Overexpression of ABCG2 Confers Resistance to MLN7243, a Ubiquitin-Activating Enzyme (UAE) Inhibitor

Overexpression of ATP-binding cassette transporter superfamily G member 2 (ABCG2), is known as a major mechanism mediating multidrug resistance (MDR) in cancer cells. MLN7243 is a small-molecule ubiquitin activating enzyme inhibitor currently under clinical investigation. The aim of the current study is to determine if MLN7243 is a substrate of MDR-related ABCG2 transporter. Our results showed that cancer cells overexpressing ABCG2 transporter were resistant to MLN7243 compared to the parental cells, while knockout of ABCG2 gene or pharmacological inhibition of ABCG2 efflux function completely reversed the drug resistance. Unexpectedly, the endogenous low expression of ABCG2 is sufficient to confer cancer cells resistance to MLN7243. The ABCG2 ATPase assay and HPLC assay suggested that MLN7243 can significantly stimulate ABCG2 ATPase activity and be pumped out from ABCG2-overexpressing cells by ABCG2. The docking analysis also implied that MLN7243 binds to ABCG2 drug-binding pocket with optimal binding affinity. However, MLN7243 did not competitively inhibit the efflux of other ABCG2 substrate drugs, indicating it may not serve as an MDR reversal agent. In conclusion, our study provides direct in vitro evidence to show that MLN7243 is a potent ABCG2 substrate. If our results can be translated to humans, it suggests that combining MLN7243 with ABCG2 inhibitors may enhance the anticancer efficacy for patients with high tumor ABCG2 level.

Solvothermal Synthesis of Multiple Dihydropyrimidinones at a Time as Inhibitors of Eg5

Solvothermal synthesis of multiple dihydropyrimidinones at a time has been developed in inexpensive and green bio-based solvent lactic acid without any additional catalysts or additives. By this method, thirty new dihydropyrimidinone derivatives were synthesized in two batches and characterized. All of the compounds were screened by Eg5 motor protein ATPase assay, and the positive compounds were tested against the Caco-2 cell line, HeLa cell line, L929 cell line and T24 cell line in vitro. Among them, compound C9 exhibited the best inhibitory activity against motor protein ATPase with an IC50 value of 30.25 μM and significant cytotoxic activity in the micromolar range against the cells above. The Lineweaver–Burk plot revealed that compound C9 was a mixed-type Eg5 inhibitor. A molecular modeling study using the Discovery Studio program was performed, where compound C9 exhibited good binding interaction with Eg5 motor protein ATPase, and this was consistent with the attained experimental results.

Novel photochromic inhibitor for mitotic kinesin Eg5 which forms multiple isomerization states

The mitotic kinesin Eg5 is a plus-end directed homotetrameric molecular motor essential for the formation of bipolar spindles during cell division. Kinesin Eg5 is overexpressed in cancer cells and hence considered as a target for cancer therapy; the inhibitors specific for Eg5 have been developed as anticancer drugs. In this study, we synthesized a novel functional photoresponsive inhibitor composed of spiropyran and azobenzene derivatives to control Eg5 function with multistage inhibitory activity accompanied by the formation of different isomerization states. The photochromic inhibitor spiropyran-sulfo-azobenzene (SPSAB) exhibited three isomerization states: spiro (SP)-trans, merocyanine (MC)-cis and MC-trans, upon exposure to visible light, ultraviolet and in the dark, respectively. SPSAB-induced reversible changes in the inhibitory activity of ATPase and motor activities correlating with photoisomerization among the three states. Among the three isomerization states of SPSAB, the SP-trans isomer showed potent inhibitory activity at an IC50 value of 30 µM in the basal ATPase assay. MC-trans and MC-cis exhibited less inhibitory activity at IC50 values of 38 and 86 µM, respectively. The results demonstrated that the novel photochromic inhibitor enabled precise control of Eg5 function at three different levels using light irradiation.

