Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA.
AIDS Res Ther. 2021 Oct 26;18(1):77. doi: 10.1186/s12981-021-00399-z.
Targeting RNA is a promising yet underdeveloped modality for the selective killing of cells infected with HIV-1. The secretory ribonucleases (RNases) found in vertebrates have cytotoxic ribonucleolytic activity that is kept in check by a cytosolic ribonuclease inhibitor protein, RI.
We engineered amino acid substitutions that enable human RNase 1 to evade RI upon its cyclization into a zymogen that is activated by the HIV-1 protease. In effect, the zymogen has an HIV-1 protease cleavage site between the termini of the wild-type enzyme, thereby positioning a cleavable linker over the active site that blocks access to a substrate.
The amino acid substitutions in RNase 1 diminish its affinity for RI by 10-fold and confer high toxicity for T-cell leukemia cells. Pretreating these cells with the zymogen leads to a substantial drop in their viability upon HIV-1 infection, indicating specific toxicity toward infected cells.
These data demonstrate the utility of ribonuclease zymogens as biologic prodrugs.
靶向 RNA 是一种有前途但尚未开发的方法，可用于选择性杀死感染 HIV-1 的细胞。脊椎动物中发现的分泌型核糖核酸酶（RNases）具有细胞毒性的核糖核酸酶活性，这种活性受到细胞质核糖核酸酶抑制剂蛋白 RI 的抑制。
我们设计了氨基酸取代，使人类 RNase 1 在环化形成受 HIV-1 蛋白酶激活的酶原时能够逃避 RI。实际上，酶原在野生型酶的末端之间具有 HIV-1 蛋白酶切割位点，从而在活性位点上方定位一个可切割的连接物，阻止底物进入。
RNase 1 中的氨基酸取代使其对 RI 的亲和力降低了 10 倍，并赋予 T 细胞白血病细胞高毒性。用酶原预处理这些细胞，会导致它们在感染 HIV-1 后活力大幅下降，表明对感染细胞具有特异性毒性。
这些数据证明了核糖核酸酶酶原作为生物前药的实用性。
