The role of chemical biology in tuberculosis drug discovery and

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The role of chemical biology in tuberculosis drug discovery and diagnosis. Katharina Kolbe, Srikumar Veleti, Emma E Johnson, Young-Woo Cho, Sangmi Oh, and Clifton E. Barry, III ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00242 • Publication Date (Web): 24 Jan 2018 Downloaded from http://pubs.acs.org on January 25, 2018

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ACS Infectious Diseases

The role of chemical biology in tuberculosis drug discovery and diagnosis.

Katharina Kolbe, Sri Kumar Veleti, Emma E. Johnson, Young-Woo Cho, Sangmi Oh, Clifton E. Barry, III*

Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Disease, NIH, Bethesda, Maryland, USA.

*- Correspondence: [email protected], Rm. 2W20D, Building 33, 9000 Rockville Pike, Bethesda, Maryland 20892.

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ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The use of chemical techniques to study biological systems (often referred to currently as “chemical biology”) has become a powerful tool for both drug discovery and the development of novel diagnostic strategies. In tuberculosis, such tools have been applied to identifying drug targets from hit compounds, matching high-throughput screening hits against large numbers of isolated protein targets, and identifying classes of enzymes with important functions. Metabolites unique to mycobacteria have provided important starting points for the development of innovative tools. For example, the unique biology of trehalose has provided both novel diagnostic strategies as well as probes of in vivo biological processes difficult to study any other way. Other mycobacterial metabolites are potentially valuable starting points and have the potential to illuminate new aspects of mycobacterial pathogenesis.

Keywords: tuberculosis, chemical biology, imaging, drug target identification

Ever since the term “chemical biology” came into wide use in the late 1990s,1 chemical principles have been successfully applied to the investigation of diverse biological phenomena. A broad definition of this research field encompasses the entire interface between chemistry and biology, with examples ranging from the discovery of RNAi pathways using chemically synthesized siRNA to the development of kinase inhibitors.1 The design and construction of chemical probes, modified from biomolecules or synthetic molecules, plays an important role in much contemporary research addressing important biological issues. This research paradigm is poised to significantly enrich drug discovery programs and imaging-based diagnostics, particularly in the field of infectious diseases. As a leading infectious cause of death, tuberculosis (TB) and its causative pathogen Mycobacterium tuberculosis (Mtb) have been the focus of considerable scientific effort to


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improve detection and treatment. Although intensive studies have made great strides toward understanding this pathogen at the molecular and cellular levels, there remain many mysteries with regards to understanding its interaction with humans and how to detect and treat the disease more efficiently. TB remains a persistent threat, causing nearly 1.6 million deaths annually with highly drug-resistant forms becoming increasingly common.2 At the heart of this persistent epidemic are antiquated detection and treatment methods that are inefficient and resource-intensive. A better understanding of the disease is required in order to develop new diagnostics and therapies. In this context, chemical biology is providing important new approaches to explore this infectious disease and Mtb, the causative bacteria. In the study of mycobacteria, chemical biology using chemical probes has provided rapid and specific imaging tools for diagnostic approaches. In addition to that, chemical tools have also enabled investigators to study this pathogen at the molecular level and sometimes, provided a rapid method to profile and analyze diverse proteomes, allowing for the identification and validation of target proteins. Therefore, designing high-quality probes to detect selective interactions of small molecules and macromolecules has facilitated TB drug discovery and a deeper understanding of the complex interactions between Mtb and its human host. Regarding the design of chemical probes, a common theme in many of these approaches is the application of bioorthogonal chemistry to enable biomolecular labeling without serious cellular perturbation. Since the Bertozzi group introduced a chemical probe based on a monosaccharide to proceed Staudinger ligation,3 many reactions including the highly useful “click chemistry” have been applied in the design of chemical probes.4 In addition, many chemical probes are designed to contain a photocrosslinking group to form a covalent bond for the identification of interacting macromolecules.5 In this review, we highlight recent studies in which chemical probes and approaches were utilized for the study of Mtb. These chemical probes can be classified into two categories: enzyme-based and metabolite-based according to their design and function (Figure 1A and B). 3 ACS Paragon Plus Environment


The first contains small-molecule probes based on the chemical structure of an enzyme substrate, which have been utilized directly not only for specific labeling of enzyme but also for the identification of target proteins so that they can provide efficient tools for elucidating mechanism of action. The second category contains chemical probes which are synthetic analogues of metabolites produced by the pathogen that can be incorporated into bacteriaspecific macromolecules by competing with their endogenous counterparts. Both approaches to incorporating analogs provide opportunities for identifying proteins that interact with various small molecules and also for visualizing Mtb bacilli as they interact with the host in cell culture and through whole-body imaging.

