Heterobifunctional Reagent Research Articles

Multiple redox switches of the SARS-CoV-2 main protease in vitro provide opportunities for drug design

Besides vaccines, the development of antiviral drugs targeting SARS-CoV-2 is critical for preventing future COVID outbreaks. The SARS-CoV-2 main protease (Mpro), a cysteine protease with essential functions in viral replication, has been validated as an effective drug target. Here, we show that Mpro is subject to redox regulation in vitro and reversibly switches between the enzymatically active dimer and the functionally dormant monomer through redox modifications of cysteine residues. These include a disulfide-dithiol switch between the catalytic cysteine C145 and cysteine C117, and generation of an allosteric cysteine-lysine-cysteine SONOS bridge that is required for structural stability under oxidative stress conditions, such as those exerted by the innate immune system. We identify homo- and heterobifunctional reagents that mimic the redox switching and inhibit Mpro activity. The discovered redox switches are conserved in main proteases from other coronaviruses, e.g. MERS-CoV and SARS-CoV, indicating their potential as common druggable sites.

New Insights on Saporin Resistance to Chemical Derivatization with Heterobifunctional Reagents.

Saporin is a type 1 ribosome-inactivating protein widely used as toxic payload in the construction of targeted toxins, chimeric molecules formed by a toxic portion linked to a carrier moiety. Among the most used carriers, there are large molecules (mainly antibodies) and small molecules (such as neurotransmitters, growth factors and peptides). Some saporin-containing targeted toxins have been used for the experimental treatment of several diseases, giving very promising results. In this context, one of the reasons for the successful use of saporin lies in its resistance to proteolytic enzymes and to conjugation procedures. In this paper, we evaluated the influence of derivatization on saporin using three heterobifunctional reagents, namely 2-iminothiolane (2-IT), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and 4-succinimidyloxycarbonyl-α-methyl-α-[2-pyridyldithio]toluene (SMPT). In order to obtain the highest number of inserted -SH groups with the lowest reduction of saporin biological activities, we assessed the residual ability of saporin to inhibit protein synthesis, to depurinate DNA and to induce cytotoxicity after derivatization. Our results demonstrate that saporin maintains an excellent resistance to derivatization processes, especially with SPDP, and permit us to define reaction conditions, in which saporin biological properties may not be altered. Therefore, these findings provide useful information for the construction of saporin-based targeted toxins, especially with small carriers.

A Novel EGFR Targeted Immunotoxin Based on Cetuximab and Type 1 RIP Quinoin Overcomes the Cetuximab Resistance in Colorectal Cancer Cells.

Cetuximab is a monoclonal antibody blocking the epidermal growth factor receptor (EGFR) in metastatic colorectal cancer (mCRC). However, cetuximab treatment has no clinical benefits in patients affected by mCRC with KRAS mutation or in the presence of constitutive activation of signalling pathways acting downstream of the EGFR. The aim of this study was to improve cetuximab's therapeutic action by conjugating cetuximab with the type 1 ribosome inactivating protein (RIP) quinoin isolated from quinoa seeds. A chemical conjugation strategy based on the use of heterobifunctional reagent succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was applied to obtain the antibody-type 1 RIP chimeric immunoconjugate. The immunotoxin was then purified by chromatographic technique, and its enzymatic action was evaluated compared to quinoin alone. Functional assays were performed to test the cytotoxic action of the quinoin cetuximab immunoconjugate against the cetuximab-resistant GEO-CR cells. The novel quinoin cetuximab immunoconjugate showed a significant dose-dependent cytotoxicity towards GEO-CR cells, achieving IC50 values of 27.7 nM (~5.0 μg/mL) at 72 h compared to cetuximab (IC50 = 176.7 nM) or quinoin (IC50 = 149.3 nM) alone assayed in equimolar amounts. These results support the therapeutic potential of quinoin cetuximab immunoconjugate for the EGFR targeted therapy, providing a promising candidate for further development towards clinical use in the treatment of cetuximab-resistant metastatic colorectal cancer.

CEBA: A new heterobifunctional reagent for plasmid DNA functionalization by click chemistry

Here, we report the synthesis of 3,6,9-trioxaundecan-1-{4-[(2-Chloroethyl)Ethylamino)]-Benzylamino},11-Azide (CEBA). CEBA alkylates the N7 of guanine of DNA thanks its chloroethyl group and can be coupled by a strain-promoted azide-alkyne cycloaddition to an alkynylated molecule. The optimization of the alkylation level of pDNA reveals that the expression of the encoded gene is preserved when it is randomly modified with at most 1 CEBA molecule per 150 bp. We show that the azido group of CEBA allows the linkage via click chemistry of CEBA-pDNA with a fluorophore or a peptide containing a dibenzocyclooctyne (DBCO) function. This new heterobifunctional reagent opens new ways to equip pDNA easily with signal molecules including peptides and nucleic acids without side products providing great interest for non-viral gene therapy.

