title: A simple three-step method for design and affinity testing of new antisense peptides: an example of erythropoietin.
abstract: Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide-receptor modulation. It is based on the fact that peptides specified by the complementary (antisense) nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope) as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense-antisense (epitope-paratope) peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s) could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines.
authors.affiliation: Center for Nuclear Magnetic Resonance, Ruđer Bošković Institute, Bijenička cesta 54, 10002 Zagreb, Croatia. stambuk@irb.hr.
authors.forename: Nikola
authors.initials: N
authors.lastname: Štambuk
authors.affiliation: Croatian Institute for Toxicology and Antidoping, Borongajska 83 g, 10000 Zagreb, Croatia. zoran.manojlovic@antidoping-hzta.hr.
authors.forename: Zoran
authors.initials: Z
authors.lastname: Manojlović
authors.affiliation: Department of Pharmacology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Domagojeva 2, 10000 Zagreb, Croatia. pturcic@pharma.hr.
authors.forename: Petra
authors.initials: P
authors.lastname: Turčić
authors.affiliation: Department for Clinical Pathophysiology, Clinical Hospital Centre Split, Šoltanska 1, 21000 Split, Croatia. roko.romast@gmail.com.
authors.forename: Roko
authors.initials: R
authors.lastname: Martinić
authors.affiliation: Center for Nuclear Magnetic Resonance, Ruđer Bošković Institute, Bijenička cesta 54, 10002 Zagreb, Croatia. pkonjev@irb.hr.
authors.forename: Paško
authors.initials: P
authors.lastname: Konjevoda
authors.affiliation: Department of General and Inorganic Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, Croatia. tweitner@pharma.hr.
authors.forename: Tin
authors.initials: T
authors.lastname: Weitner
authors.affiliation: NanoTemper Technologies GmbH, Flößergasse 4, 81369 Munich, Germany. piotr.wardega@nanotemper.de.
authors.forename: Piotr
authors.initials: P
authors.lastname: Wardega
authors.affiliation: Department of General and Inorganic Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, Croatia. mariog@pharma.hr.
authors.forename: Mario
authors.initials: M
authors.lastname: Gabričević
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004921:Erythropoietin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014158:Transcription, Genetic
chemical_list: D010455:Peptides; D012333:RNA, Messenger; D004921:Erythropoietin