Identification of Mycobacterium tuberculosis transcriptional repressor EthR inhibitors: Shape-based search and machine learning studies.

Tuberculosis has been a challenge to the world since prehistoric times, and with the advent of drug-resistant strains, it has become more challenging to treat this infection. Ethionamide (ETH), a second-line drug, acts as a prodrug and targets mycolic acid synthesis by targeting the enoyl-acyl carrier protein reductase (InhA) enzyme. Mycobacterium tuberculosis (Mtb) EthR is an ethA gene repressor required to activate prodrug ETH. Recent studies suggest targeting the EthR could lead to newer drug molecules that would help better activate the ETH or complement this process. In this report, we have attempted and successfully identified three new molecules from the drug repurposing library that can target EthR protein and function as ETH boosters. These molecules were obtained after rigorous filtering of the database for their physicochemical, toxicological properties and safety. The molecular docking, molecular dynamics simulations and binding energy studies yielded three compounds, Ethyl (2-amino-4-((4-fluorobenzyl)amino)phenyl)carbamate) (L1), 2-((2,2-Difluorobenzo [d] [1,3]dioxol-5-yl)amino)-2-oxoethyl (E)-3-(5-bromofuran-2-yl)acrylate (L2), and N-(2,3-Dihydrobenzo [b] [1,4]dioxin-6-yl)-4-(2-((4-fluorophenyl)amino)-2-oxoethoxy)-3-methoxy benzamide (L3) are potential EthR inhibitors. We applied machine learning methods to evaluate these molecules for toxicity and synthesisability, suggesting safety and ease of synthesis for these molecules. These molecules are known for other pharmacological activities and can be repurposed faster as adjuvant therapy for tuberculosis.

The multi-target aspect of an MmpL3 inhibitor: The BM212 series of compounds bind EthR2, a transcriptional regulator of ethionamide activation.

The emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) ensures that drug discovery efforts remain at the forefront of TB research. There are multiple different experimental approaches that can be employed in the discovery of anti-TB agents. Notably, inhibitors of MmpL3 are numerous and structurally diverse in Mtb and have been discovered through the generation of spontaneous resistant mutants and subsequent whole genome sequencing studies. However, this approach is not always reliable and can lead to incorrect target assignment and requires orthogonal confirmatory approaches. In fact, many of these inhibitors have also been shown to act as multi-target agents, with secondary targets in Mtb, as well as in other non-MmpL3-containing pathogens. Herein, we have investigated further the cellular targets of the MmpL3-inhibitor BM212 and a number of BM212 analogues. To determine the alternative targets of BM212, which may have been masked by MmpL3 mutations, we have applied a combination of chemo-proteomic profiling using bead-immobilised BM212 derivatives and protein extracts, along with whole-cell and biochemical assays. The study identified EthR2 (Rv0078) as a protein that binds BM212 analogues. We further demonstrated binding of BM212 to EthR2 through an in vitro tryptophan fluorescence assay, which showed significant quenching of tryptophan fluorescence upon addition of BM212. Our studies have demonstrated the value of revisiting drugs with ambiguous targets, such as MmpL3, in an attempt to find alternative targets and the study of off-target effects to understand more precisely target engagement of new hits emerging from drug screening campaigns.

Genetic Variation Putatively Associated with Mycobacterium tuberculosis Resistance to Perchlozone, a New Thiosemicarbazone: Clues from Whole Genome Sequencing and Implications for Treatment of Multidrug-Resistant Tuberculosis.

Perchlozone ([PCZ] 4-thioureido-iminomethylpyridinium perchlorate) is a new thiosemicarbazone approved for the treatment of multidrug-resistant tuberculosis (MDR-TB) in Russia and some other countries. The ethA and hadABC mutations may confer PCZ resistance. At the same time, ethA mutations are known to mediate resistance to ethionamide (ETH) and prothionamide (PTH). We aimed to study the genetic variation underlying Mycobacterium tuberculosis resistance to PCZ through whole genome sequencing (WGS) of consecutive isolates recovered during long-term treatment. This prospective study included patients admitted in 2018-2019 to the regional tuberculosis dispensary, Kaliningrad, Russia, whose treatment regimen included PCZ. Multiple M. tuberculosis isolates were recovered during PCZ treatment, and the bacterial DNA was subjected to WGS followed by bioinformatics analysis. We identified mutations in the genes putatively associated with PCZ resistance, ethA, and hadA. The most frequent one was a frameshift ethA 106 GA > G (seven of nine patients) and most of the other mutations were also likely present before PCZ treatment. In one patient, a frameshift mutation ethA 702 CT > C emerged after six months of PCZ treatment. A frequent presence of cross-resistance mutations to PCZ and ETH/PTH should be taken into consideration when PCZ is included in the treatment regimen of MDR-TB patients.

