Use of pyridazinediones as extracellular cleavable linkers through reversible cysteine conjugation

Bahou, Calise; Spears, Richard J.; Aliev, Abil E.; Maruani, Annabel; Fernández, Marcos; Javaid, Faiza; Szijj, Péter A.; Baker, James R.; Chudasama, Vijay

doi:10.1039/c9cc08362f

Cited by 10 publications

(19 citation statements)

References 32 publications

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“…In addition, we also performed stability measurements of the labelled Fab chains in the presence of 5 mM GSH. 36 The samples were kept at room temperature and in all cases no signicant change in the labelling ratio was observed aer 24 h ( Fig. 4, S18-S21 †).…”

Section: Resultsmentioning

confidence: 89%

Cysteine specific bioconjugation with benzyl isothiocyanates

et al. 2020

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Section: Resultsmentioning

confidence: 89%

Cysteine specific bioconjugation with benzyl isothiocyanates

et al. 2020

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“…The AgUox-HSA conjugates were incubated at 37 • C in PBS buffer (pH 7.4) containing 5 µM glutathione and 20 µM HSA, mimicking normal blood conditions, for five days [17]. Then, the samples were analyzed by SDS-PAGE.…”

Section: Generation Of Aguox-hsa Conjugates (Aguox-mal-hsa and Aguox-apn-hsa)mentioning

confidence: 99%

“…Given its selectivity, efficiency, and accessibility under moderate reaction conditions, thiol-maleimide chemistry has been used for the development of a variety of materials ranging from chemical drugs to therapeutics for several decades [11][12][13][14]. However, thiol-maleimides also generate the thioether group, which is unstable over a long period in aqueous media and in vivo conditions, and undergoes retro-conjugate additions and subsequent trapping with endogenous thiols, such as glutathione in vivo [15][16][17][18][19]. This major drawback of thiol-maleimide chemistry hampers the in vivo application of protein conjugates, such as antibody-drug conjugates [20][21][22].…”

Section: Introductionmentioning

confidence: 99%

Chemical Modification of Cysteine with 3-Arylpropriolonitrile Improves the In Vivo Stability of Albumin-Conjugated Urate Oxidase Therapeutic Protein

Yang

Kwon

2021

Biomedicines

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3-arylpropiolonitriles (APN) are promising alternatives to maleimide for chemo-selective thiol conjugation, because the reaction product has a remarkably hydrolytic stability compared with that of thiol-maleimide reactions in vitro. However, whether cysteine modification with APN enhances stability in vivo compared to thiol-maleimide reactions remains unclear, probably due to the too short in vivo serum half-life of a protein to observe significant cleavage of thiol-maleimide/-APN reaction products. The conjugation of human serum albumin (HSA) to a therapeutic protein reportedly prolongs the in vivo serum half-life. To evaluate the in vivo stability of the thiol-APN reaction product, we prepared HSA-conjugated Arthrobacter globiformis urate oxidase (AgUox), a therapeutic protein for gout treatment. Site-specific HSA conjugation to AgUox was achieved by combining site-specific incorporation of tetrazine containing an amino acid (frTet) into AgUox and a crosslinker containing trans-cyclooctene and either thiol-maleimide (AgUox-MAL-HSA) or -APN chemistry (AgUox-APN-HSA). Substantial cleavage of the thioester of AgUox-MAL-HSA was observed in vitro, whereas no cleavage of the thiol-APN product of AgUox-APN-HSA was observed. Furthermore, the in vivo serum half-life of AgUox-APN-HSA in the late phase was significantly longer than that of AgUox-MAL-HSA. Overall, these results demonstrate that the thiol-APN chemistry enhanced the in vivo stability of the HSA-conjugated therapeutic protein.

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“…The adduct gains no additional stereocenters and can tracelessly revert to starting materials at elevated pH, thus providing a pH-controlled catch-and-release process . Another class of maleimide isosteres, 1,2-dihydropyridazine-3,6-diones, undergo a similar retro-Michael reaction for cargo release …”

Section: Introductionmentioning

confidence: 99%

Pyrocinchonimides Conjugate to Amine Groups on Proteins via Imide Transfer

et al. 2020

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Advances in bioconjugation, the ability to link biomolecules to each other, small molecules, surfaces, and more, can spur the development of advanced materials and therapeutics. We have discovered that pyrocinchonimide, the dimethylated analog of maleimide, undergoes a surprising transformation with biomolecules. The reaction targets amines and involves an imide transfer, which has not been previously reported for bioconjugation purposes. Despite their similarity to maleimides, pyrocinchonimides do not react with free thiols. Though both lysine residues and the N-termini of proteins can receive the transferred imide, the reaction also exhibits a marked preference for certain amines that cannot solely be ascribed to solvent accessibility. This property is peculiar among amine-targeting reactions and can reduce combinatorial diversity when many available reactive amines are available, such as in the formation of antibody-drug conjugates. Unlike amides, the modification undergoes very slow reversion under high pH conditions. The reaction offers a thermodynamically controlled route to single or multiple modifications of proteins for a wide range of applications.

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Use of pyridazinediones as extracellular cleavable linkers through reversible cysteine conjugation

Abstract: Herein we report the potential use of pyridazinediones as novel extracellular cleavable linkers in the context of protein bioconjugates.

Cited by 10 publications

References 32 publications

Cysteine specific bioconjugation with benzyl isothiocyanates

Cysteine specific bioconjugation with benzyl isothiocyanates

Chemical Modification of Cysteine with 3-Arylpropriolonitrile Improves the In Vivo Stability of Albumin-Conjugated Urate Oxidase Therapeutic Protein

Pyrocinchonimides Conjugate to Amine Groups on Proteins via Imide Transfer

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