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. 2021 Feb 15;12(3):433–442. doi: 10.1021/acsmedchemlett.0c00653

Semisynthesis and Biological Evaluation of Platencin Thioether Derivatives: Dual FabF and FabH Inhibitors against MRSA

Yuling Li †, Xiang Weng †, Youchao Deng †, Jian Pan †, Saibin Zhu †, Zhongqing Wen †, Yanqiu Yuan ‡, Shaowen Li §, Ben Shen ∥, Yanwen Duan †,⊥,#,*, Yong Huang †,#,*
PMCID: PMC7957939  PMID: 33738071

Abstract

graphic file with name ml0c00653_0007.jpg

The discovery and clinical use of multitarget monotherapeutic antibiotics is regarded as a promising approach to reduce the development of antibiotic resistance. Platencin (PTN), a potent natural antibiotic initially isolated from a soil actinomycete, targets both FabH and FabF, the initiation and elongation condensing enzymes for bacterial fatty acid biosynthesis. However, its further clinical development has been hampered by poor pharmacokinetics. Herein we report the semisynthesis and biological evaluation of platencin derivatives 1–15 with potent antibacterial activity against methicillin-resistant Staphylococcus aureus in vitro. Some of these PTN analogues showed similar yet distinct interactions with FabH and FabF, as shown by molecular docking, differential scanning fluorometry, and isothermal titration calorimetry. Compounds 3, 8, 10, and 14 were further evaluated in a mouse peritonitis model, among which 8 showed in vivo antibacterial activity comparable to that of PTN. Our results suggest that semisynthetic modification of PTN is a rapid route to obtain active PTN derivatives that might be further developed as promising antibiotics against drug-resistant major pathogens.

Keywords: Platencin, FabF, FabH, antibiotics, semisynthesis, differential scanning fluorometry, isothermal titration calorimetry

The emergence of antibiotic resistance in pathogenic bacteria has become a major threat to global public health. The World Health Organization has warned that common infections might lead to considerable high mortality and morbidity in a possible post-antibiotic era.1 It is encouraging that several antibiotics classified as new molecular entities, such as cefiderocol, eravacycline, and plazomicin, have been approved by U.S. Food and Drug Administration to treat bacterial infections in recent years.2 However, resistance to these newly approved antibiotics will likely develop in the future. Therefore, the discovery and development of multitarget monotherapeutic antibiotics is regarded as an effective approach to reduce the development of high-level resistance,3 as the probability that a resistant bacterium will develop missense mutations simultaneously in multiple targets is low.4

Several natural products are dual inhibitors against the rate-limiting enzymes FabF/FabB or FabH for bacterial fatty acid biosynthesis, such as thiolactomycin, cerulenin, and platencin (PTN) (Figures 1A and S1).5−9 PTN, isolated from Streptomyces platensis MA7339 collected in Spain, was a potent inhibitor against FabB/FabF and FabH, with IC50 values of 4.6 μM and 9.2 μM, respectively.8 It was shown to be effective against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and multidrug-resistant Mycobacterium tuberculosis (MIC = 1 μg/mL; racemic PTN was used).10 PTN was effective in a MRSA-infected mice peritonitis model when given by continuous infusion, suggesting its poor pharmacokinetics.8,11 In contrast to its natural analogue platensimycin (PTM) with a tetracyclic terpene-derived cage moiety, PTN consists of a tricyclic cage unit biosynthetically derived from an ent-atiserene precursor.12−14 Both PTN and PTM contain the same 3-amino-2,4-dihydroxybenzoic acid (ADHBA) moiety, which interacts with the critical active-site residues Cys-His-His (FabF) and Cys-Asn-His (FabH), as suggested by their cocrystal structures with Escherichia coli FabF(C163Q) and docking studies with E. coli FabH and FabF.9,12,16 Therefore, the difference of the terpene cage moieties of PTM and PTN contributes significantly to their target selectivity.

Figure 1.

Figure 1

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Strategy to modify the PTN terpene cage. (A) Structures of PTN (i), thioplatencin (ii), PTM (iii), thioplatensimycin (iv), platencin 3n (v), platencin A3 (vi), platencin A4 (vii), nor-platencin (viii), iso-platencin (ix), PTN sulfa-Michael adduct (x), and 6′-nitroplatensimycin (xi). (B) Retrosynthetic analysis of platencin-6-thioether derivatives.

