Location of contact residues in pharmacologically distinct drug binding sites on P-glycoprotein

© 2016 Elsevier Inc. The multidrug resistance P-glycoprotein (P-gp) is characterised by the ability to bind and/or transport an astonishing array of drugs. This poly-specificity is imparted by at least four pharmacologically distinct binding sites within the transmembrane domain. Whether or not these sites are spatially distinct has remained unclear. Biochemical and structural investigations have implicated a central cavity as the likely location for the binding sites. In the present investigation, a number of contact residues that are involved in drug binding were identified through biochemical assays using purified, reconstituted P-gp. Drugs were selected to represent each of the four pharmacologically distinct sites. Contact residues important in rhodamine123 binding were identified in the central cavity of P-gp. However, contact residues for the binding of vinblastine, paclitaxel and nicardipine were located at the lipid-protein interface rather than the central cavity. A key residue (F978) within the central cavity is believed to be involved in coupling drug binding to nucleotide hydrolysis. Data observed in this investigation suggest the presence of spatially distinct drug binding sites connecting through to a single translocation pore in the central cavity

Identifying the location of the drug binding site(s) in P-glycoprotein

P-glycoprotein (P-gp), an ABC transporter protein, is characterized by its ability to recognize, bind, and efflux over 300 chemically and structurally unrelated compounds. P-gp overexpression in cancer cells confers multidrug resistance (MDR) by preventing sufficient accumulation of anticancer drugs within the cell, thus avoiding their cytotoxic effects. P-gp harnesses the energy from ATP hydrolysis to translocate substrates across the plasma membrane against a concentration gradient. Biochemical and structural investigations have identified the presence of a large aqueous central cavity which is likely the location for the drug binding sites (DBSs). The substrate polyspecificity displayed by P-gp is imparted by the existence of at least four pharmacologically distinct DBSs located within the transmembrane domain (TMD). However, whether these sites are spatially distinct from each other was not clear. The current investigation has identified several key contact residues of four pharmacologically distinct substrates/modulators of P-gp (nicardipine, vinblastine, rhodamine 123, and paclitaxel), known to bind at the four identified sites. Seven residues in various TMDs were mutated to a cysteine. Biochemical assays using purified, reconstituted P-gp expressing each mutant isoform was used to identify which contact residues were implicated in the binding of each drug. For rhodamine 123 binding, the identified contact residues were located within the central cavity of P-gp. However, for vinblastine, paclitaxel, and nicardipine, the implicated contact residues were located at the lipid-protein interface rather than the central cavity. A key residue (F978) located within the central cavity is believed to be involved in the interdomain communication between the TMDs and the nucleotide binding domains (NBD; site of ATP hydrolysis). Collectively, data presented here suggests the existence of at least four spatially distinct drug binding sites that are connected by a single translocation pore in the central cavity

Reversing multidrug resistance (MDR) in cancer cells by targeting p-glycoprotein (P-gp) : insights into the mechanism of MDR reversal from in silico P-gp modeling

Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), Universidade de Lisboa, Faculdade de Farmácia, 2017Multidrug resistance (MDR) in cancer is one of the major impairments in the success of chemotherapy. The main objective of this work was the identification and optimization of MDR reversers, derived from Euphorbia species, and to gain insights on the drug efflux mechanism by P-gp. The phytochemical study of Euphorbia pedroi yielded four new diterpenes, two macrocyclic lathyranes (9, 12), one jatrophane (10) and an unprecedented rearranged tigliane (13). While 9 is characterized by a rare double α,β-unsaturated ketone system, 13 has a new skeleton that may result from a pinacol rearrangement as proposed in a possible biogenetic pathway. Furthermore, a new spiroterpenoid (6) was also isolated, together with several known terpenoids (1-5, 7, 8, 14-16) and flavonoids (17-18). Molecular derivatization of compounds 15 and 17 yielded two set of new derivatives (19-24 and 25-71, respectively). In this way, reaction of 15 with hydroxylamine hydrochloride gave compound 19 that was further acylated with acyl anhydrides (20) and chlorides (21-24). Flavanone derivatives were obtained through three main approaches. Firstly, the methylation of naringenin (17) yielded compounds 46 and 47. Following, while hydrazones (25-28, 48-53) and carbohydrazides (37, 38, 40-42, 54-63) were obtained from compounds 17, 46 and 47, azines (29-36) were prepared by the reaction of 28 with aldehydes. A thiosemicarbazone derivative (39) was also prepared from 17. Other flavanone derivatives were additionally synthesized through a Mannich-type reaction (43-45) or by alkylation of compound 47 with epichlorohydrin (64, 65) followed by the reaction with amines, indole or thiophenol to yield 66-71. The chemical structures of all compounds were deduced from physical and spectroscopic data (IR, MS, 1D- and 2D-NMR experiments). The P-gp-mediated MDR reversal activity of compounds was evaluated by combining transport and chemosensitivity assays, in mouse lymphoma L5178Y-MDR (1-71) and Colo320 (1-18) cell models. While 6 showed high modulation activity even at 0.2 μM, compound 9 combined a good P-gp modulatory activity with a strong cytotoxic effect in both cell lines. When compared to the parent compound (15), the derivatives 20 and 22 and 23 were stronger efflux modulators towards the L5178Y-MDR cells. Most of the flavanone derivatives (25-71) were also more active than the parent compound (17) in L5178Y-MDR cells, being the most significant results observed for propanolamines 66-69, where compound 69 was found to be a strong P-gp modulator even at 2.0 μM. When in combination with doxorubicin, the natural compounds 6, 9, 10, 12 and 13 synergistically enhanced the cytotoxic effects of the drug. Strong synergistic effects were also observed for the derivatives 22 and 69. The ability of compounds 25-45 to modulate drug efflux by MRP1 and BCRP was also assessed, using human MRP1- and BCRP-transfected cell models. For this set of compounds, a second P-gp-transfected cell model was used. Azines (29-36) displayed significant activity towards BCRP while hydrazides (38-42) showed a good selectivity profile for MRP1. Oppositely, derivatives 35 and 36 displayed a good activity profile in both efflux pumps, when tested at 20 μM. Based on these results, new structure-activity relationships (SAR) for the selective BCRP and MRP1 inhibitors were obtained, unveiling which structural features could be directly correlated with the observed biological activity. The efflux mechanism of P-gp was studied by means of molecular dynamics and docking studies. The ‘linker’ polypeptide sequence was found to be important to absorb stronger motions and acting as a ‘damper’ between both NBDs, stabilizing the cytosolic portion of the transporter. Following, based on a previously refined P-gp structure, three distinct drugbinding sites could be identified and characterized, in a good agreement with published experimental data. Together with a new classification scheme, cross interactions between the substrate/modulator and each halve of P-gp were identified as an important mechanism in efflux modulation. Drug transit from bulk water into the DBP was also characterized as an overall free-energy downhill process, with no activation energy required for crossing the gate found between transmembrane helices 10 and 12. Furthermore, from the analysis on drug adsorption to the cytoplasmic domains in P-gp substrates and modulators were show to have different free energies of adsorption in both lipid/water and protein/ water interfaces and important differences in drug–protein interactions, protein dynamics and membrane biophysical characteristics were observed between non-substrates, substrates and modulators.A resistência a múltiplos fármacos (MDR) no cancro configura-se como um dos principais problemas que atualmente comprometem o sucesso dos regimes de quimioterapia. Dos mecanismos celulares envolvidos na MDR, um dos mais importantes consiste no aumento do efluxo de citotóxicos ou de sequestração intracelular devido à sobre-expressão de transportadores da família ABC, nomeadamente a glicoproteína-P (P-gp), a proteína associada à multirresistência 1 (MRP1) e a proteína de resistência do cancro da mama (BCRP). Envolvidas em fenómenos normais de destoxificação celular, estas bombas de efluxo encontram-se igualmente implicadas na redução da concentração intracelular de fármacos antitumorais, transportando-os contra o seu gradiente de concentração, através da utilização da energia gerada pela ligação e hidrólise do ATP. Apesar das três gerações de moduladores da P-gp já desenvolvidas, nenhum modulador foi clinicamente eficaz na reversão da MDR quando em coadministração com fármacos citotóxicos. No entanto, e uma vez que a procura de fármacos capazes de reverter a MDR continua a ser uma das abordagens mais promissoras, novas moléculas isoladas de fontes naturais são atualmente consideradas como uma possível quarta geração de moduladores de bombas de efluxo, atuando como reversores da MDR em células tumorais. Assim, um dos objetivos principais deste trabalho foi a identificação e otimização de novos reversores da MDR, isolados a partir da espécie Euphorbia pedroi ou obtidos através de derivatização química de compostos isolados em grandes quantidades. O estudo fitoquímico da E. pedroi permitiu o isolamento de quatro novos diterpenos, dois latiranos (9, 12), um jatrofano (10) e um tigliano rearranjado com um esqueleto novo (13). Enquanto a pedrodiona A (9) é caracterizada pela presença de dois sistemas α,β-insaturados, o