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Functional and structural characterization and immune response evaluation of a recombinant serine protease from Crotalus durissus collilineatus modified by PEGylation

Grant number: 16/04761-4
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): March 01, 2018
Effective date (End): August 31, 2020
Field of knowledge:Health Sciences - Pharmacy - Toxicological Analysis
Principal researcher:Eliane Candiani Arantes Braga
Grantee:Ernesto Lopes Pinheiro Junior
Home Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Associated research grant:11/23236-4 - Native and recombinant animal toxins: functional, structural and molecular analysis, AP.TEM


The snakes belonging to genus Crotalus are classified, phylogenetically, in the superfamily Colubroidea, family Viperidae and subfamily Crotalinae. This genus is comprised of approximately 29 species and, in South America, the specimens belong to Crotalus durissus species. The venom is composed mainly of crotoxin, possessing a neurotoxic action, and other toxins, such as metalloproteases, serine proteases, growth factors, among others. Snake Venom Serine Proteases (SVSPs) own the capability of affecting the prey's hemostatic system, due to the resemblance between these toxins and endogenous enzymes present in the blood coagulation, fibrinolytic and platelet aggregation pathways. In this way, they are considered promising therapeutic agents, as well as biotechnological tools. The expression of toxins in heterologous systems are an important step in the transition of one toxin to therapeutic agent, since it permits the obtainment of these proteins in large quantities, allowing their full characterization studies as well as their commercialization. On the other hand, it is well known the immunogenicity of exogenous proteins in diverse organisms, causing the destruction of the biopharmaceutical and, consequently, a drop in the therapeutic activity. The PEGylation, process in which a Polyethylene Glycol (PEG) is attached to proteins, aims to reduce the degradation process of these molecules in living organisms, as well as their innate immunogenicity. In this context, this project aims the purification of one SVSP from Crotalus durissus collilineatus venom, together with its heterologous expression and purification, followed by their PEGylation, using a PEG-derived molecule, m-PEG-Maleimide. Studies regarding the functional biochemical characterization and the determination of the specificity of enzymatic subsites will be employed as a way to elucidate the functional properties of these enzymes. The immune response triggered by the enzyme, in its recombinant and PEGylated-recombinant forms will be evaluated, in order to determine if the PEGylation process is capable of reducing in vivo immunogenicity. Additionally, in vitro studies concerning the activation of complement system by these enzymes will also be analyzed. Regarding the structural characterization, three-dimensional models will be proposed for the native and recombinant forms, as well as the determination of their glycosylation patterns. (AU)

