Small Molecules Detected by Second-Harmonic Generation Modulate the Conformation of Monomeric α-Synuclein and Reduce Its Aggregation in Cells

Ben Moree,Guowei Yin,Diana F Lázaro,Francesca Munari,Timo Strohäker,Karin Giller,Stefan Becker,Tiago F Outeiro,Markus Zweckstetter,Joshua Salafsky

doi:10.1074/jbc.m114.636027

Abstract

Proteins are structurally dynamic molecules that perform specialized functions through unique conformational changes accessible in physiological environments. An ability to specifically and selectively control protein function via conformational modulation is an important goal for development of novel therapeutics and studies of protein mechanism in biological networks and disease. Here we applied a second-harmonic generation-based technique for studying protein conformation in solution and in real time to the intrinsically disordered, Parkinson disease related protein α-synuclein. From a fragment library, we identified small molecule modulators that bind to monomeric α-synuclein in vitro and significantly reduce α-synuclein aggregation in a neuronal cell culture model. Our results indicate that the conformation of α-synuclein is linked to the aggregation of protein in cells. They also provide support for a therapeutic strategy of targeting specific conformations of the protein to suppress or control its aggregation.

Highlights

Proteins are structurally dynamic molecules that perform specialized functions through unique conformational changes accessible in physiological environments
To overcome the challenges associated with traditional small molecule screening of ␣-synuclein, we developed a novel second-harmonic generation (SHG)-based screen to identify compounds that directly modulate the conformation of monomeric ␣-synuclein
In the study presented here, we used an SHG-based fragment screen to identify novel conformational modulators of ␣-synuclein. We subsequently used these modulators, which include a compound we named BIOD303 and its analogs, to probe the biological chemistry of monomeric ␣-synuclein. We demonstrate that these molecules bind to the protein by both SHG and NMR spectroscopy and by paramagnetic relaxation enhancement (PRE)

Summary

Introduction

Proteins are structurally dynamic molecules that perform specialized functions through unique conformational changes accessible in physiological environments. Classical biochemical experiments on enzyme catalysis led to the development of the structure-function paradigm, whereby the structure of a protein determines its function [4, 5] This paradigm has been challenged by the discovery of intrinsically disordered proteins (IDPs)4 [6, 7]. IDPs as a class are estimated to comprise ϳ15– 45% of all eukaryotic proteins and include both proteins that lack a folded structure as well as those that have disordered regions, such as loops and linkers, of greater than 30 amino acids in length [8]. Irrespective of the toxic species, monomeric ␣-synuclein is an attractive therapeutic target for small molecules that modulate ␣-synuclein conformation as it is the most upstream form of the protein in the aggregation process. The intrinsic disorder of the protein makes it difficult to screen by conventional techniques and few molecules are known to bind to the protein [25,26,27,28,29,30,31,32]

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Conclusion