Novel [1,3,4]Thiadiazole[3,2-a]pyrimidin-5-ones as Promising Biofilm Dispersal Agents against Relevant Gram-Positive and Gram-Negative Pathogens.

Biofilm-associated infections pose significant challenges in healthcare settings due to their resistance to conventional antimicrobial therapies. In the last decade, the marine environment has been a precious source of bioactive molecules, including numerous derivatives with antibiofilm activity. In this study, we reported the synthesis and the biological evaluation of a new series of twenty-two thiadiazopyrimidinone derivatives obtained by using a hybridization approach combining relevant chemical features of two important classes of marine compounds: nortopsentin analogues and Essramycin derivatives. The synthesized compounds were in vitro tested for their ability to inhibit biofilm formation and to disrupt mature biofilm in various bacterial strains. Among the tested compounds, derivative 8j exhibited remarkable dispersal activity against preformed biofilms of relevant Gram-positive and Gram-negative pathogens, as well as towards the fungus Candida albicans, showing BIC50 values ranging from 17 to 40 µg/mL. Furthermore, compound 8j was in vivo assayed for its toxicity and the anti-infective effect in a Galleria mellonella model. The results revealed a promising combination of anti-infective properties and a favorable toxicity profile for the treatment of severe chronic biofilm-mediated infections.

Exploring the therapeutic potential of focal adhesion kinase inhibition in overcoming chemoresistance in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is among the leading causes of cancer-related deaths worldwide. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase often overexpressed in PDAC. FAK has been linked to cell migration, survival, proliferation, angiogenesis and adhesion. This review first highlights the chemoresistant nature of PDAC. Second, the role of FAK in PDAC cancer progression and resistance is carefully described. Additionally, it discusses recent developments of FAK inhibitors as valuable drugs in the treatment of PDAC, with a focus on diamine-substituted-2,4-pyrimidine-based compounds, which represent the most potent class of FAK inhibitors in clinical trials for the treatment of PDAC disease. To conclude, relevant computational studies performed on FAK inhibitors are reported to highlight the key structural features required for interaction with the protein, with the aim of optimizing this novel targeted therapy.

Nortopsentins as Leads from Marine Organisms for Anticancer and Anti-Inflammatory Agent Development.

The marine environment is an excellent source of molecules that have a wide structural diversity and a variety of biological activities. Many marine natural products (MNPs) have been established as leads for anticancer drug discovery. Most of these compounds are alkaloids, including several chemical subclasses. In this review, we focus on the bis-indolyl alkaloid Nortopsentins and their derivatives with antiproliferative properties. Nortopsentins A-C were found to exhibit in vitro cytotoxicity against the P388 murine leukaemia cell line. Their structural manipulation provided a wide range of derivatives with significant anti-tumour activity against human cell lines derived from different cancer types (bladder, colon, gastric, CNS, liver, lung, breast, melanoma, ovarian, pancreatic, prostate, pleural mesothelioma, renal, sarcoma, and uterus). In vivo assays on animal models also proved that Nortopsentins and related bis-indolyl compounds have potent anti-inflammatory activity. These remarks set the foundation for future investigations into the development of new Nortopsentin derivatives as new anticancer and anti-inflammatory agents.

A Combination of Polymethoxyflavones from Citrus sinensis and Prenylflavonoids from Humulus lupulus Counteracts IL-1ß-Induced Differentiated Caco-2 Cells Dysfunction via a Modulation of NF-κB/Nrf2 Activation.

We here investigated the anti-inflammatory activity of a polymethoxylated flavone-containing fraction (PMFF) from Citrus sinensis and of a prenylflavonoid-containing one (PFF) from Humulus lupulus, either alone or in combination (MIX). To this end, an in vitro model of inflammatory bowel disease (IBD), consisting of differentiated, interleukin (IL)-1ß-stimulated Caco-2 cells, was employed. We demonstrated that non-cytotoxic concentrations of either PMFF or PFF or MIX reduced nitric oxide (NO) production while PFF and MIX, but not PMFF, also inhibited prostaglandin E2 release. Coherently, MIX suppressed both inducible NO synthase and cyclooxygenase-2 over-expression besides NF-κB activation. Moreover, MIX increased nuclear factor erythroid 2-related factor 2 (Nrf2) activation, heme oxygenase-1 expression, restoring GSH and reactive oxygen and nitrogen species (RONs) levels. Remarkably, these effects with MIX were stronger than those produced by PMFF or PFF alone. Noteworthy, nobiletin (NOB) and xanthohumol (XTM), two of the most represented phytochemicals in PMFF and PFF, respectively, synergistically inhibited RONs production. Overall, our results demonstrate that MIX enhances the anti-inflammatory and anti-oxidative effects of the individual fractions in a model of IBD, via a mechanism involving modulation of NF-κB and Nrf2 signalling. Synergistic interactions between NOB and XTM emerge as a relevant aspect underlying this evidence.

