A New Antioxidant Prevents Toxicity of HIV Proteins with Methamphetamine

1. A New Antioxidant Prevents Toxicity of HIV Proteins with Methamphetamine Nuran Ercal MD PhD Department of Chemistry, Missouri University of Science and Technology, Rolla, MO & Department of Internal Medicine, St. Louis University, St. Louis, MO

2. Our Hypothesis: Toxic HIV Proteins Addictive Drugs (METH) OXIDATIVE STRESS BBB BBB integrity disrupted NEURONs are exposed to TOXINS!

3. What is Oxidative Stress? • Increased generation of ROS and/or a decrease in the antioxidant capacity of cells result in “Oxidative Stress”. • Oxidative stress compromises crucial cellular functions.

4. Sources of Reactive Oxygen Species 1) Non mitochondrial: NADPH Oxidases Microsomal cytochrome P-450 Cyclooxygenases Monoamine oxidases Peroxisomal b oxidation of fatty acids Phagocytes 2) >90% is mitochondrial electron transport chain contains several redox centers that may leak electrons to oxygen----- Superoxide radical!

6. ROS • Superoxide (O2¯•)– • No direct effects on targets • Penetrates important sites • Subsequently converted to other ROI • Hydrogen Peroxide (H2O2)– • Dismutation of superoxide radical 2H + + 2O2¯•H2O2 + O2 • Reacts with thiols • Bacteriocidal only at higher concentrations • Secondary oxidants from H2O2 responsible for killing SOD

7. Hydroxyl Radicals (OH•)– Fenton Reaction Fe 2+ +H2O2 Fe 3+ + OH¯ + OH• • OH•as a major component of neutrophil bacteriocidal arsenal is controversial • Limited radius of action

8. Oxidative Damage to Lipids Membrane peroxidation Decreased membrane fluidity PROTEIN oxo8dG Oxidative Damage to DNA Mutations Deletions H N N H O N H N N H 2 Sugar Oxidation of sulfhydryl groups Reactions with aldehydes Protein aggregation Oxidative Damage to Proteins Protein carbonyls Consequences of oxidative stress Marker F2-Isoprostanes 8-iso-PGF2a

9. Outline • Introduction (background) • HIV Associated Dementia • Methamphetamine • Blood Brain Barrier (BBB) • Proposed Research Goals • Experimental Methods • Previous Results: RBE4 • Recent Results: HBMVEC • Future Studies: immortal HBMVEC and transgenic animals • Conclusion

10. What is the problem? • Neurological disorders are serious complications of human immunodeficency virus type 1 (HIV-1).

11. What are these neurological complications? • HIV-associated dementia (HAD) • HIV-related encephalitis (HIVE) • More commonly minor neurocognitive problems • DEMENTIA is the most challenging complications of HIV infection. Avindra Nath et al International Review of Psychiatry, Feb 2008, 20 (1), 25-31.

12. What is HAD? Aneurological syndrome characterized by cognitive deficits and motor and behavioral dysfunction.

13. What are the symptoms of HAD? • Symptoms include: • Sluggish mental capabilities and motor control • Marked apathy • Loss of interest in previously enjoyable activities • Tremors and impaired balance, slow eye movement, abnormal reflexes.. • The disorder continues to escalate, eventually resulting in severe dementia • Some experience mania or even psychosis

14. How often is HAD seen? • One third of adults and half of children with HIV have dementia in the western countries • HAD is the most common case of dementia among people aged 40 or less and a significant independent risk factor for death due to AIDS! Avindra Nath et al International Review of Psychiatry, Feb 2008, 20 (1), 25-31. • HAART (Highly Active AntiRetroviral Therapy) therapy has reduced HAD in infected population some but it does not seem to be effective. • In the era of HAART, the course of HIV dementia appears to have changed. Steiner J et al, Antioxidants &Redox Signaling, 2006; McArthur JC et al, Neurology, 1993; Bouwman FH et al, Neurology, 1998

15. Treatment of AIDS/HIV • Highly active antiretroviral therapy (HAART) is introduced in the mid-90s. • HAART consists of combination of • nucleoside reverse transcriptase inhibitors • non-nucleoside RT inhibitors • and protease inhibitors • There are more than 20 (zidovudine, lamivudine, stavudine, emtriva, crixivan, kaletra and more) approved antiretroviral drugs. • HAART therapy has been shown to prolong survival in AIDS patients. Current Pharmaceutical Design, 2006, 12,2031-2055

16. How about: World Wide AIDS Statistics • Over 22 million people have died from AIDS • Over 42 million people are living with HIV/AIDS • Over 19 million women are living with HIV/AIDS • There are over 14,000 new infections every day (95% are in developing countries) http://www.until.org/statistics.shtml

17. World Wide Future Estimates • The UN estimates that, currently, there are 14 million AIDS orphans and that by 2010 there will be 25 million • By the year 2010, five countries (Ethiopia, Nigeria, China, India, and Russia) with 40% of the world’s population will add 50 to 75 million infected people to the worldwide pool of HIV disease if nothing is done to help stop the spread of HIV/AIDS http://www.until.org/statistics.shtml

18. United States AIDS Statistics • One million people are currently living with HIV in the U.S., with approximately 40,000 new infections occurring each year • 70% of these new infections occur in men and 30% occur in women • 75% of the new infections in women are heterosexually transmitted • Half of all new infections occur in people 25 years old or younger http://www.until.org/statistics.shtml

19. What is the possible mechanism of HIV dementia? • HIV envelope protein (gp120) and transregulatory protein (Tat) may play a role in the development of HAD by increasing the production of reactive oxygen species (ROS) in blood brain barrier (BBB). • Oxidative stress may be involved in HIV neuropathogenesis.

