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ABSTRACT: P-glycoprotein acts as a protective barrier against xenobiotics and cellular toxicants in the human body while playing an important role in drug transportation in many organs. Overexpression of p-glycoprotein can lead to a decrease in the absorption of many drugs. After screening, 33 phytochemicals from 25 spices were selected for docking with p-glycoprotein to detect some naturally occurring p-glycoprotein inhibitors to modulate multidrug resistance. Absorption, distribution, metabolism, excretion, and toxicity prediction and drug-like properties of those ligands were investigated from pkCSM, Molinspiration, and SwissADME software, followed by molecular docking study and molecular dynamic simulation on BIOVIA Discovery Studio. These 33 phytochemicals met the criteria of p-glycoprotein inhibitor as much as the reference drug verapamil. Pandamarilactone-31 showed the highest binding affinity for p-glycoprotein, acting as the lead p-glycoprotein inhibitor, followed by α-D-fructofuranoside methyl, sesamolinol, and nigellidine.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Simulación del Acoplamiento Molecular , Fitoquímicos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Humanos , Fitoquímicos/farmacología , Simulación por Computador , Simulación de Dinámica MolecularRESUMEN
LPS induced sepsis is a complex process involving various immune cells and signaling molecules. Dysregulation of macrophage polarization and ROS production contributed to the pathogenesis of sepsis. PGP is a transmembrane transporter responsible for the efflux of a number of drugs and also expressed in murine macrophages. Natural products have been shown to decrease inflammation and expression of efflux transporters. However, no treatment is currently available to treat LPS induced sepsis. Verapamil and Tangeretin also reported to attenuate lipopolysaccharide-induced inflammation. However, the effects of verapamil or tangeretin on lipopolysaccharide (LPS)-induced sepsis and its detailed anti-inflammatory mechanism have not been reported. Here, we have determined that verapamil and tangeretin protects against LPS-induced sepsis by suppressing M1 macrophages populations and also through the inhibition of P-glycoprotein expression via downregulating STAT1/STAT3 and upregulating SOCS3 expression in macrophages. An hour before LPS (10 mg/kg) was administered; mice were given intraperitoneal injections of either verapamil (5 mg/kg) or tangeretin (5 mg/kg). The peritoneal macrophages from different experimental groups of mice were isolated. Hepatic, pulmonary and splenic morphometric analyses revealed that verapamil and tangeretin decreased the infiltration of neutrophils into the tissues. Verapamil and tangeritin also enhanced the activity of SOD, CAT, GRX and GSH level in all the tissues tested. verapamil or tangeretin pre-treated mice shifted M1 macrophages to M2 type possibly through the inhibition of P-glycoprotein expression via downregulating STAT1/STAT3 and upregulating SOCS3 expression. Hence, both these drugs have shown protective effects in sepsis via suppressing iNOS, COX-2, oxidative stress and NF-κB signaling in macrophages. Therefore, in our study we can summarize that mice were treated with either Vera or Tan before LPS administration cause an elevated IL-10 by the macrophages which enhances the SOCS3 expression, and thereby able to limits STAT1/STAT3 inter-conversion in the macrophages. As a result, NF-κB activity is also getting down regulated and ultimately mitigating the adverse effect of inflammation caused by LPS in resident macrophages. Whether verapamil or tangeretin offers such protection possibly through the inhibition of P-glycoprotein expression in macrophages needs clarification with the bio availability of these drugs under PGP inhibited conditions is a limitation of this study.
