1. Structure and Function Laura Martin
HIV Integrase (IN)
Structure and Function
Laura Martin

2. What is Integrase? A part of Pre- Integration Complex (PIC)
Integrates viral DNA into host, essential to replication cycle of HIV
Without integrase viral DNA could not get into host genome
Carries out cutting and joining events to integrate HIV DNA (happens within the nucleus)

4. Integrase has NLS ?! - not proven yet -we do not know nuclear import pathway (not the expected pathway) - other proteins in the PIC that are also important to nuclear localization- (Matrix)
Reverse
transcription
Pre-Integration
Complex
Nuclear
import
Integration

5. Mechanism 1st step- 3’ end processing 2nd step- DNA strand transfer
3rd step- Repair or 5’ end joining
(may involve integrase)- exact pathway of how ends are cleaned up is not known
some site specificity but mainly infrequently integrated sites

7. Structure
Three domains- the catalytic core, the C- terminal and N-terminal domains
Catalytic core – contains active site responsible for catalysis of integration reaction
C- terminal- binds both viral and host DNA
N- terminal- unknown, but has zinc binding motif suggesting nucleic acid interaction

8. HIV Integrase N CORE C (1-49) (50-212) (213-288) D-X(39-58)-D-X35-E
H12 H16
C40 C43
D64
D116
E152 N
CORE C
(1-49)
(50-212)
( )

9. HIV Integrase
N-terminus
Core
C-terminus

10. Structure of Catalytic Core Domain
Responsible for catalysis
Central 5 stranded Beta- sheet with six surrounding helices
-3 highly conserved acidic residues (D64- Asp, D116-Asp, E152- Glu)

11. C- Terminal Domain -Binds DNA nonspecifically -Each monomer is
constructed of 5 stranded Beta- sheet
-Hydrophobic dimer interface formed between two Beta- sheets

12. The N-terminal Domain Bundle of 3 alpha helices with SH3 fold
-Conserved HHCC motif – zinc finger- tetramerization
-His and Cys residues that bind zinc
-Probably functions in
multimerization and stimulates catalysis
-Probably specific DNA binding

13. Sources King, Peter. Powerpoint: HIV, AIDS, Integrase and You
Pieribone, Ph.d, Vincent. "HIV Integrase: A New Therapeutic Target." Treatment Issues (1995). 8 Nov <
Robert, Cragie. "HIV Integrase, a Brief Overview from Chemistry to Therapeutics." Journal of Biological Chemistry (2001). 8 Nov <
Subhendu Chakraborti Power Point presentation: HIV Integrase Last but not least
Walsh, Matthew B., and Marcey David. "HIV- 1 Integrase The Catalytic Core Domain and Minimal DNA Binding Domain." (2001). 8 Nov <

14. HIV Integrase Inhibitors Therapeutics
Lica Abu-Esba

15. Brief History
20 years of research has produced 17 FDA approved drugs for AIDS treatment.
Drugs developed targeted viral enzymes: reverse transcriptase and protease
About 11 years ago HIV Integrase Inhibitors were reported
Diketo acids
Drugs have been effective but in 30-50% of patients resistance to the drug has developed.
New strategy of using combination therapy----combining drugs that attack at different stages in viral life cycle
Exapanding their studies to include integrase
promising because there is no human counterpart low potential for side effects because other enzymes wont be blocked
IN Inhibitors took so long to develop because
- 3D structure very hard to obtain because of low solubility
Many different inhibitors that differ in structure, most promising Diketo acids,
Two drugs in clinical trial

16. Function of Integrase 1. 3’end processing reaction
The removal of GT from the 3’end exposing the CA nucleotide
2. Strand transfer reaction
cleavage of DNA and the ligation of the 3’OH ends of viral DNA with the 5’ phosphate end of Target DNA
3. 5’ end joining reaction
nucleotides at the 5’ end of viral DNA are removed
gaps between the viral and target DNA are repaired.
In order for Inhibitors to efficiently prevent HIV Integrase from functioning they must prevent the following:

17. Inhibitor Function Competitive Inhibition
Prevent 3’ processing and strand transfer reactions which would otherwise result in the covalent linkage of viral DNA 3’ends to cellular target DNA.

18. Inhibitor binding cont…
Viral DNA unable to bind to IN
Viral DNA is accessible to metabolism by cellular recombination repair enzymes
Leads to irreversible blocking of viral replication due to unstable integration

19. Diketo Acids General Structure: 4-aryl-2-hydroxy-4-oxo-2-butenoic acid
Most promising points of Inhibitor Developement
Aryl= anything of an aromatic compound
5citep--- tetrazole- 5 membered ring of 1 carbon and 4 nitrogens

20. Inhibitor binding
Actual binding site of IN not yet understood but it is speculated that the inhibitor recognizes a conformation in the IN active site and binds to it
Catalytic core domain composed of DDE Motif
Asp 64, Asp 116, Glu 152
L-708,906 and 5CITEP binds centrally in the active site
5citep stabilized by hydrogen bonds in the active site with Glutamic acid---e 152 and with other amino acids around
No direct contact with the aspartic acids were found or with magnesium ions detected
They think its van der waals induced dipole dipole interaction with mg and mn
Magnesium=purple
Water= gold
Catalyitc a.a red
Viral DNA unable to bind to IN
Viral DNA is accessible to metabolism by cellular recombination repair enzymes
Leads to irreversible blocking of viral replication due to unstable integration

21. Results of Binding
5CITEP functions more efficiently at lower concentrations than L-708,906
5CITEP active against 3’end processing and strand transfer
IC50= Inhibition Concentration, Concentration that reduces the Integrase action by 50%

22. Compounds in Clinical Trial
S-1360 comes from Shionogi and Co. in phase two of clinical trials
derivative of the 5citep compound, with a triazole instead of a tetrazole
Merck Research Labs developed L-870,810 derived from diketo acids as the L

23. References
Johnson, Allison A.; et al. HIV-1 Integrase Inhibitors: A Decade of Research and Two Drugs in Clinical Trial. Current Topics in Medicinal Chemistry 2004,4,
Gupta, S.P; et al. Design and Development of Integrase Inhibitors as Anti-HIV Agents. Current Medicinal Chemistry, 2003, 10,
Chen, I-Jen.; et al. Structure-Based Inhibitor Design Targeting HIV-1 Integrase. Current Drug Targets, 2002,2,
Marchand, Christophe; et al. Structural Determinants for HIV-1 Integrase Inhibition by -Diketo Acids. J. Biol. Chem., 2002,277,15,
Saliva, Carlos; et al. Rational Design of Novel Diketoacid-Containing Ferrocene Inhibitors of HIV-1 Integrase, Biorg Chem, 2005,33,

24. Questions??