pedrolido (13) apresenta um rearranjo de pinacol em C-6/C-7 incomum. Foi também isolado um esteroide novo designado por spiropedroxodiol (6), contendo um esqueleto spiro raro, conjuntamente com vários terpenoides (1-5, 7, 8, 14-16) e flavonoides conhecidos (17-18). Por forma a otimizar as propriedades moduladoras do helioscopinolido E (15) e da naringenina (17), foram preparados dois conjuntos de compostos com o núcleo do ent-abietano (19-24) e da flavanona (25-71) através da derivatição molecular de vários grupos funcionais. Enquanto que no primeiro caso a reação do composto 15 com hidroxilamina deu origem à oxima 19 (C=N-OH) e posteriormente aos compostos 20-24 por acilação com anidridos ou cloretos de ácido, os derivados do núcleo da flavanona foram obtidos através de três abordagens distintas. Inicialmente, a metilação dos hidroxilos da naringenina (17) nas posições C-7 e C-4’ originou a sakuranetina (47) e a 4’-metoxisakuranetina (48). Em seguida, enquanto que as hidrazonas 25-28, 48-53 (C=N-NH-R) e as carbohidrazidas 37, 38, 40-42, 54-63 (C=N-NHCO-R) foram preparadas a partir dos compostos 17, 46 e 47, as azinas 29-36 (C=N-N=CH-R) foram sintetizadas através da reação do composto 28 (C=N-NH2) com aldeídos. Foi também sintetizada uma tiosemicarbazona (39) através da reação da naringenina (17) com a N,Ndimetiltio- semicarbazida. Foram ainda preparados outros derivados do núcleo da flavanona i) através de uma reação de Mannich nas posições C-6 e C-8 (43-45) e ii) através da alquilação do hidroxilo da posição C-4’ da sakuranetina (47) com epiclorohidrina (64, 65) seguida da reação com aminas, indole ou tiofenol para originar as correspondentes propanolaminas (66- 69) e os compostos 70-71. As estruturas químicas dos compostos foram deduzidas a partir dos seus dados físicos e espectroscópicos (IR, MS, 1D e 2D-RMN). A capacidade de reversão de MDR dos compostos foi avaliada através da combinação de ensaios funcionais com ensaios de quimiossensibilidade, utilizando como modelos as células de linfoma de rato L5178Y-MDR (1-71) e células Colo320 humanas (1-18). Enquanto que o spiropedroxodiol (6) demonstrou possuir uma elevada capacidade para modular o efluxo mesmo em concentrações submicromolares (0.2 μM), a atividade expressa pela pedrodiona A (8) combinou uma boa atividade na reversão de MDR com uma elevada citotoxicidade nas linhas celulares L5178Y-MDR (FAR 19.13, IC50 0.259 ± 1.05 μM) e Colo320 (FAR 1.52, IC50 0.822 ± 1.05 μM). Relativamente aos derivados obtidos a partir do composto 15, a acilação da oxima do heliscopinolido E (19) aumentou a capacidade moduladora do efluxo nas células de linfoma de rato na maioria dos compostos sintetizados. Nos derivados da naringenina (17), a metilação de hidroxilos fenólicos em conjunto com i) a substituição do grupo carbonilo na posição C-4 por hidrazonas (25-28, 48-53), azinas (29-36) ou carbohidrazidas (37-42, 54-63) aumentou a atividade de reversão de MDR dos compostos a 20 μM. Verificou-se também que a alquilação do hidroxilo na posição C-4’ para gerar as correspondentes propanolaminas (64-71) aumentou substancialmente as suas propriedades anti-MDR (66-69) a concentrações mais baixas (2.0 μM). Adicionalmente, e quando testados em combinação com a doxorubicina, todos os compostos testados (6, 10, 12, 13, 22, 69) exceto o 9 (efeito aditivo) e o 60 (antagonismo) potenciaram a atividade citotóxica quando em co-administração com o fármaco antitumoral. Os efeitos dos derivados 25-45 da flavanona (17) foram igualmente avaliados noutros transportadores ABC frequentemente sobre-expressos em células tumorais, como a proteína de resistência do cancro da mama (BCRP) e a proteína associada à multirresistência 1 (MRP1). Adicionalmente, estes compostos foram também testados numa linha celular alternativa, igualmente transfetada com o gene da P-gp (NIH/3T3). Assim, enquanto que as hidrazonas (25-36) demonstraram uma maior seletividade para a BCRP, os derivados de hidrazidas (37, 38, 40-42) foram seletivos para a MRP1. No entanto, importa referir que os compostos 35, 36 e 39 (uma tiosemicarbazona) demonstraram possuir uma atividade apreciável como modulador de efluxo em ambas as bombas MRP1 e BCRP (a 20 μM). Finalmente, e tendo como base os resultados acima descritos, foram desenvolvidas novas relações estrutura-atividade (SAR) em que a posição espacial do substituinte foi identificada como um dos principais fatores para a atividade registada na MRP1 e BCRP. Por fim, este estudo providenciou pela primeira vez um racional para o desenvolvimento de novos moduladores para a P-gp, BCRP e MRP1 a partir do núcleo da flavanona. A publicação da estrutura cristalográfica da P-gp murina, em 2009, colmatou uma importante falha no estudo das bombas de efluxo e permitiu um crescimento exponencial de estudos estruturais, visando um maior conhecimento sobre o mecanismo de efluxo pela P gp. Desta forma, e para evitar os problemas subjacentes ao desenvolvimento das primeiras três gerações de moduladores da MDR em que a estrutura do transportador não era conhecida, importa saber os principais passos pelos quais ocorre o efluxo de substratos e alguns detalhes específicos adicionais acerca do mecanismo de efluxo da P-gp Assim, foi estudado o mecanismo de efluxo pela P-gp através de dinâmica e docking molecular. A estrutura polipeptídica em falta (“linker”) foi determinada como essencial para a estabilização dos domínios citoplasmáticos da P-gp, atuando por forma a absorver fortes oscilações estruturais. Com base na estrutura previamente refinada da P-gp, foram identificados e caracterizados de acordo com dados experimentais publicados, três locais de ligação distintos, dois de ligação a substratos e um de ligação a moduladores. Através da publicação de um novo esquema de classificação, as interações cruzadas entre o modulador e cada domínio da P-gp (N-e C-terminais) foram identificadas como um mecanismo importante na modulação de efluxo. O processo biofísico pelo qual moléculas são capazes de permear a membrana a partir do citoplasma e a sua entrada na cavidade interna da P-gp foi também caracterizado como um processo energeticamente favorável, desprovido de barreiras energéticas, mesmo durante a passagem através das hélices transmembranares 10 e 12. Do mesmo modo, substratos e moduladores revelaram ter diferentes energias livres de adsorção em cada uma das interfaces (lípidos/água e proteína/água), tendo sido igualmente registadas diferenças importantes nas interações fármaco-proteína, nos processos dinâmicos do transportador e nas características biofísicas da membrana quando em contacto com não-substratos, substratos e moduladores

