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

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)

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

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

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

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) (213-288)

HIV Integrase N-terminus Core C-terminus

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)

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

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

Sources King, Peter. Powerpoint: HIV, AIDS, Integrase and You Pieribone, Ph.d, Vincent. "HIV Integrase: A New Therapeutic Target." Treatment Issues (1995). 8 Nov. 2005 <http://www.aegis.com>. Robert, Cragie. "HIV Integrase, a Brief Overview from Chemistry to Therapeutics." Journal of Biological Chemistry (2001). 8 Nov. 2005 <http://www.jbc.org>. 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. 2005 <http://www.clunet.edu>.

HIV Integrase Inhibitors Therapeutics Lica Abu-Esba

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

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:

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.

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

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

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

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%

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-708906

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 2004,4,1059-1077 Gupta, S.P; et al. Design and Development of Integrase Inhibitors as Anti-HIV Agents. Current Medicinal Chemistry, 2003, 10,1779-1794 Chen, I-Jen.; et al. Structure-Based Inhibitor Design Targeting HIV-1 Integrase. Current Drug Targets, 2002,2,217-234 Marchand, Christophe; et al. Structural Determinants for HIV-1 Integrase Inhibition by -Diketo Acids. J. Biol. Chem., 2002,277,15,12596-12603. Saliva, Carlos; et al. Rational Design of Novel Diketoacid-Containing Ferrocene Inhibitors of HIV-1 Integrase, Biorg Chem, 2005,33,274-284.

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