In the last 10 years, the treatment of TB has become increasingly challenging owing to the emergence of multidrug resistant- and extremely drug-resistant strains of Mycobacterium tuber-culosis. The lengthy and complex therapeutic regimens make treatment difficult and decrease patient compliance.
A small number of new anti-TB drugs are undergoing clinical trials; however, there is an urgent need for the development of new drugs that target novel biological pathways to avoid cross-resistance. In addition, drugs that target persistent and antibiotic-tolerant organisms, thought to be responsible for the requirement for extended treatment time and treatment fail-ure, are needed. There are few well-validated drug targets for M. tuberculosis, although cell wall biosynthesis and RNA polymerase are tar-geted by the first line drugs isoniazid and rifam-picin. Target validation can be difficult, since most essentiality studies are conducted in vitro, which may not reflect the in vivo setting (and vice versa) and there are a dearth of inhibitors available for chemical validation. However, an increased effort has been made to identify novel drug targets, using both genetic validation to demonstrate essentiality in vitro and in vivo, as well as chemical validation to demonstrate target vulnerability to inhibition. Along with these empirical processes, more attention is being focused on enzymes with known func-tions in other organisms and those that have been amenable to drug targeting.
Proteases play a central role in important cel-lular processes in all organisms including pro-tein turnover and the degradation of misfolded proteins, as well as gene regulation. They can have either a wide specificity and degrade many proteins or a narrow specificity and only target one or a few proteins. Proteolysis can lead to complete or partial degradation and may result in either inactivation or activation.
M. tuberculosis has more than 100 genes encoding proteases or peptidases. A number of proteases have been studied in some detail. There is increasing evidence that proteases may be good drug targets for M. tuberculosis, and for bacterial infections in general. In this review, the authors will summarize their understand-ing of key proteases of M. tuberculosis and their potential as drug targets (Table 1).
Proteases involved in secretionLepB: the sole type I signal peptidaseA large number of bacterial proteins perform a function either within the cell envelope or in the extracellular environment and, therefore, need to be shuttled from their site of synthesis to their final destination. One new pathway to target in the development of novel antibiot-ics is the bacterial protein secretion pathway, including the Sec proteins of trans location mach inery and the signal peptidases, which are important enzymes in preprotein pro-cessing and translocation across the bacterial cell membrane.
Proteases in Mycobacterium tuberculosis pathogenesis: potential as drug targets
David M Roberts1, Yoann Personne2, Juliane Ollinger1 & Tanya Parish*1,2
1TB Discovery Research, Infectious Disease Research Institute, Seattle, WA, USA 2Queen Mary University of London, Barts & the London School of Medicine & Dentistry, London, UK *Author for correspondence: [email protected]
TB is still a major global health problem causing over 1 million deaths per year. An increasing problem of drug resistance in the causative agent, Mycobacterium tuberculosis, as well as problems with the current lengthy and complex treatment regimens, lends urgency to the need to develop new antitubercular agents. Proteases have been targeted for therapy in other infections, most notably these have been successful as antiviral agents in the treatment of HIV infection. M. tuberculosis has a number of proteases with good potential as novel drug targets and developing drugs against these should result in agents that are effective against drug-resistant and drug-sensitive strains. In this review, the authors summarize the current status of proteases with potential as drug targets in this pathogen, particularly focusing on proteases involved in protein secretion (signal peptidases LepB and LspA), protein degradation and turnover (ClpP and the proteasome) and virulence (mycosins and HtrA).
Keywords
n antibiotics n drug discovery n essential genes n proteases n tuberculosis
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The type I signal peptidase (SPase I) plays a key role in the protein secretion process by cleaving the N-terminal signal peptide leading to release of the mature protein from the cytoplas-mic membrane [1]. Its activity is essential for the viability of all bacterial species tested including M. tuberculosis [2].
SPase I (LepB) has five conserved regions denoted boxes A–E [1], which include a trans-membrane domain anchoring the protein in the cytoplasmic membrane, as well as the active site composed of a Ser/Lys catalytic dyad. The cata-lytic dyad is unique to SPase I as compared with other serine proteases, which all utilize a cata-lytic triad of Ser/His/Asp [3]. The basic structure of the SPase I proteins is conserved across Gram-positive and -negative bacteria including Bacillus subtilis, Staphylococcus aureus, Streptococcus pneumonia and Escherichia coli. However, the SPase I substrates differ between species, with Gram-positive bacteria having longer and more hydrophobic signal peptides as compared with those of Gram-negative bacteria [3].
