AN EXHAUSTIVE REVIEW ON EMERGING DRUG TARGETS OF TUBERCULOSIS WITH SPECIAL EMPHASIS ON CELL WALL SYNTHESIS

Tuberculosis (TB) is one of the top 10 causes of mortality and morbidity. Worldwide, yet, it has been over 60 years since a novel drug was introduced in market to treat the disease exclusively. Increased number of drug resistant TB cases has prompted the search for novel potent anti-TB drug. Mycobacterial cell wall has unique structure which provides integrity to the cell. The future development of new potent anti-TB drug targets is associated with the synthesis of various cell wall constituents; the structural and genetic information about mycobacterial cell wall envelope is now available. In the present review, we have focused on prospective drug targets that can be optimum triumph for successful drug candidate.


INTRODUCTION
Tuberculosis (TB), the infection caused by Mycobacterium species, is the major disease across the globe. There are three species of mycobacteria which are prominently observed in major population of patients. The species are Mycobacterium tuberculosis (Mtb), Mycobacterium avium, and Mycobacterium smegmatis out of which Mtb infection is more common. TB is one of the top 10 causes of death worldwide. According to recent World Health Organization's report, over 10 million people get infected with TB. Despite widespread use of BCG vaccine, TB infection has not been reduced as compared to other diseases [1].
The disease has reemerged as growing global health problem not only because of lack of proper therapeutic agents but also due to the development of drug resistance by mycobacterial strains. The emergence of multidrug-resistant (MDR) and extremely drug resistance (XDR) strains of this lethal pathogen renders current treatment strategies very difficult, and in some cases, there is complete failure [2]. Thus, the development of new anti-TB drug is an urgent need. The unrevealing of mycobacterial cell wall envelope has given new key to successful development of anti-TB drugs. The review describes about overview of current treatment its drawback and future prospective of drug development against TB infection.

CATEGORIES OF TB INFECTIONS
There are four categories of TB infection. Primary infection Category I belongs to new cases of sputum smear-positive pulmonary or extrapulmonary TB infection. Category II belongs to patients which are defaulted, irregularly treated, or sometimes relapsed. Category III is MDR TB where the patient is resistant to rifampicin which is given orally and needs further injectables and fluoroquinolones with combination. Category IV is extensively XDR TB which is resistant to two first-line agents (isoniazid and rifampicin), to any one fluoro-quinolone, and to any one second-line drug like amikacin [3].

M. avium complex (MAC)
MAC is an atypical mycobacterial infection, that is, one with nontuberculous mycobacteria which consists of two mycobacterial species M. avium and Mycobacterium intracellulare. This infection causes respiratory illness in patients, especially in immunocompromised people. It is also called as opportunistic infection, because it develops when cell-mediated immunity is markedly depressed (CD4 count drops to <50 cells/µL).

CURRENT SCENARIO OF TB INFECTION
At present, 10.0 million people around the world acquire TB disease, out of which 1 million cases occurred in infants and children. 1.6 million TB-related deaths worldwide including 0.3 million infected with HIV. Multidrug-resistant TB (MDR-TB) remains a public health crisis, the WHO estimated that there were 0.6 million new cases with resistance to rifampicin. About 82% had MDR-TB. Globally, TB incidence is declining about 2% per year, which should be accelerated to 4-5% to reduce TB risk. The summarized view of statistical data is depicted in Fig. 1.

Group V (Drugs with unclear efficacy)
These drugs are used when the patient develops XDR-TB phase of infection. These are broad spectrum antibiotics such as broad-spectrum penicillins, sulfonamides, and macrolide antibiotics. Commonly used are amoxicillin/clavulanic acid, thiacetazone, clarithromycin, clofazemine, linezolid, and imipenem [4][5][6][7][8]. The common anti-TB drugs with their mode of action, binding site, and category are summarized as depicted below ( Fig. 2 and Table 1).
The anti-TB agents are prescribed in combination as per the guidelines given by the WHO. The Directly Observed Therapy Short-course (DOTS) program is followed for the treatment of TB. The patients are grouped according to site and severity of disease, sputum smear condition, and patient history. The DOTS therapy regimen is summarized in Table 2.

