key: cord-0866734-0kkaosbd
authors: Yang, Kai S.; Alex Kuo, Syuan-Ting; Blankenship, Lauren R.; Geng, Zhi Zachary; Li, Shuhua G.; Russell, David H.; Yan, Xin; Xu, Shiqing; Liu, Wenshe Ray
title: Repurposing Halicin as a Potent Covalent Inhibitor for the SARS-CoV-2 Main Protease
date: 2022-04-22
journal: Current Research in Chemical Biology
DOI: 10.1016/j.crchbi.2022.100025
sha: 880dffb64897135f5236d5f3a0cac3eba49468ba
doc_id: 866734
cord_uid: 0kkaosbd

The rapid spread of COVID-19 has caused a worldwide public health crisis. For prompt and effective development of antivirals for SARS-CoV-2, the pathogen of COVID-19, drug repurposing has been broadly conducted by targeting the main protease (MPro), a key enzyme responsible for the replication of virus inside the host. In this study, we evaluate the inhibition potency of a nitrothiazole-containing drug, halicin, and reveal its reaction and interaction mechanism with MPro. The in vitro potency test shows that halicin inhibits the activity of MPro an IC50 of 181.7 nM. Native mass spectrometry and X-ray crystallography studies clearly indicate that the nitrothiazole fragment of halicin covalently binds to the catalytic cysteine C145 of MPro. Interaction and conformational changes inside the active site of MPro suggest a favorable nucleophilic aromatic substitution reaction mechanism between MPro C145 and halicin, explaining the high inhibition potency of halicin towards MPro.

The ongoing pandemic of coronavirus disease 2019 has led to catastrophic impacts on the whole world. Its scale and duration have surpassed the 1918 influenza pandemic [1, 2] . As of Feb 1st, 2022, the total number of confirmed global COVID-19 cases is about 376 million, of which 5.6 million have succumbed to death (WHO data) [3] . The current available vaccines and drugs against COVID-19 have led to lots of exciting innovations [4] [5] [6] [7] . However, new SARS-CoV-2 variants that display attenuated responses or complete evasion of vaccine protection and developed drug resistant strains have been continuously emerging [8] . In the context of the disastrous damage of COVID-19 to public health, civil society and the global economy, the search for effective drugs against new strains of SARS-CoV-2 is still in urgent demand.

Given the rapid spread and high fatality of COVID-19, drug repurposing stands out as an attractive and quick access to effective antivirals. If an approved drug could be identified to treat COVID-19, it can promptly proceed to clinical trials and GMP manufacture.

Previously, encouraging results that show antiviral activity against SARS-CoV-2 were obtained from repurposing small molecule medicines including remdesivir, ritonavir/lopinavir, bepridil and nitazoxanide [9] [10] [11] . Nitazoxanide was developed as an antiparasitic agent especially against Cryptosporidium spp. A later in vitro assessment of nitazoxanide has confirmed its promising activity against SARS-CoV-2 with an EC50 value as 2.12 μM [12] . Several clinical trials that are evaluating the use of nitazoxanide for the treatment of COVID-19 are currently underway or in development. Nitazoxanide is a nitrothiazole-containing compound ( Figure 1A ). Another notable compound that belongs to the same group is halicin ( Figure 1A) . Halicin (formerly known as SU3327) is an inhibitor of the enzyme c-Jun N-terminal kinase (JNK) which regulates important cellular activities including cell proliferation, differentiation, and apoptosis. It was first predicted by molecular modeling that halicin binds at the active site of JNK with its nitrothiazole group and blocks the access of JNK to its substrates. Further mice studies also showed the ability of halicin to significantly reduce blood glucose levels and restore insulin sensitivity in a type-2 diabetes mouse model [13] . More recently, driven by a deep neural network assisted drug prediction, halicin was discovered to play a role against a wide phylogenetic spectrum of pathogens. This research suggested that halicin associates to the bacterial J o u r n a l P r e -p r o o f membrane by binding to the iron ion, and dissipates the ΔpH component of the proton motive force [14] . However, its capability to inhibit SARS-CoV-2 has not been studied yet.