SPase I is an attractive drug target for TB for several reasons. M. tuberculosis has a single SPase I that is essential for bacterial growth [2]. Inhibition of the M. tuberculosis SPase I activity using MD-3 (Figure 1), a known SPase I inhibitor [4], leads to the death of both replicating and nonreplicating bacteria [2], suggesting that drugs
targeting SPase I might be able to reduce per-sistence and shorten therapy. In addition, cross-reactivity with the human SPase I is predicted to be limited owing to differences in structure, localization and catalytic mechanism. Usually, bacterial SPases are monomers located at the cytoplasmic membrane surface with the active site facing the extracellular side. This ensures the active site is relatively accessible to poten-tial inhibitors. By contrast, eukaryotic SPases are multimeric with the active site located in the mitochondrial inner-membrane space (mito-chondrial Imp1 and Imp2 SPases) or in the endo-plasmic reticulum lumen (ER signal peptidase complex) [1,3].
Type I SPases are not inhibited by known ser-ine protease inhibitors [5], likely owing to their unique catalytic mechanism, suggesting that compounds that target SPase I activity will not affect the activity of other serine proteases. A number of promising classes of SPase I inhibitors have been reported for other bacterial species. The MD-3 inhibitor, which inhibits M. tuber-culosis growth, is not a good starting point for drug development owing to its potential to degrade into α- and β-unsaturated ketones [4]. The β-lactams were the first nonpeptide inhibi-tors reported to inhibit E. coli SPase I, suggest-ing that acylating agents have good potential as inhibitors [6]. The 5S penem stereoisomers
Table 1. Mycobacterium tuberculosis proteases with potential as drug targets.
Protease Description Protease activity
Essentiality Status as drug target
LepB Type I signal peptidase
Yes [2] In vitro [2,25] Promising; essential; sole SPase I in Mycobacterium tuberculosis; low similarity to eukaryotic SPase I proteases
Mycosins (MycP1–5)
Subtilisin-like serine protease
Yes [20] MycP3 predicted to be essential in vitro [25]
Difficult; high similarity among orthologs; low similarity with eukaryotic proteases; difficulty in developing multitarget drugs; only one predicted essential mycosin
LspA Lipoprotein signal peptidase
Yes [13] In vivo [13,25] Possible; essential; low similarity to eukaryotic proteases; difficulty in progressing virulence targets; not essential in vitro
ClpP1/P2 Caseinolytic proteases
Yes [26,27] In vitro and in vivo [25,30,31]
Promising; essential; inhibition or activation can be bactericidal; possibly difficulties with specificity
Proteasome ATP-dependent protein complex
Yes [62,63] In vivo subunits PrcA and B [65,66]
Promising; similarity with eukaryotic proteasome; virulence target; mycobacterial-specific inhibitors identified
HtrA1–3 High-temperature requirement A serine protease and chaperone
HtrA1 – NoHtrA2 – Yes [72]HtrA3 – No
In vitro [25,72] Possible for HtrA1; essential; inhibition may affect various cell wall-associated mechanisms
FtsH ATP-dependent metalloprotease
Yes [83] Predicted to be essential in vitro [25]
Difficult; essential; similarity with majority of eukaryotic proteases; conserved structure among prokaryotes
Proteases were classified as essential in vitro or in vivo when demonstrated by conditional knockdown strains or by standard genetic methods (inability to delete a gene except in the presence of another functional copy); predicted essentiality is from saturating transposon mutagenesis studies.
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are potent irreversible inhibitors identified at the same time as weak inhibitors such as the clavem systems [7]; however, the potency of these compounds is limited in pathogenic bacteria [8]. Arylomycin A and B, produced by Streptomyces, inhibit SPase I [9] with arylomycin A2 binding noncovalently to the active site of the E. coli SPase I [10,11]. Unfortunately the arylomycins are inactive against M. tuberculosis where the SPase I has a point mutation rendering it insensitive to inhibition [12]. Although none of these inhibitors are under development, the validation of the tar-get as essential for survival and persistence of the organism, as well as its vulnerability to chemical inhibition in whole cells, suggest that a search for novel scaffolds would be of utility.
