A Series of COX-2 Inhibitors Endowed with NO-Releasing Properties: Synthesis, Biological Evaluation, and Docking Analysis

Herein we report the synthesis, biological evaluation, and docking analysis of a class of cyclooxygenase-2 (COX-2) inhibitors with nitric oxide (NO)-releasing properties. In an earlier study, a number of selective COX-2 inhibitors/NO donors were developed by conjugating a diarylpyrrole scaffold endowed with selective COX-2 inhibitory properties with various nitrooxyalkyl side chains such as esters, -amino esters, amides, -amino amides, ethers, -amino ethers, inverse esters, and amides. These candidates were found to have high invitro potencies (COX-2 inhibition at 10m: 96%), great efficacy in determining NO-vasorelaxing responses, and good antinociceptive activity in an abdominal writhing test. Among the compounds synthesized in the present work, derivative 2b [2-(2-(1-(3-fluorophenyl)-2-methyl-5-(4-sulfamoylphenyl)-1H-pyrrol-3-yl)acetamido)ethyl nitrate] showed particularly outstanding activity, with efficacy similar to that of celecoxib even at very low concentrations

Synthesis, biological evaluation and docking analysis of a new series of methylsulfonyl and sulfamoyl acetamides and ethyl acetates as potent COX-2 inhibitors

We report herein the synthesis, biological evaluation and docking analysis of a new series of methylsulfonyl, sulfamoyl acetamides and ethyl acetates that selectively inhibit cyclooxygenase-2 (COX-2) isoform. Among the newly synthesized compounds, some of them were endowed with a good activity against COX-2 and a good selectivity COX-2/COX-1 in vitro as well as a desirable analgesic activity in vivo, proving that replacement of the ester moiety with an amide group gave access to more stable derivatives, characterized by a good COX-inhibition

An Optimized Workflow for the Discovery of New Antimicrobial Compounds Targeting Bacterial RNA Polymerase Complex Formation

Bacterial resistance represents a major health problem worldwide and there is an urgent need to develop first-in-class compounds directed against new therapeutic targets. We previously developed a drug-discovery platform to identify new antimicrobials able to disrupt the protein-protein interaction between the beta' subunit and the sigma(70) initiation factor of bacterial RNA polymerase, which is essential for transcription. As a follow-up to such work, we have improved the discovery strategy to make it less time-consuming and more cost-effective. This involves three sequential assays, easily scalable to a high-throughput format, and a subsequent in-depth characterization only limited to hits that passed the three tests. This optimized workflow, applied to the screening of 5360 small molecules from three synthetic and natural compound libraries, led to the identification of six compounds interfering with the beta'-sigma(70) interaction, and thus was capable of inhibiting promoter-specific RNA transcription and bacterial growth. Upon supplementation with a permeability adjuvant, the two most potent transcription-inhibiting compounds displayed a strong antibacterial activity against Escherichia coli with minimum inhibitory concentration (MIC) values among the lowest (0.87-1.56 mu M) thus far reported for beta'-sigma PPI inhibitors. The newly identified hit compounds share structural feature similarities with those of a pharmacophore model previously developed from known inhibitors

Consensus based recommendations for diagnosis and medical management of Poland syndrome (sequence)

Background Poland syndrome (OMIM: 173800) is a disorder in which affected individuals are born with missing or underdeveloped muscles on one side of the body, resulting in abnormalities that can affect the chest, breast, shoulder, arm, and hand. The extent and severity of the abnormalities vary among affected individuals. Main body The aim of this work is to provide recommendations for the diagnosis and management of people affected by Poland syndrome based on evidence from literature and experience of health professionals from different medical backgrounds who have followed for several years affected subjects. The literature search was performed in the second half of 2019. Original papers, meta-analyses, reviews, books and guidelines were reviewed and final recommendations were reached by consensus. Conclusion Being Poland syndrome a rare syndrome most recommendations here presented are good clinical practice based on the consensus of the participant experts

