1. Anti-HIV Drugs Melissa Morgan Medicinal Chemistry November 23, 2004

2. The Life Cycle of HIV HIV causes the depletion of CD4 T- cells in the immune system HIV causes the depletion of CD4 T- cells in the immune system A CD4 receptor protein and a co- receptor, such as CCR5 or CXCR4, are required for HIV to enter a cell A CD4 receptor protein and a co- receptor, such as CCR5 or CXCR4, are required for HIV to enter a cell Fusion of the viral envelope with the cell membrane allows the viral genome and proteins to enter cell Fusion of the viral envelope with the cell membrane allows the viral genome and proteins to enter cell Reverse-transcriptase produces a cDNA copy of the viral RNA Reverse-transcriptase produces a cDNA copy of the viral RNA Viral integrase incorporates viral cDNA into host DNA as provirus Viral integrase incorporates viral cDNA into host DNA as provirus Transcription and translation of viral proteins occur Transcription and translation of viral proteins occur Capsids assemble around viral genomes and enzymes Capsids assemble around viral genomes and enzymes New virus particles bud from host T- cell after assembly New virus particles bud from host T- cell after assembly Infected T-cell eventually dies Infected T-cell eventually dies

4. The Reverse-Transcriptase Enzyme The primary source of scientific results on reverse- transcriptase is conducting X- ray crystallography studies on the enzyme The primary source of scientific results on reverse- transcriptase is conducting X- ray crystallography studies on the enzyme The catalytic p66 subunit of reverse-transcriptase has 4 domains, shown here as different colored regions of the ribbon diagram The catalytic p66 subunit of reverse-transcriptase has 4 domains, shown here as different colored regions of the ribbon diagram Several classes of anti-HIV drugs target the actions of reverse-transcriptase Several classes of anti-HIV drugs target the actions of reverse-transcriptase Reverse-transcriptase active site

5. The Classes of Anti-HIV Drugs Nucleoside reverse-transcriptase inhibitors (NRTIs) Nucleoside reverse-transcriptase inhibitors (NRTIs) Nucleotide reverse-transcriptase inhibitors Nucleotide reverse-transcriptase inhibitors Non-nucleoside reverse-transcriptase inhibitors (NNRTIs) Non-nucleoside reverse-transcriptase inhibitors (NNRTIs) HIV Protease Inhibitors HIV Protease Inhibitors Entry Inhibitors – includes the chemokine receptor binders and the gp41-dependent membrane fusion inhibitors Entry Inhibitors – includes the chemokine receptor binders and the gp41-dependent membrane fusion inhibitors

6. Sites of Action of Anti-HIV Drugs

7. Nucleoside Reverse-Transcriptase Inhibitors (NRTIs) NRTIs must be phosphorylated 3 times by kinases to form nucleoside triphosphates once inside the cell NRTIs must be phosphorylated 3 times by kinases to form nucleoside triphosphates once inside the cell This causes reverse-transcriptase to incorporate the drug, rather than the natural nucleoside triphosphate, thus terminating the growth of the DNA strand This causes reverse-transcriptase to incorporate the drug, rather than the natural nucleoside triphosphate, thus terminating the growth of the DNA strand Drugs in this class include zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, and emtricitabine Drugs in this class include zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, and emtricitabine 2 mechanisms associated with HIV resistance to the NRTIs: 2 mechanisms associated with HIV resistance to the NRTIs: 1. impairment of the enzyme’s ability to incorporate an analog into DNA 2. removal of the analog from the prematurely terminated DNA strand

8. Zidovudine (AZT) AZT was originally developed in 1964 as a potential anti-cancer agent, but was found to be ineffective In the mid-1980s, AZT was found to be effective in fighting HIV as a result of a screening process aimed at identifying anti-HIV agents AZT works because it is an analog of thymidine that can be incorporated into the DNA strand Normally, the 3’ –OH group of thymidine binds to the phosphate group of the next nucleotide in the DNA strand Normally, the 3’ –OH group of thymidine binds to the phosphate group of the next nucleotide in the DNA strand However, AZT has an azido group instead of an –OH group, and the azido group cannot bind to a phosphate group However, AZT has an azido group instead of an –OH group, and the azido group cannot bind to a phosphate group As a result, reverse transcription stops once AZT is incorporated into the DNA strand and incomplete proviral DNA is produced As a result, reverse transcription stops once AZT is incorporated into the DNA strand and incomplete proviral DNA is produced

9. Nucleotide Reverse-Transcriptase Inhibitors Same mechanism of action as NRTIs, but only 2 phosphorylation events are required to convert drug to its active triphosphate form Same mechanism of action as NRTIs, but only 2 phosphorylation events are required to convert drug to its active triphosphate form These drugs compete with normal DNA substrates to act as chain termination inhibitors of reverse-transcriptase These drugs compete with normal DNA substrates to act as chain termination inhibitors of reverse-transcriptase The mutations that confer resistance to the NRTIs also confer resistance to the nucleotide reverse-transcriptase inhibitors The mutations that confer resistance to the NRTIs also confer resistance to the nucleotide reverse-transcriptase inhibitors The only FDA-approved drug in this class is tenofovir disoproxil fumarate, a prodrug that is converted to its active form, tenofovir, once in the body The only FDA-approved drug in this class is tenofovir disoproxil fumarate, a prodrug that is converted to its active form, tenofovir, once in the body Structure of tenofovir disoproxil fumarate: Structure of tenofovir disoproxil fumarate:

