Dissertations / Theses on the topic 'Patched drug efflux inhibitors'

Nous avons récemment démontré que le récepteur du morphogène Hedgehog, Patched, qui est exprimé dans de nombreux cancers, est un transporteur de multiples drogues qui contribue à la résistance des cellules cancéreuses à la chimiothérapie. Le criblage d'une banque de molécules nous a permis d'identifier deux molécules qui inhibent l'activité d'efflux de doxorubicine de Patched. Nous avons montré que ces molécules renforcent les effets cytotoxiques, proapoptotiques, antiprolifératifs et anticlonogéniques de la doxorubicine sur les cellules de cancer de la glande surrénale (surrénalome) qui expriment de façon endogène Patched. De plus, nous avons observé que l’ajout de la molécule P375 au traitement à la doxorubicine inhibe le développement des tumeurs chez des souris ayant reçu une xénogreffe de cellules de surrénalome de façon plus significative que la doxorubicine seule. Nos résultats suggèrent que l'utilisation d'un inhibiteur de l'activité d'efflux de drogues de Patched en association avec la doxorubicine est une option thérapeutique prometteuse pour le surrénalome, et très probablement pour d'autres cancers exprimant Patched. Nous avons découvert qu'une petite fraction seulement des cellules de la lignée de surrénalome exprime Patched au niveau de la membrane plasmique (cellules PM-Patched). Les cellules PM-Patched sont plus résistantes à la doxorubicine, et présentent une expression plus élevée de Patched mais aussi de la protéine ABCG2. ABCG2 étant un marqueur de cellules souches cancéreuses (CSC), nous pensons que les cellules PM-Patched pourraient être des CSC.D'autres expériences sont nécessaires pour valider cette hypothèse
We recently demonstrated that the Hedgehog receptor Patched, which is expressed in many recurrent and metastatic cancers, is a multidrug transporter contributing to chemotherapy resistance. The screening of a chemical library allowed us identifying two molecules which inhibit the doxorubicin efflux activity of Patched. We showed that these molecules enhance the cytotoxic, proapoptotic, antiproliferative and anticlonogenic effects of doxorubicin on adrenocortical carcinoma (ACC) cells which endogenously express Patched. Moreover, we reported that the addition of the drug-like molecule P375 to doxorubicin treatment prevents the development of xenograft ACC tumours in mice much more significantly than the doxorubicin alone. Our results suggest that the use of an inhibitor of Patched drug efflux activity in combination with doxorubicin is a promising therapeutic option for ACC and most likely for other Patched-expressing cancers. We discovered that only a small fraction of the ACC cell line expressed Patched at the plasma membrane (PMPatched cells). We observed that these cells are more resistant to doxorubicin treatment than ACC cells that express Patched only in intracellular compartments. Moreover, we estimated that PMPatched cells have higher expression of Patched but also of ABCG2/BCRP proteins. Based on the fact ABCG2/BCRP is a cancer stem cell (CSC) marker and that Hedgehog signaling is involved in maintenance of CSC, we think that PM-Patched cells could be CSCs. More experiments are needed to confirm this hypothesis

Nous avons récemment démontré que le récepteur du morphogène Hedgehog, Patched, qui est exprimé dans de nombreux cancers, est un transporteur de multiples drogues qui contribue à la résistance des cellules cancéreuses à la chimiothérapie. Le criblage d'une banque de molécules nous a permis d'identifier deux molécules qui inhibent l'activité d'efflux de doxorubicine de Patched. Nous avons montré que ces molécules renforcent les effets cytotoxiques, proapoptotiques, antiprolifératifs et anticlonogéniques de la doxorubicine sur les cellules de cancer de la glande surrénale (surrénalome) qui expriment de façon endogène Patched. De plus, nous avons observé que l’ajout de la molécule P375 au traitement à la doxorubicine inhibe le développement des tumeurs chez des souris ayant reçu une xénogreffe de cellules de surrénalome de façon plus significative que la doxorubicine seule. Nos résultats suggèrent que l'utilisation d'un inhibiteur de l'activité d'efflux de drogues de Patched en association avec la doxorubicine est une option thérapeutique prometteuse pour le surrénalome, et très probablement pour d'autres cancers exprimant Patched. Nous avons découvert qu'une petite fraction seulement des cellules de la lignée de surrénalome exprime Patched au niveau de la membrane plasmique (cellules PM-Patched). Les cellules PM-Patched sont plus résistantes à la doxorubicine, et présentent une expression plus élevée de Patched mais aussi de la protéine ABCG2. ABCG2 étant un marqueur de cellules souches cancéreuses (CSC), nous pensons que les cellules PM-Patched pourraient être des CSC.D'autres expériences sont nécessaires pour valider cette hypothèse
