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Prolactin Receptors in Normal Tissues and in Animal Models for Breast Cancer
Published in Nagasawa Hiroshi, Prolactin and Lesions in Breast, Uterus, and Prostate, 2020
Paul A. Kelly, David Gould, Hiroaki Okamura, Jean Dijane
The specificity of the affinity labeling technique is shown in Figure 2. The Mr ~ 59,000 band disappeared when the incubation was performed in the presence of 4 μg of oPRL, hGH, or human PRL, although ovine growth hormone (oGH), follicle-stimulating hormone (oFSH), luteinizing hormone (oLH), and insulin had no effect.
Affinity Labeling
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
Affinity labeling is a technique for the specific modification of an amino acid residue in a protein which involves both the binding of the reagent (affinity label) on the basis of biological specificity and subsequent modification of an amino acid residue through the formation of a covalent bond. There are therefore at least two separate and distinct steps in the process of affinity labeling regardless of whether one is concerned with modification of an enzyme catalytic site or a binding site on a protein not involved in catalysis: the process of specific (selective) binding and the process of covalent bond formation.
Affinity Labeling
Published in Roger L. Lundblad, Claudia M. Noyes, Chemical Reagents for Protein Modification, 1984
Roger L. Lundblad, Claudia M. Noyes
Affinity labeling is a technique for the specific modification of an amino acid residue in a protein which involves both the binding of the reagent (affinity label) on the basis of biological specificity and subsequent modification of an amino acid residue through the formation of a covalent bond. There are therefore at least two separate and distinct steps in the process of affinity labeling regardless of whether one is concerned with modification of an enzyme catalytic site or a binding site on a protein not involved in catalysis: the process of specific (selective) binding and the process of covalent bond formation.
Combretastatin-based compounds with therapeutic characteristics: a patent review
Published in Expert Opinion on Therapeutic Patents, 2019
Lalit Mohan Nainwal, Mohammad Mumtaz Alam, Mohammad Shaquiquzzaman, Akranth Marella, Ahmed Kamal
A new series of aryl azide derivatives of combretastatin A-4 were designed as molecular probe by Pinnay et al. [51] to inhibit tubulin polymerization. They observed that replacement of 3′-hydroxyl group of CA-4 with azido group delivered a compound 3 which exhibited average cytotoxicity (GI50) at 4.07 × 10−8 M against NCI 60 cell line panel and anti-tubulin polymerization potential (IC50 = 1.4 µM) comparable to that of CA-4. Whereas, the 4′-azido analogue 4 was less active than 3 that indicated that the presence of 4′-methoxy group is probably essential to derive potent tubulin polymerization inhibitors. These derivatives could be good candidates for photo-affinity labeling studies (Figure 5).
An overview of process development for antibody-drug conjugates produced by chemical conjugation technology
Published in Expert Opinion on Biological Therapy, 2021
Yutaka Matsuda, Brian A. Mendelsohn
Chemical conjugation utilizing these affinity compounds, termed affinity labeling, is one of the promising approaches for the site-specific modification of proteins [107]. Photo affinity labeling is the most commonly used technique in this field. Several groups reported their unique method using a variety of affinity compounds such as Z domain of Protein A [108,109], Protein G mimic peptide [110] or Fc III derivative peptide; however, UV irradiation poses potential risks to the protein structure. To avoid this, the use of a reactive group such as an activated ester, (i.e. 4-fluorophenyl carbamate lysine (FPheK) [111] or NHS esters [112]) have been reported. In the past decade, this conjugation strategy has been applied for site-specific ADC synthesis in several laboratories. However, no industrial research and development activity for ADC production by an affinity labeling method was reported in peer-reviewed literature until 2019 despite the potential advantages of this relatively simple conjugation procedure. In 2019, the Ajinomoto group applied a peptide reagent derived from Fc III possessing cleavable linker to traceless labeling technology, termed AJICAP®, and demonstrated sufficient in vivo efficacy of the resulting site-specific ADC [113]. This process was then rapidly applied for scale-up and a GMP strategy for the production of GLP material using AJICAP technology was established (Figure 5) [114]. The affinity peptide reagent incorporating an activated ester was conjugated to Lys 248 in the Fc region of trastuzumab to provide trastuzumab-peptide conjugates in a site-specific manner. The disulfide linker between the antibody and the peptide moiety was cleaved and re-oxidation followed to provide the thiol-incorporating antibody. The newly installed thiol groups reacted with commercially available MC-VC-MMAE to afford an AJICAP®-ADC. This site-occupancy was controlled by the affinity peptide and determined by peptide mapping analysis [115]. The total product yield for the four steps was 90%, and 1.72 g of site-specific ADC was obtained [114]. This ADC production process termed AJICAP® ‘first generation’ consists of a simple procedure with a TFF purification step obviating the need for preparative chromatographic purification.