Therapeutic potential of AMPK activators for correction of mitochondrial fatty acid ß-oxidation disorders. – THERABETAOX

Inborn enzyme defects of fatty acid ß-oxidation (FAO) form a group of genetic disorders associated to life-threatening pediatric presentations, or to milder phenotypes with later onset and severity. Advances in diagnosis revealed a large number of disease-causing genes , and a continuous increase in the number of patients. However, little progress was made in their treatment. In recent years, our group demonstrated that a target-based pharmacological strategy could result in up-regulation of deficient enzyme activity, and could be successful for correction of some FAO disorders. In line with this, the aim of this project is, based on their presumed mechanism of action, to screen new candidate molecules for the therapy of inborn FAO disorders.
AMPK (AMP activated protein kinase) is considered to be a key energy sensor of the cells activated by all metabolic stress conditions that generate an increase in the AMP/ATP ratio.
Given the central role of AMPK in energy metabolism, it was identified as a valuable therapeutic target for the treatment of several diseases like type II diabetes or obesity. In recent years, academic laboratories and pharmaceutical companies have studied the effects of many direct or indirect activators of AMPK in the field of common diseases. We hypothesize that the AMPK signaling pathway could be a highly relevant system in our research of new therapeutic targets for the treatment of FAO disorders. In line with this, our project aims at investigating if AMPK activators could represent candidate molecules for correction of FAO disorders in a large panel of deficient patients’ fibroblasts.
Cells: Control or patient fibroblasts. This project will benefit of the large cells repository and the expertise on FAO-deficiency of the service of Biochemistry (Bicêtre Hospital): we will test around 100 FAO-deficient fibroblasts.
Compounds: We will study the effects of drugs, and of experimental (collaboration with GlaxoSmithKline) or natural compounds, considered as direct or indirect activators of AMPK.
Methods: To address theses issues we will use various pharmacological approaches previously developed in our group. The initial readout for the effects of tested compounds will be the measurement of tritiated palmitate oxidation capacities, according to a standardized assay routinely used in our group. We will also analyze by western-blot the possible changes in FAO protein levels to determine if pharmacological improvements are due to increases in mutated protein level, as anticipated. Finally, quantification of mRNA levels of FAO genes will be performed to determine if the various compounds do act at the level of gene expression.
Molecular mechanisms: For compounds inducing a correction of FAO flux in cells, we will analyze the involvement of AMPK/PGC-1a axis by siRNA strategy. Indeed, the effects of AMPK activators on mitochondrial metabolism are supposed to be mediated by the activation of PGC-1a (a transcriptional co-activator) through its phosphorylation. Phosphorylation of AMPK and of its downstream target Acetyl-CoA carboxylase, and phosphorylation of PGC-1a will be studied.
Mitochondrial biogenesis: As just mentioned, activation of PGC-1a is part of the molecular mechanisms suggested to explain the effect of AMPK activators on mitochondrial energy metabolism. Since PGC-1a is a key regulator of mitochondrial biogenesis, we will determine if active compounds for the correction of FAO also induce a mitochondrial biogenesis, by the study of three different parameters.
Altogether, this project should allow identifying possible new candidate drug(s) for the treatment of mitochondrial FAO diseases, and provides a true pre-clinical evaluation of the tested compounds that could benefit shortly to patients.