帕博西尼是一种细胞周期蛋白依赖性激酶4/6的选择性抑制剂，它通过控制含无菌α基序和HD结构域蛋白1（SAMHD1）的活性来阻断HIV-1逆转录。

Palbociclib, a selective inhibitor of cyclin-dependent kinase4/6, blocks HIV-1 reverse transcription through the control of sterile α motif and HD domain-containing protein-1 (SAMHD1) activity.

作者信息

Pauls Eduardo, Badia Roger, Torres-Torronteras Javier, Ruiz Alba, Permanyer Marc, Riveira-Muñoz Eva, Clotet Bonaventura, Marti Ramón, Ballana Ester, Esté José A

机构信息

aAIDS Research Institute-IrsiCaixa and AIDS Unit, Hospital Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain bResearch Group on Neuromuscular and Mitochondrial Disorders, Vall d'Hebron Institut de Recerca, Universitat Autònoma de Barcelona and Biomedical Network Research Centre on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Valencia, Spain. *Eduardo Pauls and Roger Badia contributed equally to this work.

出版信息

AIDS. 2014 Sep 24;28(15):2213-22. doi: 10.1097/QAD.0000000000000399.

DOI:10.1097/QAD.0000000000000399

PMID:25036183

Abstract

BACKGROUND

Sterile α motif and HD domain-containing protein-1 (SAMHD1) inhibits HIV-1 reverse transcription by decreasing the pool of intracellular deoxynucleotides. SAMHD1 is controlled by cyclin-dependent kinase (CDK)-mediated phosphorylation. However, the exact mechanism of SAMHD1 regulation in primary cells is unclear. We explore the effect of palbociclib, a CDK6 inhibitor, in HIV-1 replication.

METHODS

Human primary monocytes were differentiated into macrophages with monocyte-colony stimulating factor and CD4 T lymphocytes stimulated with phytohaemagglutinin (PHA)/interleukin-2. Cells were treated with palbociclib and then infected with a Green fluorescent protein-expressing HIV-1 or R5 HIV-1 BaL. Viral DNA was measured by quantitative PCR and infection assessed by flow cytometry. Deoxynucleotide triphosphate (dNTP) content was determined using a polymerase-based method.

RESULTS

Pan-CDK inhibitors AT7519, roscovitine and purvalanol A reduced SAMHD1 phosphorylation. HIV-1 replication was blocked by AT7519 (66.4 ± 3.8%; n = 4), roscovitine (47.3 ± 3.9%; n = 4) and purvalanol A (55.7 ± 15.7%; n = 4) at subtoxic concentrations. Palbociclib, a potent and selective CDK6 inhibitor, blocked SAMHD1 phosphorylation, intracellular dNTP levels, HIV-1 reverse transcription and HIV-1 replication in primary macrophages and CD4 T lymphocytes. Notably, treatment of macrophages with palbociclib led to reduced CDK2 activation, measured as the phosphorylation of the T-loop at the Thr160. The antiviral effect was lost when SAMHD1 was degraded by Vpx, providing further evidence for a role of SAMHD1 in mediating the antiretroviral effect.

CONCLUSIONS

Our results indicate that SAMHD1-mediated HIV-1 restriction is controlled by CDK as previously suggested but point to a preferential role for CDK2 and CDK6 as mediators of SAMHD1 activation. Our study provides a new signaling pathway susceptible for the development of new therapeutic approaches against HIV-1 infection.

摘要

背景

含无菌α基序和HD结构域蛋白1（SAMHD1）通过减少细胞内脱氧核苷酸池来抑制HIV-1逆转录。SAMHD1受细胞周期蛋白依赖性激酶（CDK）介导的磷酸化调控。然而，SAMHD1在原代细胞中的调控确切机制尚不清楚。我们探究了CDK6抑制剂帕博西尼对HIV-1复制的影响。

方法

用人单核细胞集落刺激因子将人原代单核细胞分化为巨噬细胞，并用植物血凝素（PHA）/白细胞介素-2刺激CD4 T淋巴细胞。细胞用帕博西尼处理，然后用表达绿色荧光蛋白的HIV-1或R5 HIV-1 BaL感染。通过定量PCR检测病毒DNA，通过流式细胞术评估感染情况。使用基于聚合酶的方法测定三磷酸脱氧核苷酸（dNTP）含量。

结果

泛CDK抑制剂AT7519、罗斯考维汀和嘌呤霉素A降低了SAMHD1的磷酸化。在亚毒性浓度下，AT7519（66.4±3.8%；n = 4）、罗斯考维汀（47.3±3.9%；n = 4）和嘌呤霉素A（55.7±15.7%；n = 4）阻断了HIV-1复制。帕博西尼是一种强效且选择性的CDK6抑制剂，可阻断原代巨噬细胞和CD4 T淋巴细胞中SAMHD1的磷酸化、细胞内dNTP水平、HIV-1逆转录和HIV-1复制。值得注意的是，用帕博西尼处理巨噬细胞导致CDK2激活减少，以苏氨酸160处T环的磷酸化来衡量。当SAMHD1被Vpx降解时，抗病毒作用丧失，这为SAMHD1在介导抗逆转录病毒作用中的作用提供了进一步证据。