Figure 1. Using chemical tools in drug development and diagnostics. (A) Chemical probes may also be designed based on small-molecules to identify their target protein interactions. (B) Chemical probes may be used in metabolic incorporation studies for bacteria-specific labeling. (C) Affinity-based methods may also be employed to improve drug screening efficiency through the use of ALIS (Automated Ligand Identification System).


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Enzyme-specific chemical probes For the process of drug discovery and development, identification and validation of targets of lead molecules identified as growth inhibitors from screening efforts is crucial. There are several approaches to investigate new targets, including genetics, proteomics, molecular profiling, and bioinformatics.6 In the field of chemical biology, the design of chemical probes based on active site-directed small-molecules (activity-based probes, ABP’s) or substrates with affinity for a protein binding site (affinity-based probes, AfBP’s), plays an important role in exploring the relationship between small-molecule substrates and their counterpart enzymes. The application of chemical probes in protein profiling, activity-based protein profiling (ABPP) and affinity-based protein profiling (AfBPP), have been demonstrated through studies in various disease models including Mtb.7 Structurally, ABP’s are designed with a reactive functional group (also termed a “warhead”) that binds irreversibly and directly to all compatible active sites, whereas AfBP’s interact with targets based on their affinities, and only link covalently upon activation, typically by UV-irradiation of photocrosslinking groups such as benzophenones, diazirines, and aryl azides. In addition, the incorporation of a tag group, like a fluorescent molecule or biotin, directly into a chemical probe or with a tandem labeling strategy using click chemistry have been applied. Thus, following the successful incorporation of ABP’s and AfBP’s into cell extracts or live cells, protein hits can be characterized via gel electrophoresis, fluorescence microscopy, and mass spectrometry. Here we focus on chemical probes for studying specific mycobacterial enzymes as binding partners of synthetic small-molecules or biomolecules (various examples of these probes are summarized in Table 1). One of the largest and most diverse enzyme families is the serine hydrolase (SH) family with central functions on all levels of Mtb physiology including persistence. Recently, protein profiling studies applying fluorophosphonate (FP)-based ABP’s validated SH activity in both replicating and non-replicating Mtb. 78 proteins were detected which are known to possess SH activity or 5 ACS Paragon Plus Environment


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are hypothetical proteins. Thirty-four of these were found to remain active during non-replication and 3 were specifically labeled in non-replicating cells.8 Since slowly or non-replicating Mtb are connected to poor treatment outcomes and only a few drug targets are known in persisters to date, detection of SH activity might be of special interest for drug development. In support of this, lassomycin, which targets the ATP-dependent protease ClpC1P1P2, was shown to kill both replicating and non-replicating bacteria.11 Although ABPP was successfully applied to investigate a large spectrum of serine hydrolases or more specifically lipid esterases under different physiological conditions,9-10 some enzymes were missed using covalent-linking ABP’s. For example, the triacylglycerol lipase LipY, which is known to be upregulated in dormant Mtb, was not detected. An additional method relies on fluorogenic probes, which can be used to visualize esterase activity of enzymes following separation via native polyacrylamide gel electrophoresis.11-12

Several

7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)

