key: cord-0691895-0usy6rix
authors: Devkota, Kanchan; Schapira, Matthieu; Perveen, Sumera; Yazdi, Aliakbar Khalili; Li, Fengling; Chau, Irene; Ghiabi, Pegah; Hajian, Taraneh; Loppnau, Peter; Bolotokova, Albina; Satchell, Karla J.F.; Wang, Ke; Li, Deyao; Liu, Jing; Smil, David; Luo, Minkui; Jin, Jian; Fish, Paul V.; Brown, Peter J.; Vedadi, Masoud
title: Probing the SAM Binding Site of SARS-CoV-2 nsp14 in vitro Using SAM Competitive Inhibitors Guides Developing Selective bi-substrate Inhibitors
date: 2021-02-19
journal: bioRxiv
DOI: 10.1101/2021.02.19.424337
sha: 7fcaa9e0c2f1c52a72099ef14dd841ead7f81928
doc_id: 691895
cord_uid: 0usy6rix

The COVID-19 pandemic has clearly brought the healthcare systems world-wide to a breaking point along with devastating socioeconomic consequences. The SARS-CoV-2 virus which causes the disease uses RNA capping to evade the human immune system. Non-structural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small molecule inhibitors of nsp14 methyltransferase (MT) activity, we developed and employed a radiometric MT assay to screen a library of 161 in house synthesized S-adenosylmethionine (SAM) competitive methyltransferase inhibitors and SAM analogs. Among seven identified screening hits, SS148 inhibited nsp14 MT activity with an IC50 value of 70 ± 6 nM and was selective against 20 human protein lysine methyltransferases indicating significant differences in SAM binding sites. Interestingly, DS0464 with IC50 value of 1.1 ± 0.2 μM showed a bi-substrate competitive inhibitor mechanism of action. Modeling the binding of this compound to nsp14 suggests that the terminal phenyl group extends into the RNA binding site. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein methyltransferases. The structure-activity relationship provided by these compounds should guide the optimization of selective bi-substrate nsp14 inhibitors and may provide a path towards a novel class of antivirals against COVID-19, and possibly other coronaviruses.

(ORFs) 6 , encoding 16 non-structural proteins (nsp) and four main structural and accessory proteins. 7 The 16 non-structural proteins (referred to as nsp1 to nsp16) are more conserved amongst coronaviruses compared to the structural and accessory proteins. 8 These nsps in coronaviruses form a replicase-transcriptase complex and are essential for the transcription and replication of the virus. 9 Among these, nsp14 and nsp16 are RNA methyltransferases involved in RNA capping. 10 Nsp14 is a bi-functional protein with a C-terminal methyltransferase domain catalyzing N7guanosine methylation, and an N-terminal exoribonuclease domain (Supplementary Fig. 1 ). In the replicase-transcriptase complex of coronaviruses, nsp14 functions as an exoribonuclease and is involved in maintaining the fidelity of coronavirus RNA synthesis. 11 Nsp14 in complex with nsp10 can function as a proofreading exoribonuclease and removes 3′-end mismatched nucleotides from dsRNA. 12 Breaking this interaction between nsp10 and nsp14 results in a decrease in virus replication fidelity. 13 Besides nsp10, nsp14 also interacts with the nsp7-nsp8-nsp12 complex where the exonuclease function of nsp14 decreases the incidence of mismatched nucleotides 14 by erasing the mutated nucleotides. 11 While complex formation between nsp10 and nsp14 is required for enhanced exoribonuclease activity, the MT activity of nsp14 is independent of nsp10-nsp14 complex formation. 15, 16 The nsp14 SAM-dependent methyltransferase (MTase) activity is essential for viral mRNA capping. 16, 17 The cap1 structure at the 5′ -end of viral RNA helps in masking the virus from the host immune system. 18, 19 Cap (GpppN) structure in nascent RNA of coronaviruses is formed by nsp13 20,21 and a guanylyltransferase (GTase). Nsp14 methylates this cap structure at the N7 position of the guanosine, forming a cap-O (N7mGpppN). 17 Nsp16 further 2′-O-methylates the product of the nsp14 methyltransferase activity, completing the capping process (N7mGpppNm). 16, 22 Nsp14 is conserved amongst the seven coronaviruses known to infect humans to-date ( Supplementary Fig. 2 ). 23 The SARS-CoV-2 nsp14 overall amino acid sequence shows 95.1, 62.7, 57.8, 58.5, 52.9 and 53.7% identity with nsp14 from SARS-CoV, MERS-CoV, OC43, HKU1, 299E and NL63, respectively. This suggests the possibility of SARS-CoV-2 nsp14

