Human immunodeficiency virus (HIV) reverse transcriptase (RT) contains two distinct functional domains: a DNA polymerase (pol) domain and a ribonuclease H (RNase H) domain, both of which are required for viral genome replication. Over the last 3 decades, RT has been at the forefront of HIV drug discovery efforts with numerous nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) approved by the FDA. However, all these RT inhibitors target only the pol function, and inhibitors of RT-associated RNase H have yet to enter the development pipeline, which in itself manifests both the opportunity and challenges of targeting RNase H: if developed, RT RNase H inhibitors would represent a mechanistically novel class of HIV drugs that can be particularly valuable in treating HIV strains resistant to current drugs. The challenges include (1) the difficulty in selectively targeting RT RNase H over RT pol due to their close interplay both spatially and temporally and over HIV-1 integrase strand transfer (INST) activity because of their active site similarities; (2) to a larger extent, the inability of active site inhibitors to confer significant antiviral effect, presumably due to a steep substrate barrier by which the pre-existing substrate prevents access of small molecules to the active site. As a result, previously reported RT RNase H inhibitors typically lacked target specificity and significant antiviral potency. Achieving meaningful antiviral activity via active site targeting likely entails selective and ultrapotent RNase H inhibition to allow small molecules to cut into the dominance of substrates. Based on a pharmacophore model informed by prior work, we designed and redesigned a few metal-chelating chemotypes, such as 2-hydroxyisoquinolinedione (HID), hydroxypyridonecarboxylic acid (HPCA), 3-hydroxypyrimidine-2,4-dione (HPD), and N-hydroxythienopyrimidine-2,4-dione (HTPD). Analogues of these chemotypes generally exhibited improved potency and selectivity inhibiting RT RNase H over the best previous compounds and further validated the pharmacophore model. Extended structure-activity relationship (SAR) on the HPD inhibitor type by mainly altering the linkage generated a few subtypes showing exceptional potency (single-digit nanomolar) and excellent selectivity over the inhibition of RT pol and INST. In parallel, a structure-based approach also allowed us to design a unique double-winged HPD subtype to potently and selectively inhibit RT RNase H and effectively compete against the RNA/DNA substrate. Significantly, all potent HPD subtypes consistently inhibited HIV-1 in the cell culture, suggesting that carefully designed active site RNase H inhibitors with ultrapotency could partially overcome the barrier to antiviral phenotype. Overall, in addition to identifying our own inhibitor types, our medicinal chemistry efforts demonstrated the value of pharmacophore and structure-based approaches in designing active side-directed RNase H inhibitors and could provide a viable path to validating RNase H as a novel antiviral target.

中文翻译：

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切入底物主导地位：抑制人类免疫缺陷病毒逆转录酶相关的核糖核酸酶H的药理和基于结构的方法。

人类免疫缺陷病毒（HIV）逆转录酶（RT）包含两个不同的功能域：DNA聚合酶（pol）域和核糖核酸酶H（RNase H）域，这两个都是病毒基因组复制所必需的。在过去的30年中，RT已成为FDA批准的众多核苷类逆转录酶抑制剂（NRTIs）和非核苷类逆转录酶抑制剂（NNRTIs）的HIV药物开发工作的最前沿。但是，所有这些RT抑制剂仅靶向pol功能，与RT相关的RNase H的抑制剂尚未进入开发流程，这本身就表明了靶向RNase H的机遇和挑战：如果开发出来，RT RNase H抑制剂将代表一类机制新颖的HIV药物，在治疗对当前药物具有抗药性的HIV毒株中可能特别有价值。面临的挑战包括：（1）由于它们在空间和时间上的紧密相互作用以及在HIV-1整合酶链转移（INST）活性方面，由于它们的活性位点相似，因此难以选择性地将RT RNase H靶向于RT pol之上；（2）在更大程度上，活性位点抑制剂不能赋予显着的抗病毒作用，这大概是由于陡峭的底物屏障，通过该屏障先前存在的底物阻止了小分子进入活性位点。结果，先前报道的RT RNase H抑制剂通常缺乏靶标特异性和显着的抗病毒效力。通过活性位点靶向实现有意义的抗病毒活性可能需要选择性和超强的RNase H抑制作用，以允许小分子切入底物的优势。根据先前工作提供的药效团模型，我们设计和重新设计了一些金属螯合化学型，例如2-羟基异喹啉二酮（HID），羟基吡啶酮羧酸（HPCA），3-羟基嘧啶-2,4-二酮（HPD）和N-羟基硫代嘧啶-2,4-二酮（HTPD）。这些化学型的类似物通常表现出比以前最好的化合物更高的抑制RT RNase H的效能和选择性，并进一步验证了药效团模型。通过主要改变键合，在HPD抑制剂类型上扩展的结构-活性关系（SAR）产生了一些亚型，这些亚型显示出超强的效力（单位数纳摩尔），并且在抑制RT pol和INST方面具有出色的选择性。同时，基于结构的方法还允许我们设计独特的双翼HPD亚型，以有效和选择性地抑制RT RNase H并有效对抗RNA / DNA底物。重要的是，所有有效的HPD亚型在细胞培养物中均能持续抑制HIV-1，这表明精心设计的具有超强活性的活性位点RNase H抑制剂可以部分克服抗病毒表型的障碍。总体而言，除了确定我们自己的抑制剂类型外，