Catch‐and‐Release Probes Applied to Semi‐Intact Cells Reveal Ubiquitin‐Specific Protease Expression in Chlamydia trachomatis Infection

Protein ubiquitylation controls many cellular pathways, and timely removal of ubiquitin by deubiquitylating enzymes (DUBs) is essential to govern these different functions. To map endogenous expression of individual DUBs as well as that of any interacting proteins, we developed a catch‐and‐release ubiquitin probe. Ubiquitin was equipped with an activity‐based warhead and a cleavable linker attached to a biotin affinity‐handle through tandem site‐specific modification, in which we combined intein chemistry with sortase‐mediated ligation. The resulting probe is cell‐impermeable and was therefore delivered to the cytosol of perfringolysin O (PFO)‐permeabilized cells. This allowed us to retrieve and identify 34 DUBs and their interacting partners. We also noted the expression, in host cells infected with Chlamydia trachomatis, of two additional DUBs. Furthermore, we retrieved and identified chlamydial DUB1 (ChlaDUB1) and DUB2 (ChlaDUB2), demonstrating by experiment that ChlaDUB2, the presence and activity of which had not been detected in infected cells, is in fact expressed during the course of infection.     

Facile One‐Step Assembly of Bona Fide SUMO Conjugates by Chemoenzymatic Ligation

The post‐translational conjugation of the small ubiquitin‐like modifiers (SUMOs) to target proteins occurs through a complex machinery that involves sequential action of at least three enzymes. SUMOylation performs crucial regulatory functions in several cellular processes. The availability of well‐defined SUMO conjugates is necessary for untangling the mechanism of SUMOylation. However, assembly of homogeneous SUMO conjugates represents a challenge because of the multi‐step synthesis involved and the unwieldiness of the reconstituted biosynthetic systems. Here we describe a simple one‐step chemoenzymatic strategy for conjugating engineered SUMO (eSUMO) proteins to a prefabricated isopeptide‐linked SUMO target peptide. Notably, the eSUMOs were efficiently recognized by the enzymes of the SUMOylation machinery and the SUMO conjugates served as bona fide substrates for DeSUMOylating enzymes.     

Effect of pH on the drug release rate from a new polymer–drug conjugate system

The corresponding N‐hydroximide and N‐methyl‐N‐hydroximide of poly[ethylene‐alt‐(maleic anhydride)] (weight average molecular weight (M_w) of 100–500 g mol^?1) were prepared as a new oral drug delivery system. Syntheses of N‐hydroximide and N‐methylhydroxamic acid of poly[ethylene‐alt‐(maleic anhydride)] were carried out by chemical modification of polymer with hydroxylamine and N‐methylhydroxylamine, respectively, to give water‐soluble polymers. These activated polymers were immobilized with ketoprofen in the presence of dicyclohexylcarbodiimide to give the corresponding water‐insoluble ketoprofen conjugates. All products were characterized by elemental analysis as well as Fourier transform infrared and ¹H NMR spectra. In vitro release of ketoprofen was studied by measuring UV absorption at λ_max = 260 nm as a function of time. This study demonstrated the potential use of N‐hydroximide and N‐methyl‐N‐hydroxamic acid of poly[ethylene‐alt‐(maleic anhydride)] as a drug delivery system. Controlled release was studied at different pH values and at different temperatures. At physiological temperature, the amount of drug released increased with increasing pH. The copolymer‐drug adducts released the drug very slowly at the low pH found in the stomach thus protecting the drug from the action of high acid conditions and resident digestive enzymes. These N‐hydroxamic acid polymer‐drug conjugates were found to be potentially useful in the delivery of macromolecular drugs to targeted sites in the lower gastrointestinal tract and the colon area. Copyright © 2007 Society of Chemical Industry     

A Native Chemical Ligation Handle that Enables the Synthesis of Advanced Activity‐Based Probes: Diubiquitin as a Case Study

We present the development of a native chemical ligation handle that also functions as a masked electrophile that can be liberated during synthesis when required. This handle can thus be used for the synthesis of complex activity‐based probes. We describe the use of this handle in the generation of linkage‐specific activity‐based deubiquitylating enzyme probes that contain substrate context and closely mimic the native ubiquitin isopeptide linkage. We have generated activity‐based probes based on all seven isopeptide‐linked diubiquitin topoisomers and demonstrated their structural integrity and ability to label DUBs in a linkage‐specific manner.     

