Mycobacterium tuberculosis: Pathogenesis and therapeutic targets

Abstract Tuberculosis (TB) remains a significant public health concern in the 21st century, especially due to drug resistance, coinfection with diseases like immunodeficiency syndrome (AIDS) and coronavirus disease 2019, and the lengthy and costly treatment protocols. In this review, we summarize the pathogenesis of TB infection, therapeutic targets, and corresponding modulators, including first‐line medications, current clinical trial drugs and molecules in preclinical assessment. Understanding the mechanisms of Mycobacterium tuberculosis (Mtb) infection and important biological targets can lead to innovative treatments. While most antitubercular agents target pathogen‐related processes, host‐directed therapy (HDT) modalities addressing immune defense, survival mechanisms, and immunopathology also hold promise. Mtb’s adaptation to the human host involves manipulating host cellular mechanisms, and HDT aims to disrupt this manipulation to enhance treatment effectiveness. Our review provides valuable insights for future anti‐TB drug development efforts.


INTRODUCTION
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a significant global health problem, despite advances in diagnosis and treatment.Approximately 10 million new cases of TB are diagnosed each year, with over a million deaths attributed to the disease. 1 TB is particularly prevalent in developing countries, where coinfection with human immunodeficiency virus (HIV) and poor living conditions are common. 2Additionally, the emergence of drug-resistant strains of Mtb poses a signif-icant challenge to global TB control efforts. 3The current standard regimen for drug-sensitive TB involves a combination of drugs, including isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA), and ethambutol (EMB).However, treatment can be lengthy and costly, with patients often requiring 6 months of therapy. 4Moreover, the emergence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) has further complicated the treatment landscape (Figure 1).
While progress has been made in the development of new TB drugs, there remains a significant need for novel treatment options.Several drug candidates are presently in diverse phases of clinical development (Figure 2), MedComm.2023;4:e353.
wileyonlinelibrary.com/journal/mco2 1 of 39 https://doi.org/10.1002/mco2.353F I G U R E 1 Grouping of medicines recommended for use in longer multidrug-resistant TB (MDR-TB) regimens by the WHO. 2 Group A drugs are strongly recommended for inclusion in all longer MDR-TB regimens containing fluoroquinolones, bedaquiline, and linezolid.Group B drugs were recommended to add one or two to the regimen to improve outcomes.Group C drugs were recommended as a secondary option to Group A and B drugs.

F I G U R E 2
Current global clinical pipeline of new tuberculosis drugs based on information provided by the Working Group for New TB Drugs (WGND). 156a Trial of high-dose Rifampin in patients with TB.
comprising reoptimized adaptations of current TB drugs and some involving original mechanisms of action.Additionally, several new targets for drug development have been identified. 4In the context of the coronavirus disease 2019 (COVID-19) pandemic, the impact of TB on global health has become even more severe.Individuals who have recovered from COVID-19 have been found to have a higher risk of developing TB, likely due to the negative impact of COVID-19 on the immune system. 5herefore, new TB treatment drugs remain an urgent research priority.
This review aims to identify potential targets that show promise for the development of new therapeutic approaches to treat TB, based on an understanding of the pathogenesis of the disease.The ability of Mtb to survive in the microenvironment of the human host is one of the greatest challenges faced when developing new drugs.We first elucidate the pathogenesis of Mtb, which encompasses the phases of invasion, proliferation, latency, and revival of the pathogen.By understanding the mechanisms, we can identify new drug targets and advance the development of more effective TB treatments.In the pathogenic process, Mtb acts as a pathogen causing TB, so targets within the bacterium and drugs that directly kill Mtb are of great interest.We reviewed all kinds of targets and drugs targeting Mtb.Simultaneously, Mtb provokes an immunoreaction in its host, while skillfully tampering with vital cellular processes, leading to protracted treatment and unfavorable prognosis.This review will summarize the latest research identifying high-value targets in host-targeted therapies and describe the related modulaters.

PATHOGENESIS OF Mtb
Mtb complex (MTBC) has persistently accompanied our species, anatomically modern humans, during our evolutionary journey and widespread dispersion across the globe throughout the past 70 millennia.Mtb initiates its life cycle upon invasion of the respiratory tract and lungs (Figure 3), as the microorganism is classically believed to thrive only within living organisms. 6,7This initial interaction, commonly referred to as primary infection, marks the onset of contact between the pathogen and the respiratory system.There has been a contention that the coevolution between Mtb and its human host does not follow the conventional trajectory of an evolutionary arms-race.Instead, it is suggested to be characterized by mechanisms of manipulation. 7Following exposure to multiple host cell receptors (including toll-like receptors [TLRs], C-type lectin receptors [CLRs], dendritic cells [DCs], mannose receptors [MRs], and NOD-like receptors [NLRs]) and internalization by alveolar macrophages and DCs, Mtb undergoes a phase of intracellular replication.Subsequently, infected cells spread to lymph nodes while attempting to destroy the bacteria with various proteolytic enzymes and cytokines (e.g., tumor necrosis factor alpha [TNF-α] and interferon gamma [IFN-γ]), perpetuating spread of the pathogen throughout the host's lung parenchyma upon arrival. 8Activation of macrophages results in recruitment of additional innate immune cells, thereby fostering inflammatory responses that contribute to host defense against the pathogen.Neutrophils demonstrate a heightened intensity of phagocytosis compared to macrophages, coupled with elevated levels of reactive oxygen species (ROS)-mediated oxidative burst. 9Upon recruitment of lymphocytes to the site of infection, a cascade of cell-mediated immune responses is initiated, leading to the arrival of additional immune cells aimed at localizing bacterial colonization and inhibiting their proliferation. 10,11During the initial stages, a characteristic delay is observed in the T-cell response, which aids the pathogen in establishing a persistent infection. 10At this point, complete eradication of Mtb is feasible if the host immune capacity remains intact. 12In most cases, however, the immune response is not sufficient to eradicate Mtb.Monocytes accumulate in the vicinity of infected macrophages, precipitating the formation of solid granulomas, a pathological feature characteristic of TB.Additionally, Mtb can traverse the mucosa upon infection of alveolar epithelial cells and stimulation of cellular necrosis, further exacerbating pathogenesis. 13,14Hence, the gathered data suggest that local immune responses play a pivotal role in mitigating progression of TB disease. 15espite being formed with the intent of containing the spread of bacteria, granulomas can also serve as a refuge for bacterial populations, thus evading further recognition and removal by the host immune system, resulting in a clinically defined state of latent TB infection (LTBI). 12,16nsuring screening, diagnosis, and treatment of LTBI is critical in facilitating the global decline in TB incidence and ultimately achieving TB elimination.During early development, the granuloma is highly vascularized (via vascular endothelial growth factor (VEGF)) and the vessels have extensive lymphocytic cuffs.As the granuloma progresses, macrophages undergo differentiation into various morphotypes, resulting in the formation of a layered structure with a layer of lymphocytes aggregated outside a fibrous cuff surrounding a macrophage-rich layer.In this scenario, patients harbouring granulomas are asymptomatic and noninfectious. 17,18Studies have revealed that Mtb harnesses the production of mycolic acid (MA) to instigate differentiation of macrophages into foam cells.The core of the granuloma can give rise to a caseous granuloma, characterised by the accumulation of foam cells and a multitude of necrotic host immune cells. 19During F I G U R E 3 Pathophysiology of pulmonary TB.Upon entering the respiratory tract and lungs of the host, Mtb incites an innate immune response and is engulfed by pivotal immune cells such as macrophages and dendritic cells.Subsequently, Mtb replicates within these cells as more immune cells are recruited to the site of infection.Whilst it is possible for the host to completely eliminate Mtb at this stage, the formation of solid granulomas is often prompted.These granulomas are composed of foam cells derived from macrophages, as well as a multitude of necrotic immune cells, culminating in caseous granulomas that eventually rupture and release bacteria, giving way to the subsequent development of active TB.Ultimately, these Mtb bacteria are released as infectious aerosol droplets, reinstating a new cycle of infection.The elements in the figure were drawn using BioRender online tool (https://biorender.com).late-stage TB, cavitation of the caseous granuloma core may ensue, resulting in the release of bacteria and consequent progression to active TB disease. 20Consequently, the reactivation of LTBI, and ensuing progression to symptomatic disease, can enable transmission of the bacteria to a new host, perpetuating a new cycle of infection.Moreover, Mtb can disseminate through the bloodstream 21 and lymphatic endothelial cells, 22 disseminating beyond infected lungs and leading to the development of extrapulmonary TB (EPTB).EPTB can manifest in nearly any part of the body, akin to lymph nodes, pleura, genitourinary system, bones and joints, and other organs. 23ursuing an in-depth comprehension of the pathogenesis of TB may unlock novel therapeutic avenues.Despite having been identified and isolated over a century ago, Mtb has continued to inflict protracted distress and fatalities worldwide.Moreover, the latest data from the World Health Organization (WHO) regarding drug-resistant TB are alarming, with approximately 450,000 new cases of RIF-resistant TB reported in 2021. 1 The emergence of drugresistant strains of TB correlates with the epidemic of HIV, and early incidence of drug-resistant TB epidemics were witnessed primarily among HIV-Mtb coinfected patients. 24,25Furthermore, the detrimental effects of the COVID-19 pandemic have impeded the identification and management of TB cases, undermining the gains made in combatting TB in recent years. 5,26Therefore, it is imperative to recognize the urgency of discovering effective therapies for TB.
8][29] Nonetheless, promising breakthroughs in the analysis of pathogen-host interactions and evolutionary investigations of Mtb offer prospective avenues for identifying pathogenicity and virulence factors that could catalyze the development of novel therapies for TB. 30

DRUG TARGETS AND INHIBITORS TARGETING Mtb
Invasion of Mtb causes TB and its ability to replicate and maintain using host cellular mechanisms makes it a major target for killing by drugs.The publication of the whole genome sequencing of Mtb has advanced our understanding of the molecular biology of this bacterium, making it easier to identify specific targets.In fact, the most potent drugs targeting Mtb primarily aim at eradicating the pathogen currently. 31Unquestionably, novel drug discovery and development efforts concentrate predominantly on direct Mtb killing.This section reviews the drug targets and inhibitors targeting Mtb, which are classified according to the different actions associated with bacterial survival and are discussed in different subsections.

