Structural, kinetic, and thermodynamic analysis of the binding of the 40 kDa PEG-interferon-alpha2a and its individual positional isomers to the extracellular domain of the receptor IFNAR2

Type-I Interferons exert antiviral and antiproliferative activities through the binding to a common cell surface receptor comprising two subunits, IFNAR1 and IFNAR2. Human recombinant Interferon-alpha(2a) (IFNalpha(2a)) is a potent drug (Roferon-A) used to treat various cancers and viral diseases including Hepatitis B/C infections. To significantly improve the pharmacological properties of the drug, a pegylated form of IFNalpha(2a) was developed (PEGASYS). This 40 kDa PEG-conjugated IFNalpha(2a) ((40)PEG-IFNalpha(2a)) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side-chain of IFNalpha(2a). Here, we report the detailed structural, kinetic, and thermodynamic analysis of the binding to the extracellular domain of the receptor IFNAR2 of (40)PEG-IFNalpha(2a) and its isolated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively, in comparison with unmodified IFNalpha(2a). Our binding studies, using the surface plasmon resonance technique, show that the pegylation does not abolish the binding to the receptor, but significantly reduces the affinity mainly due to a change of the association rate. The results are supported by modeling and simulation of the binding, using Self-Avoiding-Walk calculations for the polymer conformations. A correlation between the structural parameters and the kinetic and thermodynamic parameters of the binding of the positional isomers could be established. For the Isomer-K31 and -K164, the PEG-polymer attachment point is located in proximity to the binding interface, and the isomers display affinity in the range 150-520 nM in an enthalpy-driven binding process. In contrast for the Isomer-K134, -K131, -K121, and -K70, the PEG-polymer is attached remotely from the binding interface, and the isomers exhibit a higher affinity (32-76 nM) in an entropy-driven binding process. This study constitutes an essential collection of knowledge on which the interaction of (40)PEG-IFNalpha(2a) and its positional isomers with its cellular receptors can be better understood.     

Isolation,structural characterization,and antiviral activity of positional isomers of monopegylated interferon alpha-2a (PEGASYS)

Interferon alpha-2a plays an essential role in the treatment of chronic hepatitis C, but it is limited in its efficacy by the short in vivo half-life. To improve the half-life and efficacy, interferon alpha-2a is conjugated with a 40-kDa branched polyethylene glycol moiety (PEG-IFN, PEGASYS). From this preparation the positional PEG-IFN isomers were isolated and characterized by different analytical methods and antiviral assay. Two chromatographic steps were used to separate and purify nine isomers. The analytical methods IE-HPLC, RP-HPLC, SE-HPLC, SDS-PAGE, and MALDI-TOF MS indicated that each of these nine isomers is conjugated to the branched polyethylene glycol chain at a specific lysine. No isomer with a modification at the amino terminus was observed. All positional isomers induced viral protection of MDBK cells in the antiviral assay. When comparing the quantitative potency of the individual isomers with the whole mixture of PEG-IFN, significant differences in the specific activities were observed: PEG-Lys(31) and PEG-Lys(134) showed higher activities than the mixture, PEG-Lys(164) was equal to the mixture, whereas the activities of PEG-Lys(49), PEG-Lys(70), PEG-Lys(83), PEG-Lys(112), PEG-Lys(121), and PEG-Lys(131) were lower.     

Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon alpha-2a for the treatment of hepatitis C

A potent, long-lasting form of interferon alpha-2a mono-pegylated with a 40 kilodalton branched poly(ethylene glycol) was designed, synthesized, and characterized. Mono-pegylated interferon alpha-2a was comprised of four major positional isomers involving Lys31, Lys121, Lys131, and Lys134 of interferon. The in vitro anti-viral activity of pegylated interferon alpha-2a was found to be only 7% of the original activity. In contrast, the in vivo antitumor activity was severalfold enhanced compared to interferon alpha-2a. Pegylated interferon alpha-2a showed no immunogenicity in mice. After subcutaneous injection of pegylated interferon alpha-2a, a 70-fold increase in serum half-life and a 50-fold increase in mean plasma residence time concomitant with sustained serum concentrations were observed relative to interferon alpha-2a. These preclinical results suggest a significantly enhanced human pharmacological profile for pegylated interferon alpha-2a. Results of Phase II/III hepatitis C clinical trials in humans confirmed the superior efficacy of pegylated interferon alpha-2a compared to unmodified interferon alpha-2a.     

