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.

Molecular Modelling Study and Antibacterial Evaluation of Diphenylmethane Derivatives as Potential FabI Inhibitors.

The need for new antibiotics has become a major worldwide challenge as bacterial strains keep developing resistance to the existing drugs at an alarming rate. Enoyl-acyl carrier protein reductases (FabI) play a crucial role in lipids and fatty acid biosynthesis, which are essential for the integrity of the bacterial cell membrane. Our study aimed to discover small FabI inhibitors in continuation to our previously found hit MN02. The process was initially started by conducting a similarity search to the NCI ligand database using MN02 as a query. Accordingly, ten compounds were chosen for the computational assessment and antimicrobial testing. Most of the compounds showed an antibacterial activity against Gram-positive strains, while RK10 exhibited broad-spectrum activity against both Gram-positive and Gram-negative bacteria. All tested compounds were then docked into the saFabI active site followed by 100 ns MD simulations (Molecular Dynamics) and MM-GBSA (Molecular Mechanics with Generalised Born and Surface Area Solvation) calculations in order to understand their fitting and estimate their binding energies. Interestingly, and in line with the experimental data, RK10 was able to exhibit the best fitting with the target catalytic pocket. To sum up, RK10 is a small compound with leadlike characteristics that can indeed act as a promising candidate for the future development of broad-spectrum antibacterial agents.

Biochemical and structural basis for Moraxella catarrhalis enoyl-acyl carrier protein reductase (FabI) inhibition by triclosan and estradiol.

The enoyl-acyl carrier protein reductase (ENR) is an established drug target and catalyzes the last reduction step of the fatty acid elongation cycle. Here, we report the crystal structures of FabI from Moraxella catarrhalis (McFabI) in the apo form, binary complex with NAD+ and ternary complex with NAD + -triclosan (TCL) determined at 2.36, 2.12 and 2.22 Å resolutions, respectively. The comparative study of these three structures revealed three different conformational states for the substrate-binding loop (SBL), including an unstructured intermediate, a structured intermediate and a closed conformation in the apo, binary and ternary complex forms, respectively; indicating the flexibility of SBL during the ligand binding. Virtual screening has suggested that estradiol cypionate may be a potential inhibitor of McFabI. Subsequently, estradiol (EST), the natural form of estradiol cypionate, was assessed for its FabI-binding and -inhibition properties. In vitro studies demonstrated that TCL and EST bind to McFabI with high affinity (KD = 0.038 ± 0.004 and 5 ± 0.06 µM respectively) and inhibit its activity (Ki = 62.93 ± 3.95 nM and 25.97 ± 1.93 µM respectively) and suppress the growth of M. catarrhalis. These findings reveal that TCL and EST inhibit the McFabI activity and thereby affect cell growth. This study suggests that estradiol may be exploited as a novel scaffold for the designing and development of more potential FabI inhibitors.

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.

In silico studies, synthesis and anticancer activity of novel diphenyl ether-based pyridine derivatives.

A series of novel 2-amino-4-(3-hydroxy-4-phenoxyphenyl)-6-(4-substituted phenyl) nicotinonitriles were synthesized and evaluated against HepG2, A-549 and Vero cell lines. Compounds 3b (IC50 16.74 ± 0.45 µM) and 3p (IC50 10.57 ± 0.54 µM) were found to be the most active compounds against A-549 cell line among the evaluated compounds. Further 3b- and 3p-induced apoptosis was characterized by AO/EB (acridine orange/ethidium bromide) nuclear staining method and also by DNA fragmentation study. A decrease in cell viability and initiation of apoptosis was clearly evident through the morphological changes in the A-549 cells treated with 3b and 3p when stained with this method. Fragmentation of DNA into nucleosomes was observed which further confirmed the cell apoptosis in cells treated with compound 3b. Flow cytometry studies confirmed the cell cycle arrest at G2/M phase in A549 cells treated with compound 3b. Further in silico studies performed supported the in vitro anticancer activity of these compounds as depicted by dock score and binding energy values.

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.

Synthesis, biological evaluation and in silico molecular modeling of pyrrolyl benzohydrazide derivatives as enoyl ACP reductase inhibitors.

