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Current Medicinal Chemistry


ISSN (Print): 0929-8673
ISSN (Online): 1875-533X

Research Article

Prothymosin α and its C-Terminal Immunoreactive Decapeptide Show No Evidence of Acute Toxicity: A Preliminary In Silico, In Vitro and In Vivo Investigation

Author(s): Anastasios I. Birmpilis*, Panagiotis Vitsos*, Ioannis V. Kostopoulos, Lillian Williams, Kyriaki Ioannou, Pinelopi Samara, Chrysoula-Evangelia Karachaliou, Ioannis F. Voutsas, Elena Alyfanti, Nikolaos Angelis, Nikolaos G. Gavalas, Themis Gkraikou, Niki Kappa, Eleftheria Klagkou, Persefoni Klimentzou, Spiridoula Nikou, Nikos E. Papaioannou, Margarita Skopeliti, David Toukli, Meletios-Athanasios Dimopoulos, Aristotelis Bamias, Evangelia Livaniou, Hubert Kalbacher, Ourania E. Tsitsilonis* and Wolfgang Voelter

Volume 29, Issue 42, 2022

Published on: 14 January, 2022

Page: [6463 - 6478] Pages: 16

DOI: 10.2174/0929867328666211117093401

Price: $65


Background: Members of the α-thymosin family have long been studied for their immunostimulating properties. Among them, the danger-associated molecular patterns (DAMPs) prothymosin α (proTα) and its C-terminal decapeptide proTα(100–109) have been shown to act as immunomodulators in vitro, due to their ability to promote T helper type 1 (Th1) responses. Recently, we verified these findings in vivo, showing that both proTα and proTα(100-109) enhance antitumor-reactive T cell-mediated responses.

Methods: In view of the eventual use of proTα and proTα(100-109) in humans, we investigated their safety profile in silico, in human leukocytes and cancer cell lines in vitro, and in immunocompetent mice in vivo, in comparison to the proTα derivative thymosin alpha 1 (Τα1), a 28-mer peptide extensively studied for its safety in clinical trials.

Results: In silico prediction via computational tools showed that all three peptide sequences likely are non-toxic or do not induce allergic regions. In vitro, pro- Tα, proTα(100-109) and Tα1 did not affect the viability of human cancer cell lines and healthy donor-derived leukocytes, did not promote apoptosis or alter cell cycle distribution. Furthermore, mice injected with proTα, proTα(100-109) and Tα1 at doses equivalent to the suggested dose regimen of Tα1 in humans, did not show signs of acute toxicity, whereas proTα and proTα(100-109) increased the levels of proinflammatory and Th1- type cytokines in their peripheral blood.

Conclusion: Our preliminary findings suggest that proTα and proTα(100-109), even at high concentrations, are non-toxic in vitro and in an acute toxicity model in vivo; moreover, we show that the two peptides retain their immunomodulatory properties in vivo and, eventually, could be considered for therapeutic use in humans.

Keywords: DAMP, prothymosin alpha, in vivo toxicity, immunomodulation, thymic peptides, thymosin alpha 1.

