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In This Article
 »  Abstract
 » Introduction
 »  Importance of Ir...
 » Iron Source
 »  Iron Acquisition...
 » Iron Storage
 » Iron Homeostasis
 »  Machineries for ...
 »  Regulation of Bi...
 » IrtA and IrtB
 »  Siderophore Recy...
 » Heme Uptake
 »  Glycolytic Enzym...
 »  New Viable Targe...
 »  Targeting Esx-3-...
 »  Inhibition of Mb...
 »  Targeting Heme I...
 » Heme Analogues
 »  Xenosiderophores...
 »  Alternative Modi...
 »  Siderophore Drug...
 »  Siderophore Drug...
 » Discussion
 » Conclusion
 »  References
 »  Article Figures
 »  Article Tables

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 Table of Contents    
REVIEW ARTICLE
Year : 2022  |  Volume : 54  |  Issue : 5  |  Page : 353-363
 

Deployment of iron uptake machineries as targets against drug resistant strains of mycobacterium tuberculosis


Assistant Professor, Department of Pharmaceutical Chemistry, Dr L H Hiranandani College of Pharmacy, Ulhasnagar, Maharashtra, India

Date of Submission12-Sep-2020
Date of Decision25-Aug-2021
Date of Acceptance12-Nov-2022
Date of Web Publication13-Dec-2022

Correspondence Address:
Kunal Mohan Gokhale
Dr L H Hiranandani College of Pharmacy, CHM Campus, Opp UNR Railway Station, - 421003, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijp.IJP_667_20

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 » Abstract 


Mycobacterium tuberculosis (MTB) requires a perpetual supply of iron for its sustenance. Iron scarcity and its limited availability in the host environment because of an encounter of various sites during the establishment of infection has led to the evolution of strategies for iron uptake, which includes biosynthesis of iron-chelating molecules called siderophores, Heme uptake pathways, recently discovered host iron transport protein receptors like glyceraldehyde-3-phosphate dehydrogenase and the development of machinery for proper storage of the acquired iron and its regulation. The components of the iron uptake machineries are viable targets in multidrug-resistant tuberculosis, some of which include the MmpL3 heme transfer protein, MbtA enzyme, and the ESX-3 system, while employment of approaches like the synthesis of siderophore drug conjugates, heme analogs, xenosiderophores as drug delivery agents, and the blockade of siderophore recycling are encouraged too. Thus, the mentioned discoveries stand as promising targets against various strains of MTB.


Keywords: Glyceraldehyde-3-phosphate dehydrogenase, heme uptake, iron, siderophore


How to cite this article:
Gokhale KM, Iyer AM. Deployment of iron uptake machineries as targets against drug resistant strains of mycobacterium tuberculosis. Indian J Pharmacol 2022;54:353-63

How to cite this URL:
Gokhale KM, Iyer AM. Deployment of iron uptake machineries as targets against drug resistant strains of mycobacterium tuberculosis. Indian J Pharmacol [serial online] 2022 [cited 2023 Jan 31];54:353-63. Available from: https://www.ijp-online.com/text.asp?2022/54/5/353/363395





 » Introduction Top


Mycobacteria have developed drug resistance because of the continuous employment of antitubercular drugs whose conventional antibacterial targets are now effective no more, because of the acquisition of adaptive and evolutionary measures like genetic mutations. Mono-resistant, poly-resistant, multidrug-resistant, extensively drug-resistant, and totally drug-resistant tuberculosis (TB) are into existence.[1] Hence, old targets such as RNA polymerase, topoisomerase II, and mycolic acid biosynthesis are discouraged, and new targets such as iron sequestrants and uptake machineries are scoped, which become potential areas for research.[2] These targets can restrict iron substantially and cut down the survival rate of the bacteria.


 » Importance of Iron in Mycobacteria Top


Mycobacterium tuberculosis (MTB) needs iron for multiplication, virulence, pathogenesis, enzyme-catalyzed/oxidoreduction reactions as a cofactor (for hydroxylases and oxygenases), for the synthesis of cytochrome and thus to live in the host cell.[3] Iron chelation appears as an onerous challenge for MTB. The pathogenic bacterium replicates and survives inside the macrophage and fights for limited iron resources with the host. It has strategized mechanisms for the sequestration of iron from the host and its utilization for the continuation of growth.[4]


 » Iron Source Top


Iron, being a transition metal, can exist in different oxidation states, prevailing in Fe+2 and Fe+3 oxidation states in human. As iron can induce oxidative stress via Fenton reaction resultant hydroxyl ion generation, its availability in the body is tightly regulated. Iron is available in the form of complexes such as transferrin, lactoferrin, protease-dependent ferritin, and hemoglobin (largest iron pool) which are iron transport proteins that become a source of iron available for the inhabiting Mycobacterium.[4],[5] Otherwise, macrophages which engulf erythrocytes after their senescence, break down heme and release iron which is stored in its cytosol, becoming an iron pool to MTB existing within.[6] For supporting the growth of mycobacterium cells, 7-64 μg of iron/gm. is required.[7] The available iron thus available is assimilated, warehoused, and used.


