Potential Inhibitors Targeting Escherichia coli UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA): An Overview

Abstract

Antibiotic resistance is one of the biggest challenges that is escalating and affecting humanity across the globe. To overcome this increasing burden of resistance, discovering novel hits by targeting the enzymes involved in peptidoglycan (murein) biosynthesis has always been considered better in antimicrobial drug discovery. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme has been identified as essential for Escherichia coli survival and catalyzes the early-stage step in bacterial cell wall synthesis. The present article gives a brief overview of the role of enzymes in peptidoglycan synthesis and MurA enzyme (previously known as MurZ in E. coli), in particular, including its structural and active site features. This review also provides an insight into the current knowledge of the reported MurA inhibitors, their mechanism of action and drawbacks of these hits that hinder their clinical trials, which would be helpful for synthesis and discovering potent molecules.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-021-00988-6.

Introduction

Gram-negative infections are the leading cause of suffering worldwide. In infectious diseases, the widespread emergence of resistance in these pathogenic strains to clinically known important drugs is one of the foremost pressing challenges [1, 2]. All this emphasizes an urgent need to develop antibacterials directed at novel targets. Among Gram-negative pathogens, E. coli has been known to cause severe intestinal and extra intestinal infections [3]. Although this bacterium is a part of normal gut flora, a variety of serotypes are responsible for systemic infections, and its pathotypes contribute to about 75% of all worldwide urinary tract infections (UTIs). At present, E. coli includes several commensal and pathogenic strains [4, 5]. Antimicrobial-resistant Escherichia coli is a major source of bloodstream and urinary tract infections (UTIs) in both community and healthcare settings around the world and is designated as a red-alert pathogen for humans [6]. E. coli has been able to spread its genes horizontally and bacteria usually acquire resistance genes from other Enterobacterales members [7]. In the list of public health threats, the emergence of multi-drug resistance is ranked among the top three [8]. Moreover, extended-spectrum β-lactamase- (ESBL) producing E. coli has outpaced the accessible options for treatment [9]. These prevailing, drastic conditions need to be addressed by applying new strategies for developing novel drugs and minimizing the drying up of the antibiotic pipeline [10].

Both Gram-positive and Gram-negative bacteria are surrounded by a protective layer—peptidoglycan (or murein)—that protects cells against external damage and osmotic stress by imparting rigidity to the bacterial cells [11]. Being unique to prokaryotic cells and important for the survival of bacteria, the peptidoglycan (PG) biosynthesis pathway is always considered the most valid target [12, 13]. Cell wall synthesis is a complex and multistage process catalyzed by a number of enzymes [14]. Broadly, three distinct stages include the cytoplasmic stage (nucleotide precursor synthesis), the membrane-associated stage (lipid-linked intermediate synthesis), and the exocytoplasmic stage (polymerization reactions) that lead to the formation of mature bacterial cell molecules [15]. The pathway starts from fructose-6-phosphate, resulting in peptidoglycan synthesis through various enzymatic steps, as shown in Fig. 1. The key precursor of bacterial cell wall synthesis is UDP-N-acetylmuramate (UDP-MurNAc), which is produced by two cytoplasmic enzymes, UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and UDP-N-acetylenolpyruvyl glucosamine reductase (MurB) in a two-step process. Targeting early-stage enzymes like MurA, which is required for viability and has no mammalian homology, is a well-proven target in drug research. Cytosolic enzyme MurA (UDP-N-acetylglucosamine enolpyruvyl transferase, EC 2.5.1.7) catalyzes the first promising step of PG biosynthesis by transferring enolpyruvate from phosphoenolpyruvate (PEP) to UDP-N-acetylglucosamine (UDP-GlcNAc/UNAG), resulting a product enolpyruvyl-UDP-N-acetylglucosamine (UDP-GlcNAc-enolpyruvate), with a release of inorganic phosphate (Pi) from the tetrahedral intermediate [16, 17] (Fig. 2). According to kinetics data, MurA follows the addition and elimination mechanism for catalyzing the reaction (Fig. 3). The binding of UDP-GlcNAc and PEP to the active site of MurA initiates the reaction. The oxocarbenium ion formed by the transfer of proton to PEP is added to UNAG, resulting in the formation of a tetrahedral intermediate (containing both the substrates attached covalently) (Fig. 3). Then, in the final step, the C-3 of the PEP proton is subtracted, leading to double bond reformation between C-2 and C-3 in PEP with the Pi elimination from the tetrahedral intermediate [18, 19]. MurA belongs to the family of enolpyruvyl transferases and the other enzyme of this family is 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase. These two proteins have less similarity in amino acid sequence (~ 20%) but high similarity at the structural level. Gram-positive bacteria harbor two genes, namely, murA and murZ, for the activity of UDP-N-acetylglucosamine enolpyruvyl transferase. These genes express two proteins called MurA and MurZ, whereas in some Gram-positive organisms the second gene, murZ, is also referred to as murA2 or murAB [20]. MurA and MurZ proteins from Staphylococcus aureus share 46.7 percent amino acid sequence identity and are roughly the same length [421 residues (MurA) versus 419 residues (MurZ)]. According to sequence analysis, two MurA enzymes in S. pneumoniae were found to be 45 percent identical at the amino acid level, and the enzymes contain the key catalytic residues previously found in E. coli MurA [21]. For Gram-positive microbes gene deletion tests further show that removing one of the genes still results in a viable organism, despite the fact that this activity is plainly required for survival. Furthermore, deletion of either gene primarily reduces PG content; for example, in S. aureus, loss of murA resulted in a 26% decrease in cellular PG content, whereas loss of murZ resulted in a 3% decrease. Five amino acids (Lys22, Cys115, Asp305, Asp369, and Leu370, numbering in E. coli) are conserved among the MurA enzymes in several bacterial strains, including Haemophilus influenzae, E. cloacae, Pseudomonas aeruginosa, Staphylococcus aureus, S. pneumoniae, and Bacillus subtilis. Finding hits against the MurA enzyme could help to restore therapeutic options that have been dwindling [22].

