key: cord-0078976-z50zrbkx
authors: Belete, Tafere Mulaw
title: Recent Progress in the Development of Novel Mycobacterium Cell Wall Inhibitor to Combat Drug-Resistant Tuberculosis
date: 2022-05-23
journal: Microbiol Insights
DOI: 10.1177/11786361221099878
sha: 789446536433f01e4cac7241381b810a7af99f5b
doc_id: 78976
cord_uid: z50zrbkx

Despite decades of research in drug development against TB, it is still the leading cause of death due to infectious diseases. The long treatment duration, patient noncompliance coupled with the ability of the tuberculosis bacilli to resist the current drugs increases multidrug-resistant tuberculosis that exacerbates the situation. Identification of novel drug targets is important for the advancement of drug development against Mycobacterium tuberculosis. The development of an effective treatment course that could help us eradicates TB. Hence, we require drugs that could eliminate the bacteria and shorten the treatment duration. This review briefly describes the available data on the peptidoglycan component structural characterization, identification of the metabolic pathway, and the key enzymes involved in the peptidoglycan synthesis, like N-Acetylglucosamine-1-phosphate uridyltransferase, mur enzyme, alanine racemase as well as their inhibition. Besides, this paper also provides studies on mycolic acid and arabinogalactan synthesis and the transport mechanisms that show considerable promise as new targets to develop a new product with their inhibiter.

This effect makes many research companies to develop a new anti-tuberculosis drug that can effectively cure both the MDR and XDR tuberculosis, reduce treatment duration, and increase patient compliance. Therefore, it is important to develop new anti-tuberculosis drugs that can inhibit both actively multiplying bacilli and a non-growing persistent population of MTB to prevent reactivation of the infection. 6, 7 Over the past 50 years 2 drugs approved by the Food and Drug Administration (FDA) for the treatment of MDR-TB. Bedaquiline is the first drug approved for the treatment of multidrug-resistant TB in 2012 and pretomanid approved in 2019 as part of a 3-drug regimen (bedaquiline, pretomanid, and linezolid (BPaL)) for the management of drug-resistant TB. Delamanid is another important nitroimidazole compound approved by EMA for the treatment of MDR-TB. 7, 8 This review briefly describes data on the mycolic acid, peptidoglycan (PG), arabinogalactan, and the key enzymes involved in these cell wall component synthesis, as well as their inhibition. This review also provides inspiring target-based approaches that are used to develop an effective inhibitor against selected enzymes from MTB.

Streptomycin was the first antibiotic isolated in 1943 with significant activity against MTB, thereby providing hope for TB management. Effective TB chemotherapy began in 1952. MTB has developed resistance to different drugs minimizing both treatment and control. 9 Between 1940 and 1980, several drugs 2 

have been developed like Isoniazid, p-Aminosalicylic acid, Ethambutol, Rifampicin, and Ofloxacin. The discovery of Rifampicin in the 1963s with a new mechanism shortened treatment duration to 9 months. When pyrazinamide was included in the treatment regimen, the management duration decreased to 6 months. Nowadays, TB management has 4 firstline drugs: Isoniazid, Pyrazinamide, Rifampin, and Ethambutol for the first 2 months followed by Isoniazid, and Rifampin for the next 4 months. 10 Antimycobacterial treatment success rates were 85%, and between 2000 and 2018, 58 million lives were saved. 11 The second-line drugs used for the management of MDR-TB include fluoroquinolones (moxifloxacin, levofloxacin, ofloxacin), aminoglycosides (kanamycin, amikacin), capreomycin, cycloserine, para-aminosalicylic acid, and thioamides (ethionamide, prothionamide) as shown in Table 1 . Suitable drugs regime are chosen based on efficacy, susceptibility, safety, and cost. The treatment duration of the intensive phase is 6 months when an injectable drug is added to the regime. The continuation phase without the injectable drug prolongs until 18 months after culture conversion. These drugs require a longer duration of treatment than first-line drugs resulting in toxic side effects and loss of adherence. As such, it is needed to develop new drugs that could solve the current challenges associated with the treatment of TB. 12, 13 The Search for Novel Drugs and the Era of Target-Based Approaches

The problem of multidrug resistance and persistent infection will be solved with the identification of new drugs with novel modes of action. The target-based screening was rendered possible by advances in the genomics and molecular genetics of mycobacteria enabled the identification, functional characterization, and genetic validation of potential targets. 39 The advantage of the molecular target-based approach is compound isolated as inhibitors modified to improve effectiveness and selectivity while decreasing side effects. 40 Because of their mycobacterial-specific metabolic processes, several cell wall synthetic enzymes appeared to be promising candidates agents. Most candidates recently identified and progressed to trials identified through high-throughput screening followed by a drug-to-target approach (eg, bedaquiline and PBTZ169); however, target-based approaches are extensively used in TB drug discovery programs. Now a day, several in silico approaches proposed novel compounds as promising anti-TB agents. 41 Ideal targets for TB drug discovery programs are proteins involved in crucial pathways of MTB that are either absent in humans or have low sequence identity to human homologs. Genomic and system biology studies are vital for novel target discovery and validation to increase the probability of successful drug discovery. 6, 41 