We recently demonstrated that the Hedgehog receptor Patched, which is expressed in many recurrent and metastatic cancers, is a multidrug transporter contributing to chemotherapy resistance. The screening of a chemical library allowed us identifying two molecules which inhibit the doxorubicin efflux activity of Patched. We showed that these molecules enhance the cytotoxic, proapoptotic, antiproliferative and anticlonogenic effects of doxorubicin on adrenocortical carcinoma (ACC) cells which endogenously express Patched. Moreover, we reported that the addition of the drug-like molecule P375 to doxorubicin treatment prevents the development of xenograft ACC tumours in mice much more significantly than the doxorubicin alone. Our results suggest that the use of an inhibitor of Patched drug efflux activity in combination with doxorubicin is a promising therapeutic option for ACC and most likely for other Patched-expressing cancers. We discovered that only a small fraction of the ACC cell line expressed Patched at the plasma membrane (PMPatched cells). We observed that these cells are more resistant to doxorubicin treatment than ACC cells that express Patched only in intracellular compartments. Moreover, we estimated that PMPatched cells have higher expression of Patched but also of ABCG2/BCRP proteins. Based on the fact ABCG2/BCRP is a cancer stem cell (CSC) marker and that Hedgehog signaling is involved in maintenance of CSC, we think that PM-Patched cells could be CSCs. More experiments are needed to confirm this hypothesis

Multidrug resistance (MDR), a phenomenon in which tumors that were initially sensitive, recur and start showing resistance not only to the initial chemotherapeutic agent but also to various anticancer drugs that are structurally and functionally different from the initial drug, constitutes one of the main reasons for the failure of chemotherapy. An important mechanism of MDR is the enhanced cellular efflux of anticancer agents due to an overexpression of ATP-binding cassette (ABC) transporters (i.e. efflux transporters), especially P-glycoprotein (Pgp), Multidrug Resistance-associated Protein 1 (MRP1) and Breast Cancer Resistance Protein (BCRP), in cancer cells. In order to reverse this resistance, there has been a lot of emphasis on the development of Pgp, MRP1 and BCRP inhibitors. Although this search has been ongoing for three decades, there are still no clinically available efflux transporter modulators. Tyrosine kinase inhibitors (TKIs) are a novel, rapidly growing class of anticancer agents that have a target-based mechanism of action, and their use transformed cancer chemotherapy due to higher specificity and enhanced safety profiles compared to conventional chemotherapeutic agents. Despite their tremendous success in treating various types of tumors, patients develop resistance to TKIs over time. Most of the FDA- approved TKIs are substrates of Pgp and/or BCRP, and as a result, these efflux transporters are also an important cause of conferred resistance against TKIs in cancer cells. Additionally, none of the 31 approved TKIs have an indication for use in brain tumors and interestingly, this may also due to the presence of Pgp and BCRP at the blood-brain barrier (BBB) and in the tumor cells, which prevent the TKI from crossing the BBB and reaching its target tumor site. Since Pgp- and BCRP- mediated TKI efflux has been shown to be involved in TKI resistance, the inhibition of these transporters could represent a potential TKI resistance reversal strategy. Over the last three decades, a large number of Pgp and/or BCRP inhibitors have been identified, but none of them have successfully made it to the clinic. It was observed that most drugs identified as inhibitors were either unable to achieve Pgp and BCRP inhibitory concentrations in-vivo without imparting severe toxicity, or did not possess adequate bioavailability and tissue distribution profiles in order to reach the tumor site. From these identified candidate inhibitors, after much thought and consideration, we chose to investigate TKIs and methylated flavones as modulators of efflux transporter-mediated TKI resistance. The overall goal of this project was to investigate the promising chemosensitizing potential of TKIs and methylated flavones in efflux transporter-mediated TKI resistance, both in-vitro and in-vivo. To identify potent efflux transporter inhibitor TKIs, we evaluated the effect of various TKIs on the accumulation of afatinib, the model TKI substrate, in Pgp- and BCRP- overexpressing cell lines. Afatinib was chosen as the model TKI substrate for our study because it undergoes very minimal metabolism in several species. Afatinib is a substrate of both Pgp and BCRP, but is not a substrate of uptake transporters. Therefore, it was anticipated that an in-vivo efflux transporter-mediated interaction with afatinib would most likely not be confounded or masked by other factors influencing its disposition. From the in-vitro cell uptake studies, we found that nilotinib is a potent inhibitor of both Pgp and BCRP, and it reversed Pgp- and BCRP- mediated afatinib efflux. Subsequently, an in-vivo study was carried out in mice to investigate the interaction between afatinib and nilotinib; and also the impact of nilotinib on the pharmacokinetics and tissue distribution of afatinib. Afatinib exposure in the plasma and in most tissues, namely liver, lung, kidney, heart, muscle, fat, and skin, was found to be significantly increased when nilotinib was coadministered with afatinib. Further, the nilotinib concentrations in most mice tissues was above that needed for Pgp and BCRP inhibition. These results showed that nilotinib could be a potent chemosensitizing agent for Pgp- and BCRP- mediated TKI resistance. Additionally, a significant increase in afatinib brain exposure was observed in the mice which were administered afatinib in combination with nilotinib. This is an interesting and important finding that could potentially be very useful in the treatment of primary and metastasized brain tumors. We also developed a physiologically based pharmacokinetic model of afatinib to characterize its tissue disposition in mice organs, and this model was then scaled up to humans. The developed model accurately predicted afatinib plasma exposure in healthy volunteers and patients with solid malignant tumors, renal impairment, and hepatic impairment. To investigate the chemosensitizing potential of methylated flavones in efflux transporter-mediated TKI resistance, the Bcrp1 inhibitory effect of 5,7-DMF and its effect on sorafenib accumulation was evaluated in-vitro. 