HESI/ILSI Health and Environmental Sciences Institute Workshop session I: Molecular and Cellular Biology Underlying TdP Co-Chairs: Craig January and Dan Roden Rapportuers: Kristy Bruse and Ying Ying Zhou Workshop participants: Blake Anson, Siham Biade, Eve Bijaoui, Albert Defelice, Michael Nabauer, Guy Salama, Peter Siegle, Steve Sorota, Antoniao Zaza, Ravikumar Peri, Karin Sipido May 25, 2019

What is the relationship between hERG ≈ QT prolongation ≈ TdP? Goal 1: Define the relationship between IKr block and risk for drug-induced QT prolongation and TdP What is the relationship between hERG ≈ QT prolongation ≈ TdP? Genetic influences Cellular changes Single channel event APD event Other mechanisms Clinical event Are there other ways to predict risk for TdP? May 25, 2019

other currents & mechanisms Goal 1. The relationship between drug-induced IKr block and risk for drug-induced QT prolongation/TdP IKr blockade other currents & mechanisms Increased APD/EADS cell Ca2+, ? Increased QT interval electrical heterogeneity, ? TdP event May 25, 2019

Goal 1. Problem: Quantifying drug-induced IKr block Potency Relative potency compared to target pharmacophore Need to compare to positive control (compare IC50 or percent channel blockade) Kinetics/voltage dependence Testing system Protocol-dependent, cell type, solutions, temperature, intrinsic drug properties, adsorption to tubing/set-up, etc Subunit interactions Transfected cell line properties Protein binding May 25, 2019

Goal 1. Future needs: Quantifying drug-induced IKr block To standardize potency/effect Standardize test systems where possible Standardize the verbiage May 25, 2019

Goal 1. IKr blockade affects on action potential IKr blockade  other mechanisms?  action potential effects Discordance: What is an EAD? There is not agreement as to the electrophysiological “shape” of an EAD. Agreement: irrespective of the shape definition, EADs can enhance dispersion of repolarization and/or cause triggered activity TdP May 25, 2019

Goal 1. IKr blockade affects on action potential Unknown: what are these “other mechanisms”? Altered channel trafficking as an alternate mechanism to reduce IKr, then how often? how important? Other inward/outward current data may be useful for predictivity Could in silico assist in assimilation of other ion current data results AP interpretation from In vitro data- need a positive control reference and understanding of the strengths/weaknesses of the assay(s) Future: should we evaluate other ion channels to better predict risk for TdP? Do not over-interpret the IKr data Note, other targets may be “protective” against the IKr blockade May 25, 2019

Goal 1. Delayed repolarization (APD/EADs) influences on QT prolongation/TdP Unknowns: How do APD/EADs perturb QT duration or morphology alterations (e.g. TdP) ? Rate dynamicity- is this important? Does it matter how you change the rate? Is there an optimal heart rate correction factor for QT interval (e.g. QTc)? Is the beat to beat variance of QT a more relevant evaluation? How are U-waves interpreted? What is QT prolongation? May 25, 2019

Goal 1. Relationship between QT prolongation and TdP Unknowns: In face of QT prolongation, what predicts TdP? What terminates TdP? What percentage of patients with QT prolongation are at risk of TdP? What percentage of patients with QT prolongation are resistant to TdP? Concerns: How is TdP defined clinically? Is there a distinct difference from polymorphic ventricular tachycardia and TdP (associated with long QT)? How is TdP identified in the clinic? How many cases are not identified and end up as syncope, sudden death or possibly resolve? May 25, 2019

Goal 2. Evolving tools to move to better predictors of drug-induced TdP In silico modeling in drug safety/mechanisms In vitro cell biology of IKr Stem cell research and more Genetic screening/other biomarkers May 25, 2019

Goal 2. in silico modeling in drug safety/mechanisms In silico- hypothesis generators Pharmacophore (QSAR) modeling-rank ordering tool for early development Purpose- to refine chemical structure design for future in vitro testing Modeling for AP/whole heart Advantages: test hypotheses not otherwise accessible Limitations: never “proves” anything. Limited to current knowledge. Structural modeling of hERG and other ion channels May 25, 2019

Goal 2. In vitro cell biology of IKr Future: further understand the regulation and dynamics of the IKr channel. Lipid/structural influences, subunits, interacting proteins, transcriptional/post-transcriptional regulation, and post-translational processing. May 25, 2019

Goal 2. Consequences of modifying IKr Altered intracellular calcium dynamics Activation of CaM Kinase II + ? Adrenergic changes/autonomic tone Transcription, translation, etc. Intracellular magnesium and potassium concentrations May 25, 2019

Goal 2. Cutting Edge Science! Stem cell research and more 5+ year deliverable Direct high throughput screening for drug effects on action potential, Ca2+i, arrythmogenicity of mammalian and human myocytes This may also include transgenic non-rodent animal models May 25, 2019

Goal 2. Need for Genetic screening/biomarkers Discovering and characterizing of sequence variants in patient populations. Role of known variants: For drug screening? (Not yet) For screening patients for TdP susceptibility Global effort- academia, industry, regulatory agencies: Concerted effort to ascertain information (e.g. DNA, serum, ECG samples) from a large number patients with drug-induced TdP. Platform for discovery. May 25, 2019