Methionine supplementation for multi-organ dysfunction in MetRS-related pulmonary alveolar proteinosis.

INTRODUCTION: Pulmonary alveolar proteinosis related to mutations in the methionine tRNA synthetase (MARS1) gene is a severe, early-onset disease that results in death before the age of 2 years in one-third of patients. It is associated with a liver disease, growth failure and systemic inflammation. As methionine supplementation in yeast models restored normal enzymatic activity of the synthetase, we studied the tolerance, safety and efficacy of daily oral methionine supplementation in patients with severe and early disease. METHODS: Four patients received methionine supplementation and were followed for respiratory, hepatic, growth and inflammation-related outcomes. Their course was compared to those of historical controls. Reactive oxygen species production by patient monocytes before and after methionine supplementation was also studied. RESULTS: Methionine supplementation was associated with respiratory improvement, clearance of the extracellular lipoproteinaceous material and discontinuation of whole-lung lavage in all patients. The three patients who required oxygen or noninvasive ventilation could be weaned off within 60 days. In addition, liver dysfunction, inflammation and growth delay improved or resolved. At a cellular level, methionine supplementation normalised the production of reactive oxygen species by peripheral monocytes. CONCLUSION: Methionine supplementation was associated with important improvements in children with pulmonary alveolar proteinosis related to mutations in the MARS1 gene. This study paves the way for similar strategies for other tRNA synthetase deficiencies.

Inhibition of protein synthesis and malaria parasite development by drug targeting of methionyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRSs) are housekeeping enzymes that couple cognate tRNAs with amino acids to transmit genomic information for protein translation. The Plasmodium falciparum nuclear genome encodes two P. falciparum methionyl-tRNA synthetases (PfMRS), termed PfMRS(cyt) and PfMRS(api). Phylogenetic analyses revealed that the two proteins are of primitive origin and are related to heterokonts (PfMRS(cyt)) or proteobacteria/primitive bacteria (PfMRS(api)). We show that PfMRS(cyt) localizes in parasite cytoplasm, while PfMRS(api) localizes to apicoplasts in asexual stages of malaria parasites. Two known bacterial MRS inhibitors, REP3123 and REP8839, hampered Plasmodium growth very effectively in the early and late stages of parasite development. Small-molecule drug-like libraries were screened against modeled PfMRS structures, and several "hit" compounds showed significant effects on parasite growth. We then tested the effects of the hit compounds on protein translation by labeling nascent proteins with (35)S-labeled cysteine and methionine. Three of the tested compounds reduced protein synthesis and also blocked parasite growth progression from the ring stage to the trophozoite stage. Drug docking studies suggested distinct modes of binding for the three compounds, compared with the enzyme product methionyl adenylate. Therefore, this study provides new targets (PfMRSs) and hit compounds that can be explored for development as antimalarial drugs.

Two-strain, cell-selective protein labeling in mixed bacterial cultures.

Cell-selective metabolic labeling of proteins with noncanonical amino acids enables the study of proteomic changes in specified subpopulations of complex multicellular systems. For example, azidonorleucine (Anl) and 2-aminooctynoic acid, both of which are activated by an engineered methionyl-tRNA synthetase (designated NLL-MetRS), are excluded from proteins made in wild-type cells but incorporated readily into proteins made in cells that carry NLL-MetRS. To expand the set of tools available for cell-selective metabolic labeling, we sought a MetRS variant capable of activating propargylglycine (Pra). Pra was chosen as the target amino acid because its alkynyl side chain can be selectively and efficiently conjugated to azide-functionalized fluorescence probes and affinity tags. Directed evolution, using active-site randomization and error-prone PCR, yielded a MetRS variant (designated PraRS) capable of incorporating Pra at near-quantitative levels into proteins made in a Met-auxotrophic strain of Escherichia coli cultured in Met-depleted media. Proteins made in E. coli strains expressing PraRS were labeled with Pra in Met-supplemented media as shown by in-gel fluorescence after conjugation to Cy5-azide. The combined use of NLL-MetRS and PraRS enabled differential, cell-selective labeling of marker proteins derived from two bacterial strains cocultured in media supplemented with Met, Anl, and Pra. Treatment of the mixed marker proteins by sequential strain-promoted and copper(I)-catalyzed cycloadditions allowed straightforward identification of the cellular origin of each protein.

Recent advances in the preclinical evaluation of the topical antibacterial agent REP8839.

REP8839 is a synthetic fluorovinylthiophene-containing diaryldiamine that inhibits bacterial methionyl tRNA synthetase (MetRS) and is a new chemical entity that represents a novel pharmacological class. The compound has potent in vitro antibacterial activity against many clinically important Gram-positive bacteria including the major skin pathogens Staphylococcus aureus and Streptococcus pyogenes. In light of the emergence of methicillin-resistant S. aureus in the community and increasing resistance to mupirocin, REP8839 is being evaluated as a topical agent for the treatment of superficial skin infections. REP8839 was active against resistant phenotypes of S. aureus and can be formulated at high concentrations to minimize the development of resistance. A formulation of REP8839 has demonstrated efficacy in a porcine partial thickness wound infection model against mupirocin-resistant S. aureus.

Activation of methionine by Escherichia coli methionyl-tRNA synthetase.

In the present work, we have examined the function of three amino acid residues in the active site of Escherichia coli methionyl-tRNA synthetase (MetRS) in substrate binding and catalysis using site-directed mutagenesis. Conversion of Asp52 to Ala resulted in a 10,000-fold decrease in the rate of ATP-PPi exchange catalyzed by MetRS with little or no effect on the Km's for methionine or ATP or on the Km for the cognate tRNA in the aminoacylation reaction. Substitution of the side chain of Arg233 with that of Gln resulted in a 25-fold increase in the Km for methionine and a 2000-fold decrease in kcat for ATP-PPi exchange, with no change in the Km for ATP or tRNA. These results indicate that Asp52 and Arg233 play important roles in stabilization of the transition state for methionyl adenylate formation, possibly directly interacting with complementary charged groups (ammonium and carboxyl) on the bound amino acid. Primary sequence comparisons of class I aminoacyl-tRNA synthetases show that all but one member of this group of enzymes has an aspartic acid residue at the site corresponding to Asp52 in MetRS. The synthetases most closely related to MetRS (including those specific for Ile, Leu, and Val) also have a conserved arginine residue at the position corresponding to Arg233, suggesting that these conserved amino acids may play analogous roles in the activation reaction catalyzed by each of these enzymes. Trp305 is located in a pocket deep within the active site of MetRS that has been postulated to form the binding cleft for the methionine side chain.(ABSTRACT TRUNCATED AT 250 WORDS)