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European Journal of Medicinal Chemistry Nov 2023New 2,5- and 1,5-disubstituted tetrazoles, and 2,5-disubstituted-1,3,4-oxadiazoles were synthesized as tariquidar and elacridar derivatives and studied as multidrug...
New 2,5- and 1,5-disubstituted tetrazoles, and 2,5-disubstituted-1,3,4-oxadiazoles were synthesized as tariquidar and elacridar derivatives and studied as multidrug resistance (MDR) reversers. Their behaviour on the three ABC transporters P-gp, MRP1 and BCRP was investigated. All compounds inhibited the P-gp transport activity in MDCK-MDR1 cells overexpressing P-gp, showing EC values even in the low nanomolar range (compounds 15, 22). Oxadiazole derivatives were able to increase the antiproliferative effect of doxorubicin in MDCK-MDR1 and in HT29/DX cells confirming their nature of P-gp modulators, with derivative 15 being the most potent in these assays. Compound 15 also displayed a dual inhibitory effect showing good activities towards both P-gp and BCRP. A computational study suggested a common interaction pattern on P-gp for most of the potent compounds. The bioisosteric substitution of the amide group of lead compounds allowed identifying a new set of potent oxadiazole derivatives that modulate MDR through inhibition of the P-gp efflux activity. If compared to previous amide derivatives, the introduction of the heterocycle rings greatly enhances the activity on P-gp, introduces in two compounds a moderate inhibitory activity on MRP1 and maintains in some cases the effect on BCRP, leading to the unveiling of dual inhibitor 15.
Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; Drug Resistance, Neoplasm; Structure-Activity Relationship; Neoplasm Proteins; Drug Resistance, Multiple; Tetrazoles; Amides
PubMed: 37573829
DOI: 10.1016/j.ejmech.2023.115716 -
Establishment and Characterization of Multi-Drug Resistant p53-Negative Osteosarcoma SaOS-2 Subline.Diagnostics (Basel, Switzerland) Aug 2023To establish a p53-negative osteosarcoma (OS) SaOS-2 cellular subline exhibiting resistance to specific chemotherapeutic agents, including topoisomerase II inhibitors,...
AIM
To establish a p53-negative osteosarcoma (OS) SaOS-2 cellular subline exhibiting resistance to specific chemotherapeutic agents, including topoisomerase II inhibitors, taxanes, and vinca alkaloids.
METHODS
The OS subline exhibiting resistance to the chemotherapeutic agents indicated above was generated by the stepwise treatment of the parental SaOS-2 cell line with increasing concentrations of doxorubicin (Dox) for 5 months. Half-inhibitory concentrations (IC) for Dox, vinblastine (Vin), and paclitaxel (PTX) were calculated by a colorimetric MTS-based assay. Crystal violet staining was used to assess cellular viability, whereas the proliferation capacities of cancer cells were monitored in real-time by the i-Celligence system. Expression of apoptotic markers (e.g., cleaved PARP and caspase-3), DNA repair proteins (e.g., ATM, DNA-PK, Nbs1, Rad51, MSH2, etc.), and certain ABC transporters (P-glycoprotein, MRP1, ABCG2, etc.) was assessed by western blotting and real-time PCR. Flow cytometry was used to examine the fluorescence intensity of Dox and ABC-transporter substrates (e.g., Calcein AM and CMFDA) and to assess their excretion to define the activity of specific ABC-transporters. To confirm OS resistance to Dox in vivo, xenograft experiments were performed.
RESULTS
An OS subline generated by a stepwise treatment of the parental SaOS-2 cell line with increasing concentrations of Dox resulted in an increase in the IC for Dox, Vin, and PTX (~6-, 4-, and 30-fold, respectively). The acquisition of chemoresistance in vitro was also evidenced by the lack of apoptotic markers (e.g., cleaved PARP and caspase-3) in resistant OS cells treated with the chemotherapeutic agents indicated above. The development of the multidrug resistance (MDR) phenotype in this OS subline was due to the overexpression of ABCB1 (i.e., P-glycoprotein) and ABCC1 (i.e., multidrug resistance protein-1, MRP-1), which was evidenced on both mRNA and protein levels. Due to increased expression of MDR-related proteins, resistant OS exhibited an excessive efflux of Dox. Moreover, decreased accumulation of calcein AM, a well-known fluorescent substrate for both ABCB1 and ABCC1, was observed for resistant OS cells compared to their parental SaOS-2 cell line. Importantly, tariquidar and cyclosporin, well-known ABC inhibitors, retained the intensity of Dox-induced fluorescence in resistant SAOS-2 cells. Furthermore, in addition to the increased efflux of the chemotherapeutic agents from Dox-resistant OS cells, we found higher expression of several DNA repair proteins (e.g., Rad51 recombinase, Mre11, and Nbs1, activated forms of ATM, DNA-PK, Chk1, and Chk2, etc.), contributing to the chemoresistance due to the excessive DNA repair. Lastly, the in vivo study indicated that Dox has no impact on the SaOS-2 Dox-R xenograft tumor growth in a nude mouse model.
