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1.
Cell Biol Int ; 48(5): 638-646, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38328902

RESUMO

The bile salt export pump (ABCB11/BSEP) is a hepatocyte plasma membrane-resident protein translocating bile salts into bile canaliculi. The sequence alignment of the four full-length transporters of the ABCB subfamily (ABCB1, ABCB4, ABCB5 and ABCB11) indicates that the NBD-NBD contact interface of ABCB11 differs from that of other members in only four residues. Notably, these are all located in the noncanonical nucleotide binding site 1 (NBS1). Substitution of all four deviant residues with canonical ones (quadruple mutant) significantly decreased the transport activity of the protein. In this study, we mutated two deviant residues in the signature sequence to generate a double mutant (R1221G/E1223Q). Furthermore, a triple mutant (E502S/R1221G/E1223Q) was generated, in which the deviant residues of the signature sequence and Q-loop were mutated concurrently to canonical residues. The double and triple mutants showed 80% and 60%, respectively, of the activity of wild-type BSEP. As expected, an increasing number of mutations gradually impair transport as an intricate network of interactions within the ABC proteins ensures proper functioning.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Nucleotídeos , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Nucleotídeos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Mutação/genética , Sítios de Ligação
2.
PLoS Genet ; 16(10): e1009016, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33031417

RESUMO

Several ABC exporters carry a degenerate nucleotide binding site (NBS) that is unable to hydrolyze ATP at a rate sufficient for sustaining transport activity. A hallmark of a degenerate NBS is the lack of the catalytic glutamate in the Walker B motif in the nucleotide binding domain (NBD). The multidrug resistance transporter ABCB1 (P-glycoprotein) has two canonical NBSs, and mutation of the catalytic glutamate E556 in NBS1 renders ABCB1 transport-incompetent. In contrast, the closely related bile salt export pump ABCB11 (BSEP), which shares 49% sequence identity with ABCB1, naturally contains a methionine in place of the catalytic glutamate. The NBD-NBD interfaces of ABCB1 and ABCB11 differ only in four residues, all within NBS1. Mutation of the catalytic glutamate in ABCB1 results in the occlusion of ATP in NBS1, leading to the arrest of the transport cycle. Here we show that despite the catalytic glutamate mutation (E556M), ABCB1 regains its ATP-dependent transport activity, when three additional diverging residues are also replaced. Molecular dynamics simulations revealed that the rescue of ATPase activity is due to the modified geometry of NBS1, resulting in a weaker interaction with ATP, which allows the quadruple mutant to evade the conformationally locked pre-hydrolytic state to proceed to ATP-driven transport. In summary, we show that ABCB1 can be transformed into an active transporter with only one functional catalytic site by preventing the formation of the ATP-locked pre-hydrolytic state in the non-canonical site.


Assuntos
Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Transporte Biológico/genética , Proteínas de Ciclo Celular/genética , Proteínas Nucleares/genética , Domínio AAA/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Transporte Biológico Ativo/genética , Domínio Catalítico/genética , Ácido Glutâmico/genética , Humanos , Hidrólise , Metionina/genética , Simulação de Dinâmica Molecular , Mutação/genética , Nucleotídeos/genética , Ligação Proteica/genética , Domínios Proteicos/genética
3.
Clin Chem Lab Med ; 59(5): 987-994, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33554519

RESUMO

OBJECTIVES: The qualitative results of SARS-CoV-2 specific real-time reverse transcription (RT) PCR are used for initial diagnosis and follow-up of Covid-19 patients and asymptomatic virus carriers. However, clinical decision-making and health management policies often are based additionally on cycle threshold (Ct) values (i.e., quantitative results) to guide patient care, segregation and discharge management of individuals testing positive. Therefore, an analysis of inter-protocol variability is needed to assess the comparability of the quantitative results. METHODS: Ct values reported in a SARS-CoV-2 virus genome detection external quality assessment challenge were analyzed. Three positive and two negative samples were distributed to participating test laboratories. Qualitative results (positive/negative) and quantitative results (Ct values) were assessed. RESULTS: A total of 66 laboratories participated, contributing results from 101 distinct test systems and reporting Ct values for a total of 92 different protocols. In all three positive samples, the means of the Ct values for the E-, N-, S-, RdRp-, and ORF1ab-genes varied by less than two cycles. However, 7.7% of reported results deviated by more than ±4.0 (maximum 18.0) cycles from the respective individual means. These larger deviations appear to be systematic errors. CONCLUSIONS: In an attempt to use PCR diagnostics beyond the identification of infected individuals, laboratories are frequently requested to report Ct values along with a qualitative result. This study highlights the limitations of interpreting Ct values from the various SARS-CoV genome detection protocols and suggests that standardization is necessary in the reporting of Ct values with respect to the target gene.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , DNA Viral/análise , Genoma Viral , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/química , COVID-19/diagnóstico , Teste de Ácido Nucleico para COVID-19/estatística & dados numéricos , Reações Falso-Negativas , Reações Falso-Positivas , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos
4.
Clin Chem Lab Med ; 59(10): 1735-1744, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34187131