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Flavonas , Lipopolisacáridos , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Proteína 3 Supresora de la Señalización de Citocinas , Verapamilo , Animales , Verapamilo/farmacología , Factor de Transcripción STAT1/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas/genética , Flavonas/farmacología , Flavonas/uso terapéutico , Ratones , Factor de Transcripción STAT3/metabolismo , Masculino , Sepsis/tratamiento farmacológico , Sepsis/inmunología , Sepsis/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Regulación hacia Abajo/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/inmunología , Células Cultivadas , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/inmunología , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The lipopolysaccharide, a microbial toxin, is one of the major causative agents of sepsis. P-gp expression and its functions are altered during inflammation. LPS has been known to impair the functions of P-gp, an efflux transporter. But the effect of LPS on P-gp expression in murine peritoneal macrophages is poorly understood. Molecular docking studies reveal that vitexin is a potent substrate and verapamil a potent inhibitor of P-gp. In the present experimental study, the curative potential of vitexin as a fruit component and verapamil treated as a control inhibitor of P-gp was examined in a murine LPS sepsis model. The effects of vitexin and verapamil on P-gp expression in macrophages correlating with changes in macrophage polarization and associated functional responses during LPS induced sepsis were studied. Peritoneal macrophages of LPS (10 mg/kg body weight) challenged mice exhibited elevated levels of H2O2, superoxide, and NO in parallel with lower antioxidant activity. LPS treatment increased P-gp expression through increased TLR4/expression. However, LPS challenged mice treated with vitexin (5 mg/kg body weight) + verapamil (5 mg/kg body weight) showed higher anti-oxidant enzyme activity (SOD, CAT and GRx) resulting in reduced oxidative stress. This combination treatment also elevated TNFR2, concomitant with down-regulation of TLR4, NF-κB and P-gp expression in murine peritoneal macrophages, resulting in a switch from M1 to M2 polarisation of macrophages and reduced inflammatory responses. In conclusion, combined vitexin and verapamil treatment could be used as a promising therapy to regulate P-gp expression and protection against LPS mediated sepsis and inflammatory damages.
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Apigenina , FN-kappa B , Sepsis , Ratones , Animales , FN-kappa B/metabolismo , Lipopolisacáridos/farmacología , Receptor Toll-Like 4/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/farmacología , Verapamilo/farmacología , Peróxido de Hidrógeno/metabolismo , Simulación del Acoplamiento Molecular , Macrófagos/metabolismo , Glicoproteínas/metabolismo , Sepsis/tratamiento farmacológico , Subfamilia B de Transportador de Casetes de Unión a ATP/farmacología , Peso CorporalRESUMEN
Background: Oral lichen planus (OLP) is an autoimmune disease primarily affecting the middle-aged females. The present study aims to determine the relation of the oestrogen receptor alpha (ERα) with OLP pathogenesis, correlating it with the possible cause of its higher prevalence among females. Materials and Methods: Clinically and histologically identified fifteen of each pre-menopausal and peri-menopausal OLP female patients were chosen for this study. The expression of ERα was analysed from the collected lesion tissue samples by using two-step semi-quantitative reverse transcriptase polymerase chain reaction (SqRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Results: mRNA and protein expression of ERα were significantly higher in both groups of OLP female patients when compared with the control. The perimenopausal OLP patients showed significantly elevated expression of ERα compared to premenopausal patients. Conclusion: Higher expression of ERα in pre- and peri-menopausal females may be a causative factor for the higher prevalence of OLP among females.
