scholarly journals Engineering a Monomeric Fc Domain Modality by N-Glycosylation for the Half-life Extension of Biotherapeutics

2013 ◽  
Vol 288 (23) ◽  
pp. 16529-16537 ◽  
Author(s):  
Tetsuya Ishino ◽  
Mengmeng Wang ◽  
Lidia Mosyak ◽  
Amy Tam ◽  
Weili Duan ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Tandem Repeat ◽  
Half Life ◽  
Fusion Proteins ◽  
Life Extension ◽  
Dependent Manner ◽  
Neonatal Fc Receptor ◽  
Dimer Interface ◽  
Ph Dependent

Human IgG is a bivalent molecule that has two identical Fab domains connected by a dimeric Fc domain. For therapeutic purposes, however, the bivalency of IgG and Fc fusion proteins could cause undesired properties. We therefore engineered the conversion of the natural dimeric Fc domain to a highly soluble monomer by introducing two Asn-linked glycans onto the hydrophobic CH3-CH3 dimer interface. The monomeric Fc (monoFc) maintained the binding affinity for neonatal Fc receptor (FcRn) in a pH-dependent manner. We solved the crystal structure of monoFc, which explains how the carbohydrates can stabilize the protein surface and provides the rationale for molecular recognition between monoFc and FcRn. The monoFc prolonged the in vivo half-life of an antibody Fab domain, and a tandem repeat of the monoFc further prolonged the half-life. This monoFc modality can be used to improve the pharmacokinetics of monomeric therapeutic proteins with an option to modulate the degree of half-life extension.

scholarly journals In vivo pharmacokinetic enhancement of monomeric Fc and monovalent bispecific designs through structural guidance

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Lu Shan ◽  
Nydia Van Dyk ◽  
Nantaporn Haskins ◽  
Kimberly M. Cook ◽  
Kim L. Rosenthal ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Half Life ◽  
Fusion Proteins ◽  
Molecular Design ◽  
Protein Therapeutics ◽  
Life Extension ◽  
Therapeutic Landscape ◽  
Exciting Potential ◽  
Shed Light

AbstractIn a biologic therapeutic landscape that requires versatility in targeting specificity, valency and half-life modulation, the monomeric Fc fusion platform holds exciting potential for the creation of a class of monovalent protein therapeutics that includes fusion proteins and bispecific targeting molecules. Here we report a structure-guided approach to engineer monomeric Fc molecules to adapt multiple versions of half-life extension modifications. Co-crystal structures of these monomeric Fc variants with Fc neonatal receptor (FcRn) shed light into the binding interactions that could serve as a guide for engineering the half-life of antibody Fc fragments. These engineered monomeric Fc molecules also enabled the generation of a novel monovalent bispecific molecular design, which translated the FcRn binding enhancement to improvement of in vivo serum half-life.

Cellular recycling-driven in vivo half-life extension using recombinant albumin fusions tuned for neonatal Fc receptor (FcRn) engagement

2018 ◽  
Vol 287 ◽  
pp. 132-141 ◽  
Author(s):  
Maja Thim Larsen ◽  
Helen Rawsthorne ◽  
Karen Kræmmer Schelde ◽  
Frederik Dagnæs-Hansen ◽  
Jason Cameron ◽  
...  
Keyword(s):  
Half Life ◽  
Fc Receptor ◽  
Life Extension ◽  
Neonatal Fc Receptor

scholarly journals Improved Antithrombotic Activity and Diminished Bleeding Side Effect of a PEGylated αIIbβ3 Antagonist, Disintegrin

Toxins ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 426
Author(s):  
Yu-Ju Kuo ◽  
Yao Tsung Chang ◽  
Ching-Hu Chung ◽  
Woei-Jer Chuang ◽  
Tur-Fu Huang
Keyword(s):  
Platelet Aggregation ◽  
Side Effect ◽  
Half Life ◽  
Platelet Rich Plasma ◽  
Drug Stability ◽  
Severe Thrombocytopenia ◽  
Bleeding Tendency ◽  
Dependent Manner