Fig. 1.

PG synthesis cytoplasmic steps [15]

Fig. 2.

Reaction catalyzed by MurA enzyme [35]

Fig. 3.

Addition–elimination mechanism exhibited by MurA. Here X, Y, and Z are amino acid side chains [32]

E. coli Cell Wall Enzymes as Drug Targets

Cell wall enzymes have always been a preferred target in drug discovery because of their essentiality in pathogen survival. There is a plethora of literature explaining the importance of these enzymes for bacterial growth. The most widely used β-lactam antibiotics are in use for over 70 years against a variety of Gram-negative and Gram-positive infections. These hydrophilic cell wall directing antibiotics cause cell wall disruption and cause bulges in the inner membrane, spheroplast formation, abnormalities in cell morphology or cell lysis [23]. β-lactam composed of cyclic amide rings is basically a terminal D-alanyl-D-alanine dipeptide analogue of peptidoglycan and in the acylation step, acts as a PBP substrate, resulting in the disruption of the normal peptidoglycan lengthening (Fig. S1). But the number of ESBL-producing organisms is challenging our presently available treatment options [24, 25]. The present article will focus on the importance of the cytosolic E. coli MurA enzyme in drug development and the reported inhibitors against the target.

Mur Enzymes in Cell Wall Synthesis

Mur enzymes are the crucial cytoplasmic enzymes in PG formation. UDP-N-acetylmuramyl (UDP-MurNAc) pentapeptide precursor for cell wall synthesis is formed by these Mur enzymes (MurA–F). Based on the type of reactions catalyzed, Mur enzymes are divided into three main groups:transferases (MurA), oxidoreductases (MurB) and ligases (MurC, MurD, MurE, MurF)[26]. As shown in Fig. 1, cell wall formation begins with fructose-6-phosphate. The glucosamine-6-phosphate synthase (GlmS), an amidotransferase (dimeric enzyme), converts this fructose-6-phosphate into glucosamine-6-phosphate following a bi–bi mechanism and utilizing L-glutamine as a nitrogen source. The second enzyme, phosphoglucosamine mutase (GlmM), helps in interconversion and forms glucosamine-1-phosphate,which is finally converted into UDP-GlcNAc by GlmU (bifunctional enzyme; glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase). The pathway for UDP-GlcNAc biosynthesis is quite different as found in eukaryotes. GlmM and GlmU enzymes are also unique to bacterial cells and have no eukaryotic homology. UDP-GlcNAc biosynthesis is essential for peptidoglycan and LPS biosynthesis in both Gram-positive and Gram-negative bacteria. Therefore, perturbing UDP-GlcNAc biosynthesis can be lethal for bacteria [27]. Finally, UDP-MurNAc pentapeptide is formed from this GlcNAc in a series of steps catalyzed by enzymes of the Mur group. MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) transfers enolpyruvate from PEP to UDP-GlcNAc (UNAG), after which MurB (UDP-N-acetylenolpyruvoyl glucosamine reductase) yields a product, UDP-N-acetylmuramate (UDP-MurNAc) by enolpyruvate moiety reduction to D-lactate. The step wise addition of pentapeptide side-chain is catalyzed by ligases (ATP-dependent)- MurC (UDP-N-acetylmuramate-L-alanineligase), MurD (UDP-N-acetylmuramoyla-lanine-D-glutamate ligase), MurE (UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase) and MurF (UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase) results in UDP-N-acetylmuramyl pentapeptide (UDP-MurNAc-pentapeptide) formation [17]. Both MurA and MurB are important enzymes as they catalyze the early steps towards peptidoglycan synthesis. Then an integral membrane enzyme MraY (phospho-MurNAc-pentapeptide translocase) attaches a 55-carbon aliphatic chain known as caprenyl pyrophosphate to pentapeptide [28]. MraY is thought to be a good target for novel antibiotics because it is an important molecular tool for building the cellular envelope and has no homologs in mammalian cells. The cytoplasmic steps are followed by inner and periplasmic steps, which complete the PG synthesis in bacteria. Inner membrane steps involve the MurG [UDP-N-acetylglucosamine-N-acetylmuramyl (pentapeptide)pyrophosphoryl-undecaprenol N-acetylglucosamine transferase] that catalyses GlcNAc addition to the MurNAc moiety of lipid I to form lipid II that is flipped over into the periplasmic space with the help of flippase enzyme. Two proteins FtsW and MurJ have been identified for translocation of lipid-linked muropeptides (cytoplasm to periplasmic space) in E. coli [29]. Finally, in periplasmic space final stages of Murein synthesis take place by the addition of a component of lipid II (GlcNAc-MurNAc pentapeptide pyrophosphoryl undecaprenol) into the murein sacculus. Peptide cross-links are catalyzed by high molecular mass penicillin-binding proteins (HMM PBPs) with a transpeptidase domain (PBP1A, PBP1B, PBP1C, PBP2 and PBP3) [30].

