FabI Inhibitors Research Articles

CRISPRi/dCpf1-mediated dynamic metabolic switch to enhance butenoic acid production in Escherichia coli.

Butenoic acid is a short-chain unsaturated fatty acid and important precursor for pharmaceutical and other applications. Heterologous thioesterases are able to convert a fatty acid biosynthesis intermediate in Escherichia coli to butenoic acid. In order to acquire high titer and yield of the product, dynamically switching the metabolic flux from fatty acid biosynthesis pathway to butenoic acid is critical after achieving enough cell mass of the host. A previous developed switch for butenoic acid fermentation is based on triclosan molecule as the FabI inhibitor in the fatty acid biosynthesis cycle. However, triclosan is toxic to human, which may limit its pharmaceutical application. Alternatively, we here purposed a nontoxic switch of carbon flux by harnessing recently developed CRISPR interference (CRISPRi) approach. In our work, we constructed a CRISPRi/dCpf1-mediated dynamic metabolic switch to separate the host growth and production phase via switching the expression of the fabI gene in fatty acid biosynthesis pathway. After optimizing the programmable targets, the CRISPRi-based switch boosted the titer of butenoic acid by 6-fold (1.41g/L) in fed-batch fermentation. Our work supported that the CRISPRi/dCpf1 switch could replace triclosan-based switch as a nontoxic switch for butenoic acid production, and outcompeted the later switch in the biomass accumulation of the host cell. Moreover, the CRISPRi/dCpf1 system was integrated into the chromosome of the host to improve its genetic stability for long-term fermentation and other applications.Key Points• A programmable metabolic switch was developed to replace the toxic chemical switch to separate the growth phase and production phase of the butenoic acid.• The programmable CRISPRi/dCpf1 switch was efficiently and stably integrated into the host genome to increase their genetic stability during fermentation.• The optimized metabolic switch simultaneously increased the host biomass and butenoic acid titer, and solved the paradox of the competition between growth and production.

The Kalimantacin Polyketide Antibiotics Inhibit Fatty Acid Biosynthesis in Staphylococcus aureus by Targeting the Enoyl‐Acyl Carrier Protein Binding Site of FabI

AbstractThe enoyl‐acyl carrier protein reductase enzyme FabI is essential for fatty acid biosynthesis in Staphylococcus aureus and represents a promising target for the development of novel, urgently needed anti‐staphylococcal agents. Here, we elucidate the mode of action of the kalimantacin antibiotics, a novel class of FabI inhibitors with clinically‐relevant activity against multidrug‐resistant S. aureus. By combining X‐ray crystallography with molecular dynamics simulations, in vitro kinetic studies and chemical derivatization experiments, we characterize the interaction between the antibiotics and their target, and we demonstrate that the kalimantacins bind in a unique conformation that differs significantly from the binding mode of other known FabI inhibitors. We also investigate mechanisms of acquired resistance in S. aureus and identify key residues in FabI that stabilize the binding of the antibiotics. Our findings provide intriguing insights into the mode of action of a novel class of FabI inhibitors that will inspire future anti‐staphylococcal drug development.

The Kalimantacin Polyketide Antibiotics Inhibit Fatty Acid Biosynthesis in Staphylococcus aureus by Targeting the Enoyl‐Acyl Carrier Protein Binding Site of FabI

The enoyl-acyl carrier protein reductase enzyme FabI is essential for fatty acid biosynthesis in Staphylococcus aureus and represents a promising target for the development of novel, urgently needed anti-staphylococcal agents. Here, we elucidate the mode of action of the kalimantacin antibiotics, a novel class of FabI inhibitors with clinically-relevant activity against multidrug-resistant S. aureus. By combining X-ray crystallography with molecular dynamics simulations, in vitro kinetic studies and chemical derivatization experiments, we characterize the interaction between the antibiotics and their target, and we demonstrate that the kalimantacins bind in a unique conformation that differs significantly from the binding mode of other known FabI inhibitors. We also investigate mechanisms of acquired resistance in S. aureus and identify key residues in FabI that stabilize the binding of the antibiotics. Our findings provide intriguing insights into the mode of action of a novel class of FabI inhibitors that will inspire future anti-staphylococcal drug development.