20. Parkinson’s Disease Alzheimer’s Disease Stroke Rheumatoid arthritis Atherosclerosis Vascular dysfunction Multiple sclerosis Inflammatory bowel disease H. pylori-associated gastritis Systemic inflammatory response syndrome And the list is growing… Autoimmune thyroid disease Cystic fibrosis Diabetes Aging Macular degeneration HIV/AIDS Cancer Septic shock Heavy metal toxicity Nanoparticle toxicity EtOH abuse Meth ALS Oxidative Stress Related Disorders

21. Is there “Oxidative Stress” in HIV Dementia? • Analyses of brain tissue and CSF of patients with HIV-1 dementia shows evidence for oxidative stress correlated with disease pathogenesis and cognitive impairment. • Evidences are: • 4-HNE is high • Protein carbonyls are high • Nitrated tyrosine residues are increased in HIV dementia brains

22. Another important concern: Methamphetamine (METH) • It is widely known that many people with HIV-1 use addictive drugs. Among them: • Alcohol • Methamphetamine • Cocaine • Nitrite Inhalants • Hallucinogens • Nearly 50% of HIV positive women in the US contract the infection via drug use!! • Methamphetamine abuse is common • 9.4 million people in the US • Midwest – 90% of all drug cases • Methamphetamine (METH) induces ROS.

23. Crystal Powder Pill

24. Missouri – Highest Rate of METH Lab Activity in the Country Picture source: US Department of Justice (www.usdoj.gov)

25. More on Methamphetamine • Street names of methamphetamine (METH) • Speed • Ice • Crystal Meth • Chalk • Consumption • Smoke • Snort • Inject • Oral Ingestion • Surge in catecholamine levels: • Dopamine (DA) • Serotonin • Norepinephrine (NE) National Drug Intelligence Center, National Drug Threat Assessment 2008, October 2007; Picture Source: http://cerhr.niehs.nih.gov/chemicals/stimulants/amphetamines/methamphetamine.gif

26. Oxidative stress has been shown to play an important role in the toxic effects of METH. METH increases levels of dopamine. Dopamine can react to form ROS through several different pathways. Josephine W.S et al., Annals of the New York Academy of Sciences (2000) Zecca, L., et al., Nature Reviews (2004) Meth and Oxidative Stress Dopamine enzymatic oxidation MAO DA + O2 + H2O------------------- 3,4,dihydroxyphenylacetic acid + NH3 + H2O2 Dopamine autoxidation DA + O2 SQ·+ O2·- + H+ DA + O2· - + 2 H+  SQ·+ H2O2

27. What are toxic HIV proteins? HIV envelope protein (gp120) and transregulatory protein (Tat) may play a role in the development of HAD by increasing the production of reactive oxygen species (ROS) in blood brain barrier (BBB).

28. What exactly is happening? • The BBB has been implicated in the development of HIV Dementia. • The BBB is a barrier between the blood and the fluid that surrounds the cells of the brain. • The BBB is selectively permeable, allowing some substances to cross and not others. It is generally more permeable to lipophilic substances. • The cells that line the capillaries of the rest of the body usually have small gaps between them, that allow substance exchange. The BBB lacks these small gaps, prohibiting much exchange.

29. Blood Brain Barrier (BBB)

30. What is the function of the BBB? • The BBB maintains that delicate balance by regulating the entry and exit of substances. • The BBB also provides protection by preventing toxic chemicals from entering the brain. • The structural integrity of the BBB is compromised in HIV/AIDs demented patients. • What is the cause of this destruction?

31. What’s causing the blood-brain barrier to fail? • Studies have found that HIV-1 envelope protein gp120 and/or Tat may have a role in the structural damage that the BBB experiences.

32. Our Hypothesis: Toxic HIV Proteins Gp120 and tat Addictive Drugs (METH) OXIDATIVE STRESS BBB BBB integrity disrupted NEURONs are exposed to TOXINS..

33. Let’s study HIV-1 and its proteins

34. gp120 • gp120 is a protein on the outer envelope of the HIV-1 virus. • It binds to a receptor on CD4 cells and aides in the injection of viral nucleic acid into the host cell. • The amino acids responsible for this binding interaction are highly conserved amongst strains of the virus.