Site of pegylation and polyethylene glycol molecule size attenuate interferon-alpha antiviral and antiproliferative activities through the JAK/STAT signaling pathway

Therapeutic pegylated interferon-alphas (IFN-alpha) are mixtures of positional isomers that have been monopegylated at specific sites on the core IFN-alpha molecule. The pegylation results in lower in vitro specific activity associated with the core IFN-alpha molecule that is related to the site of pegylation and size of polyethylene glycol (PEG) attached. We prepared purified, homogeneous, positional pegylation isomers of IFN-alpha2b that were monopegylated using 5-30-kDa linear PEG molecules attached at 7 primary reactive amino acid residues: Cys(1), His(34), Lys(31), Lys(83), Lys(121), Lys(131), and Lys(134). The isomers were evaluated for STAT translocation and antiviral and antiproliferative activity. The site of pegylation strongly influenced activity relative to an IFN-alpha2b control. The highest residual activity was observed with the His(34) positional isomers, and the lowest was observed with the Cys(1) positional isomers. The Lys positional isomers demonstrated intermediate activity, with a general order of Lys(134) > Lys(83) approximately Lys(131) approximately Lys(121) > Lys(31). The progressive relationship between decreased activity and increased PEG size suggests that pegylation may interfere with interaction and binding of IFN-alpha to the IFNAR1-IFNAR2 heterodimeric receptor. The higher specific activity associated with the His(34) positional isomer suggests that this site may be favorable for pegylating IFN-alpha2b molecules.     

Biophysical analysis of the interaction of human ifnar2 expressed in E. coli with IFNalpha2.

Type I interferons are cytokines which activate an anti-viral response by binding to two specific cell surface receptors, ifnar1 and ifnar2. Here, we report purification and refolding of the extracellular part of human ifnar2 (ifnar2-EC) expressed in Escherichia coli and its characterization with respect to its interaction with interferon alpha2 (IFNalpha2). The 25 kDa, non-glycosylated ifnar2-EC is a stable, fully active protein, which inhibits antiviral activity of IFNalpha2. The stoichiometry of binding IFNalpha2 is 1:1, as determined by gel filtration, chemical cross-linking and solid-phase detection. The affinity of this interaction is 10 nM, which is similar to the affinity measured for the cell surface-bound ifnar2 receptor. No difference in affinity was found throughout various assays using optical detection as BIAcore or reflectometric interference spectorscopy. However, the binding kinetics as measured in homogeneous phase by fluorescence de-quenching was about three times faster than that measured on a sensor surface. The rate of complex formation is relatively high compared to other cytokine-receptor interactions. The salt dependence of the association kinetics suggest a limited but significant contribution of electrostatic forces towards the rate of complex formation. The dissociation constant increases with decreasing pH according to the protonation of a base with a pKa of 6.7. The surface properties of the IFNalpha2 binding surface on ifnar2 were interpreted according to the pH and salt dependence of the interaction.     

Nitrile hydratase and amidase from Rhodococcus rhodochrous hydrolyze acrylic fibers and granular polyacrylonitriles

Rhodococcus rhodochrous NCIMB 11216 produced nitrile hydratase (320 nkat mg of protein(-1)) and amidase activity (38.4 nkat mg of protein(-1)) when grown on a medium containing propionitrile. These enzymes were able to hydrolyze nitrile groups of both granular polyacrylonitriles (PAN) and acrylic fibers. Nitrile groups of PAN40 (molecular mass, 40 kDa) and PAN190 (molecular mass, 190 kDa) were converted into the corresponding carbonic acids to 1.8 and 1.0%, respectively. In contrast, surfacial nitrile groups of acrylic fibers were only converted to the corresponding amides. X-ray photoelectron spectroscopy analysis showed that 16% of the surfacial nitrile groups were hydrolyzed by the R. rhodochrous enzymes. Due to the enzymatic modification, the acrylic fibers became more hydrophilic and thus, adsorption of dyes was enhanced. This was indicated by a 15% increase in the staining level (K/S value) for C. I. Basic Blue 9.     