Cell wall synthesis and assembly
Mtb, the causative agent of TB, possesses an atypical cellular envelope comprising primarily lipids and carbohydrates.This envelope is characterized by the complex mycolyl-arabinogalactan-peptidoglycan (mAGP) and phosphatidyl-myo-inositol-based lipoglycans.The mAGP complex is constructed from several critical components, including peptidoglycan (PG), arabinogalactan (AG), MA units, and the indispensable lipoarabinomannan (LAM). 32,33It is estimated that the cellular envelope synthesis and assembly pathway of Mtb harbors a minimum of 60 prospective enzymatic targets. 34,4,35The targets described in this section involve the synthesis of MA, AG, LAM, PG, and the transport of MA.

MA biosynthesis
MA, a crucial component of Mtb's cellular envelope, is synthesized predominantly through the fatty acid synthesis (FAS) pathway.It is noteworthy that unlike mammalian FAS-I synthase, which utilizes a multidomain protein, FAS in Mtb is carried out by a combination of FAS-I synthase and several FAS-II monofunctional enzymes.The FAS-II type system is responsible for the extension of acyl-CoA (C16:0 to C18:0), the products of de novo synthesis by FAS-I. 36This unique contrast renders the FAS pathway a viable therapeutic target for pharmacological agents aimed at combating Mtb infection.

FAS-II pathway
InhA, an enoyl-acyl carrier protein (ACP) reductase, facilitates the reduction of long-chain trans-2-enoyl-ACP by forming covalent adducts between nicotinamide cofactors and enoyl-CoA substrates in the FAS-II pathway. 37The role of several first-line anti-TB drugs such as INH, ethionamide (ETH, Group C), and prothionamide (PTH, Group C) 38,39 (Table 1) demonstrates that InhA is an important therapeutic target for the treatment of Mtb infection.
1][42][43] Mtb possesses two enzymes-KatG, a catalase-peroxidase, 44 and EthA, a monooxygenase 45 which catalyze the generation of free radical inhibitors that bind to NADH to form binary complexes. 38Recent research has revealed that during the catalytic cycle of InhA, 37 NADH cofactors and octenoyl-CoA substrates form covalent adducts, including a C2-ene adduct, which offers insight into the function of key amino acid residues at the active site of InhA.
The emergence of drug resistance due to mutations of activating enzymes is a formidable challenge in the case of INH, ETH, and PTH.7][48] Promising direct inhibitors of InhA have been identified, such as diphenyl ethers, 47,49,50 pyrrolidine carboxamides, 51 arylamides, 52 indole-5-Amides, 53 pyridones, 54 imidazopiperidines, 55 thiadiazoles, 56 diazaborines, 57 and benzimidazoles. 58ost InhA inhibitors occupy the substrate binding sites.However, pyridomycin, 59 produced by Streptomyces pyridomyceticus 60,61 or Dactylosporangium fulvum, 62 represented the first direct competitive inhibitor of NADH binding with specific activity against mycobacteria.The crystal structures of InhA, either WT (PDB: 4BII) or the INH-resistant S94A mutant (PDB: 4BGE), 63 bound to pyridomycin were determined and confirmed that pyridomycin occupied simultaneously the NADH and the substrate binding sites. 64imilar to InhA, β-ketoacyl synthase KasA also plays a pivotal role in the elongation of long-chain fatty acids by facilitating the initial step of the FAS-II pathway. 657][68][69] DG167, an indazole sulfonamide, was first identified as an antitubercular through a high-throughput screening (HTS) campaign. 70,71The Xray crystallography has been employed to determine the molecular structure of this compound while it was bound to KasA. 72,73The in vivo efficacy of transposed indazole sulfonamide derivatives based on DG167 has shown substantial improvement in an acute infection model of Mtb in mice. 74

Fatty acid degradation protein D32/Polyketide synthase 13 crosstalk
Fatty acid degradation protein D32 (FadD32) acts as a fatty acyl-AMP ligase, transferring the resulting acyl-adenylate to specific thioester acceptors. 75Polyketide synthase 13 (Pks13) is a module encoding several enzymatic and transport functions to the viability and virulence of Mtb. 76Once produced by the FadD32 enzyme, the resulting acyl-AMPs are specifically transferred to the ketosynthase domain of Pks13 after binding to the phosphopantetheinyl moiety of its N-terminal ACP domain (N-ACP(Pks13)).Together, FadD32 and N-ACP(Pks13) constitute the initiation module of the mycolic condensation system. 77Recent research reports that several quinoline-2-carboxamides effectively reduce the mycobacterial burden in mouse lungs by inhibiting FadD32 activity upon oral administration. 78AM16, a benzofuran derivative, has also been identified as a promising inhibitor of Pks13. 79Additionally, coumarin 80 and chromen-4-one derivatives 81 have likewise demonstrated Pks13 inhibition, although a continued focus on optimizing in vivo therapeutic efficacy is warranted.Delamanid (DLM, Group C) and pretomanid (Pa) are prodrugs that need to be activated by the deazaflavin F420-dependent nitroreductase enzyme. 82,83DLM and Pa, two promising nitroimidazole candidates, exert their anti-Mtb effects by selectively inhibiting the biosynthesis of specific MA, such as methoxy-and keto-MA. 84By contrast, INH inhibits the synthesis of all MA classesmethoxy-, keto-, and α-MA. 85The precise enzyme targets of nitroimidazoles in cell wall biosynthesis have not yet been identified, though current research supports their multitargeted nature.Further, Pa has been shown to act as a direct nitric oxide (NO) donor, offering insight into its mechanisms for Mtb killing under hypoxic, nonreplicating conditions. 86This is an encouraging prospect for the treatment of on LTBI.Spontaneous drug-resistant mutants of Mtb were found to carry mutations in MA methyltransferases, MmaA4 and MmaA2. 87Crystallographic studies identify the interaction of S-adenosyl-N-decyl-aminoethyl (SADAE) with MmaA4 and provide ideas for inhibitor design. 88MAs cyclopropanation contributes to virulence, antibiotic resistance, and intracellular survival and is catalyzed by enzymes of the cyclopropane MA synthase (CMAS) family. 89,90MA cyclopropane synthase PcaA (also known as UmaA2), which is essential for the nucleation morphology of Mtb 91 and is expressed at high levels during Mtb dormancy, 92 appears to be a potential target for dormant mycobacteria.In addition to PcaA, the cyclopropane synthases CmaA1 and CmaA2 are also involved in the cyclopropanation of MAs. 93,94Several skeletons have been obtained by in silico docking, 92,95,96 and subsequent biochemical validation and optimization are expected.These targets have received limited research attention and hold promising potential for further investigation and development.

AG biosynthesis
N-acetylglucosamine-1-phosphate transferase N-acetylglucosamine-1-phosphate transferase (WecA) responsible for initiating AG biosynthesis in Mtb, has been identified as a potential target for the caprazamycin derivative CPZEN-45. 97This preclinical drug candidate has shown promise as an inhalation treatment option for TB. 98,99Studies have demonstrated that transcriptional silencing of the gene encoding WecA has a bactericidal effect on Mtb both in vitro and in vivo. 100The discovery of novel WecA inhibitors has been supported by mediumto HTS methods targeting WecA.Such screening methods have contributed to the identification of potential inhibitors of WecA, which can serve as lead compounds for the development of new drugs to treat TB. 101

Arabinosyl transferase C
Arabinosyl transferases EmbA, EmbB, and EmbC are critical components of the mycobacterial cell wall biosynthesis pathway.While EmbA and EmbB are known to interact and form a heterodimeric complex, EmbC functions as a homodimeric enzyme. 102Specifically, EmbA and EmbB are involved in the formation of the terminal hexaarabinofuranoside motif in AG, 103 while EmbC is responsible for chain lengthening of LAM. 104Recent data from crystallography and overexpression studies suggest that EMB (Group C) competes with substrates for binding to the EmbB and EmbC subunits. 102,105Notably, a high-throughput virtual screening of the United States Food and Drug Administration (US FDA) library has identified two additional EmbC inhibitors, terlipressin and amikacin 106 (Group C).

PG biosynthesis d-Alanyl-d-alanine dipeptide synthesis pathway
Alanine racemase (Alr) is a crucial pyridoxal 5′-phosphatedependent amino acid racemase enzyme that facilitates the conversion of l-alanine to d-alanine, which is utilized by bacterial cell walls for PG biosynthesis. 126d-Alanyl-dalanine ligase (Ddl) is a multistructural domain protein that is dependent on adenosine triphosphate (ATP) and is involved in the biosynthesis of PG precursors.Ddl catalyzes the ligation of two d-alanine molecules into one d-alanyl-d-alanine dipeptide. 127The inhibition of both Alr and Ddl enzymes in Mtb can lead to a significant weakening of the cell wall, making these enzymes crucial targets for intervention. 128Terizidone (Group B), a compound comprising of two cycloserine (CS) moieties linked to a terephthalaldehyde molecule, undergoes in vivo hydrolysis to CS and exhibits activity against these enzymes. 129CS (Group B), a second-line medication utilized in the management of TB and MDR-TB, is effective in inhibiting the synthesis of PG by concurrently targeting the Alr and Ddl enzymes. 130,131However, CS has limited clinical utility owing to its nonspecific nature.Other Alr inhibitors, including alanine phosphonates and thiadiazolidinones that are currently underutilized in clinical practice, similarly lack specificity due to their effects on multiple other phosphate-dependent enzymes. 126Although several Ddl inhibitors have been demonstrated to be effective at the molecular and cellular level, they require further development for clinical application. 127,132d-Transpeptidase type 2 The presence of β-lactamase, BlaC, in Mtb has long impeded the development of β-lactam anti-TB antibiotics. 133However, the combination of carbapenem antibiotics and β-lactamase inhibitors has recently been revisited as a strategy in the fight against TB.Studies have shown that meropenem-clavulanate (Group C) is highly effective against extensively XDR-TB. 134,135dt Mt2 is a critical enzyme involved in cell wall synthesis, virulence and amoxicillin tolerance of Mtb. 136,137omplexation of l,d-transpeptidase type 2 (Ldt Mt2 ) and meropenem demonstrates that inactivation of Ldt Mt2 may be the main mechanism of meropenem-clavulanate effectiveness against Mtb. 138Hybrid quantum mechanics/molecular mechanics offer a potential avenue to obtain active molecules that inhibit Ldt Mt2 139 .Biapenem, a carbapenem that boasts superior stability, has been evaluated against RIF-resistant Mtb. 140,141Recent investigations have implicated both reversible reactions and nonhydrolytic offloading reactions from the cysteine transpeptidase Ldt Mt2 in the effectiveness of meropenem. 142These findings provide a direction for future optimization of next-generation anti-TB carbapenems.The development of carbapenem antibiotics is a remarkable accomplishment, and it is essential to proceed quickly with in vitro and in vivo experimental validations to establish effective treatment regimens for XDR-TB.Recently, a low-molecular-weight organoselenium compound ebselen was shown to inhibit Ldt Mt2 , suggesting that cysteine-reactive reagents may act as potential Ldt Mt2 inhibitors. 143

