Structure Based Screening for Inhibitory Therapeutics of CTLA-4 Unveiled New Insights About Biology of ACTH

  • Aghdas Ramezani
  • Alireza Zakeri
  • Maysam Mard-Soltani
  • Ali Mohammadian
  • Zahra Sadat Hashemi
  • Hemn Mohammadpour
  • Abolfazl Jahangiri
  • Saeed KhaliliEmail author
  • Mohammad Javad RasaeeEmail author


Although the biology of adrenocorticotropic hormone (ACTH) protein has already been scrutinized, some functional aspects of its biology are yet to be elucidated in the context of immunological disorders. In this regard, virtual screening of a compound library was performed against the structure of Cytotoxic T-Lymphocyte Associated Protein-4 (CTLA-4) (assessed both spatially and energetically) to discover novel biological functions for ACTH. The results of virtual screening and the MD simulation demonstrated that DB01284 has high binding energy along with proper interaction orientation against CTLA-4 (FG loop) by a clamp like structure. The employed methodology was checked using confirmatory control analyses. Intriguingly, DB01284 belongs to Tetracosactide (already prescribed protein drug for clinical conditions) which is the N-terminal region of ACTH. This is the first study to reveal that ACTH protein binds to the same amino acids of CTLA-4 (FG-loop) as B7 and anti-CTLA-4 antibody binds. In light of this finding, the molecular mechanism of ACTH function in patients suffering from Cushing’s Syndrom and the immunological bases for ACTH therapy of multiple sclerosis (MS) patients could be further delineated. Moreover, this finding suggests that ACTH could also act to block CTLA-4 in the context of anticancer immune check point blockade.


ACTH CTLA-4 Virtual screening Autoimmune diseases 



The authors wish to thank Tarbiat Modares University for supporting the conduct of this research.

Compliance with Ethical Standards

Conflict of interest

All the authors declared that they have no conflict of interest.