Structure of E. coli MurA

In E. coli murA gene is approximately 1260 base pairs long and codes for 419 amino acids of an enzyme that is approximately 44.7KDa in size. X-ray crystallographic studies by Skarzynskiet al.(1996) [31] demonstrated that this globular protein is made up of two prominent domains, which have α helices and β sheets in the same proportion and are linked together by a linker of double-stranded. Each domain possesses a similar main-chain folding pattern with three internal helices encircled by helices (3 in no.) and stranded beta sheets (3–4). One domain has catalytic site Cys-115 (22–229 residues), known as the catalytic domain, and the other one has residues from 1–21 and 230–419, respectively, known as the C-terminal domain. This catalytic site is placed in a cavity formed between these domains. It is this cys115 that is ultimately involved in the product formation. However, residues numbering from 111–121 form a loop-like structure that acts as a lid for this catalytic pocket around which are residues involved in 10 hydrogen bonds between these two domains. These include 4, 3, 1 and 2 hydrogen bonds between 46–49 and 396–400, 116–119 and 329–330, Glu188 and His299, Glu190 and Arg232 (bottom part of catalytic site) respectively. These interactions are known to be very important for the enzyme in catalyzing the reaction efficiently. Structural studies revealed that due to the flexible loop, this protein undergoes a drastic conformation change in the presence and absence of ligand (UDP-GlcNAc) and is referred to as closed (catalytically active) and open form, respectively. In closed form, this Cys115 residue is a part of the active site and a substrate, PEP, comes closer for the reaction. All this occurs by altering residues 111–121 on first substrate (UDP-GlcNAc) binding (Fig. S2) [32]. Various novel sets of side chain (intramolecular) and enzyme ligand interactions have been reported in newly staged protein conformations identified by Jackson et al. (2009) [33], during the crystallographic study of the MurA complex (MurA + UDP-MurNAc + Phosphite). Moreover, it has been found that MurA’s from Escherichia coli, Enterobacter cloacae, and Haemophilus influenzae have highly homologous amino acid sequences, and the crystal structure of MurA’s from these organisms is substantially similar. Table 1 summarizes the information on the kinetic parameters of the MurA enzyme of E. coli and various other Gram-negative pathogens. Several 3D structures of protein (E. coli MurA) submitted to the protein data bank (PDB) are given in Supp. Table1.

Table 1.

MurA enzyme’s kinetic parameters reported for E. coli in different studies and for several other Gram-negative pathogens

S. No	Organism	Parameters					References
		Km(UNAG) (μM)	Km(PEP) (μM)	kcat (S−1)	kcat/Km (UNAG) (μM−1S−1)	kcat/Km (PEP) (μM−1S−1)
1	Escherichia coli	15.0	0.4	3.8	0.25	10.0	[18]
		5.7	4.1	8.9	–	–	[16]a
		36	0.84	2.4	–	–	[60]
		200	85	-	–	–	[41]
2	Pesudomonas aeruginosa	17.8	0.45	1.8	0.101	4.0	[60]
3	Enterobacter cloacae	80.0	8.3	3.0	0.0375	0.36	[61]
4	Acinetobacter baumannii	1062 ± 90	1806 ± 230	–	–	–	[62]
5	Haemophilus influenzae	31	24	2.1 × 102 (min−1)	–	8.9 (μM−1 min−1)	[63]

^aCapillary electrophoresis-based enzyme assay

Known Inhibitors Targeting E. coli MurA

Various strategies are employed to screen compounds against this target to identify novel compounds, including the high throughput screening (HTS) approach, structure–activity relationship (SAR), etc. There are a number of inhibitors known to date that target MurA, but only the natural origin drug fosfomycin is in use clinically. Inhibitors are well studied in detail to find out their binding mechanisms and ultimately inhibit the cell wall biosynthesis process. Hits targeting the enzyme in E. coli are summarized in Supp. Table 2 and are discussed in detail below. Moreover, the inhibitors reported for MurA in other pathogenic strains are listed in supp. Table 3.

