Accessible Disulfide Research Articles

Bispecific FpFs: a versatile tool for preclinical antibody development.

We previously described FpFs 1̲ (Fab-PEG-Fab) as binding mimetics of IgGs. FpFs are prepared with di(bis-sulfone) conjugation reagents 3̲ that undergo disulfide rebridging conjugation with the accessible disulfide of each Fab (Scheme 1). We have now prepared bispecific FpFs 2̲ (bsFpF and Fab1-PEG-Fab2) as potential bispecific antibody mimetics with the intent that bsFpFs could be used in preclinical antibody development since sourcing bispecific antibodies may be challenging during preclinical research. The di(bis-sulfone) reagent 3̲ was first used to prepare a bsFpF 2̲ by the sequential conjugation of a first Fab and then a second Fab to another target (Scheme 2). Seeking to improve bsFpF synthesis, the asymmetric conjugation reagent, bis-sulfone bis-sulfide 1̲6̲, with different thiol conjugation reactivities at each terminus (Scheme 4) was examined and the bsFpFs appeared to be formed at similar conversion to the di(bis-sulfone) reagent 3̲. To explore the advantages of using common intermediates in the preparation of bsFpF families, we investigated bsFpF synthesis with a protein conjugation-ligation approach (Scheme 5). Reagents with a bis-sulfone moiety for conjugation on one PEG terminus and a ligation moiety on the other terminus were examined. Bis-sulfone PEG trans-cyclooctene (TCO) 2̲8̲ and bis-sulfone PEG tetrazine (Tz) 3̲0̲ were used to prepare several bsFpFs targeting various therapeutic targets (TNF-α, IL6R, IL17, and VEGF) and tissue affinity targets (hyaluronic acid and collagen II). Surface plasmon resonance (SPR) binding studies indicated that there was little difference between the dissociation rate constant (k d) for the unmodified Fab, mono-conjugated PEG-Fab and the corresponding Fab in a bsFpF. The Fab association rate (k a) in the bsFpF was slower than for PEG-Fab, which may be because of mass differences that influence SPR results. These observations suggest that each Fab will bind to its target independently of the other Fab and that bsFpF binding profiles can be estimated using the corresponding PEG-Fab conjugates.

Trifunctional Dibromomaleimide Reagents Built Around A Lysine Scaffold Deliver Site-selective Dual-modality Antibody Conjugation.

We describe the synthesis and application of a selection of trifunctional reagents for the dual-modality modification of native, solvent accessible disulfide bonds in trastuzumab. The reagents were developed from the dibromomaleimide (DBM) platform with two orthogonal clickable functional groups built around a lysine core. We also describe the development of an aryl diselenide additive which enables antibody disulfide reduction in 4 minutes and a rapid overall reduction-bridging-double click sequence.

Conformational perturbation of SARS-CoV-2 spike protein using N-acetyl cysteine: an exploration of probable mechanism of action to combat COVID-19

The infection caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) resulted in a pandemic with huge death toll and economic consequences. The virus attaches itself to the human epithelial cells through noncovalent bonding of its spike protein with the angiotensin-converting enzyme-2 (ACE2) receptor on the host cell. Based on in silico studies we hypothesized that perturbing the functionally active conformation of spike protein through the reduction of its solvent accessible disulfide bonds, thereby disintegrating its structural architecture, may be a feasible strategy to prevent infection by reducing the binding affinity towards ACE2 enzyme. Proteomics data showed that N-acetyl cysteine (NAC), an antioxidant and mucolytic agent been widely in use in clinical medicine, forms covalent conjugates with solvent accessible cysteine residues of spike protein that were disulfide bonded in the native state. Further, in silico analysis indicated that the presence of the selective covalent conjugation of NAC with Cys525 perturbed the stereo specific orientations of the interacting key residues of spike protein that resulted in threefold weakening in the binding affinity of spike protein with ACE2 receptor. Interestingly, almost all SARS-CoV-2 variants conserved cystine residues in the spike protein. Our finding results possibly provides a molecular basis for identifying NAC and/or its analogues for targeting Cys-525 of the viral spike protein as fusion inhibitor and exploring in vivo pharmaco-preventive and its therapeutic potential activity for COVID-19 disease. However, in-vitro assay and animal model-based experiment are required to validate the probable mechanism of action. Communicated by Ramaswamy H. Sarma

Disulfide Modified IgG1: An Investigation of Biophysical Profile and Clinically Relevant Fc Interactions.