Phospho-N-acetylmuramoyl-pentapeptide transferase
Phospho-N-acetylmuramoyl-pentapeptide transferase (MurX), also called translocase I, converts UDP-MurNAcpentapeptide into prenyl-MurNAc-pentapeptide (lipid I) in PG biosynthesis. 1446][147] The capuramycin analogue SQ641 has been shown to be effective in killing Mtb by inhibiting MurX. 148,149However, to accommodate higher drug loads, SQ641 requires a phospholipid-based nanoemulsion formulation. 150This formulation allows for increased drug delivery and improved efficacy in treating TB.The chemical properties of lipid I renders MurX enzyme assays impractical for screening and lacks reproducibility of the enzyme assays. 151However, in vitro experiments have shown that a water-soluble analogue of lipid I can be used as a substitute to quantify the inhibitory activity of library molecules against MurX. 152This approach has allowed researchers to identify potential new inhibitors of MurX that could be further developed for use in TB treatment.

Mamalian membrane protein large 3
Trehalose monomycolates (TMMs), crucial components in the robust barrier against Mtb, are transferred from the cytoplasm to the periplasm via the Mamalian membrane protein large 3 (MmpL3).Further, TMMs act as the essential building blocks for the synthesis of MAs, an indispensable feature of the protective envelope of Mtb, rendering the latter vulnerable to the effects of TMM depletion. 153he discovery of knockdown mutants has illuminated the critical role of MmpL3 in both the replication and activity of Mtb, thereby enhancing our comprehension of its multifaceted pathogenesis. 154Notably, recent advances have led to the discovery of several structurally unique MmpL3 inhibitors, including the 1,2-ethylenediamine SQ109, which has successfully advanced to clinical Phase 2 trials. 155,156[159][160]

Lipid metabolism
Complex lipids of Mtb demonstrate a striking ability to function as critical effector molecules that engage in dynamic interactions with the host.By modulating host metabolism and inciting a robust immune response, these lipids play a paramount role in shaping both the mycobacterium's own physiology and that of the host cells.This section reviews the discovery process of targeting aspartate decarboxylase (PanD).

Aspartate decarboxylase
PanD is a key enzyme implicated in the biosynthesis of β-alanine, a critical precursor molecule for pantothenate and CoA biosynthesis, derived from l-asparate. 161Pantothenate, also known as vitamin B5, serves as a crucial building block for CoA production, which is primarily involved in the synthesis of mycolic MA. 162 Notably, PanD is conspicuously absent in mammalian systems, thereby indicating the therapeutic potential of PanD inhibitors. 163anD, along with pantothenate synthase, has piqued the interest of researchers in the development of PZA, a Group C anti-TB drug. 164PZA functions as a prodrug that is enzymatically activated by nicotinamidase or pyrazinamidase (PZAse), encoded by the pncA gene in Mtb, thereby promoting its conversion into the pharmacologically active pyrazinoic acid (POA). 165PZA's discovery was the result of a therapeutic test on Mtb-infected mice. 166Although PZA exhibited limited anti-TB activity in vitro in the standard neutral pH broth medium, the exact mechanism remained an enigma. 167,168Nevertheless, years of research have resulted in significant breakthroughs in understanding PZA's mode of action.Genome sequencing data have identified mutations in the panD gene responsible for PanD production in PZA-resistant strains, independent of mutations in pncA and rpsA, which were previously presumed as targets for PZA. 169,170Here, it was discovered that while PZA was ineffective, the active metabolite POA competes with PanD, thereby disrupting the CoA biosynthetic pathway. 171,172This novel insight may help clarify why POA can effectively target Mtb under nonreplicating conditions, leading to a reduction of the treatment regimen by three months. 169he other enzymes of the pantothenate synthetase pathway, ketopantoate hydroxymethyltransferase PanB, pantothenate synthetase PanC, and ketopantoate reductase PanE, are also worthy targets for development. 161,173,174esearch on pantothenate synthetase inhibition is currently focused on two main approaches.The first approach involves the synthesis of nonreactive analogues of the reaction intermediate. 175,176The second approach involves identifying hits for pantothenate synthetase inhibition through HTS, followed by structure-based validation to determine their efficacy and safety as potential drug candidates. 177These methods contribute pantoyl adenylate analogues, 175 nafronyl oxalate, 178 actinomycin D, 179 3-phenyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine derivatives, 180 and 5-tert-butyl-N-pyrazol-4-yl-4,5,6,7tetrahydrobenzo[d]isoxazole-3-carboxamide derivatives 181 as pantothenate synthetase inhibitors.In addition to the two approaches previously mentioned, there are also groups focused on fragment growing, 177 virtual screening, 182 and molecular hybridization 183,184 aimed at providing compounds with higher whole-cell activity.These approaches can lead to the development of novel compounds with improved efficacy and safety for the treatment of TB.

Protein synthesis and breakdown
Protein synthesis represents an indispensable process vital for the survival and replication of all living organisms, taking place within the ribosomes of cells. 185This section describes the Mtb ribosome and the leucyl-tRNA synthase (LeuRS) involved in protein synthesis, as well as the caseinolytic protease P (ClpP) system and the proteasome system responsible for the degradation of intracellular damaged proteins.

Ribosome
In the instance of Mtb, the ribosomal machinery forms a large, functional 70S ribosome consisting of a 2.7 MDa complex. 186Notably, the 50S large subunit contains 37 ribosomal proteins, as well as 23S and 5S rRNAs, whereas the smaller 30S subunit is comprised of 21 ribosomal proteins and 16S rRNA. 187,188In particular, Mycobacterial-specific protein Y successfully binds to the 30S subunit, inducing ribosomal hibernation and mediating resistance to aminoglycoside antibiotics. 189This phenomenon is implicated in the development of nonreplicating Mtb, 190 highlighting the potential of ribosomal targeting to impede protein synthesis.Aminoglycosides, oxazolidinones (including TBI-223, 191 currently in phase I, sutezolid, 192 and delpazolid, 193 presently in phase II) represent classes of drugs effective for targeting ribosomes.

Leucyl-tRNA synthase
LeuRS, belonging to the class I aminoacyl-tRNA synthase subgroup, represents a critical player in intracellular transport.Specifically, GSK656 (phase II), a benzoxazole compound, has shown considerable potential as a specific inhibitor of LeuRS through its targeting of the catalytic site of hydrolysis of incorrectly ligated aminoacylated tRNA. 194nlike the initial lead compounds, GSK656 exhibits enhanced selectivity over human cytoplasmic LeuRS while effectively inhibiting protein synthesis in intact human cells. 195Upon computer screening, N-benzylidene-N'thiazol-2-yl-hydrazines 196 and 5-Phenylamino-2H-[1,2,4] triazin-3-ones 197 were successfully synthesized as promising inhibitors of MtbLeuRS.However, further elucidation of the structure-activity relationships between these molecules and their targets is necessary to gain a deeper understanding of the inhibitory mechanisms and potential structural optimization of these molecules.

Caseinolytic protease P
ClpP represents an ATP-dependent, unfolding peptidase protein vital in preserving cellular homeostasis by degrading damaged and misfolded proteins.The joint expression of Mtb's two clpP genes (ClpP1 and ClpP2) generates an active structure capable of hydrolyzing oligopeptides.However, aside from ClpP, ClpX, or ClpC1 is also necessary for the efficient hydrolysis of large, globular proteins. 198andidate compounds can selectively bind to either the catalytic active center or the chaperone-binding site of ClpP, while also partially influencing ClpC1 and ultimately affecting ClpP activity. 199-lactone derivatives, such as EZ120, represent promising inhibitors of ClpP, although further optimization is necessary to enhance their efficacy. 200The Bortezomib analog Pyr-FL-CMK displays MtbClpP selective inhibitory activity. 201Acyldepsipeptide antibiotics target the ATP-binding site to curb the activity of ClpP, 202 thereby stunting Mtb growth.In the same way, pyrrole derivatives are also targeted-ClpP1P2 regulators. 203Lassomycin, a cyclic peptide synthesized by ribosomes, as well as ecumicin, a macrocyclic tridecapeptide, eradicate Mtb by targeting the ClpC1 ATPase complex. 204,205Additionally, cyclomarin A serves to excessively activate ClpC1, thereby interfering with the usual function of ClpP, 206 eventually resulting in the development of novel strategies for regulating ClpP activity.

Proteasome
Mtb has a proteasomal degradation system that is responsible for the prompt degradation of the majority of damaged proteins.In this system, the prokaryotic ubiquitin-like protein (Pup) plays the role of ubiquitin in the degradation process. 207Mtb proteasomes consists of 1 α and 1 β subunit, encoded by genes prcA and prcB, respectively. 2089][210] Previous studies have shown that deamidase of Pup (Dop) deamidates the C-terminal glutamine of Pup to glutamate, preparing it for ligation to target proteins by proteasome accessory factor A (PafA). 211PafA can efficiently move Pup from one proteasome substrate, inositol 1-phosphate synthetase, to two different proteins, malonyl-CoA:ACP transacylase (FabD) and lonely guy (LOG). 212We recently developed a mutant of MtbPafA, purified active PafA on a large scale, and conducted HTS to identify two promising PafA inhibitors, ST1926 and bithionol. 213In addition, the computational approach also yields potential inhibitors of the proteasome. 214,215

Amino acid synthesis and metabolism
The levels of various amino acids vary throughout distinct stages of infection and disease progression, reflecting the dynamic nature of infection. 216Amino acid synthesis and metabolic processes represent crucial determinants in Mtb survival and pathogenesis, underscoring their significance in combatting TB.This section describes the Ser/Thr protein kinases (STPKs) that regulate glutamate metabolism, the shikimate pathway involved in aromatic amino acid synthesis and the tryptophan synthase (TrpAB) involved in L-tryptophan synthesis.