RESUMO

OBJECTIVES: External quality assessment (EQA) schemes provide information on individual and general analytical performance of participating laboratories and test systems. The aim of this study was to investigate the use and performance of SARS-CoV-2 virus genome detection systems in Austrian laboratories and their preparedness to face challenges associated with the pandemic. METHODS: Seven samples were selected to evaluate performance and estimate variability of reported results. Notably, a dilution series was included in the panel as a measure of reproducibility and sensitivity. Several performance criteria were evaluated for individual participants as well as in the cohort of all participants. RESULTS: A total of 109 laboratories participated and used 134 platforms, including 67 different combinations of extraction and PCR platforms and corresponding reagents. There were no false positives and 10 (1.2%) false negative results, including nine in the weakly positive sample (Ct ∼35.9, ∼640 copies/mL). Twenty (22%) laboratories reported results of mutation detection. Twenty-five (19%) test systems included amplification of human RNA as evidence of proper sampling. The overall linearity of Ct values from individual test systems for the dilution series was good, but inter-assay variability was high. Both operator-related and systematic failures appear to have caused incorrect results. CONCLUSIONS: Beyond providing certification for participating laboratories, EQA provides the opportunity for participants to evaluate their performance against others so that they may improve operating procedures and test systems. Well-selected EQA samples offer additional inferences to be made about assay sensitivity and reproducibility, which have practical applications.


Assuntos
COVID-19/diagnóstico , Genoma Viral , Garantia da Qualidade dos Cuidados de Saúde , SARS-CoV-2/isolamento & purificação , Áustria/epidemiologia , COVID-19/virologia , Humanos , Laboratórios , Técnicas de Diagnóstico Molecular/métodos , Pandemias , SARS-CoV-2/genética , Sensibilidade e Especificidade
5.
Int J Mol Sci ; 22(2)2021 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-33466755

RESUMO

The bile salt export pump (BSEP/ABCB11) is responsible for the transport of bile salts from hepatocytes into bile canaliculi. Malfunction of this transporter results in progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2) and intrahepatic cholestasis of pregnancy (ICP). Over the past few years, several small molecular weight compounds have been identified, which hold the potential to treat these genetic diseases (chaperones and potentiators). As the treatment response is mutation-specific, genetic analysis of the patients and their families is required. Furthermore, some of the mutations are refractory to therapy, with the only remaining treatment option being liver transplantation. In this review, we will focus on the molecular structure of ABCB11, reported mutations involved in cholestasis and current treatment options for inherited BSEP deficiencies.


Assuntos
Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Ácidos e Sais Biliares/metabolismo , Colestase Intra-Hepática/genética , Mutação , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Colestase Intra-Hepática/tratamento farmacológico , Colestase Intra-Hepática/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/uso terapêutico
6.
Biophys J ; 114(2): 331-342, 2018 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-29401431

