A Methyl 4-Oxo-4-phenylbut-2-enoate with in Vivo Activity against MRSA that Inhibits MenB in the Bacterial Menaquinone Biosynthesis Pathway

Abstract

4-Oxo-4-phenyl-but-2-enoates inhibit MenB, the 1,4-dihydroxyl-2-naphthoyl-CoA synthase in the bacterial menaquinone (MK) biosynthesis pathway, through the formation of an adduct with coenzyme A (CoA). Here, we show that the corresponding methyl butenoates have MIC values as low as 0.35–0.75 µg/mL against drug sensitive and resistant strains of Staphylococcus aureus. Mode of action studies on the most potent compound, methyl 4-(4-chlorophenyl)-4-oxobut-2-enoate (1), reveal that 1 is converted into the corresponding CoA adduct in S. aureus cells, and that this adduct binds to the S. aureus MenB (saMenB) with a K_d value of 2 µM. The antibacterial spectrum of 1 is limited to bacteria that utilize MK for respiration, and the activity of 1 can be complemented with exogenous MK or menadione. Finally, treatment of methicillin-resistant S. aureus (MRSA) with 1 results in the small colony variant phenotype and thus 1 phenocopies knockout of the menB gene. Taken together the data indicate that the antibacterial activity of 1 results from a specific effect on MK biosynthesis. We also evaluated the in vivo efficacy of 1 using two mouse models of MRSA infection. Notably, compound 1 increased survival in a systemic infection model and resulted in a dose-dependent decrease in bacterial load in a thigh infection model, validating MenB as a target for the development of new anti-MRSA candidates.

Introduction

There is an urgent need to identify new drug targets to combat the rising threat of antibiotic resistant bacteria including methicillin-resistant Staphylococcus aureus (MRSA), which the Center for Disease Control and Prevention (CDC) ranks as one of the top 10 “serious threats” in the United States.^1–4 S. aureus infection is the leading cause of nosocomial dermatitis, folliculitis, osteomyelitis, endocarditis and bacteremia worldwide,^5–9 and the cost of treating these infections is currently $20 billion per year.^{10, 11} Vancomycin is the drug of last resort for treating MRSA infection, but the advent of vancomycin-resistant strains of S. aureus in clinical settings,^{12, 13} mitigates the development of new chemotherapeutics to treat these highly dangerous strains.

One major hurdle to treating S. aureus infection is the ability of this organism to persist in a slow growth small colony variant (SCV) phenotype. Although many bacteria are known to form SCVs as a natural survival mechanism,^14–17 S. aureus SCVs hamper rapid clinical diagnosis and complicate routine laboratory tests, often leading to misidentification and ultimately misdiagnosis. Furthermore, due to the slow growth characteristics of S. aureus SCVs, clinical studies have shown that they are highly drug resistant, less prone to eliciting strong immune responses, and are a source of persistent infection.^18–22 Thus, it is important to treat S. aureus infection with therapeutic agents at drug concentrations where SCVs are not induced,²³ and to develop antimicrobial agents that act specifically on SCVs. The SCV phenotype can arise from genetic and metabolic defects,^24–26 environmental pressure,^{27, 28} and metabolite auxotrophism.^{25, 29, 30} Three well defined SCV auxotrophisms have been identified in which wild-type growth can be restored by supplementation with thymidine,³¹ CO₂,^{27, 32} and molecules that complement deficiencies in electron-transport.^33–35 Typically, for electron-transport deficient SCVs, the wild-type phenotype can be restored by supplementation with menaquinone (MK, Vitamin K2) or menadione (MD, Vitamin K3).^{34, 36} In some cases, this auxotrophism has been directly linked to mutations in enzymes that constitute the MK biosynthesis pathway.^{34, 36, 37}

S. aureus and other Gram-positive bacteria utilize MK during oxidative and fermentative respiration as an electron carrier in the electron transport chain (Figure S1).^{38, 39} The classical de novo MK biosynthesis pathway consists of at least nine distinct enzymes (Scheme 1), and is absent in humans, making MK biosynthesis an attractive therapeutic target. Enzymes in this pathway have been implicated in the survival and virulence of many human pathogens, and several classes of inhibitors have been developed to interrogate the pathway as a prelude to novel antibacterial discovery.^40–44 In our own studies, we have undertaken detailed characterization and inhibition studies of several enzymes in the pathway including MenB, the 1,4-dihydroxynaphthol-CoA synthase. Mutations in MenB have been shown to correlate with S. aureus strains that are auxotrophic for menadione,³⁶ and genetic approaches have demonstrated that MenB is essential for growth of Mycobacterium tuberculosis.⁴⁵

Scheme 1.

The classical pathway for menaquinone (MK) biosynthesis in organisms such as S. aureus, E. coli, and M. tuberculosis showing the reaction catalyzed by MenB. Original details of this pathway are summarized in Meganathan, 2001.⁷⁰ More recently, it has been shown that MenH converts SEPHCHC to SHCHC (SHCHC synthase).⁷¹ In addition, a dedicated DHNA-CoA thioesterase (MenI) has been identified in phylloquinone (vitamin K1) biosynthesis,⁷² and a bacterial homolog of this enzyme has potentially been identified in E. coli⁷³ The classical pathway is distinct from the pathway that proceeds via futalosine in Streptomyces⁷⁴ Also shown is the non-enzymatic decomposition of OSB-CoA to spirodilactone.