Strong and oriented conjugation of nanobodies onto magnetosomes for the development of a rapid immunomagnetic assay for the environmental detection of tetrabromobisphenol-A.

Variable domain of heavy chain antibody (nanobody, Nb) derived from camelids is an efficient reagent in monitoring environmental contaminants. Oriented conjugates of Nbs and bacterial magnetic particles (BMPs) provide new tools for the high-throughput immunoassay techniques. An anti-tetrabromobisphenol-A (TBBPA) Nb genetically integrated with an extra cysteine residue at the C terminus was immobilized onto BMPs enclosed within the protein membrane, using a heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithiol) propionate, to form a solid BMP-Nb complex. A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) based on the combination of BMP-Nb and T5-horseradish peroxidase was developed for the analysis of TBBPA, with a total assay time of 30min and a half-maximum signal inhibition concentration (IC50) of 1.04ng/mL in PBS (pH10, 10% methanol and 0.137 moL/L NaCl). This assay can even be performed in 100% methanol, with an IC50 value of 44.3ng/mL. This assay showed quantitative recoveries of TBBPA from spiked canal water (114-124%) and sediment (109-113%) samples at 1.0-10ng/mL (or ng/g (dw)). TBBPA residues determined by this assay in real canal water samples were below the limit of detection (LOD) and in real sediments were between <LOD and 23.4ng/g (dw). The BMP-Nb-based ELISA shows promising application in environmental monitoring. Graphical abstract ᅟ.

ChemInform Abstract: A Highly Stable and Versatile Heterobifunctional Fluoroalkylation Reagent for Preparation of Fluorinated Organic Compounds.

AbstractA dipolar cycloaddition of the newly prepared, highly stable fluoroalkylation reagent (II) with terminal alkynes affords 1‐(chlorofluoroiodoethyl)‐1,2,3‐triazoles.

Fluorinated Triazoles by Using a Heterobifunctional Fluoroalkylation Reagent

Fluorinated Triazoles by Using a Heterobifunctional Fluoroalkylation Reagent

Design, synthesis, and evaluation of cisplatin-containing EGFR targeting bioconjugates as potential therapeutic agents for brain tumors.

The aim of this study was to evaluate four different platinated bioconjugates containing a cisplatin (cis-diamminedichloroplatinum [cis-DDP]) fragment and epidermal growth factor receptor (EGFR)-targeting moieties as potential therapeutic agents for the treatment of brain tumors using a human EGFR-expressing transfectant of the F98 rat glioma (F98EGFR) to assess their efficacy. The first two bioconjugates employed the monoclonal antibody cetuximab (C225 or Erbitux®) as the targeting moiety, and the second two used genetically engineered EGF peptides. C225-G5-Pt was produced by reacting cis-DDP with a fifth-generation polyamidoamine dendrimer (G5) and then linking it to C225 by means of two heterobifunctional reagents. The second bioconjugate (C225-PG-Pt) employed the same methodology except that polyglutamic acid was used as the carrier. The third and fourth bioconjugates used two different EGF peptides, PEP382 and PEP455, with direct coordination to the Pt center of the cis-DDP fragment. In vivo studies with C225-G5-Pt failed to demonstrate therapeutic activity following intracerebral (ic) convection-enhanced delivery (CED) to F98EGFR glioma-bearing rats. The second bioconjugate, C225-PG-Pt, failed to show in vitro cytotoxicity. Furthermore, because of its high molecular weight, we decided that lower molecular weight peptides might provide better targeting and microdistribution within the tumor. Both PEP382-Pt and PEP455-Pt bioconjugates were cytotoxic in vitro and, based on this, a pilot study was initiated using PEP455-Pt. The end point for this study was tumor size at 6 weeks following tumor cell implantation and 4 weeks following ic CED of PEP455-Pt to F98 glioma-bearing rats. Neuropathologic examination revealed that five of seven rats were either tumor-free or only had microscopic tumors at 42 days following tumor implantation compared to a mean survival time of 20.5 and 26.3 days for untreated controls. In conclusion, we have succeeded in reformatting the toxicity profile of cis-DDP and demonstrated the therapeutic efficacy of the PEP455-Pt bioconjugate in F98 glioma-bearing rats.

