Bisubstrate analogue inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: New design with improved properties

Abstract

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a key enzyme in the folate biosynthetic pathway, catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin. The enzyme is essential for microorganisms, is absent from humans, and is not the target for any existing antibiotics. Therefore, HPPK is an attractive target for developing novel antimicrobial agents. Previously, we characterized the reaction trajectory of HPPK-catalyzed pyrophosphoryl transfer and synthesized a series of bisubstrate analog inhibitors of the enzyme by linking 6-hydroxymethylpterin to adenosine through 2, 3, or 4 phosphate groups. Here, we report a new generation of bisubstrate analog inhibitors. To improve protein binding and linker properties of such inhibitors, we have replaced the pterin moiety with 7,7-dimethyl-7,8-dihydropterin and the phosphate bridge with a piperidine linked thioether. We have synthesized the new inhibitors, measured their Kd and IC50 values, determined their crystal structures in complex with HPPK, and established their structure-activity relationship. 6-Carboxylic acid ethyl ester-7,7-dimethyl-7,8-dihydropterin, a novel intermediate that we developed recently for easy derivatization at position 6 of 7,7-dimethyl-7,8-dihydropterin, offers a much high yield for the synthesis of bisubstrate analogs than that of previously established procedure.

The identification, analysis and structure-based development of novel inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase

“…Similar strategies involving the synthesis of bisubstrate analogs (pterin-nucleotide adducts) have already demonstrated the feasibility of this approach for this target. 7, 21, 22 We recognize that these nucleotide binding sites are inherently polar and that the build-out process must eventually incorporate strategies for optimizing membrane permeability and cell penetration.…”

Bisubstrate analog inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: New lead exhibits a distinct binding mode

“…Therefore, HPPK is an attractive target for developing novel antimicrobial agents; attempts have been made for decades. 10–15 …”

Structural enzymology and inhibition of the bi‐functional folate pathway enzyme HPPK–DHPS from the biowarfare agent Francisella tularensis

“…(A) The chemical structures of the substrates and products of HPPK- and DHPS-catalyzed reactions. (B) Chemical structure of HP-18 [16]. (C) Chemical structure of HP-26 [15].…”

“…Of these four enzymes, the mechanism of HPPK action is best studied and understood [8, 9]. Attempts to inhibit the activity of this enzyme have been made for decades [10–16]. …”

“…1B) and 2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydro-pteridine-6-carboxylic acid (2-{2-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-ylmethanesulfonyl]-ethylcarbamoyl}-ethyl)-amide (HP-26, Fig. 1C) [15, 16]. As shown in Figure 1, the chemical difference between the two inhibitors is the spacer that connects the pterin and the adenosine moieties, an aminoethylpiperidine spacer in HP-18 and a glycyl aminoethyl spacer in HP-26.…”