1. Nelfinavir is effective in inhibiting the multiplication and aspartic peptidase activity of Leishmania species, including strains obtained from HIV-positive patients
Bianca S Vitório, Lívia O Santos, Claudia M d'Avila-Levy, Marta H Branquinha, Conceição M Pedroso e Silva, André L S Santos J Antimicrob Chemother . 2013 Feb;68(2):348-53. doi: 10.1093/jac/dks410.
Objectives:There is a general lack of effective and non-toxic chemotherapeutic agents for leishmaniasis and there is as yet no study about the effect of HIV peptidase inhibitors (HIV PIs) on Leishmania/HIV-coinfected patients. In the present work, we performed a comparative analysis of the spectrum of action of HIV PIs on different Leishmania spp., including strains obtained from HIV-positive patients receiving or not receiving antiretroviral treatment.Methods:The effects of nelfinavir and saquinavir on Leishmania proliferation were assessed by means of a colorimetric assay (MTT). Subsequently, the effect of nelfinavir on aspartic peptidase activity from Leishmania spp. was assessed by following the degradation of the fluorogenic substrate MCA-G-K-P-I-L-F-F-R-L-K-DNP-Arg-NH(2).Results:Nelfinavir was capable of significantly reducing the multiplication of many Leishmania reference strains and isolates obtained from HIV-positive patients receiving or not receiving antiretroviral treatment. Leishmania major growth was inhibited by ≈ 50%, while all other flagellates were strongly inhibited (at least 94%), except for a Leishmania chagasi strain obtained from an HIV-positive patient under treatment with highly active antiretroviral therapy (HAART). Culture of this isolate in the presence of nelfinavir induced a considerable reduction in the aspartic peptidase activity. In addition, nelfinavir was also capable of inhibiting the aspartic peptidase activity of all Leishmania strains tested.Conclusions:The present data contribute to the study of the effect of HIV PIs on Leishmania infection and add new insights into the possibility of exploiting aspartic peptidases as promising targets in order to generate novel medications to treat leishmaniasis.
2. A novel metalloproteinase, almelysin, from a marine bacterium, Alteromonas sp. No. 3696: purification and characterization
M Shibata, R Sato, S Takahashi, K Oda Biosci Biotechnol Biochem . 1997 Apr;61(4):710-5. doi: 10.1271/bbb.61.710.
We have discovered a novel metalloproteinase, which has high activity at low temperatures, from the culture supernatant of a marine bacterium. The strain was identified as Alteromonas sp. No. 3696. The metalloproteinase, named almelysin, was purified to homogeneity from the cultured supernatant at 10 degrees C by two column chromatographies. About 20 mg of purified almelysin was obtained from 18.4 liters of the culture supernatant. The molecular mass of almelysin was estimated to be 28 kDa by SDS-PAGE and the isoelectric point was 4.3. The optimum pH for activity of almelysin was pH 8.5-9.0 and 6.5 using casein and (7-methoxycoumarin-4-yl)acetyl(MOCAc)-Pro-Leu-Gly-Leu-(N3- [2,4-dinitrophenyl]-L-2,3-diaminopropionyl) [A2pr(Dnp)]-Ala-Arg-NH2 as substrates, respectively. Almelysin was stable between pH 7.5-8.0 and below 40 degrees C. The optimum temperature for the activity was observed to be 40 degrees C using both casein and MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2 as substrates. The activity of almelysin was inhibited by such metallo chelators as EDTA and o-phenanthroline, while talopeptin, phosphoramidon, and SMPI, typical metalloproteinase inhibitors, had no effect. Almelysin primarily cleaved the Ala14-Leu15 bond and Phe24-Phe25 bond, and secondarily the Tyr16-Leu17 bond in oxidized insulin B-chain. However, almelysin could not cleave the His5-Leu6, His10-Leu11, and Gly23-Phe24 bonds, which were cleaved by other metalloproteinases. These results indicate that the substrate specificity of almelysin is different from other metalloproteinases. Interestingly, Alteromonas sp. No. 3696 strain produced another proteinase as well as almelysin at 25 degrees C.