RESUMO

P-glycoprotein, also known as multidrug resistance protein 1 or ABCB1, can export a wide range of chemically unrelated compounds, including chemotherapeutic drugs. ABCB1 consists of two transmembrane domains that form the substrate binding and translocation domain, and of two cytoplasmic nucleotide binding domains (NBDs) that energize substrate transport by ATP binding and hydrolysis. ATP binding triggers dimerization of the NBDs, which switches the transporter from an inward facing to an outward facing transmembrane domain conformation. We performed MD simulations to study the dynamic behavior of the NBD dimer in the presence or absence of nucleotides. In the apo configuration, the NBDs were overall attractive to each other as shown in the potential of mean force profile, but the energy well was shallow and broad. In contrast, a sharp and deep energy minimum (∼-42 kJ/mol) was found in the presence of ATP, leading to a well-defined conformation. Motif interaction network analyses revealed that ATP stabilizes the NBD dimer by serving as the central hub for interdomain connections. Simulations showed that forces promoting dimerization are multilayered, dominated by electrostatic interactions between the nucleotide and conserved amino acids of the signature sequence and the Walker A motif. In addition, direct and water-bridged hydrogen bonds between NBDs provided conformation-defining interactions. Importantly, we characterized a largely unrecognized but essential contribution from hydrophobic interactions between the adenine moiety of the nucleotides and a hydrophobic surface of the X-loop to the stabilization of the nucleotide-bound NBD dimer. These hydrophobic interactions lead to a sharp energy minimum, thereby conformationally restricting the nucleotide-bound state.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Nucleotídeos/metabolismo , Multimerização Proteica , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Estrutura Quaternária de Proteína
7.
Mol Pharmacol ; 92(4): 401-413, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28784620

RESUMO

The bile salt export pump (BSEP/ABCB11) transports bile salts from hepatocytes into bile canaliculi. Its malfunction is associated with severe liver disease. One reason for functional impairment of BSEP is systemic administration of drugs, which as a side effect inhibit the transporter. Therefore, drug candidates are routinely screened for potential interaction with this transporter. Hence, understanding the functional biology of BSEP is of key importance. In this study, we engineered the transporter to dissect interdomain communication paths. We introduced mutations in noncanonical and in conserved residues of either of the two nucleotide binding domains and determined the effect on BSEP basal and substrate-stimulated ATPase activity as well as on taurocholate transport. Replacement of the noncanonical methionine residue M584 (Walker B sequence of nucleotide binding site 1) by glutamate imparted hydrolysis competency to this site. Importantly, this mutation was able to sustain 15% of wild-type transport activity, when the catalytic glutamate of the canonical nucleotide binding site 2 was mutated to glutamine. Kinetic modeling of experimental results for the ensuing M584E/E1244Q mutant suggests that a transfer of hydrolytic capacity from the canonical to the noncanonical nucleotide binding site results in loss of active and adoption of facilitative characteristics. This facilitative transport is ATP-gated. To the best of our knowledge, this result is unprecedented in ATP-binding cassette proteins with one noncanonical nucleotide binding site. Our study promotes an understanding of the domain interplay in BSEP as a basis for exploration of drug interactions with this transporter.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Ácidos e Sais Biliares/metabolismo , Colestase Intra-Hepática/metabolismo , Ácido Taurocólico/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Sítios de Ligação/fisiologia , Transporte Biológico/fisiologia , Células HEK293 , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
8.
J Hepatol ; 66(1): 95-101, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27593105

RESUMO

BACKGROUND & AIMS: Cholestasis is characterized by intrahepatic accumulation of potentially cytotoxic bile acids (BAs) subsequently leading to liver injury with disruption of hepatocellular integrity, inflammation, fibrosis and ultimately liver cirrhosis. Bile salt export pump (BSEP/ABCB11) is the main canalicular BA transporter and therefore the rate limiting step for hepatobiliary BA excretion. In this study we aimed to investigate the role of BSEP/ABCB11 in the development of acquired cholestatic liver and bile duct injury. METHODS: Wild-type (WT) and BSEP knockout (BSEP-/-) mice were subjected to common bile duct ligation (CBDL) or 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) feeding as models for cholestasis with biliary obstruction and bile duct injury. mRNA expression profile, serum biochemistry, liver histology, immunohistochemistry, hepatic hydroxyproline levels and BA composition as well as biliary pressure were assessed. RESULTS: BSEP-/- mice were protected against acquired cholestatic liver injury induced by 7days of CBDL or 4weeks of DDC feeding, as reflected by unchanged serum levels of liver transaminases, alkaline phosphatase and BAs. Notably, BSEP-/- mice were also protected from cholestasis-induced hepatic inflammation and biliary fibrosis. In line with induced BA detoxification/hydroxylation pathways in BSEP-/- mice, polyhydroxylated BAs were increased 4-fold after CBDL and 6-fold after DDC feeding in comparison with cholestatic WT mice. Finally, following CBDL, biliary pressure in WT mice increased up to 47mmH2O but remained below 11mmH2O in BSEP-/- mice. CONCLUSION: Metabolic preconditioning with subsequent changes in BA metabolism favors detoxification of potentially toxic BAs and thereby protects BSEP-/- mice from cholestatic liver and bile duct injury. LAY SUMMARY: Reduced hepatobiliary bile acid transport due to loss of BSEP function leads to increased hydroxylation of bile acids in the liver. Metabolic preconditioning with a hydrophilic bile pool protects the BSEP-/- mice from acquired cholestatic liver disease.