New insights into the chemical behavior of S-oxide derivatives of thiocarbonyl-containing antitubercular drugs and the influence on their mechanisms of action and toxicity.

OBJECTIVES: This work aims at getting more insights into the distinct behavior of S-oxide derivatives of thiocarbonyl-containing antitubercular drugs, in order to better understand their mechanism of action and toxicity. METHODS: Computational calculation of relative free energy (ΔΔG) of S-oxide tautomers (sulfine R-C [SO]NH2), sulfenic acid (R-C [S-OH]NH) and sulfoxide (R-C [SHO]NH) derived from thioamide and thiourea antitubercular drugs and an update of the literature data with a new point of view about how the structural features of oxidized primary metabolites (S-oxide) can influence the outcome of the reactions and be determinant for the mechanisms of action and of toxicity of these drugs. RESULTS: The calculated free energy of S-oxide tautomers, derived from thioamide and thiourea-type antitubercular drugs, supported by some experimental results, revealed that S-oxide derivatives could be found under sulfine and sulfenic acid forms depending on their chemical structures. Thiocarbonyl compounds belonging to the thioamide series are firstly oxidized, in the presence of H2O2, into the corresponding S-oxide derivatives that are more stable under the sulfine tautomeric form. Otherwise, S-oxides of thiourea-type (acyclic and cyclic) compounds tend to adopt the sulfenic acid tautomeric form preferentially. While the intermediate ethionamide-SO under sulfine form can be isolated and in the presence of H2O2 can undergo further oxidation by a mechanism yielding radical species that are toxic for Mycobacterium tuberculosis and human, thioacetazone-SO, found mainly into sulfenic acid form, is unstable and sufficiently reactive in biological conditions to intercept different biochemical pathways and manifests thus its toxicity. CONCLUSION: Based on experimental and theoretical data, we propose that S-oxide derivatives of thioamide and thiourea-type antitubercular drugs have preference for distinct tautomeric forms. S-oxide of ethioamide is preferentially under sulfine form whereas S-oxide of thiourea compound as thioacetazone is mainly found under sulfenic acid form. These structural features lead to individual chemical reactivities that might explain the distinct mechanism of action and toxicity observed for the thioamide and thiourea antitubercular drugs.

EthA/R-Independent Killing of Mycobacterium tuberculosis by Ethionamide.

Ethionamide (ETH) is part of the drug arsenal available to treat multi-drug resistant tuberculosis. The current paradigm of this pro-drug activation involves the mycobacterial enzyme EthA and the transcriptional repressor, EthR. However, several lines of evidence suggest the involvement of additional players. The ethA/R locus was deleted in Mycobacterium bovis BCG and three Mycobacterium tuberculosis (MTB) strains. While complete resistance to ETH was observed with BCG ethA/R KO, drug susceptibility and dose-dependent killing were retained in the ethA/R KO MTB mutants, suggesting the existence of an alternative pathway of ETH bio-activation in MTB. We further demonstrated that this alternative pathway is EthR-independent, whereby re-introduction of ethR in ethA/R KO MTB did not lead to increased resistance to ETH. Consistently, ethA KO MTB (with intact ethR expression) displayed similar ETH susceptibility profile as their ethA/R KO counterparts. To identify the alternative ETH bio-activator, spontaneous ETH-resistant mutants were obtained from ethA/R KO MTB and whole genome sequencing identified single nucleotide polymorphisms in mshA, involved in mycothiol biosynthesis and previously linked to ETH resistance. Deletion of mshA in ethA/R KO MTB led to complete ETH resistance, supporting that the role of MshA in ETH killing is EthA/R-independent. Furthermore mshA single KO MTB displayed levels of ETH resistance similar or greater than those obtained with ethA/R KO strains, supporting that mshA is as critical as ethA/R for ETH killing efficacy.

