HIV Treatment using Nucleoside Analogs

PHM142 Fall 2014 Instructor: Dr. Jeffrey Henderson

HIV Infection

42 million worldwide living with HIV , of which the majority are in poor countries with little to none therapeutic resources. RNA retroviral infection

initiates incorporation of DNA to host chromosome after activity of viral reverse transcriptase.

Two parts of the envelop; external surface proteins composed of gp120 and gp41 which anchor the viron to the cell surface

Matrix protein; p17 located on the inner membrane ensures the full inclusion of viral proteins

Capsid made up of p24 (gag protein) which contains two (+) non coding ssRNA strands, reverse transcriptase, integrase, and protease

HIVs genetic structure

HIV Virion

http://hivbook.com/tag/structure-of-hiv-1/

Antiviral agents commonly used in the therapy of immunodeficiency virus (HIV), as well as Hepatitis B, and cancer

HIV encoded RNA dependent DNA polymerase called reverse transcriptase- inhibitors for this enzyme

Zidovudine was the first drug approved which after phosphorylation competes with dTTP for a site on the transcript of the DNA

Overview of Nucleoside Analogues

Combination Drug Therapy

Amdoxovir: phase II clinical trials and is designed to be more soluble and bioavailable

It is rapidly absorbed and

deaminated to generate a second structure which is then phosphorylated

Reverset: competes with dCTP

New Players in the Field

Mechanism

HIV Infection Mechanism

Mitsuya H, Yarchoan R, Broder S (1990). "Molecular targets for AIDS therapy". Science 249 (4976): 1533–44.Slide 9:Chan DC, Kim PS (1998). "HIV entry and its inhibition". Cell 93 (5): 681–4.Slide 9: Wyatt R, Sodroski J; Sodroski (1998). "The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens". Science 280 (5371): 1884–8.

The virus has a RNA genome Once inside the cell, the virus uses its

viral reverse transcriptase to make a DNA complement strand from the RNA

DNA dependent DNA polymerase then makes opposite DNA strand and then this viral double stranded DNA molecule is incorporated into the cell’s genome

HIV Infection Mechanism

Mitsuya H, Weinhold K, Furman P, St Clair M, Li, Lars, Lehrman S, Gallo R, Bolognesi D, Barry D, Broder S (1985). "3'-Azido-3'-deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type III/lymphadenopathy-associated virus in vitro". Proc Natl Acad Sci USA 82 (20): 7096–100.

Nucleoside analogues used in HIV treatments are designed to be preferentially used by reverse transcriptase

Once inside the body’s cells, the analog has three phosphate groups added to the 5’ OH to become activated

Once bound to the growing DNA strand they prevent further elongation of the strand, since they lack a 3’ OH, and cause premature termination of strand elongation

Premature product is non-functional

Nucleoside Analogues

Azidothymidine (AZT)Thymidine

Nucleoside Analogue Mechanism

Resistance

Retroviral Reverse Transcriptase is the cause of most mutations.

No exonucleolytic proofreading

Error rate goes as high as 1/1700 nucleic acids

Development of Resistance

Nucleosides selects and destroy the virus.

This ultimately selects for resistance with the rise of resistant strains.

Development of Resistance

Insertion of amino acids at position 69 and 70 in the finger subdomain causes resistance to Nucleoside Analogues

Insertion associated with changes in flanking amino acids at position 215

T215F/Y mutation makes ATP binding to RT more effective increasing excision of zidovudine (AZT) in vivo

Specific Mutations

M184V increases susceptibility to other drugs (tenofovir, adefovir, zidovudine) and is a result of treatment with lamivudine and abacavir

K65R mutation causes resistance to most nucleoside analogues with the exception of zidovudine

Both mutations together causes an increased susceptibility to zidovudine.

