Modulation of hydrogen sulfide metabolism: new pharmacological targets in cancer and amyotrophic lateral sclerosis therapy [Tesi di dottorato]

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule with a key role in human (patho)physiology. In human cells, H2S is synthetized by cystathionine β-synthase (CBS), cystathionine γ-lysase (CSE) and 3-mercaptopyruvate sulfurtransferase (MST), and is oxidatively catabolized in the mitochondrion by sulfide:quinone oxidoreductase (SQR). H2S homeostasis relies on a fine balance between its catabolism and biosynthesis. Dysregulation of H2S metabolism has been associated to neurodegeneration and cancer. Reprogramming of H2S metabolism was shown to have pro-survival effects in cancer cells and, recently, N-acetylcysteine (NAC) was suggested to exert its antioxidant effects by increasing intracellular levels of persulfides and polysulfides (collectively known as “sulfane sulfur species”). In paper 1, the effect of NAC on the H2S metabolism of SW480 colon cancer cells was investigated. After exposing cells to 10 mM NAC for 24 hours, both MST and SQR displayed enhanced expression and activity. Moreover, NAC has proven to persist inside colon cancer cells over the 24-hour treatment and to act as a substrate for human MST, as shown working on the isolated recombinant enzyme. Altogether, these findings demonstrate that NAC stimulates H2S metabolism in colon cancer cells and serves as substrate for human MST. Hypoxia is currently recognized as a hallmark of the microenvironment of solid tumors, typically associated to malignant phenotypes. An increasing body of evidence suggests that H2S can increase cell resistance to hypoxia, while supporting angiogenesis, energy metabolism and drug resistance in cancer. Available information on the role played by H2S in the tumor microenvironment was reviewed in paper 3. In paper 2, working on SW480 colon cancer cells, the effect of hypoxia on the ability of cells to metabolize H2S was evaluated by high resolution respirometry. Hypoxia was found to decrease the mitochondrial content and the overall H2S-consuming activity of cells, while inducing a mitochondrial enrichment in SQR. These findings suggest that under hypoxic conditions cancer cells undergo adaptive changes to ensure higher intracellular H2S levels with pro-survival effects and, concomitantly, protection of cell respiration from H2S poisoning. In the central nervous system, H2S plays a central role in the regulation of numerous physiological processes, including neurotransmission and cytoprotection. Dysregulation of sulfide metabolism has been associated to cognitive disturbances like in Down’s syndrome and neurodegeneration like in Parkinson’s and Alzheimer’s disease. Recently, it was suggested an involvement of H2S in the etiology of amyotrophic lateral sclerosis (ALS). In the provisional paper 5, working on in vivo (Drosophila melanogaster) and in vitro ALS models, the effect of H2S on ALS-associated neurotoxicity was investigated. Of interest, knock down or pharmacological inhibition of the two H2S-synthetizing enzymes CBS and CSE was found to result in ameliorative phenotypes. These results triggered additional, still on-going investigations aimed at elucidating the impact of H2S in ALS and the underlined molecular mechanisms. Given the role of H2S in human (patho)physiology, there is an urgent need for inhibitors of the human H2S-synthetizing enzymes with pharmacological potential. In paper 4, a small library of newly synthesized pyridine derivatives was screened for the ability of these compounds to inhibit the human CBS, CSE and MST, as recombinantly produced in Escherichia coli and purified. By combining a wide range of biophysical and biochemical techniques, two compounds with similar molecular scaffold were found to weakly inhibit both CBS and CSE. In this study, a robust methodological platform for compound screening was set-up, and two hit compounds were identified which will be used as a starting point for future screening campaigns. The new knowledge acquired here, while deepening our understanding of the role of H2S in human (patho)physiology and, more specifically, in tumorigenesis and neurodegeneration, will hopefully set the basis for innovative diagnostic and therapeutic approaches.

diritti: info:eu-repo/semantics/embargoedAccess
In relazione con info:eu-repo/semantics/altIdentifier/hdl/11573/1354711
valutatori esterni: Paolo Ascenzi, Andrea Battistoni, Giorgio Camilloni.

Tesi di dottorato. | Lingua: Inglese. | Paese: | BID: TD20019036