(DDAO)

derivatives were successfully applied to reveal functional esterases in active, dormant, and reactivating cultures. This fluorogenic probe-based profiling was shown to be highly sensitive, but does not provide a straightforward extraction handle for detected proteins. While some studies mainly applied ABPP to investigate the breadth of SHs, the “hydrolase important for pathogenesis 1” (Hip1) has received considerable attention as a cell envelope-associated serine protease whose proteolytic activity is connected to immunomodulatory functions of Mtb.13 A peptide-based Hip1 substrate conjugated with ascaffold that binds irreversibly, allowed the identification of not only a new class of covalent inhibitors for Hip1 carrying a chloroisocoumarin scaffold, but also Hip1-specific fluorescent reporter substrates. These reagents may be used as lead compounds to develop clinically relevant drugs and specific imaging probes to visualize Mtb infections, respectively. High-throughput quantitative ABPP has further been used in Mtb to identify adenosine triphosphate (ATP)-binding proteins including enzymes, such as kinases and ATPases.14 Desthiobiotin-conjugated ATP was used to characterize ATP-binding proteins under not only 6 ACS Paragon Plus Environment

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regular aerobic growth but also under hypoxic conditions. Approximately 122 ATP-binding proteins have been identified in either metabolic state, 60 % of which are known to be crucial for Mtb survival in vitro.15 The essential roles ATP-dependent enzymes play in Mtb viability and pathogenesis make detection of this class of proteins relevant for drug discovery. In terms of druggable targets for the treatment of TB, adenylating enzymes have been considered to be better targets than ATP-binding proteins.16 Mtb putatively encodes more than 60 adenylating enzymes, which are involved in a variety of essential biochemical processes, such as glycolysis, lipid metabolism, as well as the synthesis of small molecule metabolites including iron acquiring mycobactins (Mbt). The biosynthetic pathways of these siderophores initiate from the adenylate-forming enzyme MbtA, which activates and loads salicylic acid onto the peptide synthase MbtB. Since MbtA has no mammalian homologues, it is considered an ideal drug target.17-19 5′-O-[N-(Salicyl)sulfamoyl]adenosine (Sal-AMS) was identified as a potent inhibitor of MbtA.17,

20

However, this chemical probe showed anti-TB activity under both iron-

deficient and iron-replete conditions, suggesting off-target inhibition of other

biochemical

pathways. To investigate the mechanism of action of Sal-AMS and to identify additional targets, AfBPP was applied. This work led to the design and synthesis of a Sal-AMS analogue, which is comprised of a benzophenone photoreactive cross-linker and an alkyne moiety for click ligation.21 This probe displayed suitable binding affinity towards MbtA and its protein interaction could be outcompeted by preincubation with Sal-AMS, even within mycobacteria. However, additional targets likely remain to be discovered. Since the development of the early anti-TB drugs isoniazid (INH) and ethambutol (EMB), it has been recognized that there are many potential target enzymes in the unique biosynthetic pathways involved in constructing the unique cell wall of Mtb. In this context fluorescence polarization-based high-throughput assays have been developed to identify inhibitors of critical enzymes responsible for carbohydrate incorporation. With a fluorescent uridine diphosphate (UDP) derivative, new ligands of UDP-galactopyranose mutase (UGM), which catalyzes the ring 7 ACS Paragon Plus Environment


contraction

of

UDP-galactopyranose

to

UDP-galactofuranose

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in

the

arabinogalactan

biosynthetic pathway, were identified.22 β-lactam antibiotics are a well-known antibiotic class for disruption of cell wall biosynthesis in gram-positive and gram-negative bacteria. However, βlactams were widely considered to be ineffective against Mtb due to the presence of the βlactam-insensitive L,D-transpeptidases and the activity of the β-lactamase BlaC.23-24 However, among the β-lactams currently available, carbapenems show some unique properties. Carbapenems seem to inactivate L,D-transpeptidases and are only poor substrates for BlaC, thus, bringing β-lactam antibiotics for TB treatment and chemical probe design back in focus.25 A biotinylated meropenem, for example, was recently synthesized to verify the activity of carbapenems against L,D-transpeptidases and to identify additional targets.26 Although the lactamase activity of BlaC is a big hurdle for the application of β-lactams in TB treatment, it can be exploited for TB diagnostics. Highly specific cephalosporin-based fluorogenic substrates, which generate fluorescence after BlaC cleavage, can contribute to the detection of Mtb in vitro and in vivo.27-29 This chemical biology tool was shown to rapidly and with high accuracy and sensitivity detect Mtb, even in sputum samples, and represents a promising potential tool for the clinical diagnosis of TB. Several fluorescence-based assays have also been developed for the study of enzyme activity or as diagnostic tools. One area of interest has been focused on mycobacterial sulfatase-activity not only to detect Mtb, but to discriminate between different species and lineages.30 The sulfatase-activatable probe, 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2one)-sulfate, allowed the visualization of enzyme activity in native protein gels and thereby a rapid detection of sulfatases in mycobacterial lysates. This assay revealed that mycobacterial strains have distinct sulfatase fingerprints by which they can be distinguished. We are just beginning to understand the correlation between the clinical outcome of a TB infections and the lineage affiliation of the pathogen.31-33