CONCLUSIONS
An acquired resistance of OS to the chemotherapeutic agents might be due to the several mechanisms undergoing simultaneously on the single-cell level. This reveals the complexity of the mechanisms involved in the secondary resistance of OS to chemotherapies.
PubMed: 37627905
DOI: 10.3390/diagnostics13162646 -
Frontiers in Molecular Biosciences 2024The human multidrug transporter P-glycoprotein (P-gp) is physiologically essential and of key relevance to biomedicine. Recent structural studies have shed light on the...
The human multidrug transporter P-glycoprotein (P-gp) is physiologically essential and of key relevance to biomedicine. Recent structural studies have shed light on the mode of inhibition of the third-generation inhibitors for human P-gp, but the molecular mechanism by which these inhibitors enter the transmembrane sites remains poorly understood. In this study, we utilized all-atom molecular dynamics (MD) simulations to characterize human P-gp dynamics under a potent inhibitor, tariquidar, bound condition, as well as the atomic-level binding pathways in an explicit membrane/water environment. Extensive unbiased simulations show that human P-gp remains relatively stable in tariquidar-free and bound states, while exhibiting a high dynamic binding mode at either the drug-binding pocket or the regulatory site. Free energy estimations by partial nudged elastic band (PNEB) simulations and Molecular Mechanics Generalized Born Surface Area (MM/GBSA) method identify two energetically favorable binding pathways originating from the cytoplasmic gate with an extended tariquidar conformation. Interestingly, free tariquidar in the lipid membrane predominantly adopts extended conformations similar to those observed at the regulatory site. These results suggest that membrane lipids may preconfigure tariquidar into an active ligand conformation for efficient binding to the regulatory site. However, due to its conformational plasticity, tariquidar ultimately moves toward the drug-binding pocket in both pathways, explaining how it acts as a substrate at low concentrations. Our molecular findings propose a membrane-assisted mechanism for the access and binding of the third-generation inhibitors to the binding sites of human P-gp, and offer deeper insights into the molecule design of more potent inhibitors against P-gp-mediated drug resistance.
PubMed: 38560519
DOI: 10.3389/fmolb.2024.1364494 -
Molecular Pharmaceutics Nov 2023P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two ATP-binding cassette efflux transporters that are coexpressed at the human blood-brain barrier...
P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two ATP-binding cassette efflux transporters that are coexpressed at the human blood-brain barrier (BBB) and blood-retina barrier (BRB). While pharmacological inhibition of P-gp and/or BCRP results in increased brain distribution of dual P-gp/BCRP substrate drugs, such as the tyrosine kinase inhibitor erlotinib, the effect of P-gp and/or BCRP inhibition on the retinal distribution of such drugs has hardly been investigated. In this study, we used positron emission tomography (PET) imaging to assess the effect of transporter inhibition on the distribution of [C]erlotinib to the human retina and brain. Twenty two healthy volunteers underwent two PET scans after intravenous (i.v.) injection of a microdose (<5 μg) of [C]erlotinib, a baseline scan, and a second scan either with concurrent i.v. infusion of tariquidar to inhibit P-gp ( = 5) or after oral intake of single ascending doses of erlotinib (300 mg, 650 mg, or 1000 mg, = 17) to saturate erlotinib transport. In addition, transport of [H]erlotinib to the retina and brain was assessed in mice by in situ carotid perfusion under various drug transporter inhibition settings. In comparison to the baseline PET scan, coadministration of tariquidar or erlotinib led to a significant decrease of [C]erlotinib total volume of distribution () in the human retina by -25 ± 8% ( ≤ 0.05) and -41 ± 16% ( ≤ 0.001), respectively. In contrast, erlotinib intake led to a significant increase in [C]erlotinib in the human brain (+20 ± 16%, ≤ 0.001), while administration of tariquidar did not result in any significant changes. In situ carotid perfusion experiments showed that both P-gp and BCRP significantly limit the distribution of erlotinib to the mouse retina and brain but revealed a similar discordant effect at the mouse BRB and BBB following co-perfusion with tariquidar and erlotinib as in humans. Co-perfusion with prototypical inhibitors of solute carrier transporters did not reveal a significant contribution of organic cation transporters (e.g., OCTs and OCTNs) and organic anion-transporting polypeptides (e.g., OATP2B1) to the retinal and cerebral distribution of erlotinib. In conclusion, we observed a dissimilar effect after P-gp and/or BCRP inhibition on the retinal and cerebral distribution of [C]erlotinib. The exact mechanism for this discrepancy remains unclear but may be related to the function of an unidentified erlotinib uptake carrier sensitive to tariquidar inhibition at the BRB. Our study highlights the great potential of PET to study drug distribution to the human retina and to assess the functional impact of membrane transporters on ocular drug distribution.