Ligand-induced assembling of the type I interferon receptor on supported lipid bilayers

Type I interferons (IFNs) elicit antiviral, antiproliferative and immuno-modulatory responses through binding to a shared receptor consisting of the transmembrane proteins ifnar1 and ifnar2. Differential signaling by different interferons, in particular IFNalphas and IFNbeta, suggests different modes of receptor engagement. Using reflectometric interference spectroscopy (RIfS), we studied kinetics and affinities of the interactions between IFNs and the extracellular receptor domains of ifnar1 (ifnar1-EC) and ifnar2 (ifnar2-EC). For IFNalpha2, we determined a K(D) value of 3 nM and 5 microM for the interaction with ifnar2-EC and ifnar1-EC, respectively. As compared to IFNalpha2, IFNbeta formed complexes with ifnar2-EC as well as ifnar1-EC with substantially higher affinity. For neither IFNalpha2 nor IFNbeta was stabilization of the complex with ifnar1-EC in the presence of soluble ifnar2-EC observed. We investigated ligand-induced complex formation with ifnar1-EC and ifnar2-EC being tethered onto solid-supported, fluid lipid bilayers by RIfS and total internal reflection fluorescence spectroscopy. We observed very stable binding of IFNalpha2 at high receptor surface concentrations with an apparent k(d) value approximately 200 times lower than that for ifnar2-EC alone. The apparent k(d) value was strongly dependent on the surface concentration of the receptor components, suggesting kinetic stabilization. This was corroborated by the fast exchange of labeled IFNalpha2 bound to the receptor by unlabeled IFNalpha2. Taken together, our results indicate that IFN first binds to ifnar2 and subsequently recruits ifnar1 in a transient fashion. In particular, this second step is much more efficient for IFNbeta than for IFNalpha2, which could explain differential activities observed for these IFNs.     

Identification of the major positional isomer of pegylated interferon alpha-2b

Interferons display a wide range of antiviral, antiproliferative, and immunomodulatory activities on a variety of cell types and have been used to treat many diseases including hairy-cell leukemia and hepatitis B and C and have also been applied to other therapeutic areas. To improve the pharmacological properties of interferon (IFN) alpha-2b, a long-acting pegylated form (PEG-IFN) has been developed [PEG, monomethoxy poly(ethylene glycol) with average molecular mass of 12 000 Da]. PEG-IFN is a mixture of pegylated proteins with differing sites of PEG attachment. To identify the major positional isomer in the pegylated material [PEG-IFN(His-34)], NMR studies were conducted on a subtilisin-digested N-acetylated peptide of the major positional isomer [PEG-IFN(His-34)dig], synthetic peptide analogues containing His-34, as well as unmodified IFN and PEG-IFN(His-34). Our studies reveal a novel interferon-polymer attachment site as a histidine-linked interferon conjugate. We show that the major component of PEG-IFN is pegylated in the imidazole side chain of histidine-34. Chemical shift data suggest that pegylation occurs mainly at the N(delta)(1) position in the imidazole side chain of this residue. This positional isomer, PEG-IFN(His-34), comprises approximately 47% of the total pegylated species when PEG-IFN is synthesized under the current experimental conditions at pH 6.5 with an electrophilic derivative of PEG, succinimidyl carbonate PEG. The reversibility of the histidine modification was examined. The PEG-imidazole adduct in the intact protein, PEG-IFN(His-34), is labile but much more stable than in the peptide, PEG-IFN(His-34)dig. Apparently, the tertiary structure of the intact protein protects the His(34)-imidazole ring from depegylation.     