5,7- DMF was found to be a potent inhibitor of Bcrp1 and consequently, its impact on the pharmacokinetics and tissue distribution of sorafenib was evaluated in mice. Results showed that co-administration with 5,7-DMF led to significantly greater sorafenib exposure in plasma and in most tissues collected. This indicated that 5,7-DMF may represent a promising chemosensitizing agent for Bcrp1-mediated TKI resistance due to its low toxicity and potent Bcrp1 inhibition. Our results may have important clinical implications as TKIs are currently the most widely used anticancer agents. 5,7-DMF may show great potential in reversing MDR in tumors expressing BCRP. On the other hand, TKI-TKI combination therapy, especially with nilotinib as the perpetrator, is an attractive strategy to combat both Pgp- and BCRP-mediated TKI resistance. Additionally, since nilotinib has a wide volume of distribution and can reach various tissues at concentrations sufficient enough to inhibit Pgp and BCRP; it could potentially be used as a chemosensitizer in the treatment of numerous types of cancers. Furthermore, its chemosensitizing potential could particularly be useful in the treatment of primary and metastatic brain tumors. Further studies are warranted to assess the chemosensitizing effect of nilotinib in tumor xenograft models.

Malgré des efforts continus dans le développement de nouveaux médicaments, la résistance aux chimiothérapies reste un défi majeur dans le traitement du cancer. L'un des principaux mécanismes responsables de la résistance aux médicaments est l'efflux des agents thérapeutiques hors des cellules cancéreuses par les transporteurs multidrogues.Le récepteur Hedgehog Patched1 (PTCH1), qui fait partie de la voie de signalisation Hedgehog, est surexprimé dans de nombreux cancers. Outre son rôle physiologique de transporteur de cholestérol, PTCH1 est aussi capable de transporter des agents anticancéreux hors des cellules cancéreuses, contribuant ainsi à la résistance aux médicaments avec les transporteurs ABC (ATP binding cassette). Contrairement à ces derniers, qui sont exprimés de manière endogène dans les cellules normales et jouent un rôle crucial dans la survie cellulaire, l'activité d'efflux de PTCH1 n'a lieu que dans les cellules cancéreuses. Cela fait de PTCH1 une nouvelle cible prometteuse pour le traitement du cancer. Trois inhibiteurs de son activité d'efflux ont été identifiés à ce jour : l'astémizole, la méthiothépine et un composé naturel, la panicéine A hydroquinone (PAH). Ces composés augmentent l'efficacité des chimiothérapies contre les cellules de mélanome in vitro et in vivo, prouvant que l'inhibition de PTCH1 est une stratégie anticancéreuse pertinente. Cependant, ils présentent des limites intrinsèques et doivent être optimisés. Par exemple, la PAH a une très faible stabilité métabolique, ce qui empêche d'envisager son développement en tant que médicament.Dans ce contexte, le projet a pour objectif de développer un inhibiteur de l'efflux de PTCH1, en tant que médicament “first-in-class” pour lutter contre la résistance à la chimiothérapie et améliorer la survie des patients grâce à une optimisation rationnelle de la PAH.Nous avons donc étudié l'interaction entre la PAH et PTCH1 au moyen de méthodologies in silico et effectué une comparaison structure-ligand entre les trois inhibiteurs connus. En utilisant un protocole de docking couplé à des simulations de dynamiques moléculaires, nous avons extrait des informations importantes sur la conformation active d'un inhibiteur de PTCH1 et identifié un site de liaison supposé dans le canal hydrophobe de PTCH1.Nous avons mis au point une nouvelle synthèse totale de l'E-PAH qui répond à certaines limitations importantes de la précédente, telles que sa non-stéréosélectivité et sa grande spécificité de substrat. Après optimisation, nous avons finalement obtenu un nouveau protocole de synthèse stéréosélective et robuste pour les E et Z PAH. Ceci a permis de synthétiser plusieurs analogues de PAH dont l'activité a été évaluée sur des cellules de mélanome, en combinaison avec le vemurafenib, dans le but de réaliser une étude structure-activité pour le motif hydroquinone. Nous avons également évalué certaines propriétés pharmacocinétiques de ce motif, telles que la stabilité plasmatique et le profil de sécurité.La partie hydroquinone de la PAH est importante pour son activité biologique, mais c'est aussi l'un de ses principaux inconvénients en raison de son oxydation facile en quinone. En combinant les connaissances acquises grâce aux études in silico et SAR dans la dernière partie de ce projet, nous avons cherché à synthétiser des nouvelles molécules en appliquant un remplacement "bioisostérique", une approche souvent utilisée avec succès en chimie médicinale. Tout d'abord, le remplacement de la double liaison de la PAH par des groupes chimiques biologiquement équivalents a permis une stratégie de synthèse robuste, convergente et efficace, qui s'inscrit dans les tendances modernes des approches de chimie durable. Ensuite, il a été possible de synthétiser facilement un grand nombre de composés, en remplaçant le motif hydroxyquinone, et de cribler l'activité biologique in cellulo d'un espace chimique diversifié avec des résultats biologiques très prometteurs
