Research Update: In silico screening of Chinese herbal medicines with the potential to directly inhibit 2019 novel coronavirus

1. Introduction

Toward the end of December 2019, a novel coronavirus (2019-nCoV) with human-to-human transmission and severe human infection, originating in Wuhan, China, was identified [1]. This virus has affected many persons in China and spread to other countries in a very short time. On January 30, 2020, the Director-General of the World Health Organization declared that the outbreak of 2019-nCoV constitutes a public health emergency of international concern and issued temporary recommendations under the International Health Regulations [2]. According to the Daily Report of China National Health Commission, as of this writing on February 2, 2020, 14,488 cases, including 304 deaths, have been confirmed in China; 146 cases, including 1 death, have also been reported among 23 other countries. This pandemic is still ongoing, so it is urgent to find new preventive and therapeutic agents as soon as possible. In addition, commensurate with the risk, strong measures for early detection, isolation and treatment of cases, as well as minimization of transmission through social interaction must be implemented.

While specific vaccines and antiviral agents are the most effective methods to prevent and treat viral infection, there are not yet effective treatments that target the 2019-nCoV. Development of these treatments may require months or years, meaning that a more immediate treatment or control mechanism should be found if possible. Herbs used in traditional Chinese medicine present a potentially valuable resource to this end. The effectiveness of herbal treatment to control contagious disease was demonstrated during the 2003 severe acute respiratory syndrome (SARS) outbreak [3]. As such, the Chinese government is encouraging the use of herbal plants in fighting this new viral pneumonia. However, the application of herbal treatment is mainly guided by the type of herb (based on the catalogue of classic literature on herbs) and the patient’s symptoms or signs. There is often not enough information to predetermine whether the herbs in question can directly target the viral cause, in other words, herbal usage is generally not guided by viral pathology. We think more detailed knowledge about the direct antiviral effects of different plants would be greatly helpful to the doctors selecting them.

In fact, after the outbreak of SARS, many groups dedicated themselves to finding anti-coronavirus agents, including some natural compounds that exist in traditional Chinese herbal medicines [4], [5], [6], [7], [8], [9], [10], [11], [12]. The coronavirus encodes more than one dozen proteins, some of which are essential to viral entry and replication. Among these proteins, the most well-studied are papain-like protease (PLpro), 3C-like protease (3CLpro) and spike protein. Coronavirus PLpro not only processes the viral polypeptide onto functional proteins but is also a deubiquitinating enzyme that can dampen host anti-viral response by hijacking the ubiquitin (Ub) system. For example, SARS PLpro cleaves ISG15, a two-domain Ub-like protein, and Lys48-linked polyUb chains, releasing diUbLys48 products [13], [14]. SARS-3CLpro is a cysteine protease indispensable to the viral life cycle [15]. Coronavirus spike protein uses angiotensin-converting enzyme 2 as a receptor to help the virus enter cells [16]. These three proteins make attractive targets for drug development.

Through in silico and biological processing, a series of small molecules, including those from natural compounds, have been screened and confirmed to directly inhibit these important proteins in SARS or Middle East respiratory syndrome (MERS) coronavirus [17], [18], [19], [20], [21], [22], [23]. The gene sequence of 2019-nCoV has been released, which suggests high similarities between the main proteins in this virus and those previously identified in SARS-Cov or MERS-Cov [24], [25]. In this sense, previously reported anti-SARS-Cov or anti-MERS-Cov natural compounds may become a valuable guide to finding anti-coronavirus (2019-nCoV) herbal plants among the traditional Chinese herbs used to treat viral pneumonia.