Synthesis and Biological Evaluation of 9‐Oxo‐9H‐indeno[1,2‐b]pyrazine‐2,3‐dicarbonitrile Analogues as Potential Inhibitors of Deubiquitinating Enzymes

High‐throughput screening highlighted 9‐oxo‐9H‐indeno[1,2‐b]pyrazine‐2,3‐dicarbonitrile ( 1 ) as an active inhibitor of ubiquitin‐specific proteases (USPs), a family of hydrolytic enzymes involved in the removal of ubiquitin from protein substrates. The chemical behavior of compound 1 was examined. Moreover, the synthesis and in vitro evaluation of new compounds, analogues of 1 , led to the identification of potent and selective inhibitors of the deubiquitinating enzyme USP8.     

Chemical immobilization of retinoic acid within poly(ε‐caprolactone) nanoparticles based on drug–polymer bioconjugates

All‐trans‐retinoic acid (RA) was chemically conjugated to biodegradable poly(ε‐caprolactone) (PCL₁₀; number‐average M_w ≈ 1250) via an ester linkage. The conjugation was carried out using N,N‐dicyclohexylcarbodiimide and 4‐dimethyl aminopyridine as a coupling agent. The molar ratio of the drug to the polymer was 1.11 as determined by ¹H‐NMR analysis. DSC and WAXD results showed that the formation of crystalline structures of RA was effectively suppressed by conjugation with PCL. The RA–PCL conjugates were formulated into nanoparticles by a spontaneous phase‐inversion technique. Morphological characteristics of the resultant nanoparticles and drug‐loading efficiencies were compared with those of free RA‐loaded nanoparticles. The drug‐loading efficiency of RA–PCL conjugates was almost 100%, while that of free RA was only ～12%. The majority of unconjugated RA was found to form undesirable free‐drug crystals out of nanoparticles, as observed by TEM analysis. This study demonstrates that the conjugation approach of RA to PCL can be an effective means to immobilize and encapsulate RA within nanoparticles for pharmaceutical applications. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 1631–1637, 2003     

Fabrication of size‐controllable mPEG‐decorated microparticles conjugating optically active ketoprofen based on self‐assembly of amphiphilic random copolymers

Novel size‐controllable mPEG‐decorated polymeric microparticles binding optically active ketoprofen were successfully fabricated based on chemoenzymatic synthesis and self‐assembly of amphiphilic random polymer–ketoprofen conjugates with mPEG and (S)‐ketoprofen as pendants. A series of mPEG₃₅₀‐ or mPEG₁₀₀₀‐functionalized amphiphilic random polymer–ketoprofen conjugates with drug loading capacity from 16.5% to 73.2% were easily prepared by combining enzymatic resolution with radical polymerization and characterized by Fourier Transform Infrared spectroscopy, ¹H‐NMR, and gel permeation chromatography. The formation of aggregates from the amphiphilic random polymer–ketoprofen conjugates was investigated by ultraviolet‐visible absorption spectra using pyrene as the guest molecule. Transmission electron microscopy measurement revealed that the self‐assemblies were well dispersed as spherical microparticles. The size of the self‐assemblies could be widely tuned by varying the length of mPEG chains and the content of ketoprofen in the synthetic polymer–ketoprofen conjugates, and a series of mPEG‐decorated (S)‐ketoprofen‐bound polymeric microparticles with average radius from 70 nm to 1.1 μm were obtained. The successful preparation of the microparticles containing (S)‐ketoprofen provided a new strategy for the design and fabrication of optically active drug delivery systems. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Molecular‐Target‐Based Anticancer Photosensitizer: Synthesis and in vitro Photodynamic Activity of Erlotinib–Zinc(II) Phthalocyanine Conjugates

