Abstract
Novel low molecular weight spirodiketopiperazine derivatives which potently inhibit R5 human immunodeficiency virus type 1 (HIV-1) infection through their antagonistic effects on CCR5 were identified. One such compound E913 (M(r) 484) specifically blocked the binding of macrophage inflammatory protein-1alpha (MIP-1alpha) to CCR5 (IC(50) 0.002 microm) and MIP-1alpha-elicited cellular Ca(2+) mobilization (IC(50) approximately 0.02 microm). E913 potently inhibited the replication of laboratory and primary R5 HIV-1 strains as well as various multidrug-resistant monocyte/macrophage tropic (R5) HIV-1 at IC(50) values of 0.03 to 0.06 microm. E913 was inactive against T cell tropic (X4) HIV-1; however, when combined with a CXCR4 antagonist AMD-3100, E913 potently and synergistically inhibited the replication of dualtropic HIV-1 and a 50:50 mixture of R5 and X4 HIV-1. Antagonism in anti-HIV-1 activity was not seen when E913 was combined with the reverse transcriptase inhibitor zidovudine or protease inhibitors. E913 proved to compete with the binding of antibodies to CCR5 which recognize the C-terminal half of the second extracellular loop (ECL2B) of CCR5. E913 and its analogs are acid-resistant and orally bioavailable in rodents. These data warrant that spirodiketopiperazine derivatives be further developed as potential therapeutics for HIV-1 infection.
Highlights
Active antiretroviral therapy has brought about a major impact on the AIDS epidemics in the industrially advanced nations [1, 2], eradication of HIV-11 appears to be currently impossible, in part, due to the viral reservoirs
It has been shown that HIV-1 undergoes phenotypic shift in the course of development of AIDS: nonsyncytia-inducing R5 HIV-1 predominates in early infection, while syncytia-inducing X4 HIV-1 and those which use both CCR5 and CXCR4 for cell entry (R5X4 HIV-1 or dualtropic HIV-1) emerge as the disease progresses [31, 32]
C-C chemokines appear to affect the replication of X4 HIV-1 and influence the phenotypic HIV-1 shift seen in HIV-1-infected individuals
Summary
ReagentsāSpirodiketopiperazine derivatives including E913 were newly designed and synthesized as will be described elsewhere. Chemokine Binding StudiesāCCR5-CHO cells (1.2 Ļ« 105 cells/well) were plated onto 48-well, flat-bottomed culture plates, incubated for 18 ā24 h, rinsed once with Hamās F-12 medium containing 20 mM Hepes and 0.5% BSA These adherent CCR5-CHO cells were exposed to 0.1 nM 125I-labeled MIP-1ā£ in the presence of varying concentrations of a test compound at room temperature for 40 min, washed thoroughly with cold phosphate-buffered saline (PBS), and lysed with 0.5 ml of 1 N NaOH. After a 24-h incubation, the cells were exposed to various concentrations of a test compound and HIV-1 virus in Dulbeccoās modified Eagleās medium containing 15% fetal calf serum, and were stained at 48 h of culture with chlorophenol red ā¤-D-galactopyranoside as previously described [22, 23]. In 7 days of culture, 100 ā®l of the medium was removed from
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