Mycobacterial tuberculosis (MTB) cell wall is essential to the survival of microorganisms by providing inherent resistance. This intrinsic resistance is due to the presence of thick, waxy tightly packed cell walls, and drug degrading enzymes. Cell wall synthesis gives several novel targets because synthetic enzymes do not possess homolog in humans. The current firstand second-line drugs inhibit enzymes involved in cell wall synthesis. Resistance to these drugs develops with mutations in the target enzymes or the activation pathways of the drugs, paving the way for new resistant strains. Resistance against these drugs makes the development of new drugs against the MTB cell wall. 42, 43 The MTB cell wall has 3 layers: PG, mycolic acids, and arabinogalactan covered with a capsule. The capsule consists of protein, polysaccharides, and several types of lipids (phthiocerol dimycocerosate, diacyl trehaloses, and phosphatidylethanolamine). 44 Besides these parts, different lipids components are available including non-covalently attached waxes and glycophospholipids that act as a permeability barrier against drugs and play a key role in the survival and pathogenesis of MTB. Several enzymes facilitate the formation of mycobacterial cell wall precursors in the cytoplasm and subsequent transportation into the periplasmic space and polymerization is used as a promising target to develop a new anti-mycobacterial drug. 45 These enzymes are mycobacterial-specific and do not have homologs in humans. The current available anti-TB drugs inhibit the synthesis of mycolic acids (Isoniazid and Ethionamide), arabinogalactan (Ethambutol), and PG (Cycloserine). 39, 45 PG Synthesis Inhibitor PG (murein) is an essential part of the cell wall found on the outside of the cell membrane of MTB that keeps the integrity of the cell by enduring pressure and maintains cell shape. PG also provides support to several cell components (proteins, teichoic acids), hence, it is essential for bacterial survival. 46 Enzymes that involve during PG synthesis provide a promising target to develop new drugs against TB. PG has linear glycan chains of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) moiety, cross-linked by trans peptide bridges. 39, 46 A set of recently identified PG synthesis pathways are summarized in Figure 1 .

N-Acetylglucosamine-1-phosphate uridyltransferase PG synthesis starts with the conversion of fructose-6-phosphate to UDP-N-acetylglucosamine (UDP-GlcNAc) by acetyltransferase and uridyltransferase activity of GlmU, a bifunctional enzyme that has 2 active sites. GlmU catalyzes the acetylation of glucosamine-1-phosphate by its acetyltransferase which further nucleotide activated through uridyltransferase, thus facilitating the synthesis of UDP-GlcNAc that is the starting material for PG synthesis. 47 The similarity of the GlmU uridyltransferase with human analog makes this enzyme, not a potential target. But, the lack of GlcN-1-P in humans makes the acetyltransferase domain the most promising drug target to develop a new anti-TB. 48 Currently, several groups of agents have been reported as promising inhibitors of GlmU, namely, aminoquinazolines, 2-phenylbenzofurans, arylamines, arylsulfonamides, nonspecific thiol reactive agents, GlcN-6-P and GlcN-1-P GlmU analogs, and diterpenoids extracted from medicinal plants.

sulfanyl}acetyl)-2-thienyl]acetic acid) with an IC 50 of 5.3 μM, and 2-amino-2,3-dideoxy-3-fluoro-α-d-glucopyranosyl phosphate posses excellent activity. 49, 50 The mur enzymes After the formation of UDP-GlcNAc several Mur enzymes (MurA-F) also contribute to PG synthesis. The first Mur enzyme (MurA) transfers enolypyruvate residues from phosphoenolpyruvate to UDPGlcNAc. Later, MurB catalyzes NADPH-dependent reduction of enolpyruvate residues to D-lactate forming UDPN-acetylmuramic acid (UDP-MurNAc). The muramic acid residues in MTB contain N-acetyl and N-glycolyl derivatizations. 49 Further, the synthesis of peptide part of the PG monomer unit (UDP-MurNAC-pentapeptide) is catalyzed by 4 essential Mur ligases MurC, MurD, MurE, and MurF catalyzes the addition of L-Ala, D -Glu, mDAP, and D-Ala-D-Ala to UDP-MurNAc respectively. The whole Mur enzymes are crucial for cell integrity and unique to prokaryotes that make these enzymes the most promising target to develop a new drug. 51, 52 In Gram-negative bacteria, fosfomycin inhibits the MurA enzyme, but mycobacterial MurA has no active site for fosfomycin attachment. Fosfomycin analogs that can inhibit MTB MurA are the most promising to develop a new drug. Several MurA inhibitors were identified using an in silico approach. 53 Peptidomimetic compounds and sulfonoxyanthranilic acid derivatives posses a promising inhibitors effect against MTB MurA enzyme. 54, 55 MurB enzyme is essential for the viability of bacterial cells and the absence of a homolog in eukaryotic cells makes the MurB enzyme, a potential target for several inhibitors. Several MurB inhibitors were reported using structure-based drug design approaches such as Sulfadoxine, Pyrimethamine, piperazine derivatives, and Chloropicolinate Amides. [56] [57] [58] [59] As there are significant similarities in the structures of MurC and MurD/MurE and MurF, it has a potential target to develop a drug that can block the Mur enzymes simultaneously. Glucosamine uridine analogs are inhibitors of MTB MurE enzyme. 59, 60 Alanine racemase inhibiter

