Inhibitors of the Cysteine Synthase CysM with Antibacterial Potency against Dormant <i>Mycobacterium tuberculosis</i>

Brunner, Katharina; Maric, Selma; Reshma, Rudraraju Srilakshmi; Almqvist, Helena; Seashore‐Ludlow, Brinton; Gustavsson, Anna‐Lena; Poyraz, Ömer; Yogeeswari, Perumal; Lundbäck, Thomas; Vallin, Michaela; Sriram, Dharmarajan; Schnell, R.; Schneider, Günter

doi:10.1021/acs.jmedchem.6b00674

Cited by 55 publications

(53 citation statements)

References 35 publications

(73 reference statements)

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“…l‐ Cys metabolism has also been linked to antibiotic resistance in S. Typhimurium and is actively being explored as a target for novel antimicrobial therapies and the development of antibiotic enhancers . This approach is beginning to produce some encouraging results, particularly in the case of Mycobacterium tuberculosis . Intriguingly, the CysK–CysE interaction is commonly exploited by other proteins to promote so‐called ‘moonlighting’ activities of CysK .…”

Section: Discussionmentioning

confidence: 99%

Modulation of Escherichia coli serine acetyltransferase catalytic activity in the cysteine synthase complex

Benoni

Bei

Paredi³

et al. 2017

FEBS Letters

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In bacteria and plants, serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase-A sulfhydrylase (CysK) collaborate to synthesize L-Cys from L-Ser. CysE and CysK bind one another with high affinity to form the cysteine synthase complex (CSC). We demonstrate that bacterial CysE is activated when bound to CysK. CysE activation results from the release of substrate inhibition, with the K i for L-Ser increasing from 4 mM for free CysE to 16 mM for the CSC. Feedback inhibition of CysE by L-Cys is also relieved in the bacterial CSC. These findings suggest that the CysE active site is allosterically altered by CysK to alleviate substrate and feedback inhibition in the context of the CSC. Author contributions BC, SB, and AM conceived and supervised the study; RB, ODB, GP, and NF performed experiments; CSH provided the expression vectors; BC, RB, and ODB analyzed the data; BC prepared the original draft; BC, SB, CSH, and AM reviewed and edited the manuscript. Supporting informationAdditional Supporting Information may be found online in the supporting information tab for this article. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptPlants and bacteria share a common two-reaction pathway for the synthesis of L-cysteine (LCys) from L-serine (L-Ser; Fig. 1). Serine acetyltransferase (CysE) catalyzes an acyl transfer from acetyl-CoA to L-Ser using a random-order kinetic mechanism [1]. The second reaction is catalyzed by O-acetylserine sulfhydrylase-A (CysK), a pyridoxal 5′-phosphate (PLP)-dependent enzyme that displaces the acetoxy group from O-acetylserine with bisulfide to yield L-Cys [2][3][4][5][6][7][8]. Many bacteria also encode O-acetylserine sulfhydrylase-B (CysM) [9,10] that is thought to play an important role in L-Cys biosynthesis under stress conditions [11].Kredich et al. [2,12] first discovered that CysE and CysK from Salmonella Typhimurium bind to one another with high affinity, and they called this assembly the cysteine synthase complex (CSC; Fig. 1). The CysE-CysK interaction is highly conserved across species, and the plant enzymes also form a high-affinity CSC. Although there is no experimentally solved structure available for the CSC, biochemical and spectroscopic approaches revealed that the C-terminal tail of CysE inserts into the CysK active site to anchor the interaction. CysE proteins that lack C-terminal residues are unable to bind CysK [13][14][15], and CSC formation is disrupted by millimolar O-acetylserine, which competes with CysE for binding to the CysK active site [12,16,17]. These findings are supported by crystal structures of CysE Cterminal peptides bound in the active site of CysK. These structures show that the C-terminal Ile residue of CysE engages in the same specific interactions with the active site as Oacetylserine substrate [18,19]. The stoichiometry of CysE to CysK has been determined to be 3:2 for CSCs from S. Typhimurium and Haemophilus influenzae. Because CysK forms homodimers and CysE exists as a dimer of trimers [20,21],...