1,3,4-Oxadiazole and 1,3,4-Thiadiazole Nortopsentin Derivatives against Pancreatic Ductal Adenocarcinoma: Synthesis, Cytotoxic Activity, and Inhibition of CDK1.

A new series of nortopsentin analogs, in which the central imidazole ring of the natural lead was replaced by a 1,3,4-oxadiazole or 1,3,4-thiadiazole moiety, was efficiently synthesized. The antiproliferative activity of all synthesized derivatives was evaluated against five pancreatic ductal adenocarcinoma (PDAC) cell lines, a primary culture and a gemcitabine-resistant variant. The five more potent compounds elicited EC50 values in the submicromolar-micromolar range, associated with a significant reduction in cell migration. Moreover, flow cytometric analysis after propidium iodide staining revealed an increase in the G2-M and a decrease in G1-phase, indicating cell cycle arrest, while a specific ELISA demonstrated the inhibition of CDK1 activity, a crucial regulator of cell cycle progression and cancer cell proliferation.

Structural Manipulations of Marine Natural Products Inspire a New Library of 3-Amino-1,2,4-Triazine PDK Inhibitors Endowed with Antitumor Activity in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the main aggressive types of cancer, characterized by late prognosis and drug resistance. Among the main factors sustaining PDAC progression, the alteration of cell metabolism has emerged to have a key role in PDAC cell proliferation, invasion, and resistance to standard chemotherapeutic agents. Taking into account all these factors and the urgency in evaluating novel options to treat PDAC, in the present work we reported the synthesis of a new series of indolyl-7-azaindolyl triazine compounds inspired by marine bis-indolyl alkaloids. We first assessed the ability of the new triazine compounds to inhibit the enzymatic activity of pyruvate dehydrogenase kinases (PDKs). The results showed that most of derivatives totally inhibit PDK1 and PDK4. Molecular docking analysis was executed to predict the possible binding mode of these derivatives using ligand-based homology modeling technique. Evaluation of the capability of new triazines to inhibit the cell growth in 2D and 3D KRAS-wild-type (BxPC-3) and KRAS-mutant (PSN-1) PDAC cell line, was carried out. The results showed the capacity of the new derivatives to reduce cell growth with a major selectivity against KRAS-mutant PDAC PSN-1 on both cell models. These data demonstrated that the new triazine derivatives target PDK1 enzymatic activity and exhibit cytotoxic effects on 2D and 3D PDAC cell models, thus encouraging further structure manipulation for analogs development against PDAC.

Recent Developments in the Inhibition of Bacterial Adhesion as Promising Anti-Virulence Strategy.

Infectious diseases caused by antimicrobial-resistant strains have become a serious threat to global health, with a high social and economic impact. Multi-resistant bacteria exhibit various mechanisms at both the cellular and microbial community levels. Among the different strategies proposed to fight antibiotic resistance, we reckon that the inhibition of bacterial adhesion to host surfaces represents one of the most valid approaches, since it hampers bacterial virulence without affecting cell viability. Many different structures and biomolecules involved in the adhesion of Gram-positive and Gram-negative pathogens can be considered valuable targets for the development of promising tools to enrich our arsenal against pathogens.

Discovery of the 3-Amino-1,2,4-triazine-Based Library as Selective PDK1 Inhibitors with Therapeutic Potential in Highly Aggressive Pancreatic Ductal Adenocarcinoma.

