9 research outputs found
The development of new GABA-based anticonvulsant agents
EThOS - Electronic Theses Online ServiceGBUnited Kingdo
Confounding Factors in Targeted Degradation of Short-Lived Proteins
Targeted protein degradation has recently emerged as a novel option in drug discovery. Natural protein half-life is expected to affect the efficacy of degrading agents, but to what extent it influences target protein degradation has not been systematically explored. Using simple mathematical modeling of protein degradation, we find that the natural half-life of a target protein has a dramatic effect on the level of protein degradation induced by a degrader agent which can pose significant hurdles to screening efforts. Moreover, we show that upon screening for degraders of short-lived proteins, agents that stall protein synthesis, such as GSPT1 degraders and generally cytotoxic compounds, deceptively appear as protein-degrading agents. This is exemplified by the disappearance of short-lived proteins such as MCL1 and MDM2 upon GSPT1 degradation and upon treatment with cytotoxic agents such as doxorubicin. These findings have implications for target selection as well as for the type of control experiments required to conclude that a novel agent works as a bona fide targeted protein degrader.</p
Determination of Ligand-Binding Affinity (<i>K</i><sub>d</sub>) Using Transverse Relaxation Rate (<i>R</i><sub>2</sub>) in the Ligand-Observed <sup>1</sup>H NMR Experiment and Applications to Fragment-Based Drug Discovery
High hit rates from initial ligand-observed NMR screening
can make
it challenging to prioritize which hits to follow up, especially in
cases where there are no available crystal structures of these hits
bound to the target proteins or other strategies to provide affinity
ranking. Here, we report a reproducible, accurate, and versatile quantitative
ligand-observed NMR assay, which can determine Kd values of fragments in the affinity range of low μM
to low mM using transverse relaxation rate R2 as the observable parameter. In this study, we examined the
theory and proposed a mathematical formulation to obtain Kd values using non-linear regression analysis. We designed
an assay format with automated sample preparation and simplified data
analysis. Using tool compounds, we explored the assay reproducibility,
accuracy, and detection limits. Finally, we used this assay to triage
fragment hits, yielded from fragment screening against the CRBN/DDB1
complex
Demonstrating In-Cell Target Engagement Using a Pirin Protein Degradation Probe (CCT367766)
Demonstrating
intracellular protein target engagement is an essential step in the
development and progression of new chemical probes and potential small
molecule therapeutics. However, this can be particularly challenging
for poorly studied and noncatalytic proteins, as robust proximal biomarkers
are rarely known. To confirm that our recently discovered chemical
probe <b>1</b> (CCT251236) binds the putative transcription
factor regulator pirin in living cells, we developed a heterobifunctional
protein degradation probe. Focusing on linker design and physicochemical
properties, we generated a highly active probe <b>16</b> (CCT367766)
in only three iterations, validating our efficient strategy for degradation
probe design against nonvalidated protein targets
Discovery of a Chemical Probe Bisamide (CCT251236): An Orally Bioavailable Efficacious Pirin Ligand from a Heat Shock Transcription Factor 1 (HSF1) Phenotypic Screen
Phenotypic screens, which focus on
measuring and quantifying discrete
cellular changes rather than affinity for individual recombinant proteins,
have recently attracted renewed interest as an efficient strategy
for drug discovery. In this article, we describe the discovery of
a new chemical probe, bisamide (CCT251236), identified using an unbiased
phenotypic screen to detect inhibitors of the HSF1 stress pathway.
The chemical probe is orally bioavailable and displays efficacy in
a human ovarian carcinoma xenograft model. By developing cell-based
SAR and using chemical proteomics, we identified pirin as a high affinity
molecular target, which was confirmed by SPR and crystallography