MenB is the sixth enzyme in the MK pathway and catalyzes an intramolecular Claisen condensation (Dieckmann) reaction leading to the formation of 1,4-dihydroxynaphthoyl-CoA (DHNA-CoA).⁴⁶ We previously conducted a high throughput screen on MenB from M. tuberculosis (mtMenB) and identified several classes of mtMenB inhibitors including the 4-oxo-4-phenyl-2-amino butanoates.⁴¹ We subsequently demonstrated that elimination of the 2-amino substituent occurs in aqueous solution to generate the corresponding butenoate which reacts with CoA to form an adduct that inhibits MenB (Figure S2).^46–49 We also reported that while the butanoate CoA adducts were potent mtMenB inhibitors in vitro (K_i 50 nM), the corresponding butenoic acids were poor bacterial growth inhibitors (MIC 25 µg/mL against H37Rv). In contrast, the corresponding methyl butenoates were found to have promising MICs (MIC < 1 µg/mL H37Rv), suggesting that protection of the carboxylate aids cell penetration. We now extend these studies to probe their mode of action in S. aureus. We first demonstrate that the methyl 4-oxo-4-phenyl-2-butenoates are active against both drug sensitive S. aureus (MSSA) and MRSA in vitro, and that their spectrum of activity extends to other bacteria that synthesize MK. We subsequently show that treatment of MRSA cells with the most potent methyl butenoate (1) leads to formation of the corresponding CoA adduct, and results in the SCV growth phenotype, thus phenocopying knockout of the menB gene. In addition, we demonstrate that compound 1 reduces levels of both MK as well as DHNA, a downstream product of MenB, and that the antibacterial activity of 1 can be rescued by supplementing with exogenous MK, supporting the proposed mode of action. Finally, we find that the most potent methyl ester conjugate is active against MRSA in mouse models where it can prolong survival time and reduces bacterial load.

Results and Discussion

Methyl 4-oxo-4-(4-chlorophenyl)-2-butenoate (1) has potent activity against S. aureus

Previously we identified a series of 4-oxo-4-phenyl-2-butenoate inhibitors of the MenB from M. tuberculosis and demonstrated that the corresponding methyl esters had potent antibacterial activity against replicating and non-replicating M. tuberculosis (0.6–1.5 µg/ml).⁴¹ To explore the activity and mode of action of these compounds in S. aureus, we first evaluated the whole cell antibacterial activity of several methyl butenoates against methicillin sensitive and resistant S. aureus (MSSA and MRSA, respectively). The minimum inhibitory concentrations (MIC) are summarized in Table 1 where it can be seen that the 4-chloro analog 1 has the most promising antibacterial activity with MIC values of 0.35 and 0.75 µg/mL against MSSA and MRSA, respectively. We subsequently focused our efforts on interrogating the mode of action of 1 in S. aureus.

Table 1.

In vitro antibacterial activity of the 4-oxo-4-phenyl-2-butenoic acids and methyl esters against S. aureus.

Compound	Structure	R	MIC (µg/mL)a
			MSSAb	MRSAb
1		4-Cl	0.35	0.75
2		4-F	8	12
3		4-Br	8	16
4		4-NO2	4	4
5		2,4-Cl	1	2
6		4-Cl	> 60	> 60
Oxacillin			0.1	>128
Vancomycin			2	4

^aModal value of experimental triplicates.

^bMSSA, methicillin-sensitive S. aureus; MRSA, methicillin-resistant S. aureus. Oxacillin and vancomycin were used as controls.

1 is a Specific Inhibitor for MK Utilizing Bacteria

Bacteria generally utilize structural analogs of either ubiquinone (Q) or MK as the lipid soluble redox active electron carrier in the electron transport chain. To explore the spectrum of antibacterial activity, we thus selected bacteria that use either Q or MK for respiration. The Gram-positive bacteria Bacillus subtilis and Enterococcus faecium, as well as Mycobacterium smegmatis and the Gram-negative bacterium Haemophilus influenza, use MK when grown either aerobically or anaerobically. Conversely, Pseudomonas aeruginosa uses Q under all growth conditions whereas the Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis use Q under aerobic growth conditions but switch to MK when grown anaerobically (Table 2). Antibacterial assays revealed that 1 was only active under growth conditions where MK was the primary electron carrier. Thus, 1 was active against B. subtilis, E. faecium and M. smegmatis, under aerobic, capnophilic, and anaerobic growth conditions, consistent with our previous work where 1 demonstrated MIC values of 0.64 and 1.5 µg/mL against M. tuberculosis grown aerobically or in the low oxygen recovery assay, respectively.⁴¹ In addition, whereas compound 1 showed no activity towards E. coli, P. mirabilis and K. pneumoniae under aerobic growth conditions, MIC values of 12.5 µg/mL (E. coli and P. mirabilis) and 62.5 µg/mL (K. pneumoniae) were observed under anaerobic conditions. Compound 1 also demonstrated activity towards H. influenza grown under capnophilic conditions (MIC 0.25 µg/mL) but showed no activity towards P. aeruginosa under either aerobic, capnophilic, or anaerobic conditions. Complementation studies were performed by supplementing the media with 10 µM MK-4 or MD, and in organisms for which 1 displayed an MIC, addition of MK-4 resulted in an increase in the MIC value to > 60 µg/mL.