Lau, J.L.; Dunn, M.K. Therapeutic peptides: historical perspectives, current development trends, and future directions. Bioorg. Med. Chem., 2018, 26(10), 2700-2707.
[] [PMID: 28720325]
Groß, A.; Hashimoto, C.; Sticht, H.; Eichler, J. Synthetic peptides as protein mimics. Front. Bioeng. Biotechnol., 2016, 3, 211.
[] [PMID: 26835447]
Goldstein, A.L.; Slater, F.D.; White, A. Preparation, assay, and partial purification of a thymic lymphocytopoietic factor (thymosin). Proc. Natl. Acad. Sci. USA, 1966, 56(3), 1010-1017.
[] [PMID: 5230175]
Samara, P.; Ioannou, K.; Tsitsilonis, O.E. Prothymosin alpha and immune responses: Are we close to potential clinical applications? Vitam. Horm., 2016, 102, 179-207.
[] [PMID: 27450735]
Scaffidi, P.; Misteli, T.; Bianchi, M.E. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature, 2002, 418(6894), 191-195.
[] [PMID: 12110890]
Andersson, U.; Yang, H.; Harris, H. High-mobility group box 1 protein (HMGB1) operates as an alarmin outside as well as inside cells. Semin. Immunol., 2018, 38, 40-48.
[] [PMID: 29530410]
Samara, P.; Miriagou, V.; Zachariadis, M.; Mavrofrydi, O.; Promponas, V.J.; Dedos, S.G.; Papazafiri, P.; Kalbacher, H.; Voelter, W.; Tsitsilonis, O. A fragment of the alarmin prothymosin α as a novel biomarker in murine models of bacteria-induced sepsis. Oncotarget, 2017, 8(30), 48635-48649.
[] [PMID: 28611290]
Skopeliti, M.; Kratzer, U.; Altenberend, F.; Panayotou, G.; Kalbacher, H.; Stevanovic, S.; Voelter, W.; Tsitsilonis, O.E. Proteomic exploitation on prothymosin α-induced mononuclear cell activation. Proteomics, 2007, 7(11), 1814-1824.
[] [PMID: 17474146]
Omotuyi, O.; Matsunaga, H.; Ueda, H. Evidence for ProTα-TLR4/MD-2 binding: molecular dynamics and gravimetric assay studies. Expert Opin. Biol. Ther., 2015, 15(Suppl. 1), S223-S229.
[] [PMID: 25604147]
Mosoian, A. Intracellular and extracellular cytokine-like functions of prothymosin α: implications for the development of immunotherapies. Future Med. Chem., 2011, 3(9), 1199-1208.
[] [PMID: 21806381]
Ioannou, K.; Derhovanessian, E.; Tsakiri, E.; Samara, P.; Kalbacher, H.; Voelter, W.; Trougakos, I.P.; Pawelec, G.; Tsitsilonis, O.E. Prothymosin α and a prothymosin α-derived peptide enhance T(H)1-type immune responses against defined HER-2/neu epitopes. BMC Immunol., 2013, 14, 43.
[] [PMID: 24053720]
Birmpilis, A.I.; Karachaliou, C.E.; Samara, P.; Ioannou, K.; Selemenakis, P.; Kostopoulos, I.V.; Kavrochorianou, N.; Kalbacher, H.; Livaniou, E.; Haralambous, S.; Kotsinas, A.; Farzaneh, F.; Trougakos, I.P.; Voelter, W.; Dimopoulos, M-A.; Bamias, A.; Tsitsilonis, O. Antitumor reactive T-Cell responses are enhanced in vivo by DAMP prothymosin alpha and its c-terminal decapeptide. Cancers (Basel), 2019, 11(11), 1764.
[] [PMID: 31717548]
Karachaliou, C.E.; Triantis, C.; Liolios, C.; Palamaris, L.; Zikos, C.; Tsitsilonis, O.E.; Kalbacher, H.; Voelter, W.; Loudos, G.; Papadopoulos, M.; Pirmettis, I.; Livaniou, E. In vivo biodistribution and imaging studies with a 99mTc-radiolabeled derivative of the C-terminus of prothymosin alpha in mice bearing experimentally-induced inflammation. Eur. J. Pharm. Biopharm., 2017, 113, 188-197.
[] [PMID: 28087377]
Fujita, R.; Ueda, H. Prothymosin-α1 prevents necrosis and apoptosis following stroke. Cell Death Differ., 2007, 14(10), 1839-1842.
[] [PMID: 17599097]
Halder, S.K.; Matsunaga, H.; Ueda, H. Prothymosin alpha and its mimetic hexapeptide improve delayed tissue plasminogen activator-induced brain damage following cerebral ischemia. J. Neurochem., 2020, 153(6), 772-789.
[] [PMID: 31454420]
