Extendable stapling of unprotected peptides by crosslinking two amines with o-phthalaldehyde

Peptide modification methods that do not rely on the cysteine residue are underdeveloped, and their development could greatly expand the current toolbox for peptide chemistry. During the course of preliminary investigations into the classical ortho-phthalaldehyde (OPA)-amine-thiol condensation reaction, we found that in the absence of thiol, OPA readily condenses with two primary alkyl amines to form a class of underexplored isoindolin-1-imine compounds under mild aqueous conditions. From the intramolecular version of this OPA-2amines reaction, an efficient and selective methodology using mild reaction conditions has been developed for stapling unprotected peptides via crosslinking of two amino groups in both an end-to-side and side-to-side fashion. The stapling method is superfast and broadly applicable for various peptide substrates with the reacting amino groups separated by a wide range of different amino acid units. The macrocyclization reactions of selected substrates are completed within 10 seconds at 5 mM concentration and within 2 minutes at 50 μM concentration. Importantly, the resulting cyclized peptides with an isoindolinimine linkage can be extended in a one-pot sequential addition manner with several different electron-deficient π electrophiles, thereby generating more complex structures.

2-Sulfonylpyrimidines as Privileged Warheads for the Development of S. aureus Sortase A Inhibitors

Staphylococcus aureus is one of the most frequent causes of nosocomial and community-acquired infections, with emerging multiresistant isolates causing a significant burden to public health systems. We identified 2-sulfonylpyrimidines as a new class of potent inhibitors against S. aureus sortase A acting by covalent modification of the active site cysteine 184. Series of derivatives were synthesized to derive structure-activity relationship (SAR) with the most potent compounds displaying low micromolar KI values. Studies on the inhibition selectivity of homologous cysteine proteases showed that 2-sulfonylpyrimidines reacted efficiently with protonated cysteine residues as found in sortase A, though surprisingly, no reaction occurred with the more nucleophilic cysteine residue from imidazolinium-thiolate dyads of cathepsin-like proteases. By means of enzymatic and chemical kinetics as well as quantum chemical calculations, it could be rationalized that the SNAr reaction between protonated cysteine residues and 2-sulfonylpyrimidines proceeds in a concerted fashion, and the mechanism involves a ternary transition state with a conjugated base. Molecular docking and enzyme inhibition at variable pH values allowed us to hypothesize that in sortase A this base is represented by the catalytic histidine 120, which could be substantiated by QM model calculation with 4-methylimidazole as histidine analog.

Programmed Hierarchical Radial Association of Anisotropic Foldamer Assemblies

Herein, we report the first example of a programmed radial assembly of anisotropic microparticles derived from a helical foldamer with a C-terminal cysteine residue. Surface-exposed thiols played a crucial role...

A Redox-Sensitive Cysteine Is Required for PIN1At Function

Parvulins are ubiquitous peptidyl-prolyl isomerases (PPIases) required for protein folding and regulation. Among parvulin members, Arabidopsis PIN1At, human PIN1, and yeast ESS1 share a conserved cysteine residue but differ by the presence of an N-terminal WW domain, absent in PIN1At. In this study, we have explored whether the cysteine residue of Arabidopsis PIN1At is involved in catalysis and subject to oxidative modifications. From the functional complementation of yeast ess1 mutant, we concluded that the cysteine at position 69 is mandatory for PIN1At function in vivo, unless being replaced by an Asp which is found in a few parvulin members. This result correlates with a decrease of the in vitro PPIase activity of non-functional PIN1At cysteinic variants. A decrease of PIN1At activity was observed upon H2O2 treatment. The in vitro oxidation of cysteine 69, which has an acidic pKa value of 4.9, leads to the formation of covalent dimers that are reduced by thioredoxins, or to sulfinic or sulfonic acid forms at higher H2O2 excess. These investigations highlight the importance of the sole cysteine residue of PIN1At for activity. The reversible formation of an intermolecular disulfide bond might constitute a protective or regulatory mechanism under oxidizing conditions.

Native mass spectrometry identifies the HybG chaperone as carrier of the Fe(CN)2CO group during maturation of E. coli [NiFe]-hydrogenase 2

Abstract[NiFe]-hydrogenases activate dihydrogen. Like all [NiFe]-hydrogenases, hydrogenase 2 of Escherichia coli has a bimetallic NiFe(CN)2CO cofactor in its catalytic subunit. Biosynthesis of the Fe(CN)2CO group of the [NiFe]-cofactor occurs on a distinct scaffold complex comprising the HybG and HypD accessory proteins. HybG is a member of the HypC-family of chaperones that confers specificity towards immature hydrogenase catalytic subunits during transfer of the Fe(CN)2CO group. Using native mass spectrometry of an anaerobically isolated HybG–HypD complex we show that HybG carries the Fe(CN)2CO group. Our results also reveal that only HybG, but not HypD, interacts with the apo-form of the catalytic subunit. Finally, HybG was shown to have two distinct, and apparently CO2-related, covalent modifications that depended on the presence of the N-terminal cysteine residue on the protein, possibly representing intermediates during Fe(CN)2CO group biosynthesis. Together, these findings suggest that the HybG chaperone is involved in both biosynthesis and delivery of the Fe(CN)2CO group to its target protein. HybG is thus suggested to shuttle between the assembly complex and the apo-catalytic subunit. This study provides new insights into our understanding of how organometallic cofactor components are assembled on a scaffold complex and transferred to their client proteins.