Topics: Humans; Mice; Animals; Female; Erlotinib Hydrochloride; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; Neoplasm Proteins; Brain; ATP Binding Cassette Transporter, Subfamily B; Blood-Brain Barrier; ATP-Binding Cassette Transporters; Blood-Retinal Barrier; Membrane Transport Proteins; Breast Neoplasms
PubMed: 37883694
DOI: 10.1021/acs.molpharmaceut.3c00715 -
Drug Resistance Updates : Reviews and... Nov 2023Therapy resistance has long been considered to occur through the selection of pre-existing clones equipped to survive and quickly regrow, or through the acquisition of...
Therapy resistance has long been considered to occur through the selection of pre-existing clones equipped to survive and quickly regrow, or through the acquisition of mutations during chemotherapy. Here we show that following in vitro treatment by chemotherapy, epithelial breast cancer cells adopt a transient drug tolerant phenotype characterized by cell cycle arrest, epithelial-to-mesenchymal transition (EMT) and the reversible upregulation of the multidrug resistance (MDR) efflux transporter P-glycoprotein (P-gp). The drug tolerant persister (DTP) state is reversible, as cells eventually resume proliferation, giving rise to a cell population resembling the initial, drug-naïve cell lines. However, recovery after doxorubicin treatment is almost completely eliminated when DTP cells are cultured in the presence of the P-gp inhibitor Tariquidar. Mechanistically, P-gp contributes to the survival of DTP cells by removing reactive oxygen species-induced lipid peroxidation products resulting from doxorubicin exposure. In vivo, prolonged administration of Tariquidar during doxorubicin treatment holidays resulted in a significant increase of the overall survival of Brca1;p53 mammary tumor bearing mice. These results indicate that prolonged administration of a P-gp inhibitor during drug holidays would likely benefit patients without the risk of aggravated side effects related to the concomitantly administered toxic chemotherapy. Effective targeting of DTPs through the inhibition of P-glycoprotein may result in a paradigm shift, changing the focus from countering drug resistance mechanisms to preventing or delaying therapy resistance.
Topics: Humans; Animals; Mice; Female; ATP Binding Cassette Transporter, Subfamily B, Member 1; Lipid Peroxidation; Pharmaceutical Preparations; Breast Neoplasms; ATP Binding Cassette Transporter, Subfamily B; Doxorubicin
PubMed: 37741091
DOI: 10.1016/j.drup.2023.101007 -
Scientific Reports Oct 2023Drug designing is high-priced and time taking process with low success rate. To overcome this obligation, computational drug repositioning technique is being promptly...
Drug designing is high-priced and time taking process with low success rate. To overcome this obligation, computational drug repositioning technique is being promptly used to predict the possible therapeutic effects of FDA approved drugs against multiple diseases. In this computational study, protein modeling, shape-based screening, molecular docking, pharmacogenomics, and molecular dynamic simulation approaches have been utilized to retrieve the FDA approved drugs against AD. The predicted MADD protein structure was designed by homology modeling and characterized through different computational resources. Donepezil and galantamine were implanted as standard drugs and drugs were screened out based on structural similarities. Furthermore, these drugs were evaluated and based on binding energy (Kcal/mol) profiles against MADD through PyRx tool. Moreover, pharmacogenomics analysis showed good possible associations with AD mediated genes and confirmed through detail literature survey. The best 6 drug (darifenacin, astemizole, tubocurarine, elacridar, sertindole and tariquidar) further docked and analyzed their interaction behavior through hydrogen binding. Finally, MD simulation study were carried out on these drugs and evaluated their stability behavior by generating root mean square deviation and fluctuations (RMSD/F), radius of gyration (Rg) and soluble accessible surface area (SASA) graphs. Taken together, darifenacin, astemizole, tubocurarine, elacridar, sertindole and tariquidar displayed good lead like profile as compared with standard and can be used as possible therapeutic agent in the treatment of AD after in-vitro and in-vivo assessment.