Targeted photodynamic therapy is a new promising therapeutic strategy to overcome growing problems in contemporary medicine, such as drug toxicity and drug resistance. A series of erlotinib–zinc(II) phthalocyanine conjugates were designed and synthesized. Compared with unsubstituted zinc(II) phthalocyanine, these conjugates can successfully target EGFR‐overexpressing cancer cells owing to the presence of the small molecular‐target‐based anticancer agent erlotinib. All conjugates were found to be essentially non‐cytotoxic in the absence of light (up to 50 μM ), but upon illumination, they show significantly high photo‐cytotoxicity toward HepG2 cells, with IC₅₀ values as low as 9.61–91.77 nM under a rather low light dose (λ=670 nm, 1.5 J cm^?2). Structure–activity relationships for these conjugates were assessed by determining their photophysical/photochemical properties, cellular uptake, and in vitro photodynamic activities. The results show that these conjugates are highly promising antitumor agents for molecular‐target‐based photodynamic therapy.     

A polymer–drug conjugate for doxorubicin: Synthesis and biological evaluation of pluronic F127‐doxorubicin amide conjugates

Higher molecular weight of the polymer carrier is the basis for enhanced accumulation of the pro‐drug in a solid tumor tissue due to a tumor‐related phenomenon described as the enhanced permeability and retention (EPR) effect. The anticancer drug doxorubicin was covalently bound to F127 through amide group susceptible to lysosomal hydrolysis. The in vitro and in vivo properties of F127‐DOX amide conjugates were studied. F127‐DOX amide conjugates (M_w: 13,400) were stable in neutral circumstance and showed the potency and mechanism of action of the released drug. In the in vivo experiment, results showed that F127‐DOX amide conjugates prolonged blood circulation and lowered in vivo toxicity than corresponding DOX, which illustrated the importance of molecular weight. The results demonstrated that F127‐DOX amide conjugates may be very promising and clinically suitable candidates for anticancer therapy. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Cellular Antisense Activity of PNA–Oligo(bicycloguanidinium) Conjugates Forming Self‐Assembled Nanoaggregates

A series of peptide nucleic acid–oligo(bicycloguanidinium) (PNA–BG_n) conjugates were synthesized and characterized in terms of cellular antisense activity by using the pLuc750HeLa cell splice correction assay. PNA–BG₄ conjugates exhibited low micromolar antisense activity, and their cellular activity required the presence of a hydrophobic silyl terminal protecting group on the oligo(BG) ligand and a minimum of four guanidinium units. Surprisingly, a nonlinear dose–response with an activity threshold around 3–4 μM , indicative of large cooperativity, was observed. Supported by light scattering and electron microscopy analyses, we propose that the activity, and thus cellular delivery, of these lipo‐PNA–BG₄ conjugates is dependent on self‐assembled nanoaggregates. Finally, cellular activity was enhanced by the presence of serum. Therefore we conclude that the lipo‐BG‐PNA conjugates exhibit an unexpected mechanism for cell delivery and are of interest for further in vivo studies.     

Platinum(II) as Bifunctional Linker in Antibody–Drug Conjugate Formation: Coupling of a 4‐Nitrobenzo‐2‐oxa‐1,3‐diazole Fluorophore to Trastuzumab as a Model

The potential of platinum(II) as a bifunctional linker in the coordination of small molecules, such as imaging agents or (cytotoxic) drugs, to monoclonal antibodies (mAbs) was investigated with a 4‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD) fluorophore and trastuzumab (Herceptin?) as a model antibody. The effect of ligand and reaction conditions on conjugation efficiency was explored for [Pt(en)(L‐NBD)Cl](NO₃) (en=ethylenediamine), with L=N‐heteroaromatic, N‐alkyl amine, or thioether. Conjugation proceeded most efficiently at pH 8.0 in the presence of NaClO₄ or Na₂SO₄ in tricine or HEPES buffer. Reaction of N‐coordinated complexes (20 equiv) with trastuzumab at 37 °C for 2 h, followed by removal of weakly bound complexes with excess thiourea, afforded conjugates with an NBD/mAb ratio of 1.5–2.9 that were stable in phosphate‐buffered saline at room temperature for at least 48 h. In contrast, thioether‐coordinated complexes afforded unstable conjugates. Finally, surface plasmon resonance analysis showed no loss in binding affinity of trastuzumab after conjugation.     