Structural identification and biological activity of positional isomers of long-acting and mono-PEGylated recombinant human granulocyte colony-stimulating factor with trimeric-structured methoxy polyethylene glycol N-hydroxysuccinimidyl functional group

The individual positional isomers from the mono-PEGylated recombinant human granulocyte colony-stimulating factor (rhG-CSF) were successfully isolated with additional strong cation exchange chromatography using Source 15S. The three isolated individual positional isomers were found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), analytical size exclusion high-performance liquid chromatography (SE-HPLC), and analytical cation exchange HPLC (CIE-HPLC) and were also characterized with respect to site of PEGylation by enzymatic digestion with endoproteinase Lys-C and N-terminal sequencing. In addition, in vitro biological activity was determined by cell proliferation assay. It was determined that the three isolated individual positional isomers were PEGylated at Lys35, Met(N-terminal), and Lys17 of the rhG-CSF molecule with a 23-kDa trimer-structured methoxy polyethylene glycol N-hydroxysuccinimidyl functional group (mPEG-NHS). All individual positional isomers (Lys35-PEGylated rhG-CSF, Met(N-terminal)-PEGylated rhG-CSF, and Lys17-PEGylated rhG-CSF) retained in vitro biological activity and were found to be 18.5%, 37.6%, and 7.1%, respectively, compared with the rhG-CSF molecule. The significantly different in vitro biological activities observed in the individual positional isomers could be presumably due to interference of receptor binding or active sites on the rhG-CSF molecule. In conclusion, the individual positional isomers isolated from the mono-PEGylated rhG-CSF were well characterized with respect to the site of PEGylation involving Lys35, Met(N-terminal), and Lys17. This characterization of the individual positional isomers would be critical to provide a basis for establishing consistency in the manufacturing process.     

Biochip as a potential platform of serological interferon alpha2b antibody assay

The formation of antibodies against cytokines may play a major role in the generation of the immune response and may affect treatment protocols with recombinant cytokines. Interferon (IFN) is one of the effective therapeutic agents with anti-viral and anti-tumor specific effects. The appearance of IFN antibodies in patients may limit the natural and the therapeutic effect by IFNs. In contrast to conventional ELISA techniques, we here report a simple biochip methodology that enables identification of antibodies against cytokines and peptides. The method takes advantage of a functionalized self-assembled monolayer modified by N-hydroxysuccinimide (NHS). To validate this surface, four human proteins: IFNalpha2b, leptin, growth hormone and human IgG, with molecular sizes ranging between 14 and 150 kDa, were used. A number of other parameters for protein assay conditions by array technology were evaluated concomitantly. Finally, 56 serum samples from patients treated with recombinant human IFNalpha2b were simultaneously tested on single chip. In these patients, 16.1% (9 of 56 cases) were positive for IFNalpha2b antibodies. All results were confirmed in an ELISA, specific for the identification of IFNalpha specific antibodies in human samples. The potential application of this protein biochip can be amplified rapidly and reliably to test not only IFNalpha2b, but also other cytokine specific antibodies. The clinical relevance of such assays for investigations in autoimmune disorders is expected.     

Existing and future therapeutic options for hepatitis C virus infection

Hepatitis C virus (HCV) infection is an important cause of chronic hepatitis, cirrhosis, hepatocellular carcinoma and liver failure worldwide. Chronic hepatitis C virus infection is treated with interferon-a (IFN-alpha), pegylated interferon-alpha (PEG-IFNalpha) alone or in combination with ribavirin; however, a significant fraction of patients either fail to respond or relapse after cessation of therapy. Efforts to identify and develop highly specific and potent HCV inhibitors have intensified recently. Each of the virally encoded replication enzymes has been a focus of studies as well as viral receptors and the host immune system. This review summarizes recent progress in the search for novel anti-HCV agents.     

Identification and characterization of a novel inositol hexakisphosphate kinase

The inositol pyrophosphate disphosphoinositol pentakisphosphate (PP-InsP(3)/InsP(7)) is formed in mammals by two recently cloned inositol hexakiphosphate kinases, InsP(6)K1 and InsP(6)K2 (Saiardi, A., Erdjument-Bromage, H., Snowman, A. M., Tempst, P., and Snyder, S. H. (1999) Curr. Biol. 9, 1323-1326). We now report the identification, cloning, and characterization of a third InsP(7) forming enzyme designated InsP(6)K3. InsP(6)K3 displays 50 and 45% sequence identity to InsP(6)K1 and InsP(6)K2, respectively, with a smaller mass (46 kDa) and a more basic character than the other two enzymes. InsP(6)K3 is most enriched in the brain where its localization resembles InsP(6)K1 and InsP(6)K2. Intracellular disposition discriminates the three enzymes with InsP(6)K2 being exclusively nuclear, InsP(6)K3 predominating in the cytoplasm, and InsP(6)K1 displaying comparable nuclear and cytosolic densities.     

