Design,synthesis, and evaluation of gluten peptide analogs as selective inhibitors of human tissue transglutaminase

Recent studies have implicated a crucial role for tissue transglutaminase (TG2) in the pathogenesis of Celiac Sprue, a disorder of the small intestine triggered in genetically susceptible individuals by dietary exposure to gluten. Proteolytically stable peptide inhibitors of human TG2 were designed containing acivicin or alternatively 6-diazo-5-oxo-norleucine (DON) as warheads. In biochemical and cell-based assays, the best of these inhibitors, Ac-PQP-(DON)-LPF-NH(2), was considerably more potent and selective than other TG2 inhibitors reported to date. Selective pharmacological inhibition of extracellular TG2 should be useful in exploring the mechanistic implications of TG2-catalyzed modification of dietary gluten, a phenomenon of considerable relevance in Celiac Sprue.     

Chemical proteomic probes for profiling cytochrome p450 activities and drug interactions in vivo

The cytochrome P450 (P450) superfamily metabolizes many endogenous signaling molecules and drugs. P450 enzymes are regulated by posttranslational mechanisms in vivo, which hinders their functional characterization by conventional genomic or proteomic methods. Here we describe a chemical proteomic strategy to profile P450 activities directly in living systems. Derivatization of a mechanism-based inhibitor with a "clickable" handle provided an activity-based probe that labels multiple P450s both in proteomic extracts and in vivo. This probe was used to record alterations in liver P450 activities triggered by chemical agents, including inducers of P450 expression and direct P450 inhibitors. The chemical proteomic strategy described herein thus offers a versatile method to monitor P450 activities and small-molecule interactions in any biological system and, through doing so, should facilitate the functional characterization of this large and diverse enzyme class.     

Cinnamoyl inhibitors of tissue transglutaminase

Transglutaminases (TGases) catalyze the intermolecular cross-linking of certain proteins and tissue TGases (TG2) are involved in diverse biological processes. Unregulated, high TGase activities have been implicated in several physiological disorders, but few reversible inhibitors of TG2 have been reported. Herein, we report the synthesis of a series of novel trans-cinammoyl derivatives, discovered to be potent inhibitors of guinea pig liver transglutaminase. The most effective inhibitors evaluated can be sorted into two subclasses: substituted cinnamoyl benzotriazolyl amides and the 3-(substituted cinnamoyl)pyridines, referred to more commonly as azachalcones. Kinetic evaluation of both of these subclasses revealed that they display reversible inhibition and are competitive with acyl donor TGase substrates at IC50 values as low as 18 microM. An analysis of structure-activity relationships within these series of inhibitors permitted the identification of potentially important binding interactions. Further testing of some of the most potent inhibitors demonstrated their selectivity for TG2 and their potential for further development.     

Optimization of activity-based probes for proteomic profiling of histone deacetylase complexes

Histone deacetylases (HDACs) are key enzymatic regulators of the epigenome and serve as promising targets for anticancer therapeutics. Recently, we developed a photoreactive "clickable" probe, SAHA-BPyne, to report on HDAC activity and complex formation in native biological systems. Here, we investigate the selectivity, sensitivity, and inhibitory properties of SAHA-BPyne and related potential activity-based probes for HDACs. While we identified several probes that are potent HDAC inhibitors and label HDAC complex components in native proteomic preparations, SAHA-BPyne was markedly superior for profiling HDAC activities in live cells. Interestingly, the enhanced performance of SAHA-BPyne as an in situ activity-based probe could not be solely ascribed to potency in HDAC binding, implying that other features of the molecule were key to efficient active site-directed labeling in living systems. Finally, we demonstrate the value of in situ profiling of HDACs by comparing the activity and expression of HDAC1 in cancer cells treated with the cytotoxic agent parthenolide. These results underscore the utility of activity-based protein profiling for studying HDAC function and may provide insight for the future development of click chemistry-based photoreactive probes for the in situ analysis of additional enzyme activities.     

Tissue transglutaminase inhibition

Khosla and coworkers report the synthesis of peptidic dihydroisoxazole derivatives, the in vitro evaluation of these novel compounds as inhibitors of recombinant human tissue transglutaminase (TG2), and their oral bioavailability and efficacy for the synergistic treatment of glioblastoma tumors.     

