Malonic Acid-Type Fullerene Derivatives Strongly Inhibit The SARS-Cov-2 Main Protease

COVID-19 is a disease that is causing a global pandemic. There is an urgent need to develop new drugs to treat it. In this study, we evaluated the inhibitory activities of a series of fullerene derivatives against the main protease of SARS-CoV-2, the virus that causes COVID-19. As a result, it was found that the malonic acid-type fullerene derivatives showed strong inhibitory activities.

Introduction COVID-19 (coronavirus disease 2019) is an acute respiratory disease caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). It is generally believed that the rst outbreak of COVID-19 occurred in Wuhan City, Hubei Province, People's Republic of China, in December 2019, [1] and the infection has spread all over the world. This pandemic is still ongoing, and at the time of writing this article (6:18 PM CET, 4 January 2022), the number of infected people worldwide exceeds 290,959,019, with 5,446,753 deaths. [2] Patients with COVID-19 have suffered a wide spectrum of symptoms ranging from asymptomatic/mild symptoms to severe pneumonia and death. [3] SARS-CoV-2 is a positive-sense single-stranded RNA virus, and its genome length is 29.881 kb (GenBank No. MN908947), encoding 4 structural proteins, 16 nonstructural proteins (nsp1-nsp16), and accessory proteins. The structural proteins include the spike (S) protein, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein. The N protein binds to viral genomic RNA and forms a nucleocapsid that is enclosed by a lipid membrane known as the viral envelope containing S, E, and M proteins. The S protein plays an important role in viral entry. It mediates cell infection by interacting with a receptor on the host cell surface, angiotensin-converting enzyme 2 (ACE2). [4] The E and M proteins are necessary for virus assembly and budding. Among the 16 nonstructural proteins, nsp3 (papain-like protease, PL pro ), nsp5 (main protease, M pro , or 3-chymotrypsin-like protease, 3CL pro ), nsp12 (RNA-dependent RNA polymerase), nsp13 (helicase), nsp14 (N7-methyltransferase), and nsp16 (2'-O-methyltransferase) act as enzymes. [5] Thus, while there are many potential targets for COVID-19, SARS-CoV-2 M pro plays an important role in the cleavage of viral polyproteins into functional proteins and is recognized as an attractive target for antiviral drugs. SARS-CoV-2 M pro is a cysteine protease with a catalytic dyad comprised of Cys145 and His41. In the hydrolysis reaction of polyprotein, the imidazole group of His41 activates the SH group of Cys145, which acts as a nucleophile. [6] Some inhibitors against SARS-CoV-2 have been reported, and most of them were originally drugs for other targets, [7][8][9] including anti-human immunode ciency virus (HIV) drugs such as lopinavir, ritonavir, darunavir and cobicistat, [8] and anti-hepatitis C virus (HCV) drugs such as boceprevir and narlaprevir. [9] Drugs for the treatment of COVID-19 are being developed by pharmaceutical companies around the world. For example, molnupiravir, developed by Merck, was approved for use in some countries.
Fullerene is a third carbon allotrope represented by soccer-ball-shaped C 60 discovered by Kroto et al. [10] Its spherical and condensed aromatic rings with an extended p-conjugation system are so unique that C 60 has fascinated scientists all over the world. With the aim of pharmaceutical usage, its poor water solubility is a barrier. Therefore, many water-soluble fullerene derivatives have been synthesized by chemical derivatization, and their biological activities have been investigated. [11] We have previously reported inhibitory activities of fullerene derivatives against HIV protease, [12] HIV reverse transcriptase, [12][13][14][15] HCV protease [16] and HCV RNA-dependent RNA polymerase. [13,16] As mentioned above, there have been many reports that anti-HIV and HCV drugs deserve further attention as repurposed drugs for COVID-19. Therefore, fullerene derivatives exhibiting anti-HIV and anti-HCV activities could be drug candidates for the treatment of COVID-19. The aim of the current study was to comprehensively investigate the inhibitory activities of a variety of fullerene derivatives against SARS-

Reagents
The chemical structures of the test compounds used for the present study are shown in Figure 1. We have reported the synthesis of fullerene derivatives 1 to 4 and 8 to 13. [12,[15][16][17][18] Our previous studies con rmed that 1, 2, and 8 are trans isomers and 3 is a cis isomer. Bis-adduct fullerene derivatives such as 7 and 12 are mixtures of several regioisomers. A polyhydroxylated fullerene, which is also known as fullerenol or fullerol, 5 (with 30-50 hydroxy groups) was obtained from SOLARIS CHEM (Vaudreuil-Dorion, Quebec, Canada). Compounds 6 and 7 were synthesized in the same manner as in the literature, [19] with minor modi cations. Boceprevir and malonic acid were purchased from ChemScene (Monmouth Junction, NJ, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively.

