was of 1.5 mg/L, close to its MIC, superior to those of vancomycin and erythromycin. Compound 4k eradicated the formation of biofilm through inhibiting the gene expression of branched-chain fatty acid synthesis, down-regulating the expression of quorum-sensing (QS) regulatory genes (norA, agrA, icaA, hla), decreasing the level of hemolysis in a dose-dependent manner, and inhibiting rabbit blood hemolysis by 86.9% at a concentration of 1.25 mg/L. In a mouse model of abdominal infection, compound 4k was more effective than vancomycin in reducing bacterial load. CONCLUSIONS: These results suggested that compound 4k could be developed as promising an anti-MRSA agent through affecting quorum-sensing system.

Carfilzomib as a potential inhibitor of NADH-dependent enoyl-acyl carrier protein reductases of Klebsiella pneumoniae and Mycobacterium tuberculosis as a drug target enzyme: insights from molecular docking and molecular dynamics.

Multiple antibiotic-resistant strains of Klebsiella pneumoniae can cause life-threatening infections. Bacterial enoyl-acyl carrier protein (ACP) reductases (ENRs) are considered critical targets for developing antibiotics. Our current study aims to identify inhibitors of K. pneumoniae ENRs (FabI and FabV). Due to the unavailability of experimental structures, protein models of FabI and FabV were predicted and validated in this study. Virtual screening of the 1930 FDA-approved drug database was conducted against the active site of the FabI protein with the help of the LEA3D server, and carfilzomib was chosen among the screened drugs for further docking studies. Carfilzomib, a proteasome inhibitor used in the treatment of multiple myeloma, was among the best-suited compounds obtained from the virtual screening and was found to be bactericidal in the in vitro experiment. Carfilzomib was docked against the active sites of the FabI and FabV proteins, and the ENR of Mycobacterium tuberculosis, InhA. Carfilzomib showed a high binding affinity with all three proteins. Molecular dynamics (MD) simulations were conducted following the docking studies. MD simulations revealed that carfilzomib binds strongly to the active sites of the above mentioned ENRs. Our study found that carfilzomib is a potential inhibitor of the ENRs of K. pneumoniae and M. tuberculosis. This is a possible mechanism of its bactericidal property against M. tuberculosis observed in vitro in addition to its predicted actions on zinc-dependent metalloprotease-1 and peptide deformylase, two other drug target enzymes of M. tuberculosis. Our study suggests that this drug could be used as a lead compound to develop antibiotics that can selectively act against ENRs of bacteria, without interfering with the activities of human proteasome. Communicated by Ramaswamy H. Sarma.

Targeting Mycobacterium Tuberculosis Enoyl-Acyl Carrier Protein Reductase Using Computational Tools for Identification of Potential Inhibitor and their Biological Activity.

Enoyl-acyl carrier protein reductase (InhA) of type II fatty acid synthase system is involved in the synthesis of mycolic acids which is a major component of the bacterial cell wall. Since they are the key enzymes playing a very significant role in the FASII pathway of the bacterium. In this study, we have developed a workflow for identification of InhA inhibitors by utilizing in silico virtual screening approaches based on various machine learning algorithms followed by pharmacophore based virtual screening. The hits screened from the models were further subjected to molecular docking. Further, based on the XP docking score best twenty compounds were subjected to molecular dynamics study. Finally, nine compounds were shortlisted on the basis of best stable ligand RMSD, c-alpha RMSD, and RMSF plot for biological evaluation studies. Experimental validation of the shortlisted compounds identified one compound JFD01724 having potent inhibitory activity and was able to inhibit the growth of mycobacterium tuberculosis. Further medicinal chemistry efforts may help to improve the inhibitory potency of the identified compound.

The food-grade antimicrobial xanthorrhizol targets the enoyl-ACP reductase (FabI) in Escherichia coli.