3.4.1
Ser/Thr protein kinases STPKs are phosphorylating enzymes that play essential roles in regulating various cellular functions.Within Mtb, 11 STPKs have been identified, of which PknA, B, and G (protein kinases A, B, and G) are indispensable for survival.Studies have highlighted the limited effectiveness of PknA and PknB inhibitors in targeting TB. 107 Our recent work reports a series of antitubercular compounds based on ceritinib derivatives LPX-16j, 217 of which 5a has good efficacy and safety profile.The differential scanning fluorescence, isothermal titration calorimetry and molecular docking assays suggest that PknB may be one of the targets of 5a. 218Notably, PknG represents a vital kinase regulating glutamate metabolism. 219The inhibition of PknG kinase activity was first reported in vitro with the use of AX20017, a pioneering tetrahydrobenzothiophene compound that was specifically designed for this purpose. 220The inhibition of PknG activity has also been observed with nitro-fatty acids, 221 sclerotiorin, 222 and steroidal lactones, 223 which have all been reported as effective inhibitors of this kinase.NU-6027, a dual inhibitor of PknG and PknD, has emerged as a potent inhibitor of Mtb growth in macrophages and mouse tissues. 224In recent studies, it has been suggested that PknG may additionally block autophagic flux by inhibiting phagosome maturation. 225

Shikimate pathway
The shikimate pathway comprises seven critical enzymatic steps, which are fundamental in Mtb and are connected with the synthesis of vital aromatic molecules 226 (e.g., tryptophan 227 ).Among these, inhibitors 3-dehydroquinate synthase (DHQS), shikimate dehydrogenase, and shikimate kinase have been specifically targeted and have demonstrated noteworthy activity against Mtb.One of the important targets of multitarget compound IMB-T130 is DHQS, which can effectively inhibit Mtb. 2280][231] Shikimate kinase inhibitor Compound 5631296, which was acquired through a comprehensive screening process, has demonstrated a remarkably low toxicity to HepG2 cells.Furthermore, it has exhibited synergistic activity when combined with RIF, resulting in the effective eradication of Mtb. 232

Tryptophan synthase
The indispensability of TrpAB for the sustenance of Mtb within macrophages and circumvention of host immune milieu renders it a highly auspicious therapeutic target. 227,2335][236] TrpA converts indole-3-glycerol phosphate into glyceraldehyde-3-phosphate and indole.TrpB catalyzes PLP-dependent β-replacement reaction in which indole displaces the hydroxyl group of l-Ser to produce l-Trp. 237,238An allosteric, mixed-type inhibitor BRD4592 inhibits enzyme subunits and shows in vitro antitubercular efficacy. 233The same group also reported GSK1 and GSK2, which were found to target TrpAB in 2017 by Abrahams et al., 239 both bind to TrpAB very similarly to BRD4592. 238

Nucleotide synthesis
Mtb must execute conserved DNA replication to transmit genetic information, a highly regulated process that represents a rich source of potential drug targets.This section describes the topoisomerase, the RNA polymerase (RNAP), and the transcriptional repressor of ethA (EthR).

Topoisomerase
The genome of Mtb encodes a solitary type I topoisomerase (topoI) and a single type II topoisomerase (gyrase), comprising gyrA (Rv0006) and gyrB (Rv0005). 133iochemical studies utilizing monoclonal antibodies and oligonucleotides have specifically demonstrated the site-specificity of MtbtopoI. 240Various compounds have demonstrated inhibitory activity against MtbtopoI, including m-AMSA, 241 polyamines, 242 imipramine, and norclomipramine, 243 but appear limited in their cytostatic abilities.In comparison, DNA gyrase has emerged as a promising drug target for anti-TB drug development.Several fluoroquinolone derivatives (Group A) have exhibited substantial inhibitory potential against TB, and are currently undergoing evaluation for the treatment of MDR-TB and XDR-TB. 1,244X-ray crystallography has proven to be an instrumental tool in the concerted effort to comprehend the precise mechanism by which fluoroquinolones affect DNA gyrase and to develop novel inhibitors for this crucial enzyme. 245In sharp contrast to fluoroquinolones-which chiefly target the N-terminal domain of GyrA along with the C-terminal domain of GyrB fused to GyrA-the newly developed phase II drug, SPR720, 246 selectively targets GyrB.This aminobenzimidazole is both structurally and mechanistically dissimilar to fluoroquinolones, thus significantly reducing the risk of cross-resistance.Thiazolopyridine ureas, 247 thiazole-aminopiperidine hybrid analogues, 248 substituted benzofurans, 249 and 4-aminoquinolines 250 have shown promising results as GyrB inhibitors with anti-TB activity.Undoubtedly, the triumph of fluoroquinolones and the existence of other potential ligand-binding sites 251 in topoisomerase clearly suggest that the search for new topoisomerase inhibitors is a worthwhile scientific pursuit.

RNA polymerase
RNAP is an evolutionarily conserved enzyme that plays a vital role in both transcription initiation and RNA elongation, and is subject to diverse regulatory mechanisms mediated by multiple transcription factors.In the case of Mtb, the RNAP is comprised of a central core that consists of five subunits (α2ββ'ω), with the β subunit being susceptible to inhibition by RIF. 252Several RIF analogues (e.g.rifamycin, rifalazil, and rifabutin) have been developed with the aim of enhancing the therapeutic efficacy of RIF through the same mechanistic pathway.Regrettably, a significant number of RIF-resistant TB cases have emerged in clinical settings.Gene mutations that arise in rpoB are the primary culprits behind this phenomenon, rendering RNAP an unviable target for novel drug development. 253,254Nevertheless, strategies aimed at targeting other critical transcriptional processes continue to be promising avenues for future investigations. 255

Transcriptional repressor of ethA
EthR is a repressor of ethA, a gene encoding a monooxygenase required for the activation of the prodrug ETH.Overexpression of ethR, which codes for the repressor EthR belonging to the TetR/CamR family of transcriptional regulators, has been found to induce potent inhibition of ethA. 256As previously mentioned, various thiocarbamidecontaining drugs, including ETH, rely on the activity of the monooxygenase EthA for activation.A breakthrough inhibitor of EthR, BVL-GSK098, was developed via a combination of molecular design, screening, and optimization.This compound demonstrated impressive synergy with ETH combination therapy, as evidenced by a mouse model of TB. 257,258 Notably, the molecular targeting of EthR presents a groundbreaking approach that may help reverse ETH-induced resistance.Spiroisoxazoline analogues, 258 oxadiazole derivatives, 259 and Nphenylphenoxyacetamides 260 have been discovered to possess EthR-inhibitory and ETH-enhancing properties.It is worth noting that first-line drugs such as INH also require mycobacterial enzyme activation, making the development of transcriptional regulator-targeting agents a pressing clinical need, as drug resistance often arises at this stage.

Energy metabolism
In recent years, significant attention has been devoted to Mtb's energy metabolism-particularly, the oxidative phosphorylation pathway-with the aim of identifying novel strategies for pathogen control and drug discovery.Among these promising strategies are classes of antibacterial agents that target different elements of the oxidative phosphorylation pathway, which have shown significant efficacy in controlling dormant or latent mycobacterial infections.These novel therapeutic approaches hold tremendous potential for shortening the chemotherapy regimen for TB.In oxidative phosphorylation, the respiratory chain protein complexes facilitate the generation of a proton motive force (PMF) across a biomembrane, which is then harnessed by ATP synthase to produce ATP. 261This process involves several key steps, including (a) the transfer of electrons from NADH via the type II NADH dehydrogenase (NDH-2) into the electron transport chain and (b) the acceptance of electrons by oxygen via a supercomplex comprising the cytochrome bc 1 complex and the cytochrome aa 3 -type terminal oxidase.Additionally, a cytochrome bd-type terminal oxidase can directly accept electrons from the menaquinone pool. 262lofazimine 263 (Group B), a phase III drug currently used to target NDH-2 in leprosy, is also undergoing repurposing as a treatment for TB.Additionally, clinical trials (phase I) have been initiated for TBI-166, 264 a riminophenazine analogue that may enhance the efficacy of clofazimine while reducing potential side effects.However, recent findings suggest that the activity of these drugs is not solely dependent on NDH-2. 265Other agents that inhibit NDH-2 and are commonly employed for the treatment of psychiatric disorders-such as thioridazine 266 and other phenothiazines 267 -are currently being assessed as alternatives to conventional anti-TB therapy.Telacebec (Q203), an imidazopyridine amide that targets the QcrB subunit of respiratory cytochrome bc 1 complex, disrupts ATP synthesis.Encouraging results from phase I clinical trials regarding safety and tolerability have led to the initiation of phase II clinical trials examining the efficacy of Telacebec against MDR-TB and XDR-TB strains. 268,269Another drug, PZA, affects PMF 270 and is commonly used in combination with other respiratory chain inhibitors.Although quinazoline derivatives, 271 morpholino thiophenes, 272 arylvinylpiperazine amides, 273 heterobiaryl side chain analogues, 274 and imidazo[2,1b]thiazole derivatives 275 have shown promise as inhibitors of QcrB, their metabolic stability requires further optimization.

