N-acyl-4-arylaminopiperidines: Design and synthesis of a potential antimicrobial scaffold.

We report a concise synthesis of N-acylated piperidines through a Knoevenagel-Doebner condensation/amide construction/ amination sequence. The design of the piperidines considered the pharmacophoric features found in previously reported inhibitors of FabI, an enzyme implicated in bacterial fatty acid biosynthesis. After the microbiological evaluation at 50 µM, the analogs displayed moderate activity against some pathogens from the ESKAPE group, reaching up to 42 % of growth inhibition for MRSA, 54 % for K. pneumoniae, and 37 % for P. aeruginosa (multiresistant strains). Docking studies demonstrate that almost all of them docked satisfactorily into the catalytic domain of S. aureus FabI, maintaining a similar pose as other reported inhibitors. The results shown herein propose the N-acyl-4-arylaminopiperidines as the basis for the development of more active candidates.

Unsaturated fatty acid synthesis in Enterococcus faecalis requires a specific enoyl-ACP reductase.

The Enterococcus faecalis genome contains two enoyl-ACP reductases genes, fabK and fabI, which encode proteins having very different structures. Enoyl-ACP reductase catalyzes the last step of the elongation cycle of type II fatty acid synthesis pathway. The fabK gene is located within the large fatty acid synthesis operon whereas fabI is located together with two genes fabN and fabO required for unsaturated fatty acid synthesis. Prior work showed that FabK is weakly expressed due to poor translational initiation and hence virtually all the cellular enoyl ACP reductase activity is that encoded by fabI. Since FabK is a fully functional enzyme, the question is why FabI is an essential enzyme. Why not increase FabK activity? We report that overproduction of FabK is lethal whereas FabI overproduction only slows the growth and is not lethal. In both cases, normal growth is restored by the addition of oleic acid, an unsaturated fatty acid, to the medium indicating that enoyl ACP reductase overproduction disrupts unsaturated fatty acid synthesis. We report that this is due to competition with FabO, a putative 3-ketoacyl-ACP synthase I via FabN, a dehydratase/isomerase providing evidence that the enoyl-ACP reductase must be matched to the unsaturated fatty acid synthetic genes. FabO has been ascribed the same activity as E. coli FabB and we report in vitro evidence that this is the case, whereas FabN is a dehydratase/isomerase, having the activity of E. coli FabA. However, FabN is much larger than FabA, it is a hexamer rather than a dimer like FabA.

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.

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.

Ecological Role of 6OH-BDE47: Is It a Chemical Offense Molecule Mediated by Enoyl-ACP Reductases?

Although hydroxylated polybrominated diphenyl ethers (OH-BDEs) are among the most abundant natural organobromine compounds, the fundamental biological rationale for marine organisms to produce OH-BDEs remains elusive. Herein, we demonstrated that natural OH-BDEs exerted strong antibacterial activities against Escherichia coli by inhibiting enoyl-[acyl-carrier-protein] reductase (FabI), while anthropogenic OH-BDEs were inactive. Distinct from E. coli, OH-BDE-producing marine γ-proteobacteria including Marinomonas mediterranea MMB-1 (MMB-1) and Pseudoalteromonas luteoviolacea 2ta16 (Pl2ta16) exhibited resistance to 6OH-BDE47. An alternative enoyl-[acyl-carrier-protein] (ACP) reductase, FabV, was detected in all three OH-BDE-producing marine γ-proteobacteria. Thermal stability and protein affinity purification studies revealed that 6OH-BDE47 did not bind to recombinant or endogenous FabV of MMB-1 or Pl2ta16, demonstrating that FabV was the primary mechanism for OH-BDE-producing marine γ-proteobacteria to be resistant to 6OH-BDE47. To further confirm if the laboratory results were evidenced in the field, the 16S rRNA sequencing and metagenomics data from seven field-collected marine sponges were analyzed. Notably, the two Clade 4 sponges containing high concentrations of 6OH-BDE47 exhibited a distinct microbiome community structure compared to the other analyzed clades. Correspondingly, FabV was found to be selectively enriched in the same Clade 4 sponges. The merged evidence from the laboratory experiments and field studies demonstrated that 6OH-BDE47 may act as a chemical offense molecule in marine sponges.

Triclosan is the Predominant Antibacterial Compound in Ontario Sewage Sludge.

