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

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

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

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

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

Experimental Design

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

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

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

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

Doms, et al Virology 235:

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

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

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

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

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

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 J. Neuroimmunol. 121:67-75.

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)

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

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

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

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

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

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

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

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

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