A two-state homology model of the hERG K+ channel: application to ligand binding

Ramkumar Rajamani, Brett Tongue, Jian Li, Charles H. Reynolds

J & J PRD

Biorg. Med. Chem. Lett. 2005, 15, 1737

Kiran-20060214

• There is a wide range of anticancer, antipsychotic, antihypertensive and antihistamine compounds which interact with the hERG channel.

• hERG plays a critical role in repolarization of the cardiac action potential and interference leads to serious side effects.

• The hERG K+ channel is a tetramer. Each monomer has 6 transmembrane domains. Voltage dependent opening and closing is determined by the S4 domain. The S6 domain has a cavity where drugs bind.

Biology

Defining the problem

• No crystal structure available. The closest homologous protein is the bacterial KcsA protein which has been crystallized.

• We are dealing with not one, but four protein molecules which co-assemble to form a functional channel.

• The channel has open and closed states, the issue of flexibility has to be addressed.

• Once a ligand binds, the S6 helix can close to varying degrees.

- Acronyms:

Two crystal structures were used for homology modeling: KcsA from Streptomyces

lividans and MthK from Methanobacterium thermoautrophicum

Goal and Methods

• Goal: Identify specific protein-ligand interactions responsible for affinity to hERG and predict ligand modifications to mitigate binding

• Method:

- Align sequences in the pore forming region, then predict coordinates for non-identical residues using PRIME. Add hydrogens and charges using Maestro, do a constrained minimization of the protein.

- Dock the ligand set using GLIDE. The best pose for each ligand is minimized within the protein using a conjugate gradient minimizer (0.05), the OPLS-AA force field and the GB/SA continuum water model, residues within 8 Å are also allowed to optimize.

- The ligand from the minimized ligand-receptor complex is extracted and re-minimized in GB/SA water to get reference energetics for the free state. The LIE (Linear Interaction Energy) method was used to predict binding affinities.

- It was determined that multiple homology models were needed to mimic the partially closed and open states of the channel. The KcsA and the MthK structures were used.

Results

• Initial homology modeling with the (closed) KcsA structure revealed that the S6 domain active site cavity was too small to accommodate known ligands of hERG.

• The hERG K+ structure contained a Phe656 residue in an equivalent position to the Thr residue in KcsA.

• The difference in the size of the two residues argued for a channel structure that differed in the S6 helix domain from that of the closed KcsA structure.

• This led to an examination of another known crystal structure: the MthK structure (in open state) was known.

KcsA K+ channel: two views PDB 1J95

MthK channel, open state: side & end views PDB 1LNQ

- Glycine in the hinge is conserved across species, the direction and angle of the bend

was used to derive intermediate states of helix motion

Closed

channel

Open

channel

Copyright © 1999-2005 Joyce J. Diwan. at RPI

• The S6 helix from the reference closed state was rotated to match the reference open state.

• The channel was closed in 1° increments and subjected to a protocol of heating (0.4 ps), equilibration (0.6 ps), dynamics (5 ps). A harmonic constraint of 24 kcal/mol/Å2 was applied on the C atoms. This protocol removed any unfavorable interactions arising from helix translation.

Derivation of two states for docking studies

Results

• In the dual state model, ligands were separated into bins based on their preference for a particular state (open 19° tilt, partially open 10° tilt)

• The preference was established by comparing (∆ele + ∆vdw) for each ligand in the two states and picking the lower value.

• A set of 32 ligands (for which experimental IC50s were known) was examined.

All 32, R2 = 0.2421, preference

for open state,

RMSD = 1.2

11, preference for

partially open ,

RMSD = 0.85

• Equations:

• Combine energies for each ligand from its best fit state, 5 outliers found

• Primary contribution is from the vdw term

Results

27 compounds,

R2 = 0.82

RMSD = 0.56

pIC50open = -0.166(∆vdw) + 0.002(∆ele) (1)

pIC50partiallyopen = -0.155(∆vdw) + 0.004(∆ele) (2)

pIC50combined = -0.163(∆vdw) + 0.0009(∆ele) (3)

• The combined two-state model of hERG binding affinity could reproduce pIC50 values and correct docked poses.

• This study improved upon earlier work with single state models.

• The hydrophobicity of Phe656 and Tyr652 was the best predictor of affinity.

Conclusions