Sewage treatment plants (STPs) accumulate both antibiotic and nonantibiotic antimicrobial compounds that can select for antibiotic resistant bacteria. Herein, we aimed to identify the predominant antibacterial compounds impacting E. coli from Ontario sewage sludge consisting of thousands of unknown compounds. Among the 10 extracted sludge samples, 6 extracts exerted significant growth inhibition effects in E. coli. A total of 103 compounds were tentatively detected across the 10 sludge samples by suspect screening, among which the bacterial enoyl-ACP reductase (FabI) inhibitor triclocarban was detected at the highest abundance. A hypomorphic FabI knockdown E. coli strain was highly susceptible to the sludge extracts, confirming FabI inhibitors as the primary antibacterial compounds in the sludge. Protein affinity pulldown identified triclosan as the major ligand binding to a His-tagged FabI protein from the sludge, despite the higher abundance of triclocarban in the same samples. Effect-directed analysis was used to determine the contributions of triclosan to the observed antibacterial potencies. Antibacterial effects were only detected in F17 and F18 across 20 fractions, which was consistent with the elution of triclosan and triclocarban in the same two fractions. Further, potency mass balance analysis confirmed that triclosan explained the majority (58-113%) of inhibition effects from sludge extracts. This study highlighted triclosan as the predominant antibacterial compound in sewage sludge impacting E. coli despite the co-occurrence of numerous other antibiotics and nonantibiotics.

The genome of a Bacteroidetes inhabitant of the human gut encodes a structurally distinct enoyl-acyl carrier protein reductase (FabI).

Enoyl-acyl carrier protein reductase (FabI) catalyzes a rate-controlling step in bacterial fatty-acid synthesis and is a target for antibacterial drug development. A phylogenetic analysis shows that FabIs fall into four divergent clades. Members of clades 1-3 have been structurally and biochemically characterized, but the fourth clade, found in members of phylum Bacteroidetes, is uncharacterized. Here, we identified the unique structure and conformational changes that distinguish clade 4 FabIs. Alistipes finegoldii is a prototypical Bacteroidetes inhabitant of the gut microbiome. We found that A. finegoldii FabI (AfFabI) displays cooperative kinetics and uses NADH as a cofactor, and its crystal structure at 1.72 Å resolution showed that it adopts a Rossmann fold as do other characterized FabIs. It also disclosed a carboxyl-terminal extension that forms a helix-helix interaction that links the protomers as a unique feature of AfFabI. An AfFabI·NADH crystal structure at 1.86 Å resolution revealed that this feature undergoes a large conformational change to participate in covering the NADH-binding pocket and establishing the water channels that connect the active site to the central water well. Progressive deletion of these interactions led to catalytically compromised proteins that fail to bind NADH. This unique conformational change imparted a distinct shape to the AfFabI active site that renders it refractory to a FabI drug that targets clade 1 and 3 pathogens. We conclude that the clade 4 FabI, found in the Bacteroidetes inhabitants of the gut, have several structural features and conformational transitions that distinguish them from other bacterial FabIs.

Structure-Activity Relationship Modeling and Experimental Validation of the Imidazolium and Pyridinium Based Ionic Liquids as Potential Antibacterials of MDR Acinetobacter Baumannii and Staphylococcus Aureus.

Online Chemical Modeling Environment (OCHEM) was used for QSAR analysis of a set of ionic liquids (ILs) tested against multi-drug resistant (MDR) clinical isolate Acinetobacter baumannii and Staphylococcus aureus strains. The predictive accuracy of regression models has coefficient of determination q2 = 0.66 - 0.79 with cross-validation and independent test sets. The models were used to screen a virtual chemical library of ILs, which was designed with targeted activity against MDR Acinetobacter baumannii and Staphylococcus aureus strains. Seven most promising ILs were selected, synthesized, and tested. Three ILs showed high activity against both these MDR clinical isolates.

Afabicin, a First-in-Class Antistaphylococcal Antibiotic, in the Treatment of Acute Bacterial Skin and Skin Structure Infections: Clinical Noninferiority to Vancomycin/Linezolid.