10. Non-Nucleoside Reverse- Transcriptase Inhibitors (NNRTIs) These drugs work by binding to an allosteric site, specifically a hydrophobic pocket located near the catalytic domain of reverse-transcriptase These drugs work by binding to an allosteric site, specifically a hydrophobic pocket located near the catalytic domain of reverse-transcriptase The binding of the inhibitor restricts the activity and mobility of the enzyme, thus blocking the polymerization of viral DNA The binding of the inhibitor restricts the activity and mobility of the enzyme, thus blocking the polymerization of viral DNA These drugs do not have to be activated by kinases to form phosphate esters like the NRTIs These drugs do not have to be activated by kinases to form phosphate esters like the NRTIs Increasing the amount of substrate does not displace the drug from the enzyme; therefore, NNRTIs demonstrate non- competitive inhibitory action with the enzyme Increasing the amount of substrate does not displace the drug from the enzyme; therefore, NNRTIs demonstrate non- competitive inhibitory action with the enzyme Drugs in this class include nevirapine, delavirdine, and efavirenz Drugs in this class include nevirapine, delavirdine, and efavirenz Mutations that confer resistance to NNRTIs are located in the hydrophobic pocket targeted by the drugs, and they act by reducing the affinity of the drug for the site Mutations that confer resistance to NNRTIs are located in the hydrophobic pocket targeted by the drugs, and they act by reducing the affinity of the drug for the site

11. Examples of NNRTIs NevirapineEfavirenz Delavirdine

12. HIV Protease Inhibitors The 3-dimensional structure of the HIV-1 protease was determined in 1988 when the enzyme was crystallized The 3-dimensional structure of the HIV-1 protease was determined in 1988 when the enzyme was crystallized The structure consists of a dimer demonstrating precise dual rotational C2 symmetry The structure consists of a dimer demonstrating precise dual rotational C2 symmetry The active sites are located in loops that approach the center of the dimer The active sites are located in loops that approach the center of the dimer HIV-1 protease develops the gag and gag-pol polyproteins into functional viral proteins & enzymes HIV-1 protease develops the gag and gag-pol polyproteins into functional viral proteins & enzymes The structures of the HIV protease inhibitors are derived from the natural peptidic substrates of the HIV-1 protease The structures of the HIV protease inhibitors are derived from the natural peptidic substrates of the HIV-1 protease These drugs work by binding the active site of HIV-1 protease, thereby preventing the enzyme from releasing individual viral proteins These drugs work by binding the active site of HIV-1 protease, thereby preventing the enzyme from releasing individual viral proteins HIV-1 protease with bound inhibitor

13. Examples of HIV Protease Inhibitors Ritonavir SaquinavirIndinavir Amprenavir

14. HIV Entry Inhibitors The HIV entry inhibitors class includes the chemokine receptor binders and the gp41-dependent membrane fusion inhibitors The HIV entry inhibitors class includes the chemokine receptor binders and the gp41-dependent membrane fusion inhibitors The HIV-1 envelope glycoprotein contains 2 non-covalently coupled subunits, gp120 and gp41 The HIV-1 envelope glycoprotein contains 2 non-covalently coupled subunits, gp120 and gp41 gp120 controls target cell recognition and viral tropism through interaction with a CD4 receptor and a co-receptor, such as CCR5 or CXCR4, on the target cell gp120 controls target cell recognition and viral tropism through interaction with a CD4 receptor and a co-receptor, such as CCR5 or CXCR4, on the target cell Co-receptors are members of the seven-transmembrane- spanning, G-protein-coupled receptor family, whose normal function is to bind chemokines Co-receptors are members of the seven-transmembrane- spanning, G-protein-coupled receptor family, whose normal function is to bind chemokines Thus, gp120 is the subunit involved in the mechanism of action of the chemokine receptor binding drugs Thus, gp120 is the subunit involved in the mechanism of action of the chemokine receptor binding drugs gp41 promotes fusion of the viral and cellular membranes gp41 promotes fusion of the viral and cellular membranes

15. HIV Entry Inhibitors

16. Chemokine Receptor Binders Different strains of HIV-1 employ different co-receptors for entry Different strains of HIV-1 employ different co-receptors for entry The R5 strain utilizes the CCR5 co-receptor The R5 strain utilizes the CCR5 co-receptor The X4 strain utilizes the CXCR4 co-receptor The X4 strain utilizes the CXCR4 co-receptor The R5X4 strain utilizes both CCR5 and CXCR4 co-receptors The R5X4 strain utilizes both CCR5 and CXCR4 co-receptors AMD-3100 hinders only the X4 strains of HIV-1 because it acts as a selective inhibitor of the CXCR4 co-receptor AMD-3100 hinders only the X4 strains of HIV-1 because it acts as a selective inhibitor of the CXCR4 co-receptor TAK-779 blocks only the R5 strains of HIV-1 because it demonstrates selective binding to the CCR5 co-receptor TAK-779 blocks only the R5 strains of HIV-1 because it demonstrates selective binding to the CCR5 co-receptor Both of these drugs are currently in clinical trials Both of these drugs are currently in clinical trials

