Drug targeting of heme proteins in Mycobacterium tuberculosis

Kirsty J. McLean and Andrew W. Munro

School of Chemistry, The University of Manchester, Manchester Institute of Biotechnology, 131 Princess Street, Manchester M1 7DN, UK.

Corresponding author: Munro, A. W. (Andrew.Munro@Manchester.ac.uk). Phone 0044 161 3065151

Keywords: Mycobacterium tuberculosis, hemoprotein, heme acquisition, heme oxygenase, heme sensors, truncated hemoglobin, catalase-peroxidase, cytochrome P450

Teaser: Heme enzymes are crucial to viability and pathogenicity of Mycobacterium tuberculosis, and novel strategies for their inhibition present exciting new opportunities for TB drug development.

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Abstract

The human pathogen Mycobacterium tuberculosis (Mtb) causes more deaths than any other infectious disease. Iron is crucial for Mtb to infect the host and to sustain infection, with Mtb encoding large numbers of iron-binding proteins. Many of these are hemoproteins with key roles including defense against oxidative stress, cellular signaling and regulation, host cholesterol metabolism and respiratory processes. Various heme enzymes in Mtb are validated drug targets and/or products of genes essential for bacterial viability or survival in the host. This article reviews structure, function and druggability of key Mtb heme enzymes and strategies used for their inhibition.

Introduction

Iron is among the most important nutrients, and is essential in all organisms for metabolic functions including nucleic acid synthesis and respiration [1]. Mtb requires iron for vital functions including bacterial infection, proliferation and viability in host cells, where it is scavenged by Mtb engulfed in macrophages for bacterial survival [2]. Although iron is one of the most abundant elements, it exists predominantly as insoluble salts and is thus not freely available in living cells. In addition, iron binding proteins in the human host (e.g. ferritin, lactoferritin and transferrin) sequester ferric iron (FeIII), or it is found as heme contained in hemoproteins or in other iron-binding proteins. The host also produces proteins that efflux iron (e.g. NRAMP1), further decreasing the intracellular iron pool [3,4]. Iron availability is also tightly regulated in both the host and the Mtb pathogen to protect against iron-mediated toxicity and damaging hydroxyl radicals that are generated from free iron via the Fenton reaction [5]. Iron assimilation is a challenging process in Mtb and the deprivation of iron is a critical environmental stress encountered during the infection process, where Mtb replicates within the macrophage phagosome. Included in the host defense mechanisms against mycobacterial infection are the reduction of dietary iron uptake and restriction of circulating transferrin-bound iron levels [6]. Mtb has evolved strategies for assimilation of iron that are often more efficient than those of the human host, enabling Mtb to sequester iron through various mechanisms. Mtb transcriptional regulators confer strict control over iron metabolism, transport and storage. For example, IdeR (Rv2711) is an essential iron-binding transcriptional regulator that upregulates genes involved in iron-chelating siderophore synthesis and transport (mbt and irtAB, respectively) and represses iron storage genes (bfrA and B) during conditions of iron depletion [7,8]. Iron assimilation is thus a crucial process and Mtb acquires either non-heme or heme-bound iron from the host by different mechanisms. Non-heme and heme-derived iron uptake strategies are thought to play complementary roles whereby one or both pathways are activated by environmental stresses imposed by the host or associated with a particular phase of the infection process. Heme uptake, compared with non-heme iron uptake, can tap into a much larger host iron pool source, with around three quarters of the total iron in adults bound within the heme prosthetic group [9,10].

Mtb’s ability to utilize heme as an abundant iron source has led to identification of important heme acquisition strategies involving Mtb proteins which act as heme transport enzymes that bind (Rv0203) and internalize the heme (MmpL11 and MmpL3) [9,11]. In order for iron to be released from heme, it is bound and oxidatively cleaved by a heme oxygenase (MhuD) [9,12]. Mtb can also synthesize heme de novo using the essential ferrochelatase enzyme HemZ, which catalyzes insertion

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of ferrous iron (FeII) into protoporphyrin IX to make protoheme in the terminal step of heme biosynthesis [13]. However, limited studies have been performed on the Mtb heme synthesis pathway enzymes and HemZ has low activity in vitro [14,15]. In addition to iron acquisition, it is possible that some heme imported into Mtb is also incorporated into hemoproteins, supplementing its own heme biosynthetic pathway and providing a degree of redundancy. Mtb contains several heme enzymes with important regulatory and catalytic roles, such as the heme sensors/regulators DosS and DosT, truncated hemoglobin (TrHbN), catalase-peroxidase (KatG) and cytochromes P450. Mtb can thus use heme as an iron source as well as a cofactor for its heme-binding enzymes. Here we describe selected important Mtb heme enzymes and highlight current strategies for development of inhibitors against these potential drug targets.

