Detection of esterase activity by chromogenic and fluorogenic probe based on an O-nitrobenzoxadiazole (O-NBD) unit

We designed a conjugated molecule bearing an O-nitrobenzoxadiazole (O-NBD) unit and an acetylated trimethyl lock as a chromogenic and fluorogenic probe for the detection of esterase activity. The designed molecule was briefly synthesized from a commercially available compound in two steps. Several experiments revealed that the conjugated molecule serves as a sensitive chromogenic and fluorogenic probe for the detection of porcine liver esterase activity. Mechanistic studies indicated that an intramolecular O- to N-NBD migration is involved in the chromogenic/fluorogenic phenomena. The results here would be helpful for designing other O-NBD-based chromogenic/fluorogenic probes in future.     

DICER-ARGONAUTE2 Complex in Continuous Fluorogenic Assays of RNA Interference Enzymes

Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC) have been hindered by lack of methods for continuous monitoring of enzymatic activity. “Quencherless” fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS) and hypoxia-inducible factor 1-α subunit (HIF1A). Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (K _m=74 nM) with substrate inhibition kinetics (K _i=105 nM), demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER) bound in the active site of DICER may undergo direct transfer (as AGO2 substrate) to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29), suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a strong (~2800-fold) improvement in potency for mRNA knockdown. This study lays the foundation of a systematic biochemical approach to optimize nucleic acid-based therapeutics for Dicing and ARGONAUTE2-loading for improving efficacy.     

Characterization of recombinant human renin: Kinetics,pH-stability,and peptidomimetic inhibitor binding

The kinetic behavior andpH-stability of recombinant human renin was analyzed using a new fluorogenic substrate based on the normal P₆-P_3′ renin cleavage sequence in human angiotensinogen. The design of this fluorogenic substrate makes possible, for the first time, direct monitoring of the kinetics of proteolytic conversion of prorenin to renin. ThepH-stability profile for renin, measured with the substrate at 25°C, indicated a broad plateau of stability betweenpH 6.0 and 10.0. Analysis of thepH-activity profile of renin for the substrate indicated a minimumK _m (～1.8 µM) atpH ～7.4 and a maximumV _m betweenpH 7.4 and 8.0. The thermodynamics of the binding of a novel, soluble, peptidomimetic inhibitor to renin indicated it is possible to retain the tight-binding characteristics and enthalpy contributions to binding of larger peptide-derived inhibitors, while reducing inhibitor size and entropic contributions to binding. A novel derivative of the fluorogenic substrate, containing a 3-methyl histidine substitution at the P₂ site, was used to test the recent hypothesis that renin functions by virtue of substrate-directed catalysis.     

FeCl3-catalyzed alpha-glycosidation of glycosamine pentaacetates

An efficient method for the large-scale preparation of α-N-acetylglycosaminides, such as fluorogenic T_N-antigen probes and an α-GalNAc-Ser derivative, has been achieved using FeCl₃-catalyzed one-step condensation between commercially available d-glycosamine pentaacetates and fluorogenic acceptors (or serine acceptor) in refluxing 1,2-dichloroethane. Experimental simplicity, high α-stereoselectivity, low cost, satisfactory yield, and the environmentally benign nature are major advantages of our approach.     

A new resorufin-based α-glucosidase assay for high-throughput screening

Mutations in α-glucosidase cause accumulation of glycogen in lysosomes, resulting in Pompe disease, a lysosomal storage disorder. Small molecule chaperones that bind to enzyme proteins and correct the misfolding and mistrafficking of mutant proteins have emerged as a new therapeutic approach for the lysosomal storage disorders. In addition, α-glucosidase is a therapeutic target for type II diabetes, and α-glucosidase inhibitors have been used in the clinic as alternative treatments for this disease. We have developed a new fluorogenic substrate for the α-glucosidase enzyme assay, resorufin α-d-glucopyranoside. The enzyme reaction product of this new substrate emits at a peak of 590 nm, reducing the interference from fluorescent compounds seen with the existing fluorogenic substrate, 4-methylumbelliferyl-α-d-glucopyranoside. Also, the enzyme kinetic assay can be carried out continuously without the addition of stop solution due to the lower pK_a of the product of this substrate. Therefore, this new fluorogenic substrate is a useful tool for the α-glucosidase enzyme assay and will facilitate compound screening for the development of new therapies for Pompe disease.     

