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1.
Expert Opin Drug Discov ; 19(10): 1247-1257, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39105537

RESUMO

INTRODUCTION: Determining whether a new drug is a substrate, inhibitor or inducer of efflux or uptake membrane transporters has become a routine process during drug discovery and development. In vitro assays are utilized to establish whether a new drug has the potential to be an object (substrate) or precipitant (inhibitor, inducer) in transporter-mediated clinical drug-drug interactions. The findings from these in vitro experiments are then used to determine whether further in vivo drug interaction studies are necessary for a new drug. AREAS COVERED: This article provides an update on in vitro transporter assays, focusing on new uses of transfected cells, time-dependent inhibition, transporter induction, and complex model systems. EXPERT OPINION: The newer in vitro assays add to the toolbox in defining new drugs as transporter substrates, inhibitors, or inducers. Complex models such as spheroids, organoids, and microphysiological systems require standardization and further research with model transporter substrates and inhibitors. In drug discovery, the more traditional transporter assays may be employed as substrate and inhibitor screening assays. In drug development, more complex cell models can be employed in later drug development to better understand how transporter(s) are involved in the absorption, distribution, and excretion of new drugs.


Assuntos
Desenvolvimento de Medicamentos , Descoberta de Drogas , Interações Medicamentosas , Proteínas de Membrana Transportadoras , Descoberta de Drogas/métodos , Humanos , Desenvolvimento de Medicamentos/métodos , Animais , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Transporte Biológico , Preparações Farmacêuticas/metabolismo , Modelos Biológicos
2.
J Ethnopharmacol ; 332: 118365, 2024 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-38796070

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Fuzheng Touxie Jiedu Huayu Decoction (FTJHD) is a commonly used clinical formula that has been found effective in resisting multidrug resistance-Pseudomonas aeruginosa in previous in vivo and in vitro studies. AIM OF THE STUDY: To investigate the antimicrobial effects of FTJHD and its drug-containing serum alone or in combination with ceftazidime on difficult-to-treat multidrug resistance-P. aeruginosa (DTMDR-P. aeruginosa). MATERIALS AND METHODS: The antibacterial effects of FTJHD and its drug-containing alone or in combination with ceftazidime against DTMDR-P. aeruginosa were examined by the tube dilution method and bacterial growth curves. The changes in the bacterial ultrastructure were examined by transmission electron microscopy. The biofilm formation ability of bacteria was examined by crystal violet staining and scanning electron microscopy. The expression of the MexAB-OprM efflux pump and quorum sensing system genes were validated through quantitative polymerase chain reaction. Molecular docking was used to evaluate the interaction between active components and the MexAB-OprM efflux pump. RESULTS: FTJHD-containing serums at 1-, 2-, 4-, and 8-fold concentrations reduced the minimal inhibitory concentration (MIC) of ceftazidime against DTMDR-P. aeruginosa from 128 µg/mL to 64 µg/mL. Sub-inhibitory concentrations of ceftazidime in combination with FTJHD and FTJHD-containing serum prolonged the lag period of bacterial growth and reduced bacterial numbers. Additionally, 1/2 MIC of ceftazidime combined with FTJHD-containing serum significantly inhibited the activity of the MexAB-OprM efflux pump and quorum sensing system, thus reducing biofilm formation while causing more severe damage to the bacteria. Molecular docking revealed a strong affinity of quercetin, baicalein, luteolin, kaempferol, and ß-sitosterol for the efflux pump regulatory proteins OprM and MexR. CONCLUSION: FTJHD can exert synergistic anti-DTMDR-P. aeruginosa effects with ceftazidime by inhibiting biofilm formation mediated by the MexAB-OprM efflux pump and quorum sensing.


Assuntos
Antibacterianos , Proteínas da Membrana Bacteriana Externa , Biofilmes , Farmacorresistência Bacteriana Múltipla , Medicamentos de Ervas Chinesas , Simulação de Acoplamento Molecular , Pseudomonas aeruginosa , Percepção de Quorum , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Percepção de Quorum/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Ceftazidima/farmacologia
3.
Clin Ther ; 46(6): 499-508, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38734524