Polymer polyethylene glycol (PEG), or PEGylation of polypeptides improves protein drug stability by decreasing degradation and reducing renal clearance. To produce a pharmaceutical disintegrin derivative, the N-terminal PEGylation technique was used to modify the disintegrin derivative [KGDRR]trimucrin for favorable safety, pharmacokinetic profiles, and antithrombotic efficacy. We compared intact [KGDRR]trimucrin (RR) and PEGylated KGDRR (PEG-RR) by in vitro and in vivo systems for their antithrombotic activities. The activity of platelet aggregation inhibition and the bleeding tendency side effect were also investigated. PEG-RR exhibited optimal potency in inhibiting platelet aggregation of human/mouse platelet-rich plasma activated by collagen or ADP with a lower IC50 than the intact derivative RR. In the illumination-induced mesenteric venous thrombosis model, RR and PEG-RR efficaciously prevented occlusive thrombosis in a dose-dependent manner. In rotational thromboelastometry assay, PEG-RR did not induce hypocoagulation in human whole blood even given at a higher concentration (30 μg/mL), while RR slightly prolonged clotting time. However, RR and PEG-RR were not associated with severe thrombocytopenia or bleeding in FcγRIIa-transgenic mice at equally efficacious antithrombotic dosages. We also found the in vivo half-life of PEGylation was longer than RR (RR: 15.65 h vs. PEG-RR: 20.45 h). In conclusion, injectable PEG-RR with prolonged half-life and decreased bleeding risk is a safer anti-thrombotic agent for long-acting treatment of thrombus diseases.

scholarly journals Engineering of Long-Circulating Peptidoglycan Hydrolases Enables Efficient Treatment of Systemic Staphylococcus aureus Infection

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Anna M. Sobieraj ◽  
Markus Huemer ◽  
Léa V. Zinsli ◽  
Susanne Meile ◽  
Anja P. Keller ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Human Blood ◽  
Half Life ◽  
Multidrug Resistant ◽  
Life Extension ◽  
Drug Resistant ◽  
Content Type ◽  
Peptidoglycan Hydrolases ◽  
Life Threatening

ABSTRACT Staphylococcus aureus is a human pathogen causing life-threatening diseases. The increasing prevalence of multidrug-resistant S. aureus infections is a global health concern, requiring development of novel therapeutic options. Peptidoglycan-degrading enzymes (peptidoglycan hydrolases, PGHs) have emerged as a highly effective class of antimicrobial proteins against S. aureus and other pathogens. When applied to Gram-positive bacteria, PGHs hydrolyze bonds within the peptidoglycan layer, leading to rapid bacterial death by lysis. This activity is highly specific and independent of the metabolic activity of the cell or its antibiotic resistance patterns. However, systemic application of PGHs is limited by their often low activity in vivo and by an insufficient serum circulation half-life. To address this problem, we aimed to extend the half-life of PGHs selected for high activity against S. aureus in human serum. Half-life extension and increased serum circulation were achieved through fusion of PGHs to an albumin-binding domain (ABD), resulting in high-affinity recruitment of human serum albumin and formation of large protein complexes. Importantly, the ABD-fused PGHs maintained high killing activity against multiple drug-resistant S. aureus strains, as determined by ex vivo testing in human blood. The top candidate, termed ABD_M23, was tested in vivo to treat S. aureus-induced murine bacteremia. Our findings demonstrate a significantly higher efficacy of ABD_M23 than of the parental M23 enzyme. We conclude that fusion with ABD represents a powerful approach for half-life extension of PGHs, expanding the therapeutic potential of these enzybiotics for treatment of multidrug-resistant bacterial infections. IMPORTANCE Life-threatening infections with Staphylococcus aureus are often difficult to treat due to the increasing prevalence of antibiotic-resistant bacteria and their ability to persist in protected niches in the body. Bacteriolytic enzymes are promising new antimicrobials because they rapidly kill bacteria, including drug-resistant and persisting cells, by destroying their cell wall. However, when injected into the bloodstream, these enzymes are not retained long enough to clear an infection. Here, we describe a modification to increase blood circulation time of the enzymes and enhance treatment efficacy against S. aureus-induced bloodstream infections. This was achieved by preselecting enzyme candidates for high activity in human blood and coupling them to serum albumin, thereby preventing their elimination by kidney filtration and blood vessel cells.

MAZ Is a Substrate for the Deubiquitinating Enzyme DUB1

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3913-3913
Author(s):  
Yong-Soo Kim ◽  
Dong-Mi Shin ◽  
Hongsheng Wang ◽  
Herbert Morse
Keyword(s):  
Cell Differentiation ◽  
B Cell ◽  
Half Life ◽  
Conflicts Of Interest ◽  
Immunoblot Analysis ◽  
Dependent Manner ◽  
B Cell Differentiation ◽  
Deubiquitinating Enzymes ◽  
Transfected Cells