Table 2.

Antibacterial spectrum of 1 in menaquinone (MK) and ubiquinone (Q) producing human pathogens.

Pathogen	Electron carriera	MIC aerobic (µg/ml)	MIC capnophilic (µg/ml)b	MIC anaerobic (µg/ml)b	+ MK-4c
MRSA	MK	0.75	0.37	0.37	> 60
B. subtilis	MK	8	4	2	> 60
E. faecium	MK	4	2.5	1.25	31.5
E. coli	Q / MK	> 60	15.5	15.5	> 60
P. aeruginosa d	Q	> 60	> 60	> 60
H. influenzae	dMK / MK		0.25		> 60
K. pneumoniae d	Q / MK	> 60	> 60	62.5	> 60
P. mirabilis d	Q / MK	> 60	12.5	12.5	31.5
M. smegmatis	MK	0.5	0.25		> 60
M. tuberculosis e	MK	0.64	1.5

MIC values are the modal values of technical and experimental triplicates.

^aQ, ubiquinone; MK, menaquinone; dMK, demethylMK (Scheme 1).

^bMICs determined under capnophilic (candle) or anaerobic (GasPak) conditions by culturing bacteria in a candle jar (see Materials and Methods section for complete protocol).

^cMIC value obtained following supplementation with 10 µM commercially available menaquinone-4 (MK-4).

^dActivity of 1 against P. aeruginosa, K. pneumoniae, and P. mirabilis was determined in the presence or absence (no change in MIC) of ½ MIC polymyxin B under aerobic conditions.

^eMIC values reported in Li, X., et al. ACS Med Chem Lett, 2011 under aerobic and low oxygen (LORA) conditions.

Although these studies provide strong support that the antibacterial activity of 1 results from a direct effect on MK biosynthesis, we speculated that if 1 did have additional target(s) unrelated to MK biosynthesis, then the lack of activity of 1 against the Gram-negative bacteria that were grown aerobically (i.e. where they utilize Q and not MK) might be due to an inability of the compound to penetrate the bacteria. Thus, we evaluated the activity of 1 against P. aeruginosa, K. pneumoniae, and P. mirabilis under aerobic conditions in the presence of ½ MIC polymyxin B to improve cell permeability.^{50, 51} However, this did not alter the whole cell activity of 1, suggesting that the lack of activity was not caused by uptake issues.

1 Reduces MK level in MRSA

S. aureus produces an array of MK species that differ in the length of the isoprenyl chain attached to the naphthoquinone nucleus. In agreement with previous studies, MK-8 (8 isoprenyl units) was found to be the most abundant MK in S. aureus (Figure S3).^{24, 38, 52, 53} In order to analyze the impact of 1 on MK levels in growing cells, the change in MK-8 by treatment of MRSA with 1 was quantified by tandem mass spectrometry using commercially available MK-9 as a standard (Figure 1). Treatment of MRSA cells with increasing concentrations of 1 (1/10, 1/5, 1/3, and ½ MIC) resulted in a dose dependent decrease in MK-8 (Figure 1A). In addition, whereas ½ MIC of 1 (0.375 µg/mL) reduced the relative amount of MK-8 by 75%, ½ MIC of vancomycin (2 µg/mL) only resulted in a ~10% change, again consistent with a specific effect of 1 on MK biosynthesis (Figure 1B).

Figure 1. Menaquinone-8 (MK-8) quantification in MRSA cells treated with 1.

(a) Change in MK-8 levels determined in MRSA treated with sub-MICs (1/10, 1/5, 1/3, and ½ MIC) of 1 (MIC = 0.75 µg/mL). A 75% decrease in MK-8 is seen at ½ MIC. (b) MRSA treated with vancomycin (2 µg/mL) and 1 (0.38 µg/mL). Treatment with vancomycin and 1 resulted in a 10% and 75% decrease in MK-8 levels, respectively. Ctrl (control) are untreated cells. *p < 0.01.

1 Forms a CoA Adduct in MRSA Treated Cells

Previously, we reported that the 4-oxo-4-phenyl-2-butenoates form a CoA adduct in vitro which is responsible for the inhibition of MenB.⁴¹ We thus speculated that the antibacterial activity of 1 results from the formation of this CoA adduct (8) (Table 3) in cells where it may inhibit MenB. Using high resolution LC/UV/MS we could demonstrate the accumulation of 8 (calculated m/z = 991.1392) in treated cells with peaks corresponding to [M-H]⁻ = 990.1315 and [M-2H]²⁻ = 494.5608 (Figure 2). Isotopic distribution peaks corresponding to the 2 m/z increase due to the presence of Cl in the molecule were also observed: [M-H]⁻ = 990.1315 (³⁵Cl) & 992.1300 (³⁷Cl) (Figure 2 inset, Table S1), thus clearly indicating the formation of 8 from treatment of 1 in MRSA cells.

Table 3.

