Remarkable activity of a novel cyclic seleninate ester as a glutathione peroxidase mimetic and its facile in situ generation from allyl 3-hydroxypropyl selenide

1,2-Oxaselenolane Se-oxide is a novel cyclic seleninate ester that functions as a remarkably efficient glutathione peroxidase mimetic by catalyzing the reduction of tert-butyl hydroperoxide to tert-butyl alcohol in the presence of benzyl thiol. The seleninate ester can be conveniently generated in situ by oxidation of allyl 3-hydroxypropyl selenide with tert-butyl hydroperoxide. Its catalytic activity surpasses that of several other known GPx mimetics containing cyclic selenenamide structures, which were also tested for comparison.     

A Thiol–Ene Coupling Approach to Native Peptide Stapling and Macrocyclization

We report the discovery of a peptide stapling and macrocyclization method using thiol–ene reactions between two cysteine residues and an α,ω‐diene in high yields. This new approach enabled us to selectively modify cysteine residues in native, unprotected peptides with a variety of stapling modifications for helix stabilization or general macrocyclization. We synthesized stapled Axin mimetic analogues and demonstrated increased alpha helicity upon peptide stapling. We then synthesized stapled p53 mimetic analogues using pure hydrocarbon linkers and demonstrated their abilities to block the p53‐MDM2 interaction and selectively kill p53 wild‐type colorectal carcinoma HCT‐116 cells but not p53 null cells. In summary, we demonstrated a robust and versatile peptide stapling method that could be potentially applied to both synthetic and expressed peptides.     

Analysis of phosphorylation sites on focal adhesion kinase using nanospray liquid chromatography/multiple reaction monitoring mass spectrometry

An approach based on nanospray liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) was developed in order to analyze twenty-nine phosphorylated and non-phosphorylated tryptic peptides from focal adhesion kinase (FAK). All peptides monitored were resolved and showed excellent peak shape with the exception of one doubly phosphorylated peptide. Optimization of the LC method enabled the identification and subsequent monitoring of six important tyrosine phosphorylation sites on FAK, including phosphorylated Y397 (pY397), pY407, pY576, pY577, pY861, and pY925. This technique was able to identify sites of phosphorylation on FAK as well as qualitatively differentiate between autocatalytic and Src-induced phosphorylation events. FAK was shown to have autocatalytic function, which resulted in efficient phosphorylation of Y397. FAK was also capable of autophosphorylation on residues Y407 and Y576, though apparently less effectively than autophosphorylation at Y397. Src was found to phosphorylate FAK at Y407, Y576, Y577, and Y861. The presence of Src increased the abundance of pY576 at low temperature indicating Src had particularly high kinase activity toward this residue. Furthermore, Src phosphorylated FAK at Y577 to produce FAK bis-phosphorylated at Y576 and Y577. In addition, six novel sites of phosphorylation (Y148, Y347, Y441, T503, S850, and Y1007) were identified on FAK. Interestingly, Src phosphorylated FAK to form a peptide uniquely phosphorylated on Y407, together with substantial amounts of the bis-phosphorylated pY397pY407 peptide. These findings will impact significantly on future studies of FAK activity since pY397 is often used as a measure of FAK activity and Src association.     

A photoactivable probe for calcium binding proteins

Ca2+ as a signaling molecule carries information pivotal to cell life and death via its reversible interaction with a specific site in a protein. Although numerous Ca2+-dependent activities are known, the proteins responsible for some of these activities remain unidentified. We synthesized and characterized a photoreactive reagent, azido ruthenium (AzRu), which interacts specifically with Ca2+ binding proteins and strongly inhibits their Ca2+-dependent activities, regardless of their catalytic mechanisms or functional state as purified proteins, embedded in the membrane or in intact cells. As expected from a Ca2+ binding protein-specific reagent, AzRu had no effect on Ca2+-independent and Mg2+-dependent activities. Az103Ru covalently bound, and specifically labeled, known Ca2+ binding proteins. AzRu is a photoreactive reagent that provides an approach for identification of Ca2+ binding proteins, characterization of their binding sites, and exploration of new Ca2+-dependent processes.     

