发现一种新型非核苷类抑制剂，可抑制依赖RNA的RNA聚合酶，用于对抗登革病毒

《MedComm》：Discovery of a Novel Non-Nucleoside Inhibitor of RNA-Dependent RNA Polymerase Against Dengue Virus

PN-1抑制登革病毒复制的关键机制研究。研究发现天然小分子PN-1通过共价结合DENV RdRp的E255、M387、E479和A507关键残基，诱导酶构象变化并抑制RNA合成，显著降低病毒载量及病理损伤，在ICR幼鼠和AG129小鼠模型中均显示高效低毒特性。

　　
Dengue virus (DENV) remains a critical global health threat due to its increasing incidence and complex clinical manifestations. Current therapeutic strategies face significant challenges, including limited availability of effective antivirals, the threat of drug resistance, and the need for broad-spectrum coverage against multiple DENV serotypes. This study highlights a novel approach to addressing these challenges through the identification and characterization of a natural compound, 12β-hydroxydammar-3-one-20(S)-O-β-D-glucopyranoside (PN-1), which selectively inhibits the RNA-dependent RNA polymerase (RdRp) of DENV.

### Key Findings and Mechanistic Insights
1. **Discovery of PN-1 via Targeted Screening**
The study employed a surface plasmon resonance (SPR) assay to screen natural products for RdRp interactions. Among 14 dammarane-type triterpenoid saponins isolated from *Panax notoginseng*, PN-1 emerged as a strong candidate with a dissociation constant (*K_D*) of 1.42 nM, indicating high binding affinity. This compound effectively suppressed RdRp enzymatic activity at an IC₅₀ of 6.10 μM, establishing its potential as a non-nucleoside RdRp inhibitor (NNI).

2. **Selective Targeting of RdRp Domain**
生化分析证实PN-1通过共价修饰RdRp上的关键氨基酸残基（E255, M387, E479, A507）实现特异性结合。分子动力学研究显示，PN-1诱导RdRp构象变化，尤其是手掌和拇指亚基的构象重组，导致酶活性显著下降。通过突变体实验验证，这些残基的替换完全或部分削弱了PN-1与RdRp的结合能力，证实其靶向特异性。

3. **Antiviral Efficacy in vitro and in vivo**
PN-1 demonstrated broad-spectrum antiviral activity against DENV-1, DENV-2, and DENV-3 strains in multiple cell lines (BHK-21, Huh7, HepG2). Early post-infection treatment (1 hpi) showed optimal efficacy, with PN-1 reducing viral RNA copies and protein expression (E and NS1) by >90% at 10 μM concentration. In animal models, PN-1 protected ICR suckling mice from fatal neurological complications and AG129 mice from systemic infection-induced organ damage (liver, colon, small intestine). The mice treated with PN-1 exhibited restored body weight, reduced clinical scores, and prolonged survival rates compared to controls.

4. **Mechanistic Advantages Over Existing Inhibitors**
Unlike nucleoside analogs (NIs) that face competition with host NTPs and toxic side effects, PN-1 acts as an allosteric modulator. This mechanism reduces the risk of off-target effects and drug resistance. Additionally, PN-1's structural basis (targeting conserved residues in RdRp) provides a template for rational drug design. The compound's ability to stabilize RdRp thermally and resist proteolytic degradation further underscores its unique interaction profile.

### Clinical and Therapeutic Implications
- **Serotype Agnosticism**: PN-1's efficacy across multiple DENV serotypes suggests its potential as a pan-serotype therapeutic. This property addresses a major limitation of current vaccines (e.g., CYD-TDV and TAK-003), which exhibit uneven protection against serotypes.
- **Low Toxicity Profile**: Cytotoxicity assays confirmed that PN-1 at ≤10 μM concentrations did not impair cell viability, unlike repurposed agents such as balapiravir, which failed in clinical trials due to insufficient efficacy and toxicity.
- **Multi-Organ Protection**: In AG129 mice, PN-1 mitigated liver steatosis, intestinal villus atrophy, and colonic hyperproliferation, highlighting its systemic protective effects. These findings align with recent advances in antiviral drug development, where targeting conserved viral components offers dual benefits of efficacy and safety.

### Future Directions and Challenges
While PN-1 shows promise, critical questions remain:
1. **Cross-Virus Potency**: Although RdRp is conserved across flaviviruses (e.g., HCV, Zika), testing PN-1 against these pathogens is necessary to assess its broad applicability.
2. **Resistance Monitoring**: The compound's binding to conserved residues raises concerns about potential resistance. Further mutagenesis studies and structural analysis of drug-resistant RdRp variants are required.
3. **Pharmacokinetic Optimization**: Current in vivo studies use high doses (1–4 mg/kg) for prophylaxis, which may reflect poor oral bioavailability. Research into formulation enhancements (e.g., sustained-release nanoparticles) could improve therapeutic indices.
4. **Host-Mediated Effects**: Although RdRp is host-free, PN-1 may indirectly influence host pathways via nuclear localization signal (NLS) interactions. Exploratory studies using NLS-mutated NS5 constructs are warranted.

### Conclusion
PN-1 represents a paradigm shift in DENV therapeutics by combining natural product origins, allosteric mechanism, and broad serotype coverage. Its ability to inhibit RdRp at non-toxic doses while preserving host safety offers a promising alternative to current failing strategies. The study underscores the value of integrating chemical genetics with structural biology in antiviral drug discovery, particularly for RNA viruses with high mutation rates. As the first clinical-stage NNI targeting DENV RdRp, PN-1 could serve as a lead compound for developing next-generation antivirals with enhanced durability against resistance and reduced toxicity. Collaboration between natural product chemists and virologists will be essential to advance this candidate through preclinical and clinical phases, ultimately addressing the unmet need for safe, effective DENV treatments.