Transmembrane Efflux Pump Research Articles

Phosphatidylserine-targeting bis(zinc-dipicolylamine) farnesol inhibits ATP production in cancer cells to overcome multidrug resistance

Multidrug resistance significantly impedes the efficacy of cancer chemotherapy. Resistance often arises from the reduced cellular uptake of chemotherapeutic drugs, a process crucial for their cytotoxic effects. This reduction is frequently due to transmembrane efflux pumps powered by ATP from mitochondria and the cytoplasmic matrix, leading to lower intracellular concentrations of these drugs. This study introduces an amphiphilic molecule, bis(zinc-dipicolylamine) farnesol (Bis-ZnDPA), which targets phosphatidylserine (PS) – a negatively charged phospholipid prominently displayed on the outer leaflet of cancer cell plasma membranes. Integrating the hydrophobic segment of Bis-ZnDPA into the plasma membrane disrupts its integrity, potentially leading to hole formation and facilitating the uptake of chemotherapeutic drugs. Furthermore, the binding of Bis-ZnDPA to phosphatidylserine inhibits ATP production caused by Ca2+ influx and deregulation of the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway, reducing the efflux of drugs from cells. The results indicate the potent synergistic effect of Bis-ZnDPA with chemotherapeutic agents, suggesting that targeting PS is a viable strategy for overcoming multidrug resistance in cancer chemotherapy.

Abstract 7291: Detection of a truncated form of multidrug resistance protein 1 (MRP1) in plasma sEVs derived from cancer patients

Abstract Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP dependent transmembrane efflux pump that confers to drug resistance. Overexpression of MRP1 has been observed in both pancreatic ductal adenocarcinoma (PDAC) and breast cancer tissues. Numerous proteomics studies have revealed the presence of MRP1 in small extracellular vesicles (sEVs) released from cancer cells. Cancer-derived sEVs are known to be released into the circulation, thus becoming attractive candidates for non-invasive biomarker development. However, whether the MRP1 level in plasma sEVs is indicative of human cancer has not been evaluated. This study examined the MRP1 expression in plasma sEVs derived from patients with PDAC, breast and colon cancer, and age- and gender-matched healthy subjects. The patient plasma samples were obtained primarily from the NCI-sponsored Cooperative Human Tissue Network and Stephenson Cancer Center. Healthy plasma samples were provided by the Oklahoma Blood institute. Human pancreatic cancer cell lines PANC-1, MIA PaCa-2 and BxPC-3, and the pancreatic ductal cell line hTERT-HPNE were tested for MRP1 expression prior to examination of plasma samples. Cellular and plasma sEVs were isolated using the double filtration followed by polymer precipitation method. Western blot was performed to assess the expression level of MRP1 and proteomics was applied to confirm the detection of a truncated form of MRP1 in plasma sEVs. MRP1 expression was detected in the sEVs derived from all three pancreatic cancer cell lines but absent in the sEVs from the HPNE line. Overexpression of a truncated MRP1 protein was detected in plasma sEVs derived from patients with early stage (stage I-IIA) and late stage (III-IV) PDAC, compared with that from matched healthy subjects (n=17 for each group). Similar findings were evident in plasma sEVs from patients with breast cancer (n=7), while the results remained inconclusive in plasma sEVs from patients with colon cancer (n=7). Protein structure analysis indicated that the truncated form of MRP1 likely consists of the regions between the QCRL-1 motif and the C-terminus of the protein. Proteomic analysis detected the peptides derived from the C-terminus region, consistent with the structural analysis. Furthermore, the truncated form of MRP1 can only be detected using antibodies against the epitopes in the C-terminus and QCRL-1 region of MRP1 protein. In summary, we have detected a truncated form of MRP1 protein in plasma sEVs derived from cancer patients, and the level of its expression is significantly elevated in patients with PDAC and breast cancer. Citation Format: Kritisha Bhandari, Jeng Shi Kong, Chao Xu, Ajay Jain, Wei-Qun Ding. Detection of a truncated form of multidrug resistance protein 1 (MRP1) in plasma sEVs derived from cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7291.

Evaluation of Novel Benzo-annelated 1,4-dihydropyridines as MDR Modulators in Cancer Cells.

