Fluorogenic peptide-based substrates for monitoring thrombin activity

The synthesis of a series of peptides containing C‐terminal 7‐amino‐4‐methylcoumarin (AMC) for use in the thrombin generation test (TGT) is described. The lead structure in this project was H‐Gly‐Gly‐Arg‐AMC, of which the water solubility and kinetic parameters (K_M and k_cat) are greatly improved over those of the substrate in current use in the TGT: Cbz‐Gly‐Gly‐Arg‐AMC. A series of N‐terminally substituted Gly‐Gly‐Arg‐AMC derivatives were synthesized, as well as implementation of structural changes at either the P₂ or P₃ position of the peptide backbone. Furthermore, two substrates were synthesized that have structural similarities to the chromogenic thrombin substrate SQ68 or that contain a 1,2,3‐triazole moiety in the peptide chain, mimicking an amide bond. To determine the applicability of newly synthesized fluorogenic substrates for monitoring continuous thrombin generation, the K_M and k_cat values of the conversion of these fluorogenic substrates by thrombin (FIIa) and factor Xa (FXa) were quantified. An initial selection was made on basis of these data, and suitable substrates were further evaluated as substrates in the thrombin generation assay. Assessment of the acquired data showed that several substrates, including the SQ68 derivative Et‐malonate‐Gly‐Arg‐AMC and N‐functionalized Gly‐Gly‐Arg‐AMC derivatives, are suitable candidates for replacement of the substrate currently in use.     

Rational Design,Synthesis and Biological Evaluation of Modular Fluorogenic Substrates with High Affinity and Selectivity for PTP1B

Protein‐tyrosine phosphatase 1B (PTP1B) is a key regulatory enzyme in several signal transduction pathways, and its upregulation has been associated with type‐2 diabetes, obesity and cancer. Selective determination of the functional significance of PTP1B remains a major challenge because the activity of this crucial enzyme is currently evaluated through the use of fluorescent probes that lack selectivity and are limited to biochemical assays. Here we describe the rational design, synthesis and biological evaluation of new modular PTP1B fluorogenic substrates. The self‐immolative 4‐hydroxybenzyl alcohol has been used as a key component for the design of phosphotyrosine mimics linked to a latent chromophore, which is released through an enzyme‐initiated domino reaction. Preliminary biological investigations showed that, by optimising the stereoelectronic properties and the binding interactions at the enzyme active site, it is possible to achieve substrates with high affinity and promising selectivity. Due to their modular nature, the synthesised fluorogenic probes represent versatile tools; customisation of the different subunits could widen the scope of these probes to a broader range of in vitro assays. Finally, these studies elucidate the critical role played by Asp181 in the PTP1B‐catalysed dephosphorylation mechanism: disruption of the native conformation of this key amino acid residue on the WDP loop yields fluorogenic inhibitors, rather than substrates. For this reason, our studies also represent a step forward for the development of improved PTP1B noncovalent inhibitors.     

Elucidating the Catalytic Subunit Composition of Distinct Proteasome Subtypes: A Crosslinking Approach Employing Bifunctional Activity‐Based Probes

In addition to two well‐recognized proteasome subtypes—constitutive proteasomes and immunoproteasomes—mounting evidence also suggests the existence of intermediate proteasome subtypes containing unconventional mixtures of catalytic subunits. Although they appear to play unique biological roles, the lack of practical methods for detecting distinct proteasome subtypes has limited functional investigations. Here, we report the development of activity‐based probes that crosslink two catalytic subunits within intact proteasome complexes. Identification of the crosslinked subunit pairs provides direct evidence of the catalytic subunit composition of proteasomes. Using these probes, we found that U266 multiple myeloma cells contain intermediate proteasomes comprising both β1i and β2, but not β1 and β2i, consistent with previous findings with other cell types. Our bifunctional probes can be utilized in functional investigations of distinct proteasome subtypes in various biological settings.     

