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1.
J Membr Biol ; 257(1-2): 79-89, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38436710

RESUMO

The gastric H+,K+-ATPase is an integral membrane protein which derives energy from the hydrolysis of ATP to transport H+ ions from the parietal cells of the gastric mucosa into the stomach in exchange for K+ ions. It is responsible for the acidic environment of the stomach, which is essential for digestion. Acid secretion is regulated by the recruitment of the H+,K+-ATPase from intracellular stores into the plasma membrane on the ingestion of food. The similar amino acid sequences of the lysine-rich N-termini α-subunits of the H+,K+- and Na+,K+-ATPases, suggests similar acute regulation mechanisms, specifically, an electrostatic switch mechanism involving an interaction of the N-terminal tail with the surface of the surrounding membrane and a modulation of the interaction via regulatory phosphorylation by protein kinases. From a consideration of sequence alignment of the H+,K+-ATPase and an analysis of its coevolution with protein kinase C and kinases of the Src family, the evidence points towards a phosphorylation of tyrosine-7 of the N-terminus by either Lck or Yes in all vertebrates except cartilaginous fish. The results obtained will guide and focus future experimental research.


Assuntos
ATPase Trocadora de Sódio-Potássio , Estômago , Animais , ATPase Trocadora de Sódio-Potássio/metabolismo , Transporte Biológico , ATPase Trocadora de Hidrogênio-Potássio/química , Íons/metabolismo
2.
Annu Rev Biochem ; 89: 583-603, 2020 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31874046

RESUMO

P-type ATPases are found in all kingdoms of life and constitute a wide range of cation transporters, primarily for H+, Na+, K+, Ca2+, and transition metal ions such as Cu(I), Zn(II), and Cd(II). They have been studied through a wide range of techniques, and research has gained very significant insight on their transport mechanism and regulation. Here, we review the structure, function, and dynamics of P2-ATPases including Ca2+-ATPases and Na,K-ATPase. We highlight mechanisms of functional transitions that are associated with ion exchange on either side of the membrane and how the functional cycle is regulated by interaction partners, autoregulatory domains, and off-cycle states. Finally, we discuss future perspectives based on emerging techniques and insights.


Assuntos
Trifosfato de Adenosina/química , ATPases Transportadoras de Cobre/química , ATPase Trocadora de Hidrogênio-Potássio/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , ATPase Trocadora de Sódio-Potássio/química , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Cátions Bivalentes , Cátions Monovalentes , ATPases Transportadoras de Cobre/genética , ATPases Transportadoras de Cobre/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/genética , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Humanos , Transporte de Íons , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Prótons , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Imagem Individual de Molécula , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Especificidade por Substrato
3.
Proc Natl Acad Sci U S A ; 116(33): 16332-16337, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31371510

RESUMO

Phospholipid flippases (P4-ATPases) utilize ATP to translocate specific phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thus generating and maintaining transmembrane lipid asymmetry essential for a variety of cellular processes. P4-ATPases belong to the P-type ATPase protein family, which also encompasses the ion transporting P2-ATPases: Ca2+-ATPase, Na+,K+-ATPase, and H+,K+-ATPase. In comparison with the P2-ATPases, understanding of P4-ATPases is still very limited. The electrogenicity of P4-ATPases has not been explored, and it is not known whether lipid transfer between membrane bilayer leaflets can lead to displacement of charge across the membrane. A related question is whether P4-ATPases countertransport ions or other substrates in the opposite direction, similar to the P2-ATPases. Using an electrophysiological method based on solid supported membranes, we observed the generation of a transient electrical current by the mammalian P4-ATPase ATP8A2 in the presence of ATP and the negatively charged lipid substrate phosphatidylserine, whereas only a diminutive current was generated with the lipid substrate phosphatidylethanolamine, which carries no or little charge under the conditions of the measurement. The current transient seen with phosphatidylserine was abolished by the mutation E198Q, which blocks dephosphorylation. Likewise, mutation I364M, which causes the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome, strongly interfered with the electrogenic lipid translocation. It is concluded that the electrogenicity is associated with a step in the ATPase reaction cycle directly involved in translocation of the lipid. These measurements also showed that no charged substrate is being countertransported, thereby distinguishing the P4-ATPase from P2-ATPases.