Scanning calorimetric studies of aqueous dispersions of bilayers made with cholesterol and a pair of positional isomers of 3-sn-phosphatidylcholine

Differential scanning calorimetric studies have been carried out on aqueous dispersions of cholesterol plus one of the pair of positional isomers, 1-arachidoyl-2-oleoylphosphatidylcholine (AOPC) or 1-oleoyl-2-arachidoylphosphatidylcholine (OAPC). There were some differences in the shapes of the endotherms obtained from the dispersions with the two positional isomers. These observations confirm the previous finding (Davis, P.J. and Keough, K.M.W. (1984) Biochemistry 22, 6334–6340) that positional isomers of unsaturated phosphatidylcholines may interact differently with cholesterol, at least in the gel state. The shapes of endotherms obtained here and in the previous study are consistent with the suggestion that the position of the unsaturated chain on the glycerol has a role in determining the nature of the phosphatidylcholine-cholesterol interaction. Certain features of the endotherms seen here and previously (op. cit.) suggest that factors such as effective chain depth in the bilayer or efficiency of chain packing in the bilayer may also influence this interaction.     

Raman spectroscopic study of polycrystalline mono- and polyunsaturated 1-eicosanoyl-d(39)-2-eicosenoyl-sn-glycero-3-phosphocholines: bilayer lipid clustering and acyl chain order and disorder characteristics

Polycrystalline lipid samples of a series of mono- and polyunsaturated, double bond positional isomers of 1-eicosanoyl-d(39)-2-eicosenoyl-sn-glycero-3-phosphocholines [C(20-d(39)):C(20:1 Delta(j))PC, with j = 5, 8, 11, or 13; C(20-d(39)):C(20:2 Delta(11,14))PC; and C(20-d(39)):C(20:3 Delta(11, 14,17))PC] were investigated using vibrational Raman spectroscopy to assess the acyl chain packing order-disorder characteristics and putative bilayer cluster formation of the isotopically differentiated acyl chains. Perdeuteration of specifically the saturated sn-1 acyl chains for these bilayer systems enables each chain's intra- and intermolecular conformational and organizational properties to be evaluated separately. Various saturated chain methylene CD(2) and carbon-carbon (C&bond;C) stretching mode peak height intensity ratios and line width parameters for the polycrystalline samples demonstrate a high degree of sn-1 chain order that is unaffected by either the double bond placement or number of unsaturated bonds within the sn-2 chain. In contrast, the unsaturated sn-2 chain spectral signatures reflect increasing acyl chain conformational disorder as either the cis double bond is generally repositioned toward the chain terminus or the number of double bonds increases from one to three. The lipid bilayer chain packing differences observed between the sn-1 and sn-2 chains of this series of monounsaturated and polyunsaturated 20 carbon chain lipids suggest the existence of laterally distributed microdomains predicated on the formation of highly ordered, saturated sn-1 chain clusters.     

Isolation and characterization of three positional isomers of diglucosylcyclomaltoheptaose

Three positional isomers of diglucosylcyclomaltoheptaose [(G)2-beta-cyclodextrin], 6(1),6(4)-di-O-(a-D-glucopyranosyl)-cyclomaltoheptaose (1), 6(1),6(3)-di-O-(a-D-glucopyranosyl)-cyclomaltoheptaose (2), and 6(1),6(2)-di-O-(a-D-glucopyranosyl)-cyclomaltoheptaose (3) were isolated by h.p.l.c. on a reversed-phase column from the mother liquors of a large-scale preparation of beta CD with Bacillus ohbensis cyclomaltodextrin glucanotransferase (EC 2.4.1.19) and were characterized by h.p.l.c. analysis of partial hydrolyzates and by 13C-n.m.r. spectroscopy. Their molecular weights were confirmed by f.a.b.-m.s. Their characteristic chromatographic behavior on four h.p.l.c. columns of different separation modes was found to be very useful for their identification. It is particularly noteworthy that the first application of a graphitized carbon column to CDs enabled a fine separation of all three positional isomers.     