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, called eNTRyway, which predicts compound accumulation (in E. coli) from its structure. eNTRyway, in conjunction with structure-activity relationship and x-ray data, 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 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 Escherichia 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.

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.

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.

Classification of Staphylococcus Aureus FabI Inhibitors by Machine Learning Techniques

Enoyl-acyl carrier protein reductase (FabI) is a key enzyme in the fatty acid metabolism of gram-positive bacteria and is considered a potential target for new antibacterial drugs development. Indeed, triclosan is a widely employed antibacterial and AFN-1252 is currently under phase-II clinical trials, both are known as FabI inhibitors. Nowadays, there is an urgent need for new drug discovery due to increasing antibacterial resistance. In the present study, classification models using machine learning techniques were generated to distinguish SaFabI inhibitors from non-inhibitors successfully (e.g., Mathews correlation coefficient values equal to 0.837 and 0.789 calculated with internal and external validations). The interpretation of a selected model indicates that larger compounds, number of N atoms and the distance between central amide and naphthyridinone ring are important to biological activity, corroborating previous studies. Therefore, these obtained information and generated models can be useful for design/discovery of novel bioactive ligands as potential antibacterial agents.

Ligand- and Structure-Based Approaches of Escherichia coli FabI inhibition by triclosan derivatives: from chemical similarity to protein dynamics influence

Ligand- and Structure-Based Approaches of Escherichia coli FabI inhibition by triclosan derivatives: from chemical similarity to protein dynamics influence

Determination of absolute configuration and binding efficacy of benzimidazole-based FabI inhibitors through the support of electronic circular dichroism and MM-GBSA techniques

We have previously reported benzimidazole-based compounds to be potent inhibitors of FabI for Francisella tularensis (FtFabI), making them promising antimicrobial hits. Optically active enantiomers exhibit markedly differing affinities toward FtFabI. The IC50 of benzimidazole (−)-1 is ∼100× lower than the (+)-enantiomer, with similar results for the 2 enantiomers. Determining the absolute configuration for these optical compounds and elucidating their binding modes is important for further design. Electronic circular dichroism (ECD) quantum calculations have become important in determining absolute configurations of optical compounds. We determined the absolute configuration of (−)/(+)-1 and (−)/(+)-2 by comparing experimental spectra and theoretical density functional theory (DFT) simulations of ECD spectra at the B3LYP/6-311+G(2d, p) level using Gaussian09. Comparison of experimental and calculated ECD spectra indicates that the S configuration corresponds to the (−)-rotation for both compounds 1 and 2, while the R configuration corresponds to the (+)-rotation. Further, molecular dynamics simulations and MM-GBSA binding energy calculations for these two pairs of enantiomers with FtFabI show much tighter binding MM-GBSA free energies for S-1 and S-2 than for their enantiomers, R-1 and R-2, consistent with the S configuration being the more active one, and with the ECD determination of the S configuration corresponding to (−) and the R configuration corresponding to (+). Thus, our computational studies allow us to assign (−) to (S)- and (+) to (R)- for compounds 1 and 2, and to further evaluate structural changes to improve efficacy.