Despite the continuous efforts in the design and development of new drugs with innovative mode of action, resistance to both chemo- and targeted therapy remains a major challenge in cancer treatment. One of the major mechanisms responsible for multidrug resistance is the efflux of therapeutics out of cancer cells by multidrug transporters.The Hedgehog receptor Patched1 (PTCH1), part of the Hedgehog signaling pathway, is over-expressed in many cancers. In addition to its physiological role as cholesterol transporter, PTCH1 is also able to transport anticancer agents out of cancer cells, thus contributing to multidrug resistance together with the ATP binding cassette (ABC) transporters. Unlike ABC transporters which are endogenously expressed in normal cells and play a crucial role in cell survival, PTCH1 efflux activity takes place only in cancer cells. This makes PTCH1 a new attractive target for cancer treatment. Three inhibitors of PTCH1 efflux activity have been identified to date, namely, astemizole, methiothepin and the natural compound panicein A hydroquinone (PAH). These compounds increased the efficacy of both conventional and targeted chemotherapies against melanoma cells in vitro and in vivo, showing the proof of concept of PTCH1 inhibition as a successful anticancer strategy. However, they have some intrinsic limitations and require optimization. For instance, PAH, has a very low metabolic stability which prevents its advance in a drug development pipeline towards the clinics.In that context, this PhD project is aimed at the ultimate goal of developing a PTCH1 drug efflux inhibitor as a first-in-class drug candidate to fight chemotherapy resistance and improve patient survival. In particular, it comprehends the medicinal chemistry efforts made in the direction of rational optimization of the natural compound PAH.To this aim we studied the interaction between PAH and PTCH1 by means of in silico methodologies and we performed structure- and ligand based comparison between the three known inhibitors. By using an ensemble docking protocol coupled to molecular dynamics simulations of the ligands we extracted important information about the active conformation of a PTCH1 inhibitor and we identified a putative binding site within the hydrophobic channel on PTCH1.We developed a new total synthesis for E-PAH which addresses some important limitations of the previous one, such as its non-stereoselectivity and high substrate specificity. We applied several strategies and eventually obtained a stereoselective and robust new synthesis protocol for E and Z PAH. This allowed for the synthesis of several PAH analogs whose activity was evaluated on melanoma cells, in combination with vemurafenib, with the aim to assess a structure-activity relationship for the hydroquinone scaffold. We further evaluated some PK properties of the hydroquinone scaffold such as plasma stability and safety profile.The hydroquinone moiety of PAH is important for its biological activity but it is also one of its major drawbacks due to its easy oxidation to quinone. By combining the knowledge acquired from the in silico and SAR studies on the hydroquinone, in the last part of this PhD project we aimed at the synthesis of innovative scaffolds by applying a “bioisosteric” replacement on the hydroquinone, an approach often successfully used in medicinal chemistry. First, the replacement of the double bond of PAH with biologically equivalent chemical groups allowed for a robust, convergent and efficient synthetic strategy, which is line with the modern trends of sustainable chemistry approaches. Next, it made possible to readily synthesize a large number of compounds, substituting the hydroxyquinone motif, and screen the in cellulo biological activity of a diversified chemical space with quite promising biological results

The trimethoprim and sulfamethoxazole combination, co-trimoxazole, plays a vital role in the treatment of Burkholderia pseudomallei infections. Previous studies demonstrated that the B. pseudomallei BpeEF-OprC efflux pump confers widespread trimethoprim resistance in clinical and environmental isolates, but this is not accompanied by significant resistance to co-trimoxazole. Using the excluded select-agent strain B. pseudomallei Bp82, we now show that in vitro acquired trimethoprim versus cotrimoxazole resistance is mainly mediated by constitutive BpeEF-OprC expression due to bpeT mutations or by BpeEF-OprC overexpression due to bpeS mutations. Mutations in bpeT affect the carboxy-terminal effector-binding domain of the BpeT LysR-type activator protein. Trimethoprim resistance can also be mediated by dihydrofolate reductase (FolA) target mutations, but this occurs rarely unless BpeEF-OprC is absent. BpeS is a transcriptional regulator that is 62% identical to BpeT. Mutations affecting the BpeS DNA-binding or carboxy-terminal effector-binding domains result in constitutive BpeEF-OprC