Enhancing the modularity of the modular polyketide synthases: transacylation in modular polyketide synthases catalyzed by malonyl-CoA:ACP transacylase

Selective incorporation of extender units in modular polyketide synthases is primarily controlled by acyl transferase (AT) domains. The AT domains catalyze transacylation of the extender unit from acyl-CoA to the phosphopantetheine arm of an acyl carrier protein (ACP) domain in the same module. New methods that can modulate the extender unit specificity of individual modules with minimal structural or kinetic perturbations in the engineered module are desirable for the efficient biosynthesis of novel natural product analogues. We have demonstrated that transacylation of malonyl groups onto an AT-null form of a mutant modular polyketide synthase by malonyl-CoA:ACP transacylase is an effective strategy for the engineered biosynthesis of site specifically modified polyketides. Using this strategy, 6-deoxyerythronolide B synthase was engineered to exclusively produce 2-desmethyl-6-deoxyerythronolide B. The productivity of the modified system was comparable to that of the wild-type synthase in vitro and in vivo.     

Chemoselective ligation applied to the synthesis of a biantennary N-linked glycoform of CD52

We report here a strategy for the synthesis of N-linked glycopeptide analogues that replace the glycosidic linkages extending from the core pentasaccharide with thioethers amenable to construction by chemoselective ligation. The key building block, a pentasaccharide-Asn analogue containing two thiol residues, was incorporated into CD52 by 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis. An undecasaccharide mimetic was then readily generated by alkylation of this glycopeptide with an N-bromoacetamido trisaccharide. The rapid assembly of a complex type N-linked glycopeptide mimetic was accomplished using this technique.     

Discovery of a Potent Allosteric Kinase Modulator by Combining Computational and Synthetic Methods

The rational design of allosteric kinase modulators is challenging but rewarding. The protein kinase PDK1, which lies at the center of the growth‐factor signaling pathway, possesses an allosteric regulatory site previously validated both in vitro and in cells. ANCHOR.QUERY software was used to discover a potent allosteric PDK1 kinase modulator. Using a recently published PDK1 compound as a template, several new scaffolds that bind to the allosteric target site were generated and one example was validated. The inhibitor can be synthesized in one step by multicomponent reaction (MCR) chemistry when using the ANCHOR.QUERY approach. Our results are significant because the outlined approach allows rapid and efficient scaffold hopping from known molecules into new easily accessible and biologically active ones. Based on increasing interest in allosteric‐site drug discovery, we foresee many potential applications for this approach.     

Uracil-directed ligand tethering: an efficient strategy for uracil DNA glycosylase (UNG) inhibitor development

Uracil DNA glycosylase (UNG) is an important DNA repair enzyme that recognizes and excises uracil bases in DNA using an extrahelical recognition mechanism. It is emerging as a desirable target for small-molecule inhibitors given its key role in a wide range of biological processes including the generation of antibody diversity, DNA replication in a number of viruses, and the formation of DNA strand breaks during anticancer drug therapy. To accelerate the discovery of inhibitors of UNG we have developed a uracil-directed ligand tethering strategy. In this efficient approach, a uracil aldehyde ligand is tethered via alkyloxyamine linker chemistry to a diverse array of aldehyde binding elements. Thus, the mechanism of extrahelical recognition of the uracil ligand is exploited to target the UNG active site, and alkyloxyamine linker tethering is used to randomly explore peripheral binding pockets. Since no compound purification is required, this approach rapidly identified the first small-molecule inhibitors of human UNG with micromolar to submicromolar binding affinities. In a surprising result, these uracil-based ligands are found not only to bind to the active site but also to bind to a second uncompetitive site. The weaker uncompetitive site suggests the existence of a transient binding site for uracil during the multistep extrahelical recognition mechanism. This very general inhibitor design strategy can be easily adapted to target other enzymes that recognize nucleobases, including other DNA repair enzymes that recognize other types of extrahelical DNA bases.     

A chemoenzymatic approach enables the site‐specific conjugation of recombinant proteins

Many biotechniques including protein microarray, drug screening, biosensors rely on the immobilization of recombinant proteins on the solid supports. It is well known that random orientation of the immobilized proteins could impair their biologic functions. Thus, it is very important to develop new site‐specific immobilization approach. In this study, we presented a chemoenzymatic approach for site‐specific conjugation of recombinant proteins onto solid support. In this strategy, the affinity tag on recombinant protein was enzymatically cleaved to expose the N‐terminal serine, which was oxidized to carry an aldehyde group and was then covalently coupled to hydrazide resin through hydrazone ligation. As this approach takes advantage of the most frequently used TEV protease, it requires no further sequence design on recombinant protein. This method was validated by site specific coupling of a synthetic peptide and a recombinant protein onto solid supports. It was found that the site specific immobilized SH2 domain is functional and could be used to enrich tyrosine phosphorylated peptides.     

