Novel 5-oxo-hexahydroquinoline derivatives: design, synthesis, in vitro P-glycoprotein-mediated multidrug resistance reversal profile and molecular dynamics simulation study

Overexpression of the efflux pump P-glycoprotein (P-gp) is one of the important mechanisms of multidrug resistance (MDR) in many tumor cells. In this study, 26 novel 5-oxo-hexahydroquinoline derivatives containing different nitrophenyl moieties at C-4 and various carboxamide substituents at C-3 were designed, synthesized and evaluated for their ability to inhibit P-gp by measuring the amount of rhodamine 123 (Rh123) accumulation in uterine sarcoma cells that overexpress P-gp (MES-SA/Dx5) using flow cytometry. The effect of compounds with highest MDR reversal activities was further evaluated by measuring the alterations of MES-SA/Dx5 cells' sensitivity to doxorubicin (DXR) using MTT assay. The results of both biological assays indicated that compounds bearing 2-nitrophenyl at C-4 position and compounds with 4-chlorophenyl carboxamide at C-3 demonstrated the highest activities in resistant cells, while they were devoid of any effect in parental nonresistant MES-SA cells. One of the active derivatives, 5c, significantly increased intracellular Rh123 at 100 mu M, and it also significantly reduced the IC50 of DXR by 70.1% and 88.7% at 10 and 25 mu M, respectively, in MES-SA/Dx5 cells. The toxicity of synthesized compounds against HEK293 as a noncancer cell line was also investigated. All tested derivatives except for 2c compound showed no cytotoxicity. A molecular dynamics simulation study was also performed to investigate the possible binding site of 5c in complex with human P-gp, which showed that this compound formed 11 average H-bonds with Ser909, Thr911, Arg547, Arg543 and Ser474 residues of P-gp. A good agreement was found between the results of the computational and experimental studies. The findings of this study show that some 5-oxo-hexahydroquinoline derivatives could serve as promising candidates for the discovery of new agents for P-gp-mediated MDR reversal

Multiplex touchdown PCR assay to enhance specificity and sensitivity for concurrent detection of four foodborne pathogens in raw milk

Aims: The aim of this study was to develop a multiplex touchdown PCR (multiplex TD-PCR) for rapid and simultaneous detection of four major foodborne pathogens to avoid mispriming and unwanted production during gene amplification. Touchdown PCR is the modified form of standard PCR, which enhances specificity, sensitivity. Methods and Results: For this reason, a multiplex TD-PCR assay with a pre-enrichment step was developed to detect four foodborne pathogens namely Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, and Salmonella enterica serovar Enteritidis in pure culture and raw milk samples. The results showed that this protocol can eliminate the unwanted band or reduce significantly. The detection sensitivity of the single and multiplex TD-PCR was one cell per ml in pure culture. Furthermore, the detection limit of multiplex TD-PCR was one cell per 25 ml for artificially contaminated raw milk. We obtained similar results for detection of aforementioned pathogens in raw milk, after comparing the multiplex TD-PCR method with the traditional culture, except in one or two samples. Conclusions: Hence, the proposed multiplex TD-PCR method could be confirmed as an effective way for rapid optimization of PCR reactions to increase specificity, sensitivity during gene amplification. Significance and Impact of the Study: Hence, due to its simplicity, cost-effectiveness and being time-saving, it seems that this method is reasonable and economical for rapid optimization of PCR reaction

Oxytocinergic system mediates the proconvulsant effects of sildenafil: The role of calcineurin

Sildenafil is a phosphodiesterase type 5 inhibitor used to treat male erectile dysfunction and pulmonary hypertension. A potential side effect of sildenafil is a noticeable decrease in seizure threshold. Oxytocin (OXT) secretion and the subsequent cAMP-responsive element-binding (CREB) phosphorylation are involved in proconvulsant effects of sildenafil in experimental models. The aim of the present study was to investigate the potential role of OXT receptors and their downstream calcineurin (CN)/inducible nitric oxide synthase (iNOS) pathways in proconvulsant effects of sildenafil. The pentylenetetrazole (PTZ)-induced seizure was used as a standard convulsion model in this study. Cortical CN activity, hippocampal nitrite levels, and proinflammatory cytokine content were measured. Our results indicated that following PTZ administration, sildenafil significantly increased CN activity at 40 mg/kg, respectively, in the control group. The combination of sildenafil and OXT receptor antagonist, atosiban (10 μg/kg, i.c.v) 30 min before sildenafil administration significantly reduced the CN activity. Also, the subeffective dose of CN inhibitor cyclosporine (5 mg/kg) 30 min before the administration of effective dose of sildenafil (40 mg/kg) reversed proconvulsant actions of sildenafil. This effect was iNOS-dependent because pretreatment of a low dose of aminoguanidine (20 mg/kg) 15 min before the administration of a low dose of cyclosporine (1 mg/kg) reversed the proconvulsant action of sildenafil (40 mg/kg). Finally, sildenafil induced the elevation of tumor necrosis factor alpha (TNF-α) and the nitrite level was blocked by the administration of cyclosporine in PTZ-treated mice. Collectively, our data provide insights into the role of OXT receptor/CN/iNOS pathway in the proconvulsant aspect of sildenafil. © 202

Tetrahydroquinolinone derivatives as potent P-glycoprotein inhibitors: design, synthesis, biological evaluation and molecular docking analysis