It is a challenge to screen out the herbs containing anti-coronavirus (2019-nCoV) compounds from the large number of those possibly being used for patients infected with this pathogen, especially in very short time. Here, we propose two principles to guide such work: oral effectiveness and traditional usage compatibility. The first principle refers to the fact that most Chinese herbal plants are orally ingested after boiling with water, meaning that the anti-coronavirus (2019-nCoV) ingredients in selected plants should be absorbable via oral preparation. The second principle recognizes that candidate plants should be consistent with the type classifications for traditional herbal usage, since type-guided applications are integral to herbal use, as mentioned above. Following these two principles, we used a 6-step selection process (3 for each principle), including drug-likeness, evaluation of oral bioavailability, molecular docking, network pharmacology analysis and other methods to identify herbs that have both a high possibility of containing effective anti-coronavirus (2019-nCoV) compounds and are classified as treating virus-caused respiratory infection.

2. Materials and methods

3. Results

3.1. Overlapping of natural compounds biologically confirmed to be anti-SARS or anti-MERS coronavirus in literature and in Chinese herbal database

We received 261 hits from conducting our search in the PubMed database. After careful evaluation of the abstracts from these citations we downloaded and carefully analyzed the full text of 23 highly relevant papers. The natural compounds reported to have biologically confirmed anti-coronavirus activity were identified and then compared to the ingredients listed in three Chinese herbal databases. The result was 115 overlapping ingredients, which we used for further testing (Fig. 1).

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Fig. 1. Workflow scheme. The work is divided into two main parts, natural compound selection and herbal plant selection. Each part consists of three steps. As detailed in the text, oral effectiveness is important in compound selection, while in the plant selection portion, the selected herbs should be compatible with the classic usages of herbal treatment in traditional Chinese medicine. TCMSP: Traditional Chinese Medicine Systems Pharmacology.

3.3. Docking between selected compounds and their reported targets

To perform the docking analysis, the 3D structure files of 2019-nCoV PLpro, 3CLpro and spike proteins were built based on the corresponding SARS-CoV templates, i.e., PDB 5e6j, 1uj1 and 6cad, respectively. Then, molecule-protein docking was carried out between the molecules and their reported targets. If the molecules were reported to inhibit viral entry, they were docked with spike proteins (Table 1). Each separate analysis returned positive results (Table 1, Fig. 2 and online supplementary Fig. S1), indicating the natural compounds we selected might directly inhibit 2019-nCoV. The molecules selected to target PLpro (M2, M3, M7, M9, M10, M11 and M13) mainly bound in the region between the thumb and palm domains, which might interfere with substrate entering this enzyme’s active sites, located at the bottom of the two domains [29]. The molecules reported to inhibit 3CLpro (M1, M2, M3, M4, M5, M7, M8, M10, M11, M12 and M13) mainly entered the region between domains 2 and 3, and this region is important for 3CLpro to form a dimmer [30]. M6 was reported to inhibit viral entry, accordingly it bound the fusion cone of spike protein; this cone structure is important for viral membrane fusion [31] (Fig. 2 and online supplementary Fig. S1).

Table 1. The molecules and their docking proteins, binding energy (kcal/mol).

No.	Molecular name	Targets or inhibition	Reference	Docking (binding energy)
				PLpro	3CLpro	Spike
M1	Betulinic acid	Replication, 3CLpro	[16]	Undo	−4.23	Undo
M2	Coumaroyltyramine	PLpro and 3CLpro	[11], [20]	−3.22	−4.18	Undo
M3	Cryptotanshinone	PLpro and 3CLpro	[18]	−5.25	−6.23	Undo
M4	Desmethoxyreserpine	Replication, 3CLpro, and entry	[6]	Undo	−3.52	Undo
M5	Dihomo-γ-linolenic acid	3CLpro	[7]	Undo	−3.88	Undo
M6	Dihydrotanshinone Ⅰ	Entry, and spike protein	[28]	Undo	Undo	−5.16
M7	Kaempferol	PLpro and 3CLpro	[11]	−2.15	−6.01	Undo
M8	Lignan	Replication, 3CLpro	[16]	Undo	−4.27	Undo
M9	Moupinamide	PLpro	[20]	−3.05	Undo	Undo
M10	N-cis-feruloyltyramine	PLpro and 3CLpro	[11], [20]	−3.11	−4.31	Undo
M11	Quercetin	PLpro and 3CLpro	[20]	−4.62	−6.25	Undo
M12	Sugiol	Replication, 3CLpro	[16]	Undo	−6.04	Undo
M13	Tanshinone IIa	PLpro and 3CLpro	[18]	−5.02	−5.17	Undo

3CLpro: 3C-like protease; PLpro: papain-like protease.