Afabicin (formerly Debio 1450, AFN-1720) is a prodrug of afabicin desphosphono, an enoyl-acyl carrier protein reductase (FabI) inhibitor, and is a first-in-class antibiotic with a novel mode of action to specifically target fatty acid synthesis in Staphylococcus spp. The efficacy, safety, and tolerability of afabicin were compared with those of vancomycin/linezolid in the treatment of acute bacterial skin and skin structure infections (ABSSSI) due to staphylococci in this multicenter, parallel-group, double-blind, and double-dummy phase 2 study. Randomized patients (1:1:1) received either low-dose (LD) afabicin (intravenous [i.v.] 80 mg, followed by oral 120 mg, twice a day [BID]), high-dose (HD) afabicin (i.v. 160 mg, followed by oral 240 mg, BID), or vancomycin/linezolid (i.v. vancomycin 1 g or 15 mg/kg, followed by oral linezolid 600 mg, BID). The most frequent baseline pathogen was Staphylococcus aureus (97.5% of microbiological intent-to-treat [mITT] population), and 50.4% of patients had methicillin-resistant S. aureus Clinical response rates at 48 to 72 h postrandomization in the mITT population were comparable among treatment groups (94.6%, 90.1%, and 91.1%, respectively). Both LD and HD afabicin were noninferior to vancomycin/linezolid (differences, -3.5% [95% confidence interval {CI}, -10.8%, 3.9%] and 1.0% [95% CI, -7.3%, 9.2%], respectively). Most common treatment-emergent adverse events were mild and were headache (9.1% and 16.8%) and nausea (6.4% and 8.4%) with LD and HD afabicin, respectively. Afabicin was efficacious and well tolerated in the treatment of ABSSSI due to staphylococci, and these data support further development of afabicin for the treatment of ABSSSI and potentially other types of staphylococcal infections. (This study has been registered at ClinicalTrials.gov under identifier NCT02426918.).

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.

[The study of patterns of development of resistance of Staphylococcus aureus to triclosan].

The article presents the results of studying molecular genetic mechanisms of development of resistance to antiseptic Triclosan in strain Staphylococcus aureus АТСС25923. The modifcations of strain S. aureus АТСС25923 (Tr1, Tr2, Tr1С и Tr2С) are obtained resistant to 64 mg/l of Triclosan and stably preserving the given characteristic under cultivation in absence of selective pressure. The strain S. aureus Tr1was characterized by slightly delayed growth and the strain S. aureus Tr2 was characterized by growth velocity comparable with initial strain. In the Triclosan-resistant strains a mutation C284T in gene fabI was detected resulting in amino-acid replacement A95V in enzyme enoyl-acyl protein reductase FabI, triclosan target. Besides, in these strains a stably inheriting mutation was detected in genes associated with transport of substances in cell: hypothetical transport protein HlyC/CorC family transporter, protein-transporter of ions of Na+, K+, Li+ and alkali of Na+/H+ antiporter subunit F, membrane hypothetical protein and ATP-binding protein. It is demonstrated that resistance to triclosan in staphylococci is associated with acquirement of point mutations in genes of enoyl-acyl protein reductase and also in other genes related to transport of substances in bacterial cell.

Structural insights into the dimer-tetramer transition of FabI from Bacillus anthracis.

Enoyl-ACP reductase (ENR, also known as FabI) has received considerable interest as an anti-bacterial target due to its essentiality in fatty acid synthesis. All the FabI structures reported to date, regardless of the organism, are composed of homo-tetramers, except for two structures: Bacillus cereus and Staphylococcus aureus FabI (bcFabI and saFabI, respectively), which have been reported as dimers. However, the reason for the existence of the dimeric form in these organisms and the biological meaning of dimeric and tetrameric forms of FabI are ambiguous. Herein, we report the high-resolution crystal structure of a dimeric form of Bacillus anthracis FabI (baFabI) and the crystal structures of tetrameric forms of baFabI in the apo state and in complex with NAD+ and with NAD+-triclosan, at 1.7 Å, 1.85 Å, 1.96 Å, and 1.95 Å, respectively. Interestingly, we found that baFabI with a His6-tag at its C-terminus exists as a dimer, whereas untagged-baFabI exists as a tetramer. The His6-tag may block the dimer-tetramer transition, since baFabI has relatively short-length amino acids (255LG256) after the 310-helix η7 compared to those of FabI of other organisms. The dimeric form of baFabI is catalytically inactive, because the α-helix α5 occupies the NADH-binding site. During the process of dimer-tetramer transition, this α5 helix rotates about 55° toward the tetramer interface and the active site is established. Therefore, tetramerization of baFabI is required for cofactor binding and catalytic activity.

Genomic and transcriptome analysis of triclosan response of a multidrug-resistant Acinetobacter baumannii strain, MDR-ZJ06.

During the last decade, an increasing amount of attention has focused on the potential threat of triclosan to both the human body and environmental ecology. However, the role of triclosan in the development of drug resistance and cross resistance is still in dispute ascribed to largely unknown of triclosan resistance mechanism. In this work, Acinetobacter baumannii MDR-ZJ06, a multidrug-resistant strain, was induced by triclosan, and the genomic variation and transcriptional levels were investigated, respectively. The comparative transcriptomic analysis found that several general protective mechanisms were enhanced under the triclosan condition, including responses to reactive oxygen species and cell membrane damage. Meanwhile, all of the detected fifteen single nucleotide polymorphisms were not directly associated triclosan tolerance. In summary, this work revealed the crucial role of the general stress response in A. baumannii under a triclosan stress condition, which informs a more comprehensive understanding of the role of triclosan in the spread of drug-resistant bacteria.