Assuntos
Adenosina Trifosfatases/genética , Transporte Biológico/genética , Lipídeos de Membrana/genética , Proteínas de Transferência de Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Ataxia Cerebelar/genética , Citoplasma/genética , Citoplasma/metabolismo , Fenômenos Eletrofisiológicos/genética , ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/genética , Humanos , Deficiência Intelectual/genética , Lipídeos de Membrana/metabolismo , Mutação/genética , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/genética , Especificidade por Substrato/genética
4.
Bioorg Chem ; 81: 389-395, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30199841

RESUMO

Hydrazone compounds were considered as a useful moiety in drug design development. Therefore, these studies were aimed at the synthesis of new dihydrazones and were screened for their in vitro H+/K+-ATPase and anti-inflammatory activities. The results revealed that compounds 9 (22 ±â€¯0.62 µg/mL), 10 (26 ±â€¯0.91 µg/mL), 15 (24 ±â€¯0.44 µg/mL), 16 (28 ±â€¯0.63 µg/mL), 17 (12 ±â€¯0.38 µg/mL), 18 (14 ±â€¯0.47 µg/mL), 19 (26 ±â€¯0.54 µg/mL), 20 (16 ±â€¯0.41 µg/mL), 25 (06 ±â€¯0.68 µg/mL) and 26 (08 ±â€¯0.43 µg/mL) showed excellent H+/K+-ATPase activity and their IC50 value were lower than the standard drug Omerazole (48 ±â€¯0.12 µg/mL). Compounds 5 (28 ±â€¯0.65 µg/mL), 6 (24 ±â€¯0.61 µg/mL), 7 (28 ±â€¯0.64 µg/mL), 8 (26 ±â€¯0.45 µg/mL), 11 (30 ±â€¯0.74 µg/mL), 12 (28 ±â€¯0.40 µg/mL), 13 (32 ±â€¯0.24 µg/mL), 14 (30 ±â€¯0.55 µg/mL) and 21 (08 ±â€¯0.47 µg/mL), 22 (12 ±â€¯0.47 µg/mL), 23 (10 ±â€¯0.51 µg/mL) and 24 (14 ±â€¯0.84 µg/mL) showed better anti-inflammatory activity compared to standard indomethacin (44 ±â€¯0.15 µg/mL). The structure activity relationship (SAR) showed that, electron donating groups (OH, OCH3) favored the H+/K+-ATPase and antioxidants activity, whereas, electron withdrawing groups (F, Cl, Br and NO2) favored the anti-inflammatory activity. Furthermore, molecular docking study was performed to investigate the binding interactions of the most active analogs with the active site of H+/K+-ATPase enzyme. Compounds 25 (G-score = -9.063) and 26 (G-score = -8.977) showed the highest docking G-scores for H+/K+-ATPase inhibition activity.


Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Hidrazonas/química , Hidrazonas/farmacologia , Inibidores da Bomba de Prótons/química , Inibidores da Bomba de Prótons/farmacologia , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Domínio Catalítico/efeitos dos fármacos , Células Cultivadas , Inibidores de Ciclo-Oxigenase 2/química , Inibidores de Ciclo-Oxigenase 2/farmacologia , Desenho de Fármacos , ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Humanos , Simulação de Acoplamento Molecular , Ovinos , Relação Estrutura-Atividade
5.
Sci Rep ; 8(1): 12732, 2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-30143663

RESUMO

The H+, K+-ATPase (HKA) uses ATP to pump protons into the gastric lumen against a million-fold proton concentration gradient while counter-transporting K+ from the lumen. The mechanism of release of a proton into a highly acidic stomach environment, and the subsequent binding of a K+ ion necessitates a network of protonable residues and dynamically changing protonation states in the cation binding pocket dominated by five acidic amino acid residues E343, E795, E820, D824, and D942. We perform molecular dynamics simulations of spontaneous K+ binding to all possible protonation combinations of the acidic amino acids and carry out free energy calculations to determine the optimal protonation state of the luminal-open E2P state of the pump which is ready to bind luminal K+. A dynamic pKa correlation analysis reveals the likelihood of proton transfer events within the cation binding pocket. In agreement with in-vitro measurements, we find that E795 is likely to be protonated, and that E820 is at the center of the proton transfer network in the luminal-open E2P state. The acidic residues D942 and D824 are likely to remain protonated, and the proton redistribution occurs predominantly amongst the glutamate residues exposed to the lumen. The analysis also shows that a lower number of K+ ions bind at lower pH, modeled by a higher number of protons in the cation binding pocket, in agreement with the 'transport stoichiometry variation' hypothesis.