Assuntos
Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Ácidos e Sais Biliares/metabolismo , Ductos Biliares , Colestase Intra-Hepática/metabolismo , Hepatócitos/metabolismo , Ligadura/métodos , Oclusão Terapêutica/métodos , Animais , Canalículos Biliares , Ductos Biliares/fisiopatologia , Ductos Biliares/cirurgia , Colestase Intra-Hepática/prevenção & controle , Camundongos
9.
Mol Pharm ; 13(1): 163-71, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26642869

RESUMO

The bile salt export pump (BSEP) is an ABC-transporter expressed at the canalicular membrane of hepatocytes. Its physiological role is to expel bile salts into the canaliculi from where they drain into the bile duct. Inhibition of this transporter may lead to intrahepatic cholestasis. Predictive computational models of BSEP inhibition may allow for fast identification of potentially harmful compounds in large databases. This article presents a predictive in silico model based on physicochemical descriptors that is able to flag compounds as potential BSEP inhibitors. This model was built using a training set of 670 compounds with available BSEP inhibition potencies. It successfully predicted BSEP inhibition for two independent test sets and was in a further step used for a virtual screening experiment. After in vitro testing of selected candidates, a marketed drug, bromocriptin, was identified for the first time as BSEP inhibitor. This demonstrates the usefulness of the model to identify new BSEP inhibitors and therefore potential cholestasis perpetrators.


Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Bromocriptina/farmacologia , Animais , Células CHO , Linhagem Celular , Colestase/prevenção & controle , Simulação por Computador , Cricetulus , Suínos
10.
FASEB J ; 28(10): 4335-46, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25016028

RESUMO

For a primary active pump, such as the human ATP-binding-cassette (ABC) transporter ABCB1, coupling of drug-binding by the two transmembrane domains (TMDs) to the ATP catalytic cycle of the two nucleotide-binding domains (NBDs) is fundamental to the transport mechanism, but is poorly understood at the biochemical level. Structure data suggest that signals are transduced through intracellular loops of the TMDs that slot into grooves on the NBDs. At the base of these grooves is the Q loop. We therefore mutated the eponymous glutamine in one or both NBD Q loops and measured the effect on conformation and function by using a conformation-sensitive antibody (UIC2) and a fluorescent drug (Bodipy-verapamil), respectively. We showed that the double mutant is trapped in the inward-open state, which binds the drug, but cannot couple to the ATPase cycle. Our data also describe marked redundancy within the transport mechanism, because single-Q-loop mutants are functional for Bodipy-verapamil transport. This result allowed us to elucidate transduction pathways from twin drug-binding cavities to the Q loops using point mutations to favor one cavity over the other. Together, the data show that the Q loop is the central flexion point where the aspect of the drug-binding cavities is coupled to the ATP catalytic cycle.


Assuntos
Trifosfato de Adenosina/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico Ativo , Células HEK293 , Humanos , Dados de Sequência Molecular , Mutação Puntual , Ligação Proteica , Verapamil/farmacologia
11.
Mol Pharmacol ; 85(3): 420-8, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24366667