RESUMO

PURPOSE: This analysis aimed to provide mechanistic understanding and clinical relevance of pharmacokinetic drug-drug interactions (DDIs) associated with drugs approved by the Food and Drug Administration in 2022. METHODS: Drug metabolism, transport, and DDI data available in New Drug Applications (NDAs) of small molecular drugs approved (n = 22) was analyzed. The mechanism and clinical magnitude of these interactions were characterized based on in vitro, in silico, and clinical data. FINDINGS: As victims, 10 drugs were identified as clinical substrates. Of these, 7 drugs were substrates of CYP3A, including the sensitive substrates daridorexant and mitapivat. As perpetrators, 3 drugs (adagrasib, lenacapavir, and vonoprazan) were clinical inhibitors of CYP enzymes, and 2 drugs (mavacamten and mitapivat) showed induction. Regarding transporter data, abrocitinib and deucravacitinib were found to be substrates of OAT3 and P-gp/BCRP, respectively, and 4 drugs (abrocitinib, adagrasib, lenacapavir, and oteseconazole) were found to inhibit P-gp and/or BCRP. As expected, all clinical DDIs with AUC changes ≥ 2-fold triggered label recommendations. Over half of DDIs with an AUC change < 2 also had label recommendations, pertaining most often to the concomitant use of drugs with a narrow therapeutic index. Overall, CYP3A played a major role in the drug disposition of the drugs approved in 2022, mediating all strong drug interactions. IMPLICATIONS: The mechanistic information obtained from studying these new therapeutics with marker compounds can be extrapolated to common concomitant medications sharing the same pharmacokinetic properties, enhancing the safe and effective administration of these products in situations of polytherapy.


Assuntos
Aprovação de Drogas , Interações Medicamentosas , United States Food and Drug Administration , Humanos , Estados Unidos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Preparações Farmacêuticas/metabolismo , Preparações Farmacêuticas/administração & dosagem
4.
Curr Drug Res Rev ; 16(3): 349-368, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38288795

RESUMO

Multidrug Resistance mechanisms in microorganisms confer the slackness of the existing drugs, leading to added difficulty in treating infections. As a consequence, efficient novel drugs and innovative therapies to treat MDR infections are necessarily required. One of the primary contributors to the emergence of multidrug resistance in gram-negative bacteria has been identified as the efflux pumps. These transporter efflux pumps reduce the intracellular concentration of antibiotics and aid bacterial survival in suboptimal low antibiotic concentration environments that may cause treatment failure. The reversal of this resistance via inhibition of the efflux mechanism is a promising method for increasing the effectiveness of antibiotics against multidrug-resistant pathogens. Such EPI, in combination with antibiotics, can make it easier to reintroduce traditional antibiotics into clinical practice. This review mostly examines efflux-mediated multidrug resistance in critical gram-negative bacterial pathogens and EPI of plant origin that have been reported over previous decades.


Assuntos
Antibacterianos , Farmacorresistência Bacteriana Múltipla , Bactérias Gram-Negativas , Proteínas de Membrana Transportadoras , Bactérias Gram-Negativas/efeitos dos fármacos , Antibacterianos/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Humanos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/microbiologia
5.
Biomolecules ; 13(6)2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37371573

RESUMO

BACKGROUND: The carnitine/acylcarnitine carrier (CAC) represents the route of delivering acyl moieties to the mitochondrial matrix for accomplishing the fatty acid ß-oxidation. The CAC has a couple of Cys residues (C136 and C155) most reactive toward ROS and redox signaling compounds such as GSH, NO, and H2S. Among physiological compounds reacting with Cys, itaconate is produced during inflammation and represents the connection between oxidative metabolism and immune responses. The possible interaction between the CAC and itaconate has been investigated. METHODS: the modulatory effects of itaconate on the transport activity of the native and recombinant CAC were tested using the proteoliposome experimental model together with site-directed mutagenesis and computational analysis. RESULTS: Itaconate reacts with the CAC causing irreversible inhibition. Dose-response experiment performed with the native and recombinant protein showed IC50 for itaconate of 11 ± 4.6 mM and 8.4 ± 2.9 mM, respectively. The IC50 decreased to 3.8 ± 1.0 mM by lowering the pH from pH 7.0 to pH 6.5. Inhibition kinetics revealed a non-competitive type of inhibition. C136 is the main target of itaconate, as demonstrated by the increased IC50 of mutants in which this Cys was substituted by Val. The central role of C136 was confirmed by covalent docking. Administration of dimethyl itaconate to HeLa cells inhibited the CAC transport activity, suggesting that itaconate could react with the CAC also in intact cells.