In order to assess the antiviral activity of halicin against SARS-CoV-2, a specific target from SARS-CoV-2 needs to be chosen. A quick molecular docking of halicin against main protease (M Pro ), one of the promising drug targets of SARS-CoV-2, shows that halicin could well fit into the active site of M Pro with a reasonable binding energy as -6.1 kcal/mol ( Figure S1 ). This interesting result encourage us to further explore the inhibition of halicin on M Pro . M Pro has been widely targeted to conduct drug repurposing for SARS-CoV-2 because of its essential role in virus replication and pathogenesis [10] . It is a peptide fragment of pp1a and pp1ab, two translation products from the SARS-CoV-2 RNA genome after the virus infects human cells [15] . Both pp1a and pp1ab are very large polypeptides that need to undergo proteolytic hydrolysis to form a number of nonstructural proteins (Nsps) which are essential for the virus to replicate its genome in host cells, evade from the host immune system, and package new virions for infection of new host cells [15] . M Pro processes 13 out of the total 16 Nsps. The crystal structure of M Pro was resolved recently [16] . The structure shows M Pro comprising of three domains, among which the first two have an antiparallel β-barrel structure, and the third has five α-helices that form an antiparallel conglomerate and connect to the first two domains by a long loop region. In its active site it has a C145-H41 catalytic dyad with the substrate-binding site located between domains [17] . M Pro exits as a homodimer in solution, and the dimer form is highly active compared to the monomer form [18] . Therefore, small-molecule medicines that specifically target SARS-CoV-2 M Pro should be potentially effective treatment options for COVID-19 [19] . Inspired by the above ideas and also taking advantage of previous drug repurposing efforts on M Pro of SARS-CoV-2 in our lab [10] , we herein aim to assess the potency of halicin against M Pro of SARS-CoV-2 and identify the inhibition mechanism between them.

In this article, in vitro potency analysis was conducted and shows that halicin has a potent 

The potency assessment of halicin on M Pro started by testing its IC50 in vitro and using nitazoxanide as a comparison. First, the compounds were incubated with 50 nM M Pro at 37 °C for 30 min. Then the reaction was initiated by adding 100 µM of Sub3, a fluorescent substrate of M Pro [10] . The assay was monitored by a plate reader with Ex336/Em455 for 30 min. The first 10 min was fitted with linear regression by GraphPad Prism. The initial slope value was used as normalized activity. As shown in Figure 2 , halicin has a determined IC50 value as 181.7 nM, while nitazoxanide with the concentration up to 200 µM did not show any inhibition of M Pro activity. This IC50 value of halicin is lower than most of the noncovalent inhibitors of M Pro [10] . Considering the relatively large active pocket size of M Pro , halicin is not supposed to be big enough to fully occupy the active site to achieve this level of potency as a noncovalent inhibitor. Based on the above assumption, halicin was proposed to be a covalent inhibitor for M Pro .

In order to verify the covalent binding assumption for halicin, a sample of M Pro incubated with halicin was subjected to native mass spectrometry analysis. Interestingly, binding between M Pro and halicin was observed as shown in the native mass spectra ( Figure   3 ). The M Pro -halicin complex was identified on the basis of the measured and calculated mass shifts. Notably, Figure 3A shows that both the monomer and the dimer of the main protease can bind to halicin. The binding patterns were distinct from a reported inhibitor, GC376, which only binds to the dimeric form of M Pro [20] , suggesting the N-finger may not be essential for halicin to bind with the main protease. A mass shift of 127 Da was identified in the monomeric region ( Figure 3B ), corresponding to only half of halicin with 1 Da deviation (Table 1, Figure S2 ). In the dimeric region, the M Pro was found to bind to J o u r n a l P r e -p r o o f two half halicin fragments, resulting in a 256 Da mass shift ( Figure 3C ). These results indicate that halicin can bind to M Pro in both catalytically inert monomeric and active dimeric forms via a mode of action of covalent binding.

To further characterize the interactions between M Pro and halicin, we crystallized M Pro in its apo form, soaked apo-M Pro crystals with halicin, and then determined structures of M Pro bound with halicin using X-ray crystallography. The complex structure was determined to a resolution of 1.85 Å with an R/Rfree value of 24.9/27.2 (Table S2) . 2Fo-Fc electron density map around C145, C156 and C300 clearly indicates a ligand fragment covalently bound to these three cysteines respectively ( Figure 4A ). Ligand building inside the 2Fo-Fc electron density map shows that the nitrothiazole fragment of halicin fits perfectly. This is consistent with the mass spectrometry analysis that showed the addition of 127 Da to the monomeric M Pro . In the M Pro -halicin crystal structure, three nitrothiazole fragments rather than one were observed on an M Pro monomer. A possible reason for this result is the high concentration of halicin that was used to soak apo-M Pro crystals and the exposure of C156 and C300 of M Pro . Since the active site C145 is the most active one and the only one responsible for the activity of M Pro [16] , only interactions around C145 will be further discussed.

