An approach for identifying in silico peptides against authentic metabolites: in vitro characterization of thymosin β4 metabolites

다국어 초록 (Multilingual Abstract)

Purpose Thymosin β4 is a highly active protein that exerts multiple biological activities such as tissue repair, anti-inflammation, and cell maturation. Thymosin β4 has also been listed as a prohibited drug by the World Anti-doping Agency (WADA).
Based on its biological activities, thymosin β4 has a high potential of abuse for the performance enhancement among athletes.
This study aimed to investigate and characterize the metabolism of thymosin β4 in vitro system.
Methods TB4 protein was metabolized in six different enzyme–buffer systems in vitro. After TB4 was metabolized with an appropriate buffer system, the resulting metabolites were detected by high resolution LC–MS/MS. The mass spectrum data of the observed metabolites were characterized in silico, and confirmed the structures based on synthesized authentic standards.
Results Total 13 new metabolites, some of which were detected in more than one enzyme system, were found. This study characterized all of the detected metabolites according to their in silico m/z ions and compared our findings with synthesized standards. Finally, metabolites M1, M5, M7, M11, M12, and M13 were confirmed based on their synthesized authentic standards.
Conclusion By using an approach for metabolizing a protein to detect, characterize and identify new peptide metabolites, 6 metabolites are identified among 13 expected potential metabolites. Newly detected metabolites may have the potential for biological activities after further screening compared to their parent protein.

참고문헌 (Reference)

1 Malinda KM, "Thymosin beta4 accelerates wound healing" 113 : 364-368, 1999

2 Goldstein AL, "Thymosin beta4 : actin-sequestering protein moonlights to repair injured tissues" 11 : 421-429, 2005

3 Scarth JP, "The use of in vitro technologies coupled with high resolution accurate mass LC–MS for studying drug metabolism in equine drug surveillance" 2 : 1-10, 2010

4 Wrighton SA, "The use of in vitro metabolism techniques in the planning and interpretation of drug safety studies" 23 : 199-208, 1995

5 Charman SA, "Systemic availability and lymphatic transport of human growth hormone administered by subcutaneous injection" 89 : 168-177, 2000

6 Di L, "Strategic approaches to optimizing peptide ADME properties" 17 : 134-143, 2015

7 Motyan JA, "Research applications of proteolytic enzymes in molecular biology" 3 : 923-942, 2013

8 Hasan M, "Quantitative proteome analysis of brain subregions and spinal cord from experimental autoimmune encephalomyelitis mice by TMT-based mass spectrometry" 19 : e1800355-, 2019

9 Sosne G, "Primary mechanisms of thymosin beta4repair activity in dry eye disorders and other tissue injuries" 56 : 5110-5117, 2015

10 Yu X, "Metabolite identification of therapeutic peptides and proteins by top-down differential mass spectrometry and metabolite database matching" 92 : 8298-8305, 2020

11 Matthews RA, "Medical progress depends on animal models—doesn’t it?" 101 : 95-98, 2008

12 Bagchi S, "In-vitro blood-brain barrier models for drug screening and permeation studies : an overview" 13 : 3591-3605, 2019

13 Palermo A, "In vitro evaluation of the effects of anti-fungals, benzodiazepines and non-steroidal anti-inflammatory drugs on the glucuronidation of 19-norandrosterone : implications on doping control analysis" 8 : 930-939, 2016

14 Jyrkas J, "Hepatic in vitro metabolism of peptides;Comparison of human liver S9, hepatocytes and Upcyte hepatocytes with cyclosporine A, leuprorelin, desmopressin and cetrorelix as model compounds" 196 : 113921-, 2021

15 Zhu T, "Effects of thymosin beta4 on wound healing of rat palatal mucosa" 34 : 816-821, 2014

16 Ho EN, "Doping control analysis of TB-500, a synthetic version of an active region of thymosin beta(4), in equine urine and plasma by liquid chromatography-mass spectrometry" 1265 : 57-69, 2012

17 Crockford D, "Development of thymosin beta4 for treatment of patients with ischemic heart disease" 1112 : 385-395, 2007

18 Zvereva I, "Comparison of various in vitro model systems of the metabolism of synthetic doping peptides : proteolytic enzymes, human blood serum, liver and kidney microsomes and liver S9 fraction" 149 : 85-97, 2016

19 Ezan E, "Assessment of the metabolism of therapeutic proteins and antibodies" 10 : 1079-1091, 2014

20 Ooka M, "Application of in vitro metabolism activation in high-throughput screening" 21 : 8182-, 2020

21 Philp D, "Animal studies with thymosin beta, a multifunctional tissue repair and regeneration peptide" 1194 : 81-86, 2010

22 Prueksaritanont T, "ADME of biologics—what have we learned from small molecules?" 14 : 410-419, 2012