Association of MiR-126 with Soluble Mesothelin-Related Peptides, a Marker for Malignant Mesothelioma

BACKGROUND: Improved detection methods for diagnosis of malignant pleural mesothelioma (MPM) are essential for early and reliable detection as well as treatment. Since recent data point to abnormal levels of microRNAs (miRNAs) in tumors, we hypothesized that a profile of deregulated miRNAs may be a marker of MPM and that the levels of specific miRNAs may be used for monitoring its progress. METHODS AND RESULTS: miRNAs isolated from fresh-frozen biopsies of MPM patients were tested for the expression of 88 types of miRNA involved in cancerogenesis. Most of the tested miRNAs were downregulated in the malignant tissues compared with the normal tissues. Of eight significantly downregulated, three miRNAs were assayed in cancerous tissue and adjacent non-cancerous tissue sample pairs collected from 27 formalin-fixed, paraffin-embedded MPM tissues by quantitative RT-PCR. Among the miRNAs tested, only miR-126 significantly remained downregulated in the malignant tissues. Furthermore, the performance of the selected miR-126 as biomarker was evaluated in serum samples of asbestos-exposed subjects and MPM patients and compared with controls. MiR-126 was not affected by asbestos exposure, whereas it was found strongly associated with VEGF serum levels. Levels of miR-126 in serum, and its levels in patients' serum in association with a specific marker of MPM, SMRPs, correlate with subjects at high risk to develop MPM. CONCLUSIONS AND SIGNIFICANCE: We propose miR-126, in association with SMRPs, as a marker for early detection of MPM. The identification of tumor biomarkers used alone or, in particular, in combination could greatly facilitate the surveillance procedure for cohorts of subjects exposed to asbestos

Antibiotic compounds targeting bacterial RNA polymerase holoenzyme assembly and other protein-protein interaction inhibitiors identified with an in vivo-bret drug discovery platform

The research and development of a new drug is a lengthy and costly process, which requires considerable investment by the pharmaceutical industries, with a very low success rate and a constant need for innovative approaches. The so-called “reverse approach” (or target-based drug discovery) is based on the screening of small molecule libraries, to identify "hit compounds" capable of interacting and modulating the biological activity of the target of interest. In recent years, protein-protein interactions (PPI) have emerged as promising new targets, especially considering their key role in most cellular processes, under both physiological and pathological conditions. The aim of this thesis work is the development of an in vivo high-throughput platform based on the BRET (Bioluminescence Resonance Energy Transfer) technology as a tool to monitor PPI and to screen compound libraries for the identification of new potential PPI inhibitors. To this end, I reproduced various interactions of marked biomedical interest in the unicellular eukaryote Saccharomyces cerevisiae and monitored them using the yeast-BRET (yBRET) assay. The set up and validation of yBRET was carried out using the HDM2-p53 interaction, involved in cell-cycle control, as a proof of concept case study, given the availability of effective inhibitors targeting this particular PPI. The same yBRET technology was subsequently applied, and further optimized, for the study of the interaction between the ' subunit of bacterial RNA polymerase (RNAP) and the 70 specificity factor of Escherichia coli, as a new target for the discovery of antibiotics capable of interfering with transcription complex assembly, thus blocking pathogen viability and propagation. The screening of a subset of computationally pre-selected molecules plus a collection of small-molecule libraries of different origin was performed on this interaction, for a total of over 18,000 different compounds. Hit compounds emerging from yBRET were validated with a dedicated ELISA, to confirm the ability of the hits to disrupt the RNAP-70 interaction, and with the use of an in vitro transcription assay to verify their ability to inhibit RNAP activity. Finally, I demonstrated that the validated lead compounds are in fact endowed with antimicrobial activity against both Gram-positive (Bacillus subtilis) and Gram-negative (E. coli) bacteria, displaying growth inhibition starting from 200 μM. This led to the identification of new potential antibiotics, some of which have chemical structures similar to those of already known 70-' interaction inhibitors (a class of indole-containing compounds), while others have completely new scaffolds. I also applied the yBRET screening to other interactions of biomedical interest, such as receptor-ligand interactions involved in human immuno-modulatory pathway. I focused, in particular, on the extracellular domains of 2B4-CD48, whose immuno-inhibitory interaction is involved in T cell exhaustion, and CD40-CD40L, whose immuno-stimulatory interaction is associated to various autoimmune inflammatory pathologies of the central nervous system and in lymphomas. For the latter interaction, I developed a surface exposed version of yeast BRET (named, syBRET), which allows the expression of the interacting partner proteins on the yeast cell wall. This allowed the reconstruction of the pentameric CD40-CD40L complex, formed by the CD40L trimeric ligand associated with two monomers of CD40 receptor, each containing eight disulfide bridges. The syBRET was validated with Suramin, a known inhibitor of this interaction. Overall the yBRET methodology proved to be a fairly robust, versatile and high-throughput tool for PPI-orineted drug discovery

