rBaltMIP, a recombinant alpha-type myotoxin inhibitor from Bothrops alternatus (Rhinocerophis alternatus) snake, as a potential candidate to complement the antivenom therapy.
Phospholipase A2inhibitors (PLIs) are important targets in the search and development of new drugs. This study aimed at evaluating the potential of an alpha-type phospholipase A2inhibitor from Bothrops alternatus (Rhinocerophis alternatus) snake in its recombinant form (rBaltMIP) to complement the conventional antivenom therapy. Biochemical experiments showed that rBaltMIP presented pI 5.8 and molecular masses of ∼21 kDa by SDS-PAGE and 19.57 kDa by MALDI/TOF MS. After tryptic peptides sequencing, the results were compared with other PLIs available in databases, showing 100% identity between rBaltMIP and its native inhibitor BaltMIP and from 92% to 96% identity with other inhibitors. Myotoxic activities of BthTX-I and BthTX-II toxins were measured via plasma CK levels, showing myotoxic effective concentrations (EC50) of 0.1256 μg/μL and 0.6183 μg/μL, respectively. rBaltMIP neutralized the myotoxicity caused by these two toxins up to 65%, without promoting primary antibody response against itself. Nevertheless, this recombinant PLI was immunogenic when standard immunization protocol with Freud's adjuvant was used. In paw edema assays, EC50 of 0.02581 μg/μL and 0.02810 μg/μL, respectively, were observed with edema reductions of up to 40% by rBaltMIP, suggesting its use as an additional antivenom. In addition, myotoxicity neutralization experiments with the myotoxin BthTX-I showed that rBaltMIP was more effective in inhibiting muscle damage than the conventional antivenom. Thus, considering the severity of envenomations due to Bothrops alternatus (Rhinocerophis alternatus) and the low neutralization of their local effects (such as myotoxicity) by the current antivenoms, rBaltMIP is a promising molecule for the development of novel therapeutic strategies for clinical applications.
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil; Departamento de Bioquímica e Tecnologia Química, Instituto de Química, Univ. Estadual Paulista, Araraquara, SP, Brazil.
Norival A
NA
Santos-Filho
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil.
Tiago S
TS
Sousa
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil.
Johara
J
Boldrini-França
Departamento de Genética e Evolução, Universidade Federal de São Carlos, UFScar, São Carlos, SP, Brazil.
Ludier K
LK
Santos-Silva
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil.
Danilo L
DL
Menaldo
Departamento de Genética e Evolução, Universidade Federal de São Carlos, UFScar, São Carlos, SP, Brazil.
Flávio
F
Henrique-Silva
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil.
Adélia C O
AC
Cintra
Departamento de Biologia Celular e Molecular e Bioagentes Patogénicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, FMRP-USP, Ribeirão Preto, SP, Brazil.
Helen J
HJ
Laure
Departamento de Ciências Fisiológicas, Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Uberlândia, MG, Brazil.
Carla C N
CC
Mamede
Departamento de Ciências Fisiológicas, Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Uberlândia, MG, Brazil.
Fábio
F
Oliveira
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil.
Thalita B
TB
Riul
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil.
Marcelo
M
Dias-Baruffi
Departamento de Biologia Celular e Molecular e Bioagentes Patogénicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, FMRP-USP, Ribeirão Preto, SP, Brazil.
José C
JC
Rosa
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto, SP, Brazil. Electronic address: suvilela@usp.br.
Suely V
SV
Sampaio
D000818:Animals; D000997:Antivenins / Q000737:chemistry / Q000627:therapeutic use*; D017837:Bothrops; D051379:Mice; D008807:Mice, Inbred BALB C; D064801:Phospholipase A2 Inhibitors / Q000302:isolation & purification / Q000627:therapeutic use*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000627:therapeutic use*; D030162:Reptilian Proteins; D012909:Snake Bites / Q000188:drug therapy* / Q000473:pathology; D018675:Toxicity Tests
D000997:Antivenins; D064801:Phospholipase A2 Inhibitors; D011994:Recombinant Proteins; D030162:Reptilian Proteins
Bothrops alternatus; Phospholipase A(2) inhibitor; rBaltMIP; αPLI