Screening for new peptide substrates for the development of albumin binding anticancer pro-drugs that are cleaved by prostate-specific antigen (PSA) to improve the anti tumor efficacy

Several attempts have been made over the past decade to explore the concept of prodrug strategies that exploit PSA as a molecular target for the release of anticancer drugs in prostate tumors using various prostate specific antigen (PSA)-cleavable peptide linkers, but the desired antitumor and antimetastatic efficacy has not yet been fully achieved. We set out to look for new PSA-cleavable peptide substrates that could be cleaved more rapidly and efficiently than the previously used peptides. To look for the most susceptible PSA-cleavable peptide substrates, we used the so-called spot technology. With the following general formula, we designed 25 different fluorogenic heptapeptides; Cellulose-P5-P4-P3-P2-P1-P1′-P2’ (Fluorophore). The increase of the fluorescence in the supernatant of the reaction mixture was monitored using a 96-well fluorometric plate reader with excitation of λ_ex 485 nm and λ_em 535 nm. Three sequences showed a high fluorogenic liberation after incubation with PSA, i.e., Arg-Arg-Leu-His-Tyr-Ser-Leu (7), Arg-Arg-Leu-Asn-Tyr-Ser-Leu (8) and Arg-Ser-Ser-Tyr-Arg-Ser-Leu (23). Future incorporation of these optimized substrates in the PSA-cleavable prodrug formulations could further optimize the cleavage pattern and so the release characteristics of these prodrugs to rapidly and efficiently liberate the free cytotoxic agents inside the tumor tissues.     

Cyclosis-mediated transfer of H2O2 elicited by localized illumination of Chara cells and its relevance to the formation of pH bands

Cytoplasmic streaming occurs in most plant cells and is vitally important for large cells as a means of long-distance intracellular transport of metabolites and messengers. In internodal cells of characean algae, cyclosis participates in formation of light-dependent patterns of surface pH and photosynthetic activity, but lateral transport of regulatory metabolites has not been visualized yet. Hydrogen peroxide, being a signaling molecule and a stress factor, is known to accumulate under excessive irradiance. This study was aimed to examine whether H₂O₂ produced in chloroplasts under high light conditions is released into streaming fluid and transported downstream by cytoplasmic flow. To this end, internodes of Chara corallina were loaded with the fluorogenic probe dihydrodichlorofluorescein diacetate and illuminated locally by a narrow light beam through a thin optic fiber. Fluorescence of dihydrodichlorofluorescein (DCF), produced upon oxidation of the probe by H₂O₂, was measured within and around the illuminated cell region. In cells exhibiting active streaming, H₂O₂ first accumulated in the illuminated region and then entered into the streaming cytoplasm, giving rise to the expansion of DCF fluorescence downstream of the illuminated area. Inhibition of cyclosis by cytochalasin B prevented the spreading of DCF fluorescence along the internode. The results suggest that H₂O₂ released from chloroplasts under high light is transported along the cell with the cytoplasmic flow. It is proposed that the shift of cytoplasmic redox poise and light-induced elevation of cytoplasmic pH facilitate the opening of H⁺/OH^?-permeable channels in the plasma membrane.     

TAMRA- and Cy5-labeled probe for efficient kinetic characterization of caspase-3

Our objective was to create a novel fluorogenic substrate for efficient in vitro kinetic assays on caspase-3. We designed a TAMRA (5′-tetramethylrhodamine-5(6)-carboxamide)- and Cy5 (cyanine 5)-labeled probe that allowed us to evaluate the caspase-3 activity via the changes in fluorescence intensity and wavelength. The prepared probe was found to be an efficient and selective substrate of caspase-3, with V_max of 41.4 ± 3.3 nM/min and K_M of 1.60 ± 0.23 μM. The strategy used in the design of this fluorogenic substrate can be applied in future endeavors to development of substrates for caspase-3 inhibitor screening assays or for real-time detection of apoptosis in living cells.     