Assuntos
ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Potássio/metabolismo , Prótons , Animais , Sítios de Ligação , Células HEK293 , Humanos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Conformação Proteica , Suínos , Termodinâmica
6.
J Membr Biol ; 251(5-6): 653-666, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30056551

RESUMO

The catalytic α-subunits of both the Na+,K+-ATPase and the gastric H+,K+-ATPase possess lysine-rich N-termini which project into the cytoplasm. Due to conflicting experimental results, it is currently unclear whether the N-termini play a role in ion pump function or regulation, and, if they do, by what mechanism. Comparison of the lysine frequencies of the N-termini of both proteins with those of all of their extramembrane domains showed that the N-terminal lysine frequencies are far higher than one would expect simply from exposure to the aqueous solvent. The lysine frequency was found to vary significantly between different vertebrate classes, but this is due predominantly to a change in N-terminal length. As evidenced by a comparison between fish and mammals, an evolutionary trend towards an increase of the length of the N-terminus of the H+,K+-ATPase on going from an ancestral fish to mammals could be identified. This evolutionary trend supports the hypothesis that the N-terminus is important in ion pump function or regulation. In placental mammals, one of the lysines is replaced by serine (Ser-27), which is a target for protein kinase C. In most other animal species, a lysine occupies this position and hence no protein kinase C target is present. Interaction with protein kinase C is thus not the primary role of the lysine-rich N-terminus. The disordered structure of the N-terminus may, via increased flexibility, facilitate interaction with another binding partner, e.g. the surrounding membrane, or help to stabilise particular enzyme conformations via the increased entropy it produces.


Assuntos
ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Lisina/química , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , ATPase Trocadora de Hidrogênio-Potássio/classificação , Modelos Moleculares , Conformação Proteica , Proteína Quinase C/química , Proteína Quinase C/classificação , Proteína Quinase C/metabolismo , Análise de Sequência de Proteína , ATPase Trocadora de Sódio-Potássio/classificação
7.
Nature ; 556(7700): 214-218, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29618813

RESUMO

The gastric proton pump-the H+, K+-ATPase-is a P-type ATPase responsible for acidifying the gastric juice down to pH 1. This corresponds to a million-fold proton gradient across the membrane of the parietal cell, the steepest known cation gradient of any mammalian tissue. The H+, K+-ATPase is an important target for drugs that treat gastric acid-related diseases. Here we present crystal structures of the H+, K+-ATPase in complex with two blockers, vonoprazan and SCH28080, in the luminal-open state, at 2.8 Å resolution. The drugs have partially overlapping but clearly distinct binding modes in the middle of a conduit running from the gastric lumen to the cation-binding site. The crystal structures suggest that the tight configuration at the cation-binding site lowers the pK a value of Glu820 sufficiently to enable the release of a proton even into the pH 1 environment of the stomach.


Assuntos
ATPase Trocadora de Hidrogênio-Potássio/química , Estômago/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cátions Monovalentes/metabolismo , Cristalografia por Raios X , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Imidazóis/química , Imidazóis/farmacologia , Modelos Moleculares , Potássio/metabolismo , Ligação Proteica , Inibidores da Bomba de Prótons/química , Inibidores da Bomba de Prótons/farmacologia , Prótons , Pirróis/química , Pirróis/farmacologia , Coelhos , Sulfonamidas/química , Sulfonamidas/farmacologia , Suínos
8.
J Mol Graph Model ; 78: 129-138, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29055186

RESUMO

Acid suppressant SCH28080 and its derivatives reversibly reduce acid secretion activity of the H+,K+-ATPase in a K+ competitive manner. The results on homologation of the SCH28080 by varying the linker chain length suggested the improvement in efficacy. However, the pharmacokinetic studies reveal that the hydrophobic nature of the CH2 linker units may not help it to function as a better acid suppressant. We have exploited the role of linker unit to enhance the efficacy of such reversible acid suppressant drug molecules using hetero linker, i.e., disulfide and peroxy linkers. The logarithm of partition coefficient defined for a drug molecule relates to the partition coefficient, which allows the optimum solubility characteristics to reach the active site. The logarithm of partition coefficient calculated for the designed inhibitors suggests that inhibitors would possibly reach the active site in sufficient concentration like in the case of SCH28080. The steered molecular dynamics studies have revealed that the Inhibitor-1 with disulfide linker unit is more stable at the active site due to greater noncovalent interactions compared to the SCH28080. Centre of mass distance analysis suggests that the Cysteine-813 amino acid residue selectively plays an important role in the inhibition of H+,K+-ATPase for Inhibitor-1. Furthermore, the quantum chemical calculations with M11L/6-31+G(d,p) level of theory have been performed to account the noncovalent interactions responsible for the stabilization of inhibitor molecules in the active site gorge of the gastric proton pump at different time scale. The hydrogen bonding and hydrophobic interaction studies corroborate the center of mass distance analysis as well. Well-tempered metadynamics free energy surface and center of mass separation analysis for the Inhibitor-1 is in good agreement with the steered molecular dynamics results. The torsional angle of the linker units seems to be crucial for better efficacy of drug molecules. The torsional angle of linker units of SCH28080 (COCH2C) and of Inhibitor 1 (CSSC) prefers to lie within ∼60°-90° for a longer time during the simulations, whereas, the peroxy linker (COOC) of Inhibitor 2 prefers to adopt ∼120-160°. Therefore, it appears that the smaller torsion angle of linker units can achieve better interactions with the active site residues of H+,K+-ATPase to inhibit the acid secretion activity. The reversible drug molecules with disulfide linker unit would be a promising candidate as proton pump antagonist to H+,K+-ATPase.