LspA: lipoprotein signal peptidase IIThe M. tuberculosis genome encodes approxi-mately 70 lipoproteins of which many are impor-tant for virulence by mediating host–pathogen interactions. Lipoproteins contain an N-terminal signal sequence followed by a cysteine residue, which directs preproteins to the Sec or twin argi-nine translocation machineries for export. Signal peptidase II (LspA) mediates cleavage of the preproteins allowing transport across the mem-brane. SPase II are membrane-bound proteases with their active site located on the extracellular side of the cytoplasmic membrane making them accessible to small molecule inhibitors. In Gram-positive and Gram-negative bacteria, SPase II have five conserved regions (Box A–E) similar to those of the SPase I [1]. They are absent in eukaryotic cells making them an attractive tar-get for the development of new antimicrobial treatments.
LspA activity is not essential for growth in vitro and an lspA-deficient strain has no discernible effects in colony morphology or cell wall struc-ture [13]. However, inactivation of lspA resulted in a marked reduction of replication in the lung and an inability to disseminate to the spleen in the mouse infection model. In addition, lspA mutants exhibit a reduced capacity to replicate in mouse macrophages [13]. There is increasing interest in targeting virulence factors, which are only required during infection, and not in vitro, despite the obvious difficulties in finding appro-priate models for optimizing compounds. Since no human analog of this protein exists, LspA might be an attractive target for the development of this type of ‘antivirulence’ drugs.
Globomycin and its derivatives are active in both Gram-negative [14] and Gram-positive bacteria [15] with activity in Gram-negative
Cl
N
O
Cl
NH
O
N
O
O
NN
O
O
O
NH
F F
O
HN
O
N
HONH
O
NH
O
N O
HN
ONH
NH
O
N O
MeO
O
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O
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OO
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S
Figure 1. Structures of known mycobacterial protease inhibitors or activators. (A) MD-3 inhibitor of SPase I. (B) ADEP2 activator of ClpP. (C) Cyclomarin A inhibitor of ClpC1 ATPase. (D) Oxathiazol-2-one inhibitor of proteasome.
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bacteria being LspA-dependent [14]. However, globomycin activity in M. tuberculosis is LspA-independent [16], therefore, inhibitors targeting its SPase II function are yet to be identified.
Mycosins: subtilisin-like serine proteases involved in protein secretionM. tuberculosis has five type VII secretion sys-tems encoded in the esx1-5 loci, named after one of the major antigens secreted by the sys-tem, ESAT-6 [17]. Each locus contains proteins involved in the formation of a dedicated protein secretion system including proteins of the PE, PPE and CFP-10 protein families, proteases and ATP-binding proteins. The encoded proteases are members of the subtilisin-like serine prote-ase family, commonly called mycosins, which are found in bacteria and eukaryotes [18]. These proteases are proposed to be secreted or cell wall-associated, although they may also be present in the cytoplasmic membrane. The mycosins have a conserved catalytic Asp–His–Ser triad, typical for this protease family [18]. Unlike LepB sub-strates, type VII secretion substrates do not con-tain a signal sequence as a target for proteolytic activity [19], although the mycosins are proposed to modify their substrates upon secretion.
Mycosins are important for M. tuberculosis vir-ulence [20]. For example, MycP1 is required for growth in mice and loss of this protease results in reduced virulence in the acute infection model; furthermore, the loss of proteolytic activ-ity results in reduced virulence in the chronic phase of infection [20]. The role of MycP1 in secretion is complex, since its deletion leads to a lack of secretion of ESX-1 substrates, whereas mutant strains expressing proteolytically inac-tive MycP3 show increased substrate secretion. MycP1 cleaves periplasmic EspB and it has been proposed that the cleaved product of EspB inhibits secretion, thus its loss leads to increased secretory activity. Concurrently, it was proposed that MycP1 is required to generate the secretion complex and, therefore, bacteria carrying proteo-lytically inactive MycP1 can form the complex, but have full length EspB, which promotes secre-tion of ESX-1 substrates [20]. This could be an unusual way of targeting bacteria during infec-tion, since inhibition of protease activity would be expected to lead to hypersecretion, increased macrophage response and decreased virulence in the chronic stage of infection. Inhibitors targeting this pathway could be of interest for disrupting latent infection.
