Recent advancements in the development of anti-tuberculosis drugs

Modern chemotherapy has significantly improved patient outcomes against drug-sensitive tuberculosis. However, the rapid emergence of drug-resistant tuberculosis, together with the bacterium’s ability to persist and remain latent present a major public health challenge. To overcome this problem, research into novel anti-tuberculosis targets and drug candidates is thus of paramount importance. This review article provides an overview of tuberculosis highlighting the recent advances and tools that are employed in the field of anti-tuberculosis drug discovery. The predominant focus is on anti-tuberculosis agents that are currently in the pipeline, i.e. clinical trials.     

Mutagenic effect of orally given AF-2 on embryonic cells in pregnant Syrian hamsters

Pregnant hamsters were given various doses of AF-2 by stomach tube; then the cells of their embryos were isolated and cultured in normal medium. Chromosome preparations were made within 24 h after the start of primary culture, and examined for chromosomal aberrations. Marked chromosomal abnormalities were observed in cells of embryos of animals treated with AF-2 at over 20 mg/kg. Samples of surviving cells were also cultured in normal medium for 48 h, and then selected in medium containing 8AG or 6TG. This treatment with AF-2 caused marked dose-dependent induction of 8AG- or 6TG-resistant mutations: mutant colonies were even obtained after a single treatment with 2 mg of AF-2 per kg. These results show that this is a sensitive and useful mammalian system for detecting environmental mutagens.     

Promoter polymorphisms in trefoil factor 2 and trefoil factor 3 genes and susceptibility to gastric cancer and atrophic gastritis among Chinese population

The polymorphisms in trefoil factor (TFF) gene family that protect gastrointestinal epithelium might influence individual vulnerability to gastric cancer (GC) and atrophic gastritis. We used the Sequenom MassARRAY platform to identify the genotypes of TFF2 rs3814896 and TFF3 rs9981660 polymorphisms in 478 GC patients, 652 atrophic gastritis patients, and 724 controls. For the TFF2 rs3814896 polymorphism, in the subgroup aged ≤ 50 years, we found that AG + GG genotypes were associated with a 0.746-fold decreased risk of atrophic gastritis [p = 0.023, 95% confidence interval (CI) = 0.580–0.960], a 0.626-fold decreased risk of GC (p = 0.005, 95% CI = 0.451–0.868), and a 0.663-fold decreased risk of diffuse-type GC (p = 0.034, 95% CI = 0.452–0.970) compared with the common AA genotype. For the TFF3 rs9981660 polymorphism, in the male subgroup, individuals with variant AG + AA genotype were associated with a 0.761-fold decreased risk of diffuse-type GC compared with the common GG genotype (p = 0.043, 95% CI = 0.584–0.992). Additionally, we found that in subjects aged ≤ 50 years compared with common AA genotype, TFF2 rs3814896 AG + GG genotypes were associated with increased TFF2 mRNA levels in the total gastric cancer specimens and in the diffuse-type gastric cancer specimens; and in males aged ≤ 50 years compared with common GG genotype, TFF3 rs9981660 AA + AG genotypes were associated with TFF3 mRNA levels in diffuse-type gastric cancer tissues and their corresponding non-cancerous tissues. To our knowledge, this is the first report of an association between the TFF2 rs3814896 AG + GG genotypes and decreased risks of GC, diffuse-type GC, and atrophic gastritis in younger people aged ≤ 50 years, and an association between TFF3 rs9981660 AG + AA genotype and decreased risk of diffuse-type GC in men. Moreover, we found that TFF2 rs3814896 AG + GG genotypes in people aged ≤ 50 years and TFF3 rs9981660 AG + AA genotypes in younger males with diffuse-type GC were associated with higher levels of TFF2 and TFF3 mRNA respectively.     