Heme transport enzymes

Mtb’s heme iron uptake pathway was identified through investigation of mycobacterial strains with attenuated iron-chelating siderophore biosynthetic pathways, but which still grew in the absence of this iron uptake machinery, suggesting that alternative pathway(s) for iron acquisition existed [11,16,17]. The addition of heme or hemoglobin supplemented growth of these iron deficient Mtb strains and was also essential for growth of wild-type Mtb cultured in iron depleted media [11,16]. Furthermore, the identification and characterization of the heme degrading oxidase MhuD (see below) also demonstrated that Mtb utilizes heme as an iron source. The Mtb H37Rv gene cluster rv0201c-rv0207c was shown to be involved in Mtb heme uptake and its intracellular transfer [11]. To date, three proteins within this cluster have been assigned roles in heme transport across the mycobacterial membrane.

Rv0203 is a small, secreted hemoprotein specific to Mtb. Rv0203 was first identified using a heme pull-down assay, where Mtb culture filtrate was washed through heme-agarose beads to identify a single protein using mass spectrometry. Targeted gene deletion studies confirmed Rv0203’s role in heme uptake [11]. Spectroscopic characterization of Rv0203 demonstrated important roles for His89 (and/or His63) and a tyrosine residue (Tyr59) in heme binding [18]. The crystal structure of apo-Rv0203 revealed a novel -helical fold with the heme-binding site close to the protein surface, likely facilitating the capture and release of heme consistent with its role as an extracellular or periplasmic heme transfer protein. The genes surrounding rv0203 are predicted to encode transmembrane proteins, with two particular candidates within this gene cluster, rv0202c (mmpL11) and rv0206c (mmpL3), identified to encode heme transmembrane transporters [11]. MmpL11 and MmpL3 are members of the Mycobacterial membrane protein Large (MmpL) family, consisting of 13 inner membrane proteins predicted to have key roles in transportation of molecules across the mycobacterial membrane. The MmpL proteins are also mycobacterial members of the designated Resistance Nodulation cell Division (RND) permease superfamily of efflux pumps that are involved in transporting a variety of substrates, such as drugs, bile salts, fatty acids and (in Mtb) phthiocerol dimycocerosate (MmpL7) and sulfatide precursors (MmpL8) [19]. The mmpL3 is the only gene of this family essential for Mtb growth, indicating a vital functional role. MmpL3 and MmpL11, located distinct from the other Mtb MmpL family members, share 25% amino acid sequence identity and are predicted to have similar roles. Gene knockout studies on mmpL11, combined with its overexpression and analysis of heme binding to the soluble periplasmic (D1) domains of both MmpL

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proteins, support their roles as heme transporters. Fast reaction spectroscopic studies have shown that secreted Rv0203 can directly transport heme to the soluble D1 domains of the MmpL transmembrane proteins [11,19,20]. It is thought that Rv0203 binds heme, likely scavenged from host hemoproteins (e.g. hemoglobin), and transports it across the bacterial periplasm to the MmpL3 and MmpL11 proteins, which then efficiently carry heme across the inner membrane to the cytoplasm (Figure 1A).