Evaluating thermodynamic integration performance of the new amber molecular dynamics package and assess potential halogen bonds of enoyl-ACP reductase (FabI) benzimidazole inhibitors.

Thermodynamic integration (TI) can provide accurate binding free energy insights in a lead optimization program, but its high computational expense has limited its usage. In the effort of developing an efficient and accurate TI protocol for FabI inhibitors lead optimization program, we carefully compared TI with different Amber molecular dynamics (MD) engines (sander and pmemd), MD simulation lengths, the number of intermediate states and transformation steps, and the Lennard-Jones and Coulomb Softcore potentials parameters in the one-step TI, using eleven benzimidazole inhibitors in complex with Francisella tularensis enoyl acyl reductase (FtFabI). To our knowledge, this is the first study to extensively test the new AMBER MD engine, pmemd, on TI and compare the parameters of the Softcore potentials in the one-step TI in a protein-ligand binding system. The best performing model, the one-step pmemd TI, using 6 intermediate states and 1 ns MD simulations, provides better agreement with experimental results (RMSD = 0.52 kcal/mol) than the best performing implicit solvent method, QM/MM-GBSA from our previous study (RMSD = 3.00 kcal/mol), while maintaining similar efficiency. Briefly, we show the optimized TI protocol to be highly accurate and affordable for the FtFabI system. This approach can be implemented in a larger scale benzimidazole scaffold lead optimization against FtFabI. Lastly, the TI results here also provide structure-activity relationship insights, and suggest the parahalogen in benzimidazole compounds might form a weak halogen bond with FabI, which is a well-known halogen bond favoring enzyme.

Rational design of broad spectrum antibacterial activity based on a clinically relevant enoyl-acyl carrier protein (ACP) reductase inhibitor.

Determining the molecular basis for target selectivity is of particular importance in drug discovery. The ideal antibiotic should be active against a broad spectrum of pathogenic organisms with a minimal effect on human targets. CG400549, a Staphylococcus-specific 2-pyridone compound that inhibits the enoyl-acyl carrier protein reductase (FabI), has recently been shown to possess human efficacy for the treatment of methicillin-resistant Staphylococcus aureus infections, which constitute a serious threat to human health. In this study, we solved the structures of three different FabI homologues in complex with several pyridone inhibitors, including CG400549. Based on these structures, we rationalize the 65-fold reduced affinity of CG400549 toward Escherichia coli versus S. aureus FabI and implement concepts to improve the spectrum of antibacterial activity. The identification of different conformational states along the reaction coordinate of the enzymatic hydride transfer provides an elegant visual depiction of the relationship between catalysis and inhibition, which facilitates rational inhibitor design. Ultimately, we developed the novel 4-pyridone-based FabI inhibitor PT166 that retained favorable pharmacokinetics and efficacy in a mouse model of S. aureus infection with extended activity against Gram-negative and mycobacterial organisms.

Comparison of radii sets, entropy, QM methods, and sampling on MM-PBSA, MM-GBSA, and QM/MM-GBSA ligand binding energies of F. tularensis enoyl-ACP reductase (FabI).

To validate a method for predicting the binding affinities of FabI inhibitors, three implicit solvent methods, MM-PBSA, MM-GBSA, and QM/MM-GBSA were carefully compared using 16 benzimidazole inhibitors in complex with Francisella tularensis FabI. The data suggests that the prediction results are sensitive to radii sets, GB methods, QM Hamiltonians, sampling protocols, and simulation length, if only one simulation trajectory is used for each ligand. In this case, QM/MM-GBSA using 6 ns MD simulation trajectories together with GB(neck2) , PM3, and the mbondi2 radii set, generate the closest agreement with experimental values (r(2) = 0.88). However, if the three implicit solvent methods are averaged from six 1 ns MD simulations for each ligand (called "multiple independent sampling"), the prediction results are relatively insensitive to all the tested parameters. Moreover, MM/GBSA together with GB(HCT) and mbondi, using 600 frames extracted evenly from six 0.25 ns MD simulations, can also provide accurate prediction to experimental values (r(2) = 0.84). Therefore, the multiple independent sampling method can be more efficient than a single, long simulation method. Since future scaffold expansions may significantly change the benzimidazole's physiochemical properties (charges, etc.) and possibly binding modes, which may affect the sensitivities of various parameters, the relatively insensitive "multiple independent sampling method" may avoid the need of an entirely new validation study. Moreover, due to large fluctuating entropy values, (QM/)MM-P(G)BSA were limited to inhibitors' relative affinity prediction, but not the absolute affinity. The developed protocol will support an ongoing benzimidazole lead optimization program.