Thermodynamic Constants for the Binding of CoA and the CoA Adducts of 1 and 6 to saMenB

Compound	IC50 (µM)a	Kd (µM)b	ΔΔGc (kcal/mol)
CoA		25 ± 4
	4.2 ± 1.6	2.8 ± 0.2	1.3
	7.2 ± 3.0	2.8 ± 0.7	1.3

^aIC₅₀ values were determined using the MenE-MenB coupled assay where MenB was rate limiting. In contrast, no inhibition was observed with concentrations of up to 50 µM of 7 or 8 when MenE was rate limiting in the coupled assay.

^bK_d values for the interaction of compounds with saMenB were determined using isothermal titration calorimetry (ITC). In contrast, no binding could be detected with up to 200 µM of 7 or 8 when ITC was used to analyze the interaction of the compounds with MenE.

^cΔΔG was determined by calculating ΔG of compound binding vs ΔG of CoA binding (see Table S2 for complete thermodynamic values). All IC₅₀ measurements were performed in triplicate and ITC experiments were performed in duplicate.

Figure 2. Formation of CoA Adduct 8 in MRSA Cells Treated with 1.

High Resolution LC/UV/MS spectrum of an extract from MRSA cells treated with 8 µg/mL of 1. Found-by-Formula (FBF) search clearly indicates the presence of 8, which is the CoA adduct of 1 (m/z = 991.1329) but not of 7, which is the CoA adduct of the corresponding acid (6) (m/z = 977.1235). Observed peaks correspond to 8 [M-H] = 990.1315 and [M-2H]⁻² = 494.5608. The inset (red and green box) shows isotopic distribution of ³⁵Cl/³⁷Cl present in 8 with calculated relative abundance of m/z ions = 100, 39, 48, and 16 % respectively among the 4 peaks shown for [M-H] (see Table S1 for complete analysis of theoretical and observed isotopic distribution). Y-axis is relative intensity and x-axis is Counts vs. Mass-to-Charge (m/z).

Inhibition of saMenB by 8

Although the presence of 8 in MRSA treated cells was not unsurprising, we initially hypothesized that the active species would be the acid form 7 since this is a potent inhibitor of mtMenB (Figure S2). This hypothesis stemmed from the knowledge that esterification is used as a strategy to protect free-carboxylates to facilitate cell penetration after which hydrolysis by endogenous esterases liberates the active pharmacophore.^{54, 55} However, using high resolution LC/UV/MS, we were unable to detect 7 in cell extracts (Figure 2). We thus explored the ability of 8 and the corresponding acid (7) to bind to and inhibit saMenB in vitro. Using the MenE-MenB coupled assay,⁵⁶ we discovered that both 7 and 8 inhibit saMenB with similar IC₅₀ values (4–7 µM, Table 3). In addition we also used ITC to quantify binding of 7 and 8 to saMenB. The data shown in Figure S4 were fit to a one-site binding model yielding K_d values of 2.8 µM for each ligand (Table 3). In comparison, CoA has a K_d value of 25 µM for saMenB showing that the acyl group provides an additional 1.3 kcal/mol in binding energy equivalent to the formation of one hydrogen bond between the acyl group and the enzyme (ΔΔG = 1.3 kcal/mol) (Table S2). Since the inhibition of saMenB was assessed using a coupled assay which involved generation of the MenB substrate using MenE, we also verified that 7 and 8 did not bind to nor inhibit MenE (Table 3).

1 Reduces the Level of DHNA in MRSA Cells

To further probe if 1 directly targets saMenB in MRSA, we posited that the substrate of MenB, OSB-CoA, would accumulate in treated cells. The mass spectrum of OSB-CoA has peaks at [M-H]⁻ = 970.1492 and [M-2H]²⁻ = 484.5686 (Figure S5). However, using this as a standard, we were not able to detect OSB-CoA in extracts from untreated cells or those treated with 1. We posited that our inability to detect OSB-CoA might be related to the relative instability of this metabolite which can undergo a non-enzymatic rearrangement leading to the formation of spirodilactone (Scheme 1). However, we were also unable to detect spirodilactone in either treated or untreated cells. We then searched for the downstream product of MenB and determined that levels of DHNA were substantially reduced in MRSA cells treated with 1 (Figure 3). Commercially available DHNA was used as a standard for method development and MS detection. The retention time of DHNA was determined to be 12.22 min with a [M+H]⁺ of 205.0499. DHNA undergoes facile MS fragmentation with the loss of CO₂ to give a m/z =159.0443 ion, and also yields ions with m/z =227.0321 ([M+Na]⁺), m/z=131.0493 (loss of CO) and 103.0546 (loss of CO) (Figure S6). Extracts from untreated cells were shown to contain DHNA (retention time 12.26 min) (Figure 3A) characterized by ESI⁺ peaks at m/z=205.0499 ([M+H]⁺), 159.0439, 131.049 and 103.055 (Figure 3B). However, DHNA could not be detected in cells treated with 1 (Figure 3C), supporting a specific effect of 1 on saMenB.

Figure 3. Effect of 1 on the Level of DHNA in MRSA cells.