Core-Clickable PEG-Branch-Azide Bivalent-Bottle-Brush Polymers by ROMP: Grafting-Through and Clicking-To

The combination of highly efficient polymerizations with modular "click" coupling reactions has enabled the synthesis of a wide variety of novel nanoscopic structures. Here we demonstrate the facile synthesis of a new class of clickable, branched nanostructures, polyethylene glycol (PEG)-branch-azide bivalent-brush polymers, facilitated by "graft-through" ring-opening metathesis polymerization of a branched norbornene-PEG-chloride macromonomer followed by halide-azide exchange. The resulting bivalent-brush polymers possess azide groups at the core near a polynorbornene backbone with PEG chains extended into solution; the structure resembles a unimolecular micelle. We demonstrate copper-catalyzed azide-alkyne cycloaddition (CuAAC) "click-to" coupling of a photocleavable doxorubicin (DOX)-alkyne derivative to the azide core. The CuAAC coupling was quantitative across a wide range of nanoscopic sizes (～6-～50 nm); UV photolysis of the resulting DOX-loaded materials yielded free DOX that was therapeutically effective against human cancer cells.     

Transglutaminase 2 inhibits apoptosis induced by calciumoverload through down-regulation of Bax

An abrupt increase of intracellular Ca²⁺ is observed in cells under hypoxic or oxidatively stressed conditions. The dysregulated increase of cytosolic Ca²⁺ triggers apoptotic cell death through mitochondrial swelling and activation of Ca²⁺-dependent enzymes. Transglutaminase 2 (TG2) is a Ca²⁺-dependent enzyme that catalyzes transamidation reaction producing cross-linked and polyaminated proteins. TG2 activity is known to be involved in the apoptotic process. However, the pro-apoptotic role of TG2 is still controversial. In this study, we investigate the role of TG2 in apoptosis induced by Ca²⁺-overload. Overexpression of TG2 inhibited the {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187-induced apoptosis through suppression of caspase-3 and -9 activities, cytochrome c release into cytosol, and mitochondria membrane depolarization. Conversely, down-regulation of TG2 caused the increases of cell death, caspase-3 activity and cytochrome c in cytosol in response to Ca²⁺-overload. Western blot analysis of Bcl-2 family proteins showed that TG2 reduced the expression level of Bax protein. Moreover, overexpression of Bax abrogated the anti-apoptotic effect of TG2, indicating that TG2-mediated suppression of Bax is responsible for inhibiting cell death under Ca²⁺-overloaded conditions. Our findings revealed a novel anti-apoptotic pathway involving TG2, and suggested the induction of TG2 as a novel strategy for promoting cell survival in diseases such as ischemia and neurodegeneration.     

Ortho‐Stabilized 18F‐Azido Click Agents and their Application in PET Imaging with Single‐Stranded DNA Aptamers

Azido ¹⁸F‐arenes are important and versatile building blocks for the radiolabeling of biomolecules via Huisgen cycloaddition (“click chemistry”) for positron emission tomography (PET). However, routine access to such clickable agents is challenged by inefficient and/or poorly defined multistep radiochemical approaches. A high‐yielding direct radiofluorination for azido ¹⁸F‐arenes was achieved through the development of an ortho‐oxygen‐stabilized iodonium derivative (OID). This OID strategy addresses an unmet need for a reliable azido ¹⁸F‐arene clickable agent for bioconjugation reactions. A ssDNA aptamer was radiolabeled with this agent and visualized in a xenograft mouse model of human colon cancer by PET, which demonstrates that this OID approach is a convenient and highly efficient way of labeling and tracking biomolecules.     

Synthesis of glycopolymers via click reactions

This mini-review describes recent work in the field of glycopolymer synthesis, with a focus on methods that have employed “click chemistry” and controlled polymerization methodology. A variety of carbohydrates with clickable groups such as azide, alkyne, and thiol moieties provide new routes to glycopolymers. Several studies use copper catalyzed azide-alkyne cycloaddition (CuAAC) reactions to synthesize glycomonomers or to incorporate carbohydrates into a clickable polymeric backbone. Alternatively, there are many thiol based click reactions which provide metal-free synthesis, which are discussed in details.     

