m6A modification inhibits miRNAs' intracellular function, favoring their extracellular export for intercellular communication.

Epitranscriptomics represents a further layer of gene expression regulation. Specifically, N6-methyladenosine (m6A) regulates RNA maturation, stability, degradation, and translation. Regarding microRNAs (miRNAs), while it has been reported that m6A impacts their biogenesis, the functional effects on mature miRNAs remain unclear. Here, we show that m6A modification on specific miRNAs weakens their coupling to AGO2, impairs their function on target mRNAs, determines their delivery into extracellular vesicles (EVs), and provides functional information to receiving cells. Mechanistically, the intracellular functional impairment is caused by m6A-mediated inhibition of AGO2/miRNA interaction, the EV loading is favored by m6A-mediated recognition by the RNA-binding protein (RBP) hnRNPA2B1, and the EV-miRNA function in the receiving cell requires their FTO-mediated demethylation. Consequently, cells express specific miRNAs that do not impact endogenous transcripts but provide regulatory information for cell-to-cell communication. This highlights that a further level of complexity should be considered when relating cellular dynamics to specific miRNAs.

(Heteroarylmethyl)benzoic Acids as a New Class of Bacterial Cystathionine γ-Lyase Inhibitors: Synthesis, Biological Evaluation, and Molecular Modeling.

Antibiotic resistance is one of the most serious global health threats. Therefore, there is a need to develop antimicrobial agents with new mechanisms of action. Targeting of bacterial cystathionine γ-lyase (bCSE), an enzyme essential for bacterial survival, is a promising approach to overcome antibiotic resistance. Here, we described a series of (heteroarylmethyl)benzoic acid derivatives and evaluated their ability to inhibit bCSE or its human ortholog hCSE using known bCSE inhibitor NL2 as a lead compound. Derivatives bearing the 6-bromoindole group proved to be the most active, with IC50 values in the midmicromolar range, and highly selective for bCSE over hCSE. Furthermore, none of these compounds showed significant toxicity to HEK293T cells. The obtained data were rationalized by ligand-based and structure-based molecular modeling analyses. The most active compounds were also found to be an effective adjunct to several widely used antibacterial agents against clinically relevant antibiotic-resistant strains of such bacteria as Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The most potent compounds, 3h and 3i, also showed a promising in vitro absorption, distribution, metabolism, and excretion (ADME) profile. Finally, compound 3i manifested potentiating activity in pneumonia, sepsis, and infected-wound in vivo models.

Detergents with Scalable Properties Identify Noncanonical Lipopolysaccharide Binding to Bacterial Inner Membrane Proteins.

Lipopolysaccharide (LPS) is vital for maintaining the outer membrane barrier in Gram-negative bacteria. LPS is also frequently obtained in complex with the inner membrane proteins after detergent purification. The question of whether or not LPS binding to inner membrane proteins not involved in outer membrane biogenesis reflects native lipid environments remains unclear. Here, we leverage the control of the hydrophilic-lipophilic balance and packing parameter concepts to chemically tune detergents that can be used to qualitatively differentiate the degree to which proteins copurify with phospholipids (PLs) and/or LPS. Given the scalable properties of these detergents, we demonstrate a detergent fine-tuning that enables the facile investigation of intact proteins and their complexes with lipids by native mass spectrometry (nMS). We conclude that LPS, a lipid that is believed to be important for outer membranes, can also affect the activity of membrane proteins that are currently not assigned to be involved in outer membrane biogenesis. Our results deliver a scalable detergent chemistry for a streamlined biophysical characterization of protein-lipid interactions, provide a rationale for the high affinity of LPS-protein binding, and identify noncanonical associations between LPS and inner membrane proteins with relevance for membrane biology and antibiotic research.

A host-directed oxadiazole compound potentiates antituberculosis treatment via zinc poisoning in human macrophages and in a mouse model of infection.