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Background: Oral lichen planus (OLP) is a chronic inflammatory disease. Apoptosis of the basal keratinocytes is a causative factor for OLP pathogenesis but the detailed mechanism of apoptosis among nondysplastic and dysplastic OLP lesions is yet unraveled. Aims: This study aims to evaluate the involvement of cellular DNA fragmentation and alteration in the expression of Bcl-2 and B-cell lymphoma extra-large (Bcl-xL), p53, p21 and heat shock protein 70 (HSP70) in nondysplastic and dysplastic OLP lesions. Materials and Methods: Untreated, fifteen OLP patients each with nondysplastic and dysplastic lesions were enrolled for this study. Their DNA fragmentation was analyzed by the agarose gel electrophoresis method. The mRNA expression of Bcl-2, Bcl-xL, p53, p21 and HSP70 were measured using semi-quantitative reverse transcription-polymerase chain reaction. Results: Elevated DNA fragmentations were found in dysplastic lesions compared to nondysplastic type. Significantly higher expression of Bcl-2, Bcl-xL, p53 and p21 were found in both types of OLP lesion compared to the control. Expression of Bcl-2 and Bcl-xL were significantly elevated in nondysplastic lesions, whereas significantly overexpression of p53 and p21 were found in dysplastic lesions. Anti-stress protein HSP70 was overtly expressed in dysplastic lesions compared to other groups. Conclusion: Reduced expression of Bcl-2 and Bcl-xL, with elevated DNA fragmentation, may be associated with increased apoptosis in dysplastic lesions which aid in the resolution of the chronic inflammatory process. Higher expression of p53 and p21 in dysplastic lesions reflect its malignant potentiality. Overexpression of HSP70 in dysplastic lesions is a useful marker for higher cellular stress.
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BACKGROUND: Yogasana improves both mental and physical health. There is sparse systematic research on the growth of yogasana practising adolescents. OBJECTIVES: This study aims to assess the differential skeletal growth pattern among pre-adolescent and adolescent girls as a result of yoga practice. METHODS: A cross-sectional anthropometric study was conducted on stature (height), sitting height and leg length of 757 school-going girls (4-15 years old), divided in two groups, Yoga Group (YG) (n=380) and age matched Control Group (CG) (n=377) participants participating in recreational games other than yoga. Descriptive and inferential statistical analyses were applied. Unpaired t-test was performed for assessment of level of significance and Pearson's correlation (r) test was performed to to identify the association between growth pattern of stature and leg length at specific ages. RESULTS: The physical growth showed an ascending trend in both Yoga group (YG) participants and control group (CG). At the onset of adolescence (10-12 years) the mean stature and leg length of YG participants were retarded (p < 0.05). Sitting height in YG was significantly (p < 0.05) low only in 10-year-olds. The similar trends were observed in stature and leg length in YG participants at 10 years (5th and 10th percentile) and 12 years (90th and 95th percentile). There was strong positive relationship between stature and leg length of YG participants (10 years, r = 0.86, p < 0.01; 11 years, r = 0.86, p < 0.01; 12 years, r = 0.72, p < 0.01). The stunted growth in YG participants during adolescence may be related to retarded growth of leg length. CONCLUSIONS: Intense yogasana practice with greater skeletal stress possibly hinders stature in adolescent girls from 10 to 12 years. This may compromise with the natural growth pattern, necessitating special care during yoga training among adolescents while selecting the type, intensity and duration of yogasanas practice.
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BACKGROUND: Lichen planus is a chronic inflammatory mucocutaneous disease which frequently involves the oral mucosa. The most common types of oral lichen planus (OLP) are reticular (asymptomatic) and erosive (ulcerative) with malignant potentiality. The aims of the present study are to assess the cellular stress level in both types of OLP lesions with respect to oxidative stress, DNA damage and inflammation. MATERIALS AND METHODS: Freshly diagnosed untreated 25 OLP reticular type and 25 OLP erosive (OLP-E) type patients aged 35-55 years were enrolled in the study along with age and sex-matched 25 healthy subjects as control. Tissue antioxidant enzymes were measured biochemically, single-cell DNA damage was measured by comet assay and the molecular markers for inflammation were assessed by using semi-quantitative reverse transcriptase-polymerase chain reaction. Statistical analyses were performed using one-way ANOVA and Tukey's post hoc test. RESULTS: Oxidative stress was significantly greater in OLP-E type compared to the reticular. mRNA expression of cyclooxygenase-2 was significantly elevated (P < 0.0001) in erosive form, but such expression of nuclear factor kappa beta, tumor necrosis factor-alpha, Interleukin-6 and inducible nitric oxide synthase did not significantly differ between the two disease groups. Comet assay revealed a higher degree of DNA strand breakage in erosive lesions. CONCLUSIONS: The unhandled free radicals may imbalance the homeostatic network toward pro-inflammatory, DNA damaging responses, creating a vicious cell-damaging spree resulting in stress. Molecular analyses showed that erosive lichen planus is more under stress than the reticular form.