Multidrug resistance (MDR) is the main problem in anticancer therapy today. Causative transmembrane efflux pumps in cancer cells have been reconsidered as promising anticancer target structures to restore anticancer drug sensitivity by various strategies, including MDR modulators. MDR modulators interfere with the efflux pumps and improve the cellular efficiency of chemotherapeutics. So far, only a few candidates have gone through clinical trials with disappointing results because of low specificity and toxic properties. This study aimed to find novel MDR modulators to effectively combat multidrug resistance in cancer cells. We synthesized various novel benzo-annelated 1,4-dihydropyridines to evaluate them as MDR modulators towards ABCB1 in cancer cells. Synthesized compounds were purified by column chromatography. The MDR modulation of ABCB1 was determined in cellular efflux assays using the flow cytometry technique and cellular fluorescent measurements by the use of each fluorescent substrate. Compounds were yielded in a two-step reaction with structurally varied components. Further, substituent- dependent effects on the determined MDR inhibiting properties towards ABCB1 were discussed. Cellular studies prove that there is no toxicity and restoration of cancer cell sensitivity towards the used anticancer drug. Novel MDR modulators could be identified with favorable methoxy and ester group functions. Their use in both ABCB1 non-expressing and overexpressing cells proves a selective toxicity-increasing effect of the applied anticancer agent in the ABCB1 overexpressing cells, whereas the toxicity effect of the anticancer drug was almost unchanged in the non-expressing cells. These results qualify our novel compounds as perspective anticancer drugs compared to MDR modulators with nonselective toxicity properties.

Modulatory Effect of Croton heliotropiifolius Kunth Ethanolic Extract on Norfloxacin Resistance in Staphylococcus aureus

The high frequency of infectious diseases has spurred research into effective tactics to combat microorganisms that are resistant to several drugs. The overproduction of the transmembrane efflux pump protein NorA, which may export hydrophilic fluoroquinolones, is a common mechanism of resistance in S. aureus strains. This work evaluated the antimicrobial activity of the ethanolic extract from the leaves of Croton heliotropiifolius (EECH) against different bacterial and fungal strains, as well as investigating its modulating effect on the resistance to norfloxacin in a Staphylococcus aureus SA1199B overproducing the NorA efflux pump. Microdilution assays were used to assess the EECH’s antibacterial efficacy. The MIC of norfloxacin or ethidium bromide (EtBr) against the SA1199B strain was determined in the presence or absence of the EECH in order to assess the modifying influence on drug resistance. The EECH showed no activity against the Gram-positive and Gram-negative bacterial strains tested. The EECH also showed no antifungal activity against Candida albicans ATCC 10231. On the other hand, the extract reduced the MIC values for norfloxacin against SA1199B at subinhibitory concentrations. In addition, the EECH also reduced the MIC values of EtBr at subinhibitory concentrations, suggesting the occurrence of phytochemicals that inhibit efflux pumps. Molecular docking showed that retusin, a flavonoid found in the extract, could compete with norfloxacin at the orthosteric site of the NorA, indicating that it could be a potential efflux pump inhibitor. However, isolated retusin did not enhance the activity of norfloxacin or EtBr and it did not inhibit the EtBr efflux, showing that it is not a NorA inhibitor. Even though C. heliotropiifolius is a source of phytochemicals that function as adjuvants for norfloxacin, isolated retusin cannot be used in conjunction with norfloxacin to treat infections brought on by S. aureus that overproduces NorA.

Influence of CYP2C19, CYP2D6, and ABCB1 Gene Variants and Serum Levels of Escitalopram and Aripiprazole on Treatment-Emergent Sexual Dysfunction: A Canadian Biomarker Integration Network in Depression 1 (CAN-BIND 1) Study.

