The human muscle isoform of lactate dehydrogenase (hLDH5) is one of the key enzymes of the glycolytic process. It is overexpressed in metastatic cancer cells and is linked to the vitality of tumors in hypoxic conditions. With the aim of identifying new hLDH5 inhibitors, a fully automated docking-based virtual screening platform was developed by considering different protein conformations and the consensus docking strategy. In order to verify the reliability of the reported platform, a small database of about 10,000 compounds was filtered by using this method, and the top-ranked compounds were tested for their hLDH5 inhibition activity. Enzymatic assays revealed that, among the ten selected compounds, two proved to efficiently inhibit enzyme activity with IC50 values in the micromolar range. These results demonstrate the validity of the methodologies we followed, encouraging the application of larger virtual screening studies and further refinements of the platform. Furthermore, the two active compounds herein described may be considered as interesting leads for the development of new and more efficient LDH inhibitors.

Therapeutic options for diffuse malignant peritoneal mesothelioma (DMPM) are limited to surgery and locoregional chemotherapy. Despite improvements in survival rates, patients eventually succumb to disease progression. We investigated splicing deregulation both as molecular prognostic factor and potential novel target in DMPM, while we tested modulators of SF3b complex for antitumor activity.

Starting from the analysis of the hypothetical binding mode of our previous furan-based hit (I), we successfully achieved our objective to replace the nitro moiety, leading to the disclosure of a new lead exhibiting a strong activity against MbtI. Our best candidate 1 h displayed a Ki of 8.8 µM and its antimycobacterial activity (MIC99 = 250 µM) is conceivably related to mycobactin biosynthesis inhibition. These results support the hypothesis that 5-phenylfuran-2-carboxylic derivatives are a promising class of MbtI inhibitors.

There is increasing evidence of a constitutive activation of Akt in pancreatic ductal adenocarcinoma (PDAC), associated with poor prognosis and chemoresistance. Therefore, we evaluated the expression of phospho-Akt in PDAC tissues and cells, and investigated molecular mechanisms influencing the therapeutic potential of Akt inhibition in combination with gemcitabine.

Fyn tyrosine kinase inhibitors are considered potential therapeutic agents for a variety of human cancers. Furthermore, the involvement of Fyn kinase in signalling pathways that lead to severe pathologies, such as Alzheimer's and Parkinson's diseases, has also been demonstrated. In this study, starting from 3-(benzo[d][1,3]dioxol-5-ylamino)-6-methyl-1,2,4-triazin-5(2H)-one (VS6), a hit compound that showed a micromolar inhibition of Fyn (IC50 = 4.8 μM), we computationally investigated the binding interactions of the 3-amino-1,2,4-triazin-5(2H)-one scaffold and started a preliminary hit to lead optimisation. This analysis led us to confirm the hypothesised binding mode of VS6 and to identify a new derivative that is about 6-fold more active than VS6 (compound 3, IC50 = 0.76 μM).

In non-small cell lung cancer, sensitizing mutations in epidermal growth factor receptor (EGFR) or cMET amplification serve as good biomarkers for targeted therapies against EGFR or cMET, respectively. Here we aimed to determine how this different genetic background would affect the interaction between the EGFR-inhibitor erlotinib and the cMET-inhibitor crizotinib. To unravel the mechanism of synergy we investigated the effect of the drugs on various parameters, including cell cycle arrest, migration, protein phosphorylation, kinase activity, the expression of drug efflux pumps, intracellular drug concentrations, and live-cell microscopy. We observed additive effects in EBC-1, H1975, and HCC827, and a strong synergism in the HCC827GR5 cell line. This cell line is a clone of the HCC827 cells that harbor an EGFR exon 19 deletion and has been made resistant to the EGFR-inhibitor gefitinib, resulting in cMET amplification. Remarkably, the intracellular concentration of crizotinib was significantly higher in HCC827GR5 compared to the parental HCC827 cell line. Furthermore, live-cell microscopy with a pH-sensitive probe showed a differential reaction of the pH in the cytoplasm and the lysosomes after drug treatment in the HCC827GR5 in comparison with the HCC827 cells. This change in pH could influence the process of lysosomal sequestration of drugs. These results led us to the conclusion that lysosomal sequestration is involved in the strong synergistic reaction of the HCC827GR5 cell line to crizotinib-erlotinib combination. This finding warrants future clinical studies to evaluate whether genetic background and lysosomal sequestration could guide tailored therapeutic interventions.

