Triclosan Derivatives: Towards Potent Inhibitors of Drug‐Sensitive and Drug‐Resistant Mycobacterium tuberculosis

Overcoming resistance : Isoniazid (INH) is a frontline antitubercular drug that inhibits the enoyl acyl carrier protein reductase InhA. Novel inhibitors of InhA that are not cross‐resistant to INH represent a significant goal in antitubercular chemotherapy. The design, synthesis, and biological activity of a series of triclosan‐based inhibitors is reported, including their promising efficacy against INH‐resistant strains of M. tuberculosis.

     

Recent Advances in the Research of Heterocyclic Compounds as Antitubercular Agents

Tuberculosis (TB) is a major health problem, with approximately one‐third of the world′s population infected with Mycobacterium tuberculosis, eight million people in the active disease state, and two million dying annually. Furthermore, the prevalence of TB/HIV co‐infection, and the emergence of multidrug‐resistant tuberculosis (MDR‐TB) and extensively drug‐resistant tuberculosis (XDR‐TB) have further aggravated the spread of this disease and thus mortality by it. There is an urgent need for novel antitubercular agents with improved properties, such as lower toxicity, shortened duration of therapy, rapid bactericidal action, and enhanced activity against MDR strains. Fortunately, a number of new potential antitubercular candidate drugs with heterocyclic rings, which are most likely to be effective against resistant strains, have entered clinical trials in recent years. This review highlights recent advances in the research of novel heterocyclic compounds, with particular focus on their antimycobacterial activity, mechanisms of action, toxicity, and structure–activity relationships (SARs).     

Time‐Dependent Diaryl Ether Inhibitors of InhA: Structure–Activity Relationship Studies of Enzyme Inhibition,Antibacterial Activity,and in vivo Efficacy

The diaryl ethers are a novel class of antituberculosis drug candidates that inhibit InhA, the enoyl‐ACP reductase involved in the fatty acid biosynthesis (FASII) pathway, and have antibacterial activity against both drug‐sensitive and drug‐resistant strains of Mycobacterium tuberculosis. In the present work, we demonstrate that two time‐dependent B‐ring modified diaryl ether InhA inhibitors have antibacterial activity in a mouse model of TB infection when delivered by intraperitoneal injection. We propose that the efficacy of these compounds is related to their residence time on the enzyme, and to identify structural features that modulate drug–target residence time in this system, we have explored the inhibition of InhA by a series of B‐ring modified analogues. Seven ortho‐substituted compounds were found to be time‐dependent inhibitors of InhA, where the slow step leading to the final enzyme–inhibitor complex (EI*) is thought to correlate with closure and ordering of the InhA substrate binding loop. A detailed mechanistic understanding of the molecular basis for residence time in this system will facilitate the development of InhA inhibitors with improved in vivo activity.     

Novel 1,4‐Substituted‐1,2,3‐Triazoles as Antitubercular Agents

Tuberculosis (TB) remains a pressing unmet medical need, particularly with the emergence of multidrug‐resistant and extensively drug‐resistant tuberculosis. Here, a series of 1,4‐substituted‐1,2,3‐triazoles have been synthesized and evaluated as potential antitubercular agents. These compounds were assembled via click chemistry in high crude purity and in moderate to high yield. Of the compounds tested, 12 compounds showed promising antitubercular activity with six possessing minimum inhibitory concentration (MIC) values <10 μg mL^?1, and total selectivity for Mycobacterium tuberculosis (Mtb) growth inhibition. A second set of 21 compounds bearing variations on ring C were synthesized and evaluated. This second library gave an additional six compounds displaying MIC values ≤10 μg mL^?1 and total selectivity for Mtb growth inhibition. These compounds serve as an excellent starting point for further development of antitubercular therapies.     