High resolution LC/UV/MS spectra of untreated cells and cells treated with 8 µg/mL of 1. Commercially available DHNA has a retention time of 12.22 min with [M+H]⁺= 205.0499 and also m/z = 227.0321, [M+Na]⁺, m/z =159.0443 (loss of CO₂), m/z=131.0493 (CO loss) and 103.0546 (CO loss) (see Figure S6 for analysis of commercially available DHNA). (A) In untreated cells, a peak is observed with a retention time of 12.26 min. (B) The mass spectrum of the peak 12.26 min is consistent with DHNA: ESI⁺ = 205.0499, 159.0439, 131.049 and 103.055. (C) Using the same protocol, no evidence of DHNA is seen in cells treated with 1.

MRSA Treated with 1 Forms SCVs and Phenocopies Genetic Disruption of menB

Previous reports have shown that genetic defects in the menB gene as well as knockout/knockdown of MenB result in the SCV phenotype, and that supplementation with exogenous MK-4 or MD can restore the wild-type phenotype.^{24, 36} We thus investigated the morphology of MRSA colonies grown on tryptic soy agar (TSA) plates in the presence or absence of 1 (Figure 4). The agar MIC of 1 was found to be 8 µg/mL, which was higher than the broth MIC, a difference often observed for antibacterial compounds.⁵⁷ Growth of MRSA on TSA containing 4 µg/mL of 1 (½ agar MIC) showed no colonies after 24 hr, in contrast to cells grown in the absence of 1, while small colonies appeared in the presence of 1 after 48 hr, consistent with the SCV phenotype. Addition of 10 µM MK-4 to the TSA plates together with 16 µg/mL 1 (2x agar-MIC) showed wild-type growth, indicating that MK-4 can abrogate the antibacterial activity of 1. Figure 4 also contains data on the growth of a menB^−/− strain,²⁴ showing formation of small, pigmentless, slow-growing colonies after 48 hr. Thus, treatment of MRSA with sub-MIC concentrations of 1 phenocopies disruption of the menB gene.

Figure 4. SCV formation following treatment of MRSA cells with 1 or by knocking out menB.

A) The normal growth phenotype of wt MRSA cells showing defined individual colonies after 24 hr at 37 °C. B) MRSA plated on TSA containing 4 µg/mL 1 and incubated for 48 hr shows SCV morphology that leads to overgrowth after 72 hr (C). D) MRSA plated on TSA containing 16 µg/mL 1 (2x MIC) and 10 µg/mL MK-4. This plate shows over growth after 24 hr incubation, indicating that MK complements the antibacterial activity of 1. E) and F) show no growth even after 72 hr incubation on TSA with 8 µg/mL 1, making this concentration the agar-MIC. G) and H) compares S. aureus growth upon knockout of the menB gene.²⁴ Consistent with other studies, the menB^−/− strain²⁴ forms SCVs characterized by loss of pigmentation and small/slow growing colonies.

Complementation of defects in MK biosynthesis by MK-4 or MD has been hypothesized to occur either by directly replacing MK in the electron transport chain or by acting as substrates for the de novo synthesis of MK. To explore these possibilities, we analyzed the MK distribution in MRSA cells treated with 1 and supplemented with either MK-4 or MD (Figure 5). Although MK-4 supplementation did not lead to recovery of the natural MK distribution, addition of MD to the media resulted in a distribution of MK species that closely resembled that observed in the absence of 1 (Figure 5). Thus for the first time, we can conclude that MD can be used as a precursor for the synthesis of MK by enzyme(s) that are downstream of MenB, possibly as a substrate for MenA which adds the isoprenyl side chain to the naphthoquinone ring. MK-4 in contrast can be directly used in oxidative respiration.

Figure 5.

Menaquinone (MK) quantification in MRSA cells treated with 1 and supplemented with menaquinone-4 (MK-4) or menadione (MD). Untreated cells show a distribution of MKs that vary in the length of the polyisoprene chain and in which MK-8 is the dominant species. Treatment with 1 (0.38 µg/mL) results in a decrease in all MK species. Cells supplemented with MD (10 µM) but not MK-4 (10 µM) recover the MK distribution observed in untreated cells. This indicates that MD may act as a substrate for MK biosynthesis downstream of the enzyme inhibited by 1. Presumably MK4 directly replaces the function of MK-8 and the other major MK species in the electron transport chain. Since MK-4 and MD were supplemented at 10 µM, the levels of these molecules were > 400% relative to MK-8 levels in untreated cells.

1 Shows Promising Antibacterial Activity Against MRSA in Vivo

We evaluated the antibacterial activity of 1 in both systemic and thigh MRSA infection mouse models (Figure 6). Oxacillin served as a negative control for both models since this drug does not have antibacterial activity against MRSA in vitro, whereas vancomycin was used as a positive control (Table 1). In the systemic infection group, control animals which received only vehicle or oxacillin died within two days post-infection with an average survival rate of 1.5–1.6 days. In contrast, vancomycin increased the survival rate up to 5.8 days post infection. Promisingly, treatment with 100 mg/kg of 1 resulted in a survival rate of 4.6 days (p < 0.005) (Figure 6A). In addition, increasing concentrations of 1 resulted in a dose dependent reduction in bacterial burden in the thigh model of infection, with 100 mg/kg reducing bacterial load by 3.1 log CFU/gram tissue (Figure 6B). This contrasted with only a minimal effect of oxacillin on bacterial load, whereas vancomycin caused a reduction of 4.2 log CFU/gram of tissue. Thus, 1 demonstrates promising in vivo efficacy using both systemic and thigh models of MRSA infection.

