Biochemical Potential of α-L-Arabinofuranosidase as Anti-Tuberculosis Candidate

Mycobacterium tuberculosis (MTB), the main causative organism of tuberculosis (TB), is a successful pathogen that overcomes the numerous challenges presented by the immune system of the host. The situation regarding control of tuberculosis has significantly worsened over the last decade with the spread of strains resistant to multiple antimycobacterial agents. Mycobacterial cell wall has potential as a target for developing protein therapeutic enzybiotic to overcome resistancy case in TB. One of the compiler of mycobacterial cell wall is arabinogalactan which is compiled by arabinose with unusual stereochemical. α-L-arabinofuranosidase (Abfa) is an enzyme that can breakdown arabinofuranosidic bond. However, its ability to breakdown the arabinofuranosidic from D-arabinose as monomer is still unknown. Here we present protein engineering through computational design to improve the specificity of Abfa by replacing discriminate amino acid residue. The effort is done to analyze the ability of Abfa in hydrolyzed mycobacterial cell wall and also to analyze the anti-TB assay of Abfa.     

Design,Synthesis, and in vitro Antimycobacterial Activity of Propylene‐tethered Isatin Dimmers

A set of propylene‐tethered isatin dimmers 2a–i was synthesized via click chemistry and screened for their in vitro antimycobacterial activities against Mycobacterium tuberculosis (MTB) H₃₇Rv and multidrug‐resistant TB. In spite of all dimmers (minimum inhibitory concentration: 25– > 128 μg/mL) only exhibited weak to moderate activities against the tested MTB H₃₇Rv and multidrug‐resistant TB, the structure–activity relationship was enriched and the results warrant further development of the anti‐TB properties of this kind of dimmers.     

Synthesis of Ethylene Tethered Isatin‐Coumarin Hybrids and Evaluation of Their in vitro Antimycobacterial Activities

A series of novel isatin‐coumarin derivatives tethered through ethylene were designed, synthesized, and evaluated for their in vitro antimycobacterial activities against Mycobacterium tuberculosis (MTB) H37Rv and multidrug‐resistant tuberculosis (MDR‐TB). All hybrids exhibited potential antimycobacterial activities against MTB H37Rv and MDR‐TB with minimum inhibitory concentration (MIC) ranging from 32 to 256 μg/mL. In particular, the hybrid 4h (MIC: 50 and 32 μg/mL) was most active against MTB H₃₇Rv and MDR‐TB strains, which was 2 and >4 folds more potent than the first‐line antitubercular agents rifampicin (MIC: 64 μg/mL) and isoniazid (MIC: >128 μg/mL) against MDR‐TB, warrant further optimization.     

Design,Synthesis and In Vitro Antitubercular Evaluation of Isatin‐ciprofloxacin Hybrids with Hydrogen Bonding Capacity

A series of novel isatin‐ciprofloxacin hybrids inhaling oxime, semicarbazone, and thiosemicarbazone groups with hydrogen bonding capacity were designed, synthesized, and evaluated for their in vitro antitubercular activities against Mycobacterium tuberculosis (MTB) H37Rv and multidrug‐resistant‐TB (MDR‐TB). All hybrids endowed with potential activities against the tested MTB H37Rv and MDR‐TB strains with minimum inhibitory concentration (MIC) in a range of 0.20 to 128 μg/mL. In particular, the most active hybrid 5e (MIC: 0.20 and 0.5 μg/mL) was four and two times more active than the parent ciprofloxacin (MIC: 0.78 μg/mL) and rifampicin (MIC: 0.39 μg/mL) against MTB H37Rv, and 4–>256 times more potent than the three references ciprofloxacin (MIC: 2.0 μg/mL), rifampicin (MIC: 32 μg/mL), and isoniazid (>128 μg/mL) against MDR‐TB. Thus, this kind of hybrids holds great promise as future anti‐TB agents against both drug‐sensitive and drug‐resistant MTB strains infection.     

Rapid detection of Mycobacterium tuberculosis cells by using microtip-based immunoassay

This paper describes a microtip-based approach of concentrating target analytes for a highly sensitive bioassay. As an example, rapid screening of bacterial whole cells is presented to detect Mycobacterium tuberculosis (MTB), a pathogenic bacterium for human tuberculosis (TB). The concentration and detection is performed with three sequential steps of (1) attracting bacterial whole cells in the vicinity of a microtip by alternating current electroosmotic flow; (2) capturing the cells onto the microtip by capillary action; (3) binding fluorophore-labeled polyclonal antibodies to the cells followed by fluorescence measurement (immunofluorescence). Through this mechanism, MTB cells have been detected to the concentration of 8,000 cells/mL within 10 min. This sensitivity is comparable to that of Ziehl–Neelsen smear microscopy, a common culture-free screening method for diagnosis of TB. For comparison, Escherichia coli O157:H7 cells have also been detected to the concentration of 30,000 cells/mL in the same way.     

