Research Article: The molecular determinants of R-roscovitine block of hERG channels

Date Published: September 3, 2019

Publisher: Public Library of Science

Author(s): Bryan Cernuda, Christopher Thomas Fernandes, Salma Mohamed Allam, Matthew Orzillo, Gabrielle Suppa, Zuleen Chia Chang, Demosthenes Athanasopoulos, Zafir Buraei, Tomohiko Ai.


Human ether-à-go-go-related gene (Kv11.1, or hERG) is a potassium channel that conducts the delayed rectifier potassium current (IKr) during the repolarization phase of cardiac action potentials. hERG channels have a larger pore than other K+channels and can trap many unintended drugs, often resulting in acquired LQTS (aLQTS). R-roscovitine is a cyclin-dependent kinase (CDK) inhibitor that induces apoptosis in colorectal, breast, prostate, multiple myeloma, other cancer cell lines, and tumor xenografts, in micromolar concentrations. It is well tolerated in phase II clinical trials. R-roscovitine inhibits open hERG channels but does not become trapped in the pore. Two-electrode voltage clamp recordings from Xenopus oocytes expressing wild-type (WT) or hERG pore mutant channels (T623A, S624A, Y652A, F656A) demonstrated that compared to WT hERG, T623A, Y652A, and F656A inhibition by 200 μM R-roscovitine was ~ 48%, 29%, and 73% weaker, respectively. In contrast, S624A hERG was inhibited more potently than WT hERG, with a ~ 34% stronger inhibition. These findings were further supported by the IC50 values, which were increased for T623A, Y652A and F656A (by ~5.5, 2.75, and 42 fold respectively) and reduced 1.3 fold for the S624A mutant. Our data suggest that while T623, Y652, and F656 are critical for R-roscovitine-mediated inhibition, S624 may not be. Docking studies further support our findings. Thus, R-roscovitine’s relatively unique features, coupled with its tolerance in clinical trials, could guide future drug screens.

Partial Text

Human ether-à-go-go-related gene, or hERG [Kv11.1], is a voltage-gated potassium channel critical for nerve and cardiac function [1,2]. In the heart, hERG channels initially open during the depolarization phase of the cardiac action potential (cAP) but immediately inactivate. Upon cAP repolarization, hERG channels quickly recover from inactivation and reopen, which allows the ensuing large K+ efflux to speed cAP repolarization [1], limit cardiac excitability, and maintain normal QT intervals [3]. The cAP repolarization ultimately leads to hERG channel closing (deactivation). Mutations in hERG are one of the leading causes of congenital long QT syndrome (cLQTS), with a neonatal incidence rate of up to 1 in 2,500 [4]; the abnormal cardiac phenotypes are usually triggered during exercise, arousal, or rest [5].

In this study we investigated the mechanisms of R-roscovitine-mediated inhibition of hERG channels in Xenopus oocytes and made several contributions: 1) we determined that the IC50 for R-roscovitine is ~200 μM (in low K+), 2) mutation of the F656 residue nearly abolishes inhibition, and the T623 residue seems critical for inhibition, 3) the Y652A mutation moderately reduces inhibition, and 4) S624 is not required for inhibition, and instead, S624A unexpectedly enhances inhibition. Docking analyses further supports our experimental data. These results, collectively, suggest a comparatively unique binding orientation for R-roscovitine within hERG’s pore.