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Large scale efforts to characterize the mycobacterial proteome have led to the availability of purified samples of many of the proteins of Mtb. Application of a high-throughput automated ligand identification system (ALIS) that directly measures ligand binding by mass spectrometry has recently been validated against a large pool of mycobacterial proteins (636 targets) screened against a large pool of whole-cell actives (55,000 unique compounds) (Figure 1C).34 This validation included the correlation of known drug classes with known targets and also unknown structural classes with whole-cell activity with the known target dihydrofolate reductase. This kind of genome-scale chemical biology approach engages the strengths of whole-cell activity (known cell penetration and target sensitivity) while offering a direct translation to enzyme targets and therefore the strength of target-based drug discovery tools.



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Table 1. Representative enzyme-specific chemical probes and their applications Probe target

Structure

Applications

Ref

Serine Hydrolases (SHs)

A FP-probe was used for labeling of SHs in Mtb.


DDAO was used for labeling of esterases in Mtb.

11−12


A Chloroisocoumarin-derived ABP was used for labeling of Hip1 in Mtb

13

Kinases / ATPases

An ATP-derived tag-free alkyne probe was used for labeling of Kinases / ATPases in Mtb

14

Kinases / ATPases

Desthiobiotin-conjugated ATP was used for labeling of Kinases / ATPases in Mtb

15

Adenylating Enyzmes

Sal-AMS ABP was used for targeting the adenylating enzyme MbtA and possibly off-target proteins in mycobacteria.

21

UDPGalactopyranose Mutase (UGM)

An UDP-based fluorescent probe was used in fluorescent polarization assays to identify ligands of UGM.

22

Transpeptidase

Biotinylated carbapenem was designed to verify and identify new targets of β-lactam antibiotics in Mtb.

26

Blac (β-lactamase family)

Cephalosporin-based fluorogenic probes were used for BlaC-specific detection of Mtb.

27−29

DDAO-Sulfatase was used for detecting sulfatase activity and thereby differentiation of mycobacteria species and strains.

30

Cl

Sulfatases

O3SO

O

N

Cl


8

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Metabolite-based chemical probes The success of Mtb is due in part to the organisms ability to phenotypically adapt within a changing and adverse environment during infection.35 Its unique cell wall and ability to reprogram metabolism are thought to be crucial towards withstanding a range of ambient conditions including low-nutrient, acidic, hypoxic, nitrosative, and redox stresses. Hypoxia, for example, leads to an accumulation of intermediates in the early stages of glycolysis, the pentose phosphate pathway and aminosugar biosynthesis, including the peptidoglycan precursor N-acetylglucosamine phosphate.36 In addition to an accumulation of peptidoglycan precursors, a strong shift in lipid biosynthesis was detected, including an upregulation of cholesterol catabolism, intracellular accumulation of triacylglycerides (TAG), and the rapid increase of free mycolates accompanied by a decrease in the levels of the mycolic acidcontaining glycolipids, trehalose dimycolate (TDM) and trehalose monomycolate (TMM), in the Mtb cell wall.37-38 This metabolic adaptation coincides with a phenotypic tolerance towards nearly all clinically relevant TB drugs.39 Thus, in the context of drug development the specific physiologic conditions have to be taken into consideration as does the metabolic responses of the bacterium to treatment with the lead molecule under consideration. Indeed, comparative metabolomics have been successfully applied as an “activity-based guide” to identifying biologically relevant target proteins and pathways. Metabolic profiling not only contributes to a deeper understanding of the pathway, in which the target protein acts, but further provides novel insights into additional targets that could synergize with, or even replace the original target.40 One impressive example, based on two independent studies, identified and further probed the metabolic mechanisms of accumulation and secretion of succinate in Mtb under hypoxia. By feeding cells