Assuntos
ATPase Trocadora de Hidrogênio-Potássio/química , Imidazóis/química , Inibidores da Bomba de Prótons/química , Sequência de Aminoácidos , Sítios de Ligação/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Imidazóis/uso terapêutico , Simulação de Dinâmica Molecular , Inibidores da Bomba de Prótons/uso terapêutico
9.
Cell Physiol Biochem ; 43(3): 1037-1051, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28968600

RESUMO

BACKGROUND/AIMS: Glucose-stimulated insulin secretion (GSIS) of pancreatic ß-cells involves glucose uptake and metabolism, closure of KATP channels and depolarization of the cell membrane potential (Vmem), activation of voltage-activated Ca2+ currents (ICav) and influx of Ca2+, which eventually triggers hormone exocytosis. Beside this classical pathway, KATP-independent mechanisms such as changes in intracellular pH (pHi) or cell volume, which also affect ß-cell viability, can elicit or modify insulin release. In ß-cells the regulation of pHi is mainly accomplished by Na+/H+ exchangers (NHEs). To investigate if other proton extrusion mechanisms than NHEs are involved in pH regulation, we tested for the presence of the non-gastric H+/K+ ATPase in rat insulinoma cells and assessed effects of the H+/K+ ATPase inhibitor SCH-28080 on insulin secretion, cell viability and apoptosis. METHODS: In INS-1E cell cultures, H+/K+ ATPase gene and protein expression was analyzed by reverse transcription PCR and Western blotting. Intracellular pH (pHi) recovery after acute acidic load was measured by NH4Cl prepulsing using BCECF. Insulin secretion was determined by ELISA from the cell culture supernatant. Vmem, K+ and Ca2+ currents were recorded using patch clamp. Overall cell responses were determined using resazurin (viability) and cytotoxicity assays. The mean cell volume (MCV), cell granularity (side-scatter; SSC), phosphatidylserine (PS) exposure, cell membrane integrity, caspase activity and the mitochondrial membrane potential (ΔΨm) were measured by flow cytometry. RESULTS: We found that the α-subunit of the non-gastric H+/K+ ATPase (HKα2) is expressed on mRNA and protein level. However, compared to rat colon tissue, in INS-1E cells mRNA abundance was very low. In NH4Cl prepulsing experiments no K+-dependent pHi recovery was observed under Na+-free extracellular conditions. Nonetheless within 1 h, 20 µM SCH-28080 inhibited GSIS by ∼50%, while basal release was unaffected. The L-type ICav blocker nifedipine caused a full inhibition of GSIS at 10 and 20 µM. At 20 µM, SCH-28080 inhibited ICav comparable to 20 µM nifedipine and in addition augmented IKATP recorded at -60 mV and hyperpolarized Vmem by ∼15 mV. Cell viability 2 and 24 h post treatment with SCH-28080 was dose-dependently inhibited with IC50 values of 22.9 µM and 15.3 µM, respectively. At 20 µM the percentages of Annexin-V+, caspase+ and propidium iodide+ cells were significantly increased after 24 and 48 h. Concurrently, the MCV was significantly decreased (apoptotic volume decrease, AVD) and the SSC signal was increased. At concentrations >40-50 µM, SCH-28080 became progressively cytotoxic causing a steep increase in necrotic cells already 2 h post treatment and a breakdown of ΔΨm within 4 h under 50 and 100 µM while 10 and 20 µM had no effect on ΔΨm within 24 h. CONCLUSION: We demonstrate expression of HKα2 in rat INS-1E cells. However, the pump is apparently non-functional under the given conditions. Nonetheless the H+/K+ ATPase blocker SCH-28080 inhibits insulin secretion and induces cell death. Importantly, we show that SCH-28080 inhibits ICav - and activates KATP channels identifying them as novel "off-targets" of the inhibitor, causing hyperpolarization of Vmem and inhibition of insulin secretion.


