16th International HIV Drug Resistance Workshop 12-16 June 2007, Barbados

IN VITRO ACTIVITY OF A NON-CONVENTIONAL (FOLDING) PROTEASE INHIBITOR ON HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 REPLICATION

Antivir Ther. 2007; 12:S19 (abstract no. 17)

M Lo Cicero¹, AE Laface², S Ferramosca¹, F Sirianni¹, E Cesana², D Provasi³, G Tiana³, M Galli¹, M Moroni¹, A Clivio², RA Broglia^3,4 and S Rusconi^1
1Dipartimento di Scienze Cliniche “Luigi Sacco”, Sezione di Malattie Infettive e Immunopatologia, Università degli Studi, Milano, Italy; ²Dipartimento di Scienze Precliniche LITA Vialba, Università degli Studi, Milano, Italy; ³Dipartimento di Fisica, Università degli Studi, Milano, Italy; ⁴The Niels Bohr Institute, University of Copenaghen, Denmark

BACKGROUND: It is possible to destabilize the active conformation of HIV protease by a synthetic peptide able to interact with a folding key sequence corresponding to amino acid residues 23–32 of the enzyme. We tested the toxicity and the antiviral efficacy of this peptide and its capacity to inhibit processing of the viral Pr55 HIV-1 Gag- Pol precursor and yield of HIV structural protein p24.

METHODS: Peripheral blood mononuclear cells (PBMC) were infected with a PHI HIV-1 with 0.001 m.o.i., and cultured for 7 days in the presence or absence of 83–92 (5–20 µM) or Atazanavir (ATV). Supernatants (SN) were collected, analysed with a p24Ag ELISA such as to define IC₅₀ ranges. Cell toxicity assays were maintained for the entire duration of the experiments. SN were centrifuged in a refrigerated microfuge at maximum speed obtaining a virus-enriched pellets which were resuspended in SDS and electrophoresed in a 15% acrylamide-polyacrylamide gel. By transfer to a nitrocellulose filter, proteins were detected using HIV-specific primary antibodies (Ab) against p24 and secondary Ab labelled with alkaline phosphatase.

RESULTS: Mass spectrophotometry indicated that our peptide was able to cross the cell membrane. The residue 83–92 was not toxic in PBMC (5% cytotoxicity at the highest concentration). The IC₅₀ was in the µM range (5.8–12.13 µM) either for wild-type (wt) or multidrug resistant isolates (MDR). We evidenced a reactive band at MW 55 kD and a reduction of that one at MW 24kD by western blot. The results showed that 83–92 was able to inhibit HIV protease with lower efficiency as compared to ATV regarding wt isolates, but maintained the same activity against MDR, differently to what happened with ATV.

CONCLUSIONS: An excellent therapeutic/toxic ratio was evidenced, with antiviral levels well below toxic concentrations. We noted a correspondence between the p24Ag ELISA and western blot. Different p24 production was evident, particularly between the infected control and the treatment with 83–92. The peptide inhibited a multiresistant isolate as compared to a conspicuous loss of ATV, which was not effective on the patient infected with this MDR virus. These evidences stand for a possible new approach for the inhibition of HIV-1 infection.

2007-06-12
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