Assuntos
Proteínas de Membrana Transportadoras , Mitocôndrias , Humanos , Carnitina/metabolismo , Cisteína/metabolismo , Células HeLa/efeitos dos fármacos , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Moduladores de Transporte de Membrana/farmacologia
6.
Clin Transl Med ; 12(1): e660, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35075807

RESUMO

OBJECTIVE: To explore the therapeutic potential and the underlying mechanism of metformin, an adenosine monophosphate-activated kinase (AMPK) activator, in ocular melanoma. METHODS: CCK8, transwell, and colony formation assays were performed to detect the proliferation and migration ability of ocular melanoma cells. A mouse orthotopic xenograft model was built to detect ocular tumor growth in vivo. Western blot, immunofluorescence, and electron microscopy were adopted to evaluate the autophagy levels of ocular melanoma cells, and high-throughput proteomics and CUT & Tag assays were performed to analyze the candidate for autophagy alteration. RESULTS: Here, we revealed for the first time that a relatively low dose of metformin induced significant tumorspecific inhibition of the proliferation and migration of ocular melanoma cells both in vitro and in vivo. Intriguingly, we found that metformin significantly attenuated autophagic influx in ocular melanoma cells. Through high-throughput proteomics analysis, we revealed that optineurin (OPTN), which is a key candidate for autophagosome formation and maturation, was significantly downregulated after metformin treatment. Moreover, excessive OPTN expression was associated with an unfavorable prognosis of patients. Most importantly, we found that a histone deacetylase, SIRT1, was significantly upregulated after AMPK activation, resulting in histone deacetylation in the OPTN promoter. CONCLUSIONS: Overall, we revealed for the first time that metformin significantly inhibited the progression of ocular melanoma, and verified that metformin acted as an autophagy inhibitor through histone deacetylation of OPTN. This study provides novel insights into metformin - guided suppression of ocular melanoma and the potential mechanism underlying the dual role of metformin in autophagy regulation.


Assuntos
Autofagia/efeitos dos fármacos , Proteínas de Ciclo Celular/efeitos dos fármacos , Histona Desmetilases/efeitos dos fármacos , Melanoma/tratamento farmacológico , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Metformina/agonistas , Animais , Proteínas de Ciclo Celular/metabolismo , Modelos Animais de Doenças , Olho/efeitos dos fármacos , Olho/metabolismo , Melanoma/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Metformina/uso terapêutico , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo
7.
Plant Physiol ; 187(4): 2071-2091, 2021 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-34618047

RESUMO

Most land plants live in close contact with beneficial soil microbes: the majority of land plant species establish symbiosis with arbuscular mycorrhizal fungi, while most legumes, the third largest plant family, can form a symbiosis with nitrogen-fixing rhizobia. These microbes contribute to plant nutrition via endosymbiotic processes that require modulating the expression and function of plant transporter systems. The efficient contribution of these symbionts involves precisely controlled integration of transport, which is enabled by the adaptability and plasticity of their transporters. Advances in our understanding of these systems, driven by functional genomics research, are rapidly filling the gap in knowledge about plant membrane transport involved in these plant-microbe interactions. In this review, we synthesize recent findings associated with different stages of these symbioses, from the pre-symbiotic stage to nutrient exchange, and describe the role of host transport systems in both mycorrhizal and legume-rhizobia symbioses.


Assuntos
Fabaceae/microbiologia , Fabaceae/fisiologia , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Micorrizas/fisiologia , Fixação de Nitrogênio/fisiologia , Rhizobium/fisiologia , Simbiose/fisiologia , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia
8.
Pharmacol Res Perspect ; 9(6): e00870, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34664792

RESUMO

Zanubrutinib is a highly selective, potent, orally available, targeted covalent inhibitor (TCI) of Bruton's tyrosine kinase (BTK). This work investigated the in vitro drug metabolism and transport of zanubrutinib, and its potential for clinical drug-drug interactions (DDIs). Phenotyping studies indicated cytochrome P450 (CYP) 3A are the major CYP isoform responsible for zanubrutinib metabolism, which was confirmed by a clinical DDI study with itraconazole and rifampin. Zanubrutinib showed mild reversible inhibition with half maximal inhibitory concentration (IC50 ) of 4.03, 5.69, and 7.80 µM for CYP2C8, CYP2C9, and CYP2C19, respectively. Data in human hepatocytes disclosed induction potential for CYP3A4, CYP2B6, and CYP2C enzymes. Transport assays demonstrated that zanubrutinib is not a substrate of human breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP)1B1/1B3, organic cation transporter (OCT)2, or organic anion transporter (OAT)1/3 but is a potential substrate of the efflux transporter P-glycoprotein (P-gp). Additionally, zanubrutinib is neither an inhibitor of P-gp at concentrations up to 10.0 µM nor an inhibitor of BCRP, OATP1B1, OATP1B3, OAT1, and OAT3 at concentrations up to 5.0 µM. The in vitro results with CYPs and transporters were correlated with the available clinical DDIs using basic models and mechanistic static models. Zanubrutinib is not likely to be involved in transporter-mediated DDIs. CYP3A inhibitors and inducers may impact systemic exposure of zanubrutinib. Dose adjustments may be warranted depending on the potency of CYP3A modulators.