Biochemical Investigations of Two 6‐DMATS Enzymes from Streptomyces Reveal New Features of L‐Tryptophan Prenyltransferases

Two putative prenyltransferase genes, SAML0654 and Strvi8510, were identified in Streptomyces ambofaciens and Streptomyces violaceusniger, respectively. Their deduced products share 63 % sequence identity. Biochemical investigations with recombinant proteins demonstrated that L ‐tryptophan and derivatives, including D ‐tryptophan, 4‐, 5‐, 6‐ and 7‐methyl‐dl ‐tryptophan, were well accepted by both enzymes in the presence of DMAPP. Structural elucidation of the isolated products revealed regiospecific prenylation at C‐6 of the indole ring and proved unequivocally the identification of two very similar 6‐dimethylallyltryptophan synthases (6‐DMATS). Detailed biochemical investigations with SAML0654 proved L ‐tryptophan to be the best substrate (K_m 18 μm, turnover 0.3 s^?1). Incubation with different prenyl donors showed that they also accepted GPP and catalyzed the same specific prenylation. Utilizing GPP as a prenyl donor has not been reported for tryptophan prenyltransferases previously. Both enzymes also catalyzed prenylation of some hydroxynaphthalenes; this has not previously been described for bacterial indole prenyltransferases. Interestingly, SAML0654 transferred prenyl moieties onto the unsubstituted ring of hydroxynaphthalenes.     

A Chimeric Styrene Monooxygenase with Increased Efficiency in Asymmetric Biocatalytic Epoxidation

The styrene monooxygenase (SMO) system from Pseudomonas sp. consists of two enzymes (StyA and StyB). StyB catalyses the reduction of FAD at the expense of NADH. After the transfer of FADH₂ from StyB to StyA, reaction with O₂ generates FAD‐OOH, which is the epoxidising agent. The wastage of redox equivalents due to partial diffusive transfer of FADH₂, the insolubility of recombinant StyB and the impossibility of expressing StyA and StyB in a 1:1 molar ratio reduce the catalytic efficiency of the natural system. Herein we present a chimeric SMO (Fus‐SMO) that was obtained by genetic fusion of StyA and StyB through a flexible linker. Thanks to a combination of: 1) balanced and improved expression levels of reductase and epoxidase units, and 2) intrinsically higher specific epoxidation activity of Fus‐SMO in some cases, Escherichia coli cells expressing Fus‐SMO possess about 50 % higher activity for the epoxidation of styrene derivatives than E. coli cells coexpressing StyA and StyB as discrete enzymes. The epoxidation activity of purified Fus‐SMO was up to three times higher than that of the two‐component StyA/StyB (1:1, molar ratio) system and up to 110 times higher than that of the natural fused SMO. Determination of coupling efficiency and study of the influence of O₂ pressure were also performed. Finally, Fus‐SMO and formate dehydrogenase were coexpressed in E. coli and applied as a self‐sufficient biocatalytic system for epoxidation on greater than 500 mg scale.     

Synthesis of aryl-substituted naphthalene-linked pyrrolobenzodiazepine conjugates as potential anticancer agents with apoptosis-inducing ability

A library of new aryl‐substituted naphthalene C8‐linked pyrrolo[2,1‐c][1,4]benzodiazepine (PBD) conjugates with various linker architectures were designed, synthesized, and evaluated for their anticancer activity against a panel of 11 human cancer cell lines. All 32 conjugates show anticancer potential, with some of them exhibiting particularly high activity (0.01–0.19 μM ). Thermal denaturation studies showed effective DNA binding capacity relative to DC‐81. In assays for biological activity relating to cell‐cycle distribution, these PBD conjugates induce G₀/G₁‐phase arrest and also cause an increase in the levels of p53 and caspase‐9 proteins, followed by apoptotic cell death. One conjugate in particular is the most promising candidate of the series, with the potential to be selected for further studies, either alone or in combination with existing anticancer therapies.     

