Chemical Kinetic Strategies for High‐Throughput Screening of Protein Aggregation Modulators

Abstract

Insoluble aggregates staining positive to amyloid dyes are known histological hallmarks of different neurodegenerative disorders and of type II diabetes. Soluble oligomers are smaller assemblies whose formation prior to or concomitant with amyloid deposition has been associated to the processes of disease propagation and cell death. While the pathogenic mechanisms are complex and differ from disease to disease, both types of aggregates are important biological targets subject to intense investigation in academia and industry. Here we review recent advances in the fundamental understanding of protein aggregation that can be used on the development of anti‐amyloid and anti‐oligomerization drugs. Specifically, we pinpoint the chemical kinetic aspects that should be attended during the development of high‐throughput screening assays and in the hit validation phase. The strategies here devised are expected to establish a connection between basic research and pharmaceutical innovation.

MIRRAGGE – Minimum Information Required for Reproducible AGGregation Experiments

“…When nucleation is a rare event, the emergence of measurable amounts of protein aggregates is preceded by a lag phase whose variable duration reflects the probability distribution of a successful event (Crespo et al, 2012;Michaels et al, 2016). During amyloid fibril formation, prior addition of > 1% of preformed fibrils (or seeds) is often sufficient to eliminate the lag phase and the intrinsic uncertainty associated to primary nucleation (Sárkány et al, 2019). Conversely, protein samples containing residual, yet variable, amounts of preformed seeds may not be suitable for "unseeded" assays due to irreproducible lag times (Crespo et al, 2012(Crespo et al, , 2016.…”

Exploiting flow cytometry for the unbiased quantification of protein inclusions in Caenorhabditis elegans

“…Since the formation of insoluble proteinaceous inclusion bodies is a common feature among proteinopathies, it is thought that preventing this process is likely to be a beneficial strategy for halting the progression of these diseases (Bose & Cho 2017). Thus, multiple studies have focused on high‐throughput screening for genetic or chemical modulators of protein aggregation in various model systems (Cockburn et al, 2011; Ikenaka et al, 2019; Sarkany et al, 2019; Silva et al, 2011). These high‐throughput screens often rely on having a quantifiable readout of aggregation dynamics that can be determined rapidly and at low cost.…”

“…Thus, multiple studies have focused on high-throughput screening for genetic or chemical modulators of protein aggregation in various model systems (Cockburn et al, 2011;Ikenaka et al, 2019;Sarkany et al, 2019;Silva et al, 2011). These high-throughput screens often rely on having a quantifiable readout of aggregation dynamics that can be determined rapidly and at low cost.…”

Major Improvements in Robustness and Efficiency during the Screening of Novel Enzyme Effectors by the 3-Point Kinetics Assay

“…A minimum of 3-point readouts are required to probe the initial, intermediate, and final phases of screening reactions 12 ( Fig. 1A ).…”