Assuntos
Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Piperidinas/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Cães , Interações Medicamentosas , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Concentração Inibidora 50 , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Piperidinas/farmacocinética , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacocinética , Pirazóis/farmacologia , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Ratos
9.
Toxicol Appl Pharmacol ; 431: 115729, 2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34592323

RESUMO

Rosemary (Salvia Rosmarinus) is a rich source of dietary diterpenes with carnosol as one of the major polyphenols used to standardize rosemary extracts approved as a food preservative, however, at present there is not any information on the murine pharmacokinetic profile of carnosol or its potential for drug interactions. The present study utilizes cell-free, cell-based, and animal-based experiments to define the pharmacokinetic profile of the food based phytochemical carnosol. Mice were administered carnosol (100 mg/kg body weight) by oral gavage and plasma levels were analyzed by LC-MS/MS to establish a detailed pharmacokinetic profile. The maximum plasma concentration exceeded 1 µM after a single administration. The results are significant as they offer insights on the potential for food-drug interactions between carnosol from rosemary and active pharmaceutical ingredients. Carnosol was observed to inhibit selected CYP450 enzymes and modulate metabolic enzymes and transporters in in vitro assays.


Assuntos
Abietanos/farmacocinética , Inibidores das Enzimas do Citocromo P-450/farmacocinética , Sistema Enzimático do Citocromo P-450/metabolismo , Conservantes de Alimentos/farmacocinética , Abietanos/administração & dosagem , Abietanos/sangue , Abietanos/isolamento & purificação , Administração Oral , Animais , Disponibilidade Biológica , Óleo de Sementes de Algodão/química , Inibidores das Enzimas do Citocromo P-450/administração & dosagem , Inibidores das Enzimas do Citocromo P-450/sangue , Inibidores das Enzimas do Citocromo P-450/isolamento & purificação , Estabilidade de Medicamentos , Conservantes de Alimentos/administração & dosagem , Conservantes de Alimentos/isolamento & purificação , Células HT29 , Células Hep G2 , Humanos , Isoenzimas , Masculino , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Camundongos Endogâmicos C57BL , Rosmarinus/química , Temperatura
10.
J Ethnopharmacol ; 280: 114408, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34252529

RESUMO

ETHNOPHARMACOLOGY RELEVANCE: Suxiao jiuxin pill (SJP) is a Chinese medical drug with anti-inflammatory, anti-apoptotic, and vasodilatory function. It is widely used in combination with other drugs for the treatment of coronary heart disease (CHD) and angina. Nevertheless, the effect of SJP on Cytochrome P450 (CYP450) enzymes and transporters' activity related to drug metabolism is rarely studied. OBJECTIVE: The aim of this study was to investigate the effect of SJP on the activity of drug-metabolizing enzyme CYP450 and transporters. MATERIALS AND METHODS: Human primary hepatocytes were used in present study. Probe substrates of CYP450 enzymes were incubated in human liver microsomes (HLMs) with and without SJP while IC50 values were calculated. The inhibitory effect of SJP on the activity of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 was evaluated. The inducing effect of SJP on the activity of CYP1A2, 2B6 and 3A4 was accessed. The inhibition of SJP on human OATP1B1 was investigated through cell-based assay. The inhibition of SJP on human MDR1 and BCRP was also estimated by means of the vesicles assay. RESULTS: The results showed that the SJP under the concentration of 1000 µg/mL could inhibit the activity of CYP1A2, 2B6, 2C19, and 3A4, with IC50 values of 189.7, 308.2, 331.2 and 805.7 µg/mL, respectively. There was no inhibitory effect found in the other 3 liver drug enzyme subtypes. In addition, SJP showed no induction effect on CYP1A2, 2B6 and 3A4, however it had a significant inhibitory effect on human-derived OATP1B1 at the concentration of 100 and 1000 µg/mL, with the IC50 value of 21.9 µg/mL. Simultaneously, the SJP inhibited BCRP at high concentration of 1000 µg/mL but did not affect human MDR1. CONCLUSIONS: Based on these research results above, it is suggested that the SJP can affect some of the CYP450 enzymes and transporters' activity. When used in combination with related conventional drugs, potential herb-drug interactions should be considered.