Structural and catalytic insights into the algal prostaglandin H synthase reveal atypical features of the first non-animal cyclooxygenase

Prostaglandin H synthases (PGHSs) have been identified in the majority of vertebrate and invertebrate animals, and most recently in the red alga Gracilaria vermiculophylla. Here we report on the cloning, expression and characterization of the algal PGHS, which shares only about 20% of the amino acid sequence identity with its animal counterparts, yet catalyzes the conversion of arachidonic acid into prostaglandin-endoperoxides, PGG₂ and PGH₂. The algal PGHS lacks structural elements identified in all known animal PGHSs, such as epidermal growth factor-like domain and helix B in the membrane binding domain. The key residues of animal PGHS, like catalytic Tyr-385 and heme liganding His-388 are conserved in the algal enzyme. However, the amino acid residues shown to be important for substrate binding and coordination, and the target residues for nonsteroidal anti-inflammatory drugs (Arg-120, Tyr-355, and Ser-530) are not found at the appropriate positions in the algal sequences. Differently from animal PGHSs the G. vermiculophylla PGHS easily expresses in Escherichia coli as a fully functional enzyme. The recombinant protein was identified as an oligomeric (evidently tetrameric) ferric heme protein. The preferred substrate for the algal PGHS is arachidonic acid with cyclooxygenase reaction rate remarkably higher than values reported for mammalian PGHS isoforms. Similarly to animal PGHS-2, the algal enzyme is capable of metabolizing ester and amide derivatives of arachidonic acid to corresponding prostaglandin products. Algal PGHS is not inhibited by non-steroidal anti-inflammatory drugs. A single copy of intron-free gene encoding for PGHS was identified in the red algae G. vermiculophylla and Coccotylus truncatus genomes.     

Antigen 85C-mediated acyl-transfer between synthetic acyl donors and fragments of the arabinan

Antigen 85 (ag85) is a complex of acyltransferases (ag85A–C) known to play a role in the mycolation of the d-arabino-d-galactan (AG) component of the mycobacterial cell wall. In order to better understand the chemistry and substrate specificity of ag85, a trehalose monomycolate mimic p-nitrophenyl 6-O-octanoyl-β-d-glucopyranoside (1) containing an octanoyl moiety in lieu of a mycolyl moiety was synthesized as an acyl donor. Arabinofuranoside acceptors, methyl α-d-arabinofuranoside (2), methyl β-d-arabinofuranoside (3), and methyl 2-O-β-d-arabinofuranosyl-α-d-arabinofuranoside (9) were synthesized to mimic the terminal saccharides found on the AG. The acyl transfer reaction between acyl donor 1 and acceptors 2, 3, and 9 in the presence of ag85C from Mycobacterium tuberculosis (M. tuberculosis) resulted in the formation of esters, methyl 2, 5-di-O-octanoyl-α-d-arabinofuranoside (10), methyl 5-O-octanoyl-β-d-arabinofuranoside (11), and methyl 2-O-(5-O-octanoyl-β-d-arabinofuranosyl)-5-O-octanoyl-α-d-arabinofuranoside (12) in 2 h, 2 h and 8 h, respectively. The initial velocities of the reactions were determined with a newly developed assay for acyltransferases. As expected, the regioselectivity corresponds to mycolylation patterns found at the terminus of the AG in M. tuberculosis. The study shows that d-arabinose-based derivatives are capable of acting as substrates for ag85C-mediated acyl-transfer and the acyl glycoside 1 can be used in lieu of TMM extracted from bacteria to study ag85-mediated acyl-transfer and inhibition leading to the better understanding of the ag85 protein class. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Clones of Chinese hamster cells cultivated in vitro not permanently resistant to azaguanine

The frequency of clones not permanently resistant to azaguanine (AG) was measured in Chinese hamster ovary cells (CHO) grown in vitro by plating them in 7.5 μg/ml AG and isolating a number of clones in the course of 5 experiments. Such isolated clones were propagated to a point at which their resistance to both AG and the reverse selective medium, HAT, could be determined. Out of a total of 13 clones isolated, 4 of these could not be distinguished from the parent CHO line, either on the basis of their growth in a gradient of AG concentrations or the reverse selective HAT medium or on the basis of their mutation frequency to resistance to 30 μg/ml AG. All four of the apparent phenocopies were isolated from plates in which although lower numbers of cells were seeded, a higher frequency of clones able to grow in AG was yielded. This suggests that the higher “mutation” frequencies obtained at lower cell densities are due to the appearance of phenocopies which occur only under these conditions. It is concluded that under low plating density conditions, the lower levels of AG (7.5 μg/ml) are not satisfactory for mutagenesis and mutation rate studies.     