Tetraethylene Glycol Tethered Heteronuclear Bis‐isatin Derivatives: Design,Synthesis, and In Vitro Anti‐mycobacterial Activities

A series of novel tetraethylene glycol tethered heteronuclear bis‐isatin derivatives 7a – l were designed, synthesized, and evaluated for their in vitro anti‐mycobacterial activities against Mycobacterium tuberculosis (MTB) H37Rv and multidrug‐resistant TB (MDR‐TB) as well as cytotoxicity in VERO cell line. All hybrids exhibited potential anti‐mycobacterial activities against MTB H37Rv and MDR‐TB, and acceptable cytotoxicity. Among them, the heteronuclear bis‐isatin 7l [minimum inhibitory concentration (MIC): 16 and 16 μg/mL] was found to be most active against MTB H37Rv and MDR‐TB strains, which was 2‐fold and >8‐fold, respectively, more potent than were the first‐line anti‐tubercular agents rifampicin (MIC: 32 μg/mL) and isoniazid (MIC: >128 μg/mL) against MDR‐TB, also demonstrated acceptable cytotoxicity profile (CC₅₀: 62.5 μg/mL), could act as a starting point for further optimization.     

Quinoline Derivatives with Potential Activity Against Multidrug‐resistant Tuberculosis

Tuberculosis (TB), which affects primarily the lungs (pulmonary TB) apart from other vital organs, is a life‐threatening chronic deadliest infectious disease caused by members of Mycobacterium tuberculosis (MTB) complex and mainly by MTB itself. The emergence of MTB new virulent forms that are resistant to some or all first‐line and second‐line anti‐TB agents, including multidrug‐resistant (MDR), extensively drug‐resistant, and totally drug‐resistant strains has further aggravated the spread of this disease and was increased up to an alarming level in the recent decades. More than ever, it is imperative to develop novel, high effective, and fast acting anti‐TB drugs to prevent the spread of TB, particularly in its hard‐to‐kill MDR‐TB, extensively drug‐resistant‐TB, and totally drug‐resistant‐TB strains. In recent years, numerous compounds have been synthesized for this purpose, but only a handful of compounds have entered human trials after the discovery of rifampicin, reflecting the inherent difficulties of developing new anti‐TB agents. Despite of bedaquiline and delamanid have received approval from many countries for treatment of MDR‐TB infected patients, both drugs are associated with serious side effects and are only recommended for those MDR‐TB patients without other treatment options. All these aforementioned facts make it an urgent need to develop novel drugs. Quinoline‐based derivatives including quinolones ex biological activities, and some of them displayed excellent in vitro and in vivo activities against MDR‐TB. This review outlines the recent developments of quinoline‐based derivatives with potential activity against MDR‐TB as well as the structure–activity relationship.     

Design,Synthesis, and In Vitro Anti‐mycobacterial Activities of Propylene‐Tethered Gatifloxacin‐Isatin Hybrids

A series of propylene‐tethered mono‐/bis‐isatin‐gatifloxacin hybrids 3a–f and 4a–f were designed, synthesized, and evaluated for their in vitro anti‐mycobacterial activities against Mycobacterium tuberculosis (MTB) H37Rv and multidrug‐resistant tuberculosis (MDR‐TB) as well as cytotoxicity against VERO cell line. The results indicated that all hybrids exhibited promising anti‐mycobacterial activities against MTB H37Rv and MDR‐TB with MIC ranging from 0.25 to 16 μg/mL. In particular, the mono‐isatin‐gatifloxacin hybrid 3e (MIC: 0.25 and 0.25 μg/mL) was found to be most active against MTB H₃₇Rv and MDR‐TB strains, which was twofold more active than the parent gatifloxacin (MIC: 0.5 μg/mL) and comparable with rifampicin ( RIF ) (MIC: 0.25 μg/mL) against MTB H₃₇Rv, and 4‐ > 512 times more potent than the three references gatifloxacin (MIC: 1.0 μg/mL), RIF (MIC: 64 μg/mL), and isoniazid (>128 μg/mL) against MDR‐TB, could act as a starting point for further optimization.     

