Expression and PEGylation of Ts15, an alpha-KTx from Tityus serrulatus venom, and evaluation of its immunosuppressive action

• Grant number: 23/11311-9
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: December 01, 2023
• End date: May 31, 2026
• Field of knowledge: Health Sciences - Pharmacy - Toxicological Analysis
• Principal Investigator: Eliane Candiani Arantes Braga
• Grantee: Francielle Almeida Cordeiro
• Host Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 21/11936-3 - Center for Translational Science and Biopharmaceutical Development, AP.CCD

Abstract

Venomous animals have several bioactive compounds of pharmacological interest. In the the venom of the yellow scorpion Tityus serrulatus (Tsv), several molecules have already been identified, such as: enzymes (hyaluronidases and metalloproteases), antimicrobial peptides (AMPs) and hypotensive peptides, named hypotensins. In addition to these components, Tsv is mostly composed of neurotoxins that interact with ion channels, such as voltage-gated sodium, potassium, and calcium channels. Among the most studied voltage-gated potassium ion channels is KV1.3, which is overexpressed in autoimmune diseases. Kv1.3 is a novel target for immunomodulation of self-reactive effector memory T cells, which play an important role in the pathogenesis of autoimmune diseases. Several animal toxins specific for KV1.3 potassium channels already demonstrate immunosuppressive effects for several diseases caused by T cells, such as delayed hypersensitivity, arthritis, multiple sclerosis, contact dermatitis, type I and II diabetes and obesity. KV1.3 is highly expressed on effector memory T cells (TEM). The bioprospecting of KV1.3 blocking neurotoxins can lead to the development of new drugs, if allied to the use of biotechnological tools, such as heterologous expression and PEGlation. In this context, the present proposal aims at the larger scale heterologous expression (Pichia pastoris) of Ts15. Previous unpublished studies (under confidentiality) by our group have already demonstrated immunosuppressive activity in a model for rheumatoid arthritis of these toxin, which have already been electrophysiologically characterized, demonstrating specificity for blocking voltage-sensitive potassium channels (Kv). Purified recombinant toxins will be subjected to electrophysiological assays to confirm that they are capable of blocking Kv13 channels and their immunosuppressive roles will also be further investigated in in vitro functional tests on subsets of CD4+ cells: naive, effector (TEF), central memory (TCM ) and effector memory (TEM); and in vivo T-cell immunosuppression, especially using the animal model of collagen-induced rheumatoid arthritis, to prove its possible therapeutic actions. In addition, a biotinylation assay will be performed to identify the therapeutic target of the toxin, as well as whether it has immunogenicity. Finally, the recombinant toxins will be PEGlated with methoxy-PEG-propionaldehyde (mPEG-ALD), aiming to improve their potential for biotechnological application and will be produced on a larger scale with bioreactors.