M184V and K65R

Nucleoside Analogues as HIV Treatment

An example - Zidovudine (AZT)

Zidovudine (or azidothymidine, AZT), was the first antiretroviral drug approved for HIV treatment

First breakthrough in AIDS treatment, reducing virus replication significantly

AZT AZT took 25 months from the first indications of its effectiveness against

HIV to approval, the shortest time in recent history

Phase II studies were modified mid-trial

• 6 months: 16 deaths in placebo, 1 in AZT

• initially approved for small subset - Pneumocycstis carinii, 60%

first trials were in patients with advanced AIDS, median survival rate was increased by a year

after approval for this group of patients, trials began to test effectiveness in early stages of HIV infection

Wright, Karen. “AIDS Therapy: First Tentative Signs of Therapeutic Promise.” Nature 323.6086 (1986):283.

Mitsuya, H., Yarchoan, R., and Broder, S. “AIDS Therapies.” Scientific American 259.4 (1988):110-119.

AZT originally unclear whether resistance to AZT could develop,

or the long-term toxic effects

short-term toxic effects were known

• toxic to bone marrow, patients often developed anemia, and low numbers of WBC and platelets

• this could limit the amount of drug administered

initial dosing was once every 4 hours, all day and night

HAART HIV did eventually develop resistance to AZT alone

currently used as part of highly active antiretroviral therapy - HAART

• can reduce viral load to below the limit of detection

• in countries where patients have access to current HIV therapies, lifespans are significantly increased

chronic illness vs death sentence

Hurtley, Stella. “Take HAART.” Science 303.5664 (2004):1585

References 1. Boyer, P., Sarafianos, S., Arnold, E., & Hughes, S. (n.d.). Nucleoside Analog Resistance Caused by Insertions in the Fingers of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Involves ATP-Mediated Excision. U.S. National Library of Medicine National Institutes of Health. Retrieved October 6, 2014, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC136461/2. JD, R., K, B., & TA, K. (n.d.). Result Filters. U.S. National Library of Medicine. Retrieved October 6, 2014, from http://www.ncbi.nlm.nih.gov/pubmed/24609253. Hurtley, Stella. “Take HAART.” Science 303.5664 (2004):1585 4. Laurence, B and Chabner, B. Goodman and Gilman’s The Pharmacological Basis of Therapeutics, eleventh edition. 2010. pp. 1309-13155. Mitsuya, H., Yarchoan, R., and Broder, S. “AIDS Therapies.” Scientific American 259.4 (1988):110-119.6. Mitsuya H, Yarchoan R, Broder S (1990). "Molecular targets for AIDS therapy". Science 249 (4976): 1533–44.Slide 9:Chan DC, Kim PS (1998). "HIV entry and its inhibition". Cell 93 (5): 681–4.Slide 9: Wyatt R, Sodroski J; Sodroski (1998). "The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens". Science 280 (5371): 1884–8.7. Moyle, G. (n.d.). How the HIV-1 Reverse Transcriptase Mutations K65R, M184V and K65R + M184V Produce Resistance to NRTIs. TheBodyPRO.com. Retrieved October 6, 2014, from http://www.thebodypro.com/content/art38455.html8. V,Vivet-Boudou, J. Didierjean, C. Isel and R. Marquet. Nucleoside and nucleotide inhibitors of HIV-1 replication. Cell. Mol. Life Sci. 63 (2006) 163-186. 9. Wright, Karen. “AIDS Therapy: First Tentative Signs of Therapeutic Promise.” Nature 323.6086 (1986):283.

Summary Slide

• Antiviral agents used in HIV, Hepatitis B and cancer. HIV encoded RNA dependent DNA polymerase called reverse transcriptase.

• The virus has a RNA genome• Once inside the cell, the virus uses its viral reverse transcriptase to make a

DNA complement strand from the RNA• DNA dependent DNA polymerase then makes opposite DNA strand and then

this viral double stranded DNA molecule is incorporated into the cell’s genome

• Once inside the body’s cells, the analog has three phosphate groups added to the 5’ OH to become activated

• Once bound to the growing DNA strand they prevent further elongation of the strand, since they lack a 3’ OH, and cause premature termination of strand elongation

• Premature product is non-functional • Retroviral Reverse Transcriptase is the cause of most mutations. No

exonucleolytic proofreading• short-term toxic effects were known• toxic to bone marrow, patients often developed anemia, and low numbers

of WBC and platelets• HIV did eventually develop resistance to AZT alone - currently used as part

of highly active antiretroviral therapy – HAART -can reduce viral load to below the limit of detection