A Selective Fluorogenic Peptide Substrate for the Human Mitochondrial ATP-Dependent Protease Complex ClpXP

The goal of this work is to identify differences in the substrate determinants of two human mitochondrial matrix ATP-dependent proteases, human ClpXP (hClpXP) and human Lon (hLon). This information allows the generation of protease-specific peptide substrates that can be used as chemical biology tools to investigate the physiological functions of hClpXP. These enzymes play a role in protein quality control, but currently the physiological functions of human ClpXP are not well defined. In this study, the degradation profile of casein, an alanine positional scanning decapeptide library, and a specific peptide sequence found in an endogenous substrate of bacterial ClpXP by hClpXP as well as hLon were examined. Based on our findings, we generated a specific fluorogenic peptide substrate, FR-Cleptide, for hClpXP with a k_cat of 2.44±0.15 s⁻¹ and K_m=262±43 μM, respectively. The FR-Cleptide substrate was successfully used to identify a leucine methyl ketone as a potent lead inhibitor, and to detect endogenous hClpXP activity in HeLa cell lysate. We propose that the fluorogenic peptide substrate is a valuable tool for quantitatively monitoring the activity of hClpXP in cell lysate, as well as mechanistic characterization of hClpXP. The peptide-based chemical tools developed in this study will complement the substrates developed for human Lon in aiding the investigation of the physiological functions of the respective protease.     

Evaluation of Pseudoenantiomeric Mixed Carbonates as Efficient Fluorogenic Probes for Enantioselectivity Screening

We report mixed carbonates as enzyme substrates and demonstrate their application as fluorogenic probes for lipase and esterase enantiopreference screening. By the application of pseudoenantiomers with distinct fluorescence behaviors, it is possible to evaluate the activity and enantiopreference of hydrolytic enzymes. In order to validate our method, enantioselectivities calculated from fluorometric measurements were compared with the results obtained from larger‐scale kinetic resolution.     

MgO-Al2O3-SiO2系硬盘基板用微晶玻璃析晶动力学研究

MgO-A l2O3-SiO2系微晶玻璃是一种优良的磁盘基板备选材料,该材料具有良好的力学性能,能够满足高性能硬盘基板用材料的要求。采用差热分析方法(DTA),得到了以TiO2作为晶核剂的MgO-A l2O3-SiO2系微晶玻璃的析晶动力学参数,研究了该体系微晶玻璃的析晶动力学;利用X射线衍射技术(XRD)确定了经合适热处理后得到的样品的主晶相。结果表明:引入TiO2降低了该体系玻璃的析晶活化能Ea,当TiO2质量分数达到7%时,Ea达到最小值,玻璃析晶晶化指数n达到最大值,这表明TiO2在一定范围内促进了该系统玻璃的析晶。     

Experimental Method for a Dynamic Biaxial Flexural Strength Test of Thin Ceramic Substrates

A dynamic piston-on-three-ball experimental technique has been developed for a biaxial flexural strength test of thin ceramic substrates at high loading rates. Analytical modeling of the technique guided the experimental design and was used to judge the validity of experimental results. Thin ceramic substrates made of 1-mol%-Al₂O₃-doped 8-mol%-yttria-stabilized zirconia (8YSZ) were tested using this experimental technique, as well as its standard counterpart—the quasi-static piston-on-three-ball technique. Results show that the mean biaxial flexural strength increased from ∼322 to ∼465 MPa as the average loading rate was increased from quasi-static to dynamic (830 GPa/s) level.     

Structural Elucidation of a Nonpeptidic Inhibitor Specific for the Human Immunoproteasome

Selective inhibition of the immunoproteasome is a promising approach towards the development of immunomodulatory drugs. Recently, a class of substituted thiazole compounds that combine a nonpeptidic scaffold with the absence of an electrophile was reported in a patent. Here, we investigated the mode of action of the lead compound by using a sophisticated chimeric yeast model of the human immunoproteasome for structural studies. The inhibitor adopts a unique orientation perpendicular to the β5i substrate‐binding channel. Distinct interactions between the inhibitor and the subpockets of the human immunoproteasome account for its isotype selectivity.     

Decoupled Roles for the Atypical,Bifurcated Binding Pocket of the ybfF Hydrolase

Serine hydrolases have diverse intracellular substrates, biological functions, and structural plasticity, and are thus important for biocatalyst design. Amongst serine hydrolases, the recently described ybfF enzyme family are promising novel biocatalysts with an unusual bifurcated substrate‐binding cleft and the ability to recognize commercially relevant substrates. We characterized in detail the substrate selectivity of a novel ybfF enzyme from Vibrio cholerae (Vc‐ybfF) by using a 21‐member library of fluorogenic ester substrates. We assigned the roles of the two substrate‐binding clefts in controlling the substrate selectivity and folded stability of Vc‐ybfF by comprehensive substitution analysis. The overall substrate preference of Vc‐ybfF was for short polar chains, but it retained significant activity with a range of cyclic and extended esters. This broad substrate specificity combined with the substitutional analysis demonstrates that the larger binding cleft controls the substrate specificity of Vc‐ybfF. Key selectivity residues (Tyr116, Arg120, Tyr209) are also located at the larger binding pocket and control the substrate specificity profile. In the structure of ybfF the narrower binding cleft contains water molecules prepositioned for hydrolysis, but based on substitution this cleft showed only minimal contribution to catalysis. Instead, the residues surrounding the narrow binding cleft and at the entrance to the binding pocket contributed significantly to the folded stability of Vc‐ybfF. The relative contributions of each cleft of the binding pocket to the catalytic activity and folded stability of Vc‐ybfF provide a valuable map for designing future biocatalysts based on the ybfF scaffold.     

Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. We describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged β-tubulin in cell lysates. In addition, we have characterized a fast-labeling variant of SNAP-tag, termed SNAP(f), which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAP(f) and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.     

Identifying the Minimal Enzymes Required for Biosynthesis of Epoxyketone Proteasome Inhibitors

Epoxyketone proteasome inhibitors have attracted much interest due to their potential as anticancer drugs. Although the biosynthetic gene clusters for several peptidyl epoxyketone natural products have recently been identified, the enzymatic logic involved in the formation of the terminal epoxyketone pharmacophore has been relatively unexplored. Here, we report the identification of the minimal set of enzymes from the eponemycin gene cluster necessary for the biosynthesis of novel metabolites containing a terminal epoxyketone pharmacophore in Escherichia coli, a versatile and fast‐growing heterologous host. This set of enzymes includes a non‐ribosomal peptide synthetase (NRPS), a polyketide synthase (PKS), and an acyl‐CoA dehydrogenase (ACAD) homologue. In addition to the in vivo functional reconstitution of these enzymes in E. coli, in vitro studies of the eponemycin NRPS and ¹³C‐labeled precursor feeding experiments were performed to advance the mechanistic understanding of terminal epoxyketone formation.     

Solid-Phase Synthesis and Application of a Clickable Version of Epoxomicin for Proteasome Activity Analysis

Degradation of proteins by the proteasome is an essential cellular process and one that many wish to study in a variety of disease types. There are commercially available probes that can monitor proteasome activity in cells, but they typically contain common fluorophores that limit their simultaneous use with other activity-based probes. In order to exchange the fluorophore or incorporate an enrichment tag, the proteasome probe likely has to be synthesized which can be cumbersome. Here, we describe a simple synthetic procedure that only requires one purification step to generate epoxomicin, a selective proteasome inhibitor, with a terminal alkyne. Through a copper-catalyzed cycloaddition, any moiety containing an azide can be incorporated into the probe. Many fluorophores are commercially available that contain an azide that can be “clicked”, allowing this proteasome activity probe to be included into already established assays to monitor both proteasome activity and other cellular activities of interest.     

Characterization of Material for Low-Temperature Sintered Multilayer Ceramic Substrates

The sintering temperature of multilayer ceramic substrates must decrease to 1000° or below to avoid melting the conductors (Pd-Ag, Au, or Cu) during sintering. In this study, SiO₂, CaO, B₂O₃, and MgO were used as additives to Al₂O₃ to decrease the firing temperature by liquid-phase sintering. Compositions with 18.0 and 22.5 wt% B₂O₃ were sintered at around 1000° in an air atmosphere to yield dense ceramics with good properties: relative dielectric contant between 6 to 7 (1 MHz), tan δ≤× 3 × 10⁻⁴ (1 MHz), insulating resistivity > 10¹⁴ω cm, coefficient of thermal expansion ∼ 7.0 × 10⁻⁶/°, and thermal conductivity ∼ 4.1 W/(m · K).     

Rational Structure-Based Design of Fluorescent Probes for Amyloid Folds

Amyloid fibrils are pathological hallmarks of various human diseases, including Parkinson's, Alzheimer's, amyotrophic lateral sclerosis (ALS or motor neurone disease), and prion diseases. Treatment of the amyloid diseases are hindered, among other factors, by timely detection and therefore, early detection of the amyloid fibrils would be beneficial for treatment against these disorders. Here, a small molecular fluorescent probe is reported that selectively recognize the fibrillar form of amyloid beta(1–42), α-synuclein, and HET-s(218–289) protein over their monomeric conformation. The rational design of the reporters relies on the well-known cross-β-sheet repetition motif, the key structural feature of amyloids.