overexpression, leading to trimethoprim and sulfamethoxazole efflux and thus to cotrimoxazole resistance. The majority of laboratory-selected co-trimoxazole-resistant mutants often also contain mutations in folM, encoding a pterin reductase. Genetic analyses of these mutants established that both bpeS mutations and folM mutations contribute to co-trimoxazole resistance, although the exact role of folM remains to be determined. Mutations affecting bpeT, bpeS, and folM are common in co-trimoxazole-resistant clinical isolates, indicating that mutations affecting these genes are clinically significant. Cotrimoxazole resistance in B. pseudomallei is a complex phenomenon, which may explain why resistance to this drug is rare in this bacterium. IMPORTANCE Burkholderia pseudomallei causes melioidosis, a tropical disease that is difficult to treat. The bacterium's resistance to antibiotics limits therapeutic options. The paucity of orally available drugs further complicates therapy. The oral drug of choice is co-trimoxazole, a combination of trimethoprim and sulfamethoxazole. These antibiotics target two distinct enzymes, FolA (dihydrofolate reductase) and FolP (dihydropteroate synthase), in the bacterial tetrahydrofolate biosynthetic pathway. Although co-trimoxazole resistance is minimized due to two-target inhibition, bacterial resistance due to folA and folP mutations does occur. Co-trimoxazole resistance in B. pseudomallei is rare and has not yet been studied. Co-trimoxazole resistance in this bacterium employs a novel strategy involving differential regulation of BpeEF-OprC efflux pump expression that determines the drug resistance profile. Contributing are mutations affecting folA, but not folP, and folM, a folate pathway-associated gene whose function is not yet well understood and which has not been previously implicated in folate inhibitor resistance in clinical isolates.

Thesis (MScMedSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Central dogma suggests that mutations in target genes is the primary cause of resistance to first and second-line anti-TB drugs in Mycobacterium tuberculosis. However, it was previously reported that approximately 5% of Rifampicin mono-resistant clinical M. tuberculosis did not harbor mutations in the rpoB gene. The present study hypothesized that active efflux plays a contributory role in the level of intrinsic resistance to different anti-TB drugs (Isoniazid, Ethionamide, Pyrazinamide, Ethambutol, Ofloxacin, Moxifloxacin, Ciprofloxacin, Streptomycin, Amikacin and Capreomycin in RIF mono-resistant clinical M. tuberculosis isolates with a rpoB531 (Ser-Leu) mutation. This study aimed to define the role of Efflux pump inhibitors (verapamil, carbonylcyanide m-chlorophenylhydrazone and reserpine) in enhancing the susceptibility to different anti-TB drugs in the RIF mono-resistant clinical isolates. The isolates were characterized by determining the level of intrinsic resistance to structurally related/unrelated anti-TB drugs; determining the effect of EPIs on the level of intrinsic resistance in the isolates and comparing the synergistic properties of the combination of EPIs and anti-TB drugs. To achieve this, genetic characterization was done by PCR and DNA sequencing. Phenotyping was done by the MGIT 960 system EpiCenter software to determine the MICs of the different anti-TB drugs and the effect of verapamil and carbonylcyanide m-chlorophenylhydrazone on determined MICs. Due to inability to test reserpine in a MGIT, a different technique (broth microdilution) was used for the reserpine experiment. Additionally; fractional inhibitory concentrations (FIC) indices were calculated for each of these drugs. The FIC assess the anti-TB drugs/inhibitor interactions. STATISTICA Software: version 11 was used for statistical analysis. Results revealed that the RIF mono-resistant isolates were sensitive at the critical concentrations of all 10 drugs tested, with the exception of Pyrazinamide. This could be explained by the technical challenges of phenotypic Pyrazinamide testing. A significant growth inhibitory effect was observed between the combination of EPI and anti-TB drug exposure in vitro. This suggests that verapamil, carbonylcyanide m-chlorophenylhydrazone and reserpine play a significant role in restoring the susceptibility (decrease in intrinsic resistance level) of the RIF mono-resistant isolates to all anti-TB drugs under investigation. Additionally, a synergistic effect was observed by the combination treatment of the anti-TB drugs with the different EPIs. Based on these findings, we proposed a model suggesting that efflux pumps are activated by the presence of anti-TB drugs. The activated pumps extrude multiple or specific anti-TB drugs out of the cell, this in turn decrease the intracellular drug concentration, thereby causing resistance to various anti-TB drugs. In contrast, the addition of EPIs inhibits efflux pump activity, leading to an increase in the intracellular drug concentration and ultimate cell death. This is the first study to investigate the effect of different efflux pumps inhibitors on the level of intrinsic resistance to a broad spectrum of anti-TB drugs in drug resistant M. tuberculosis clinical isolates from different genetic backgrounds. The findings are of clinical significance as the combination of treatment with EPI and anti-TB drugs or use of EPIs as adjunctives could improve MDR-TB therapy outcome.