Biological Evaluation of Potent Triclosan‐Derived Inhibitors of the Enoyl–Acyl Carrier Protein Reductase InhA in Drug‐Sensitive and Drug‐Resistant Strains of Mycobacterium tuberculosis

New triclosan (TRC) analogues were evaluated for their activity against the enoyl–acyl carrier protein reductase InhA in Mycobacterium tuberculosis (Mtb). TRC is a well‐known inhibitor of InhA, and specific modifications to its positions 5 and 4′ afforded 27 derivatives; of these compounds, seven derivatives showed improved potency over that of TRC. These analogues were active against both drug‐susceptible and drug‐resistant Mtb strains. The most active compound in this series, 4‐(n‐butyl)‐1,2,3‐triazolyl TRC derivative 3 , had an MIC value of 0.6 μg mL^?1 (1.5 μM ) against wild‐type Mtb. At a concentration equal to its MIC, this compound inhibited purified InhA by 98 %, and showed an IC₅₀ value of 90 nM . Compound 3 and the 5‐methylisoxazole‐modified TRC 14 were able to inhibit the biosynthesis of mycolic acids. Furthermore, mc²4914, an Mtb strain overexpressing inhA, was found to be less susceptible to compounds 3 and 14 , supporting the notion that InhA is the likely molecular target of the TRC derivatives presented herein.     

A fast and efficient metal-mediated oxidation of isoniazid and identification of isoniazid-NAD(H) adducts

It is currently believed that isoniazid (INH) is oxidised inside Mycobacterium tuberculosis to generate, by covalent attachment to the nicotinamide ring of NAD(H) (beta-nicotinamide adenine dinucleotide), a strong inhibitor of InhA, an enzyme essential for mycolic acid biosynthesis. This work was carried out to characterise the InhA inhibitors (named INH-NAD(H) adducts) which are generated, in the presence of the nicotinamide coenzyme NAD+, by oxidation of INH with manganese(III) pyrophosphate, a nonenzymatic and efficient oxidant used to mimic INH activation by the catalase-peroxidase KatG inside M. tuberculosis. The oxidation process is almost complete in less than 15 minutes (in comparison to the slow activation obtained in the KatG-dependent process (2.5 hours) or in the nonenzymatic O2/Mn(II)-dependent activation (5 hours)). The alkylation of NAD+ by the postulated isonicotinoyl radical generates, in solution, a family of INH-NAD(H) adducts. Analyses with liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) and experiments performed with 18O- and 2H-labelled substrates allowed us to propose two open and four hemiamidal cyclised dihydropyridine structures as the main forms present in solution; these result from the combination of the isonicotinoyl radical and the nicotinamide part of NAD+. A small amount of a secondary oxidation product was also detected. Structural data on the forms present in solution should help in the design of inhibitors of enzymes involved in the biosynthesis of mycolic acids to act as potential antituberculosis drugs.     

Inhibition Studies of Mycobacterium tuberculosis Salicylate Synthase (MbtI)

Mycobacterium tuberculosis salicylate synthase (MbtI), a member of the chorismate‐utilizing enzyme family, catalyses the first committed step in the biosynthesis of the siderophore mycobactin T. This complex secondary metabolite is essential for both virulence and survival of M. tuberculosis, the etiological agent of tuberculosis (TB). It is therefore anticipated that inhibitors of this enzyme may serve as TB therapies with a novel mode of action. Herein we describe the first inhibition study of M. tuberculosis MbtI using a library of functionalized benzoate‐based inhibitors designed to mimic the substrate (chorismate) and intermediate (isochorismate) of the MbtI‐catalyzed reaction. The most potent inhibitors prepared were those designed to mimic the enzyme intermediate, isochorismate. These compounds, based on a 2,3‐dihydroxybenzoate scaffold, proved to be low‐micromolar inhibitors of MbtI. The most potent inhibitors in this series possessed hydrophobic enol ether side chains at C3 in place of the enol‐pyruvyl side chain found in chorismate and isochorismate.     