OTS964, a TOPK Inhibitor, Is Susceptible to ABCG2-Mediated Drug Resistance

OTS964 is a potent T-LAK cell-originated protein kinase (TOPK) inhibitor. Herein, we investigated the interaction of OTS964 and multidrug resistance (MDR)-associated ATP-binding cassette sub-family G member 2 (ABCG2). The cell viability assay indicated that the effect of OTS964 is limited in cancer drug-resistant and transfected cells overexpressing ABCG2. We found that the known ABCG2 transporter inhibitor has the ability to sensitize ABCG2-overexpressing cells to OTS964. In mechanism-based studies, OTS964 shows inhibitory effect on the efflux function mediated by ABCG2, and in turn, affects the pharmacokinetic profile of other ABCG2 substrate-drugs. Furthermore, OTS964 upregulates ABCG2 protein expression, resulting in enhanced resistance to ABCG2 substrate-drugs. The ATPase assay demonstrated that OTS964 stimulates ATPase activity of ABCG2 in a concentration-dependent manner. The computational molecular docking analysis combined with results from ATPase assay suggested that OTS964 interacts with drug-binding pocket of ABCG2 and has substrate-like behaviors. Thus, OTS964 is an MDR-susceptible agent due to its interactions with ABCG2, and overexpression of ABCG2 transporter may attenuate its therapeutic effect in cancer cells.

Antitumor Effects of Docetaxel in Truncated Basic Fibroblast Growth Factor- Functionalized Liposomes Delivered by d-α-tocopheryl Polyethylene Glycol 2000 Succinate

Background: PEGylation of stealth liposomes elevates their stability and prolongs plasma half-life. Stealth liposomes modified with targeting ligands are expected to be ideal drug delivery carriers. Objective: To encapsulate docetaxel in tbFGF (truncated basic fibroblast growth factor)-functionalized liposomes with mPEG2000-VE (d-α-tocopheryl polyethylene glycol succinate, TPGS2K) and measure their antitumor effects in vitro and in vivo. Methods: TPGS2K and COOH-PEG2000-VE were synthesized, and tbFGF was conjugated to COOH-PEG2000-VE to prepare tbFGF-PEG2000-VE. Then, tbFGF-functionalized liposomes (DTX-tbFGF-LPs) were prepared by inserting tbFGF-PEG2000-VE into docetaxel liposomes comprising TPGS2K (DTX-PEG-LPs). The stabilities and drug release profiles of the formulation were evaluated. P-glycoprotein (P-gp) inhibition was measured by ATPase assay. MTT and cell uptake were measured with B16 cells. A B16 C57BL/6 mouse model was used to evaluate in vivo antitumor efficacy. Results: Both DTX-PEG-LPs and DTX-tbFGF-LPs exhibited good stability and sustained drug release. MTT, flow cytometry, and fluorescence microscopy of B16 cells revealed higher antitumor activity and more efficient cell uptake for DTX-tbFGF-LPs compared with DTX-PEG-LPs and DTX-LPs. The P-gp ATPase assay showed that both PEG-LP and tbFGF-PEG-LP formulations inhibited P-gp pump activity in vitro. DTX-tbFGF-LPs had the highest antitumor efficacy and lowest toxicity in vivo. Conclusion: Truncated basic fibroblast growth factor-functionalized liposomes with TPGS2K as drug delivery nanocarriers were effective chemotherapy agents targeting FGFR-overexpressing tumors.

Isolating Compounds that Inhibit EV 71 Virus

Enterovirus 71, or EV 71, is responsible for causing Hand, Foot, and Mouth disease in humans. In particular, it is especially deadly when children and small infants are exposed. The objective of this research paper is to address the possibility of a novel antiviral drug that can be used once infection of EV 71 has occurred. The methods for this research include transformation of E. coli with the genetic information from Enterovirus 71, growth of the E. coli colonies in the lab setting, 2C protein purification, and ATPase assays with drug testing. Of the 364 drugs tested in the ATPase assay, a combination of two of them (Mitrofudil and N6- Benzyladenosine) indicated a stoppage in activity of ATPase, signaling no further activity of the enzyme and viral proliferation.