13

C-glucose and studying its fate under normoxic and hypoxic conditions it was



found that metabolic adaptation to anoxia involved reversal of the tricarboxylic acid (TCA) pathway41. Other studies have attributed the phenomenon to slowed TCA cycle activity and increased succinate production through isocitrate lyase (ICL),42 an enzyme previously considered only within the context of fatty acid metabolism. In addition, the conversion of succinate to fumarate was connected to the capability of Mtb to sustain both membrane potential and ATP synthesis at varying oxygen levels. Based on these metabolic studies, enzymes of the succinate metabolism, such as malate dehydrogenase and succinate dehydrogenase, were identified as potential drug targets. Metabolic profiling mainly relies on isotopic tracing and subsequent mass spectrometry, with the resulting complex data sets typically requiring specialized expertise and software for relevant analyses. An alternative strategy is to use synthetic, functionalized derivatives of known metabolites to specifically target and label pathways of interest, as well as to allow direct visualization of metabolite incorporation into target macromolecules through, for example, fluorophore conjugation. Furthermore, interacting proteins and associated metabolic pathways can be isolated and identified using functionalized crosslinking moieties. In addition, targeted approaches capable of enriching specific subsets of small molecules from biological systems, would have the advantage of reducing the molecular complexity of samples under analysis and, at the same time, facilitate the detection of lower-abundance metabolites.43 Based on its essentiality and uniqueness, the carbohydrate metabolism of Mtb presents many promising drug targets. Furthermore, external environmental conditions and stressors are known to influence carbohydrate biosynthesis, as well as the carbohydrate composition of the Mtb cell wall. For instance, nutrient depletion was shown to be associated with a lower level of the trehalose derivatives TMM and TDM, and an increase in the ratio of arabinose to mannose in the cell envelope polysaccharide lipoarabinomannan (LAM).44 Recently, several trehalose derivatives were successfully applied to target trehalose metabolism and metabolically label the pathogen specific glycolipids TMM and TDM in the Mtb cell wall. Because of the mycobacterial 12 ACS Paragon Plus Environment

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specificity of these glycolipids and their important role in the survival of the pathogen, trehalose and its related biomolecules have been considered as good starting points to design new chemical tools for not only imaging the bacteria, but also potentially treating the disease.45-47 Mtb possesses enzymes to assemble trehalose de novo. In addition, trehalose transporters are present in the cell wall to re-uptake trehalose, released during external glycolipid biosynthesis. Based on this recycling pathway, exogenously added

14

C-trehalose is taken up by

Mtb whereupon it is incorporated into TMM and TDM, both by axenically cultured bacteria as well as selectively in intracellular bacteria during macrophage infection.48 Proof of concept that this could translate into a whole-body imaging modality was recently achieved using deoxy[19F]fluoro-D-trehalose ([19F]-FDTre).49 This group set up an efficient synthetic procedure of regioisomeric

19

F-FDTre analogues, and confirmed that there was a negligible effect on the

conformation of the molecule after fluorine substitution and finally, that these

19

F-FDTre probes

were imported into Mycobacterium smegmatis (Msm) via the trehalose-specific transporter SugABC-LpqY. These results suggest the potential to use

18

F-FDTre in animals and potentially

humans as a new class of nuclear imaging probe to directly detect infecting bacteria. The first trehalose probe used for cellular imaging was a fluorescein-modified trehalose (FITC-trehalose).50 In these studies a structurally diverse library, primarily of disaccharides related to trehalose, was constructed and it was shown that the antigen 85 (Ag85) complex of trehalose metabolism displays surprisingly broad substrate specificity, including FITC-trehalose. By labeling bacteria with this unnatural trehalose, selective and sensitive detection of Mtb was demonstrated even within infected macrophages. This was expanded in scope with the introduction of regioisomeric analogues of fluorescein-trehalose (FlTre).51 By following the metabolic incorporation of FITre, trehalose probes were used to investigate cellular dynamics by quantifying the mobility of trehalose-containing glycolipids and studying the relationship between diffusion kinetics and mycolic acid chain structure. A drawback of the fluorescein derivatives, however, is a high background signal in microscopy. In an ingenious solution to this problem, a 13 ACS Paragon Plus Environment