Because of their unique structures, dual mode of action, and potent antibacterial activities, PTN and several of its derivatives have been isolated from producing strains or prepared through total synthesis and semisynthesis (Figures 1A, S1, and S2).14,17−22 In addition, we also obtained various PTN analogues from fermentation by pathway engineering, heterologous expression, and precursor-directed biosynthesis.23−26 However, compared with PTN, most of the PTN analogues showed drastically reduced antibacterial activities, except thioplatencin (ii), iso-platencin (ix), and the thioether adduct of PTN (x) (Figure S2 and Table S1).14,20,22 Compounds ii and x may form PTN in aqueous buffer through hydrolysis or β-elimination and are potential prodrugs to treat bacterial infections. Platencin A3 (vi) had a minimum inhibitory concentration (MIC) of 8–32 μg/mL against the tested MRSA strains, while more than 30 ADHBA-modified PTN analogues, such as compound v and 5′-chloroplatencin completely lost their antibiotic activities.16,20,24 Therefore, the structure–activity relationship (SAR) of the ADHBA moiety in PTN is quite tight, while modification of the cage unit might be a viable route to generate active PTN derivatives. The reason is that the ADHBA moiety closely interacts with the active-site amino acid residues of FabB/FabF or FabH, while most of the ketolide portion of PTN is exposed to the solvent.15

Here we report the semisynthesis and antibacterial activities of PTN derivatives 1–15 (Figure 2). Among the synthesized compounds, 8, 10, 13, and 14 showed antibacterial activity comparable to that of PTN against the tested S. aureus strains in vitro. Molecular docking of 1–15 with E. coli FabF(C163Q) and FabH revealed that they have similar yet distinct interactions with PTN, as suggested by differential scanning fluorometry (DSF) experiments. In contrast, isothermal titration calorimetry (ITC) showed that 1 and 15 had reduced binding with E. coli FabF(C163Q) in comparison with PTN, while 14 had a much weaker interaction. Our study not only supports the previous SAR study of PTN but presents an efficient strategy to prepare active PTN thioether analogues. In addition, our work shows that the method of determining the interactions of FabH and FabF with their inhibitors by DSF or ITC may be used to evaluate other potential inhibitors.

Figure 2.

Figure 2

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Synthesis of PTN derivatives. (A) Reaction of hydrogen peroxide with PTN to give 1. Conditions: 1–2 equiv of LiOH (2 M), tetrabutylammonium fluoride (TBAF) (catalytic amount), 30% H2O2/DCM (1:1); 90% yield from PTN. (B) Reaction of 1 with thiophenols or thiols to give 2–15. Conditions: R–SH (1.1 equiv), LiOH (2 M) to pH 9.

We previously prepared a focused library of PTM thioether derivatives through the synthesis of a key PTM oxirane intermediate.27 In the current study, we hypothesized that a similar PTN oxirane compound could be synthesized from PTN and further transformed to PTN thioether derivatives (Figure 1B).28 We first prepared PTN oxirane (1) from PTN in 90% yield using H2O2 (Figure 2A). The high regioselectivity of the oxidation reaction was likely due to the influence of the positions of the double bonds, with much higher reactivity at C6–C7 than that at the exo position (C15–C16) in PTN. Notably, 1 was obtained as a single isomer on the basis of its high-resolution mass spectrometry (HRMS) and 1D and 2D NMR data (Figures S3–S5). The extremely high stereoselectivity was likely due to the presence of the stereohindered PTN cage structure. We previously observed similar high stereoselectivity in the sulfa-Michael addition of thioacids or thiols to C7 of PTN, which was also supported by density functional theory calculations.22

Fourteen PTN thioether analogues were next prepared through thiolysis of 1 followed by dehydration under basic conditions (Figure 2B). Most of these analogues were obtained in moderate to high yields (46–94%). Compounds 2–15 contain various sulfur-containing substituents at C6 of PTN, including halogen-substituted phenyl groups, cyclohexane, and heterocycles (thiophene, furan, pyridine, pyrimidine) as well as a hydrophilic aliphatic alcohol (Figures S6–S19). When several aliphatic thiols (e.g., ethanethiol) were used as substrates, the corresponding sulfone derivatives were also observed and complicated the isolation of the thioether products. In addition, the reactions of the selected thiols or thiophenols with 1 were apparently slower than those in the previous PTM thioether synthesis.27 This is likely influenced by the less rigid structure of the PTN terpene cage compared with the PTM cage.

