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Drugs that target human thymidylate synthase (hTS), a dimeric enzyme, are widely used in anticancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces over-expression of this protein and development of drug resistance. We thus pursued an alternative strategy that led us to the discovery of TS-dimer destabilizers. These compounds bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. A structural, spectroscopic, and kinetic investigation has provided evidence and quantitative information on the effects of the interaction of these small molecules with hTS. Focusing on the best among them, E7, we have shown that it inhibits hTS in cancer cells and accelerates its proteasomal degradation, thus causing a decrease in the enzyme intracellular level. E7 also showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers.

Abstract Dysregulation of the kynurenine pathway (KP) is implicated in many human diseases and disorders, from immunological, metabolic, neurodegenerative, and neuropsychiatric conditions to cancer, and represents an appealing target for new therapeutic approaches. In this intricate scenario, invertebrates, like Lymnaea stagnalis (LS), provide a flexible tool to unravel the complexity of the KP. Starting from the available LS genome and transcriptome, we identified putative transcripts of all KP enzymes containing an ORF; each predicted protein possessed a high degree of sequence conservation to known orthologues of other invertebrate and vertebrate model organisms. Sequences were confirmed by qualitative PCR and sequencing. At the same time, the qRT-PCR analysis revealed that Lym IDO-like, Lym TDO-like, Lym AFMID-like, Lym KMO-like, Lym AADAT-like, Lym KYAT I/III-like, Lym KYNU-like, Lym HAAO-like, and Lym ACMSD-like showed widespread tissue expression. Then, tryptophan, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxy-kynurenine, xanthurenic acid, picolinic acid, and quinolinic acid were identified in the hemolymph of LS by UHPLC-Q exactive mass spectrometer. Our study provides the most thorough characterization to date of the KP in an invertebrate model, supporting the value of LS for future functional studies of this pathway at the cellular, synaptic, and behavioral levels.

Background Understanding the precise mechanisms of ketamine is crucial for replicating its rapid antidepressant effects without inducing psychomimetic changes. Here, we explore whether the antidepressant-like effects of ketamine enantiomers are underscored by protection against cytokine-induced reductions in hippocampal neurogenesis and activation of the neurotoxic kynurenine pathway in our well-established in vitro model of depression in a dish. Methods We used the fetal hippocampal progenitor cell line (HPC0A07/03C) to investigate ketamine's impact on cytokine-induced reductions in neurogenesis in vitro. Cells were treated with interleukin- 1beta (IL-1b) (10 ng/mL) or IL-6 (50 pg/mL), alone or in combination with ketamine enantiomers arketamine (R-ketamine, 400 nM) or esketamine (S-ketamine, 400 nM) or antidepressants sertraline (1 mM) or venlafaxine (1 mM). Results Resembling the effect of antidepressants, both ketamine enantiomers prevented IL-1b– and IL-6–induced reduction in neurogenesis and increase in apoptosis. This was mediated by inhibition of IL-1b–induced production of IL-2 and IL-13 by R-ketamine and of IL-1b–induced tumor necrosis factor-alpha by S-ketamine. Likewise, R-ketamine inhibited IL-6–induced production of IL-13, whereas S-ketamine inhibited IL-6–induced IL-1b and IL-8. Moreover, both R- and S-ketamine prevented IL-1b–induced increases in indoleamine 2,3-dioxygenase expression as well as kynurenine production, which in turn was shown to mediate the detrimental effects of IL-1b on neurogenesis and apoptosis. In contrast, neither R- nor S-ketamine prevented IL-6–induced kynurenine pathway activation. Conclusions Results suggest that R- and S-ketamine have pro-neurogenic and anti-inflammatory properties; however, this is mediated by inhibition of the kynurenine pathway only in the context of IL-1b. Overall, this study enhances our understanding of the mechanisms underlying ketamine's antidepressant effects in the context of different inflammatory phenotypes, ultimately leading to the development of more effective, personalized therapeutic approaches for patients suffering from depression. [ABSTRACT FROM AUTHOR]

Vaccination has drastically decreased mortality due to coronavirus disease 19 (COVID-19), but not the rate of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Alternative strategies such as inhibition of virus entry by interference with angiotensin-I-converting enzyme 2 (ACE2) receptors could be warranted. Cyclodextrins (CDs) are cyclic oligosaccharides that are able to deplete cholesterol from membrane lipid rafts, causing ACE2 receptors to relocate to areas devoid of lipid rafts. To explore the possibility of reducing SARS-CoV-2 entry, we tested hydroxypropyl-β-cyclodextrin (HPβCD) in a HEK293T-ACE2hi cell line stably overexpressing human ACE2 and Spike-pseudotyped SARS-CoV-2 lentiviral particles. We showed that HPβCD is not toxic to the cells at concentrations up to 5 mM, and that this concentration had no significant effect on cell cycle parameters in any experimental condition tested. Exposure of HEK293T-ACEhi cells to concentrations of HPβCD starting from 2.5 mM to 10 mM showed a concentration-dependent reduction of approximately 50% of the membrane cholesterol content. In addition, incubation of HEK293T-ACEhi cells with HIV-S-CoV-2 pseudotyped particles in the presence of increasing concentrations of HPβCD (from 0.1 to 10 mM) displayed a concentration-dependent effect on SARS-CoV-2 entry efficiency. Significant effects were detected at concentrations at least one order of magnitude lower than the lowest concentration showing toxic effects. These data indicate that HPβCD is a candidate for use as a SARS-CoV-2 prophylactic agent. [ABSTRACT FROM AUTHOR]

Abstract Interleukin (IL)-18 is a cytokine isolated as an important modulator of immune responses and subsequently shown to be pleiotropic. IL-18 and its receptors are expressed in the central nervous system (CNS) where they participate in neuroinflammatory/neurodegenerative processes but also influence homeostasis and behavior. Work on IL-18 null mice, the localization of the IL-18 receptor complex in neurons and the neuronal expression of decoy isoforms of the receptor subunits are beginning to reveal the complexity and the significance of the IL-18 system in the CNS. This review summarizes current knowledge on the central role of IL-18 in health and disease.