Two-prong inhibitors for human carbonic anhydrase II

The enzyme inhibitors are usually designed by taking into consideration the overall dimensions of the enzyme's active site pockets. This conventional approach often fails to produce desirable affinities of inhibitors for their cognate enzymes. To circumvent such constraints, we contemplated enhancing the binding affinities of inhibitors by attaching tether groups, which would interact with the surface exposed amino acid residues. This strategy has been tested for the inhibition of human carbonic anhydrase II. Benzenesulfonamide serves as a weak inhibitor for the enzyme, but when it is conjugated to iminodiacetate-Cu2+ (which interacts with the surface-exposed His residues) via a spacer group, its binding affinity is enhanced by about 2 orders of magnitude. This "two-prong" approach is expected to serve as a general strategy for converting weak inhibitors of enzymes into tight-binding inhibitors.     

Recent approaches for clickable poly(2-oxazoline)-based functional stimuli-responsive polymers and related applications

Abstract

Poly(2-oxazoline)s (POxs) are well-known thermo-responsive polymers that exhibit reversible hydrophilic–hydrophobic phase transitions at the lower critical solution temperature (LCST). Using living cationic ring-opening polymerisation, various functional groups can be introduced into POxs. Several clickable POxs with propargyl or azide end groups have been designed and subsequently reacted with various functional groups to prepare multifunctional POxs that respond to stimuli such as temperature, pH, chemicals and light. In this article, we briefly review recent approaches for clickable POx-based functional stimuli-responsive polymers and related applications.     

Biotinylated thermoresponsive core cross-linked nanoparticles via RAFT polymerization and "click" chemistry

A straightforward approach to the synthesis of "clickable" thermoresponsive core cross-linked (CCL) nanoparticles was developed. This approach was based on reversible addition-fragmentation chain transfer (RAFT) radical cross-linking polymerization of styrene and divinylbenzene with azide-functionalized poly(N-isopropylacrylamide) (PNIPAM-N(3)) as macro chain transfer agent in a selective solvent. Spherical nanoparticles with a diameter of 12nm were obtained after 24h polymerization. When the lyophilized CCL nanoparticles were dispersed in THF, spherical nanoparticles were observed, confirming the stability of CCL nanoparticles. The transmission electron microscopy (TEM) studies demonstrated that spherical nanoparticles and wormlike structure coexisted in the aqueous solution. The CCL nanoparticles have a lower critical solution temperature (LCST) at about 29.6°C, a little lower than that of PNIPAM homopolymer. Biotin molecules were conjugated to the surface of CCL nanoparticles via "click" chemistry in aqueous media. After bioconjugation, the LCST shifted to 28.3°C. The bioavailability of biotin to protein avidin was evaluated by a 4'-hydroxyazobenzene-2-carboxylic acid/avidin (HABA/avidin) binding assay and TEM.     

Labeling substrates of protein arginine methyltransferase with engineered enzymes and matched S-adenosyl-L-methionine analogues

Elucidating physiological and pathogenic functions of protein methyltransferases (PMTs) relies on knowing their substrate profiles. S-adenosyl-L-methionine (SAM) is the sole methyl-donor cofactor of PMTs. Recently, SAM analogues have emerged as novel small-molecule tools to efficiently label PMT substrates. Here we reported the development of a clickable SAM analogue cofactor, 4-propargyloxy-but-2-enyl SAM, and its implementation to label substrates of human protein arginine methyltransferase 1 (PRMT1). In the system, the SAM analogue cofactor, coupled with matched PRMT1 mutants rather than native PRMT1, was shown to label PRMT1 substrates. The transferable 4-propargyloxy-but-2-enyl moiety of the SAM analogue further allowed corresponding modified substrates to be characterized through a subsequent click chemical ligation with an azido-based probe. The SAM analogue, in combination with a rational protein-engineering approach, thus shows potential to label and identify PMT targets in the context of a complex cellular mixture.     

