Structure-Based Secondary Structure-Independent Approach To Design Protein Ligands:  Application to the Design of Kv1.2 Potassium Channel Blockers

Abstract

We have developed a structure-based approach to the design of protein ligands. This approach is based on the transfer of a functional binding motif of amino acids, often referred as to the "hot spot", on a host protein able to reproduce the functional topology of these residues. The scaffolds were identified by a systematic in silico search in the Protein Data Bank for proteins possessing a group of residues in a topology similar to that adopted by the functional motif in a reference ligand of known 3D structure. In contrast to previously reported studies, this search is independent of the particular secondary structure supporting the functional motif. To take into account the global properties of the host protein, two additional criteria were taken into account in the selection process: (1) Only those scaffolds sterically compatible with the positioning of the functional motif as observed in a reference complex model were retained. (2) Host proteins displaying electrostatic potentials, in the region of the transferred functional motif, similar to that of the reference ligand were selected. This approach was applied to the development of protein ligands of the Kv1.2 channel using BgK, a small protein isolated from the sea anemone Bunodosoma granulifera, as the reference ligand. Four proteins obtained by this approach were produced for experimental evaluation. The X-ray structure of one of these proteins was determined to check for similarity of the transferred functional motif with the structure it adopts in the reference ligand. Three of these protein ligands bind the Kv1.2 channel with inhibition constants of 0.5, 1.5, and 1.6 microM. Several mutants of these designed protein ligands gave binding results consistent with the presumed binding mode. These results show that protein ligands can be designed by transferring a binding motif on a protein host selected to reproduce the functional topology of this motif, irrespective to the secondary structure supporting the functional motif, if the host protein possesses steric and electrostatic properties compatible with the binding to the target. This result opens the way to the design of protein ligands by taking advantage of the considerable structural repertoire of the Protein Data Bank.

RASMOT-3D PRO: a 3D motif search webserver

“…These works clearly demonstrated the value of the approach to design protein ligands. However, one conclusion of our previous work (7) was that the success of the method depends on the number of identified scaffolds. Indeed, after topological in silico scaffold selection, several steps must be overcome.…”

“…RASMOT-3D PRO was initially elaborated to identify platforms to transfer a functional motif by systematic examination of the structures deposited in the PDB. In a previous work (7) we used this approach to design a Kv1.2 potassium channel blocker and we obtained several micromolar blockers for this channel. With a similar method, using Cα and Cβ inter-atomic distances, RMSD and steric filtering, Liu and coworkers designed the pleckstrin homology domain PLCδ 1 -PH to bind the human erythropoietin receptor by grafting the key interacting residues of the human erythropoietin (8).…”

Calix[4]arene-based conical-shaped ligands for voltage-dependent potassium channels

“…Amber* force field was chosen to model the systems. Each complex was relaxed, following a protocol already reported by Magis et al (24) in the design of Kv1.2 potassium channel blockers. The protocol consisted of an energy minimization, followed by molecular dynamics simulation applying positional restraints.…”

Triazolyl Azabicyclo[3.1.0]hexanes: a Class of Potent and Selective Dopamine D3 Receptor Antagonists

“…An open-state model was also built by exploiting the rat Kv1.2 structure as a template. [27] The same S5 glutamate, pore GYG, and S6 glycine residues were used to generate the alignment. Use was made of the S5-SS1 loop model constructed for the closed-state structure, and the same CHARMm conditions were used for final refinement.…”