ATP synthase
ATP synthase is another crucial factor in Mtb's energy metabolism and, as such, represents a critical target for drug development.Bedaquiline (BDQ) [276][277][278] (Group A), a diarylquinoline compound, exerts potent antimycobacterial activity by binding to the c and Ɛ subunits of F-ATP synthase, leading to the blockade of its proton pumping function.BDQ has been granted approval by the US FDA as a crucial component of short-term XDR-TB treatment regimens (BPaL regimen). 279This achievement confirms that ATP synthesis is a prime vulnerability in Mtb and that impairing the energy metabolism holds significant promise for shortening the duration of TB treatment.The successful initiation of phase I clinical trials for two dialkoxypyridine analogues 280 (TBAJ-876 and TBAJ-587), which exhibit higher potency, significantly reduced lipophilicity, and pose a lower risk of cardiotoxicity, is a notable breakthrough following the clinical triumph of BDQ.Sudapyridine (WX-081), a novel compound displaying similar efficacy to BDQ in the TB mouse model, boasts superior pharmacokinetic and toxicological profiles when compared with BDQ.WX-081 is currently undergoing investigations in phase 2 clinical trials involving patients. 281In addition, squaramides 282 and pyrazolopyrimidines 283 are being investigated in preclinical studies.

Virulence
Mtb is an opportunistic slow-growing intracellular organism whose multifaceted virulence mechanisms support the establishment of infection, persistence and reactivation. 284onsequently, efforts to develop Mtb virulence inhibitors are gaining increasing attention as a potential avenue for advancing TB control programs.This section describes the two-component system (TCS) and the ESX-1 secretion system of Mtb.

Two-component system
The TCS, a key pathogen-host signaling pathway constituted by two proteins responsible for transducing environmental cues into physiological responses, has emerged as a potent target for TB therapy.The canonical two-component signaling pathway is comprised of a sensor kinase (SK) that detects specific environmental cues, and a cognate response regulator (RR) that mobilizes the necessary biological response in return. 285Several TCSs, including PhoPR, 286 DosRST, 287,288 PdtaRS, 289 and MtrAB, 290 have demonstrated significant contributions to in vivo virulence and are, therefore, particularly attractive targets for future TB drug development.

PhoPR
PhoPR is recognized as a central regulator of pathogenic traits in MTBC strains, influencing the secretion of the virulence factor ESAT-6, biosynthesis of acyltrehalosebased lipids, and modulation of antigen export. 291Studies have revealed that low pH causes PhoPR phosphorylation, which, in turn, triggers the activation of the cytosolic redox sensor WhiB3. 292Given its role in Mtb virulence, PhoPR represents a compelling target candidate for TB therapy. 293thoxzolamide, a drug commonly used to manage duodenal ulcers, has been shown to inhibit PhoPR and significantly reduce Mtb load in both infected macrophages and mice. 294

DosRST
DosRS was initially discovered to play a critical role in the survival and virulence of Mycobacterial spp.under hypoxic conditions. 295,296Another SK, DosT, also contributes to sensing hypoxia and NO, alongside DosRS. 297tb exploits DosRST to establish and maintain nonreplicating persistence in response to hypoxia, NO, acid stress, or starvation. 298A recent whole-cell phenotypic HTS campaign identified three inhibitors of DosRST, including artemisinin, HC102A, and HC103A.Artemisinin functions by disrupting heme-based SKs DosS and DosT via the oxidation of ferrous heme, and subsequent heme-artemisinin adduct formation.In contrast, HC102A and HC103A do not regulate DosS/T heme, but have been found to inhibit SK autophosphorylation. 299,300 3.7.2ESX-1 secretion system ESAT-6 secretion system-1 (ESX-1) is a sophisticated type VII secretion system which is encoded by the RD1 locus. 3013][304] In addition, recent studies have proven that PhoPR inhibitors are highly effective in regulating ESX-1 due to the fact that PhoPR serves as an essential mediator in activating ESX-1 secretion. 305Leveraging the EsxAdependent cytolytic activity of Mtb, HTS has yielded two promising compounds.Notably, BBH7 and BTP15 not only significantly reduce intracellular bacterial load, but also promote phagolysosomal fusion in Mtb-infected THP-1 macrophages. 306More recently, HTS has produced new lead compound 3,5-dinitrobenzamide. 307

Dihydrofolate reductase
At the core of the folate pathway lies the pivotal role of dihydrofolate reductase (DHFR), responsible for catalyzing the transformation of dihydrofolate (DHF) into tetrahydrofolate (THF) using NADPH as an electron donor.Notably, existing DHFR inhibitors have shown limited efficacy against MtbDHFR or are only weakly effective in inhibiting Mtb.However, P-aminosalicylic acid (PAS), classified as a Group C drug, serves a dual role as both a substrate and prodrug within the folate pathway, with DHFR serving as one of its key targets. 308,309Further analysis of the mechanism of PAS' antifolate action has highlighted the potential benefits of utilizing compounds that can target multiple targets within the same pathway, thereby simplifying treatment regimens. 310Our team acquired the DHFR inhibitor AF-353 through virtual screening and confirmed its selectivity between MtbDHFR and hDHFR. 311In addition, we has predicted that ceritinib, a classical antilung cancer drug and its derivatives, may hold significant promise in combatting Mtb by serving as an effective DHFR inhibitor. 217

Iron-dependent regulator
In response to the stress imposed by the host, Mtb employs iron chelators known as siderophores, notably the mycobactin, to acquire iron.Meanwhile, Mtb has developed highly sophisticated intracellular iron sensing mechanisms, which are tightly regulated by the Fur (ferric uptake regulator) or DtxR (diphtheria toxin regulator) families. 312Among these, the mycobacterial iron-dependent regulator (IdeR), a crucial metal binding transcriptional regulator of the DtxR family, plays a key role in maintaining mycobacterial iron homeostasis and facilitating virulence. 313Through virtual screening, numerous IdeR inhibitors, including acid alizarin violet N derivatives 314 and short peptides, 315 have been identified, though further analysis pertaining to structure-activity relationships is necessary to identify even more potent candidates.

Cholesterol metabolism
Adenylyl cyclase GSK2556286 (GSK-286, phase I) is a pyrimidine-2,4-dione derivative that was uncovered from a HTS library of Mtb-infected macrophages. 316Intriguingly, several indepth analyses of the Mtb survival cycle have revealed that cholesterol metabolism plays a pivotal role in facilitating Mtb's survival within macrophages. 317In fact, in the absence of cholesterol utilization, Mtb is unable to establish an effective infection in macrophages and cannot effectively elicit pathogenesis. 318Notably, the MtbAC possesses a specific ATP pocket that is distinct from its mammalian counterpart, thus playing a key role in converting NTPs into respective 3′,5′-cyclic nucleoside monophosphates. 319Through direct adenylyl cyclase (AC) activation, GSK-286 induces the generation of high levels of 3′,5′-cyclic AMP (cAMP), thereby disrupting the Mtb cAMP signaling network. 320Furthermore, the agonist V-58 is known to operate via a similar mechanism, thereby modulating cAMP signaling and inhibiting cholesterol metabolism by Mtb. 321It is worth noting that cAMP signaling is multifaceted in terms of its impact on Mtb pathogenesis, with studies indicating that it can regulate TNF-α by macrophages. 322igure 4 provides a comprehensive summary of the main targets of anti-TB compounds targeting pathogens.Indirect effects of GSK-286 and V-58 on host macrophages get us thinking.Notably, the interaction between pathogens and their host is a crucial factor in determining bacterial pathogenicity and virulence.
It is true that all drugs in the current clinical pipeline target the pathogen directly.In fact, by targeting cell wall synthesis and assembly, protein synthesis, nucleotide synthesis, energy metabolism, folic acid, and cholesterol metabolic pathways, existing drugs have already shown great promise in achieving high clinical outcomes. 323urrent trends in drug development involve bi-directional screening for both cellular and target-based activity, with a focus on multitarget candidates.Finding new targets and their inhibitors in Mtb remains a promising strategy for combating drug resistance and developing potent lead molecules, though it is important to acknowledge the potential drawbacks such as the lengthy investment required for development and their limitations in treating both active and latent TB.Due to these challenges, an increasing number of studies have recently devoted their efforts towards host-directed therapies for TB treatment.

HOST-DIRECTED THERAPY
The infection process of Mtb is highly dependent on host cells and requires the utilization of multiple strategies to persist within infected cells.Although host-directed therapy (HDT) is often viewed as an adjunctive regimen, recent clinical studies have shown that it can lead to rapid anti-TB effects and improved prognosis. 324The complex immune events that occur during Mtb infection and pathogenesis offer numerous opportunities for HDT, and ongoing discoveries pertaining to the involved pathways and molecular participants continue to expand the list of potential molecules that can serve as anti-TB treatments.Currently, TB HDT strategies are focused on three main objectives: (a) enhancing host immune defense, (b) interfering with the use of host mechanism, and (c) limiting immunopathology.