Treatment-emergent sexual dysfunction is frequently reported by individuals with major depressive disorder (MDD) on antidepressants, which negatively impacts treatment adherence and efficacy. We investigated the association of polymorphisms in pharmacokinetic genes encoding cytochrome-P450 drug-metabolizing enzymes, CYP2C19 and CYP2D6, and the transmembrane efflux pump, P-glycoprotein (i.e., ABCB1), on treatment-emergent changes in sexual function (SF) and sexual satisfaction (SS) in the Canadian Biomarker Integration Network in Depression 1 (CAN-BIND-1) sample. A total of 178 adults with MDD received treatment with escitalopram (ESC) from weeks 0-8 (Phase I). At week 8, nonresponders were augmented with aripiprazole (ARI) (i.e., ESC + ARI, n = 91), while responders continued ESC (i.e., ESC-Only, n = 80) from weeks 8-16 (Phase II). SF and SS were evaluated using the sex effects (SexFX) scale at weeks 0, 8, and 16. We assessed the primary outcomes, SF and SS change for weeks 0-8 and 8-16, using repeated measures mixed-effects models. In ESC-Only, CYP2C19 intermediate metabolizer (IM) + poor metabolizers (PMs) showed treatment-related improvements in sexual arousal, a subdomain of SF, from weeks 8-16, relative to CYP2C19 normal metabolizers (NMs) who showed a decline, F(2,54) = 8.00, p < 0.001, q = 0.048. Specifically, CYP2C19 IM + PMs reported less difficulty with having and sustaining vaginal lubrication in females and erection in males, compared to NMs. Furthermore, ESC-Only females with higher concentrations of ESC metabolite, S-desmethylcitalopram (S-DCT), and S-DCT/ESC ratio in serum demonstrated more decline in SF (r = -0.42, p = 0.004, q = 0.034) and SS (r = -0.43, p = 0.003, q = 0.034), respectively, which was not observed in males. ESC-Only females also demonstrated a trend for a correlation between S-DCT and sexual arousal change in the same direction (r = -0.39, p = 0.009, q = 0.052). CYP2C19 metabolizer phenotypes may be influencing changes in sexual arousal related to ESC monotherapy. Thus, preemptive genotyping of CYP2C19 may help to guide selection of treatment that circumvents selective serotonin reuptake inhibitor-related sexual dysfunction thereby improving outcomes for patients. Additionally, further research is warranted to clarify the role of S-DCT in the mechanisms underlying ESC-related changes in SF and SS. This CAN-BIND-1 study was registered on clinicaltrials.gov (Identifier: NCT01655706) on 27 July 2012.

Heterologous expression of a yeast lipid transporter promotes accumulation of storage compounds in Chlamydomonas

Microalgae offer renewable and sustainable sources for the bioindustrial production of next-generation biofuels and biomaterials. In this work, we heterologously expressed a lipid transporter gene (MMF1) from the major facilitator superfamily in the model green microalga Chlamydomonas reinhardtii, under the influence of a strong light and temperature-inducible promoter. The full-length cDNA sequence of MMF1 from Moesziomyces antarcticus T-34 (MaMMF1) encodes for a 542 amino acid polypeptide with transmembrane efflux pump activity. M. antarcticus is a fungus that can produce and secrete large amounts of glycolipid biosurfactants known as mannosylerythritol lipids (MELs) under nitrogen starvation conditions. Heterologous expression of MaMMF1 in C. reinhardtii resulted in a slight increase in cell biomass, with mutants exhibiting on average about 1.8-fold larger cell sizes. Total lipid content almost doubled in cells expressing MaMMF1 relative to the control strain when cultures were grown for two days under nitrogen starvation. Moreover, average starch content was over 2-fold higher in the mutants. Our results showed that algal strains engineered with the MaMMF1 MEL transporter were capable of possibly redirecting the carbon flux, which resulted in improved growth and accumulation of high-value storage compounds.

Structural and functional mapping of ars gene cluster in Deinococcus indicus DR1

Deinococcus indicus DR1 is a novel Gram-negative bacterium, isolated from the Dadri wetlands in Uttar Pradesh, India. In addition to being radiation-resistant, the rod-shaped, red-pigmented organism shows extraordinary resistance to arsenic. The proteins of the corresponding ars gene cluster involved in arsenic extrusion in D. indicus DR1 have not yet been characterized. Additionally, how these proteins regulate each other providing arsenic resistance is still unclear. Here, we present a computational model of the operonic structure and the corresponding characterization of the six proteins of the ars gene cluster in D. indicus DR1. Additionally, we show the expression of the genes in the presence of arsenic using qRT-PCR. The ars gene cluster consists of two transcriptional regulators (ArsR1, ArsR2), two arsenate reductases (ArsC2, ArsC3), one metallophosphatase family protein (MPase), and a transmembrane arsenite efflux pump (ArsB). The transcriptional regulators are trans-acting repressors, and the reductases reduce arsenate (As5+) ions to arsenite (As3+) ions for favourable extrusion. The proteins modelled using RoseTTAFold, and their conformationally stable coordinates obtained after MD simulation indicate their various functional roles with respect to arsenic. Excluding ArsB, all the proteins belong to the α + β class of proteins. ArsB, being a membrane protein, is fully α-helical, with 12 transmembrane helices. The results show the degree of similarity or divergence of the mechanism utilized by these proteins of ars gene cluster in D. indicus DR1 to confer high levels of arsenic tolerance. This structural characterization study of the ars genes will enable new and deeper insights of arsenic tolerance.