Using Chemical Probes to Assess the Feasibility of Targeting SecA for Developing Antimicrobial Agents against Gram‐Negative Bacteria

With the widespread emergence of drug resistance, there is an urgent need to search for new antimicrobials, especially those against Gram‐negative bacteria. Along this line, the identification of viable targets is a critical first step. The protein translocase SecA is commonly believed to be an excellent target for the development of broad‐spectrum antimicrobials. In recent years, we developed three structural classes of SecA inhibitors that have proven to be very effective against Gram‐positive bacteria. However, we have not achieved the same level of success against Gram‐negative bacteria, despite the potent inhibition of SecA in enzyme assays by the same inhibitors. In this study, we use representative inhibitors as chemical probes to gain an understanding as to why these inhibitors were not effective against Gram‐negative bacteria. The results validate our initial postulation that the major difference in effectiveness against Gram‐positive and Gram‐negative bacteria is in the additional permeability barrier posed by the outer membrane of Gram‐negative bacteria. We also found that the expression of efflux pumps, which are responsible for multidrug resistance (MDR), have no effect on the effectiveness of these SecA inhibitors. Identification of an inhibitor‐resistant mutant and complementation tests of the plasmids containing secA in a secAts mutant showed that a single secA‐azi‐9 mutation increased the resistance, providing genetic evidence that SecA is indeed the target of these inhibitors in bacteria. Such results strongly suggest SecA as an excellent target for developing effective antimicrobials against Gram‐negative bacteria with the intrinsic ability to overcome MDR. A key future research direction should be the optimization of membrane permeability.     

Thiazolopyrimidine Inhibitors of 2‐Methylerythritol 2,4‐Cyclodiphosphate Synthase (IspF) from Mycobacterium tuberculosis and Plasmodium falciparum

A library of 40 000 compounds was screened for inhibitors of 2‐methylerythritol 2,4‐cyclodiphosphate synthase (IspF) protein from Arabidopsis thaliana using a photometric assay. A thiazolopyrimidine derivative resulting from the high‐throughput screen was found to inhibit the IspF proteins of Mycobacterium tuberculosis, Plasmodium falciparum, and A. thaliana with IC₅₀ values in the micromolar range. Synthetic efforts afforded derivatives that inhibit IspF protein from M. tuberculosis and P. falciparum with IC₅₀ values in the low micromolar range. Several compounds act as weak inhibitors in the P. falciparum red blood cell assay.     

Repurposing the Chemical Scaffold of the Anti‐Arthritic Drug Lobenzarit to Target Tryptophan Biosynthesis in Mycobacterium tuberculosis

The emergence of extensively drug‐resistant strains of Mycobacterium tuberculosis (Mtb) highlights the need for new therapeutics to treat tuberculosis. We are attempting to fast‐track a targeted approach to drug design by generating analogues of a validated hit from molecular library screening that shares its chemical scaffold with a current therapeutic, the anti‐arthritic drug Lobenzarit (LBZ). Our target, anthranilate phosphoribosyltransferase (AnPRT), is an enzyme from the tryptophan biosynthetic pathway in Mtb. A bifurcated hydrogen bond was found to be a key feature of the LBZ‐like chemical scaffold and critical for enzyme inhibition. We have determined crystal structures of compounds in complex with the enzyme that indicate that the bifurcated hydrogen bond assists in orientating compounds in the correct conformation to interact with key residues in the substrate‐binding tunnel of Mtb‐AnPRT. Characterising the inhibitory potency of the hit and its analogues in different ways proved useful, due to the multiple substrates and substrate binding sites of this enzyme. Binding in a site other than the catalytic site was found to be associated with partial inhibition. An analogue, 2‐(2‐5‐methylcarboxyphenylamino)‐3‐methylbenzoic acid, that bound at the catalytic site and caused complete, rather than partial, inhibition of enzyme activity was found. Therefore, we designed and synthesised an extended version of the scaffold on the basis of this observation. The resultant compound, 2,6‐bis‐(2‐carboxyphenylamino)benzoate, is a 40‐fold more potent inhibitor of the enzyme than the original hit and provides direction for further structure‐based drug design.     