Modification of immunoglobulin G (IgG) 1 proteins in cancer treatment is a rapidly growing field of research. Antibody-drug conjugates (ADCs) exploit the targeted nature of this immunotherapy by conjugating highly potent drugs to antibodies, allowing for effective transport of cargo(s) to cancerous cells. Of the many bioconjugation strategies now available for the formation of highly homogeneous ADCs, disulfide modification is considered an effective, low-cost, and widely accepted method for modifying IgG1s for improved clinical benefit. However, little is known about how disulfide modification impacts clinically relevant fragment crystallizable (Fc) region interactions. Although often overlooked as a secondary ADC function, Fc interactions could prove key in the rational design of cancer cell-targeting ADCs through consideration of potent mechanisms such as antibody-dependent cellular cytotoxicity (ADCC). This work explores different IgG1 disulfide modification techniques and the effect they have on quantifiable secondary IgG1 Fc interactions (e.g., CD16a and FcRn). The solvent accessible disulfide residues of trastuzumab, a clinically relevant IgG1, were modified to provide a range of bioconjugates with differing amounts of interchain covalent linkages. It was found that by natively rebridging the IgG1 model, all tested Fc functionalities were not significantly affected. Additionally, in non Fc-specific biophysical experiments (e.g., thermal stability/aggregation), the natively rebridged species provided an exceptional profile, showing no significant change from the tested native antibody. Conjugates with significant disruption of the covalent connectivity of IgG1 chains resulted in a suboptimal Fc profile (CD16a kinetics or ADCC activity), in addition to substandard non Fc-specific attributes (thermal stability). These results advocate native disulfide rebridging as an excellent synthetic strategy for forming homogeneous IgG1 bioconjugates, with no reported negative impact on biophysical profile relative to the native antibody.

S-linked protein homocysteinylation: identifying targets based on structural, physicochemical and protein–protein interactions of homocysteinylated proteins

An elevated level of homocysteine, a thiol-containing amino acid is associated with a wide spectrum of disease conditions. A majority (>80%) of the circulating homocysteine exist in protein-bound form. Homocysteine can bind to free cysteine residues in the protein or could cleave accessible cysteine disulfide bonds via thiol disulfide exchange reaction. Binding of homocysteine to proteins could potentially alter the structure and/or function of the protein. To date only 21 proteins have been experimentally shown to bind homocysteine. In this study we attempted to identify other proteins that could potentially bind to homocysteine based on the criteria that such proteins will have significant 3D structural homology with the proteins that have been experimentally validated and have solvent accessible cysteine residues either with high dihedral strain energy (for cysteine-cysteine disulfide bonds) or low pKa (for free cysteine residues). This analysis led us to the identification of 78 such proteins of which 68 proteins had 154 solvent accessible disulfide cysteine pairs with high dihedral strain energy and 10 proteins had free cysteine residues with low pKa that could potentially bind to homocysteine. Further, protein-protein interaction network was built to identify the interacting partners of these putative homocysteine binding proteins. We found that the 21 experimentally validated proteins had 174 interacting partners while the 78 proteins identified in our analysis had 445 first interacting partners. These proteins are mainly involved in biological activities such as complement and coagulation pathway, focal adhesion, ECM-receptor, ErbB signalling and cancer pathways, etc. paralleling the disease-specific attributes associated with hyperhomocysteinemia.

Suppression of the plasma membrane H+-conductance on the background of high H+-pump activity in dithiothreitol-treated Chara cells

Photosynthesizing cells of characean algae exposed to light are able to produce pH bands corresponding to alternate areas with dominant H+-pump activity and high H+-conductance of the cell membrane. The action potential generation temporally arrests the counter-directed H+ fluxes, which gives rise to opposite pH shifts in different cell regions and represents a suitable indicator for activities of the plasma membrane H+-transporting systems. Measurements of pH near the cell surface by means of microelectrodes and microspectrophotometry in the presence of pH-indicating dye thymol blue have shown that the treatment of cells with dithiothreitol (SH-group reducing agent) suppresses pH changes induced by the action potential generation in the alkaline cell areas and considerably increases the concurrent pH changes in the acid regions. Measurements of plasma membrane resistance in the alkaline zones revealed that dithiothreitol inhibits the light-dependent conductance of the resting cell and diminishes the conductance inactivation caused by the action potential generation. The data suggest that the reduction of accessible disulfide bonds results in the decrease of H+-conductance, whereas the activity of plasma membrane H+-pump remains unimpaired or is even enhanced.

