RESUMO
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.
Assuntos
Antimaláricos , Difosfotransferases , Plasmodium falciparum , Antimaláricos/química , Difosfotransferases/antagonistas & inibidores , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Simulação de Acoplamento Molecular , Plasmodium falciparum/efeitos dos fármacosRESUMO
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a promising antimicrobial target involved in the folate biosynthesis pathway. Although, the results from crystallographic studies of HPPK have attracted a great interest in the design of novel HPPK inhibitors, the mechanism of action of HPPK due to inhibitor binding remains questionable. Recently, mercaptoguanine derivatives were reported to inhibit the pyrophosphoryl transfer mechanism of Staphylococcus aureus HPPK (SaHPPK). The present study is an attempt to understand the SaHPPK-inhibitors binding mechanism and to highlight the key residues that possibly involve in the complex formation. To decipher these questions, we used the state-of-the-art advanced insilico approach such as molecular docking, molecular dynamics (MD), molecular mechanics-generalized Born surface area approach. Domain cross correlation and principle component analysis were applied to the snapshots obtained from MD revealed that the compounds with high binding affinity stabilize the conformational dynamics of SaHPPK. The binding free energy estimation showed that the van der Waals and electrostatic interactions played a vital role for the binding mechanism. Additionally, the predicted binding free energy was in good agreement with the experimental values (R2 = .78). Moreover, the free energy decomposition on per-residue confirms the key residues that significantly contribute to the complex formation. These results are expected to be useful for rational design of novel SaHPPK inhibitors.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/química , Difosfotransferases/química , Guanina/análogos & derivados , Mercaptopurina/análogos & derivados , Staphylococcus aureus/química , Motivos de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Domínio Catalítico , Cristalografia por Raios X , Difosfotransferases/antagonistas & inibidores , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Análise de Componente Principal , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Staphylococcus aureus/enzimologia , Relação Estrutura-Atividade , Especificidade por Substrato , TermodinâmicaRESUMO
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a member of the folate biosynthesis pathway found in prokaryotes and lower eukaryotes that catalyzes the pyrophosphoryl transfer from the ATP cofactor to a 6-hydroxymethyl-7,8-dihydropterin substrate. We report the chemical synthesis of a series of S-functionalized 8-mercaptoguanine (8MG) analogues as substrate site inhibitors of HPPK and quantify binding against the E. coli and S. aureus enzymes (EcHPPK and SaHPPK). The results demonstrate that analogues incorporating acetophenone-based substituents have comparable affinities for both enzymes. Preferential binding of benzyl-substituted 8MG derivatives to SaHPPK was reconciled when a cryptic pocket unique to SaHPPK was revealed by X-ray crystallography. Differential chemical shift perturbation analysis confirmed this to be a common mode of binding for this series to SaHPPK. One compound (41) displayed binding affinities of 120 nM and 1.76 µM for SaHPPK and EcHPPK, respectively, and represents a lead for the development of more potent and selective inhibitors of SaHPPK.
Assuntos
Difosfotransferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Staphylococcus aureus/enzimologia , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Difosfotransferases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), an enzyme from the folate biosynthesis pathway, catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin and is a yet-to-be-drugged antimicrobial target. Building on our previous discovery that 8-mercaptoguanine (8MG) is an inhibitor of Staphylococcus aureus HPPK (SaHPPK), we have identified and characterized the binding of an S8-functionalized derivative (3). X-ray structures of both the SaHPPK/3/cofactor analogue ternary and the SaHPPK/cofactor analogue binary complexes have provided insight into cofactor recognition and key residues that move over 30 Å upon binding of 3, whereas NMR measurements reveal a partially plastic ternary complex active site. Synthesis and binding analysis of a set of analogues of 3 have identified an advanced new lead compound (11) displaying >20-fold higher affinity for SaHPPK than 8MG. A number of these exhibited low micromolar affinity for dihydropteroate synthase (DHPS), the adjacent, downstream enzyme to HPPK, and may thus represent promising new leads to bienzyme inhibitors.
Assuntos
Difosfotransferases/antagonistas & inibidores , Difosfotransferases/química , Ácido Fólico/biossíntese , Guanina/química , Staphylococcus aureus/enzimologia , Trifosfato de Adenosina/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Di-Hidropteroato Sintase/química , Íons , Cinética , Espectroscopia de Ressonância Magnética , Conformação Molecular , Ligação Proteica , Conformação Proteica , Pterinas/química , Relação Estrutura-Atividade , Ressonância de Plasmônio de SuperfícieRESUMO
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.
