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The His41 site of 3CLpro is a new target for designing broad-spectrum antiviral agents against coronaviruses via a non-covalent pan-binding mode

This study identifies the conserved His41 site of coronavirus 3CLpro as a novel non-covalent pan-binding target, leading to the development of the optimized derivative ISL-221, which demonstrates broad-spectrum antiviral efficacy against multiple coronaviruses comparable to Paxlovid.

Original authors: Dongbo Sun, Xiaoxu Xing, Letian Li, Jiongze Cheng, Haixin Liu, Bin Xu, Xin Yin, Jun Wang, Feiyu Zhao, Jiawei Xiao, Ming Liu, Limin Jiang, Hansong Li, Zhen Li, Xinglin Wang, Wei Zhou, Fanbo Shen, Xiaox

Published 2026-07-22

📖 6 min read🧠 Deep dive

CC BY 4.0

Original authors: Dongbo Sun, Xiaoxu Xing, Letian Li, Jiongze Cheng, Haixin Liu, Bin Xu, Xin Yin, Jun Wang, Feiyu Zhao, Jiawei Xiao, Ming Liu, Limin Jiang, Hansong Li, Zhen Li, Xinglin Wang, Wei Zhou, Fanbo Shen, Xiaoxi Yu, Hao Yuan, Yining Song, Qinghe Zhu, Chunqiu Li, Donghua Guo, Hui-Xin Li, Da Shi, Baochao Fan, Jialin Zhang, Bin Li, Shengwang Liu, Li Feng, Yung-Fu Chang, Ningyi Jin, Mingjun Su, Chang Li

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ✨ This is an AI-generated explanation of the paper below. It is not written or endorsed by the authors. For technical accuracy, refer to the original paper. Read full disclaimer

Imagine the world of viruses as a bustling, chaotic city where tiny invaders are constantly trying to break into buildings (our cells) to steal resources and build more copies of themselves. To stop them, scientists usually try to lock the front doors (the virus's entry points) or send in police officers (vaccines) to recognize the invaders' uniforms. But viruses are tricky; they wear disguises that change every day, making it hard for vaccines to keep up. This is why scientists are also looking for a "master key" that can jam the virus's internal machinery no matter what disguise it's wearing.

The machine in question is a tiny pair of scissors inside the virus called 3CLpro. Think of the virus as a long, tangled string of beads that needs to be cut into specific pieces to work. The 3CLpro scissors do the cutting. If you can jam these scissors, the virus can't build itself, and the infection stops. For a long time, scientists tried to jam these scissors by gluing a chemical "wedge" directly onto the cutting blade. But the virus is good at changing the shape of its blade to pop the wedge off. This paper explores a different, smarter strategy: instead of gluing a wedge, what if we find a way to gently but firmly hold the scissors' handle still, so they can't move at all? This is the story of how researchers found a natural compound that does exactly that, and how they built a super-powered version of it to fight a whole family of viruses.

The Search for the "Handle" of the Viral Scissors

Coronaviruses are like shape-shifters. They jump between animals and humans, mutating constantly, which makes them hard to defeat with vaccines alone. Scientists have been hunting for a "broad-spectrum" antiviral—a single drug that can fight many different types of coronaviruses, from the ones that cause the common cold to the ones that cause severe pneumonia.

The target they chose is the 3CL protease (or 3CLpro), the virus's essential pair of scissors. Most drugs try to stop these scissors by sticking a chemical "glue" to the cutting edge (a cysteine atom). But the virus can mutate to change that edge, breaking the glue. This paper suggests a new approach: instead of gluing to the cutting edge, why not grab the handle?

The handle of these viral scissors is a specific part of the protein called His41. It's like the pivot point that makes the scissors work. The researchers discovered that a natural plant compound called Isoliquiritigenin (ISL)—found in licorice and other plants—doesn't glue itself to the scissors. Instead, it floats into the scissors' pocket and locks onto the His41 handle using a special magnetic-like force called π–π stacking.

Think of π–π stacking like two flat, round magnets (or two slices of bread) that naturally want to sit perfectly parallel to each other. The ISL molecule is flat and aromatic (like a slice of bread), and the His41 handle is also flat and aromatic. When they meet, they snap together in a stable, non-covalent embrace. This isn't a permanent glue; it's a strong, reversible hug that stops the scissors from moving.

