Letter pubs.acs.org/journal/aidcbc
Antibacterial FabH Inhibitors with Mode of Action Validated in Haemophilus influenzae by in Vitro Resistance Mutation Mapping David C. McKinney,*,†,Δ Charles J. Eyermann,†,Δ Rong-Fang Gu,†,Δ Jun Hu,‡,Δ Steven L. Kazmirski,‡,Δ Sushmita D. Lahiri,†,Δ Andrew R. McKenzie,#,Δ Adam B. Shapiro,†,Δ and Gloria Breault†,Δ †
Infection Innovative Medicines, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States Structure and Biophysics and #Chemistry Innovation Center, Discovery Sciences, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States
‡
S Supporting Information *
ABSTRACT: Fatty acid biosynthesis is essential to bacterial growth in Gram-negative pathogens. Several small molecules identified through a combination of high-throughput and fragment screening were cocrystallized with FabH (β-ketoacyl-acyl carrier protein synthase III) from Escherichia coli and Streptococcus pneumoniae. Structure-based drug design was used to merge several scaffolds to provide a new class of inhibitors. After optimization for Gram-negative enzyme inhibitory potency, several compounds demonstrated antimicrobial activity against an efflux-negative strain of Haemophilus influenzae. Mutants resistant to these compounds had mutations in the FabH gene near the catalytic triad, validating FabH as a target for antimicrobial drug discovery. KEYWORDS: structure-based drug design, FasII, antibacterial, fatty acid biosynthesis, FabH, β-ketoacyl-acyl carrier protein synthase III
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platensimycin,10 and cerulenin.11 Synthetic inhibitors include triclosan, AFN-1252, CG400549, and MUT056399,12,13 which target FabI (enoyl-acyl carrier protein reductase). FabI inhibitors triclosan,14 the diazaborines,15 ANF-1252,12 and CG40046216 have validated FabI as a target by the isolation of resistant mutants with modification in the gene for FabI synthesis. Whereas to the best of our knowledge no FabH inhibitors have been validated by this means, the existence of natural products and synthetic compounds that exert antimicrobial activity by inhibition of FAB supports the essentiality of the pathway.17 Inhibitors of the initiating step of FAB, the decarboxylative condensation of malonyl-ACP and acetyl-CoA catalyzed by FabH, have been reported.18 Several representative examples that have previously been profiled against various FabH isozymes biochemically, microbiologically, and crystallographically19 are shown in Figure 1 (compounds 1,21 2,21 and 35). Although these inhibitors have helped define how to inhibit FabH, two important questions need to be addressed. First, is the observed antibacterial activity via inhibition of FabH or some combination of nonspecific mechanism(s) seen with the general class of lipophilic acids? Second, given the lipophilic
ne strategy to develop effective treatments against the ever-increasing threat of drug-resistant bacteria is to identify and develop inhibitors of essential biological pathways that are not targeted by current therapies. The fatty acid biosynthesis (FAB) pathway has the potential to lead to clinically relevant antibacterial agents.1,2 In addition, the set of discrete enzymes involved in FAB are sufficiently different from the human fatty acid synthase complex to provide specificity for bacterial versus the human homologues, although specificity against mitochondrial FAB may prove a more significant challenge.3 However, the structural diversity across bacterial species presents a challenge to the development of a broadspectrum antimicrobial.4 Nie and co-workers5 have shown as much as a 1000-fold difference in potency between isozymes to an optimized small-molecule inhibitor, despite largely conserved protein sequences. FAB has been reported to be essential to bacterial growth,1 particularly in Gram-negative pathogens, due to the need to synthesize lipid A, which contains a 3-hydroxy fatty acid that is not available from the host environment.6,7 The essentiality of the FAB pathway, including FabH, in Gram-positive bacteria is an ongoing debate due to potential for supplementation of fatty acids from the environment.8,9 Natural product inhibitors of the FAB pathway include the FabF (β-ketoacyl-ACP synthase II) inhibitors platencin, © XXXX American Chemical Society
Received: March 31, 2016
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Figure 1. Compounds previously documented (1−3) or observed in this study (4−8), with inhibitory activity to FabH.
