Phospholipase D inhibitors screening: Probing and evaluation of ancient and novel molecules.

Phospholipase D (PLD) is a ubiquitous enzyme that cleaves the distal phosphoester bond of phospholipids generating phosphatidic acid (PA). In plants, PA is involved in numerous cell responses triggered by stress. Similarly, in mammals, PA is also a second messenger involved in tumorigenesis. PLD is nowadays considered as a therapeutic target and blocking its activity with specific inhibitors constitutes a promising strategy to treat cancers. Starting from already described PLD inhibitors, this study aims to investigate the effect of their structural modifications on the enzyme's activity, as well as identifying new potent inhibitors of eukaryotic PLDs. Being able to purify the plant PLD from Vigna unguiculata (VuPLD), we obtained a SAXS model of its structure. We then used a fluorescence-based test suitable for high-throughput screening to review the effect of eukaryotic PLD inhibitors described in the literature. In this regard, we found that only few molecules were in fact able to inhibit VuPLD and we confirmed that vanadate is the most potent of all with an IC50 around 58 µM. Moreover, the small-scale screening of a chemical library of 3120 compounds allowed us to optimize the different screening's steps and paved the way towards the discovery of new potent inhibitors.

The multifaceted contribution of α-ketoglutarate to tumor progression: An opportunity to exploit?

The thriving field that constitutes cancer metabolism has unveiled some groundbreaking facts over the past two decades, at the heart of which is the TCA cycle and its intermediates. As such and besides its metabolic role, α-ketoglutarate was shown to withstand a wide range of physiological reactions from protection against oxidative stress, collagen and bone maintenance to development and immunity. Most importantly, it constitutes the rate-limiting substrate of 2-oxoglutarate-dependent dioxygenases family enzymes, which are involved in hypoxia sensing and in the shaping of cellular epigenetic landscape, two major drivers of oncogenic transformation. Based on literature reports, we hereby review the benefits of this metabolite as a possible novel adjuvant therapeutic opportunity to target tumor progression. This article is part of the special issue "Mitochondrial metabolic alterations in cancer cells and related therapeutic targets".

Direct and Continuous Measurement of Phospholipase D Activities Using the Chelation-Enhanced Fluorescence Property of 8-Hydroxyquinoline.

Phospholipase D (PLD) hydrolyzes phospholipids to form phosphatidic acid (PA) and the corresponding headgroup. To date, PLD has been linked to several pathologies, such as cancer, making this enzyme an important therapeutic target. However, most PLD assays developed so far are either discontinuous or based on the indirect determination of choline released upon phosphatidylcholine (PC) hydrolysis. Therefore, we designed a PLD assay that is based on the chelation-enhanced fluorescence property of 8-hydroxyquinoline. This assay exhibits a strong fluorescence signal upon Ca2+ complexation with the PLD-generated PA and is not limited to PC as the substrate but allows the use of natural phospholipids with various headgroups. Besides, this easy-to-handle assay allows to monitor prokaryotic and eukaryotic PLD activities in a continuous way and on a microplate scale.

The Oncojanus Paradigm of Respiratory Complex I.

Mitochondrial respiratory function is now recognized as a pivotal player in all the aspects of cancer biology, from tumorigenesis to aggressiveness and chemotherapy resistance. Among the enzymes that compose the respiratory chain, by contributing to energy production, redox equilibrium and oxidative stress, complex I assumes a central role. Complex I defects may arise from mutations in mitochondrial or nuclear DNA, in both structural genes or assembly factors, from alteration of the expression levels of its subunits, or from drug exposure. Since cancer cells have a high-energy demand and require macromolecules for proliferation, it is not surprising that severe complex I defects, caused either by mutations or treatment with specific inhibitors, prevent tumor progression, while contributing to resistance to certain chemotherapeutic agents. On the other hand, enhanced oxidative stress due to mild complex I dysfunction drives an opposite phenotype, as it stimulates cancer cell proliferation and invasiveness. We here review the current knowledge on the contribution of respiratory complex I to cancer biology, highlighting the double-edged role of this metabolic enzyme in tumor progression, metastasis formation, and response to chemotherapy.