Enhancing host immune defense
Innate immune responses include cells and mechanisms that are either constantly present or are activated within minutes to hours following an infection to suppress the replication and spread of the invading Mtb.Multiple immune cells are endowed with a repertoire of pattern recognition receptors, including TLRs, NLRs, and CLRs, each of which has been implicated in the recognition and internalization of Mtb. 325,326These innate mechanisms act as a first line of defense against pathogenic microorganisms and are essential in shaping the subsequent adaptive immune response. 327Phagocytic DCs present Mtb antigen to T lymphocytes and promote activation and differentiation of naïve CD4 T cells and naïve CD8 T cells. 328,329his process necessitates the presentation of antigen in the context of major histocompatibility complex (MHC), costimulatory molecules, and the necessary cytokines. 11he immunity-centered HDT approach emphasizes the development of IFN-α and IFN-γ supplementation regimens, which, when utilized in conjunction with antimycobacterial therapy, may potentially influence the progression of pulmonary TB. [330][331][332] Nonetheless, we are predominantly concerned with regulating the abundance of them to influence the immune process.Several methods have been devised to enhance the endogenous IFN response, depending mainly on the activation of TLRs, but their use in the treatment of TB has not been reported. 333,334etformin (MET), the most widely administered diabetes drug, has been proposed as a candidate adjunctive HDT for TB. 335In humans, MET exhibits a multitude of effects, such as the production of TNF-α, IFN-γ, and interleukin 1β (IL-1β), augmented phagocytosis activity, and increased production of ROS. 336Myeloid-derived suppressor cells (MDSCs) are increasingly recognized as a critical driver of TB pathogenesis and represent an immunosuppressive cell population. 337In fact, elevated levels of MDSCs have been observed in both blood and sputum of patients with active TB in studies, 338 and have also been induced in healthy individuals after exposure to Mtb. 339 These cells play a detrimental role in diminishing protective T-cell responses and may contribute to the inability of hosts to eradicate the infection, which subsequently leads to the development of TB disease. 340Tasquinimod, 341 an experimental quinoline-3-carboxamide, has demonstrated tremendous promise in inhibiting tumor growth in murine cancer models and has recently been shown to deplete MDSCs and reduce the relative bacterial burden in the lung and spleen of murine models of TB 342 (Table 2).
Notably, studies have shown that enhancing phagocytosis can effectively limit the intracellular growth of Mtb. 343,344Phagocytosis serves as the fundamental link between the innate and adaptive branches of the immune system. 345In addition to effectively isolating and eliminating pathogens, the phagocytic process also plays a critical role in triggering the activation of adaptive immune responses.Following the ingestion of pathogens, phagosomes must undergo a sequence of dynamic transformations involving both the membrane and internal components, ultimately allowing for their maturation and subsequent fusion with lysosomes. 346Pathogens frequently resort to various cunning strategies to evade capture, including evading detection, disrupting signaling pathways, or disabling the machinery that drives the phagocytic process. 347Imatinib, a chemical inhibitor of tyrosine kinase (TK), has been found to promote Mtb killing through the activation of cathepsin D and phagolysosomal acidification. 348Gamma-aminobutyric acid, an inhibitory neurotransmitter, is known to regulate the Ca2+-AMPK (adenosine monophosphate-activated protein kinase) pathway, thereby enhancing phagosomal maturation. 349n the context of adaptive immune responses, antigenpresenting cells (APCs) play a pivotal role in phagocytosing antigens and attaching them to MHC class I or II molecules, thereby presenting the antigens to T cells to initiate adaptive T cell responses.Facilitating the proper activation of APCs is an effective strategy to help hosts recognize Mtb.G1-4A, a polysaccharide derived from Tinospora cordifolia and a reported TLR inhibitor, has shown promising results in improving host defense against Mtb.In fact, G1-4A has been found to increase NO and proinflammatory cytokine secretion (such as TNF-α, ILβ, IL-6, IL-12, IFN-γ) through upregulating MHC-II, thus leading to reduced intracellular survival of Mtb. 350Additionally, vitamin D, which is required for TLR production, has been recognized as a key molecule in host defense against TB. 351However, more research is necessary to fully elucidate the role of vitamin D in the prevention and treatment of TB. 352 In fact, Mtb devotes considerable energy to directing the induction of the cellular response to infection. 353Mtb induces the expression of indoleamine 2,3-dioxygenase (IDO), which degrades tryptophan and attenuates T cell and NK cell proliferation to suppress immunity. 354The specific inhibitor of IDO activity, D-1MT, has been shown to improve clinical outcomes by increasing the entry of CD4 T cells into granulomas. 355Heme oxygenase-1 (HO-1), an antioxidant enzyme, is induced by Mtb to be expressed in the lung.The inhibition of HO-1 activity with tin protoporphyrin XI (SnPPIX) was found to enhance iNOS expression and NO production by Mtb-infected macrophages following activation by IFNγ produced by T lymphocytes, consequently allowing for a more efficient control of bacterial replication by host cells. 356he epigenetic changes elicited by Mtb infection play a pivotal role in circumventing the immune response of the host and thereby inducing bacterial persistence and dissemination. 357Epigenetic modifications used by Mtb to evade host immune responses include histone acetylation, 358 noncoding RNA expression 359,360 and DNA methylation. 361Targeting epigenetics works have been undertaken for the management of TB.Studies have shown that Trichostatin A impedes host histone deacetylases (HDACs) and, as a result, augments both in vitro and in vivo antimycobacterial efficacy in human macrophages. 362Tubastatin A, an inhibitor of HDAC6, fortifies the immune response and curbs the growth of mycobacteria in an Mtb-infected mouse model. 363Valproic acid and suberoylanilide hydroxamic acid (vorinostat), both inhibitors of HDACs, have supplementary potential to INH and RIF regimens. 364Also as an adjunct to standard TB treatment, the combination of 4-phenyl butyrate (a nonspecific HDACs inhibitor) with vitamin D3 administered orally has shown beneficial effects on clinical recovery. 365,366DNA methylation inhibitors, such as 5-azacytidine, belong to another class of compounds that target host epigenetics. 367Bristol-Myers Squibb has previously submitted a phase Ib/IIa open label, nonrandomized clinical trial to investigate whether the use of injectable azacitidine affects DNA methylation levels and immune signaling pathways during the treatment of pulmonary TB.Unfortunately this study has now been withdrawn. 368eveloping methods to utilize small-molecule drugs to influence the course of immune events is a promising avenue of research, especially given the fact that most of events are still being explored.

Interfering with the use of host mechanism
Mtb is an intracellular parasitic bacterium that relies on host cell mechanisms in order to proliferate and persist.Its replication and persistence is determined by a multitude of cellular processes, including autophagy, granuloma formation, and the specific type of cell death that occurs within infected cells (such as apoptosis, necrosis, and pyroptosis). 369The majority of novel HDT strategies operate by disrupting these processes, thereby inhibiting the survival potential of Mtb.

Regulation of autophagy
Autophagy is a key mediator responsible for the degradation of damaged macromolecules and organelles.Autophagy is regulated primarily by the mammalian target of rapamycin (mTOR) complex 1 and the AMPK. 370,371OR/AMPK The 5′-adenosine AMPK plays a crucial role in maintaining cellular material and energy homeostasis via phosphorylation. 372The mammalian target of mTOR is a serine/threonine kinase that operates through two distinct complexes (mTORC1 and mTORC2) and regulates cellular metabolism in response to environmental cues. 373utophagic signaling is typically divided into mTORindependent and mTOR-dependent pathways, with the latter serving as a negative regulatory pathway through which Mtb inhibits host autophagy. 374Inhibition of mTOR has therefore emerged as a viable strategy for counteracting the low autophagic state observed in Mtb infections.One classical example of an mTOR inhibitor is rapamycin, which is capable of inducing in situ autophagy in lung macrophages and has been found to effectively alleviate Mtb burden when utilized in conjunction with INH or RIF via inhalation.This effect is mediated by the phosphorylation of S6 kinase. 375It is worth noting that the use of rapamycin is currently limited by its potential for immunosuppression. 376In contrast, the antiprotozoal drug nitazoxanide has been found to strongly stimulate autophagy while inhibiting mTOR signaling. 377Additionally, the antidepressant amoxapine has demonstrated the ability to induce autophagy and protect macrophages during infection. 378The anticonvulsant drugs carbamazepine and valproic acid, meanwhile, can induce mTOR-independent autophagy through AMPK activation, a unique characteristic that sets them apart from other drugs. 379MET, the antidiabetic drug, has been reported to significantly reduce intracellular Mtb growth in an AMPK-dependent manner. 380

TNF-α
The TNF-α signaling pathway has proved amenable for therapy of autoimmune and other chronic inflammatory noninfectious diseases. 381,382Multiple cells synthesize this cytokine in response to mycobacterial infection to induce a phagocytosis program, [383][384][385][386][387] and the regulatory process involves IFN-γ, 384 enzymes, 388 and lipid mediators. 389NF-deficient mice infected with Mtb exhibit delayed chemokine induction and immune cell recruitment. 390aintaining its normal level is essential to activate autophagy. 391TNF-α is also associated with granuloma biogenesis and integrity, driving the formation of durable solid granulomas. 392Since none of the approved TNF modulators are small-molecule drugs, they are not discussed in this review.Pasakbumin A, which is extracted from Eurycoma longifolia Jack, effectively inhibits intracellular Mtb killing by inducing both autophagy and TNF-α production through the extracellular regulated protein kinase ERK1/2-and nuclear factor NF-κB-mediated signaling pathways in Mtb-infected cells. 393It is noteworthy to mention that the studies have reported a higher incidence of active TB among patients receiving TNFneutralizing therapy. 394,395Notwithstanding its beneficial effects on granuloma formation, overexpression of TNFα can promote inflammation while contributing to the hyperactivation of infected macrophages in the granuloma and provoke programmed necrosis 396,397 (as detailed below).

Protein kinase
Kinases are central to mammalian signaling pathways.The screening process for identifying compounds that suppress the proliferation of Mtb in macrophages yielded two promising candidates, namely imatinib, which was previously mentioned, and gefitinib, an inhibitor of the epidermal growth factor receptor (EGFR). 398The therapeutic application of gefitinib in Mtb-infected macrophages has been demonstrated to effectively restrain the STAT3 signaling pathway, a transcription factor that has been found to impede effective immune responses in vivo. 399Additionally, gefitinib treatment was observed to stimulate the expression of genes associated with lysosomal biogenesis and function, resulting in an increased production of functional lysosomes with enhanced autophagy. 400ccording to recent research, the TK inhibitor, ibrutinib, which is commonly used in the treatment of chronic lymphocytic leukemia, has been identified to have efficacy against Mtb infection by inhibiting the BTK/Akt/mTOR signaling pathway and inducing autophagy.This treatment has demonstrated a significant reduction in bacterial load in Mtb-infected mice models. 401n addition to the aforementioned drugs, there is growing interest in the development of modulators that regulate autophagy through alternative pathways.Autophagyrelated proteins are regulated by the transcription factor EB (TFEB). 4024][405] Meanwhile, the activation of host sirtuin 1 (SIRT1) has also been shown to reduce intracellular growth of drugsusceptible and drug-resistant strains of Mtb, inducing phagosome-lysosome fusion and autophagy through a SIRT1-dependent mechanism. 406In recent studies, it has been demonstrated that the sirtuin 3 (SIRT3), coordinates mitochondrial function and autophagy activation to facilitate anti-Mtb responses via peroxisome proliferator activated receptor alpha (PPARA). 407Natural activators such as resveratrol 406 and honokiol 407 activate SIRT1 and SIRT3, respectively, exerting their inhibitory effects on intracellular growth of Mtb.Additionally, the use of 4phenyl butyrate in combination with vitamin D shows potential in counteracting the inhibition of human antimicrobial peptide LL-37 expression by bacilli, thereby promoting autophagy. 408This finding suggests that regulation of antimicrobial peptides could serve as an approach to promote autophagy. 408Imiquimod (IMQ), a TLR7 agonist, has been shown to act as a radiosensitizer for melanoma by inducing autophagy. 409Recent studies indicate that IMQ can stimulate TLR7 and activate autophagy by increasing the production of ROS through the p38-or MEK/ERK1/2mediated signaling pathways during the early phase. 410urther research has outlined the two E3-Ubiquitin (E3-Ub) ligases, PRKN and SMURF1, that specifically target phagophores by tagging poly-Ub, 411 highlighting the efficacy of targeting ubiquitin machinery in regulating autophagy.