Figure 2. (a) E. coli FabH bound to coenzyme A (yellow carbons) overlaid with E. coli FabH bound to 1 (cyan carbons). (b) Structures of coenzyme A and compound 1.
acid nature of these compounds,20 can a suitable clinical candidate be developed with the appropriate in vivo profile? Herein, we report the initial results of a multipronged screening approach against Escherichia coli FabH, which, combined with structural biology and structure-based drug-design, led to the identification of a novel class of FabH inhibitors with good physicochemical properties and microbiological activity via FabH inhibition.
A carboxybenzoyl-protected amino acid 4 had a half-maximal inhibitory concentration (IC50) of 150 μM against E. coli FabH. Exploration of a variety of substituted phenyl rings (Table 1) showed nearly 20-fold improvement in biochemical potency by the introduction of an o-chloro substituent (9) or phydroxymethyl group (14), whereas other substitution patterns resulted in significantly less potent analogues (e.g., 10−12) with the 3-hydroxymethyl substitution showing a moderate potency increase (13). Alteration of the carbamate linkage to the more stable urea or amide linkers, or unsaturation of the cyclohexyl group to a phenyl ring all resulted in substantially less potent compounds, as did the introduction of simple carboxamides in place of the free acid (data not shown). Extension of the carboxylic acid by either methine (15) or methylene (16) units gave significant increases in potency against Staphylococcus aureus FabH, but with little change in Gram-negative potency. Combining all of these features in a single compound provided carbamate 17, which had a submicromolar IC50 against E. coli FabH, as well as increased potency against Haemophilus influenzae FabH, while retaining inhibition of S. aureus FabH. Activity against S. pneumoniae FabH by this chemical series was not observed at tested concentrations. The published structure of E. coli FabH with indole 1 bound22 (Figure 2) suggested that this preference for ortho-substituted phenyl groups may be due to binding near the acetyl end of acetyl-coenzyme A (site 1). Binding of this series of carbamates to FabH was demonstrated by 2D proteinobserved NMR with compound 9 (Supporting Information).
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RESULTS AND DISCUSSION Screening. Two AstraZeneca compound collections, both the standard high throughput screening (HTS) library and a more fragment-like high content recognition library (HCRL), were screened using an E. coli FabH scintillation proximity assay to find inhibitors of the Claisen condensation of biotinylated malonyl-acyl carrier protein with radiolabeled acetyl-CoA.21 A diverse set of chemotypes were represented in the observed hits as exemplified by compounds 4−8 (Figure 1). The available crystal structures of FabH bound to CoA4 and compound 121 (Figure 2) were used in docking studies to generate putative binding modes for each of these various chemotypes. Docking models suggesting a given chemotype could mimic CoA in some manner guided the selection of compounds for protein-observe NMR experiments for competition with CoA binding as a secondary confirmatory assay. Finally, X-ray cocrystal structures of E. coli FabH were obtained for a small subset of confirmed CoA-competitive inhibitors. B
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Table 1. Synthetic Inhibitors, Enzyme Potencya
a
Human plasma protein binding (ppb); E. coli (Eco); H. influenzae (Hin); S. pneumoniae (Spn); S. aureus (Sau); not determined (ND). C
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Figure 3. Compound 9 (a), compound 6 (b), compound 23 (c), and compound 36 (d) bound to E. coli FabH.
Figure 4. Fragment-linking rationale and synthesis of hybrid molecule 22.
When 15N-labeled E. coli FabH was treated with excess acetylCoA, there was significant ordering of the enzyme indicative of binding. A similar effect was observed when compound 9 was added to labeled FabH. Crystal structures of both 9 (Figure 3a) and 14 (not shown) bound to E. coli FabH showed that the majority of the compound occupies the binding site of the substrate acetylCoA.19 The carboxylic acid of 9 interacts with Arg36 and Arg249 (site 3), whereas the carbonyl of the carbamate moiety forms a water-mediated hydrogen bond with Asn247 (site 2).