 » Iron Acquisition, Storage, Homeostasis, and Utilization Top


For iron acquisition, mycobacteria have developed machineries to synthesize low molecular weight iron-binding molecules called siderophores which contain chelating groups that capture iron. These are either salicylate derivatives-mycobactin and carboxymycobactin or peptide derivatives-exochelin. Mycobactin and carboxymycobactin differ in terms of polarity, solubility, and length of the alkyl chain, synthesized in extremely iron-deprived conditions. Carboxymycobactin and exochelin chelate iron from hydrophilic, aqueous medium, whereas mycobactins prevail at the lipophilic cell wall of mycobacteria and transfer and internalize iron already sequestered by carboxymycobactin.[4] The chelating groups found are catecholates, hydroxamates, and carboxylates, which can chelate ferric ion.[3],[5],[7] A resultant hexadentate octahedral complex is formed.[5] After internalization, iron from the ferrated siderophore complex is extracted by reduction to Fe (II) by exposing it to a pH as low as 5.5 facilitated by ATP-dependent pump. The Fe (II) form has little affinity to these ligands.[3] The ferric mycobactin reductase enzyme via an NADPH-dependent process can bring about the reduction of Fe (III) to Fe (II).[3]

In the absence of mycobactin, carboxymycobactin is involved in the transport of iron through mycobactin-independent mechanisms using IrtA and IrtB transporters.[8]

Excochelin is the siderophore commonly produced in Mycobacterium neoaurum and Mycobacterium smegmatis, like non-pathogenic strains.[4] Exochelin MN has been found to exist in M. neoaurum and Mycobacterium leprae. Mycobacterium avium is the only organism where lipid-soluble exochelin is found.[9] Exochelins are more polar than mycobactin, as they contain shorter alkyl side chains terminating in methyl ester moieties.[10] Siderophores have a very high affinity, Ka >1030 M−1 for iron.[5] Heme uptake pathway is another mechanism prevalent for iron procurement.[5]


 » Iron Storage Top


The iron gained by the bacteria is required to be stored intracellularly. Excess iron can induce oxidative damage. Hence, two types of iron storage proteins exist in mycobacteria-bacterioferritin and heme-free ferritins. Next, iron in its Fe+2 state is acquired by storage proteins, whereas stored in Fe+3 form.[11] The absence of iron storage machinery in MTB causes iron-mediated toxicity, increasing its susceptibility to antibiotics.[4],[11]

Very little intracellularly acquired iron remains bound with the siderophore once the iron transfer process is complete. The iron acquired is stored in ferritin, bacterioferritin, and bacterioferritin-associated ferredoxins.[7]


 » Iron Homeostasis Top


Homeostasis and regulation of iron levels are important in MTB, which is brought about by iron-dependent regulator protein (IdeR), an iron sensor belonging to the diphtheria toxin repressor family. It regulates the transcription of siderophore biosynthesis-specific genes.[3]


 » Machineries for Iron Uptake in Mycobacterium tuberculosis Top


Biosynthesis of siderophores of pathogenic mycobacteria

Mycobactin and carboxymycobactin are siderophores in pathogenic strains of MTB. Their structure has a characteristic central lysine core modified at α and ε amino terminals with a hydroxyaryloxazoline group and an alkyl group, respectively. In mycobactin, the alkyl group varies from C10 to C21 and may sometimes contain a cis double bond. However, it is shorter in carboxymycobactin and carries a free carboxyl group which adds to its polar nature.[12] Carboxymycobactin comprises an alkyl chain of 29 carbons, whereas mycobactin consists of an alkyl chain of 1021 carbons.[4]

A cluster of 10 genes encoded by mbt1, namely A to J encodes enzymes (as listed in [Table 1]) responsible for the biosynthesis of mycobactin (mbt). The cluster K to N is encoded by mbt2 and this cluster is involved in assembling of A to F.[4] During iron deprivation, IdeR increases the gene expression for siderophore biosynthesis.
Table 1: Genes and the encoded protein