Amplification of tumor oxidative stresses by Poly(disulfide acetal) for multidrug resistance reversal

Discovering new strategies to overcome multidrug resistance (MDR) is still urgently needed. MDR is associated with the overexpression of transmembrane efflux pumps, and adenosine triphosphate (ATP) is indispensable for its function. Herein, we developed a pH- and glutathione (GSH)-responsive amphiphilic poly(disulfide acetal) (PCS) containing cinnamaldehyde (CA) and disulfide groups that amplify oxidative stress for anticancer drug delivery and simultaneously overcome drug resistance in cancer cells. Reactive oxygen species (ROS)-generating CA and the disulfide groups to deplete GSH and synergize to amplify oxidative stress in cancer cells by oxidizing nicotinamide adenine dinucleotide with hydrogen (NADH) to nicotinamide adenine dinucleotide (NAD+). The production of ATP is preferentially inhibited, leading to the malfunction of efflux pumps due to the lack of ATP and making resistant cells more impressionable to anticancer drugs. The in vitro and in vivo experiments confirmed that PCS could induce amplified oxidative stress and efficiently overcome MDR in cancer cells. We believe that the polymer with amplified oxidative stress in cancer cells holds great promise in developing polymer-based drug delivery systems to reverse MDR for cancer therapy.

Novel Symmetrical Cage Compounds as Inhibitors of the Symmetrical MRP4-Efflux Pump for Anticancer Therapy.

Within the last decades cancer treatment improved by the availability of more specifically acting drugs that address molecular target structures in cancer cells. However, those target-sensitive drugs suffer from ongoing resistances resulting from mutations and moreover they are affected by the cancer phenomenon of multidrug resistance. A multidrug resistant cancer can hardly be treated with the common drugs, so that there have been long efforts to develop drugs to combat that resistance. Transmembrane efflux pumps are the main cause of the multidrug resistance in cancer. Early inhibitors disappointed in cancer treatment without a proof of expression of a respective efflux pump. Recent studies in efflux pump expressing cancer show convincing effects of those inhibitors. Based on the molecular symmetry of the efflux pump multidrug resistant protein (MRP) 4 we synthesized symmetric inhibitors with varied substitution patterns. They were evaluated in a MRP4-overexpressing cancer cell line model to prove structure-dependent effects on the inhibition of the efflux pump activity in an uptake assay of a fluorescent MRP4 substrate. The most active compound was tested to resentisize the MRP4-overexpressing cell line towards a clinically relevant anticancer drug as proof-of-principle to encourage for further preclinical studies.

Discovery of Novel Symmetrical 1,4-Dihydropyridines as Inhibitors of Multidrug-Resistant Protein (MRP4) Efflux Pump for Anticancer Therapy.

Despite the development of targeted therapies in cancer, the problem of multidrug resistance (MDR) is still unsolved. Most patients with metastatic cancer die from MDR. Transmembrane efflux pumps as the main cause of MDR have been addressed by developed inhibitors, but early inhibitors of the most prominent and longest known efflux pump P-glycoprotein (P-gp) were disappointing. Those inhibitors have been used without knowledge about the expression of P-gp by the treated tumor. Therefore the use of inhibitors of transmembrane efflux pumps in clinical settings is reconsidered as a promising strategy in the case of the respective efflux pump expression. We discovered novel symmetric inhibitors of the symmetric efflux pump MRP4 encoded by the ABCC4 gene. MRP4 is involved in many kinds of cancer with resistance to anticancer drugs. All compounds showed better activities than the best known MRP4 inhibitor MK571 in an MRP4-overexpressing cell line assay, and the activities could be related to the various substitution patterns of aromatic residues within the symmetric molecular framework. One of the best compounds was demonstrated to overcome the MRP4-mediated resistance in the cell line model to restore the anticancer drug sensitivity as a proof of concept.

