Bioorthogonal Chemical Reporters for Selective In Situ Probing of Mycomembrane Components in Mycobacteria

The global pathogen Mycobacterium tuberculosis and other species in the suborder Corynebacterineae possess a distinctive outer membrane called the mycomembrane (MM). The MM is composed of mycolic acids, which are either covalently linked to an underlying arabinogalactan layer or incorporated into trehalose glycolipids that associate with the MM non‐covalently. These structures are generated through a process called mycolylation, which is central to mycobacterial physiology and pathogenesis and is an important target for tuberculosis drug development. Current approaches to investigating mycolylation rely on arduous analytical methods that occur outside the context of a whole cell. Herein, we describe mycobacteria‐specific chemical reporters that can selectively probe either covalent arabinogalactan mycolates or non‐covalent trehalose mycolates in live mycobacteria. These probes, in conjunction with bioorthogonal chemistry, enable selective in situ detection of the major MM components.     

Cephem-Pyrazinoic Acid Conjugates: Circumventing Resistance in Mycobacterium tuberculosis

Tuberculosis (TB) is a leading source of infectious disease mortality globally. Antibiotic-resistant strains comprise an estimated 10 % of new TB cases and present an urgent need for novel therapeutics. β-lactam antibiotics have traditionally been ineffective against M. tuberculosis (Mtb), the causative agent of TB, due to the organism's inherent expression of β-lactamases that destroy the electrophilic β-lactam warhead. We have developed novel β-lactam conjugates, which exploit this inherent β-lactamase activity to achieve selective release of pyrazinoic acid (POA), the active form of a first-line TB drug. These conjugates are selectively active against M. tuberculosis and related mycobacteria, and activity is retained or even potentiated in multiple resistant strains and models. Preliminary mechanistic investigations suggest that both the POA “warhead” as well as the β-lactam “promoiety” contribute to the observed activity, demonstrating a codrug strategy with important implications for future TB therapy.     

Near-infrared fluorescence activation probes based on disassembly-induced emission cyanine dye

Currently most of the fluorogenic probes are designed for the detection of enzymes which work by converting the non-fluorescence substrate into the fluorescence product via an enzymatic reaction. On the other hand, the design of fluorogenic probes for non-enzymatic proteins remains a great challenge. Herein, we report a general strategy to create near-IR fluorogenic probes, where a small molecule ligand is conjugated to a novel γ-phenyl-substituted Cy5 fluorophore, for the selective detection of proteins through a non-enzymatic process. Detail mechanistic studies reveal that the probes self-assemble to form fluorescence-quenched J-type aggregate. In the presence of target analyte, bright fluorescence in the near-IR region is emitted through the recognition-induced disassembly of the probe aggregate. This Cy5 fluorophore is a unique self-assembly/disassembly dye as it gives remarkable fluorescence enhancement. Based on the same design, three different fluorogenic probes were constructed and one of them was applied for the no-wash imaging of tumor cells for the detection of hypoxia-induced cancer-specific biomarker, transmembrane-type carbonic anhydrase IX.     

In Situ Localization of Enzyme Activity in Live Cells by a Molecular Probe Releasing a Precipitating Fluorochrome

Current enzyme‐responsive, fluorogenic probes fail to provide in situ information because the released fluorophores tend to diffuse away from the reaction sites. The problem of diffusive signal dilution can be addressed by designing a probe that upon enzyme conversion releases a fluorophore that precipitates. An excited‐state intramolecular proton transfer (ESIPT)‐based solid‐state fluorophore HTPQ was developed that is strictly insoluble in water and emits intense fluorescence in the solid state, with λ _ex/em=410/550 nm, thus making it far better suited to use with a commercial confocal microscope. HTPQ was further utilized in the design of an enzyme‐responsive, fluorogenic probe (HTPQA), targeting alkaline phosphatase (ALP) as a model enzyme. HTPQA makes possible diffusion‐resistant in situ detection of endogenous ALP in live cells. It was also employed in the visualizing of different levels of ALP in osteosarcoma cells and tissue, thus demonstrating its interest for the diagnosis of this type of cancer.     