Click here to view


The encoded enzymes are homologous to nonribosomal peptide synthases (NRPSs), which are enzymes that handle the biosynthesis of other siderophores and peptide-derived secondary metabolites. Amino acids are converted into their acyl adenylates via an activating domain using enzymes and, further, get covalently linked to the enzyme via a phosphopantetheinyl thioester present at a peptide carrier protein domain. The condensation of two such activated amino acids to form a peptide linkage takes place at the condensation domain. The order of such activation domains decides the sequence of the final peptide. There are various other domains that perform other activities. The L to D-amino acid epimerization happens at the domain reserved for epimerization. Similarly, the formation of macrocyclic amides or esters and oxazoline/thiazoline containing smaller rings takes place at the cyclization domain. The thioesterase domain helps in cleaving and yielding the final product peptide from the enzyme.[7]

The aryl carrier protein ArCP happens to be the site for siderophore chain initiation.

The formation of activated salicylic acid product Salicyl-S-ArCP is the result of the active MbtA gene and phosphopantetheinyl transferase (PptT) gene.[4]

The MbtA, which is a domain salicylation enzyme, catalyzes a pathway involving a two-step process; (i) generation of acyl adenylate, by the attack of salicylate on α phosphate of ATP wherein the salicyl-AMP is formed, which remains temporarily bound to MbtA, (ii) the transesterification involving–SH of arolyl carrier protein (ArCP) and salicyl-AMP carrier protein (ArCP). This results in the release of AMP and the linking of the salicyl group to MbtB. MbtB is an enzyme that brings about siderophore chain initiation by causing the coupling of activated salicylic acid with serine or threonine and causing cyclization of this precursor into phenyloxazoline ring system.[3] Further, MbtE and MbtF genes encode NRPSs required for activation, condensation, and peptide carrier domains for the donation of two lysine-derived moieties. The MbtF gene causes the lactamization of terminal hydroxylysine residue, causing the release of mycobactin. The ε-N-hydroxylysines formation is encoded by MbtG gene. The lysine produced is esterified with a β-hydroxy acid at the α-carboxyl group, and the product forms peptide linkage with a second ε-N-hydroxylysine (cyclized as seven-membered lactam). The hydroxamic acid moieties formed have excellent iron-chelating power imparting siderophore a nature suitable for its purpose.[7]

The precursor for e-N-hydroxylysines is said to be L-lysine. The acyl moiety at R5 is derived from acetate. Mycobactin S gains aromatic moiety from the shikimic acid pathway. This moiety containing a methyl group is due to the conversion of 6-methylsalicylate. The β-hydroxy acid (2-methyl-3-hydroxypentanoic acid) moiety is derived from two propionate molecules condensation. 6-methylsalicylate is not a shikimate metabolite but rather a polyketide formed due to four acetate unit condensation. Two distinct pathways are used to produce the hydroxyaromatic acid. The origin of oxazoline is presumably attributed to serine and threonine as precursors of the unsubstituted and methyl-bearing rings, respectively.[7]

The structure of carboxymycobactin is like that of mycobactin with slight variations which include R1 = H, R2 = H/CH3, R3 = CH3, R4 = H, R5 = C2 to C8 [Figure 1].[13]
Figure 1: Chemical structures, (a) Carboxymycobactin (b) Exochelin MN (c) Exochelin MS (d) Mycobactin

Click here to view


The biosynthesis of siderophore is regulated by IdeR.


 » Regulation of Biosynthesis of Siderophores Top


The IdeR is a homodimer comprising two monomers identical to each other. Each monomer has three functional domains where namely DNA-binding domain, metal-binding domain, and Src homology domain, which is similar to SH3, fold of the diphtheria toxin repressor. The latter, in combination with the DNA-binding domain and metal-binding domain, yields two metal-binding sites. Site one has a higher affinity toward iron (II), so the occupancy of this site causes dimerization of protein, whereas occupancy of iron (II) at the low-affinity site (site two) causes stabilization of DNA-binding conformation, and eventually, DNA transcription is induced.[4],[3]

IdeR in mycobacteria is a functional homolog of DtxR (diphtheria toxin repressor protein), which exhibits 88% sequence similarity with DtxR within the first 140 residues of the 230 residue protein.[14]

Considering the crystal structure of IdeR, out of three functional domains, the third domain lies between domains one and two. The interaction of these domains with each other is also similar to that in DtxR. Furthermore, the position of domain three in one monomer of IdeR differs slightly from that in the other monomer of the same IdeR. During high iron levels within the bacterium, IdeR binds to DNA. This prevents transcription of genes coding for siderophore biosynthesis (mbt A to J) and stimulation of transcription of brfA genes for regulating iron storage, by stimulating the synthesis of bacterial iron storage proteins. Low iron level within the bacterium results in the dissociation of IdeR from DNA, transcription of genes coding for siderophore biosynthesis (mbt A to J), and repression of transcription of brfA genes and inhibiting the production of iron storage proteins.[3]