Reversal of Ovarian Cancer Cell Lines Multidrug Resistance Phenotype by the Association of Apiole with Chemotherapies.

Multidrug resistance (MDR) is the main obstacle in anticancer therapy. The use of drug combinations to circumvent tumor resistance is a well-established principle in the clinic. Among the therapeutic targets, glycoprotein-P (P-gp), an energy-dependent transmembrane efflux pump responsible for modulating MDR, is highlighted. Many pharmacological studies report the ability of calcium channel blockers to reverse tumor resistance to chemotherapy drugs. Isolated for the first time from parsley, the phenylpropanoid apiole is described as a potent calcium channel inhibitor. Taking this into account, herein, the ability of apiole to potentiate the action of well-established chemotherapeutics in the clinic, as well as the compound’s relationship with the reversal of the resistance phenomenon by blocking P-gp, is reported. The association of apiole with both chemotherapeutic drugs doxorubicin and vincristine resulted in synergistic effect, in a concentration-dependent manner, as evaluated by the concentration reduction index. Molecular docking analysis demonstrated the affinity between apiole and the active site of P-gp, corroborating the inhibitory effect. Moreover, apiole demonstrated druglikeness, according to ADME analysis. In conclusion, apiole possibly blocks the active P-gp site, with strong binding energy, which, in turn, inhibits doxorubicin and vincristine efflux, increasing the antiproliferative response of these chemotherapeutic agents.

A Sequentially Responsive Nanosystem Breaches Cascaded Bio-barriers and Suppresses P-Glycoprotein Function for Reversing Cancer Drug Resistance.

Cancer chemotherapy is challenged by multidrug resistance (MDR) mainly attributed to overexpressed transmembrane efflux pump P-glycoprotein (P-gp) in cancer cells. Improving drug delivery efficacy while co-delivering P-gp inhibitors to suppress drug efflux is an often-used nanostrategy for combating MDR, which is however challenged by cascaded bio-barriers en route to cancer cells and P-gp inhibitors' adverse effects. To effectively breach the cascaded bio-barriers while avoiding P-gp inhibitors' adverse effects, a stealthy, sequentially responsive doxorubicin (DOX) delivery nanosystem (RCMSNs) is fabricated, composed of an extracellular-tumor-acidity-responsive polymer shell (PEG-b-PLLDA), pH/redox dual-responsive mesoporous silica nanoparticle-based carriers (MSNs-SS-Py), and cationic β-cyclodextrin-PEI (CD-PEI) gatekeepers. The PEG-b-PLLDA corona makes RCMSNs stealthy with prolonged blood circulation time. Once tumors are reached, extracellular acidity degrades PEG-b-PLLDA, reversing nanosystem's surface charges to be positive, which drastically improves RCMSNs' tumor accumulation, penetration, and cellular internalization. Within cancer cells, CD-PEI gatekeepers detach to allow DOX unloading in response to intracellular acidity and glutathione and functionally act as a P-gp inhibitor, dampening P-gp's efflux activity by impairing ATP production. Thus, the resultant high-efficacy drug delivery along with reduced P-gp function cooperatively reverses MDR in vitro. Importantly, in preclinical tumor models, DOX@RCMSNs potently suppress MDR tumor growth without eliciting systemic toxicity, demonstrating their potential of clinical translation.

