# Picomolar bivalent inhibitors of protein kinase CK2 active against β-coronavirus replication

Dylan Grenier, Dennis Salomon Lopez Molina, Muriel Gelin, Angélique Mularoni, Jean-François Guichou, Jean-Guy Delcros, Charlotte Martinasso, Yinshan Yang, Nazim Ahnou, Jean-Michel Pawlotsky, et al.

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## To cite this version:

Dylan Grenier, Dennis Salomon Lopez Molina, Muriel Gelin, Angélique Mularoni, Jean-François Guichou, et al. Picomolar bivalent inhibitors of protein kinase CK2 active against β-coronavirus replication. European Journal of Medicinal Chemistry, 2025, 296 (12), pp.117826. 10.1016/j.ejmech.2025.117826. hal-05347267

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Submitted on 4 Nov 2025

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Distributed under a Creative Commons Attribution 4.0 International License
Picomolar bivalent inhibitors of protein kinase CK2 active against β-
coronavirus replication

Dylan Grenier<sup>a#</sup>, Dennis Salomon Lopez Molina<sup>b#</sup>, Muriel Gelin<sup>c</sup>, Angélique Mularoni<sup>a</sup>, Jean-François Guichou<sup>c</sup>, Jean-Guy Delcros<sup>a</sup>, Charlotte Martinasso<sup>a</sup>, Yinshan Yang<sup>c</sup>, Nazim Ahnou<sup>b</sup>, Jean-Michel Pawlotsky<sup>b,d</sup>, Abdelhakim Ahmed-Belkacem<sup>b</sup> and Isabelle Krimm<sup>a*</sup>

\# Contributed equally to this work

* Corresponding author

<footnote><sup>a</sup> University Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Centre de recherche en cancérologie de Lyon, Institut Convergence Plascan, 69373 Lyon, France</footnote>

<footnote><sup>b</sup> Équipe "Virus, Hépatologie, Cancer", INSERM U955, IMRB, Université Paris-Est, Créteil, France.</footnote>

<footnote><sup>c</sup> Centre de Biologie Structurale (CBS), University of Montpellier, CNRS UMR 5048, INSERM U1054, Montpellier, France.</footnote>

<footnote><sup>d</sup> Department of Virology, Hôpital Henri Mondor, AP-HP, Université Paris-Est, Créteil, France.</footnote>
## ABSTRACT

The protein casein kinase 2 (CK2) has been shown to be upregulated in SARS-CoV-2 infections. However, few inhibitors have been tested for antiviral activity against coronaviruses. In this study, highly potent and selective bivalent inhibitors targeting the protein kinase CK2 were developed. The chemical structure of the well-known inhibitor CX-4945 was used as an anchor for the ATP-binding site and was linked by polar and aliphatic linkers to chemical structures that bind the cryptic αD pocket of CK2. The bivalent inhibitor **Biv5**, which demonstrated a sub-nanomolar kinase inhibition, showed potent antiviral activity against SARS-CoV-2 in HEK-ACE2-TMPRSS2 and Vero cells. In addition, **Biv5** significantly reduced viral replication over time in an ex vivo model of primary human nasal epithelial cells. The selectivity of **Biv5** was tested against 16 kinases targeted by CX-4945, confirming that targeting the αD pocket confers high selectivity for CK2 to such inhibitors.

## Keywords

Casein kinase 2 ; bivalent inhibitor ; coronavirus ; linker ; cryptic pocket

## 1. Introduction

Protein kinase CK2 is a constitutively active Ser/Thr protein kinase that phosphorylates hundreds of substrates, regulates multiple signalling pathways and is implicated in a wide range of human diseases [1,2]. Its most well-documented role is in cancer, where it controls almost all malignant hallmarks [3,4]. CK2 activity has also been implicated in cardiovascular disease, such as cardiac ischaemia-reperfusion injury, as well as neurological disorders, inflammation, autoimmune diseases, various ophthalmic pathologies, diabetes and obesity. In addition, CK2 plays a key role in human infections, with several viruses using host cell CK2 for their life cycle. More specifically, CK2 has been
identified as a key host protein kinase involved in the infection process of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [5,6].

