17 research outputs found
Biochemical Characterization of Selective Inhibitors of Human Group IIA Secreted Phospholipase A<sub>2</sub> and Hyaluronic Acid-Linked Inhibitor Conjugates
We explored the inhibition mode of group IIA secreted
phospholipase
A<sub>2</sub> (GIIA sPLA<sub>2</sub>) selective inhibitors and tested
their ability to inhibit GIIA sPLA<sub>2</sub> activity as chemical
conjugates with hyaluronic acid (HA). Analogues of a benzo-fused indole
sPLA<sub>2</sub> inhibitor were developed in which the carboxylate
group on the inhibitor scaffold, which has been shown to coordinate
to a Ca<sup>2+</sup> ligand in the enzyme active site, was replaced
with other functionality. Replacing the carboxylate group with amine,
amide, or hydroxyl groups had no effect on human GIIA (hGIIA) sPLA<sub>2</sub> inhibition potency but dramatically lowered inhibition potency
against hGV and hGX sPLA<sub>2</sub>s. An alkylation protection assay
was used to probe active site binding of carboxylate and noncarboxylate
inhibitors in the presence and absence of Ca<sup>2+</sup> and/or lipid
vesicles. We observed that carboxylate-containing inhibitors bind
the hGIIA sPLA<sub>2</sub> active site with low nanomolar affinity,
but only when Ca<sup>2+</sup> is present. Noncarboxylate, GIIA sPLA<sub>2</sub> selective inhibitors also bind the hGIIA sPLA<sub>2</sub> active site in the nanomolar range. However, binding for GIIA sPLA<sub>2</sub> selective inhibitors was dependent on the presence of a lipid
membrane and not Ca<sup>2+</sup>. These results indicate that GIIA
sPLA<sub>2</sub> selective inhibitors exert their inhibitory effects
by binding to the hGIIA sPLA<sub>2</sub> active site. An HA-linked
GIIA inhibitor conjugate was developed using peptide coupling conditions
and found to be less potent and selective against hGIIA sPLA<sub>2</sub> than the unconjugated inhibitor. Compounds reported in this study
are some of the most potent and selective GIIA sPLA<sub>2</sub> active
site binding inhibitors reported to date
The chemical biology of coronavirus hostācell interactions
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current coronavirus disease 2019 (COVID-19) pandemic that has led to a global economic disruption and collapse. With several ongoing efforts to develop vaccines and treatments for COVID-19, understanding the molecular interaction between the coronavirus, host cells, and the immune system is critical for effective therapeutic interventions. Greater insight into these mechanisms will require the contribution and combination of multiple scientific disciplines including the techniques and strategies that have been successfully deployed by chemical biology to tease apart complex biological pathways. We highlight in this review well-established strategies and methods to study coronavirusāhost biophysical interactions and discuss the impact chemical biology will have on understanding these interactions at the molecular level
A second-generation phosphohistidine analog for production of phosphohistidine antibodies
Protein histidine phosphorylation plays a crucial role in cell signaling and central metabolism. However, its detailed functions remain elusive due to technical challenges in detecting and isolating proteins bearing phosphohistidine (pHis), a labile posttranslational modification (PTM). To address this issue, we previously developed the first pHis-specific antibodies using stable, synthetic triazole-based pHis analogs. A second-generation, pyrazole-based pHis analog that enabled the development of a pan-pHis antibody with much improved pHis specificity is now reported.close2
Near-Infrared Photoredox Catalyzed Tryptophan Functionalization for Peptide Stapling and Protein Labeling in Complex Tissue Environments
The chemical transformation of aromatic amino acids has emerged as an attractive alternative to non-selective lysine or cysteine labeling for the modification of biomolecules. However, this strategy has largely been limited by the scope of functional groups and biocompatible reaction conditions available. Herein, we report the implementation of near-infrared-activatable photocatalysts, TTMAPP and n-Pr-DMQA+, capable of generating fluoroalkyl radicals for selective tryptophan functionalization within simple and complex biological systems. At the peptide level, a diverse set of iodo-perfluoroalkyl reagents were used to install bioorthogonal handles for downstream applications or link inter- or intramolecular tryptophan residues for peptide stapling. We also found this photoredox transformation amenable to biotinylation of intracellular proteins in live cells for downstream confocal imaging and mass spectrometry-based analysis. Given the inherent tissue penetrant nature of near-infrared light we further demonstrated the utility of this technology to achieve photocatalytic protein fluoroalkylation in physiologically relevant tissue and tumor environments
Ī¼Map Photoproximity Labeling Enables Small Molecule Binding Site Mapping
The characterization of ligand binding modes is a crucial
step
in the drug discovery process and is especially important in campaigns
arising from phenotypic screening, where the protein target and binding
mode are unknown at the outset. Elucidation of target binding regions
is typically achieved by X-ray crystallography or photoaffinity labeling
(PAL) approaches; yet, these methods present significant challenges.
X-ray crystallography is a mainstay technique that has revolutionized
drug discovery, but in many cases structural characterization is challenging
or impossible. PAL has also enabled binding site mapping with peptide-
and amino-acid-level resolution; however, the stoichiometric activation
mode can lead to poor signal and coverage of the resident binding
pocket. Additionally, each PAL probe can have its own fragmentation
pattern, complicating the analysis by mass spectrometry. Here, we
establish a robust and general photocatalytic approach toward the
mapping of protein binding sites, which we define as identification
of residues proximal to the ligand binding pocket. By utilizing a
catalytic mode of activation, we obtain sets of labeled amino acids
in the proximity of the target protein binding site. We use this methodology
to map, in vitro, the binding sites of six protein targets, including
several kinases and molecular glue targets, and furthermore to investigate
the binding site of the STAT3 inhibitor MM-206, a ligand with no known
crystal structure. Finally, we demonstrate the successful mapping
of drug binding sites in live cells. These results establish Ī¼Map
as a powerful method for the generation of amino-acid- and peptide-level
target engagement data