35. Gp120 (red) in complex with a CD4 surface protein (yellow)

36. More on gp120 • gp120 is composed of an outer and inner domain made of α helices and β sheets. • The negatively charged central cavity contains the binding site for a positively charged region of a surface protein on CD4 (electrostatic interactions).

37. Molecular Structure of gp120 gp120 changes confirmation when bound to different proteins Picture Source: Tongqing Zhou et al. Nature 2007

38. Transregulatory Protein (Tat) It is a viral protein released from HIV-1-infected T cells and monocytes/macrophages Transactivator of Transcription (Tat) protein consists of between 86 and 101 amino acids depending on the subtype Jeang, K. T. (1996) In: Human Retroviruses and AIDS: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Los Alamos National Laboratory (Ed.) pp. III-3–III-18; Picture Source: Grant R. Campbell et al. J. Biol. Chem. 2004

39. Tat and gp120-Induced Oxidative Stress Although the mechanism is not explicitly known, it has been shown that both gp120 and tat increase oxidative stress levels in cells. Tat Decreases GSH by inhibiting GSH synthetase Increases NO secretion which induces apoptosis Increases calcium uptake resulting in production of ROS gp120 Decreases GSH by inhibiting GSH synthetase Increases cell permeability Promotes calcium overload Oxyradical production Mitochondrial dysfunction by altering ion channels Choi J et al, J Biol Chem 2000; Toneatto S et al, AIDS 1999; Visalli, Valeria, Neuroscience 2007

40. H2O2 Quinones O2•¯, OH• Oxidation by MAO Dopamine ROS Autooxidation METH RNS Glutamate mobilization by NMDAR activation Ca2+ overload O2•¯ NOO¯ Gp120 TAT nNOS Microglial activation Cytokines & Chemokines IL-1B,IL-8,TNF-a CCL2, CCL4,CXCL8 iNOS eNOS ERK1/2 •NO MAPK Oxidative Stress DNA oxidation Protein oxidation Lipid Peroxidation Oxidative Stress: Possible Pathways Antioxidants

41. Oxidative Stress in HIV Dementia Analyses of brain tissue and CSF of patients with HIV-1 dementia shows evidence for oxidative stress correlated with disease pathogenesis and cognitive impairment. Evidences are: 4-HNE is high Protein carbonyls are high Nitrated tyrosine residues are increased in HIV dementia brains

42. HIV Associated Dementia and METH • Overlap of pathways leading to neurodegeneration in HAD • METH use enhances gp120 and Tat mediated neurotoxicity • Oxidative stress common to viral protein and METH mediated toxicity • Therapeutic approaches • Block rise in intracellular calcium • Antagonize NMDA receptors • Target oxidative damage

43. Disruption of Blood Brain Barrier • Disruption of the BBB is seen more in AIDS/HIV dementia patients then in non-demented AIDS/HIV patients. • The disruption of the BBB may be due to an increase in ROS by viral proteins • Tat and gp120 induce oxidative stress • Stimulation of iNOS and production of NO • Glutamate mediated excitotoxicity • Disruption of calcium homeostasis • METH may potentiate Tat and gp120 induced oxidative stress on BBB.

44. Antioxidants in Neurodegeneration • Highly active antiretroviral therapy (HAART) does not prevent BBB disruption nor does it decrease ROS production. • Therefore, ANTIOXIDANTS should be included in the treatment to prevent HAD. • Dealing with BBB • Most antioxidants (AO) unable to cross BBB • Design AO capable of crossing BBB • Low MW thiol antioxidants with ability to cross BBB

45. Thiols as Antioxidants • The most important biological thiol, Glutathione (GSH), protects cells against ROS. • GSH deficiency has been associated with various neurodegenerative diseases. • A significant decrease in GSH levels in patients with AIDS/HIV in various biological samples (blood, liver, brain). • Thiol antioxidants in HAD: • NAC (decreased the mortality rate of HIV-infected patients) • NAC analogs (N-acetyl-L-cysteinyl)-S-acetylcysteamine (NACA, possible candidate) Pocernich CB et al., Brain Research Reviews, 2005 .

46. Structures of NAC and NACA NAC NACA

47. NACA related projects • NACA in radiation damage • NACA in neurological complications of • HIV Dementia • Lead poisoning (possibly via Glu) • NACA in medicinal and abusive drug complications • Acetaminophen poisoning • METH neurotoxicity • NACA in macular degeneration

48. 30-day percentage survival rates of SD-rats after irradiation with pre-treatment of NAC or NACA and post-treatment of NAC or NACA. A: XRT only; B: XRT + NAC (pre-treated); C: XRT + NACA (pre-treated);D: Control (no XRT and any treatment); E:NAC only; F: NACA only; G:XRT + NAC (post-treated); H: XRT + NACA (post-treated).

49. Control NACA only (750 μM) Glutamate (10 mM) + NACA (750 μM) Glutamate only (10 mM) PC12 cells were plated at a density 25 x 103 cells/well in a 24 well plate and grown for 24 h in culture medium; then they were treated or not (control) with 10 mM Glu with or without NACA.. Twenty four h later, cells were examined and photographed.

50. Immortalized HBMVEC