The chloro group provides a hydrophobic interaction, and the carbamate is close to the side chain of Met207. Two series of biphenyl inhibitors were found. Biphenyl acid 5 showed moderate inhibition of all four isozymes tested (Table 1), with 4-fold variation in potency across isozymes. A few analogues of this acid were prepared, and the more lipophilic dichloro derivative 18 showed slight potency increases against most isozymes. The slightly smaller and less lipophilic o-fluoro-containing 19 showed somewhat weaker activity against all isozymes. Gram-negative isozyme inhibitory D
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Table 2. Hybrid Inhibitor Variation, Left-Hand Sides
s
Human plasma protein binding (ppb); E. coli (Eco); H. influenzae (Hin); S. pneumoniae (Spn); S. aureus (Sau); not determined (ND).
was merged to design novel inhibitors intended to have improved potency and broad-spectrum FabH inhibition while being straightforward to synthesize. The biaryl moiety with a mhydroxymethyl group on the left-hand side (site 1) was merged with a hydrogen-bond-forming central group (site 2) and acidic right-hand side (site 3) to engage the CoA-binding portion of the active site. The resulting hybrid molecule 22 was synthesized in three steps (Figure 4). Initial compounds inhibited all four FabH isozymes examined (Table 2) while retaining high solubility and acceptable plasma protein binding. Substitution of the left-hand side with fluoro in the ortho position was again advantageous and resulted in 23, which was cocrystallized with E. coli FabH (Figure 3c). This structure shows the intended interactions of the compound with the enzyme. Water-mediated hydrogen bonds include the pyridine nitrogen−water−Asn247 and the carboxylic acid−water−Arg36 bonds. The hydroxymethyl group interacts with the catalytic triad His244−Cys122−Asn274. The m-hydroxymethyl group gave greater potency than the p-hydroxymethyl group (24). Aryl groups lacking the hydroxymethyl group (compare 27 and 28) showed significantly reduced potency across all isozymes. The location of the o-fluoro substituent relative to the mhydroxymethyl group had little effect on potency (compare 23 and 25). The dimethyl analogue 28 increased inhibitory potency significantly. Replacement of the o-fluoro group with an o-chloro group (compare 23 and 29) increased potency against the S. pneumoniae isozyme while retaining potency against the other isozymes. Compound 29 was crystallized with S. pneumoniae FabH in an attempt to understand some of the differences present between isozymes (Figure 5a). Inhibitor 29
potency only was greatly improved by the introduction of a 5hydroxymethyl group in compound 20. The similar biphenyl sulfonamide 6 and analogues had similar SAR, suggesting that the biphenyl rings may bind in a similar fashion in both series. Co-crystal structures of E. coli FabH with the biphenyl sulfonamide 6 (Figure 3b), the hydroxymethyl phenylcontaining carbamate 14, and hydroxymethyl biphenyl acid 20 (structures not shown) showed similar orientations to these compounds, with little order in the phenyl sulfonamide region of 6 apparent in the structure. The biphenyl portion makes mostly liphophilic interactions, with the sulfonamide too far from the many arginines in the right side of the pocket to form favorable interactions. The hydroxymethyl group engages with the His244−Asn274−Cys112 catalytic triad1 in a complex of hydrogen bonding. As with other scaffolds, the o-fluorohydroxymethyl substitution 21 was slightly more potent than the hydroxymethyl substitution alone (Table 1). A crystal structure of 5 bound to E. coli FabH (not shown) showed that the position of the chlorophenyl group of 5 overlaid with the position of the chlorophenyl group of 9. The hydroxymethyl moiety of 20 bound to the catalytic triad in near superposition with the same group in sulfonamide 6. The hydroxymethyl group, binding to the catalytic triad, represented an opportunity to introduce polarity into the fatty acid portion of the binding pocket (site 1). This was considered advantageous for the development of small-molecule inhibitors of FabH with physical properties suited to support Gram-negative cell permeability. Hybrid Inhibitor Design. Information gathered from the structure−activity relationship of the scaffolds described above E
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Figure 5. (a) S. pneumoniae FabH (E. coli numbering) with compound 29. (b) S. pneumoniae FabH (cyan ribbons) with compound 29 (gold carbons) overlaid with E. coli FabH (green ribbons) with compound 23 (yellow carbons).