In Situ Proteome Profiling and Bioimaging Applications of Small‐Molecule Affinity‐Based Probes Derived From DOT1L Inhibitors

DOT1L is the sole protein methyltransferase that methylates histone H3 on lysine 79 (H3K79), and is a promising drug target against cancers. Small‐molecule inhibitors of DOT1L such as FED1 are potential anti‐cancer agents and useful tools to investigate the biological roles of DOT1L in human diseases. FED1 showed excellent in vitro inhibitory activity against DOT1L, but its cellular effect was relatively poor. In this study, we designed and synthesized photo‐reactive and “clickable” affinity‐based probes (AfBPs), P1 and P2 , which were cell‐permeable and structural mimics of FED1 . The binding and inhibitory effects of these two probes against DOT1L protein were extensively investigated in vitro and in live mammalian cells (in situ). The cellular uptake and sub‐cellular localization properties of the probes were subsequently studied in live‐cell imaging experiments, and our results revealed that, whereas both P1 and P2 readily entered mammalian cells, most of them were not able to reach the cell nucleus where functional DOT1L resides. This offers a plausible explanation for the poor cellular activity of FED1 . Finally with P1 / P2 , large‐scale cell‐based proteome profiling, followed by quantitative LC‐MS/MS, was carried out to identify potential cellular off‐targets of FED1 . Amongst the more than 100 candidate off‐targets identified, NOP2 (a putative ribosomal RNA methyltransferase) was further confirmed to be likely a genuine off‐target of FED1 by preliminary validation experiments including pull‐down/Western blotting (PD/WB) and cellular thermal shift assay (CETSA).     

Thiol‐ene clickable hyperscaffolds bearing peripheral allyl groups

Radical catalyzed thiol‐ene reaction has become a useful alternative to the Huisgen‐type click reaction as it helps to expand the variability in reaction conditions as well as the range of clickable entities. Thus, direct generation of hyperbranched polymers bearing peripheral allyl groups that could be clicked using a variety of functional thiols would be of immense value. A specifically designed AB₂ type monomer, that carries two allyl benzyl ethers groups and one alcohol functionality, was shown to undergo self‐condensation under acid‐catalyzed melt‐transetherification to yield a hyperbranched polyether that carries numerous allyl end‐groups. Importantly, it was shown that the kinetics of polymerization is not dramatically affected by the change of the ether unit from previously studied methyl benzyl ether to an allyl benzyl ether. The peripheral allyl groups were readily clicked quantitatively, using a variety of thiols, to generate an hydrocarbon‐soluble octadecyl‐derivative, amphiphilic systems using 2‐mercaptoethanol and chiral amino acid (N‐benzoyl cystine) derivatized hyperbranched structures; thus demonstrating the versatility of this novel class of clickable hyperscaffolds. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Photoinduced Electron Transfer in Clicked Ferrocene-BODIPY-Fullerene Conjugates

Ferrocene-BODIPY (Fc-BDP) conjugates in which one or two ferrocene entities are linked to the β-positions of the BODIPY core by an ethynyl bridge have been developed. These derivatives were easily and efficiently grafted onto a dual-clickable fullerene platform using CuAAC reactions, leading to a clickable Fc-BDP-C₆₀ triad and a clickable [Fc]₂-BDP-C₆₀ tetrad which can be used for further derivatization with complex structures. Due to the extended π-conjugation and the presence of an intramolecular charge transfer band from Fc to BDP, all these conjugates display a broad absorption in the visible region, which is bathochromically shifted when two Fc are appended to the BDP core. Ultrafast multistep electron transfers leading to charge stabilization were demonstrated in the Fc-BDP-C₆₀ triad and [Fc]₂-BDP-C₆₀ tetrad by femtosecond transient absorption studies.     