RESUMO

The multispecific efflux transporter, P-glycoprotein, plays an important role in drug disposition. Substrate translocation occurs along the interface of its transmembrane domains. The rotational C2 symmetry of ATP-binding cassette transporters implies the existence of two symmetry-related sets of substrate-interacting amino acids. These sets are identical in homodimeric transporters, and remain evolutionary related in full transporters, such as P-glycoprotein, in which substrates bind preferentially, but nonexclusively, to one of two binding sites. We explored the role of pore-exposed tyrosines for hydrogen-bonding interactions with propafenone type ligands in their preferred binding site 2. Tyrosine 953 is shown to form hydrogen bonds not only with propafenone analogs, but also with the preferred site 1 substrate rhodamine123. Furthermore, an accessory role of tyrosine 950 for binding of selected propafenone analogs is demonstrated. The present study demonstrates the importance of domain interface tyrosine residues for interaction of small molecules with P-glycoprotein.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Hidrogênio/metabolismo , Propafenona/metabolismo , Tirosina/genética , Tirosina/metabolismo , Sítios de Ligação/genética , Linhagem Celular , Células HEK293 , Humanos , Ligação de Hidrogênio , Ligantes , Mutação/genética , Estrutura Terciária de Proteína/genética
12.
Pharmacol Res ; 83: 63-73, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24316454

RESUMO

SLC6 family members and ABC transporters represent two extremes: SLC6 transporters are confined to the membrane proper and only expose small segments to the hydrophilic milieu. In ABC transporters the hydrophobic core is connected to a large intracellular (eponymous) ATP binding domain that is comprised of two discontiguous repeats. Accordingly, their folding problem is fundamentally different. This can be gauged from mutations that impair the folding of the encoded protein and give rise to clinically relevant disease phenotypes: in SLC6 transporters, these cluster at the protein-lipid interface on the membrane exposed surface. Mutations in ABC-transporters map to the interface between nucleotide binding domains and the coupling helices, which provide the connection to the hydrophobic core. Folding of these mutated ABC-transporters can be corrected with ligands/substrates that bind to the hydrophobic core. This highlights a pivotal role of the coupling helices in the folding trajectory. In contrast, insights into pharmacochaperoning of SLC6 transporters are limited to monoamine transporters - in particular the serotonin transporter (SERT) - because of their rich pharmacology. Only ligands that stabilize the inward facing conformation act as effective pharmacochaperones. This indicates that the folding trajectory of SERT proceeds via the inward facing conformation. Mutations that impair folding of SLC6 family members can be transmitted as dominant or recessive alleles. The dominant phenotype of the mutation can be rationalized, because SLC6 transporters are exported in oligomeric form from the endoplasmic reticulum (ER). Recessive transmission requires shielding of the unaffected gene product from the mutated transporter in the ER. This can be accounted for by a chaperone-COPII (coatomer protein II) exchange model, where proteinaceous ER-resident chaperones engage various intermediates prior to formation of the oligomeric state and subsequent export from the ER. It is likely that the action of pharmacochaperones is contingent on and modulated by these chaperones.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Descoberta de Drogas , Proteínas de Membrana Transportadoras/química , Dobramento de Proteína/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Humanos , Ligantes , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Mutação , Conformação Proteica/efeitos dos fármacos
13.
Bioorg Med Chem ; 22(7): 2311-9, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24613626

RESUMO

P-glycoprotein (P-gp) is an ATP-dependent multidrug resistance efflux transporter that plays an important role in anticancer drug resistance and in pharmacokinetics of medicines. Despite a large number of structurally and functionally diverse compounds, also flavonoids and chalcones have been reported as inhibitors of P-gp. The latter share some similarity with the well studied class of propafenones, but do not contain a basic nitrogen atom. Furthermore, due to their rigidity, they are suitable candidates for 3D-QSAR studies. In this study, a set of 22 new chalcone derivatives were synthesized and evaluated in a daunomycin efflux inhibition assay using the CCRF.CEM.VCR1000 cell line. The compound 10 showed the highest activity (IC50=42nM), which is one order of magnitude higher than the activity for an equilipohillic propafenone analogue. 2D- and 3D-QSAR studies indicate the importance of H-bond acceptors, methoxy groups, hydrophobic groups as well as the number of rotatable bonds as pharmacophoric features influencing P-gp inhibitory activity.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Chalcona/farmacologia , Relação Quantitativa Estrutura-Atividade , Chalcona/síntese química , Chalcona/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
14.
Drug Discov Today Technol ; 12: e87-94, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25027379

