rd
23 Congress of the International Union for Biochemistry and Molecular Biology
th
44 Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology
th
th
Foz do Iguaçu, PR, Brazil, August 24 to 28 , 2015
DIFFERENTIAL PROTEASE PRODUCTION BY BRAZILIAN CLINICAL STRAINS OF
PSEUDOMONAS AERUGINOSA: EFFECTS OF 1,10-PHENANTHROLINE AND ITS
DERIVATIVES ON ELASTASE B ACTIVITY
Galdino, A.C.M.1,2,; Silva, L.V.2; Ziccardi, M.3, Ramalho, T5.;Castro5, A.A.;Nunes,
A.P.F4; Santos, K.R.N.2; Branquinha, M.H.2; Santos, A.L.S.1,2
1
Programa de Pós-Graduação em Bioquímica, Instituto de Química, Universidade Federal
do Rio de Janeiro, Rio de Janeiro, Brazil; 2 Departamento de Microbiologia Geral, Instituto
de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro,
Brazil; 3Fundação Oswaldo Cruz, Rio de Janeiro, Brazil; 4 Programa de Pós-Graduação
em Doenças Infecciosas, Universidade Federal do Espírito Santo, Espírito Santo, Brazil; 5
Universidade Federal de Lavras, Minas Gerais, Brazil.
Pseudomonas aeruginosa is an opportunistic human pathogen responsible for
acute and chronic infections with significant morbidity and mortality. Its pathogenicity
comes from genetic/metabolic plasticity, antimicrobial resistance and expression of
virulence factors. Elastase B is a metalloprotease that play a pivotal role in
pseudomonal infection. In this context, our group is focused on detection of bioactive
compounds that inhibit bacterial growth and virulence traits of P. aeruginosa. The aim of
the present study was to (i) analyzed the production/enzymatic activity of LasB in 96
clinical samples of P. aeruginosa, (ii) evaluate the in silico interactions among 1,10phenantroline
and
its
derivates,
1,10-phenanthroline-5,6-dione
(phendio),
[Cu(phendio)3](ClO4)2.4H2O (Cu-phendio) and [Ag(phendio)2]ClO4 (Ag-fendio) and the
active site of LasB, and (iii) investigate the in vitro effects of these compounds on the
LasB hydrolytic activity. By zymography, the proteolytic profile I, composed by two
metalloproteases (118 and 50 kDa), was the most prevalent, being observed in 72% of
cellular extracts, 62% of planktonic culture supernatants and 76% of biofilm
supernatants from P. aeruginosa strains. The average of elastinolytic activity was 5.36 ±
3.21 AU regarding the cellular extracts, 12.95 ± 4.89 AU to planktonic culture
supernatants and 25.13 ± 9.89 AU to the biofilm culture supernatants. Also, we detected
the presence of lasB gene in 97.9% of our sample collection. Molecular docking assays
revealed that 1,10-phenanthroline and its derivatives are capable of interacting with the
active site of lasB, especially Cu-phendio that showed more favorable interaction
energy value. Similarly the in vitro assays revealed that these compounds were
effective inhibitors of the LasB activity, particularly Cu-phendio that was able to inhibit
95% of its activity at 10mM. Collectively, our data highlight the production/activity of
lasB as a potential therapeutic target. In addition, the 1,10-phenanthroline derivative
compounds emerged as potential anti-virulence drugs against P. aeruginosa.
Financial Support: CNPq, FAPERJ, FAPES and CAPPES .
Brazilian Society for Biochemistry and
Molecular Biology (SBBq)