Among the Mtb heme acquisition enzymes, the genetically essential MmpL3 is considered the most druggable target. As well as having roles in heme transport, the MmpL proteins were shown to transport other substrates across the inner membrane in the opposite direction (from cytoplasm to periplasm). MmpL3 mediates transport of mycolates in the form of trehalose monomycolate (TMM) [21]. MmpL11 was also shown to export monomeromycolyl diacylglycerol (mmDAG) and mycolate ester wax in M. smegmatis. The Mtb mmpL11 gene can also compensate for its M. smegmatis counterpart in mutant strains, suggesting a common role in both species [22]. In vivo studies showed that mice infected with mmpL11 deletion strains survive considerably longer than those infected with wild-type Mtb, suggesting roles in infection and virulence. Furthermore, a MmpL3 homolog was shown to co-localize with fatty acid synthase-II (FAS-II) complexes that produce meromycolate at the poles and septa of growing M. smegmatis cells [23]. These studies suggest that MmpL3 not only has dual roles in heme and TMM transport, but that its dynamic localization may indicate a function relating to the coordination of cell envelope assembly and processes of cell elongation and division, with FAS-II derived synthesis of mycolic acids at the active poles and septa enabling lipid transport by MmpL3 across the dividing membrane [24]. The MmpL family is predicted to share a similar structural topology, with twelve transmembrane helices and two extra-periplasmic domains located between helices 1 and 2 (periplasmic domain D1) and helices 7 and 8. MmpL3 and MmpL11 are phylogenetically distinct from the other family members, with a shorter second periplasmic domain (D2) between helices 7 and 8, in contrast to the large ‘porter’ (or ‘pore’) domains composed of three subdomains (a docking domain and two sub-porter domains) present in the remaining Mtb MmpL’s and the larger RND family transporters. Recently, the crystal structure of the soluble second periplasmic D2 domain of MmpL11 was solved, revealing its structural similarity to a single sub-porter domain of the larger RND periplasmic porter. Due to amino acid similarities and through homology modelling, it is presumed that this D2 domain is similar in MmpL3, as well as resembling the first D1 domain (shown to bind heme), and may provide insights into periplasmic interactions of this class of Mtb MmpL protein. MmpL3 and MmpL11 also possess an additional extended cytoplasmic C-terminal domain (termed D3) that is proposed to mediate interactions with biosynthetic or accessory membrane proteins involved in transport of mycolates [25,26]. It is interesting to speculate that this cytoplasmic domain, specific to MmpL3 and MmpL11, may also be involved in heme transfer, perhaps interacting transiently with the cytosolic heme oxygenase MhuD and facilitating delivery of heme from host hemoproteins through to its ultimate breakdown to release iron essential for Mtb viability.

MmpL3 inhibitors have emerged over the last few years and were shown to inhibit mycolic acid synthesis and disrupt Mtb growth. These inhibitors have different structural scaffolds and may offer new routes to inhibiting mycolic acid transport and disrupting cell envelope structure [24,27]. Examples of MmpL3 inhibitors are SQ109, a 1,2-diamine related to ethambutol [28]; BM212, a 1,5-diarylpyrrole derivative [29,30]; THPPs, the tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamides [31,32]; AU1235, an adamantly urea based compound [33]; C215, a benzimidazole derivative [34];

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indolecarboxamides [35,36], and N-benzyl-6’,7’-dihydrospiro[piperidine-4,4’-thieno[3,2-c]pyran [31] (Figure 1B). Most of these inhibitors were identified by whole cell screening of compound libraries against Mtb and were found to abolish the proton gradient that supplies energy for the majority of transmembrane transport processes. However, further studies revealed that these compounds also targeted multiple enzymes with roles in menaquinone biosynthesis, respiration and ATP synthesis. In addition, SQ109, BM212 and the THPPs target non-replicating Mtb, but also have a broad spectrum of inhibition, with activity against bacterial and fungal organisms devoid of mycolates and MmpL3 orthologs [24,26,27]. MmpL3 is a promiscuous drug target and it remains unknown whether its inhibitors will specifically target heme transport and iron acquisition. However, a multi-target, multi-pathway drug approach, although challenging, may provide a route to avoid long treatment regimens and associated resistance issues.

