Project 1:Anti-HIV Mechanisms of NK-1R Antagonists PI: Wenzhe HoCo-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 98:3970-3975NK-1R Antagonist (CP96,345) Inhibits HIV Infection of Macrophage

A B C

Fig. 5

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

Bal

ADA

CSF-6

BL-6

89.6

UG024

HIV RT Activity (% of Control)

0

35

70

105

140

R5

R5X4

X4

Experimental Design

Overall Experimental Plan for Aims 1 and 2NK-1R AntagonistsHIV replicationHIV entryHIV activationHIV infectionPBMC from HIV+ subjects (Project 4)Acute infectionLatent infectionPBMC 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)

L733060 (Merck)

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

Chart2

24.8

3.79

5.63

6.02

7.91

5.43

Control Aprepitant CJ12,255 CP-96,345 RP67,580 L-733060 10-6M 10-6M 10-6M 10-6M 10-6M

HIV RT Activity (103 cpm)

Sheet1

7/30/04

MDM (Doug)Day 8

TreatmentRT Activity (cpm)% of ControlHIV RT Activity (103 cpm)

1Control24802.510024.8

2Aprepitant 10-6M379115.33.79

3CJ-12,255 10-6M562522.75.63

4CP96,345 10-6M602024.36.02

5RP67,580 10-6 M790731.97.91

6L733060 10-6 M542821.95.43

Sheet1

24.8

3.79

5.63

6.02

7.91

5.43

Control Aprepitant CJ12,255 CP-96,345 RP67,580 L-733060 10-6M 10-6M 10-6M 10-6M 10-6M

HIV RT Activity (103 cpm)

Sheet2

24.824.8

3.79

3.79

5.63

6.025.63

7.91

6.02

5.43

7.91

5.43

Sheet2

24.8

3.79

5.63

6.02

7.91

5.43

Control Aprepitant CJ12,255 CP-96,345 RP67,580 L-733060 10-6M 10-6M 10-6M 10-6M 10-6M

HIV RT Activity (103 cpm)

Sheet3

Effect of SP Receptor Antagonist on HIV (BAL) Infection in Macrophage

Sheet3

24.8

3.79

5.63

6.02

7.91

5.43

Control Aprepitant CJ12,255 CP-96,345 RP67,580 L-733060 10-6M 10-6M 10-6M 10-6M 10-6M

HIV RT Activity (103 cpm)

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

T-tropic strains

Dual tropic strains

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

Aim 1To 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 PBMCM-, T-, and dual tropic HIV strainsHIV-1 p24 (Core B) Day 0Day 4AssayWith or without NK-1R antagonistsPBMC from normal subjects

MacrophagesNK-1R AntagonistsControlPseudotyped HIV infectionLuciferase activity at 72 h post-infectionAim 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 primersAim 1c: To determine whether NK-1R antagonists act upon a specific step of HIV replicationPBMCR/U5 pairEarly12h post-infectionGag pairLate intermediate36h post-infectionU3/U5 pairEarly Intermediate24h post-infectionU5/gag pairLate48h 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 subjectsCtl.SPCtl.SPCtl.SPCtl.SPCtl.SPCtl.SPDay 2Day 5Day 2Day 5Day 2Day 5Patient 1Patient 2Patient 3gagb-actin500 bp1000 bp+MarkersHIV-1Li, et al 2001. J. Neuroimmunol. 121:67-75.

Aim 2: To examine the anti-HIV activity of NK-1R antagonist using PBMC from HIV-infected subjectsHIV+ Subjects (Project 4)PBMCControl (no treatment)SPNK-1R Antagonistsand/orHIV RT Day 9, 12, 16 post-treatmentCo-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-infectedindividuals harbor drug-resistant virus, witha rapidly growing subgroup (5-10%) exhibiting resistance to all classes of RT andprotease 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 3PBMC from normal subjectsAntiretroviral drugs (RT and protease inhibitors)and/orHIV p24 (Core B)M- and T-tropic strainsw or w/o NK-1R antagonistsw or w/o NK-1R antagonistsdrug 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 gp120HIV (Bal, JR-PL)With or without NK-1R antagonistsAim 4: To determine whether NK-1Rantagonists have a neuroprotective effectNT2-N MicrogliaCytotoxicityInflammatory Factors

Interaction with other Projects and CoresProject 1 (Ho)Core B (BBI)Core C (Biostatistics)Project 3 (Lackner)Project 2 (Douglas)Project 4 (Tebas)Design and Data analysisHIV isolatesHIV tropismHIV infectivity assayAnti-HIV effectAnti-HIV effectSubjects for Aim 2HIV entyNK-1R-CCR5 interaction

Timelines Performance Schedule of the Research Plan

TasksYear 1Year 2Year 3Year 4Aim 1Aim 2Aim 3Aim 4