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Section: Discussionmentioning

confidence: 99%

Modulation of Escherichia coli serine acetyltransferase catalytic activity in the cysteine synthase complex

Benoni

Bei

Paredi³

et al. 2017

FEBS Letters

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“…Compounds arising from whole cell screening are presumably taken up into the cell to exert bactericidal activity, without any preconceptions about suitable targets. An alternate approach is to postulate which enzymes play a role in nonreplicating persistence based on informatics or biochemical or genetic studies, set up relevant biochemical assays, identify inhibitors, and then assay these inhibitors for whole-cell activity in nonreplicating models (100,(106)(107)(108)(109)(110)(111)(112)(113)(114)(115). The limitation of biochemical screening, however, is that the majority of enzyme inhibitors so identified lack activity against intact M. tuberculosis due to poor uptake, the sufficiency of residual enzyme activity for cell survival, intracellular metabolism, or redundant pathways (116).…”

Section: Designing High-throughput Screens To Target Phenotypically Tmentioning

confidence: 99%

Targeting Phenotypically TolerantMycobacterium tuberculosis

Gold

Nathan

2017

Tuberculosis and the Tubercle Bacillus

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While the immune system is credited with averting tuberculosis in billions of individuals exposed to Mycobacterium tuberculosis, the immune system is also culpable for tempering the ability of antibiotics to deliver swift and durable cure of disease. In individuals afflicted with tuberculosis, host immunity produces diverse microenvironmental niches that support suboptimal growth, or complete growth arrest, of M. tuberculosis. The physiological state of nonreplication in bacteria is associated with phenotypic drug tolerance. Many of these host microenvironments, when modeled in vitro by carbon starvation, complete nutrient starvation, stationary phase, acidic pH, reactive nitrogen intermediates, hypoxia, biofilms, and withholding streptomycin from the streptomycinaddicted strain SS18b, render M. tuberculosis profoundly tolerant to many of the antibiotics that are given to tuberculosis patients in a clinical setting. Targeting nonreplicating persisters is anticipated to reduce the duration of antibiotic treatment and rate of post-treatment relapse. Some promising drugs to treat tuberculosis, such as rifampicin and bedaquiline, only kill nonreplicating M. tuberculosis in vitro at concentrations far greater than their minimal inhibitory concentrations against replicating bacilli. There is an urgent demand to identify which of the currently used antibiotics, and which of the molecules in academic and corporate screening collections, have potent bactericidal action on nonreplicating M. tuberculosis. With this goal, we review methods of high throughput screening to target nonreplicating M. tuberculosis and methods to progress candidate molecules. A classification based on structures and putative targets of molecules that have been reported to kill nonreplicating M. tuberculosis revealed a rich diversity in pharmacophores. However, few of these compounds were tested under conditions that would exclude the impact of adsorbed compound acting during the recovery phase of the assay, and few were tested under more than one condition imposing nonreplication. That nonreplicating mycobacteria are metabolically active was corroborated by their susceptibility to several antibiotics and tool compounds that target the synthesis of lipids, RNA, DNA, proteins, and peptidoglycan.

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“…The biosynthesis of cysteine is needed in the oxidative defense and for dormant mycobacteria to persist inside infected macrophages. Therefore, we selected cysQ and cysM, two critical enzymes of this pathway [65,66]. In addition, we chose cdh, a predicted membrane protein and CDP-diacylglycerol pyrophosphatase, which is involved in the biosynthesis of phospholipids, and whose M .…”

Section: Resultsmentioning

confidence: 99%

Identification of novel antigen candidates for a tuberculosis vaccine in the adult zebrafish (Danio rerio)

et al. 2017

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Tuberculosis (TB) remains a major global health challenge and the development of a better vaccine takes center stage in fighting the disease. For this purpose, animal models that are capable of replicating the course of the disease and are suitable for the early-stage screening of vaccine candidates are needed. A Mycobacterium marinum infection in adult zebrafish resembles human TB. Here, we present a pre-clinical screen for a DNA-based tuberculosis vaccine in the adult zebrafish using an M. marinum infection model. We tested 15 antigens representing different types of mycobacterial proteins, including the Resuscitation Promoting factors (Rpf), PE/PPE protein family members, other membrane proteins and metabolic enzymes. The antigens were expressed as GFP fusion proteins, facilitating the validation of their expression in vivo. The efficiency of the antigens was tested against a low-dose intraperitoneal M. marinum infection (% 40 colony forming units), which mimics a primary M. tuberculosis infection. While none of the antigens was able to completely prevent a mycobacterial infection, four of them, namely RpfE, PE5_1, PE31 and cdh, led to significantly reduced bacterial burdens at four weeks post infection. Immunization with RpfE also improved the survival of the fish against a high-dose intraperitoneal injection with M. marinum (% 10.000 colony forming units), resembling the disseminated form of the disease. This study shows that the M. marinum infection model in adult zebrafish is suitable for the pre-clinical screening of tuberculosis vaccines and presents RpfE as a potential antigen candidate for further studies.

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Inhibitors of the Cysteine Synthase CysM with Antibacterial Potency against Dormant Mycobacterium tuberculosis

Cited by 55 publications

References 35 publications

Modulation of Escherichia coli serine acetyltransferase catalytic activity in the cysteine synthase complex

Modulation of Escherichia coli serine acetyltransferase catalytic activity in the cysteine synthase complex

Targeting Phenotypically TolerantMycobacterium tuberculosis

Identification of novel antigen candidates for a tuberculosis vaccine in the adult zebrafish (Danio rerio)

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