Fosfomycin

Fosfomycin [(1R, 2S)-1, 2-epoxypropylphosphonic acid], an epoxide compound also known as phosphonomycin, was firstly produced in Spain by using Streptomyces fradiae ATCC 21096 [34]. In 1996, it was approved in the US for use in treating UTIs caused by E. coli and Enterococcus faecalis [35]. It is also produced by some other strains, including S. wedmorensis ATCC 21,239 and S. viridochromogenes ATCC 21,240. This broad-spectrum bactericidal antibiotic belongs to aminoglycoside drug class and is also produced on an industrial scale by synthetic processes as an acid derivative of polar phosphonic (cis–1,2-epoxypropyl phosphonic acid) and is among the first-line treatment options for bacterial UTI infections. The drug act as a PEP analog and competes while MurA reaction. There is a covalent binding via thio ether bond between the drug and Cys115 residue of MurA and it follows an irreversible mode of inhibition [36]. While binding fosfomycin gets packed between the substrate UDP-GlcNAc and MurA enzyme, it results in several hydrogen bonds with polypeptide chains at different sites (Fig. 4). Mainly, strong electrostatic interactions are created by the surrounding phosphonate group and three MurA residues (Lys22, Arg120 and Arg397) that are positively charged. UDP-N-acetylglucosamine presence during the reaction capable of inducing changes in the active site was found to be an important factor that increased fosfomycin inactivation in atime-dependent manner. According to fluorescence spectroscopy data, structural variations caused by the drug in UDP-GlcNAc liganded MurA (closed form) in time dependence are the same as the inactivation process [17]. IC₅₀ of the drug is 8.8 µM which decreased to 0.40 µM in pre-incubation conditions with UNAG. Fosfomycin entry in whole-cell bacterium is by hexose-6-phosphate transporter and L-alphaglycerophosphate system. E. coli MurA crystal structure with substrate UNAG and drug fosfomycin is provided by Skarzynski and their coworkers [31]. Antibiotic is active against a variety of bacteria, including Citrobacter spp., Klebsiella pneumonia, Salmonella typhi, Enterobacter spp., Proteus mirabilis, Staphylococcus epidermidis and Streptococcus pneumonia. In vitro and in vivo activity of Fosfomycin (Monurol) has been reported against MDR and XDR species of the family Enterobacteriaceae [37]. No cross-resistance of this drug is reported with known antibiotics due to its uniqueness in the mechanism of action and structure.

Fig. 4.

Inhibition of MurA by fosfomycin [17]

Cyclic Disulphide, Pyrazolopyrimidine and Purine Analogue

Three compounds were identified while library screening, where RWJ-3981 (Cyclic disulphide), RWJ-110192 (Pyrazolopyrimidine) and RWJ-140998(Purine analogue) showed IC₅₀ values of0.20 µM, 0.30 µM and 0.90 µM respectively. These compounds have no structural resemblance to fosfomycin. Based on structure–activity relationships (SARs) of hits, it was found that for structural analogs of RWJ-3981 and RWJ-140998, presence of thiazocine ring and for RWJ-3981, chlorine on the ring having disulfide were important for inactivation of protein. These three compounds showed a decrease in IC₅₀ values during pre-incubation with UNAG (same as fosfomycin). Cyclic disulphide and purine analogue are irreversible inhibitors and bind very tightly and non-covalently to the binding site/near the protein's active site. In contrast, Pyrazolopyrimidine is reversible and binds to the site/near active site weakly through non-covalent interactions. Modelling studies revealed that in RWJ-3981carbonyl oxygen may form strong hydrogen bonds with Arg residues (Arg120 and Arg397) whereas the sulfur atoms form hydrophobic contacts with Met90 and Arg91, while the centroid of the rings in RWJ-110192 and RWJ-140998 is predicted to bind farther away from the catalytic site than RWJ-3981. Only compound RWJ-3981 showed an MIC of 8 µg/ml while the other two compounds showed > 32 µg/ml values against the whole cell of E. coli [22].

