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Bisubstrate inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: Transition state analogs for high affinity binding.

ABSTRACT: 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a key enzyme in the folate biosynthesis pathway. It catalyzes pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). HPPK is essential for microorganisms but absent in mammals; therefore, it is an attractive target for developing novel antimicrobial agents. Previously, based on our studies of the structure and mechanism of HPPK, we created first-generation bisubstrate inhibitors by linking 6-hydroxymethylpterin to adenosine through phosphate groups, and developed second-generation inhibitors by replacing the phosphate bridge with a linkage that contains a piperidine moiety. Here, we report third-generation inhibitors designed based on the piperidine-containing inhibitor, mimicking the transition state. We synthesized two such inhibitors, characterized their protein-binding and enzyme inhibition properties, and determined their crystal structures in complex with HPPK, advancing the development of such bisubstrate analog inhibitors.

SUBMITTER: Shi G

PROVIDER: S-EPMC7855645 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description: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 K(d) and IC(50) 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.

| S-EPMC3257516 | biostudies-literature

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

Project description:6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a key enzyme in the folate biosynthesis pathway catalyzing the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin, is an attractive target for developing novel antimicrobial agents. Previously, we studied the mechanism of HPPK action, synthesized bisubstrate analog inhibitors by linking 6-hydroxymethylpterin to adenosine through phosphate groups, and developed a new generation of bisubstrate inhibitors by replacing the phosphate bridge with a piperidine-containing linkage. To further improve linker properties, we have synthesized a new compound, characterized its protein binding/inhibiting properties, and determined its structure in complex with HPPK. Surprisingly, this inhibitor exhibits a new binding mode in that the adenine base is flipped when compared to previously reported structures. Furthermore, the side chain of amino acid residue E77 is involved in protein-inhibitor interaction, forming hydrogen bonds with both 2' and 3' hydroxyl groups of the ribose moiety. Residue E77 is conserved among HPPK sequences, but interacts only indirectly with the bound MgATP via water molecules. Never observed before, the E77-ribose interaction is compatible only with the new inhibitor-binding mode. Therefore, this compound represents a new direction for further development.

| S-EPMC3389233 | biostudies-literature

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

Project description:6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is an essential enzyme in the microbial folate biosynthetic pathway. This pathway has proven to be an excellent target for antimicrobial development, but widespread resistance to common therapeutics including the sulfa drugs has stimulated interest in HPPK as an alternative target in the pathway. A screen of a pterin-biased compound set identified several HPPK inhibitors that contain an aryl substituted 8-thioguanine scaffold, and structural analyses showed that these compounds engage the HPPK pterin-binding pocket and an induced cryptic pocket. A preliminary structure activity relationship profile was developed from biophysical and biochemical characterizations of derivative molecules. Also, a similarity search identified additional scaffolds that bind more tightly within the HPPK pterin pocket. These inhibitory scaffolds have the potential for rapid elaboration into novel lead antimicrobial agents.

| S-EPMC4269157 | biostudies-literature

Role of protein conformational dynamics in the catalysis by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase.

Project description:Enzymatic catalysis has conflicting structural requirements of the enzyme. In order for the enzyme to form a Michaelis complex, the enzyme must be in an open conformation so that the substrate can get into its active center. On the other hand, in order to maximize the stabilization of the transition state of the enzymatic reaction, the enzyme must be in a closed conformation to maximize its interactions with the transition state. The conflicting structural requirements can be resolved by a flexible active center that can sample both open and closed conformational states. For a bisubstrate enzyme, the Michaelis complex consists of two substrates in addition to the enzyme. The enzyme must remain flexible upon the binding of the first substrate so that the second substrate can get into the active center. The active center is fully assembled and stabilized only when both substrates bind to the enzyme. However, the side-chain positions of the catalytic residues in the Michaelis complex are still not optimally aligned for the stabilization of the transition state, which lasts only approximately 10(-13) s. The instantaneous and optimal alignment of catalytic groups for the transition state stabilization requires a dynamic enzyme, not an enzyme which undergoes a large scale of movements but an enzyme which permits at least a small scale of adjustment of catalytic group positions. This review will summarize the structure, catalytic mechanism, and dynamic properties of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase and examine the role of protein conformational dynamics in the catalysis of a bisubstrate enzymatic reaction.

| S-EPMC3482336 | biostudies-literature

Crystallization and preliminary X-ray analysis of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Staphylococcus aureus.