De Novo Sequencing of RCB-1 to -3: Peptide Biomarkers from the Castor Bean PlantRicinus communis

Ricinus communis (also know as the castor bean plant) whose forbears escaped from suburban gardens or commercial cultivation grow wild in many countries. In temperate and tropical climates seeds will develop to maturity, and plants may be perennial. In Australia these plants have become widespread and are regarded as noxious weeds in many localities. The seeds of R. communis contain ricin, a protein toxin which can easily be extracted into an aqueous solution. Ricin is toxic by ingestion, inhalation, and injection. The history of terrorist and anarchist interest in the use of seeds from R. communis has driven the development of strategies for determination of cultivar and geographic location of the source of an extract of wild-grown castor bean seed. This forensic information is of considerable interest to law enforcement and intelligence organizations. During forensic studies of both the metabolome and proteome of extracts from eight specimens of six different cultivars of R. communis ("zanzibariensis" collected from Kenya and Tanzania, "gibsonii", "impala", "dehradun", "carmencita", and "sanguineus" collected from Spain and Tanzania), three peptide biomarkers (designated Ricinus communis biomarkers, or RCB) were identified in both the MALDI and electrospray LC-MS spectra. Two of these peptides (RCB-1 and RCB-2) were present in varying amounts in all cultivars, while RCB-3 was present only in the "carmencita" cultivar. The amino acid sequences of RCB-1 to -3 were determined using LC-MS(n) fragmentation and de novo sequencing on both the intact and the carbamidomethyl modified peptides. The connectivity of the two disulfide bonds that were present in all three RCB were determined using a strategy of partial reduction and differential alkylation using tris-(2-carboxyethyl)phosphine with N-ethylmaleimide to reduce and alkylate the most accessible disulfide bond, followed by reduction and alkylation of the remaining disulfide bond with dithiolthreitol and iodoacetamide. The possible functional role of RCB-1 to -3 in R. communis seeds is also discussed.

Identification and insertion of 3-carbon bridges in protein disulfide bonds: a computational approach

More than 42,000 3D structures of proteins are available on the Internet. We have shown that the chemical insertion of a 3-carbon bridge across the native disulfide bond of a protein or peptide can enable the site-specific conjugation of PEG to the protein without a loss of its structure or function. For success, it is necessary to select an appropriate and accessible disulfide bond in the protein for this chemical modification. We describe how to use public protein databases and molecular modeling programs to select a protein rationally and to identify the optimum disulfide bond for experimental studies. Our computational approach can substantially reduce the time required for the laboratory-based chemical modification. Identification of solvent-accessible disulfides using published structural information takes approximately 2 h. Predicting the structural effects of the disulfide-based modification can take 3 weeks.

PEGylation of native disulfide bonds in proteins

PEGylation has turned proteins into important new biopharmaceuticals. The fundamental problems with the existing approaches to PEGylation are inefficient conjugation and the formation of heterogeneous mixtures. This is because poly(ethylene glycol) (PEG) is usually conjugated to nucleophilic amine residues. Our PEGylation protocol solves these problems by exploiting the chemical reactivity of both of the sulfur atoms in the disulfide bond of many biologically relevant proteins. An accessible disulfide bond is mildly reduced to liberate the two cysteine sulfur atoms without disturbing the protein's tertiary structure. Site-specific PEGylation is achieved with a bis-thiol alkylating PEG reagent that sequentially undergoes conjugation to form a three-carbon bridge. The two sulfur atoms are re-linked with PEG selectively conjugated to the bridge. PEGylation of a protein can be completed in 24 h and purification of the PEG-protein conjugate in another 3 h. We have successfully applied this approach to PEGylation of cytokines, enzymes, antibody fragments and peptides, without destroying their tertiary structure or abolishing their biological activity.

Ab initio structure solution of a dimeric cytochrome c3 from Desulfovibrio gigas containing disulfide bridges.

The 1.2 A resolution crystal structure of the 29 kDa di-tetrahaem cytochrome c3 from the sulfate reducing bacterium Desulfovibrio gigas was solved by ab initio methods, making this the largest molecule to be solved by this procedure. The actual refined model of the cysteine-linked dimeric molecule reveals that this molecule is very similar to the non-covalently linked symmetrical dimer of the di-tetrahaem cytochrome c3 from Desulfomicrobium norvegicum. Each monomer has the typical polypeptide fold, haem arrangement and iron coordination found for the tetrahaem cytochrome c3 molecules. The interface between the covalently linked monomers in the asymmetric unit of the crystal shows a pseudo two-fold arrangement, disturbed from symmetry by crystal packing forces. The fact that D. gigas contains a dimeric tetrahaem cytochrome with solvent accessible disulfide bridges and that this cytochrome specifically couples hydrogen oxidation to thiosulfate reduction in bacterial extracts provides an interesting aspect related to disulfide exchange reactions in this microorganism.

