The molecular determinants of R-roscovitine block of hERG channels

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.

170 as multiplicity adjusted p values. Statistical significance was designated when p < 0.05 and all p-171 values were subsequently reported. 175 To determine the appropriate concentration of R-roscovitine for comparing different 176 hERG constructs, we wanted to establish the effectiveness of R-roscovitine inhibition on wild-177 type (WT) hERG channels in Xenopus oocytes. The half-maximal inhibitory concentration (IC 50 ) 178 of tail currents for R-roscovitine with WT hERG was determined previously in HEK-293 cells 179 (27 µM), but Xenopus oocytes are known to require higher drug doses [25]. Therefore, we 180 injected cRNA for WT hERG into Xenopus oocytes and recorded potassium currents 2-7 days 181 later (see methods). The currents were elicited using a 2-second pulse protocol consisting of a 1-182 second depolarization to +40 mV followed by a 1-second repolarization to -50 mV, during which 183 tail currents were measured (Fig 1B). This pulse protocol was repeated either every 5 seconds for 184 R-roscovitine concentrations applied in a randomized order (100, 300, 30, and 10 µM) or every 185 12 seconds for ascending R-roscovitine concentrations (10, 30, 100, 300, and 1000 µM), with the 186 various drug concentrations being repeatedly applied and washed off (Fig 1C  Next, we examined currents from hERG mutants to establish their baseline properties 256 before applying R-roscovitine. hERG block is commonly associated with residues located near 257 the selectivity filter (e.g. S624) and on the S6 helices (e.g. Y652) [32,33]. Therefore, single-258 mutant channels S624A and Y652A were expressed in Xenopus oocytes and subjected to a step 259 depolarization protocol (Fig 3A, top).
337 Furthermore, the Y652A IC 50 (567 ± 122 µM) was ~ 2.75-fold larger than WT (196 ± 12 µM; p 338 = 0.0005 for WT vs. Y652A, n ≥ 8, one-way ANOVA; Fig 5F). The attenuated step and tail 339 current inhibition of Y652A with 200 µM R-roscovitine, as well as the difference in IC 50 values 340 between Y652A and WT hERG, seemed to indicate that Y652 is involved in R-roscovitine 341 binding. This was disimiliar to the results for S624A hERG (Fig 4), and made us question 342 whether additional residues in the pore region were involved in R-roscovitine inhibition, and 343 whether they would be more critical.

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With R-roscovitine binding to the hERG pore, it was likely that the large influx of K + ions 371 and the change in external solution would alter the WT hERG IC 50 . To determine the dose-372 response curves for R-roscovitine from WT tail currents, a pulsing protocol was applied, 373 comprised of a 1 second depolarization to +40 mV and a 1 second repolarization to -120 mV; 374 peak tail currents were measured in various R-roscovitine concentrations (Figs 6A, B). Fitting 511 intermolecular interactions. This was evident with the T623A hERG mutant, which exhibited an 512 overall weak tail current inhibition by R-roscovitine (Fig 7A). Upon analysis the shift in E rev in 513 the presence of R-roscovitine, it was apparent that the drug was not altering permeation ( Fig 7B).
514 Furthermore, the larger IC 50 value for T623A hERG versus WT hERG suggested that the drug 515 efficacy was reduced by the mutation (Fig 7D). Collectively, our data suggest the drug was no 516 longer able to bind in its original pore location. This level of inhibition loss could represent R-517 roscovitine's requirement of hydrogen-bonding to the hydroxyl group of T623 for maintaining 518 normal potency.
519 Surprisingly, step and tail current inhibition were not impacted by mutation to the S624 520 residue (Figs 4B, 4D). In fact, inhibition levels increased in the S624A hERG mutant when 521 compared to WT inhibition at the same voltages (Figs 4C, 4E), and no general increase in IC 50 522 was apparent for S624A ( Fig 4F)