Gastrointestial absorption of pimozide is enhanced by inhibition of P-glycoprotein

Following the death due to cardiac arrest of a patient taking pimozide, sertraline and aripiprazole antipsychotic/antidepressant combination therapy, a role of drug-drug interaction was suggested. Here, we investigated P-glycoprotein (P-gp)-mediated interaction among the three drugs using in vitro methods. Sertraline or aripiprazole significantly increased the permeability of pimozide in Caco-2 cell monolayers. ATPase assay indicated that pimozide is a P-gp substrate, and might act as a P-gp inhibitor at higher concentrations. The values of the kinetic parameters of carrier-mediated efflux, calculated from the concentration dependence of pimozide efflux from LLC-GA5-COL150 cells expressing human P-gp, were as follows: maximum transport rate (Jmax) = 84.9 ± 8.9 pmol/min/mg protein, half-saturation concentration (Kt) = 10.6 ± 4.7 μM, first-order rate constant (kd) = 0.67 ± 0.14 pmol/min/mg protein. Further, the efflux ratio of pimozide in LLC-GA5-COL150 cells was significantly decreased in the presence of sertraline or aripiprazole. These results indicate that pimozide is a substrate of P-gp, and its efflux is inhibited by sertraline and aripiprazole. Thus, P-gp inhibition by sertraline and/or aripiprazole may alter the gastrointestinal permeability of co-administered pimozide, resulting in an increased blood concentration of pimozide, which may increase the likelihood of pimozide’s known life-threatening side effect of QT prolongation.

Similar Safety Profile of the Enantiomeric N-Aminoalkyl Derivatives of Trans-2-Aminocyclohexan-1-ol Demonstrating Anticonvulsant Activity

Epilepsy is one of the most common neurological disorder in the world. Many antiepileptic drugs cause multiple adverse effects. Moreover, multidrug resistance is a serious problem in epilepsy treatment. In the present study we evaluated the safety profile of three (1–3) new chiral N-aminoalkyl derivatives of trans-2-aminocyclohexan-1-ol demonstrating anticonvulsant activity. Our aim was also to determine differences between the enantiomeric compounds with respect to their safety profile. The results of the study indicated that compounds 1–3 are non-cytotoxic for astrocytes, although they exhibit cytotoxic activity against human glioblastoma cells. Moreover, 1–3 did not affect the viability of HepG2 cells and did not produce adducts with glutathione. Compounds 1–3 demonstrated no mutagenic activity either in the Salmonella typhimurium or in Vibrio harveyi tests. Additionally, the compounds displayed a strong or moderate antimutagenic effect. Finally, the P-glycoprotein (P-gp) ATPase assay demonstrated that both enantiomers are potent P-gp inhibitors. To sum up, our results indicate that the newly synthesized derivatives may be considered promising candidates for further research on anticonvulsant drug discovery and development. Our study indicated the similar safety profile of the enantiomeric N-aminoalkyl derivatives of trans-2-aminocyclohexan-1-ol, although in the previous studies both enantiomers differ in their biotransformation pathways and pharmacological activity.

Development and Optimization of Expression, Purification, and ATPase Assay of KaiC for Medium-Throughput Screening of Circadian Clock Mutants in Cyanobacteria

The slow but temperature-insensitive adenosine triphosphate (ATP) hydrolysis reaction in KaiC is considered as one of the factors determining the temperature-compensated period length of the cyanobacterial circadian clock system. Structural units responsible for this low but temperature-compensated ATPase have remained unclear. Although whole-KaiC scanning mutagenesis can be a promising experimental strategy, producing KaiC mutants and assaying those ATPase activities consume considerable time and effort. To overcome these bottlenecks for in vitro screening, we optimized protocols for expressing and purifying the KaiC mutants and then designed a high-performance liquid chromatography system equipped with a multi-channel high-precision temperature controller to assay the ATPase activity of multiple KaiC mutants simultaneously at different temperatures. Through the present protocol, the time required for one KaiC mutant is reduced by approximately 80% (six-fold throughput) relative to the conventional protocol with reasonable reproducibility. For validation purposes, we picked up three representatives from 86 alanine-scanning KaiC mutants preliminarily investigated thus far and characterized those clock functions in detail.