 » IrtA and IrtB Top


Rv1348 and Rv1349 are complete and incomplete ABC transporters, respectively, and are also known as irtA and irtB. They are regulated by IdeR. Rv2895c is a siderophore-binding protein. SBD and ATPase are the two domains on IrtA. IrtB contains a permease domain and an ATPase domain. The genes (Rv1348 and Rv1349) that code for IrtA and IrtB are operonic. Both are present on a single operon. IrtA, IrtB, and Rv2895c are membranous and are stimulated in iron-deprived conditions. Rv2895c binds more to ferrated siderophores (Fe-cMBT), whereas SBD (part of irtA) binds more to nonferrated siderophores (cMBT). The irtB permease domain binds to Rv2895c, which is bound to ferrated siderophore already. IrtA participates in the export of nonferrated siderophores, while IrtB and Rv2895c participate in the importing of ferrated siderophores. Thus, they involve themselves in iron trafficking.[3],[8]

The siderophore, which continuously carries out its major function of iron transfer to mycobacteria, is recycled through specialized machinery for its reuse by the bacterium which is described as siderophore recycling.


 » Siderophore Recycling Top


MTB has developed recycling as a process against de novo synthesis of siderophore for preventing hefty investment of energy.

In this process, Mycobactin and carboxymycobactin loaded with iron are acquired by the “ESX-3” secretion system-dependent process involving a siderophore receptor.[15]

The IrtAB transporter causes the dissociation of iron from cMBT, whereas iron dissociated from MBT is via a reductive process (reduction by FADH2).[13]

The deferrated siderophore ready for another round of iron acquisition is exported along with the de novo synthesized Siderophores via transmembrane transport proteins MmpL4 and MmpL5.[15]

Heme is another major source of iron for uptake in MTB, which is mediated via a separate system called the heme uptake system.


 » Heme Uptake Top


Rv0203 has been predicted to behave as a heme-binding protein that is dimeric. It is called a hemophore which is periplasmic and extracellular, and it is called so, as it is the transporter of heme.[5],[16] It is an α-helical protein with each monomer containing five α helices arranged as a structural fold. Tyr59, His63, and His89 are believed to be involved in heme-binding. This was proved by scientists by comparing heme uptake in wild-type Rv0203 with that in mutants, where mutants failed to mediate heme uptake. The mutants were Y59A-Rv0203 and H63A/H89A-Rv0203. The heme-binding affinity is Ka = 1.9 × 109 M − 1 for Rv0203.[5],[17]

MmpL3 or trehalose monomycolate transporter is a multifunctional transmembrane protein belonging as one of the 13 members of MmpL (mycobacterial membrane protein large) family. MmpL3 and MmpL11 are huge proteins with 944 and 966 amino acids, respectively. Both proteins comprise one intracellular domain-C1 and two extracellular domains – E1 and E2. The extracellular domains bind to heme; however, the intracellular domain does not, and the heme-binding kinetics for each protein at the E1 domain has been different.[5] The heme-binding affinity of E1 of MmpL3 = 8.1 × 109 M−1 whereas that of E1 of MmpL11 = 1.6 × 108 M−1; thus, MmpL3-E1 site being more tightly bound with heme when compared to MmpL11–E1.[16]

Both Rv0203 and MmpL11 are essential for the growth of MTB whenever siderophore-mediated iron uptake is deprived and heme is the only source of iron.[5]

The acquired heme is donated to the extracellular domains of MmpL3 and MmpL11. The process of heme transfer between Rv0203 and E1 domains is resultant of transient protein-protein interactions.[16] Within the cytosol, the MhuD enzyme degrades heme to release iron and protoporphyrin IX (PPIX) which is used or stored within the bacterial cell. Two isomers of mycobilin are formed during this process of degradation, with no carbon monoxide being produced.[5]

A report describes MmpL11 as the major heme transporter.[16] [Figure 2] illustrates heme and non-heme uptake mechanisms in MTB.
Figure 2: Heme and non-heme uptake in MTB

Click here to view


Mycobacteria have advancement as far as fulfillment of iron needs is concerned, where additionally glycolytic enzymes have a pivotal role in acquiring iron from host transferrin.