Design, synthesis, 3D pharmacophore, QSAR, and docking studies of some new (6-methoxy-2-naphthyl) propanamide derivatives with expected anti-bacterial activity as FABI inhibitor

Antibiotic resistance is a major health problem; so there is a emerging need for developing new antibiotic agents. A novel series of (6-methoxy-2-naphthyl) propanamide derivatives were synthesized and evaluated for their potential antibacterial activity. The minimum inhibitory concentration of these compounds were determined by microdilution technique against five known strains of bacteria. Test strains included three Gram-positive strains (Streptococcus pneumonia, Bacillus subtilis and Staphylococcus aureus) and two Gram-negative strains (Escherichia coli and Salmonella typhimurium). According to the observed antibacterial activity results showed that compounds N-(4-(2-(5-bromo-2-hydroxybenzylidene)hydrazine carbonyl) phenyl)-2-(6-meth oxynaphthalen-2-yl)propanamide 2d and N-(4-(2-(2,4-dichlorobenzylidene)hydrazine carbonyl)phenyl)-2-(6-methoxy-naphthalen-2-yl)propanamide 2j has potent antibacterial activity against B. subtilis (minimal inhibitory concentrations 1.95 µg/ml). Also 2-(6-methoxynaphthalen-2-yl)-N-(4-(2-(4-oxopentan-2-ylidene)hydrazinecarbonnyl)phenyl) propanamide 6 showed potent antibacterial activity against S. pneumonia (minimal inhibitory concentrations 1.95 µg/ml) compared to Naproxen (7.81, 15.63 µg/ml) and the reference drug Ampicillin (7.81 µg/ml). Two of the synthesized compounds, namely, 2j and 2k exhibited more potent antibacterial activity than the reference drugs (Gentamicin and Ampicillin) against all the test strains of bacteria. Molecular docking simulation was also carried out for Enoyl-aceyl carrier protein reductase enzyme, which is responsible for catalyzing the final step of bacterial fatty acid biosynthesis and is an attractive target for the development of novel antibacterial agents. In addition, generation of 3D pharmacophore model and quantitative structure–activity relationship models were combined to explore the structural requirements controlling the observed antibacterial properties.

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

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. In the present study, holographic structure-activity relationship (HQSAR) analyses were employed for determining structural contributions of a series containing 105 FabI inhibitors. 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.

Docking and molecular dynamics studies on triclosan derivatives binding to FabI.

FabI, enoyl-ACP reductase (ENR), is the rate-limiting enzyme in the last step for fatty acids biosynthesis in many bacteria. Triclosan (TCL) is a commercial bactericide, and as a FabI inhibitor, it can depress the substrate (trans-2-enoyl-ACP) binding with FabI to hinder the fatty acid synthesis. The structure-activity relationship between TCL derivatives and FabI protein has already been acknowledged, however, their combination at the molecular level has never been investigated. This paper uses the computer-aided approaches, such as molecular docking, molecular dynamics simulation, and binding free energy calculation based on the molecular mechanics/Poisson-Bolzmann surface area (MM/PBSA) method to illustrate the interaction rules of TCL derivatives with FabI and guide the development of new derivatives. The consistent data of the experiment and corresponding activity demonstrates that electron-withdrawing groups on side chain are better than electron-donating groups. 2-Hydroxyl group on A ring, promoting the formation of hydrogen bond, is vital for bactericidal effect; and the substituents at 4-position of A ring, 2'-position and 4'-position of B ring benefit antibacterial activity due to forming a hydrogen bond or stabilizing the conformation of active pocket residues of receptor. While the substituents at 3'-position and 5'-position of B ring destroy the π-π stacking interaction of A ring and NAD+ which depresses the antibacterial activity. This study provides a new sight for designing novel TCL derivatives with superior antibacterial activity.

Benzimidazole-Based FabI Inhibitors: A Promising Novel Scaffold for Anti-staphylococcal Drug Development.