AFRIKAANSE OPSOMMING: Sentrale dogma beweer dat mutasies in teiken gene die primêre oorsaak van die weerstandheid teen anti-TB-middels in Mycobacterium tuberculosis is. Vorige studies het getoon dat ongeveer 5% van Rifampisien enkelweerstandige kliniese M. tuberculosis isolate nie ‘n mutasie in die rpoB geen het nie. Die hipotese van die huidige studie was dat aktiewe pompe 'n bydraende rol speel in die vlak van intrinsieke weerstandheid teen 10 verskillende anti-TB-middels (Isoniasied, Ethionamied, Pyrazinamied, Ethambutol, Ofloxacin, Moxifloxacin, Siprofloksasien, Streptomisien, Amikasien and Capreomycin) in RIF enkelweerstandige kliniese M . tuberculosis isolate met 'n rpoB531 (Ser-Leu) mutasie. Die doel van hierdie studie was om die rol van uitpomp inhibeerders (verapamil, carbonylcyanide m-chlorophenylhydrazone en reserpien) te definieer in die verbetering van die werking vir verskillende anti-TB-middels in die RIF enkelweerstandige kliniese isolate. Die doelstellings van die studie was om die vlak van intrinsieke weerstandigheid teen struktureel verwante/onverwante anti-tuberkulose middels asook die effek van die EPIs op die vlak van intrinsieke weerstand in die isolate is bepaal. Verder is sinergistiese eienskappe van die kombinasie van EPIs en anti-TB-middels ondersoek. Hierdie doelstellings is bereik deur genetiese karakterisering deur PKR en DNS volgorde bepaling. Fenotipering is gedoen deur gebruik te maak van MGIT 960 EpiCenter sagteware om die Minimum Inhibisie Konsentrasie (MIC) van die verskillende anti-TB-middels en die effek van verapamil en carbonylcyanide m-chlorophenylhydrazone op die MIC te bepaal. Reserpien kan nie in die MGIT sisteem getoets word nie, and daarom is 'n ander tegniek (mikro-verdunning) is gebruik om die effek van reserpien te toets. Fraksionele inhiberende konsentrasies (FIC) is bereken vir elk van hierdie middels die anti-TB-middels / inhibeerder interaksies te bepaal. STATISTICA v11 sagteware is gebruik vir alle statistiese analises. Resultate van hierdie studie toon dat die RIF enkelweerstandige isolate sensitief is teen kritieke konsentrasies van al die middels, met die uitsondering van Pyrazinamied. Weerstandigheid van Pyrazinamied kan wees as gevolg van welbekende tegniese probleme met die standaard fenotipiese pyrazinamied toets. ‘n Beduidende groei inhiberende effek is waargeneem tussen die kombinasie van EPI en anti-TB middel blootstelling in vitro. Dit dui daarop dat verapamil, CCCP en reserpine 'n belangrike rol speel in die herstel van die sensitiwiteit (afname in intrinsieke weerstand vlak) van die RIF enkelweerstandige isolate aan alle anti-TB-middels wat ondersoek is. Daarbenewens is 'n sinergistiese effek waargeneem deur die kombinasie van die verskillende anti-TB-middels en die verskillende EPIs. Op grond van hierdie bevindinge het ons ‘n model voorgestel wat toon dat uitvloei pompe geaktiveer word deur die teenwoordigheid van anti-TB-middels en die geaktiveerde pompe dan verskeie of spesifieke anti-TB-middels uit die sel pomp. Dus verminder die intrasellulêre konsentrasie van die middel en veroorsaak daardeur weerstandigheid teen verskeie anti-TB-middels. Die byvoeging van EPIs inhibeer uitvloei pompe se werking en lei tot 'n toename in die intrasellulêre konsentrasie van die middels en uiteindelik die dood van die selle. Hierdie is die eerste studie wat die effek van verskillende uitvloei pompe inhibeerders op die vlak van intrinsieke weerstand teen 'n breë spektrum van anti-TB-middels in die middel-weerstandige kliniese isolate ondersoek. Die bevindinge kan van belangrike kliniese belang wees aangesien die kombinasie van behandeling met EPI en anti-TB-middels die uitkoms MDR-TB terapie kan verbeter.

Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2004.
"A dissertation in pharmaceutical science and chemistry." Advisor: Ashim K. Mitra. Typescript. Vita. Description based on contents viewed Feb. 27, 2006; title from "catalog record" of the print edition. Includes bibliographical references (leaves 175-199). Online version of the print edition.