Assuntos
Apoptose/efeitos dos fármacos , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Imidazóis/toxicidade , Insulina/análise , Inibidores da Bomba de Prótons/toxicidade , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colo/metabolismo , Ensaio de Imunoadsorção Enzimática , Glucose/farmacologia , ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/genética , Concentração de Íons de Hidrogênio , Insulina/metabolismo , Secreção de Insulina , Insulinoma/metabolismo , Insulinoma/patologia , Canais KATP/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Nifedipino/toxicidade , Técnicas de Patch-Clamp , Fosfatidilserinas/farmacologia , RNA Mensageiro/metabolismo , Ratos
10.
Sci Rep ; 7(1): 6632, 2017 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-28747707

RESUMO

The gastric proton pump H+,K+-ATPase acidifies the gastric lumen, and thus its inhibitors, including the imidazo[1,2-a]pyridine class of K+-competitive acid blockers (P-CABs), have potential application as acid-suppressing drugs. We determined the electron crystallographic structure of H+,K+-ATPase at 6.5 Å resolution in the E2P state with bound BYK99, a potent P-CAB with a restricted ring structure. The BYK99 bound structure has an almost identical profile to that of a previously determined structure with bound SCH28080, the original P-CAB prototype, but is significantly different from the previously reported P-CAB-free form, illustrating a common conformational change is required for P-CAB binding. The shared conformational changes include a distinct movement of transmembrane helix 2 (M2), from its position in the previously reported P-CAB-free form, to a location proximal to the P-CAB binding site in the present BYK99-bound structure. Site-specific mutagenesis within M2 revealed that D137 and N138, which face the P-CAB binding site in our model, significantly affect the inhibition constant (K i) of P-CABs. We also found that A335 is likely to be near the bridging nitrogen at the restricted ring structure of the BYK99 inhibitor. These provide clues to elucidate the binding site parameters and mechanism of P-CAB inhibition of gastric acid secretion.


Assuntos
Microscopia Crioeletrônica , ATPase Trocadora de Hidrogênio-Potássio/química , Inibidores da Bomba de Prótons/química , Piridinas/química , Animais , Ligação Proteica , Conformação Proteica , Inibidores da Bomba de Prótons/metabolismo , Piridinas/metabolismo , Suínos
11.
Bioorg Med Chem ; 25(14): 3719-3735, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28522264

RESUMO

With the aim to discover a gastric antisecretory agent more potent than the existing proton pump inhibitors, novel 3,4-dihydro-1H-spiro(naphthalene-2,2'-piperidin)-1-one derivatives, which could occupy two important lipophilic pockets (described as LP-1 and LP-2) of H+,K+-ATPase and can strongly bind to the K+-binding site, were designed based on a docking model. Among the compounds synthesized, compound 4d showed a strong H+,K+-ATPase-inhibitory activity and a high stomach concentration in rats, resulting in potent inhibitory action on histamine-stimulated gastric acid secretion in rats. Furthermore, 4d exerted significant inhibitory action on histamine-stimulated gastric-acid secretion in rats with a rapid onset and moderate duration of action after the administration. These findings may lead to a new insight into the drug design of potassium-competitive acid blockers.


Assuntos
ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Piperidinas/química , Potássio/metabolismo , Inibidores da Bomba de Prótons/síntese química , Compostos de Espiro/química , Administração Intravenosa , Animais , Área Sob a Curva , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Ácido Gástrico/metabolismo , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/química , Meia-Vida , Histamina/toxicidade , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Naftalenos/química , Piperidinas/síntese química , Piperidinas/farmacocinética , Potássio/química , Inibidores da Bomba de Prótons/química , Inibidores da Bomba de Prótons/farmacocinética , Curva ROC , Ratos , Compostos de Espiro/síntese química , Compostos de Espiro/farmacocinética , Relação Estrutura-Atividade
12.
Bioorg Med Chem Lett ; 27(14): 3048-3054, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28554871

RESUMO

A series of new of furan derivatised [1,4] benzothiazepine analogues were synthesized starting from 1-(furan-2-yl)ethanone. 1-(Furan-2-yl)ethanone was converted into chalcones by its reaction with various aromatic aldehydes, then were reacted with 2-aminobenzenethiol in acidic conditions to obtain the title compounds in good yields. The synthesized new compounds were characterized by 1H NMR, 13C NMR, Mass spectral studies and elemental analyses. All the new compounds were evaluated for their in vitro VRV-PL-8a and H+/K+ ATPase inhibitor properties. Preliminary studies revealed that, some molecules amongst the designed series showed promising VRV-PL-8a and H+/K+ ATPase inhibitor properties. Further, rigid body docking studies were performed to understand possible docking sites of the molecules on the target proteins and the mode of binding. This finding presents a promising series of lead molecules that can serve as prototypes for the treatment of inflammatory related disorder that can mitigate the ulcer inducing side effect shown by other NSAIDs.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Fosfolipases A2 do Grupo II/antagonistas & inibidores , ATPase Trocadora de Hidrogênio-Potássio/química , Tiazepinas/síntese química , Tiazepinas/farmacologia , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Sítios de Ligação , Chalconas/química , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Furanos/química , Fosfolipases A2 do Grupo II/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Conformação Molecular , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Inibidores da Bomba de Prótons/síntese química , Inibidores da Bomba de Prótons/química , Inibidores da Bomba de Prótons/metabolismo , Inibidores da Bomba de Prótons/farmacologia , Tiazepinas/química , Tiazepinas/metabolismo
13.
Proc Natl Acad Sci U S A ; 114(2): 316-321, 2017 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-28028214