Intervention of granuloma formation
Pathogenic mycobacteria have been shown to induce the formation of complex cellular aggregates known as granulomas, which are a hallmark of TB.These granulomas are composed of various immune cells, including macrophages, DCs, and T cells, which surround and attempt to contain the mycobacteria through the formation of a barrier.However, the bacteria can persist within the granuloma, which can lead to recurrent or chronic infections.If uncontrolled, the formation of granulomas can lead to the development of active TB in human patients. 412

Angiogenesis obstruction
In tumors, the hypoxic environment induces VEGF expression and stimulates angiogenesis. 413Significantly high levels of serum VEGF were also found in patients with active TB. 414In 2015, Oehlers et al. reported that intercepting VEGF pathway signaling by pazopanib (a VEGF receptor inhibitor 415 ) or SU5416 (a prototypical TK receptor 416 ) inhibits granuloma-associated angiogenesis, reduces Mtb burden and limits the spread. 417Subsequent studies have demonstrated that angiopoietin-2 (ANG-2) expression is prominently upregulated within granulomas.Specifically, pharmacological inhibition of vascular endothelial-protein tyrosine phosphatase 418 (AKB-9785, a structurally similar and equivalent inhibitor to AKB-9778, patented from Aerpio Therapeutics 419,420 ) interrupts the ANG-2/TIE2/VE-PTP axis and severs the stabilizing effects of this pathway, culminating in vascular permeability alterations that effectively limit the growth of Mtb. 421

Metabolism blockage
Previous studies have reviewed the strategies by which Mtb influences host-pathogen interactions by regulating metabolism. 422The metabolic alterations culminating due to stressors present in the microenvironment of granuloma-namely a paucity of nutrients, hypoxia, and acidic pH-hold great significance.The metabolic transition from oxidative phosphorylation to aerobic glycolysis of cells in the presence of sufficient oxygen, 423 a process known as the Warburg effect, 424 is present in tumors 425 and has parallels within TB granulomas. 426As a consequence, regulation of glycolysis, glucose transport and glucose homeostasis are important strategies to limit granuloma metabolism.Treatment to limit aerobic glycolysis can limit granuloma as well as Mtb replication.Mtb-infected macrophages in granuloma undergo metabolic changes, including upregulation of hypoxiainducible factor-1α (HIF-1α) and using glutamine as an important carbon and nitrogen source. 427,428Insufficient cytoplasmic aspartate delivery leads to cell death when TCA circulating carbon is reduced after glutamine inhibition. 429Foamy macrophages, a granuloma-specific cell population characterized by its high lipid content, constitute a long-term reservoir for Mtb in the human host. 430Lipid bodies serve as both a source of nutrients and a secure niche for the bacterium. 431Formation of Mtb-dependent lipid bodies is mediated through the G protein-coupled receptor GPR109A.Inhibition of the GPR109A leads to a reduced bacterial load and the reduction in alveolar macrophage lipid bodies in vivo, which are associated with granuloma caseation. 431The significance of glycolysis and lipid metabolism in the advancement of Mtb infection highlights the potential for HDT.In addition, metabolic alterations within granulomas can lead to the accumulation of advanced glycation end products (AGEs).These AGEs are known to affect macrophage death and activation, potentially playing a role in the progression of TB. 432

Regulation of cell death
Cell death can be classified into two main categories based on molecular mechanisms, morphological characteristics, and signal dependency: programmed cell death (PCD) and nonprogrammed cell death (n-PCD). 433The interaction between Mtb and the host in relation to cell death is complex, with Mtb initially inhibiting host cell apoptosis to ensure replication niches, and subsequently inducing necroptosis and pyroptosis after successful replication to release and infect new cells. 434

Apoptosis activation
Numerous studies 435,436 have demonstrated that Mtb has the ability to regulate proapoptotic proteins to inhibit the intrinsic pathway of host cell apoptosis, and this escape ability is thought to be a critical virulence factor. 437Currently, much work is being done to regulate antiapoptotic genes and controlling apoptotic factors with the aim of enhancing host cell apoptosis. 438Peroxisome proliferatoractivated receptor gamma (PPARγ), a member of the nuclear receptor superfamily, is a transcriptional factor that governs inflammation. 439This protein exhibits elevated expression within activated alveolar macrophages and macrophage-derived foam cells, each of which plays a significant role in the pathogenesis of TB. 440,441 Mtb and its cell wall mannose-capped lipoprotein mannan activate PPARγ and stimulate MRs to enhance their intracellular survival. 442To prevent apoptosis, Mtb differentially regulates the Bcl-2 (B-cell lymphoma protein 2) family members Bax (proapoptotic) and Mcl-1 (prosurvival) expression through PPARγ. 443Therapeutics aimed at Mcl-1, alongside other Bcl-2 prosurvival proteins, is currently under development as potential cancer treatments. 444The Mcl-1 inhibitors sabutoclax, TW-37, A-1210477, and MIM-1 have been shown to significantly decrease Mtb survival within human macrophages via the activation of apoptosis. 443hese promising results indicate the feasibility of Mcl-1 and other antiapoptotic proteins as viable targets for HDT.

Necrosis suppression
Conversely, another idea that is being explored is the reversing of cell necrosis.The necrosis of infected macrophages represents a crucial event in the pathogenesis of TB as it results in the release of mycobacteria into the extracellular environment, which is permissive for the growth and spread of the pathogen.Excess TNF has been shown to trigger programmed necrosis. 397Recent research has elucidated the mechanism by which excess TNF induces mitochondrial ROS (mROS) production in TB.It has been found that excess TNF in macrophages infected with mycobacterium triggers an increase in mROS production through the reverse electron transport process, which occurs through complex I. MET may inhibit complex I and thus prevent TNF-induced mROS and macrophage necrosis. 396Moreover, excess TNF-α following infection has been found to upregulate mROS and induce the formation of the mitochondrial permeability transition pore complex, subsequently triggering necrosis via cyclophilin D (CypD).The inhibitory peptide alisporivir of CypD has been found to synergistically inhibit Mycobacterium zebrafish infection when used in combination with the ceramide inhibitor desipramine. 445Interestingly, it has been found that ceramide is induced to be produced by TNF-α. 445Studies have demonstrated a significant correlation between Mtb-induced host cell necrosis and ferroptosis, with decreased lung bacterial load observed in mice with acute TB infection following ferroptosis inhibitor, Ferrostatin-1 treatment. 446Furthermore, corticosteroids have been found to inhibit necrosis by targeting mitochondrial membrane stability through the inhibition of p38 mitogen-activated protein kinase (MAPK) in addition to their known actions through glucocorticoid receptors (GRs). 447

Pyroptosis suppression
Pyroptosis is a proinflammatory PCD pathway distinct from other forms of cell death, mediated by pyroptosomes, dependent on N-terminal fragment of the caspase-1-cleaved gasdermin D (GSDMD) and causes plasma membrane rupture. 448,449Two known mechanisms for initiating pyroptosis are through (a) damage to the plasma membrane of host cell by the ESX-1 system, which activates the NLRP3 (domain-like receptor protein 3) inflammasome, 450 and (b) interaction between EST12, a protein that induces cell pyroptosis, and active C kinase 1, leading to activation of the macrophage NLRP3. 451ltimately, both mechanisms result in the activation of the inflammasome-caspase-1-GSDMD pyroptosis-IL-1β immune pathway.Also, upon internalization in the phagosome, Mtb create phagosomal pores that allow for the passage of bacterial factors, ultimately activating the cGAS/STING pathway and resulting in an IFN-γ response. 452Research shows that baicalin reduced pyroptosis by inhibiting the PERK/TXNIP/NLRP3 axis. 453dditionally, Tanshinone IIA inhibited upstream signals of NLRP3 inflammasome activation in Mtb-infected macrophages. 454Despite this understanding of the mechanisms behind pyroptosis induction in Mtb, there are currently no known modulators for the pyroptosis suppression for the clearance of Mtb.

