Assay and Drug Development Technologies - 4

monitoring PROTAC-mediated ubiquitination of target proteins
on a microtiter plate-based assay called PROTAC Plate.''
PROTAC discoveryisalsohamperedbyalargegap in understanding
the fine link between ubiquitination and degradation.
''Current methods to quantify this process are of limited
utility in drug discovery,'' Kadimisetty points out. ''We addressed
these gaps utilizing Tandem Ubiquitin Binding Entities (TUBES)
in high-throughput biochemical and cell-based assays.''
TUBEs are specifically designed to isolate polyubiquitinated
proteins from cell lysates and tissues. Kadimesetty explains,
''These high-affinity capture reagents bind polyubiquitin
chains. One can perform quantitative pulldown ofubiquitinated
proteins and establish correlations between PROTAC-mediated
ubiquitination and degradation of proteins. Further, mass
spectrometry ubiquitin proteomics has facilitated PROTACmediated
ubiquiti-nation of target proteins. These and other
methods have clarified PROTAC mechanisms of action.''
''TUBEs will help cell and molecular biologists better understand
the role of novel polyubiquitin chains,'' Kadimisetty
predicts. ''Further, if we understand the role of other polyubiquitin
chains and E3 ligases responsible for novel functions,
new nondegradative PROTACs can be designed to
broaden their therapeutic potential.''
Protein Interference Technology Expands Target Space
Over the past two decades, large-scale omics-based initiatives have
revolutionized our understanding of disease. In particular, functional
genomic screens that utilize CRISPR and RNAi have identified many
target genes and pathways as potential drivers of disease. This has catalyzed
significant drug discovery activity across industry and academia,
leading to an improved landscape for development of novel therapeutics.
However, blind spots in the reach of these tools are now becoming
apparent, according to Christian Dillon, PhD, vice president of biology
at PhoreMost. Program failures due to poor target selection are
common, and significant challenges remain in early drug discovery for
targets with limited precedent, he explains, adding that these failures
are often exacerbated by a selection bias for ''easier to prosecute''
targets, highlighting the urgent need for new approaches to inform
target-based drug discovery.
''A promising and highly differentiated technology to systematically
open up new 'druggable' target space is PROTEINi (protein interference).
PROTEINi utilizes computationally derived coded peptide fragments
in the context of a functional phenotypic screen, allowing for
the discovery of novel disease-relevant targets that may be missed
through gene editing or knockdown approaches,'' Dillon tells GEN.
''Moreover, peptide fragment hits from a PROTEINi screen provide
valuable mechanistic insight that can help to truncate, inform, and
derisk the path from target to drug discovery.''
PROTEINi screening has now been successfully employed across a range
of areas to identify novel targets for drug discovery, continues Dillon.
''One important example is the identification of targets that can
LifeSensors develops technology to accelerate the design, synthesis,
and testing of PROteolytic TArgeting Chimeras (PROTACs), which are
heterobifunctional molecules that hijack ubiquitin ligase and target
cellular proteins for degradation. For example, the company has developed
a microtiter plate-based assay called PROTAC Plate that incorporates
Tandem Ubiquitin Binding Entities (TUBEs) to specifically
isolate polyubiquitylated proteins from cell lysates and tissues. This
image shows how the plate can be used to evaluate functional ternarycomplex-and
ubiquitin-mediated degradation.
specifically modulate oncogenic KRAS signaling, a key cancer-driving
oncogene that has traditionally been considered to be 'undruggable,''' he
says. ''In a further application, PROTEINi screens have been configured to
identify novel functional degrading E3 ligases that can be developed for
Targeted Protein Degradation (TPD) applications, such as PROTACs.
Alongside this we have a network of industry collaborations applying
PROTEINi to develop a pipeline of validated targets across different areas
of disease where treatment options are currently severely limited.''
4 Genetic Engineering & Biotechnology News
ª 2022 GEN PUBLISHING
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