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Oral lichen planus (OLP) is a stress induced inflammatory condition with malignant potency. The mdr1 (multidrug resistance) is a stress gene overexpressed in cancerous conditions and its translated form, the p-glycoprotein efflux transporter is usually overexpressed with chemotherapy, leading to chemoresistance. OLP, a lesion with carcinogenic potency, is broadly classified into the asymptomatic reticular form and the aggressive erosive form. The objective of the study was to verify the expression level of p-glycoprotein in antifungal-treated and untreated reticular OLP, in untreated erosive OLP and erosive OLP patients treated with corticosteroid. Semi-quantitative reverse transcriptase polymerase chain reaction (SQ-RTPCR) and ELISA were performed on biopsy tissue samples to evaluate the mdr1 mRNA and protein expression of p-glycoprotein, respectively. The present study shows for the first time that mdr1 mRNA as well as its translated form p-glycoprotein are overexpressed in OLP subjects compared to healthy individuals. This overexpression is significantly higher in erosive than in reticular OLP patients, further confirming that the erosive form has higher risk for multidrug resistance. A higher expression is also observed in corticosteroid-treated erosive cases than similar untreated ones. The gradation of expression is in conformity with severity of the disease.
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Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Corticoesteroides/uso terapéutico , Antifúngicos/uso terapéutico , Liquen Plano Oral/tratamiento farmacológico , Liquen Plano Oral/metabolismo , Adulto , Análisis de Varianza , Biopsia , Farmacorresistencia Fúngica Múltiple , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Liquen Plano Oral/patología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Índice de Severidad de la Enfermedad , Piel/patologíaRESUMEN
Abstract: Oral lichen planus (OLP) is a stress induced inflammatory condition with malignant potency. The mdr1 (multidrug resistance) is a stress gene overexpressed in cancerous conditions and its translated form, the p-glycoprotein efflux transporter is usually overexpressed with chemotherapy, leading to chemoresistance. OLP, a lesion with carcinogenic potency, is broadly classified into the asymptomatic reticular form and the aggressive erosive form. The objective of the study was to verify the expression level of p-glycoprotein in antifungal-treated and untreated reticular OLP, in untreated erosive OLP and erosive OLP patients treated with corticosteroid. Semi-quantitative reverse transcriptase polymerase chain reaction (SQ-RTPCR) and ELISA were performed on biopsy tissue samples to evaluate the mdr1 mRNA and protein expression of p-glycoprotein, respectively. The present study shows for the first time that mdr1 mRNA as well as its translated form p-glycoprotein are overexpressed in OLP subjects compared to healthy individuals. This overexpression is significantly higher in erosive than in reticular OLP patients, further confirming that the erosive form has higher risk for multidrug resistance. A higher expression is also observed in corticosteroid-treated erosive cases than similar untreated ones. The gradation of expression is in conformity with severity of the disease.