High-grade serous ovarian cancer (HGSOC) is one of the major life-threatening cancers in women, with a survival rate of less than 50%. So far, chemotherapy is the main therapeutic tool to cure this lethal disease; however, in many cases, it fails to cure HGSOC even with severe side effects. Self-therapeutic nanomaterials could be an effective alternative to chemotherapy, facilitated by their diverse physicochemical properties and the ability to generate reactive species for killing cancer cells. Herein, inorganic cobalt hydroxide nanosheets (Co(OH)2 NS) were synthesized by a simple solution process at room temperature, and morphological, spectroscopic, and crystallographic analyses revealed the formation of Co(OH)2 NS with good crystallinity and purity. The as-prepared Co(OH)2 NS showed excellent potency, comparable to the FDA-approved cisplatin drug to kill ovarian cancer cells. Flow cytometric analysis (nnexin V) revealed increased cellular apoptosis for Co(OH)2 NS than cobalt acetate (the precursor). Tracking experiments demonstrated that Co(OH)2 NS are internalized through the lysosome pathway, although relocalization in the cytoplasm has been observed. Hence, Co(OH)2 NS could be an effective self-therapeutic drug and open up an area for the optimization of self-therapeutic properties of cobalt nanomaterials for cancer treatment.

Sirtuin 1 (SIRT1) is a NAD+-dependent deacetylase implicated in various biological and pathological processes, including cancer, diabetes, and cardiovascular diseases. In recent years, SIRT1-activating compounds have been demonstrated to exert cardioprotective effects. Therefore, this enzyme has become a feasible target to treat cardiovascular diseases, and many SIRT1 activators, of a natural or synthetic origin, have been identified. In the present work, we developed thiazole-based SIRT1 activators, which showed remarkably higher SIRT1 activation potencies compared with those of the reference compound resveratrol when tested in enzymatic assays. Thiazole 8, a representative compound of this series, was also subjected to further pharmacological investigations, where it was proven to reduce myocardial damage induced by an in vivo occlusion/reperfusion event, thus confirming its cardioprotective properties. In addition, the cardioprotective effect of compound 8 was significantly higher than that of resveratrol. Molecular modeling studies suggest the binding mode of these derivatives within SIRT1 in the presence of the p53-AMC peptide. These promising results could pave the way to further expand and optimize this chemical class of new and potent SIRT1 activators as potential cardioprotective agents.

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.

Cyclin-dependent kinase 5 (Cdk5) is an atypical proline-directed serine/threonine protein kinase well-characterized for its role in the central nervous system rather than in the cell cycle. Indeed, its dysregulation has been strongly implicated in the progression of synaptic dysfunction and neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), and also in the development and progression of a variety of cancers. For this reason, Cdk5 is considered as a promising target for drug design, and the discovery of novel small-molecule Cdk5 inhibitors is of great interest in the medicinal chemistry field. In this context, we employed a machine learning-based virtual screening protocol with subsequent molecular docking, molecular dynamics simulations and binding free energy evaluations. Our virtual screening studies resulted in the identification of two novel Cdk5 inhibitors, highlighting an experimental hit rate of 50% and thus validating the reliability of the in silico workflow. Both identified ligands, compounds CPD1 and CPD4, showed a promising enzyme inhibitory activity and CPD1 also demonstrated a remarkable antiproliferative activity in ovarian and colon cancer cells. These ligands represent a valuable starting point for structure-based hit-optimization studies aimed at identifying new potent Cdk5 inhibitors.

The unmet need for novel therapeutic options for ovarian cancer (OC) deserves further investigation. Among the different novel drugs, small interfering RNAs (siRNAs) are particularly attractive because of their specificity of action and efficacy, as documented in many experimental setups. However, the fragility of these molecules in the biological environment necessitates the use of delivery materials able to protect them and possibly target them to the cancer cells. Among the different delivery materials, those based on polymers and lipids are considered very interesting because of their biocompatibility and ability to carry/deliver siRNAs. Despite these features, polymers and lipids need to be engineered to optimize their delivery properties for OC. In this review, we concentrated on the description of the therapeutic potential of siRNAs and polymer-/lipid-based delivery systems for OC. After a brief description of OC and siRNA features, we summarized the strategies employed to minimize siRNA delivery problems, the targeting strategies to OC, and the preclinical models available. Finally, we discussed the most interesting works published in the last three years about polymer-/lipid-based materials for siRNA delivery.

Conjugation of known biologically active molecules to carbohydrate frameworks represents a valuable option for the preparation of hybrid, structurally-related families of compounds with the aim of modulating their biological response. Therefore, we present here a study on the preparation of d-galacto, d-manno, d-gluco, and d-lactose glycoconjugates of an established N-hydroxyindole-based (NHI) inhibitor of lactated dehydrogenase (LDH). Structural variations involved the sugar stereochemistry and size as well as the anchoring point of the NHI on the carbohydrate frame (either C-1 or C-6). In the case of the galactose anomeric glycoconjugate (C-1), intriguing solvent-dependent effects were observed in the glycosylation stereochemical outcome. The biological activity of the deprotected glycoconjugates in contrasting lactate formation and cancer cell proliferation are described.

