Cryo-EM structure of P-glycoprotein bound to triple elacridar inhibitor molecules.

P-glycoprotein (P-gp) is an ATP-binding cassette transporter known for its roles in expelling xenobiotic compounds from cells and contributing to cellular drug resistance through multidrug efflux. This mechanism is particularly problematic in cancer cells, where it diminishes the therapeutic efficacy of anticancer drugs. P-gp inhibitors, such as elacridar, have been developed to circumvent the decrease in drug efficacy due to P-gp efflux. An earlier study reported the cryo-EM structure of human P-gp-Fab (MRK-16) complex bound by two elacridar molecules, at a resolution of 3.6 Å. In this study, we have obtained a higher resolution (2.5 Å) structure of the P-gp- Fab (UIC2) complex bound by three elacridar molecules. This finding, which exposes a larger space for compound-binding sites than previously acknowledged, has significant implications for the development of more selective inhibitors and enhances our understanding of the compound recognition mechanism of P-gp.

Enhancement of transcription efficiency by TAR-Tat system increases the functional expression of human olfactory receptors.

Humans have approximately 400 different olfactory receptors (hORs) and recognize odorants through the repertoire of hOR responses. Although the cell surface expression of hORs is critical to evaluate their response, hORs are poorly expressed on the surface of heterologous cells. To address this problem, previous studies have focused on hOR transportation to the membrane. Nevertheless, the response pattern of hORs to odorants has yet to be successfully linked, and the response sensitivity still remains to be improved. In this study, we demonstrate that increasing the transcriptional level can result in a significant increase in cell surface and functional expression of hORs. We used the TAR-Tat system, which increases the transcription efficiency through positive feedback, and found that OR1A1, OR6N2, and OR51M1 exhibited robust expression. Moreover, this system induces enhanced hOR responses to odorants, thus defining four hORs as novel n-hexanal receptors and n-hexanal is an inverse agonist to one of them. Our results suggested that using the TAR-Tat system and increasing the transcriptional level of hORs can help understanding the relationship between hORs and odorants that were previously undetectable. This finding could facilitate the understanding of the sense of smell by decoding the repertoire of hOR responses.

Non-invasive hERG channel screening based on electrical impedance tomography and extracellular voltage activation (EIT-EVA).

hERG channel screening has been achieved based on electrical impedance tomography and extracellular voltage activation (EIT-EVA) to improve the non-invasive aspect of drug discovery. EIT-EVA screens hERG channels by considering the change in extracellular ion concentration which modifies the extracellular resistance in cell suspension. The rate of ion passing in cell suspension is calculated from the extracellular resistance Rex, which is obtained from the EIT measurement at a frequency of 500 kHz. In the experiment, non-invasive screening is applied by a novel integrated EIT-EVA printed circuit board (PCB) sensor to human embryonic kidney (HEK) 293 cells transfected with the human ether-a-go-go-related gene (hERG) ion channel, while the E-4031 antiarrhythmic drug is used for hERG channel inhibition. The extracellular resistance Rex of the HEK 293 cells suspension is measured by EIT as the hERG channels are activated by EVA over time. The Rex is reconstructed into extracellular conductivity distribution change Δσ to reflect the extracellular K+ ion concentration change Δc resulting from the activated hERG channel. Δc is increased rapidly during the hERG channel non-inhibition state while Δc is increased slower with increasing drug concentration cd. In order to evaluate the EIT-EVA system, the inhibitory ratio index (IR) was calculated based on the rate of Δc over time. Half-maximal inhibitory concentration (IC50) of 2.7 nM is obtained from the cd and IR dose-response relationship. The IR from EIT-EVA is compared with the results from the patch-clamp method, which gives R2 of 0.85. In conclusion, EIT-EVA is successfully applied to non-invasive hERG channel screening.