MAPN: First-in-Class Reagent for Kinetically Resolved Thiol-to-Thiol Conjugation

Thiols are among the most frequently used functional groups in the field of bioconjugation. While there exists a variety of heterobifunctional reagents that allow for coupling thiols to other functions (e.g., amines, carboxylic acids), there is no specific reagent for creating heteroconjugates using two different thiols. In response to the ever-increasing demand for bioconjugation tools, we have developed p-(maleimide)-phenylpropionitrile (MAPN)-an efficient reagent for kinetically resolved thiol-to-thiol coupling. In a comparative study with its closest commercially available analogue, p-phenylenedimaleimide, MAPN has shown substantial advantages for the preparation of thiol-thiol heteroconjugates. Namely, an antibody-drug conjugate (ADC) with mertansine (DM1), conjugated to the cysteine residues of Trastuzumab, was prepared for the first time.

CBTF: new amine-to-thiol coupling reagent for preparation of antibody conjugates with increased plasma stability.

Amine-to-thiol coupling is the most common route for the preparation of antibody-drug conjugates (ADC). It is usually achieved by using heterobifunctional reagents possessing an activated ester at one end and a maleimide group at the other. However, maleimide-based conjugates were recently revealed to have limited stability in blood circulation, which can compromise therapeutic efficacy of the conjugate. To address this issue, we have developed a heterobifunctional reagent, sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF), for amine-to-thiol coupling. It comprises a recently described 3-arylpropionitrile (APN) function in replacement of maleimide and allows for the preparation of remarkably stable conjugates. A series of antibody-dye conjugates have been prepared using this reagent and shown superior stability in human blood plasma compared to maleimide-derived conjugates.

Mass spectrometry as a tool for structural biology: crosslinking‐MS and native‐MS methods to study the RNA Pol II ‐ Mediator holoenzyme (778.6)

The Mediator complex plays a central role in transcriptional regulation by relaying gene specific regulatory signals to the general transcriptional machinery. The yeast holoenzyme between mediator and RNA pol II consists of 33 subunits totaling over 1.5 MDa in size. The large size precludes atomic resolution crystallography of the entire assembly. Hence hybrid methods of structure determination are necessary, integrating various techniques including: crystallography of stable subassemblies, cryoEM of the intact complex, and mass spectrometry (MS). Mass spectrometry based structural techniques including chemical crosslinking and native‐MS provide spatial restraints that guide and constrain the modeling process. Crosslinking measurements map interacting protein‐protein surfaces through the identication of amino acid residues that have been covalently modified by heterobifunctional reagents. Native MS permits the determination of stably associated assemblies of proteins in the gas phase. We focus on the development of these new MS based technologies including the design of robust bioinformatics strategies, enrichment methods, and reagents for optimizing mass spectrometric analysis of crosslinked peptides, as well as deconvolution algorithms and sample preparation for native MS analysis. We discuss the application of these methods to determining the structure of the Mediator holoenzyme.Grant Funding Source: Supported by: NIH P41GM103481

Evaluation of immunocastration conjugates based on GnRH linked to carrier molecules in a male rodent model

Gonadotropin-releasing hormone (GnRH) stimulates the pituitary gland to secrete sex hormones. In the present study, we evaluated different conjugates of GnRH to abrogate sex hormone secretion in a male rat model. Firstly, GnRH-I was conjugated to keyhole limpet haemocyanin (KLH) using either whole-sequence GnRH-I or an analogue of GnRH-I (CHWSYGLRPG-NH2) using glutaraldehyde cross linkage. Six-week-old Sprague-Dawley male rats (n = 6) were immunized intramuscularly with the conjugates adsorbed onto alum, equivalent to 50 μg of GnRH peptide and administered in weeks 1, 3, 5 and 7. The study was concluded in week 15. Comparison was made with untreated controls and previously established anti-fertility conjugates: CHWSYGLRPG-NH2 (GnRH-I) or CHWSHDWKPG-NH2 (analogue of lamprey GnRH-III, lGnRH-III) linked to tetanus toxoid (using a heterobifunctional reagent to achieve cross linkage). Antibody production, hormone levels and testicular diameter changes were assessed, together with, sperm movement and effects on organ weights. Similar high levels of antibody secretion were observed in all the immunized groups, although whole GnRH-I-KLH produced a sustained level of production for an additional week. Similarly, testosterone levels were significantly (p < 0.05) reduced in all immunized groups. There were no significant changes observed in body weight and testicular diameter of immunized animals compared with the untreated controls. However, in terms of sperm motility and sperm number, the best anti-fertility effects were observed with lGnRH-III-TT and GnRH-I-KLH and to a lesser extent whole GnRH-I-KLH. These groups also showed significant increase in kidney weight. Finally, considering all the above-mentioned subjects in addition to availability and easier and cheaper way of preparation, we came to this conclusion that whole GnRH-I-KLH satisfactorily met most of our favourite criteria and could be used in immunocastration vaccine production purposes successfully.