Topics: Humans; Drug Repositioning; Molecular Docking Simulation; Alzheimer Disease; Prognosis; Astemizole; Tubocurarine; Molecular Dynamics Simulation
PubMed: 37865690
DOI: 10.1038/s41598-023-45347-1 -
Biomedicine & Pharmacotherapy =... Dec 2023Despite extensive efforts and ongoing progress in personalized anticancer approaches, chemotherapy remains the first line or the only treatment for some tumors that may...
Despite extensive efforts and ongoing progress in personalized anticancer approaches, chemotherapy remains the first line or the only treatment for some tumors that may develop resistance to chemotherapeutics in time due to inter alia overexpression of ATP-binding cassette transporters. Using clinically-relevant resistant models of triple negative breast cancer (MDA-MB-231; TNBC) as well as non-small cell lung cancer (A549; NSCLC), we tested the efficacy of I-CBP112 - CBP/EP300 bromodomain inhibitor to overcome drug resistance by declining ABC gene transcription. I-CBP112 significantly reduced ABCB1, ABCC1, ABCC2, ABCC3, ABCC5 and ABCG2 in all resistant lines, as well as ABCC10 in TNBC and ABCC4 in paclitaxel-resistant NSCLC, thereby increasing intracellular drug accumulation and cytotoxicity in 2D and 3D cultures. This was phenocopied only by the joint effect of ABC inhibitors such as tariquidar (ABCB1 - P-glycoprotein and ABCG2) and MK-571 (ABCC), whereas single inhibition of ABCB1/ABCG2 or ABCC proteins did not affect drug accumulation, thereby implying the need of simultaneous deficiency in activity of majority of drug pumps for enhanced drug retention. I-CBP112 failed to directly inhibit activity of ABCB1, ABCG2 and ABCC subfamily members at the same time. Importantly, I-CBP112 treated cancer cells polarized human macrophages into proinflammatory phenotypes. Moreover, I-CBP112 remained non-toxic to primary cell lines, nor did it enhance anticancer drug toxicity to blood-immune cells. In silico assay of ADMET properties confirmed the desired pharmacokinetic features of I-CBP112. The results suggest that the CBP/p300 inhibitor is a promising co-adjuvant to chemotherapy in drug-resistant cancer phenotypes, capable of decreasing ABC transporter expression.
Topics: Humans; ATP-Binding Cassette Transporters; Drug Resistance, Multiple; Carcinoma, Non-Small-Cell Lung; A549 Cells; Triple Negative Breast Neoplasms; Drug Resistance, Neoplasm; Lung Neoplasms; Cell Line, Tumor; Antineoplastic Agents
PubMed: 37913733
DOI: 10.1016/j.biopha.2023.115798 -
Bioorganic Chemistry Apr 2024Being aware of the need to develop more efficient therapies against cancer, herein we disclose an innovative approach for the design of selective antiproliferative...
Being aware of the need to develop more efficient therapies against cancer, herein we disclose an innovative approach for the design of selective antiproliferative agents. We have accomplished the conjugation of a coumarin fragment with lipophilic cations (triphenylphosphonium salts, guanidinium) for providing mitochondriotropic agents that simultaneously target also carbonic anhydrases IX and XII, involved in the development and progression of cancer. The new compounds prepared herein turned out to be strong inhibitors of carbonic anhydrases IX and XII of human origin (low-to-mid nM range), also endowed with high selectivity, exhibiting negligible activity towards cytosolic CA isoforms. Key interactions with the enzyme were analysed using docking and molecular dynamics simulations. Regarding their in vitro antiproliferative activities, an increase of the tether length connecting both pharmacophores led to a clear improvement in potency, reaching the submicromolar range for the lead compounds, and an outstanding selectivity towards tumour cell lines (S.I. up to >357). Cytotoxic effects were also analysed on MDR cell lines under hypoxic and normoxic conditions. Chemoresistance exhibited by phosphonium salts, and not by guanidines, against MDR cells was based on the fact that the former were found to be substrates of P-glycoprotein (P-gp), the pump responsible for extruding foreign chemicals; this situation was reversed by administrating tariquidar, a third generation P-gp inhibitor. Moreover, phosphonium salts provoked a profound depolarization of mitochondria membranes from tumour cells, thus probably compromising their oxidative metabolism. To gain insight into the mode of action of title compounds, continuous live cell microscopy was employed; interestingly, this technique revealed two different antiproliferative mechanisms for both families of mitocans. Whereas phosphonium salts had a cytostatic effect, blocking cell division, guanidines led to cell death via apoptosis.