Xanthorrhizol, isolated from the Indonesian Java turmeric Curcuma xanthorrhiza, displays broad-spectrum antibacterial activity. We report herein the evidence that mechanism of action of xanthorrhizol may involve FabI, an enoyl-(ACP) reductase, inhibition. The predicted Y156V substitution in the FabI enzyme promoted xanthorrhizol resistance, while the G93V mutation originally known for triclosan resistance was not effective against xanthorrhizol. Two other mutations, F203L and F203V, conferred FabI enzyme resistance to both xanthorrhizol and triclosan. These results showed that xanthorrhizol is a food-grade antimicrobial compound targeting FabI but with a different mode of binding from triclosan.

FabI (enoyl acyl carrier protein reductase) - A potential broad spectrum therapeutic target and its inhibitors.

Development of new anti-bacterial agents acting upon underexploited targets and thus evading known mechanisms of resistance is the need of the hour. The highly conserved and distinct bacterial fatty acid biosynthesis pathway (FAS-II), presents a validated and yet relatively underexploited target for drug discovery. FabI and its isoforms (FabL, FabK, FabV and InhA) are essential enoyl-ACP reductases present in several microorganisms. In addition, the components of the FAS-II pathway are distinct from the multi-enzyme FAS-I complex found in mammals. Thus, inhibition of FabI and its isoforms is anticipated to result in broad-spectrum antibacterial activity. Several research groups from industry and academic laboratories have devoted significant efforts to develop effective FabI-targeting antibiotics, which are currently in various stages of clinical development for the treatment of multi-drug resistant bacterial infections. This review summarizes all the natural as well as synthetic inhibitors of gram-positive and gram-negative enoyl ACP reductases (FabI). The knowledge of the reported inhibitors can aid in the development of broad-spectrum antibacterials specifically targeting FabI enzymes from S. aureus, S. epidermidis, B. anthracis, B. cereus, E. coli, P. aeruginosa, P. falciparum and M. tuberculosis.

In silico evaluation and in vitro growth inhibition of Plasmodium falciparum by natural amides and synthetic analogs.

Malaria, caused by protozoa of the genus Plasmodium, is a disease that infects hundreds of millions of people annually, causing an enormous social burden in many developing countries. Since current antimalarial drugs are starting to face resistance by the parasite, the development of new therapeutic options has been prompted. The enzyme Plasmodium falciparum enoyl-ACP reductase (PfENR) has a determinant role in the fatty acid biosynthesis of this parasite and is absent in humans, making it an ideal target for new antimalarial drugs. In this sense, the present study aimed at evaluating the in silico binding affinity of natural and synthetic amides through molecular docking, in addition to their in vitro activity against P. falciparum by means of the SYBR Green Fluorescence Assay. The in vitro results revealed that the natural amide piplartine (1a) presented partial antiplasmodial activity (20.54 µM), whereas its synthetic derivatives (1m-IC50 104.45 µM), (1b, 1g, 1k, and 14f) and the natural amide piperine (18a) were shown to be inactive (IC50 > 200 µM). The in silico physicochemical analyses demonstrated that compounds 1m and 14f violated the Lipinski's rule of five. The in silico analyses showed that 14f presented the best binding affinity (- 13.047 kcal/mol) to PfENR and was also superior to the reference inhibitor triclosan (- 7.806 kcal/mol). In conclusion, we found that the structural modifications in 1a caused a significant decrease in antiplasmodial activity. Therefore, new modifications are encouraged in order to improve the activity observed.

Virtual screening of antibacterial compounds by similarity search of Enoyl-ACP reductase (FabI) inhibitors.

Aim: Antibiotic resistance is an alarming issue, as multidrug-resistant bacteria are growing worldwide, hence the decrease of therapeutic potential of available antibiotic arsenal. Among these bacteria, Staphylococcus aureus was pointed by the WHO in the pathogens list to be prioritized in drug development. Methods: We report the use of chemical similarity models for the virtual screening of new antibacterial with structural similarity to known inhibitors of FabI. The potential inhibitors were experimentally evaluated for antibacterial activity and membrane disrupting capabilities. Results & conclusion: These models led to the finding of four new compounds with antibacterial activity, one of which having antimicrobial activity already reported in the literature.