Sesquiterpene Lactones (SLs)

Natural origin sesquiterpene lactones (SLs) which include cnicin, cynaropicrin, parthenolide, eupatoriopicrin, helenine, costunolide, dehydrocostuslactone, α-cyclocostunolide, melampolide are reported to be tested against the target [38]. Among them, only cnicin and cynaropicrin irreversibly bind to the target and produce good IC₅₀ values of 16.7 µM and 19.5 µM, respectively, whereas eupatoriopicrin shows an IC₅₀ of 32.7 µM and the remaining all were > 50 µM. Based on SAR studies’ prediction, it was found that in cnicin and cynaropicrin, C8-position side chain ester (unsaturated) was involved in MurA inhibition. SL’s Michael reaction with the thiol group of Cys115 is shown in Fig. 5. The crystal structure of MurA protein in complex with the substrates UNAG and cnicin reveals a non-covalent suicide mode of inhibition in which the adduct of UNAG cnicin behaves identically to the intermediate adduct formed in the native reaction [39]. For SLs, it was also concluded that antibacterial activity was due to exocyclic methylene groups present either on the electrophile side chain or on the macrocycle, attacking nucleophilic binding sites in a bacterial cell. SLs are also reported to be active for Pseudomonas aeruginosa MurA. Data is shown in supp. Table 3.

Fig. 5.

Side chain of Cys115 reaction with SLs [38]

2-Aminotetralone Derivatives

Seven compounds were identified by applying the HTS approach by a multinational company. Among all the tested 2-aminotetralonederivatives, only four compounds were active against the target, exhibiting an IC₅₀in the range of 3.1–22.5 µM. The C115D E. coli mutant was generated and its protein was purified. Upon SAR result evaluation, it was found that C115 is responsible for inhibition due to an attack on the ketone moiety by the thiol group to give a thiohemiketal adduct (Fig. S3). In the whole cell-based assay, only 4 and 6 coded compounds showed MIC of 128 µg/ml. The binding of compound 4 near the crucial C115 residue in the active site of E. coli MurA was revealed by docking it into the active site. The paper also reported the loss of activity of compounds (3–6) against chymotrypsin and malate dehydrogenase (MDH) in selective assays and therefore called them not ‘promiscuous’ inhibitors. These compounds were also tested against MurA and MurZ enzymes of S. aureus [40]. Most active hit in S. aureus was C-4 against MurA and C-6 against MurZ with IC₅₀ of 17.35 µM and 16.09 µM respectively shown in Supp. Table 3.

Tulipalines, Tuliposides and their Derivatives

Natural origin based tuliposides (sugar esters) are produced as secondary metabolites by Tulips and are made up of D-Glc and 4’-hydroxy-2’-methylenebutanoyl and/or (3’S)-3’,4’-dihydroxy-2’-methylenebutanoyl side chains. E. coli MurA is inhibited by tulips and their derivatives in a time-dependent manner. It is also a non-covalent suicide inhibitor and binds to both the active site Cys115 residue and UNAG, the same as cnicin [41]. Synthesis of 6-tuliposide B (1) and (-) -tulipalin B (2) analogues by Shigetomi et al. [42] and SAR studies confirmed that 3’, 4’ -dihydroxy-2'-methylene butanoate structure is responsible for the activities. Like the known drug fosfomycin, cnicin and derivatives of tulipaline possessa bacteriostatic action on cells at low concentrations and a bactericidal action at higher concentrations. In another study, the hydroxylated analogues (±)-tulipaline B and 1-tuliposide B effectively inhibited MurA compared to 1-tuliposide A and tulipaline A. But MurA was not the only target for these tulipalines, as these inhibitors targeted wild type E. coli cells as well as C115D mutant E. coli. It was concluded that the inhibitor’s reactivity towards sulfhydryl nucleophiles is the most likely factor for inactivation, and it could also be linked to MurA binding [41].

Benzothioxalone Derivatives

High-throughput screening of approximately 650, 000 library compounds led to the identification of a series of benzothioxalone derivatives having IC₅₀ values in the range of 0.25–18.54 µM. These inhibitors showed irreversible inhibition mode and bonded covalently to MurA enzyme. Molecular docking radio labeling studies revealed an extra binding pocket in fosfomycin-inhibited enzyme. The proposed mechanism of thiazolone inhibitor inhibition is depicted in Fig. S4, which suggests that forming a disulfide bond with MurA by attacking the thioxalone ring carbonyl group with an active site cysteine is followed by ring opening to release an S-acylated protein (S-acylated covalent adduct).

A ¹⁴C-labelled version of compound 18 (methoxy derivative) was prepared to test the hypothesis of a covalent mechanism of inhibition by benzothioxalones on the MurA enzyme. [¹⁴C]18 demonstrated approximately stoichiometric binding to the wild-type protein in radio labeling assays up to a concentration of 1 mM, which was not identified with the C115D mutant form of the protein. The results were consistent with the benzothioxalone inhibitors binding to the enzymes via a covalent mechanism involving the site corresponding to C115 in E. coli MurA, since mass spectrometry showed that compound coded 1 remained stable in the presence of 0.1 M DTT for several days. Also, prior incubation of the enzyme with benzothioxalone 1 effectively prevented the binding of [¹⁴C] 18 to wild-type MurA, demonstrating that both inhibitors bind at the same location (s). Fosfomycin, on the other hand, had no effect on this binding. Although various results, including modelling studies, revealed the possibility of an extra binding pocket in fosfomycin-inhibited enzymes, in docking studies of compounds coded as 1 into the MurA-UDP-GlcNAc crystal structure, which contains a fosfomycin fragment covalently connected to C115, it was found that the C115-linked fosfomycin fragment occupied the original binding pocket. In contrast, inhibitor 1 binding occurred in a second pocket on the other side of UDP-GlcNAc. These compounds were also tested against Gram-positive MurA enzymes (Supp. Table 3). MIC against E. coli was in the range of 128 to > 256 µg/ml [43].

