Novel aza peptide inhibitors and active-site probes of papain-family cysteine proteases

Recent characterization of multiple classes of functionalized azapeptides as effective covalent inhibitors of cysteine proteases prompted us to investigate O-acyl hydroxamates and their azapeptide analogues for use as activity-based probes (ABPs). We report here a new class of azaglycine-containing O-acylhydroxamates that form stable covalent adducts with target proteases. This allows them to be used as ABPs for papain family cysteine proteases. A second class of related analogues containing a novel O-acyl hydroxyurea warhead was found to function as covalent inhibitors of papain-like proteases. These inhibitors can be easily synthesized on solid support, which allows rapid optimization of compounds with improved selectivity and potency for a given target enzyme. We present here one such optimized inhibitor that showed selective inhibition of falcipain 1, a protease of the malaria-causing parasite, Plasmodium falciparum.     

Novel Peptidyl Aryl Vinyl Sulfones as Highly Potent and Selective Inhibitors of Cathepsins L and B

Herein we present the design, synthesis, and evaluation of a structurally novel library of 20 peptidyl 3‐aryl vinyl sulfones as inhibitors of cathepsins L and B. The building blocks, described here for the first time, were synthesized in a highly efficient and enantioselective manner, starting from 3‐aryl‐substituted allyl alcohols. The corresponding vinyl sulfones were prepared by a new approach, based on a combination of solid‐phase peptide synthesis using the Fmoc/tBu strategy, followed by solution‐phase coupling to the corresponding (R)‐3‐amino‐3‐aryl vinyl sulfones as trifluoroacetate salts. The inhibitory activity of the resulting compounds against cathepsins L and B was evaluated, and the compound exhibiting the best activity was selected for enzymatic characterization. Finally, docking studies were performed in order to identify key structural features of the aryl substituent.     

Microbial Transglutaminase and c‐myc‐Tag: A Strong Couple for the Functionalization of Antibody‐Like Protein Scaffolds from Discovery Platforms

Antibody‐like proteins selected from discovery platforms are preferentially functionalized by site‐specific modification as this approach preserves the binding abilities and allows a side‐by‐side comparison of multiple conjugates. Here we present an enzymatic bioconjugation platform that targets the c‐myc‐tag peptide sequence (EQKLISEEDL) as a handle for the site‐specific modification of antibody‐like proteins. Microbial transglutaminase (MTGase) was exploited to form a stable isopeptide bond between the glutamine on the c‐myc‐tag and various primary‐amine‐functionalized substrates. We attached eight different functionalities to a c‐myc‐tagged antibody fragment and used these bioconjugates for downstream applications such as protein multimerization, immobilization on surfaces, fluorescence microscopy, fluorescence‐activated cell sorting, and in vivo nuclear imaging. The results demonstrate the versatility of our conjugation strategy for transforming a c‐myc‐tagged protein into any desired probe.     

The importance of the active site histidine for the activity of epoxide- or aziridine-based inhibitors of cysteine proteases

In the present study the importance of the active site histidine residue (His) for the activity of epoxide- or aziridine-based cysteine protease inhibitors is examined theoretically. To account for all important effects, QM/MM hybrid approaches are employed which combine quantum mechanical (QM) methods that are necessary to describe bond-breaking and formation processes, with molecular mechanics (MM) methods that incorporate the influence of the protein environment. Using various model systems, the computations exclude a direct proton shift from the active site His residue to the inhibitor, but show that one water molecule is sufficient to establish a very efficient relay system. This relay system allows an easy proton transfer from the active site His residue to the inhibitor and is thus essential for the activity of both types of inhibitors. Differences between the epoxides and the aziridines are discussed, along with some implications for the rational design of optimized inhibitors. The work presented herein represents the first QM/MM study into the mode of action of these important inhibitor classes.     

Activity-based probes for the study of proteases: recent advances and developments

Proteases are important targets for the treatment of human disease. Several protease inhibitors have failed in clinical trials due to a lack of in vivo specificity, indicating the need for studies of protease function and inhibition in complex, disease-related models. The tight post-translational regulation of protease activity complicates protease analysis by traditional proteomics methods. Activity-based protein profiling is a powerful technique that can resolve this issue. It uses small-molecule tools-activity-based probes-to label and analyze active enzymes in lysates, cells, and whole animals. Over the last twelve years, a wide variety of protease activity-based probes have been developed. These synthetic efforts have enabled techniques ranging from real-time in vivo imaging of protease activity to high-throughput screening of uncharacterized proteases. This Review introduces the general principles of activity-based protein profiling and describes the recent advancements in probe design and analysis techniques, which have increased the knowledge of protease biology and will aid future protease drug discovery.     