The heme degrading oxidase MhuD

An important enzyme in the heme and heme iron acquisition process is MhuD ( Mycobacterial heme utilization, Degrader), an unusual heme oxygenase (HOX) that binds and degrades heme [37] (Figure 1A). Mammalian heme oxygenases are well-characterized enzymes that typically yield ferrous iron, carbon monoxide (CO) and biliverdin though three successive monooxygenation steps, using heme as both a substrate and an oxygen activating cofactor. HOXs have important physiological roles in cellular heme homeostasis, with links to cell signaling processes, antioxidants and biosynthesis of light-sensing bilins, as well as functioning in iron uptake in pathogenic bacteria [38]. MhuD is a non-canonical HOX that produces iron and mycobilins (the isomers mycobilin–a and –b), mycobacteria-specific tetrapyrrole products of heme catabolism that possess an aldehyde moiety at the meso-heme cleavage site. MhuD is mechanistically distinct from conventional HOXs and does not release CO during heme degradation and subsequent iron release [12]. MhuD’s ability to cleave heme without releasing CO has important physiological implications, as CO has crucial roles in latency and persistence through activation of the two-component DosS/DosR signal transduction system (see below) [39], thus enabling acquisition of iron without the onset of dormancy. The crystal structure of MhuD has been solved, revealing a single active site with heme bound in a distorted, non-planar conformation described as “ruffled” [12]. MhuD can also accommodate a second heme molecule in an inactive state of the enzyme [37]. Heme ruffling in MhuD is proposed to contribute to its unusual reaction mechanism that involves both monooxygenation and dioxygenation steps in the active site at different stages of catalysis [40]. Similar heme ruffling is observed in the MhuD orthologs IsdG and IsgI from Staphylococcus aureus. These are also evolutionarily and structurally distinct from the canonical HOXs, and also yield different products to MhuD (staphylobilins and formaldehyde, in addition to iron) through heme oxidative degradation, indicating an important mechanistic difference [41]. MhuD-bound heme iron is ligated through a histidine residue (His75) with a tryptophan (Trp66) located 3.67 Å from the heme -meso carbon and postulated to contribute to steric interactions that influence dynamics of heme ruffling [42]. A combination of spectroscopic studies suggest that MhuD primarily hydroxylates heme in the - or -meso positions forming a transient hydroxyheme intermediate with a porphyrin radical, possibly induced and modulated by heme ruffling, that reacts with oxygen to facilitate dioxygenation and subsequent ring cleavage to produce mycobilins and liberate ferrous iron [40].

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MhuD is non-essential for Mtb growth in vitro. However, its role in heme degradation resulting in iron release, coupled with its importance in iron acquisition from the aforementioned heme uptake systems, presents it as a potential drug target for heme analog-based compounds. Targeting heme uptake (e.g. Rv0203, MmpL3 and MmpL11) and heme metabolizing enzymes (MhuD) may enable intracellular delivery of molecules that are toxic to Mtb. Non-iron metalloporphyrins are a group of heme analogues that have other metal ions substituted for the iron, and these molecules were shown to inhibit the growth of many species of bacteria in conditions of iron-depletion [43]. Exposure to the heme analogues gallium protoporphyrin (GaPPIX) and zinc protoporphyrin (ZnPPIX) was shown to be effective in inhibiting Staphylococcus aureus growth, and these molecules are thought to target heme uptake and aerobic respiration. In addition, selenium, palladium and zinc modified porphyrins (e.g. mesoporphyrins) are active against the protozoan parasite Trypanosoma cruzi through inhibition of heme transporters [9]. Furthermore, GaPPIX is lethal to M. smegmatis [43] and was recently shown to be a potent inhibitor of M. abscessus growth, including antibiotic-resistant clinical isolates [44], with Ga nitrates also able to inhibit growth of Mtb and M. avium [45]. The exploitation of Mtb heme uptake systems to transport, deliver and release toxic components such as gallium (that targets iron-dependent pathways and proteins) is an interesting antibiotic strategy. Determination of crystal structures of these discrete Mtb heme acquisition proteins and the interrogation of their mechanisms, as has been achieved for MhuD, may facilitate design of molecules that specifically target the heme binding sites of essential Mtb enzymes, while avoiding the human heme uptake machinery. The simultaneous targeting of multiple systems important for infection, but which have certain levels of redundancy, such as heme and iron uptake, is a challenging proposition. Further research is needed in this area, but inhibiting several key enzymes involved in Mtb heme acquisition and degradation appears a good strategy that might avoid development of drug resistance.