Terreic Acid

Fungal Secondary metabolite terreic acid has already been reported in the literature as a potent antibacterial agent, although its cellular target is still debatable. It is primarily produced by Aspergillus terreusand is a quinine epoxide. Kinetic studies revealed that protein inactivation by terreic acid require a closed-state configuration of protein (presence of substrate UDP-N-acetylglucosamine in preincubation conditions). To explain the mechanism of inhibition of MurA at the molecular level, Han and his co-workers did crystallographic studies for MurA enzyme of E. cloacae in the presence of UNAG (substrate) and terreic acid with the determination of structure at 2.25 Å resolution and found that due to steric hindrance in the active site of the protein, UNAG was released from the terreic acid complexed MurA protein that adapts to open form confirmation. This was in contrast to fosfomycin–UNAG-enzyme complex formation, which results in the closed conformation of the enzyme. In the binary complex (terreic acid–MurA), the adduct (cys115-terreic acid) was mainly solvent exposed, whereas in the fosfomycin ternary complex (fosfomycin–UNAG–MurA), the adduct (Cys-115-fosfomycin) was deep seated in the enzyme cavity [44]. In another study, the MIC of terreic acid against E. coli ATCC25922 was reported to be 23 µg/ml and has been reported to target GlmU enzyme in E. coli [45].

Nitrovinylfuran Derivatives

Derivatives of nitrovinylfuran are known for their antimicrobial activities for a long time. Scholz et al. (2013) [46], using a bromonitrovinyl scaffold, designed inhibitors that mainly interact with the residues of cysteine in target proteins. In their study, they found di-bromo substituted Nitrovinylfuran [2-bromo-5-(2-bromo-2-nitrovinyl) coded as C-1] and bromonitromethane substituted derivative were the most potent MurA inhibitors with IC₅₀ values of 2.8 (± 0.2) µM and 4.6 (± 2.8) µM, respectively. SAR revealed that either in isolated form or in conjugation state with vinylfuran structure of bromonitromethyl moiety was responsible for inhibitory activity against MurA. For compound coded 1,it was reported that there could be a nitrovinyl moiety and various other congeners that lead to Michael addition to nucleophilic structures in proteins (targeted one). It was also concluded that compound 1 and its descendants involved thiol functionalities via oxidative processes or addition reactions and included the non-native disulfide bond formation that causes stability impairment and is responsible for bacterial protein catalytic functions [46]. Inhibition data of inhibitors against Pseudomonas aeruginosa is shown in Supp. Table 3.

Avenaciolides

Four derivatives of avenaciolidewere isolated from Neosartorya fischeri. Among them,the IC₅₀ for compounds coded 1, 2 and 3 were 0.9 ± 1.11 µM 2.8 ± 1.22 µM and 10.8 ± 1.13 µM respectively. And the C-4 compound was not active against the target. Three active avenaciolide molecules have α, β unsaturated carbonyl moieties showing inhibitory activity against E. coli MurA, probably by reacting with the cysteine present at the active site through the Michael addition reaction. This mechanism is similar to that of known natural products that inhibit MurA, such as some acrylic acid derivatives (tulipalines and tuliposides) and sesquiterpene lactones (cnicin and cynaropicrin). However, mass spectrometry analysis did not support the hypothesis for these avenaciolide compounds.

Moreover, C-3 and C-4 compounds werediscovered for the first time as novel natural products. MIC against E. coli ATCC25922 was found to be 128 µg/ml for compound 1 and 2 whereas compound 3 had no antibacterial activity. Chang and his co-workers performed ³¹P NMR spectroscopy for characterization of the inhibitory activity of tested derivatives of avenaciolide using MurA of methicillin-resistant S. aureus (MRSA), and it was evaluated that three compounds were irreversible inhibitors that caused modifications in catalytic residue in the wild type strain MRSA MurA (Supp. Table 3). Among the three compounds, C-1 and C-2 showed very good efficacy against wild type and a fosfomycin resistant mutant (MurA C119D) of MRSA. It has been reported that the formation of a covalent adduct of catalytic Cys119 with the first, α, β-unsaturated carbonyl moiety in WT MurA (MRSA) was responsible for inhibition. However, isolated compounds were more potent for E. coli MurA as compared to MRSA MurA (WT) and mutant MRSA MurA (C119B) [47].