SwissADME, a popular tool to predict the physiochemical properties and pharmacokinetics of small molecules, was used to evaluate the properties of the newly synthesized PTN derivatives.29 Most of these PTN derivatives show increased flexibility and lipophilicity compared with PTN as a result of the newly introduced hydrophobic moiety (Figure S20). Compared with PTN, 1–15 have low gastrointestinal absorption and varying skin permeability, and most of them would be metabolized in the liver similarly (Table S2).

The antibacterial activities of 1–15 were determined against S. aureus ATCC 29213 as well as E. coli, methicillin-sensitive S. aureus (MSSA), and MRSA strains isolated from local hospitals using PTN, PTM, and linezolid as controls (Table 1). Several PTN analogues, including 3, 5, 8, 10, 13, and 14, had MICs of 1–2 μg/mL against the tested S. aureus strains. In contrast to many previous inactive PTN analogues (Table S1 and Figure S2), these compounds showed activities comparable to that of PTN against S. aureus. Most of the remaining PTN derivatives showed attenuated antibacterial activity against S. aureus, with MICs in the range of 4–16 μg/mL. Compounds 1–15 had no antibacterial activity against the Gram-negative pathogen E. coli when evaluated at 64 μg/mL. The data suggest that modification of C6 in PTN is an effective approach to generate active PTN analogues.

Table 1. MICs (in μg/mL) of PTN Derivatives against S. aureus ATCC 29213, Two Clinical MSSA and MRSA Strains, E. coli ΔtolC, E. faecalis ATCC 29212, MRSA in the Presence of 10% Human Serum, S. aureus FabH (A81V, A111T), and S. aureus FabH (A81V)30 (Data Are from at Least Two Independent Experiments).

compound S. aureus ATCC 29213a MSSAa MRSAa S. aureus FabH(A81V, A111T)b S. aureus FabH (A81V)b MRSA + serumb E. coli ΔtolCa E. faecalis ATCC 29212a
linezolid 0.5 0.5 0.5 –c – – – 0.5
polymyxin B – – – – – – 4 –
ampicillin – – – 32 16 – – –
PTN 1 1 1 32 >32 8 4 2
PTM 1 0.5 1 >32 >32 2 32 2
1 8 4 4 >32 >32 16 4 2
2 4 2 1 16 16 >32 >32 2
3 2 2 2 – – – – –
4 8 4 4 – – – – –
5 2 2 2 – – – – –
6 4 2 2 – – – – –
7 16 8 8 – – – – –
8 2 1 1 – – – – –
9 8 4 4 – – – – –
10 2 1 1 8 8 16 >32 2
11 16 8 8 – – – – –
12 4 1 1 4 8 16 >32 2
13 1 1 1 4 4 32 >32 2
14 2 1 1 4 8 16 >32 2
15 8 32 >32 >32 >32 >32 >32 –
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a

Plate dilution method.

b

Broth dilution method.

c

Not tested.

Next, the antibacterial activities of PTN derivatives 2, 10, and 12–15 were further evaluated against Enterococcus faecalis ATCC 29212, two S. aureus strains bearing point mutations in the FabH active site, the clinical MRSA strain with the addition of human serum, and the E. coli ΔtolC mutant lacking of the TolC efflux pump (Table 1).30,31 All of the tested PTN derivatives had the same MIC as PTN and PTM against E. faecalis ATCC 29212 (2 μg/mL). Interestingly, compounds 2, 10, and 12 – 14 were able to inhibit S. aureus strains bearing FabH mutations (e.g., A81V and A111T or A81V only) close to the substrate binding site (MIC = 4–16 μg/mL), in contrast to PTN and PTM (MIC ≥ 32 μg/mL) (Table 1).30 In addition, the tested PTN derivatives showed attenuated antibacterial activity against the tested MRSA strain in the presence of human serum, consistent with the previous report.8 PTN and 1 were active against E. coli ΔtolC, while the other tested PTN derivatives were inactive, suggesting more rapid efflux of these compounds by alternative efflux pumps.8 One alternative explanation is that the more hydrophobic and larger PTN derivatives may not be able to enter the treated E. coli through the water-filled channels provided by their outer membrane porins.

