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

Preventing Atomicity Violations with Contracts

Software developers are expected to protect concurrent accesses to shared regions of memory with some mutual exclusion primitive that ensures atomicity properties to a sequence of program statements. This approach prevents data races but may fail to provide all necessary correctness properties.The composition of correlated atomic operations without further synchronization may cause atomicity violations. Atomic violations may be avoided by grouping the correlated atomic regions in a single larger atomic scope. Concurrent programs are particularly prone to atomicity violations when they use services provided by third party packages or modules, since the programmer may fail to identify which services are correlated. In this paper we propose to use contracts for concurrency, where the developer of a module writes a set of contract terms that specify which methods are correlated and must be executed in the same atomic scope. These contracts are then used to verify the correctness of the main program with respect to the usage of the module(s). If a contract is well defined and complete, and the main program respects it, then the program is safe from atomicity violations with respect to that module. We also propose a static analysis based methodology to verify contracts for concurrency that we applied to some real-world software packages. The bug we found in Tomcat 6.0 was immediately acknowledged and corrected by its development team

From business process modeling to data model : a systematic approach

Business process modeling and management approaches are increasingly used and disclosed between organizations as a means of optimizing and streamlining the business activities. Among the various existing modeling languages, we stress the Business Process Model and Notation (BPMN), currently in version 2.0. BPMN is a widespread OMG standard that is actually used either in academia and in organizations. BPMN enables business process modeling, but does not facilitate the modeling of the information infrastructure involved in the process. However, interest in the data and its preservation has increased in BPMN’s most recent version. The aim of this paper is to study BPMN 2.0, particularly on the usage and persistence of data, and present an approach for obtaining an early data model from the business process modeling, which may then be used as a starting data model in the software development process

Adaptable control for electrical generation at irregural wind speeds

The main aims of this work are the development and the validation of one generic algorithm to provide the optimal control of small power wind generators. That means up to 40 kW and blades with fixed pitch angle. This algorithm allows the development of controllers to fetch the wind generators at the desired operational point in variable operating conditions. The problems posed by the variable wind intensity are solved using the proposed algorithm. This is done with no explicit measure of the wind velocity, and so no special equipment or anemometer is required to compute or measure the wind velocity

Carbon (CI) and energy intensity (EI) dataset for retail stores

Transparency data associated with this article can be found in the online version at: https://doi.org/10.1016/j.dib.2018.10.080This data article presents data collected from the 250 highest revenue retailers around the world, assessed according to publicly available data from the fiscal year 2016, in order to determine retailer?s overall carbon intensity (CI) and energy intensity (EI). Data collection included additional variables such as retailers? revenue rank, operational typology, number of stores, store sales area and number of workers. CI and EI benchmarks were calculated for food and non-food retailers, applying the statistic function first quartile (Q1) for the best practice, second (Q2) and third (Q3) quartiles for conventional practice and fourth quartile (Q4) for worst practice. Correlations were tested between the variables "EI", "CI" and "retailer revenue", applying the statistic function CORREL. Finally, a cluster analysis was performed for food and non-food retailers, to identify possible segmentation patterns between the variables ?EI?, ?CI? and ?retailer revenue?. The information provided in this data article is useful for furthering research developments on the influence of isolated variables on retail EI and CI and in assisting retailers, architects, engineers, and policy makers in establishing optimal energy performance goals for the design and operation of retail stores. For further data interpretation and discussion, see the article ?Combined carbon and energy intensity benchmarks for sustainable retail stores?[1], of the same authors.This work was supported by FCT - Fundação para a Ciência e Tecnologia [grant number PD/BD/127852/2016] under the Doctoral Program EcoCoRe - Eco-Construction and Rehabilitation.info:eu-repo/semantics/publishedVersio

Combined carbon and energy intensity benchmarks for sustainable retail stores

Retail stores are amongst the building typologies with the highest carbon (CI) and energy intensities (EI). However, previous studies have only explored the EI of food and non-food retailers. This study is the first of its kind to examine the link between CI and EI. Establishing the nature of this link will allow a deeper understanding of how to decarbonize the retail sector. Here, we hypothesised whether in retail low EI correlated with low CI and how corporate revenue affected these variables. ?Best practice? and ?conventional practice? benchmarks were then developed to assess retail buildings' sustainability. These represent missing and highly desirable tools in retail sustainable management. Average EI and CI of food retailers were twice that of non-food retailers (EI-548 vs 238?kWh/m2/y; CI266 vs 132?kg CO?eq/m2/y). The correlation found between EI and CI indicates that low energy consumption leads to low greenhouse gas (GHG) emissions. CI variability resulted mostly of energy-efficiency strategies, of the energy production process and of GHG emissions from refrigeration systems. EI variability resulted mostly from store typology, volume and usage. The proposed benchmarks help to set energy and carbon reference performance levels in retail buildings and to stimulate best sustainable practice amongst retailers.This work was supported by FCT - Fundação para a Ciência e Tecnologia [grant number PD/BD/127852/2016] under the Doctoral Program EcoCoRe - Eco-Construction and Rehabilitation. Support from CERIS and Instituto Superior Técnico is also acknowledged.info:eu-repo/semantics/publishedVersio

Overcoming inertia : drivers of the outsourcing process

Almost all managers have directly or indirectly been involved in the practice of outsourcing in recent years. But as they know, outsourcing is not straightforward. Outsourcing inertia, when companies are slow to adapt to changing circumstances that accommodate higher outsourcing levels, may undermine a firm’s performance. This article investigates the presence of outsourcing inertia and the factors that help managers overcome it. Using statistical evidence, we show that positive performance effects related to outsourcing can accumulate when circumstances change. This is then followed by rapid increases in outsourcing levels (i.e. outsourcing processes). We investigate what gives rise to these outsourcing processes through follow-up interviews with sourcing executives, which suggest five drivers behind outsourcing processes: managerial initiative (using outside experience); hierarchy (foreign headquarters); imitation (of competitors and of similar firms); outsider advice (from external institutions); knowledge sources (using external information). These five drivers all offer scope for managerial action. We tie them to academic literatures and suggest ways of investigating their presence and impact on the outsourcing process. Overall, we conclude that while economizing factors play a key role in explaining how much firms outsource, it is socializing factors that tend to drive outsourcing processes

A 802.11p prototype implementation

This paper presents an IEEE 802.11p full-stack prototype implementation to data exchange among vehicles and between vehicles and the roadway infrastructures. The prototype architecture is based on FPGAs for Intermediate Frequency (IF) and base band purposes, using 802.11a based transceivers for RF interfaces. Power amplifiers were also addressed, by using commercial and in-house solutions. This implementation aims to provide technical solutions for Intelligent Transportation Systems (ITS) field, namely for tolling and traffic management related services, in order to promote safety, mobility and driving comfort through the dynamic and real-time cooperation among vehicles and/or between vehicles and infrastructures. The performance of the proposed scheme is tested under realistic urban and suburban driving conditions. Preliminary results are promising, since they comply with most of the 802.11p standard requirements