Monoacylglycerol lipase (MAGL) is an important enzyme of the endocannabinoid system that catalyzes the degradation of the major endocannabinoid 2-arachidonoylglycerol (2-AG). MAGL is associated with pathological conditions such as pain, inflammation and neurodegenerative diseases like Parkinson's and Alzheimer's disease. Furthermore, elevated levels of MAGL have been found in aggressive breast, ovarian and melanoma cancer cells. Due to its different potential therapeutic implications, MAGL is considered as a promising target for drug design and the discovery of novel small-molecule MAGL inhibitors is of great interest in the medicinal chemistry field. In this context, we developed a pharmacophore-based virtual screening protocol combined with molecular docking and molecular dynamics simulations, which showed a final hit rate of 50% validating the reliability of the in silico workflow and led to the identification of two promising and structurally different reversible MAGL inhibitors, VS1 and VS2. These ligands represent a valuable starting point for structure-based hit-optimization studies aimed at identifying new potent MAGL inhibitors.

Introduction: Colorectal cancer (CRC) is a common cancer associated with poor outcomes, underscoring a need for the identification of novel prognostic and therapeutic targets to improve outcomes. This study aimed to identify genetic variants and differentially expressed genes (DEGs) using genome-wide DNA and RNA sequencing followed by validation in a large cohort of patients with CRC. Methods: Whole genome and gene expression profiling were used to identify DEGs and genetic alterations in 146 patients with CRC. Gene Ontology, Reactom, GSEA, and Human Disease Ontology were employed to study the biological process and pathways involved in CRC. Survival analysis on dysregulated genes in patients with CRC was conducted using Cox regression and Kaplan-Meier analysis. The STRING database was used to construct a protein-protein interaction (PPI) network. Moreover, candidate genes were subjected to ML-based analysis and the Receiver operating characteristic (ROC) curve. Subsequently, the expression of the identified genes was evaluated by Real-time PCR (RT-PCR) in another cohort of 64 patients with CRC. Gene variants affecting the regulation of candidate gene expressions were further validated followed by Whole Exome Sequencing (WES) in 15 patients with CRC. Results: A total of 3576 DEGs in the early stages of CRC and 2985 DEGs in the advanced stages of CRC were identified. ASPHD1 and ZBTB12 genes were identified as potential prognostic markers. Moreover, the combination of ASPHD and ZBTB12 genes was sensitive, and the two were considered specific markers, with an area under the curve (AUC) of 0.934, 1.00, and 0.986, respectively. The expression levels of these two genes were higher in patients with CRC. Moreover, our data identified two novel genetic variants-the rs925939730 variant in ASPHD1 and the rs1428982750 variant in ZBTB1-as being potentially involved in the regulation of gene expression. Conclusions: Our findings provide a proof of concept for the prognostic values of two novel genes-ASPHD1 and ZBTB12-and their associated variants (rs925939730 and rs1428982750) in CRC, supporting further functional analyses to evaluate the value of emerging biomarkers in colorectal cancer.

Malignant mesothelioma (MM) is a highly aggressive and resistant tumor. The prognostic role of key effectors of glycolytic metabolism in MM prompted our studies on the cytotoxicity of new inhibitors of glucose transporter type 1 (GLUT-1) and lactate dehydrogenase-A (LDH-A) in relation to ATP/NAD+ metabolism, glycolysis and mitochondrial respiration. The antiproliferative activity of GLUT-1 (PGL13, PGL14) and LDH-A (NHI-1, NHI-2) inhibitors, alone and in combination, were tested with the sulforhodamine-B assay in peritoneal (MESO-II, STO) and pleural (NCI-H2052 and NCI-H28) MM and non-cancerous (HMEC-1) cells. Effects on energy metabolism were measured by both analysis of nucleotides using RP-HPLC and evaluation of glycolysis and respiration parameters using a Seahorse Analyzer system. All compounds reduced the growth of MM cells in the µmolar range. Interestingly, in H2052 cells, PGL14 decreased ATP concentration from 37 to 23 and NAD+ from 6.5 to 2.3 nmol/mg protein. NHI-2 reduced the ATP/ADP ratio by 76%. The metabolic effects of the inhibitors were stronger in pleural MM and in combination, while in HMEC-1 ATP reduction was 10% lower compared to that of the H2052 cells, and we observed a minor influence on mitochondrial respiration. To conclude, both inhibitors showed cytotoxicity in MM cells, associated with a decrease in ATP and NAD+, and were synergistic in the cells with the highest metabolic modulation. This underlines cellular energy metabolism as a potential target for combined treatments in selected cases of MM.

