Potent Inhibition of E. coli DXP Synthase by a gem-Diaryl Bisubstrate Analog

Abstract

New antimicrobial strategies are needed to address pathogen resistance to currently used antibiotics. Bacterial central metabolism is a promising target space for the development of agents that selectively target bacterial pathogens. 1-Deoxy-D-xylulose 5-phosphate synthase (DXPS) converts pyruvate and D-glyceraldehyde 3-phosphate (D-GAP) to DXP, which is required for synthesis of essential vitamins and isoprenoids in bacterial pathogens. Thus, DXPS is a promising antimicrobial target. Toward this goal, our lab has demonstrated selective inhibition of Escherichia coli DXPS by alkyl acetylphosphonate (alkylAP)based bisubstrate analogs that exploit the requirement for ternary complex formation in the DXPS mechanism. Here, we present the first DXPS structure with a bisubstrate analog bound in the active site. Insights gained from this cocrystal structure guided structure−activity relationship studies of the bisubstrate scaffold. A low nanomolar inhibitor (compound 8) bearing a gem-dibenzyl glycine moiety conjugated to the acetylphosphonate pyruvate mimic via a triazole-based linker emerged from this study. Compound 8 was found to exhibit slow, tight-binding inhibition, with contacts to E. coli DXPS residues R99 and R478 demonstrated to be important for this behavior. This work has discovered the most potent DXPS inhibitor to date and highlights a new role of R99 that can be exploited in future inhibitor designs toward the development of a novel class of antimicrobial agents.

An activity-based probe for antimicrobial target DXP synthase, a thiamin diphosphate-dependent enzyme

“…As PLThDP formation is reversible (Sanders et al, 2017), a crosslinking group is required to ensure that active DXPS can be labeled irreversibly. Sterically demanding substituents can be incorporated into the phosphonyl ester group without significant loss in inhibitor potency (Morris et al, 2013;Sanders et al, 2017;Bartee and Meyers, 2018a;Coco et al, 2024), due to the large active site volume of DXPS, whereas modifications to the reactive acetyl group are not tolerated (Smith, Vierling, and Meyers, 2012). Based on this, we designed alkylAP-based probe 1 bearing the commonly-used 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethyl moiety (Li et al, 2013), capable of crosslinking to the DXPS active site and presenting a biorthogonal handle for introduction of a fluorophore or biotin.…”

“…The phosphonolactylThDP (PLThDP) adduct formed via reaction of an alkylAP with ThDP can be detected by circular dichroism (CD) on pyruvate decarboxylase enzymes, including DXPS (Jordan et al, 2003;Nemeria et al, 2009Nemeria et al, , 2010Heflin, 2015;Zhou et al, 2017;Coco et al, 2024). ThDP bound to DXPS exists in the 4′-aminopyrimidine (AP) form (Figure 4A) with a characteristic negative CD signal at 320 nm (Figure 4B, blue line) (Patel et al, 2012).…”

“…Using the DXPS-IspC coupled assay (Coco et al, 2024) to measure initial velocity of DXP formation (Supplementary Figure S2), we assessed the ability of ABP 1 to inhibit DXPS-catalyzed DXP formation. Consistent with the observed formation of PLThDP, ABP 1 inhibits EcDXPS with a K i of 1.60 ± 0.22 μM (Figure 4C), comparable to the observed low micromolar potencies of other first-generation alkylAPs (Smith et al, 2014;Sanders et al, 2017).…”

“…To gain additional evidence that ABP 1 engages the DXPS active site, we conducted competition experiments using previously studied pyruvate-competitive alkylAP-based inhibitors known to act via PLThDP formation on DXPS. Three inhibitors with varying potencies were selected (Figure 7A), including butylacetylphosphonate (BAP, 7), methylacetylphosphonate (MAP, 8), and dibenzylglycine triazole acetylphosphonate (DBGlyTrAP, 9) (Smith, Vierling, and Meyers, 2012;Sanders et al, 2017;Coco et al, 2024). BAP (7) and MAP (8) are first-generation alkylAPs that display low micromolar and submicromolar potencies, respectively, against DXPS enzymes.…”

“…Kluger and Tsui, 1986), and they react with ThDP in a similar manner to form a stable C2α-phosphonolactylthiamin diphosphate (PLThDP) adduct. We have advanced alkylAP inhibitor development to target the large DXPS active site and its gated mechanism requiring ternary complex formation (Smith, Vierling, and Meyers, 2012;Morris et al, 2013;Sanders et al, 2017;Bartee and Meyers, 2018a;Coco et al, 2024). Here, we describe the first activity-based probe for DXPS, ABP 1, based on first-generation alkylAP inhibitors.…”