A highly atom economic, chemo-, regio- and stereoselective synthesis, and discovery of spiro-pyrido-pyrrolizines and pyrrolidines as antimycobacterial agents

The 1,3-dipolar cycloaddition of azomethine ylides derived from acenaphthenequinone and α-amino acids viz. proline, phenylglycine and sarcosine to a series of 1-methyl-3,5-bis[(E)-arylmethylidene]tetrahydro-4(1H)-pyridinones afforded novel spiro-pyrido-pyrrolizines and pyrrolidines chemo-, regio- and stereoselectively in quantitative yields. These compounds were screened for their in vitro activity against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant Mycobacterium tuberculosis (MDR-TB) and Mycobacterium smegmatis using agar dilution method. Among the synthesized compounds, spiro-[2.2″]acenaphthene-1″-one-spiro[3.3′]-5′-(2-chlorophenylmethylidene)-1′-methyltetrahydro-4′(1H)-pyridinone-4-(2-chlorophenyl)hexahydro-1H-pyrrolizine (3e) was found to be the most active with a minimum inhibitory concentration (MIC) of 0.4 μg/mL against MTB and MDR-TB.     

Design,Synthesis, and Antimycobacterial Activities of Diethylene Glycol Tethered Moxifloxacin–Isatin Hybrids

A new class of diethylene glycol tethered moxifloxacin–isatin hybrids 5a–l was designed, synthesized, and evaluated for their in vitro antimycobacterial activity against Mycobacterium tuberculosis (MTB) H37Rv and multidrug‐resistant tuberculosis (MDR‐TB) strains. Our results showed that all hybrids with higher lipophilicity than the parent moxifloxacin exhibited promising activity against the tested strains with minimum inhibitory concentration (MIC) in a range of 0.2–16 μg/mL. In particular, hybrid 5h (MIC: 0.20 and 0.5 μg/mL), which was found to be most active against MTB H37Rv and MDR‐TB, was twofold more potent than isoniazid (MIC: 0.39 μg/mL) against MTB H37Rv and ≥64‐fold more active than isoniazid and rifampicin (MIC: >128 and 32 μg/mL, respectively) against MDR‐TB.     

Isatin‐(thio)semicarbazide/oxime‐1H‐1,2,3‐triazole‐coumarin Hybrids: Design,Synthesis, and in vitro Anti‐mycobacterial Evaluation

Isatin and coumarin derivatives with potential anti‐tubercular activity, while (thio)semicarbazide/oxime and 1H‐1,2,3‐triazole moieties exhibited favorable properties such as hydrogen bonding and/or metal chelation capability, so integration of the four pharmacophores into one molecule may provide more effective anti‐tubercular candidates. Based on the consideration earlier, 12 isatin‐(thio)semicarbazide/oxime‐1H‐1,2,3‐triazole‐coumarin hybrids 8a–l were designed, synthesized, and evaluated for their in vitro anti‐mycobacterial activities against M. tuberculosis (MTB) H₃₇Rv and MDR‐TB. The results showed that all the hybrids (MIC: 50–>200 μg/mL) exhibited weak to moderate inhibitory activity against MTB H₃₇Rv and MDR‐TB, which were far less potent than the references isoniazid (MIC: 0.05 μg/mL) and rifampicin (MIC: 0.39 μg/mL) against MTB H₃₇Rv. The most active hybrid 8h (MIC: 50 μg/mL) was comparable with rifampicin (MIC: 32 μg/mL) and more active than isoniazid (MIC: >128 μg/mL) against MDR‐TB, could be act as a lead for further optimization. Moreover, the enriched structure–activity relationship paved the way to the further rational development of this kind of hybrids.     

Development of novel LpxC inhibitors: chiral-pool synthesis of C-triazolyl glycosides

The Zn²⁺-dependent deacetylase LpxC plays an important role in the biosynthesis of the cell wall of Gram-negative bacteria and therefore represents an interesting target for the development of novel antibiotics. In a 10-step, chiral pool synthesis starting from d-mannose (3), a series of C-aryl furanosidic hydroxamic acids bearing a 1,4-disubstituted triazole ring in α-configuration at the furanose moiety was stereoselectively synthesized and tested for inhibitory activity against LpxC. The key step of the synthesis comprises a Cu(I) catalyzed Huisgen cycloaddition of terminal alkyne 10 with various azides to introduce diversity to the potential LpxC inhibitors. The X-ray crystal structure of the click product 11e proves the stereochemistry at the anomeric center and the substitution pattern of the triazole ring. The synthesized compounds did not inhibit LpxC.     

