large molecules and hERG assay

4 posts / 0 new
Last post
lazy's picture
large molecules and hERG assay

I am wondering if a large molecule can inhibit hERG channel or not.
My understanidng is that the hERG inhibition is caused by sticking the molecule to the cavity inside the channel structure. If so, the large molecule that cannot enter into the cavity will not inhibit the hERG channel. Also the large molecule that cannot cross the plasma membrane will not give influence to trafficking.
ICHS7B guideline madates hERG assay prior to human trial to all new chemical entities. Should we plan an assay even for the large molecule such like biologics? 

Bluejay's picture
Your logic appears to be

Your logic appears to be correct.   However, there are porbably scenarios where the drug could bind to the herg channel or get into the cell and cause problems.   A common progression would be to test in a binding assay, then a medium throughput electrophysiology experiment, and then before human tests, a complete patch clamp with 37C would be appropriate.  (However, this is general guidance:  see FDA publications for exact guidance.)  This is also the order of increasing complexity and cost.   It would be good to follow Dr. Craig January and his group which have published many key articles in the hERG area.

Fraser Moss
Fraser Moss's picture
here is the mechanism of hERG

here is the mechanism of hERG block by a peptide toxin
Biophys J. 2007 Jun 1;92(11):3915-29.
Mechanism of block of the hERG K+ channel by the scorpion toxin CnErg1.
Hill AP, Sunde M, Campbell TJ, Vandenberg JI.
Mark Cowley Lidwill Research Program in Electrophysiology and Biophysics, Victor Chang Cardiac Research Institute, New South Wales, Australia.
The scorpion toxin CnErg1 binds to human ether-a-go-go related gene (hERG) K(+) channels with a 1:1 stoichiometry and high affinity. However, in contrast to other scorpion toxin-ion channel interactions, the inhibition of macroscopic hERG currents by high concentrations of CnErg1 is incomplete. In this study, we have probed the molecular basis for this incomplete inhibition. High concentrations of CnErg1 had only modest effects on hERG gating that could not account for the incomplete block. Furthermore, the residual current in the presence of 1 microM CnErg1 had normal single channel conductance. Analysis of the kinetics of CnErg1 interaction with hERG indicated that CnErg1 binding is not diffusion-limited. A bimolecular binding scheme that incorporates an initial encounter complex and permits normal ion conduction was able to completely reproduce both the kinetics and steady-state level of CnErg1-hERG binding. This scheme provides a simple kinetic explanation for incomplete block; that is, relatively fast backward compared to forward rate constants for the interconversion of the toxin-channel encounter complex and the blocked toxin-channel complex. We have also examined the temperature-dependence of CnErg1 binding to hERG. The dissociation constant, K(d), for CnErg1 increases from 7.3 nM at 22 degrees C to 64 nM at 37 degrees C (i.e., the affinity decreases as temperature increases) and the proportion of binding events that lead to channel blockade decreases from 70% to 40% over the same temperature range. These temperature-dependent effects on CnErg1 binding correlate with a temperature-dependent decrease in the stability of the putative CnErg1 binding site, the amphipathic alpha-helix in the outer pore domain of hERG, assayed using circular dichroism spectropolarimetry. Collectively, our data provides a plausible kinetic explanation for incomplete blockade of hERG by CnErg1 that is consistent with the proposed highly dynamic conformation of the outer pore domain of hERG.
also here is  the mapping of a toxin binding site

J Biol Chem. 2002 May 10;277(19):16403-11. Epub 2002 Feb 25.
Mapping the binding site of a human ether-a-go-go-related gene-specific peptide toxin (ErgTx) to the channel's outer vestibule.
Pardo-Lopez L, Zhang M, Liu J, Jiang M, Possani LD, Tseng GN.
Department of Physiology, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
The goals of this study are to investigate the mechanism and site of action whereby a human ether-a-go-go-related gene (HERG)-specific scorpion peptide toxin, ErgTx, suppresses HERG current. We apply cysteine-scanning mutagenesis to the S5-P and P-S6 linkers of HERG and examine the resulting changes in ErgTx potency. Data are compared with the characteristics of charybdotoxin (ChTx, or its analogs) binding to the Shaker channel. ErgTx binds to the outer vestibule of HERG but may not physically occlude the pore. In contrast to ChTx.Shaker interaction, elevating [K](o) (from 2 to 98 mm) does not affect ErgTx potency, and through-solution electrostatic forces only play a minor role in influencing ErgTx.HERG interaction. Cysteine mutations of three positions in S5-P linker (Trp-585, Gly-590, and Ile-593) and 1 position in P-S6 linker (Pro-632) induce profound changes in ErgTx binding (DeltaDeltaG > 2 kcal/mol). We propose that the long S5-P linker of the HERG channel forms an amphipathic alpha-helix that, together with the P-S6 linker, forms a hydrophobic ErgTx binding site. This study paves the way for future mutant cycle analysis of interacting residues in the ErgTx.HERG complex, which, in conjunction with NMR determination of the ErgTx solution structure, will yield information about the topology of HERG's outer vestibule.

griffrat's picture
I was looking on pub med

I was looking on pub med today for new insights on the BeMK-1 (a purified short protien~4kDa from an Asian scorpion venom) that also has hERG effects and came across these journal articles as well.  It is similar to the CnErg-1 Fraser cites in his reply as well.  Both BeMK and CnErg are the most studied in terms of peptides for the hERG assay. 

APETx1 from sea anemone Anthopleura elegantissima is a gating modifier peptide toxin of the human ether-a-go-go- related potassium channel.

Zhang M, Liu XS, Diochot S, Lazdunski M, Tseng GN.

Mol Pharmacol. 2007 Aug;72(2):259-68. Epub 2007 May 1.

PMID: 17473056 [PubMed - indexed for MEDLINE]

Related articles Free article

A novel toxin from the venom of the scorpion Tityus trivittatus, is the first member of a new alpha-KTX subfamily.

Abdel-Mottaleb Y, Coronas FV, de Roodt AR, Possani LD, Tytgat J.

FEBS Lett. 2006 Jan 23;580(2):592-6. Epub 2006 Jan 4.

PMID: 16405970 [PubMed - indexed for MEDLINE]

Related articles

  As to the ICH7B I would plan on not only doing hERG but also a telemetriezed animal model prior to clinical trials as well.  The hERG is a reliable test/assay but in my experiences with large molecules there could be a binding event to a pore adjacent to the K+ channel.  So, while there could be a reduction in the tail current of the hERG from the large molecule whereas this interference can usually be elucidated with the whole animal assay.

Hope that helps out as well...