Banca de QUALIFICAÇÃO: FÁBIO THIMOTEO DE MENDONÇA

Uma banca de QUALIFICAÇÃO de MESTRADO foi cadastrada pelo programa.
STUDENT : FÁBIO THIMOTEO DE MENDONÇA
DATE: 08/12/2023
TIME: 09:00
LOCAL: Câmpus de São Bernardo do Campo da UFABC
TITLE:

STUDY OF THE INTERACTION BETWEEN SERINO PROTEASE INHIBITORS WITH TMPRSS2, A TRANSMEMBRANAR SERINE PROTEASE INVOLVED IN SARS-COV-2 ENDOCYTOSIS IN HUMAN CELLS

PAGES: 104
BIG AREA: Ciências Biológicas
AREA: Bioquímica
SUBÁREA: Biologia Molecular
SUMMARY:

The COVID-19 pandemic has resulted in nearly 7 million deaths to date, and it has also been responsible for numerous cases of patients experiencing sequelae from the disease. COVID-19 is caused by the SARS-CoV-2 virus, whose mechanisms of entry, replication, and action in human and animal cells have been extensively investigated.

One of the mechanisms involved in the entry of the SARS-CoV-2 virus is the action of an enzyme present in the cell membrane, TMPRSS2, a serine protease. This enzyme is responsible for hydrolyzing specific peptide bonds of the viral envelope protein (spike), which is associated with the binding of the angiotensin-converting enzyme 2 (ACE2) enzyme. This activation facilitates the internalization mechanism of the virus into host cells.

Different research groups have been conducting searches for TMPRSS2 inhibitors in an attempt to identify molecules with therapeutic potential against SARS-CoV-2 infection.

This project aims to evaluate the inhibitory potential of the recombinant serine protease inhibitor rBmTI-A on the TMPRSS2 enzyme.

The rBmTI-A serine protease inhibitor was produced in the Pichia pastoris yeast system, its purification was carried out through affinity chromatography using a trypsin-sepharose column, followed by gel filtration chromatography, the Ki was determined in nM range against trypsin.

Transfection assays were also conducted on A549 cells with the TMPRSS2-addgene plasmid, containing the genetic information of TMPRSS2. As a result, there was a slight increase in proteolytic activity on the z-ala-arg-arg-mca substrate in transfected cells, indicating the presence of proteolytic activity corresponding to TMPRSS2.

Assays using rBmTI-A on A549 cells transfected with the TMPRSS2-addgene plasmid showed a slight inhibitory effect on proteolytic activity compared to cells transfected without inhibitor incubation.

Recombinant TMPRSS2 enzyme construction was also produced in the form of XXA-TMPRSS2 in E. coli BL21-DE3 bacteria. The recombinant enzyme is fused with a protein called XXA, known for increasing the solubility of the recombinant protein. The chimeric protein was named XXA-TMPRSS2. The purification system used was affinity chromatography with a nickel column and gel filtration. The protein was identified on SDS-PAGE and exhibited proteolytic activity on the z-ala-arg-arg-mca substrate.

As the next steps of the work, the expression of XXA-TMPRSS2 will be optimized to obtain the protein with a high degree of homogeneity, and inhibition assays with rBmTI-A will be conducted.

COMMITTEE MEMBERS:
Presidente - Interno ao Programa - 2605490 - SERGIO DAISHI SASAKI
Membro Titular - Examinador(a) Externo à Instituição - MARCELO BERGAMIN ZANI
Membro Titular - Examinador(a) Externo à Instituição - LÍVIA DE MORAES BOMEDIANO CAMILLO
Membro Suplente - Examinador(a) Externo à Instituição - GRAZIELE CRISTINA FERREIRA - UFABC