Research Involving Human and Animal Rights

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Abdel-Malek Z (2001) Melanocortin receptors: their functions and regulation by physiological agonists and antagonists. Cell Mol Life Sci 58:434–441Google Scholar
  2. Alegre M-L, Shiels H, Thompson CB, Gajewski TF (1998) Expression and function of CTLA-4 in Th1 and Th2 cells. J Immunol 161:3347–3356Google Scholar
  3. Anderson DE, Bieganowska KD, Bar-Or A, Oliveira EM, Carreno B, Collins M, Hafler DA (2000) Paradoxical inhibition of T-cell function in response to CTLA-4 blockade; heterogeneity within the human T-cell population. Nat Med 6:211–214Google Scholar
  4. Arnason BG, Berkovich R, Catania A, Lisak RP, Zaidi M (2013) Mechanisms of action of adrenocorticotropic hormone and other melanocortins relevant to the clinical management of patients with multiple sclerosis. Mult Scler J 19:130–136Google Scholar
  5. Bazmara H, Rasooli I, Jahangiri A, Sefid F, Astaneh SDA, Payandeh Z (2019) Antigenic properties of iron regulated proteins in Acinetobacter baumannii: an in silico approach. Int J Pept Res Ther 25:205–213Google Scholar
  6. Berkovich R, Agius MA (2014) Mechanisms of action of ACTH in the management of relapsing forms of multiple sclerosis. Ther Adv Neurol Disord 7:83–96Google Scholar
  7. Boasberg P, Hamid O, O’Day S (2010) Ipilimumab: unleashing the power of the immune system through CTLA-4 blockade. In: Seminars in oncology, vol 5. Elsevier, Amsterdam, pp 440–449Google Scholar
  8. Brod SA, Hood ZM (2011) Ingested (oral) ACTH inhibits EAE. J Neuroimmunol 232:131–135Google Scholar
  9. Brzoska T, Luger TA, Maaser C, Abels C, Böhm M (2008) α-Melanocyte-stimulating hormone and related tripeptides: biochemistry, antiinflammatory and protective effects in vitro and in vivo, and future perspectives for the treatment of immune-mediated inflammatory diseases. Endocr Rev 29:581–602Google Scholar
  10. Catania A, Gatti S, Colombo G, Lipton JM (2004) Targeting melanocortin receptors as a novel strategy to control inflammation. Pharmacol Rev 56:1–29Google Scholar
  11. Colao A et al (2000) Increased prevalence of thyroid autoimmunity in patients successfully treated for Cushing’s disease. Clin Endocrinol 53:13–19Google Scholar
  12. Cross AH et al (1995) Long-term inhibition of murine experimental autoimmune encephalomyelitis using CTLA-4-Fc supports a key role for CD28 costimulation. J Clin Investig 95:2783Google Scholar
  13. da Mota F, Murray C, Ezzat S (2011) Overt immune dysfunction after Cushing’s syndrome remission: a consecutive case series and review of the literature. J Clin Endocrinol Metab 96:E1670–E1674Google Scholar
  14. Dallakyan S, Olson AJ (2015) Small-molecule library screening by docking with PyRx. In: Chemical biology. Springer, New York, pp 243–250Google Scholar
  15. Egen JG, Kuhns MS, Allison JP (2002) CTLA-4: new insights into its biological function and use in tumor immunotherapy. Nat Immunol 3:611–618Google Scholar
  16. Fareau GG, Vassilopoulou-Sellin R (2007) Hypercortisolemia and infection. Infect Dis Clin 21:639–657Google Scholar
  17. Fong L et al (2009) Potentiating endogenous antitumor immunity to prostate cancer through combination immunotherapy with CTLA4 blockade and GM-CSF. Cancer Res 69:609–615Google Scholar
  18. Fukazawa T et al (1999) CTLA-4 gene polymorphism may modulate disease in Japanese multiple sclerosis patients. J Neurol Sci 171:49–55Google Scholar
  19. Gimmi CD, Freeman GJ, Gribben JG, Gray G, Nadler LM (1993) Human T-cell clonal anergy is induced by antigen presentation in the absence of B7 costimulation. Proc Natl Acad Sci 90:6586–6590Google Scholar
  20. Grohmann U et al (2002) CTLA-4–Ig regulates tryptophan catabolism in vivo. Nat Immunol 3:1097Google Scholar
  21. Jahangiri A, Amani J, Halabian R (2017) In silico analyses of staphylococcal enterotoxin B as a DNA vaccine for cancer therapy. Int J Pept Res Ther 24(1):131–142. Google Scholar
  22. Jahangiri A, Rasooli I, Owlia P, Fooladi AAI, Salimian J (2018a) Highly conserved exposed immunogenic peptides of Omp34 against Acinetobacter baumannii: an innovative approach. J Microbiol Methods 144:79–85Google Scholar