Coronaviruses (CoVs) are a diverse group of enveloped, positive-sense RNA viruses that infect a wide range of hosts. Human can be infected by the alphacoronaviruses (α-CoV) and betacoronaviruses (β-CoV) sub-families, with three highly pathogenic β-CoVs having emerged in recent years. Severe acute respiratory syndrome coronavirus (SARS-CoV) was first identified in 2002, followed by Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, and the novel SARS-CoV-2, which caused the COVID-19 pandemic, in 2019 [7].

Although effective vaccines and direct-acting antivirals have been rapidly developed, the continuous evolution of β-CoV, the inevitable rise of drug resistance, and the potential emergence of new zoonotic SARS-like β-CoVs emphasize the urgent need for effective broad-spectrum oral antiviral therapies to combat infections. Moreover, the emergence of these new viruses highlights the critical need to deepen our understanding of their mechanisms of action during host cell infection.

A key step in the viral life cycle is the hijacking of host proteins and cellular machinery, which is essential for viral replication and spread. Recent proteomic studies have shown that coronaviruses extensively target the human protein kinase family. For instance, casein kinase 2 (CK2) has been shown to be upregulated in SARS-CoV-2 infections, where it forms complexes with the viral nucleocapsid protein [5,6]. Interestingly, similar interactions have been observed in both SARS-CoV and MERS-CoV, suggesting that this mechanism may be conserved across other β-CoVs [8].

Despite these findings, few studies have investigated the effects of CK2 inhibition on SARS-CoV-2 replication. One such study by Bouhaddou *et al.* [5] demonstrated that the CK2 inhibitor CX-4945 [9] exhibits antiviral activity in VeroE6 cells infected with SARS-CoV-2. In 2022, Ramon *et al.* [10] explored the antiviral potential of the anti-CK2 peptide CIGB-325 against bovine coronavirus (BCoV) using various *in vitro* assays. Their results showed that CIGB-325 reduces cytopathic effects and plaque-forming units and significantly reduces intracellular viral protein levels. Additionally, Wilson's group [11] described a novel ATP-competitive CK2 inhibitor, derived from the pyrazolo[1,5-
a]pyrimidine chemical probe SGC-CK2-1. This inhibitor was shown to suppress replication of SARS-CoV-2, MHV, and two bat coronaviruses (SHC014-CoV and WIV1-CoV) in vitro.

To further investigate the role of CK2 and the impact of CK2 inhibition on SARS-CoV-2 infection and the broader coronavirus life cycle, we aimed to develop bivalent inhibitors. We hypothesize that this approach will result in the development of potent and selective inhibitors suitable for use as chemical tools. We selected the chemical structure of CX-4945 as an anchor for the ATP-binding site, while taking advantage of the αD pocket. Bivalent CK2 inhibitors were generated by linking the modified CX-4945 moiety to chemical structures that have been previously identified as αD pocket binders [12,13].

## 2. Results

The design strategy involved replacing the chlorophenyl group of CX-4945 with a linker, enabling the attachment of a small molecule that specifically binds to the αD pocket (Fig. 1). We hypothesized that this modification would result in a more potent and selective CK2 inhibitor compared to CX-4945. To test this, we first modified CX-4945 into compound 5 by substituting the chlorophenyl group with a methyl group (Fig. 1). The synthesis of compound 5, based on the previous work of Pierre et al. [9], is detailed in Scheme 1. The synthesis is a six-step sequence starting with a Suzuki-Miyaura coupling using the corresponding boronic acid 1 and bromide 2 derivatives. The compound was then treated with neat phosphoryl trichloride to undergo the first step of the Vilsmeier-Haack reaction and convert the pyridinone into the corresponding 2-chloropyridine 3. Microwave treatment in the presence of methylamine afforded compound 4 and compound 5 was obtained by hydrolysis of 4.
<figure><img src="image_3.png" /><figcaption>Fig. 1. Inhibitor design strategy. Crystal structures of (a) CX-4945 (code pdb 3NGA) and (b) compound 5 (pdb code 9QSV) bound to CK2α. Overlay of compound 5 with (c) CX-4945 and (d) KN2 (pdb code 7AT5) as the reference bivalent inhibitor.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Scheme 1. Synthesis of compound 5</figcaption></figure>