Chalcones identify cTXNPx as a potential antileishmanial drug target

With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2’,6’-dihydroxy-4’-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2’,4’,6’- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.

Modification of Glyceraldehyde-3-Phosphate Dehydrogenase with Nitric Oxide: Role in Signal Transduction and Development of Apoptosis

This review focuses on the consequences of GAPDH S-nitrosylation at the catalytic cysteine residue. The widespread hypothesis according to which S-nitrosylation causes a change in GAPDH structure and its subsequent binding to the Siah1 protein is considered in detail. It is assumed that the GAPDH complex with Siah1 is transported to the nucleus by carrier proteins, interacts with nuclear proteins, and induces apoptosis. However, there are several conflicting and unproven elements in this hypothesis. In particular, there is no direct confirmation of the interaction between the tetrameric GAPDH and Siah1 caused by S-nitrosylation of GAPDH. The question remains as to whether the translocation of GAPDH into the nucleus is caused by S-nitrosylation or by some other modification of the catalytic cysteine residue. The hypothesis of the induction of apoptosis by oxidation of GAPDH is considered. This oxidation leads to a release of the coenzyme NAD+ from the active center of GAPDH, followed by the dissociation of the tetramer into subunits, which move to the nucleus due to passive transport and induce apoptosis. In conclusion, the main tasks are summarized, the solutions to which will make it possible to more definitively establish the role of nitric oxide in the induction of apoptosis.

A Simple Method to Dual Site-Specifically Label a Protein Using Tryptophan Auxotrophic Escherichia coli

Site-specifically labeling proteins with multiple dyes or molecular moieties is an important yet non-trivial task for many research, such as when using Föster resonance energy transfer (FRET) to study dynamics of protein conformational change. Many strategies have been devised, but usually done on a case-by-case basis. Expanded genetic code provided a general platform to incorporate non-canonical amino acids (ncAA), which can also enable multiple site-specific labeling, but it’s technically complicated and not suitable for some applications. Here we present a streamlined method that could enable dual site-specific protein labeling by using a tryptophan auxotroph of Escherichia coli to incorporate a naturally found tryptophan analog, 5-hydroxytryptophan into a recombinant protein. As a demonstration, we incorporated 5-hydroxytryptophan into E. coli release factor 1 (RF1), a protein known to possess two different conformations, and site-specifically attached two different fluorophores, one on 5-hydroxytryptophan and another on a cysteine residue. This method is simple, generally applicable, efficient, and can serve as an alternative way for researchers who want to install an additional labeling site in their proteins.

Class-II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis

Background In most fungi, quinone-dependent Class-II dihydroorotate dehydrogenases (DHODs) are essential for pyrimidine biosynthesis. Coupling of these Class-II DHODHs to mitochondrial respiration makes their in vivo activity dependent on oxygen availability. Saccharomyces cerevisiae and closely related yeast species harbor a cytosolic Class-I DHOD (Ura1) that uses fumarate as electron acceptor and thereby enables anaerobic pyrimidine synthesis. Here, we investigate DHODs from three fungi (the Neocallimastigomycete Anaeromyces robustus and the yeasts Schizosaccharomyces japonicus and Dekkera bruxellensis) that can grow anaerobically but, based on genome analysis, only harbor a Class-II DHOD. Results Heterologous expression of putative Class-II DHOD-encoding genes from fungi capable of anaerobic, pyrimidine-prototrophic growth (Arura9, SjURA9, DbURA9) in an S. cerevisiae ura1Δ strain supported aerobic as well as anaerobic pyrimidine prototrophy. A strain expressing DbURA9 showed delayed anaerobic growth without pyrimidine supplementation. Adapted faster growing DbURA9-expressing strains showed mutations in FUM1, which encodes fumarase. GFP-tagged SjUra9 and DbUra9 were localized to S. cerevisiae mitochondria, while ArUra9, whose sequence lacked a mitochondrial targeting sequence, was localized to the yeast cytosol. Experiments with cell extracts showed that ArUra9 used free FAD and FMN as electron acceptors. Expression of SjURA9 in S. cerevisiae reproducibly led to loss of respiratory competence and mitochondrial DNA. A cysteine residue (C265 in SjUra9) in the active sites of all three anaerobically active Ura9 orthologs was shown to be essential for anaerobic activity of SjUra9 but not of ArUra9. Conclusions Activity of fungal Class-II DHODs was long thought to be dependent on an active respiratory chain, which in most fungi requires the presence of oxygen. By heterologous expression experiments in S. cerevisiae, this study shows that phylogenetically distant fungi independently evolved Class-II dihydroorotate dehydrogenases that enable anaerobic pyrimidine biosynthesis. Further structure–function studies are required to understand the mechanistic basis for the anaerobic activity of Class-II DHODs and an observed loss of respiratory competence in S. cerevisiae strains expressing an anaerobically active DHOD from Sch. japonicus.

Microwave Heating Promotes the S-Alkylation of Aziridine Catalyzed by Molecular Sieves: A Post-Synthetic Approach to Lanthionine-Containing Peptides

Aziridine derivatives involved in nucleophilic ring-opening reactions have attracted great interest, since they allow the preparation of biologically active molecules. A chemoselective and mild procedure to convert a peptide cysteine residue into lanthionine via S-alkylation on aziridine substrates is presented in this paper. The procedure relies on a post-synthetic protocol promoted by molecular sieves to prepare lanthionine-containing peptides and is assisted by microwave irradiation. In addition, it represents a valuable alternative to the stepwise approach, in which the lanthionine precursor is incorporated into peptides as a building block.