Exploring Non‐obvious Hydrophobic Binding Pockets on Protein Surfaces: Increasing Affinities in Peptide–Protein Interactions

A 42‐residue polypeptide conjugated to a small‐molecule organic ligand capable of targeting the phosphorylated side chain of Ser15 was shown to bind glycogen phosphorylase a (GPa) with a K_D value of 280 nm . The replacement of hydrophobic amino acids by Ala reduced affinities, whereas the incorporation of l ‐2‐aminooctanoic acid (Aoc) increased them. Replacing Nle5, Ile9 and Leu12 by Aoc reduced the K_D value from 280 to 27 nm . “Downsizing” the 42‐mer to an undecamer gave rise to an affinity for GPa an order of magnitude lower, but the undecamer in which Nle5, Ile9 and Leu12 were replaced by Aoc showed a K_D value of 550 nm , comparable with that of the parent 42‐mer. The use of Aoc residues offers a convenient route to increased affinity in protein recognition as well as a strategy for the “downsizing” of peptides essentially without loss of affinity. The results show that hydrophobic binding sites can be found on protein surfaces by comparing the affinities of polypeptide conjugates in which Aoc residues replace Nle, Ile, Leu or Phe with those of their unmodified counterparts. Polypeptide conjugates thus provide valuable opportunities for the optimization of peptides and small organic compounds in biotechnology and biomedicine.     

SARS-CoV-2 Papain-Like Protease Potential Inhibitors—In Silico Quantitative Assessment

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes the papain-like protease (PLpro). The protein not only plays an essential role in viral replication but also cleaves ubiquitin and ubiquitin-like interferon-stimulated gene 15 protein (ISG15) from host proteins, making it an important target for developing new antiviral drugs. In this study, we searched for novel, noncovalent potential PLpro inhibitors by employing a multistep in silico screening of a 15 million compound library. The selectivity of the best-scored compounds was evaluated by checking their binding affinity to the human ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), which, as a deubiquitylating enzyme, exhibits structural and functional similarities to the PLpro. As a result, we identified 387 potential, selective PLpro inhibitors, from which we retrieved the 20 best compounds according to their IC₅₀ values toward PLpro estimated by a multiple linear regression model. The selected candidates display potential activity against the protein with IC₅₀ values in the nanomolar range from approximately 159 to 505 nM and mostly adopt a similar binding mode to the known, noncovalent SARS-CoV-2 PLpro inhibitors. We further propose the six most promising compounds for future in vitro evaluation. The results for the top potential PLpro inhibitors are deposited in the database prepared to facilitate research on anti-SARS-CoV-2 drugs.     

Characterization of Carboxylic Acid Reductases for Biocatalytic Synthesis of Industrial Chemicals

Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids into aldehydes, which can serve as common biosynthetic precursors to many industrial chemicals. This work presents the systematic biochemical characterization of five carboxylic acid reductases from different microorganisms, including two known and three new ones, by using a panel of short‐chain dicarboxylic acids and hydroxy acids, which are common cellular metabolites. All enzymes displayed broad substrate specificities. Higher catalytic efficiencies were observed when the carbon chain length, either of the dicarboxylates or of the terminal hydroxy acids, was increased from C₂ to C₆. In addition, when substrates of the same carbon chain length are compared, carboxylic acid reductases favor hydroxy acids over dicarboxylates as their substrates. Whole‐cell bioconversions of eleven carboxylic acid substrates into the corresponding alcohols were investigated by coupling the CAR activity with that of an aldehyde reductase in Escherichia coli hosts. Alcohol products were obtained in yields ranging from 0.5 % to 71 %. The de novo stereospecific biosynthesis of propane‐1,2‐diol enantiomer was successfully demonstrated with use of CARs as the key pathway enzymes. E. coli strains accumulated 7.0 mm (R)‐1,2‐PDO (1.0 % yield) or 9.6 mm (S)‐1,2‐PDO (1.4 % yield) from glucose. This study consolidates carboxylic acid reductases as promising enzymes for sustainable synthesis of industrial chemicals.     