To investigate the detailed binding modes of 1–15, they were docked to E. coli FabF and FabH using the Molecular Operating Environment platform.32−34 The protein template ecFabF(C163Q) (PDB ID 2GFX) with PTM cocrystallization has been previously used for the docking of PTN.15 The template ecFabH (PDB ID 5BNR) was recently shown to cocrystallize with a designed hybrid inhibitor containing a biaryl moiety, a hydrogen-bond-forming group, and a carboxylic acid.35 One hundred docking iterations and force field optimization were performed for each compound.

Consistent with the previous docking experiments and the X-ray crystallographic study of PTN and platencin A1 with ecFabF(C163Q),15 compounds 1–15 were all docked into the active site of ecFabF(C163Q) (Figures 3A and S21). The carboxylic acid group of their ADHBA moiety interacts with His303 and His340, while the amide bond forms hydrogen-bonding interactions with Thr270 and Thr307 in the FabF active site.9 Compared with PTN, the ketolide portion of compounds 1–15 has lost some hydrophobic interactions with FabF, such as Ala205, Ala207, His268, or Ser271, probably because of the presence of the additional thioether moiety. However, several new interactions, such as a cation−π interaction at Arg206 in 2 and 4 as well as certain hydrophobic interactions at Asp227 in 3, 8, 9, 11, and 12, could also be observed in the docked structures.

Figure 3.

Figure 3

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Predicted docking modes of 8 in ecFabF and ecFabH. (A) Binding mode of 8 with ecFabF(C163Q). The 2D model shows interactions with amino acid residues in the enzyme active site, such as His303, His340, Thr270, and Thr307. (B) Binding mode of 8 with ecFabH. The 2D model shows that 8 interacts with various amino acid residues in the ecFabH active site, such as His244, Asn274, Cys112, Met207, Ala246, and Arg249.

Compounds 1–15 could also be docked to the active site of ecFabH (Figures 3B and S21). Extensive hydrogen-bonding interactions of the ADHBA moiety of these compounds with the conserved FabH active-site residues Cys112, His244, and Asn274 as well as Met207 were identified, while several of them, including 4, 9, 14, showed no hydrogen-bonding interaction with either His244 or Asn274 in ecFabH. A π-stacking interaction between Ala246 and the aminobenzoate as well as a hydrogen-bonding interaction between Arg249 and the carbonyl oxygen of the α,β-unsaturated ketone could also be observed in the docking of 8 and ecFabH. Interestingly, several new water-mediated long-range interactions of 6 and 14 with the amino acids in the entrance to the FabH active site could also be observed (Figure S21). Taken together, these results show that compounds 1–15 exhibit similar yet distinct binding modes with both ecFabF(C163Q) and ecFabH through these docking studies.

Differential scanning fluorometry (DSF) has been extensively used to study protein and small-molecule interactions because of the small amounts and low concentrations of protein required.36−41 In this assay, the presence of protein-interacting ligands affects the binding of a hydrophobic fluorescent dye with the denaturing protein at elevated temperatures, which results in a thermal shift (ΔTm) of the protein melting temperature (Tm). In general, addition of ligand molecules can enhance the stability of the protein, although there are reports that the stability of the protein is reduced with increasing ligand concentration.37

Therefore, we used DSF to further study the interaction of selected PTN derivatives with FabF or FabH. The overexpression and purification of S. aureus FabF and E. coli FabH were based on previous reports.42 We first identified the optimal DSF buffer for FabF or FabH by screening of seven different buffers using PTM, PTN, and previously synthesized inactive 6′-nitroplatensimycin (xi) as controls (Figures S22 and S23). Next, the interactions of compounds 1–14 with FabF or FabH were measured under the same assay conditions (Figure 4). Surprisingly, the addition of PTN and their derivatives led to decreases in Tm of FabF or FabH, suggesting that the protein–ligand interactions destabilize the proteins. Compounds 2–4 might have reduced binding toward FabH, while 1, 8, and 10 could interact with FabH more strongly than PTN. Compounds 12 and 13 might also interact more strongly with FabF than both PTN and PTM.

