Investigation of connexin 43 uncoupling and prolongation of the cardiac QRS complex in preclinical and marketed drugs

Abstract

BACKGROUND AND PURPOSEProlongation of the cardiac QRS complex is linked to increased mortality and may result from drug-induced inhibition of cardiac sodium channels (hNaV1.5). There has been no systematic evaluation of preclinical and marketed drugs for their additional potential to cause QRS prolongation via gap junction uncoupling. EXPERIMENTAL APPROACHUsing the human cardiac gap junction connexin 43 (hCx43), a dye transfer 'parachute' assay to determine IC50 values for compound ranking was validated with compounds known to uncouple gap junctions. Uncoupling activity (and hNaV1.5 inhibition by automated patch clamp) was determined in a set of marketed drugs and preclinical candidate drugs, each with information regarding propensity to prolong QRS. KEY RESULTSThe potency of known gap junction uncouplers to uncouple hCx43 was ranked (according to IC50) as phorbol ester>digoxin>meclofenamic acid>carbenoxolone>heptanol. Among the drugs associated with QRS prolongation, 29% were found to uncouple hCx43 (IC50 < 50 μM), whereas no uncoupling activity was observed in drugs not associated with QRS prolongation. In preclinical candidate drugs, hCx43 and hNaV1.5 IC50 values were similar (within threefold). No consistent margin over preclinical Cmax (free) was apparent for QRS prolongation associated with Cx43 inhibition. However, instances were found of QRS prolonging compounds that uncoupled hCx43 with significantly less activity at hNaV1.5. CONCLUSION AND IMPLICATIONSThese results demonstrate that off-target uncoupling activity is apparent in drug and drug-like molecules. Although the full ramifications of Cx inhibition remain to be established, screening for hCx43 off-target activity could reduce the likelihood of developing candidate drugs with a risk of causing QRS prolongation. AbbreviationsCx, connexin; PDBu, phorbol 12,13-dibutyrate

Quantitative Automated Assays in Living Cells to Screen for Inhibitors of Hemichannel Function

“…Surprisingly, carbenoxolone displayed inconsistent effects in the two assays, potentially correlating with its previously described relative potency on Cx43. 41,42 The two assays have been described as dependent upon Cx43 HC function; 17,43 this has been further demonstrated in the present study using RNA silencing in U251, and elsewhere in Ln215 using CRISPR-Cas9 KO. 39 In addition, the Yo-Pro incorporation assay notably depends on the transfer of Yo-Pro from the cytoplasm to the nucleus after uptake, while the YFP quenching assay notably requires an active quenching of YFP by iodide after iodide uptake.…”

Up‐regulated Cx43 phosphorylation at Ser368 prolongs QRS duration in myocarditis

“…Replacement of Cx43 by Cx31 in the heart leads to significant prolongation of QRS duration . In addition, some preclinical and marketed drugs have been shown to cause QRS prolongation via Cx43 uncoupling . Therefore, as a principal conductor of intercellular current in the ventricle, Cx43 is one of the molecular determinants for the prolongation of QRS duration.…”

Baseline Framingham risk score does not predict future ECG-derived QRS duration over an average of 3.3 years

“…Additionally, we do not have strict diagnosis of whether bundle branch block was evident at higher QRS durations in our population. Cardiac myocyte gap junction uncoupling as a consequence of LV hypertrophy or fibrosis can give rise to a wide QRS complex with morphological features that are similar to ECGderived LBBB but are not LBBB [25,26].…”