Site-selective enzymatic chemistry for polymer conjugation to protein lysine residues: PEGylation of G-CSF at lysine-41

Mero, Anna; Grigoletto, Antonella; Maso, Katia; Yoshioka, Hiroki; Rosato, Antonio; Pasut, Gianfranco

doi:10.1039/c6py01616b

Cited by 30 publications

(32 citation statements)

References 21 publications

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“…Among the different established methods for protein PEGylation ( 19 – 22 , 37 ), we selected an approach based on the three-dimensional structure of the target proteins (Fig. S1 ).…”

Section: Resultsmentioning

confidence: 99%

A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease

et al. 2017

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PurposeBecause of the evolutionary loss of the uricolytic pathway, humans accumulate poorly soluble urate as the final product of purine catabolism. Restoration of uricolysis through enzyme therapy is a promising treatment for severe hyperuricemia caused by deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). To this end, we studied the effect of PEG conjugation on the activity and stability of the enzymatic complement required for conversion of urate into the more soluble (S)-allantoin.MethodsWe produced in recombinant form three zebrafish enzymes required in the uricolytic pathway. We carried out a systematic study of the effect of PEGylation on the function and stability of the three enzymes by varying PEG length, chemistry and degree of conjugation. We assayed in vitro the uricolytic activity of the PEGylated enzymatic triad.ResultsWe defined conditions that allow PEGylated enzymes to retain native-like enzymatic activity even after lyophilization or prolonged storage. A combination of the three enzymes in an appropriate ratio allowed efficient conversion of urate to (S)-allantoin with no accumulation of intermediate metabolites.ConclusionsPharmaceutical restoration of the uricolytic pathway is a viable approach for the treatment of severe hyperuricemia.Electronic supplementary materialThe online version of this article (doi:10.1007/s11095-017-2167-6) contains supplementary material, which is available to authorized users.

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“…Among the different established methods for protein PEGylation ( 19 – 22 , 37 ), we selected an approach based on the three-dimensional structure of the target proteins (Fig. S1 ).…”

Section: Resultsmentioning

confidence: 99%

A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease

et al. 2017

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“…Transglutaminase (TGase) is another enzyme well suited to selectively mediate polymer conjugation at specific glutamines but, as shown recently, also to lysine [29]. There are several transglutaminases from different sources with different site specificities and they work via a single step reaction on many native proteins without the need to add specific substrate sequences [30].…”

Section: Discussionmentioning

confidence: 99%

“…In addition, in this case, the requirement of flexibility is important. This reaction can be exploited to add other polymers to a protein as well [29].…”

Section: Discussionmentioning

confidence: 99%

Nanomedicines for the Delivery of Biologics

et al. 2019

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A special symposium of the Academy of Pharmaceutical Sciences Nanomedicines Focus Group reviewed the current status of the use of nanomedicines for the delivery of biologics drugs. This meeting was particularly timely with the recent approval of the first siRNA-containing product Onpattro™ (patisiran), which is formulated as a lipid nanoparticle for intravenous infusion, and the increasing interest in the use of nanomedicines for the oral delivery of biologics. The challenges in delivering such molecules were discussed with specific emphasis on the delivery both across and into cells. The latest developments in Molecular Envelope Technology® (Nanomerics Ltd, London, UK), liposomal drug delivery (both from an academic and industrial perspective), opportunities offered by the endocytic pathway, delivery using genetically engineered viral vectors (PsiOxus Technologies Ltd, Abingdon, UK), Transint™ technology (Applied Molecular Transport Inc., South San Francisco, CA, USA), which has the potential to deliver a wide range of macromolecules, and AstraZeneca’s initiatives in mRNA delivery were covered with a focus on their uses in difficult to treat diseases, including cancers. Preclinical data were presented for each of the technologies and where sufficiently advanced, plans for clinical studies as well as early clinical data. The meeting covered the work in progress in this exciting area and highlighted some key technologies to look out for in the future.

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“…In one case, a PEG polymer was linked to N-benzyloxycarbonyl-L-glutaminylglycine (ZQG), a well-studied glutamine-bearing dipeptide substrate for MTG, Similar to observations of glutamine labelling, selectivity was also achieved with several proteins. K41 on GSCF 149 and K164 on IFN α−2b 223 were found to be modified by MTG despite the presence of other lysine residues on the proteins.…”

Section: Enzymatic Protein Labelling Strategiesmentioning

confidence: 94%

Recent progress in enzymatic protein labelling techniques and their applications

et al. 2018

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Protein-based conjugates are valuable constructs for a variety of applications. Conjugation of proteins to fluorophores is commonly used to study their cellular localization and the protein-protein interactions. Modification of therapeutic proteins with either polymers or cytotoxic moieties greatly enhances their pharmacokinetics and potency. To label a protein of interest, conventional direct chemical reaction with the side-chains of native amino acids often yields heterogeneously modified products. This renders their characterization complicated, requires difficult separation steps and may impact protein function. Although modification can also be achieved via the insertion of unnatural amino acids bearing bioorthogonal functional groups, these methods can have lower protein expression yields, limiting large scale production. As a site-specific modification method, enzymatic protein labelling is highly efficient and robust under mild reaction conditions. Significant progress has been made over the last five years in modifying proteins using enzymatic methods for numerous applications, including the creation of clinically relevant conjugates with polymers, cytotoxins or imaging agents, fluorescent or affinity probes to study complex protein interaction networks, and protein-linked materials for biosensing. This review summarizes developments in enzymatic protein labelling over the last five years for a panel of ten enzymes, including sortase A, subtiligase, microbial transglutaminase, farnesyltransferase, N-myristoyltransferase, phosphopantetheinyl transferases, tubulin tyrosin ligase, lipoic acid ligase, biotin ligase and formylglycine generating enzyme.

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Site-selective enzymatic chemistry for polymer conjugation to protein lysine residues: PEGylation of G-CSF at lysine-41

Cited by 30 publications

References 21 publications

A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease

A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease

Nanomedicines for the Delivery of Biologics

Recent progress in enzymatic protein labelling techniques and their applications

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