Phosphine-Activated Lysine Analogues for Fast Chemical Control of Protein Subcellular Localization and Protein SUMOylation

The Staudinger reduction and its variants have exceptional compatibility with live cells but can be limited by slow kinetics. Herein we report new small-molecule triggers that turn on proteins through a Staudinger reduction/self-immolation cascade with substantially improved kinetics and yields. We achieved this through site-specific incorporation of a new set of azidobenzyloxycarbonyl lysine derivatives in mammalian cells. This approach allowed us to activate proteins by adding a nontoxic, bioorthogonal phosphine trigger. We applied this methodology to control a post-translational modification (SUMOylation) in live cells, using native modification machinery. This work significantly improves the rate, yield, and tunability of the Staudinger reduction-based activation, paving the way for its application in other proteins and organisms.     

Quantitative Determination of Cellular Farnesyltransferase Activity: Towards Defining the Minimum Substrate Reactivity for Biologically Relevant Protein Farnesylation

Prenylation is a post‐translational modification wherein an isoprenoid group is attached to a protein substrate by a protein prenyltransferase. Hundreds of peptide sequences are in vitro substrates for protein farnesyltransferase (FTase), but it remains unknown which of these sequences can successfully compete for in vivo prenylation. Translating in vitro studies to predict in vivo protein farnesylation requires determining the minimum reactivity needed for modification by FTase within the cell. Towards this goal, we developed a reporter protein series spanning several orders of magnitude in FTase reactivity as a calibrated sensor for endogenous FTase activity. Our approach provides a minimally invasive method to monitor changes in cellular FTase activity in response to environmental or genetic factors. Determining the reactivity “threshold” for in vivo prenylation will help define the prenylated proteome and identify prenylation‐dependent pathways for therapeutic targeting.     

Cell-Permeable Ubiquitin and Histone Tools for Studying Post-translational Modifications

Protein post-translational modifications (PTMs) regulate nearly all biological processes in eukaryotic cells, and synthetic PTM protein tools are widely used to detect the activity of the related enzymes and identify the interacting proteins in cell lysates. Recently, the study of these enzymes and the interacting proteome has been accomplished in live cells using cell-permeable PTM protein tools. In this concept, we will introduce cell penetrating techniques, the syntheses of cell-permeable PTM protein tools, and offer some future perspective.     

The role played by the alpha-helix in the unfolding pathway and stability of azurin: switching between hierarchic and nonhierarchic folding

The role played by the alpha-helix in determining the structure, the stability and the unfolding mechanism of azurin was addressed by studying a helix-depleted azurin variant produced by site-directed mutagenesis. The protein structure was investigated by CD, 1D (1)H NMR, fluorescence spectroscopy measurements and MD simulations, whilst EPR, UV-visible and cyclic voltammetry experiments were carried out to investigate the geometry and the properties of the Cu(II) site. The effects of the alpha-helix depletion on the thermal stability and the unfolding pathway of the protein were determined by DSC, UV/visible and fluorescence measurements at increasing temperature. The results show that, in the absence of the alpha-helix segment, the overall protein structure is maintained, and that only the Cu site is slightly modified. In contrast, the protein stability is diminished by about 60% with respect to the wild-type azurin. Moreover, the unfolding pathway of the mutant azurin involves the presence of detectable intermediates. In comparison with previous studies concerning other small beta-sheet cupredoxins, the results as a whole support the hypothesis that the presence of the alpha-helix can switch the folding of azurin from a hierarchic to a nonhierarchic mechanism in which the highly conserved beta-sheet core provides a scaffold for cooperative folding of the wild-type protein.     