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Fig. 2. Docking between selected natural compounds and their reported targets. A. PLpro and natural compound M2; B. 3CL and M1; C. Spike and M6. Docking is performed with AutoDock 4 which is detailed in Materials and Methods. The protein structure files are listed in Table 1. Protein domains are shown in different colors, while natural compounds are shown in dark yellow. The amino acids labeled were those interacting with compound. 3CLpro: 3C-like protease; PLpro: papain-like protease.

3.5. Network analysis of possible effects or mechanisms

Each of the potentially effective herbal remedies contains many ingredients in addition to the antiviral ones found here. Thus, the general effects of each plant should be examined by combining the effects of all of the orally absorbable and biologically active ingredients in it. To evaluate the possible general in vivo effects of each of our identified herbs, we used the ADME indices listed above to examine each of the orally absorbable and drug-like ingredients recorded in the TCMSP database for each plant. We then extracted the target proteins for each ingredient which had passed the screening process. All proteins belonging to a single plant were combined as input on the online protein–protein interaction analysis server, String, to find the pathway enrichment.

For the 26 herbs, about 1/3 of the top 30 KEGG-enriched pathways (mean = 11) were related to regulating viral infection, immune/inflammatory reactions and hypoxia response, indicating that they are potentially effective treatments for viral respiratory infection (Fig. 3 and online supplementary Fig. S2). Note that some of the herbal plants selected here had been reported to be effective for SARS-CoV infection in 2003 (online supplementary Table S2).

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Fig. 3. Kyoto Encyclopedia of Genes and Genomes pathways enriched for the herb Forsythiae Fructus in network pharmacology analysis. The top 30 pathways are shown. The blue, red and green bars represent the pathways related to viral infection, immune/inflammation response, and hypoxia response, respectively. EGFR: epidermal growth factor receptor; AGE: advanced glycation end product; RAGE: receptor for AGE.

4. Discussion

In this work, we undertook a multiple step selection process and screened out 26 herbal plants with a high probability of directly inhibiting the novel coronavirus (2019-nCoV), possibly providing instant help in the prevention and treatment of the pneumonia that it can cause. While mainly in China at this point, viral spread is ongoing and has affect persons worldwide.

Two principles guided our screening work. The first is that the anti-coronavirus (2019-nCoV) components contained in the source plants should be absorbable via oral prescription. This principle requires that the herbs selected should contain biologically proven anti-coronavirus (2019-nCoV) ingredients, and that these natural compounds should pass the drug-likeness and oral bioavailability evaluations. Therefore, we conducted a three-step screening process. First, we extracted natural compounds verified in PubMed as being effective in treating SARS or MERS coronavirus and then cross-checked these compounds in the Chinese herbal databases. There were 115 overlapping compounds. This method was an expeditious way to identify natural components both pre-existing in Chinese herbal treatment and having a high possibility of anti-coronavirus (2019-nCoV) activity. This is important, as the anti-coronavirus effects of the selected compounds have been biologically confirmed, and the genetic similarities between coronavirus (2019-nCoV) and SARS or MERS coronavirus are high [24], [25].

The anti-coronavirus effects of the natural compounds screened by the above method have been mainly confirmed in vitro by direct loading onto cultured cells, thus it does not guarantee their effectiveness in vivo, especially with oral preparation—the principal way in which Chinese herbals are administered. Therefore, to meet the first principle, we ran ADME filters on the natural compounds selected by 4 indices, as used by Hu et al. [26]. Among the 115 compounds highlighted by our first step, only 13 passed this screening, showing the necessity of such a test.