Characterization of FabG and FabI of the Streptomyces coelicolor dissociated fatty acid synthase.

Streptomyces coelicolor produces fatty acids for both primary metabolism and for biosynthesis of the secondary metabolite undecylprodiginine. The first and last reductive steps during the chain elongation cycle of fatty acid biosynthesis are catalyzed by FabG and FabI. The S. coelicolor genome sequence has one fabI gene (SCO1814) and three likely fabG genes (SCO1815, SCO1345, and SCO1846). We report the expression, purification, and characterization of the corresponding gene products. Kinetic analyses revealed that all three FabGs and FabI are capable of utilizing both straight and branched-chain ß-ketoacyl-NAC and enoyl-NAC substrates, respectively. Furthermore, only SCO1345 differentiates between ACPs from both biosynthetic pathways. The data presented provide the first experimental evidence that SCO1815, SCO1346, and SCO1814 have the catalytic capability to process intermediates in both fatty acid and undecylprodiginine biosynthesis.

Discovery of a potent enoyl-acyl carrier protein reductase (FabI) inhibitor suitable for antistaphylococcal agent.

We report the discovery, synthesis, and biological activities of phenoxy-4-pyrone and phenoxy-4-pyridone derivatives as novel inhibitors of enoyl-acyl carrier protein reductase (FabI). Pyridone derivatives showed better activities than pyrone derivatives against FabI and Staphylococcus aureus strains, including methicillin-resistant Staphylococcus aureus (MRSA). Among the pyridone derivatives, compound 16l especially exhibited promising activities against the MRSA strain and good pharmacokinetic profiles.

Structural and biological evaluation of a novel series of benzimidazole inhibitors of Francisella tularensis enoyl-ACP reductase (FabI).

Francisella tularensis, the causative agent of tularemia, presents a significant biological threat and is a Category A priority pathogen due to its potential for weaponization. The bacterial FASII pathway is a viable target for the development of novel antibacterial agents treating Gram-negative infections. Here we report the advancement of a promising series of benzimidazole FabI (enoyl-ACP reductase) inhibitors to a second-generation using a systematic, structure-guided lead optimization strategy, and the determination of several co-crystal structures that confirm the binding mode of designed inhibitors. These compounds display an improved low nanomolar enzymatic activity as well as promising low microgram/mL antibacterial activity against both F. tularensis and Staphylococcus aureus and its methicillin-resistant strain (MRSA). The improvements in activity accompanying structural modifications lead to a better understanding of the relationship between the chemical structure and biological activity that encompasses both enzymatic and whole-cell activity.

Resistance to AFN-1252 arises from missense mutations in Staphylococcus aureus enoyl-acyl carrier protein reductase (FabI).

AFN-1252 is a potent antibiotic against Staphylococcus aureus that targets the enoyl-acyl carrier protein reductase (FabI). A thorough screen for AFN-1252-resistant strains was undertaken to identify the spectrum of mechanisms for acquired resistance. A missense mutation in fabI predicted to encode FabI(M99T) was isolated 49 times, and a single isolate was predicted to encode FabI(Y147H). AFN-1252 only bound to the NADPH form of FabI, and the close interactions between the drug and Met-99 and Tyr-147 explained how the mutations would result in resistant enzymes. The clone expressing FabI(Y147H) had a pronounced growth defect that was rescued by exogenous fatty acid supplementation, and the purified protein had less than 5% of the enzymatic activity of FabI. FabI(Y147F) was also catalytically defective but retained its sensitivity to AFN-1252, illustrating the importance of the conserved Tyr-147 hydroxyl group in FabI function. The strains expressing FabI(M99T) exhibited normal growth, and the biochemical properties of the purified protein were indistinguishable from those of FabI. The AFN-1252 Ki(app) increased from 4 nm in FabI to 69 nm in FabI(M99T), accounting for the increased resistance of the corresponding mutant strain. The low activity of FabI(Y147H) precluded an accurate Ki measurement. The strain expressing FabI(Y147H) was also resistant to triclosan; however, the strain expressing FabI(M99T) was more susceptible. Strains with higher levels of AFN-1252 resistance were not obtained. The AFN-1252-resistant strains remained sensitive to submicromolar concentrations of AFN-1252, which blocked growth through inhibition of fatty acid biosynthesis at the FabI step.