4-N,N-dimethylamino-1,8-naphthalimide-conjugated trehalose (DMN-Tre) was synthesized that displayed solvatochromic behavior showing over 700-fold fluorescence increase in the hydrophobic environments of the bacterial cell wall compared to aqueous media.52 This trehalose probe has a great potential to illuminate Mtb in sputum samples and improve TB diagnostics. In terms of application of bioorthogonal chemical reporters, a series of azide-modified trehalose (TreAz) analogues were utilized to investigate the “trehalome” in live bacteria.53 After metabolic labeling with this unnatural trehalose, bioorthogonal chemistry using an alkyne-dye was performed to specifically visualize cell wall incorporation. Besides cellular imaging with trehalose probes, this disaccharide has also been used for the design and construction of a selective mechanism-based inhibitor of Ag85. During catalysis of mycolate transfer to trehalose, Ag85 forms a mycolyl-enzyme complex via tetrahedral transition state. Fluorophosphonate-conjugated trehalose was an effective mimic of this tetrahedral transition state and formed a covalent bond with Ser124 in the active site.48 This compound proved to be a potent and highly selective covalent inhibitor probe of Ag85c without cross reactivity toward other serine acyltransferases. Apart from trehalose, arabinofuranosides are unique components of cell surface polysaccharides and lipoglycans of Corynebacterineae. In order to detect cell wall arabinosylation, azido pentoses were recently designed, including 5-azido-5-deoxy-Darabinofuranose (5AraAz) and 3-azido-3-deoxy-D-arabinose (3AraAz).54 These azido derivatives were successfully used to metabolically label Mtb. However, inconsistent with the known arabinose metabolism,55 5AraAz was most efficiently incorporated, ending up in a yet uninvestigated glycoconjugate of the Mtb cell wall. Incorporation of 5AraAz indicates the presence of unknown pentose transporters in the Mtb cell envelope, putative novel arabinose biosynthesis pathways and potentially a new cell wall component. Thus, this azido pentose may shed light on mycobacterial arabinan metabolism in greater detail. However, metabolic labeling 14 ACS Paragon Plus Environment

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of arabinosides in LAM and the structuring cell wall polysaccharide arabinogalactan (AG) has still to be developed. Furthermore, based on the higher required concentration of 5AraAz for incorporation compared to trehalose derivatives, it might not be applicable as an in vivo imaging tool. The strong influence of ambient stresses on Mtb lipid metabolism, as well as the association of cell wall lipids with the pathogenicity of mycobacteria, makes lipid-based metabolic probes of particular relevance. One known lipid-based derivative applied is the TMM analogue O-AlkTMM, containing an alkyne tagged short alkyl chain in place of the mycolic acid. The biosynthesis of TDM, as described above, is catalyzed by one of several enzymes constituting Ag85 complex, through transfer of a mycolyl group from TMM to the primary hydroxyl group of a second TMM. In addition, the Ag85 complex is also involved in mycolylation of AG. Using O-AlkTMM revealed labeling of TDM and, as anticipated, of the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex in Msm and Corynebacterium glutamicum (Cg).56 The incorporation was visualized with an azido fluorophore using click chemistry. This metabolic labeling method might reveal new insights into mAGP metabolism and might be used to analyze alterations of mycolation under varying environmental conditions or drug treatment. For example, O-AlkTMM was recently successfully applied to detect defects in cell wall mycolylation in genetically modified strains deficient in Ag85A and the lipid transport protein G (LprG).57 Based on the specificity of O-AlkTMM for Corynebacterineae, it might be even applicable to detect cell wall mycolylation in vivo and visualize Mtb in sputum samples or during infections. Mycolic acids are a hallmark of the Corynebacterineae and are intimately associated with the complex cell envelope of these organisms. These complex fatty acids are not only present on the cell wall and their function in other aspects of the bacterial lifecycle are only now being explored with the tools of chemical biology. Mycolyltransferases, as recently discovered in Cg, C. efficiens, and C. diphtheriae, can also play a role in the O-mycolylation of serine residues