In the cytoplasm, Alanine racemase enzymes catalyze the pyridoxal phosphate-dependent conversion of the L-alanine to D-alanine, which is essential in PG biosynthesis as shown in Figure 2 . D-Cycloserine is an analog of d-alanine used as a second-line drug for TB treatment. D-Cycloserine blocks the synthesis of PG by inhibiting Alanine racemase and D-alanine-D-alanine ligase. 60 Besides, D-cycloserine inhibits several PLP-dependent enzymes, inducing a severe adverse effect, and enters into the central nervous system to induce severe neurological side effects like headaches, drowsiness, depression, dizziness. So that, several studies are undertaken to develop a non-alanine analog inhibitor of Alanine racemase to minimize the off-target effects of D-cycloserine. Thiazolidinediones inhibit Alanine racemase with IC 50 lower than 1 μM. But, their inhibition of MTB growth was not related only to Alanine racemase inhibition, since their effects could not be attenuated by providing exogenous d-alanine. 61, 62 

Overall, 80% of the peptides are cross-linked in 2 types of linkages to keep the mycobacterial cell envelope during growth and non-replicating conditions. PG structure cross-linking is catalyzed by d,d-transpeptidases (penicillin-binding proteins) and typically by the combined action of non-classical l,d-transpeptidases (Ldts) and d,d-carboxypeptidases. PG structure cross-linked mainly by 4→3 transpeptidase linkages by D, D-transpeptidase enzyme, inhibited by β-lactam antibiotic. But, there are non-classical 3→3 transpeptide linkage predominate in the PG layer of non-replicating M. tuberculosis. L,Dtranspeptidase (LdtMt2) is responsible for 3 to 3 transpeptide linkages in the PG layer. 63 Carbapenem antibiotics inhibit d,dtranspeptidases, l,d-transpeptidases and d,d-carboxypeptidases enzyme. Ldt Mt2 is essential for M. tuberculosis virulence and intrinsic resistance to β-lactam. Disruption of the ldt Mt2 gene 

Microbiology Insights abolishes bacterial virulence, alters colony morphology, and increased susceptibility to amoxicillin-clavulanate. Hence, LdtMt2 is considered as a potential anti-tuberculosis drug target that is inhibited by carbapenems and has attracted considerable attention as a potential anti-tuberculosis target. 64, 65 The next step catalyzed by Phospho-MurNAc-pentapeptide translocase (MurX/MraY, translocase I) that links the UDP-MurNAc/Glyc-L-Ala-D-Glu-meso-DAP-D-Ala-D-Ala to a decaprenyl phosphate (C50-P) to form lipid I. MraY is a promising target because it is vital in MTB survival. Several MraY inhibitor including Capuramycin, tunicamycin, and muraymycin D2 have promising effects to design new inhibitors. 66 Sansanmycin and its analogs have been reported to inhibit mraY and murX. 67 MurG catalyzes the transfer of GlcNAc from UDP-GlcNAc to MurNAc or MurNGlyc of Lipid I to produce Lipid II. MurG activity is blocked by a lipoglycodepsipeptide antibiotic like ramoplanin and enduracidin that binds the lipid component. 68 Then, the MurT-GatD complex and AsnB amidate the a-carboxyl group of D-glutamate and the D-carboxyl group of meso-DAP to synthesize amidated Lipid II, respectively. These amidations are essential for PG cross-linking, and as such, the MurT-GatD complex and AsnB are the most promising targets to develop PG targeting antibiotics. 69 The soluble UDP-MurNAc-pentapeptide translocate into the periplasmic space. The penicillin-binding proteins (PBPs) carry out transglycosylation and transpeptidation reactions to form mature PG in the periplasmic space. The MurJ Translocat Lipid II from the cytoplasm into the periplasmic space has a promising target to develop new agents including humimycin, b-lactam potentiators. 70 But, the MurJ enzyme in MTB must be characterized and further study will help to develop a new MTB MurJ inhibitor. Ramoplanin, teixobactin, malacidin, and nisin bind Lipid II but the glycopeptides teicoplanin and vancomycin bind D-Ala-D-Ala terminus of lipid II blocking lipid II elongation. 69, 71 Once the lipid II is transported into the periplasmic space transglycosylase enzyme catalyzes the addition of the Lipid II to PG chains. 69 The moenomycin blocks the transglycosylase activity of PBPs while b-lactam blocks the transpeptidase effect of PBPs. The b-lactam antibiotics have no activity against mycobacteria due to b-lactamase-BlaC that breaks down the b-lactam ring. carbapenem could not be broken down by BlaC and combined with b-lactamase inhibitor are effective in killing Mycobacteria. Due to this effect, meropenem plus clavulanic acid with amoxicillin (Mero/Clv/Amx) WHO endorsed the use of this regimen for the treatment of DR-TB. Therefore, developing effective anti-tuberculosis carbapenem agent continued. 33, 72 Arabinogalactan synthesis inhibitor Arabinogalactan (AG) is a heteropolysaccharide that is covalently tethered to ~10% of the muramic acid residues of PG. AG consist of galactose and arabinose that constitute ~35% of the cell wall. Both sugars exist as d-arabinofuranose (Araf) and d-galactofuranose (Galf) that are scare in nature. AG has 3 parts: the linker unit (LU), the galactan chain, and the arabinan chain. 73 The d-galactofuranose posses 30 linear Galf residues linked to the rhamnosyl residue of the LU. The arabinofuranose is a branched polysaccharide with 3 tricosamer domains, each contains 23 Araf residues, linked to 8th, 10th, and 12th Galf residues of the galactan domain as shown in Figure 3 . 73, 74 GlcNAc-1-P transferase AG synthesis starts in the cytoplasm by forming the linker unit (LU) that connects PG to AG.