RESUMO

Na+,K+-ATPase and H+,K+-ATPase are electrogenic and nonelectrogenic ion pumps, respectively. The underlying structural basis for this difference has not been established, and it has not been revealed how the H+,K+-ATPase avoids binding of Na+ at the site corresponding to the Na+-specific site of the Na+,K+-ATPase (site III). In this study, we addressed these questions by using site-directed mutagenesis in combination with enzymatic, transport, and electrophysiological functional measurements. Replacement of the cysteine C932 in transmembrane helix M8 of Na+,K+-ATPase with arginine, present in the H+,K+-ATPase at the corresponding position, converted the normal 3Na+:2K+:1ATP stoichiometry of the Na+,K+-ATPase to electroneutral 2Na+:2K+:1ATP stoichiometry similar to the electroneutral transport mode of the H+,K+-ATPase. The electroneutral C932R mutant of the Na+,K+-ATPase retained a wild-type-like enzyme turnover rate for ATP hydrolysis and rate of cellular K+ uptake. Only a relatively minor reduction of apparent Na+ affinity for activation of phosphorylation from ATP was observed for C932R, whereas replacement of C932 with leucine or phenylalanine, the latter of a size comparable to arginine, led to spectacular reductions of apparent Na+ affinity without changing the electrogenicity. From these results, in combination with structural considerations, it appears that the guanidine+ group of the M8 arginine replaces Na+ at the third site, thus preventing Na+ binding there, although allowing Na+ to bind at the two other sites and become transported. Hence, in the H+,K+-ATPase, the ability of the M8 arginine to donate an internal cation binding at the third site is decisive for the electroneutral transport mode of this pump.


Assuntos
Substituição de Aminoácidos , Arginina , Cisteína , ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Sódio-Potássio/química , Sítios de Ligação , Ligação Competitiva , Cátions , Membrana Celular/enzimologia , ATPase Trocadora de Hidrogênio-Potássio/genética , Hemiplegia , Humanos , Canais Iônicos , Transporte de Íons , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fenilalanina , Potássio/metabolismo , Conformação Proteica , Subunidades Proteicas/química , Bombas de Próton , Alinhamento de Sequência , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética
14.
J Phys Chem B ; 120(51): 13031-13038, 2016 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-27992223

RESUMO

Acid-related gastric diseases are associated with disorder of digestive tract acidification due to the acid secretion by gastric proton pump, H+,K+-ATPase. Omeprazole is one of the persuasive irreversible inhibitor of the proton pump H+,K+-ATPase. However, the reports on the mechanistic pathway of irreversible proton pump inhibitors (PPIs) on the acid activation and formation of disulfide complex are scarce in the literature. We have examined the acid activation PPIs, i.e., timoprazole, S-omeprazole and R-omeprazole using M062X/6-31++G(d,p) in aqueous phase with SMD solvation model. The proton pump inhibitor is a prodrug and activated in the acidic canaliculi of the gastric pump H+,K+-ATPase to sulfenic acid which can either form another acid activate intermediate sulfenamide or a disulfide complex with cysteine amino acid of H+,K+-ATPase. The quantum chemical calculations suggest that the transition state (TS5) for the disulfide complex formation is the rate-determining step of the multistep acid inhibition process by PPIs. The free energy barrier of TS5 is 5.5 kcal/mol higher for timoprazole compared to the S-omeprazole. The stability of the transition state for the formation of disulfide bond between S-omeprazole and cysteine amino acid of H+,K+-ATPase is governed by inter- and intramolecular hydrogen bonding. The disulfide complex for S-omeprazole is thermodynamically more stable by 4.5 kcal/mol in aqueous phase compared to disulfide complex of timoprazole, which corroborates the less efficacy of timoprazole as irreversible PPI for acid inhibition process. It has been speculated that sulfenic acid can either form sulfenamide or a stable disulfide complex with cysteine amino acid residue of H+,K+-ATPase. The M062X/6-31++G(d,p) level of theory calculated results reveal that the formation of tetra cyclic sulfenamide is unfavored by ∼17 kcal/mol for S-omeprazole and 11.5 kcal/mol for timoprazole compared to the disulfide complex formation in each case. The DFT calculations have further shed light on the acid activation process of R- and S-isomers of omeprazole. The calculated results suggest that the efficacy of these isomers lie on their metabolic pathway and excretion from human body.


