Empowering drug off-target discovery with metabolic and structural analysis.

Elucidating intracellular drug targets is a difficult problem. While machine learning analysis of omics data has been a promising approach, going from large-scale trends to specific targets remains a challenge. Here, we develop a hierarchic workflow to focus on specific targets based on analysis of metabolomics data and growth rescue experiments. We deploy this framework to understand the intracellular molecular interactions of the multi-valent dihydrofolate reductase-targeting antibiotic compound CD15-3. We analyse global metabolomics data utilizing machine learning, metabolic modelling, and protein structural similarity to prioritize candidate drug targets. Overexpression and in vitro activity assays confirm one of the predicted candidates, HPPK (folK), as a CD15-3 off-target. This study demonstrates how established machine learning methods can be combined with mechanistic analyses to improve the resolution of drug target finding workflows for discovering off-targets of a metabolic inhibitor.

Adaptation to mutational inactivation of an essential gene converges to an accessible suboptimal fitness peak.

The mechanisms of adaptation to inactivation of essential genes remain unknown. Here we inactivate E. coli dihydrofolate reductase (DHFR) by introducing D27G,N,F chromosomal mutations in a key catalytic residue with subsequent adaptation by an automated serial transfer protocol. The partial reversal G27- > C occurred in three evolutionary trajectories. Conversely, in one trajectory for D27G and in all trajectories for D27F,N strains adapted to grow at very low metabolic supplement (folAmix) concentrations but did not escape entirely from supplement auxotrophy. Major global shifts in metabolome and proteome occurred upon DHFR inactivation, which were partially reversed in adapted strains. Loss-of-function mutations in two genes, thyA and deoB, ensured adaptation to low folAmix by rerouting the 2-Deoxy-D-ribose-phosphate metabolism from glycolysis towards synthesis of dTMP. Multiple evolutionary pathways of adaptation converged to a suboptimal solution due to the high accessibility to loss-of-function mutations that block the path to the highest, yet least accessible, fitness peak.

Cofactors and metabolites as protein folding helpers in metabolic diseases.

In the past few decades, improved early diagnosis methods, technological developments and an increasing crosstalk between clinicians and researchers has led to the identification of an increasing number of inborn metabolic diseases. In these disorders, missense mutations are the most frequent type of genetic defects, frequently resulting in defective protein folding. A better understanding at the molecular level of protein misfolding and its role in disease has prompted the emergence of therapies based in the use of small molecules that have the ability to correct protein folding defects. Well-known cases are reported for phenylketonuria and Gaucher's disease. Most of these compounds have a specific mechanism of action interacting directly with a particular protein, the so called pharmacological chaperones. Among such small molecules are protein ligands, either natural substrates or synthetic derivatives, cofactors, competitive inhibitors, and agonist/antagonists. In this review we will start by briefly overviewing the mechanisms through which such ligands exert a stabilizing action, and then move on to an extended discussion on therapeutic approaches and use of vitamins and substrates to correct protein misfolding in metabolic disorders. Examples of vitamins that have been successfully prescribed to rescue some cases of inborn errors of metabolism will be presented. In particular, the role of riboflavin supplementation in the treatment of fatty acid ß-oxidation disorders will be thoroughly analyzed, focusing on recent reports that shed light on the molecular basis of vitamin responsiveness. Moreover, we will highlight the latest studies that point to a synergistic effect of cofactors and metabolites in the rescue of defective fatty acid ß-oxidation enzymes. The synergism of multiple small molecules may underlie a promising general pharmacological strategy for the treatment of metabolic diseases in general.

Role of flavinylation in a mild variant of multiple acyl-CoA dehydrogenation deficiency: a molecular rationale for the effects of riboflavin supplementation.

Mutations in the genes encoding the alpha-subunit and beta-subunit of the mitochondrial electron transfer flavoprotein (ETF) and the electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO) cause multiple acyl-CoA dehydrogenation deficiency (MADD), a disorder of fatty acid and amino acid metabolism. Point mutations in ETF, which may compromise folding, and/or activity, are associated with both mild and severe forms of MADD. Here we report the investigation on the conformational and stability properties of the disease-causing variant ETFbeta-D128N, and our findings on the effect of flavinylation in modulating protein conformational stability and activity. A combination of biochemical and biophysical methods including circular dichroism, visible absorption, flavin, and tryptophan fluorescence emission allowed the analysis of structural changes and of the FAD moiety. The ETFbeta-D128N variant retains the overall fold of the wild type, but under stress conditions its flavin becomes less tightly bound. Flavinylation is shown to improve the conformational stability and biological activity of a destabilized D128N variant protein. Moreover, the presence of flavin prevented proteolytic digestion by avoiding protein destabilization. A patient homozygous for the ETFbeta-D128N mutation developed severe disease symptoms in association with a viral infection and fever. In agreement, our results suggest that heat inactivation of the mutant may be more relevant at temperatures above 37 degrees C. To mimic a situation of fever in vitro, the flavinylation status was tested at 39 degrees C. FAD exerts the effect of a pharmacological chaperone, improving ETF conformation, and yielding a more stable and active enzyme. Our results provide a structural and functional framework that could help to elucidate the role that an increased cellular FAD content obtained from riboflavin supplementation may play in the molecular pathogenesis of not only MADD, but genetic disorders of flavoproteins in general.