 » Glycolytic Enzyme-Glyceraldehyde-3- Phosphate Dehydrogenase Top


A recent study made revelations that a mycobacterial recombinant strain BCG (mbtB) 30, which cannot synthesize siderophores, was found to still gain iron and survive, which validated the existence of an alternative iron capture mechanism in MTB.[18],[19] These were surface proteins later on identified as glycolytic glyceraldehyde-3-phosphate dehydrogenase. This is an enzyme known to catalyze the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate accompanied by the release of 1 molecule of NADH.[18]

It is a transferrin receptor on the macrophage. It is nearly 40 kDa, varying in molecular weight by 10 kDa from mammalian GAPDH. Iron depletion increases the levels of GAPDH and increases transferrin binding and its internalization within the macrophage. It has a probable role in virulence. It specifically binds to holo transferrin. Iron is released from the internalized transferrin by ferrireductases.[18],[20],[21]

Scientists confirmed its presence in M. TB (H37Rv) and M. smegmatis, that is, in both virulent and nonvirulent strains.[18]

All the above machineries can be potentially targeted via strategies as follows.


 » New Viable Targets and Various Approaches for Drug Delivery Top


Blockade of siderophore recycling

De novo synthesis of siderophores is metabolically expensive. Hence, MTB has developed siderophore recycling as a process to reduce metabolic burden.

Through this process, the ferrated siderophore is deferrated, and the deferrated siderophore ready for another round of iron acquisition is exported along with de novo synthesized siderophores via MmpL4 and MmpL5. MTB mutant △ mmpS4/S5 in which the transporters mmpS4/mmpL4 and mmpS5/mmpL5 are dysfunctional and lacks the recycling mechanism but still possess de novo synthesis of siderophores. However, deferrated siderophores remain accumulated, causing toxicity. Exogenous siderophores have experimentally proven to be bactericidal in such mutants. In such mutants, growth is inhibited, although iron can still be gained through alternative iron sources such as heme, which suggests that iron availability does not affect this inhibitory mechanism. The minimum inhibitory concentration of exogenous mycobactin was determined to be 0.06 μg/ml, whereas that for carboxymycobactin was 0.4 μg/ml in mutants lacking siderophore recycle machinery. It has been reported that the siderophore export mutant, which lacks siderophore recycling, shows a strong virulence defect in mice; thus, blockade of recycling is promising as a viable and valid drug target.[15]


 » Targeting Esx-3-blockade of Siderophore Bound Iron Transport Top


Esx-3 is a secretion system working in harmony with the ABC transport system comprising irtA and irtB. Mutations in this secretion system or its repression cause severe impairment of the growth of Mycobacterium within the macrophage, suggesting that Esx-3 is valid as a potential target.[1],[11] Anhydrotetracycline causes downregulation of the Esx-3 gene cluster in MTB.[22]


 » Inhibition of Mbta Enzyme-Inhibition of Siderophore Biosynthesis Top


The MbtA enzyme, which is essential for encoding peptide synthetase enzyme and carrying out adenylation (generation of acyl adenylate), was found to be inhibited by some molecules. One of these molecules was salicyl AMS, chemically 5'-O-(N-salicylsulfamoyl) adenosine which was found to be an adenylation inhibitor of MbtA. The IC50 was found to be 11 ± 2 nM. The molecule exhibited inhibition in the iron-deprived condition at an MIC50 of 2.2 ± 0.3 μM. and in the iron-rich condition at an MIC50 of 40 ± 8 μM.[3],[4]

The bisubstrate moieties that consist of adenosine riboside and salicylate group were also tested against MbtA as per a report. Salicyl sulfamide derivative was found to be the most efficient and the most potent with MIC50 of 0.08 ± 0.02 μM. Its MIC99 matched with that of isoniazid.[3]

On similar grounds inducing mutations (deletion) of gene encoding MbtB and MbtE (Rv2380c) can also retard the growth of MTB. The mutant of MbtE (Mtb △ mbtE), as per experimentation, could not produce siderophores and this caused alterations in their growth patterns, such as elevated aerial growth, changed cell wall permeability with increased staining, and no fixed boundaries of the cell wall. Lack of ability to biosynthesize siderophores induced iron starvation. This significantly reduced the extent of growth and the ability to infect host macrophage THP1.[23],[24]

Para-aminosalicylic acid is also reportedly known to have antitubercular activity since it inhibits the conversion of salicylate to mycobactin by blocking salicylate AMP kinase. This is the enzyme involved in the formation of salicyloyl-AMP from salicylate in the first step of the synthesis of mycobactin and carboxymycobactin in Mycobacterium. It was tested on Mycobacterium smegmatis.[5],[6],[13],[25]