makes the same interactions with S. pneumoniae FabH (Figure 5a) as inhibitor 23 does with E. coli FabH in sites 1 and 2. The carboxylic acid appears to make interactions with Arg249 and Arg36, as observed with 9 (Figure 3a), displaying a different vector than observed with 23. Overlaying the two structures (Figure 5b) highlights the overall structural homology observed between FabH inhibitors. A variety of analogues involving alternate right-hand pieces were investigated (Table 3). Altering the 4-carboxy piperidine (23) to 3-carboxy piperidine(30) or 4-carboxymethyl piperidine (31) resulted in slight improvements in potency, particularly in S. aureus, possibly by improving the angles for the water-mediated hydrogen bond to Arg151 and by displacing the water to make a direct hydrogen bond to Arg36. Shrinking the ring to a carboxypyrrolidine (32) or removing the carboxylate group (30) was tolerated, albeit with reduced potency. Amides linked at the acid group either through carboxy or carboxymethyl (34−36) or through 4amino piperidines (37) were tolerated, as were sulfonamides off of the 4-amino piperidines (38), with the larger analogues even showing some improvements in Gram-positive potency. There were, however, no clear advantages to these modifications. Piperazine sulfonamides (e.g., 39), ureas, and amides all showed reduced potency (not all data shown). Whereas many of these analogues were relatively potent, there was no clear advantage gained by the larger substituents. In attempts to reach into the adenine portion of the binding site, the larger quinoline-bearing amide 36 was synthesized, which showed a significant increase in E. coli potency. A cocrystal structure of 36 in complex with E. coli FabH showed the quinoline ring stacked between Trp32 and Arg151 (Figure 3d), analogous to the adenine of acetyl-CoA (Figure 2), with minimal shifting of the remainder of the molecule relative to 23. The smaller and more soluble (138 μM vs 64 μg/mL in both a wild-type E. coli strain W3110 and in an isogenic efflux mutant strain of E. coli (ATCC 27325 ΔtolC::Tn10). Spontaneous resistant mutants of H. influenzae KM460 were isolated on growth medium containing inhibitory concentrations of compound 41. Seven of the mutants were characterized for their level of resistance relative to the parental MIC.23 In addition, DNA fragments containing the FabH and FabF gene, including 250 nucleotides upstream and downstream, were PCR-amplified from each mutant and were sequenced and analyzed for mutations relative to the parent sequence. The MICs for compound 41 were elevated 4−8-fold in the resistant mutants relative to the parent strain (Table 4), whereas the MICs for control inhibitor levofloxacin, which targets type II topoisomerases, was unaffected. DNA sequencing identified missense mutations in FabH for all seven H. influenzae mutants. Interestingly, FabF inhibitor platensimycin showed an increased MIC against one of these mutants. This may have been due to compensatory effects in fatty acid regulation, analogous24 to that seen by Rock and coworkers in S. aureus.25 In that study, resistance mutants to FabF F
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Table 3. Hybrid Inhibitor Variation, Right-Hand Sidea
a
Human plasma protein binding (ppb); E. coli (Eco); H. influenzae (Hin); S. pneumoniae (Spn); S. aureus (Sau); not determined (ND).
Table 4. Characterization of H. influenzae Mutants Resistant to Compound 41 MIC (μg/mL) (fold change) mutant strain KM460 (parent) Mut1a Mut1b Mut2 Mut3
no. of mutants 1 4 1 1
changed residuea
41b
platensimycin
levofloxacin
A215V A215S A245S S275I
8 32 (4×) 32−64 (4−8×) 64 (8×) 64 (8×)
4 32 (8×) NDc 4 4
0.004 0.002 ND 0.002 0.002
a
Location of missense mutation in the coding sequence of H. influenzae FabH. bMICs performed in duplicate on two separate occasions. cND, not determined.
retained sensitivity to FabF inhibitor platensimycin. In five of the seven mutants isolated, changes were located to Ala215 (Ala216 in E. coli numbering). The other two mutations were found at Ala245 (Ala246 in E. coli numbering) and Ser275 (Ser276 in E. coli numbering). Mapping of the changed amino acids onto the E. coli structure of 23 (Figure 2c) revealed that all of the mutations were located within close proximity to the
inhibitors were isolated, and all of the resulting mutants were found to contain single-point mutations, all of which resided in FabH. Several of those mutants were shown to be catalytically defective, with the resulting increase in malonyl-CoA likely protecting FabF from inhibition by raising the concentration of one of its substrates. Compound 41 yielded mutants that did not appear to be catalytically defective, as both Mut2 and Mut3 G
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active site catalytic triad (Cys112−His243−Asn273 in H. influenza numbering) (Figure 6). This result is consistent with the mode of action of these small molecules in H. influenzae being through FabH.
