Structure-based drug discovery in the lipoic acid salvage pathway against MRSA

SCATTOLINI, A; GRAMMATOGLO, K; WINDSHÜGEL, B; JIRGENSONS, A; MANSILLA, MC

Mendoza

Congreso; 58 Reunión Anual SAIB; 2022

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of healthcare-related infection worldwide. The increasing emergence of multidrug resistant strains urgently requires novel therapeutic approaches in order to keep the drug discovery pipeline filled. Lipoic acid (LA) is a universally conserved sulfur-containing cofactor required for intermediary metabolism, that is either synthesized de novo or acquired from environmental sources. In the model Gram-positive bacterium Bacillus subtilis LA synthesis involves four protein activities, instead of the two enzymes necessary in the Gram-negative bacterium Escherichia coli . First, the octanoyl-acyl carrier protein (ACP): protein-N-octanoyltransferase, LipM, transfers the octanoyl moieties to GcvH, the H subunit of the glycine cleavage system. Then, the lipoate synthase LipA inserts sulfur atoms into C6 and C8 of the octanoyl moieties. Finally, the amidotransferase LipL transfers the lipoyl side chain from GcvH to the E2 subunits of dehydrogenase complexes. Since lipoate ligase, LplJ, can only transfer exogenous lipoate to GcvH and E2o (the lipoylable subunit of oxoglutarate dehydrogenase), LipL is also required for modification of the remaining E2s. The bacterial pathogen S. aureus also employs this ?lipoyl-relay? pathway for de novo biosynthesis and salvage. Furthermore, it encodes two additional proteins: a secondary lipoate ligase, LplA2, and a protein similar to GcvH, named GcvH-L, that which are sufficient for LA salvage during infection. Due to its essentiality for cell viability and virulence, interfering with LA synthesis represents a promising approach for treating S. aureus infections. In this work, we performed a phenotypic screen of different molecules that wereidentified by a virtual screen against S. aureus enzymes involved in LA salvage, LplA1 and LplA2. We selected a compound, lpl-004, that caused a marked growth inhibition of the WT strain. This effect was less severe in ΔlplA1 or ΔlplA2 single mutants. Furthermore, growth of the double mutant ΔlplA1 ΔlplA2 was not affected in the presence of the compound. Similar results were obtained using the LA analogue selenolipoate, a compound reported to block dehydrogenase activity in E. coli . Using protein extracts of different mutants of S. aureus , deficient in LA synthesis and uptake, we determined that lpl-004 would be bound to E2s. We can conclude that, by the sequential action of staphylococcal lipoate ligase and amidotransferase, lpl-004 would be accepted as a substrate and transferred to E2s, eventually impairing dehydrogenase activity. This compound would be useful for further drug development against this pathogenic bacterium.