Assuntos
Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Interações Ervas-Drogas , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Inibidores das Enzimas do Citocromo P-450/administração & dosagem , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/administração & dosagem , Células HEK293 , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Concentração Inibidora 50 , Proteínas de Membrana Transportadoras/metabolismo , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/metabolismo
11.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946818

RESUMO

Since 2010, several treatment options have been available for men with metastatic castration-resistant prostate cancer (mCRPC), including immunotherapeutic agents, although the clinical benefit of these agents remains inconclusive in unselected mCRPC patients. In recent years, however, immunotherapy has re-emerged as a promising therapeutic option to stimulate antitumor immunity, particularly with the use of immune checkpoint inhibitors (ICIs), such as PD-1/PD-L1 and CTLA-4 inhibitors. There is increasing evidence that ICIs may be especially beneficial in specific subgroups of patients with high PD-L1 tumor expression, high tumor mutational burden, or tumors with high microsatellite instability/mismatch repair deficiency. If we are to improve the efficacy of ICIs, it is crucial to have a better understanding of the mechanisms of resistance to ICIs and to identify predictive biomarkers to determine which patients are most likely to benefit. This review focuses on the current status of ICIs for the treatment of mCRPC (either as monotherapy or in combination with other drugs), mechanisms of resistance, potential predictive biomarkers, and future challenges in the management of mCRPC.


Assuntos
Adenocarcinoma/secundário , Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/métodos , Neoplasias de Próstata Resistentes à Castração/terapia , Adenocarcinoma/terapia , Antineoplásicos Imunológicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores , Ensaios Clínicos como Assunto , Reparo do DNA/genética , Resistencia a Medicamentos Antineoplásicos , Previsões , Humanos , Inibidores de Checkpoint Imunológico/administração & dosagem , Masculino , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Estudos Multicêntricos como Assunto , Proteínas de Neoplasias/antagonistas & inibidores , Compostos Organoplatínicos/administração & dosagem , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Medicina de Precisão/métodos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Taxoides/administração & dosagem
12.
PLoS Pathog ; 17(3): e1009338, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33647048

RESUMO

Host defense proteins (HDPs), aka defensins, are a key part of the innate immune system that functions by inserting into the bacterial membranes to form pores to kill invading and colonizing microorganisms. To ensure survival, microorganism such as S. aureus has developed survival strategies to sense and respond to HDPs. One key strategy in S. aureus is a two-component system (TCS) called GraRS coupled to an efflux pump that consists of a membrane permease VraG and an ATPase VraF, analogous to the BceRS-BceAB system of Bacillus subtilis but with distinct differences. While the 9 negatively charged amino acid extracellular loop of the membrane sensor GraS has been shown to be involved in sensing, the major question is how such a small loop can sense diverse HDPs. Mutation analysis in this study divulged that the vraG mutant phenocopied the graS mutant with respect to reduced activation of downstream effector mprF, reduction in surface positive charge and enhanced 2 hr. killing with LL-37 as compared with the parental MRSA strain JE2. In silico analysis revealed VraG contains a single 200-residue extracellular loop (EL) situated between the 7th and 8th transmembrane segments (out of 10). Remarkably, deletion of EL in VraG enhanced mprF expression, augmented surface positive charge and improved survival in LL-37 vs. parent JE2. As the EL of VraG is rich in lysine residues (16%), in contrast to a preponderance of negatively charged aspartic acid residues (3 out of 9) in the EL of GraS, we divulged the role of charge interaction by showing that K380 in the EL of VraG is an important residue that likely interacts with GraS to interfere with GraS-mediated signaling. Bacterial two-hybrid analysis also supported the interaction of EL of VraG with the EL of GraS. Collectively, we demonstrated an interesting facet of efflux pumps whereby the membrane permease disrupts HDP signaling by inhibiting GraS sensing that involves charged residues in the EL of VraG.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Aminoaciltransferases/genética , Peptídeos Catiônicos Antimicrobianos/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Staphylococcus aureus/genética
13.
Am J Physiol Cell Physiol ; 320(5): C916-C925, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33760662