Letter
AUTHOR INFORMATION
Corresponding Author
*(D.C.M.) E-mail:
[email protected]. Present Address Δ
(D.C.M.) The Broad Institute, Cambridge, MA, USA. (C.J.E.) University of Cape Town, South Africa. (R-F.G.) Biogen idec, Cambridge, MA, USA. (J.H.) Shire Pharmaceuticals, Lexington, MA, USA. (S.L.K.) Third Rock Ventures, Boston, MA, USA. (S.D.L.) Macrolide Pharmaceuticals, Lexington, MA, USA. (A.R.M.) Moderna Therapeutics, Cambridge, MA, USA. (A.B.S.) Entasis Therapeutics, Waltham, MA, USA. (G.B.) Framingham State University, Framingham, MA, USA. Author Contributions
D.C.M., A.B.S., A.R.M., and G.B. drafted the manuscript. S.D.L. and S.L.K. performed crystallography work. R.-F.G. and A.B.S. performed the bulk of the biochemical screening. D.C.M., C.J.E., A.R.M., and G.B. designed compounds, D.C.M., G.B., and A.R.M. performed compound synthesis. J.H. provided NMR support for protein binding. All authors were involved in the review of the manuscript. Notes
The authors declare the following competing financial interest(s): The authors are current or former employees of AstraZeneca and may possess AstraZeneca stock and/or stock options.
Figure 6. E. coli FabH structure with compound 23 bound. Residues corresponding to those observed in H. influenzae resistant mutants are highlighted in red.
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In summary, screening for inhibitors of FabH identified a number of different chemotypes. Many of these were cocrystallized with E. coli FabH. By merging several chemical scaffolds with the aid of structure-guided design, a novel series was identified that demonstrated potent inhibition of FabH and had antibacterial activity against an efflux-deficient strain of H. influenzae. Mutants resistant to this series of compounds were isolated, all of which showed mutations in the FabH gene. This result is consistent with the mode of action being through FabH inhibition and supports the essentiality of the FAB pathway in H. influenzae in vitro. Further optimization of this scaffold, utilizing other portions of the substrate binding pocket highlighted by the crystal structures presented, such as the adenine pocket, provides opportunities to identify novel antimicrobial compounds that inhibit this step of the essential bacterial lipid metabolism pathway in different pathogens. These optimized inhibitors should be able to provide further insights into the continuing questions surrounding the potential for bacteria to circumvent the de novo synthesis of fatty acids by complementation from the environment. The coordinates and structure factors deposited into the Protein Data Bank are under the following codes: 4Z8D of E. coli FabH with compound 9, 5BNM of E. coli FabH with compound 6, 5BNR of E. coli FabH with compound 23, 5BNS of E. coli FabH with compound 36, 5BQS of S. pneumoniae FabH with compound 29.
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ACKNOWLEDGMENTS We thank Art Patten and Paul Fleming for their encouragement and support in data analysis and compound design, Beth Andrews for her work in assay development and execution, Oluyinka Green for her help in compound synthesis, and Kathy MacCormack for her isolation and characterization of resistant mutants.
