Acylated sulfonamide adenosines as potent inhibitors of the adenylate-forming enzyme superfamily

Abstract

The superfamily of adenylate-forming enzymes all share a common chemistry. They activate a carboxylate group, on a specific substrate, by catalyzing the formation of a high energy mixed phosphoanhydride-linked nucleoside intermediate. Members of this diverse enzymatic family play key roles in a variety of metabolic pathways and therefore many have been regarded as drug targets. A generic approach to inhibit such enzymes is the use of non-hydrolysable sulfur-based bioisosteres of the adenylate intermediate. Here we compare the activity of compounds containing a sulfamoyl and sulfonamide linker respectively. An improved synthetic strategy was developed to generate inhibitors containing the latter that target isoleucyl-(IleRS) and seryl-tRNA synthetase (SerRS), two structurally distinct representatives of class I and II aminoacyl-tRNA synthetases (aaRSs). These enzymes attach their respective amino acid to its cognate tRNA and are indispensable for protein translation. Evaluation of the ability of the two similar isosteres to inhibit serRS revealed a remarkable difference, with an almost complete loss of activity for seryl-sulfonamide 15 (SerSoHA) compared to its sulfamoyl analogue (SerSA), while inhibition of IleRS was unaffected. To explain these observations, we have determined a 2.1 Å crystal structure of Klebsiella pneumoniae SerRS in complex with SerSA. Using this structure as a template, modelling of 15 in the active site predicts an unfavorable eclipsed conformation. We extended the same modelling strategy to representative members of the whole adenylate-forming enzyme superfamily, and were able to disclose a new classification system for adenylating enzymes, based on their protein fold. The results suggest that, other than for the structural and functional orthologues of the class II aaRSs, the O to C substitution within the sulfur-sugar link should generally preserve the inhibitory potency.

Structural Insights into the Binding of Natural Pyrimidine-Based Inhibitors of Class II Aminoacyl-tRNA Synthetases

“…For all three ligand-bound structures the calculated electron density map permitted unambiguous modelling of compounds 5a-5c (Supplementary Figure 4A). Comparison of the seryl, sulfamoyl and ribose moieties in the three ligandbound structures shows that they make the same interactions as observed in the previously published structure of Kp-serRS in complex with SSA 27 (Supplementary Figures 3B and 4B). Therefore, the relative affinity of the pyrimidine-containing compounds can be solely attributed to base-protein interactions (Figure 3A).…”

“…We opted for the stable acylated sulfamate linkage as a bioisoster for the acylphosphate where nature opted for the phosphoramidate as in McC 13 . The adenine-containing variants of such compounds have been shown to be potent inhibitors with a Ki app of 0.18 nM and 0.052 nM for serRS and aspRS, respectively and therefore act as a benchmark to compare activity 26,27 .…”

“…The adenine-containing variants of such compounds have been shown to be potent inhibitors with a K i app of 0.18 nM and 0.052 nM for serRS and aspRS, respectively, and therefore act as a benchmark to compare activity. 26,27 Using the respective recombinant aaRS from E. coli the inhibitory activities of the six different compounds were evaluated in an in vitro aminoacylation assay (Table 1 and Supplementary Figure 1A and B). For both enzymes the uridine substituted aminoacyl sulfamoyl 5b and 6b proved to be the least active compounds, with a greater than 10-fold increase in the determined IC 50 value when compared to the best pyrimidine-containing inhibitor targeting the same aaRS.…”

Aminoacyl-tRNA Synthetases as Valuable Targets for Antimicrobial Drug Discovery

“…Unfortunately, the majority of these aaSoA congeners are not able to efficiently inhibit their target aaRS. Subsequently, we substituted 5 -oxygen with a carbon atom to generate two similar isosteres [153] (aaSoHA, Figure 10a) targeting class I IleRS and class II SerRS, respectively. Evaluation of their in vitro enzyme inhibitory activity showed that the isoleucyl-sulfonamide analog retained its potency compared to the parent sulfamoyl derivative (ISA), while an almost complete loss of activity was observed for the seryl congener.…”

One Pot Use of Combilipases for Full Modification of Oils and Fats: Multifunctional and Heterogeneous Substrates

“…There are many examples of homo-multifunctional substrates that are modified using biocatalytic approaches, such as polyhydroxy [117][118][119][120][121][122][123][124][125][126][127][128], polycarboxylic [129][130][131], and polyamine Effect of the product inhibition on the reaction courses using two hypothetic enzymes, one with a very high initial activity but showing a strong inhibition by the product, the other with a lower activity but without product inhibition.…”