Structure-based discovery of novel inhibitors of Mycobacterium tuberculosis CYP121 from Indonesian natural products

Tuberculosis (TB) continues to be a serious global health threat with the emergence of multidrug-resistant tuberculosis (MDR-TB) and extremely drug-resistant tuberculosis (XDR-TB). There is an urgent need to discover new drugs to deal with the advent of drug-resistant TB variants. This study aims to find new M. tuberculosis CYP121 inhibitors by the screening of Indonesian natural products using the principle of structure-based drug design and discovery. In this work, eight natural compounds isolated from Rhoeo spathacea and Pluchea indica were selected based on their antimycobacterial activity. Derivatives compound were virtually designed from these natural molecules to improve the interaction of ligands with CYP121. Virtual screening of ligands was carried out using AutoDock Vina followed by 50 ns molecular dynamics simulation using YASARA to study the inhibition mechanism of the ligands. Two ligands, i.e., kaempferol (KAE) and its benzyl derivative (KAE3), are identified as the best CYP121 inhibitors based on their binding affinities and adherence to the Lipinski’s rule. Results of molecular dynamics simulation indicate that KAE and KAE3 possess a unique inhibitory mechanism against CYP121 that is different from GGJ (control ligand). The control ligand alters the overall dynamics of the receptor, which is indicated by changes in residue flexibility away from CYP121 binding site. Meanwhile, the dynamic changes caused by the binding of KAE and KAE3 are isolated around the binding site of CYP121. These ligands can be developed for further potential biological activities.     

Synthesis and characterization of phenolic antioxidants with surfactant properties: glucosyl- and glucuronosyl alkyl gallates

In the search of better antioxidants for different applications, we have designed and synthesized two series of antioxidants that possess surfactant properties: glucosyl- and glucuronosyl alkyl gallates. They display better surface-active efficiency that alkyl gallates and some show critical micelle concentration (CMC) and surfactant effectiveness (γ_cmc) in the same range of worldwide known surfactants, such as Brij-30 or Tween-20. Moreover, they exhibit a high antioxidant activity due to the di-ortho phenolic moiety present in their structure. Nevertheless, glucosyl- and glucuronosyl alkyl gallates are worse antioxidants than the corresponding alkyl gallates.     

Chemical Validation of Mycobacterium tuberculosis Phosphopantetheine Adenylyltransferase Using Fragment Linking and CRISPR Interference**

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a K_D <20 μM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.     

Rapid diagnosis of mycobacterium tuberculosis with electrical impedance spectroscopy in suspensions using interdigitated microelectrode

A Mycobacterium tuberculosis (MTB) bacilli are still widely spreading and have to be diagnosed fast and efficiently. Therefore, a new simple and rapid method was proposed to detect MTB by the impedance properties of MTB suspensions using interdigitated microelectrodes. As a result, MTB suspensions in deionized (DI) water with different cell concentrations generated different electrical impedance spectral responses. Whereas MTB suspensions in 0.9 wt. % NaCl solution did not produce any significant differences in the impedance spectra in response to different cell concentrations. In DI water suspensions, the impedance at 1 kHz decreased with increasing cell concentrations. The impedance of MTB suspension in DI water has been discussed; it was found to be resulted from the cell wall charges and release of ions from the cells. There was a linear relationship between the impedance and logarithmic value of the cell concentration in the cell concentration range of 10² to 10⁸ cfu/mL, which can be expressed by the regression equation of Z (kΩ) =–456lnN (cfu/mL) + 9717 with R² = 0.99. Detection limit was calculated as 10⁴ cfu/mL, which is comparable with many label-free immunosensors for detecting pathogenic bacteria reported in the literature. This work demonstrated that MTB concentration can be determined through measuring the impedance of MTB suspensions in DI water. This new detection mechanism can be an alternative for current impedance methods available for detecting bacterial cells.     

Immune modulation properties of herbal plant leaves: Phyllanthus niruri aqueous extract on immune cells of tuberculosis patient - in vitro study

Disease progression in Tuberculosis (TB) is dependent on host’s immune system. Phyllanthus niruri, a traditional herb, has long been used to boost immune system in Indonesian society. This study aimed to observe the potential role of P. niruri in inducing immune cells activity in TB patients by in vitro approach. Peripheral blood mononuclear cells (PBMCs) and macrophages were collected from active pulmonary TB patients. After stimulation with graded doses of P. niruri aqueous extract, cell proliferation, phagocytic activity and nitric oxide (NO) release were analysed. P. niruri aqueous extract induced proliferation of PBMCs, increased NO release, and improved macrophages phagocytic activity. These effects were observed in a dose-dependent manner. This may lead to further research for the potential role of P. niruri as immunomodulatory adjuvant therapy for TB patients.     