Interestingly, MycP3 is the only one in this group of proteases predicted to be essential for
the growth of M. tuberculosis, suggesting that there is no redundancy in their function; how-ever, this remains to be proven [18]. Since myco-sins are involved in growth and virulence and are highly conserved, they could be concurrently targeted using one inhibitor. Thus the myco-sins could pose an interesting new approach to development [17].
The ESX systems do not complement each other [21], which might be due to differences in their regulation leading to expression under different environments or in different metabolic states; therefore, targeting all mycosins might be an advantage. For example, the ESX-1 genes are downregulated under starvation conditions, while the ESX-2 genes are upregulated [22]. The ESX-3 genes are regulated by the availability of iron and zinc [23,24]. Furthermore, transposon mutagenesis showed that the ESX-2 and the ESX-4 gene clusters can be deleted, but not the ESX-3 gene clusters, suggesting that the latter genes are essential for the growth of M. tuber-culosis [25]. Work on ESX-5 is less conclusive. The inactivation of single components of the ESX-5 system was possible, but impacted the cell wall integrity of M. tuberculosis and increased sensitivity to detergents and antibiotics [26]. However, the disruption of large portions of the ESX-5 secretion apparatus resulted in the total inhibition of M. tuberculosis growth [27].
Targeting mycosins for the development of novel antibiotics is attractive owing to the high homology of their protein sequence and the conservation of the active residues across all mycosins, increasing the probability that inhibitors active against one mycosin may also impact the activity of the other mycosins. There is precedent for antitubercular agents having multiple targets – ethambutol inhibits all three arabino syl transferases involved in the synthe-sis of the arabinogalactan and lipoarabinoman-nan components of the cell wall. However, in this case, both arabinogalactan and lipoarabi-nomannan are essential for M. tuberculosis, as are all three genes (embA, embB and embC ). By contrast, since not all of the mycosins are essential, the organisms may develop resistance easily by upregulation of a redundant enzyme. In addition, additional difficulties are posed in development of drug candidates, as optimiza-tion for all targets simultaneously might not be possible.
A homolog of the mycosin 1 protein is found in humans. Although the amino acid identity between the human and M. tuberculosis protein is low and the active sites are not conserved [28],
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there may still be problems with attaining speci-ficity. For those reasons, the mycosins are of less interest than other proteases.
Proteases involved in protein turnoverClpP1/P2Clp proteases are highly conserved serine prote-ases present in a wide range of bacteria as well as in plants and mammals. Clp proteases par-ticipate in protein quality control by degrading misfolded and aggregated proteins potentially toxic to the cell, which contributes to cellular protein homeostasis (Figure 2). Degradation of these nonfunctional proteins is useful to protect the cell, but also serves as a way to efficiently recycle resources that are limited in the cell. In addition Clp proteases play a role in various regulatory processes via controlled proteolysis of key regulatory proteins [26,27].
The Clp holoenzyme is structurally similar to the eukaryotic 26S proteasomal complex and is composed of a catalytic and a regulatory subunit. The central proteolytic core assembles into a tetradecamer of ClpP subunits consist-ing of two heptameric rings stacked on top of each other forming a cavity where protein deg-radation occurs. Inside the proteolytic chamber, each monomer contains an active site, which is formed by a Ser–His–Asp catalytic triad typical of serine proteases [28]. Axial pores of 1–2 nm are present at either end of the ClpP tetradecamer where substrates enter [29]. These pores are only large enough for small polypeptides and unfolded proteins to enter, thus cytoplasmic proteins appear to be protected from acciden-tal degradation [27]. ClpP alone displays limited peptidase activity; it can degrade small peptides and is capable of hydrolyzing proteins, but at a very slow rate. In order to function as an effec-tive protease, ClpP associates with Clp ATPases ClpC1, ClpC2 or ClpX (which confer substrate specificity), at either one or both ends, to make an active complex (Figure 2).
