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.

Structural and Functional Modeling of Artificial Bioactive Proteins

“…the protein virtual spectroscopy technique, i.e., the informational spectrum method (ISM), based on the amino acid electron-ion interaction potential (EIIP) [40][41][42][43][44]; 2.…”

“…This virtual spectroscopy method is useful for structure/function analysis of proteins and the identification of functional protein domains [40][41][42][43][44]. The method is also applicable for the assessment of biological effects of mutations.…”

“…To address this problem, we analyzed 15 SynRescue protein sequences using the informational spectrum method (ISM) [40][41][42][43][44]. This is a virtual spectroscopy method for structure-function analysis of proteins based on the amino acid electron-ion interaction potential (EIIP) in the Rydberg energy units [40][41][42][43][44]. According to the underlying theory, the highest peaks found using this type of analysis represent hot spots, i.e., bioactive parts, of the protein molecule [40][41][42][43][44].…”

“…From a practical standpoint, it would be desirable not only to construct and test a large number of structural patterns in proteins, but also to predict the functional characteristics of the newly-designed molecules. To address this problem, we analyzed 15 SynRescue protein sequences using the informational spectrum method (ISM) [40][41][42][43][44]. This is a virtual spectroscopy method for structure-function analysis of proteins based on the amino acid electron-ion interaction potential (EIIP) in the Rydberg energy units [40][41][42][43][44].…”

“…This is a virtual spectroscopy method for structure-function analysis of proteins based on the amino acid electron-ion interaction potential (EIIP) in the Rydberg energy units [40][41][42][43][44]. According to the underlying theory, the highest peaks found using this type of analysis represent hot spots, i.e., bioactive parts, of the protein molecule [40][41][42][43][44]. Table 2 presents the results of virtual spectroscopy for seven Hecht_α SynRescue proteins that save Escherichia coli auxotrophs with disrupted amino acid enzyme pathways for serine (SerB), glutamate/glutamic acid (GltA) and isoleucine (IlvA).…”