Exocyclic Olefinic Maleimides: Synthesis and Application for Stable and Thiol‐Selective Bioconjugation

Michael addition reactions between biological thiols and endocyclic olefinic maleimides are extensively used for site‐specific bioconjugation. The resulting thio‐succinimidyl linkages, however, lack stability because of their susceptibility to thiol exchange. Reported herein is that in contrast to their endocyclic counterparts, exocyclic olefinic maleimides form highly stable thio‐Michael adducts which resist thiol exchange at physiological conditions. A high‐yielding approach for synthesizing a variety of exocyclic olefinic maleimides, by 4‐nitrophenol‐catalyzed solvent‐free Wittig reactions, is reported. Mechanistic studies reveal that the catalyst facilitates the formation of the Wittig ylide intermediate through sequential proton donation and abstraction. Overall, this report details an improved thiol bioconjugation approach, a facile method for synthesizing exocyclic olefinic maleimides, and demonstrates that phenolic compounds can catalyze ylide formation.     

Discovery of cryptic allosteric sites using reversed allosteric communication by a combined computational and experimental strategy

Allostery, which is one of the most direct and efficient methods to fine-tune protein functions, has gained increasing recognition in drug discovery. However, there are several challenges associated with the identification of allosteric sites, which is the fundamental cornerstone of drug design. Previous studies on allosteric site predictions have focused on communication signals propagating from the allosteric sites to the orthosteric sites. However, recent biochemical studies have revealed that allosteric coupling is bidirectional and that orthosteric perturbations can modulate allosteric sites through reversed allosteric communication. Here, we proposed a new framework for the prediction of allosteric sites based on reversed allosteric communication using a combination of computational and experimental strategies (molecular dynamics simulations, Markov state models, and site-directed mutagenesis). The desirable performance of our approach was demonstrated by predicting the known allosteric site of the small molecule MDL-801 in nicotinamide dinucleotide (NAD⁺)-dependent protein lysine deacetylase sirtuin 6 (Sirt6). A potential novel cryptic allosteric site located around the L116, R119, and S120 residues within the dynamic ensemble of Sirt6 was identified. The allosteric effect of the predicted site was further quantified and validated using both computational and experimental approaches. This study proposed a state-of-the-art computational pipeline for detecting allosteric sites based on reversed allosteric communication. This method enabled the identification of a previously uncharacterized potential cryptic allosteric site on Sirt6, which provides a starting point for allosteric drug design that can aid the identification of candidate pockets in other therapeutic targets.

Using reversed allosteric communication, we performed MD simulations, MSMs, and mutagenesis experiments, to discover allosteric sites. It reproduced the known allosteric site for MDL-801 on Sirt6 and uncovered a novel cryptic allosteric Pocket X.     

Phosphopeptide selective coordination complexes as promising SRC homology 2 domain mimetics

Src Homology 2 (SH2) domains are the paradigm of phosphotyrosine (pY) protein recognition modules and mediate numerous cancer-promoting protein-protein complexes. Effective SH2 domain mimicry with pY-binding coordination complexes offers a promising route to new and selective disruptors of pY-mediated protein-protein interactions. We herein report the synthesis and in vitro characterization of a library of coordination complex SH2 domain proteomimetics. Compounds were designed to interact with phosphopeptides via a two-point interaction, principally with pY, and to make secondary interactions with pY+2/3, thereby achieving sequence-selective discrimination. Here, we report that lead mimetics demonstrated high target phosphopeptide affinity (K(a) ～ 10(7) M(-1)) and selectivity. In addition, biological screening in various tumor cells for anticancer effects showed a high degree of variability in cytotoxicity among receptors, which supported the proposed two-point binding mode. Several receptors potently disrupted cancer cell viability in breast cancer, prostate cancer, and acute myeloid leukemia cell lines.     

Solid‐Phase Synthesis of a 1‐Thio‐β‐d‐GlcNAc Carbohydrate Mimetic Library

The solid phase synthesis of N‐acetyl‐2‐deoxy‐1‐thio‐β‐d‐glucopyranoside derivatives by reacting an immobilized sugar thiol with Michael acceptors and α‐chloroketones, followed by ketone reductions, reductive aminations, acylations and alkylations was developed to yield a library of 1088 compounds. Such carbohydrate mimetic libraries are synthesized efficiently on the solid phase without the need for protection of the sugar hydroxyl groups. The library was designed for the identification of potential inhibitors of β‐d‐GlcNAc binding proteins.     

