2. How do the Anti-HIV drugs woks? By Tawitch Suriyo Master degree of Toxicology Mahidol University

3. How do anti-HIV drugs work? Content The step of HIV life cycle that might be stopped. The types of anti-HIV drugs that we have now Reverse Transcriptase Inhibitors Integrase Inhibitors Protease Inhibitors

4. The step of HIV life cycle that might be stopped. 1111 2222 3333 5555 6666 4444 1.Attachment 2.Reverse transcriptase 3.Integrase & transcription 4.Translation 5.viral protease 6.assembly & budding

5. 1.Attachment The step of HIV life cycle that might be stopped.

6. 2.Reverse Transcriptase

7. The step of HIV life cycle that might be stopped. 3.Integration,Transcription Viral DNA joins host DNAMaking multiple viral RNAs

8. The step of HIV life cycle that might be stopped. 4.Translation

9. 5.Viral Protease

10. The step of HIV life cycle that might be stopped. 6.Assembly & Budding

11. The step of HIV life cycle that might be stopped.

12. Types of anti-HIV drugs that we have now. Reverse Transcriptase Inhibitors Integrase Inhibitors (clinical trial) Protease Inhibitors

13. Reverse Transcriptase Inhibitor Nucleoside analog E.g. AZT,ddI,ddC,d4T Non-nucleoside analog E.g. Nevirapine,Delavirdine

14. Reverse Transcriptase Inhibitor Nucleoside analog These agents are both inhibitors and substrates of RT, broad in family of 2’-3’-dideoxynucleoside Need metabolism before functional Competitive inhibition with dNTP Incorperation into the viral DNA lead to DNA termination

15. Reverse Transcriptase Inhibitor Nucleoside analog need metabolize

16. Reverse Transcriptase Inhibitor Nucleoside analog + Incorporation lead to DNA chain termination

17. Reverse Transcriptase Inhibitor Nucleoside analog : Zidovudine or AZT + Thymidine analog + 3’-azido-2’,3’-dideoxythymidine + MW 267.24 + fomular C 10 H 13 N 5 O 4

18. Reverse Transcriptase Inhibitor Nucleoside analog : Zidovudine or AZT The mechanism of action of AZT +Metabolize of AZT

19. Reverse Transcriptase Inhibitor Nucleoside analog : Zidovudine or AZT AZT-DP bind with NDP-K

20. Reverse Transcriptase Inhibitor Nucleoside analog : Zidovudine or AZT AZT-TP +competitive inhibit RT with respect to TTP +incorporate into growing chain of DNA +AZT reduce the amount of dNTP (decreasing competition for AZT-TP)

21. Reverse Transcriptase Inhibitor Nucleoside analog : Zidovudine or AZT AZT-TP +Low concentration onhibit DNA polymerase +distrub growing of cellular DNA chain

22. Reverse Transcriptase Inhibitor Non-nucleoside analog Structurally heterogenous Active for HIV-1 only Not need metabolism before functional Interact with a nonsubstrate binding site Noncompetitive inhibition

23. Reverse Transcriptase Inhibitor Non-nucleoside analog : Nevirapine Viramune (trade name) MW 266.3 fumular C 15 H 14 N 4 O

24. Reverse Transcriptase Inhibitor Non-nucleoside analog : Nevirapine The machanism action of nevirapine +bind directly to RT +block RT activity (RNA dependent and DNA-dependent DNA polymerase) by distrub the RT’s catalytic site (110 Asp,185Asp and 186 Asp triad)

25. Reverse Transcriptase Inhibitor Non-nucleoside analog : Nevirapine +not compete with template or nucleoside tritposphate +not inhibit HIV-2 RT and cellular DNA polymerase

26. Integrase Inhibitor HIV integrase is a viable therapeutic strategy that will abort completion of the viral life cycle The commercial drug of this type not have yet now. Some drug just under clinical trial. E.g. Zintevir currently in Phase I/II clinical trial.

