Rational design of drugs that induce human immunodeficiency virus replication

Abstract

Drugs that induce human immunodeficiency virus type 1 (HIV-1) replication could be used in combination with highly active antiretroviral therapy (HAART) to reduce the size of the latent reservoir that is in part responsible for viral persistence. Protein kinase C (PKC) is a logical target for such drugs because it activates HIV-1 transcription through multiple mechanisms. Here we show that HIV-1 gene expression can be induced by potent synthetic analogues of the lipid second messenger diacylglycerol (DAG) synthesized on a five-member ring platform that reduces the entropy of binding relative to that of the more flexible DAG template. By varying the alkyl side chains of these synthetic DAG lactones, it was possible to maximize their potency and ability to render latently infected T cells sensitive to killing by an anti-HIV-1 immunotoxin while minimizing the side effects of CD4 and CXCR4 downregulation and tumor necrosis factor alpha upregulation. The two lead compounds, LMC03 and LMC07, regulated a series of PKC-sensitive genes involved in T-cell activation and induced viral gene expression in peripheral blood mononuclear cells from HIV-1-infected individuals. These studies demonstrate the potential for the rational design of agents that, in conjunction with HAART and HIV-specific toxins, can be used to decrease or eliminate the pool of latently infected reservoirs by forcing viral expression.

FIG. 1.

Synthesis scheme for DAG lactones. For details, see references , , and .

FIG. 2.

Biological activities of DAG compounds compared to that of DPP. (A) ACH-2 cells were grown in the presence of various concentrations of the indicated compounds for 48 h, and p24 antigen production was measured by ELISA. (B) ACH-2 cells were grown in the presence of various concentrations of the indicated compounds for 16 h, and cell surface CD4 expression was measured by antibody staining and fluorescence-activated cell sorting; results were normalized to 100% for no added compound. (C) ACH-2 cells were grown in the presence of various concentrations of the indicated compounds for 16 h, and cell surface CXCR4 expression was measured by antibody staining and fluorescence-activated cell sorting; results were normalized to 100% for no added compound. (D) PBMC were grown in the presence of various concentrations of the indicated compounds for 18 h, and TNF-α secretion was measured by ELISA.

FIG. 3.

Induction of immunotoxin sensitivity. ACH-2 cells were grown with no inducer or with 100 nM concentrations of the indicated compounds in the presence or absence of 5 nM 3B3:N31H/Q100eY(dsFv)-PE for 5 days. (A) Cell viability was assayed by the MTT oxidation procedure, and the percentage of cell killing was calculated relative to that with untreated cells. (B) Annexin V binding was assayed by fluorescence-activated cell sorting.

FIG. 4.

Microarray analysis. cDNA prepared from A3.01 cells grown for 24 h with 100 nM LMC01, DPP, LMC03, or LMC07 was labeled with Cy3 (red). Each sample was mixed with Cy5 (green)-labeled cDNA from uninduced cells and hybridized to a microarray. The control lane contains Cy3-labeled cDNA from uninduced cells. Shown are those genes that were induced or repressed at least twofold by LMC03 and LMC07.

FIG. 5.

Induction of HIV-1 ex vivo. CD8-depleted (patients 1 and 2) or highly purified CD4⁺ T cells from HIV-1-infected individuals were cultured for 3 weeks with no inducer, anti-CD3 (1:4,000), DPP (100 nM), or DAG lactones (100 nM). HIV-1 p24 levels in the cell-free culture fluid were measured by ELISA; the detection limit of the assay was 50 pg/ml.