Fluorogenic Probes with Substitutions at the 2 and 7 Positions of Cephalosporin are Highly BlaC‐Specific for Rapid Mycobacterium tuberculosis Detection

Current methods for the detection of Mycobacterium tuberculosis (Mtb) are either time consuming or require expensive instruments and are thus are not suitable for point‐of‐care diagnosis. The design, synthesis, and evaluation of fluorogenic probes with high specificity for BlaC, a biomarker expressed by Mtb, are described. The fluorogenic probe CDG‐3 is based on cephalosporin with substitutions at the 2 and 7 positions and it demonstrates over 120 000‐fold selectivity for BlaC over TEM‐1 Bla, the most common β‐lactamase. CDG‐3 can detect 10 colony‐forming units of the attenuated Mycobacterium bovis strain BCG in human sputum in the presence of high levels of contaminating β‐lactamases expressed by other clinically prevalent bacterial strains. In a trial with 50 clinical samples, CDG‐3 detected tuberculosis with 90 % sensitivity and 73 % specificity relative to Mtb culture within one hour, thus demonstrating its potential as a low‐cost point‐of‐care test for use in resource‐limited areas.     

Acute Modulation of Mycobacterial Cell Envelope Biogenesis by Front‐Line Tuberculosis Drugs

Front‐line tuberculosis (TB) drugs have been characterized extensively in vitro and in vivo with respect to gene expression and cell viability. However, little work has been devoted to understanding their effects on the physiology of the cell envelope, one of the main targets of this clinical regimen. Herein, we use metabolic labeling methods to visualize the effects of TB drugs on cell envelope dynamics in mycobacterial species. We developed a new fluorophore–trehalose conjugate to visualize trehalose monomycolates of the mycomembrane using super‐resolution microscopy. We also probed the relationship between mycomembrane and peptidoglycan dynamics using a dual metabolic labeling strategy. Finally, we found that metabolic labeling of both cell envelope structures reports on drug effects on cell physiology in two hours, far faster than a genetic sensor of cell envelope stress. Our work provides insight into acute drug effects on cell envelope biogenesis in live mycobacteria.     

Intramolecular Long‐Distance Nucleophilic Reactions as a Rapid Fluorogenic Switch Applicable to the Detection of Enzymatic Activity

Long‐distance intramolecular nucleophilic reactions are promising strategies for the design of fluorogenic probes to detect enzymatic activity involved in lysine modifications. However, such reactions have been challenging and hence have not been established. In this study, we have prepared fluorogenic peptides that induce intramolecular reactions between lysine nucleophiles and electrophiles in distal positions. These peptides contain a lysine and fluorescence‐quenched fluorophore with a carbonate ester, which triggers nucleophilic transesterification resulting in fluorogenic response. Transesterification occurred under mild aqueous conditions despite the presence of a long nine‐amino‐acid spacer between the lysine and fluorophore. In addition, one of the peptides showed the fastest reaction kinetics with a half‐life time of 3.7 min. Furthermore, the incorporation of this fluorogenic switch into the probes allowed rapid fluorogenic detection of histone deacetylase (HDAC) activity. These results indicate that the transesterification reaction has great potential for use as a general fluorogenic switch to monitor the activity of lysine‐targeting enzymes.     

Comparative study of Factor Xa fluorogenic substrates and their influence on the quantification of LMWHs

Low molecular weight heparins (LMWHs) are recognised as the preferred anticoagulants in the prevention and treatment of venous thromboembolism. Anti-Factor Xa (anti-FXa) levels are used to monitor the anticoagulant effect of LMWHs and such assays are routinely employed in hospital diagnostic laboratories. In this study, a fluorogenic anti-FXa assay was developed using a commercially available fluorogenic substrate with an attached 6-amino-1-naphthalene-sulfonamide (ANSN) fluorophore and was used for the determination of two LMWHs, enoxaparin and tinzaparin and the heparinoid, danaparoid. The assay was based on the complexation of heparinised plasma with 100 nM exogenous FXa and 25 μM of the fluorogenic substrate Mes-D-LGR-ANSN (C₂H₅)₂ (SN-7). The assay was tested with pooled plasma samples spiked with anticoagulant concentrations in the range 0–1.6 U mL⁻¹. The statistically sensitive assay range was 0–0.4 U mL⁻¹ for enoxaparin and tinzaparin and 0–0.2 U mL⁻¹ for danaparoid, with assay variation typically below 10.5%. This assay was then compared with a previously published fluorogenic anti-FXa assay developed with the peptide substrate, methylsulfonyl-d-cyclohexylalanyl-glycyl-arginine-7-amino-4-methylcoumarin acetate (Pefafluor FXa). Both assays were compared in terms of fluorescence intensity, lag times and sensitivity to anticoagulants.     