The enoyl-ACP reductase (FabI) enzyme is a well validated target for anti-staphylococcal drug discovery and development. With the goal of finding alternate therapeutics for drug-resistant strains of Staphylococcus aureus, such as methicillin-resistant S. aureus (MRSA), our previously published series of benzimidazole-based inhibitors of the FabI enzyme from Francisella tularensis (FtFabI) have been evaluated against FabI from S. aureus (SaFabI). We report here the preliminary structure-activity relationship of this series and the prioritization of compounds toward lead optimization. Mutational studies have identified key residues that contribute toward stabilizing the inhibitors in the active site of FabI. Mutations that do not significantly impact enzyme function but destabilize inhibitor binding are more likely to occur in nature as organisms evolve to evade the action of antibiotics leading to resistance. Identifying these residues provides guidance for minimizing susceptibility to resistance. Additionally, we have identified compounds that elicit antibacterial activity through off-target effects and observe that close analogs can display differing modes of action (on-target vs off-target) and need to be individually evaluated early on to prioritize compounds for lead optimization. Overall, our data suggest that the benzimidazole scaffold is a promising scaffold for anti-staphylococcal drug development.

Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes.

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes.

An Unbiased Chemical Proteomics Method Identifies FabI as the Primary Target of 6-OH-BDE-47

Determination of the physical interactions of environmental chemicals with cellular proteins is important for characterizing biological and toxic mechanism of action. Yet despite the discovery of numerous bioactive natural brominated compounds, such as hydroxylated polybrominated diphenyl ethers (OH-PBDEs), their corresponding protein targets remain largely unclear. Here, we reported a systematic and unbiased chemical proteomics assay (Target Identification by Ligand Stabilization, TILS) for target identification of bioactive molecules based on monitoring ligand-induced thermal stabilization. We first validated the broad applicability of this approach by identifying both known and unexpected proteins bound by diverse compounds (anticancer drugs, antibiotics). We then applied TILS to identify the bacterial target of 6-OH-BDE-47 as enoyl-acyl carrier protein reductase (FabI), an essential and widely conserved enzyme. Using affinity pull-down and in vitro enzymatic assays, we confirmed the potent antibacterial activity of 6-OH-BDE-47 occurs via direct binding and inhibition of FabI. Conversely, overexpression of FabI rescued the growth inhibition of Escherichia coli by 6-OH-BDE-47, validating it as the primary in vivo target. This study documents a chemical proteomics strategy for identifying the physical and functional targets of small molecules, and its potential high-throughput application to investigate the modes-of-action of environmental compounds.

A Pathogen-Selective Antibiotic Minimizes Disturbance to the Microbiome.

Broad-spectrum antibiotic therapy decimates the gut microbiome, resulting in a variety of negative health consequences. Debio 1452 is a staphylococcus-selective enoyl-acyl carrier protein reductase (FabI) inhibitor under clinical development and was used to determine whether treatment with pathogen-selective antibiotics would minimize disturbance to the microbiome. The effect of oral Debio 1452 on the microbiota of mice was compared to the effects of four commonly used broad-spectrum oral antibiotics. During the 10 days of oral Debio 1452 treatment, there was minimal disturbance to the gut bacterial abundance and composition, with only the unclassified S24-7 taxon reduced at days 6 and 10. In comparison, broad-spectrum oral antibiotics caused ∼100- to 4,000-fold decreases in gut bacterial abundance and severely altered the microbial composition. The gut bacterial abundance and composition of Debio 1452-treated mice were indistinguishable from those of untreated mice 2 days after the antibiotic treatment was stopped. In contrast, the bacterial abundance in broad-spectrum-antibiotic-treated mice took up to 7 days to recover, and the gut composition of the broad-spectrum-antibiotic-treated mice remained different from that of the control group 20 days after the cessation of antibiotic treatment. These results illustrate that a pathogen-selective approach to antibiotic development will minimize disturbance to the gut microbiome.

