High-throughput screens identify genotype-specific therapeutics for channelopathies
Christian L. Egly, Alex Shen, Tri Q. Do, Carlos Tellet Cabiya, Paxton A. Ritschel, Suah Woo, Matthew Ku, Brian P. Delisle, Brett M. Kroncke, Björn C. Knollmann
Christian L. Egly, Alex Shen, Tri Q. Do, Carlos Tellet Cabiya, Paxton A. Ritschel, Suah Woo, Matthew Ku, Brian P. Delisle, Brett M. Kroncke, Björn C. Knollmann
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Research Article Cardiology Genetics

High-throughput screens identify genotype-specific therapeutics for channelopathies

Abstract

Genetic diseases such as ion channelopathies substantially burden human health. Existing treatments are limited and not genotype specific. Here, we report a 2-step high-throughput approach to rapidly identify drug candidates for repurposing as genotype-specific therapy. We first screened 1,680 medicines using a thallium-flux trafficking assay against Kv11.1 gene variants causing long QT syndrome (LQTS), an ion channelopathy associated with fatal cardiac arrhythmia. We identified evacetrapib as a suitable drug candidate that improves membrane trafficking and activates channels. We then used deep mutational scanning to prospectively identify all Kv11.1 missense variants in an LQTS hotspot region responsive to treatment with evacetrapib. Combining high-throughput drug screens with deep mutational scanning establishes a paradigm for mutation-specific drug discovery translatable to personalized treatment of carriers with rare genetic disorders.

Authors

Christian L. Egly, Alex Shen, Tri Q. Do, Carlos Tellet Cabiya, Paxton A. Ritschel, Suah Woo, Matthew Ku, Brian P. Delisle, Brett M. Kroncke, Björn C. Knollmann

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Figure 6

Evacetrapib rescues trafficking-deficient Kv11.1 channel missense variants.

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Evacetrapib rescues trafficking-deficient Kv11.1 channel missense varian...
(A) Schematic of multiplexed assay of variant effect to evaluate trafficking-deficient Kv11.1 variants rescued by evacetrapib. Plasmids containing all possible Kv11.1 variants in a LQTS2 hotspot region (residues 536–628) were transfected into HEK293T cells. Pooled cell lines were treated with drugs for 24 hours and then sorted using FACS into 4 groups based on cell-surface Kv11.1 expression: (1) negative, (2) low, (3) medium, or (4) high. Cells were cultured, PCR purified, and analyzed using next-generation sequencing to generate trafficking scores for each variant. (B) Heatmaps detailing 1,878 Kv11.1 variants for residues 536–628, displaying trafficking-deficient variants (black) defined as trafficking score ≤10% WT or variants with >10% WT trafficking (white). Colored heatmap displays variant-specific response to overnight incubation with E-4031 (10 μmol/L) or evacetrapib (10 μmol/L). (C) Venn diagram depicting Kv11.1 variant trafficking response to pharmacological chaperones E-4031 and evacetrapib. (D) 3D structural model of a Kv11.1 channel tetramer assembly (side view). Black region highlights residues 536–628 of Kv11.1 channels that we mutagenized. Red dotted residues show trafficking increased in at least 4 variants of that residue with both E-4031 and evacetrapib. Yellow highlights residues rescued in at least 4 variants by evacetrapib only.