High-throughput screening methods to identify small molecule targets

Inventors

YOUNGER, David • Lopez, Randolph • Sen, Arpita • Emerson, Ryan

Assignees

A Alpha Bio Inc

Abstract

Provided herein are methods for identifying pairs of protein binding partners, mutations of which may inform the discovery of pharmaceutically useful small molecules. The methods disclosed herein may allow for the adaptation of the native protein degradation system to modulate specific disease targets at the protein level, in particular, for targets that have long been considered undruggable.

Core Innovation

The patent describes a method for assaying protein-protein interactions using yeast cell display libraries of polypeptide ubiquitin ligase species and polypeptide substrate species. The ubiquitin ligase library comprises wild-type polypeptide ubiquitin ligase species and mutant polypeptide ubiquitin ligase species modified at one or more amino acid residue positions by mutagenesis. The substrate library comprises wild-type polypeptide substrate species and mutant polypeptide substrate species modified at one or more amino acid residue positions by mutagenesis. Both libraries are expressed and displayed on the surface of recombinant haploid yeast cells.

The method combines the first plurality of recombinant haploid yeast cells and the second plurality of recombinant haploid yeast cells in a liquid medium to produce a culture. The culture is grown for a time and under conditions such that one or more interactions between one or more ubiquitin ligase species and one or more substrate species mediates one or more mating events to produce diploid yeast cells. The number of mating events in the culture is used to determine the strength of the interactions between the ubiquitin ligase species and the substrate species. Interaction strength is expressed in terms of the strength of the interaction (K_D).

Based on the determined interaction strengths, the method identifies pairs of polypeptides where one or both of the ubiquitin ligase species and the substrate species have been modified by mutagenesis and where the strength of the interaction (K_D) is stronger or weaker than the interaction between the corresponding wild-type polypeptide species by at least 10%. The patent further describes additional constraints and refinements, including interaction-strength thresholds, substrate features such as degron motifs, computational interface modeling to determine the structure of the interface, and determining how interaction strength changes in the presence of chemical entities.

Claims Coverage

The independent claim recites an assay method with a plurality of mutagenesis-modified ubiquitin ligase polypeptide species and a plurality of mutagenesis-modified substrate polypeptide species, where mating events in yeast are used to determine interaction strength (K_D). Across the independent claim and dependent claims, the coverage focuses on multiple mutagenized libraries, identifying interaction-strength changes versus wild-type by quantitative thresholds, and refinements that specify E3 ubiquitin ligase usage, degron motifs, computational interface modeling, and modulation by chemical entities.

Overall claim coverage centers on a mating-event based yeast assay that uses mutagenesis-modified ubiquitin ligase and substrate polypeptide display libraries to quantify interaction strength (K_D), followed by identification of polypeptide pairs showing stronger or weaker K_D relative to corresponding wild-type interactions by specified quantitative thresholds. Dependent claim coverage further narrows and extends the assay to E3 ubiquitin ligases, substrate degron motifs, computational interface modeling, and interaction modulation by chemical entities.

Stated Advantages

Documented Applications