Safety, tolerability and pharmacokinetics of multiple oral doses of AFN-1252 administered as immediate release (IR) tablets in healthy subjects

Background: AFN-1252 is a novel inhibitor of FabI, which is essential in Staphylococcus spp. This study evaluated the safety, tolerability and pharmacokinetics of multiple oral doses of AFN-1252 immediate-release tablets.Methods: Part I evaluated AFN-1252 as a single 200 mg dose in fed versus fasted subjects. Part II evaluated 200, 300 and 400 mg doses of AFN-1252 administered once-daily for 10 days.Results: Pharmacokinetics indicated good absorption with a median Tmax of 2–3 hours, and a mean t1/2 of 7–10 hours, for all doses. Cmax and AUC responses were non-linear. A high-fat meal reduced AUC0–t and Cmax values by 62% and 48%, respectively, and delayed Tmax by 2.5 hours. All adverse events, including possibly drug-related headache and nausea, were mild or moderate.Conclusions: Multiple ascending doses of AFN-1252 were safe and well tolerated. AFN-1252 has potential for once- or twice-daily dosing in the treatment of staphylococcal infections.

Minimum structural requirements for cell membrane leakage-mediated anti-MRSA activity of macrocyclic bis(bibenzyl)s

Macrocyclic bis(bibenzyl)-type phenolic natural products, found exclusively in bryophytes, exhibit potent antibacterial activity towards methicillin-resistant Staphylococcus aureus (anti-MRSA activity). Here, in order to identify the minimum essential structure for cell membrane leakage-mediated anti-MRSA activity of these compounds, we synthesized acyclic fragment structures and evaluated their anti-MRSA activity. The activities of all of the acyclic fragments tested exhibited similar characteristics to those of the macrocycles, i.e., anti-MRSA bactericidal activity, an enhancing effect on influx and efflux of ethidium bromide (EtBr: fluorescent DNA-binder) in Staphylococcus aureus cells, and bactericidal activity towards a Staphylococcus aureus strain resistant to 2-phenoxyphenol (4). The latter result suggests that they have a different mechanism of action from 4, which is a FabI inhibitor previously proposed to be the minimum active fragment of riccardin-type macrocycles. Thus, cyclic structure is not a necessary condition for cell membrane leakage-mediated anti-MRSA activity of macrocyclic bis(bibenzyl)s.

Resistance Mechanisms and the Future of Bacterial Enoyl-Acyl Carrier Protein Reductase (FabI) Antibiotics.

Missense mutations leading to clinical antibiotic resistance are a liability of single-target inhibitors. The enoyl-acyl carrier protein reductase (FabI) inhibitors have one intracellular protein target and drug resistance is increased by the acquisition of single-base-pair mutations that alter drug binding. The spectrum of resistance mechanisms to FabI inhibitors suggests criteria that should be considered during the development of single-target antibiotics that would minimize the impact of missense mutations on their clinical usefulness. These criteria include high-affinity, fast on/off kinetics, few drug contacts with residue side chains, and no toxicity. These stringent criteria are achievable by structure-guided design, but this approach will only yield pathogen-specific drugs. Single-step acquisition of resistance may limit the clinical application of broad-spectrum, single-target antibiotics, but appropriately designed pathogen-specific antibiotics have the potential to overcome this liability.

Correlating Drug-Target Kinetics and In vivo Pharmacodynamics: Long Residence Time Inhibitors of the FabI Enoyl-ACP Reductase.

Drug-target kinetics enable time-dependent changes in target engagement to be quantified as a function of drug concentration. When coupled to drug pharmacokinetics (PK), drug-target kinetics can thus be used to predict in vivo pharmacodynamics (PD). Previously we described a mechanistic PK/PD model that successfully predicted the antibacterial activity of an LpxC inhibitor in a model of Pseudomonas aeruginosa infection. In the present work we demonstrate that the same approach can be used to predict the in vivo activity of an enoyl-ACP reductase (FabI) inhibitor in a model of methicillin-resistant Staphylococcus aureus (MRSA) infection. This is significant because the LpxC inhibitors are cidal, whereas the FabI inhibitors are static. In addition P. aeruginosa is a Gram-negative organism whereas MRSA is Gram-positive. Thus this study supports the general applicability of our modeling approach across antibacterial space.