Study on the Administration Method of Combining Tetrandrine with Adriamycin to Reverse Drug-Resistance of Leukemia

Increasing drug efflux pumps (P-gp) on the cell membrane due to the overexpression of MDR1 gene in tumor cells is the main mechanism of multidrug resistance of cancer cells. P-gp belongs to the ATP-binding cassette family and functions as a transmembrane efflux pump that translocates chemotherapy drugs from an intracellular to an extracellular domain; thus, it is impossible to achieve efficient drug accumulation in tumor cells. Tetrandrine (TTD) has been shown in both in vitro and in animal experiments to reverse MDR by reducing the expression of MDR1 mRNA and P-gp. However, the effect of administration of TTD one week before chemotherapy is not ideal in the current clinical trials. In view of the previous in vitro and animal experiments have confirmed the effect of TTD in reversing the drug resistance of leukemia, this study returned to the cell experiment, selected K562 and K562/ADM cell lines as the research object, through different ADM combined with TTD administration sequences, to detect the inhibitory effect of different administration sequences on cell proliferation and whether it affected the intracellular ADM concentration, P- gp ATPase activity, MDR1 mRNA and P-gp expression levels, comparing the effect of TTD combined with ADM different administration sequences on the reversal of MDR in K562/ADM cells. We found that TTD can significantly antagonize P-gp-mediated ADM resistance by competitively binding P-gp and down-regulating P-gp expression and we, therefore, conclude that the co-administration of TTD, as a drug resistance reversal agent with ADM may be a better administration in the clinic.

Novel Nonsymmetrical 1,4-Dihydropyridines as Inhibitors of Nonsymmetrical MRP-Efflux Pumps for Anticancer Therapy.

Cancer is a strong global burden with increasing numbers of diseases and ongoing anticancer drug resistance. The number of structurally novel anticancer drugs is strongly limited. They cause high costs for the social health systems. Most critical so-called multidrug resistances (MDR) are caused by transmembrane efflux pumps that transport drugs with various structures out of the cancer cells. Multidrug resistance proteins (MRPs) type 1 and 2 are found overexpressed in various kinds of cancer. There is a strong need for inhibitors of those efflux pumps. We developed novel nonsymmetrical 1,4-dihydropyridines as novel inhibitors of cancer relevant MRP types 1 and 2. The structure-dependent activities of the differently substituted derivatives were evaluated in cellular assays of respective cancer cells and are discussed. Promising candidates were identified. One candidate was demonstrated to resensitize a cisplatin resistant cancer cell line and thus to overcome the anticancer drug resistance.

Antioxidant, Anti-Inflammatory, and Multidrug Resistance Modulation Activity of Silychristin Derivatives.

Silychristin A is the second most abundant compound of silymarin. Silymarin complex was previously described as an antioxidant with multidrug resistance modulation activity. Here, the results of a classical biochemical antioxidant assay (ORAC) were compared with a cellular assay evaluating the antioxidant capacity of pure silychristin A and its derivatives (anhydrosilychristin, isosilychristin and 2,3-dehydrosilychristin A). All the tested compounds acted as antioxidants within the cells, but 2,3-dehydro- and anhydro derivatives were almost twice as potent as the other tested compounds. Similar results were obtained in LPS-stimulated macrophages, where 2,3-dehydro- and anhydrosilychristin inhibited NO production nearly twice as efficiently as silychristin A. The inhibition of P-glycoprotein (P-gp) was determined in vitro, and the respective sensitization of doxorubicin-resistant ovarian carcinoma overproducing P-gp was detected. Despite the fact that the inhibition of P-gp was demonstrated in a concentration-dependent manner for each tested compound, the sensitization of the resistant cell line was observed predominantly for silychristin A and 2,3-dehydrosilychristin A. However, anhydrosilychristin and isosilychristin affected the expression of both the P-gp (ABCB1) and ABCG2 genes. This is the first report showing that silychristin A and its 2,3-dehydro-derivative modulate multidrug resistance by the direct inhibition of P-gp, in contrast to anhydrosilychristin and isosilychristin modulating multidrug resistance by downregulating the expression of the dominant transmembrane efflux pumps.

Dually Acting Nonclassical 1,4-Dihydropyridines Promote the Anti-Tuberculosis (Tb) Activities of Clofazimine.

The number of effective antituberculotic drugs is strongly limited to four first-line drugs in standard therapy. In case of resistances second-line antibiotics are used with a poor efficacy and tolerability. Therefore, novel antituberculotic drugs are urgently needed. We synthesized novel nonclassical 1,4-dihydropyridines and evaluated their antituberculotic properties depending on substituent effects. Preferred substituents could be identified. As related classical 1,4-dihydropyridines are known as inhibitors of the transmembrane efflux pump ABCB1 in cancer cells, we wondered whether a use of our compounds may be of favour to enhance the antituberculotic drug efficacy of the second-line antituberculotic drug clofazimine, which is a known substrate of ABCB1 by a suggested inhibition of a corresponding efflux pump in Mycobacterium tuberculosis (Mtb). For this, we determined the ABCB1 inhibiting properties of our compounds in a mouse T-lymphoma cell line model and then evaluated the drug-enhancing properties of selected compounds in a co-application with clofazimine in our Mtb strain. We identified novel enhancers of clofazimine toxicity which could prevent clofazimine resistance development mediated by an efflux pump activity.