inhibitors also inhibiting nsp14 methyltransferase activity of other coronaviruses. Such pan inhibitors would be priceless for developing pan anti-viral therapeutics for COVID-19 that would be also effective on future coronaviruses which may jump to humans. In this study, we first developed a radiometric high throughput activity assay for SARS-CoV-2 nsp14 methyltransferase activity and screened a library of 161 S-adenosylmethionine (SAM) competitive methyltransferase inhibitors we previously synthesized, and SAM analogs. We identified seven reproducible hits which we mapped on the active site of SARS-CoV-2 nsp14. The data shows for the first time a clear path towards the development of potent and selective bi-substrate nsp14 inhibitors that may lead to a novel class of therapeutics for COVID-19 and possibly other coronaviruses to come.

Developing therapeutics for COVID-19 and other coronaviruses requires reliable high throughput screening assays. Radiometric assays have been widely used for developing potent substrate and SAM competitive inhibitors for human methyltransferases within the last decade. 24 Using biotinylated RNA substrate, a radiometric nsp14 methyltransferase (MTase) activity assay was developed with 3 H-SAM as a methyl donor (Fig. 1) . Nsp14 MTase activity was evaluated in various buffers, pH and additives (Supplementary Fig. 3 ). The highest MTase activity was observed in Tris HCl buffer at pH 7.5. No significant effect was observed for DTT up to 10 mM and it was included in the assay reaction mixture at 5 mM to maintain reducing conditions. Triton X-100 at 0.01% was added to the reaction buffer to minimize binding of proteins and compounds to plates. DMSO at concentrations up to 10% had little effect on nsp14 MTase activity. However, MgCl2 was only tolerated at concentrations below 1 mM (Supplementary Fig. 3) . The assay optimization resulted in selecting 20 mM Tris HCl pH 7.5, 250 µM MgCl2, 5 mM DTT and 0.01 % Triton X-100 for testing nsp14 MTase activity and determining its kinetic parameters.

Using optimized assay conditions, linearity of initial velocities (activity versus time) was assessed at various concentrations of RNA at fixed SAM concentration (1 µM) (Supplementary Fig. 4a Fig 1a) and 52 ± 1 h -1 (SAM ; Fig 1b) , were reasonably close. Addition of nsp10 at various molar ratios from 1 (nsp10):1 (nsp14) to 20 (nsp10):1 (nsp14) did not have any significant effect on nsp14 methyltransferase activity (Supplementary Fig. 5 ).

In small molecule screening campaigns, typically the assays are performed at Km of the substrates to allow potential inhibitors to compete with the substrates and allow their binding to be detected.

However, the activity of the enzyme should be linear during the assay period. As the radiometric methyltransferase assays are endpoint assays, lack of linearity may mask inhibition of some compounds. Testing the activity of nsp14 at 50 µM RNA and 250 nM SAM indicated that the assay can be run for at least 20 minutes while maintaining the linearity (Fig. 1c) . To determine the reproducibility of such conditions for high throughput screening, the assay was performed in the presence (1 µM) and absence of sinefungin, a pan methyltransferase inhibitor that inhibits nsp14

MTase activity with an IC50 value of 0.019 ± 0.01 µM (Supplementary Fig. 6) . A Z′-Factor of 0.69 was calculated for nsp14 screening indicating suitability of the assay for high throughput screening (Fig. 1d) .