LU synthesis begins on decaprenyl-phosphate (C 50 -P) where GlcNAc-1-P transferase transfers GlcNAc-1-P to C50-P to form C 50 -P-P-GlcNAc referred to as glycolipid 1 (GL-1). GlcNAc-1-P transferase (WecA) enzyme is a promising target to develop a new drug. It is inhibited by tunicamycin and caprazamycin derivatives, such as CPZEN-45. 39, 75 Several In-vitro studies revealed that CPZEN-45 is active in replicating and non-replicating bacteria with potent MIC (3 µg/mL). 76

Then rhamnosyltransferase (WbbL) catalyzes the transfer of L-rhamnose (L-Rhap) from dTDP-L-Rha to 3-position of the GlcNAc of GL-1 forming glycolipid 2 (GL-2) or the linker unit (C50-P-P-GlcNAc-L-Rha). The formation of GL-2 by using rhamnosyltransferase is a promising novel target to develop a new drug. 77 

The linker unit provides an attachment site for the polymerization of the galactan chain in the cytoplasm. The bifunctional galactofuranosyltransferases (GlfT1 and GlfT2) catalyze the synthesis of the galactan chain. Initially, GlfT1 transfers Galf from UDP-Galf to the C-4 position of L-Rha, and then adds a second Galf residue to the C-5 position of the primary Galf, generating C50-P-P-GlcNAc-L-Rha-Galf1-Galf2. GlfT2 add more Galf residues to the growing galactan chain with alternating β (1→5) and β (1→6) glycosidic linkages. 78 The galactan chains form C50-PP-GlcNAc-L-Rha-Galf. GlfT1 and GlfT2 are promising targets to develop a new drug. UDP-Galf derivatives are suitable inhibitors of these enzymes, whereby they cause premature galactan chain termination. 79 

Arabinofuranosyl transferase catalyzes the addition of the first Araf residues into galactan core (C50-P-P-GlcNAc-L-Rha-Galf ) to form decaprenylphosphoryl-D-arabinose (DPA). DPA synthesis occurs from phospho-α-D-ribosyl-1pyrophosphate (pRpp). The pRpp synthetase transfers pyrophosphate from ATP into C-1 of ribose-5-phosphate to produce pRpp. A decaprenyl moiety is added, catalyzed by UbiA (decaprenol-1-phosphate 5-phosphoribosyltransferase) to form decaprenol-1-monophosphate 5-phosphoribose. Then phospholipid phosphatase catalyzes C-5 dephosphorylation to form decaprenol-1-phosphoribose (DPR). Finally, DPA is formed by epimerization of the ribose C-2 hydroxyl, catalyzed by decaprenylphosphoribose-2-epimerase consisting of subunits DprE1 andDprE2. 80, 81 Several compounds effectively inhibit epimerase activity of MTB'S DprE1 as shown in Table  2 . Nitrobenzothiazinone (BTZ-043) has a MIC of 1 ng/mL (0.23 nM) against replicating and non-replicating MTB and entered Phase1b/IIa clinical trials. BTZ-043 showed selective effect against MTB activity against both MDR and XDR-MTB strains, synergy with drug bedaquilin, and safety profile.

The derivative of BTZ-043, PBTZ169 is in Phase II with MIC 0.19 ng/mL. PBTZ169 has better advantages over its predecessor including, a simple synthetic route and better pharmacodynamics. 82, 83 Recent studies identified the Rv3789 gene that transports DPA to the periplasmic space to donate arabinose for the synthesis of AG. Several ArafTs contribute to the assembly of arabinan into AG. AftA transfers 3 single Araf residues to the 8th, 10th, and 12th Galf residues of AG. Further α (1→5) polymerization of the arabinan domain catalyzed by EmbA and EmbB. The AftC and AftD responsible for the α (1→3) branching of the internal arabinan domain of AG. AftB catalyzes the transfer of β (1→2) Araf residues from DPA to the hexa-arabinofuranoside motif of AG. Ethambutol is the first line of anti-TB drug that inhibits AG synthesis by blocking the effect of arabinosyl transferase. 84 

Rhamnose is a saccharide component essential for the virulence of pathogenic bacteria. The synthesis of rhamnose is vital for mycobacterial cell growth. Inhibiting rhamnose sugar synthesis has potential drug targets because rhamnose sugar synthesis is absent in humans. l-Rhamnose synthesis comes from glucose-1-phosphate and dTTP (deoxythymidine triphosphate) by 4 different enzymes. 93 Glucose-1-phosphate thymidyl transferase (RmlA), couples the glucose-1-phosphate moiety to deoxythymidine triphosphate. dTDP-d glucose 4,6-dehydratase (RmlB), then oxidizes the 4 hydroxyls and dehydrates the 6 hydroxyls. dTDP-6-deoxy-d-xylo-4-hexulose 3,5-epimerase (RmlC), inverts the 3 and 5 hydroxyls, creating an unstable ring structure that flips. Finally, dTDP-6-deoxyd-xylo-4-hexulose reductase (RmlD) reduces the 4 ketones to form dTDP-rhamnose. Currently, the RmlC is the best promising drug target, because RmlC is specific, unique, and does not need cofactor binding. 94 Triazinoindol-benzimidazolones inhibit RmlC enzyme. 95