In efforts to develop lead anti-TB compounds, a novel series of 19 pyrrolyl benzohydrazides were synthesized and screened to target enoyl-ACP reductase enzyme, which is one of the important enzymes involved in type II fatty acid biosynthetic pathway of M. tuberculosis. Pharmacophores were constructed using GALAHAD to generate alignment of data sets and calculated by Pareto ranking. The pharmacophore features were then filtered by Surflex-dock study using enoyl ACP reductase from M. tuberculosis. Compounds 5b and 5d showed H-bonding interactions with Tyr158, Thr196 and co-factor NAD+ that fitted well within the binding pocket of InhA. All the synthesized compounds were screened for preliminary antibacterial activities against Gram-positive S. aureus and Gram-negative E. coli and M. tuberculosis H37Rv to evaluate their antitubercular activities. Some representative compounds were further tested for mammalian cell toxicity using human lung cancer cell-line (A549) that was found to be nontoxic. These compounds exhibited moderate inhibition activities against InhA.

Selectivity of Pyridone- and Diphenyl Ether-Based Inhibitors for the Yersinia pestis FabV Enoyl-ACP Reductase.

The enoyl-ACP reductase (ENR) catalyzes the last reaction in the elongation cycle of the bacterial type II fatty acid biosynthesis (FAS-II) pathway. While the FabI ENR is a well-validated drug target in organisms such as Mycobacterium tuberculosis and Staphylococcus aureus, alternate ENR isoforms have been discovered in other pathogens, including the FabV enzyme that is the sole ENR in Yersinia pestis (ypFabV). Previously, we showed that the prototypical ENR inhibitor triclosan was a poor inhibitor of ypFabV and that inhibitors based on the 2-pyridone scaffold were more potent [Hirschbeck, M. (2012) Structure 20 (1), 89-100]. These studies were performed with the T276S FabV variant. In the work presented here, we describe a detailed examination of the mechanism and inhibition of wild-type ypFabV and the T276S variant. The T276S mutation significantly reduces the affinity of diphenyl ether inhibitors for ypFabV (20-fold → 100-fold). In addition, while T276S ypFabV generally displays an affinity for 2-pyridone inhibitors higher than that of the wild-type enzyme, the 4-pyridone scaffold yields compounds with similar affinity for both wild-type and T276S ypFabV. T276 is located at the N-terminus of the helical substrate-binding loop, and structural studies coupled with site-directed mutagenesis reveal that alterations in this residue modulate the size of the active site portal. Subsequently, we were able to probe the mechanism of time-dependent inhibition in this enzyme family by extending the inhibition studies to include P142W ypFabV, a mutation that results in a gain of slow-onset inhibition for the 4-pyridone PT156.

Structural Basis for Cyclopropanation by a Unique Enoyl-Acyl Carrier Protein Reductase.

The natural product curacin A, a potent anticancer agent, contains a rare cyclopropane group. The five enzymes for cyclopropane biosynthesis are highly similar to enzymes that generate a vinyl chloride moiety in the jamaicamide natural product. The structural biology of this remarkable catalytic adaptability is probed with high-resolution crystal structures of the curacin cyclopropanase (CurF ER), an in vitro enoyl reductase (JamJ ER), and a canonical curacin enoyl reductase (CurK ER). The JamJ and CurK ERs catalyze NADPH-dependent double bond reductions typical of enoyl reductases (ERs) of the medium-chain dehydrogenase reductase (MDR) superfamily. Cyclopropane formation by CurF ER is specified by a short loop which, when transplanted to JamJ ER, confers cyclopropanase activity on the chimeric enzyme. Detection of an adduct of NADPH with the model substrate crotonyl-CoA provides indirect support for a recent proposal of a C2-ene intermediate on the reaction pathway of MDR enoyl-thioester reductases.

Design, synthesis, and biological activity of diaryl ether inhibitors of Toxoplasma gondii enoyl reductase.

Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme for parasite survival. In view of triclosan's poor druggability, which limits its therapeutic use, a new set of B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display improved solubility, permeability and a comparable MIC50 value to that of triclosan. Modeling of these inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the B-ring modifications have additional interactions with the strongly conserved Asn130.