Implementation of permeation rules leads to a FabI inhibitor with activity against Gram-negative pathogens.

Gram-negative bacterial infections are a significant public health concern, and the lack of new drug classes for these pathogens is linked to the inability of most drug leads to accumulate inside Gram-negative bacteria1-7. Here, we report the development of a web application-eNTRyway-that predicts compound accumulation (in Escherichia coli) from its structure. In conjunction with structure-activity relationships and X-ray data, eNTRyway was utilized to re-design Debio-1452-a Gram-positive-only antibiotic8-into versions that accumulate in E. coli and possess antibacterial activity against high-priority Gram-negative pathogens. The lead compound Debio-1452-NH3 operates as an antibiotic via the same mechanism as Debio-1452, namely potent inhibition of the enoyl-acyl carrier protein reductase FabI, as validated by in vitro enzyme assays and the generation of bacterial isolates with spontaneous target mutations. Debio-1452-NH3 is well tolerated in vivo, reduces bacterial burden in mice and rescues mice from lethal infections with clinical isolates of Acinetobacter baumannii, Klebsiella pneumoniae and E. coli. This work provides tools for the facile discovery and development of high-accumulating compounds in E. coli, and a general blueprint for the conversion of Gram-positive-only compounds into broad-spectrum antibiotics.

Ligand- and Structure-Based Approaches of Escherichia coli FabI Inhibition by Triclosan Derivatives: From Chemical Similarity to Protein Dynamics Influence.

Enoyl-acyl carrier protein reductase (FabI) is the limiting step to complete the elongation cycle in type II fatty acid synthase (FAS) systems and is a relevant target for antibacterial drugs. E. coli FabI has been employed as a model to develop new inhibitors against FAS, especially triclosan and diphenyl ether derivatives. Chemical similarity models (CSM) were used to understand which features were relevant for FabI inhibition. Exhaustive screening of different CSM parameter combinations featured chemical groups, such as the hydroxy group, as relevant to distinguish between active/decoy compounds. Those chemical features can interact with the catalytic Tyr156. Further molecular dynamics simulation of FabI revealed the ionization state as a relevant for ligand stability. Also, our models point the balance between potency and the occupancy of the hydrophobic pocket. This work discusses the strengths and weak points of each technique, highlighting the importance of complementarity among approaches to elucidate EcFabI inhibitor's binding mode and offers insights for future drug discovery.

Small-Molecule Inhibition of the C. difficile FAS-II Enzyme, FabK, Results in Selective Activity.

Clostridioides difficile infection (CDI) is a leading cause of significant morbidity, mortality, and healthcare-related costs in the United States. After standard therapy, recurrence rates remain high, and multiple recurrences are not uncommon. Causes include treatments employing broad-spectrum agents that disrupt the normal host microbiota, as well as treatment-resistant spore formation by C. difficile. Thus, novel druggable anti-C. difficile targets that promote narrow-spectrum eradication and inhibition of sporulation are desired. As a critical rate-limiting step within the FAS-II bacterial fatty acid synthesis pathway, which supplies precursory component phospholipids found in bacterial cytoplasmic and spore-mediated membranes, enoyl-acyl carrier protein (ACP) reductase II (FabK) represents such a target. FabK is essential in C. difficile (CdFabK) and is structurally and mechanistically distinct from other isozymes found in gut microbiota species, making CdFabK an attractive narrow-spectrum target. We report here the kinetic evaluation of CdFabK, the biochemical activity of a series of phenylimidazole analogues, and microbiological data suggesting these compounds' selective antibacterial activity against C. difficile versus several other prominent gut organisms. The compounds display promising, selective, low micromolar CdFabK inhibitory activity without significantly affecting the growth of other gut organisms, and the series prototype (1b) is shown to be competitive for the CdFabK cofactor and uncompetitive for the substrate. A series analogue (1g) shows maintained inhibitory activity while also possessing increased solubility. These findings represent the basis for future drug discovery efforts by characterizing the CdFabK enzyme while demonstrating its druggability and potential role as a narrow-spectrum antidifficile target.