Biofunctionalization of a "clickable" organic layer photochemically grafted on titanium substrates

We have developed a general method combining photochemical grafting and copper-catalyzed click chemistry for biofunctionalization of titanium substrates. The UV-activated grafting of an α,ω-alkenyne onto TiO(2)/Ti substrates provided a "clickable" thin film platform. The selective attachment of the vinyl end of the molecule to the surface was achieved by masking the alkynyl end with a trimethylgermanyl (TMG) protecting group. Subsequently, various oligo(ethylene glycol) (OEG) derivatives terminated with an azido group were attached to the TMG-alkynyl modified titanium surface via a one-pot deprotection/click reaction. The films were characterized by X-ray photoelectron spectroscopy (XPS), contact angle goniometry, ellipsometry, and atomic force microscopy (AFM). We showed that the titanium surface presenting click-immobilized OEG substantially suppressed the nonspecific attachment of protein and cells as compared to the unmodified titanium substrate. Furthermore, glycine-arginine-glycine-aspartate (GRGD), a cell adhesion peptide, was coimmobilized with OEG on the platform. We demonstrated that the resultant GRGD-presenting thin film on Ti substrates can promote the specific adhesion and spreading of AsPC-1 cells.     

Chemistry and biology of dihydroisoxazole derivatives: selective inhibitors of human transglutaminase 2

3-halo-4,5-dihydroisoxazoles are attractive warheads for the selective inhibition of nucleophilic active sites in biological systems. A series of 3-bromo-4,5-dihydroisoxazole compounds were prepared and tested for their ability to irreversibly inhibit human transglutaminase 2 (TG2), an enzyme that plays an important role in the pathogenesis of diverse disorders including Celiac Sprue and certain types of cancers. Several compounds showed high specificity for human TG2 (k(inh)/K(I) > 2000 min(-1)M(-1)) but essentially no reactivity (k < 1 min(-1)M(-1)) toward physiological thiols such as glutathione. The pharmacokinetic and pharmacodynamic properties of a prototype dihydroisoxazole inhibitor, 1b, were evaluated; in mice the compound showed good oral bioavailability, short serum half-life and efficient TG2 inhibition in small intestinal tissue, and low toxicity. It also showed excellent synergism with N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU, carmustine) against refractory glioblastoma tumors in mice. A fluorescent dihydroisoxazole inhibitor 5 facilitated microscopic visualization of TG2 endocytosis from the extracellular surface of HCT-116 cells. Together, these findings demonstrate the promise of dihydroisoxazole compounds as probes for the biology of TG2 and its role in human disease.     

Clickable inverse opal: a useful platform for fabrication of stimuli-responsive photonic materials

Based on azide-containing clickable inverse opal, a strategy for efficiently fabricating functional photonic materials was developed. By using three types of ethynylated compounds as model molecules, it is found that different functional groups can be facilely introduced into the prepared inverse opal via click reaction to access various inverse opaline materials.     

Preparation and orthogonal postmodification of dual‐clickable polymer precursors bearing both aldehyde and alkyne groups

A new styrenic monomer 2‐propargyloxy‐5‐vinylbenzaldehyde (PVB) containing both aldehyde and alkyne reactive groups was designed for the synthesis and subsequent orthogonal postfunctionalization of dual‐clickable polymer precursor. Reversible addition‐fragmentation chain transfer polymerization of PVB afforded a structurally well‐defined polymer poly(2‐propargyloxy‐5‐vinylbenzaldehyde) (PPVB) bearing alkyne and aldehyde functionalities that are reactive towards azide ‐ and aminooxy‐containing molecules, respectively. Therefore, the resulting PPVB can be served as a dual‐clickable polymer scaffold for construction of multiple functional polymers via orthogonal alkyne–azide and aldehyde–aminooxy click reactions. Postpolymerization modification of PPVB sequentially with aminooxy‐terminated poly(ethylene oxide)s (H₂NO‐PEO) and azide‐functionalized imidazolium‐type ionic liquid (N₃‐IL·TFSI, having bis(trifluoromethane)sulfonamide, TFSI, counter‐anion) yielded an interesting multicomponent graft polymer PPVB‐g‐(PEO‐and‐IL·TFSI). After anion exchange of hydrophobic TFSI counter‐anion by bromide (Br) anion, the resulting graft copolymer PPVB‐g‐(PEO‐and‐IL·Br) becomes soluble in water, and its imidazolium units can capture negatively charged tetraphenylethylene disulfonate derivative (TPE‐2 ) guest molecule via electrostatic complexation to form in situ self‐assembled fluorescent nanoaggregates with colloidal stability imparted by hydrophilic PEO chains. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 2650–2656