Biomedical subjects

Dingyi Wen

Publications and source records attributed to Dingyi Wen.

9 recordsLinked to original sources

PubMed2005

Disulfide structure of the leucine-rich repeat C-terminal cap and C-terminal stalk region of Nogo-66 receptor.

Nogo-66 receptor (NgR1) is a leucine-rich repeat (LRR) protein that forms part of a signaling complex modulating axon regeneration. Previous studies have shown that the entire LRR region of NgR1, including the C-terminal cap of the LRR, LRRCT, is needed for ligand binding, and that the adjacent C-terminal region (CT stalk) of the NgR1 contributes to interaction with its coreceptors. To provide structure-based information for these interactions, we analyzed the disulfide structure of full-length NgR1. Our analysis revealed a novel disulfide structure in the C-terminal region of the NgR1, wherein the two Cys residues, Cys-335 and Cys-336, in the CT stalk are disulfide-linked to Cys-266 and Cys-309 in the LRRCT region: Cys-266 is linked to Cys-335, and Cys-309 to Cys-336. The other two Cys residues, Cys-264 and Cys-287, in the LRRCT region are disulfide-linked to each other. The analysis also showed that Cys-419 and Cys-429, in the CT stalk region, are linked to each other by a disulfide bond. Although published crystal structures of a recombinant fragment of NgR1 had revealed a disulfide linkage between Cys-266 and Cys-309 in the LRRCT region and we verified its presence in the corresponding fragment, this is artificially caused by the truncation of the protein, since this linkage was not detected in intact NgR1 or a slightly larger fragment containing Cys-335 and Cys-336. A structural model of the LRRCT with extended residues 311-344 from the CT stalk region is proposed, and its function in coreceptor binding is discussed.

Alkylation↗

PubMed2005

Selectivity of BAFF/BLyS and APRIL for binding to the TNF family receptors BAFFR/BR3 and BCMA.

BAFF (B cell activating factor of the TNF family, also known as BlyS and TALL-1), a TNF family cytokine critical for the development and function of B cells, has been reported to bind to three receptors, BCMA (B cell maturation protein), TACI (transmembrane activator and CAML [calcium-modulator and cyclophilin ligand] interactor), and BAFFR (BAFF receptor), but with widely conflicting values for the affinity and selectivity of binding. BCMA and TACI additionally bind APRIL (a proliferation-inducing ligand), the TNF family ligand most homologous to BAFF. Using soluble, monomeric forms of the receptors, we demonstrate that BAFFR binds BAFF with K(D) approximately 16 nM, while BCMA binds with K(D) approximately 1.6 microM, indicating a approximately 100-fold selectivity for binding to BAFFR over BCMA. APRIL shows the opposite selectivity, binding to BCMA with K(D) approximately 16 nM while showing no detectable affinity for BAFFR (K(D) > 3 microM). The binding of BAFF or APRIL to these receptors is highly sensitive to assay-dependent avidity effects, likely explaining the widely ranging affinity values reported in the literature. Binding of BAFF to BCMA-Fc, a bivalent fusion protein consisting of the extracellular domain of BCMA fused to the hinge and CH1 and CH2 domains of human IgG1, in solution or coated onto an ELISA plate gave apparent binding affinities of approximately 0.63 and approximately 0.15 nM, respectively, compared to values of K(D(app)) <or= 30 and approximately 100 pM for the corresponding BAFFR/IgG1 fusion protein, BAFFR-Fc. The high selectivity of BAFF for BAFFR versus BCMA is thus partly obscured in these multivalent assays. The intrinsically high selectivity inferred from the measurements with monomeric receptor correlates well with in vivo data from knockout mice, providing a possible explanation for the observations that interruption of the BAFFR gene in the A/WySnJ mouse produces a phenotype similar to the BAFF knockout mouse, while the BCMA knockout mouse has no discernible B cell phenotype.

Animals↗

PubMed2005

Mechanistic studies of inactivation of glutathione S-transferase Pi isozyme by a haloenol lactone derivative.