Pyruvate dehydrogenase kinases (PDKs) are serine/threonine kinases, that are directly involved in altered cancer cell metabolism, resulting in cancer aggressiveness and resistance. Dichloroacetic acid (DCA) is the first PDK inhibitor that has entered phase II clinical; however, several side effects associated with weak anticancer activity and excessive drug dose (100 mg/kg) have led to its limitation in clinical application. Building upon a molecular hybridization approach, a small library of 3-amino-1,2,4-triazine derivatives has been designed, synthesized, and characterized for their PDK inhibitory activity using in silico, in vitro, and in vivo assays. Biochemical screenings showed that all synthesized compounds are potent and subtype-selective inhibitors of PDK. Accordingly, molecular modeling studies revealed that a lot of ligands can be properly placed inside the ATP-binding site of PDK1. Interestingly, 2D and 3D cell studies revealed their ability to induce cancer cell death at low micromolar doses, being extremely effective against human pancreatic KRAS mutated cancer cells. Cellular mechanistic studies confirm their ability to hamper the PDK/PDH axis, thus leading to metabolic/redox cellular impairment, and to ultimately trigger apoptotic cancer cell death. Remarkably, preliminary in vivo studies performed on a highly aggressive and metastatic Kras-mutant solid tumor model confirm the ability of the most representative compound 5i to target the PDH/PDK axis in vivo and highlighted its equal efficacy and better tolerability profile with respect to those elicited by the reference FDA approved drugs, cisplatin and gemcitabine. Collectively, the data highlights the promising anticancer potential of these novel PDK-targeting derivatives toward obtaining clinical candidates for combatting highly aggressive KRAS-mutant pancreatic ductal adenocarcinomas.

1,2,4-Amino-triazine derivatives as pyruvate dehydrogenase kinase inhibitors: Synthesis and pharmacological evaluation.

Among the different hallmarks of cancer, deregulation of cellular metabolism turned out to be an essential mechanism in promoting cancer resistance and progression. The pyruvate dehydrogenase kinases (PDKs) are well known as key regulators in cells metabolic process and their activity was found to be overexpressed in different metabolic alerted types of cancer, including the high aggressive pancreatic ductal adenocarcinoma (PDAC). To date few PDK inhibitors have been reported, and the different molecules developed are characterized by structural chemical diversity. In an attempt to find novel classes of potential PDK inhibitors, the molecular hybridization approach, which combine two or more active scaffolds in a single structure, was employed. Herein we report the synthesis and the pharmacological evaluation of the novel hybrid molecules, characterized by the fusion of three different pharmacophoric sub-units such as 1,2,4-amino triazines, 7-azaindoles and indoles, in a single structure. The synthesized derivatives demonstrated a promising ability in hampering the enzymatic activity of PDK1 and 4, further confirmed by docking studies. Interestingly, these derivatives retained a strong antiproliferative activity against pancreatic cancer cells either in 2D and 3D models. Mechanistic studies in highly aggressive PDAC cells confirmed their ability to hamper PDK axis and to induce cancer cell death by apoptosis. Moreover, in vivo translational studies in a murine syngeneic solid tumor model confirmed the ability of the most representative compounds to target the PDK system and highlight the ability to reduce the tumor growth without inducing substantial body weight changes in the treated mice.

Development of a new indole derivative dry powder for inhalation for the treatment of biofilm-associated lung infections.

The aim of this work was to produce an inhalable dry powder formulation of a new anti-biofilm compound (SC38). For this purpose, chitosan was used as a polymeric carrier and l-leucine as a dispersibility enhancer. SC38 was entrapped by spray-drying into previously optimized chitosan microparticles. The final formulation was fully characterized in vitro in terms of particle morphology, particle size and distribution, flowability, aerodynamic properties, anti-biofilm activity and effects on lung cell viability. The SC38-loaded chitosan microparticles exhibited favorable aerodynamic properties with emitted and respirable fractions higher than 80 % and 45 % respectively. The optimized formulation successfully inhibited biofilm formation at microparticle concentrations starting from 20 µg/mL for methicillin-sensitive and 100 µg/mL for methicillin-resistant Staphylococcus aureus and showed a relatively safe profile in lung cells after 72 h exposure. Future in vivo tolerability and efficacy studies are needed to unravel the potential of this novel formulation for the treatment of difficult-to-treat biofilm-mediated lung infections.

Corrigendum: Overcome Chemoresistance: Biophysical and Structural Analysis of Synthetic FHIT-Derived Peptides.