Antituberculosis drugs, mostly developed over 60 years ago, combined with a poorly effective vaccine, have failed to eradicate tuberculosis. More worryingly, multiresistant strains of Mycobacterium tuberculosis (MTB) are constantly emerging. Innovative strategies are thus urgently needed to improve tuberculosis treatment. Recently, host-directed therapy has emerged as a promising strategy to be used in adjunct with existing or future antibiotics, by improving innate immunity or limiting immunopathology. Here, using high-content imaging, we identified novel 1,2,4-oxadiazole-based compounds, which allow human macrophages to control MTB replication. Genome-wide gene expression analysis revealed that these molecules induced zinc remobilization inside cells, resulting in bacterial zinc intoxication. More importantly, we also demonstrated that, upon treatment with these novel compounds, MTB became even more sensitive to antituberculosis drugs, in vitro and in vivo, in a mouse model of tuberculosis. Manipulation of heavy metal homeostasis holds thus great promise to be exploited to develop host-directed therapeutic interventions.

Uracil- and Pyridine-Containing HDAC Inhibitors Displayed Cytotoxicity in Colorectal and Glioblastoma Cancer Stem Cells.

Cancer stem cells (CSCs) are a niche of highly tumorigenic cells featuring self-renewal, activation of pluripotency genes, multidrug resistance, and ability to cause cancer relapse. Seven HDACi (1-7), showing either hydroxamate or 2'-aminoanilide function, were tested in colorectal cancer (CRC) and glioblastoma multiforme (GBM) CSCs to determine their effects on cell proliferation, H3 acetylation levels and in-cell HDAC activity. Two uracil-based hydroxamates, 5 and 6, which differ in substitution at C5 and C6 positions of the pyrimidine ring, exhibited the greatest cytotoxicity in GBM (5) and CRC (6) CSCs, followed by the pyridine-hydroxamate 2, with 2- to 6-fold higher potency than the positive control SAHA. Finally, increased H3 acetylation as well as HDAC inhibition directly in cells by selected 2'-aminoanilide 4 and hydroxamate 5 confirmed target engagement. Further investigation will be conducted into the broad-spectrum anticancer properties of the most potent derivatives and their effects in combination with approved, conventional anticancer drugs.

Specific Inhibitors of Mitochondrial Deacylase Sirtuin 4 Endowed with Cellular Activity.

Sirtuins are NAD+-dependent protein lysine deacylases implicated in aging-related diseases. Mammalian Sirtuin 4 (Sirt4) is located in mitochondria and a potential therapeutic target for cancer and metabolic diseases, but no potent and selective Sirt4 inhibitors have been reported. Here, we describe the identification of potent Sirt4-specific small-molecule inhibitors. Testing hits from a target-based virtual screen revealed 12 active compounds. A focused screen based on two top compounds, followed by structure-assisted design of derivatives, yielded four first-in-class potent Sirt4 inhibitors. Kinetic analyses indicate compound competition with the acyl peptide substrate, consistent with the docking models and implicating Sirt4's unique acyl binding site. The compounds indeed show preference for Sirt4 over other isoforms, with one of them (69) being highly isoform selective, and they are active in cells. Our results provide first lead compounds and mechanistic insights for optimization toward Sirt4-specific inhibitors useful as experimental tools and potential therapeutics.

Management of collagenous gastritis in children: Case series and literature review.

Collagenous gastritis (CG) is a rare histopathological finding on gastric biopsies in children. It is associated with abdominal pain and iron deficiency anemia, usually not respondent to oral iron supplements. The aim of this study was to describe our experience in the management of pediatric patients with CG. Moreover, we propose to review the literature on this topic. We retrospectively reviewed all pediatric patients diagnosed with CG at our centre from January 2014 to January 2019. Three pediatric patients (2 F, mean age 12.3) were diagnosed with CG during the study period. Two presented with moderate and one with severe anemia. Symptoms were abdominal pain, asthenia and headache in two and asthenia and abdominal pain in one. All underwent upper and lower gastrointestinal endoscopy. All were firstly started with oral iron supplements with no benefit, principally due to poor compliance secondary to the worsening of the epigastric pain and proton pump inhibitor resistance. Therefore, they underwent ferric carboxymaltose (FCM) infusion with good clinical and laboratory response. Patients received a mean of two infusions/year, with stable hemoglobin levels and no adverse outcomes. Our review failed to identify a consistent response to specific treatments. Considering the apparent benign nature of the disease, symptomatic and supportive treatments are advisable. Iron deficiency anemia is largely present and therapy with oral iron supplements is not always successful. In our study, FCM infusion was effective in increasing the key blood indices in patients who poorly tolerated oral supplements.