Covalent modification of actin by 4-hydroxy-trans-2-nonenal (HNE): LC-ESI-MS/MS evidence for Cys374 Michael adduction

We demonstrate for the first time, by a combined mass spectrometric and computational approach, that G- and F-actin can be covalently modified by the lipid-derived aldehyde, 4-hydroxy-trans-2-nonenal, providing information on the molecular mass of modified protein and the mechanism and site of adduction.ESI-MS analysis of actin treated with different molar ratios of HNE (1 : 1 to 1 : 20) showed the formation of a protein derivative in which there was an increase of 156 Da (42028 Da) over native actin (41872 Da), consistent with the adduction of one HNE residue through Michael addition. To identify the site of HNE adduction, G- and F-actin were stabilized by NaBH(4) reduction and digested with trypsin. LC-ESI-MS/MS analysis in data-dependent scan mode of the resulting peptides unequivocally indicated that Cys374 is the site of HNE adduction. Computational studies showed that the reactivity of Cys374 residue is due to a significant accessible surface and substantial thiol acidity due to the particular microenvironment surrounding Cys374.     

Structural mimicry of retroviral tat proteins by constrained beta-hairpin peptidomimetics: ligands with high affinity and selectivity for viral TAR RNA regulatory elements

An approach is described to the design of beta-hairpin peptidomimetic ligands for bovine immunodeficiency virus (BIV) Tat protein, which inhibit binding to its transactivator response element (TAR) RNA. A library of peptidomimetics was derived by grafting onto a hairpin-inducing d-Pro-l-Pro template sequences related to the RNA recognition element in Tat. One hairpin mimetic was identified that binds tightly (K(d) approximately 150 nM) to BIV TAR, and another that binds also to HIV-1 TAR RNA (K(d) approximately 1-2 microM). (In the same assay, the wild-type BIV Tat(65-81) peptide binds to BIV TAR with K(d) approximately 50 nM.) The high-affinity BIV-Tat mimetic was shown to adopt a stable beta-hairpin conformation in free solution by NMR methods. Amino acid substitutions in this mimetic were shown to impact on the hairpin structure and to disrupt binding to the RNA. This family of conformationally constrained peptidomimetics affords insights into the structural requirements for binding to TAR RNA and provides a basis for the design of new ligands with increased inhibitory activity and specificity to both BIV and HIV TAR RNAs.     

Internal Spin Trapping of Thiyl Radical during the Complexation and Reduction of Cobalamin with Glutathione and Dithiothrietol

The activation of cobalamin requires the reduction of Cbl(III) to Cbl(II). The reduction by glutathione and dithiothreitol was followed using visible spectroscopy and electron paramagnetic resonance. In addition the oxidation of glutathione was monitored. Glutathione first reacts with oxidized Cbl(III). The binding of a second glutathione required for the reduction to Cbl(II) is presumably located in the dimethyl benzimidazole ribonucleotide ligand cavity. The reduction of Cbl(III) by dithiothreitol, which contains two thiols, is much faster even though no stable Cbl(III) complex is formed. The reduction, by both thiol reagents, results in the formation of thiyl radicals, some of which are released to form oxidized thiol products and some of which remain associated with the reduced cobalamin. In the reduced state the intrinsic lower affinity for the benzimidazole base, coupled with a trans effect from the initial GSH bound to the β-axial site and a possible lowering of the pH results in an equilibrium between base-on and base-off complexes. The dissociation of the base facilitates a closer approach of the thiyl radical to the Co(II) α-axial site resulting in a complex with ferromagnetic exchange coupling between the metal ion and the thiyl radical. This is a unique example of 'internal spin trapping' of a thiyl radical formed during reduction. The finding that the reduction involves a peripheral site and that thiyl radicals produced during the reduction remain associated with the reduced cobalamin provide important new insights into our understanding of the formation and function of cobalamin enzymes.     

Gel-based fluorescent proteomic tools for investigating cell redox signaling. A mini-review

The specific chemical reactivity of thiol groups makes protein cysteines susceptible to reactions with reactive oxygen species (ROS) and reactive nitrogen species (RNS) resulting in the formation of various reversible and irreversible oxidative post-translational modifications (oxPTMs). This review highlights a number of gel-based redox proteomic approaches to detect protein oxPTMs, with particular emphasis on S-nitrosylation, which we believe are currently one of the most accurate way to analyze changes in the redox status of proteins. The information collected in this review relates to the recent progress regarding methods for the enrichment and identification of redox-modified proteins, with an emphasis on fluorescent gel proteomics. Gel-based fluorescent proteomic strategies are low-cost and easy-to-use tools for investigating the thiol proteome and can provide substantial information on redox signaling.