Thesis (Ph. D.)--School of Pharmacy. University of Missouri--Kansas City, 2007.
"A dissertation in pharmaceutical science and pharmacology." Advisor: Ashim K. Mitra. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed July 16, 2008. Includes bibliographical references (leaves 231-248). Online version of the print edition.

Cutaneous melanoma is a highly metastatic and drug-resistant skin cancer type, responsible for a disproportionate number of skin cancer deaths. Targeted therapies, in the form of BRAF inhibitors (BRAFis), have been effective at treating BRAFV600 mutant melanomas. However, majority of the melanoma patients fail to respond to BRAFis due to intrinsic or acquired resistance within one year of treatment commencement. Multiple mechanisms that contribute to BRAFi resistance in melanoma cells have been identified, as discussed in the review in Chapter 1. Overexpression of ATP-binding cassette (ABC) transporters has been linked to multidrug resistance in numerous cancer types. These transporters expel the anti-cancer drugs out of the cell, thereby decreasing the intracellular concentration of the drug. In melanoma, the ATP-binding cassette B5 transporter (a member of ABC superfamily) has been linked to chemoresistance by drug extrusion. Moreover, overexpression of ABCB5 has been observed in BRAFV600 melanoma cells after short-term BRAFi treatment. In this study we investigated the role of the ABCB5 transporter as potential mediators of resistance to BRAFis by drug expulsion. In Chapter 2, we showed increased ABCB5 expression in melanoma cell lines after short-term treatment with the BRAFis accompanied by an increased expression of melanocytic signature. Gene expression of fluorescent activated cell sorted melanoma cells into ABCB5high and ABCB5low populations, revealed an increased melanocytic signature in the ABCB5high population. Moreover, analysis of single-cell RNA sequencing (scRNAseq) data of two BRAFV600 melanoma cell lines, A2058 and 451Lu, revealed a strong association between ABCB5 expression and melanocytic signature. Based on these initial observations, the capacity of the ABCB5 transporter to efflux BRAFis was evaluated indirectly through an in-silico approach using molecular docking simulations (Chapter 3 and 4), and directly through in vitro experiments using an ABCB5 overexpressing melanoma BRAFV600 cell line (Chapter 5). In Chapter 3, a full-length ABCB5 model was generated, based on mouse ATP-binding cassette B1 transporter (ABCB1; Pgp1), a close homologue of ABCB5. Molecular dynamics simulations were performed in 2 model cell membranes and the dominant conformation was identified. Docking simulations of known ABCB5 substrates such as taxanes, anthracyclines, camptothecin and etoposide enabled the identification of at least three putative substrate binding sites in ABCB5. The overlap of these three binding sites with validated binding sites for these chemotherapeutic drugs in Pgp1 corroborate our findings. In Chapter 4, docking simulations revealed at least one overlapping binding site for BRAFis and chemotherapeutic drugs on ABCB5, suggesting that BRAFis could potentially act as a substrate for ABCB5. In Chapter 5, we generated an ABCB5 overexpressing BRAFV600E melanoma cell line. However, no differences in sensitivity to BRAF inhibition was observed as a result of ABCB5 overexpression. Intracellular drug accumulation analyses revealed no reduction in vemurafenib or dabrafenib concentrations, indicating that BRAFis do not act as substrates for ABCB5. Altogether, our studies suggest that ABCB5 expression is linked to the melanocytic program. However, despite the molecular docking evidence that BRAFis may be substrates of ABCB5, in vitro studies failed to demonstrate direct efflux of BRAFis by ABCB5.