TARGETS OF ANTI-TB TREATMENT
Although the disease having wide range of drugs serving the treatment, still there is a need to develop new drug candidate as the current treatment is having two major problems one is discontinuation of therapy by patient due to longer duration of time and second is the development of resistance to maximum drug candidates by mycobacterial strains over the period. For the development of drugs, the research has new dimension of genomics which provide information about the nature of enzymes and metabolites of the pathogens, which will help to design scaffold for potential drug targets. To develop new anti-TB drug, mycobacterial cell envelope is attractive core. Mycobacteria have unique cell wall structure, which regulates major cell functions. The mycobacterial cell has been explored for enzymatic and genomic pathways which can be targeted for drug discovery in the past few years numerous gene targets have been explored for the anti-tubercular drug development across the globe. Out of which cell wall synthesis/cell envelope was the prime target for researchers. The targets are classified based on its presence in the cell wall [9,10].
To understand the targets, study of components of cell membrane of the mycobacteria is to be done. The mycobacterial cell wall primarily comprises arabinogalactan, peptidoglycan, and mycolic acid. Other than these components, it contains carrier proteins, carbohydrates, and lipids. The detailed structure of mycobacterial cell membrane is described in the diagram below (Fig. 3).

COMPONENTS OF MYCOBACTERIAL CELL WALL
Chiaradia et al. examined the mycobacterial cell membrane by transmission electron microscopy and biochemical analysis. The results determined composition of the mycobacterial cell wall. It contains more than 2100 proteins between plasma membrane and mycobacterial cell wall. Among these, the mannosyl transferase (PimB), galactofuranosyl transferase (GlfT2), Cytochrome p450, and ABC transporter (YjfF) were most abundant in the plasma membrane. Plasma membrane also contains lipoglycans, phospholipids including phosphatidylinositol mannosides, and other glycolipids [11]. Antigen 85 complex proteins, porins, putative transporters, and mammalian cell entry protein family were mostly found in mycobacterial cell wall fraction that contains mycolic acid esterifying arabinogalactan constituting the inner leaflet of mycomembrane, along with glycolipids, phospholipids, lipoglycans, and proteins [12].
The cell wall of mycobacteria contains outermost layer (OL) which constitutes proteins with carbohydrate and negligible amount of lipids. After the OL layer of mycolic acid, mycomembrane (MM), arabinogalactan (AG), and peptidoglycan (PG) are present, respectively. The inner leaflet of the MM is made of very long-chain fatty acids (mycolic acids) esterifying (AG), covalently attached to (PG). The outer leaflet of the MM is presumably composed of lipids extractable with organic solvents, which includes phospholipids, trehalose mycolates, glycopeptidolipids, and lipoglycans. A periplasmic space separates the cell wall from the conventional lipid bilayer of plasma membrane (PM) of phospholipids and proteins. The cell envelop has different components regulated by various enzymes. The enzymes are primary targets for cell wall inhibition. Mycolic acid metabolism is considered as upcoming target related to mycolic acid [13].

Peptidoglycan biosynthesis
Peptidoglycan is the essential cell wall component of mycobacterial cell wall which gives rigidity to cell. Peptidoglycan consists of N-Acetyl Glucosamine (NAG), N-Glycolyl muramic acid (NAM), and alanine (Al). The peptidoglycan biosynthesis is targeted for anti-TB drug development from decades but still any drug candidate other than D-clycoserine has not shown promising effect, D-cycloserine inhibits peptidoglycan biosynthesis by inhibiting essential enzymes alanine racemase (Alr) and D-Ala-D-Ala ligase. It was predicted that bacitracin antibiotic also targets peptidoglycan biosynthesis but exact mechanism is not known. Another target in peptidoglycan synthesis is glutamate racemase for which β-chloro-D-alanine and its derivatives are under study, the exact mechanism is not identified but the enzyme is considered as potential target. The peptidoglycan biosynthesis can be inhibited by alanine like small molecules which can act as competitive inhibitor of alanine inhibiting attachment of alanine to NAG and NAM to form peptidoglycan [14][15][16][17][18].

Arabinogalactan biosynthesis
The arabinogalactan layer is present just next to the peptidoglycan layer. It contains sugars like rhamnose, galactose, phospholipids, and carrier proteins. The next important target in the mycobacterial cell wall is arabinogalactan synthesis. Ethambutol is the one of the extensively used anti-TB drugs which act through inhibition of arabinogalactan synthesis through inhibition of arabinosyltransferase enzyme.
Uridine diphosphate-galactopyranose (UDP-galactopyranose) is also identified as important enzyme in this pathway which involves in attachment of galactose in the arabinogalactan. Recent studies reported that the biosynthesis of the cell wall galactan of mycobacteria through Rv3808c protein can be inhibited through galactosyl transferase enzymes. Decaprenyl phosphorylase-2-epimerases are challenging target in the mycobacterial synthesis because targeting this enzyme has many challenges due to its oxidoreductive property responsible for degeneration of chemical structure of drug. A prodrug approach toward the drug development can be considered as novel way to target the enzyme, benzothiazinone which inhibits the enzyme and is under clinical investigation. From the description of enzymes, one can predict small cyclic molecules with structural similarity to galactose with hetero-atoms as potent candidates to target the pathway [19][20][21].