Precise and comparative pegylation analysis by microfluidics and mass spectrometry

Standard SDS-PAGE analysis of a pegylated protein was able to confirm an increase in its molecular size after reaction with an activated polyethylene glycol (PEG) but could do little to identify the extent of pegylation or to support characterization of the consistency of the modified protein. In this article, we demonstrate the utility of the capillary electrophoresis technology (using a microfluidic system) in analyzing the pegylation pattern of a recombinant protein over a range of 1-12 PEGs per polypeptide. Confirmatory data from mass spectrometry analysis of pegylated adducts are also presented. These allowed independent confirmation of the extent of pegylation. This electrophoretic analysis gives a robust, reproducible, and direct characterization of PEG adducts. We found that traditional estimation of PEG adducts by an indirect colorimetric (trinitrobenzene sulfonic acid) reaction, which detects loss of free amino groups, was quite erroneous for the recombinant protein in our study as well as several commercially available pegylated proteins. These results support the use of this capillary electrophoresis device for precise characterization of pegylated proteins.     

Correlations between in vitro potency of polyethylene glycol-protein conjugates and their chromatographic behavior

Pegylation is the most widely used and accepted methodology for half-life extension of biopharmaceutical drugs that also improves physicochemical and biological characteristics of proteins considerably. Most of the positive pharmacological effects of pegylated proteins are believed to be related to an increased hydrodynamic volume and molecular size. To explore the size impact of polyethylene glycol (PEG) on in vitro potency, a series of well-defined conjugates of interferon α-2b (IFN) were prepared with PEGs of different lengths and shapes specifically attached to the N-terminal amino group of the protein. Specificity of the attachment was confirmed by peptide mapping and mass spectroscopy. When potency values determined by reporter gene assay were correlated with methods for molecular weight and size characterization, such as size exclusion chromatography and dynamic light scattering, rough parallels were found. Unexpectedly, the retention times on cation exchange chromatography showed much higher correlation with experimentally determined in vitro potency. It appears that in a series of N-terminally pegylated IFNs, their in vitro potency could be predicted from the retention times on the cation exchange chromatography columns, probably because both methods reflect not only the influence of molecular size but also the impact of protein masking exerted by attached PEG moiety.     

Isolation and structural analyses of positional isomers of 6(1),6(n)-di-O-(N-acetyl-beta-D-glucosaminyl)cyclomaltoheptaose (n=2, 3, and 4) and 6-O-[6-O-(N-acetyl-beta-D-glucosaminyl)-N-acetyl-beta-D-glucosaminyl]cyclomaltoheptaose.

6-O-[6-O-(N-acetyl-beta-D-glucosaminyl)-N-acetyl-beta-D-glucosaminyl]cyclomaltoheptaose (beta CD) and three positional isomers of 6(1),6(n)-di-O-(N-acetyl-beta-D-glucosaminyl)cyclomaltoheptaose (n=2, 3, and 4) in a mixture of products from beta CD and N-acetylglucosamine by the reversed reaction of beta-N-acetylhexosaminidase from jack bean were isolated and purified by HPLC. The structures of four isomers of di-N-acetylglucosaminyl-beta CDs were determined by FABMS and NMR spectroscopy. The degree of polymerization of the branched oligosaccharides produced by enzymatic degradation with bacterial saccharifying alpha-amylase (BSA) was established by LC-MS methods.     

Isolation and structure determination of enzymatically formed styrene oxide glutathione conjugates.

When styrene oxide was incubated with glutathione in the presence of rat liver cytosolic fraction, two conjugates were formed. Structural investigation by mass spectrometry (MS), proton magnetic resonance (PMR) analysis and chemical fragmentation showed the presence of two positional isomers, namely S-(1-phenyl-2-hydroxyethyl)glutathione and S-(2-phenyl-2-hydroxyethyl)glutathione in a ratio of approx. 60 : 40.