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsinfecdis.6b00053. Full experimental details for synthesis of compound 23, description of protein 2D NMR work, descriptions of biological assays, and crystal structure tables and procedures (PDF) H
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(9) Parsons, J. B., Frank, M. W., Subramanian, C., Saenkham, P., and Rock, C. O. (2011) Metabolic basis for the differential susceptibility of Gram-positive pathogens to fatty acid synthesis inhibitors. Proc. Natl. Acad. Sci. U.S.A. 108, 15378−15383. (10) Peterson, R. M., Huang, T., Rudolf, J. D., Smanski, M. J., and Shen, B. (2014) Mechanisms of Self-Resistance in the Platensimycinand Platencin-Producing Streptomyces platensis MA7327 and MA7339 Strains. Chem. Biol. 21, 389−397. (11) Trajtenberg, F., Altabe, S., Larrieux, N., Ficarra, F., de Mendoza, D., Buschiazzo, A., and Schujman, G. E. (2014) Structural insights into bacterial resistance to cerulenin. FEBS J. 281, 2324−2338. (12) Yao, J., Maxwell, J. B., and Rock, C. O. (2013) Resistance to AFN-1252 Arises from Missense Mutations in Staphylococcus aureus Enoyl-acyl Carrier Protein Reductase (FabI). J. Biol. Chem. 288, 36261−36271. (13) Yao, J., Abdelrahman, Y. M., Robertson, R. M., Cox, J. V., Belland, R. J., White, S. W., and Rock, C. O. (2014) Type II Fatty Acid Synthesis Is Essential for the Replication of Chlamydia trachomatis. J. Biol. Chem. 289, 22365−22376. (14) McMurry, L. M., Oetinger, M., and Levy, S. B. (1998) Triclosan targets lipid synthesis. Nature 394, 531−532. (15) Baldock, C., de Boer, G. J., Rafferty, J. B., Stuitje, A. R., and Rice, D. W. (1998) Mechanism of action of diazaborines. Biochem. Pharmacol. 55, 1541−1550. (16) Park, H. S., Yoon, Y. M., Jung, S. J., Yun, I. N., Kim, C. M., Kim, J. M., and Kwak, J. H. (2007) CG400462, a new bacterial enoyl-acyl carrier protein reductase (FabI) inhibitor. Int. J. Antimicrob. Agents 30, 446−451. (17) Wang, Yi, and Ma, S. (2013) Recent Advances in Inhibitors of Bacterial Fatty Acid Synthesis Type II (FASII) System Enzymes as Potential Antibacterial Agents. ChemMedChem 8, 1589−1608. (18) Campbell, J. W., and Cronan, J. E., Jr. (2001) Bacterial Fatty Acid Biosynthesis: Targets for Antibacterial Drug Discovery. Annu. Rev. Microbiol. 55, 305−332. (19) Qiu, X., Janson, C. A., Smith, W. W., Head, M., Lonsdale, J., and Konstantinidis, A. K. (2001) Refined structures of beta-ketoacyl carrier protein synthase III. J. Mol. Biol. 307, 341−356. (20) Lu, S., Jessen, B., Strock, C., and Will, Y. (2012) The contribution of physicochemical properties to multiple in vitro cytotoxicity endpoints. Toxicol. In Vitro 26, 613−620. (21) He, X., Mueller, J. P., and Reynolds, K. A. (2000) Development of a scintillation proximity assay for beta-Ketoacyl-acyl carrier protein synthase III. Anal. Biochem. 282, 107−114. (22) Daines, R. A., Pendrak, I., Sham, K., Van Aller, G. S., Konstantinidis, A. K., Lonsdale, J. T., Janson, C. A., Qiu, X., Brandt, M., Khandekar, S. S., Silverman, C., and Head, M. S. (2003) First X-ray Cocrystal Structure of a Bacterial FabH Condensing Enzyme and a Small Molecule Inhibitor Achieved Using Rational Design and Homology Modeling. J. Med. Chem. 46, 5−8. (23) Buurman, E. T., Johnson, K. D., Kelly, R. K., and MacCormack, K. (2006) Different Modes of Action of Naphthyridones in GramPositive and Gram-Negative Bacteria. Antimicrob. Agents Chemother. 50, 385−387. (24) FabH mutations in S. aureus, e.g. (Sau-Ala240Val corresponding to Hin-Ala245 and Sau-Ala210Thr corresponding to Hin Ala215), were observed in mutants resistant to FabF inhibitors platencin, platensimycin, and thiolactomycin (16−32-fold). This resistance appears to be due to the regulatory function of FabH as it applies to FAB and general fatty acid regulation. The mutated FabH proteins were found to be significantly catalytically defective, and by impairing FabH, its substrate malonyl-ACP levels are likely raised, effectively out competing the FabF inhibitors.25 (25) Parsons, J. B., Yao, J., Frank, M. W., and Rock, C. O. (2015) FabH Mutations Confer Resistance to FabF-Directed Antibiotics in Staphylococcus aureus. Antimicrob. Agents Chemother. 59, 849−858.
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