Synthesis of 2′-deoxycytidine and its triphosphate bearing tryptophan-based imidazolinone fluorophore for environment sensitive fluorescent labelling of DNA

We synthesized 2′-deoxycytidine and the corresponding nucleoside triphosphate bearing (indol-3-yl)methylene-2-methyl-5-oxo-4,5-dihydroimidazol-1-yl group (a tryptophan-based fluorophore from cyan fluorescent protein) linked through a propargyl group at position 5. The fluorophore is weakly solvatochromic, sensitive to pH, and, as a molecular rotor, it is highly sensitive to viscosity. In low viscosity solvents, the fluorescence is very weak, whereas in more viscous environment it lights up. Primer extension or PCR using the modified dC^TrpTP and KOD XL DNA polymerase was used for construction of labelled oligonucleotides and DNA. Preliminary study showed a 2-fold increase of fluorescence of labelled ON probe in presence of single strand-binding protein indicating a potential of this label for sensing of protein-DNA interactions.     

An ESIPT-Dependent AIE Fluorophore Based on HBT Derivative: Substituent Positional Impact on Aggregated Luminescence and its Application for Hydrogen Peroxide Detection

Aiming to develop the facile organic fluorophore possessing excited state intramolecular proton transfer (ESIPT) and aggregation-induced emission (AIE), we designed and synthesized two isomers with different linkage site between hydroxyl of 2-(2-hydroxyphenyl) benzothiazole (HBT) and a benzothiazole substituent (para position refers to p-BHBT and ortho position refers to o-BHBT). Fluorescence emission properties of p-BHBT and o-BHBT in THF/water mixtures with different water volume fractions indicated an opposite luminescence in aggregates, in which p-BHBT showed an ESIPT-dependent AIE properties while o-BHBT displayed ESIPT effect and aggregation-caused quenching (ACQ) qualities. A possible mechanism for molecular actions to illustrate the aggregating luminescence alteration of these two isomers had been proposed and verified by theoretical and experimental studies. More importantly, Probe-1, generated from dual ESIPT-AIE fluorophore p-BHBT, was successfully used as a ratiometric fluorescent chemosensor for highly selective (above 15-fold over other ROS) and sensitive (69-fold fluorescence enhancement with 0.22 μM of detection limit) detection of hydrogen peroxide in aqueous solution and living cells, respectively.     

Mechanism‐Based Fluorogenic trans‐Cyclooctene–Tetrazine Cycloaddition

The development of fluorogenic reactions which lead to the formation of fluorescent products from two nonfluorescent starting materials is highly desirable, but challenging. Reported herein is a new concept of fluorescent product formation upon the inverse electron‐demand Diels–Alder reaction of 1,2,4,5‐tetrazines with particular trans ‐cyclooctene (TCO) isomers. In sharp contrast to known fluorogenic reagents the presented chemistry leads to the rapid formation of unprecedented fluorescent 1,4‐dihydropyridazines so that the fluorophore is built directly upon the chemical reaction. Attachment of an extra fluorophore moiety is therefore not needed. The photochemical properties of the resulting dyes can be easily tuned by changing the substitution pattern of the starting 1,2,4,5‐tetrazine. We support the claim with NMR measurements and rationalize the data by computational study. Cell‐labeling experiments were performed to demonstrate the potential of the fluorogenic reaction for bioimaging.     

A Novel Fluorescence Sensor for Cocaine with Signal Amplification through Cycling Exo-Cleaving with a Hairpin Probe

A novel fluorescence assay, based on the signal amplification through cycling reactions of aptameric recognition and nucleic acid exo-cleaving, was established for the sensitive detection of cocaine. In this assay, a new single fluorophore-labeled DNA-hairpin probe was used. This smart probe was established based on the significant nucleobase quenching between the nucleobases and the fluorophore. In the presence of cocaine and its aptamer, the structure of the smart probe changed to recover the fluorescence. In order to enhance the sensitivity of this assay, exonuclease III was introduced to enable the inputted cocaine to react with multiple probes in a recycling manner. Under the optimal conditions, a linear range for cocaine from 4.0 × 10^?9 to 8.0 × 10^?8 M with a detection limit of 1.76 × 10^?9 M (3σ, n = 11) was obtained.     