  23. Jahangiri A, Rasooli I, Owlia P, Fooladi AAI, Salimian J (2018b) An integrative in silico approach to the structure of Omp33-36 in Acinetobacter baumannii. Comput Biol Chem 72:77–86Google Scholar
  24. Kantarci OH et al (2003) CTLA4 is associated with susceptibility to multiple sclerosis. J Neuroimmunol 134:133–141Google Scholar
  25. Kazemi Moghaddam E, Owlia P, Jahangiri A, Rasooli I, Rahbar MR, Aghajani M (2017a) Conserved OprF as a selective immunogen against Pseudomonas aeruginosa. Iran J Pathol 12:86–93Google Scholar
  26. Kazemi Moghaddam E, Owlia P, Jahangiri A, Rasooli I, Rahbar MR, Aghajani M (2017b) Conserved OprF as a selective immunogen against Pseudomonas aeruginosa. Iran J Pathol 12:165–170Google Scholar
  27. Khalili S, Rasaee M, Bamdad T (2017a) 3D structure of DKK1 indicates its involvement in both canonical and non-canonical Wnt pathways. Mol Biol 51:155–166Google Scholar
  28. Khalili S, Rasaee MJ, Mousavi SL, Amani J, Jahangiri A, Borna H (2017b) In silico prediction and in vitro verification of a novel multi-epitope antigen for HBV detection. Mol Genet Microbiol Virol 32:230–240Google Scholar
  29. Khalili S, Zakeri A, Hashemi ZS, Masoumikarimi M, Manesh MRR, Shariatifar N, Sani MJ (2017c) Structural analyses of the interactions between the thyme active ingredients and human serum albumin. Turk J Biochem 42(4):459–467Google Scholar
  30. Khalili S, Jahangiri A, Hashemi ZS, Khalesi B, Mardsoltani M, Amani J (2017d) Structural pierce into molecular mechanism underlying Clostridium perfringens Epsilon toxin function. Toxicon 127:90–99Google Scholar
  31. Khoury SJ, Akalin E, Chandraker A, Turka LA, Linsley PS, Sayegh MH, Hancock WW (1995) CD28-B7 costimulatory blockade by CTLA4Ig prevents actively induced experimental autoimmune encephalomyelitis and inhibits Th1 but spares Th2 cytokines in the central nervous system. J Immunol 155:4521–4524Google Scholar
  32. Kovalovsky D, Refojo D, Holsboer F, Arzt E (2000) Molecular mechanisms and Th1/Th2 pathways in corticosteroid regulation of cytokine production. J Neuroimmunol 109:23–29Google Scholar
  33. Krummel MF, Allison JP (1995) CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. J Exp Med 182:459–465Google Scholar
  34. Krummel MF, Allison JP (1996) CTLA-4 engagement inhibits IL-2 accumulation and cell cycle progression upon activation of resting T cells. J Exp Med 183:2533–2540Google Scholar
  35. Krummel MF, Sullivan TJ, Allison JP (1996) Superantigen responses and co-stimulation: cD28 and CTLA-4 have opposing effects on T cell expansion in vitro and in vivo. Int Immunol 8:519–523Google Scholar
  36. Kwon ED et al (2014) Ipilimumab versus placebo after radiotherapy in patients with metastatic castration-resistant prostate cancer that had progressed after docetaxel chemotherapy (CA184-043): a multicentre, randomised, double-blind, phase 3 trial. Lancet Oncol 15:700–712Google Scholar
  37. Kyi C, Postow MA (2014) Checkpoint blocking antibodies in cancer immunotherapy. FEBS Lett 588:368–376Google Scholar
  38. Lee JY et al (2016) Structural basis of checkpoint blockade by monoclonal antibodies in cancer immunotherapy. Nat Commun 7:13354Google Scholar
  39. Lenschow DJ, Walunas TL, Bluestone JA (1996) CD28/B7 system of T cell costimulation. Annu Rev Immunol 14:233–258Google Scholar
  40. Linsley PS, Greene JL, Brady W, Bajorath J, Ledbetter JA, Peach R (1994) Human B7-1 (CD80) and B7-2 (CD86) bind with similar avidities but distinct kinetics to CD28 and CTLA-4 receptors. Immunity 1:793–801Google Scholar
  41. Manzotti CN, Tipping H, Perry LC, Mead KI, Blair PJ, Zheng Y, Sansom DM (2002) Inhibition of human T cell proliferation by CTLA-4 utilizes CD80 and requires CD25 + regulatory T cells. Eur J Immunol 32:2888–2896Google Scholar
  42. McCoy KD, Le Gros G (1999) The role of CTLA-4 in the regulation of T cell immune responses. Immunol Cell Biol 77:1–10Google Scholar
  43. Metzler WJ et al (1997) Solution structure of human CTLA-4 and delineation of a CD80/CD86 binding site conserved in CD28. Nat Struct Biol 4:527–531Google Scholar
  44. Mocellin S, Nitti D (2013) CTLA-4 blockade and the renaissance of cancer immunotherapy. Biochimica et Biophysica Acta (BBA) 1836:187–196Google Scholar