The Discovery: A Natural Lockpick

The team started by screening 6,000 natural products to see which one could jam the PEDV (a pig coronavirus) scissors. They found ISL. When they tested it, they saw something surprising: ISL bound tightly to the normal scissors, but if they changed the His41 handle (mutated it), the ISL just slid right off. This proved that ISL relies entirely on that specific handle to work.

It wasn't just a one-hit wonder. The researchers tested ISL against nine different coronaviruses from four different families (alpha, beta, gamma, and delta). In every case, ISL latched onto the His41 handle. It successfully stopped the scissors from working and reduced the amount of virus in cells. In piglets infected with a deadly strain of PEDV, giving them ISL before infection saved most of them from dying, and giving it after they got sick also helped them survive.

Leveling Up: From "Good" to "Great" with ISL-221

While ISL was effective, the scientists wanted to make it even better. They realized that the "magnetic hug" (the π–π stacking) between ISL and the His41 handle was the key. They asked: "What if we make the ISL molecule slightly more magnetic?"

They designed a new version called ISL-221. By adding a tiny electron-donating group to the molecule, they strengthened the π–π stacking interaction. Imagine making the magnets slightly stronger so they snap together even tighter.

The results were impressive. ISL-221 bound to the scissors 23 times tighter than the original ISL. In the lab, it was 11 times more effective at stopping the virus. But the real magic happened in the living animals.

In Piglets: When treated with ISL-221, 83.33% of piglets infected with PEDV survived, compared to 66.67% with the original ISL. For TGEV and PDCoV (two other pig viruses), ISL-221 saved 100% of the treated piglets.
In Mice: They tested ISL-221 against SARS-CoV-2 (the virus that causes COVID-19). The drug reduced the amount of virus in the mice's lungs to 1.65 ± 0.68 Log10 copies/g. This is a huge drop, and the paper notes that its performance was comparable to Paxlovid, a leading clinical drug.

Why This Matters

The paper argues that targeting the His41 handle with a non-covalent "hug" is a brilliant strategy for two reasons:

It's Hard to Cheat: The His41 handle is so essential to the scissors' function that the virus can't easily change it without breaking its own tools. This makes it harder for the virus to develop resistance.
It's Broad: Because this handle is almost identical across all four families of coronaviruses, a drug designed to hug it can fight a wide range of threats, from pig viruses to human pandemics.

The researchers didn't just guess this would work; they used computer simulations to watch the molecules dance, confirmed the binding with high-tech sensors, and proved it in living animals. While ISL-221 isn't a finished medicine yet (it still needs more safety testing), this study lights a bright path forward. It shows that by understanding the tiny, conserved "handles" of viral machinery, we can design smart, broad-spectrum shields that might just be the key to stopping the next coronavirus outbreak before it starts.

Technical Summary: Targeting the Conserved His41 Site of Coronavirus 3CLpro via Non-Covalent $\pi$ – $\pi$ Stacking

Problem Statement
Coronaviruses (CoVs) pose a persistent threat to global public health and animal agriculture due to their high mutation rates, recombination capabilities, and broad host range across $\alpha$ -, $\beta$ -, $\gamma$ -, and $\delta$ -genera. While vaccines are central to control, their antigen specificity often fails to keep pace with viral evolution. Current antiviral strategies face significant limitations: covalent inhibitors targeting the catalytic cysteine of the main protease (3CLpro) may suffer from off-target reactivity, pharmacokinetic issues, and variable efficacy across divergent CoV strains; monoclonal antibodies targeting the Spike protein are highly susceptible to mutational escape; and traditional broad-spectrum nucleoside analogs often lack specificity, leading to cytotoxicity. There is an urgent need for broad-spectrum antivirals that target highly conserved viral functions to mitigate the risk of emerging and re-emerging threats.

