Aim: On the basis of theoretical approaches, we designed a peptide (BRU 83-92) able to interact with a highly conserved folding key sequence of HIV-1 protease. Because of that, the peptide should destabilize the active conformation of the enzyme in a way which is unlikely to bring life to drug resistance. To establish the pattern/rate of emergence of resistance to BRU, we passaged a wild-type clinical isolate in human peripheral blood mononuclear cells (PBMCs) in the presence of drug for up to 9 months. Methods: In vitro “long-term” susceptibility was performed by passaging in PBMCs of a wild-type HIV-1 isolate in the presence of BRU 83-92 or Atazanavir (ATV). Cultures in the absence of drug were maintained as control. The p24 yield was monitored every 3 or 4 days by ELISA assay. In case of viral breakthrough, the concentration of drug was increased and the HIV-RNA at that time-point was extracted from the supernatant and used to detect appearance of mutations leading to resistance by Nested PCR and sequencing of the protease gene. Results: After 11 months of in vitro passage, the experiments showed that the peptide was able to steadily inhibit the replication of HIV, whereas the ATV pressure caused increases of p24 production at different time-points. Thus, the amount of azapeptide was increased several times in order to lower the replication of the virus. By genotype sequencing, it was seen that BRU 83-92 did not select for any mutation that led to resistance. On the contrary, it was noted appearance of primary (I50L) and/or secondary mutations (L10I and Q58E) on the protease gene of the isolate under ATV. Conclusions: The capacity to escape selection of resistance after several in vitro passages suggests that primary and/or secondary mutations on the protease gene able to overcome the inhibition of BRU 83-92 are likely to be incompatible with the regular activity of the enzyme. Resistance to protease inhibitors require specific or multiple mutations in discrete regions of the protease and BRU seems to avoid them so far. These data support the investigation of this peptide in the clinical setting.

In vitro efficacy of a non-conventional (folding) HIV-1 protease inhibitor without selection of resistance / S. Ferramosca, M. Lo Cicero, A.E. Laface, F. Sirianni, E. Cesana, D. Provasi, G. Tiana, M. Galli, M. Moroni, A. Clivio, R.A. Broglia, S. Rusconi. - In: INFECTION. - ISSN 0300-8126. - 37:suppl. 2(2009 May), pp. 23-23. ((Intervento presentato al convegno ICAR - Italian Conference on AIDS and Retroviruses tenutosi a Milano nel 2009 [10.1007/s15010-009-1002-3].

In vitro efficacy of a non-conventional (folding) HIV-1 protease inhibitor without selection of resistance

S. Ferramosca;M. Lo Cicero;A.E. Laface;E. Cesana;D. Provasi;G. Tiana;M. Galli;M. Moroni;A. Clivio;R.A. Broglia;S. Rusconi
2009-05

Abstract

Aim: On the basis of theoretical approaches, we designed a peptide (BRU 83-92) able to interact with a highly conserved folding key sequence of HIV-1 protease. Because of that, the peptide should destabilize the active conformation of the enzyme in a way which is unlikely to bring life to drug resistance. To establish the pattern/rate of emergence of resistance to BRU, we passaged a wild-type clinical isolate in human peripheral blood mononuclear cells (PBMCs) in the presence of drug for up to 9 months. Methods: In vitro “long-term” susceptibility was performed by passaging in PBMCs of a wild-type HIV-1 isolate in the presence of BRU 83-92 or Atazanavir (ATV). Cultures in the absence of drug were maintained as control. The p24 yield was monitored every 3 or 4 days by ELISA assay. In case of viral breakthrough, the concentration of drug was increased and the HIV-RNA at that time-point was extracted from the supernatant and used to detect appearance of mutations leading to resistance by Nested PCR and sequencing of the protease gene. Results: After 11 months of in vitro passage, the experiments showed that the peptide was able to steadily inhibit the replication of HIV, whereas the ATV pressure caused increases of p24 production at different time-points. Thus, the amount of azapeptide was increased several times in order to lower the replication of the virus. By genotype sequencing, it was seen that BRU 83-92 did not select for any mutation that led to resistance. On the contrary, it was noted appearance of primary (I50L) and/or secondary mutations (L10I and Q58E) on the protease gene of the isolate under ATV. Conclusions: The capacity to escape selection of resistance after several in vitro passages suggests that primary and/or secondary mutations on the protease gene able to overcome the inhibition of BRU 83-92 are likely to be incompatible with the regular activity of the enzyme. Resistance to protease inhibitors require specific or multiple mutations in discrete regions of the protease and BRU seems to avoid them so far. These data support the investigation of this peptide in the clinical setting.
HIV-1 ; protease ; folding inhibitor ; gag
Settore MED/17 - Malattie Infettive
Settore FIS/03 - Fisica della Materia
Settore FIS/04 - Fisica Nucleare e Subnucleare
Settore BIO/13 - Biologia Applicata
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/72773
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