Growth inhibition of the cereal root pathogens Rhizoctonia solani AG8, R. oryzae and Gaeumannomyces graminis var. tritici by a recombinant 42-kDa endochitinase from Trichoderma harzianum

The objective of this study was to determine the effectiveness of a 42-kDa endochitinase coded by the ThEn42 gene from Trichoderma harzianum as a potential source of transgenic resistance to Rhizoctonia root rot of barley caused by Rhizoctonia solani AG8 and/or R. oryzae. The gene cThEn42 was codon optimized (GC content increased from 53.3 to 65.1%) and then synthesized to produce the modified cThEn42GC in Pichia pastoris for in vitro tests. Two expression vectors were constructed: one with the fungal signal peptide and the fungal activation peptide [FSP-FAP-cThEn(GC)] and the other with barley chitinase 26 signal peptide followed by the fungal signal and activation peptides [SP(HVChi26)-FSP-FAP-cThEn(GC)]. N-terminal sequencing showed that, of two proteins secreted into liquid medium, FSP was cleaved off faithfully in one protein and both FSP and FAP were cleaved from the other protein. Purified endochitinase provided strong in vitro inhibition of both R. solani AG8 and R. oryzae. The enzyme had an intermediate inhibitory activity against Gaeumannomyces graminis var. tritici, and no inhibitory activity against Fusarium graminearum, F. pseudograminearum, and F. culmorum.     

Sustained EKR inhibition by EGFR targeting therapies is a predictive factor for synergistic cytotoxicity with PDT as neoadjuvant therapy

Background

Tyrosin kinase inhibitors (TKIs) and monoclonal antibodies aimed to target epidermal growth factor receptor (EGFR) have shown limited effect as monotherapies and drug resistance is a major limitation for therapeutic success. Adjuvant therapies to EGFR targeting therapeutics are therefore of high clinical relevance.

Methods

Three EGFR targeting drugs, Cetuximab, Erlotinib and Tyrphostin AG1478 were used in combination with photodynamic therapy (PDT) in two EGFR positive cell lines, A-431 epidermoid skin carcinoma and WiDr colorectal adenocarcinoma cells. The amphiphilic meso-tetraphenylporphine with 2 sulphonate groups on adjacent phenyl rings (TPPS_2a) was utilized as a photosensitizer for PDT. The cytotoxic outcome of the combined treatments was evaluated by cell counting and MTT. Cellular signalling was explored by Western blotting.

Results

PDT as neoadjuvant to Tyrphostin in A-431 cells as well as to Tyrphostin or Erlotinib in WiDr cells revealed synergistic cytotoxicity. In contrast, Erlotinib or Cetuximab combined with neoadjuvant PDT induced an antagonistic effect on cell survival of A-431 cells. Neoadjuvant PDT and EGFR targeting therapies induced a synergistic inhibition of ERK as well as synergistic cytotoxicity only when the EGFR targeting monotherapies caused a prolonged ERK inhibition. There were no correlation between EGFR inhibition by the EGFR targeting monotherapies or the combined therapies and the cytotoxic outcome combination-therapies.

Conclusions

The results suggest that sustained ERK inhibition by EGFR targeting monotherapies is a predictive factor for synergistic cytotoxicity when combined with neoadjuvant PDT.

General significance

The present study provides a rationale for selecting anticancer drugs which may benefit from PDT as adjuvant therapy.