Structure-based drug design and in vitro testing reveal new inhibitors of enoyl-acyl carrier protein reductases.

The need for new antibacterial agents is increasingly becoming of great importance as bacterial resistance to current drugs is quickly spreading. Enoyl-acyl carrier protein reductases (FabI) are important enzymes for fatty acid biosynthesis in bacteria and other micro-organisms. In this project, we conducted structure-based virtual screening against the FabI enzyme, and accordingly, 37 compounds were selected for experimental testing. Interestingly, five compounds were able to demonstrate antimicrobial effect with variable inhibition activity against various strains of bacteria and fungi. Minimum inhibitory concentrations of the active compounds were determined and showed to be in low to medium micromolar range. Subsequently, enzyme inhibition assay was carried out for our five antimicrobial hits to confirm their biological target and determine their IC50 values. Three of these tested compounds exhibited inhibition activity for the FabI enzyme where our best hit MN02 had an IC50 value of 7.8 µM. Furthermore, MN02 is a small bisphenolic compound that is predicted to have all required features to firmly bind with the target enzyme. To sum up, hits discovered in this work can act as a good starting point for the future development of new and potent antimicrobial agents.

Tryptanthrin Analogues as Inhibitors of Enoyl-acyl Carrier Protein Reductase: Activity against Mycobacterium tuberculosis, Toxicity, Modeling of Enzyme Binding.

BACKGROUND: High numbers of infection with resistant forms of Micobacterium tuberculosis (Mtb) contribute to a constant growing demand in new highly active and effective therapeutics. Current drug discovery efforts directed towards new antituberculosis agents include the development of new inhibitors of enoyl-acyl carrier protein reductase (InhA) that do not require activation by the specific enzymes. Tryptanthrin is a known inhibitor of Mtb InhA and its analogues are investigated as potential agents with antimycobacterial efficiency. OBJECTIVE: The main objective of the presented research was to develop a new group of tryptanthrin analogues with good inhibition properties against Mtb. METHODS: Synthesis of new derivatives of 5H-[1,3,4]thiadiazolo[2,3- b]quinazolin-5-one and evaluation of their activity against Mtb, as well as acute and chronic toxicity studies were carried out. Molecular modeling studies were performed to investigate the binding mechanisms of the synthesized ligands with InhA. Binding energies and non-covalent interactions stabilizing the ligand-receptor complexes were obtained from the results of molecular docking. RESULTS: The most active compound in the obtained series, 2-(propylthio)-5H-[1,3,4]thiadiazolo[2,3- b]quinazolin-5-one, exhibited the superior inhibition activity (up to 100%) against mycobacterial growth at MIC 6.5 µg/mL, showed good affinity to the InhA enzyme in docking studies and demonstrated a very low per oral toxicity in animals falling under the category 5 according to GHS classification. CONCLUSION: 2-(propylthio)-5H-[1,3,4]thiadiazolo[2,3-b]quinazolin-5-one can be further explored for the development of a new series of compounds active against Mtb.

The Fatty Acid Synthesis Protein Enoyl-ACP Reductase II (FabK) is a Target for Narrow-Spectrum Antibacterials for Clostridium difficile Infection.

