1. Project 1: Anti-HIV Mechanisms of NK-1R Antagonists PI: Wenzhe Ho Co-PI: Steven D. Douglas

2. Objective To examine the anti-HIV activity of NK-1R antagonists using in vitro and ex vivo cell models To determine the mechanisms involved in the anti-HIV action of NK-1R antagonists

3. Lai et al. 2001, PNAS 98:3970-3975 NK-1R Antagonist (CP96,345) Inhibits HIV Infection of Macrophage

4. Effect of CP-96,345 on HIV Infection Lai et al. 2001, PNAS 98:3970-3975

5. Experimental Design

6. Overall Experimental Plan for Aims 1 and 2 NK-1R Antagonists HIV replicationHIV entryHIV activation HIV infection PBMC from HIV+ subjects (Project 4) Acute infectionLatent infection PBMC from normal subjects

7. NK-1R Antagonists to be Tested Aprepitant (Merck) CJ-12,255 (Pfizer) CP-96,345 (Pfizer) RP-67,580 (Rhone-Poulenc Rorer) L733060 (Merck)

8. Inhibition of HIV (Bal) Infection of Macrophages by the SP Receptor Antagonists

9. HIV Isolates to be Used (Provided by Core B) M-tropic strains T-tropic strains Dual tropic strains

10. Doms, et al 1997. Virology 235:179-90.

11. Aim 1 To examine the in vitro anti-HIV activity and mechanism of NK-1R antagonists using PBMC from normal subjects

12. Aim 1a: To determine whether the NK-1R antagonists inhibit HIV infection of PBMC M-, T-, and dual tropic HIV strains HIV-1 p24 (Core B) Day 0 Day 4 Assay With or without NK-1R antagonists PBMC from normal subjects

13. Macrophages NK-1R AntagonistsControl Pseudotyped HIV infection Luciferase activity at 72 h post-infection Aim 1b: To determine the impact of the NK-1R antagonists on HIV entry using pseudotyped viruses

14. NK-1R AntagonistsControl (no treatment) HIV infection (Bal, NL43) RT-PCR using 4 pairs of primers Aim 1c: To determine whether NK-1R antagonists act upon a specific step of HIV replication PBMC R/U5 pair Early 12h post-infection Gag pair Late intermediate 36h post-infection U3/U5 pair Early Intermediate 24h post-infection U5/gag pair Late 48h post-infection

15. Aim 2 To determine the anti-HIV effects of NK-1R antagonists using PBMC from HIV-infected subjects (prior to aprepitant treatment, Project 4).

16. Effect of SP on HIV-1 gag Gene Expression in PBMC from HIV- Infected subjects Ctl. SP Ctl. SP Ctl. SP Ctl. SP Ctl. SP Ctl. SP Day 2Day 5Day 2Day 5Day 2Day 5 Patient 1Patient 2Patient 3 gag  -actin 500 bp 1000 bp + Markers HIV-1 Li, et al 2001. J. Neuroimmunol. 121:67-75.

17. Aim 2: To examine the anti-HIV activity of NK-1R antagonist using PBMC from HIV-infected subjects HIV + Subjects (Project 4) PBMC Control (no treatment) SPNK-1R Antagonists and/or HIV RT Day 9, 12, 16 post-treatment Co-culture Assay for Co-receptor usage and Drug susceptibility (Core B)

18. Aim 3 To examine whether NK-1R antagonists inhibit drug-resistant HIV strains and have a synergistic anti-HIV effect with commonly used antiretrovirals.

19. It is estimated that up to 45% of HIV-infected individuals harbor drug-resistant virus, with a rapidly growing subgroup (5-10%) exhibiting resistance to all classes of RT and protease inhibitors. Antiretroviral Drugs and Drug-Resistant Virus

20. Drug-resistant Strains to be used (Provided by Core B) NRTIs resistant NNRTIs resistant Protease Inhibitor resistant

21. Overall Experimental Plan for Aim 3 PBMC from normal subjects Antiretroviral drugs (RT and protease inhibitors) and/or HIV p24 (Core B) M- and T-tropic strains w or w/o NK-1R antagonists w or w/o NK-1R antagonists drug resistant strains

22. Aim 4 To determine whether NK-1R antagonists have anti-HIV activity in microglia and a neuroprotective effect in neuronal cells

23. Rationale HIV not only attacks the immune system but also the CNS. Microglia is the primary target cells for HIV infection in CNS. SP is a major mediator involved in inflammation and immunomodulatory activities within the CNS

24. HIV TAT and/or gp120 HIV (Bal, JR-PL) With or without NK-1R antagonists Aim 4: To determine whether NK-1R antagonists have a neuroprotective effect NT2-N Microglia Cytotoxicity Inflammatory Factors

25. Interaction with other Projects and Cores Project 1 (Ho) Core B (BBI)Core C (Biostatistics) Project 3 (Lackner)Project 2 (Douglas)Project 4 (Tebas) Design and Data analysis HIV isolates HIV tropism HIV infectivity assay Anti-HIV effect Subjects for Aim 2 HIV enty NK-1R-CCR5 interaction

26. Timelines – Performance Schedule of the Research Plan TasksYear 1Year 2Year 3Year 4 Aim 1 Aim 2 Aim 3 Aim 4