A rapid method for determining Mycobacterium tuberculosis based on a bulk acoustic wave impedance biosensor

The bulk acoustic wave impedance biosensor was set up and used to monitor the growth of Mycobacterium tuberculosis (M.TB). This sensor is rapid, simple, sensitive (lower limit is 2×10³ cells ml⁻¹) and cheap (easy to generalize). The typical response curve was different from other bacteria's, such as Escherichia coli, Staphylococcus aureus, Proteus mirabilis. The frequency detection time was used to quantitatively determine M.TB. It was proportional to logarithm of the initial concentration of M.TB in the range of 2×10³-3×10⁷ cells ml⁻¹. The set up sensor was applied to the direct diagnosis of M.TB samples. The interference of other bacteria was eliminated by pretreatment. Our results confirmed that the use of the set up biosensor was reliable, sensitive. It gives out the potential use for determining M.TB.     

Analysis of DNA relaxation and cleavage activities of recombinant Mycobacterium tuberculosis DNA topoisomerase I from a new expression and purification protocol

Background  

Mycobacterium tuberculosis DNA topoisomerase I is an attractive target for discovery of novel TB drugs that act by enhancing the accumulation of the topoisomerase-DNA cleavage product. It shares a common transesterification domain with other type IA DNA topoisomerases. There is, however, no homology between the C-terminal DNA binding domains of Escherichia coli and M. tuberculosis DNA topoisomerase I proteins.     

Comparison of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> isocitrate dehydrogenases (ICD-1 and ICD-2) reveals differences in coenzyme affinity,oligomeric state,pH tolerance and phylogenetic affiliation

Background  

M.tb icd-1 and M.tb icd-2, have been identified in the Mycobacterium tuberculosis genome as probable isocitrate dehydrogenase (ICD) genes. Earlier we demonstrated that the two isoforms can elicit B cell response in TB patients and significantly differentiate TB infected population from healthy, BCG-vaccinated controls. Even though immunoassays suggest that these proteins are closely related in terms of antigenic determinants, we now show that M.tb icd-1 and M.tb icd-2 code for functional energy cycle enzymes and document the differences in their biochemical properties, oligomeric assembly and phylogenetic affiliation.     

A Fluorescence Quenching Method for the Determination of Bromate Ion with 4,5-Dibromophenylfluorone and Cetyltrimethylammonium Bromide

Abstract

A highly sensitive and selective fluorescence quenching method was developed to determine the bromate ion (BrO₃ ^?) with 4,5-dibromophenylfluorone (DB-PF) as fluorogenic reagent. BrO₃ ^? reacts with potassium bromide in sulfuric acid solution (0.6M) giving bromine (Br₂) which is estimated with fluorescence quenching method using DB-PF as fluorogenic reagent. Bromine reacts with DB-PF to produce a compound, whose maximum excitation wavelength and emission wavelength are 543nm, 560nm respectively. The linear calibration range is 0.05–0.5 μg/25ml. The detection limit is 0.05μg/25ml. The method may be used to determine microamounts of BrO₃ ^? in potassium chlorate with satisfactory results. The method offers the advantages of simplicity, rapidity and sensitivity.     

A New Bacteriophage-Modified Piezoelectric Sensor for Rapid and Specific detection of Mycobacterium

Detection of tuberculosis and related diseases caused by mycobacteria is costly, time-consuming, and labor-intensive. Here a new phage-modified piezoelectric system for rapid and specific detection of mycobacteria was developed. In this system, interdigital gold electrode immobilized with lytic phage was used as a probe in place of a steel electrode in the multi-channel series piezoelectric quartz crystal (MSPQC) system. The probe was directly connected to the piezoelectric detection system. Mycobacterium was specifically captured to the phage-modified electrode and then lysed by immobilized phage, which caused the electrode electrical properties change. This change can be sensitively monitored by the piezoelectric detection system. The detection time of Mycobacterium smegmatis was less than 2 hours and a detection limit of 10³cfu mL^?1 was obtained. Additionally, it was successfully used to detect Mycobacterium tuberculosis. The developed system using phage-modified interdigital electrode showed high specificity and reproducibility for mycobacterium detection. Compared with the MSPQC system, the proposed system was faster and more specific.     