Figure 4.

Figure 4

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Biochemical analysis of the interactions of FabH and FabF proteins with PTN derivatives. (A–D, F–I) Melting temperature curves of (A–D) FabH and (F–I) FabF (n = 4). (E, J) ΔTm values for compounds 1–14 for (E) FabF and (J) FabH (E–J). FabF assay buffer: 20 mM HEPES, 180 mM NaCl, pH 7.5; FabH assay buffer: chosen 1.0 M Tris-HCl, pH 6.8.

Isothermal titration calorimetry (ITC) is a direct approach for studying ligand–receptor interactions by determining the heat generated from their combination.43,44 The enthalpy (ΔH), entropy (ΔS), Gibbs free energy (ΔG), binding constant (Kd), and stoichiometry (n) in the reaction system can be calculated. Therefore, we next evaluated the interaction of PTN and selected PTN derivatives 1, 14, and 15 with E. coli FabF(C163Q) using PTM and xi as controls (Figure 5). Both PTM and PTN bind to E. coli FabF(C163Q) strongly in a 1:1 ratio, with Kd values of 17.28 and 26.97 nM, respectively, while xi shows no detectable binding to E. coli FabF(C163Q). In contrast, 1 and 15 have weaker interactions with E. coli FabF(C163Q), with Kd values of 101.7 and 223.2 nM, consistent with their reduced MICs. Compound 14 might have only a weak interaction with E. coli FabF(C163Q), as a significantly larger amount of heat was generated than with xi (Figure S24). Taken together, these data suggest that 14 may interact more strongly with the other targeted enzymes, such as FabH.

Figure 5.

Figure 5

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Binding of PTN derivatives with E. coli FabF(C163Q) studied by isothermal titration calorimetry. PTM, PTN, 6′-nitroplatensimycin (xi), 1, 14, and 15 were dissolved in PBS buffer (10 mM, pH 7.4) and used to titrate E. coli FabF(C163Q) in the same buffer. The concentrations of the protein and the tested compounds were 12 or 14 μM and 250 μM, respectively. The experiments were performed at least three times, and one representative measurement was used.

Both FabF and FabH catalyze Claisen condensation to form the C16 fatty acid chain using acyl–acyl carrier proteins as substrates, whereas FabH requires acetyl-CoA as a primer through a ping-pong mechanism.42 PTM can bind only to the so-called “pong” state of transient acyl-FabF, as shown in the binding of PTM to ecFabF(C163Q), the mimic of the acyl-enzyme intermediate. On the basis of the X-ray structures of PTN with ecFabF(C163Q), Burkholderia vietnamiensis FabF, and Brucella melitensis FabB (PDB IDs 3HO2, 4F32, and 4JV3, respectively) as well as that of platencin A1 with ecFabF(C163Q) (PDB ID 3HO9), it is likely that these PTN derivatives may also interact with FabF or FabH.15,45,46 Interestingly, binding of PTN and 1–14 decreases the Tm of FabH and FabF, which suggests that the binding interactions destabilize both enzymes. It would be likely that they interact with the “pong” state of these two enzymes, which are in their high-energy transition states. S. aureus and E. coli mutants resistant to PTN have been previously isolated and contain several point mutations in the active sites of FabF in S. aureus and FabH in E. coli. These PTN thioether analogues showing variable binding modes with either FabF or FabH and increased antibacterial activity over PTN, such as 10–14, may be further developed as drug leads against pathogens with mutations in FabB/FabF or FabH active sites.