SUN-113 COMBINATION OF *1/*1 WILD VARIANT OF CYP3A5 AND TT/AA MUTANT OF MDR G2677T/A GENE POLYMORPHISM REQUIRES HIGHER TACROLIMUS DOSE TO ACHIEVE TARGET TROUGH LEVEL

Tacrolimus (Tac) is the most commonly used immunosuppressive drug in transplantation. Tac disposition has very narrow therapeutic range and great inter-individual variability in its pharmacokinetics. Achieving the target blood concentration is of crucial importance in the early stage after transplantation and to attain good transplant outcome. This differential bioavailability in trough Tac concentration may be due to expression of P-gp varying between individuals and/or differential metabolizing capacity of CYP450.Tac share CYP3A5 enzymes for their hepatic elimination and P-gp, a product of MDR-1 gene expressed on gut wall, renal tubular cells, hepatocytes, lymphocytes etc and works as transmembrane efflux pump which exports xenobiotics from inside the cell to outside. Hence we aimed this study to analyse the influence of the combination of MDR-1 and CYP3A5 SNPs on daily Tac requirement to achieve trough level post-transplantation. We recruited 248 renal transplant recipients (mean age 36.48±10.54; male (83.47%),all on Tac; MMF and prednisone in the study. Of the 248 patients; 110 had CGN; 65 had CIN; 7 had ADPKD; 21 had DKD; 8 had CKDu; 7 had DGGS; 3 had Alport's syndrome and 27 had other native kidney diseases. The mean creatinine level at the time of discharge was 1.15±0.27(0.48-2.31). P-gp expression was analyzed by flow cytometry and polymorphism was performed by PCR-RFLP. Of the 248; 123(49.59%) were CYP3A5*3/*3(non-expressers homozygous); 94(37.90%) CYP3A5*1/*3; and 31(12.5%) were CYP3A5*1/*1 (expressers). In MDR G2677T/A gene of 248 patients; 99(39.91%) were GG (wild type homozygous); 104(41.92%) were GT/GA (heterozygous) and 45(18.13%) were TT/AA (mutant homozygous) of MDR Gene. Tac trough concentration was high in *3/*3 as compare to *1*1(p=0.001) and *1*3 (p=0.022) respectively. CYP3A5*1/*1 patients had lower levels of dose-adjusted TAC (55.69±26.84 ng/mL/mg/kg/day) to achieve target blood level and required higher daily dose per weight (0.142±0.028mg/kg/day) than CYP3A5*1/*3 patients; 111.12±8.93ng/mL/mg/kg/day and 0.121±0.035mg/kg/day(p=0.022) and CYP3A5*3/*3 patients; 142.21±75.23ng/mL/mg/kg/day and 0.109±0.032mg/kg/day(p<0.001). Tac Co was low in TT/AA as compare to GG(p<0.001) and GT/GA(p<0.001) respectively and daily Tac dose requirement was high in TT/AA as compare to GG(p<0.001) and GT/GA(p=0.027) genotype to achieve target blood concentration of MDR G2677T/A gene. The P-gp expression was also high in TT/AA as compare to GG(p<0.001) and GT/GA(p<0.001). Genotype C1236T and C3435T of MDR-1 gene had no significant effect on Tac dose requirement. Patients having CYP3A5*1/*1(expresser) and TT/AA (homozygous mutant) of MDRG2677T/A genotype had highest Tac daily dose requirement (0.147mg/kg/day) and having CYP3A5*3/*3 (non-expresser) and GG of MDRG2677T/A (wild type) genotype requires lowest Tac dose (0.101 mg/kg/day) to achieve target Tac tough concentration (8-12ng/ml). Daily Tac dose requirement is highest in patients having CYP3A5 expresser and MDR G2677T/A homozygous mutant gene together. Genetic variants may predicts the optimal dose of Tac in renal transplant recipients.