Structural Determinants for the Non‐Canonical Substrate Specificity of the ω‐Transaminase from Paracoccus denitrificans

Substrate binding pockets of ω‐transaminase (ω‐TA) consist of a large (L) pocket capable of dual recognition of hydrophobic and carboxyl substituents, and a small (S) pocket displaying a strict steric constraint that permits entry of a substituent no larger than an ethyl group. Despite the unique catalytic utility of ω‐TA enabling asymmetric reductive amination of carbonyl compounds, the severe size exclusion occurring in the S pocket has limited synthetic applications of ω‐TA to access structurally diverse chiral amines and amino acids. Here we report the first example of an ω‐TA whose S pocket shows a non‐canonical steric constraint and readily accommodates up to an n‐butyl substituent. The relaxed substrate specificity of the (S)‐selective ω‐TA, cloned from Paracoccus denitrificans (PDTA), afforded efficient asymmetric syntheses of unnatural amino acids carrying long alkyl side chains such as L ‐norvaline and L ‐norleucine. Molecular modeling using the recently released X‐ray structure of PDTA could pinpoint an exact location of the S pocket which had remained dubious. Entry of a hydrophobic substituent in the L pocket was found to have the S pocket accept up to an ethyl substituent, reminiscent of the canonical steric constraint. In contrast, binding of a carboxyl group to the L pocket induced a slight movement of V153 away from the small‐pocket‐forming residues. The resulting structural change elicited excavation of the S pocket, leading to formation of a narrow tunnel‐like structure allowing accommodation of linear alkyl groups of carboxylate‐bearing substrates. To verify the active site model, we introduced site‐directed mutagenesis to six active site residues and examined whether the point mutations alleviated the steric constraint in the S pocket. Consistent with the molecular modeling results, the V153A variant assumed an elongated S pocket and accepted even an n‐hexyl substituent. Our findings provide precise structural information on substrate binding to the active site of ω‐TA, which is expected to benefit rational redesign of substrate specificity of ω‐TA.

     

Flexible and general synthesis of functionalized phosphoisoprenoids for the study of prenylation in vivo and in vitro

Protein modification with isoprenoid lipids affects hundreds of signaling proteins in eukaryotic cells. Modification of isoprenoids with reporter groups is the main approach for the creation of probes for the analysis of protein prenylation in vitro and in vivo. Here, we describe a new strategy for the synthesis of functionalized phosphoisoprenoids that uses an aminederivatized isoprenoid scaffold as a starting point for the synthesis of functionalized phosphoisoprenoid libraries. This overcomes a long-standing problem in the field, where multistep synthesis had to be carried out for each individual isoprenoid analogue. The described approach enabled us to synthesize a range of new compounds, including two novel fluorescent isoprenoids that previously could not be generated by conventional means. The fluorescent probes that were developed using the described approach possess significant spectroscopic advantages to all previously generated fluorescent isoprenoid analogue. Using these analogues for flow cytometry and cell imaging, we analyzed the uptake of isoprenoids by mammalian cells and zebrafish embryos. Furthermore, we demonstrate that derivatization of the scaffold can be coupled in a one-pot reaction to enzymatic incorporation of the resulting isoprenoid group into proteins. This enables rapid evaluation of functional groups for compatibility with individual prenyltransferases and identification of the prenyltransferase specific substrates.     

Position‐Specific Incorporation of Fluorescent Non‐natural Amino Acids into Maltose‐Binding Protein for Detection of Ligand Binding by FRET and Fluorescence Quenching

FRETting about MBP : Position‐specific incorporation of fluorescent groups is a useful method for analysis of the functions and structures of proteins. Here we demonstrate that position‐specific incorporation of fluorescent non‐natural amino acids in response to expanded codons enables us to detect ligand‐binding activity of maltose‐binding protein (MBP) through fluorescence resonance energy transfer (FRET) and ligand‐dependent fluorescence quenching.

     

The search for novel human pancreatic alpha-amylase inhibitors: high-throughput screening of terrestrial and marine natural product extracts

Specific inhibitors of human pancreatic alpha-amylase (HPA) have potential as oral agents for the control of blood glucose levels in the treatment of diabetes and obesity. In a search for novel inhibitors, a library of 30 000 crude biological extracts of terrestrial and marine origin has been screened. A number of inhibitory extracts were identified, of which the most potent was subjected to bioassay-guided purification. A family of three glycosylated acyl flavonols, montbretins A-C, was thereby identified and characterized as competitive amylase inhibitors, with K(i) values ranging from 8.1-6100 nM. Competitive inhibition by myricetin, which corresponds to the flavone core, and noncompetitive inhibition by a second fragment, ethyl caffeiate, suggest a binding mode for these inhibitors.