Search

Targeted pathogen-selective approach to antibiotic development holds promise to minimize collateral damage to the beneficial microbiome. The AB5-topology pertussis toxin (PtxS1-S5, 1:1:1:2:1) is a major virulence factor ofBordetella 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 truncated but highly active recombinantB. pertussisPtxS1 fromEscherichia coliand anin vitrohigh 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 thein vitroNAD+consumption assay that also were potent in an independentin vitroassay monitoring conjugation of ADP-ribose to Gαi. 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 AB5holotoxin-catalyzed ADP-ribosylation of Gαi in living human cells with a low micromolar IC50-value (2.4 µM). NSC228155 and NSC29193 might prove useful hit compounds in targetedB. pertussis-selective drug development.

Pseudomonasare a common cause of hospital acquired infections that may be lethal. ADP-ribosyltransferase activities ofPseudomonasexotoxin-S and -T depend on 14-3-3 proteins inside the host cell. By binding in the 14-3-3 phosphopeptide binding groove, a hydrophobic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3β:ExoS and -ExoT complexes presented here reveal an extensive novel binding interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the hydrophobic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the hydrophobic C-terminus with a fragment fromVibrioVis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, we show that 14-3-3 stabilizes ExoS against thermal aggregation. Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the hydrophobic C-terminal segment.Short summaryCrystal structures of Pseudomonas exotoxins-S and –T identify a novel hydrophobic interface with 14-3-3 proteins, and we show that 14-3-3 activates these toxins independent of their phosphopeptide groove binding C-termini, by preventing their aggregation.

A DNA-encoded chemical library (DECL) with 1.2 million compounds was synthesized by combinatorial reaction of seven central scaffolds with two sets of 343×492 building blocks. Library screening by affinity capture revealed that for some target proteins, the chemical nature of building blocks dominated the selection results, whereas for other proteins, the central scaffold also crucially contributed to ligand affinity. Molecules based on a 3,5-bis(aminomethyl)benzoic acid core structure were found to bind human serum albumin with a Kd value of 6 nm, while compounds with the same substituents on an equidistant but flexible l-lysine scaffold showed 140-fold lower affinity. A 18 nm tankyrase-1 binder featured l-lysine as linking moiety, while molecules based on d-Lysine or (2S,4S)-amino-l-proline showed no detectable binding to the target. This work suggests that central scaffolds which predispose the orientation of chemical building blocks toward the protein target may enhance the screening productivity of encoded libraries.

Muscarinic toxin α (MTα), a peptide isolated from the venom of the African black mamba, was recently found to selectively antagonize the human α 2B -adrenoceptor. To gain more information about the binding of this peptide toxin, we studied the properties of the [ 3 H]UK14,304 agonist and the [ 3 H]MK-912 antagonist binding to the α 2B -adrenoceptor in the presence of MTα. In equilibrium binding experiments, MTα decreased the binding of the orthosteric ligands, but failed to completely displace these. This effect of MTα was due to noncompetitive inhibition of B max without change in radioligand affinity. On the contrary, cellular signaling via the α 2B -adrenoceptor could be titrated to zero despite the incomplete receptor blockade. To locate binding sites for MTα on the receptor protein, we generated chimeric receptors of α 2B - and α 2A - or α 2C -adrenoceptors. Data based on these constructs revealed the extracellular loop two (ECL2) as the structural entity that enables MTα binding. Cumulative exchange of parts of ECL2 of α 2B for α 2A -adrenoceptor sequence resulted in a gradual decrease in the affinity for MTα, indicating that MTα binds to the α 2B -adrenoceptor through multiple sites dispersed over the whole ECL2. Together the results suggest that binding of MTα to the α 2B -adrenoceptor occludes orthosteric ligand access to the binding pocket. Putative homomeric receptor complexes as factors underlying the apparent noncompetitivity are also discussed.

Muscarinic toxins (MTs) are snake venom peptides found to selectively target specific subtypes of G-protein-coupled receptors. In here, we have attached a glycosylphosphatidylinositol (GPI) tail to three different toxin molecules and evaluated their receptor-blocking effects in a heterologous expression system. MT7–GPI remained anchored to the cell surface and selectively inhibited M 1 muscarinic receptor signaling expressed in the same cell. To further demonstrate the utility of the GPI tail, we generated MT3- and MTα-like gene sequences and fused these to the signal sequence for GPI attachment. Functional assessment of these membrane-anchored toxins on coexpressed target receptors indicated a prominent antagonistic effect. In ligand binding experiments the GPI-anchored toxins were found to exhibit similar selection profiles among receptor subtypes as the soluble toxins. The results indicate that GPI attachment of MTs and related receptor toxins could be used to assess the role of receptor subtypes in specific organs or even cells in vivo by transgenic approaches.