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Identification of undecylenic acid as EAG channel inhibitor using surface plasmon resonance-based screen of KCNH channels.
Wang ZJ
,
Tiwari PB
,
Üren A
,
Brelidze TI
.
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BACKGROUND: KCNH family of potassium channels is responsible for diverse physiological functions ranging from the regulation of neuronal excitability and cardiac contraction to the regulation of cancer progression. KCNH channels contain a Per-Arn-Sim (PAS) domain in their N-terminal and cyclic nucleotide-binding homology (CNBH) domain in their C-terminal regions. These intracellular domains shape the function of KCNH channels and are important targets for drug development.
METHODS: Here we describe a surface plasmon resonance (SPR)-based screening method aimed in identifying small molecule binders of PAS and CNBH domains for three KCNH channel subfamilies: ether-à-go-go (EAG), EAG-related gene (ERG), and EAG-like K+ (ELK). The method involves purification of the PAS and CNBH domains, immobilization of the purified domains on the SPR senor chip and screening small molecules in a chemical library for binding to the immobilized domains using changes in the SPR response as a reporter of the binding. The advantages of this method include low quantity of purified PAS and CNBH domains necessary for the implementation of the screen, direct assessment of the small molecule binding to the PAS and CNBH domains and easiness of assessing KCNH subfamily specificity of the small molecule binders.
RESULTS: Using the SPR-based method we screened the Spectrum Collection Library of 2560 compounds against the PAS and CNBH domains of the three KCNH channel subfamilies and identified a pool of small molecules that bind to the PAS or CNBH domains. To further evaluate the effectiveness of the screen we tested the functional effect of one of the identified mEAG PAS domain specific small molecule binders on currents recorded from EAG channels. Undecylenic acid inhibited currents recorded from EAG channels in a concentration-dependent manner with IC50 of ~ 1 μM.
CONCLUSION: Our results show that the SPR-based method is well suited for identifying small molecule binders of KCNH channels and can facilitate drug discovery for other ion channels as well.
R01 GM124020 National Institute of General Medical Sciences, S10 OD019982 Office of the Director, R01 GM124020 NIGMS NIH HHS , S10 OD019982 ODCDC CDC HHS
Fig. 1. Description of the SPR-based screening method. a Isolated PAS and C-linker/CNB(CNBH) domains of HCN and KCNH channels were purified with affinity and size-exclusion chromatography. b The purified domains were immobilized on the NTA sensor chip. The NTA chip contained 20 spots for protein immobilization, five spots (S1-S5) per each of the four flow cells (FCs). For each FC, S3 was used as a control spot for non-specific binding, S1 and S2 were used to immobilize the same protein at high density (HD) and low density (LD), respectively, and similarly, S4 and S5 were used to immobilize the same protein at LD density for S4 and HD for S5. Only spots with HD of the immobilized protein are depicted in the figure. For FC1 only spots S4 and S5 had immobilized protein. c The Spectrum Library compounds at 50 μM concentration distributed into 384-well plates were injected to the seven immobilized proteins simultaneously. In addition to the library compounds cAMP was also injected at the beginning and end of each plate screening. Binding of cAMP to the C-linker/CNB of hHCN4 channels was used as a positive control for the screen. The pdb IDs of the PAS and C-linker/CNB(CNBH) domain structures are: 3OTF (HCN4 CNB), 4HOI (EAG PAS), 4F8A (EAG CNBH), 1BYW (ERG PAS), 2N7G (ERG CNBH), 4HP4 (ELK PAS) and 3UKN (ELK CNBH)
Fig. 2. SPR screen results for a 96-well Spectrum Collection chemical library plate. Plots of the SPR responses in % of the maximal expected response (% Rmax) versus the Spectrum Collection library compound number in the 96-well plate for the indicated PAS and C-linker/CNB(CNBH) domains of KCNH and HCN channels. Library compounds with the % Rmax < 30 were excluded from further analysis. The compounds were applied at 50 μM concentration
Fig. 3. Undecylenic acid is a novel small molecule binder of the PAS domain of mEAG channels. a Chemical structure of undecylenic acid identified as the mEAG PAS domain specific binder with the SPR-based screening method. SPR sensorgrams recorded for the immobilized PAS domains of mEAG (b), hERG (c) and hELK (d), and C-linker/CNB(CNBH) domains of hHCN4 (e), mEAG (f), hERG (g) and hELK (h) channels recorded with the indicated concentration of undecylenic acid
Fig. 4. Determination of the binding affinity of undecylenic acid for the mEAG PAS domain. Plots of the SPR response at 55 s after the start of the injection versus total undecylenic acid concentration for sensorgrams shown in Fig. 3b. The lines represents fit of the data with Hill equation. The binding affinity for undecylenic acid was ~ 11 μM
Fig. 5. Undecylenic acid inhibits currents from mEAG channels. a Representative mEAG current traces recorded in the inside-out configuration in the absence (black) and presence (red) of 50 μM undecylenic acid. b The tail current recorded at − 100 mV after a voltage step to + 70 mV in the absence (black) and presence (red) of 50 μM undecylenic acid. The grey lines represent fits of the tail currents with a single exponential function with the time constants of deactivation of 3.1 ms in the absence and 2.1 ms in the presence of undecylenic acid. c Plots of the percent inhibition of tail currents versus the undecylenic acid concentration. Tail currents were recorded at − 100 mV after a voltage step to 70 mV. The line represents fit of the data with Hill equation with the IC50 of 1.06 ± 0.03 μM. d Plot of the averaged percent inhibition of tail currents versus voltage obtained in the presence of 50 μM undecylenic acid (n = 4). e Conductance-voltage relationship in the absence (black) and presence (red) of 50 μM undecylenic acid. The black and red lines represent fits of the conductance-voltage relationships in the absence (black) and presence (red) of 50 μM undecylenic acid with a Boltzmann function with V1/2 of − 30.9 ± 1.5 mV and − 30.3 ± 1.6 mV, respectively
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