 » Targeting Heme Iron Transport by Blockade of mmpl3 Top


MmpL is a family of 13 members. Some researchers targeted members of the MmpL family for inhibiting and blocking the heme uptake pathway. As per the findings, in mice studies of MTB, it was inferred that MmpL11 and MmpL8 deletions helped the longer mice survival. The mutants of MmpL4 and MmpL7 are avirulent. MmpL10 and MmpL4 are pivotal in the survival of MTB in the lungs of mice, whereas MmpL7 and MmpL5 indulge in drug efflux. While MmpL4 and MmpL5 are a part of the siderophore export machinery, MmpL3, MmpL7, and MmpL8 are involved in the export of dimycocerosate, trehalose monomycolate, and sulfolipid-1 across the bacterial inner membrane, respectively. As among these, MmpL3 is also involved in heme transfer, it is considered a bifunctional entity.[5]

BM212 is a 1,5-diarylpyrrole derivative chemically, 1-{ [1,5-bis (4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl] methyl}-4-methylpiperazine, that targets MmpL3. Its MIC is 1.5 μg/ml in MTB H37Rv.[5],[26] MIC, in this case, was defined to be the minimum concentration required to prevent the formation of colonies. However, a single mutation at position 215 within MmpL3 can induce resistance against this compound, and the resistance developed is through some other mechanism and not due to BM212 efflux mediated by MmpL5 and MmpL7. The MIC in the mutant strain of MTB was found to be 20 μg/ml as against the parent strain (1.5 μg/ml).[5]

On similar grounds, adamantyl ureas are a group of molecules found to be active against MTB with MIC >0.1 μg/ml. However, they have poor solubility and poor bioavailability, which are barriers to the therapeutic approach. Out of 1600 adamantyl urea compounds, the most effective one with MIC of 0.01 μg/ml was (1-(2-adamantyl)-3-(2, 3, 4-trifluorophenyl) urea (AU1235).[5],[27]

Some adamantly ureas were ineffective or less effective in supporting the survival of mice infected with MTB and the reason being a mutation in MmpL3.[5]

The benzimidazole derivative C215 inhibits the growth of MTB by targeting MmpL3 at an MIC of 24 μg/ml. However, several amino acid mutations developed resistance against the same.[5]

SQ109 is a 1, 2 diammine moiety related to ethambutol. It is another compound acting on MmpL3 with an MIC of 0.5 μg/ml with low cytotoxicity. Its mechanism of action is through the accumulation of TMM, an important component of the mycobacterial cell wall whose export is managed by MmpL3. However, two MmpL3 mutations, A700T and Q40R, build up resistance against SQ109. It shows a synergistic effect when combined with TMC207 and PNU-100480, which are the other two antimycobacterial compounds.[5]

SQ109 is active against fungi and bacteria that do not possess mycolic acid. It is also an inhibitor of menaquinone synthesis, cellular respiration, and ATP synthesis. MmpL3 inhibitors are quite lipophilic, causing problems related to distribution.[28] AU1235, THPP, spiro, NITD-304, and indole carboxamides are among other molecules having potent antitubercular activity.[29]

SQ109 is currently in phase 2b-3 clinical trials. Both SQ109 and BM212 show activity against hypoxic nonreplicating forms of MTB. AU1235 and SQ109 show synergism with rifampicin. SQ109 increases its accumulation in MTB.[30] [Figure 3] depicts the chemical structures of some of these molecules.
Figure 3: Chemical structures of molecules with antitubercular activity

Click here to view


A single molecule that can target most of the members of the MmpL family is desired.[28]


 » Heme Analogues Top


Diversities and differences in terms of heme coordination and its binding sites concerning Mycobacterium-specific heme uptake machinery and host heme transport machinery help many compounds with antitubercular activity, target heme uptake machinery specifically without influencing the host. Similar is the scenario for siderophore receptors which are said to be highly specific, which are susceptible to siderophore-like analogs which have anti-TB activity, for example-mycobactin T, scandium, or iridium-loaded siderophore analogs[5],[31],[32],[33]

Heme analogs are prepared by replacing the central metal atom Fe from porphyrins with another transition metal atom or bringing about alterations of substitutions on the tetrapyrrole ring. Such analogs can be used as antimicrobial agents.[5]

Once these analogs reach the cytosol of the bacterial cell, the MhuD enzyme catalyzes their degradation that releases mycobilin and the toxic transition metal that accumulates.[5],[34],[35]

