New antiviral and immunoactivation compounds. Study of molecular mechanisms.

Search and study of new antivirals

List of viruses

Collection of drug-resistant HIV-1 mutants MutantID 50,  M ID 90,  M ISThe level of reduction of sensitivity to drug ddI- resistant > d4T- resistant N 12.23> d4T- resistant N 2* AZT- resistant N 19.0> AZT- resistant N AZT- resistant N 34.5> AZT- resistant N AZT- resistant N

Methods for estimation of anti-HIV activity

Anti-HIV activity of the selected derivatives of AZT and d4T (tested compounds were added after virus absorption)

Dependence of Anti-HIV activity on time addition of the compounds

CONCLUSION 1:

FIC 2 indicates antagonism

The recombinant integrase protein of HIV-1 (isolate Bru) was prepared by constructing plasmid based on pET15b encoding the integrase gene. We purified IN of HIV-1 by using a bacterial expression system (Escherichia coli).

The purified protein was examined using oligonucleotide-based model DNA substrates for the catalytic activities and the kinetic analysis in 3’-processing reaction: Km =(3,7 ± 0,2) М, k kat = (1,2 ± 0,3) /s.

We have now studied the activity of recombinant HIV-1 integrase on linear 636 bp and 1270 bp double-stranded DNA, containing long terminal repeat (LTR) of HIV-1. The LTR- based model DNA substrates were constructed using circular DNA forms.

Specific 3'-end proccessing activity of the 2-LTR substrate by HIV-1 integrase. Specific 3'-end proccessing activity of the 2-LTR substrate by HIV-1 integrase. The 2-LTR substrate was incubated with recombinant HIV- 1 IN for min at 37oC. The reaction products were analyzed after separation on 20 % urea- polyacrylamide gel. The removal of the labeled 3'-dinucleotide from the 2-LTR substrate by integrase were visualized by autoradiography.

Analysis of insertion 1-LTR substrate.

Inhibition of recombinant HIV-1 integrase by triterpene’s derivatives

Inhibition HIV-1 integrase by triterpene derivatives in 3’-proccessing reaction derivatives in 3’-proccessing reaction

M.Stevenson, Immunology, 2003 RNA interference susceptible targets in the HIV-1 replication cycle RNA intermediates during replication of virus, translation of mRNAs and RNA packaging can potentially be targeted by RNA interference (in nuclei ?)

Silencing of HIV genes by siRNA % of reduction p24 antigen in HIV-1 infected MT-4 cells Time after transfection 12344* 72 hours 043,230,837,7н.о 96 hours 0-24,715,593 1 –МТ-4 cells transfectd by plasmid Id-hp-pEGFP-NI 2 – МТ-4 cells transfectd by plasmid pr-hp-pEGEP-NI 3 – МТ-4 cells transfectd by plasmid tat-hp-pEGFP-NI 4 – МТ-4 cells transfectd by plasmid RT- hp-pEGFP-NI

Antiviral activity of TEDU 5 ’ -phosphonates

Study of immunomodulatory activity

Effect of niglizin on production of interferon-  (by ELISPOT)

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