An in-house library of 161 SAM competitive methyltransferase inhibitors and SAM analogs was screened against nsp14 at 50 µM, and 19 compounds were identified that inhibited nsp14 MTase activity more than 75% (Fig. 1e) . Compounds that interfered with the readout signal or were not reproducible were eliminated. The remaining seven compounds ( Fig. 2 Supplementary Fig. 7 ).

This limited and focused screening exercise provided important insights on the structural chemistry of SARS CoV-2 nsp14 inhibition ( Table 1) . First, we noted that the de-methylated Table 1 , Fig 3) . Interestingly, compound DS0464, where the amino-acid moiety is replaced with a physico-chemically more favorable phenyl-ethyl-urea, retains significant inhibitory activity (IC50: 1.1 ± 0.2 µM). Since all residues lining SAH and the substrate RNA cap GpppA in the SARS-CoV-1 nsp14 structure are conserved in SARS-CoV-2 ( Supplementary Fig.   8 ), the SARS-CoV-1 structure was used to dock DS0464 (Fig. 4) . The model revealed a possible arrangement where the adenosine end of the inhibitor overlays with the adenosine of the bound cofactor, and the terminal phenyl group recapitulates stacking interactions observed between the guanine ring of the RNA cap and surrounding residues (Y420, F426, F506, N386) ( Fig. 4) . Such a binding mode suggests a mechanism of action where DS0464 behaves as a bi-substrate inhibitor.

This possibility was further tested by performing mechanism of action (MOA) studies with DS0464 which revealed that DS0464 competes against both SAM and RNA and can act as a bifunctional inhibitor (Fig. 5, Supplementary Fig. 9 ).

The selectivity of all seven compounds was tested against the human RNA methyltransferases BCDIN3D, and METTL3-METTL14 complex (METTL3-14), the RNA demethylase ALKBH5, and protein lysine methyltransferases G9a and SETD3 ( Table 1 , Supplementary Fig. 10-14) .

Interestingly, none of the compounds inhibited G9a or ALKBH5 activities indicating some level of selectivity. SS148 and DS0464 potently inhibited BCDIN3D with IC50 values of 0.03 ± 0.002 and 46 ± 9 µM, respectively, but not G9a, SETD3 or ALKBH5 ( Table 1) . G9a and SETD3 are SET domain methyltransferases that are structurally distinct from class I methyltransferases such as nsp14, BCDIN3D or METTL3-METTL14, which could explain the observed specificity profile. 25 For instance, the channel separating the substrate and cofactor binding sites is wide in nsp14 but narrow in G9a. Additionally, a cavity that can accommodate the nitrile group of SS148 in nsp14 and BCDIN3D is absent in G9a, in agreement with the obtained IC50 values (Fig 6) . To further characterize our nsp14 inhibitors, the selectivity of SS148 and DS0464 was evaluated against a larger panel of lysine, arginine, DNA and RNA methyltransferases ( Table 2 , Fig. 7 ). As expected, while SS148 inhibited arginine, DNA and RNA methyltransferases (all class I methyltransferases), it did not inhibit any of the 20 SET domain lysine methyltransferases (Fig. 7 , Table 2 , Supplementary Fig. 15 ). DS0464 was even more selective and inhibited only PRMT4, PRMT5, PRMT7, DOT1L and BCDIN3D, but none of the protein lysine methyltransferases tested.

( Table 2 , Supplementary Fig. 16 ).