Cancer chemotherapy often fails due to acquired drug resistance. One of the most critical biochemical changes observed in drug-resistant tumor cells is over-expression of glutathione S-transferase Pi isozyme (GSTP1). Glutathione S-transferase inhibitors have been used as potentiating agents of chemotherapeutic drugs. Earlier we reported haloenol lactone 1 as a site-directed GSTP1 inactivator. We proposed that enzymatic hydrolysis of the haloenol lactone may be the initial step of GSTP1 chemical modification, resulting in the inactivation of the enzyme. Enzyme inactivation is initiated through addition of Cys-47 to the lactone ring, which is opened in the process to form an alpha-bromoketone adduct. The acidity of Cys-47 confers good leaving group properties, and rapid hydrolysis occurs to generate an alpha-bromoketoacid intermediate. The reaction may proceed via alkylation of the transient thioester to form a six-membered ring episulfonium ion intermediate which would be yet more reactive toward hydrolysis, with either process leading to the observed mass increase of 230 Da. To probe the importance of the bromine of the lactone in GST inactivation, we designed and synthesized compound 2. Unlike lactone 1, lactone 2 did not show time-dependent inhibitory effect on GSTP1. Incubation of compounds 1 and 2 with excess of N-acetyl cysteine produced the corresponding di-N-acetyl cysteine conjugate and mono-N-acetyl cysteine conjugate, respectively. To probe the role of Cys-47 in the inactivation of GSTP1 by compound 1, we prepared mutant C47A GSTP1. The mutant GSTP1 still showed good activity toward CDNB, but it lost susceptibility to the inactivation by compound 1. In addition, LC-MS/MS technique allowed us to identify the modified Cys-47 after the enzyme was exposed to compound 1.

Enzyme Activation↗

PubMed2004

Design, synthesis, and structure-activity relationships of haloenol lactones: site-directed and isozyme-selective glutathione S-transferase inhibitors.

Overexpression of glutathione S-transferase (GST), particularly the GST-pi isozyme, has been proposed to be one of the biochemical mechanisms responsible for drug resistance in cancer chemotherapy, and inhibition of overexpressed GST has been suggested as an approach to combat GST-induced drug resistance. 3-Cinnamyl-5(E)-bromomethylidenetetrahydro-2-furanone (1a), a lead compound of site-directed GST-pi inactivator, has been shown to potentiate the cytotoxic effect of cisplatin on tumor cells. As an initial step to develop more potent and more selective haloenol lactone inactivators of GST-pi, we examined the relationship between the chemical structures of haloenol lactone derivatives and their GST inhibitory activity. A total of 16 haloenol lactone derivatives were synthesized to probe the effects of (1) halogen electronegativity, (2) electron density of aromatic rings, (3) molecular size and rigidity, (4) lipophilicity, and (5) aromaticity on the potency of GST-pi inactivation. The inhibitory potency of each compound was determined by time-dependent inhibition tests, and recombinant human GST-pi was used to determine their inhibitory activity. Our structure-activity relationship studies demonstrated that (1) reactivity of the halide leaving group plays a weak role in GST inactivation by the haloenol lactones, (2) aromatic electron density may have some influence on the potency of GST inactivation, (3) high rigidity likely disfavors enzyme inhibition, (4) lipophilicity is inversely proportional to enzyme inactivation, and (5) an unsaturated system may be important for enzyme inhibition. This work facilitated understanding of the interaction of GST-pi with haloenol lactone derivatives as site-directed and isozyme-selective inactivators, possibly potentiating cancer chemotherapy.

Drug Design↗

PubMed2004

Expression, purification, and characterization of rat interferon-beta, and preparation of an N-terminally PEGylated form with improved pharmacokinetic parameters.

