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 method expresses on the surface of haploid yeast cells one or both of wild-type targeting proteins and/or modified targeting proteins with at least one amino acid difference from the wild-type targeting protein, and expresses on the surface of haploid yeast cells one or both of wild-type target proteins and/or modified target proteins with at least one amino acid difference from the wild-type target protein. The first culture and the second culture are combined in a single liquid culture so that binding of a targeting protein with a target protein leads to the formation of diploid yeast cells.

The approach determines, based on the number of mating events between the haploid yeast cells of the first and second cultures, the amino acid sequence mutations of the targeting protein and/or target protein, or both, that result in a dissociation constant between the targeting protein and the target protein of about ten-fold less than the interaction between the wild-type target protein and the wild-type targeting protein. The interaction between the wild-type target protein and the wild-type targeting protein has a dissociation constant (Kd) greater than about 100 nM.

The method includes an explicit exclusion in which neither the targeting protein nor target protein is a ubiquitin ligase. In the disclosed embodiments, determination based on mating events is used to identify amino acid sequence mutations that change protein binding strength, including comparisons to wild-type interactions and quantitative relationships based on Kd and relative weaker or stronger thresholds.

Claims Coverage

The provided partial claim set includes one independent claim and multiple dependent claims that further specify quantitative thresholds, mutation generation modes, library scale constraints, optional structural determination, and refinement of comparative interaction strength and weakness definitions. The inventive features mainly cover yeast-surface display of targeting protein and target protein variants, mating-event-based readout for interaction strength changes, and constrained selection criteria using Kd relationships and an exclusion of ubiquitin ligases.

Across the independent claim and its dependents, the claim coverage focuses on a haploid yeast mating assay where binding between surface-expressed targeting and target protein variants produces diploid formation, and mating-event counts are used to identify amino acid mutations that achieve an about ten-fold lower Kd than the wild-type interaction under an explicit wild-type Kd threshold. The dependent claims further specify additional quantitative comparison thresholds, weak-interaction Kd thresholds, library scale, mutagenesis modes, and an option for crystallography-based structure determination, while maintaining the independent claim’s restriction that neither protein is a ubiquitin ligase.

Stated Advantages

Documented Applications