Reagents for Rapid Reduction of Native Disulfide Bonds in Proteins

Bis(2-mercaptoethyl)sulfone (BMS) and N,N′-dimethyl-N,N′-bis(mercaptoacetyl)hydrazine (DMH) reduce native disulfide bonds in proteins at pH 7 significantly faster than does dithiothreitol (DTT). The accessible disulfide bonds in immunoglobulin and trypsinogen are reduced under nondenatoring conditions at pH 7 faster using BMS and DMH than using DTT by a factor of ∼5 to 7. The relatively less accessible disulfide bond in α-chymotrypsinogen A is also reduced faster using BMS and DMH than using DTT by a factor of 2.3. Although both BMS and DMH reduce disulfides at similar rates, we recommend BMS because it is commercially available and has superior physical characteristics and a higher reduction potential than DMH.

Disulfide arrangement and functional domains of beta-1,4-endoglucanse E5 from Thermomonospora fusca.

Thermomonospora fusca cellulase E5 contains six cysteine residues. The number and location of the disulfide bonds and the effect of reduction of the disulfides and modification of the resulting half-cystine residues on enzymatic activity were determined. No free sulfhydryl groups were found in E5. Reduction and subsequent labeling with iodoacetamide of E5 and of an enzymatically active 32-kDa proteolytic derivative of E5 (E5cd) showed that one of the three disulfides is accessible to reduction under nondenatured conditions while the other two are not accessible. Full reduction of the disulfides and complete carboxymethylation of the six cysteines decrease the specific activity of E5 on CMC by more than half, but reduction of only the exposed disulfide bond does not affect enzymatic activity or binding of E5 to cellulose. A 14-kDa proteolytic fragment of E5 containing 120 amino acids from the N-terminus of the protein was shown to bind to crystalline cellulose. This confirms earlier evidence that the cellulose binding domain of E5 is located at the N-terminus of the protein. This 14-kDa fragment contains the accessible disulfide bond involving Cys93 and Cys100. The location of the two disulfide bonds in the other fragment (E5cd) was determined by cleaving it with cyanogen bromide under conditions that left the disulfide bonds intact. The resulting peptides were separated under both nonreducing and reducing conditions using RP-HPLC. Amino acid analysis of peptide peaks indicated that one disulfide linkage in E5cd joins Cys138 to Cys143 while the other joins Cys166 to Cys406.

The Fab/c fragment of IgG produced by cleavage at cyanocysteine residues

The intra- and inter-heavy chain disulfides of rabbit IgG were cleaved by mild reduction with either dithiothreitol or sulfite and cyanocysteines generated by treatment with either 2-nitro-5-thiocyanobenzoic acid or KCN, respectively. When cleavage occurs at a cyanocysteine residue in the hinge region of one heavy chain alone the Fab/c fragment is produced. Fab/c was also produced by papain digestion of IgG. Fab/c made by papain digestion was able to active complement in haemolytic assays; this activity was lost after cleavage of its accessible disulfide bonds. Fab/c made by cyanylysis of sulfite-reduced IgG was also active in these assays, but Fab/c made by cyanylysis of dithiothreitol-reduced IgG was not. Treatment of the latter fragment with cysteine and cystine resulted in partial reformation of cleaved disulfide bonds. Fab/c was also made from human IgG and from murine IgG2a and IgG2b.

Determination of the sulfhydryl and disulfide groups in proteins by methylation, treatment with raney nickel and infrared spectrometry

A method is described for the determination of the total and accessible free SH-groups in proteins, based on the methylation of the SH-groups in non-hydrolysed proteins with dimethyl sulfate, followed by hydrolysis, additional methylation (for the total free SH-groups) and treatment with Raney nickel. The methane released is measured by infrared spectroscopy. Methionine must be determined separately and a correction made. The relative error is 3%, and the time necessary for one determination is about 2 h (excluding the time for hydrolysis). The method may also be applied to the determination of accessible SH-groups of homogenates of internal organs. If the protein hydrolysate and the non-hydrolysed protein are reduced before methylation, the total and accessible disulfide groups can also be determined.