Exploiting Chemical Protein Synthesis to Study the Role of Tyrosine Sulfation on Anticoagulants from Hematophagous Organisms.

Tyrosine sulfation is a post-translational modification (PTM) that modulates function by mediating key protein-protein interactions. One of the early proteins shown to possess this PTM was hirudin, produced in the salivary glands of the medicinal leech Hirudo medicinalis, whereby tyrosine sulfation led to a ∼10-fold improvement in α-thrombin inhibitory activity. Outside of this pioneering discovery, the involvement of tyrosine sulfation in modulating the activity of salivary proteins from other hematophagous organisms was unknown. We hypothesized that the intrinsic instability of the tyrosine sulfate functionality, particularly under the acidic conditions used to isolate and analyze peptides and proteins, has led to poor detection during the isolation and/or expression of these molecules.Herein, we summarize our efforts to interrogate the functional role of tyrosine sulfation in the thrombin inhibitory and anticoagulant activity of salivary peptides and proteins from a range of different blood feeding organisms, including leeches, ticks, mosquitoes, and flies. Specifically, we have harnessed synthetic chemistry to efficiently generate homogeneously sulfated peptides and proteins for detailed structure-function studies both in vitro and in vivo.Our studies began with the leech protein hirudin P6 (from Hirudinaria manillensis), which is both sulfated on tyrosine and O-glycosylated at a nearby threonine residue. Synthetically, this was achieved through solid-phase peptide synthesis (SPPS) with a late-stage on-resin sulfation, followed by native chemical ligation and a folding step to generate six differentially modified variants of hirudin P6 to assess the functional interplay between O-glycosylation and tyrosine sulfation. A one-pot, kinetically controlled ligation of three peptide fragments was used to assemble homogeneously sulfoforms of madanin-1 and chimadanin from the tick Haemaphysalis longicornis. Dual tyrosine sulfation at two distinct sites was shown to increase the thrombin inhibitory activity by up to 3 orders of magnitude through a novel interaction with exosite II of thrombin. The diselenide-selenoester ligation developed by our lab provided us with a means to rapidly assemble a library of different sulfated tick anticoagulant proteins: the andersonins, hyalomins, madanin-like proteins, and hemeathrins, thus enabling the generation of key structure-activity data on this family of proteins. We have also confirmed the presence of tyrosine sulfation in the anticoagulant proteins of Anopheles mosquitoes (anophelins) and the Tsetse fly (TTI) via insect expression and mass spectrometric analysis. These molecules were subsequently synthesized and assessed for thrombin inhibitory and anticoagulant activity. Activity was significantly improved by the addition of tyrosine sulfate modifications and led to molecules with potent antithrombotic activity in an in vivo murine thrombosis model.The Account concludes with our most recent work on the design of trivalent hybrids that tandemly occupy the active site and both exosites (I and II) of α-thrombin, with a TTI-anophelin hybrid (Ki = 20 fM against α-thrombin) being one of the most potent protease inhibitors and anticoagulants ever generated. Taken together, this Account highlights the importance of the tyrosine sulfate post-translational modification within salivary proteins from blood feeding organisms for enhancing anticoagulant activity. This work lays the foundation for exploiting native or engineered variants as therapeutic leads for thrombotic disorders in the future.

Site-selective photocatalytic functionalization of peptides and proteins at selenocysteine.

The importance of modified peptides and proteins for applications in drug discovery, and for illuminating biological processes at the molecular level, is fueling a demand for efficient methods that facilitate the precise modification of these biomolecules. Herein, we describe the development of a photocatalytic method for the rapid and efficient dimerization and site-specific functionalization of peptide and protein diselenides. This methodology, dubbed the photocatalytic diselenide contraction, involves irradiation at 450 nm in the presence of an iridium photocatalyst and a phosphine and results in rapid and clean conversion of diselenides to reductively stable selenoethers. A mechanism for this photocatalytic transformation is proposed, which is supported by photoluminescence spectroscopy and density functional theory calculations. The utility of the photocatalytic diselenide contraction transformation is highlighted through the dimerization of selenopeptides, and by the generation of two families of protein conjugates via the site-selective modification of calmodulin containing the 21st amino acid selenocysteine, and the C-terminal modification of a ubiquitin diselenide.

Synthesis of Protein-Oligonucleotide Conjugates.

Nucleic acids and proteins form two of the key classes of functional biomolecules. Through the ability to access specific protein-oligonucleotide conjugates, a broader range of functional molecules becomes accessible which leverages both the programmability and recognition potential of nucleic acids and the structural, chemical and functional diversity of proteins. Herein, we summarize the available conjugation strategies to access such chimeric molecules and highlight some key case study examples within the field to showcase the power and utility of such technology.