Heme Sensors and Regulators

The Dormancy survival heme sensor proteins DosS and DosT (Rv3132c and Rv2027c, also called DevS and DevT) are histidine kinases that are members of two-component systems along with their DNA binding response regulator partner protein DosR (Rv3133c, DevR) (Figure 2) [46,47]. These sensor/regulator systems play important roles in detecting and adapting to environmental stresses from the human host during infection. Mtb can survive for extended periods within the granuloma in a non-replicating, latent state that is unresponsive to most antibiotics [39]. Diverse conditions such as starvation, hypoxia, nitric oxide (NO) exposure, and increased acidity are associated with and thought to trigger dormancy (also known as persistence), as well as activating host immune responses [48]. The DosS/DosT-DosR system is induced by multiple gaseous stresses, in particular hypoxia or exposure to NO and CO, as well as by vitamin C that leads to hypoxia [47]. The activation of DosS/T enables autophosphorylation of a conserved histidine residue (His395/His392 in DosS/DosT) and phosphate transfer to Asp54 of DosR. Phosphorylated DosR binds to DNA regulatory elements upstream of hypoxic response genes, leading to upregulation of their expression. The DosR regulon includes ~47 genes, many of which are still uncharacterized but predicted to have roles in the suppression of metabolic activity and adaptation of Mtb to hypoxia and gas exposure, triggering the transition into the non-replicating, persistent state (Figure 2A). Induction of the DosR regulon is essential to adjustment and survival of Mtb under these dormancy-related conditions and during

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exposure to host defense mechanisms [39,49]. DosS/DosT are closely related (61% amino acid identity) and have similar structural architecture consisting of two N-terminal GAF (cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA) domains, followed by histidine kinase and ATPase domains. Only the GAF-A domain binds heme The DosS GAF-A domain structure shows heme ligation by a proximal histidine (His149) and a distal hydrogen bonding network that extends from a water coordinated to the heme iron through the side chains of Tyr171, Glu87 and His89 [50,51]. It was postulated that DosT (which is expressed constitutively in anaerobic dormancy and in aerobic growth) is a gas sensor, whereas DosS (which is induced by hypoxia, CO and NO, as well by menaquinone and ascorbate in aerobic conditions) is both a gas and intracellular redox sensor [39,51].

DosS is the most druggable component of the heme sensor/regulator system and is essential for Mtb survival in mice [52], as well as being the key protein involved in CO-mediated initiation of dormancy. Recent studies have highlighted DosS to have a dual role, with kinase activity to trigger phosphorylation-dependent transition to the dormant state under hypoxia, as well as phosphatase activity towards DosR under aerobic conditions, thus blocking expression of the DosR regulon [53]. These opposing roles reveal DosS to be an important bifunctional heme sensor protein with a critical role in tuning the response of the dormancy regulon to its environment. Peptides that bind to DosS were shown to impair its kinase function and to prevent Mtb survival in hypoxic conditions [54]. In addition, peptides have been identified which bind to DosS surfaces that interact with DosR, inhibiting phosphorylation of both DosR/S (Figure 2B). These were effective in preventing the DosS/R cascade and interrupting the transition of Mtb to the dormant state. A phenylcoumarin derivative molecule (Figure 2C) was also shown to lock DosR in an inactive conformation, inhibiting binding to its target DNA and exhibiting activity against non-replicating Mtb [55]. The use of mimetic peptides as inhibitors may have significant advantages, including increased target specificity coupled with lower accumulation in host tissues and associated toxicity. Such peptides can be cost effective alternatives to conventional compounds, particularly in light of improved peptide synthesis routes [56]. Targeting both DosS and DosR could effectively inhibit the transition of Mtb into the latent state, leaving active Mtb that are more susceptible to antibiotics and more readily killed.

