New antiviral and immunoactivation compounds. Study of molecular mechanisms.

1. New antiviral and immunoactivation compounds. Study of molecular mechanisms.

2. Search and study of new antivirals

3. List of viruses

4. Mutant ID50, M ID90, M IS The level of reduction of sensitivity to drug ddI- resistant 131.23 >380.98 13 10.3 d4T- resistant N 1 2.23 >4.46 234 7.2 d4T- resistant N 2* 1.33 3.57 392 70 AZT- resistant N 1 9.0 >37.5 17 160.7 AZT- resistant N 2 6.36 31.81 24 113.6 AZT- resistant N 3 4.5 >37.5 33 80.4 AZT- resistant N 4 1.42 26.2 105 25.4 AZT- resistant N 5 0.37 3.74 405 6.6 Collection of drug-resistant HIV-1 mutants

5. Methods for estimation of anti-HIV activity

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

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

8. CONCLUSION 1:

9. FIC<1 indicatessynergism, FIC<2 indicates additive interaction and FIC>2 indicates antagonism

11. 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) .

12. 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) 10-10 М, k kat = (1,2 ± 0,3) 10-7 1/s.

13. linear viral DNA env gag pol U3 U3 R U5 R U5 LTR LTR n 1 n 1 U3 U5 U3 R U5 R 2 n n 2 5 U U 3 R R 3 U U 5 s1 circular viral DNA 2-LTR substrate (1270 bp) 1-LTR substrate (636 bp) + U5 U5 U5 U3 U3 R U3 R R Schematic representation of the construction LTR- based substrates. 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.

14. 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 10 - 60 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.

15. Analysis of insertion 1-LTR substrate.

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

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

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

21. % of reduction p24 antigen in HIV-1 infected MT-4 cells Time after transfection 1 2 3 4 4* 72 hours 0 43,2 30,8 37,7 н.о 96 hours 0 - 24,7 15,5 93 Silencing of HIV genes by siRNA 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

22. Antiviral activity of TEDU 5’-phosphonates

23. Study of immunomodulatory activity

24. Effect of niglizin on production of interferon-g (by ELISPOT)

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