ZnMPIX, SnPPIX, and Pd-mesoporphyrin IX (MPIX) inhibit the growth of the liquid culture of Trypanosoma cruzi. The analogs SnPPIX and PdMPIX bring about the inhibition of ABC transporters. CoPPIX or FePPIX-dimethylester are other heme analogs that mediate the inhibition of the heme uptake pathway in Staphyllococcus aureus bacterium. GaPPIX is quite lethal to M. smegmatis because of the release of toxic gallium in place of iron in the cytosol or because of the inactivation of hemoproteins attributed to the incorporation of non-Fe heme. Furthermore, it is assumed that GaPPIX may efficiently bring disruption to the heme transfer pathway by targeting MmpL3. Gallium induces the generation of reactive oxygen radicals and produces oxidative stress in the bacterium.[5],[13],[36],[37]

The drawbacks of heme analogs are their insolubility and harmful oxidative stress to the host. Drug encapsulation is a remedy for resolving these issues in some analogs such as ZnPPIX.[5]


 » Xenosiderophores as Drug Delivery Agents Top


Xenosiderophores are siderophores synthesized by other organisms that can be taken up by the organism under evaluation. Siderophores that can permeate through the thick lipoidal mycobacterial envelope and permit no ligand exchange are selected as candidates for drug delivery. It has been found that mycobacterial strains can take up ferrirhodotorulic acid found in and obtained from Rhodotorula strains, rhizoferrin from Rhizopus, and ferricrocin from Aspergillus and Neurospora species. These siderophores can be chosen as suitable candidates for developing drug conjugates.[38]

There is a report on different xenosiderophores taken up by mycobacteria, according to which, ferrirubin, ferrichrysin, coprogen, and myxochelin E are the xenosiderophores that are not used by Mycobacterium smegmatis mc2 155. Ferricrocin, corynebactin, ferrioxamine B, and ferrioxamine D1 are xenosiderophores whose uptake depends on ligand exchange with exochelin and mycobactin. Strain B3, in which exochelin and mycobactin are not synthesized, and strain U3, devoid of mycobactin and exochelin permease, cannot use them. Rhodotorulic acid and ferrioxamin G also require exochelin for ligand exchange. It can also bring about mycobactin-dependent utilization. Strains lacking exochelin permease or exochelin biosynthesis cannot make use of ferrioxamine D2 and ferrioxamine E. All strains except U3 respond to amycolachrom and ferrithiocin utilization. Iron bound to ferrichrome, enterobactin, ferrichrome A, aerobactin, arthrobactin, myxochelin C, rhizoferrin, omibactin, and triacetylfusarinine is used by all strains with different effectiveness.[38]


 » Alternative Modified Forms of Mycobactin (Siderophore Analogues) Top


Alternative modified forms of siderophores can compete with native siderophores and function as growth inhibitors. By substituting α-hydrogen of the main chain with a boc-protected amine, an analog of Mycobactin is generated which has an MIC less than equal to 0.2 μg/ml, causing 98% growth inhibition in MTB. Scandium or iridium-loaded siderophore analogs cause inhibition of MTB growth by competing with native siderophores for siderophore receptors.[3],[5]


 » Siderophore Drug Conjugates Top


Iron– Siderophore– Linker/Spacer– Drug

[Figure 4] illustrates the general structure of drug conjugate.[39]
Figure 4: Siderophore drug conjugate

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The stability of the drug conjugate is influenced and decided by the nature of the linker group and the compatibility of the drug with the conjugation process. The choice of a drug depends upon the target.[39]

Siderophore to be synthesized has to satisfy the following criteria.[39]

It should contain moieties such as hydroxamic acid, catechols, or α-hydroxycarboxylic acid as the sites for metal binding. Preferably a bidentate ligand is a requisite, as it can form a stable octahedral complex with the metal.

Enterobactin, mycobactin, and ferrichrome are a few examples as depicted in [Figure 5].
Figure 5: Examples of siderophores suitable for preparing siderophore-drug conjugates

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 » Siderophore Drug Conjugate Synthesis Top


Considering the concept of peptide linkage, Miller and Malaouin selected β lactam antibiotic as the candidate. Acidic hydrogenolysis gave the desired drug conjugate. They treated E. coli with this drug conjugate, and the conjugate was observed targeting the penicillin-binding protein and producing large inhibition of the growth of E. coli. It was inferred that the drug conjugates were using an iron transport mechanism for reaching the target.[39]

Desferri-Exochelin (D-Exo) is the most well-studied siderophore drug vehicle useful in treatment for postangioplasty restenosis, breast cancer, iron overload in thalassemia, and removal of excess of iron from heart and liver. It is also less cytotoxic.[40] It has been known to obtain iron from host transferrin, lactoferrin, and ferritin, and the D-Exo s can donate iron to mycobactins present in the cell wall of mycobacteria.[41]


 » Discussion Top


TB is a huge health threat, attributed mainly to the generation of drug resistance. The causative agent has established enormous mechanisms for its survival within the host environment. XDR strains of Mycobacterium are resistant to even the first-line agents used for the therapy. Hence, there arises an immense need to overcome these shortcomings by establishing new targets.