Design,synthesis, and in vitro antimycobacterial activities of butylene tethered 7‐fluoroisatin‐isatin scaffolds

In this work, a series of butylene tethered heteronuclear 7‐fluoroisatin‐isatin scaffolds 4a to 4h were designed and synthesized, and the antimycobacterial activity profiles, cytotoxicity together with inhibitory activity against MTB DNA gyrase, were also investigated. All the synthesized heteronuclear scaffolds were active against MTB H₃₇Rv and MDR‐MTB strains, and some of them were more potent than isoniazid ( INH ), rifampicin ( RIF ), ethambutol ( EMB ) against MDR‐MTB strain. The structure‐activity relationship demonstrated that the substituents on C‐3 position of 7‐fluoroisatin and isatin moieties were closely related with the activity, and hydrogen bond donors were favorable to the activity. Scaffolds with different substituents showed higher activity than the analogs with the same substituents at this position of the two isatin motifs, which may be attributed to the synergistic effect. Among them, the most active scaffold 4e (MIC: 1 and 4 μg/mL) was comparable with the first‐line anti‐TB agent EMB against MTB H₃₇Rv, and ≥16‐fold more potent than INH , RIF , and EMB against MDR‐MTB strain, demonstrating its potential for fighting against infections caused by both drug‐sensitive and MDR MTB strains. Moreover, scaffold 4e also possessed excellent toxicological profiles and promising inhibitory activity against MTB DNA gyrase. Thus, scaffold 4e could act as an ideal platform for further optimization.     

Genome-wide analysis of Excretory/Secretory proteins in root-knot nematode,Meloidogyne incognita provides potential targets for parasite control

The root-knot nematode, Meloidogyne incognita causes significant damage to various economically important crops. Infection is associated with secretion of effector proteins into host cytoplasm and interference with host innate immunity. To combat this infection, the identification and functional annotations of Excretory/Secretory (ES) proteins serve as a key to produce durable control measures. The identification of ES proteins through experimental methods are expensive and time consuming while bioinformatics approaches are cost-effective by prioritizing the experimental analysis of potential drug targets for parasitic diseases. In this study, we predicted and functionally annotated the 1889 ES proteins in M. incognita genome using integration of several bioinformatics tools. Of these 1889 ES proteins, 473 (25%) had orthologues in free living nematode Caenorhabditis elegans, 825(67.8%) in parasitic nematodes whereas 561 (29.7%) appeared to be novel and M. incognita specific molecules. Of the C. elegans homologues, 17 ES proteins had “loss of function phenotype” by RNA interference and could represent potential drug targets for parasite intervention and control. We could functionally annotate 429 (22.7%) ES proteins using Gene Ontology (GO) terms, 672 (35.5%) proteins to protein domains and established pathway associations for 223 (11.8%) sequences using Kyoto Encyclopaedia of Genes and Genomes (KEGG). The 162 (8.5%) ES proteins were also mapped to several important plant cell-wall degrading CAZyme families including chitinase, cellulase, xylanase, pectate lyase and endo-β-1,4-xylanase. Our comprehensive analysis of M. incognita secretome provides functional information for further experimental study.     

Gas-phase conformations and exciton couplings in 5,6,11,12-tetrahydrodibenzo[a,e]cyclooctene

The conformations and exciton couplings in 5,6,11,12-tetrahydrodibenzo[a,e]cyclooctene (THDC) have been studied using resonance-enhanced two-photon ionization spectroscopy in a supersonic jet expansion. It has been estimated from the spectral analysis that 90% of THDC exists in the twist-boat (TB) conformation; the chair (C) conformer constitutes the remaining 10%. Most of the vibronic activity in the spectrum of THDC is associated with the symmetric flapping of the aromatic rings of the TB conformer. The observed S₁/S₂ exciton splitting of the TB conformer is 100 cm^?1. The S₁/S₂ transition of the C conformer is found to be forbidden. The exciton splittings of the C and TB conformers were estimated by the spectral analysis of two deuterated isotopomers of THDC. The estimated exciton splittings of the C and TB conformers are 14.7 and 101.9 cm^?1, respectively. The supramolecular model of bichromophores with identical chromophores at the CIS/6-31+G(d)//HF/6-31+G(d) level of theory predicted electronic coupling energies that are very close to the experimental exciton coupling energies.