Subsequently, the in vivo antibacterial activities of 3, 8, 10, and 14 were evaluated in a mouse peritonitis model using PTN and vancomycin as controls (Figures 6, S26, and S27). C57BL/6J female mice were inoculated intraperitoneally with 1.5–2.0 × 107 colony-forming units (CFUs) of MRSA in combination with 5% w/v hog gastric mucin.27,33,47 The infected mice (n = 5 per group) were then intraperitoneally treated with 3 or 8 at 10 mg/kg 1 and 5 h after bacterial injection, with PTN (10 mg/kg) and vancomycin (10 mg/kg) as controls (Figures 6 and S26). None of the infected mice survived in the 3-treated group, while 80% of infected mice survived in the 8-treated group. All of the infected mice in the PTN-treated group survived. The bacterial loads in kidneys of mice treated with PTN and 8 were similar. When higher dosages of MRSA (2.5 × 107 CFUs) were used to infect the mice, most of the infected mice were not rescued by either PTN, 10, or 14 (Figure S27). These in vivo data suggest that compound 8 may have in vivo activity comparable to that of PTN.

Figure 6.

Figure 6

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In vivo antibacterial activities of 3 and 8 in a mouse peritonitis model. (A) General procedure for the mouse peritonitis model (n = 5 per group). (B) Survival rates of MRSA-infected mice after treatment with PTN, 3, 8 (10 mg/kg), or vancomycin (positive control, 10 mg/kg). (C) Weights of surviving mice in the period of 7 days. (D) Total bacterial loads in kidneys.

With the development of drug-resistant bacteria, it is urgent to develop new antibiotics with promising drug targets.1,3 In sum, the current study suggests that modification of C6 of PTN with a thioether group might be a useful strategy to generate active PTN analogues. Molecular docking revealed variation of the interactions of PTN analogues with FabF and FabH, which was further confirmed by newly developed DSF and ITC methods. However, although four of the PTN derivatives were evaluated in a MRSA-infected mouse peritonitis model, none of them were superior to PTN, which suggests that further optimization of the PTN scaffold or utilization of efficient drug delivery systems is needed to improve their in vivo efficacy. Considering the significant differences between bacteria and mammalian fatty acid biosynthetic enzymes,4−7 some of the dual targeting PTN thioether analogues need to be further developed as new antibacterial leads. The discovery and development of PTN derivatives may inspire future efforts to discover and develop new dual-targeting inhibitors against drug-resistant bacteria.

Acknowledgments

This work was supported in part by the National Natural Science Foundation of China (Grants 81473124 and 81530092) and the Chinese Ministry of Education 111 Project (B0803420). We are grateful to the Center for Advanced Research at CSU, including the NMR facility, for the NMR experiments.

Glossary

Abbreviations

PTN

platencin

PTM

platensimycin

FabF

ketoacyl-acyl carrier protein synthase II

FabH

ketoacyl-acyl carrier protein synthase III

ADHBA

3-amino-2,4-dihydroxybenzoic acid

MRSA

methicillin-resistant Staphylococcus aureus

MSSA

methicillin-sensitive Staphylococcus aureus

VRE

vancomycin-resistant Enterococci

SAR

structure–activity relationship

DSF

differential scanning fluorometry

TBAF

tetrabutylammonium fluoride

MIC

minimum inhibitory concentration

EtOAc

ethyl acetate

PE

petroleum ether

Tm

melting temperature

ΔTm

thermal shift

CFU

colony-forming unit

HCl

hydrochloric acid

ITC

isothermal titration calorimetry

ΔH

enthalpy

ΔS

entropy

ΔG

Gibbs free energy

Kd

binding constant

n

stoichiometry

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00653.

  • General experimental procedures, HRMS and NMR spectra of 1–15, molecular docking, DSF buffer screening, and raw ITC data (PDF)

Author Contributions

∇ Y.L. and X.W. contributed equally. Y.H. and Y. Duan conceived the project; Y.L., X.W., Y. Deng, and Z.W. performed the experiments; J.P., S.Z., Y.Y., and S.L. contributed reagents; Y.L. and Y.H. analyzed the data and wrote the manuscript with help from all of the coauthors.

All animal protocols were approved by the Animal Care and Use Committee of Central South University. All experimental procedures complied with the Guide for the Care and Use of Laboratory Animals (1996).

The authors declare no competing financial interest.

Supplementary Material

ml0c00653_si_001.pdf (8.7MB, pdf)

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