The binding of compound 5 to CK2α was assessed by a Thermal Shift Assay (TSA), which revealed a significant shift (ΔTm = 11.2°C) (Table 1). The inhibition of CK2α kinase activity by compound 5 was further confirmed using an ADP-Glo assay (Fig. S1). Compared to CX-4945, compound 5 showed reduced activity ($IC_{50}$ = 5.5 ± 0.7 nM), likely due to the loss of hydrophobic contacts, leading
to an approximately 10-fold decrease in potency. As expected, the crystal structure of compound **5** bound to CK2α showed a binding mode very similar to that of CX-4945 (Fig. 1 and Fig. S2).

Our goal was to generate bivalent CK2 inhibitors where compound **5** serves as the ATP-site binder and is linked to the 3,4-dichlorophenethylamine moiety that anchors to the αD pocket (see Table 1 in red). The 3,4-dichlorophenethylamine moiety was first reported by the groups of Spring and Hyvönen [12] and was latter incorporated in the bivalent CK2 inhibitor KN2 [13]. The overlay of the crystal structures of compounds KN2 and **5** bound to CK2α suggested that a bivalent inhibitor could be created by linking compound **5** to the 3,4-dichlorophenethylamine moiety (Figure 1D). To test this hypothesis, we synthesized compounds **Biv1** and **Biv2**, which incorporated linkers with 11 and 12 atoms, respectively (Table 1).

<table><caption>**Table 1.** Compound names, chemical structures, and results for binding assay (TSA) and enzymatic assays (ADP-Glo). The 3,4-dichlorophenethylamine and the (2-chloro-[1,1'-biphenyl]-4-yl)methanamine moieties are highlighted in red and blue, respectively.</caption><thead><tr><th>Nom</th><th>Structure</th><th>TSA ΔTm (°C)</th><th>Enzymologie IC<sub>50</sub> (pM)</th></tr></thead><tbody><tr><td>CX-4945</td><td><img src="cx4945_structure.png" alt="Chemical structure of CX-4945"></td><td>13.0 ± 0.2</td><td>656 ± 160</td></tr><tr><td><b>5</b></td><td><img src="5_structure.png" alt="Chemical structure of 5"></td><td>10.2 ± 0.8</td><td>5520 ± 700</td></tr><tr><td><b>Biv1</b></td><td><img src="biv1_structure.png" alt="Chemical structure of Biv1"></td><td>14.3 ± 0.3</td><td>180 ± 23</td></tr><tr><td><b>Biv2</b></td><td><img src="biv2_structure.png" alt="Chemical structure of Biv2"></td><td>16.7 ± 0.2</td><td>89 ± 9</td></tr><tr><td><b>Biv3</b></td><td><img src="biv3_structure.png" alt="Chemical structure of Biv3"></td><td>15.8 ± 0.7</td><td>46 ± 4</td></tr></tbody></table>
<table><tr><td>Biv4</td><td><img src="biv4.png" alt="Chemical structure of Biv4"></td><td>20.3 ± 1.2</td><td>65 ± 7</td></tr><tr><td>Biv5</td><td><img src="biv5.png" alt="Chemical structure of Biv5"></td><td>19.6 ± 0.7</td><td>51 ± 10</td></tr><tr><td>Biv6</td><td><img src="biv6.png" alt="Chemical structure of Biv6"></td><td>22.2 ± 0.1</td><td>258 ± 27</td></tr><tr><td>Biv7</td><td><img src="biv7.png" alt="Chemical structure of Biv7"></td><td>21.8 ± 0.3</td><td>221 ± 20</td></tr></table>

The synthesis of these compounds is outlined in Scheme 2. The synthesis followed a convergent strategy starting with the reaction of compound **3** with tert-butyl (3-aminopropyl)carbamate and tert-butyl (4-aminobutyl)carbamate respectively under microwave condition, followed by Boc-deprotection with trifluoroacetic acid to give compounds **6** and **7**, respectively. In parallel, compound **8** was synthesized using 2-(3,4-dichlorophenyl)ethanamine and monoprotected succinic acid, followed by a deprotection step using TIPS and TFA. Ammonium salts **6** and **7** were coupled with compound **8** using HATU agent and then the corresponding esters were hydrolyzed using lithium hydroxide to give **Biv1** and **Biv2**.