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Scientific publications (9)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
BOLDRINI-FRANCA, JOHARA; PINHEIRO-JUNIOR, ERNESTO LOPES; PEIGNEUR, STEVE; PUCCA, MANUELA BERTO; CERNI, FELIPE AUGUSTO; BORGES, RAFAEL JUNQUEIRA; COSTA, TASSIA RAFAELLA; IMAI CARONE, SANTE EMMANUEL; DE MATTOS FONTES, MARCOS ROBERTO; SAMPAIO, SUELY VILELA; et al. Beyond hemostasis: a snake venom serine protease with potassium channel blocking and potential antitumor activities. SCIENTIFIC REPORTS, v. 10, n. 1, . (16/04761-4, 15/17286-0, 11/23236-4, 17/13485-3, 17/14035-1, 15/00740-0, 18/14158-9, 15/18432-0, 16/24191-8, 14/16182-3, 15/16714-8)
FIGUEIREDO BORDON, KARLA DE CASTRO; COLOGNA, CAMILA TAKENO; FORNARI-BALDO, ELISA CORREA; PINHEIRO-JUNIOR, ERNESTO LOPES; CERNI, FELIPE AUGUSTO; AMORIM, FERNANDA GOBBI; PINO ANJOLETTE, FERNANDO ANTONIO; CORDEIRO, FRANCIELLE ALMEIDA; WIEZEL, GISELE ADRIANO; CARDOSO, IARA AIME; et al. From Animal Poisons and Venoms to Medicines: Achievements, Challenges and Perspectives in Drug Discovery. FRONTIERS IN PHARMACOLOGY, v. 11, . (16/04761-4, 18/21233-7, 13/26619-7, 13/26200-6, 17/04724-4, 19/10173-6, 17/14035-1, 18/14158-9, 17/03580-9, 17/00586-6)
AMORIM, FERNANDA GOBBI; CORDEIRO, FRANCIELLE ALMEIDA; PINHEIRO-JUNIOR, ERNESTO LOPES; BOLDRINI-FRANCA, JOHARA; ARANTES, ELIANE CANDIANI. Microbial production of toxins from the scorpion venom: properties and applications. Applied Microbiology and Biotechnology, v. 102, n. 15, p. 6319-6331, . (14/16182-3, 16/04761-4, 12/14996-8, 11/12317-3)
PINHEIRO-JUNIOR, ERNESTO LOPES; KALINA, RIMMA; GLADKIKH, IRINA; LEYCHENKO, ELENA; TYTGAT, JAN; PEIGNEUR, STEVE. Tale of Toxin Promiscuity: The Versatile Pharmacological Effects of Hcr 1b-2 Sea Anemone Peptide on Voltage-Gated Ion Channel. ARINE DRUG, v. 20, n. 2, . (16/04761-4)
MITCHELL, MICHELA L.; HOSSAIN, MOHAMMED AKHTER; LIN, FENG; PINHEIRO-JUNIOR, ERNESTO L.; PEIGNEUR, STEVE; WAI, DOROTHY C. C.; DELAINE, CARLIE; BLYTH, ANDREW J.; FORBES, BRIONY E.; TYTGAT, JAN; et al. Identification, Synthesis, Conformation and Activity of an Insulin-like Peptide from a Sea Anemone. BIOMOLECULES, v. 11, n. 12, . (16/04761-4)
ABREU, CAIO B.; BORDON, KARLA C. F.; CERNI, FELIPE A.; OLIVEIRA, ISADORA S.; BALENZUELA, CARLA; ALEXANDRE-SILVA, GABRIEL M.; ZOCCAL, KARINA F.; REIS, MOUZARLLEM B.; WIEZEL, GISELE A.; PEIGNEUR, STEVE; et al. Pioneering Study onRhopalurus crassicaudaScorpion Venom: Isolation and Characterization of the Major Toxin and Hyaluronidase. FRONTIERS IN IMMUNOLOGY, v. 11, . (17/00586-6, 16/04761-4, 17/04724-4, 17/03580-9, 19/10173-6, 17/14035-1, 18/21233-7)
GLADKIKH, IRINA; PEIGNEUR, STEVE; SINTSOVA, OKSANA; LOPES PINHEIRO-JUNIOR, ERNESTO; KLIMOVICH, ANNA; MENSHOV, ALEXANDER; KALINOVSKY, ANATOLY; ISAEVA, MARINA; MONASTYRNAYA, MARGARITA; KOZLOVSKAYA, EMMA; et al. Kunitz-Type Peptides from the Sea Anemone Heteractis crispa Demonstrate Potassium Channel Blocking and Anti-Inflammatory Activities. BIOMEDICINES, v. 8, n. 11, . (16/04761-4)
EMAM, AYA M.; PEIGNEUR, STEVE; DEPUYDT, ANNE-SOPHIE; IBRAHIM, SAMY M.; PINHEIRO-JUNIOR, ERNESTO LOPES; EL-SADEK, MOHAMED; HENDRICKX, LOUISE; TYTGAT, JAN; KOTHAYER, HEND. Quinazolinone dimers as a potential new class of safer Kv1 inhibitors: Overcoming hERG, sodium and calcium channel affinities. BIOORGANIC CHEMISTRY, v. 115, . (16/04761-4)
Academic Publications
(References retrieved automatically from State of São Paulo Research Institutions)
PINHEIRO JUNIOR, Ernesto Lopes. Functional and structural characterization and immune response evaluation of a recombinant serine protease from Crotalus durissus collilineatus modified by PEGylation. 2021. Doctoral Thesis - Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC) Ribeirão Preto.

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