Ueda, H. Prothymosin α and cell death mode switch, a novel target for the prevention of cerebral ischemia-induced damage. Pharmacol. Ther., 2009, 123(3), 323-333.
[] [PMID: 19500618]
Zisblatt, M.; Goldstein, A.L.; Lilly, F.; White, A. Acceleration by thymosin of the development of resistance to murine sarcoma virus-induced tumor in mice. Proc. Natl. Acad. Sci. USA, 1970, 66(4), 1170-1174.
[] [PMID: 5273447]
Talmadge, J.E.; Benedict, K.L.; Uithoven, K.A.; Lenz, B.F. The effect of experimental conditions on the assessment of T cell immunomodulation by biological response modifiers (thymosin fraction five). Immunopharmacology, 1984, 7(1), 17-26.
[] [PMID: 6232241]
King, R.; Tuthill, C. Immune modulation with thymosin alpha 1 treatment. Vitam. Horm., 2016, 102, 151-178.
[] [PMID: 27450734]
Dillman, R.O.; Beauregard, J.; Royston, I.; Zavanelli, M.I. Phase II trial of thymosin fraction 5 and thymosin alpha 1. J. Biol. Response Mod., 1987, 6(3), 263-267.
[PMID: 3598602]
Schulof, R.S.; Lloyd, M.J.; Ueno, W.M.; Green, L.D.; Stallings, J.J. Phase II trial of thymosin fraction 5 in advanced renal cancer. J. Biol. Response Mod., 1984, 3(2), 151-159.
[PMID: 6610022]
Danielli, R.; Cisternino, F.; Giannarelli, D.; Calabrò, L.; Camerini, R.; Savelli, V.; Bova, G.; Dragonetti, R.; Di Giacomo, A.M.; Altomonte, M.; Maio, M. Long-term follow up of metastatic melanoma patients treated with Thymosin alpha-1: investigating immune checkpoints synergy. Expert Opin. Biol. Ther., 2018, 18(sup1), 77-83.
[] [PMID: 30063847]
Dominari, A.; Hathaway Iii, D.; Pandav, K.; Matos, W.; Biswas, S.; Reddy, G.; Thevuthasan, S.; Khan, M.A.; Mathew, A.; Makkar, S.S.; Zaidi, M.; Fahem, M.M.M.; Beas, R.; Castaneda, V.; Paul, T.; Halpern, J.; Baralt, D. Thymosin alpha 1: a comprehensive review of the literature. World J. Virol., 2020, 9(5), 67-78.
[] [PMID: 33362999]
Camerini, R.; Garaci, E. Historical review of thymosin α 1 in infectious diseases. Expert Opin. Biol. Ther., 2015, 15(Suppl. 1), S117-S127.
[] [PMID: 26098768]
Romani, L.; Tomino, C.; Puccetti, P.; Garaci, E. Off-label therapy targeting pathogenic inflammation in COVID-19. Cell Death Discov., 2020, 6, 49.
[] [PMID: 32547788]
Wu, M.; Ji, J.J.; Zhong, L.; Shao, Z.Y.; Xie, Q.F.; Liu, Z.Y.; Wang, C.L.; Su, L.; Feng, Y.W.; Liu, Z.F.; Yao, Y.M. Thymosin α1 therapy in critically ill patients with COVID-19: A multicenter retrospective cohort study. Int. Immunopharmacol., 2020, 88, 106873.
[] [PMID: 32795897]
Liu, X.; Liu, Y.; Wang, L.; Hu, L.; Liu, D.; Li, J. Analysis of the prophylactic effect of thymosin drugs on COVID-19 for 435 medical staff: a hospital-based retrospective study. J. Med. Virol., 2021, 93(3), 1573-1580.
[] [PMID: 32897543]
Liu, Y.; Pan, Y.; Hu, Z.; Wu, M.; Wang, C.; Feng, Z.; Mao, C.; Tan, Y.; Liu, Y.; Chen, L.; Li, M.; Wang, G.; Yuan, Z.; Diao, B.; Wu, Y.; Chen, Y. Thymosin alpha 1 reduces the mortality of severe Coronavirus Disease 2019 by restoration of lymphocytopenia and reversion of exhausted T cells. Clin. Infect. Dis., 2020, 71(16), 2150-2157.
[] [PMID: 32442287]
Gupta, S.; Kapoor, P.; Chaudhary, K.; Gautam, A.; Kumar, R.; Raghava, G.P. In silico approach for predicting toxicity of peptides and proteins. PLoS One, 2013, 8(9), e73957.
[] [PMID: 24058508]
Dimitrov, I.; Bangov, I.; Flower, D.R.; Doytchinova, I. AllerTOP v.2--a server for in silico prediction of allergens. J. Mol. Model., 2014, 20(6), 2278.
[] [PMID: 24878803]
Dimitrov, I.; Naneva, L.; Doytchinova, I.; Bangov, I. AllergenFP: allergenicity prediction by descriptor fingerprints. Bioinformatics, 2014, 30(6), 846-851.
[] [PMID: 24167156]
