Discovery of novel fragments inhibiting O-acetylserine sulphhydrylase by combining scaffold hopping and ligand-based drug design.

Several bacteria rely on the reductive sulphur assimilation pathway, absent in mammals, to synthesise cysteine. Reduction of virulence and decrease in antibiotic resistance have already been associated with mutations on the genes that codify cysteine biosynthetic enzymes. Therefore, inhibition of cysteine biosynthesis has emerged as a promising strategy to find new potential agents for the treatment of bacterial infection. Following our previous efforts to explore OASS inhibition and to expand and diversify our library, a scaffold hopping approach was carried out, with the aim of identifying a novel fragment for further development. This novel chemical tool, endowed with favourable pharmacological characteristics, was successfully developed, and a preliminary Structure-Activity Relationship investigation was carried out

Discovery of Substituted (2-Aminooxazol-4-yl)Isoxazole-3-carboxylic Acids as Inhibitors of Bacterial Serine Acetyltransferase in the Quest for Novel Potential Antibacterial Adjuvants

Many bacteria and actinomycetales use L-cysteine biosynthesis to increase their tolerance to antibacterial treatment and establish a long-lasting infection. In turn, this might lead to the onset of antimicrobial resistance that currently represents one of the most menacing threats to public health worldwide. The biosynthetic machinery required to synthesise L-cysteine is absent in mammals; therefore, its exploitation as a drug target is particularly promising. In this article, we report a series of inhibitors of Salmonella thyphimurium serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of L-cysteine biosynthesis. The development of such inhibitors started with the virtual screening of an in-house library of compounds that led to the selection of seven structurally unrelated hit derivatives. A set of molecules structurally related to hit compound 5, coming either from the original library or from medicinal chemistry efforts, were tested to determine a preliminary structure–activity relationship and, especially, to improve the inhibitory potency of the derivatives, that was indeed ameliorated by several folds compared to hit compound 5 Despite these progresses, at this stage, the most promising compound failed to interfere with bacterial growth when tested on a Gram-negative model organism, anticipating the need for further research efforts

Discovery of Substituted (2-Aminooxazol-4-yl)Isoxazole-3-carboxylic Acids as Inhibitors of Bacterial Serine Acetyltransferase in the Quest for Novel Potential Antibacterial Adjuvants

Many bacteria and actinomycetales use L-cysteine biosynthesis to increase their tolerance to antibacterial treatment and establish a long-lasting infection. In turn, this might lead to the onset of antimicrobial resistance that currently represents one of the most menacing threats to public health worldwide. The biosynthetic machinery required to synthesise L-cysteine is absent in mammals; therefore, its exploitation as a drug target is particularly promising. In this article, we report a series of inhibitors of Salmonella thyphimurium serine acetyltransferase (SAT), the enzyme that catalyzes the rate-limiting step of L-cysteine biosynthesis. The development of such inhibitors started with the virtual screening of an in-house library of compounds that led to the selection of seven structurally unrelated hit derivatives. A set of molecules structurally related to hit compound 5, coming either from the original library or from medicinal chemistry efforts, were tested to determine a preliminary structure–activity relationship and, especially, to improve the inhibitory potency of the derivatives, that was indeed ameliorated by several folds compared to hit compound 5 Despite these progresses, at this stage, the most promising compound failed to interfere with bacterial growth when tested on a Gram-negative model organism, anticipating the need for further research efforts

Prikazi i recenzije knjiga i časopisa

Prikazi i recenzije knjiga i časopis

An original phylogenetic approach identified mitochondrial haplogroup T1a1 as inversely associated with breast cancer risk in BRCA2 mutation carriers

Introduction: Individuals carrying pathogenic mutations in the BRCA1 and BRCA2 genes have a high lifetime risk of breast cancer. BRCA1 and BRCA2 are involved in DNA double-strand break repair, DNA alterations that can be caused by exposure to reactive oxygen species, a main source of which are mitochondria. Mitochondrial genome variations affect electron transport chain efficiency and reactive oxygen species production. Individuals with different mitochondrial haplogroups differ in their metabolism and sensitivity to oxidative stress. Variability in mitochondrial genetic background can alter reactive oxygen species production, leading to cancer risk. In the present study, we tested the hypothesis that mitochondrial haplogroups modify breast cancer risk in BRCA1/2 mutation carriers. Methods: We genotyped 22,214 (11,421 affected, 10,793 unaffected) mutation carriers belonging to the Consortium of Investigators of Modifiers of BRCA1/2 for 129 mitochondrial polymorphisms using the iCOGS array. Haplogroup inference and association detection were performed using a phylogenetic approach. ALTree was applied to explore the reference mitochondrial evolutionary tree and detect subclades enriched in affected or unaffected individuals. Results: We discovered that subclade T1a1 was depleted in affected BRCA2 mutation carriers compared with the rest of clade T (hazard ratio (HR) = 0.55; 95% confidence interval (CI), 0.34 to 0.88; P = 0.01). Compared with the most frequent haplogroup in the general population (that is, H and T clades), the T1a1 haplogroup has a HR of 0.62 (95% CI, 0.40 to 0.95; P = 0.03). We also identified three potential susceptibility loci, including G13708A/rs28359178, which has demonstrated an inverse association with familial breast cancer risk. Conclusions: This study illustrates how original approaches such as the phylogeny-based method we used can empower classical molecular epidemiological studies aimed at identifying association or risk modification effects.Peer reviewe

Inhibition of cysteine biosynthesis for the development of enhancers of antibiotic therapy

