Inhibition of Tat-mediated HIV-1 replication and neurotoxicity by novel GSK3-beta inhibitors

Abstract

The HIV-1 protein Tat is a critical regulator of viral transcription and has also been implicated as a mediator of HIV-1 induced neurotoxicity. Here using a high throughput screening assay, we identified the GSK-3 inhibitor 6BIO, as a Tat-dependent HIV-1 transcriptional inhibitor. Its ability to inhibit HIV-1 transcription was confirmed in TZM-bl cells, with an IC50 of 40 nM. Through screening 6BIO derivatives, we identified 6BIOder, which has a lower IC50 of 4 nM in primary macrophages and 0.5 nM in astrocytes infected with HIV-1. 6BIOder displayed an IC50 value of 0.03 nM through in vitro GSK-3β kinase inhibition assays. Finally, we demonstrated 6BIO and 6BIOder have neuroprotective effects on Tat induced cell death in rat mixed hippocampal cultures. Therefore 6BIO and its derivatives are unique compounds which, due to their complex mechanisms of action, are able to inhibit HIV-1 transcription as well as to protect against Tat induced neurotoxicity.

Novel Neuroprotective GSK-3β Inhibitor Restricts Tat-Mediated HIV-1 Replication

“…We used VEEV, since the 6BIOder compound was previously shown by our group to significantly inhibit viral replication (95). Quantification of previously obtained data (33,95) in Fig. 1B indicated that 18BIOder was effective in inhibiting Tat-dependent HIV-1 transcription (Ͼ75% inhibition) but ineffective in VEEV infection (no inhibition).…”

“…We previously identified the GSK-3␤ inhibitor 6BIO as a potent HIV-1 LTR transcriptional inhibitor by performing a high-throughput screen for Tat-dependent transcription inhibitors from LopacSigma-Aldrich (1,280 compounds) and Spectrum-Microsource (2,000 compounds) (33). In order to identify more potent 6BIO analogs, a Hit2Lead compound query based on chemical structure yielded 38 commercially available derivatives.…”

“…The levels of 18BIOder inhi- Twenty-four hours postinfection, the viral supernatants were collected and assayed for viral replication by plaque assays. For HIV-1 studies, TZM-bl cells were processed as previously described (33). The data are representative of three technical triplicates and at least 2 biological replicates.…”

“…Similarly, gp120 treatment induced injury to primary rat dopamine cells and reduced dopamine transport, suggesting a neuropathological consequence of gp120 (27), while this negative effect was prevented by gp120 antibody. Moreover, studies carried out over the last 2 decades link extracellular Tat to HIV-1 HAND, where Tat functions as a neuroexcitatory toxin that plays a role in virus-mediated neuronal dysfunction (29)(30)(31)(32)(33). Postulated mechanisms of Tat neurotoxicity include altered calcium homeostasis and calcium dependence in fetal neurons (27,34,35), increased oxidative stress resulting from direct injection of Tat into the intrastriatal space (36), increased gliosis and glial infiltration (36)(37)(38), stimulation of the glutamatergic system (39), increased nitric oxide production in microglial cultures (40), and increased apoptosis from cell damage and death following Tat exposure (34,41).…”

“…For instance, robust glycogen synthase kinase-3␤ (GSK-3␤) inhibitors, such as lithium and valproic acid, have been shown to protect against Tat-and gp120-mediated neurotoxicity (59,76,77). Recently, small chemical molecules have taken the spotlight due to their capacity for conferring both potent Tatdependent transcriptional inhibition and cytoprotection from Tat-induced neurotoxicity through mechanisms that remain to be determined (33,78).…”