Mycolic acids (MAs), 2-alkyl, 3-hydroxy long-chain fatty acids (FAs), are the hallmark of the cell envelope of Mycobacterium tuberculosis. MAs are characterized by very hydrophobic C54 to C63 fatty acids with C22 to C24 α side chain. MA in M. tuberculosis exists as α, methoxy, and keto-mycolic acids. The α-mycolic acid is the most abundant (70%), whereas methoxy and ketone mycolic acids are the minor components (10%-15%). MA synthesis inhibiter has a promising anti-tubercular effect because it enhances the permeability and sensitivity of the organism to antibiotics. 96, 97 The synthesis of mycolic acids occurs in different stages as shown in Figure 4 . The first is the de novo fatty acids (C16-26) that occur in the FAS-I pathway. These fatty acids help to form the a-alkyl branch (C24) or further elongated by the FAS II pathway to give fatty acids up to C56 to produce mycolic acids that are essential for the growth and survival of mycobacterium. The mammalian fatty acid synthesis is similar to the FAS I pathway, making the FAS II pathway unique to microbes. 98 FAS-II pathway begins with a condensation reaction catalyzed by FabH (β-ketoacyl-ACP synthase III) using malonyl-AcpM and palmitoyl-CoA to produce a β-ketoacyl-AcpM intermediate. FabH enzyme links the FAS-I and FAS-II pathways. Then β-ketoacyl-AcpM is reduced by MabA (β-ketoacyl-ACP reductase), yielding β-hydroxy acyl-AcpM that is dehydrated by HadAB/BC (β-hydroxy acyl-ACP dehydratase) to produce a trans-2-enoyl-AcpM intermediate. This is then reduced by InhA (enoyl-ACP reductase) to produce an acyl-AcpM elongated by 2 carbons. The FAS-II cycle continues with further condensation, catalyzed by KasA/B (β-ketoacyl-ACP synthase), repeating the above cycle via MabA, HadAB/BC, and InhA and continues until the acyl chain reaches C42-62, producing the saturated long-chain meromycolate. 97, 98 Modification of long-chain meromycolate introduces functional groups including cis-/trans-cyclopropanation, keto, and methoxy groups by CmaA1-2-, MmaA1-4-, and PcaA-type enzymes. The meromycolic acid chain (C42-62) is activated for condensation by generation of a meromycolyl-AMP by fatty acid adenylating enzymes (FadD32). The meromycolyl-AMP linked to α-alkyl short-chain (C22-24) in a reaction catalyzed by polyketide synthase (PKS13) to produce α-alkyl-β-ketomycolic acid that subsequently reduced. The mycolates in the cytoplasm are converted to trehalose monomycolate (TMM), which are effluxed out through mycobacterial membrane proteins large (MmpL3). The mycolate on the translocated TMM is then coupled to AG through the mycolyltransferase Antigen 85 complex completing the mAGP assembly. Antigen 85 complex also catalyzes the synthesis of membrane unbound mycolates such as trehalose dimycolate (TDM) from TMM. 99, 100 Mycolic acids targets and their inhibitors β-Ketoacyl-acyl carrier protein synthase III (KAS III, FabH) inhibitor. FabH controls the initial step of FAS II by catalyzing the condensation of acetyl-CoA with a malonyl-acyl carrier protein (ACP) to form β-ketoacyl-ACP. The significance of FabH for bacterial viability makes it an ideal target for the development of novel bactericides. 101 In the past decades, several FabH inhibitors were reported such as platensimycin, platencin, thiazole, and pyrazol-benzimidazole amide derivatives. 102, 103 Enoyl-ACP reductase (InhA) inhibitors. InhA is a clinically validated MTB target, as highlighted by the success of Isoniazid in treating patients with TB. Triclosan is one of the first InhA inhibitors identified, had a submicromolar (0.2 μM) IC 50 toward the target, but its antimycobacterial activity was weak with a MIC of 64 μM. 102 Isoniazid and Ethionamide inhibit enoyl-ACP reductase (InhA) that is involved in the synthesis of mycolic acids that is crucial for growth and virulence. Isoniazid is prodrugs that are activated by catalase-peroxidase KatG enzyme-producing reactive oxygen species that inhibit InhA and this bioactivation step is where MTB primarily develops resistance by inactivating this enzyme. Therefore, direct InhA inhibitors targeting MDR-TB would solve the risk of crossresistance. 104, 105 Currently, several direct InhA inhibitors have been identified such as thiadiazoles-based chemical inhibitors (GSK693), Pyridomycin, Pyrrolidine, Carboxamides, Pyrroles, Acetamides, Thiadiazoles, and Triazole act without prodrug activation. Pyrrolidine and carboxamides were the best in inhibiting InhA with good bioavailability and better MIC. 106 KasA and KasB inhibiter. The enzyme β-Ketoacyl ACP synthases (KasA and KasB) is a promising drug target in the mycolic acid pathway of Mycobacterium tuberculosis (MTB). 98 KasA inhibitor causes cell lysis signifying this enzyme plays a vital role in the MA pathway. 107 MmpL3 inhibitors. MmpL3 export trehalose-monomycolates (TMM), precursors for trehalose-dimycolates and mycolic acids that build the mycobacterial cell wall. MmpL3 inhibitors weaken the mycobacterial cell wall and causes cell death. This highlights the promising novel target of MmpL3. 108 Several compounds were identified as MmpL3 inhibitors. SQ109 is in Phase 2b-3 trial and reported safe and effective. BM212/ BM635, NITD-304, and NITD-349 showed good preclinical safety. 109, 110 Polyketide synthase 13. Polyketide synthase 13 (Pks13) plays a key role in the mycolic acid synthesis and is essential in MTB survival. Pks13 condenses 2 fatty acyl chains to produce α-alkyl β-ketoesters that serve as the precursors for the synthesis of mycolic acids. 102 Therefore, Pks13 is a promising target for a new antituberculosis drug. Currently, several Pks13 inhibitors have been reported including Cerulenin, plastensimycin, thiolactomycin, thiophene compounds, β-lactones, benzofuran derivatives (MIC 2.0 µM) and coumestan compounds. 61, 111, 112 Antigen 85 complex inhibitors. Antigen 85 complex enzyme has 3 homologous (Ag85A, Ag85B, Ag85C). The Ag85C homolog catalyzes the conversion of TMMs to trehalose dimycolates (TDMs) before being attached to AG. Ag85C also helps the mycobacteria to evade the host immune response by inhibiting the formation of phagolysosomes to eradicate the infection. It has a key role in MTB virulence. 113 The Ag85C emerged as a promising novel drug target due to its key role in forming major parts of the mycobacterial membrane. Mutation of the Ag85C gene reveals ∼40% decrease in the mycolic acid amount in the cell wall. Currently, several compounds like CyC (CyC 7 β, CyC 8 β, CyC 17 , Phosphonic acids, ebselen, Cyclipostins, and cyclophostin inhibition Ag85C causing the incorporation of MAs into the mycobacterial cell wall. 114, 115 FadD32 inhibitor. FadD32, a fatty acyl-AMP ligase (FAAL32) involved in the synthesis of mycolic acids. FadD32 catalyzes the conversion of fatty acid to acyl-adenylate (acyl-AMP) in the presence of adenosine triphosphate. It is conserved in all the mycobacterial species. Thus, FadD32 represents a promising target for the development of novel antituberculous drugs. 116 Coumarin compounds showed a good antimycobacterial activity (MIC 90 = 0.24-5.6 µM) against drug-susceptible and drug-resistant TB, species that provided a starting point to validate FadD32 as a promising target for the treatment of TB. But, this class of agents has short half-lives. Modification of the metabolically labile coumarin core with a 2-quinolone core achieved high antimycobacterial activity, with MIC value of 0.5 µg/mL against mycobacterial TB. 117