Optochemical Control of Therapeutic Agents through Photocatalyzed Isomerization.

A Ru(bpy)3 Cl2 photocatalyst is applied to the rapid trans to cis isomerization of a range of alkene-containing pharmacological agents, including combretastatin A-4 (CA-4), a clinical candidate in oncology, and resveratrol derivatives, switching their configuration from inactive substances to potent cytotoxic agents. Selective in cellulo activation of the CA-4 analog Res-3M is demonstrated, along with its potent cytotoxicity and inhibition of microtubule dynamics.

Expressed Protein Selenoester Ligation.

Herein, we describe the development and application of a novel expressed protein selenoester ligation (EPSL) methodology for the one-pot semi-synthesis of modified proteins. EPSL harnesses the rapid kinetics of ligation reactions between modified synthetic selenopeptides and protein aryl selenoesters (generated from expressed intein fusion precursors) followed by in situ chemoselective deselenization to afford target proteins at concentrations that preclude the use of traditional ligation methods. The utility of the EPSL technology is showcased through the efficient semi-synthesis of ubiquitinated polypeptides, lipidated analogues of the membrane-associated GTPase YPT6, and site-specifically phosphorylated variants of the oligomeric chaperone protein Hsp27 at high dilution.

Biosupramolecular networks: Taking inspiration from nature to create powerful synthetic platforms.

Nature is predicated on the ability to process large number of parallel signals to produce specific downstream outputs. Biosupramolecular networks are beginning to allow such processing power in synthetic systems, particularly through harnessing the recognition power of biomolecules. Such systems can be summarised through the reductionist view of containing inputs, circuitry motifs and functional outputs, with each of these elements able to be readily combined in a modular approach. Through the inherent 'plug and play' nature of these systems the field continues to rapidly expand, providing a wealth of new smart diagnostic and therapeutic systems.

Expressed Protein Selenoester Ligation.

Herein, we describe the development and application of a novel expressed protein selenoester ligation (EPSL) methodology for the one-pot semi-synthesis of modified proteins. EPSL harnesses the rapid kinetics of ligation reactions between modified synthetic selenopeptides and protein aryl selenoesters (generated from expressed intein fusion precursors) followed by in situ chemoselective deselenization to afford target proteins at concentrations that preclude the use of traditional ligation methods. The utility of the EPSL technology is showcased through the efficient semi-synthesis of ubiquitinated polypeptides, lipidated analogues of the membrane-associated GTPase YPT6, and site-specifically phosphorylated variants of the oligomeric chaperone protein Hsp27 at high dilution.

Total Synthesis of the Spider-Venom Peptide Hi1a.

Hi1a is a venom peptide from the Australian funnel-web spider Hadronyche infensa with a complex tertiary structure. Hi1a has neuroprotective and cardioprotective properties due to its potent inhibition of acid-sensing ion channel 1a (ASIC1a) and is currently being pursued as a novel therapy for acute ischemic events. Herein, we describe the total synthesis of Hi1a using native chemical ligation. The synthetic peptide was successfully folded and exhibited similar inhibitory activity on ASIC1a to recombinant Hi1a.

Biosupramolecular Systems: Integrating Cues into Responses.

Life is orchestrated by biomolecules interacting in complex networks of biological circuitry with emerging function. Progress in different areas of chemistry has made the design of systems that can recapitulate elements of such circuitry possible. Herein we review prominent examples of networks, the methodologies available to translate an input into various outputs, and speculate on potential applications and directions for the field. The programmability of nucleic acid hybridization has inspired applications beyond its function in heredity. At the circuitry level, DNA provides a powerful platform to design dynamic systems that respond to nucleic acid input sequences with output sequences through diverse logic gates, enabling the design of ever more complex circuitry. In order to interface with more diverse biomolecular inputs and yield outputs other than oligonucleotide sequences, an array of nucleic acid conjugates have been reported that can engage proteins as their input and yield a turn-on of enzymatic activity, a bioactive small molecule, or morphological changes in nanoobjects. While the programmability of DNA makes it an obvious starting point to design circuits, other biosupramolecular interactions have also been demonstrated, and harnessing progress in protein design is bound to deliver further integration of macromolecules in artificial circuits.

Potent Trivalent Inhibitors of Thrombin through Hybridization of Salivary Sulfopeptides from Hematophagous Arthropods.

