Probing the Robustness of Inhibitors of Tuberculosis Aminoglycoside Resistance Enzyme Eis by Mutagenesis

Abstract

Each year, millions of people worldwide contract tuberculosis (TB), the deadliest infection. The spread of infections with drug-resistant strains of Mycobacterium tuberculosis (Mtb) that are refractory to treatment poses a major global challenge. A major cause of resistance to antitubercular drugs of last resort, aminoglycosides, is overexpression of the Eis (enhanced intracellular survival) enzyme of Mtb, which inactivates aminoglycosides by acetylating them. We showed previously that this inactivation of aminoglycosides could be overcome by our recently reported Eis inhibitors that are currently in development as potential aminoglycoside adjunctive therapeutics against drug-resistant TB. To interrogate the robustness of the Eis inhibitors, we investigated the enzymatic activity of Eis and its inhibition by Eis inhibitors from three different structural families for nine single-residue mutants of Eis, including those found in the clinic. Three engineered mutations of the substrate binding site, D26A, W36A, and F84A, abolished inhibitor binding while compromising Eis enzymatic activity 2-to 3-fold. All other Eis mutants, including clinically observed ones, were potently inhibited by at least one inhibitor. This study helps position us one step ahead of Mtb resistance to Eis inhibitors as they are being developed for TB therapy.

Inhibition of Aminoglycoside 6′-N-acetyltransferase Type Ib (AAC(6′)-Ib): Structure–Activity Relationship of Substituted Pyrrolidine Pentamine Derivatives as Inhibitors

“…Unfortunately, such a successful alternative has not yet been fully developed for aminoglycosides. Despite significant efforts, no formulations that combine an aminoglycoside and an inhibitor of the resistance have been approved for human use [4,38]. Although there are many mechanisms and variations by which bacteria resist aminoglycosides, the presence of AAC( 6)-Ib in the majority of Gram-negative amikacin-resistant clinical strains implies that the search to find inhibitors that permit their use in a significant number of infections may not be as insurmountable as it seems [4,8,39].…”

Inhibition of Aminoglycoside 6’-N-Acetyltransferase Type Ib [Aac(6′)-Ib]: Structure-Activity Relationship of Substituted Pyrrolidine Pentamine Derivatives as Inhibitors

“…Unfortunately, such a successful alternative has not yet been 15 fully developed for aminoglycosides. Despite significant efforts, no formulations that 16 combine an aminoglycoside and an inhibitor of the resistance have been approved for 17 human use [4,35]. Although there are many mechanisms and variations by which bacteria 18 resist aminoglycosides, the presence of AAC(6′)-Ib in the majority of Gram-negative 19 amikacin-resistant clinical strains implies that the search to find inhibitors that permit 20 their use in a significant number of infections may not be as insurmountable as it seems 21 [4,8,36].…”

An Outline of the Latest Crystallographic Studies on Inhibitor-Enzyme Complexes for the Design and Development of New Therapeutics against Tuberculosis

“…In a previous manuscript, Green and co-authors [38] used three compounds (89-91, Figure 40) belonging to different structural families to demonstrate how Eis mutations can influence the potency of the inhibitors. Compound 89 belongs to the same family of analogue 87 [36] and its crystal structure showed the same interactions illustrated above for 87 and 88.…”

“…An NMRbased activity assay showed that the activity of MenD at a concentration of 5 μM was Starting from this information, seven residues (Asp26, Trp36, Arg37, Leu63, Met65, Ser83, and Phe84) not directly involved in the acetyl transfer function of Eis, but observed to interact with the inhibitors, were mutated into alanine residues to determine the effects on aminoglycoside acetylation and on inhibitor binding. The results showed that mutations in only three amino acids (Asp26Ala, Trp36Ala, and Phe84Ala) rendered all inhibitors inactive, suggesting that the future design of inhibitors should not depend on interactions with these residues [38].…”

“…In a previous manuscript, Green and co-authors [ 38 ] used three compounds ( 89 – 91 , Figure 40 ) belonging to different structural families to demonstrate how Eis mutations can influence the potency of the inhibitors.…”