To identify potential new clinical uses and routes of administration for human interferon-beta-1a (IFN-beta-1a), we have developed an expression and purification procedure for the preparation of highly purified rat interferon-beta (IFN-beta) suitable for testing in rat models of human disease. An expression vector containing the rat IFN-beta signal sequence and structural gene was constructed and transfected into Chinese hamster ovary (CHO) cells. The protein was purified from CHO cell conditioned medium and purified to > 99.5% purity using standard chromatographic techniques. Analytical characterization indicated that the protein was a heavily glycosylated monomeric protein, with two of the four predicted N-glycosylation sites occupied. Analysis of the attached oligosaccharides showed them to be a complex mixture of bi-antennary, tri-antennary, and tetra-antennary structures with a predominance of sialylated tri-antennary and tetra-antennary structures. Peptide mapping, N-terminal sequencing, and mass spectrometry confirmed the identity and integrity of the purified protein. The purified protein had a specific activity of 2.1x10(8)U/mg when assayed on rat RATEC cells, which is similar in magnitude to the potencies observed for murine IFN-beta and human IFN-beta-1a assayed on murine and human cells, respectively. We also prepared an N-terminally PEGylated form of rat IFN-beta in which a 20 kDa methoxy polyethylene glycol (PEG)-propionaldehyde was attached to the N-terminal alpha-amino group of Ile-1. The PEGylated protein, which retained essentially full in vitro antiviral activity, had improved pharmacokinetic parameters in rats as compared to the unmodified protein. Both the unmodified and PEGylated forms of rat IFN-beta will be useful for testing in rat models of human disease.

Animals↗

PubMed2003

Expression of Sonic hedgehog-Fc fusion protein in Pichia pastoris. Identification and control of post-translational, chemical, and proteolytic modifications.

We have investigated the suitability of Pichia pastoris as an expression system for the candidate therapeutic protein, Sonic hedgehog fused to an immunoglobulin Fc domain (Shh-Fc). Sonic hedgehog is a morphogen protein involved in the patterning of a wide range of tissues during animal embryogenesis. The presence of Sonic hedgehog and its receptor, Patched, in adult nervous tissue suggests possible applications for the protein in the treatment of neurodegenerative disease and injury. We have engineered the Shh-Fc fusion protein in order to improve binding affinity and increase systemic exposure in animals. N-terminal sequencing, peptide mapping, mass spectrometry, and other biochemical and biological methods were used to characterize the purified protein. These analyses revealed several unanticipated problems, including thiaproline modification of the N-terminal cysteine, cleavage by a Kex2-like protease at a site near the N-terminus, proteolysis at sites near the hinge, addition of a hexose in the CH3 domain of the Fc region, and several sites of methionine oxidation. Sequence modifications to the protein and changes in fermentation conditions resulted in increased potency and greater consistency of the product. The final product was shown to be biologically active in animal studies.

Amino Acid Sequence↗

PubMed2003

The CRIPTO/FRL-1/CRYPTIC (CFC) domain of human Cripto. Functional and structural insights through disulfide structure analysis.

The disulfide structure of the CRIPTO/FRL-1/CRYPTIC (CFC) domain of human Cripto protein was determined by a combination of enzymatic and chemical fragmentation, followed by chromatographic separation of the fragments, and characterization by mass spectrometry and N-terminal sequencing. These studies showed that Cys115 forms a disulfide bond with Cys133, Cys128 with Cys149, and Cys131 with Cys140. Protein database searching and molecular modeling revealed that the pattern of disulfide linkages in the CFC domain of Cripto is the same as that in PARS intercerebralis major Peptide C (PMP-C), a serine protease inhibitor, and that the EGF-CFC domains of Cripto are predicted to be structurally homologous to the EGF-VWFC domains of the C-terminal extracellular portions of Jagged 1 and Jagged 2. Biochemical studies of the interactions of ALK4 with the CFC domain of Cripto by fluorescence-activated cell sorter analysis indicate that the CFC domain binds to ALK4 independent of the EGF domain. A molecular model of the CFC domain of Cripto was constructed based on the nuclear magnetic resonance structure of PMP-C. This model reveals a hydrophobic patch in the domain opposite to the presumed ALK4 binding site. This hydrophobic patch may be functionally important for the formation of intra or intermolecular complexes.

Activin Receptors, Type I↗

PubMed2002

Long-acting forms of Sonic hedgehog with improved pharmacokinetic and pharmacodynamic properties are efficacious in a nerve injury model.