Characterization of a new fluorogenic substrate for microsomal glutathione transferase 1

A new thiol-reactive electrophilic, disubstituted rhodamine-based fluorogenic probe (bis-2,4-dinitrobenzenesulfonyl rhodamine [BDR]) with very high quantum yield was synthesized and described recently [A. Shibata et al., Bioorg. Med. Chem. Lett. 18 (2008) 2246-2249]. Because hydrophobic electrophiles are often conjugated by glutathione transferases, the BDR or monosubstituted rhodamine derivatives (2,4-dinitrobenzenesulfonyl rhodamine [DR]) were tested with microsomal glutathione transferase 1 (MGST1) and shown to function as substrates. The kinetic parameters for purified enzyme and DR were k_cat = 0.075 ± 0.005 s⁻¹ and K_m = 21 ± 3 μM (k_cat/K_m = 3.6 × 10³ ± 5.6 × 10² M⁻¹ s⁻¹), giving a rate enhancement of 10⁶ compared with the nonenzymatic reaction. In cells overexpressing MGST1, the addition of BDR caused a time-dependent increase of fluorescence compared with control cells. Preincubating the cells with a thiol reagent (N-ethylmaleimide) abolished the fluorescent signal. By using DR, we could determine the MGST1 activity in whole cell extracts with high sensitivity. In addition, the activity could be increased by thiol reagents (a hallmark of MGST1). Thus, we have identified a new fluorogenic substrate for MGST1 that will be a useful tool in the study of this enzyme and related enzymes.     

Rational design of a SHP-2 targeted,fluorogenic peptide substrate

Protein tyrosine phosphatase (PTP) targeted, peptide based chemical probes are valuable tools for studying this important family of enzymes, despite the inherent difficulty of developing peptides targeted towards an individual PTP. Here, we have taken a rational approach to designing a SHP-2 targeted, fluorogenic peptide substrate based on information about the potential biological substrates of SHP-2. The fluorogenic, phosphotyrosine mimetic phosphocoumaryl aminopropionic acid (pCAP) provides a facile readout for monitoring PTP activity. By optimizing the amino acids surrounding the pCAP residue, we obtained a substrate with the sequence Ac-DDPI-pCAP-DVLD-NH₂ and optimized kinetic parameters (k_cat = 0.059 ± 0.008 s⁻¹, K_m = 220 ± 50 µM, k_cat/K_m of 270 M⁻¹s⁻¹). In comparison, the phosphorylated coumarin moiety alone is an exceedingly poor substrate for SHP-2, with a k_cat value of 0.0038 ± 0.0003 s⁻¹, a K_m value of 1100 ± 100 µM and a k_cat/K_m of 3 M⁻¹s⁻¹. Furthermore, this optimized peptide has selectivity for SHP-2 over HePTP, MEG1 and PTPµ. The data presented here demonstrate that PTP-targeted peptide substrates can be obtained by optimizing the sequence of a pCAP containing peptide.     

Km values of influenza virus neuraminidases for a new fluorogenic substrate, 4-methylumbelliferone N-acetyl neuraminic acid ketoside.

A fluorogenic substrate of neuraminidase, 4-methylumbelliferone N-acetylneuraminic acid ketoside (MUN), was synthesized. K_m values were obtained for Clostridium perfringens neuraminidase and strains of A- and B-type influenza neuraminidase from chicken red blood cell influenza virus eluates.     

3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures

DNA probes with conjugated minor groove binder (MGB) groups form extremely stable duplexes with single-stranded DNA targets, allowing shorter probes to be used for hybridization based assays. In this paper, sequence specificity of 3′-MGB probes was explored. In comparison with unmodified DNA, MGB probes had higher melting temperature (T_m) and increased specificity, especially when a mismatch was in the MGB region of the duplex. To exploit these properties, fluorogenic MGB probes were prepared and investigated in the 5′-nuclease PCR assay (real-time PCR assay, TaqMan assay). A 12mer MGB probe had the same T_m (65°C) as a no-MGB 27mer probe. The fluorogenic MGB probes were more specific for single base mismatches and fluorescence quenching was more efficient, giving increased sensitivity. A/T rich duplexes were stabilized more than G/C rich duplexes, thereby leveling probe T_m and simplifying design. In summary, MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.     