New var reconstruction algorithm exposes high var sequence diversity in a single geographic location in Mali.

BACKGROUND: Encoded by the var gene family, highly variable Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) proteins mediate tissue-specific cytoadherence of infected erythrocytes, resulting in immune evasion and severe malaria disease. Sequencing and assembling the 40-60 var gene complement for individual infections has been notoriously difficult, impeding molecular epidemiological studies and the assessment of particular var elements as subunit vaccine candidates. METHODS: We developed and validated a novel algorithm, Exon-Targeted Hybrid Assembly (ETHA), to perform targeted assembly of var gene sequences, based on a combination of Pacific Biosciences and Illumina data. RESULTS: Using ETHA, we characterized the repertoire of var genes in 12 samples from uncomplicated malaria infections in children from a single Malian village and showed them to be as genetically diverse as vars from isolates from around the globe. The gene var2csa, a member of the var family associated with placental malaria pathogenesis, was present in each genome, as were vars previously associated with severe malaria. CONCLUSION: ETHA, a tool to discover novel var sequences from clinical samples, will aid the understanding of malaria pathogenesis and inform the design of malaria vaccines based on PfEMP1. ETHA is available at: https://sourceforge.net/projects/etha/ .

Determination of Ethanol and Toluene Residual Solvent in Diphenhydramine Hydrochloride Raw Materia by Headspace Capillary Gas Chromatography / 中国药房

OBJECTIVE:To establish a method for the determination of residual solvent of ethanol and toluene in diphenhydr-amine hydrochloride raw material. METHODS:Headspace capillary gas charmatography and butanone as internal standard were used. The column was Agilent DB-624 capillary column,inlet temperature was 200 ℃,hydrogen flame ionization detector was 250 ℃,the carried gas was high purity nitrogen,flow rate was 3.0 ml/min with temperature programmed,the splitting-ratio was 20∶1,the containers of headspace injector were in equilibrium at 80 ℃ for 30 min,and the injection time was 1 min. RESULTS:With this chromatographic condition,ethanol,toluene and internal standard peak were well separated;there was a good linear rela-tionship of ethanol and toluene in the range of 0.02-0.8 mg/ml (r=0.999 8 and r=0.999 4);RSDs of precision and stability test were lower than 3%;recoveries were 95.50%-103.50%(RSD=2.6%,n=9) and 96.91%-103.74%(RSD=2.2%,n=9). CON-CLUSIONS:The method is simple,sensitive and accurate,and can be used for the determination of residual solvent of ethanol and toluene in diphenhydramine hydrochloride raw material.

Compounds for use in the treatment of mycobacterial infections: a patent evaluation (WO2014049107A1).

Tuberculosis is one of the main causes of mortality with 1.5 million deaths a year worldwide. The growing emergence of multi- and extremely resistant strains highlights the urgent need of novel antibiotic strategies. Ethionamide, interfering with the mycobacterial membrane biosynthesis, is used in second-line treatment. This molecule is a prodrug, which requires activation by EthA. The patent described in this evaluation (WO2014049107A1) claimed a new family of molecules and their use as antibiotic treatment against mycobacteria such as Mycobacterium tuberculosis, M. leprae and atypical mycobacteria, either as a single active agent or in combination with antibiotics activable by EthA pathway.

The new tuberculosis drug Perchlozone® shows cross-resistance with thiacetazone.

Perchlozone(®) (PCZ), a new thiosemicarbazone developed by JSC Pharmasyntez (Moscow, Russia) for the treatment of tuberculosis (TB), was approved for use against multidrug-resistant disease in Russia in 2012. The mechanism of action of the drug is unknown. A well-studied thiosemicarbazone is the old TB drug thiacetazone (TAC). It has a narrow spectrum and inhibits the FASII dehydratase complex HadABC, which is involved in cell wall biosynthesis in Mycobacterium tuberculosis. TAC is a prodrug, requiring activation by the monooxygenase EthA. In this study, a comparative in vitro analysis of both drugs was performed. The two compounds had an identical spectrum of activity, spontaneous resistant mutants showed cross-resistance, and resistance was mapped to HadABC and EthA. These results suggest that PCZ, like TAC, is a prodrug and that both drugs share EthA as an activating enzyme and HadABC as their principal target.