17. gp41-Dependent Membrane Fusion Inhibitors Fusion of the viral and cellular membranes elicits several conformational changes that lead to the formation of the trimer-of- hairpins structure in gp41 Fusion of the viral and cellular membranes elicits several conformational changes that lead to the formation of the trimer-of- hairpins structure in gp41 The drugs in this class prevent membrane fusion by interfering with the development of the trimer-of-hairpins structure The drugs in this class prevent membrane fusion by interfering with the development of the trimer-of-hairpins structure It is believed that a mechanism of dominant-negative inhibition, involving C-peptides, prevents this development It is believed that a mechanism of dominant-negative inhibition, involving C-peptides, prevents this development One drug in this sub-class is enfuvirtide One drug in this sub-class is enfuvirtide

18. Enfuvirtide (Fuzeon) Enfuvirtide is the only FDA-approved drug in the sub- class of gp41-dependent membrane fusion inhibitors Enfuvirtide is the only FDA-approved drug in the sub- class of gp41-dependent membrane fusion inhibitors Enfuvirtide is a 36-amino acid peptide derived from the HR2 region of gp41 Enfuvirtide is a 36-amino acid peptide derived from the HR2 region of gp41 During membrane fusion, HR2, a distal hydrophobic region of gp41, folds onto HR1, a proximal hydrophobic region, in order to shorten the molecule. During membrane fusion, HR2, a distal hydrophobic region of gp41, folds onto HR1, a proximal hydrophobic region, in order to shorten the molecule. Enfuvirtide binds to HR1 and inhibits the formation of the gp41 conformation necessary for fusion by interfering with the interaction between the COOH- and NH 2 -terminal repeat Enfuvirtide binds to HR1 and inhibits the formation of the gp41 conformation necessary for fusion by interfering with the interaction between the COOH- and NH 2 -terminal repeat Resistance to enfuvirtide arises as a result of mutations in a 10-amino acid motif in the HR1 region of gp41 Resistance to enfuvirtide arises as a result of mutations in a 10-amino acid motif in the HR1 region of gp41

19. Combination Therapy - HAART Combinations of antiretroviral drugs are also used for the treatment of HIV Combinations of antiretroviral drugs are also used for the treatment of HIV Highly active antiretroviral therapy, or HAART, enables the pairing of different types of drugs that may control or prevent the emergence of drug-resistant HIV strains Highly active antiretroviral therapy, or HAART, enables the pairing of different types of drugs that may control or prevent the emergence of drug-resistant HIV strains HAART regimens typically include 3 drugs, usually 2 nucleoside reverse-transcriptase inhibitors and either a protease inhibitor or a non-nucleoside reverse- transcriptase inhibitor HAART regimens typically include 3 drugs, usually 2 nucleoside reverse-transcriptase inhibitors and either a protease inhibitor or a non-nucleoside reverse- transcriptase inhibitor

20. Future Anti-HIV Drug Targets One step of the HIV life cycle that may be a future drug target is the RNase H activity of reverse-transcriptase. Screenings of compounds may enable researchers to discover a drug that can attack that target. One step of the HIV life cycle that may be a future drug target is the RNase H activity of reverse-transcriptase. Screenings of compounds may enable researchers to discover a drug that can attack that target. Another potential drug target relates to the HIV virion infectivity factor (VIF). It is believed that the VIF prevents the activity of a cellular factor that normally stops the creation of infectious virions. Another potential drug target relates to the HIV virion infectivity factor (VIF). It is believed that the VIF prevents the activity of a cellular factor that normally stops the creation of infectious virions. A final stage of the HIV life cycle that may be targeted by future drugs is the import of nucleic acids. Nuclear uptake is facilitated by virion proteins that may function with importin B and other nuclear import receptors A final stage of the HIV life cycle that may be targeted by future drugs is the import of nucleic acids. Nuclear uptake is facilitated by virion proteins that may function with importin B and other nuclear import receptors These potential drug targets will undoubtedly be exploited in the coming years These potential drug targets will undoubtedly be exploited in the coming years

21. Summary of Anti-HIV Drugs

22. Conclusion Current drugs provide many options to patients, through both monotherapy and HAART Current drugs provide many options to patients, through both monotherapy and HAART HAART produces particularly promising clinical results HAART produces particularly promising clinical results Future HIV drug treatment options must continue to focus on the development of drugs that are less likely to encourage mutations that confer resistance Future HIV drug treatment options must continue to focus on the development of drugs that are less likely to encourage mutations that confer resistance Hopefully, more successful drugs to fight HIV and a vaccine will be among the developments in the future of HIV research Hopefully, more successful drugs to fight HIV and a vaccine will be among the developments in the future of HIV research