Benzoic Acid Derivatives

Three novel MurA inhibitors were discovered by structure-based virtual screening and validated by in vitro screening. Rozman et al. (2017) [48] used the ProBiS-CHARMMing web server for induced fit simulation in their study. The first two compounds were benzoic acid derivatives and had three rings. Compound 1[(E)-3-(5-(2-carboxy-2-((3-methyl-1H-1,2,4-triazol-5-yl)thio)vinyl)furan-2-yl]benzoic acid had two 5-membered rings (furan and triazole) with additional acidic moiety (acrylic acid). Compound 2(quinazolinone) had imidazole as well as dihydroquinazolinone rings. The third compound (pyrrolopyridine) had four rings containing pyrazole, dihydropyridine, pyrrolopyridine, and benzene connected by short-linkers. Among them, compound 1 was entirely a novel MurA inhibitor based on chemical structure and showed better inhibitory activity than the remaining two compounds. As per pre-incubation assay, compound 1 was a reversible inhibitor and exhibited an IC₅₀of1 ± 2 µM and for compounds coded 2 and 3, it was 82 µM and 109 µM, respectively. No antibacterial activity was reported for these three compounds against E. coli and other Gram positive strains. According to them, this may be due to any of the following factors, i.e. efflux pumps transportation of these compounds from the cytoplasm, less penetration through the cell wall or the requirement of a high dose to exhibit whole cell activity by inhibiting the target.

Benzohydrazide Compounds

In a study reported by our group, screening of approximately 52,026 compounds (Chembrige, Chemdiv and in- house synthetic compounds) using a pharmacophore screening approach resulted in the identification of two novel hits in the benzohydrazide class. Compounds coded IN00152 and IN00156 exhibited IC₅₀ of 14.03 ± 2.991 µM and 32.30 ± 1.547 µM, respectively. In docking studies, it was found that these compounds had Arg91 involvement in binding UNAG substrate and open closed-transition state, the same as 5-Sulfonyloxy-anthranilic acid derivatives (previously reported compound). Moreover, using outer membrane permeabilizer ethylenediaminetetra-acetic acid (EDTA), we observed increased potency of these compounds in E. coli cells, resulting in a decrease in MIC values from > 128 µg/ml to 32 µg/ml [49].

Some Potential Reported Inhibitors for Other Mur Enzymes

Several inhibitors were reported against other Mur enzymes that include 3, 5-Dioxopyrazolidines (4 compounds) tested against Mur enzymes (MurA, MurC, MurD, MurE, MurF) of E. coli and S. aureus MurB. It was found that 3, 5-dioxopyrazolidine4-carboxamides were potent inhibitors of E. coli MurB and exhibited an IC₅₀ in the range of 4.1–35 µM. Though these compounds also inhibited E. coli MurA (IC₅₀ ~ 6.8–29.4 µM). Compound coded as 2 inhibited Mur C to a small extent. And in the case of S. aureus, the range of IC₅₀ was 4.3–24.5 µM for the enzyme MurB [50]. Inhibition of different Mur enzymes by the same compounds is expected, as MurA catalyzed reaction product acts as a substrate for MurB. The same is the case for MurC, where MurB product acts as a substrate. In the same study crystal structure of compound coded 4 with MurB was also resolved for determining interactions of compound and enzyme. In another study, an analogue of phosphinate transition-state has been reported to inhibit E. coli MurC enzyme (IC50 = 49 nM) [51]. There is a report of a compound from athiazolidin-4-one-based structure inhibiting MurD and MurE ligases of E. coli as well as S. aureus [52]. Diarylquinoline compounds DQ1 and DQ2 are reported to inhibit Mur F in E. coli and belong to the same class as the anti-TB inhibitor TMC207 (R207910) targeting ATP synthase. Both compounds (DQ and TMC207) have different actions on respective bacteria [53].

Drawbacks of Reported Inhibitors Against the MurA Enzyme

Despite the identified number of inhibitors from natural and synthetic sources by applying various approaches, none have their clinical use except the old drug fosfomycin. It has been almost 51 years since the discovery of fosfomycin, no other identified inhibitor replaces the drug. Most importantly, to act on a target, a compound must pass the cell membrane barrier because biosynthesis of the cell wall starts in the cytosol. Most of the reported inhibitors of MurA targeted isolated protein but hits failed to exhibit good antibacterial activity due to the complex cell wall structure in E. coli. The impermeability issue of the compounds can be overcome by using different outer membrane permeabilizers that can sensitize the cells (gram-negative strains) to the molecules [49]. Although purine analogs, cyclic disulphide, and pyrazolopyrimidine were all more efficient than fosfomycin against E. coli MurA, investigations employing [³H] NAG incorporation in S. aureus demonstrated that, in addition to suppressing cell wall formation, chemicals also inhibit DNA, RNA, and protein synthesis [17, 22]. The non-specificity of the reported inhibitors is another drawback that stopped their further development. Moreover, fosfomycin resistance in many Gram-negative strains, including E. coli, has been observed [54, 55]. So, there is a need to discover novel inhibitors, and that drug should be more potent than fosfomycin, exhibiting a good safety profile and antibacterial properties.