6-Aminoquinoline as a fluorogenic leaving group in peptide cleavage reactions: A new fluorogenic substrate for chymotrypsin

A new fluorogenic substrate capable of measuring the amidolytic activity of chymotrypsin and based upon the enzyme-catalyzed release of a highly fluorescent aromatic amine, 6-aminoquinoline, was prepared. The substrate, 6-(N-glutaryl-l-phenylalanylamido)quinoline, was found to have at pH 8.0 and 25°C K_m = 1.77 mm and k_cat = 1.4 × 10^?1 s^?1. The aminoquinoline is a unique leaving group in that its appearance can be measured fluorometrically at its excitation and emission maxima, while, under these conditions, fluorescence associated with unhydrolyzed substrate is negligible.     

A small compound targeting the interaction between nonstructural proteins 2B and 3 inhibits dengue virus replication

The non-structural protein NS2B/NS3 serine-protease complex of the dengue virus (DENV) is required for the maturation of the viral polyprotein. Dissociation of the NS2B cofactor from NS3 diminishes the enzymatic activity of the complex. In this study, we identified a small molecule inhibitor that interferes with the interaction between NS2B and NS3 using structure-based screening and a cell-based viral replication assay. A library containing 661,417 small compounds derived from the Molecular Operating Environment lead-like database was docked to the NS2B/NS3 structural model. Thirty-nine compounds with high scores were tested in a secondary screening using a cell-based viral replication assay. SK-12 was found to inhibit replication of all DENV serotypes (EC₅₀ = 0.74–4.92 μM). In silico studies predicted that SK-12 pre-occupies the NS2B-binding site of NS3. Steady-state kinetics using a fluorogenic short peptide substrate demonstrated that SK-12 is a noncompetitive inhibitor against the NS2B/NS3 protease. Inhibition to Japanese encephalitis virus by SK-12 was relatively weak (EC₅₀ = 29.81 μM), and this lower sensitivity was due to difference in amino acid at position 27 of NS3. SK-12 is the promising small-molecule inhibitor that targets the interaction between NS2B and NS3.     

Systematic reconstruction of binding and stability landscapes of the fluorogenic aptamer spinach

Fluorogenic RNAs that are based on the complex formed by 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) derivatives and the RNA aptamer named Spinach were used to engineer a new generation of in vitro and in vivo sensors for bioanalytics. With the resolved crystal structure of the RNA/small molecule complex, the engineering map becomes available, but comprehensive information regarding the thermodynamic profile of the molecule is missing. Here, we reconstructed the full thermodynamic binding and stability landscapes between DFHBI and a truncated sequence of first-generation Spinach. For this purpose, we established a systematic screening procedure for single- and double-point mutations on a microfluidic large-scale integrated chip platform for 87-nt long RNAs. The thermodynamic profile with single base resolution was used to engineer an improved fluorogenic spinach generation via a directed rather than evolutional approach.     

Fluorogenic probe for measuring high-mannose type glycan-specific endo-β-N-acetylglucosaminidase H activity

We synthesized a fluorogenic probe with a high-mannose type heptasaccharide structure to detect the hydrolytic activity of endo-β-N-acetylglucosaminidase from Streptomyces plicatus (Endo-H). The heptasaccharide derivative (1) was labeled with an N-methylanthraniloyl group as a reporter dye at the branching point of the β-mannoside residue and 2,4-dinitrophenyl group as a quencher molecule at the reducing end, which was hydrolyzed by Endo-H, resulting in increased fluorescence intensity. Thus, Endo-H activities could be evaluated easily and quantitatively by measuring the fluorescence signal. Using both this probe (1) and a previously synthesized pentasaccharide probe, the hydrolysis activity of Endo-H and Endo-M were investigated. The results clearly showed a correlation with the substrate specificity of each enzyme.     