Topics: Humans; Carbonic Anhydrases; Salts; Structure-Activity Relationship; Antigens, Neoplasm; Antineoplastic Agents; Coumarins; Guanidines; Carbonic Anhydrase Inhibitors; Molecular Structure; Organophosphorus Compounds
PubMed: 38354500
DOI: 10.1016/j.bioorg.2024.107168 -
Biomedicines Jul 2023Despite disadvantages, such as high cost and their poor predictive value, animal experiments are still the state of the art for pharmaceutical substance testing. One...
Despite disadvantages, such as high cost and their poor predictive value, animal experiments are still the state of the art for pharmaceutical substance testing. One reason for this problem is the inability of standard cell culture methods to emulate the physiological environment necessary to recapitulate in vivo processes. Microphysiological systems offer the opportunity to close this gap. In this study, we utilize a previously employed microphysiological system to examine the impact of pressure and flow on the transportation of substances mediated by multidrug resistance protein 1 (MDR1) across an artificial cell-based tubular barrier. By using a miniaturized fluorescence measurement device, we could continuously track the MDR1-mediated transport of rhodamine 123 above the artificial barrier over 48 h. We proved that applying pressure and flow affects both active and passive transport of rhodamine 123. Using experimental results and curve fittings, the kinetics of MDR1-mediated transport as well as passive transport were investigated; thus, a kinetic model that explains this transport above an artificial tubular barrier was identified. This kinetic model demonstrates that the simple Michaelis-Menten model is not an appropriate model to explain the MDR1-mediated transport; instead, Hill kinetics, with Hill slope of = 2, is a better fit. The kinetic values, , , and apparent permeability (P), obtained in this study are comparable with other in vivo and in vitro studies. Finally, the presented proximal tubule-on-a-chip can be used for pharmaceutical substance testing and to investigate pharmacokinetics of the renal transporter MDR1.
PubMed: 37509683
DOI: 10.3390/biomedicines11072045 -
IBRO Neuroscience Reports Dec 2023P-glycoprotein (Pgp) overexpressed in blood brain barrier (BBB) is hypothesized to lower brain drug concentrations and thus inhibit anticonvulsant effects in...
P-glycoprotein (Pgp) overexpressed in blood brain barrier (BBB) is hypothesized to lower brain drug concentrations and thus inhibit anticonvulsant effects in drug-resistant epilepsy. Pluronic P85 (P85) was proved to enhance the delivery of drugs into the brain by inhibition of Pgp. To determine whether the surfactant P85 [versus Pgp inhibitor tariquidar (TQD)] enhance phenytoin (PHT) into the brain in drug-resistant rats with chronic mesial temporal lobe epilepsy (MTLE) induced by lithium-pilocarpine, in brain of which Pgp were overexpressed, then direct verification of PHT transport via measurement of PHT concentration in brain using microdialysis. The drug-resistant model rats were randomly divided into three groups, which were treated with PHT, 1%P85 + PHT, or PHT+TQD, respectively. 1%P85 + PHT treatment displayed a lower ratio of the area under the curve (AUC) of the PHT concentration in the brain/plasma even than that of the PHT treatment in model rats (p < 0.05), while PHT+TQD showed the highest ratio of the AUC of all treatments. However, the ratio of the PHT concentration in the liver/plasma was similar in three model groups (p > 0.05). For the ratio of the kidney/plasma, PHT+TQD treatment model group had the highest ratio of the other treatments in model rats. Thus, P85 oppositely decreased PHT concentration in brain in drug-resistant model rats with Pgp overexpressed MTLE while TQD could increase PHT distribution in brain.
PubMed: 37485299
DOI: 10.1016/j.ibneur.2023.06.009