RESUMO

The endoplasmic reticulum (ER) quality control system distinguishes between correctly and incorrectly folded proteins to prevent processing of aberrantly folded conformations along the secretory pathway. Non-synonymous mutations can lead to misfolding of ABC proteins and associated disease phenotypes. Specific phenotypes may at least partially be corrected by small molecules, so-called pharmacological chaperones. Screening for folding correctors is expected to open an avenue for treatment of diseases such as cystic fibrosis and intrahepatic cholestasis.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Colestase Intra-Hepática/tratamento farmacológico , Fibrose Cística/tratamento farmacológico , Deficiências na Proteostase/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Transportadores de Cassetes de Ligação de ATP/genética , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Animais , Benzodioxóis/farmacologia , Benzodioxóis/uso terapêutico , Colestase Intra-Hepática/metabolismo , Ensaios Clínicos como Assunto , Fibrose Cística/metabolismo , Descoberta de Drogas , Humanos , Ligação Proteica , Dobramento de Proteína , Transporte Proteico , Deficiências na Proteostase/metabolismo , Bibliotecas de Moléculas Pequenas/uso terapêutico
15.
J Comput Aided Mol Des ; 27(2): 161-71, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23400406

RESUMO

The ATP-binding cassette efflux transporter P-glycoprotein (P-gp) is notorious for contributing to multidrug resistance in antitumor therapy. Due to its expression in many blood-organ barriers, it also influences the pharmacokinetics of drugs and drug candidates and is involved in drug/drug- and drug/nutrient interactions. However, due to lack of structural information the molecular basis of ligand/transporter interaction still needs to be elucidated. Towards this goal, a series of Benzopyranes and Benzopyrano[3,4b][1,4]oxazines have been synthesized and pharmacologically tested for their ability to inhibit P-gp mediated daunomycin efflux. Both quantitative structure-activity relationship (QSAR) models using simple physicochemical and novel GRID-independent molecular descriptors (GRIND) were established to shed light on the structural requirements for high P-gp inhibitory activity. The results from 2D-QSAR showed a linear correlation of vdW surface area (Å(2)) of hydrophobic atoms with the pharmacological activity. GRIND (3D-QSAR) studies allowed to identify important mutual distances between pharmacophoric features, which include one H-bond donor, two H-bond acceptors and two hydrophobic groups as well as their distances from different steric hot spots of the molecules. Activity of the compounds particularly increases with increase of the distance of an H-bond donor or a hydrophobic feature from a particular steric hot spot of the benzopyrane analogs.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Antibióticos Antineoplásicos/farmacologia , Benzopiranos/farmacologia , Resistência a Múltiplos Medicamentos , Oxazinas/química , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Relação Quantitativa Estrutura-Atividade , Benzopiranos/química , Transporte Biológico , Daunorrubicina/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Estrutura Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Células Tumorais Cultivadas
16.
Cell Mol Life Sci ; 69(1): 129-48, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21671119

RESUMO

An alternatively spliced form of human sulfonylurea receptor (SUR) 1 mRNA lacking exon 2 (SUR1Δ2) has been identified. The omission of exon 2 caused a frame shift and an immediate stop codon in exon 3 leading to translation of a 5.6-kDa peptide that comprises the N-terminal extracellular domain and the first transmembrane helix of SUR1. Based on a weak first splice acceptor site in the human SUR1 gene (ABCC8), RT-PCR revealed a concurrent expression of SUR1Δ2 and SUR1. The SUR1Δ2/(SUR1 + SUR1Δ2) mRNA ratio differed between tissues, and was lowest in pancreas (46%), highest in heart (88%) and negatively correlated with alternative splice factor/splicing factor 2 (ASF/SF2) expression. In COS-7 cells triple transfected with SUR1Δ2/SUR1/Kir6.2, the SUR1Δ2 peptide co-immunoprecipitated with Kir6.2, thereby displacing two of four SUR1 subunits on the cell surface. The ATP sensitivity of these hybrid ATP-sensitive potassium channels (K(ATP)) channels was reduced by about sixfold, as shown with single-channel recordings. RINm5f rat insulinoma cells, which genuinely express SUR1 but not SUR1Δ2, exhibited a strongly increased K(ATP) channel current upon transfection with SUR1Δ2. This led to inhibition of glucose-induced depolarization, calcium flux, insulin release and glibenclamide action. A non-mutagenic SNP on nucleotide position 333 (Pro69Pro) added another exonic splicing enhancer sequence detected by ASF/SF2, reduced relative abundance of SUR1Δ2 and slightly protected from non-insulin dependent diabetes in homozygotic individuals. Thus, SUR1Δ2 represents an endogenous K(ATP)-channel modulator with prodiabetic properties in islet cells. Its predominance in heart may explain why high-affinity sulfonylurea receptors are not found in human cardiac tissue.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Processamento Alternativo/fisiologia , Diabetes Mellitus Tipo 2 , Canais KATP/metabolismo , Polimorfismo de Nucleotídeo Único , Canais de Potássio Corretores do Fluxo de Internalização , Receptores de Droga , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Células COS , Cálcio/metabolismo , Chlorocebus aethiops , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Éxons/fisiologia , Glibureto/farmacologia , Humanos , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Canais KATP/efeitos dos fármacos , Miocárdio/metabolismo , Especificidade de Órgãos/genética , Pâncreas/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores de Droga/genética , Receptores de Droga/metabolismo , Especificidade da Espécie , Receptores de Sulfonilureias
17.
PLoS Comput Biol ; 7(5): e1002036, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21589945