碩士
國立臺灣海洋大學
食品科學系
106
Multi-drug resistance (MDR) of Gram-negative bacteria constitutes a major obstacle in the antibacterial fight worldwide. The AcrAB-TolC efflux pump is the most well-studied resistance nodulation division (RND) pump and responsible for most intrinsic drug resistance in Escherichia coli. To combat MDR, several efflux pump inhibitors (EPIs) that act against the AcrAB-TolC efflux pump have already been described. Seaweeds are considered as a source of bioactive compounds as they are able to produce a great variety of secondary metabolites which characterized by a broad spectrum of biological activities. This study aimed to evaluate the potential of macroalgae extracts which inhibit the level of secondary active drug transporter AcrB from Escherichia coli. Four seaweeds were extracted by 95% ethanol and combined with serveral antibiotics against E. coli Kam3 pSYC and pSYC-acrB which determined by antibacterial activity assay, modulation assay, time-kill assay, ethidium bromide (EtBr) accumulation and finally identified four algae extracts by GC-MS. The result of modulation showed that Laminaria japonica, Sargassum horneri and Gracilaria sp. significantly reduced IC50 of E. coli harboring pSYC in erythromycin and clarithromycin by at least 2 to 8-fold. Laminaria japonica, Sargassum horneri, Gracilaria sp. and Porphyra dentata also significantly reduced IC50 of E. coli harboring pSYC-acrB in erythromycin and clarithromycin by at least 2 to 16-fold. Four macroalgae in different concentrations could modulate with clarithromycin to reduce log10 cfu below the initial log10 cfu at 0 h in pSYC-acrB. All seaweed extracts in pSYC-acrB slightly increased the accumulation of EtBr in relation to the EtBr control, in which L. japonica and Gracilaria sp. increased the level of EtBr accumulation higher than S. horneri and P. dentata. Most components in four algae were identified by using GC-MS and they contained the same structure of benzene. In conclusion, four macroalgae potentiated antibiotic activity by inhibiting AcrB efflux pump in E. coli. They could be applied as a potential efflux pump inhibitor to restore antibiotic activity. Keywords : Escherichia coli, AcrB, Efflux pump inhibitor, Algae extracts, Antibacterial activity assay

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lucia Dziaková Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on interactions of PARP inhibitors with ABC drug efflux transporters. ATP-binding cassette (ABC) transporters are integral membrane proteins that use the energy obtained from ATP to carry transport of numerous endogenous substrances out of the cells, but attention is drawn primarily to the fact that they transfer also xenobiotics. Their overexpression in tumor tissue contributes to multidrug resistance (MDR), which in most cases leads to therapy failure. Poly(ADP-ribose)polymerase inhibitors (PARPi) represent a promising therapeutic approach in the treatment of cancers that exhibit defects in homologous recombination (HR). This work focuses on four selected PARPi (olaparib, rucaparib, niraparib, veliparib) and their interaction potential towards ABC drug efflux transporters (ABCB, ABCC1, ABCG2). In our work, we worked with MDCKII cells (parent, transduced by the transporters of interest) and utilized the principle of accumulation studies based on the measurement of fluorescence intensity of specific model substrates (hoechst33342, calcein AM, daunorubicin, mitoxantrone). We used established inhibitors of studied...

4 ABSTRACT Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Dimitrios Vagiannis Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Evaluation of antiproliferative effect of selected tyrosine kinase inhibitors in MDCKII cell lines Tyrosine kinases are important enzymes regulating crucial cellular processes including differentiation, proliferation, apoptosis, transcription, metabolism, and intercellular communication. Deregulation of these enzymes is the cause of various types of cancers. The blockade of their function by tyrosine kinase inhibitors (TKis) is considered a promising approach especially in antitumor pharmacotherapy. ATP-binding cassette (ABC) drug efflux transporters are a family of transmembrane proteins that pump a variety of structurally unrelated compounds out of the cell in an energy-dependent manner. They play an important role in pharmacokinetics (affect absorption, distribution, elimination) and, at the same time, can negatively influence efficacy of chemotherapy (participate in multidrug resistance phenomenon). In our research, we evaluated antiproliferative properties of four selected TKis, namely alectinib, brivanib, osimertinib and selumetinib, in MDCKII cell lines (parent one and those transduced with human...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Simona Suchá Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study of ABC drug efflux transporter inhibition by selected tyrosine kinase inhibitors using accumulation methods with cytostatic substrates ATP-binding cassette (ABC) drug efflux transporters are transmembrane proteins that utilize the energy from ATP hydrolysis to drive transport of endogenous and exogenous compounds out of the cell. The overexpression of ABC transporters plays a crucial role in the development of multidrug resistance (MDR), a phenomenon responsible for the failure of chemotherapy. Tyrosine kinase inhibitors (TKI) represent novel beneficial therapeutic approach in cancer treatment. TKI block tyrosine kinases which regulate important cellular processes. Deregulation of these enzymes can lead to various types of cancers. In the present work, we investigated interaction potential of selected TKI (alectinib, brivanib, osimertinib, selumetinib) in MDCKII parent cell line and those transduced with human efflux transporters ABCB1, ABCC1 and ABCG2. Using the accumulation studies, we determined the amount of accumulated model substrates (daunorubicin, mitoxantrone) and evaluated the inhibitory effect of...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Martina Sýkorová Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on impact of selected tyrosine kinase inhibitors on multidrug resistance mediated by ABC drug efflux transporters Tyrosine kinases are an important class of enzymes controlling cell proliferation, carcinogenesis, apoptosis and cell differentiation. Deregulation of these enzymes can transform normal cell into a cancerous one. Blocking their function by tyrosine kinase inhibitors (TKi) is considered a promising treatment for various types of cancer. ATP-binding cassette (ABC) transporters form a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes via ATP-dependent drug efflux pumps. They modulate drug pharmacokinetics, but on the other hand, lead to therapy failure due to overexpression in cancer cells. In our previous study, we evaluated inhibition properties of two selected TKi (alectinib, brivanib) in MDCKII cell lines (parent one and those transduced with human ABCB1, ABCC1 and ABCG2). Alectinib significantly inhibited ABCB1, ABCG2 but not ABCC1 transporter. Brivanib showed triple inhibition of all studied transporters. In the present work, we...