Abstract Abstract 3913 Ubiquitination is a fundamental mechanism of signal transduction that regulates immune responses and many other biological processes. Similar to phosphorylation, ubiquitination is a reversible process that is counter-regulated by ubiquitinating enzymes and deubiquitinating enzymes (DUBs). DUB1 has been identified as a member of a subfamily of deubiquitinating enzymes that are induced by Interleukin-3 (IL-3) in Ba/F3 cells. Recently, we've known that DUB1 is expressed in some pro-B and pre-B cell lines and is differentially regulated during normal B cell differentiation with highest expression in small pre-B cells. To understand the functional role of DUB1 in early B cell development, we identified a transcription factor, MAZ, as an interacting partner or substrate of DUB1 in the Abelson-transformed 220-8 pro-B cell line by using a retrovirus-based protein-fragment complementation assay (RePCA) screen. MAZ has been identified as a critical regulator of p21 gene induction, which controls cell cycle progression in synovial fibroblast cells. Immunoprecipitation and immunoblot analysis confirmed that MAZ and DUB1 interact with each other in cells. DUB1 expression significantly increased the steady-state cellular levels of MAZ. Notably, the half-life of MAZ in the pEGFP-DUB1-transfected cells was significantly increased by DUB1 expression, whereas the half-life of MAZ in the mock-transfected cells was less than 1 hr. These data therefore demonstrate that DUB1 specifically stabilizes MAZ in vivo. We found that the overexpressed MAZ was polyubiquitinated and that the polyubiquitinated MAZ was the substrate for proteasome inhibitor MG132 caused a robust increase of MAZ polyubiquitination. Overexpression of DUB1 in 293T cells caused a decrease of MAZ polyubiquitination in a DUB1 dose-dependent manner. Taken together, these results indicate that DUB1 mediate the ubiquitination-dependent degradation of MAZ. Other study shows that the DUB1 targets both K-48 and K-63 linked ubiquitination suggesting that it may be involved in both protein degradative and non-degradative functions during early B cell differentiation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Residue R113 Is Essential for PhoP Dimerization and Function: a Residue Buried in the Asymmetric PhoP Dimer Interface Determined in the PhoPN Three-Dimensional Crystal Structure

2003 ◽  
Vol 185 (1) ◽  
pp. 262-273 ◽  
Author(s):  
Yinghua Chen ◽  
Catherine Birck ◽  
Jean-Pierre Samama ◽  
F. Marion Hulett
Keyword(s):  
Crystal Structure ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Salt Bridge ◽  
Pho Regulon ◽  
Dimer Interface ◽  
And Function ◽  
Negative Phenotype

ABSTRACT Bacillus subtilis PhoP is a member of the OmpR/PhoB family of response regulators that is directly required for transcriptional activation or repression of Pho regulon genes in conditions under which Pi is growth limiting. Characterization of the PhoP protein has established that phosphorylation of the protein is not essential for PhoP dimerization or DNA binding but is essential for transcriptional regulation of Pho regulon genes. DNA footprinting studies of PhoP-regulated promoters showed that there was cooperative binding between PhoP dimers at PhoP-activated promoters and/or extensive PhoP oligomerization 3′ of PhoP-binding consensus repeats in PhoP-repressed promoters. The crystal structure of PhoPN described in the accompanying paper revealed that the dimer interface between two PhoP monomers involves nonidentical surfaces such that each monomer in a dimer retains a second surface that is available for further oligomerization. A salt bridge between R113 on one monomer and D60 on another monomer was judged to be of major importance in the protein-protein interaction. We describe the consequences of mutation of the PhoP R113 codon to a glutamate or alanine codon and mutation of the PhoP D60 codon to a lysine codon. In vivo expression of either PhoPR113E, PhoPR113A, or PhoPD60K resulted in a Pho-negative phenotype. In vitro analysis showed that PhoPR113E was phosphorylated by PhoR (the cognate histidine kinase) but was unable to dimerize. Monomeric PhoPR113E∼P was deficient in DNA binding, contributing to the PhoPR113E in vivo Pho-negative phenotype. While previous studies emphasized that phosphorylation was essential for PhoP function, data reported here indicate that phosphorylation is not sufficient as PhoP dimerization or oligomerization is also essential. Our data support the physiological relevance of the residues of the asymmetric dimer interface in PhoP dimerization and function.

scholarly journals The structure of a class 3 nonsymbiotic plant haemoglobin fromArabidopsis thalianareveals a novel N-terminal helical extension

2014 ◽  
Vol 70 (5) ◽  
pp. 1411-1418 ◽  
Author(s):  
Brandon J. Reeder ◽  
Michael A. Hough
Keyword(s):  
Crystal Structure ◽  
Protein Sequence ◽  
Disulfide Bonds ◽  
External Environment ◽  
Tryptophan Residue ◽  
Central Cavity ◽  
Dimer Interface ◽  
Iron Coordination