Figure 6. In vivo antibacterial efficacy of compound 1 against MRSA.

(a) Survival of infected mice treated with vehicle (●, n=5), oxacillin (100 mg/kg, ■, n=5), compound 1 (100 mg/kg, , n=10) or vancomycin (100 mg/kg, , n=10). (b) Decrease of bacterial burden in thigh muscle after treatment with vehicle (n=3), oxacillin (Oxa, 100 mg/kg, n=3), compound 1 (25, 50 and 100 mg/kg, n=5), and vancomycin (Vanc, 100 mg/kg, n=3). Statistical significance was determined using a one tail T-test where * p < 0.05 and ** p < 0.005.

Outlook and Conclusions

Previously we performed an HTS to identify inhibitors of MenB, the 1,4-dihydroxynaphthoyl-CoA synthetase in the bacterial MK pathway. This HTS yielded several classes of inhibitors including a series of 2-amino-4-oxo-4-phenylbutanoates. We subsequently demonstrated that the 2-aminobutanoates eliminate the amino group, yielding the corresponding butenoate which then reacts with CoA to form an adduct that inhibits MenB. Although the butenoic acids have poor antibacterial activity, the methyl ester derivatives have potent activity, suggesting that the lack of activity of the butenoic acids results from poor penetration into the cell. In the present work, we have expanded our studies to explore the mode of action of the methyl butenoates in S. aureus. Methyl 4-oxo-4-(4-chlorophenyl)-2-butenoate 1 has potent activity against the human pathogen M. tuberculosis (MIC 0.64 µg/mL). Here, we show that 1 also has potent antibacterial activity against MSSA and MRSA, as well as other MK-utilizing bacteria. We also provide compelling evidence that the activity of 1 results from a direct effect on MK biosynthesis through inhibition of saMenB (Figure 7). We show that 1 can penetrate into MRSA cells and form a CoA adduct that is an inhibitor of saMenB. Treatment with 1 depletes DHNA levels, reduces MK, and ultimately results in the formation of the SCV growth phenotype, thus phenocopying a menB^−/− knockout strain.²⁴ The growth defects caused by 1 can be recovered by addition of MK-4 or MD to the media, and we demonstrate that complementation with MD results from conversion of this compound into MK. Finally, given the potent in vitro activity of 1, we evaluated the in vivo efficacy of this compound in two mouse models of MRSA infection and show that 1 increases survival time and reduces bacterial load. These studies thus validate the MK biosynthesis pathway as a target for the development of novel antibacterial agents.

Figure 7.

Proposed mode of action of the 4-oxo-4-phenyl-2-butenoyl methyl esters. Upon penetration into the cell, the corresponding CoA adduct is formed, leading to inhibition of MenB and a decrease in the levels of DHNA and MK.

Whilst 1 is a useful chemical tool for exploring the role of MenB and MK biosynthesis in bacterial growth and survival, it is likely that 1 also reacts with other nucleophiles in the cell. Improvements in the selectivity of compounds derived from 1 can be envisaged based on elegant approaches that have been applied to the generation of selective covalent kinase inhibitors including compounds that target Bruton’s tyrosine kinase (BTK)⁵⁸ such as ibrutinib,⁵⁹ and CC-292,⁶⁰ as well as the reversible covalent cyanoacrylamides developed by Taunton and co-workers.^{61, 62} In this regard, the observation that CoA can bind to MenB suggests that modifications that reduce the electrophilicity of the Michael acceptor whilst increasing the affinity of 1 for the enzyme might lead to compounds that inhibit MenB through the formation of the CoA adduct on the enzyme. In addition, it is known that variation in the substitution of the phenyl ring in the 4-oxo-4-phenyl-2-amino butanoates alters the rate at which the amine is eliminated from these compounds,⁴¹ and such changes are also envisaged to alter the stability of the corresponding thiol adducts. Thus, the reversible covalent formation of CoA adducts that are stabilized by MenB might also provide a strategy for improving the selectivity of molecules that target MenB.

Materials and Methods

Compounds

Chemicals were obtained from commercial vendors and used without further purification. The compounds tested for activity were available from previous studies.⁴¹ The CoA adduct of methyl 4-oxo-4-(4-chlorophenyl)-2-butenoate (8) was prepared as follows: a solution of CoA (0.1 mmol) in water (1.5 mL) was added to a solution of methyl 4-oxo-4-(4-chlorophenyl)-2-butenoate (0.5 mmol) in anhydrous DMSO (1 mL) at RT. The resulting mixture was stirred at RT for 4 hr after which the reaction was quenched by addition of formic acid (0.1%, 0.5 mL). The product was purified by HPLC using a Shimadzu 20AB instrument with a Phenomenex C18 5µm 250×10 mm semi-preparative column and by running a gradient of 0 to 40% acetonitrile in water over 40 min at a flow rate of 4 mL. The product eluted at 15 min and was obtained by lyophilization. ESI⁻ calculated for [M-H]⁻ C₃₂H₄₅ClN₇O₁₉P₃S (compound 8) 990.13, found 990.2 (Figure S7).