in proteins.58-59 O-AlkTMM appears to be capable of installing an alkyne onto any mycolyltransferase acceptor substrate, including proteins.60 Using this strategy mycolylation of the known pore forming proteins PorA and PorH, as well as a novel protein of unknown function has been demonstrated. Furthermore, the anion-selective channel protein PorB and a variety of other proteins were identified as being putatively mycoloylated. Recently, an alkynyl-fatty acid analog (17-ODYA) has been used for the same purpose. This study revealed that derivatives of long chain carboxylic acids are incorporated via the mycolic acid biosynthesis pathway and are sufficient to label cell wall structures. Mycolylation of the porins PorA and PorH was confirmed and PorB and PorC were identified for the first time.61 Protein mycoloylation pathways have not been detected in other Corynebacterineae, such as mycobacteria, to date, and the mycobacterial mycolyltransferases differ significantly from the mycolyltransferase mytC of Cg. However, the importance of protein mycolylation of Mtb remains to be analyzed. While 17ODYA has been used to study O-mycolylation of proteins, it can be assumed that similar fatty acid-based probes might be useful to study lipidation of other cell wall structures and to target and enrich metabolites of the lipid metabolism under varying ambient conditions. Distinct metabolic pathways can also interact with each other, and understanding these interactions can reveal important aspects of cellular metabolism, as well as aid in identifying new drug targets and efficient novel drug combinations. Recently, a chemical biology approach has been successfully applied to identify the direct interplay between two lipid biosynthesis pathways. De novo fatty acid synthesis in mycobacteria is performed by the fatty acid synthase type I complex (Fas I), which uses acetyl-CoA and malonyl-CoA to produce saturated short chain fatty acids (C18). The Fas II multi-enzyme system can further elongate and modify these lipids up to C45/56 in length, which are used in mycolic acid biosynthesis. In addition, the PpsAE enzymes and the multi-domain enzyme Mas catalyze the conversion of the short chain fatty acids to phthiocerol and mycerosic acid, respectively. Phthiocerol and mycerosic acid are the subunits of the cell wall lipid phthiocerol dimycocerosate (PDIM), which is only present in 16 ACS Paragon Plus Environment

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pathogenic mycobacteria where it constitutes an important virulence factor.62 To identify connections between the Fas II and PDIM biosynthetic pathways, other work has exploited the shared ability of lipid metabolizing enzymes to bind and shuttle 4′-phosphopantetheine (Ppant)linked substrates.63 Two synthetic CoA analogs, either carrying a biotin or a benzophenone moiety, were tested, enabling the direct detection of lipid transfer and interactions between the studied enzymes. This study verified the interaction between the previously unrelated polyketide synthase PpsB, involved in PDIM biosynthetic pathway, and β-ketoacyl-ACP synthase (KasA) of the Fas II pathway, and potentially signaled the existence of other yet unknown metabolic pathway cross-talk. The presence of Ppant prosthetic groups in carrier protein units of enzymes can not only be utilized to identify metabolic pathways, but represents a promising drug target. In lipid metabolism the biosynthetic intermediates are tethered as thioesters onto the terminal thiol of Ppant. This acylation is catalyzed by an adenylating enzyme, which uses acyladenylate as a substrate. Analogues that mimic the essential features of the acyladenylate, but carry a reactive functional group (e.g. Michael acceptor), which can either reversibly or irreversibly bind the terminal thiol of Ppant and thereby inhibit natural acyl transfer have been synthesized.64 These acyladenylate analogs, however, have not been tested for their antibacterial activity to date. A natural compound, that shows a comparable mode of action, is Platensimycin, a secondary metabolite from Streptomyces platensis. This natural product showed anti-bacterial activity inhibiting KasA and KasB of the mycobacterial Fas metabolism and provides a new lead for anti-TB drug design.65-66 Besides the use of carbohydrate and lipid derivatives to metabolically label Mtb, amino acid analogs have been applied to visualize peptidoglycan (PG) biosynthesis. PG is a dense polymer mesh of the Mtb cell envelope comprised of glycan chains crosslinked by oligopeptides. Because PG integrity dictates cell viability, further investigation of its structure and synthesis may point to new drug targets. It was previously found that PG’s terminal D-ala-D-ala are subject to highly promiscuous endogenous editing mechanisms, permitting the incorporation of 17 ACS Paragon Plus Environment