Molecular insights into RmcA-mediated c-di-GMP consumption: Linking redox potential to biofilm morphogenesis in Pseudomonas aeruginosa.

The ability of many bacteria to form biofilms contributes to their resilience and makes infections more difficult to treat. Biofilm growth leads to the formation of internal oxygen gradients, creating hypoxic subzones where cellular reducing power accumulates, and metabolic activities can be limited. The pathogen Pseudomonas aeruginosa counteracts the redox imbalance in the hypoxic biofilm subzones by producing redox-active electron shuttles (phenazines) and by secreting extracellular matrix, leading to an increased surface area-to-volume ratio, which favors gas exchange. Matrix production is regulated by the second messenger bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) in response to different environmental cues. RmcA (Redox modulator of c-di-GMP) from P. aeruginosa is a multidomain phosphodiesterase (PDE) that modulates c-di-GMP levels in response to phenazine availability. RmcA can also sense the fermentable carbon source arginine via a periplasmic domain, which is linked via a transmembrane domain to four cytoplasmic Per-Arnt-Sim (PAS) domains followed by a diguanylate cyclase (DGC) and a PDE domain. The biochemical characterization of the cytoplasmic portion of RmcA reported in this work shows that the PAS domain adjacent to the catalytic domain tunes RmcA PDE activity in a redox-dependent manner, by differentially controlling protein conformation in response to FAD or FADH2. This redox-dependent mechanism likely links the redox state of phenazines (via FAD/FADH2 ratio) to matrix production as indicated by a hyperwrinkling phenotype in a macrocolony biofilm assay. This study provides insights into the role of RmcA in transducing cellular redox information into a structural response of the biofilm at the population level. Conditions of resource (i.e. oxygen and nutrient) limitation arise during chronic infection, affecting the cellular redox state and promoting antibiotic tolerance. An understanding of the molecular linkages between condition sensing and biofilm structure is therefore of crucial importance from both biological and engineering standpoints.

The role of structural biology in the design of sirtuin activators.

Sirtuins are NAD+-dependent protein lysine deacylases and mono-ADP-ribosylases whose activity regulates different pathways, including DNA damage repair, cell survival and metabolism, reactive oxygen species (ROS) detoxification, inflammation, cardiac function, and neuronal signaling. Considering the beneficial effects of specific sirtuin isoforms on health and lifespan, the past two decades have seen a mounting interest in the development of sirtuin activators. The availability of enzyme-activator co-crystal structures has proven significant throughout the years for elucidating the mechanisms of action of activators and designing more potent and selective molecules. In this review, we highlight the most interesting examples of sirtuin activators and provide comprehensive coverage of the role that structural biology played in their discovery and characterization.

NTRK gene aberrations in triple-negative breast cancer: detection challenges using IHC, FISH, RT-PCR, and NGS.

Triple-negative breast cancer (TNBC) is usually an aggressive disease with a poor prognosis and limited treatment options. The neurotrophic tyrosine receptor kinase (NTRK) gene fusions are cancer type-agnostic emerging biomarkers approved by the Food and Drug Administration (FDA), USA, for the selection of patients for targeted therapy. The main aim of our study was to investigate the frequency of NTRK aberrations, i.e. fusions, gene copy number gain, and amplification, in a series of TNBC using different methods. A total of 83 TNBCs were analyzed using pan-TRK immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), real-time polymerase chain reaction (RT-PCR), and RNA-based next-generation sequencing (NGS). Of 83 cases, 16 showed pan-TRK positivity although no cases had NTRK-fusions. Indeed, FISH showed four cases carrying an atypical NTRK1 pattern consisting of one fusion signal and one/more single green signals, but all cases were negative for fusion by NGS and RT-PCR testing. In addition, FISH analysis showed six cases with NTRK1 amplification, one case with NTRK2 copy number gain, and five cases with NTRK3 copy number gain, all negative for pan-TRK IHC. Our data demonstrate that IHC has a high false-positive rate for the detection of fusions and molecular testing is mandatory; there is no need to perform additional molecular tests in cases negativity for NTRK by IHC. In conclusion, the NTRK genes are not involved in fusions in TNBC, but both copy number gain and amplification are frequent events, suggesting a possible predictive role for other NTRK aberrations.

First-in-Class Selective Inhibitors of the Lysine Acetyltransferase KAT8.

