5 research outputs found
Intracellular Generation of a Diterpene-Peptide Conjugate that Inhibits 14-3-3-Mediated Interactions
Synthetic agents that disrupt intracellular
protein–protein
interactions (PPIs) are highly desirable for elucidating signaling
networks and developing new therapeutics. However, designing cell-penetrating
large molecules equipped with the many functional groups necessary
for binding to large interfaces remains challenging. Here, we describe
a rational strategy for the intracellular oxime ligation-mediated
generation of an amphipathic bivalent inhibitor composed of a peptide
and diterpene natural product, fusicoccin, which binds 14-3-3 protein
with submicromolar affinity. Our results demonstrate that co-treatment
of cells with small module molecules, the aldehyde-containing fusicoccin <b>1</b> and the aminooxy-containing peptide <b>2</b>, generates
the corresponding conjugate <b>3</b> in cells, resulting in
significant cytotoxicity. In contrast, chemically synthesized <b>3</b> is not cytotoxic, likely due to its inability to penetrate
cells. Compound <b>3</b>, but not <b>1</b> or <b>2</b>, disrupts endogenous 14-3-3/cRaf interactions, suggesting that cell
death is caused by inhibition of 14-3-3 activity. These results suggest
that intracellular generation of large-sized molecules may serve as
a new approach for modulating PPIs
Going beyond Binary: Rapid Identification of Protein–Protein Interaction Modulators Using a Multifragment Kinetic Target-Guided Synthesis Approach
Kinetic target-guided synthesis (KTGS) is a powerful
screening
approach that enables identification of small molecule modulators
for biomolecules. While many KTGS variants have emerged, a majority
of the examples suffer from limited throughput and a poor signal/noise
ratio, hampering reliable hit detection. Herein, we present our optimized
multifragment KTGS screening strategy that tackles these limitations.
This approach utilizes selected reaction monitoring liquid chromatography
tandem mass spectrometry for hit detection, enabling the incubation
of 190 fragment combinations per screening well. Consequentially,
our fragment library was expanded from 81 possible combinations to
1710, representing the largest KTGS screening library assembled to
date. The expanded library was screened against Mcl-1, leading to
the discovery of 24 inhibitors. This work unveils the true potential
of KTGS with respect to the rapid and reliable identification of hits,
further highlighting its utility as a complement to the existing repertoire
of screening methods used in drug discovery
Going beyond Binary: Rapid Identification of Protein–Protein Interaction Modulators Using a Multifragment Kinetic Target-Guided Synthesis Approach
Kinetic target-guided synthesis (KTGS) is a powerful
screening
approach that enables identification of small molecule modulators
for biomolecules. While many KTGS variants have emerged, a majority
of the examples suffer from limited throughput and a poor signal/noise
ratio, hampering reliable hit detection. Herein, we present our optimized
multifragment KTGS screening strategy that tackles these limitations.
This approach utilizes selected reaction monitoring liquid chromatography
tandem mass spectrometry for hit detection, enabling the incubation
of 190 fragment combinations per screening well. Consequentially,
our fragment library was expanded from 81 possible combinations to
1710, representing the largest KTGS screening library assembled to
date. The expanded library was screened against Mcl-1, leading to
the discovery of 24 inhibitors. This work unveils the true potential
of KTGS with respect to the rapid and reliable identification of hits,
further highlighting its utility as a complement to the existing repertoire
of screening methods used in drug discovery
Going beyond Binary: Rapid Identification of Protein–Protein Interaction Modulators Using a Multifragment Kinetic Target-Guided Synthesis Approach
Kinetic target-guided synthesis (KTGS) is a powerful
screening
approach that enables identification of small molecule modulators
for biomolecules. While many KTGS variants have emerged, a majority
of the examples suffer from limited throughput and a poor signal/noise
ratio, hampering reliable hit detection. Herein, we present our optimized
multifragment KTGS screening strategy that tackles these limitations.
This approach utilizes selected reaction monitoring liquid chromatography
tandem mass spectrometry for hit detection, enabling the incubation
of 190 fragment combinations per screening well. Consequentially,
our fragment library was expanded from 81 possible combinations to
1710, representing the largest KTGS screening library assembled to
date. The expanded library was screened against Mcl-1, leading to
the discovery of 24 inhibitors. This work unveils the true potential
of KTGS with respect to the rapid and reliable identification of hits,
further highlighting its utility as a complement to the existing repertoire
of screening methods used in drug discovery
Going beyond Binary: Rapid Identification of Protein–Protein Interaction Modulators Using a Multifragment Kinetic Target-Guided Synthesis Approach
Kinetic target-guided synthesis (KTGS) is a powerful
screening
approach that enables identification of small molecule modulators
for biomolecules. While many KTGS variants have emerged, a majority
of the examples suffer from limited throughput and a poor signal/noise
ratio, hampering reliable hit detection. Herein, we present our optimized
multifragment KTGS screening strategy that tackles these limitations.
This approach utilizes selected reaction monitoring liquid chromatography
tandem mass spectrometry for hit detection, enabling the incubation
of 190 fragment combinations per screening well. Consequentially,
our fragment library was expanded from 81 possible combinations to
1710, representing the largest KTGS screening library assembled to
date. The expanded library was screened against Mcl-1, leading to
the discovery of 24 inhibitors. This work unveils the true potential
of KTGS with respect to the rapid and reliable identification of hits,
further highlighting its utility as a complement to the existing repertoire
of screening methods used in drug discovery