The frequent emergence in the last two decades of novel coronaviruses as human pathogens, highlighted by the current COVID-19 pandemic, urgently needs to be addressed, preferably with pan-coronavirus drugs. Nsp14 is an essential methyltransferase in RNA cap formation which is required for protecting viral RNA and proper replication of coronaviruses. Therefore, targeting nsp14 methyltransferase activity would be a viable option towards developing anti-viral therapeutics. 26 Methyltransferases are druggable. 27 In the last decade, a significant number of selective and cell-active small molecules (chemical probes) have been discovered for human methyltransferases 24, 28, 29 and some are in clinical trials for various cancers. 28, 29 Key to a successful discovery campaign of such chemical probes is the availability of reliable screening methods that could enable medium to high throughput screening with low false-positive and false-negative rates. Various assays including mass spectrometry 30-32 , fluorescence 33 , and radiometric assays 24 have been used for screening libraries of compounds. Mass spectrometrybased assays require more expensive instrumentation and expertise. Fluorescence assays can be performed in any lab, however, many fluorescent compounds in chemical libraries may increase the background and lead to high numbers of false positives to triage following screening large libraries. Radiometric assays are typically more reliable and have fewer false positives leading to identifying more reliable screening hits. 24 In this study, we have developed a radiometric assay for nsp14 and employed this assay for screening a small library of selected SAM competitive inhibitors and analogs.

Targeting the SAM binding site has successfully led to the discovery of chemical probes for human methyltransferases such as DOT1L 34, 35 , EZH2/EZH1 36 , and SMYD2 37 . SS148 (nsp14 IC50: 70 ± 6 nM) was reported as a DOT1L inhibitor with a nitrile as a non-traditional replacement for heavy halogen atoms. 38 This is consistent with the selectivity of SS148 against all other protein lysine methyltransferases (PKMTs) due to narrower active sites that could not fit the added nitrile group ( Fig. 4) . Keeping this substitution in designing future nsp14 inhibitors will provide selectivity against PKMTs. WZ16 39 

In this study, we developed a radiometric assay for nsp14 methyltransferase activity, determined the kinetic parameters and optimized the assay for high throughput screening. Through limited screening of SAM competitive inhibitors, we identified seven confirmed hits that we used to probe the active site of nsp14. Our study revealed a path towards developing selective bi-substrate inhibitors for nsp14.

S-adenosylhomocysteine (SAH) and sinefungin were purchased from Sigma-Aldrich. S-adenosyl-L-methionine, 3 

Expression and purification of SARS-CoV-2 nsp14 is provided as supplementary data.

Methyltransferase activity of nsp14 was measured using a radiometric assay. The transfer of 3 

To evaluate the effectiveness of the nsp14 assay for screening purposes, the Z′-factor was 

Nsp14 was screened against the in-house library of 161 compounds at 50 µM in 1% DMSO.

Compounds with inhibition of more than 75% were selected as screening hits for further analysis.

The hits were tested for assay signal quenching at 50 µM. The signal was generated using 0. 

Selectivity assays were performed as previously described. 24 Briefly, compounds were tested at 50 µM in duplicate using radiometric assays. IC50 values were determined for compounds with higher than 50% inhibitory effect, as described above.

KD values for initial screening hits for nsp14 was determined by Surface Plasmon Resonance (SPR) using a Biacore T200 from GE Healthcare. N-terminally biotinylated nsp14 (aa 1-527) and

C-terminally biotinylated SETD3 (aa 1-605, as control), were coupled on a CM5 SPR Sensor chip (GE healthcare). Compounds were injected into the sensitised chip at 5 concentrations (0.6, 1. 

Table1: Confirmation and selectivity of nsp14 screening hits. The screening hits were tested for binding to nsp14 by SPR and for inhibition of methyltransferase activity of nsp14 and selected methyltransferases by activity assays. All values are from experiments presented in Figure 3 , and Supplementary Fig. 7, 10 Table 2 . Table 1 . 

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Supplementary Figure 16: Selectivity of DS0464. Dose response analysis of DS0464 against selected methyltransferases. Experiments were performed in duplicate (n=2)

+ESI Scan (rt: 6.616-7.392 min, 48 scans) Frag=250.0V Internal_Walkup-C3-10kD-100kDnsp140612