PDF(Pubmed)
Abstract：
The \"block and lock\" strategy is one approach that might elicit a sterilizing cure for HIV-1 infection. The \"block\" refers to a compound\'s ability to inhibit latent HIV-1 proviral transcription, while the \"lock\" refers to its capacity to induce permanent proviral silencing. We previously identified PF-3758309, a pan-isoform inhibitor of p21-activated kinases (PAKs), as a potent inhibitor of HIV-1 latency reversal. The goal of this study was to define the mechanism(s) involved. We found that both 24ST1NLESG cells (a cell line model of HIV-1 latency) and purified CD4+ naïve and central memory T cells express high levels of PAK2 and lower levels of PAK1 and PAK4. Knockdown of PAK1 or PAK2, but not PAK4, in 24ST1NLESG cells resulted in a modest, but statistically significant, decrease in the magnitude of HIV-1 latency reversal. Overexpression of PAK1 significantly increased the magnitude of latency reversal. A phospho-protein array analysis revealed that PF-3758309 down-regulates the NF-κB signaling pathway, which provides the most likely mechanism by which PF-3758309 inhibits latency reversal. Finally, we used cellular thermal shift assays combined with liquid chromatography and mass spectrometry to ascertain whether PF-3758309 off-target binding contributed to its activity. In 24ST1NLESG cells and in peripheral blood mononuclear cells, PF-3758309 bound to mitogen-activated protein kinase 1 and protein kinase A; however, knockdown of either of these kinases did not impact HIV-1 latency reversal. Collectively, our study suggests that PAK1 and PAK2 play a key role in the maintenance of HIV-1 latency.
摘要：
“阻断和锁定”策略是一种可能引发HIV-1感染的绝育治疗方法。“阻断”是指化合物抑制潜伏HIV-1前病毒转录的能力，而“锁”是指它诱导永久性原沉默的能力。我们先前鉴定了PF-3758309，p21活化激酶（PAKs）的泛同工型抑制剂，作为HIV-1潜伏期逆转的有效抑制剂。这项研究的目的是确定所涉及的机制。我们发现24ST1NLESG细胞（HIV-1潜伏期的细胞系模型）和纯化的CD4+初始和中枢记忆T细胞表达高水平的PAK2和较低水平的PAK1和PAK4。在24ST1NLESG细胞中敲除PAK1或PAK2，但不敲除PAK4，导致适度的，但具有统计学意义，降低HIV-1潜伏期逆转的幅度。PAK1的过表达显着增加了潜伏期逆转的幅度。磷蛋白阵列分析显示，PF-3758309下调NF-κB信号通路，这提供了PF-3758309抑制延迟逆转的最可能机制。最后,我们使用细胞热转移分析结合液相色谱和质谱来确定PF-3758309脱靶结合是否有助于其活性.在24ST1NLESG细胞和外周血单核细胞中，PF-3758309与丝裂原活化蛋白激酶1和蛋白激酶A结合；然而，这两种激酶的敲除均不影响HIV-1潜伏期逆转.总的来说，我们的研究提示PAK1和PAK2在HIV-1潜伏期的维持中起关键作用.