RESUMO

Niemann-Pick C1 Like-1 (NPC1L1) mediates the uptake of micellar cholesterol by intestinal epithelial cells and is the molecular target of the cholesterol-lowering drug ezetimibe (EZE). The detailed mechanisms responsible for intracellular shuttling of micellar cholesterol are not fully understood due to the lack of a suitable NPC1L1 substrate that can be traced by fluorescence imaging and biochemical methods. 27-Alkyne cholesterol has been previously shown to serve as a substrate for different cellular processes similar to native cholesterol. However, it is not known whether alkyne cholesterol is absorbed via an NPC1L1-dependent pathway. We aimed to determine whether alkyne cholesterol is a substrate for NPC1L1 in intestinal cells. Human intestinal epithelial Caco2 cells were incubated with micelles containing alkyne cholesterol in the presence or absence of EZE. Small intestinal closed loops in C57BL/6J mice were injected with micelles containing alkyne cholesterol with or without EZE. Alkyne cholesterol esterification in Caco2 cells was significantly inhibited by EZE and by inhibitor of clathrin-mediated endocytosis Pitstop 2. The esterification was similarly reduced by inhibitors of the acyl-CoA cholesterol acyltransferase (ACAT). Alkyne cholesterol efficiently labeled the apical membrane of Caco2 cells and the amount retained on the membrane was significantly increased by EZE as judged by accessibility to exogenous cholesterol oxidase. In mouse small intestine, the presence of EZE reduced total alkyne cholesterol uptake by ∼75%. These data show that alkyne cholesterol acts as a substrate for NPC1L1 and may serve as a nonradioactive tracer to measure cholesterol absorption in both in vitro and in vivo models.


Assuntos
Colesterol/metabolismo , Células Epiteliais/metabolismo , Absorção Intestinal , Mucosa Intestinal/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Anticolesterolemiantes/farmacologia , Transporte Biológico , Células CACO-2 , Colesterol/análogos & derivados , Endocitose , Células Epiteliais/efeitos dos fármacos , Ezetimiba/farmacologia , Humanos , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Camundongos Endogâmicos C57BL
14.
Biomed Pharmacother ; 137: 111331, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33578235

RESUMO

SCOPE: To investigate the effect of Qingjie Fuzheng Granule (QFG) on lymphangiogenesis and lymphatic metastasis in colorectal cancer. METHODS: The effects of QFG on the expression and secretion of vascular endothelial growth factor-C (VEGF-C) in HCT-116 cells were investigated both in vitro and in vivo. HCT-116 cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. The VEGF-C expression level was determined using RT-qPCR and western blotting, and the VEGF-C concentration in supernatant was measured by ELISA. Tumor xenograft models of HCT-116 cells were generated using BALB/c nude mice, and the mice were randomly divided into a control group (gavaged with normal saline) and QFG group (gavaged with 2 g/kg QFG). The effect of QFG on tumor growth was evaluated by comparing the volume and weight of tumors between two groups. Immunohistochemistry (IHC) and RT-qPCR were performed to detect the expression levels of VEGF-C, vascular endothelial growth factor receptor 3 (VEGFR-3), and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1). ELISA was performed to measure the concentration of serum VEGF-C. TMT proteomics technology and Reactome pathway analysis were used to explore the mechanism of QFG inhibiting lymphangiogenesis in tumor. The VEGF-C (5 ng/mL)-stimulated human lymphatic endothelial cell (HLEC) model was conducted to evaluate the effect of QFG on lymphangiogenesis in vitro. The model cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. Cell viability was then determined using an MTT assay. The cell migration, invasion, and tube-formation ability were analyzed using transwell migration, matrigel invasion and tube formation assays, respectively. The underlying mechanism was uncovered, the levels of VEGFR-3, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), p-PI3K/PI3K, p-AKT/AKT and p-mTOR/ mTOR were detected using western blotting. RESULTS: QFG significantly reduced VEGF-C expression and secretion in HCT-116 cells. QFG evidently suppressed in vivo tumor growth and the expression of VEGF-C, VEGFR-3, and LYVE-1. The serum VEGF-C level was also reduced by QFG. Moreover, TMT proteomics technology and Reactome pathway analysis identified 95 differentially expressed protein and multiple enriched pathway about matrix metalloproteinase and extracellular matrix, which is direct associate with lymphangiogenesis. In vitro experiment, QFG inhibited the viability, migration, invasion and tube formation of HLECs. Additionally, QFG reduced the VEGFR-3, MMP-2, MMP-9 expression levels, and the p-PI3K/PI3K, p-AKT/AKT, p-mTOR/ mTOR ratios. CONCLUSION: QFG can exert its effect on both tumor cells and HLECs, exhibiting ani- lymphangiogenesis in colorectal cancer via the VEGF-C/VEGFR-3 dependent PI3K/AKT pathway pathway.