<figure><img src="image_5.png" /></figure>

Scheme 2. Synthesis of compounds **Biv1** and **Biv2**. Yields marked with an asterisk (*) are indicated for two steps.
TSA and enzymatic assays showed that linking the ATP-site binder (compound 5) to the dichlorophenethylamine moiety improved binding strength, resulting in sub-nanomolar CK2 inhibition, with IC₅₀ values of 180 pM and 90 pM for **Biv1** and **Biv2**, respectively (Table 1 and Fig. S1). X-ray crystallography confirmed the binding modes of **Biv1** and **Biv2** (Fig. 2 and Fig. S2). In both crystal structures, the chlorine atoms of the dichlorophenethylamine moiety deeply interact within the αD pocket. Additionally, the linker's amide groups form hydrogen bonds with residues V162 and H160, a feature also observed in the structure of KN2 bound to CK2α (pdb code 7AT5). In the crystal structure of **Biv1**, a hydrogen bond is also observed between the linker and residue N118.

<figure><img src="image_6.png" /><figcaption>Fig. 2. X-ray crystallographic structures of (a) **Biv1** (pdb code 9QRH), (b) **Biv2** (pdb code 9QRI) and (c) **KN2** (pdb code 7AT5) bound to CK2α.</figcaption></figure>

To explore other linking strategies, we tested a different αD pocket binder: the (2-chloro-[1,1'-biphenyl]-4-yl)methanamine moiety (See Table 1 in blue). This sub-structure was used in the very first reported CK2 bivalent inhibitor, CAM4066 [12], and more recently, in bivalent inhibitors where it was linked to small ATP-mimicking moieties with a variety of linkers [14]. To link compound 5 to the biphenyl-methanamine moiety, we used two types of linkers: an aliphatic linker containing 6, 7, or 8 methylene groups (**Biv3**, **Biv4**, and **Biv5**) and a more polar linker containing one amide group (**Biv6**), as described in CAM4066. The linker length for **Biv6** was determined based on enzymatic data for **Biv1** and **Biv2**, resulting in a 4-atom linker between compound 5's amine and the amide group.
Compounds **Biv3**, **Biv4**, and **Biv5** were synthesized as shown in Scheme 3, while **Biv6** was synthesized as outlined in Scheme 4. **Biv3**, **Biv4**, and **Biv5** were prepared starting from compound **3**. Compound **3** was reacted with three different Boc-diamine linkers of 6, 7 and 8 carbon lengths and Boc-deprotected with trifluoroacetic acid to give compounds 9, 10 and 11 as trifluoroacetic acid salts. Reductive amination was then carried out in the presence of NaBH₃CN and 2-chloro-[1,1'-biphenyl]-4-carbaldehyde. The final step was a conventional ester hydrolysis using lithium hydroxide to give **Biv3**, **Biv4**, and **Biv5**, respectively.

<figure><img src="image_7.png" /></figure>

Scheme 3. Synthesis of compounds **Biv3**, **Biv4** and **Biv5**. Yields marked with an asterisk (*) are indicated for two steps.

TSA and enzymatic assays demonstrated that linking compound **5** to the (2-chloro-[1,1'-biphenyl]-4-yl)methanamine moiety significantly enhanced binding strength, leading to potent CK2 inhibitors. IC₅₀ values of 46 ± 4 pM, 65 ± 7 pM, and 51 ± 10 pM were obtained for inhibitors **Biv3**, **Biv4**, and **Biv5**, respectively (Table 1). The inhibitor **Biv6**, which contains an amide group in the linker, showed a larger thermal shift (ΔTm = 22.2°C) compared to **Biv3** (ΔTm = 15.3°C), **Biv4** (ΔTm = 20.3°C), and **Biv5** (ΔTm = 19.6°C) (Table 1). However, the ADP-Glo assay indicated that the CK2 inhibition activity of **Biv6** (IC₅₀ = 258 pM) was slightly lower than expected based on TSA results.