Chemodynamic therapy (CDT) holds great promise in achieving cancer therapy through Fenton and Fenton-like reactions, which generate highly toxic reactive species. However, CDT is limited by the lower amount of catalyst ions that can decompose already existing intracellular H2O2 and produce reactive oxygen species (ROS) to attain a therapeutic outcome.

A cross talk between the Estrogen Receptor (ESR1) and the Retinoblastoma (pRb) pathway has been demonstrated to influence the therapeutic response of breast cancer patients but the full mechanism remains poorly understood. Here we show that the N-terminal domain of pRb interacts with the CD domain of ESR1 to allow for the assembly of intermediate complex chaperone proteins HSP90 and p23. We demonstrated that a loss of pRb in human/mouse breast cells decreases the expression of the ESR1 protein through the proteasome pathway. Our work reveals a novel regulatory mechanism of ESR1 basal turnover and activity and an unanticipated relationship with the pRb tumor suppressor.

Colorectal cancer is the third most common cancer in the world, a small fraction of which is represented by locally advanced rectal cancer (LARC). If not medically contraindicated, preoperative chemoradiotherapy, represent the standard of care for LARC patients. Unfortunately, patients shows a wide range of response rates in which approximately 20% has a complete pathological response, whereas in 20 to 40% the response is poor or absent.

G protein-coupled receptors (GPCRs) are integral cell-surface proteins having a central role in tumor growth and metastasis. However, several GPCRs retain an atypical intracellular/nuclear location in various types of cancer. The pathological significance of this is currently unknown. Here we extend this observation by showing that the bradykinin B2R (BK-B2R) is nuclearly expressed in the human triple-negative breast cancer (TNBC) cell line MDA-MB-231 and in human clinical specimens of TNBC. We posited that these "nuclearized" receptors could be involved in oncogenic signaling linked to aberrant growth and survival maintenance of TNBC. We used cell-penetrating BK-B2R antagonists, including FR173657 and novel transducible, cell-permeable forms of the peptide B2R antagonist HOE 140 (NG68, NG134) to demonstrate their superior efficacy over impermeable ones (HOE 140), in blocking proliferation and promoting apoptosis of MDA-MB-231 cells. Some showed an even greater antineoplastic activity over conventional chemotherapeutic drugs in vitro. The cell-permeable B2R antagonists had less to no anticancer effects on B2R shRNA-knockdown or non-B2R expressing (COS-1) cells, indicating specificity in their action. Possible mechanisms of their anticancer effects may involve activation of p38kinase/p27Kip1 pathways. Together, our data support the existence of a possible intracrine signaling pathway via internal/nuclear B2R, critical for the growth of TNBC cells, and identify new chemical entities that enable to target the corresponding intracellular GPCRs.

A number of pharmacogenetic studies have been carried out in non-small-cell lung cancer (NSCLC) to identify and characterize genes involved in chemotherapy activity. However, the results obtained so far are controversial and no reliable biomarker is currently used to predict clinical benefit from platinum-based chemotherapy, which represents the cornerstone of treatment of advanced NSCLC. This study investigated the expression levels of ERCC1 and of six genes (RRM1, RRM2, hENT1, dCK, cN-II and CDA) involved in gemcitabine metabolism in locally/advanced NSCLC patients treated with gemcitabine/platinum combination. Gene expression was assessed by quantitative-PCR in laser-microdissected specimens and correlated with tumor response. Frequency distribution of responses above and below the median expression level of biomarkers was compared using a two-sided Fisher's test. 5'-nucleotidase (cN-II) was the only gene differently expressed (p = 0.016) in the responders (complete/partial-response) compared to non-responders (stable/progressive disease). In the multivariate analysis, overexpression of this catabolic enzyme of gemcitabine remained a significant negative predictive factor. Patients with low cN-II had a modest trend toward increased survival, while both survival and progression-free survival were significantly longer in a more homogenous validation cohort of 40 advanced NSCLC (8.0 vs. 5.1 months, p = 0.026). Moreover, in vitro studies showed that silencing or pharmacological inhibition of cN-II increased the cytotoxicity of gemcitabine. This is the first study demonstrating the role of cN-II as a predictor of response to gemcitabine/platinum combinations in NSCLC. Its validation in prospective studies may improve clinical outcome of selected patients.