[This corrects the article DOI: 10.3389/fmolb.2021.715263.].

Metabolomics-assisted discovery of a new anticancer GLS-1 inhibitor chemotype from a nortopsentin-inspired library: From phenotype screening to target identification.

The enzyme glutaminase-1 (GLS-1) has shown a clear and coherent implication in the progression and exacerbation of different aggressive tumors such as glioblastoma, hepatocarcinoma, pancreas, bone, and triple-negative breast cancer. Few chemotypes are currently available as selective GLS-1 inhibitors, and still, fewer of them are at the clinical stage. In the present paper, starting from a naturally-inspired antitumor compound library, metabolomics has been used to putatively identify the molecular mechanism underlying biological activity. GLS-1 was identified as a potential target. Biochemical analysis confirmed the hypothesis leading to the identification of a new hit compound acting as a GLS-1 selective inhibitor (IC50 = 3.96 ± 1.05 µM), compared to the GLS-2 isoform (IC50 = 12.90 ± 0.87 µM), with remarkable antitumor potency over different aggressive tumor cell lines. Molecular modelling studies revealed new insight into the drug-target interaction providing robust SAR clues for the rational hit-to-lead development. The approach undertaken underlines the wide potential of metabolomics applied to drug discovery, particularly in target identification and hit discovery following phenotype screening.

Interrelationship between miRNA and splicing factors in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers because of diagnosis at late stage and inherent/acquired chemoresistance. Recent advances in genomic profiling and biology of this disease have not yet been translated to a relevant improvement in terms of disease management and patient's survival. However, new possibilities for treatment may emerge from studies on key epigenetic factors. Deregulation of microRNA (miRNA) dependent gene expression and mRNA splicing are epigenetic processes that modulate the protein repertoire at the transcriptional level. These processes affect all aspects of PDAC pathogenesis and have great potential to unravel new therapeutic targets and/or biomarkers. Remarkably, several studies showed that they actually interact with each other in influencing PDAC progression. Some splicing factors directly interact with specific miRNAs and either facilitate or inhibit their expression, such as Rbfox2, which cleaves the well-known oncogenic miRNA miR-21. Conversely, miR-15a-5p and miR-25-3p significantly downregulate the splicing factor hnRNPA1 which acts also as a tumour suppressor gene and is involved in processing of miR-18a, which in turn, is a negative regulator of KRAS expression. Therefore, this review describes the interaction between splicing and miRNA, as well as bioinformatic tools to explore the effect of splicing modulation towards miRNA profiles, in order to exploit this interplay for the development of innovative treatments. Targeting aberrant splicing and deregulated miRNA, alone or in combination, may hopefully provide novel therapeutic approaches to fight the complex biology and the common treatment recalcitrance of PDAC.

Nobiletin and Xanthohumol Sensitize Colorectal Cancer Stem Cells to Standard Chemotherapy.

Colorectal cancer (CRC) mortality is mainly caused by patient refractoriness to common anti-cancer therapies and consequent metastasis formation. Besides, the notorious toxic side effects of chemotherapy are a concurrent obstacle to be tackled. Thus, new treatment approaches are needed to effectively improve patient outcomes. Compelling evidence demonstrated that cancer stem cells (CSCs) are responsible for treatment failure and relapse. New natural treatment approaches showed capabilities to selectively target the CSC subpopulation by rendering them targetable by standard cytotoxic compounds. Herein we show the anti-cancer properties of the polymethoxyflavones and prenylflavonoids extracted from Citrus sinensis and Humulus lupulus, respectively. The natural biofunctional fractions, singularly and in combination, reduced the cell viability of CRC stem cells (CR-CSCs) and synergized with 5-fluorouracil and oxaliplatin (FOX) chemotherapy. These phenomena were accompanied by a reduced S and G2/M phase of the cell cycle and upregulation of cell death-related genes. Notably, both phytoextracts in combination with FOX thwarted stemness features in CR-CSCs as demonstrated by the impaired clonogenic potential and decreased Wnt pathway activation. Extracts lowered the expression of CD44v6 and affected the expansion of metastatic CR-CSCs in patients refractory to chemotherapy. Together, this study highlights the importance of polymethoxyflavones and prenylflavonoids as natural remedies to aid oncological therapies.