Assuntos
Difosfotransferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Tioguanina/farmacologia , Cristalografia por Raios X , Difosfotransferases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Tioguanina/análogos & derivados , Tioguanina/químicaRESUMO
As the second essential enzyme of the folate biosynthetic pathway, the potential antimicrobial target, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase), catalyzes the Mg(2+-)dependant transfer of pyrophosphate from the cofactor (ATP) to the substrate, 6-hydroxymethyl-7,8-dihydropterin. Recently, we showed that 8-mercaptoguanine (8-MG) bound at the substrate site (KD â¼13 µM), inhibited the S. aureus enzyme (SaHPPK) (IC50 â¼ 41 µM), and determined the structure of the SaHPPK/8-MG complex. Here we present the synthesis of a series of guanine derivatives, together with their HPPK binding affinities, as determined by SPR and ITC analysis. The binding mode of the most potent was investigated using 2D NMR spectroscopy and X-ray crystallography. The results indicate, firstly, that the SH group of 8-MG makes a significant contribution to the free energy of binding. Secondly, direct N(9) substitution, or tautomerization arising from N(7) substitution in some cases, leads to a dramatic reduction in affinity due to loss of a critical N(9)-H···Val46 hydrogen bond, combined with the limited space available around the N(9) position. The water-filled pocket under the N(7) position is significantly more tolerant of substitution, with a hydroxyl ethyl 8-MG derivative attached to N(7) (compound 21a) exhibiting an affinity for the apo enzyme comparable to the parent compound (KD â¼ 12 µM). In contrast to 8-MG, however, 21a displays competitive binding with the ATP cofactor, as judged by NMR and SPR analysis. The 1.85 Å X-ray structure of the SaHPPK/21a complex confirms that extension from the N(7) position towards the Mg(2+)-binding site, which affords the only tractable route out from the pterin-binding pocket. Promising strategies for the creation of more potent binders might therefore include the introduction of groups capable of interacting with the Mg(2+) centres or Mg(2+)-binding residues, as well as the development of bitopic inhibitors featuring 8-MG linked to a moiety targeting the ATP cofactor binding site.
Assuntos
Vias Biossintéticas/efeitos dos fármacos , Difosfotransferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Ácido Fólico/biossíntese , Guanina/análogos & derivados , Guanina/farmacologia , Sítios de Ligação , Difosfotransferases/química , Difosfotransferases/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/química , Guanina/química , Ligantes , Modelos Moleculares , Conformação Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , TermodinâmicaRESUMO
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.
Assuntos
Difosfotransferases/antagonistas & inibidores , Inibidores Enzimáticos/química , Pterinas/química , Adenina/química , Sítios de Ligação , Cristalografia por Raios X , Difosfotransferases/metabolismo , Inibidores Enzimáticos/síntese química , Ligação de Hidrogênio , Ligação Proteica , Estrutura Terciária de Proteína , Pterinas/síntese química , Especificidade por SubstratoRESUMO
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.
Assuntos
Difosfotransferases/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/química , Pterinas/química , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Difosfotransferases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Cinética , Conformação Molecular , Estrutura Terciária de Proteína , Pterinas/síntese química , Pterinas/farmacologia , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyses the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HMDP), and is an essential enzyme in the biosynthesis of folic acid. It is also a potential target for antimicrobial drugs. HPPK from Escherichia coli, which has been the most intensively investigated, is a monomeric protein with a molecular mass of about 18,000. Structures of the enzyme, determined by X-ray crystallography and NMR, have shown that it adopts an alpha/beta fold with a substrate-binding cleft on the surface. Three loop regions surround the enzyme active site and form intimate contacts with the substrates. The enzyme has a fixed order of substrate binding, with ATP binding first, followed by HMDP. Binding of ATP causes a shift in the conformations of the loop regions, which completes formation of the HMDP-binding site. Two magnesium ions bind within the active site, bridging between the phosphate groups in ATP and the enzyme. Both ions appear to play an integral role in ATP recognition and stabilization of the transition state of the reaction. Ligand binding and kinetic studies have shown that the overall rate of the reaction is not limited by the rate of substrate transformation into products on the enzyme, which is relatively fast, but is more likely caused by a slow step associated with product release. These fundamental studies open up the potential for exploitation through the design of specific HPPK inhibitors.
Assuntos
Difosfotransferases/metabolismo , Antibacterianos/farmacologia , Domínio Catalítico , Difosfotransferases/antagonistas & inibidores , Difosfotransferases/química , Difosfotransferases/genética , Escherichia coli/enzimologia , Modelos Moleculares , Conformação ProteicaRESUMO
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a key enzyme in the folate-biosynthetic pathway and is essential for microorganisms but absent from mammals. HPPK catalyzes Mg(2+)-dependent pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). Previously, three-dimensional structures of Escherichia coli HPPK (EcHPPK) have been determined at almost every stage of its catalytic cycle and the reaction mechanism has been established. Here, the crystal structure of Yersinia pestis HPPK (YpHPPK) in complex with HP and an ATP analog is presented together with thermodynamic and kinetic characterizations. The two HPPK molecules differ significantly in a helix-loop area (alpha2-Lp3). YpHPPK has lower affinities than EcHPPK for both nucleotides and HP, but its rate constants for the mechanistic steps of both chemical transformation and product release are comparable with those of EcHPPK. Y. pestis, which causes plague, is a category A select agent according to the Centers for Disease Control and Prevention (CDC). Therefore, these structural and biochemical data are valuable for the design of novel medical countermeasures against plague.