The emerging drug-resistant MTB to the current drugs makes the eradication of TB remains a global challenge. Therefore, it is urgent to develop a new drug with a novel target that does not suffer from cross-resistance with the existing drugs. It is also interesting to identify novel targets that are vital for replicating and non-replicating mycobacteria. Understanding the mechanism of action of drugs may shed light on structural elements that are needed for multi-targeting strategies. Mycobacterial cell wall synthesis inhibiter is a cornerstone for tuberculosis management. Several studies showed that enzymes are involved in cell wall synthesis and proceed to be potential drug targets in antimycobacterial drug discovery. The whole cell phenotypic screening and target-based development approaches facilitate new groups of drug candidates being identified as mycobacterial cell wall inhibitors with excellent antimycobacterial activity. This increased a ray of hope in the discovery of new drugs that will control the problem of drug resistance.

Future TB drug regimens should include inhibitors having non-canonical targets or binding different regions of the same target or blocking all alternate paths may be more effective, less prone to resistance, and may shorten therapy duration. The scientific community should try attaining a cumulative treatment outcome from different approaches, fill gaps that exist within a strategy, pursue newer treatment strategies, and continuously engage in the socio-political establishment to achieve a holistic control strategy against MTB.

Global tuberculosis report 2020 -reflections on the Global TB burden, treatment and prevention efforts

How COVID-19 can instruct TB research: ensuring the safety of researchers exposed to infectious disease

Increasing prevalence of resistance to second-line drugs among multidrug-resistant Mycobacterium tuberculosis isolates in Kuwait

Comparing costeffectiveness of standardised tuberculosis treatments given varying drug resistance

Regimens to treat multidrug-resistant tuberculosis: past, present and future perspectives

Tuberculosis: past, present and future of the treatment and drug discovery research

Curving tuberculosis: current trends and future needs

An update on multidrug-resistant tuberculosis

Antibiotics from natural sources

Molecular Epidemiology Study of Mycobacterium Tuberculosis Complex

Post-tuberculosis lung disease: clinical review of an under-recognised global challenge

World Health Organization recommendations on the treatment of drug-resistant tuberculosis, 2020 update

Classifying new anti-tuberculosis drugs: rationale and future perspectives

Resistance to rifampicin: a review

du Preez I. Metabolomics describes previously unknown toxicity mechanisms of isoniazid and rifampicin

Two novel katG mutations conferring isoniazid resistance in Mycobacterium tuberculosis