Assuntos
2-Piridinilmetilsulfinilbenzimidazóis/química , Ácido Gástrico/química , ATPase Trocadora de Hidrogênio-Potássio/química , Omeprazol/química , Pró-Fármacos/química , Inibidores da Bomba de Prótons/química , 2-Piridinilmetilsulfinilbenzimidazóis/metabolismo , Sítios de Ligação , Dissulfetos/química , Dissulfetos/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Omeprazol/metabolismo , Pró-Fármacos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Inibidores da Bomba de Prótons/metabolismo , Sulfamerazina/química , Sulfamerazina/metabolismo , Ácidos Sulfênicos/química , Ácidos Sulfênicos/metabolismo , Termodinâmica , Água/metabolismo
15.
PLoS One ; 11(10): e0163696, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27783698

RESUMO

The stomach, which is characterized by acid peptic digestion in vertebrates, has been lost secondarily multiple times in the evolution of the teleost fishes. The Cypriniformes are largely seen as an agastric order; however, within the superfamily Cobitoidea, the closely related sister groups Nemacheilidae and Balitoridae have been identified as gastric families. The presence of these most recently diverged gastric families in an otherwise agastric clade indicates that either multiple (>2-3) loss events occurred with the Cyprinidae, Catostomidae and Cobitidae, or that gastric reinvention arose in a recent ancestor of the Nemacheilidae/Balitoridae sister clade. In the present study, the foregut regions of Cobitidae, Nemacheilidae/Balitoridae and the ancestral Botiidae family members were examined for the presence of gastric glands and gastric proton pump (Atp4a) α subunit expression by histology and immunohistochemistry respectively. Atp4a gene expression was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Gastric glands expressing apical H+/K+-ATPase α subunit and isolated partial sequences of atp4a, identified using degenerate primers showing clear orthology to other vertebrate atp4a sequences, were detected in representative species from Nemacheilidae/ Balitoridae and Botiidae, but not Cobitidae (Misgurnus anguillicaudatus). In summary, we provide evidence for an uninterrupted gastric evolutionary lineage in the Cobitoidea, making it highly improbable that the stomach was reinvented in the Nemacheilidae/Balitoridae clade consistent with Dollo's principle. These results also indicate that the gastric trait may be present elsewhere in the Cobitoidea.


Assuntos
Cipriniformes/metabolismo , Mucosa Gástrica/metabolismo , Sequência de Aminoácidos , Animais , Cipriniformes/classificação , Evolução Molecular , Expressão Gênica , ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/genética , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Intestinos/patologia , Dados de Sequência Molecular , Fenótipo , Filogenia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA/isolamento & purificação , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Estômago/patologia
16.
Mol Biosyst ; 12(6): 1772-80, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26952080

RESUMO

Affinity probes are useful tools for determining molecular targets and elucidating mechanism of action for novel, bioactive compounds. In the case of covalent inhibitors, activity based probes are particularly valuable for ensuring acceptable selectivity margins. However, there is a variety of bioorthogonal chemistry reactions available for modifying compounds of interest with clickable tags. Here, we describe a direct comparison of tetrazine ligation and strain promoted azide-alkyne cycloaddition using benzimidazole based probes to bind their known target, the gastric proton pump, ATP4A. This study validates the use of chemical probes for target identification and illustrates the superior efficiency of tetrazine ligation for copper-free click systems. In addition, we have identified several novel binding partners of benzimidazole probes: Isoform 2 of deleted in malignant brain tumors 1 protein (DMBT1) and three uncharacterized proteins.


Assuntos
Benzimidazóis/química , ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Sondas Moleculares/química , Inibidores da Bomba de Prótons/química , Benzimidazóis/síntese química , Cobre/química , Corantes Fluorescentes/química , Espectrometria de Massas , Microscopia de Fluorescência , Sondas Moleculares/síntese química , Estrutura Molecular , Inibidores da Bomba de Prótons/síntese química , Coloração e Rotulagem
17.
Biochim Biophys Acta ; 1858(6): 1228-35, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26922883