Regulation of Hepatic Drug Metabolizing Enzymes in Chronic Kidney Disease

Chronic kidney disease (CKD) occurs as a result of declining renal function for 3 or more months. CKD effects 1 in 10 Canadians and is associated with a number of co-morbidities including diabetes and cardiovascular disease. To manage CKD and associated co-morbidities, patients take an average of 12 medications with a median pill burden of 19. Indeed, renal drug elimination is compromised in CKD, as declining glomerular filtration reduces drug excretion into urine. More recently, studies have provided evidence of altered non-renal drug clearance in CKD. The majority of drug clearance occurs in the liver by CYP2C and CYP3A drug metabolizing enzymes. Hepatic CYP2C and CYP3A drug metabolizing enzymes are tightly regulated by nuclear receptors. The majority of CKD patients have mild to moderate degrees of CKD and the potential for altered hepatic drug metabolism at these earlier stages is unknown. As renal function declines, patients begin to experience the uremic condition, which consists of metabolic waste product accumulation in the blood. A number of studies suggest that these uremic toxins may mediate the downregulation of hepatic CYP2C and CYP3A; however, the mechanism by which this occurs remains to be determined. My overall hypothesis is that hepatic drug metabolism is altered in CKD. Herein, we evaluate the effects of moderate CKD on hepatic drug metabolism and determine a possible mechanism of CYP2C and CYP3A downregulation in rats with CKD. In a rat model of moderate CKD, hepatic CYP2C and CYP3A function and expression were significantly decreased demonstrating a negative exponential correlation with kidney function. Transcriptional activation in both the CYP2C and CYP3A promoters was reduced as a result of decreased nuclear receptor binding and histone acetylation. Untargeted metabolomics was utilized to identify potential uremic mediators of hepatic CYP2C and CYP3A altered expression and function. Gut-derived uremic toxins accounted for the most significant metabolic signatures defining plasma and liver tissue in CKD, leading to the evaluation of these toxin effects on hepatic CYP2C and CYP3A. Reduction of gut-derived uremic toxins did not recover CYP2C and CYP3A function and expression in CKD. In conclusion, these studies further our understanding of hepatic drug metabolizing enzyme downregulation in the setting of CKD