unnatural D-amino acids to monitor Mtb cell wall synthesis.67

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Metabolic labeling of the

mycobacterial PG layer has been achieved by employing the alkyne-azide click ligation between the D-alanine analog D-propargylglycine (alkDala) and the fluorescent dye azido-Fluor 488.68 In addition, the gram-positive bacterium Listeria monocytogenes could be specifically labeled with this alkDala within macrophages. Although not shown yet, using this technique to visualize mycobacterial PG biosynthesis during infection should also be possible. D-Alanine analogs have further been used to identify subpolar localization of new cell wall biosynthesis,69 and to study the spatiotemporal dynamics of peptidoglycan formation.70 Using super-resolution imaging and metabolic labeling of PG have demonstrated morphological differences between meropenemtreated Msm and Mtb bacteria, and identified that this drug does not influence the formation of the dividing septum, but rather its closure. Metabolic labeling with amino acid analogs will facilitate a better definition of the PG biosynthesis in mycobacteria, which will help to rationalize the modes of action of the growing number of β-lactam antimicrobials under consideration for use in TB therapy.

Figure 2. Overview of metabolite-based chemical probes used in Mtb. Previous strategies of metabolic profiling primarily applied isotopic tracing using

13

C-labeled metabolite derivatives (A). The structures of


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representative probes for labeling the various metabolic pathways and destinations of Mtb are shown above: TreAz (B) for TDM biosynthesis, 5-AraAz (C) for arabinose metabolic pathways, O-AlkTMM (D) for mycomembrane biosynthesis, Ppant-linked lipid metabolizing enzymes (E) for PDIM biosynthesis,17ODYA (F) for mycolic acid biosynthesis, and finally alkDala (G) for peptidoglycan biosynthesis. Colored stars indicate the expected area of probe incorporation in the cytosol or along the mycobacterial cell wall.

Perspective for future studies Chemical biology as a field has matured sufficiently to make major contributions to controlling the TB epidemic. For example, the vast majority of the world diagnoses TB today using differential stain retention under acidic conditions – the so-called “acid-fast” stain that was first described in the late 19th century – and surely an efficient replacement for that which is more reliable and effective is close at hand. Nuclear imaging techniques such as Positron Emission Tomography (PET) are being used to monitor TB treatment responses in TB patients in large scale clinical trials yet the available probes only examine inflammation. TB-specific probes, that would give real-time readouts of viable bacteria offer the potential for quantitative measures of burden of disease and treatment efficacy. Large screening campaigns have been undertaken to develop whole-cell actives to prime the pipeline of TB drug development yet the methods for determining the mechanism of action of such compounds largely remain laborious and inefficient. Applying the concepts and techniques of bioorthogonal chemical handles onto existing hit and lead optimization programs will dramatically improve our ability to move from activity to mechanism. Chemical biology is also poised to make major contributions to our basic understanding of the organism that causes TB. A large fraction of the genome still includes proteins of unknown function and regulatory systems we have not decoded. As the initial studies with serine hydrolases and ATP-binding proteins illustrates, clever application of appropriate probes can significantly boost our ability to experimentally annotate and understand proteins of unknown function. Other appropriately functionalized substrates and coenzyme analogs will no doubt 19 ACS Paragon Plus Environment


reveal surprising new and interesting biochemical connections in Mtb. Major unresolved issues in even the most basic steps of the TB life cycle remain and these questions only become more difficult when considering how this pathogen interacts with humans. The tools of chemical biology will find broad utility in starting to answer many of these questions at a molecular level.

ACKNOWLEDGMENTS This work was funded by the Intramural Research Program of the National Institute of Allergy and Infectious Disease, National Institutes of Health.

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