M. tuberculosis has two ClpP subunits, ClpP1 and ClpP2, both essential for growth and dur-ing infection [30,31]. When ClpP1 and ClpP2 were expressed in E. coli, the proteins were enzymatically inactive [28]; proteolytic activity was only detected when the two ClpP proteins were present together in the presence of small activating molecules [32,33]. This suggests that ClpP1 and ClpP2 form a mixed, active com-plex; furthermore, there is evidence that the tet-radecamer complex is composed of ClpP1 and ClpP2 hepta meric rings [32]. ClpP1 and clpP2 are highly expressed in both aerobic and hypoxic
environments and are further upregulated dur-ing re-aeration from anaerobic conditions, sug-gesting they are important for survival during latency and reactivation [34,35]. A ClpP1–ClpP2 knockdown strain exhibited reduced growth and virulence in a macrophage infection model, fur-ther confirming the importance of ClpP1 and ClpP2 for growth and infection [36].
Clp proteases have a broad substrate range. More than 50 Clp substrate proteins have been identified in E. coli; several are transcriptional regulators including FnR and an IscR, while oth-ers are involved in cell division (GTPase, and FtsZ). Clp proteases have also been implicated in the degradation of antitoxins of several toxin–antitoxin complexes [37–39]. In B. subtilis, general protein turnover depends almost exclusively on the Clp proteases [40]. B. subtilis ClpP mutants fail to develop competence, display defects in sporulation [41], are unable to grow at high temperatures and are nonmotile [41]. To date, only one substrate protein has been identified in M. tuberculosis: RseA, the SigE anti-σ-factor [42]. In the presence of vancomycin, RseA becomes phosphorylated and the ClpC1P2 complex degrades phosphorylated RseA allowing SigE to become activated. This cleavage is specific to the phosphorylated state of the protein, as degradation was blocked when the protein was dephosphorylated. Furthermore, it is exclusive to the ClpC1P2 complex as ClpP1 could not substitute ClpP2 and ClpX could not substitute ClpC1 [42].
ClpP is an unusual drug target as both inhi-bition or activation of the protease can induce cell death. In Caulobacter crescentus, where ClpP is essential, cyclic peptides were found
Figure 2. Protein degradation by the Clp proteases. In the presence of ATP, ClpP and Clp ATPases form a complex. Clp ATPases recognize and bind to substrate proteins. The protein is unfolded and translocated into ClpP for degradation. Degraded peptides of approximately seven to eight residues are released into the cytosol.
Clp ATPase
ClpP1
ClpP2
ATP
ATP
ClpP/ATPase associationand substrate
recognition
Protein unfoldingand delivery to
proteolytic chamber
Degredation andpeptide release
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to be bactericidal by preventing degradation of ClpXP substrates by binding to the ClpXP–sub-strate complex [43]. In addition compounds of the β-lactone family have been identified to attenuate the production of extracellular viru-lence factors in S. aureus and Listeria monocyto-genes by inhibiting ClpP [44,45], suggesting that β-lactones could be used as antivirulence agents.
On the other hand, acyldepsi peptides (ADEPs) are activators rather than inhibitors of the protease function of ClpP [46]. ADEPs are active against M. tuberculosis (Figure 1) and also have potent activity against various Gram-positive bacteria, including multidrug resistant isolates such as methicillin-resistant S. aureus [30,46,47]. ADEPs prevent the interaction of ClpP with its ATPase partner by competing for the same binding site and trigger a conformational change in ClpP that widens the entrance pores [48,49]. ADEPs induce oligomerization of ClpP monomers and activate the resulting tetra-decamer to bind and degrade unfolded, nascent polypeptides [50]. This unregulated proteolysis by the protease ultimately leads to cell death caused by either the shortage of essential cellu-lar proteins or by the accumulation of consider-able levels of protein fragments. Additionally, ADEP treatment induces FtsZ degradation and prevents the formation of FtsZ rings, thus inhibiting cell division in Gram-positive bac-teria [51]. Gram-negative bacteria are resistant to ADEP due to efflux pumps that remove the drug from the cell [46]. ADEP activity against M. tuberculosis is enhanced by the addition of efflux pump inhibitors, demonstrating that export occurs [30]. Resistant strains can arise by deletion of the clpP gene in species where clpP is not essential, such as B. subtilis [46]. Resistance was also observed in S. lividans, which contains five ClpP proteins, ClpP3 is insensitive to ADEP and can substitute for ClpP1; other resistance mechanisms are also involved but remain to be characterized [52]. Non-ADEP compounds that also activate ClpP have recently been identified through high-throughput screening and were named activators of self-compartmentalizing proteases [53].