Itraconazole inhibits nuclear delivery of extracellular vesicle cargo by disrupting the entry of late endosomes into the nucleoplasmic reticulum.

Extracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pathological conditions, including the induction of pro-metastatic traits, but it is not yet known how and where functional cargoes of EVs are delivered to their targets in host cell compartments. We have described that after endocytosis, EVs reach Rab7+ late endosomes and a fraction of these enter the nucleoplasmic reticulum and transport EV biomaterials to the host cell nucleoplasm. Their entry therein and docking to outer nuclear membrane occur through a tripartite complex formed by the proteins VAP-A, ORP3 and Rab7 (VOR complex). Here, we report that the antifungal compound itraconazole (ICZ), but not its main metabolite hydroxy-ICZ or ketoconazole, disrupts the binding of Rab7 to ORP3-VAP-A complexes, leading to inhibition of EV-mediated pro-metastatic morphological changes including cell migration behaviour of colon cancer cells. With novel, smaller chemical drugs, inhibition of the VOR complex was maintained, although the ICZ moieties responsible for antifungal activity and interference with intracellular cholesterol distribution were removed. Knowing that cancer cells hijack their microenvironment and that EVs derived from them determine the pre-metastatic niche, small-sized inhibitors of nuclear transfer of EV cargo into host cells could find cancer therapeutic applications, particularly in combination with direct targeting of cancer cells.

CHK1 inhibitor sensitizes resistant colorectal cancer stem cells to nortopsentin.

Limited therapeutic options are available for advanced colorectal cancer (CRC). Herein, we report that exposure to a neo-synthetic bis(indolyl)thiazole alkaloid analog, nortopsentin 234 (NORA234), leads to an initial reduction of proliferative and clonogenic potential of CRC sphere cells (CR-CSphCs), followed by an adaptive response selecting the CR-CSphC-resistant compartment. Cells spared by the treatment with NORA234 express high levels of CD44v6, associated with a constitutive activation of Wnt pathway. In CR-CSphC-based organoids, NORA234 causes a genotoxic stress paralleled by G2-M cell cycle arrest and activation of CHK1, driving the DNA damage repair of CR-CSphCs, regardless of the mutational background, microsatellite stability, and consensus molecular subtype. Synergistic combination of NORA234 and CHK1 (rabusertib) targeting is synthetic lethal inducing death of both CD44v6-negative and CD44v6-positive CRC stem cell fractions, aside from Wnt pathway activity. These data could provide a rational basis to develop an effective strategy for the treatment of patients with CRC.

SF3B1 modulators affect key genes in metastasis and drug influx: a new approach to fight pancreatic cancer chemoresistance.

Aim: Because mutations of splicing factor 3B subunit-1 (SF3B1) have been identified in 4% of pancreatic ductal adenocarcinoma (PDAC) patients, we investigated the activity of new potential inhibitors of SF3B1 in combination with gemcitabine, one of the standard drugs, in PDAC cell lines. Methods: One imidazo[2,1-b][1,3,4]thiadiazole derivative (IS1) and three indole derivatives (IS2, IS3 and IS4), selected by virtual screening from an in-house library, were evaluated by the sulforhodamine-B and wound healing assay for their cytotoxic and antimigratory activity in the PDAC cells SUIT-2, Hs766t and Panc05.04, the latter harbouring the SF3B1 mutations. The effects on the splicing pattern of proto-oncogene recepteur d'origine nantais (RON) and the gemcitabine transporter human equilibrative nucleoside transporter-1 (hENT1) were assessed by PCR, while the ability to reduce tumour volume was tested in spheroids of primary PDAC cells. Results: The potential SF3B1 modulators inhibited PDAC cell proliferation and prompted induction of cell death. All compounds showed an interesting anti-migratory ability, associated with splicing RON/ΔRON shift in SUIT-2 cells after 24 h exposure. Moreover, IS1 and IS4 potentiated the sensitivity to gemcitabine in both conventional 2D monolayer and 3D spheroid cultures, and these results might be explained by the statistically significant increase in hENT1 expression (P < 0.05 vs. untreated control cells), potentially reversing PDAC chemoresistance. Conclusion: These results support further studies on new SF3B1 inhibitors and the role of RON/hENT1 modulation to develop effective drug combinations against PDAC.