Mycolic acid biosynthesis and metabolism
Mycolic acid is unique feature of mycobacteria it is fatty acid containing carbon chain of 70 carbons. It makes the mycobacteria more resistant to chemical damage and dehydration. It also helps mycobacteria to grow inside the macrophages by effectively hiding from host immune system.

Fatty acid synthesis I pathway (FAS I)
The pathway includes synthesis of C 26 straight chain fatty acids, which involves enzymes such as Ketoacyl-ACP synthases (CasA) and Catalaseperoxidase (KatG). The enzymes are inhibited by popular drugs such as isoniazid, pyrazinamide, and ethionamide. The drugs are susceptible to resistance due to mutations in genes regulating the enzyme. The ketoacyl-ACP synthases enzyme is indirectly involved in mechanism of many drugs but direct target for the enzyme is under study. The KatG is involved in activation of isoniazid into isoform to the anion of nicotinic acid, which further inhibits the cell all synthesis by inhibiting FAS II pathway competitively. FAS I pathway can be inhibited by chemical moieties such as thiazolidine linezolid and heterocyclic five and sixmembered derivatives [22,23].

Fatty acid synthesis II pathway (FAS II)
The pathway further involves in chain elongation and addition of functional groups to the chain. It involves enzymes such as enoyl acyl carrier protein reductase (InhA) and EthA gene. The well-known drug isoniazid inhibits (InhA) enzymes after activation by KatG. EthA gene is another target on the FAS II pathway which is inhibited by ethionamide.

Fatty acid synthesis condensing pathway
The pathways involve condensation and attachment of mycolic acid to arabinogalactan layer. The enzymes involved in pathway are polyketide synthase (Pks13) and fatty acid desaturase (Fad13/Fad32). The pathways are not having any current potential anti-TB targets. However, there are numerous drugs and enzyme pathways such as acyl-adenylate monophosphate ligase (acyl-AMP ligase), FadD32, acyl-coenzyme A (CoA) carboxylase. Benzofuran, thiophene, and coumarin derivative inhibit the fatty acid synthesis condensing pathway [26].

ANTI-TB SCAFFOLDS IN PIPELINE
The era of anti-TB drug development is rapidly changing from phenotypic to genotypic research. Till date, numerous scaffolds are explored for this purpose. The scaffolds screened are ranging from NCE's obtained from natural sources, biological extracts, and enzymes from genetically modified microorganisms and from synthetic sources. In the past 10 years, various scaffolds from synthetic source such as β-chloro-D-alanine, ethers, dinitrobenzamide (benzothiazinone), linezolid, thiocarbamides, cinnamic acid, benzofuran, and quinazolinones derivatives are used. Few natural products such as epigallocatechin, quinines, and coumarins are also explored for the development of new drug.

CONCLUSION
Today, it is very important to develop new anti-TB drugs. With the help of enzymatic and genetic pathways, we can achieve target-based drug development which will be helpful to overcome the problems associated with existing anti-TB therapy. The unique structure of mycobacterial cell wall makes it ideal for target-based drug development. The architecture of the mycobacterial cell wall is complex and represents a substantial permeability barrier that is made up of a wide variety of compounds including mycolic acids, free lipids, polysaccharides, glycolipids, and lipoarabinomannan. Many of these components are genera or species specific. Most of the existing anti-TB drug candidates targets cell wall to inhibit bacterial growth. Nevertheless, none of the known cell wall synthesis inhibitors are endowed with rapidly acting sterilizing activity, hence, these drugs are urgently needed in TB control programs. All apparently require actively replicating bacteria for efficacy. A problematic issue one addresses latent TB, a condition in which mycobacteria are not actively dividing or synthesizing cell wall component which is apparently prevalent in about one-third of the global population. At present, the anti-TB drug development should be focused on potent drug candidates who can withstand to MDR TB. The novel drug development includes targeting unexplored cell wall components for drug development, shortening duration of existing drug therapy, development of potent anti-TB drugs, and repurposing of old drugs.