Clostridium difficile infection (CDI) is an antibiotic-induced microbiota shift disease of the large bowel. While there is a need for narrow-spectrum CDI antibiotics, it is unclear which cellular proteins are appropriate drug targets to specifically inhibit C. difficile. We evaluated the enoyl-acyl carrier protein (ACP) reductase II (FabK), which catalyzes the final step of bacterial fatty acid biosynthesis. Bioinformatics showed that C. difficile uses FabK as its sole enoyl-ACP reductase, unlike several major microbiota species. The essentiality of fabK for C. difficile growth was confirmed by failure to delete this gene using ClosTron mutagenesis and by growth inhibition upon gene silencing with CRISPR interference antisense to fabK transcription or by blocking protein translation. Inhibition of C. difficile's FASII pathway could not be circumvented by supply of exogenous fatty acids, either during fabK's gene silencing or upon inhibition of the enzyme with a phenylimidazole-derived inhibitor (1). The inability of fatty acids to bypass FASII inhibition is likely due to the function of the transcriptional repressor FapR. Inhibition of FabK also inhibited spore formation, reflecting the enzyme's role in de novo fatty acid biosynthesis for the formation of spore membrane lipids. Compound 1 did not inhibit growth of key microbiota species. These findings suggest that C. difficile FabK is a druggable target for discovering narrow-spectrum anti- C. difficile drugs that treat CDI but avoid collateral damage to the gut microbiota.

Bone and Joint Tissue Penetration of the Staphylococcus-Selective Antibiotic Afabicin in Patients Undergoing Elective Hip Replacement Surgery.

Afabicin (formerly Debio 1450, AFN-1720) is a prodrug of afabicin desphosphono (Debio 1452, AFN-1252), a novel antibiotic in development which targets the staphylococcal enoyl-acyl carrier protein reductase (FabI) and exhibits selective potent antibacterial activity against staphylococcal species, including methicillin-resistant Staphylococcus aureus As part of clinical development in bone and joint infections, a distribution study in bone was performed in 17 patients who underwent elective hip replacement surgery. Patients received 3 doses of 240 mg afabicin orally (every 12 h) at various time points before surgery. Afabicin desphosphono concentrations were measured by liquid chromatography-tandem mass spectrometry in plasma, cortical bone, cancellous bone, bone marrow, soft tissue, and synovial fluid collected during surgery at 2, 4, 6, or 12 h after the third afabicin dose. The study showed good penetration of afabicin desphosphono into bone tissues, with mean area under the curve ratios for cortical bone-, cancellous bone-, bone marrow-, soft tissue-, and synovial fluid-to-total plasma concentrations of 0.21, 0.40, 0.32, 0.35, and 0.61, respectively. When accounting for the free fraction in plasma (2%) and synovial fluid (9.4%), the mean ratio was 2.88, which is indicative of excellent penetration and which showed that the afabicin desphosphono concentration was beyond the MIC90 of S. aureus over the complete dosing interval. These findings, along with preclinical efficacy data, clinical efficacy data for skin and soft tissue staphylococcal infection, the availability of both intravenous and oral formulations, and potential advantages over broad-spectrum antibiotics for the treatment of staphylococcal bone or joint infections, support the clinical development of afabicin for bone and joint infections. (This study has been registered at ClinicalTrials.gov under identifier NCT02726438.).

Identification of PfENR inhibitors: A hybrid structure-based approach in conjunction with molecular dynamics simulations.

In the current study, we have constructed receptor-based pharmacophore models by exploiting the Plasmodium falciparum enoyl-acyl carrier protein reductase (PfENR) structural proteome. The derived models were subjected to a series of validation procedures to list the representative hypotheses that can be used for the screening of the Drug-like Diverse Database. A set of 739 molecules was retrieved and analyzed for the adsorption, distribution, metabolism, excretion and toxicity (ADMET) and drug-likeness attributes. The filtered drug-like molecules (64) were then subjected to molecular docking and HYDE assessment studies. The hybrid structure-based approach yielded 4 molecules, UKR1308259, ENA1096786, UKR403454, and ASI51224, as PfENR inhibitors. The stability of these inhibitors was assessed using molecular mechanics-generalized born surface area approach-based free binding energy calculations and molecular dynamics simulations. Molecular mechanics-generalized born surface area calculations and molecular dynamics simulations showed that UKR1308259, ENA1096786, and ASI51224 were more potent PfENR inhibitors. The rationale behind the current work was to identify orally available inhibitor molecules with diverse scaffolds that could serve as initial leads for the drug design against PfENR.