The molecular basis of pyrazinamide activity on Mycobacterium tuberculosis PanD

The high incidence of severe adverse events due to pyrazinamide in elderly patients with tuberculosis

Irreversible neuropathy in extremely-drug resistant tuberculosis: an unfortunate clinical conundrum

Detecting ethambutol resistance in Mycobacterium tuberculosis isolates in China: a comparison between phenotypic drug susceptibility testing methods and DNA sequencing of embAB

Molecular analysis of streptomycin-resistance associating genes in Mycobacterium tuberculosis isolates from Nepal

Prevalence and molecular characterization of amikacin resistance among Mycobacterium tuberculosis clinical isolates from southern China

Molecular characterization of amikacin, kanamycin and capreomycin resistance in M/ XDR-TB strains isolated in Thailand

Overview of anti-tuberculosis (TB) drugs and their resistance mechanisms

Fluoroquinolones, tuberculosis, and resistance

Resistance to isoniazid and ethionamide in Mycobacterium tuberculosis: genes, mutations, and causalities

Genetic mutations associated with isoniazid resistance in Mycobacterium tuberculosis in Mongolia

Molecular characterization of prothionamide-resistant Mycobacterium tuberculosis isolates in southern China

Identification of novel mutations associated with cycloserine resistance in Mycobacterium tuberculosis

Mycobacterium tuberculosis folate metabolism and the mechanistic basis for para-aminosalicylic acid susceptibility and resistance. Antimicrob Agents Chemother

Systematic review of mutations associated with resistance to the new and repurposed Mycobacterium tuberculosis drugs bedaquiline, clofazimine, linezolid, delamanid and pretomanid

Linezolid: a review of its properties, function, and use in critical care

Carbapenems against Mycobacterium tuberculosis: a review of the evidence

Mutation in an unannotated protein confers carbapenem resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother

Synthesis, antitubercular activity and mechanism of resistance of highly effective thiacetazone analogues

Delamanid resistance: update and clinical management

The role of delamanid in the treatment of drug-resistant tuberculosis

Mutations in f biD (Rv2983) as a novel determinant of resistance to pretomanid and delamanid in Mycobacterium tuberculosis

Mycobacterial cell wall biosynthesis: a multifaceted antibiotic target

The future for early-stage tuberculosis drug discovery

Progress in targeting cell envelope biogenesis in Mycobacterium tuberculosis

Cell wall peptidoglycan in Mycobacterium tuberculosis: an Achilles' heel for the TB-causing pathogen

Antibiotics and resistance: the twosided coin of the mycobacterial cell wall

Identification of inhibitors targeting Mycobacterium tuberculosis cell wall biosynthesis via dynamic combinatorial chemistry

Inhibition of the first step in synthesis of the mycobacterial cell wall core, catalyzed by the glcnac-1-phosphate transferase WecA, by the novel caprazamycin derivative CPZEN-45

New agents for the treatment of drugresistant Mycobacterium tuberculosis

Encyclopedia of Microbiology

Exploring MDR-TB inhibitory potential of 4-Aminoquinazolines as Mycobacterium tuberculosis N-Acetylglucosamine-1-Phosphate uridyltransferase (GlmUMTB) inhibitors

Mechanism and potential inhibitors of GlmU: a novel target for antimicrobial drug discovery

Design and synthesis of novel cell wall inhibitors of Mycobacterium tuberculosis GlmM and GlmU

The mur enzymes chink in the armour of Mycobacterium tuberculosis cell wall

Screening of antitubercular compound library identifies inhibitors of Mur enzymes in Mycobacterium tuberculosis

Molecular modelling and dynamic simulation of UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA) from Mycobacterium tuberculosis using in silico approach

Identification of peptidomimetic compounds as potential inhibitors against MurA enzyme of Mycobacterium tuberculosis

Comprehensive structural and functional characterization of Mycobacterium tuberculosis UDP-NAG enolpyruvyl transferase (Mtb-MurA) and prediction of its accurate binding affinities with inhibitors

Synthesis, in-vitro and in-silico study of novel thiazoles as potent antibacterial agents and MurB inhibitors

Repurposing of FDAapproved drugs to target MurB and MurE enzymes in Mycobacterium tuberculosis

Design and synthesis of "chloropicolinate amides and urea derivatives" as novel inhibitors for Mycobacterium tuberculosis

Design and synthesis of various 5′-Deoxy-5′-(4-substituted-1,2,3-triazol-1-yl)-uridine analogues as inhibitors of Mycobacterium tuberculosis Mur ligases

Mechanism-based inhibition of the Mycobacterium tuberculosis branched-chain aminotransferase by d-and l-cycloserine

Inhibition of D-Ala:D-Ala ligase through a phosphorylated form of the antibiotic D-cycloserine

Cell wall: a versatile fountain of drug targets in Mycobacterium tuberculosis

Revisiting anti-tuberculosis therapeutic strategies that target the peptidoglycan structure and synthesis

The 1-β-methyl group confers a lower affinity of l,d-transpeptidase LdtMt2 for ertapenem than for imipenem

The Mycobacterium tuberculosis protein LdtMt2 is a nonclassical transpeptidase required for virulence and resistance to amoxicillin

Therapeutic efficacy of SQ641-NE against Mycobacterium tuberculosis

Synthetic sansanmycin analogues as potent Mycobacterium tuberculosis translocase I inhibitors