Heme enzymes with important roles in Mtb

Truncated hemoglobin (TrHbN)

The Mtb truncated hemoglobin N (TrHbN) is implicated in NO detoxification in the macrophage. Its crystal structure shows that TrHbN is a homodimer and has a histidine-coordinated heme iron with a dioxygen ligand at the opposite face of the heme [57]. A tyrosine highly conserved in TrHbs stabilizes the dioxygen, and addition of NO was shown to produce nitrate as the major product in studies on M. bovis TrhbN, highlighting a dioxygenase mechanism by which TrHbN protects mycobacteria from nitrosative stress imposed by the host [58]. Hydrophobic tunnels to the heme were identified in the Mtb TrHbN and other TrHbs, and proposed to mediate O2, NO and other gas transport to the catalytic site [59]. Molecular simulations indicated that two orthogonal channels facilitate O2 (short channel) and NO (longer channel) transport to the active site, and that a Phe15 “gate” modulates NO diffusion in its channel and may regulate activity [60]. Related studies predicted a similarly (Phe62) gated access for O2 in a distinct Mtb TrHbN channel [61]. Other recent studies indicate that TrHbN is

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post-translationally glycosylated in Mtb and is retained on the cell membrane and wall, with glycan modification by a mannose. A new role was thus suggested for TrHbN in modulating host pathogen interactions and the host immune response [62].

Catalase-peroxidase (KatG)

The Mtb KatG enzyme is a His-coordinated, heme b-containing catalase-peroxidase enzyme. Mutants of KatG can confer bacterial resistance to the front-line drug isoniazid (INH), with the S315T mutant found in 70% of INH-resistant Mtb strains. It is thought that KatG converts INH into an isonicotinoyl radical, which in turn binds to NAD(H) or NAD radical and forms an isonicotinoyl-NAD adduct that inhibits InhA and disrupt synthesis of the Mtb cell wall [63,64]. Decreased catalase activity is a feature of clinical isolates showing resistance to INH [63,65]. KatG was also reported to have peroxynitritase activity, revealing an important role in detoxifying reactive nitrogen species and possibly helping facilitate Mtb survival in the macrophage [66]. Similarly, KatG was shown to catabolize peroxides produced by the macrophage NADPH oxidase [67]. KatG is negatively regulated by FurA, which is encoded immediately upstream of the katG gene [68,69]. Mutations that downregulate katG expression were also identified in the furA-katG intergenic region, and are associated with INH resistance in Mtb [70]. KatG thus plays a central role in combating oxidative/nitrosative stress, as well as being crucial to the efficacy of INH.

Cytochromes P450

Cytochrome P450 enzymes (P450s or CYPs) are a large family of monooxygenases that typically catalyze the activation of molecular oxygen, and the regio- and stereo-specific insertion of an oxygen atom into a wide variety of substrates. The CYPs are heme b-binding proteins with a cysteine thiolate ligation that gives them distinctive spectral properties. They display a diagnostic absorption peak at ~450 nm in the ferrous, CO-bound form, hence ‘pigment at 450 nm’ or ‘P450’ [71]. Mtb has genes encoding 20 CYP proteins. This large number suggests important biochemical roles within Mtb [72]. Characterization of selected Mtb CYPs has highlighted diverse and unexpected roles in the metabolism of sterols (particularly cholesterol) and lipids, the production of secondary metabolites (cyclic dipeptides) and the oxidative modification of respiratory menaquinone [73]. The Mtb CYP125A1 (product of the rv3545c gene) is a cholesterol oxidase that is arguably the most druggable P450. Cholesterol is the major carbon source during early infection and is essential for Mtb survival in the macrophage phagosome. It accumulates in Mtb-induced foamy macrophages and is important during late infection within necrotic granulomas [74,75]. Studies in mice demonstrated that the cyp125A1 gene is required for Mtb survival in the mammalian host, despite being non-essential for growth in vitro [76]. The cyp125A1 gene is also induced in Mtb-infected human macrophages [77,78]. A second non-essential cholesterol oxidase CYP142A1 (product of the rv3518c gene) was shown to compensate for defects in cyp125A1 in certain Mtb strains [79]. However, CYP125A1 is Mtb’s major cholesterol oxidase, playing roles in the catabolism of host cholesterol for energy, detoxification of cellular cholest-4-en-3-one (cholestenone, a ketone derivative), and potentially in the cholesterol-derived synthesis of the cell wall lipid phthiocerol dimycoserate (PDIM) [79-82]. CYP125A1 and CYP142A1 both catalyze C26 ω-hydroxylation(s) of the side-chain of cholesterol and