A novel series of enoyl reductase inhibitors targeting the ESKAPE pathogens, Staphylococcus aureus and Acinetobacter baumannii.

S. aureus and A. baumannii are among the ESKAPE pathogens that are increasingly difficult to treat due to the rise in the number of drug resistant strains. Novel therapeutics targeting these pathogens are much needed. The bacterial enoyl reductase (FabI) is as potentially significant drug target for developing pathogen-specific antibiotics due to the presence of alternate FabI isoforms in many other bacterial species. We report the identification and development of a novel N-carboxy pyrrolidine scaffold targeting FabI in S. aureus and A. baumannii, two pathogens for which FabI essentiality has been established. This scaffold is unrelated to other known antibiotic families, and FabI is not targeted by any currently approved antibiotic. Our data shows that this scaffold displays promising enzyme inhibitory activity against FabI from both S. aureus and A. baumannii, as well as encouraging antibacterial activity in S. aureus. Compounds also display excellent synergy when combined with colistin and tested against A. baumannii. In this combination the MIC of colistin is reduced by 10-fold. Our first generation compound displays promising enzyme inhibition, targets FabI in S. aureus with a favorable selectivity index (ratio of cytotoxicity to MIC), and has excellent synergy with colistin against A. baumannii, including a multidrug resistant strain.

Rationalizing the Binding Kinetics for the Inhibition of the Burkholderia pseudomallei FabI1 Enoyl-ACP Reductase.

There is growing awareness of the link between drug-target residence time and in vivo drug activity, and there are increasing efforts to determine the molecular factors that control the lifetime of a drug-target complex. Rational alterations in the drug-target residence time require knowledge of both the ground and transition states on the inhibition reaction coordinate, and we have determined the structure-kinetic relationship for 22 ethyl- or hexyl-substituted diphenyl ethers that are slow-binding inhibitors of bpFabI1, the enoyl-ACP reductase FabI1 from Burkholderia pseudomallei. Analysis of enzyme inhibition using a two-dimensional kinetic map demonstrates that the ethyl and hexyl diphenyl ethers fall into two distinct clusters. Modifications to the ethyl diphenyl ether B ring result in changes to both on and off rates, where residence times of up to ∼700 min (∼11 h) are achieved by either ground state stabilization (PT444) or transition state destabilization (slower on rate) (PT404). By contrast, modifications to the hexyl diphenyl ether B ring result in residence times of 300 min (∼5 h) through changes in only ground state stabilization (PT119). Structural analysis of nine enzyme:inhibitor complexes reveals that the variation in structure-kinetic relationships can be rationalized by structural rearrangements of bpFabI1 and subtle changes to the orientation of the inhibitor in the binding pocket. Finally, we demonstrate that three compounds with residence times on bpFabI1 from 118 min (∼2 h) to 670 min (∼11 h) have in vivo efficacy in an acute B. pseudomallei murine infection model using the virulent B. pseudomallei strain Bp400.

Studies of Staphylococcus aureus FabI inhibitors: fragment-based approach based on holographic structure-activity relationship analyses.

AIM: FabI is a key enzyme in the fatty acid metabolism of Gram-positive bacteria such as Staphylococcus aureus and is an established drug target for known antibiotics such as triclosan. However, due to increasing antibacterial resistance, there is an urgent demand for new drug discovery. Recently, aminopyridine derivatives have been proposed as promising competitive inhibitors of FabI. METHODS: In the present study, holographic structure-activity relationship (HQSAR) analyses were employed for determining structural contributions of a series containing 105 FabI inhibitors. RESULTS & CONCLUSION: The final HQSAR model was robust and predictive according to statistical validation (q2 and r2pred equal to 0.696 and 0.854, respectively) and could be further employed to generate fragment contribution maps. Then, final HQSAR model together with FabI active site information can be useful for designing novel bioactive ligands.