The therapeutic effects of the Sonic hedgehog (Shh) have been difficult to evaluate because of its relatively short serum half-life. To address this issue polyethylene glycol modification (PEGylation) was investigated as an approach to improve systemic exposure. Shh was PEGylated by a targeted approach using cysteines that were engineered into the protein by site-directed mutagenesis as the sites of attachment. Sixteen different versions of the protein containing one, two, three, or four sites of attachment were characterized. Two forms were selected for extensive testing in animals, Shh A192C, which provided a single site for PEGylation, and Shh A192C/N91C, which provided two sites. The PEGylated proteins were evaluated for reaction specificity by SDS-PAGE and peptide mapping, in vitro potency, pharmacokinetic and pharmacodynamic properties, and efficacy in a sciatic nerve injury model. Targeted PEGylation was highly selective for the engineered cysteines and had no deleterious effect on Shh function in vitro. Systemic clearance values in rats decreased from 117.4 mL/h/kg for unmodified Shh to 29.4 mL/h/kg for mono-PEGylated Shh A192C that was modified with 20 kDa PEG-maleimide and to 2.5 mL/h/kg for di-PEGylated Shh A192C/N91C modified with 2, 20 kDa PEG vinylsulfone adducts. Serum half-life increased from 1 h for unmodified Shh to 7.0 and 12.6 h for the mono- and di-PEGylated products. These changes in clearance and half-life resulted in higher serum levels of Shh in the PEG-Shh-treated animals. In Ptc-LacZ knock-in mice expressing lacZ under regulation of the Shh receptor Patched, about a 10-fold lower dose of PEG-Shh was needed to induce beta-galactosidase than for the unmodified protein. Therapeutic treatment of mice with PEG-Shh enhanced the regeneration of injured sciatic nerves. These studies demonstrate that targeted PEGylation greatly alters the pharmacokinetic and pharmacodynamic properties of Shh, resulting in a form with improved pharmaceutical properties.

Animals↗

PubMedDate not supplied

N-terminally PEGylated human interferon-beta-1a with improved pharmacokinetic properties and in vivo efficacy in a melanoma angiogenesis model.

PEGylation of IFN-alpha has been used successfully to improve the pharmacokinetic properties and efficacy of the drug. To prepare a PEGylated form of human interferon-beta-1a (IFN-beta-1a) suitable for testing in vivo, we have synthesized 20 kDa mPEG-O-2-methylpropionaldehyde and used it to modify the N-terminal alpha-amino group of the cytokine. The PEGylated protein retained approximately 50% of the activity of the unmodified protein and had significantly improved pharmacokinetic properties following intravenous administration in rats. The clearance and volume of distribution at steady state were reduced approximately 30-fold and approximately 4-fold, respectively, resulting in a significant increase in systemic exposure as determined by the area under the curve. The elimination half-life of the PEGylated protein was approximately 13-fold greater than for the unmodified protein. The unmodified and PEGylated proteins were tested for their ability to inhibit the formation of radially oriented blood vessels entering the periphery of human SK-MEL-1 melanoma tumors in athymic nude homozygous (nu/nu) mice. In a single dose comparison study, administration of 1 x 10(6) units of unmodified IFN-beta-1a resulted in a 29% reduction in vessel number, while 1 x 10(6) units of PEGylated IFN-beta-1a resulted in a 58% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle (control)-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. In a multiple versus single dose comparison study, daily administration of 1 x 10(6) units of unmodified IFN-beta-1a for 9 days resulted in a 51% reduction in vessel number, while a single dose of 1 x 10(6) units of the PEGylated protein resulted in a 66% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. Therefore, the improved pharmacokinetic properties of the modified protein translated into improved efficacy. Since unmodified IFN-beta is used for the treatment of multiple sclerosis and hepatitis C virus infection, a PEGylated form of the protein such as 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-beta-1a may serve as a useful adjunct for the treatment of these diseases. In addition, the antiangiogenic effects of PEGylated IFN-beta-1a may be harnessed for the treatment of certain cancers, either as a sole agent or in combination with other antitumor drugs.

Aldehydes↗