RESUMO

The gastric proton pump (H(+),K(+)-ATPase) consists of a catalytic α-subunit (αHK) and a glycosylated ß-subunit (ßHK). ßHK glycosylation is essential for the apical trafficking and stability of αHK in gastric parietal cells. Here, we report the properties of sialic acids at the termini of the oligosaccharide chains of ßHK. Sialylation of ßHK was found in LLC-PK1 cells stably expressing αHK and ßHK by staining of the cells with lectin-tagged fluorescent polymeric nanoparticles. This sialylation was also confirmed by biochemical studies using sialic acid-binding lectin beads and an anti-ßHK antibody. The sialic acids of ßHK are cleaved enzymatically by neuraminidase (sialidase) and nonenzymatically by an acidic solution (pH5). Interestingly, the enzymatic activity of H(+),K(+)-ATPase was significantly decreased by cleavage of the sialic acids of ßHK. In contrast, ßHK was not sialylated in the gastric tubulovesicles prepared from the stomach of fed hogs. The H(+),K(+)-ATPase activity in these tubulovesicles was not significantly altered by neuraminidase. Importantly, the sialylation of ßHK was observed in the gastric samples prepared from the stomach of famotidine (a histamine H2 receptor antagonist)-treated rats, but not histamine (an acid secretagogue)-treated rats. The enzymatic activity of H(+),K(+)-ATPase in the samples of the famotidine-treated rats was significantly higher than in the histamine-treated rats. The effects of famotidine were weakened by neuraminidase. These results indicate that ßHK is sialylated at neutral or weakly acidic pH, but not at acidic pH, suggesting that the sialic acids of ßHK positively regulate the enzymatic activity of αHK.


Assuntos
ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Estômago/enzimologia , Animais , Famotidina/farmacologia , ATPase Trocadora de Hidrogênio-Potássio/química , Células LLC-PK1 , Ácido N-Acetilneuramínico/metabolismo , Estômago/efeitos dos fármacos , Suínos
18.
J Comput Aided Mol Des ; 30(1): 27-37, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26667240

RESUMO

The interaction mechanism of triazolyl substituted tetrahydrobenzofuran derivatives (compound 1 (N, N-Dipropyl-1-(2-phenyl-4,5,6,7-tetrahydrobenzofuran-4-yl)-1H-1,2,3-triazole-4-methanamine) and 2 (1-(2-Phenyl-4,5,6,7-tetrahydrobenzofuran-4-yl)-4-(morpholin-4-ylmethyl)-1H-1,2,3-triazole)) with H(+),K(+)-ATPase at different pH were studied by induced-fit docking, QM/MM optimization and MM/GBSA binding free energy calculations of two forms (neutral and protonated form) of compounds. The inhibition activity of compound 1 is measured and almost unchanged at different pH, while the activity of compound 2 increases significantly with pH value decreased. This phenomenon could be explained by their protonated form percentages and the calculated binding free energies of protonated and neutral mixture of compounds at different pH. The binding free energy of protonated form is higher than that of neutral form of compound, and the protonated form could be a powerful inhibitor of H(+),K(+)-ATPase. By the decomposed energy comparisons of residues in binding sites, Asp137 should be the key binding site to protonated form of compound because of the hydrogen bond and electrostatic interactions. These calculation results could help for further rational design of novel H(+),K(+)-ATPase inhibitors.


Assuntos
Benzofuranos/química , Benzofuranos/farmacologia , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Inibidores da Bomba de Prótons/química , Inibidores da Bomba de Prótons/farmacologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , ATPase Trocadora de Hidrogênio-Potássio/química , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Alinhamento de Sequência , Suínos , Termodinâmica
19.
Methods Mol Biol ; 1377: 19-27, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26695019

RESUMO

Gastric H(+),K(+)-ATPase is an ATP-driven proton pump responsible for the acid secretion. Here, we describe the procedure for the isolation of H(+),K(+)-ATPase-enriched membrane vesicle fractions by Ficoll/sucrose density gradient centrifugation. Further purification by SDS treatment of membrane fractions is also introduced. These procedures allow us to obtain purified protein preparations in a quantity of several tens of milligrams, with the specific activity of ~480 µmol/mg/h. High purity and stability of H(+),K(+)-ATPase in the membrane preparation enable us to evaluate its detailed biochemical properties, and also to obtain 2D crystals for structural analysis.


Assuntos
Membrana Celular/enzimologia , ATPase Trocadora de Hidrogênio-Potássio/isolamento & purificação , Biologia Molecular/métodos , Estômago/enzimologia , Animais , Centrifugação com Gradiente de Concentração , ATPase Trocadora de Hidrogênio-Potássio/química , Suínos
20.
Methods Mol Biol ; 1377: 281-91, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26695040

RESUMO

Voltage clamp fluorometry has become a powerful tool to compare partial reactions of P-type ATPases such as the Na(+),K(+)-ATPase and H(+),K(+)-ATPase with conformational dynamics of these ion pumps. Here, we describe the methodology to heterologously express membrane proteins in X. laevis oocytes and site-specifically label these proteins with one or more fluorophores. Fluorescence changes are measured simultaneously with current measurements under two-electrode voltage clamp conditions.


Assuntos
Fluorometria/métodos , ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Técnicas de Patch-Clamp/métodos , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , ATPase Trocadora de Hidrogênio-Potássio/genética , Oócitos/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , ATPase Trocadora de Sódio-Potássio/genética , Xenopus laevis/genética
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