2‐Substituted 6‐(Het)aryl‐7‐deazapurine Ribonucleosides: Synthesis,Inhibition of Adenosine Kinases,and Antimycobacterial Activity

A series of 6‐(hetero)aryl‐ or 6‐methyl‐7‐deazapurine ribonucleosides bearing a substituent at position 2 (Cl, F, NH₂, or CH₃) were prepared by cross‐coupling reactions at position 6 and functional group transformations at position 2. Cytostatic, antiviral, and antimicrobial activity assays were performed. The title compounds were observed to be potent and selective inhibitors of Mycobacterium tuberculosis adenosine kinase (ADK), but not human ADK; moreover, they were found to be non‐cytotoxic. The antimycobacterial activities against M. tuberculosis, however, were only moderate. The reason for this could be due to either poor uptake through the cell wall or to parallel biosynthesis of adenosine monophosphate by the salvage pathway.     

Synthesis and Evaluation of 1‐(1‐(Benzo[b]thiophen‐2‐yl)cyclohexyl)piperidine (BTCP) Analogues as Inhibitors of Trypanothione Reductase

Thirty two analogues of phencyclidine were synthesised and tested as inhibitors of trypanothione reductase (TryR), a potential drug target in trypanosome and leishmania parasites. The lead compound BTCP ( 1 , 1‐(1‐benzo[b]thiophen‐2‐yl‐cyclohexyl) piperidine) was found to be a competitive inhibitor of the enzyme (K_i=1 μM ) and biologically active against bloodstream T. brucei (EC₅₀=10 μM ), but with poor selectivity against mammalian MRC5 cells (EC₅₀=29 μM ). Analogues with improved enzymatic and biological activity were obtained. The structure–activity relationships of this novel series are discussed.     

Development of Potent Inhibitors of the Mycobacterium tuberculosis Virulence Factor Zmp1 and Evaluation of Their Effect on Mycobacterial Survival inside Macrophages

The enzyme Zmp1 is a zinc‐containing peptidase that plays a critical role in the pathogenicity of Mycobacterium tuberculosis. Herein we describe the identification of a small set of Zmp1 inhibitors based on a novel 8‐hydroxyquinoline‐2‐hydroxamate scaffold. Among the synthesized compounds, N‐(benzyloxy)‐8‐hydroxyquinoline‐2‐carboxamide ( 1 c ) was found to be the most potent Zmp1 inhibitor known to date, and its binding mode was analyzed both by kinetics studies and molecular modeling, identifying critical interactions of 1 c with the zinc ion and residues in the active site. The effect of 1 c on intracellular Mycobacterium survival was assayed in J774 murine macrophages infected with M. tuberculosis H37Rv or M. bovis BCG and human monocyte‐derived macrophages infected with M. tuberculosis H37Rv. Cytotoxicity and genotoxicity were also assessed. Overall, inhibitor 1 c displays interesting in vitro antitubercular properties worthy of further investigation.     

A Second,Druggable Binding Site in UDP‐Galactopyranose Mutase from Mycobacterium tuberculosis?

UDP‐galactopyranose mutase (UGM), a key enzyme in the biosynthesis of mycobacterial cell walls, is a potential target for the treatment of tuberculosis. In this work, we investigate binding models of a non‐substrate‐like inhibitor, MS‐208, with M. tuberculosis UGM. Initial saturation transfer difference (STD) NMR experiments indicated a lack of direct competition between MS‐208 and the enzyme substrate, and subsequent kinetic assays showed mixed inhibition. We thus hypothesized that MS‐208 binds at an allosteric binding site (A‐site) instead of the enzyme active site (S‐site). A candidate A‐site was identified in a subsequent computational study, and the overall hypothesis was supported by ensuing mutagenesis studies of the A‐site. Further molecular dynamics studies led us to propose that MS‐208 inhibition occurs by preventing complete closure of an active site mobile loop that is necessary for productive substrate binding. The results suggest the presence of an A‐site with potential druggability, opening up new opportunities for the development of novel drug candidates against tuberculosis.     