Iron is essential for virulence and sustenance in mycobacteria. The organism gets it through diverse mechanisms. Siderophores are iron-chelating molecules that are produced by mycobacteria for iron uptake. These are located extracellularly as well as at the lipophilic cell wall and are synthesized during iron-deprived conditions. The internalized iron is stored in bacterioferritin and used periodically. Iron levels inside Mycobacterium are monitored and regulated by IdeR.

Restriction of iron uptake by disabling siderophore does not alone guarantee death of the pathogen, as other mechanisms of iron uptake prevail, such as the heme uptake pathway and recently discovered glycolytic glyceraldehyde-3-phosphate dehydrogenase enzyme, which is a transferrin receptor on the macrophage. This enzyme differs from the mammalian counterpart in terms of molecular weight.

Iron, being a survival kit, can be restricted via means of various approaches. The first approach includes the blockade of siderophore recycling. Recycling of siderophore reduces the burden on the organism, in contrast to de novo synthesis of siderophore. The blockade of the recycling of siderophore is characterized by the repression of transmembrane transport proteins MmpL4 and MmpL5. This allows only de novo synthesis. However, the intracellular accumulation of deferrated siderophore is bactericidal. Similarly, the induction of mutations in the Esx-3 secretion system, involved in siderophore import-export, causes severe impairment of growth. Furthermore, the inhibition of the MbTA enzyme essential for siderophore biosynthesis (de novo synthesis), by molecules like salicyl AMS, can give an overall punch to the virulence in the pathogen.

On similar grounds, heme uptake is blocked by inhibition of MmpL3 by molecules like BM212, SQ109, C215, and adamantyl ureas like AU1235. Predominantly, heme analogs appear promising, which can be prepared by replacing the central iron atom from the porphyrins with another transition metal, such as zinc. These analogs, on reaching cytosol, are degraded to release toxic transition metal, which can induce oxidative stress.

Xenosiderophores, precisely, siderophores synthesized by other organisms, are being tested as drug delivery agents, some of which include ferrirhodotorulic acid and rhizoferrin. Alternative modified forms of siderophores, on the other hand, compete with native siderophores and inhibit growth. D-Exo (D-Exo) is a very well-known siderophore drug vehicle for synthesizing siderophore drug conjugates.


 » Conclusion Top


TB continues to be a huge menace to public health. The thirst for extensive research continues. In this never-ending race of finding new alternatives in the therapy of TB, this review puts forth a view on how naïve, hitherto unseen approaches be used to give an overall punch to the drug-resistant MTB. It reflects the existence of iron-chelating molecules called siderophore and its associated pathway, heme uptake mechanism, and recently discovered glyceraldehyde-3-phosphate dehydrogenase, which are all involved in iron trafficking and their viability to be targeted. The various approaches it signifies are the synthesis of siderophore drug conjugates, heme analogs, xenosiderophores as drug delivery agents, and the blockade of siderophore recycling. The said avenues are attractive but require further exploitation for enjoying complete utilization.

Research prospect

Introspection of the current scenario as far as the discovery of new targets and drug delivery techniques is concerned, many developments are scoped.

The MhuD enzyme in the heme uptake pathway that releases iron from heme remains an avenue for further study. The absence or inactivation of this enzyme can restrict the release of iron from heme, contributing to the iron deficit.

The existence of glyceraldehyde-3-phosphate dehydrogenase enzyme has recently been confirmed. Molecules targeting the same and their effect on retarding virulence can be discovered and tested.

Esx-3 Secretion system and the ABC transporter system, siderophore recycle blockade, and IdeR provide avenues and scope for the development of new molecules.

As targeting a single iron uptake pathway may not be sufficient to arrest the growth of bacteria because of the continuation of other existing mechanisms for iron acquirement, targeting iron storage proteins, namely ferritins and bacterioferritins, to inhibit storage may give an overall punch to the virulence in Mycobacterium by causing iron overload because of the accumulation, irrespective of the pathway involved for iron uptake. The absence of the iron storage machinery in MTB may cause iron-mediated toxicity, increasing its susceptibility to antibiotics.

Acknowledgment

The authors are thankful to the Principal, Dr. L. H. Hiranandani College of Pharmacy, Ulhasnagar, India, and the HSNC Board for their support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1]



 

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