Project description:6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the Mg(2+)-dependent transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HMDP), forming 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, which is a critical step in the de novo folic acid-biosynthesis pathway. Diffraction-quality crystals of HPPK from the medically relevant species Staphylococcus aureus were grown in the presence of ammonium sulfate or sodium malonate and diffracted to better than 1.65 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 36.8, b = 76.6, c = 51.5 A, alpha = gamma = 90.0, beta = 100.2 degrees . The crystals contained two molecules per asymmetric unit, with a volume per protein weight (V(M)) of 2.04 A(3) Da(-1) and an estimated solvent content of 39.6%.

| S-EPMC2864696 | biostudies-literature

Crystal structure of the 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase•dihydropteroate synthase bifunctional enzyme from Francisella tularensis.

Project description:The 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) enzymes catalyze sequential metabolic reactions in the folate biosynthetic pathway of bacteria and lower eukaryotes. Both enzymes represent validated targets for the development of novel anti-microbial therapies. We report herein that the genes which encode FtHPPK and FtDHPS from the biowarfare agent Francisella tularensis are fused into a single polypeptide. The potential of simultaneously targeting both modules with pterin binding inhibitors prompted us to characterize the molecular details of the multifunctional complex. Our high resolution crystallographic analyses reveal the structural organization between FtHPPK and FtDHPS which are tethered together by a short linker. Additional structural analyses of substrate complexes reveal that the active sites of each module are virtually indistinguishable from those of the monofunctional enzymes. The fused bifunctional enzyme therefore represents an excellent vehicle for finding inhibitors that engage the pterin binding pockets of both modules that have entirely different architectures. To demonstrate that this approach has the potential of producing novel two-hit inhibitors of the folate pathway, we identify and structurally characterize a fragment-like molecule that simultaneously engages both active sites. Our study provides a molecular framework to study the enzyme mechanisms of HPPK and DHPS, and to design novel and much needed therapeutic compounds to treat infectious diseases.

| S-EPMC2994781 | biostudies-literature

Structure of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase-dihydropteroate synthase from Plasmodium vivax sheds light on drug resistance.

Project description:The genomes of the malaria-causing Plasmodium parasites encode a protein fused of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) domains that catalyze sequential reactions in the folate biosynthetic pathway. Whereas higher organisms derive folate from their diet and lack the enzymes for its synthesis, most eubacteria and a number of lower eukaryotes including malaria parasites synthesize tetrahydrofolate via DHPS. Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) HPPK-DHPSs are currently targets of drugs like sulfadoxine (SDX). The SDX effectiveness as an antimalarial drug is increasingly diminished by the rise and spread of drug-resistant mutations. Here, we present the crystal structure of PvHPPK-DHPS in complex with four substrates/analogs, revealing the bifunctional PvHPPK-DHPS architecture in an unprecedented state of enzymatic activation. SDX's effect on HPPK-DHPS is due to 4-amino benzoic acid (pABA) mimicry, and the PvHPPK-DHPS structure sheds light on the SDX-binding cavity, as well as on mutations that effect SDX potency. We mapped five dominant drug resistance mutations in PvHPPK-DHPS: S382A, A383G, K512E/D, A553G, and V585A, most of which occur individually or in clusters proximal to the pABA-binding site. We found that these resistance mutations subtly alter the intricate enzyme/pABA/SDX interactions such that DHPS affinity for pABA is diminished only moderately, but its affinity for SDX is changed substantially. In conclusion, the PvHPPK-DHPS structure rationalizes and unravels the structural bases for SDX resistance mutations and highlights architectural features in HPPK-DHPSs from malaria parasites that can form the basis for developing next-generation anti-folate agents to combat malaria parasites.

| S-EPMC6166723 | biostudies-literature

Kinetic and structural characterization of a product complex of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Escherichia coli.

