Comparative analysis of pyrimidine substituted aminoacyl-sulfamoyl nucleosides as potential inhibitors targeting class I aminoacyl-tRNA synthetases

Abstract

Aminoacyl-tRNA synthetases (aaRSs) catalyze the ATP-dependent coupling of an amino acid to its cognate tRNA. Being vital for protein translation aaRSs are considered a promising target for the development of novel antimicrobial agents. 5'-O-(N-aminoacyl)-sulfamoyl adenosine (aaSA) is a non-hydrolysable analog of the aaRS reaction intermediate that has been shown to be a potent inhibitor of this enzyme family but is prone to chemical instability and enzymatic modification. In an attempt to improve the molecular properties of this scaffold we synthesized a series of base substituted aaSA analogues comprising cytosine, uracil and N 3-methyluracil targeting leucyl-, tyrosyl-and isoleucyl-tRNA synthetases. In in vitro assays seven out of the nine inhibitors demonstrated K i app values in the low nanomolar range. To complement the biochemical studies, X-ray crystallographic structures of Neisseria gonorrhoeae leucyl-tRNA synthetase and Escherichia coli tyrosyl-tRNA synthetase in complex with the newly synthesized compounds were determined. These highlighted a subtle interplay between the base moiety and the target enzyme in defining relative inhibitory activity. Encouraged by this data we investigated if the pyrimidine congeners could escape a natural resistance mechanism, involving acetylation of the amine of the aminoacyl group by the bacterial Nacetyltransferases RimL and YhhY. With RimL the pyrimidine congeners were less susceptible to inactivation compared to the equivalent aaSA, whereas with YhhY the converse was true. Combined the various insights resulting from this study will pave the way for the further rational design of aaRS inhibitors.

Synthesis and Biological Evaluation of Lipophilic Nucleoside Analogues as Inhibitors of Aminoacyl-tRNA Synthetases

“…Out of six tested derivatives, four showed IC 50 in the nanomolar range in the radiolabel transfer assay (6a,b and 6e,f; Figure 2A and Table 1). However, for all four a 2-or 3-fold decrease in inhibitory activity was observed versus ISA in analogy with which was reported in the past [20,33]. A general trend of decrease in inhibitory activity was observed with increasing alkyl chain length.…”

“…But the feasibility of selective targeting has been shown previously by the availability of two marketed drugs inhibiting an aaRS, with mupirocin (likewise equipped with a long aliphatic tail) and tavaborole, and in using heterocyclic sulfonamide scaffolds [41,42]. Our extensive 3D structural work [33] on various aaRS in complex with inhibitory ligands (including unpublished work) will further pave the way for improved selectivity.…”

“…The cloning, expression, and purification of E. coli IleRS was performed as described before 33 . To examine the inhibitory effect of the various compounds, the purified E. coli IleRS was used.…”

Synthesis and Biological Evaluation of 1,3-Dideazapurine-Like 7-Amino-5-Hydroxymethyl-Benzimidazole Ribonucleoside Analogues as Aminoacyl-tRNA Synthetase Inhibitors

“…Nevertheless, IleS7HMDDA ( 32f ) is only five times less inhibitory than the well-known inhibitor IleSA, which surprisingly is 10-fold better compared to the 3-deaza derivative IleS3DA [ 3 ]. The compound also outperforms the pyrimidine analogues previously reported [ 25 ]. By contrast, the inhibitory activity for the leucine analogue 32e is analogous to its pyrimidine congeners, but for LeuRS, the LeuS3DA congener almost matched the strong activity of LeuSA.…”

“…For class II aaRSs, the adenine makes numerous interactions with active site residues, with the N 1 , N 3 and N 6 all making H-bond interactions with conserved features shared amongst all class II aaRS members. The interaction between the N 3 of adenine with the conserved water molecule for class II aaRS enzymes is crucial for the recognition of aaSAs by the corresponding enzymes [ 25 ]. It was hypothesized that the flipped exocyclic amine moiety of HMDDA should be able to mimic this specific interaction.…”