27. Integrase Inhibitor Overview of integration the formation of the ISC the 3’-processing in reaction the DNA strand transfer

28. Integrase Inhibitor HIV-Integrase a protein of 32 kDa function for intergrate viral cDNA into human DNA recognizes specific sequence in LTR in viral cDNA composed of 3 functional domain active form is oligomer ( by oligomerization)

29. Integrase Inhibitor Different approches to interfering the integration triple helix-mediated inhibition inhibition IN by peptides derived from combinatirial peptides chemistry. Screening of chemical libraries and natural compounds. Inhibition by G-quartet forming oligonucleotides

30. Integrase Inhibitor Triple helix-mediated inhibition oligonucleotide readly form stable complex with viral DNA triplex formation prevent catalytic function of IN

31. Integrase Inhibitor Inhibition IN by peptides derived from combinatirial peptides chemistry.find critical potion of IN by change amino acid that make IN loss its functional

32. Integrase Inhibitor Screening of chemical libraries and natural compounds..find the chemical ( oligonucleotide-base) that inhibit function of IN. Device into 3 categories +DNA binding agent +Polyhydroxylated aromatic compound +Nucleotides

33. Integrase Inhibitor Inhibition by G-quartet forming oligonucleotides oligonucleotides composed of base deoxyguanosine and thymidine in presence of K+, Its form G-quartet G-quartet can inhibit HIV replication by +inhibit viral entry into cell +and/or inhibition HIV IN

34. Integrase Inhibitor stable oligonucleotides of 17 nucleotides composed only deoxyguanosine and thymidine,with single phosphorothioate internucleoside linkages at 5’ and 3’ end. Fumular 5’-GTGGTGGGTGGGTGGGT-3’ Zintevir or AR-177

35. Integrase Inhibitor Form a highly stable intramolecular four- stranded DNA contain two stacked G-quartet Zintevir (G-quartet) inhibit HIV-1 IN activity Zintevir or AR-177

36. Integrase Inhibitor The machanism of action of Zintevir its inhibit integration in only step of the formation of ISC by +G-quartet interact with IN +it not bind to DNA binding domain but its interact with zinc finger domain of IN +IN can’t form active form (oligomer) +IN can’t bind with viral DNA Zintevir or AR-177

37. Protease Inhibitor Protease enzyme As aspartyl protease. Consist of 2 symmetric subunit each subunit consist 99 amino acid single active site catalytic site conserve catalytic triads ( Asp-Thr-Gly)

38. Protease Inhibitor Protease enzyme Cleavage polypeptide to functional enzyme and structural protein muation in catalytic site (Asp->Ala) cause immature virus (can’t infect new cell) HIV protease no cross-reactivity with human cellular protease

39. Protease Inhibitor Activities of HIV protease enzyme

40. Protease Inhibitor Protease inhibitor Slow down the action of HIV protease e.g. Ritonavir, Indinavir,Saquinavir

41. Protease Inhibitor Protease inhibitor Base on transitionstate mimetics of peptide substrate interact with catalytic residues and displace a structural water molecule the interaction protease and protease inhibitor complex cause enzyme not functional properly.

42. Protease Inhibitor Native HIV Protease HIV Protease with inhibitor

43. Protease Inhibitor Protease inhibitor : Ritonavir Norvir (trade name) or ABT-583 a white-to light-tan powder formular C 37 H 48 N 6 O 5 S 2 MW 720.95

44. Protease Inhibitor Protease inhibitor : Ritonavir A peptidomimetic inhibitor for HIV- 1,2 protease Its directly interact with HIV protease that cause this enzyme not function the interaction is hydrophobic interaction

45. Protease Inhibitor Protease inhibitor : Ritonavir hydrophobic interaction of P’3 isopropylthiazolyl group of ritonavir with active side chain of valine-82 (V82) of HIV protease mutation in V82 to Ala,Phe or Thr cause ritonavir resistance

46. Protease Inhibitor Protease inhibitor : Ritonavir Its metabolized by CYP 3A4 and also a potent inhibitor of CYP 3A4 inhibition of CYP by the unhindered nitrogen atom on the unsubatituted P2’ 5-thiazolyl group with bind directly to the heme in the CYP active site

47. Protease Inhibitor Protease inhibitor : Ritonavir P3’ isopropylthiazoly group P2’ 5- thiazoly group

48. Protease Inhibitor Protease - Ritonavir inhibitor complex.

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