SARS-CoV-2 M pro Inhibition Assay
The SARS-CoV-2 M pro inhibitory activities were examined in a similar manner to the HIV protease inhibition assay established by our laboratory, [12] although the incubation conditions were modi ed in accordance with the report by Jang et al., [20] in which the inhibitory activities of polyphenols against M pro were evaluated. Boceprevir, which has already been reported to have M pro inhibitory activity, was used as a positive control. ng/mL M pro . After incubation at 37 ºC for 1 h, 20 mL of 10% tri uoroacetic acid solution was added to stop the reaction. After centrifugation (1,000 rpm, 5 min), 60 mL of the supernatant was transferred to a 96-well plate, and the quantity of the cleaved peptides (SGFRKM) was measured by Agilent 1260 High Performance Liquid Chromatography (HPLC) (Agilent Technologies, Santa Clara, CA, USA) connected to an Agilent 6120 mass spectrometer (Agilent Technologies). Chromatographic separations were performed by an InertSustain®C18 3.0 mm (4.6 × 150 mm) column (GL Sciences, Tokyo, Japan) at a ow rate of 0.50 mL/min under isocratic 15% acetonitrile containing 0.1% formic acid for 10 min. The eluent was introduced directly into the mass spectrometer via electrospray ionization using the positive ion mode.
Among the assessed compounds, malonic acid-type derivatives, 6 and 7, especially exhibited the most potent inhibitory activities. Proline-type derivatives (1-3) moderately inhibited M pro , but their inhibitory activities were obviously weaker than those of the malonic acid-type derivatives. Fullerenol (5) showed no inhibitory activity against M pro at 1.0 mM, indicating that a number of hydroxy groups attached to the fullerene surface lost the inhibitory activity. Compound 4, which has a hydroxymethylcarbonyl moiety, is a transition-state mimic isostere of HIV protease substrate processing and showed potent e cacy against HIV protease in our previous report, [12] and it exhibited weak inhibition against M pro . Given the structural difference between the two proteases, this result might be reasonable. HIV protease is a homodimeric aspartyl protease composed of two identical subunits. The active site is located at the interface between the two monomers and contains catalytic Asp-Thr-Gly. In contrast, M pro is a cysteine protease that has a Cys-His catalytic dyad at its active site. Among the six cationic fullerene derivatives, only 8 and 12 showed moderate activities.
Further experiments with different concentrations con rmed that the half-maximal inhibitory concentrations (IC 50 ) for the proline-type derivative 2 and cationic fullerene derivative 8 in addition to the most potent derivatives 6 and 7 were 0.98, 1.3, 0.53 and 0.20 mM, respectively ( Table 1). The IC 50 of boceprevir, an HCV NS3/4A protease inhibitor that has been reported to exhibit M pro inhibitory activity, was 28 mM in the current experiment, indicating that these fullerene derivatives have stronger activity than the existing drug that has been reported to be active. In particular, derivative 7 showed extremely strong activity with an IC 50 value lower than that of boceprevir by two orders of magnitude. Furthermore, it was con rmed that malonic acid corresponding to the exo-substituent on the most potent derivatives (6 and 7) lost the activity (data not shown). From this result, it was shown that the inhibitory activity against SARS-CoV-2 M pro is dependent on the fullerene backbone, which is similar to our previous report that this is also the case for HCV protease and HIV protease.

Conclusions
In summary, it was found that malonic acid-type fullerene derivative 7 showed strong inhibitory activity against SARS-CoV-2 M pro . Since 7 has been previously reported to be nontoxic to HeLa cells in the range of 64 mM or less [21] and is expected to be applied to pharmaceuticals, 7 may be a potential drug candidate for the treatment of SARS-CoV-2. However, this compound is a bisadduct derivative of fullerene that contains multiple regioisomers, and it will be necessary to isolate each isomer and evaluate individual inhibitory activity to discover a more active compound. Additionally, it was found that there were remarkable differences in inhibitory activities among fullerene derivatives. To elucidate more detailed structure-activity relationships, further investigation will be required. Revised the manuscript: Yasuda, Takahashi, Nakamura, Mashino and Ohe. Figure 1