Assuntos
Inibidores da Angiogênese/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Linfangiogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Células Endoteliais/efeitos dos fármacos , Humanos , Inibidores de Metaloproteinases de Matriz/farmacologia , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Oncogênica v-akt/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fator C de Crescimento do Endotélio Vascular/efeitos dos fármacos , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacos
15.
Nat Commun ; 12(1): 172, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420032

RESUMO

The dimeric transporter, EmrE, effluxes polyaromatic cationic drugs in a proton-coupled manner to confer multidrug resistance in bacteria. Although the protein is known to adopt an antiparallel asymmetric topology, its high-resolution drug-bound structure is so far unknown, limiting our understanding of the molecular basis of promiscuous transport. Here we report an experimental structure of drug-bound EmrE in phospholipid bilayers, determined using 19F and 1H solid-state NMR and a fluorinated substrate, tetra(4-fluorophenyl) phosphonium (F4-TPP+). The drug-binding site, constrained by 214 protein-substrate distances, is dominated by aromatic residues such as W63 and Y60, but is sufficiently spacious for the tetrahedral drug to reorient at physiological temperature. F4-TPP+ lies closer to the proton-binding residue E14 in subunit A than in subunit B, explaining the asymmetric protonation of the protein. The structure gives insight into the molecular mechanism of multidrug recognition by EmrE and establishes the basis for future design of substrate inhibitors to combat antibiotic resistance.


Assuntos
Antiporters/química , Antiporters/efeitos dos fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/efeitos dos fármacos , Bicamadas Lipídicas/química , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/farmacologia , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica
16.
PLoS One ; 16(1): e0239353, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33481781

RESUMO

The phenoxyalkylimidazoles (PAI) are an attractive chemical series with potent anti-tubercular activity targeting Mycobacterium tuberculosis respiration. Our aim was to determine if the PAI compounds are subject to efflux. Two analogs containing an oxadiazole had improved potency in the presence of the efflux inhibitors reserpine and carbonyl cyanide m-chlorophenylhydrazine, whereas the potency of analogs with a diazole was not affected.


Assuntos
Mycobacterium tuberculosis/efeitos dos fármacos , Oxidiazóis/farmacologia , Fenóis/farmacologia , Antituberculosos/farmacologia , Proteínas de Bactérias/efeitos dos fármacos , Carbonil Cianeto m-Clorofenil Hidrazona/metabolismo , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Humanos , Isoniazida/química , Isoniazida/farmacologia , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Oxidiazóis/química , Fenóis/química , Reserpina/metabolismo , Reserpina/farmacologia
17.
Curr Drug Metab ; 22(7): 523-531, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33397250

RESUMO

Membrane transporters play an important role in intestinal absorption, distribution and clearance of drugs. Additionally transporters along with enzymes regulate tissue exposures (e.g. liver, kidney and brain), which are important for safety and efficacy considerations. Early identification of transporters involved guides generation of in vitro and in vivo data needed to gain mechanistic understanding on the role of transporters in organ clearance, tissue exposures and enables development of physiological-based pharmacokinetic (PBPK) models. A lot of progress has been made in developing several in vitro assay systems and mechanistic in silico models to determine kinetic parameters for transporters, which are incorporated into PBPK models. Although, intrinsic clearance and inhibition data from in vitro systems generally tend to underpredict in vivo clearance and magnitude of drug-drug interactions (DDIs), empirical scaling factors derived from a sizable dataset are often used to offset underpredictions. PBPK models are increasing used to predict the impact of transporters on intestinal absorption, clearance, victim and perpetrator DDIs prior to first in human clinical trials. The models are often refined when clinical data is available and are used to predict pharmacokinetics in untested scenarios such as the impact of polymorphisms, ontogeny, ethnicity, disease states and DDIs with other perpetrator drugs. The aim of this review is to provide an overview of (i) regulatory requirements around transporters, (ii) in vitro systems and their limitations in predicting transporter mediated drug disposition and DDIs, (iii) PBPK modelling tactics and case studies used for internal decision making and/or for regulatory submissions.


Assuntos
Vias de Eliminação de Fármacos , Interações Medicamentosas , Absorção Intestinal , Proteínas de Membrana Transportadoras/metabolismo , Farmacocinética , Animais , Humanos , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Modelos Biológicos
18.
Curr HIV Res ; 19(2): 128-137, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33032513