KAT8 is a lysine acetyltransferase primarily catalyzing the acetylation of Lys16 of histone H4 (H4K16). KAT8 dysregulation is linked to the development and metastatization of many cancer types, including non-small cell lung cancer (NSCLC) and acute myeloid leukemia (AML). Few KAT8 inhibitors have been reported so far, none of which displaying selective activity. Based on the KAT3B/KDAC inhibitor C646, we developed a series of N-phenyl-5-pyrazolone derivatives and identified compounds 19 and 34 as low-micromolar KAT8 inhibitors selective over a panel of KATs and KDACs. Western blot, immunofluorescence, and CETSA experiments demonstrated that both inhibitors selectively target KAT8 in cells. Moreover, 19 and 34 exhibited mid-micromolar antiproliferative activity in different cancer cell lines, including NSCLC and AML, without impacting the viability of nontransformed cells. Overall, these compounds are valuable tools for elucidating KAT8 biology, and their simple structures make them promising candidates for future optimization studies.

Native mass spectrometry-directed drug discovery: Recent advances in investigating protein function and modulation.

Native mass spectrometry (nMS) is a biophysical method for studying protein complexes and can provide insights into subunit stoichiometry and composition, protein-ligand, and protein-protein interactions (PPIs). These analyses are made possible by preserving non-covalent interactions in the gas phase, thereby allowing the analysis of proteins in their native state. Consequently, nMS has been increasingly applied in early drug discovery campaigns for the characterization of protein-drug interactions and the evaluation of PPI modulators. Here, we discuss recent developments in nMS-directed drug discovery and provide a timely perspective on the possible applications of this technology in drug discovery.

Antihistamines, phenothiazine-based antipsychotics, and tricyclic antidepressants potently activate pharmacologically relevant human carbonic anhydrase isoforms II and VII.

Carbonic anhydrases (CAs) are important regulators of pH homeostasis and participate in many physiological and pathological processes. CA activators (CAAs) are becoming increasingly important in the biomedical field since enhancing CA activity may have beneficial effects at neurological level. Here, we investigate selected antihistamines, phenothiazine-based antipsychotics, and tricyclic antidepressants (TCAs) as potential activators of human CAs I, II, IV, and VII. Our findings indicate that these compounds are more effective at activating hCA II and VII compared to hCA I and IV. Overall, hCA VII was the most efficiently activated isoform, particularly by phenothiazines and TCAs. This is especially relevant since hCA VII is the most abundant isoform in the central nervous system (CNS) and is implicated in neuronal signalling and bicarbonate balance regulation. This study offers additional insights into the pharmacological profiles of clinically employed drugs and sets the ground for the development of novel optimised CAAs.

Emerging Roles of SIRT5 in Metabolism, Cancer, and SARS-CoV-2 Infection.

Sirtuin 5 (SIRT5) is a predominantly mitochondrial enzyme catalyzing the removal of glutaryl, succinyl, malonyl, and acetyl groups from lysine residues through a NAD+-dependent deacylase mechanism. SIRT5 is an important regulator of cellular homeostasis and modulates the activity of proteins involved in different metabolic pathways such as glycolysis, tricarboxylic acid (TCA) cycle, fatty acid oxidation, electron transport chain, generation of ketone bodies, nitrogenous waste management, and reactive oxygen species (ROS) detoxification. SIRT5 controls a wide range of aspects of myocardial energy metabolism and plays critical roles in heart physiology and stress responses. Moreover, SIRT5 has a protective function in the context of neurodegenerative diseases, while it acts as a context-dependent tumor promoter or suppressor. In addition, current research has demonstrated that SIRT5 is implicated in the SARS-CoV-2 infection, although opposing conclusions have been drawn in different studies. Here, we review the current knowledge on SIRT5 molecular actions under both healthy and diseased settings, as well as its functional effects on metabolic targets. Finally, we revise the potential of SIRT5 as a therapeutic target and provide an overview of the currently reported SIRT5 modulators, which include both activators and inhibitors.

Rationalizing the Optimization of Detergents for Membrane Protein Purification.