Fosfomycin Resistance in Pathogenic Strains

Fosfomycin resistance has already gained a lot of attention, and currently, three resistance mechanisms have been observed. First is the gene mutation that encodes glycerol-3-phosphate transporter/glucose-6-phosphate transporter, which causes a reduction in the uptake of the drug (fosfomycin) by bacteria. The second is the point mutations at the binding site of the MurA enzyme, and the third is the fosfomycin inactivation due to epoxide ring cleavage or phosphonate group phosphorylation [56, 57]. The single-residue mutation Cys115 to Asp (numbering in E. coli MurA) renders MurA resistant to fosfomycin, which is a significant disadvantage. Various dangerous bacteria, such as M. tuberculosis, are intrinsically resistant to fosfomycin due to the expression of the Asp115 variation of MurA. Other factors, such as the fosA gene in Gram-negative bacteria and the fosB gene in Gram-positive bacteria, contribute to fosfomycin resistance, though only the Cys-to-Asp mutation on MurA causes complete fosfomycin resistance. However, the single amino acid mutation (cysteine-to-aspartate) in the active region of the MurA enzyme has also been reported in several other harmful bacteria, such as Chlamydia trachomatis and Vibrio fischeri. Avenaciolide inhibitors discussed above have been suggested to be the best alternative to treat fosfomycin-resistant pathogens as the cysteine residue involved in blocking enzyme function is different than Cyst115. However, more research is needed before the hits may be employed in combination therapy against many resistant bacteria. In some clinical isolates of Serratia marcescens, E. cloacae, Klebsiella pneumoniae, and Staphylococcus epidermidis, plasmid-encoded fosfomycin resistance comes from enzymatic alteration of the antibiotic. In human clinical E. coli isolates, the presence of plasmid-mediated fosA3 genes has been reported. Many other Gram-negative bacteria, including Klebsiella oxytoca, Enterobacter aerogenes, Pseudomonas aeruginosa, and Morganella morganii, are reported to develop intrinsic resistance to fosfomycin due to a chromosomal fosA gene, which encodes a dimeric K⁺ and Mn²⁺ dependent GST [34]. Synthesis of FosA, a glutathione S-transferase (GST), adds a glutathione residue to fosfomycin to render it inactive (Fig. S5). Because it is disseminative and commonly associated with ESBL-producing E coli, this pathway is particularly important. FosA inhibition could be a potential way to extend fosfomycin usage to Gram-negative bacteria. In a recent study by Tomich et al. 2019 [58]two inhibitors ANY1 (3-bromo-6-[3-(3-bromo-2-oxo-1H-pyrazolo[1,5-a]pyrimidin-6-yl)-4-nitro-1H-pyrazol-5-yl] and 3-bromo-6-(4-nitro-1H-pyrazol-5-yl)-1H-pyrazolo[1,5-a]pyrimidin-2-one (ANY2) has been reported forK. pneumoniae FosA, exhibiting an IC₅₀ of 5.1 + 2 and 8.4 + 3.1 µM respectively [58] (Fig. S6). ANY1 binds to FosA's active region and interacts with highly conserved amino acid residues throughout the FosA superfamily. Due to the worrisome rise in multidrug resistance, treating infections with a combination of drugs has become an inevitable trend. In another study,researchers combined fosfomycin with various other medications to see if they could kill P. aeruginosa. Fosfomycin monotherapy was effective in killing fosfomycin-susceptible isolates but resulted in rapid regrowth. Fosfomycin combined with polymyxin B or tobramycin against sensitive isolates, and ciprofloxacin + fosfomycin against fosfomycin resistant bacteria, inhibits regrowth of resistant mutants [59]. To summarise, we might infer that new tactics are urgently needed to tackle the resistance problem.

Conclusions

E. coli MurA enzyme is one of the promising targets in drug discovery. Its structural properties are studied in detail as well. Except fosfomycin, many other inhibitors that have been reported recently are devoid of good antibacterial activity. However, because of the protein's conformational variations, finding inhibitors for this target is difficult. For a potent molecule to target the MurA enzyme active site, the inhibitor should be small in size while binding adjacent to substrate UNAG just like the known drug fosfomycin or mimic the interaction of the first substrate with the protein in open conformation. It has been reported that closed conformation is the catalytically active form and necessary for a reaction to proceed, and identifying inhibitors that prevent close state formation by blocking closure of the domain would be the best alternative in drug discovery. The detailed discussed criteria of inhibitors and their interaction modes could be considered while developing a strategy for identifying MurA inhibitors.

Supplementary Information

Below is the link to the electronic supplementary material.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.