Reconstitution of P‐glycoprotein in Nanodiscs using the MSP1D1 Scaffold Protein for Biochemical Inhibitor Screens

Many cancers become multidrug resistant due to overexpression of the transmembrane efflux pump P‐glycoprotein (P‐gp). Compounds inhibiting P‐gp function may therefore have the potential to improve the effectiveness of chemotherapeutic drugs. Using in silico methods, we have been able to successfully identify a number of putative P‐gp inhibitor compounds. In cell culture assays, many of these compounds were shown to re‐sensitize cancers to chemotherapeutics. However, when tested with purified P‐gp, some of these compounds showed little effect on the catalytic activity of the isolated protein. The relatively high concentrations of detergents needed to solubilize P‐gp likely reduced the potency of the inhibitor compounds. To overcome this issue, purified P‐gp was inserted into nanostructures (nanodiscs) which much more closely resemble natural cell membrane. We and others have observed that this stabilizes P‐gp and stimulates its catalytic activity. However, previous assays of inhibitor compounds with medium size MSP1E3D1 nanodiscs still did not show clear effects on catalytic activity of the protein despite the fact that the inhibitors were effective in cell culture. We hypothesize that the previously used MSP1E3D1 nanodiscs still may have been too large, leaving hydrophobic space into which the inhibitors could partition, again reducing the effective concentration of the inhibitors. To improve on this protocol, we here show our efforts to use a shorter membrane scaffold protein, known as MSP1D1, for the assembly of smaller nanodiscs. After optimization of conditions for assembly and insertion, and tests for biological activity, we will assay our potential inhibitor hit compounds for their ability to affect catalytic ATP hydrolysis activities of P‐gp.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM094771‐02] to John G. Wise, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars Program, SMU Undergraduate Research Assistantships, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Relation of polymorphism C1236T and C3435T in ABCB1 gene with bone marrow suppression in chemotherapy-treated breast cancer patients

ABCB1 is agene that encoded P-glycoprotein (P-gp), a transmembrane active efflux pump for a variety of carcinogens and cytostatics.ABCB1 polymorphisms C1236T and C3435T contribute to the variability oftherapeutic outcome and side effects.The present study was conducted to investigatethe relation of C1236T and C3435T polymorphisms in ABCB1 gene with bone marrow suppression in breast cancer patients treated withchemotherapy72 Indonesian womens isolated DNA sampleswere amplified using the PCR method. The analysis process of ABCB1 C1236T and C3435T polymorphism was by using thePCR-RFLP method. The frequencies of ABCB1 C1236T genotype for homozygous CC,heterozygous CT and variant TT was 11(15.28%), 42(58.33%), 19(26.39%), respectively. No associationwas between ABCB1 C1236T and C3435T polymorphisms in both individually and haplotypes with bone marrow suppression event (p > 0.05). There was no specific deviation of allele and genotype frequency from Hardy-Weinberg Equilibrium. There was a linkage between heterozygous CT-heterozygous CT in position 1236 and 3435 within 25 people (35%).

Targeted production of reactive oxygen species in mitochondria to overcome cancer drug resistance

Multidrug resistance is a major challenge to cancer chemotherapy. The multidrug resistance phenotype is associated with the overexpression of the adenosine triphosphate (ATP)-driven transmembrane efflux pumps in cancer cells. Here, we report a lipid membrane-coated silica-carbon (LSC) hybrid nanoparticle that targets mitochondria through pyruvate, to specifically produce reactive oxygen species (ROS) in mitochondria under near-infrared (NIR) laser irradiation. The ROS can oxidize the NADH into NAD+ to reduce the amount of ATP available for the efflux pumps. The treatment with LSC nanoparticles and NIR laser irradiation also reduces the expression and increases the intracellular distribution of the efflux pumps. Consequently, multidrug-resistant cancer cells lose their multidrug resistance capability for at least 5 days, creating a therapeutic window for chemotherapy. Our in vivo data show that the drug-laden LSC nanoparticles in combination with NIR laser treatment can effectively inhibit the growth of multidrug-resistant tumors with no evident systemic toxicity.