Beet molasses based exponential feeding strategy for thermostable glucose isomerase production by recombinant Escherichia coli BL21 (DE3)

BACKGROUND: The effects of pretreated beet molasses based feeding strategies on thermostable glucose isomerase (GI) production by recombinant Escherichia coli BL21 (DE3) pLysS were investigated. RESULTS: The thermostable GI encoding gene of Thermus thermophilus (xyl_A) was recombined with pRSETA vector, and the pRSETA::xyl_A obtained was transferred into E.coli BL21 (DE3) pLysS and used for GI production. The highest soluble GI activity was obtained at t = 30 h, as A = 16 400 U L^?1 (20.6 U mg^?1 protein) under molasses based fed‐batch operation, with a specific growth rate µ = 0.1 h^?1 (M‐0.1); on the other hand, the highest cell concentration was obtained at µ = 0.15 h^?1 operation as 9.6 g L^?1 at t = 32 h. The highest oxygen uptake was 4.57 mol m^?3 s^?1 at M‐0.1 operation. CONCLUSIONS: Molasses based fed‐batch operations were more successful in terms of cell concentration and thermostable enzyme production due to the existence of a natural sugar inducer, galactose, in the molasses composition. This study demonstrates the significance of proper feeding strategy development for over‐production of enzymes by recombinant E. coli strains. © 2012 Society of Chemical Industry     

Polypeptide conjugate binders that discriminate between two isoforms of human carbonic anhydrase in human blood

Two binder candidates 4‐C37L34‐B and 3‐C15L8‐B from a 16‐membered set of 42‐residue polypeptide conjugates designed to bind human carbonic anhydrase II (HCAII), were shown to bind HCAII with high affinity in a fluorescence‐based screening assay. Two carbonic anhydrase isoforms with 60 % homology exist in human blood with HCAI being present in five‐ to sevenfold excess over HCAII. The ability of the binders to discriminate between HCAI and HCAII was evaluated with regard to what selectivity could be achieved by the conjugation of polypeptides from a 16‐membered set to a small organic molecule that binds both isoforms with similar affinities. The polypeptide conjugate 4‐C37L34‐B bound HCAII with a K_D of 17 nM and HCAI with a K_D of 470 nM , that is, with a 30‐fold difference in affinity. The corresponding dissociation constants for the complexes formed from 3‐C15L8‐B and the two carbonic anhydrases were 60 and 390 nM , respectively. This demonstration of selectivity between two very similar proteins is striking in view of the fact that the molecular weight of each one of the conjugate molecules is little more than 5000, the fold is unordered, and the polypeptide sequences were designed de novo and have no prior relationship to carbonic anhydrases. The results suggest that synthetic polypeptide conjugates can be prepared from organic molecules that are considered to be weak binders with low selectivity, yielding conjugates with properties that make them attractive alternatives to biologically generated binders in biotechnology and biomedicine.     

Genomic structure,cloning and expression of two phospholipase D isoenzymes from white cabbage

Phospholipase D (PLD) from cabbage is interesting as biocatalyst in phospholipid transformation. To provide the basis for genetic engineering of the enzyme, gene cloning and sequencing were carried out. We have recently identified two isoenzymes, PLD1 and PLD2, on the basis of their cDNAs, here we describe their genomic structure consisting of 3404 and 3614 bp, respectively. Based on their sequence, PLD1 and PLD2 can be assigned to the α‐type of plant PLDs, they contain two HKD motifs and the C2 domain with a phosphatidylinositol 4,5‐bisphosphate (PIP₂) binding motif. Starting from pld1 cDNA, expression studies were carried out. Whereas expression using constructs with StrepTactin or Glutathion S‐transferase tags was not successful, soluble active enzyme was produced from constructs without tag. pld2 was expressed accordingly. Both enzymes were purified by Ca²⁺‐mediated hydrophobic interaction chromatography to high purity. N‐terminal sequencing of PLD1 and PLD2 revealed that the Met‐free N‐termini of both enzymes correspond to sequences derived from the coding region of the pld1 and pld2 genes, respectively. Both recombinant enzymes showed highest hydrolytic activities at pH 5.5 to 5.6, independent of Ca²⁺ concentration (10—100 mM). The optimum Ca²⁺ concentration was 45 mM for PLD1 and PLD2. Both enzymes showed comparable activities in hydrolysis and transphosphatidylation of phospholipids.