In addition to ADEP-mediated death, other compounds act via the Clp complex. The natu-ral product cyclomarin A kills both growing and nonreplicating mycobacteria by binding to the Clp ATPase ClpC1 [54]. The frequency of spontaneous mutations rendering the bacteria resistant was very low (<10-9) suggesting that the Clp ATPases, as well as the proteolytic subunits, may represent attractive drug targets.
Clp proteases are found in the mitochondria, but not in the cytoplasm of human cells and the mycobacterial proteases differ significantly from the mitochondrial counterpart limiting the risk of toxicity from inhibition of human ClpP. In support of this, second-generation lactone com-pounds have low cytotoxicity against eukaryotic cell lines [45] while activator compounds (ADEP2 and ADEP4) are cytotoxic for eukaryotic cells only at very high concentrations [46].
These unusual properties of the Clp complex make it an attractive target for further develop-ment, especially since options for both activation and inhibition are presented, since they are not present in the cytoplasm of human cells and they differ significantly from their mitochondrial counterpart.
The proteasomeProteasomes are present in eukaryotes and archaea, but are only found in some bacteria of the order actinomycetales, including M. tuber-culosis. The proteasome is generally comprised of two complexes: the 20S core, where proteins are degraded, and the 19S regulatory cap that binds substrates to be degraded. The M. tuber-culosis 20S core is composed of seven α-type and seven β-type subunits (encoded by prcA and prcB, respectively) forming a barrel shaped struc-ture, which contains the active sites for protein degradation [55].
In eukaryotes ubiquitin is a marker for protea-somal degradation [56]. M. tuberculosis contains a similar tagging system with the ubiquitin analog Pup [7]. Pup is synthesized with a terminal gluta-mine, which is deamidated to glutamate by Dop prior to its attachment to the substrate acceptor lysine by PafA, in a process named pupylation [57–59]. Mpa unfolds Pup-tagged substrates, which are translocated into the proteasome for degradation [60,61]. To date, seven proteasome substrates have been identified in M. tuberculo-sis: FabD, PanB, MpA, PhoH2, Icl, MtrA and Ino1 [62,63]; however, numerous proteins with various cellular functions have been identified as targets for pupylation in M. smegmatis and M. tuberculosis [62,64].
Proteasomal proteolysis is dispensable for M. tuberculosis growth in vitro; nevertheless, prcBA deletion mutants exhibit growth defects and are more sensitive to reactive nitrogen inter-mediates [65,66]. However, an active site mutant proteasome can complement in vitro growth defects and nitric oxide susceptibility, suggest-ing that the proteasome also has a proteolysis-independent function [66]. By contrast, prcA
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and prcB are essential for persistence as deletion strains and active site mutant strains are unable to survive prolonged stationary phase and nutri-ent starvation in vitro, or in the chronic mouse infection [65,66]. Similarly the accessory proteins Dop, MpA and PafA are not essential for growth but are essential for virulence in the mouse and resistance to reactive nitrogen intermediates, which cause cell death in vivo [63,67,68].
The main concern for the development of proteasome inhibitors is their inherent toxic-ity owing to the high degree of conservation of the mycobacterial proteasome with the mam-malian homolog. A class of oxathiazol-2-one compounds were found to inhibit M. tubercu-losis growth effectively and to be selective for the mycobacterial proteasome over the human counterpart (>1000-fold less effective against human proteasome) [69]. These compounds induce a conformational change in the myco-bacterial proteasome and act as irreversible inhibitors. Importantly, these compounds kill persistent M. tuberculosis and could therefore be used to clear latent TB. The natural product compound, a lipopeptide aldehyde, fellutamide B is the most potent inhibitor of the M. tuber-culosis proteasome tested so far (1000-fold more potent than other peptide aldehydes tested); however, this compound was found to be cytotoxic [70,71].
Despite the structural homology between the eukaryotic and mycobacterial proteasome, Dop and PafA proteins do not appear to have homologs in eukaryotes and could be alternative targets to inhibiting the core protease.