Project description:HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase) catalyses the transfer of pyrophosphate from ATP to HMDP (6-hydroxymethyl-7,8-dihydropterin), to form AMP and DHPPP (6-hydroxymethyl-7,8-dihydropterin pyrophosphate). This transformation is a key step in the biosynthesis of folic acid, and HPPK is consequently a target for antimicrobial drugs. The substrates are known to bind to HPPK in an ordered manner, with ATP binding first followed by HMDP. In the present study we show by isothermal titration calorimetry that the product, DHPPP, can bind to the HPPK apoenzyme with high affinity (equilibrium dissociation constant, K(d)=0.2 microM), but without the enhancement of pterin fluorescence that occurs on binding of HMDP. The transient kinetics of the enzyme can be monitored by measuring the change in the fluorescence of the pterin ring using stopped-flow methods. The fluorescence exhibits a pronounced biphasic behaviour: it initially rises and then declines back to its original level. This behaviour is in agreement with a two-state kinetic model, with the first phase of fluorescence increase associated with HMDP binding to the enzyme, and the second phase with a slow event that occurs after the reaction has taken place. The HPPK-DHPPP and HPPK-DHPPP-AMP complexes were examined by NMR, and the binding site for DHPPP partially mapped from changes in chemical shifts identified from two dimensional 1H/15N heteronuclear single-quantum coherence spectra. The results demonstrate that DHPPP, in contrast to HMDP, is able to bind to the HPPK apoenzyme and suggest that the pyrophosphate moieties on the ligand play an important role in establishment of a high affinity binding site for the pterin ring.

| S-EPMC1224222 | biostudies-other

Folate biosynthesis in higher plants: purification and molecular cloning of a bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase localized in mitochondria.

Project description:In pea leaves, the synthesis of 7,8-dihydropteroate, a primary step in folate synthesis, was only detected in mitochondria. This reaction is catalyzed by a bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase enzyme, which represented 0.04-0.06% of the matrix proteins. The enzyme had a native mol. wt of 280-300 kDa and was made up of identical subunits of 53 kDa. The reaction catalyzed by the 7,8-dihydropteroate synthase domain of the protein was Mg2+-dependent and behaved like a random bireactant system. The related cDNA contained an open reading frame of 1545 bp and the deduced amino acid sequence corresponded to a polypeptide of 515 residues with a calculated M(r) of 56,454 Da. Comparison of the deduced amino acid sequence with the N-terminal sequence of the purified protein indicated that the plant enzyme is synthesized with a putative mitochondrial transit peptide of 28 amino acids. The calculated M(r) of the mature protein was 53,450 Da. Southern blot experiments suggested that a single-copy gene codes for the enzyme. This result, together with the facts that the protein is synthesized with a mitochondrial transit peptide and that the activity was only detected in mitochondria, strongly supports the view that mitochondria is the major (unique?) site of 7,8-dihydropteroate synthesis in higher plant cells.

| S-EPMC1169695 | biostudies-other

Assay Development and Identification of the First <i>Plasmodium falciparum</i> 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase Inhibitors.

Project description:In the fight towards eradication of malaria, identifying compounds active against new drug targets constitutes a key approach. Plasmodium falciparum 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (PfHPPK) has been advanced as a promising target, as being part of the parasite essential folate biosynthesis pathway while having no orthologue in the human genome. However, no drug discovery efforts have been reported on this enzyme. In this study, we conducted a three-step screening of our in-house antifolate library against PfHPPK using a newly designed PfHPPK-GFP protein construct. Combining virtual screening, differential scanning fluorimetry and enzymatic assay, we identified 14 compounds active against PfHPPK. Compounds' binding modes were investigated by molecular docking, suggesting competitive binding with the HMDP substrate. Cytotoxicity and in vitro ADME properties of hit compounds were also assessed, showing good metabolic stability and low toxicity. The most active compounds displayed low micromolar IC50 against drug-resistant parasites. The reported hit compounds constitute a good starting point for inhibitor development against PfHPPK, as an alternative approach to tackle the malaria parasite.

| S-EPMC9182141 | biostudies-literature

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