Novel insights into treatment strategies for hyperammonemia-associated urea cycle disorders and organic acidurias.

Investigador: Margarida F.B. e Silva
Ph.D., Aux Prof FFUL; Metabolism and Genetics Group at iMed.UL

Hyperammonemia in paediatric patients is mostly related to inborn errors of metabolism (IEM) including urea cycle disorders and certain organic acidurias. The rise of toxic ammonia levels may have devastating consequences on neurodegeneration and neurodevelopment. Preventive strategies of metabolic deterioration and more effective therapeutic options are definitely needed. Few studies have assessed the efficacy of novel therapies, especially tailored to the various phenotypes and IEM. This research proposal puts forward the hypotheses that: 1) hyperammonemia is linked to changes in cofactors or redox status affecting energy metabolism and resulting in mitochondrial dysfunction; 2) drug design profits of associated small molecules to improve their efficacy, reducing levels of toxic ammonia and surpassing the metabolic imbalances associated with the genetic underlying cause of IMD.

Abstract

To this end, combinatorial strategies of known nitrogen-scavenging agents, cofactors or substrates will be investigated in vitro using a human hepatoma cell model or using animal models of HA. Bioassays using targeted mass spectrometry-based techniques will be optimized for patient-derived fluids, cultured cells or exometabolome. Through the use of stable-isotope labelled standards, bioanalytical approaches will be devised and validated to trace metabolism and to evaluate enzyme activity. The metabolic modulation of target pathways affecting ammonia metabolism will be investigated using small molecules and precursors to evaluate activation/inhibitory effects. The proposed study will provide novel insights into the molecular mechanisms of ammonia pathogenesis linked to the major pathways of energy dysregulation focused on proximal or distal reactions of urea cycle, krebs cycle and fatty acid oxidation. Expected results will lead to a deeper understanding of pharmacodynamics of tested strategies, predicted to ameliorate mitochondrial function and ammonia detoxification.

Something stinks’: impaired hydrogen sulfide and cysteine persulfide production by cystathionine β-synthase variants identified in classical homocystinuria patients

Investigador: João B. Vicente
Auxiliary Investigator, ITQB-NOVA

This project aims to shed new lights onto the molecular basis of classical homocystinuria (CHU) due to cystathionine-β-synthase (CBS) deficiency, resulting from CBS gene mutations. The CBS canonical role in the transsulfuration branch of methionine metabolism has been increasingly extended to a major role as an endogenous source of hydrogen sulfide (H2S) source and, secondarily, cysteine persulfide (CysSSH). H2S regulates many physiological processes as a multifaceted ubiquitous signaling molecule. Disturbed H2S metabolism due to aberrant CBS expression is growingly established as an etiologic factor of several pathologies, some with clinical presentations common to CHU, namely cardiovascular disease and neurological impairment. Given that elevated plasma homocysteine does not fully explain the clinical presentations of CHU patients, impaired H2S and/or CysSSH production are likely to underlie the pathogenicity of various CBS mutations.

Abstract

Therefore, this project will comparatively characterize purified recombinant wild-type CBS and 14 pathogenic variants (located in the three structural domains) by a combination of biochemical and enzymatic assays, to compare their functional impairment in terms of the ‘canonical’, H2S-, and CysSSH-producing reactions, in the absence or presence of the exogenous pyridoxal-5’-phosphate cofactor and S-adenosyl-L-methionine (activator). Accomplishment of the proposed goals is neatly supported by the expertise of the Team on i) the production of recombinant WT CBS and pathogenic variants and ii) on their enzymatic characterization, both regarding its canonical and H2S-synthesizing reactions. Indeed, JBVicente and PLeandro have thus far published five articles focused on mechanistic aspects of CBS in relation with different pathophysiological conditions. Altogether, this project will contribute to a deeper understanding of the molecular basis of classical homocystinuria, to be further extended into cellular and in vivo studies, while supporting revising therapeutic options (currently based on dietary methionine restriction and vitamin B6 supplementation) and devising alternatives based on H2S-releasing drugs and/or dietary sources rich in polysulfides.