Development of Computational Antibiotic Screening Platform Across Bacterial Outer Membrane Proteins

Antibiotics are medicines used to treat bacterial infections by either killing bacteria or stopping them from reproducing. Throughout the use of antibiotics, bacteria has developed a variety of defense mechanisms against antibiotics and thus diminishing their effectiveness. Antibiotic resistance is a growing threat and becomes a global crisis as it is able to constantly evolve and rapidly spread. In the face of increasing bacterial resistance to all known antibiotics, there is an urgent need to accelerate the antibiotic discovery pipeline and discover new classes of antibiotics. A major bottleneck in the discovery of novel antibiotics is the limited permeability of potent drug molecules across the bacterial envelope to reach their target, and thus hindering their activities in vivo. With the aid of state-of-the-art computational methods and tools, we developed a computational platform to automate and study the translocation of small molecule drugs across bacterial outer membrane proteins, with a goal of accelerating the antibiotic discovery process. We applied all-atom and coarse-grained molecular modeling, enhanced sampling techniques, and a parallel computing environment to maximize the performance. We further demonstrate the efficacy of this platform with a comprehensive study of a benchmark case. Key findings include free energy profile, translocation kinetics and thermodynamics, and molecular interactions between drug molecules and protein residues. Ultimately, this approach is designed to screen small molecule libraries with a fast turnaround time to yield structure-property relationships to discover antibiotics with high permeability. Furthermore, this work is expected to provide insights in inverse engineering and mutation design during drug development

Digestion, blood-brain barrier transposition and neuroprotective effect

"This thesis is organized in five parts: introduction, three chapters of experimental work and a general discussion. In the introduction section, major topics necessary for a broad comprehension of this thesis is described. Major aspects about neurodegenerative diseases and (poly)phenols importance in the disease prevention are present. State of art of major cellular models used for studying (poly)phenols role in Parkinson’s disease (PD) is also described. Emphasis to the knowledge gaps of (poly)phenols potential to act inside the brain was considered such as their ability to cross and/or interact with the blood-brain barrier (BBB). Finally, a thesis rational and major work objectives are concluding the introductory section. Part of this section was included in a review published in Current Neuropharmacology.(...)"N/

Antioxidant and DPPH-Scavenging Activities of Compounds and Ethanolic Extract of the Leaf and Twigs of Caesalpinia bonduc L. Roxb.

Antioxidant effects of ethanolic extract of Caesalpinia bonduc and its isolated bioactive compounds were evaluated in vitro. The compounds included two new cassanediterpenes, 1α,7α-diacetoxy-5α,6β-dihydroxyl-cass-14(15)-epoxy-16,12-olide (1)and 12α-ethoxyl-1α,14β-diacetoxy-2α,5α-dihydroxyl cass-13(15)-en-16,12-olide(2); and others, bonducellin (3), 7,4’-dihydroxy-3,11-dehydrohomoisoflavanone (4), daucosterol (5), luteolin (6), quercetin-3-methyl ether (7) and kaempferol-3-O-α-L-rhamnopyranosyl-(1Ç2)-β-D-xylopyranoside (8). The antioxidant properties of the extract and compounds were assessed by the measurement of the total phenolic content, ascorbic acid content, total antioxidant capacity and 1-1-diphenyl-2-picryl hydrazyl (DPPH) and hydrogen peroxide radicals scavenging activities.Compounds 3, 6, 7 and ethanolic extract had DPPH scavenging activities with IC50 values of 186, 75, 17 and 102 μg/ml respectively when compared to vitamin C with 15 μg/ml. On the other hand, no significant results were obtained for hydrogen peroxide radical. In addition, compound 7 has the highest phenolic content of 0.81±0.01 mg/ml of gallic acid equivalent while compound 8 showed the highest total antioxidant capacity with 254.31±3.54 and 199.82±2.78 μg/ml gallic and ascorbic acid equivalent respectively. Compound 4 and ethanolic extract showed a high ascorbic acid content of 2.26±0.01 and 6.78±0.03 mg/ml respectively.The results obtained showed the antioxidant activity of the ethanolic extract of C. bonduc and deduced that this activity was mediated by its isolated bioactive compounds