The properties conferred upon triple-helical collagen-mimetic peptides by the presence of cysteine residues

Recently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated great interest. A triple-helical peptide known as collagen-related peptide (CRP) contains the sequence (Gly-Pro-Hyp)10. With Gly-Pro-Cys triplets appended to both of its termini, designated CRPcys, chemical cross-linking using heterobifunctional reagents generates CRPcys-XL, a potent, widely used, polymeric agonist for platelet Glycoprotein VI, whereas non-cross-linked, monomeric CRPcys antagonizes Glycoprotein VI. Here, we describe how cysteine in these triplets may also undergo random air-induced oxidation, especially upon prolonged storage or repeated freeze–thawing, to form disulphide bonds, resulting in a lesser degree of polymerization than with chemical cross-linking. We investigated the monomeric and polymeric states of these and other cysteine-containing collagen-derived peptides, using gel filtration and dynamic light scattering, allowing the size of a CRP-XL aggregate to be estimated. The effect of cysteine thiols upon peptide adsorption to surfaces and subsequent platelet responses was investigated. This demonstrated that cysteine is required for strong binding to glass coverslips and to plastic plates used in ELISA assays.

Chemoselective cross-linking of alginate with thiol-terminated peptides for tissue engineering applications

In this study we chemospecifically functionalized alginate with thiol-ended peptides for tissue engineering (TE); a carbodiimide linker and the disulfide exchange scheme were used. First carboxyls of alginate were activated by introducing N-hydroxysuccinimide (NHS) ester groups; these react with primary amines of the heterobifunctional reagent 2-(2-pyridyldithio)ethyleneamine (PDEA). Thiol-reactive alginate (alginate-S-S-py) forms as cross-linking intermediate. The degree of pyridyldithio-functionalization is modified through variable PDEA concentration and it was determined using UV–VIS spectroscopy and proton nuclear magnetic resonance. The applicability of the alginate-S-S-py as platform for the chemoselective coupling of thiol-ended peptides was tested with glutathione as model peptide. Other SH-terminal peptides were successfully cross-linked. Moreover, the peptide–alginate keeps its gel-forming ability when treated with Ca2+ cations, leading to functionalized hydrogels that can be further used to obtain different multicomponent systems. The developed chemoselective strategy of functionalizing alginate with thiol-ended peptides could enhance the potential of this polymer for TE.

Surface Modification of Natural Rubber Latex Film Using Photocrosslinkable Chitosan

Surface modifications of natural rubber latex films were carried out to improve the hydrophilicity by UV-induced immobilization of water-soluble butyryl derivative of chitosan using the photosensitive hetero-bifunctional crosslinking reagent, 4-azidobenzoic acid. The effects of the concentration of modified chitosan solution and UV irradiation time on the water contact angle values of natural rubber latex films were studied. ATR-FTIR analyses confirmed that the O-butylrylchitosan molecules were covalently bonded onto the surface of natural rubber latex film. The water contact angle value of natural rubber latex film which is about 95o reduces to 55o significantly with the introduction of O-butylrylchitosan. The results indicate that the hydrophilic behaviour of natural rubber latex film has been improved by immobilization of O-butylrylchitosan.

Preparation and identification of a novel immunomodulator composed of muramyl dipeptide and anti-CD10 monoclonal antibody for treatment of minimal residual disease in acute leukemia children

Targeted therapy is a potentially useful approach for antileukemic therapy, in particular eliminating minimal residual disease (MRD) to prevent tumor relapse. In this study, the immunomodulator (MDP-Ab) was constructed by coupling anti-CD10 monoclonal antibody (MAb) and muramyl dipeptide (MDP) using heterobifunctional reagent SPDP and the activity of MDP-Ab through dendritic cells (DCs)-based immunotherapy was identified in targeted therapy for leukemia. Results showed that the molecular ratio of purified MDP-Ab immunomodulator was about 2:1 according to electrospray ionization mass spectrometry (ESI-MS). The immunoreactivity and specificity of the new immunomodulator on CD10 antigen was identical to that of unconjugated native anti-CD10 MAb. The immunomodulatory effect of MDP-Ab immunoconjugate on peripheral blood dendritic cells (PBDCs) from children with acute lymphoblastic leukemia (ALL) exhibited upregulated expression of HLA-DR, co-stimulatory marker (CD80 and CD86) and maturity marker (CD83), increased cytokine secretion (interleukin-12, IL-12) and enhanced autostimulatory activity. These results in vitro suggested that MDP-Ab immunoconjugate may be a suitable candidate for targeting trials and support the further development of vaccination with the new immunomodulator-triggered DCs as a post-remission treatment to prevent relapse in ALL children.