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cholestenone; primary steps towards the breakdown of the side-chain [83-85]. One of the CYP125A1/CYP142A1 products is 3-oxo-4-cholestenoic acid, produced by three successive oxidations of cholestenone, which was shown to be an inducer of one of the two TetR-type transcriptional repressors (KstR) that regulates part of the cholesterol catabolic gene cluster in M. smegmatis [86-88]. These studies highlight a further important role of the cholesterol oxidase P450s, although it cannot be ruled out that other oxidized products from the cholesterol pathway may also act as inducers. Moreover, cholestenone was shown to inhibit the growth of Mtb when generated intracellularly or added exogenously to strains with defective cyp125A1 (and cyp142A1) genes [82]. The crystal structures of CYP125A1 (Figure 3A) and CYP142A1 have been determined at high resolution and show significant differences in their active site heme distal pockets, despite their ability to perform the same reactions, suggestive of evolutionary differences between these enzymes [82,84,85]. CYP125A1 exhibited an additional cholesterol oxidase activity, with the production of unusual cholesterol ester deformylation products derived from the cholesterol side-chain aldehyde intermediate of C26 oxidations. Further studies are required to understand the functional relevance of these ester molecules. However, cholesteryl esters are known to accumulate in Mtb infected human macrophages [89]. The redundancy of the Mtb cholesterol oxidase CYPs, with CYP142A1 able to compensate for CYP125A1 in certain Mtb strains, presents a challenge for effective drug development and highlights the adaptability of Mtb and its self-preservation mechanisms to protect important metabolic pathways. However, targeting multiple enzymes and their associated pathways may again offer new routes to prevent resistance. A number of cholesterol/one derivatives have proven effective in inhibiting Mtb growth (in addition to cholestenone that is ultimately catabolized by Mtb and is thus an unsuitable drug candidate). The hydroxylated sterol (25R)-cholest-5-en-3,16,26-triol (Figure 3B) and its keto derivative are non-degrading analogues of cholestenone that are potent inhibitors of Mtb growth regardless of the presence or absence of CYP125A1/CYP142A1. Since these 16-hydroxylated sterols are no longer substrates for the cholesterol oxidase CYPs (with no hydroxylation of the propionate side-chain), they are not catabolized by Mtb and accumulate in the cell with toxic effects [90]. Furthermore, difluoromethyl cholesterol analogues with truncated side chains (Figure 3C) (longer side chains are poorly metabolized by CYP125A1/CYP142A1 with slow conversion rates) were also shown to prevent Mtb growth in culture [90]. These molecules are promising inhibitors and demonstrate the use of inactive substrate analogs of Mtb CYP enzymes as useful agents that overcome effects of genetic redundancy where compensatory enzymes (such as CYP142A1) have likely evolved to facilitate adaptation of Mtb to utilize different carbon sources during infection. It is possible that these cholesterol analogs not only act upon the CYP-mediated cholesterol oxidation steps, but also at other target sites in cholesterol pathways, with pleiotropic effects. The efficacy of cholesterol analogs awaits in vivo studies, but if able to act at more than one site they may have therapeutic potential.

CYP121A1 (product of gene rv2276) is an essential Mtb P450 that produces the metabolite mycocyclosin through the oxidative biaryl coupling of its cyclo-L-tyrosine-L-tyrosine (cYY) substrate [91]. The physiological role of mycocyclosin is unknown, though members of this diketopiperazine class of compounds were found to have several important activities (e.g. immunosuppression, blockage of cation channels, and possibly as toxins) [92-94]. A number of high resolution structures of CYP12A1 in complex with substrate and with azole inhibitors have facilitated the application of fragment based screening and drug design methods (Figure 4) (see Targeting tuberculosis using

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structure-guided fragment-based drug design for a description of the technique) [95-97]. Substrate analogs have also been utilized to explore CYP121A1 substrate specificity and to confirm its preference for cYY over other cyclic dipeptides, leading to the design of potential inhibitors specific for CYP121A1 [98,99]. In addition, high throughput compound library screening identified several ligands that inhibit CYP121A1 (Figure 4). However, these molecules are large and very diverse in structure. Further development and validation of these inhibitors is required in order to confirm their antibacterial potency and their potential in the development of CYP121A1 as a viable anti-TB drug target.