Blood feeding arthropods, such as leeches, ticks, flies and mosquitoes, provide a privileged source of peptidic anticoagulant molecules. These primarily operate through inhibition of the central coagulation protease thrombin by binding to the active site and either exosite I or exosite II. Herein, we describe the rational design of a novel class of trivalent thrombin inhibitors that simultaneously block both exosites as well as the active site. These engineered hybrids were synthesized using tandem diselenide-selenoester ligation (DSL) and native chemical ligation (NCL) reactions in one-pot. The most potent trivalent inhibitors possessed femtomolar inhibition constants against α-thrombin and were selective over related coagulation proteases. A lead hybrid inhibitor possessed potent anticoagulant activity, blockade of both thrombin generation and platelet aggregation in vitro and efficacy in a murine thrombosis model at 1 mg kg-1 . The rational engineering approach described here lays the foundation for the development of potent and selective inhibitors for a range of other enzymatic targets that possess multiple sites for the disruption of protein-protein interactions, in addition to an active site.

Total Synthesis of Glycosylated Human Interferon-γ.

Interferon-γ (IFN-γ) is a glycoprotein that is responsible for orchestrating numerous critical immune induction and modulation processes and is used clinically for the treatment of a number of diseases. Herein, we describe the total chemical synthesis of homogeneously glycosylated variants of human IFN-γ using a tandem diselenide-selenoester ligation-deselenization strategy in the C- to N-terminal direction. The synthetic glycoproteins were successfully folded, and the structures and antiviral functions were assessed.

Diselenide-selenoester ligation for chemical protein synthesis.

Chemoselective peptide ligation methods have provided synthetic access to numerous proteins, including those bearing native post-translational modifications and unnatural labels. This protocol outlines the chemical synthesis of proteins using a recently discovered reaction (diselenide-selenoester ligation (DSL)) in a rapid, additive-free manner. After ligation, the products can be chemoselectively deselenized to produce native peptide and protein products. We describe methods for the synthesis of suitably functionalized peptide diselenide and peptide selenoester fragments via Fmoc-solid-phase peptide synthesis (SPPS) protocols, fusion of these fragments by DSL, and the chemoselective deselenization of the ligation products to generate native synthetic proteins. We demonstrate the method's utility through the total chemical synthesis of the post-translationally modified collagenous domain of the hormone adiponectin via DSL-deselenization at selenocystine (the oxidized form of selenocysteine) and the rapid preparation of two tick-derived thrombin-inhibiting proteins by DSL-deselenization at ß-selenoaspartate and γ-selenoglutamate. This method should find widespread use for the rapid synthesis of proteins, including cases in which other peptide ligation methods cannot be used (or cannot be used efficiently), e.g., at sterically hindered or deactivated acyl donors. The method's speed and efficiency may render it useful in the generation of synthetic protein libraries. Each protein discussed can be synthesized within 15 working days from resin loading and can be readily produced by practitioners with master's-level experience in organic chemistry. Each synthesis using these protocols was performed independently by two labs (one academic and one industrial), which attained comparable yields of the protein products.

Rapid assembly and profiling of an anticoagulant sulfoprotein library.

Hematophagous organisms produce a suite of salivary proteins which interact with the host's coagulation machinery to facilitate the acquisition and digestion of a bloodmeal. Many of these biomolecules inhibit the central blood-clotting serine proteinase thrombin that is also the target of several clinically approved anticoagulants. Here a bioinformatics approach is used to identify seven tick proteins with putative thrombin inhibitory activity that we predict to be posttranslationally sulfated at two conserved tyrosine residues. To corroborate the biological role of these molecules and investigate the effects of amino acid sequence and sulfation modifications on thrombin inhibition and anticoagulant activity, a library of 34 homogeneously sulfated protein variants were rapidly assembled using one-pot diselenide-selenoester ligation (DSL)-deselenization chemistry. Downstream functional characterization validated the thrombin-directed activity of all target molecules and revealed that posttranslational sulfation of specific tyrosine residues crucially modulates potency. Importantly, access to this homogeneously modified protein library not only enabled the determination of key structure-activity relationships and the identification of potent anticoagulant leads, but also revealed subtleties in the mechanism of thrombin inhibition, between and within the families, that would be impossible to predict from the amino acid sequence alone. The synthetic platform described here therefore serves as a highly valuable tool for the generation and thorough characterization of libraries of related peptide and/or protein molecules (with or without modifications) for the identification of lead candidates for medicinal chemistry programs.