Proteases involved in virulence & other mechanisms
HtrA proteasesM. tuberculosis encodes several proteases belong-ing to the HtrA family of serine proteases; HtrA1 (Rv1223), HtrA2 (Rv0983) and HtrA3 (Rv0125) [72]. Nearly all forms of life, including humans, have HtrA proteases [73,74]. This large family of proteases is responsible for maintaining the pro-teome of a cell by degrading misfolded or dam-aged membrane proteins and by acting as chap-erones for others. The overall structures of HtrA proteases are similar; they consist of a proteo lytic domain containing a highly conserved active-site catalytic triad (Ser–His–Asp), and a PDZ protein–protein interaction domain to facilitate substrate binding [74]. While similar in structure, the functions and substrates of HtrA proteases are quite diverse and are largely driven by dif-ferences not in the conserved domains, but in
the conformation of domains that make up the active site [74].
The first htrA gene was described in E. coli and studies performed since have identified several homologs that are either membrane bound or in the periplasmic space and function to regu-late protein turnover in response to stress. This suggests that HtrA proteases play a vital role in adaptation of E. coli to changing environmental conditions. In other bacterial species, HtrA prote-ases play a role in virulence [75,76]. An htrA mutant of Klebsiella pneumonia made less capsule, bound more serum complement protein, was hypersensi-tive to oxidative stress and elevated temperatures, and displayed reduced virulence in a mouse model [76]. These data re inforce the fact that HtrA proteases are critical to cellular viability.
Evidence suggests that the HtrA proteases play a significant role in the physiology of M. tubercu-losis and pathology of infection. M. tuberculosis htrA1 is essential, as determined by both trans-poson mutagenesis and attempts to knock-out the gene [25,72]. HtrA1 is found in the membrane fraction [77], and while HtrA2 and HtrA3 are also predicted to be membrane bound, evidence suggests that they actually may be secreted [78–80]. An M. tuberculosis htrA2 mutant was viable showing that the protease is not essen-tial, but the mutant was attenuated in a mouse model, causing less lung pathology and increas-ing mouse survival times [72]. In addition, HtrA2 had chaperone activity, while a knockout strain of htrA3 had no phenotypic defects and did not demonstrate chaperone activity [74].
HtrA proteases have been targeted for drug development in humans, owing to a suggested link to certain cancers [81]. In prokaryotes, HtrA has been considered a viable target in Helicobacter pylori [82]. Of the M. tuberculosis HtrA proteases, only the essential protease HtrA1 seems prom-ising as a drug target. While HtrA2 has been shown to have a role in virulence, the fact that it may be secreted into its environment suggests that there may be difficulties in targeting its activity and the membrane-located HtrA1 seems a more attractive target to pursue. However, structural data obtained from the crystallization of HtrA2 provides valuable information regarding the active site of the M. tuberculosis HtrA proteases, allowing for strategies to find molecules that can bind and block these active sites in target-based, biochemical screens [72]. Structural information will be key in identifying compounds with speci-ficity that do not cross-react with HtrA prote-ase homologs found in other species, including humans.
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FtsH proteasesM. tuberculosis FtsH is a membrane-bound, ATP-dependent, zinc metalloprotease belonging to the AAA protease superfamily (ATPases associated with diverse cellular activities). Most prokaryotes and eukaryotes, including humans, harbor homo-logs of FtsH proteases [83]. Conserved regions in all FtsH proteins include two regions of approxi-mately 200 amino acids and two ATP binding domains. Sequence similarity of these domains is greater than 40% among bacteria, yeast and humans [84,85]. While the human and yeast AAA proteases are localized to mitochondria, prokary-otic AAA proteases are localized to the plasma membrane with the catalytic subunits in the cytoplasm of the cell [84].
The functions of FtsH proteases are diverse and their broad spectrum of activity is believed to aid cells in adaptation to the different envi-ronments they may encounter [83]. M. tuberculo-sis has an active FtsH [86], which is predicted to be essential by transposon mutagenesis studies [25]. ftsH is expressed in the guinea pig model of infection and is required for cholesterol catabo-lism, which M. tuberculosis can use as a carbon source in vivo [87,88]. In other organisms, the role of FtsH has been proposed to regulate the cell membrane, much like HtrA, by degrading mistranslated or misfolded proteins that com-promise membrane integrity. ftsH knockouts in Pseudomonas aeruginosa and Caulobacter cresen-tus led to increased sensitivity to aminoglycoside antibiotics and tobramycin, respectively [89,90].
