High-Throughput Quantitative Assay Technologies for Accelerating the Discovery and Optimization of Targeted Protein Degradation Therapeutics

Abstract

The aberrant regulation of protein expression and function can drastically alter cellular physiology and lead to numerous pathophysiological conditions such as cancer, inflammatory diseases, and neurodegeneration. The steady-state expression levels of endogenous proteins are controlled by a balance of de novo synthesis rates and degradation rates. Moreover, the levels of activated proteins in signaling cascades can be further modulated by a variety of posttranslational modifications and protein–protein interactions. The field of targeted protein degradation is an emerging area for drug discovery in which small molecules are used to recruit E3 ubiquitin ligases to catalyze the ubiquitination and subsequent degradation of disease-causing target proteins by the proteasome in both a dose- and time-dependent manner. Traditional approaches for quantifying protein level changes in cells, such as Western blots, are typically low throughput with limited quantification, making it hard to drive the rapid development of therapeutics that induce selective, rapid, and sustained protein degradation. In the last decade, a number of techniques and technologies have emerged that have helped to accelerate targeted protein degradation drug discovery efforts, including the use of fluorescent protein fusions and reporter tags, flow cytometry, time-resolved fluorescence energy transfer (TR-FRET), and split luciferase systems. Here we discuss the advantages and disadvantages associated with these technologies and their application to the development and optimization of degraders as therapeutics.

Unifying Catalysis Framework to Dissect Proteasomal Degradation Paradigms

“…In order to drive SAR optimization of degraders, low- to high-throughput quantitative methods are routinely used. 85 Of these methods, luminescence-based workflows developed by Promega surfaced as very powerful tools to kinetically characterize protein degradation events. 86 In these experiments, POIs can be labeled with HiBiT tags at endogenous levels to interrogate competing biosynthesis and degradation rates.…”

A Phenotypic Approach for the Identification of New Molecules for Targeted Protein Degradation Applications

“…Additionally, even before a functional PROTAC can be constructed from an E3 ligase screening hit, a medicinal chemistry effort is typically required to improve E3 ligase binding affinity and to prepare and test multiple putative PROTACs with various linkers and linker attachment configurations. In practice, this reductionist single-target screening approach for E3 ligases is costly, inefficient, and slow, and can realistically only be conducted on a small fraction of the total number of potential E3 ligases that might have utility in PROTAC design 28 .…”

“…In practice, this reductionist single-target screening approach for E3 ligases is costly, inefficient, and slow, and can realistically only be conducted on a small fraction of the total number of potential E3 ligases that might have utility in PROTAC design. 28 To address the inherent inefficiencies posed by serial screening of individual E3 ligases, we have developed and implemented an alternative strategy that combines a cellbased targeted protein degradation phenotypic screen with efficient 384-well high-throughput chemical synthesis, shown schematically in Figure 1. We based our screening design strategy on the previously described HaloPROTAC method for the evaluation of Von Hippel-Lindau (VHL) E3 ligase binders in a cellular context.…”

“…In practice, this reductionist single-target screening approach for E3 ligases is costly, inefficient, and slow, and can realistically only be conducted on a small fraction of the total number of potential E3 ligases that might have utility in PROTAC design. 28…”

Enabling high throughput target-based drug discovery in 3D cell cultures through novel bioprinting workflow

“…Small and large molecule drug discovery projects, particularly high throughput screening, are widely using high sensitivity protein assays such as Alpha (amplified luminescence proximity homogeneous assay) [14][15][16] . Alpha is a bead-based assay platform employing oxygen-channelling chemistry for the detection and quantification of various biomolecules, from small analytes such as cAMP and cytokines to intracellular and membrane bound protein complexes and biomolecular interactions 17 .…”