S. aureus MenB (saMenB)

The gene encoding MenB was cloned from MRSA (Rosenbach, ATCC BAA-1762) into a pET28a⁺ plasmid so that a 6x-HIS tag was encoded at the N-terminus. The expression and purification of saMenB followed a similar method to that described previously for E. coli MenB.⁴⁶ Briefly, protein expression was induced with 0.5 mM IPTG when the OD₆₀₀ of the culture reached 0.55, after which the culture was shaken overnight at 22 °C. Cells were collected by centrifugation, re-suspended in 20 mM sodium phosphate buffer pH 8.5 containing 250 mM NaCl, 1 mM MgCl₂ and protease inhibitor cocktail, and then lysed by passing the suspension twice through a cell disruptor (35 kpsi). Following ultracentrifugation, saMenB was purified from the supernatant by affinity chromatography with HIS bind resin and by running an imidazole gradient from 20–500 mM. Fractions containing saMenB were pooled and chromatographed on a Sephadex 75 column using 20 mM sodium phosphate buffer pH 8.5 containing 250 mM NaCl and 1 mM MgCl₂ as the eluent to rapidly remove imidazole. The protein was concentrated to ~40 µM (ɛ₂₈₀ = 33,140 M⁻¹cm⁻¹) and stored at −80 °C.

Kinetic Assays

Enzyme inhibition studies were performed in 20 mM sodium phosphate buffer pH 8.5 containing 250 mM NaCl and 1 mM MgCl₂ using a MenE-MenB coupled assay in which saMenB was rate-limiting.⁴⁷ IC₅₀ values were determined in reaction mixtures containing o-succinylbenzoate (60 µM), ATP (120 µM), CoA (120 µM), saMenB (0.1 µM), and varying inhibitor concentrations (0.05−250 µM). Reactions were initiated by addition of ecMenE (5 µM) with or without 1 hr incubation in 37 °C and the production of DHNA-CoA was monitored at 392 nm (ε₃₉₂ = 4,000 M⁻¹ cm⁻¹).

Isothermal Titration Calorimetry (ITC)

ITC was used to quantify the affinity of CoA and the CoA adducts 7 and 8 for saMenB using a Micro-Cal VP-ITC instrument. Aliquots of 2 mM ligand (4 or 8 µL) were titrated into a solution of 25 µM saMenB (1.8 mL) using a 12–20 s titration period with 300 s between each titration. Lyophilized ligands were dissolved in the same buffer as that used for the enzyme (20 mM sodium phosphate buffer pH 8.5 containing 250 mM NaCl and 1 mM MgCl₂). ITC experiments were performed at 22 °C.

Bacterial Strains and Antibacterial Assays

Bacterial strains used in this study included methicillin-sensitive (MSSA, ATCC 25923), and -resistant (MRSA, BAA 1762) S. aureus, E. coli (ATCC 25922), Bacillus subtilis (ATCC 6051), Haemophilus influenzae (ATCC 49427), Enterococcus faecium (ATCC 19434), Klebsiella pneumoniae (ATCC 13883), Proteus mirabilis (ATCC 35659), Pseudomonas aeruginosa (ATCC 27853), and Mycobacterium smegmatis (ATCC 700084). Minimum inhibitory concentrations (MICs) were determined using visual inspection of cells grown in transparent 96-well plates. Bacteria were grown to mid-log phase (OD₆₀₀ of 0.6–0.8) in Mueller Hinton broth, tryptic soy broth, or 7H9 media at 37 °C in an orbital shaker. A final inoculum concentration of 10⁶ CFU/mL per well was treated with inhibitor in a 2-fold dilution series at concentrations ranging from 0.06–125 µg/mL. MIC values under capnophilic conditions were obtained in a humidified candle jar and using a candle to obtain (~8% O₂). GasPak EZ Anaerobe Sachets (BD 260001) were used to obtain anaerobic conditions (< 0.1% O₂). The MIC was defined as the minimum concentration (modal value) at which no obvious growth was observed after 24–72 hr incubation were used at 37 °C. Complementation studies were performed by supplementing synthetic broth with 10 µM menaquinone-4 (MK4) or 10 µM menadione (MD).

Quantification of MK Levels

MK was extracted from S. aureus cells using a liquid-liquid extraction method.^{63, 64} A 100 mL starter culture of MRSA in Mueller Hinton-II (MH-II) broth was treated with ½ MIC of vancomycin (2 µg/mL) or 1 (0.38 µg/mL). Cultures were incubated at 37 °C in an orbital shaker to late-log phase (OD₆₀₀ between 0.9 and 1.0) and then diluted with MH-II broth to 10⁹ cells/mL. Cells were isolated from 100 mL of culture by centrifugation (5,000 rpm, 30 minutes) and then re-suspended in 30 mL phosphate buffer (50 mM KH₂PO₄, pH 7.0). The suspension was extracted with 20:15 mL methanol:chloroform. The lower organic layer was collected, washed with brine (30 mL) and dried with MgSO₄ prior to MS/MS analysis. MK quantification was conducted using a flow injection analysis (FIA)-atmospheric-pressure chemical ionization (APCI)-MS/MS system and TSQ Quantum Access triple Quadruple mass spectrometer. Briefly, 5 µL of sample was loop injected and the flow was directed to the APCI source. Mass spectrometry was performed in positive ion mode with 3.5 kV, gas pressure 30 psi, and a capillary temperature 350 °C. Multiple Reaction Monitoring (MRM).^{65, 66} transitions were detected at 100 ms dwell times. Multiple injections were performed over a 5 min time frame. MK-9 was used as a standard for generating a calibration curve. Complementation studies were performed by supplementing synthetic broth with 10 µM menaquinone-4 (MK4) or 10 µM menadione (MD).