Assuntos
Difosfotransferases/química , Inibidores Enzimáticos/farmacologia , Peste/tratamento farmacológico , Yersinia pestis/enzimologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Substituição de Aminoácidos , Animais , Sítios de Ligação , Catálise , Cristalografia por Raios X , Dimerização , Difosfotransferases/antagonistas & inibidores , Difosfotransferases/genética , Desenho de Fármacos , Inibidores Enzimáticos/uso terapêutico , Humanos , Cinética , Modelos Moleculares , Peste/microbiologia , Conformação Proteica , Pterinas/química , Proteínas Recombinantes/química , Termodinâmica , Yersinia pestis/efeitos dos fármacosRESUMO
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), leading to the biosynthesis of folate cofactors. Like other enzymes in the folate pathway, HPPK is an ideal target for the development of antimicrobial agents because the enzyme is essential for microorganisms but is absent from human and animals. Three bisubstrate analogues have been synthesized for HPPK and characterized by biochemical and X-ray crystallographic analyses. All three bisubstrate analogues consist of a pterin, an adenosine moiety, and a link composed of 2-4 phosphoryl groups. P(1)-(6-Hydroxymethylpterin)-P(2)-(5'-adenosyl)diphosphate (HP(2)A, 5) shows little affinity and inhibitory activity for E. coli HPPK. P(1)-(6-Hydroxymethylpterin)-P(3)-(5'-adenosyl)triphosphate (HP(3)A, 6) shows moderate affinity and inhibitory activity with K(d) = 4.25 microM in the presence of Mg(2+) and IC(50) = 1.27 microM. P(1)-(6-Hydroxymethylpterin)-P(4)-(5'-adenosyl)tetraphosphate (HP(4)A, 7) shows the highest affinity and inhibitory activity with K(d) = 0.47 microM in the presence of Mg(2+) and IC(50) = 0.44 microM. The affinity of MgHP(4)A for HPPK is approximately 116 and 76 times higher than that of MgADP and 6-hydroxymethylpterin, respectively. The crystal structure of HPPK in complex with 7 (HPPK.MgHP(4)A) has been determined at 1.85 A resolution with a crystallographic R factor of 0.185. The crystal structure shows that 7 occupies both HP- and ATP-binding sites and induces significant conformational changes in HPPK. The biochemical and structural studies of the bisubstrate analogues indicate that the bisubstrate analogue approach can produce more potent inhibitors for HPPK and the minimum length of the link for a bisubstrate analogue is approximately 7 A.
Assuntos
Trifosfato de Adenosina/síntese química , Antibacterianos/síntese química , Difosfotransferases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Pterinas/síntese química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Antibacterianos/química , Cristalografia por Raios X , Inibidores Enzimáticos/química , Escherichia coli/química , Fluorometria , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , Ligação Proteica , Pterinas/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Folates are essential for life. Unlike mammals, most microorganisms must synthesize folates de novo. 6-Hydroxymethyl-7, 8-dihydropterin pyrophosphokinase (HPPK) catalyzes pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), the first reaction in the folate pathway, and therefore is an ideal target for developing novel antimicrobial agents. HPPK from Escherichia coli is a 158-residue thermostable protein that provides a convenient model system for mechanistic studies. Crystal structures have been reported for HPPK without bound ligand, containing an HP analog, and complexed with an HP analog, two Mg(2+) ions, and ATP. RESULTS: We present the 1.25 A crystal structure of HPPK in complex with HP, two Mg(2+) ions, and AMPCPP (an ATP analog that inhibits the enzymatic reaction). This structure demonstrates that the enzyme seals the active center where the reaction occurs. The comparison with unligated HPPK reveals dramatic conformational changes of three flexible loops and many sidechains. The coordination of Mg(2+) ions has been defined and the roles of 26 residues have been derived. CONCLUSIONS: HPPK-HP-MgAMPCPP mimics most closely the natural ternary complex of HPPK and provides details of protein-substrate interactions. The coordination of the two Mg(2+) ions helps create the correct geometry for the one-step reaction of pyrophosphoryl transfer, for which we suggest an in-line single displacement mechanism with some associative character in the transition state. The rigidity of the adenine-binding pocket and hydrogen bonds are responsible for adenosine specificity. The nonconserved residues that interact with the substrate might be responsible for the species-dependent properties of an isozyme.