Current AIDS therapies are mainly based on the use of inhibitors targeting the active site of enzymes which are crucial for the HIV-1 replication cycle. However, due to the HIV-1 high mutation frequency and the small target size, escape mutants emerge in the long term which show resistance to such inhibitors. In the attempt to overcome this problem, it is therefore desirable to switch the attention to target enzymes which are crucial for HIV-1 survival. My attention was drawn in particular to the HIV-1 protease precursor maturation process as a possible pharmacological target. The currently available tests for the screening of putative inhibitors of enzyme activity are based on the use of HIV-1-infected cell cultures which make use of live virus and therefore are cumbersome and expensive. I therefore decided to try to set up alternative methods based on the inhibition of specific target functions by developing in vitro as well as in vivo assays based on both prokaryotic and eukaryotic cells, thus avoiding the use of live virus. These assays might be proposed as a first-line screening test which might subsequently be confirmed by performing the live-virus assay only on pre-selected promising candidates. In order to meet the objectives it was necessary to clone and to express the HIV-1 protease precursor, a molecule endowed with intrinsic folding and self-processing features, with the aim of studying its properties in vitro on isolated molecules, as well an in vivo, inside bacterial as well as eukaryotic cells. The first aim was the production and purification of the HIV-1 protease precursor in a prokaryotic cell. The purified protein would be used in immuno-enzyme assays for a detailed study of its folding process, thus gaining access to the possibility of checking the activity of putative folding inhibitors. The precursor was in fact purified making use of the His tag. However, the extraction procedures required to stabilize such a hydrophobic and maturation-prone molecule turned out to be too harsh and led to the loss of maturation capacity. Therefore, I decided to postpone this part of the project and to concentrate on the in vivo assay, which looked more promising in the short term. The second target was more quickly met: the aim was to develop a quick and reliable microbiological assay suitable to screen for folding inhibitors and for inhibitors of HIV-1 protease activity. The bacterial strain carrying the precursor gene can be induced to express the precursor which quickly maturates into protease, thus being toxic for the bacterial host. This growth arrest can be released by treatment with inhibitors of the folding process, which limit the amount of mature enzyme produced, and by inhibitors of the enzyme activity, thus resulting in bacterial cell survival. This test can be modified for large-scale application and has been used as the starting point for the development of a similar test making use of a human lymphoblastoid cell line expressing the protease precursor.
MESSA A PUNTO DI TEST ALTERNATIVI PER LO SCREENING DI POTENZIALI INIBITORI DELLA PROTEASI DI HIV-1 / D. Parolin ; tutor: A. Clivio ; direttore del dottorato: M. Clerici. - Milano : Università degli studi di Milano. UNIVERSITA' DEGLI STUDI DI MILANO, 2014 Feb 03. ((25. ciclo, Anno Accademico 2013.
|Titolo:||MESSA A PUNTO DI TEST ALTERNATIVI PER LO SCREENING DI POTENZIALI INIBITORI DELLA PROTEASI DI HIV-1|
|Data di pubblicazione:||3-feb-2014|
|Parole Chiave:||TEST ; INIBITORI ; PROTEASI ; HIV-1|
|Settore Scientifico Disciplinare:||Settore BIO/13 - Biologia Applicata|
|Citazione:||MESSA A PUNTO DI TEST ALTERNATIVI PER LO SCREENING DI POTENZIALI INIBITORI DELLA PROTEASI DI HIV-1 / D. Parolin ; tutor: A. Clivio ; direttore del dottorato: M. Clerici. - Milano : Università degli studi di Milano. UNIVERSITA' DEGLI STUDI DI MILANO, 2014 Feb 03. ((25. ciclo, Anno Accademico 2013.|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.13130/parolin-debora_phd2014-02-03|
|Appare nelle tipologie:||Tesi di dottorato|