Cervical organoids go viral.

In this issue of Cell Stem Cell, Lõhmussaar et al. (2021) describe a versatile platform for generating long-term patient-derived organoids from hysterectomies and Pap brush cells. They establish malignant and benign cervix tissue organoids from both endo- and ectocervix that have applications ranging from precision medicine to infection modeling

Dry and wet approaches for genome-wide functional annotation of conventional and unconventional transcriptional activators

Transcription factors (TFs) are master gene products that regulate gene expression in response to a variety of stimuli. They interact with DNA in a sequence-specific manner using a variety of DNA-binding domain (DBD) modules. This allows to properly position their second domain, called “effector domain”, to directly or indirectly recruit positively or negatively acting co-regulators including chromatin modifiers, thus modulating preinitiation complex formation as well as transcription elongation. At variance with the DBDs, which are comprised of well-defined and easily recognizable DNA binding motifs, effector domains are usually much less conserved and thus considerably more difficult to predict. Also not so easy to identify are the DNA-binding sites of TFs, especially on a genome-wide basis and in the case of overlapping binding regions. Another emerging issue, with many potential regulatory implications, is that of so-called “moonlighting” transcription factors, i.e., proteins with an annotated function unrelated to transcription and lacking any recognizable DBD or effector domain, that play a role in gene regulation as their second job. Starting from bioinformatic and experimental high-throughput tools for an unbiased, genome-wide identification and functional characterization of TFs (especially transcriptional activators), we describe both established (and usually well affordable) as well as newly developed platforms for DNA-binding site identification. Selected combinations of these search tools, some of which rely on next-generation sequencing approaches, allow delineating the entire repertoire of TFs and unconventional regulators encoded by the any sequenced genome

Moonlighting transcriptional activation function of a fungal sulfur metabolism enzyme

Moonlighting proteins, including metabolic enzymes acting as transcription factors (TF), are present in a variety of organisms but have not been described in higher fungi so far. In a previous genome-wide analysis of the TF repertoire of the plant-symbiotic fungus Tuber melanosporum, we identified various enzymes, including the sulfur-assimilation enzyme phosphoadenosine-phosphosulfate reductase (PAPS-red), as potential transcriptional activators. A functional analysis performed in the yeast Saccharomyces cerevisiae, now demonstrates that a specific variant of this enzyme, PAPS-red A, localizes to the nucleus and is capable of transcriptional activation. TF moonlighting, which is not present in the other enzyme variant (PAPS-red B) encoded by the T. melanosporum genome, relies on a transplantable C-terminal polypeptide containing an alternating hydrophobic/hydrophilic amino acid motif. A similar moonlighting activity was demonstrated for six additional proteins, suggesting that multitasking is a relatively frequent event. PAPS-red A is sulfur-state-responsive and highly expressed, especially in fruitbodies, and likely acts as a recruiter of transcription components involved in S-metabolism gene network activation. PAPS-red B, instead, is expressed at low levels and localizes to a highly methylated and silenced region of the genome, hinting at an evolutionary mechanism based on gene duplication, followed by epigenetic silencing of this non-moonlighting gene variant