Limiting immunopathology
The immunopathological response seen in TB, characterized by excessive inflammation, can present a significant challenge in managing the disease. 455Research has confirmed that immune cells utilize distinct strategies to control Mtb invasion, with proinflammatory mechanisms playing a crucial role in slowing, sequestering, and ultimately eliminating the pathogen.The successful host response, however, relies on balancing the timing and expression levels of pro-and anti-inflammatory responses. 456Consequently, regulation of inflammation to balance protective and pathological elements of the immune response is an integral component of HDT.
Cavitary pathology is associated with host-protease imbalance driven by Mtb. 457Unbiased investigations conducted by various research groups, using a diversity of methodological approaches, have consistently identified matrix metalloproteinases (MMPs) as one of the most highly upregulated proteins in TB. [458][459][460][461][462][463][464] MMPs depend on zinc ions to carry out the hydrolysis of protein substrates, and are highly associated with TB cavity in patients. 465The original ribosome inhibitor, doxycycline, has been found to decrease MMP activity in cellular models and suppress mycobacterial growth in vitro and in guinea pigs, as evidenced by clinical trials. 466Marimastat, an antitumor drug specifically designed to inhibit MMP, was not ultimately approved for use due to musculoskeletal toxicity, recent studies have demonstrated its efficacy in reducing granuloma formation and Mtb bacterial load when combined with RIF or INH. 467Other MMP inhibitors that have been reported to improve granuloma production are batimastat, prinomastat, Sb-3ct, 467 and some specific antibodies. 468owever, the MMP-7 inhibitor cipemastat increased the frequency of lung cavitation in mice with TB. 469 Additional research is necessary to comprehend the potential of MMP inhibitors as supplementary therapeutic interventions for pulmonary TB.
Host lipid metabolism plays an essential role in nodulehost interactions. 470Lipid peroxidation, a process that causes oxidative damage to lipids in host cell membranes, has been found to induce tissue necrosis and facilitate the transmission of Mtb. 471In vivo experiments have shown that lung necrosis in acutely Mtb-infected mice is associated with reduced glutathione peroxidase-4 (Gpx4) expression as well as increased lipid peroxidation. 446Nacetylcysteine restores the reduced form of glutathione and counteracts TB-induced oxidative stress. 472The addition of N-acetylcysteine to the standard drug regimens for TB has been demonstrated to increase the levels of glutathione peroxidase in TB patients. 473As a result of this finding, clinical trials assessing the efficacy of Nacetylcysteine-assisted treatment for TB patients are currently underway. 474Host eicosanoids, lipid mediators of inflammation, have been shown to respond to the severity of TB and TB-diabetes comorbidity, 475,476 and have a significant impact on the outcome of Mtb infection through their correlation with two major counter-regulatory classes of inflammatory cytokines: IL-1 and type I IFNs. 477Currently, modulation of lipid metabolic pathways (5-lipoxygenase, 5-LOX modulators) has been reported for the treatment of TB infection.Zileuton, a 5-LOX inhibitor originally developed for asthma, has been found to improve survival, reduce bacterial burden, and alleviate lung injury in Mtb-infected mice. 477In oncology studies, cyclooxygenase-2 (COX) has been shown to be associated with MDSC activation, and COX-2 inhibitors such as aspirin and ibuprofen have been utilized to test their efficacy in treating mouse models of infection, where they demonstrated an increase in bacterial clearance when combined with PZA. 478,479However, recent studies have reported that COX-2 inhibitor treatment significantly reduces Type-1 helper (Th1) differentiation and downregulates IFN-γ expression, which exacerbates Mtb aerosol infection. 480As such, more experimental evidence is required to determine the appropriate role of nonsteroidal anti-inflammatory drugs (NSAIDs) in adjuvant anti-TB therapy. 481orticosteroid therapy probably improves neurological outcomes of, and decreases mortality due to, tuberculous meningitis of moderate severity. 482Due to their potent anti-inflammatory properties, these drugs are capable of suppressing the host's immune response, thereby reducing the potential for immunopathology.A recent study has demonstrated a differential expression of GR pathwayregulated genes in patients with TB. 483 Promisingly, the corticosteroids dexamethasone and prednisolone have shown efficacy in reducing mortality from all forms of TB, including pulmonary TB. 484 A randomized, double-blind, placebo-controlled trial has demonstrated that the administration of prednisone is effective in reducing the need for hospitalization and therapeutic procedures, while also accelerating improvements in symptoms, performance, and quality of life. 485oly(ADP-ribose) Polymerase 1 (PARP1), a member of the PARP family and a regulatory factor, is known to induce further activation of itself through the actions of inflammatory mediators.486 In light of this, PARP1 inhibitors PJ-34 have recently been proposed as an HDT approach to TB, due to their ability to reduce inflammation and alleviate lung disease.456,487 The phosphodiesterase-4 (PDE-4) inhibitor Dovramilast (CC-11050) has been found to effectively downregulate TNF-α production in macrophages through the increase of intracellular cAMP, thereby reducing inflammation in rabbits with pulmonary TB during INH treatment.488 When administered in combination with INH, roflumilast, another PDE-4 inhibitor, was shown to effectively decrease Mtb bacterial load in mice.489 Notably, the role of the Janus kinases (JAKs)/signal transducers and activators of transcription (STATs) pathway in the development of TB has received interest.The JAK/STAT pathway regulates multiple TB-related cytokines, including IL-27 490 and IFN-γ, 491 affecting T cell subset differentiation, T cell activation and generation of memory.492 LINC00870, an upregulated long, noncoding RNAs in Mtb-infected peripheral blood mononuclear cells (PBMCs), promoted p-STAT5 and p-JAK2 protein expression, thus activating JAK/STAT signaling in PBMCs. 493Blocking STAT7 or IL-10 signaling led to a diminished bacterial load in the lungs of infected mice, yet without a significant alteration in their inflammatory response.494 Figure 5 provides a comprehensive summary of the primary cellular processes and prospective targets implicated in HDT.The administration of HDT medications to enhance TB outcomes represents a propitious approach, particularly for MDR-and XDR-TB.Despite this fact, the available clinical data regarding HDT are grossly insufficient. For thenascent HDT, forthcoming research must prioritize drug repositioning alongside validating efficacy.Due to the intricacy of the diverse cellular processes in the host and the inconsistency in response to the identical medication, HDT necessitates comprehensive analysis across multiple models to reveal the implications of novel treatment opportunities.

DISCUSSION AND PERSPECTIVE
Although TB is a malady that can generally be treated and even eradicated, the process often entails a long and financially demanding regimen.This underscores the urgent need for the discovery and development of novel therapeutic agents against TB.It is disappointing that the occurrence of drug-resistant genes and multidrug-resistant strains of Mtb is distressingly common.This not only complicates the treatment and management of TB but also poses a significant threat to global health. 495Consequently, any new therapeutic strategy must prioritize the circumvention of any crosstalk or interaction with first-line drugs whilst simultaneously minimizing the risk of inducing resistance to existing therapies.Achieving this goal necessitates not only exhaustive characterization of current resistance mechanisms and the establishment of effective strategies to circumvent any potential cross-resistance, but also the identification and validation of novel biological targets, and action mechanisms and potent lead compounds with novel scaffold to guide subsequent preclinical research and development initiatives.Thanks to advances in pathogenomics, structurefunction analysis, and systems biology, the endeavor of identifying targets in infectious agents has been accelerated and refined.Historically, disruption of crucial cellular pathways in Mtb, such as cell wall synthesis and assembly, protein synthesis, and energy metabolism, has been regarded as a potent strategy for combating TB.However, current research has shifted its focus to newly uncovered targets within Mtb, such as DNA gyrase, ATP synthase, DprE1, and MmpL3, which offer a promising alternative approach to subvert existing drug resistance mechanisms and have yielded remarkable outcomes.Structural determination of enzyme-ligand complexes lays the foundation for identifying key binding sites and designing highaffinity therapeutics.Nonetheless, these drug candidates remain challenged in their ability to sufficiently engage both active and latent forms of Mtb, thus leaving behind a vicious cycle of recurrence.
As known, Mtb has coevolved with humans and has developed a remarkable degree of adaptation to the human host.Meanwhile, the latent form of the disease evades host immunity and retains the ability to cause disease upon reactivation.HDT seeks to achieve enhanced therapeutic outcomes and prognoses by judiciously manipulating host cellular processes.In healthy individuals, the foremost response upon exposure to Mtb is immunization: a cascade that begins with uptake of the pathogen by innate immune cells, which trigger the activation of adaptive immunity.Approaches aimed at optimizing this sequence represent an efficacious strategy for harnessing HDT.Interventions aimed at augmenting autophagy, dampening granuloma formation, and regulating host cell death to interfere with pathogen replication and retention have emerged as promising avenues for influencing the outcome of TB treatment.Active infection with Mtb elicits a potent immune response that often results in host-mediated tissue damage.Combining other treatments with modulation of the inflammatory response to decrease tissue damage and cavitation can enhance the effectiveness of TB treatment.
In the context of comorbidities such as HIV infection or diabetes, HDT offers the opportunity to tailor TB management to each patient's unique clinical circumstances.Such patient-specific tailoring of TB management cannot be readily achieved with antibiotics alone.Furthermore, the administration of combination HDT has consistently yielded improved therapeutic efficacy and abbreviated treatment durations across a diverse array of clinical trials.Additional benefits include the modulation of immunopathology and the facilitation of immune memory development.However, the toxicity potential of HDT must still be vigilantly monitored and actively assessed, and the combination strategies and underlying mechanisms of action must be subjected to rigorous scrutiny across multiple experimental models.
Undoubtedly, delving more deeply into the pathogenesis of TB will unveil additional targets for therapeutic intervention.These newly identified targets demand urgent attention and intensive investment to identify and optimize suitable drug candidates.For instance, target-based virtual library HTS can be a potent and efficient method for expeditiously identifying leading compound candidates while substantially mitigating the time and cost of drug development.Drug repositioning strategies likewise hold great promise, as numerous US FDA-approved drugs are currently under active investigation for their potential utility in the management of TB.It is clear that the successful control of TB represents a shared goal of both governments and civic society, and we must remain unwavering in their efforts to attain this objective.

A U T H O R C O N T R I B U T I O N S
Y. L. and J. Y. conceived the study and wrote the paper.L. Z. revised the figures.Y. L. and W. Q. revised the article.All authors read and approved the final manuscript.

A C K N O W L E D G M E N T S
This study was funded by National Natural Science Foundation of China (No. 81973368, 82173861, and 81473253), Open Project of State Key Laboratory of Respiratory Disease (No. SKLRD-OP-202113)

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors have no conflicts of interest to declare.

F I G U R E 4
Overview of antituberculosis targets aimed at Mtb. Disruption of crucial pathways in Mtb, such as cell wall synthesis and assembly, protein synthesis and breakdown, and energy metabolism, has been regarded as a potent strategy for combating tuberculosis.Recently, there has been a growing interest in interventions focused on lipid metabolism, amino acid synthesis and metabolism, nucleotide synthesis and virulence.The elements in the figure were drawn using BioRender online tool (https://biorender.com).

F I G U R E 5
Overview of the host-directed therapies (HDT) addressed.At the level of the host, targeting important processes such as immune defense, the use of host mechanism by Mtb, and inflammation regulation are invigorated to address and overcome drug resistance.The elements in the figure were drawn using BioRender online tool (https://biorender.com).

TA B L E 1 (Continued) Mechanism of action Target Typical compound Drug stage for TB
Biological targets and inhibitors targeting Mtb.
TA B L E 1 Targets, drug candidates, and actions of host-directed therapeutics for tuberculosis.