Chromophore‐Linked Substrate (CLS405): Probing Metallo‐β‐Lactamase Activity and Inhibition

Serine‐ and metallo‐β‐lactamases present a threat to the clinical use of nearly all β‐lactam antibiotics, including penicillins, cephalosporins, and carbapenems. Efforts to develop metallo‐β‐lactamase (MBL) inhibitors require suitable screening platforms to allow the rapid determination of β‐lactamase activity and efficient inhibition. Unfortunately, the platforms currently available are not ideal for this purpose. Further progress in MBL inhibitor identification requires inexpensive and widely applicable assays. Herein the identification of an inexpensive and stable chromogenic substrate suitable for use in assays of clinically relevant MBLs is described. (6R,7R)‐3‐((4‐Nitrophenoxy)methyl)‐8‐oxo‐7‐(2‐phenylacetamido)‐5‐thia‐1‐azabicyclo[4.2.0]oct‐2‐ene‐2‐carboxylic acid 5,5‐dioxide (CLS405) was synthesised in a three‐step protocol. CLS405 was then characterised spectroscopically, and its stability and kinetic properties evaluated. With a Δλ_max value of 100 nm between the parent and hydrolysis product, a higher analytical accuracy is possible with CLS405 than with commonly used chromogenic substrates. The use of CLS405 in assays was validated by MBL activity measurements and inhibitor screening that resulted in the identification of N‐hydroxythiazoles as new inhibitor scaffolds for MBLs. Further evaluation of the identified N‐hydroxythiazoles against a panel of clinically relevant MBLs showed that they possess inhibitory activities in the mid‐ to low‐micromolar range. The findings of this study provide both a useful tool compound for further inhibitor identification, and novel scaffolds for the design of improved MBL inhibitors with potential as antibiotics against resistant strains of bacteria.     

Diaryl‐Substituted Azolylthioacetamides: Inhibitor Discovery of New Delhi Metallo‐β‐Lactamase‐1 (NDM‐1)

The emergence and spread of antibiotic‐resistant pathogens is a global public health problem. Metallo‐β‐lactamases (MβLs) such as New Delhi MβL‐1 (NDM‐1) are principle contributors to the emergence of resistance because of their ability to hydrolyze almost all known β‐lactam antibiotics including penicillins, cephalosporins, and carbapenems. A clinical inhibitor of MBLs has not yet been found. In this study we developed eighteen new diaryl‐substituted azolylthioacetamides and found all of them to be inhibitors of the MβL L1 from Stenotrophomonas maltophilia (K_i<2 μM ), thirteen to be mixed inhibitors of NDM‐1 (K_i<7 μM ), and four to be broad‐spectrum inhibitors of all four tested MβLs CcrA from Bacteroides fragilis, NDM‐1 and ImiS from Aeromonas veronii, and L1 (K_i<52 μM ), which are representative of the B1a, B1b, B2, and B3 subclasses, respectively. Docking studies revealed that the azolylthioacetamides, which have the broadest inhibitory activity, coordinate to the Zn^II ion(s) preferentially via the triazole moiety, while other moieties interact mostly with the conserved active site residues Lys224 (CcrA, NDM‐1, and ImiS) or Ser221 (L1).     