Production of the ramoplanin activity analogue by double gene inactivation

Cell surface biosynthesis and remodeling pathways in mycobacteria reveal new drug targets

Chemistry of peptidoglycan in Mycobacterium tuberculosis life cycle: an off-the-wall balance of synthesis and degradation

Recent development of antibacterial agents to combat drug-resistant Gram-positive bacteria

Efficacy of β-lactam/β-lactamase inhibitor combination is linked to WhiB4-mediated changes in redox physiology of Mycobacterium tuberculosis

The mycobacterial cell wallpeptidoglycan and Arabinogalactan

Lcp1 is a phosphotransferase responsible for ligating arabinogalactan to peptidoglycan in Mycobacterium tuberculosis

Liposidomycin, the first reported nucleoside antibiotic inhibitor of peptidoglycan biosynthesis translocase I: the discovery of liposidomycin and related compounds with a perspective on their application to new antibiotics

N-acetylglucosamine-1-phosphate transferase, WecA, as a validated drug target in Mycobacterium tuberculosis

NDP-rhamnose biosynthesis and rhamnosyltransferases: building diverse glycoconjugates in nature

Biosynthesis of galactan in Mycobacterium tuberculosis as a viable TB drug target?

Potential inhibitors of galactofuranosyltransferase 2 (Glf T2): molecular docking, 3D-QSAR, and in silico ADMETox studies

Decaprenyl-phosphoryl-ribose 2′-epimerase (DprE1): challenging target for antitubercular drug discovery

The DprE1 enzyme, one of the most vulnerable targets of Mycobacterium tuberculosis

Afroz Bakht M. Synthetic molecules as DprE1 inhibitors: a patent review

Overview of the development of DprE1 inhibitors for combating the menace of tuberculosis

New tuberculosis drug targets, their inhibitors, and potential therapeutic impact

Identification of a new series of benzothiazinone derivatives with excellent antitubercular activity and improved pharmacokinetic profiles

The main results of clinical trials of the efficacy, safety and pharmacokinetics of the perspective anti-tuberculosis drug makozinone (PBTZ169)

OPC-167832, a novel carbostyril derivative with potent antituberculosis activity as a dpre1 inhibitor

Development of macozinone for TB treatment: an update

High-dose rifampicin, moxifloxacin, and SQ109 for treating tuberculosis: a multi-arm, multi-stage randomised controlled trial

Early bactericidal activity of AZD5847 in patients with pulmonary tuberculosis

The Medicines Patent Pool Announces First License for Tuberculosis Treatment. UNITAID

The downfall of TBA-354 -a possible explanation for its neurotoxicity via mass spectrometric imaging

RmlC, the third enzyme of dtdp-L-rhamnose pathway, is a new class of epimerase

Mycobacterium tuberculosisRmlC epimerase (Rv3465): a promising drug-target structure in the rhamnose pathway

Identification of triazinoindol-benzimidazolones as nanomolar inhibitors of the Mycobacterium tuberculosis enzyme TDP-6-deoxy-d-xylo-4-hexopyranosid-4-ulose 3,5-epimerase (RmlC)

Assembly of the mycobacterial cell wall

The genetics of cell wall biosynthesis in Mycobacterium tuberculosis

Mycolic acids: structures, biosynthesis, and beyond

Mycolyltrans ferase from Mycobacterium tuberculosis in covalent complex with tetrahydro lipstatin provides insights into antigen 85 catalysis

MmpL3 is the flippase for mycolic acids in mycobacteria

Discovery of natural FabH inhibitors using an immobilized enzyme column and their antibacterial activity against Xanthomonas oryzae pv. oryzae

Platensimycin and platencin: promising antibiotics for future application in human medicine

The use of platensimycin and platencin to fight antibiotic resistance

In vitro antimycobacterial activity and physicochemical characterization of diaryl ether triclosan analogues as potential InhA reductase inhibitors

Triclosan and its derivatives as antimycobacterial active agents

Enoyl-acyl carrier protein reductase (INHA): a remarkable target to exterminate tuberculosis

Identification of KasA as the cellular target of an anti-tubercular scaffold

MmpL3 inhibitors as antituberculosis drugs

MmpL3 inhibitors: diverse chemical scaffolds inhibit the same target

Lipid transporters: from bacteria, protozoa, fungi and plants, to mice and men

Benzofuran derivatives and their anti-tubercular, anti-bacterial activities

Development of a novel lead that targets M. tuberculosis polyketide synthase 13

Inactivation of the Mycobacterium tuberculosis antigen 85 complex by covalent, allosteric inhibitors

Identification of potent antigen 85C inhibitors of Mycobacterium tuberculosis via in-house lichen library and binding free energy studies Part-II

Mycobacterial antigen 85 complex (Ag85) as a target for ficolins and mannose-binding lectin

Discovery of heterocyclic replacements for the coumarin core of anti-tubercular FadD32 inhibitors

A review of current and promising nontuberculous mycobacteria antibiotics

I would like to acknowledge Mrs Fasika Abu for editing the paper.

The author confirms sole responsibility for the design, data collection, Drafting, and critical revision of the article, and final approval of the version to be published.

Tafere Mulaw Belete https://orcid.org/0000-0002-6634-5971

All data are provided in the manuscript or found from published papers as cited.