Targeted pathogen-selective approach to drug development holds promise to minimize collateral damage to the beneficial microbiome. The AB5-topology pertussis toxin (PtxS1-S5) is a major virulence factor of Bordetella pertussis, the causative agent of the highly contagious respiratory disease whooping cough. Once internalized into the host cell, PtxS1 ADP-ribosylates α-subunits of the heterotrimeric Gαi-superfamily, thereby disrupting G-protein-coupled receptor signaling. Here, we report the discovery of the first small molecules inhibiting the ADP-ribosyltransferase activity of pertussis toxin. We developed protocols to purify mg-levels of active recombinant B. pertussis PtxS1 from Escherichia coli and an in vitro high throughput-compatible assay to quantify NAD + consumption during PtxS1-catalyzed ADP-ribosylation of Gαi. Two inhibitory compounds (NSC228155 and NSC29193) with low micromolar IC50-values (3.0 µM and 6.8 µM) were identified in the in vitro NAD + consumption assay that also were potent in an independent in vitro assay monitoring conjugation of ADP-ribose to Gi. Docking and molecular dynamics simulations identified plausible binding poses of NSC228155 and in particular of NSC29193, most likely owing to the rigidity of the latter ligand, at the NAD +-binding pocket of PtxS1. NSC228155 inhibited the pertussis AB5 holotoxin-catalyzed ADP-ribosylation of Gi in living human cells with a low micromolar IC50-value (2.4 µM). NSC228155 and NSC29193 might prove useful hit compounds in targeted B. pertussis-selective drug development.
Edited by Eric R. Fearon P. A. Jänne has received consulting fees from AstraZeneca, Boehringer Ingelheim, Pfizer, Merrimack Pharmaceuticals, Roche/Genentech, Chugai Pharmaceuticals, ACEA Biosciences, and Ariad Pharmaceuticals and sponsored research funding from Astellas Pharmaceuticals, AstraZeneca, Daiichi Sankyo, and PUMA and receives post-marketing royalties on DFCI-owned intellectual property on EGFR mutations licensed to Lab Corp. K. Elenius has a research agreement with Boehringer Ingelheim and ownership interest in Abomics, Orion, and Roche. This article was selected as one of our Editors' Picks. This article contains Figs. S1-S10.
The emergence of the COVID-19 outbreak at the end of 2019, caused by the novel coronavirus SARS-CoV-2, has, to date, led to over 13.6 million infections and nearly 600,000 deaths. Consequently, there is an urgent need to better understand the molecular factors triggering immune defense against the virus and to develop countermeasures to hinder its spread. Using in silico analyses, we showed that human major histocompatibility complex (MHC) class I cell-surface molecules vary in their capacity for binding different SARS-CoV-2-derived epitopes, i.e., short sequences of 8-11 amino acids, and pinpointed five specific SARS-CoV-2 epitopes that are likely to be presented to cytotoxic T-cells and hence activate immune responses. The identified epitopes, each one of nine amino acids, have high sequence similarity to the equivalent epitopes of SARS-CoV virus, which are known to elicit an effective T cell response in vitro. Moreover, we give a structural explanation for the binding of SARS-CoV-2-epitopes to MHC molecules. Our data can help us to better understand the differences in outcomes of COVID-19 patients and may aid the development of vaccines against SARS-CoV-2 and possible future outbreaks of novel coronaviruses.
Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor important in diverse biological processes including cell proliferation and survival. Upregulation of EGFR activity due to over-expression or mutation is widely implicated in cancer. Activating somatic mutations of the EGFR kinase are postulated to affect the conformation and/or stability of the protein, shifting the EGFR inactive-active state equilibrium towards the activated state. Here, we examined a common EGFR deletion mutation, Δ746ELREA750, which is frequently observed in non-small cell lung cancer patients. By using molecular dynamics simulation, we investigated the structural effects of the mutation that lead to the experimentally reported increases in kinase activity. Simulations of the active form wild-type and ΔELREA EGFRs revealed the deletion stabilizes the αC helix of the kinase domain, which is located adjacent to the deletion site, by rigidifying the flexible β3-αC loop that accommodates the ELREA sequence. Consequently, the αC helix is stabilized in the “αC-in” active conformation that would prolong the time of the activated state. Moreover, in the mutant kinase, a salt bridge between E762 and K745, which is key for EGFR activity, was also stabilized during the simulation. Additionally, the interaction between EGFR and ATP was favored by ΔELREA EGFR over wild-type EGFR, as reflected by the number of hydrogen bonds formed and the free energy of binding. Simulation of inactive EGFR suggested the deletion would promote a shift from the inactive conformation towards active EGFR, which is supported by the inward movement of the αC helix. The MDS results also align with the effects of tyrosine kinase inhibitors on ΔELREA and wild-type EGFR lung cancer cell lines, where more pronounced inhibition was observed against ΔELREA than for wild-type EGFR by inhibitors recognizing the active kinase conformation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.