Methodology
The study employed a multi-disciplinary approach combining structure-based virtual screening, biophysical characterization, computational modeling, and in vivo validation:

Screening and Identification: A library of 6,000 natural products was virtually screened against PEDV 3CLpro. Isoliquiritigenin (ISL), a chalcone flavonoid, was identified as a high-ranking candidate.
Binding Mechanism Elucidation:
- Mutagenesis & Binding Kinetics: Bio-layer interferometry (BLI) was used to assess binding affinity of ISL against wild-type (WT) and mutant (H41A, C144A, L164A, D186A) 3CLpro enzymes from various CoVs.
- Covalent vs. Non-covalent: LC–MS and UV–visible spectroscopy were employed to determine if ISL formed covalent adducts or induced spectral shifts indicative of $\pi$ -electron system perturbation.
- Molecular Dynamics (MD): All-atom MD simulations (10 ns) and interaction fingerprint analysis were conducted to characterize the stability and specific interactions (e.g., $\pi$ – $\pi$ stacking) between ISL and the His41 residue.
Structure-Guided Optimization: Based on the identified $\pi$ – $\pi$ stacking mechanism, electron-donating groups were introduced to the ISL B-ring to enhance interaction with His41, yielding derivative ISL-221.
In Vitro and In Vivo Validation:
- Enzymatic & Antiviral Assays: FRET-based assays measured 3CLpro inhibition ( $IC_{50}$ ), and TCID $_{50}$ assays quantified viral load reduction in cell lines (Vero, ST, HRT-18) for six CoVs (PEDV, TGEV, BCoV, IBV, PDCoV, SARS-CoV-2).
- Animal Models: Prophylactic and therapeutic efficacy were evaluated in lethal piglet models (PEDV, TGEV, PDCoV) and a SARS-CoV-2 mouse model. Pharmacokinetic (PK) profiles were established in piglets.

Key Contributions and Results

Discovery of a Non-Covalent Pan-Binding Mode: The study identified that ISL binds to the 3CLpro of six representative coronaviruses (spanning four genera) via a non-covalent mechanism. Crucially, this binding is strictly dependent on the conserved His41 residue; the H41A mutation abolished binding ( $K_D$ shifted from $\sim 10^{-7}$ M to $> 10^{-3}$ M), whereas mutations at other catalytic pocket residues (Cys144, Leu164, Asp186) had minimal impact.
Mechanism of Action: ISL engages His41 through a $\pi$ – $\pi$ stacking interaction between its conjugated scaffold and the imidazole ring of His41. This was confirmed by UV–visible spectral shifts and MD simulations showing persistent $\pi$ – $\pi$ stacking distances (4.12–4.25 Å). This mode allows for competitive inhibition of the protease without irreversible covalent modification.
Rational Optimization to ISL-221: By reinforcing the $\pi$ – $\pi$ stacking interaction through structural modification, the derivative ISL-221 was developed. ISL-221 demonstrated a $\sim$ 23-fold improvement in affinity for PEDV 3CLpro ( $K_D = 2.18 \times 10^{-8}$ M) compared to parent ISL.
Broad-Spectrum Potency:
- In Vitro: ISL-221 exhibited enhanced inhibition of 3CLpro and viral replication across $\alpha$ -, $\beta$ -, $\gamma$ -, and $\delta$ -CoVs. Notably, it showed 78.5-fold and 74.1-fold greater potency against SARS-CoV-2 Beta and Omicron variants, respectively, compared to ISL.
- In Vivo: In piglet models, ISL-221 provided 83.33%, 100%, and 100% protection against lethal PEDV, TGEV, and PDCoV infections, respectively, significantly reducing intestinal viral loads and pathology. In the SARS-CoV-2 mouse model, ISL-221 reduced lung viral loads to $1.65 \pm 0.68$ Log $_{10}$ copies/g, achieving efficacy comparable to Paxlovid, while demonstrating favorable pharmacokinetic properties (oral bioavailability of 119.34% relative to ISL).

Significance
The paper establishes the highly conserved His41 site within the 3CLpro catalytic dyad as a tractable target for broad-spectrum antiviral design. It challenges the prevailing reliance on covalent cysteine-targeting strategies by demonstrating that non-covalent $\pi$ – $\pi$ stacking with His41 can yield potent, pan-coronavirus inhibitors. The study validates a mechanism-guided optimization strategy where reinforcing this specific interaction leads to significant gains in affinity and antiviral efficacy. By targeting a residue essential for catalysis and structural integrity, the approach potentially offers a higher genetic barrier to resistance. The findings support the development of ISL-221 as a lead scaffold for orally available, broad-spectrum therapeutics capable of addressing both current and future coronavirus threats across human and animal populations.

The Search for the "Handle" of the Viral Scissors

The Discovery: A Natural Lockpick

Leveling Up: From "Good" to "Great" with ISL-221

Why This Matters

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