Sample Analyses by LC/UV/MS

DHNA

High resolution LC/UV/MS analysis was performed on samples prepared as follows.^{67, 68} MRSA was grown to an OD₆₀₀ of 1 after which 1 mL of the culture was filtered using a sterile Millipore filter to capture the cells. The filter was transferred to MH-II agar plates where the cells were cultured with or without 8 µg/mL of 1 for 18 hr. Cells were harvested from the filter, lysed by bead-beating and centrifuged in a SpinX column. Samples of the supernatant were analyzed by high resolution LC/UV/MS (ESI⁺, m/z=100–3200) using an Agilent LC-UV-TOF mass spectrometer (G6224A oaTOF). The LC was performed using a Kinetex XB-C18 column (100Å, 2.6 µm, 150 × 2.1 mm) with 0.1% HOAc/H₂O (solvent A) and CH₃OH (solvent B) at 45°C. The flow rate was 0.40 mL/min and the amount of solvent B was: 0–2 min 5% B, 2–32 min 5–95% B, 32–35 min 95–96% B. Sample volumes were 5 µL for the synthetic standards and 20 µL for the sample supernatants.

CoA-derivatives

High resolution LC/UV/MS analysis was performed on samples prepared as described above. The supernatant was centrifuged using a SpinX column and the filtrate was lyophilized. Samples were analyzed by high resolution LC/UV/MS (ESI⁻, m/z=100–3200) using an Agilent LC-UV-TOF mass spectrometer (G6224A oaTOF). The LC was performed using a Kinetex HILIC column (100Å, 2.6 µm, 150 × 2.1 mm) with H₂O:CH₃CN 1:1 (10mM AmAc, 0.02% Ac) (solvent A) and CH₃CN:H₂O, 95:5 (10mM AmAc, 0.02% Ac) (solvent B) at 30 °C. The flow rate was 0.50 mL/min and the amount of solvent B was: 0–2 min 96% B, 2–22.5 min 96–0% B, 32–35 min 0% B. Sample volumes were 5 µL for the synthetic standards and 25 µL for the sample supernatants.

Colony Morphology

The colony morphology was analyzed by plating MRSA cells on tryptic soy agar (TSA) media with or without 1. A single colony of MRSA was streaked on a 20 mL TSA plate containing 0, 4, or 8 µg/mL 1. For the rescue experiments, TSA plates contained 16 µg/mL 1 and 10 µM MK-4 or MD. Plates were incubated for up to 84 hr at 37 °C. In addition to the slow growth phenotype, SCV colonies were also non-pigmented (colorless) in contrast to rapidly growing wild-type yellow colonies.⁶⁹

In Vivo Antibacterial Activity of 1

All animals were maintained in accordance with the American Association for Accreditation of Laboratory Animal Care criteria and was reviewed and approved by SBU IACUC. Experiments were conducted under BSL-2 conditions in the Division of Laboratory Animal Resources at Stony Brook University. AVMA guidelines on euthanasia after the experiments were followed at the end of each experiment. Six week old, male Swiss Webster mice (Taconic) were used. Systemic infection was induced by intraperitoneal (IP) injection of 2×10⁷ CFU MRSA in 200 µL of saline. Drug doses, including vancomycin and oxacillin, were prepared in a vehicle consisting of 40% saline, 40% ethanol, 20% PEG-400, and were delivered by subcutaneous injection (SC) at 1 hr, 12 hr and 24 hr post infection at a dose of 100 mg/kg. Mortality was checked every 12 hr for 7 days. For the thigh infection model, mice were rendered neutropenic by injecting cyclophosphamide (CPA) 4 days (IP, 150 mg/kg) and 1 day (IP, 100 mg/kg) before infection. Thigh infections were performed by injecting MRSA cells (10⁶ CFU in 50 µL of saline) into the left thigh muscle (intramuscular, IM). All drugs, including vancomycin and oxacillin, were administered by SC injection 1 hr and 12 hr post infection at a dose of 100 mg/kg (vancomycin and oxacillin) or 100, 50 and 25 mg/kg (compound 1). Infected thigh muscles were collected, weighed and homogenized in 2 mL of saline. Serial dilutions of each homogenized sample were plated on Mueller Hinton agar supplemented with 5% sheep blood. The number of colony-forming units (CFU) was determined following overnight incubation and the bacterial burden was determined. Statistical significance was determined using a one tail T-test.

ABBREVIATIONS

MRSA

Methicillin-resistant Staphylococcus aureus

MSSA

Methicillin-sensitive Staphylococcus aureus

MK

Menaquinone

DHNA

1,4-Dihydroxy-2-naphthoic acid

OSB-CoA

o-Succinylbenzoyl-Coenzyme A

MIC

Minimum inhibitory concentration

SCV

small-colony variant

MD

menadione

TSA/B

Tryptic Soy Agar/Broth

CFU

Colony Forming Unit