Rapid drug haptenization procedure: application to gentamicin and quinidine.

A rapid and simple drug conjugation procedure which utilizes the heterobifunctional reagent N-hydroxysuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate is described. With this protocol two drugs of diverse structure and solubility have been successfully haptenized. Rabbits immunized with these conjugates yielded drug-specific antibodies as judged by a solid phase radioimmunoassay. Furthermore, using this technique, no cross-reactivity could be detected. Such haptens also elicited an immune response in mice thus further extending their potential for the production of drug specific monoclonal antibodies.

Ultrasensitive quantum dots-based DNA detection and hybridization kinetics analysis with evanescent wave biosensing platform

Ultrasensitive DNA detection was achieved using a new biosensing platform based on quantum dots (QDs) and total internal reflection fluorescence, which featured an exceptional detection limit of 3.2 amol of bound target DNA. The reusable sensor surface was produced by covalently immobilizing streptavidin onto a self-assembled alkanethiol monolayer of fiber optic probe through a heterobifunctional reagent. Streptavidin served as a versatile binding element for biotinylated single-strand DNA (ssDNA). The ssDNA-coated fiber probe was evaluated as a nucleic acid biosensor through a DNA–DNA hybridization assay for a 30-mer ssDNA, which were the segments of the uidA gene of Escherichia coli and labeled by QDs using avidin–biotin interaction. Several negative control tests revealed the absence of significant non-specific binding. It also showed that bound target DNA could easily be eluted from the sensor surface using SDS solution (pH 1.9) without any significant loss of performance after more than 30 assay cycles. A quantitative measurement of DNA binding kinetics was achieved with high accuracy, indicating an association rate of 1.38 × 10 6 M −1 s −1 and a dissociation rate of 4.67 × 10 −3 s −1. The proposed biosensing platform provides a simple, cheap, fast, and robust solution for many potential applications including clinical diagnosis, pathology, and genetics.

Evaluation of Synthetic Cell-Penetrating Peptides, Pro-Rich Peptide and Octaargine Derivatives, as Adenovirus Vector Carrier

Two cell-penetrating peptides, a Pro-rich peptide derivative, acetyl-(Val-Arg-Leu-Pro-Pro-Pro)(3)-Gly-Cys amide, and an octaarginine derivative, acetyl-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Cys amide, were prepared by the solid phase method. Each peptide was coupled to the heterobifunctional cross-linking reagent, 6-maleimidohexanoic acid N-hydroxysuccinimide ester, and then conjugated to the Adenovirus vector containing luciferase gene. Peptide-modified Ad, as compared with wild-type Ad, exhibited excellent luciferase activity in B16BL6 cells.

Optimization of IgG conjugation with R-phycoerythrin from Porphyra yezoensis

To optimize the chemical conjugation between R-phycoerythrin (R-PE) and antibody, different molar ratios of the heterobifunctional reagent N-succinimidyl-3-2-pyridyldithio propionate (SPDP) to R-PE were tested for R-PE derivation into R-PE-PDP, and different molar ratios of dithiothreitol (DTT) to IgG were tested for IgG thiolation. The results showed that in terms of best product yields determined by ultraviolet (UV) spectrophotometry, the optimal molar ratio of SPDP to R-PE was 40:1 for PE derivation, and that of DTT to IgG was 500:1 for thiolation. R-PE-labeled secondary antibody was produced by cross-linking PE-PDP and thiolated IgG. After further purification, UV spectra and native polyacrylamide gel electrophoresis determined its high purity and molecular weight. Finally, in conjunction with antigen-specific first antibodies, the R-PE-labeled IgG was applied in fluorescence immunoassays as secondary antibody and successfully detected antigens spotted on nitrocellulose membrane as well as intracellular antigen in SMCC-7721 cells. This study provides a feasible method of fluorescence antibody preparation from R-PE of Porphyra yezoensis and demonstrates high fluorescent labeling efficiency and good immunologic reactivity of the product.