This project frames into the quest for antibiotics with novel scaffolds and mechanism of action, and for novel compounds with anti-inflammatory and anti-oxidative activity. We will evaluate the biological properties of compounds that include analogues/derivatives of L-glutamate, L-cysteine and L-homocysteine (HCys).
The mechanism through which compounds containing C-P-H bonds (phosphinic), patented as antibiotics, exert their activity still needs to be elucidated. The approach will be mostly metabolomic and proteomic.
The C-S containing compounds will be tested for anti-inflammatory, anti-oxidative and antiangiogenic activities. Some of these compounds include the oxidized forms of HCys and their derivatives that accumulate in patients when the methionine recycling pathway becomes impaired, as in cardiovascular diseases, neurological/psychiatric disorders and cancer. We synthesized chemically homocysteinsulfinic acid and established that it can be converted to homohypotaurine (HHT), a compound commercially not available, via an enzymatic, bio-based route. The best conditions for the bio-based synthesis and purification of HHT will be established and the compound tested on cardiomyocytes, HUVEC cells and neutrophils.
Furthermore, we will investigate the metabolic role of C-S-containing biomolecules related to taurine. Particular emphasis will be on thiotaurine, a biomolecule releasing hydrogen sulfide (H2S), and on the role of these molecules in controlling inflammation. The specific signaling pathways involved will be dissected and the enzymatic route to its synthesis will be investigated. We aim to identify the proteins that change their expression level or undergo post-translational modifications, including nitrosylation/nitration, persulfidation. Finally, the effect of H2S will be assessed in pathologies characterized by an increase of oxidative stress, as it occurs in some respiratory diseases such as Chronic Obstructive Pulmonary Disease.
The innovation of the proposed research resides in the identification of novel compounds for treating different pathologies. We want to contribute to the discovery of new antibiotics and of molecules, prepared in our laboratory and not available commercially, with anti-angiogenic, anti-oxidative and anti-inflammatory activity.
As part of a patent deposited by Sapienza (https://www.uniroma1.it/it/brevetto/102016000098005), De Biase's group demonstrated that Glu-gamma-PH, alpha-ketoglutarate-PH and the L-Leu-Glu-gamma-PH dipeptide exert a significant bacteriostatic effect, with MIC90 ranging 7-0.5 µg/ml on laboratory E. coli MG1655 strain. In the attempt to understand the enzymes/proteins that are targeted by Glu-gamma-PH and/or alpha-ketoglutarate-PH, we will carry out MIC experiments on E. coli MG1655 KO strains, some of which are already available in the lab while others will be constructed as part of this work. The mutants will be on enzymes that use glutamate or alpha-ketoglutarate as substrates in E. coli metabolic pathways. Interestingly, the three molecules above display antimicrobial effects (disk diffusion assay) also when tested against E. coli pathogenic strains, such as a nosocomial extended spectrum beta-lactamase (ESBL) strain. Although preliminary interesting results have been obtained that allowed the patenting of our findings, a complete and exhaustive comprehension of the enzymatic targets of Glu-gamma-PH and alpha-ketoglutarate-PH, along with the possible molecular strategies adopted by the cell to counteract the inhibitory activities of these molecules, are still lacking. These are the goals of this project and will be fundamental to provide a detailed picture of the molecular targets of the compounds above. We are developing this part of the project in collaboration with researchers at ITQB (Lisbon) to apply a "multi-omics" approach (see Eventuali altri partner esterni e ruolo nel progetto).
While a great deal of work is carried out on taurine and homotaurine (the latter, free or derivatized, is in widespread pharmaceutical and laboratory use), an extensive search of the literature retrieved more limited information on the properties of homohypotaurine, the decarboxylation product of HCSA. The properties of this latter molecule, structurally the closest homologue of GABA, the major inhibitory neurotransmitters in the CNS, respectively, need a more in-depth investigation. To date it is known that HCSA acts as an agonist of the metabotropic glutamate receptors (mGluR) [17], and that it increases glucose uptake in skeletal muscle via stimulation of AMP-activated protein kinase [18]. These findings point to a role of HCSA in diabetes. With this work De Biase's group will set out the basis for a deeper understanding of the possible biological activity of homohypotaurine as compared to homotaurine.
Endothelial cells (HUVEC) and mice cardiomyocytes will be treated with HCSA and HHT in conditions mimicking oxidative stress, hypoxia and hyperglycemia, respectively. The phenotypic changes that will be monitored will include the assessment of angiogenesis in HUVEC and viability assays in both cell types.
Non-steroidal anti-inflammatory drugs are among the most commonly used drugs. Despite efforts to produce non-steroidal anti-inflammatory drugs that do not cause gastrointestinal ulceration and bleeding, these adverse effects remain major limitations to their use. On the contrary it has been reported that H2S donors can increase the resistance of the gastric mucosa to injury and accelerate damaged tissue repair [19]. These observations suggest that anti-inflammatory molecules that are chemically modified to release H2S will exhibit improved efficacy and reduced toxicity. In this context thiotaurine biomolecule structurally related to taurine and to hypotaurine, particularly abundant in neutrophil granulocytes, has a thiosulfonate (RSO2SH) group which can potentially release H2S. This biocompound could mimic the role of H2S in inflammation, but also play a protective role on the gastric mucosa when administered with an anti-inflammatory drug. The study of anti-inflammatory effect of thiotaurine and its derivatives will also lead to new therapeutic applications for the treatment of inflammatory lung diseases.
The mechanisms involved in the interaction of the reactive oxygen (ROS) and nitrogen species (RNS) with sulfinates have only recently been investigated [20]. Interestingly, the oxidation of sulfinates by radicals is accompanied by the generation of highly reactive sulfonyl radicals which can promote oxidative reactions. In light of this, establishing the pathophysiological role of sulfonyl radicals will be an outcome of this work.
References
17. Shi Q. et al. (2003) J. Pharmacol. Exp. Ther. 305, 131
18. Kim J.H. et al. (2011) J. Biol. Chem. 286, 7567
19. Wallace JL (2007) Trends Pharmacol Sci, 28, 501
20. Baseggio Conrado A. et al. (2014) Free Radic. Res. 48, 1300.