Membrane protein purification by means of detergents is key to isolating membrane-bound therapeutic targets. The role of the detergent structure in this process, however, is not well understood. Detergents are optimized empirically, leading to failed preparations, and thereby raising costs. Here we evaluate the utility of the hydrophilic-lipophilic balance (HLB) concept, which was introduced by Griffin in 1949, for guiding the optimization of the hydrophobic tail in first-generation, dendritic oligoglycerol detergents ([G1] OGDs). Our findings deliver qualitative HLB guidelines for rationalizing the optimization of detergents. Moreover, [G1] OGDs exhibit strongly delipidating properties, regardless of the structure of the hydrophobic tail, which delivers a methodological enabling step for investigating binding strengths of endogenous lipids and their role for membrane protein oligomerization. Our findings will facilitate the analysis of challenging drug targets in the future.

Malignant trichilemmal carcinoma misdiagnosed with parotid gland malignant tumor.

Malignat proliferating trichilemmal tumor (MPTT) are rare tumors usually presenting in photo-exposed areas, especially on the face, scalp, neck,and dorsal part of the hand. Differential diagnosis include squamous cell carcinoma, basal-cell carcinoma, keratoacanthoma and malignant nodular melanoma, so that only incisional biopsy can lead to pre-operative diagnosis. We present case MPTT misdiagnosed with parotid gland malignant tumor that underwent radical surgical excision and adijuvant radiotherapy.

METTL3 from Target Validation to the First Small-Molecule Inhibitors: A Medicinal Chemistry Journey.

RNA methylation is a critical mechanism for regulating the transcription and translation of specific sequences or for eliminating unnecessary sequences during RNA maturation. METTL3, an RNA methyltransferase that catalyzes the transfer of a methyl group to the N6-adenosine of RNA, is one of the key mediators of this process. METTL3 dysregulation may result in the emergence of a variety of diseases ranging from cancer to cardiovascular and neurological disorders beyond contributing to viral infections. Hence, the discovery of METTL3 inhibitors may assist in furthering the understanding of the biological roles of this enzyme, in addition to contributing to the development of novel therapeutics. Through this work, we will examine the existing correlations between METTL3 and diseases. We will also analyze the development, mode of action, pharmacology, and structure-activity relationships of the currently known METTL3 inhibitors. They include both nucleoside and non-nucleoside compounds, with the latter comprising both competitive and allosteric inhibitors.

[18F]FDG PET/CT in a Case of Mycosis Fungoides Showing an Unusual Adverse Reaction to Mogamulizumab: Correlation Between Imaging and Histological Findings.

A 73-year-old female patient, affected by mycosis fungoides (MF), discontinued mogamulizumab, after initial clinical benefit, due to the onset of generalized erythema. Follow-up positron emission computed tomography (PET/CTs) carried out at 3 weeks and 6 months after therapy discontinuation showed, with respect to baseline PET/CT scan, a progressively increasing number of hypermetabolic enlarged lymph nodes suspected for a neoplastic involvement, but with histology indicative of an inflammatory reaction. After sequential therapy with corticosteroids and methotrexate, a complete remission was registered at 18F-fluorodeoxyglucose ([18F]FDG) PET/CT performed at 12 months after mogamulizumab interruption. The case we describe highlights the usefulness of serial examinations with [18F]FDG PET/CT in an MF patient presenting an unusual adverse reaction to mogamulizumab.

Systematic Design of Adenosine Analogs as Inhibitors of a Clostridioides difficile-Specific DNA Adenine Methyltransferase Required for Normal Sporulation and Persistence.

Antivirulence agents targeting endospore-transmitted Clostridioides difficile infections are urgently needed. C. difficile-specific DNA adenine methyltransferase (CamA) is required for efficient sporulation and affects persistence in the colon. The active site of CamA is conserved and closely resembles those of hundreds of related S-adenosyl-l-methionine (SAM)-dependent methyltransferases, which makes the design of selective inhibitors more challenging. We explored the solvent-exposed edge of the SAM adenosine moiety and systematically designed 42 analogs of adenosine carrying substituents at the C6-amino group (N6) of adenosine. We compare the inhibitory properties and binding affinity of these diverse compounds and present the crystal structures of CamA in complex with 14 of them in the presence of substrate DNA. The most potent of these inhibitors, compound 39 (IC50 ∼ 0.4 µM and KD ∼ 0.2 µM), is selective for CamA against closely related bacterial and mammalian DNA and RNA adenine methyltransferases, protein lysine and arginine methyltransferases, and human adenosine receptors.