The novel coronavirus has some mutations when compared to SARS or MERS coronavirus, so the natural compounds effectiveness against the two previous coronaviruses might not be present in the new virus. To reduce this risk, and as the third step of our first principle, we reconstructed the 3D structure of the new coronavirus using the reported structures of SARS and MERS coronavirus proteins as a guide, and then used molecular docking technology to simulate whether the 13 natural compounds selected could combine with the structures we constructed for the new coronavirus proteins. All 13 compounds could bind to the proteins as predicted for the new coronavirus. We believe that the high success rate of our docking screening was due to the high genetic similarity between the new coronavirus and the SARS or MERS virus [24], [25].

Our second principle for screening should also be emphasized and elaborated upon. It states that the selected herbal plants must conform to traditional usages. There are many kinds of Chinese herbs that have been used for thousands of years. Based on this rich history and experience, Chinese herbal medicines are divided into different types, each type dedicated to certain kinds of diseases. Ignoring these grouping guidelines can lead to serious side effects. Therefore, as a further condition for the medicine screened here, we verified that they have been routinely used to treat viral pneumonia. To meet this principle, we conducted another three-step screening process for the herbal plants.

First, we searched the Chinese medicine database for herbs containing the 13 natural compounds identified. Herbs containing at least 2 of these potentially useful compounds were selected, and a total of 125 herbal plants were identified. The second step in targeted plant selection was based on type classification. Of the 125 results, only 26 herbs were found to be routinely used in treating viral respiratory infection.

Finally, network pharmacological analysis was performed to predict the possible therapeutic effects of these 26 plants. Because Chinese herbal medicines contain many ingredients, and multiple absorbable ingredients might exert their effects on the body, the general effects of herbs may be dictated by all of the absorbable ingredients they contain. With this consideration in mind, we extracted the recorded ingredients of each of the plants selected from the Chinese medicine database and screened these ingredients for drug-likeness and oral availability (via ADME filter) [26], [32]. The target proteins of all ingredients passing ADME selection were used for network enrichment to predict the general effects of the herbal plants. For all the plants analyzed, nearly half of the top 30 pathways enriched in KEGG are related to antiviral, immune/inflammatory responses and hypoxia response indicating that these herbs are suitable for anti-viral usage. In fact, some of the herbal plants selected here had been reported effective in against SARS-CoV infection in 2003 (online supplementary Table S2). We thought that the general antiviral and immune/inflammation effects predicted for the 26 plants are correlated with the fact that these plants were selected according to Chinese herbal type classifications.

Of course, it should be pointed out that Chinese herbs that have not been identified through this screening process may still have beneficial effects. Further, considering that the biologically validated natural compounds reported in the literature cannot cover all antiviral natural compounds, and the natural compounds included in the Chinese medicine database are not complete, the process that we have followed may have excluded herbs that would be well suited to this treatment. Nevertheless, the purpose of this screening was to provide a rational approach for selecting Chinese herbal medicines with a high potential efficacy in treating 2019-nCoV and related viruses. The specific dosage and usage of each herb should be determined based on patients’ manifestations. Finally, the key step in this screening was molecular docking. The 3D structures of the proteins used here are based on reported gene sequences. If the virus mutates during transmission, a new screening is recommended.

In conclusion, this work has identified several Chinese medicinal plants classified as antiviral/pneumonia-effective that might directly inhibit the novel coronavirus, 2019-nCoV. Additionally, we propose screening principles and methods which may provide guidance in screening antiviral drugs from other natural drug databases.

Authors’ contributions

DZ conceived the study, participated in its design, coordination, and all the work processes. KW participated in herbal selection. XZ participated in data collection and network pharmacology analysis. SD helped to collect data. BP helped to draft the manuscript.

Funding

This work is supported by Shanghai Leading Talent Grants in Medicine (No. 2019LG26), Shanghai Traditional Chinese Medicine Content Construction Innovation Project (No. ZY3-CCCX-3-7001) and Postdoctoral Funding of Shanghai Gongil Hospital (No. GLBH2017002).