Conclusion

Heme as both a substrate (catabolized to liberate iron essential for Mtb growth) and an enzyme cofactor (essential to the function of several Mtb proteins) is a molecule with diverse and important roles in Mtb. Various hemoproteins are crucial to survival of Mtb in the human host. Novel approaches targeting hemoproteins such as key regulatory proteins (DosS/R), heme transporters (MmpL3 and 11), P450s (CYP121A1 and cholesterol oxidases CYP125A1 and CYP142A1) and heme oxygenase (MhuD) have the potential to produce effective drugs acting on new pathways pivotal to Mtb viability. Indeed, potent inhibitors have already been developed for most of these Mtb proteins. Such new strategies are desperately needed for the development of new anti-TB drugs that help combat the spread of drug-resistant Mtb strains and improve current treatment regimes.

Acknowledgements

The authors acknowledge financial support for their research from the UK Biotechnology and Biological Sciences Research Council (BBSRC, grant numbers BB/N006275/1 and BB/I019227/1).

Figures

Figure 1. Heme uptake pathway. A. A representation of the Mtb heme uptake pathway with free heme (shown in sticks colored by element) likely derived from host hemoproteins, e.g. hemoglobin is taken up by the tetrameric (dimer of dimers) heme binding enzyme Rv0203 (PDB code 3MAY) in the macrophage. Heme is internalized via the large transmembrane transporter proteins MmpL3 and MmpL11 (model structure of MmpL11 taken from [25]). The unusual heme degrading oxidase MhuD (PDB 4NL5) releases the porphyrin cleavage products mycobilins and ferrous iron intracellularly for utilization by Mtb for a variety of important roles. B. Selected inhibitors against MmpL3 with good activity against Mtb [24,28,31,34,36].

Figure 2. The two-component system DosS-DosT/DosR. A. A schematic of the two-component system containing the autophosphorylating heme sensor proteins DosS and DosT (also known as DevS and DevT) and their regulatory DNA binding protein partner DosR (DevR). Conditions conferred from the human host such as hypoxia and the presence of gases (NO and CO) activate DosS/DosT, which autophosphorylate conserved histidine residues and transfer phosphate to DosR (Asp54).

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Phosphorylated DosR binds to DNA regulatory elements and thus controls the expression of members of the DosR gene regulon that are essential for Mtb to enter the latent, dormant state [47]. B. DosS mimetic peptides that are thought to bind within DosS-DosR interacting surfaces, preventing autophosphorylation and subsequently inhibiting DosR-dependent transcriptional activity, and restricting the survival of Mtb under hypoxia [54]. C. A phenylcoumarin derivative with activity against non-replicating Mtb that inhibits binding of DosR to the target DNA [55].

Figure 3. Structures of CYP125A1 and substrate analog inhibitors. A. The active site region (upper panel) of CYP125A1 in complex with cholest-4-en-3-one (cyan sticks) (PBD 2X5W) [82] with a close up of selected amino acid residues in the substrate binding region (lower panel). The heme is shown in atom colored spheres. B. The 16-hydroxylated cholesterol analog (25R)-cholest-5-en-3,16,26-triol and its keto derivative. C. Difluoromethyl cholesterol analogues with truncated side chains, n = 1 (compound 2) cannot be metabolized by CYP125A1, n = 3 (compound 3) with the longer side chain is poorly metabolized by CYP125A1/CYP142A1 with slow conversion rates, but is still an inhibitor of Mtb growth [90].

Figure 4. Structure of CYP121A1 and development of potential inhibitor molecules. The active site region of CYP121A1 is shown in complex with overlapping substrate and fragment molecules. Diverse CYP121A1 inhibitors are shown, including ligands developed through fragment based screening techniques, highlighting the progression of molecules from a fragment with Kd = 1.7 mM to a CYP121A1 inhibitor with Kd = 0.15 nM (LE = ligand efficiency) [95]. Also illustrated are molecules identified through high throughput screening, and substrate analogs derived by fragmentation of the natural substrate cYY to develop substrate building blocks for the construction of new inhibitors [91,98,99].

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