Conservation of FtsH structure and function among a broad group of bacterial organisms is a hallmark of an attractive drug target. In fact, switching of the M. tuberculosis ftsH gene into an E. coli ftsH3 mutant not only rescued growth, but was also able to rescue its function – the normal substrates of the E. coli protease were processed by M. tuberculosis FtsH [83]. Combined with evidence that FtsH is essential and involved in adaptation to survive in vivo, these data indicate that FtsH may be a suitable target for drug development. However, consider-ing the sequence similarity of the active domains with that of human AAA proteases, finding a molecule that specifically acts on the M. tuber-culosis FtsH protease may present a significant challenge.
Future perspective on M. tuberculosis proteases as drug targets
A number of proteases have been proposed as drug targets, although target validation is more robust for some than others. Proteases with the greatest potential and those under the most scrutiny are involved in essential cellular pro-cesses such as secretion and protein turnover. Of these, SPase I, the proteasome and the ClpP complex have the greatest potential. Proteases involved in virulence are of biological signifi-cance and of interest as drug targets; however, given the difficulties posed by the drug develop-ment pathway, more resources and longer time-scales to reach drug candidates are potentially
Executive summary
TB drug discovery�n There is an urgent need for novel drugs against TB. Current therapy is lengthy and complex, requiring multiple drugs for extended
periods of time. There is a need to shorten the time length of drug therapy to increase patient compliance and reduce costs.�n There is a dearth of compounds in the TB drug discovery pipeline and a corresponding lack of validated drug targets.�n Drugs that target novel processes are required to overcome the increasing occurrence of drug resistance.�n Targeting cellular processes required for both replication and survival under nonreplicating conditions is likely to shorten therapy by
eradicating persister organisms more rapidly.
Proteases as drug targets�n Proteases are validated as drug targets in viral infections, where HIV protease inhibitors have been successfully developed.�n Mycobacterium tuberculosis has a relatively small number of proteases, representing all of the major families, of which several have
been identified as essential for growth in vitro or in vivo.�n Signal peptidases involved in protein secretion have been identified; the type I signal peptidase is essential in vitro and the type II signal
peptidase is essential in vivo.�n M. tuberculosis has two major systems involved in proteolytic turnover, the Clp complex and the proteasome, which are essential
in vitro and in vivo, respectively.�n Specialized type VII secretion systems include the mycosin proteases, which may be essential in vivo.�n M. tuberculosis proteases have been validated in vitro using genetic and chemical inactivation – these include the sole type I signal
peptidase and the two proteolytic subunits of the Clp complex.�n Several M. tuberculosis proteases hold promise as virulence targets, with loss of function resulting in disruption of the infection cycle,
including the proteasome and the type II (lipoprotein) signal peptidase.
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Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the sub-ject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
required. However, targeting proteases appears to be a legitimate enterprise and may eventually lead to shortened and effective treatments for drug-resistant TB.
Future perspectiven�Proteases that play key cellular roles and are
involved in the processing or degradation of multiple cellular substrates are likely to be good targets, since disruption is pleiotropic. More effort towards characterizing the role of these proteases, their substrates and the condi-tions that they are active under is required. Such information will lead to increased under-standing of several areas of host–pathogen biology;
n�Current inhibitors of proteases are not amen-able to drug development owing to lack of specificity for mycobacterial enzymes, toxicity for mammalian cells or lack of whole-cell activity against M. tuberculosis. The identifica-tion of novel chemical scaffolds which inhibit key proteases will likely generate lead com-pounds for drug development. An increased effort towards screening large compound
libraries could yield multiple series for progression;
n�The validation of additional proteases as drug targets, both in the classical sense as essential for viability and in the more novel approaches as virulence targets, holds promise for the identification of new antimicrobial agents;
n�Identification of inhibitors of the signal pepti-dase could lead to therapy-shortening drugs, since they are likely to target both replicating and persistent organisms;
n�Identification of activators of the ClpP protease could result in an unique class of drugs.
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