Plant nonsymbiotic haemoglobins fall into three classes, each with distinct properties but all with largely unresolved physiological functions. Here, the first crystal structure of a class 3 nonsymbiotic plant haemoglobin, that fromArabidopsis thaliana, is reported to 1.77 Å resolution. The protein forms a homodimer, with each monomer containing a two-over-two α-helical domain similar to that observed in bacterial truncated haemoglobins. A novel N-terminal extension comprising two α-helices plays a major role in the dimer interface, which occupies the periphery of the dimer–dimer face, surrounding an open central cavity. The haem pocket contains a proximal histidine ligand and an open sixth iron-coordination site with potential for a ligand, in this structure hydroxide, to form hydrogen bonds to a tyrosine or a tryptophan residue. The haem pocket appears to be unusually open to the external environment, with another cavity spanning the entrance of the two haem pockets. The final 23 residues of the C-terminal domain are disordered in the structure; however, these domains in the functional dimer are adjacent and include the only two cysteine residues in the protein sequence. It is likely that these residues form disulfide bondsin vitroand it is conceivable that this C-terminal region may act in a putative complex with a partner moleculein vivo.

scholarly journals Siderophore-mediated zinc acquisition enhances enterobacterial colonization of the inflamed gut

2020 ◽  
Author(s):  
Hui Zhi ◽  
Judith Behnsen ◽  
Allegra Aron ◽  
Vivekanandan Subramanian ◽  
Janet Z. Liu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Local Environment ◽  
Probiotic Bacterium ◽  
Zinc Transporters ◽  
Bacterial Metabolism ◽  
Dependent Manner ◽  
Metal Affinity ◽  
Ph Dependent

ABSTRACTZinc is an essential cofactor for bacterial metabolism, and many Enterobacteriaceae express the zinc transporters ZnuABC and ZupT to acquire this metal in the host. Unexpectedly, the probiotic bacterium Escherichia coli Nissle 1917 exhibited appreciable growth in zinc-limited media even when these transporters were deleted. By utilizing in vitro and in vivo studies, as well as native spray metal infusion mass spectrometry and ion identity molecular networking, we discovered that Nissle utilizes yersiniabactin as a zincophore. Indeed, yersiniabactin enables Nissle to scavenge zinc in zinc-limited media, to resist calprotectin-mediated zinc sequestration, and to thrive in the inflamed gut. Moreover, we discovered that yersiniabactin’s affinity for iron or zinc changes in a pH-dependent manner, with higher affinity for zinc as the pH increased. Altogether, we demonstrate that siderophore metal affinity can be influenced by the local environment and reveal a mechanism of zinc acquisition available to many commensal and pathogenic Enterobacteriaceae.

scholarly journals The Application of Reversible Intramolecular Sulfonamide Ligation to Modulate Reactivity in Organometallic Ruthenium(II) Diamine Complexes

Molecules ◽  
2020 ◽  
Vol 25 (2) ◽  
pp. 244
Author(s):  
Samuel A. Kemp ◽  
Timothy J. Prior ◽  
Huguette Savoie ◽  
Ross W. Boyle ◽  
Benjamin S. Murray
Keyword(s):  
Healthy Tissue ◽  
Central Metal Atom ◽  
Dependent Manner ◽  
Central Metal ◽  
Coordination Environment ◽  
Typical Cell ◽  
Arene Ligand ◽  
Ph Conditions ◽  
Ph Dependent

Metallation of biomacromolecular species forms the basis for the anticancer activity of many metallodrugs. A major limitation of these compounds is that their reactivity is indiscriminate and can, in principle, occur in healthy tissue as well as cancerous tissue, potentially leading to side effects in vivo. Here we present pH-dependent intramolecular coordination of an arene-tethered sulfonamide functionality in organometallic ruthenium(II) ethylenediamine complexes as a route to controlling the coordination environment about the central metal atom. Through variation of the sulfonamide R group and the length of the tether linking it to the arene ligand the acidity of the sulfonamide NH group, and hence the pH-region over which regulation of metal coordination occurs, can be modulated. Intramolecular sulfonamide ligation controlled the reactivity of complex 4 within the physiologically relevant pH-region, rendering it more reactive towards 5ʹ-GMP in mildly acidic pH-conditions typical of tumour tissue compared to the mildly alkaline pH-conditions typical of healthy tissue. However, the activation of 4 by ring-opening of the chelate was found to be a slow process relative to the timescale of typical cell culture assays and members of this series of complexes were found not to be cytotoxic towards the HT-29 cell line. These complexes provide the basis for the development of analogues of increased potency where intramolecular sulfonamide ligation regulates reactivity and therefore cytotoxicity in a pH-dependent, and potentially, tissue-dependent manner.

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