Baxevanis, C.N.; Spanakos, G.; Voutsas, I.F.; Gritzapis, A.D.; Tsitsilonis, O.E.; Mamalaki, A.; Papamichail, M. Increased generation of autologous tumor-reactive lymphocytes by anti-CD3 monoclonal antibody and prothymosin α. Cancer Immunol. Immunother., 1999, 48(2-3), 71-84.
[] [PMID: 10414460]
Skopeliti, M.; Iconomidou, V.A.; Derhovanessian, E.; Pawelec, G.; Voelter, W.; Kalbacher, H.; Hamodrakas, S.J.; Tsitsilonis, O.E. Prothymosin α immunoactive carboxyl-terminal peptide TKKQKTDEDD stimulates lymphocyte reactions, induces dendritic cell maturation and adopts a β-sheet conformation in a sequence-specific manner. Mol. Immunol., 2009, 46(5), 784-792.
[] [PMID: 18976813]
Samara, P.; Karachaliou, C.E.; Ioannou, K.; Papaioannou, N.E.; Voutsas, I.F.; Zikos, C.; Pirmettis, I.; Papadopoulos, M.; Kalbacher, H.; Livaniou, E.; Tsitsilonis, O.E.; Voelter, W. Prothymosin alpha: an alarmin and more. Curr. Med. Chem., 2017, 24(17), 1747-1760.
[] [PMID: 28521686]
Samara, P.; Ioannou, K.; Neagu, M.; Arnogiannaki, N.; Ardavanis, A.; Voelter, W.; Tsitsilonis, O. The C-terminal decapeptide of prothymosin α is responsible for its stimulatory effect on the functions of human neutrophils in vitro. Int. Immunopharmacol., 2013, 15(1), 50-57.
[] [PMID: 23201434]
Qin, Y.; Chen, F.D.; Zhou, L.; Gong, X.G.; Han, Q.F. Proliferative and anti-proliferative effects of thymosin α1 on cells are associated with manipulation of cellular ROS levels. Chem. Biol. Interact., 2009, 180(3), 383-388.
[] [PMID: 19442654]
Nair, A.B.; Jacob, S. A simple practice guide for dose conversion between animals and human. J. Basic Clin. Pharm., 2016, 7(2), 27-31.
[] [PMID: 27057123]
Iino, S.; Toyota, J.; Kumada, H.; Kiyosawa, K.; Kakumu, S.; Sata, M.; Suzuki, H.; Martins, E.B. The efficacy and safety of thymosin alpha-1 in Japanese patients with chronic hepatitis B; results from a randomized clinical trial. J. Viral Hepat., 2005, 12(3), 300-306.
[] [PMID: 15850471]
Fan, Y.Z.; Chang, H.; Yu, Y.; Liu, J.; Wang, R. Thymosin alpha1 suppresses proliferation and induces apoptosis in human leukemia cell lines. Peptides, 2006, 27(9), 2165-2173.
[] [PMID: 16644063]
Spangelo, B.L.; Pompilius, M.; Farrimond, D.D.; Stevens, N.; Nieva, R.; Shroff, S.; Badamchian, M.; Johnson, C.R.; Jarvis, W.D. Presence of a peptide component of thymosin fraction-5 manifesting discrete cytostatic properties in HL-60 human promyelocytic leukemia cells. Int. Immunopharmacol., 2005, 5(7-8), 1317-1329.
[] [PMID: 15914336]
Ioannou, K.; Samara, P.; Livaniou, E.; Derhovanessian, E.; Tsitsilonis, O.E. Prothymosin α: a ubiquitous polypeptide with potential use in cancer diagnosis and therapy. Cancer Immunol. Immunother., 2012, 61(5), 599-614.
[] [PMID: 22366887]
Dhingra, N.; Guttman-Yassky, E. A possible role for IL-17A in establishing Th2 inflammation in murine models of atopic dermatitis. J. Invest. Dermatol., 2014, 134(8), 2071-2074.
[] [PMID: 25029321]
Nakajima, S.; Kitoh, A.; Egawa, G.; Natsuaki, Y.; Nakamizo, S.; Moniaga, C.S.; Otsuka, A.; Honda, T.; Hanakawa, S.; Amano, W.; Iwakura, Y.; Nakae, S.; Kubo, M.; Miyachi, Y.; Kabashima, K. IL-17A as an inducer for Th2 immune responses in murine atopic dermatitis models. J. Invest. Dermatol., 2014, 134(8), 2122-2130.
[] [PMID: 24480880]
Papanastasiou, M.; Baxevanis, C.N.; Papamichail, M. Promotion of murine antitumor activity by prothymosin α treatment: I. Induction of tumoricidal peritoneal cells producing high levels of tumour necrosis factor α. Cancer Immunol. Immunother., 1992, 35(2), 145-150.
[] [PMID: 1596938]
Baxevanis, C.N.; Gritzapis, A.D.; Spanakos, G.; Tsitsilonis, O.E.; Papamichail, M. Induction of tumor-specific T lymphocyte responses in vivo by prothymosin α. Cancer Immunol. Immunother., 1995, 40(6), 410-418.