RESUMO

Overexpression of the xenotoxin transporter P-glycoprotein (P-gp) represents one major reason for the development of multidrug resistance (MDR), leading to the failure of antibiotic and cancer therapies. Inhibitors of P-gp have thus been advocated as promising candidates for overcoming the problem of MDR. However, due to lack of a high-resolution structure the concrete mode of interaction of both substrates and inhibitors is still not known. Therefore, structure-based design studies have to rely on protein homology models. In order to identify binding hypotheses for propafenone-type P-gp inhibitors, five different propafenone derivatives with known structure-activity relationship (SAR) pattern were docked into homology models of the apo and the nucleotide-bound conformation of the transporter. To circumvent the uncertainty of scoring functions, we exhaustively sampled the pose space and analyzed the poses by combining information retrieved from SAR studies with common scaffold clustering. The results suggest propafenone binding at the transmembrane helices 5, 6, 7 and 8 in both models, with the amino acid residue Y307 playing a crucial role. The identified binding site in the non-energized state is overlapping with, but not identical to, known binding areas of cyclic P-gp inhibitors and verapamil. These findings support the idea of several small binding sites forming one large binding cavity. Furthermore, the binding hypotheses for both catalytic states were analyzed and showed only small differences in their protein-ligand interaction fingerprints, which indicates only small movements of the ligand during the catalytic cycle.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Propafenona/química , Propafenona/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Análise por Conglomerados , Descoberta de Drogas , Humanos , Camundongos , Modelos Moleculares , Ligação Proteica , Homologia Estrutural de Proteína , Relação Estrutura-Atividade
18.
Parasitol Res ; 110(6): 2289-95, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22215188

RESUMO

Malaria is still a major threat in many parts of the world with resistance spreading to almost all classes of antimalarials. The limited arsenal of available antimalarial drugs emphasizes the urgent need for novel antimalarial compounds. Owing to the fact that novel leads from nature have traditionally played a pivotal role in the development of various classes of antimalarials, we investigated a set of eight naturally occurring dietary flavonoids and their analogues for their antiplasmodial activity on clinical field isolates in southeastern Bangladesh and culture-adapted chloroquine-sensitive and chloroquine-resistant parasite clones. Except for taxifolin, all the other flavonoids had 50% inhibitory concentrations below 14 µM, both in the field and laboratory-adapted parasites. Neither of the flavonoids showed any activity correlation with chloroquine. The quercetin analogue rutin (7.10 ± 10.32 µM) was the most active substance in field isolates as well as laboratory-adapted cultures (3.53 ± 13.34 µM in 3D7 and 10.38 ± 15.08 µM in K1), providing the first evidence of its activity against Plasmodium falciparum parasites. Thus, our results provide important evidence of the antimalarial activity of flavonoids in traditional use and thus warrant further investigation of these compounds as potential antiplasmodial agents.


Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Flavonóis/química , Flavonóis/farmacologia , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Adolescente , Adulto , Idoso , Animais , Bangladesh , Criança , Feminino , Humanos , Concentração Inibidora 50 , Masculino , Pessoa de Meia-Idade , Testes de Sensibilidade Parasitária , Plasmodium falciparum/isolamento & purificação , Adulto Jovem
19.
Mol Pharmacol ; 79(3): 443-52, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21177413

RESUMO

The human multispecific drug efflux transporter P-glycoprotein (P-gp) causes drug resistance and modulates the pharmacological profile of systemically administered medicines. It has arisen from a homodimeric ancestor by gene duplication. Crystal structures of mouse MDR1A indicate that P-gp shares the overall architecture with two homodimeric bacterial exporters, Sav1866 and MsbA, which have complete rotational symmetry. For ATP-binding cassette transporters, nucleotide binding occurs in two symmetric positions in the motor domains. Based on the homology with entirely symmetric half-transporters, the present study addressed the key question: can biochemical evidence for the existence of dual drug translocation pathways in the transmembrane domains of P-gp be found? P-gp was photolabeled with propafenone analogs, purified, and digested proteolytically, and peptide fragments were identified by high-resolution mass spectrometry. Labeling was assigned to two regions in the protein by projecting data into homology models. Subsequently, symmetric residue pairs in the putative translocation pathways were identified and replaced by site-directed mutagenesis. Transport assays corroborated the existence of two pseudosymmetric translocation pathways. Although rhodamine123 has a preference to take one path, verapamil, propafenones, and vinblastine preferentially use the other. Two major findings ensued from this study: the existence of two solute translocation pathways in P-gp as a reflection of evolutionary origin from a homodimeric ancestor and selective but not exclusive use of one of these pathways by different P-gp solutes. The pseudosymmetric behavior reconciles earlier kinetic and thermodynamic data, suggesting an alternative concept of drug transport by P-gp that will aid in understanding the off-target quantitative structure activity relationships of P-gp interacting drugs.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/fisiologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Transporte Proteico , Rodamina 123/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
20.
Bioorg Med Chem ; 19(7): 2190-8, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21419632

RESUMO

Aim of this study was to label the potent dual P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) inhibitor elacridar (1) with (18)F to provide a positron emission tomography (PET) radiotracer to visualize Pgp and BCRP. A series of new 1- and 2-halogen- and nitro-substituted derivatives of 1 (4a-e) was synthesized as precursor molecules and reference compounds for radiolabelling and shown to display comparable in vitro potency to 1 in increasing rhodamine 123 accumulation in a cell line overexpressing human Pgp (MDCKII-MDR1). 1-[(18)F]fluoroelacridar ([(18)F]4b) was synthesized in a decay-corrected radiochemical yield of 1.7±0.9% by a 1-step no-carrier added nucleophilic aromatic (18)F-substitution of 1-nitro precursor 4c. Small-animal PET imaging of [(18)F]4b was performed in naïve rats, before and after administration of unlabelled 1 (5 mg/kg, n=3), as well as in wild-type and Mdr1a/b((-/-))Bcrp1((-/-)) mice (n=3). In PET experiments in rats, administration of unlabelled 1 increased brain activity uptake by a factor of 9.5 (p=0.0002, 2-tailed Student's t-test), whereas blood activity levels remained unchanged. In Mdr1a/b((-/-))Bcrp1((-/-)) mice, the mean brain-to-blood ratio of activity at 60 min after tracer injection was 7.6 times higher as compared to wild-type animals (p=0.0002). HPLC analysis of rat brain tissue extracts collected at 40 min after injection of [(18)F]4b revealed that 93±7% of total radioactivity in brain was in the form of unchanged [(18)F]4b. In conclusion, the in vivo behavior of [(18)F]4b was found to be similar to previously described [(11)C]1 suggesting transport of [(18)F]4b by Pgp and/or BCRP at the rodent BBB. However, low radiochemical yields and a significant degree of in vivo defluorination will limit the utility of [(18)F]4b as a PET tracer.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Transportadores de Cassetes de Ligação de ATP/análise , Acridinas/química , Neoplasias da Mama/diagnóstico por imagem , Radioisótopos de Flúor/química , Proteínas de Neoplasias/análise , Compostos Radiofarmacêuticos/síntese química , Tetra-Hidroisoquinolinas/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Barreira Hematoencefálica/diagnóstico por imagem , Barreira Hematoencefálica/metabolismo , Neoplasias da Mama/sangue , Neoplasias da Mama/metabolismo , Resistência a Múltiplos Medicamentos , Feminino , Humanos , Marcação por Isótopo , Camundongos , Modelos Moleculares , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/química , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual
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