Carbohydrates and Activity of Natural and Recombinant Tissue Factor

The effect of glycosylation on tissue factor (TF) activity was evaluated, and site-specific glycosylation of full-length recombinant TF (rTF) and that of natural TF from human placenta (pTF) were studied by liquid chromatography-tandem mass spectrometry. The amidolytic activity of the TF·factor VIIa (FVIIa) complex toward a fluorogenic substrate showed that the catalytic efficiency (V_max) of the complex increased in the order rTF_1–243 (Escherichia coli) < rTF_1–263 (Sf9 insect cells) < pTF for the glycosylated and deglycosylated forms. Substrate hydrolysis was unaltered by deglycosylation. In FXase, the K_m of FX for rTF_1–263-FVIIa remained unchanged after deglycosylation, whereas the k_cat decreased slightly. A pronounced decrease, 4-fold, in k_cat was observed for pTF·FVIIa upon deglycosylation, whereas the K_m was minimally altered. The parameters of FX activation by both rTF_1–263D-FVIIa and pTF_D-FVIIa were identical and similar to those for rTF_1–243-FVIIa. In conclusion, carbohydrates significantly influence the activity of TF proteins. Carbohydrate analysis revealed glycosylation on asparagines 11, 124, and 137 in both rTF_1–263 and pTF. The carbohydrates of rTF_1–263 contain high mannose, hybrid, and fucosylated glycans. Natural pTF contains no high mannose glycans but is modified with hybrid, highly fucosylated, and sialylated sugars.     

Detection of Ralstonia solanacearum Strains with a Quantitative, Multiplex, Real-Time, Fluorogenic PCR (TaqMan) Assay

A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5′ nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to ≥10² cells ml⁻¹ was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum.     

Theoretical studies of clonal selection: minimal antibody repertoire size and reliability of self-non-self discrimination.

Viewing the immune system as a molecular recognition device designed to identify “foreign shapes”, we estimate the probability that an immune system with N_Ab monospecific antibodies in its repertoire can recognize a random foreign antigen. Furthermore, we estimate the improvement in recognition if antibodies are multispecific rather than monospecific. From our probabilistic model we conclude: (1) clonal selection is feasible, i.e. with a finite number of antibodies an animal can recognize an effectively infinite number of antigens; (2) there should not be great differences in the specificities of antibody molecules among different species; (3) the region of a foreign molecule recognized by an antibody must be severely limited in extent; (4) the probability of recognizing a foreign molecule, P, increases with the antibody repertoire size N_Ab; however, below a certain value of N_Ab the immune system would be very ineffectual, while beyond some high value of N_Ab further increases in N_Ab yield diminishing small increases in P; (5) multispecificity is equivalent to a modest increase (probably less than 10) in the antibody repertoire size N_Ab, but this increase can substantially improve the probability of an immune system recognizing a foreign molecule.Besides recognizing foreign molecules, the immune system must distinguish them from self molecules. Using the mathematical theory of reliability we argue that multisite recognition is a more reliable method of distinguishing between molecules than single site recognition. This may have been an important evolutionary consideration in the selection of weak non-covalent interactions as the basis of antigen-antibody bonds.     

Identification and evaluation of universal epitopes in Plasmodium vivax Duffy binding protein

Selected PvDBP-derived synthetic peptides were tested in competition assays with HLA molecules in order to identify and evaluate their binding to a wide range of MHC class II molecules. Binding was evaluated as the peptide’s ability to displace the biotinylated control peptide (HA_306-318) and was detected by a conventional ELISA. Thus, one epitope for the HLA-DR1 molecule, two epitopes for the HLA-DR4 molecule, six epitopes for the HLA-DR7 molecule and three epitopes for the HLA-DR11 molecule displaying a high binding percentage (above 50%) were experimentally obtained. The in vitro results were compared with the epitope prediction results. Two peptides behaved as universal epitopes since they bound to a larger number of HLA-DR molecules. Given that these peptides are located in the conserved PvDBP region II, they could be considered good candidates to be included in the design of a synthetic vaccine against Plasmodium vivax malaria.