[] [PMID: 7543022]
Goldstein, A.L.; Low, T.L.; Thurman, G.B.; Zatz, M.M.; Hall, N.; Chen, J.; Hu, S-K.; Naylor, P.B.; McClure, J.E. Current status of thymosin and other hormones of the thymus gland. Recent Prog. Horm. Res., 1981, 37, 369-415.
[PMID: 7025134]
Chadwick, D.; Pido-Lopez, J.; Pires, A.; Imami, N.; Gotch, F.; Villacian, J.S.; Ravindran, S.; Paton, N.I. A pilot study of the safety and efficacy of thymosin α 1 in augmenting immune reconstitution in HIV-infected patients with low CD4 counts taking highly active antiretroviral therapy. Clin. Exp. Immunol., 2003, 134(3), 477-481.
[] [PMID: 14632754]
Li, J.; Liu, C.H.; Wang, F.S. Thymosin alpha 1: biological activities, applications and genetic engineering production. Peptides, 2010, 31(11), 2151-2158.
[] [PMID: 20699109]
Cordero, O.J.; Sarandeses, C.S.; López, J.L.; Cancio, E.; Regueiro, B.J.; Nogueira, M. Prothymosin α enhances interleukin 2 receptor expression in normal human T-lymphocytes. Int. J. Immunopharmacol., 1991, 13(8), 1059-1065.
[] [PMID: 1814846]
Grünberg, E.; Eckert, K.; Maurer, H.R.; Immenschuh, P.; Kreuser, E.D. Prothymosin alpha1 effects on IL-2-induced expression of LFA-1 on lymphocytes and their adhesion to human umbilical vein endothelial cells. J. Interferon Cytokine Res., 1997, 17(3), 159-165.
[] [PMID: 9085941]
Heidecke, H.; Eckert, K.; Schulze-Forster, K.; Maurer, H.R. Prothymosin alpha 1 effects in vitro on chemotaxis, cytotoxicity and oxidative response of neutrophils from melanoma, colorectal and breast tumor patients. Int. J. Immunopharmacol., 1997, 19(8), 413-420.
[] [PMID: 9568546]
Voutsas, I.F.; Pistamaltzian, N.; Tsiatas, M.L.; Skopeliti, M.; Katsila, T.; Mavrothalassiti, I.; Spyrou, S.; Dimopoulos, M.A.; Tsitsilonis, O.E.; Bamias, A. Ovarian malignant ascites-derived lymphocytes stimulated with prothymosin α or its immunoactive decapeptide lyse autologous tumour cells in vitro and retard tumour growth in SCID mice. Eur. J. Cancer, 2013, 49(7), 1706-1714.
[] [PMID: 23276721]
Rinaldi-Garaci, C.; Baldassarre, A.M.; Pesce, A.; Frati, L.; Lazdins, J.K. In vitro effect of thymosin α 1 on the expression of peanut agglutinin binding by murine thymocytes. Cell. Immunol., 1986, 101(1), 8-14.
[] [PMID: 3488822]
Yang, X.; Qian, F.; He, H.Y.; Liu, K.J.; Lan, Y.Z.; Ni, B.; Tian, Y.; Fu, X.L.; Zhang, J.; Shen, Z.G.; Li, J.; Yin, Y.; Li, J.T.; Wu, Y.Z. Effect of thymosin alpha-1 on subpopulations of Th1, Th2, Th17, and regulatory T cells (Tregs) in vitro. Braz. J. Med. Biol. Res., 2012, 45(1), 25-32.
[] [PMID: 22245858]
Baxevanis, C.N.; Gritzapis, A.D.; Dedoussis, G.V.; Papadopoulos, N.G.; Tsolas, O.; Papamichail, M. Induction of lymphokine-activated killer activity in mice by prothymosin α. Cancer Immunol. Immunother., 1994, 38(4), 281-286.
[] [PMID: 8168124]
Chien, R.N.; Liaw, Y.F.; Chen, T.C.; Yeh, C.T.; Sheen, I.S. Efficacy of thymosin alpha1 in patients with chronic hepatitis B: A randomized, controlled trial. Hepatology, 1998, 27(5), 1383-1387.
[] [PMID: 9581695]
Zhou, Y.; He, C.; Wang, L.; Ge, B. Post-translational regulation of antiviral innate signaling. Eur. J. Immunol., 2017, 47(9), 1414-1426.
[] [PMID: 28744851]
Yang, H.; Antoine, D.J.; Andersson, U.; Tracey, K.J. The many faces of HMGB1: molecular structure-functional activity in inflammation, apoptosis, and chemotaxis. J. Leukoc. Biol., 2013, 93(6), 865-873.
[] [PMID: 23446148]
Freire, M.; Sarandeses, C.S.; Covelo, G.; Díaz-Jullien, C. Phosphorylation of prothymosin α. An approach to its biological significance. Vitam. Horm., 2016, 102, 73-99.

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