Understanding the Key Factors that Control the Inhibition of Type II Dehydroquinase by (2R)‐2‐Benzyl‐3‐dehydroquinic Acids

The binding mode of several substrate analogues, (2R)‐2‐benzyl‐3‐dehydroquinic acids 4 , which are potent reversible competitive inhibitors of type II dehydroquinase (DHQ2), the third enzyme of the shikimic acid pathway, has been investigated by structural and computational studies. The crystal structures of Mycobacterium tuberculosis and Helicobacter pylori DHQ2 in complex with one of the most potent inhibitor, p‐methoxybenzyl derivative 4 a , have been solved at 2.40 Å and 2.75 Å, respectively. This has allowed the resolution of the M. tuberculosis DHQ2 loop containing residues 20–25 for the first time. These structures show the key interactions of the aromatic ring in the active site of both enzymes and additionally reveal an important change in the conformation and flexibility of the loop that closes over substrate binding. The loop conformation and the binding mode of compounds 4 b – d has been also studied by molecular dynamics simulations, which suggest that the benzyl group of inhibitors 4 prevent appropriate orientation of the catalytic tyrosine of the loop for proton abstraction and disrupts its basicity.     

6,7‐Dimethoxy‐2‐{2‐[4‐(1H‐1,2,3‐triazol‐1‐yl)phenyl]ethyl}‐1,2,3,4‐tetrahydroisoquinolines as Superior Reversal Agents for P‐Glycoprotein‐Mediated Multidrug Resistance

P‐glycoprotein (P‐gp)‐mediated multidrug resistance (MDR) is a major obstacle for successful cancer chemotherapy. Based on our previous study, 17 novel compounds with the 6,7‐dimethoxy‐2‐{2‐[4‐(1H‐1,2,3‐triazol‐1‐yl)phenyl]ethyl}‐1,2,3,4‐tetrahydroisoquinoline scaffold were designed and synthesized. Among them, 2‐[(1‐{4‐[2‐(6,7‐dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)ethyl]phenyl}‐1H‐1,2,3‐triazol‐4‐yl)methoxy]‐N‐(p‐tolyl)benzamide (compound 7 h ) was identified as a potent modulator of P‐gp‐mediated MDR, with high potency (EC₅₀=127.5±9.1 nM ), low cytotoxicity (TI>784.3), and long duration (>24 h) in reversing doxorubicin (DOX) resistance in K562/A02 cells. Compound 7 h also enhanced the effects of other MDR‐related cytotoxic agents (paclitaxel, vinblastine, and daunorubicin), increased the accumulation of DOX and blocked P‐gp‐mediated rhodamine 123 efflux function in K562/A02 MDR cells. Moreover, 7 h did not have any effect on cytochrome (CYP3A4) activity. These results indicate that 7 h is a relatively safe modulator of P‐gp‐mediated MDR that has good potential for further development.     

Identification of the Target Protein of Agelasine D,a Marine Sponge Diterpene Alkaloid,as an Anti‐dormant Mycobacterial Substance

One of the major reasons for the wide epidemicity of tuberculosis and for the necessity for extensive chemotherapeutic regimens is that the causative agent, Mycobacterium tuberculosis, has an ability to become dormant. Therefore, new lead compounds that are anti‐bacterial against M. tuberculosis in both active and dormant states are urgently needed. Marine sponge diterpene alkaloids, agelasines B, C, and D, from an Indonesian marine sponge of the genus Agelas were rediscovered as anti‐dormant‐mycobacterial substances. Based on the concept that the transformants over‐expressing targets of antimicrobial substances confer drug resistance, strains resistant to agelasine D were screened from Mycobacterium smegmatis transformed with a genomic DNA library of Mycobacterium bovis BCG. Sequence analysis of the cosmids isolated from resistant transformants revealed that the responsible gene was located in the genome region between 3475.051 and 3502.901 kb. Further analysis of the transformants over‐expressing the individual gene contained in this region indicated that BCG3185c (possibly a dioxygenase) might be a target of the molecule. Moreover, agelasine D was found to bind directly to recombinant BCG3185c protein (K_D 2.42 μm), based on surface plasmon resonance (SPR). This evidence strongly suggests that the BCG3185c protein is the major target of agelasine D, and that the latter is the anti‐mycobacterial substance against dormant bacilli.