P-glycoprotein (P-gp) is a transmembrane efflux pump that has been associated with ineffective cancer chemotherapy and multidrug resistance (MDR). Chemical inhibitors of P-gp could have potential cancer therapeutic applications by preventing or reversing MDR. To exploit this, we designed twenty-five tetrahydroquinolinone analogs bearing pyridyl methyl carboxylate at C3 and different substituents at C4 as MDR reversal agents. The inhibitory effects of the synthesized compounds against P-gp were assessed by flow cytometric determination of rhodamine 123 accumulation in P-gp over-expressing MES-SA/DX5 cells. Fluorescence imaging of intracellular rhodamine 123 accumulation in MES-SA/DX5 cells was also performed. Furthermore, the effect of active derivatives on the reduction of doxorubicin's IC50 in MES-SA/DX5 cells was evaluated using MTT assay. Molecular docking was used to confirm the binding mode of some of the synthesized compounds. Five compounds in group A, bearing a 2-pyridyl methyl ester substituent at the C3 position, significantly increased rhodamine accumulation at 25 μM comparable to verapamil, a well-established P-gp inhibitor, while only 2 compounds in group B bearing 3-pyridyl methyl ester at the same position had this effect. This study shows that tetrahydroquinolinones containing methyl pyridine esters could represent an attractive scaffold for the discovery of P-gp inhibitors as MDR reversal agents in cancer cells

Novel 5-oxo-hexahydroquinoline derivatives: design, synthesis, in vitro P-glycoprotein-mediated multidrug resistance reversal profile and molecular dynamics simulation study

Omolbanin Shahraki,1,2 Najmeh Edraki,1 Mehdi Khoshneviszadeh,1,2 Farshid Zargari,1 Sara Ranjbar,1,2 Luciano Saso,3 Omidreza Firuzi,1 Ramin Miri1 1Medicinal and Natural Products Chemistry Research Center, 2Department of Medicinal Chemistry, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran; 3Department of Physiology and Pharmacology “Vittorio Ersparmer”, Sapienza University of Rome, Rome, Italy Abstract: Overexpression of the efflux pump P-glycoprotein (P-gp) is one of the important mechanisms of multidrug resistance (MDR) in many tumor cells. In this study, 26 novel 5-oxo-hexahydroquinoline derivatives containing different nitrophenyl moieties at C4 and various carboxamide substituents at C3 were designed, synthesized and evaluated for their ability to inhibit P-gp by measuring the amount of rhodamine 123 (Rh123) accumulation in uterine sarcoma cells that overexpress P-gp (MES-SA/Dx5) using flow cytometry. The effect of compounds with highest MDR reversal activities was further evaluated by measuring the alterations of MES-SA/Dx5 cells’ sensitivity to doxorubicin (DXR) using MTT assay. The results of both biological assays indicated that compounds bearing 2-nitrophenyl at C4 position and compounds with 4-chlorophenyl carboxamide at C3 demonstrated the highest activities in resistant cells, while they were devoid of any effect in parental nonresistant MES-SA cells. One of the active derivatives, 5c, significantly increased intracellular Rh123 at 100 µM, and it also significantly reduced the IC50 of DXR by 70.1% and 88.7% at 10 and 25 µM, respectively, in MES-SA/Dx5 cells. The toxicity of synthesized compounds against HEK293 as a noncancer cell line was also investigated. All tested derivatives except for 2c compound showed no cytotoxicity. A molecular dynamics simulation study was also performed to investigate the possible binding site of 5c in complex with human P-gp, which showed that this compound formed 11 average H-bonds with Ser909, Thr911, Arg547, Arg543 and Ser474 residues of P-gp. A good agreement was found between the results of the computational and experimental studies. The findings of this study show that some 5-oxo-hexahydroquinoline derivatives could serve as promising candidates for the discovery of new agents for P-gp-mediated MDR reversal. Keywords: cancer, P-glycoprotein, multidrug resistance, 1,4-dihydropyridine, molecular dynamics simulatio

5-Oxo-hexahydroquinoline derivatives as modulators of P-gp, MRP1 and BCRP transporters to overcome multidrug resistance in cancer cells

Multidrug resistance (MDR) in cancer cells is often associated with overexpression of ATP-binding cassette (ABC) transporters, including P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1) and breast cancer resistance protein (BCRP/ABCG2). Modulators of these transporters might be helpful in overcoming MDR. Moreover, exploiting collateral sensitivity (CS) could be another approach for efficient treatment of cancer. Twelve novel 5-oxo-hexahydroquinoline derivatives bearing different aromatic substitutions at C4, while having 2-pyridyl alkyl carboxylate substituents at the C3 were synthesized and evaluated for MDR reversal activity by flow cytometric determination of rhodamine 123, calcein and mitoxantrone accumulations in P-gp, MRP1 and BCRP-overexpressing cell lines, respectively. Furthermore, to confirm the P-gp inhibitory activity, the effect of compounds on the reduction of doxorubicin's IC50 of drug-resistant human uterine sarcoma cell line, MES-SA/DX5, was evaluated. Compounds D6, D5 and D3 (bearing 3-chlorophenyl, 2,3-dichlorophenyl and 4-chlorophenyl substituents at C4 position of 5-oxo-hexahydroquinoline core) were the most potent P-gp, MRP1 and BCRP inhibitors, respectively, causing significant MDR reversal at concentrations of 1–10 μM. Additionally, D4 (containing 3-flourophenyl) was the most effective MRP1-dependent CS inducing agent. Overall, chlorine containing compounds D6, C4 and D3 were capable of significant inhibition of all 3 important efflux pumps in cancer cells. Moreover, D6 also induced CS triggered by reducing glutathione efflux. In conclusion, some of the 5-oxo-hexahydroquinoline derivatives are effective efflux pump inhibitors capable of simultaneously blocking 3 important ABC transporters involved in MDR, and represent promising agents to overcome MDR in cancer cells. © 2018 Elsevier Inc