Berg Christian, Spiess Katja, Lüttichau Hans R, Rosenkilde Mette M
Department of Neuroscience and Pharmacology Faculty of Health and Medical Sciences The Panum Institute University of Copenhagen Copenhagen Denmark.
Department of Neuroscience and Pharmacology Faculty of Health and Medical Sciences The Panum Institute University of Copenhagen Copenhagen Denmark; Department of Medicine Infectious Disease Unit Herlev Hospital Copenhagen Denmark.
Pharmacol Res Perspect. 2016 Oct 18;4(6):e00262. doi: 10.1002/prp2.262. eCollection 2016 Dec.
Since the discovery of HIV's use of CCR5 as the primary coreceptor in fusion, the focus on developing small-molecule receptor for inhibition hereof has only resulted in one single drug, Maraviroc. We therefore investigated the possibility of using small-molecule CCR5 as HIV-1 fusion inhibitors. A virus-free cell-based fusion reporter assay, based on mixing "effector cells" (expressing HIV and luciferase activator) with "target cells" (expressing CD4, CCR5 wild type or a selection of well-described mutations, and luciferase reporter), was used as fusion readout. Receptor expression was evaluated by ELISA and fluorescence microscopy. On CCR5 WT, Maraviroc and Aplaviroc inhibited fusion with high potencies (EC values of 91 and 501 nM, respectively), whereas removal of key residues for both antagonists (Glu283Ala) or Maraviroc alone (Tyr251Ala) prevented fusion inhibition, establishing this assay as suitable for screening of HIV entry inhibitors. Both ligands inhibited HIV fusion on signaling-deficient CCR5 mutations (Tyr244Ala and Trp248Ala). Moreover, the steric hindrance CCR5 mutation (Gly286Phe) impaired fusion, presumably by a direct hindrance of gp120 interaction. Finally, the efficacy switch mutation (Leu203Phe) - converting small-molecule antagonists/inverse agonists to full agonists biased toward G-protein activation - uncovered that also small-molecule can function as direct HIV-1 cell entry inhibitors. Importantly, no agonist-induced receptor internalization was observed for this mutation. Our studies of the pharmacodynamic requirements for HIV-1 fusion inhibitors highlight the possibility of future development of biased ligands with selective targeting of the HIV-CCR5 interaction without interfering with the normal functionality of CCR5.
自从发现HIV利用CCR5作为融合过程中的主要共受体以来，致力于开发用于抑制此过程的小分子受体的研究仅产生了一种药物，即马拉维若。因此，我们研究了使用小分子CCR5作为HIV-1融合抑制剂的可能性。基于将“效应细胞”（表达HIV和荧光素酶激活剂）与“靶细胞”（表达CD4、CCR5野生型或一系列已充分描述的突变体以及荧光素酶报告基因）混合的无病毒细胞融合报告基因检测，被用作融合读数。通过酶联免疫吸附测定（ELISA）和荧光显微镜评估受体表达。在CCR5野生型上，马拉维若和阿普洛韦能高效抑制融合（EC值分别为91和501 nM），而去除两种拮抗剂的关键残基（Glu283Ala）或仅去除马拉维若的关键残基（Tyr251Ala）会阻止融合抑制，这表明该检测适用于筛选HIV进入抑制剂。两种配体均能抑制信号缺陷型CCR5突变体（Tyr244Ala和Trp248Ala）上的HIV融合。此外，空间位阻CCR5突变体（Gly286Phe）会损害融合，可能是通过直接阻碍gp120相互作用。最后，功效转换突变体（Leu203Phe）——将小分子拮抗剂/反向激动剂转变为偏向G蛋白激活的完全激动剂——揭示了小分子也可作为直接的HIV-1细胞进入抑制剂。重要地是，未观察到该突变体有激动剂诱导的受体内化现象。我们对HIV-1融合抑制剂的药效学要求的研究突出了未来开发偏向性配体的可能性，这种配体可选择性靶向HIV-CCR5相互作用而不干扰CCR5的正常功能。