From the surface binding model ( Figure 4B ), we observed a nitrothiazole fragment of halicin forms a covalent bond with C145 and fits into the area between P1 and P2 subpockets. The shortest distance from the thiazole ring of nitrothiazole to the imidazole ring of H41 was measured as 3.4 Å ( Figure 4C ). This indicated a Van der Waals interaction between nitrothiazole and the side chain of H41. Interestingly, a comparison between structure of nitrothiazole-bound M Pro and apo-M Pro indicates that the side chain of H41 flips 90º to allow nitrothiazole to fit into the binding pocket, and the Van der Waals interaction further stabilizes the binding of nitrothiazole to M Pro ( Figure S3 ). Structure superposition of nitrothiazole-bound M Pro and apo-M Pro also shows an unfolding of α-helix in the P2 subpocket, which could be another adaptation of the active site pocket to the binding of nitrothiazole ( Figure S3 ).

The native mass spectrometry analysis that detected halicin-bound M Pro and X-ray crystallography analysis of the M Pro -halicin complex provide solid evidence to support the mode of action of covalent binding. A possible mechanism is shown in Figure 1B 

Researchers have predicted that additional coronavirus diseases may emerge with higher frequencies. For both combating the current pandemic and preparing to contain future coronavirus disease outbreaks, it is imperative to discover antivirals that can be applied generally to inhibit coronaviruses, and drug repurposing turns out to be an efficient way to achieve. Due to its conserveness among coronaviruses, M Pro is an attractive drug target for broad-spectrum antivirals. Inspired by recent drug repurposing efforts on SARS-CoV-2 with nitazoxanide, we identified another nitrothiazole-containing drug, halicin, to successfully inhibit the activity of M Pro in vitro. Our mass spectrometry analysis indicated that halicin binds covalently to M Pro with a nitrothiazole fragment at a 1:1 ratio. A The first 0-300 seconds were analyzed by linear regression for initial slope analyses. Then, the initial slopes were normalized and IC50 values were determined by inhibitor vs response -Variable slope (four parameters).

The main protease was desalted into a 200 mM ammonium acetate using a Bio-Spin column (BIO-RAD) with 6k cut-off. After desalting, the main protease was mixed with halicin. The final concentration of protein and halicin was 2 µM and 10 µM, respectively.

The protein/halicin solution was incubated for 15 minutes at room temperature prior to mass spectrometry analysis. Native mass spectrometry (nMS) analysis was performed on a ThermoFisher Q-Exactive Plus UHMR with spray voltage set to 1.0-1.3 kV. Desolvation and removal of non-specific adducts were performed using a capillary temperature of 120oC, the in-source trapping energy of -10 V, and the HCD cell collision energies of 30

V.

The production of crystals of halicin bound M Pro complexes was following the previous protocols [19] . The data was collected on a Bruker Photon II detector. The diffraction data were indexed, integrated and scaled with PROTEUM3. All the structures were determined by molecular replacement using the structure model of the free enzyme of the SARS-CoV-summarized in Table S2 . All structural figures were generated with PyMOL (https://www.pymol.org).

We followed a previously established procedure strictly to characterize the cellular inhibition potency of halicin. [24] Cells transfected with the pLVX-M Pro -eGFP-2 plasmid were cultured together with different concentrations of halicin as 10, 2, 0.4, 0.08, 0.016, 0.0032 or 0 μM and analyzed by flow cytometry. MPI8 was used as a positive control. [19, 25] The Docking parameters and Methods

In addition to receptor and ligand preparations, AutoDockTools-1.5.7 was also used for J o u r n a l P r e -p r o o f

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This work was supported in part by the Welch Foundation grants A-1715 and A-2089, the Texas A&M X-Grants mechanism and the U.S. National Institutes of Health grants R35GM143047, P41GM128577 and R01GM138863.

The authors declare no competing financial interests.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.