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Humanos , Masculino , Femenino , Adulto , Corticoesteroides/uso terapéutico , Liquen Plano Oral/metabolismo , Liquen Plano Oral/tratamiento farmacológico , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Antifúngicos/uso terapéutico , Piel/patología , Biopsia , Índice de Severidad de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Análisis de Varianza , Liquen Plano Oral/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Farmacorresistencia Fúngica Múltiple , Persona de Mediana EdadRESUMEN
This scoping study investigated how evidence-based practice (EBP) principles are taught in Australian speech-language pathology (SLP) teaching and learning contexts. It explored how Australian SLP university programs: (1) facilitate student learning about the principles of EBP in academic and clinical settings, and (2) self-evaluate their curricula in relation to EBP. The research involved two surveys. Survey 1 respondents were 131 academic staff, program coordinators, and on-campus and off-campus clinical educators. This survey gathered information about EBP teaching and learning in SLP programs as well as future EBP curriculum plans. Survey 2 investigated how clinical educators incorporated EBP into the way they taught clinical decision-making to students. Surveys responses from 85 clinical educators were analysed using descriptive and non-parametric statistics and thematic grouping of open-ended qualitative responses. Both surveys revealed strengths and gaps in integrating EBP into Australian SLP curricula. Perceived strengths were that respondents were positive about EBP, most had EBP training and access to EBP resources. The perceived gaps included the academic staff's perceptions of students' understanding and application of EBP, respondents' understanding of research methodologies, communication and collaboration between academic staff and clinical educators, and a lack of explicit discussion by clinical educators and students of EBP in relation to clients.
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Educación Profesional/métodos , Práctica Clínica Basada en la Evidencia/educación , Patología del Habla y Lenguaje/educación , Enseñanza/métodos , Actitud del Personal de Salud , Australia , Comunicación , Comprensión , Curriculum , Docentes , Conocimientos, Actitudes y Práctica en Salud , Humanos , Aprendizaje , Estudiantes , Encuestas y Cuestionarios , UniversidadesRESUMEN
The drug transport function of human P-glycoprotein (Pgp, ABCB1) can be inhibited by a number of pharmacological agents collectively referred to as modulators or reversing agents. In this study, we demonstrate that certain thioxanthene-based Pgp modulators with an allosteric mode of action induce a distinct conformational change in the cytosolic domain of Pgp, which alters susceptibility to proteolytic digestion. Both cis and trans-isomers of the Pgp modulator flupentixol confer considerable protection of an 80 kDa Pgp fragment against trypsin digestion, that is recognized by a polyclonal antibody specific for the NH(2)-terminal half to Pgp. The protection by flupentixol is abolished in the Pgp F983A mutant that is impaired in modulation by flupentixols, indicating involvement of the allosteric site in generating the conformational change. A similar protection to an 80 kDa fragment is conferred by ATP, its nonhydrolyzable analog ATPgammaS, and by trapping of ADP-vanadate at the catalytic domain, but not by transport substrate vinblastine or by the competitive modulator cyclosporin A, suggesting different outcomes from modulator interaction at the allosteric site and at the substrate site. In summary, we demonstrate that allosteric interaction of flupentixols with Pgp generates conformational changes that mimic catalytic transition intermediates induced by nucleotide binding and hydrolysis, which may play a crucial role in allosteric inhibition of Pgp-mediated drug transport.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Flupentixol/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Regulación Alostérica/genética , Sitio Alostérico/genética , Sustitución de Aminoácidos , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Humanos , Ratones , Células 3T3 NIH , Mutación Puntual , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/genética , Vinblastina/químicaRESUMEN
ATP-dependent drug transport by human P-glycoprotein (Pgp, ABCB1) involves a coordinated communication between its drug-binding site (substrate site) and the nucleotide binding/hydrolysis domain (ATP sites). It has been demonstrated that the two ATP sites of Pgp play distinct roles within a single catalytic turnover; whereas ATP binding or/and hydrolysis by one drives substrate translocation and dissociation, the hydrolytic activity of the other resets the transporter for the subsequent cycle (Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515-2520; Sauna, Z. E., and Ambudkar, S. V. (2001) J. Biol. Chem. 276, 11653-11661). Trapping of ADP (or 8-azido-ADP) and vanadate (ADP.Vi or 8-azido-ADP.Vi) at the catalytic site, following nucleotide hydrolysis, markedly reduces the affinity of Pgp for its transport substrate [125I]iodoarylazidoprazosin ([125I]IAAP), resulting in dissociation of the latter. Regeneration of the [125I]IAAP site requires an additional round of nucleotide hydrolysis. In this study, we demonstrate that certain thioxanthene-based allosteric modulators, such as cis-(Z)-flupentixol and its closely related analogs, induce regeneration of [125I]IAAP binding to vanadate-trapped (or fluoroaluminate-trapped) Pgp without any further nucleotide hydrolysis. Regeneration was facilitated by dissociation of the trapped nucleotide and vanadate. Once regenerated, the substrate site remains accessible to [125I]IAAP even after removal of the modulator from the medium, suggesting a modulator-induced relaxation of a constrained transition state conformation. Consistent with this, limited trypsin digestion of vanadate-trapped Pgp shows protection by cis-(Z)-flupentixol of two Pgp fragments (approximately 60 kDa) recognizable by a polyclonal antiserum specific for the NH2-terminal half. No regeneration was observed in the Pgp mutant F983A that is impaired in modulation by flupentixols, indicating involvement of the allosteric modulator site in the phenomenon. In summary, the data demonstrate that in the nucleotide-trapped low affinity state of Pgp, the allosteric site remains accessible and responsive to modulation by flupentixol (and its closely related analogs), which can reset the high affinity state for [125I]IAAP binding without any further nucleotide hydrolysis.