Emerging antibiotic resistance represents a major hazard for public health. The necessity for development of new antibiotics especially against Gram-negative bacteria is high, although many issues must be faced, such as bacterial intrinsic protection by a complex outer membrane, deficit of knowledge regarding its permeability, low number of identified targets and lack of molecule diversity in libraries used for screening. Unsuccessful outcomes of projects focused on identification of new antibiotics led several big pharmaceutical companies to completely abolish their antimicrobial research. A promising strategy to fight bacterial resistance is to use enhancers of antibiotic therapy. Enhancers either block the main bacterial resistance mechanism or potentiate the action of a chosen antibiotic. Cysteine is a multifunctional amino acid and its auxothrops are not able to grow on minimal medium. It is the organic source of sulfur, which is donated for biosynthesis of sulfur containing molecules, while cysteine itself stabilizes protein tertiary structure by formation of bisulfide bonds and maintains intracellular redox status. It has been shown that deletion mutants of cysteine biosynthetic pathway have attenuated virulence, elevated levels of intracellular oxidative stress and they have been linked to decreased antibiotic resistance. Oxidative stress is one of the common mechanisms by which antibiotics affect bacteria, and therefore inhibition of bacterial enzymes involved in biosynthesis of cysteine, that are absent in mammals, presents promising strategy for the development of enhancers of antibiotic therapy. The project was focused on the inhibition of the last two enzymes in this pathway, serine acetyltransferase (SAT) and O-acetylserine sulfhydrilase (OASS) that, in enteric bacteria, exists in two isoforms, A and B. The chosen proteins were from Salmonella enterica serovar Typhimurium, which presents a major health risk all over the world and has been listed as a high priority pathogen by World Health Organization for the development of novel inhibiting compounds. In the course of the project, we optimized the conditions for expression and purification of target enzymes as recombinant proteins in E. coli. Expression constructs for OASS were prepared during the secondments at the University of Cambridge (supervisor Prof. Martin Welch). The proteins were later used for in vitro testing of novel reversible and irreversible inhibitors. For OASS, potent reversible inhibitors were identified previously. A cocrystal between OASS-A and the most potent inhibitor UPAR-415 was prepared that allowed us to confirm binding interactions and understand the molecular basis of enzyme inhibition. Since existing inhibitors presented issues in the permeability through the Gram-negative membrane, several derivatives were prepared in Prof. Costantino’s group (University of Parma) in order to improve their drug-likeness and penetration in bacteria. We determined their binding affinity and mechanism of action via enzyme assays and fluorimetric titrations. We identified novel potent inhibitors and the most promising ones were later selected for microbiological testing. In a collaboration with Prof. Jirgensons’ group (Latvian Institute of Organic Synthesis), we investigated covalent modification of OASS by mechanism-based inactivators. Fluoroalanine derivatives were assayed in order to determine their inactivation potency, mechanism of action and structure-activity relationship. Inhibitors of SAT were tested in our laboratory for the first time. For this purpose, we optimized and validated an indirect activity assay that can be used for inhibitors testing on small and large scale. We screened novel SAT inhibitors, selected by Prof. Costantino’s group (University of Parma) based on the in silico screening of an in house compound library and high-throughput screening of ChemDiv libraries, evaluated their potency and determined their mechanism of action. With this approach we identified the most potent SAT inhibitors reported so far. Promising protein inhibitors were later tested in microbiological assays to evaluate their effect on bacterial viability. This part of the project was partially carried out during the secondments in Aptuit Verona (supervisor Dr. Antonio Felici). We found out that they have issues in the permeability through the bacterial membrane, although in the presence of the permeability enhancer they were able to interfere with bacterial growth. To investigate their mechanism of action when inside bacteria, we developed a method for the measurement of the bacteria intracellular reduced thiols as a marker of cysteine availability

A review on immunomodulatory effects of BPA analogues

Bisphenol A (BPA) is a known endocrine disruptor found in many consumer products that humans come into contact with on a daily basis. Due to increasing concerns about the safety of BPA and the introduction of new legislation restricting its use, industry has responded by adopting new, less studied BPA analogues that have similar polymer-forming properties. Some BPA analogues have already been shown to exhibit effects similar to BPA, for example, contributing to endocrine disruption through agonistic or antagonistic behaviour at various nuclear receptors such as estrogen (ER), androgen (AR), glucocorticoid (GR), aryl hydrocarbon (AhR), and pregnane X receptor (PXR). Since the European Food Safety Authority (EFSA) issued a draft re-evaluation of BPA and drastically reduced the temporary tolerable daily intake (t-TDI) of BPA from 4 mg/kg body weight/day to 0.2 ng/kg body weight/day due to increasing concern about the toxic properties of BPA, including its potential to disrupt immune system processes, we conducted a comprehensive review of the immunomodulatory activity of environmentally abundant BPA analogues. The results of the review suggest that BPA analogues may affect both the innate and acquired immune systems and can contribute to various immune-mediated conditions such as hypersensitivity reactions, allergies, and disruption of the human microbiome

TBP, PPIA, YWHAZ and EF1A1 are the most stably expressed genes during osteogenic differentiation

RT-qPCR is the gold standard and the most commonly used method for measuring gene expression. Selection of appropriate reference gene(s) for normalization is a crucial part of RT-qPCR experimental design, which allows accurate quantification and reliability of the results. Because there is no universal reference gene and even commonly used housekeeping genes’ expression can vary under certain conditions, careful selection of an appropriate internal control must be performed for each cell type or tissue and experimental design. The aim of this study was to identify the most stable reference genes during osteogenic differentiation of the human osteosarcoma cell lines MG-63, HOS, and SaOS-2 using the geNorm, NormFinder, and BestKeeper statistical algorithms. Our results show that TBP, PPIA, YWHAZ, and EF1A1 are the most stably expressed genes, while ACTB, and 18S rRNA expressions are most variable. These data provide a basis for future RT-qPCR normalizations when studying gene expression during osteogenic differentiation, for example, in studies of osteoporosis and other bone diseases