  45. Mohammadpour H, Khalili S, Hashemi ZS (2015) Kremen is beyond a subsidiary co-receptor of Wnt signaling: an in silico validation. Turk J Biol 39:501–510Google Scholar
  46. Mohammadpour H, Pourfathollah AA, Zarif MN, Khalili S (2016) Key role of Dkk3 protein in inhibition of cancer cell proliferation: an in silico identification. J Theor Biol 393:98–104Google Scholar
  47. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30:2785–2791. Google Scholar
  48. O’Boyle NM, Banck M, James CA, Morley C, Vandermeersch T, Hutchison GR (2011) Open Babel: an open chemical toolbox. J Cheminform 3:33Google Scholar
  49. Pauken KE, Wherry EJ (2015) Overcoming T cell exhaustion in infection and cancer. Trends Immunol 36:265–276Google Scholar
  50. Payandeh Z, Rajabibazl M, Mortazavi Y, Rahimpour A, Taromchi AH, Dastmalchi S (2019) Affinity maturation and characterization of the ofatumumab monoclonal antibody. J Cell Biochem 120:940–950Google Scholar
  51. Pender MP, Greer JM (2007) Immunology of multiple sclerosis. Curr Allergy Asthma Rep 7:285–292Google Scholar
  52. Pivonello R, De Martino MC, De Leo M, Tauchmanovà L, Faggiano A, Lombardi G, Colao A (2007) Cushing’s syndrome: aftermath of the cure. Arquivos Brasileiros de Endocrinol Metab 51:1381–1391Google Scholar
  53. Postow MA, Callahan MK, Wolchok JD (2015) Immune checkpoint blockade in cancer therapy. J Clin Oncol 33:1974–1982Google Scholar
  54. Rahbar MR et al (2019) Trimeric autotransporter adhesins in Acinetobacter baumannii, coincidental evolution at work. Infect Genet Evol 71:116–127Google Scholar
  55. Schwartz RH (1992) Costimulation of T lymphocytes: the role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy. Cell 71:1065–1068Google Scholar
  56. Selby M, Engelhardt J, Quigley M, Henning K, Chen T, Srinivasan M (2013) Korman A (2013) Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells. Cancer Immunol Res 1(1):32–42. Google Scholar
  57. Stamper CC et al (2001) Crystal structure of the B7-1/CTLA-4 complex that inhibits human immune responses. Nature 410:608–611Google Scholar
  58. Suvannang N, Nantasenamat C, Isarankura-Na-Ayudhya C, Prachayasittikul V (2011) Molecular docking of aromatase inhibitors. Molecules 16(5):3597–3617Google Scholar
  59. Takasu N, Ohara N, Yamada T, Komiya I (1993) Development of autoimmune thyroid dysfunction after bilateral adrenalectomy in a patient with Carney’s complex and after removal of ACTH-producing pituitary adenoma in a patient with Cushing’s disease. J Endocrinol Investig 16:697–702Google Scholar
  60. Topalian SL, Sharpe AH (2014) Balance and imbalance in the immune system: life on the edge. Immunity 41:682–684Google Scholar
  61. Torino F, Barnabei A, De Vecchis L, Salvatori R, Corsello SM (2012) Hypophysitis induced by monoclonal antibodies to cytotoxic T lymphocyte antigen 4: challenges from a new cause of a rare disease. Oncologist 17:525–535Google Scholar
  62. Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31:455–461Google Scholar
  63. Ueda H et al (2003) Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 423:506Google Scholar
  64. Wolchok JD et al (2013) Development of ipilimumab: a novel immunotherapeutic approach for the treatment of advanced melanoma. Ann N Y Acad Sci 1291:1–13Google Scholar
  65. Wykes MN, Lewin SR (2017) Immune checkpoint blockade in infectious diseases. Nat Rev Immunol 18(2):91Google Scholar

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© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Medical Biotechnology, Faculty of Medical SciencesTarbiat Modares UniversityTehranIran
  2. 2.Department of Biology SciencesShahid Rajaee Teacher Training UniversityTehranIran
  3. 3.Department of Clinical Biochemistry, Faculty of Medical SciencesDezful University of Medical SciencesDezfulIran
  4. 4.Department of Medical Biotechnology, School of Advanced Medical TechnologiesTehran University of Medical ScienceTehranIran
  5. 5.Department of ImmunologyRoswell Park Cancer InstituteBuffaloUSA
  6. 6.Applied Microbiology Research Center, Systems Biology and Poisonings InstituteBaqiyatallah University of Medical SciencesTehranIran

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