Prediction of potential 3CL pro -targeting anti-SARS-CoV-2 compounds from Chinese medicine

The recent outbreak of coronavirus disease 2019 caused by the new coronavirus, SARS-CoV-2, has become an international emergency. Since there is no effective therapy for the treatment of this disease, drugs or vaccine that can prevent or cure the SARS-CoV-2 infection are urgently needed. The viral 3-chymotrypsin-like cysteine protease (3CL pro ), which plays a key role in the replication of coronavirus, is a potential drug target for the development of anti-SARS-CoV-2 drugs. With the crystal structure of 3CL pro , we performed virtual screening from a small chemical library of a Traditional Chinese Medicine recipe- FuFang Zhenzhu Tiaozhi (FTZ). Five compounds with the best scores were screened and could be considered as potential hit compounds to be investigated further with bioassays for their anti-virus effects.


Introduction
In December 2019, a new coronavirus, SARS-CoV-2 was reported to cause pneumonia illness in Wuhan City, Hubei Province, China. In January 2020, this coronavirus disease 2019 (COVID-19) was confirmed human-to-human transmission and spread in China [1]. The World Health Organization (WHO) has declared this a Public Health Emergency of International Concern (PHEIC) on 31 January 2020. Until now, there are 81,024 confirmed cases reported in mainland China, with 3181 deaths. The WHO reported that there are more than one hundred thousand confirm cases all over the world.
Apart from China, the situation is very serious in Italy, Korea and Iran. Therefore, there is an urgent need to discover new drug or vaccine to prevent or cure the COVID-19.
3CL protease (3CL Pro , also known as M Pro ) is the main protease produced by the coronavirus and plays a key role in the virus' replication. Most of the functional proteins of coronaviruses are encoded by specific genes, which are first translated into polyproteins and then cleave by 3CL Pro into multiple proteins with different activities for virus replication [2][3][4]. 3CL Pro is therefore considered to be a potential drug target against COVID-19.
Traditional Chinese Medicine (TCM) has been used to prevent pestilence in China for thousands of years [5]. In the treatment of SARS, TCM was confirmed to reduce hormone consumption, alleviate fever symptoms and complications effectively [6]. A retrospective analysis of COVID-19 patients newly published in the Lancet showed that 8 of the 41 infected patients had diabetes mellitus, suggesting that patients with diabetes are likely to be highly susceptible to the SARS-CoV-2 [7]. Our research group has focused on the treatment of glycolipid metabolic disease for decades and has developed a prescription called FuFang Zhenzhu Tiaozhi (FTZ) [8], which can effectively regulate glycolipid metabolic disorder. Since the development of a new drug or vaccine to fight SARS-CoV-2 could be time-consuming, screening active compounds from TCM targeting 3CL Pro could be a potential strategy for treating COVID-19. In the present study, we used molecular docking to screen bioactive molecules from FTZ against 3CL Pro and five natural compounds including lithospermic acid B, specnuezhenide, neonuezhenide, rutin and neodiosmin ( Figure 1) were predicted to target 3CL Pro .

Lithospermic acid B
Lithospermic acid B is one of polyhydroxy phenolic acid compounds isolated from Salvia miltiorrhiza (Chinese name: Dan shen). This bioactive compound has many pharmacological activities, such as improving renal function, preventing and curing cardiovascular disease, anti-oxidation, anti-fibrosis, anti-inflammatory and so on [9].  respectively. The 2D ligand interaction diagram ( Figure 3B) shows the predicted interactions between specnuezhenide and the 3CL Pro residues. The binding of specnuezhenide with THR26, GLY143, MET165, GLU166 and GLN189 of 3CL Pro protein are through conventional hydrogen bonds. Carbon hydrogen bonds can be found between THR25/PHE140/LEU141/GLY143/GLU166/GLN189 and specnuezhenide.
In addition, specnuezhenide can also interact with HIS41, CYS145 and HIS172 through hydrophobic interaction. Moreover, van der Waals force is found for specnuezhenide interacting with a few amino acids such as SER46, SER144 and PRO168 around the binding pocket. Since the molecular structure of neonuezhenide is very similar to specnuezhenide, the predicted interactions between neonuezhenide and 3CL Pro residues are comparable with specnuezhenide ( Figure 3C)  (B) The predicted interactions from specnuezhenide and the amino acids residues of 3CL Pro ; (C) The predicted interactions from neonuezhenide and the amino acids residues of 3CL Pro .

Rutin
Rutin is also known as quercetin-3-O-rutinoside and is a flavonol glycoside widely found in plants. It has been found to have antioxidant, anti-inflammatory and anti-free radical effects [12]. Our docking results suggested that rutin also targets 3CL Pro with a CDOCKER interaction energy of -69.38 kcal/mol. The 2D interaction diagram ( Figure   4B) shows that the flavonol groups of rutin formed hydrophobic bonds with Rutin can also form van der Waals interactions with 17 amino acid residues such as THR25, LEU27, TYR54 and THR190. Based on the prediction, we hypothesize that rutin is possibly active to inhibit SARS-CoV-2 through inhibiting 3CL Pro . However, it needs further investigation with in vivo assays.

Neodiosmin
Neodiosmin is a flavonoid glycoside isolated from limes and it can also be produced by the dehydrogenation of neohesperidin. Neodymium is mainly used to inhibit the bitter taste produced by naringin and limononin in the industrial production of orange juice [13]. So far, no report about its pharmacological activity has been found in the  Figure 5B). The results may suggest that neodiosmin may bind to 3CL Pro and possibly affect the activity of SARS-CoV-2.

Summary
Since the SARS-CoV-2 is spreading at a rate and scale much worse than previous coronaviral epidemics, there is an urgent need to find a feasible way to fight against COVID-19. In this study, five compounds including lithospermic acid B,

Methods
Discovery Studio (DS) 2016 was used to perform the molecular modeling study. The X-ray crystal structure of 3CL Pro was downloaded from the PDB database (PDB entry: 6LU7) [14]. This structure is in complex with a SARS-CoV inhibitor, N3. Since the identity of 3CL Pro between SARS-CoV-2 and SARS-CoV is 96%, it is feasible to screening anti-SARS-CoV-2 compounds with N3 binding pocket [4]. The protein and the ligands were prepared for this study using the own tools in DS. And then, the CDocker protocol was used to predict the binding poses with default parameters [15].
The top 3% scoring poses were containing 21 docking poses, and these poses were generated by five compounds. The top-scoring pose for each of the five compounds was selected to visually inspect.