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Subfamilia B de Transportador de Casetes de Unión a ATP/química , Adenosina Trifosfato/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/química , Sitio Alostérico , Animales , Azidas/química , Sitios de Unión , Dominio Catalítico , Línea Celular , Membrana Celular/metabolismo , Flupentixol/química , Humanos , Hidrólisis , Insectos , Ligandos , Conformación Molecular , Prazosina/análogos & derivados , Prazosina/química , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Tioxantenos/química , Factores de Tiempo , Tripsina/química , Vanadatos/químicaRESUMEN
The human P-glycoprotein (Pgp, ABCB1) is an ATP-dependent efflux pump for structurally unrelated hydrophobic compounds, conferring simultaneous resistance to and restricting bioavailability of several anticancer and antimicrobial agents. Drug transport by Pgp requires a coordinated communication between its substrate binding/translocating pathway (substrate site) and the nucleotide binding domains (NBDs or ATP sites). In this study, we demonstrate that certain thioxanthene-based Pgp modulators, such as cis-(Z)-flupentixol and its closely related analogues, effectively disrupt molecular cross talk between the substrate, and the ATP, sites without affecting the basic functional aspects of the two domains, such as substrate recognition, binding, and hydrolysis of ATP and dissociation of ADP following ATP hydrolysis. The allosteric modulator cis-(Z)-flupentixol has no effect on [alpha-(32)P]-8-azido-ATP binding to Pgp under nonhydrolytic conditions or on the K(m) for ATP during ATP hydrolysis. Both hydrolysis of ATP and vanadate-induced [alpha-(32)P]-8-azido-ADP trapping (following [alpha-(32)P]-8-azido-ATP breakdown) by Pgp are stimulated by the modulator. However, the ability of Pgp substrates (such as prazosin) to stimulate ATP hydrolysis and facilitate vanadate-induced trapping of [alpha-(32)P]-8-azido-ADP is substantially affected in the presence of cis-(Z)-flupentixol. Substrate recognition by Pgp as determined by [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) binding both in the presence and in the absence of ATP is facilitated by the modulator, whereas substrate dissociation in response to vanadate trapping is considerably affected in its presence. In the Pgp F983A mutant, which is impaired in modulation by cis-(Z)-flupentixol, the modulator has a minimal effect on substrate-stimulated ATP hydrolysis as well as on substrate dissociation coupled to vanadate trapping. Finally, cis-(Z)-flupentixol has no effect on dissociation of [alpha-(32)P]-8-azido-ADP (or ADP) from vanadate-trapped Pgp, which is essential for subsequent rounds of ATP hydrolysis. Taken together, our results demonstrate a distinct mechanism of Pgp modulation that involves allosteric disruption of molecular cross talk between the substrate, and the ATP, sites without any direct interference with their individual functions.