Novel type II fatty acid biosynthesis (FAS II) inhibitors as multistage antimalarial agents.

Malaria is a potentially fatal disease caused by Plasmodium parasites and poses a major medical risk in large parts of the world. The development of new, affordable antimalarial drugs is of vital importance as there are increasing reports of resistance to the currently available therapeutics. In addition, most of the current drugs used for chemoprophylaxis merely act on parasites already replicating in the blood. At this point, a patient might already be suffering from the symptoms associated with the disease and could additionally be infectious to an Anopheles mosquito. These insects act as a vector, subsequently spreading the disease to other humans. In order to cure not only malaria but prevent transmission as well, a drug must target both the blood- and pre-erythrocytic liver stages of the parasite. P. falciparum (Pf) enoyl acyl carrier protein (ACP) reductase (ENR) is a key enzyme of plasmodial type II fatty acid biosynthesis (FAS II). It has been shown to be essential for liver-stage development of Plasmodium berghei and is therefore qualified as a target for true causal chemoprophylaxis. Using virtual screening based on two crystal structures of PfENR, we identified a structurally novel class of FAS inhibitors. Subsequent chemical optimization yielded two compounds that are effective against multiple stages of the malaria parasite. These two most promising derivatives were found to inhibit blood-stage parasite growth with IC(50) values of 1.7 and 3.0 µM and lead to a more prominent developmental attenuation of liver-stage parasites than the gold-standard drug, primaquine.

Structure-based design of a novel class of potent inhibitors of InhA, the enoyl acyl carrier protein reductase from Mycobacterium tuberculosis: a computer modelling approach.

The NADH-dependent Enoyl-ACP reductase (InhA) of Mycobacterium tuberculosis has been shown to be the primary target of the frontline drug isoniazid (INH). However, INH must be first activated by katG gene, mutations in which have mediated resistance to INH. Recently, direct inhibitors of InhA have been reported. Using a structure-based approach, we have identified a tripeptide inhibitor with the sequence WYW, which is 100 times more potent than the existing inhibitors. It is therefore, a potential lead compound for the development of new anti-TB drugs.

Design and synthesis of aryl ether inhibitors of the Bacillus anthracis enoyl-ACP reductase.

The problem of increasing bacterial resistance to the current generation of antibiotics is well documented. Known resistant pathogens such as methicillin-resistant Staphylococcus aureus are becoming more prevalent, while the potential exists for developing drug-resistant pathogens for use as bioweapons, such as Bacillus anthracis. The biphenyl ether antibacterial agent, triclosan, exhibits broad-spectrum activity by targeting the fatty acid biosynthetic pathway through inhibition of enoyl-acyl carrier protein reductase (ENR) and provides a potential scaffold for the development of new, broad-spectrum antibiotics. We used a structure-based approach to develop novel aryl ether analogues of triclosan that target ENR, the product of the fabI gene, from B. anthracis (BaENR). Structure-based design methods were used for the expansion of the compound series including X-ray crystal structure determination, molecular docking, and QSAR methods. Structural modifications were made to both phenyl rings of the 2-phenoxyphenyl core. A number of compounds exhibited improved potency against BaENR and increased efficacy against both the Sterne strain of B. anthracis and the methicillin-resistant strain of S. aureus. X-ray crystal structures of BaENR in complex with triclosan and two other compounds help explain the improved efficacy of the new compounds and suggest future rounds of optimization that might be used to improve their potency.

Structure of fungal fatty acid synthase and implications for iterative substrate shuttling.

We report crystal structures of the 2.6-megadalton alpha6beta6 heterododecameric fatty acid synthase from Thermomyces lanuginosus at 3.1 angstrom resolution. The alpha and beta polypeptide chains form the six catalytic domains required for fatty acid synthesis and numerous expansion segments responsible for extensive intersubunit connections. Detailed views of all active sites provide insights into substrate specificities and catalytic mechanisms and reveal their unique characteristics, which are due to the integration into the multienzyme. The mode of acyl carrier protein attachment in the reaction chamber, together with the spatial distribution of active sites, suggests that iterative substrate shuttling is achieved by a relatively restricted circular motion of the carrier domain in the multifunctional enzyme.