RESUMO

BACKGROUND: Ethanol has been shown to increase oxidative stress, drug efflux transporter expression, and promote HIV progression. Macrophages, which express drug efflux transporters, serve as an essential sanctuary site for HIV. The antiretroviral drug lopinavir, a protease inhibitor, is a substrate of the drug efflux transporters P-glycoprotein and multidrug resistance-associated protein 1. The NF-κB signaling pathway is associated with inflammation and drug efflux transporter expression. OBJECTIVE: To examine the effects of ethanol on drug efflux transporters and HIV replication of macrophages and develop strategies to increase the efficacy of the protease inhibitor. METHODS: The expression of PGP and MRP1 was examined with western blot. The NF- κB inhibition was assessed with nuclear western blot. LC-MS/MS and p24 ELISA were used to assess intracellular LPV and viral replication. RESULTS: Ethanol at 40mM slightly increased drug efflux transporter PGP and MRP1 expression in activated macrophages. IKK-16, an NF- κB inhibitor, counteracted the increased transporter expression caused by ethanol exposure. MK571, an MRP1 inhibitor, and IKK-16 significantly increased intracellular LPV concentration with or without ethanol treatment. MK571 significantly increased LPV efficacy in suppressing viral replication with or without ethanol treatment. A decreasing trend and a significant decrease were observed with IKK-16+LPV treatment compared with LPV alone in the no ethanol treatment and ethanol treatment groups, respectively. CONCLUSION: In activated macrophages, inhibiting drug efflux transporter MRP1 activity and reducing its expression may represent a promising approach to suppress viral replication by increasing intracellular antiretroviral concentrations. However, different strategies may be required for ethanolrelated vs. untreated groups.


Assuntos
Fármacos Anti-HIV/farmacologia , Etanol/efeitos adversos , Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/farmacologia , Lopinavir/farmacologia , Macrófagos/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Fármacos Anti-HIV/uso terapêutico , Células Cultivadas/efeitos dos fármacos , Feminino , Inibidores da Protease de HIV/uso terapêutico , Humanos , Lopinavir/uso terapêutico , Masculino , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Pessoa de Meia-Idade
19.
Invest New Drugs ; 39(1): 131-141, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32915418

RESUMO

Today, pancreatic cancer (PC) is a major health problem in the United States. It remains a challenge to develop efficacious clinically useful PC therapies. New avenues, based on translational approaches and innovative validated biomarkers could be a preclinical option to evaluate PC drug candidates or drug combinations before clinical trials. Herein, we describe evaluation of combination therapies by incorporating a novel pathway modulator, p53-Activator Wnt Inhibitor-2 (PAWI-2) with other FDA-approved cancer drugs that have been used in PC clinical trials. PAWI-2 is a potent inhibitor of drug-resistant PC cells that has been shown to selectively ameliorate human pancreatic cancer stem cells (i.e., hPCSCs, FGß3 cells). In the present study, we showed PAWI-2 produced therapeutic synergism with certain types of anti-cancer drugs. These drugs themselves oftentimes do not ameliorate PC cells (especially PCSCs) due to high levels of drug-resistance. PAWI-2 has the ability to rescue the potency of drugs (i.e., erlotinib, trametinib) and inhibit PC cell growth. Key molecular regulators of PAWI-2 could be used to predict synergistic/antagonistic effects between PAWI-2 and other anti-cancer drugs. Anti-cancer results showed potency could be quite accurately correlated to phosphorylation of optineurin (OPTN) in PC cells. Synergism/antagonism was also associated with inhibition of PCSC marker SOX2 that was observed in FGß3 cells. Synergism broadens the potential use of PAWI-2 as an adjunct chemotherapy in patients with PC that have developed resistance to first-line targeted therapies or chemotherapies.


Assuntos
Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Pancreáticas/patologia , Quinoxalinas/farmacologia , Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Quinoxalinas/administração & dosagem , Fatores de Transcrição SOXB1/efeitos dos fármacos
20.
Pharmacol Rep ; 73(1): 1-16, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32946075

RESUMO

The discovery of antibiotics ought to have ended the issue of bacterial infections, but this was not the case as it has led to the evolution of various mechanisms of bacterial resistance against various antibiotics. The efflux pump remains one of the mechanisms through which organisms develop resistance against antibiotics; this is because organisms can extrude most of the clinically relevant antibiotics from the interior cell environment to the exterior environment via the efflux pumps. Efflux pumps are thought to contribute significantly to biofilm formation as highlighted by various studies. Therefore, the inhibition of these efflux pumps can be a potential way of improving the activity of antibiotics, particularly now that the discovery of novel antibiotics is becoming tedious. Efflux pump inhibitors (EPIs) are molecules that can inhibit efflux pumps; they have been considered potential therapeutic agents for rejuvenating the activity of antibiotics that have already lost their activity against bacteria. However, studies are yet to determine the specific substrates for such pumps; the effect of altered efflux activity of these pumps on biofilm formation is still being investigated. A clear knowledge of the involvement of efflux pumps in biofilm development could aid in developing new agents that can interfere with their function and help to prevent biofilms formation; thereby, improving the outcome of treatment strategies. This review focuses on the novel update of EPIs and discusses the evidence of the roles of efflux pumps in biofilm formation; the potential approaches towards overcoming the increasing problem of biofilm-based infections are also discussed.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Animais , Bactérias/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Humanos
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