A simple linearization method unveils hidden enzymatic assay interferences.

Enzymes are among the most important drug targets in the pharmaceutical industry. The bioassays used to screen enzyme modulators can be affected by unaccounted interferences such as time-dependent inactivation and inhibition effects. Using procaspase-3, caspase-3, and α-thrombin as model enzymes, we show that some of these effects are not eliminated by merely ignoring the reaction phases that follow initial-rate measurements. We thus propose a linearization method (LM) for detecting spurious changes of enzymatic activity based on the representation of progress curves in modified coordinates. This method is highly sensitive to signal readout distortions, thereby allowing rigorous selection of valid kinetic data. The method allows the detection of assay interferences even when their occurrence is not suspected a priori. By knowing the assets and liabilities of the bioassay, enzymology results can be reported with enhanced reproducibility and accuracy. Critical analysis of full progress curves is expected to help discriminating experimental artifacts from true mechanisms of enzymatic inhibition.

Native chemical ligation in protein synthesis and semi-synthesis.

Native chemical ligation (NCL) provides a highly efficient and robust means to chemoselectively link unprotected peptide and protein segments to generate proteins. The ability to incorporate non-proteinogenic amino acids (e.g.d-amino acids or fluorescent labels) and post-translational modifications into proteins by stitching together peptide fragments has driven extremely important developments in peptide and protein science over the past 20 years. Extensions of the original NCL concept (including the development of thiol- and selenol-derived amino acids and desulfurisation and deselenisation methods), improved access to peptide thioesters, and the use of the methodology in combination with recombinantly expressed polypeptide fragments (termed Expressed Protein Ligation, EPL) have helped to further expand the utility of the methodology. Over the past five years, there has been a dramatic increase in the number of proteins that have been accessed by total chemical synthesis and semi-synthesis, including a large range of modified proteins; new records have also been set with regards to the size of proteins that can now be accessed via ligation chemistry. Together these efforts have not only contributed to a better understanding of protein structure and function, but have also driven innovations in protein science. In this tutorial review, we aim to provide the reader with the latest developments in NCL- and EPL-based ligation technologies as well as illustrated examples of using these methods, together with synthetic logic, to access proteins and modified proteins for biological study.

Mosquito-Derived Anophelin Sulfoproteins Are Potent Antithrombotics.

The anophelins are small protein thrombin inhibitors that are produced in the salivary glands of the Anopheles mosquito to fulfill a vital role in blood feeding. A bioinformatic analysis of anophelin sequences revealed the presence of conserved tyrosine residues in an acidic environment that were predicted to be post-translationally sulfated in vivo. To test this prediction, insect cell expression of two anophelin proteins, from Anopheles albimanus and Anopheles gambiae, was performed, followed by analysis by mass spectrometry, which showed heterogeneous sulfation at the predicted sites. Homogeneously sulfated variants of the two proteins were subsequently generated by chemical synthesis via a one-pot ligation-desulfurization strategy. Tyrosine sulfation of the anophelins was shown to significantly enhance the thrombin inhibitory activity, with a doubly sulfated variant of the anophelin from A. albimanus exhibiting a 100-fold increase in potency compared with the unmodified homologue. Sulfated anophelins were also shown to exhibit potent in vivo anticoagulant and antithrombotic activity.

Solid-phase synthesis of peptide selenoesters via a side-chain anchoring strategy.

Peptide selenoesters have recently emerged as key building blocks for the ligation-based assembly of large polypeptides and proteins. Herein, we report an efficient solid-phase method for the high yielding and epimerisation-free synthesis of peptide selenoesters using a side-chain immobilisation strategy.

Sansanmycin natural product analogues as potent and selective anti-mycobacterials that inhibit lipid I biosynthesis.

Tuberculosis (TB) is responsible for enormous global morbidity and mortality, and current treatment regimens rely on the use of drugs that have been in use for more than 40 years. Owing to widespread resistance to these therapies, new drugs are desperately needed to control the TB disease burden. Herein, we describe the rapid synthesis of analogues of the sansanmycin uridylpeptide natural products that represent promising new TB drug leads. The compounds exhibit potent and selective inhibition of Mycobacterium tuberculosis, the etiological agent of TB, both in vitro and intracellularly. The natural product analogues are nanomolar inhibitors of Mtb phospho-MurNAc-pentapeptide translocase, the enzyme responsible for the synthesis of lipid I in mycobacteria. This work lays the foundation for the development of uridylpeptide natural product analogues as new TB drug candidates that operate through the inhibition of peptidoglycan biosynthesis.