Communication: Synthesis of Me α-L-Rhap-(1↠3)-2-O-Me-α-L-Rhap And Me 2,3,4-Tri-O-Me-α-L-Fucp-(1↠3)-α-L-Rhap-(1↠3)-2-O-Me-α-L-Rhap : Oligosaccha-Ride Segments of Phenolic Glycolipids in Mycobacterium Bovis Bcg and Tuberculosis Strain Canetti

The revived interest in phenolic glycolipids of pathogenic mycobacteria is evoked by a widespread^1-3 use of Mycobacterium (M.) leprae specific antigen for serodiagnosis of leprosy patients. As a consequence, a few phenolic glycolipids of other mycobacterias were isolated and structurally elucidated. Recently Vercellone and Puzo⁴ reported the isolation of new phenolic glycolipids not yet described in M. bovis BCG. The combination of sugars in one of the glycolipids was identified as 3-O-(α-L-rhamnopyranosyl)-2-O-methyl-α-L-rhamnopyranoside (1) which is closely related to the trisaccharide segment (2) of M. tuberculosis strain Canetti.⁵ The latter contains 2,3,4-tri-O-methyl-L-fucopyranosyl monosaccharide α-linked to 3′ position of 1. It has been emphasised that new found glycolipids of M. bovis BCG could share common epitopes with those of M. tuberculosis, thus leading to false positive immunoabsorbent assay tests during screening of tuberculosis patients. In addition, there is concern regarding the involvement of one of the new found glycolipids in the stimulation of T suppressor cells, thus adding to the conflicting results noted in the protection of M. tuberculosis by M. bovis BCG. In essence the new found phenolic glycolipids of M. bovis BCG are associated with interesting but unclear biological profiles. We now report the synthesis of these closely related oligosaccharides, 1 (R = Me) and 2 (R = Me).     

Design,Synthesis, and in vitro Anti‐mycobacterial Activities of Propylene‐tethered Heteronuclear Bis‐isatin Derivatives

A series of novel propylene‐tethered heteronuclear bis‐isatin derivatives were designed, synthesized, and assessed for their in vitro and anti‐mycobacterial activities. All hybrids exhibited considerable antibacterial and anti‐mycobacterial activities against Mycobacterium tuberculosis H37Rv and multi‐drug‐resistant tuberculosis (MDR‐TB) with minimum inhibitory concentration (MIC) ranging from 16 to 256 μg/mL. In particular, the heteronuclear bis‐isatin 4i (MIC: 25 and 16 μg/mL) was most active against M. tuberculosis H₃₇Rv and MDR‐TB strains, which was fourfold and greater than eightfold more potent than the first‐line anti‐tubercular agents rifampicin (MIC: 64 μg/mL) and isoniazid (MIC: >128 μg/mL) against MDR‐TB, could act as a lead for further optimization.     

Design,synthesis and biological evaluation of some 2-(6-nitrobenzo[d]thiazol-2-ylthio)-N-benzyl-N-(6-nitrobenzo[d]thiazol-2-yl)acetamide derivatives as selective DprE1 inhibitors

Abstract

Tuberculosis (TB) is an infectious disease and caused by various strains of mycobacteria. In the present study, pharmacophore model was developed using single ligand by ligand-based drug discovery approach. The key features responsible for DprE1 inhibitory activity were taken into consideration for developing pharmacophore. After the virtual screening, top 1000 hits were further subjected to docking study using GLIDE module, Schrödinger. Docking studies have shown promising interaction with amino residues with better glide score. Ligand-based drug design approach yielded a series of 15, 2-(6-nitrobenzo[d]thiazol-2-ylthio)-N-benzyl-N-(6-nitrobenzo[d]thiazol-2-yl)acetamide derivatives. All synthesized derivatives were characterized using NMR, mass, CHN analysis. The synthesized compounds were screened for In vitro antitubercular activity against Mycobacterium tuberculosis (H₃₇Rv). Four compounds, 5g (MIC-1.01?μM); 5i (MIC-0.91?μM); 5k (MIC-0.82?μM); and 5o (MIC-1.04?μM) has shown promising activity compared to MIC of standard isoniazid (INH) and DprE1 enzyme inhibition was compared to BTZ043. Two halogen-substituted compounds have exhibited drastic enzyme inhibition.