The binding modes of **Biv3**, **Biv4**, **Biv5**, and **Biv6** were further confirmed by X-ray crystallography (Fig. 3 and Fig. S2). In all the crystallographic structures, the amine group of the biphenyl-methanamine moiety formed hydrogen bonds with the backbone of residue V162. In **Biv6**, similar to **Biv2**, the amide group forms a hydrogen bond with residue H160.
Using KLIFS [15], distinct biphenyl-like moieties that bind to the αD pocket of CK2 have been identified from the PDB (communication from Albert J. Kooistra, University of Copenhagen). These substructures feature monosubstitutions - such as CH₃, OCH₃, Cl, or CF₃ - on the first ring or di-chloro substitutions, while the second ring is substituted with aliphatic groups, halogens, alcohols, or ethers (Fig. S3). However, the binding affinities of these substructures for the protein remain largely unpublished. We aimed to explore a more polar biphenyl moiety and selected the 2'-methoxy-[1,1'-biphenyl] moiety to obtain **Biv7** (Table 1).

Compounds **Biv6** and **Biv7** were synthesised as reported in Scheme 4. First, compound **12** was prepared using compound **7** and Boc-β-alanine and amide coupling conditions followed by trifluoroacetic acid Boc-deprotection conditions. Compounds **Biv6** and **Biv7** were then obtained by reductive amination with 2-chloro-[1,1'-biphenyl]-4-carbaldehyde and 2'-methoxy-[1,1'-biphenyl]-4-carbaldehyde respectively, followed by ester-lithium hydroxide hydrolysis (Scheme 4).

<figure><img src="image_8.png" /><figcaption>Scheme 4. Synthesis of compounds **Biv6** and **Biv7**. Yields marked with an asterisk (*) are indicated for two steps.</figcaption></figure>

The inhibitor **Biv7** demonstrated similar binding potency to **Biv6**, retaining its sub-nanomolar kinase inhibition activity, with an IC₅₀ of 221 ± 20 pM (Table 1 and Fig. S1). The crystallographic structure of **Biv7** confirmed that polar interactions between the linker and the kinase were conserved, similarly to those in **Biv6** (Fig. 3 and Fig. S2).
<figure><img src="image_9.png" /><figcaption>Fig. 3. Crystallographic structures of (a) **Biv3** (pdb code 9QRJ), (b) **Biv4** (pdb code 9QSR), (c) **Biv5** (pdb code 9QSS), (d) **Biv6** (pdb code 9QST) and (e) **Biv7** (pdb code 9QSU). (f) Overlay of biphenyl moieties of **Biv3-Biv7** inhibitors and CAM4066 (pdb 5CU4). The colour code is similar to the one used in a-e.</figcaption></figure>

The crystallographic structures also showed that the binding mode of the biphenyl-methanamine moiety is not strictly conserved. In most structures, the chlorine atom of the biphenyl-methanamine moieties interacts with residues P159, V162, and M221, while in one monomer of CK2 bound to **Biv5**, the chlorine atom is closer to residues F121, L128, and M225 as observed in the structure of CAM4066, suggesting that the biphenyl-methanamine moiety can adopt different orientations within the αD pocket.
To assess the potential of bivalent CK2 inhibition as a therapeutic strategy against SARS-CoV-2 and related coronaviruses, we evaluated the antiviral activity of the seven CK2-targeting compounds previously characterized for their kinase inhibitory potency.

Initial screening at 20 µM in both Vero and HEK-ACE2-TMPRSS2 cells (Fig. 4A and 4B, respectively) showed that only **Biv1** and **Biv2** exhibited limited or no antiviral activity. This lack of efficacy may be attributed to poor cellular penetration of the compounds. In contrast, **Biv5** demonstrated high potency, reducing intracellular viral RNA production by approximately three logs at 20 µM—a stronger effect than that observed with CX4945 and Remdesivir. Based on these promising results, **Biv5** was selected for further antiviral characterization.

We next evaluated the antiviral activity of **Biv5** at various concentrations (1, 5, 10, and 20 µM) in both Vero and HEK-ACE2-TMPRSS2 cells (Fig. 4C and 4D, respectively). **Biv5** showed a dose-dependent reduction in both intracellular and extracellular viral RNA levels, with a $CC_{50}$ higher than the highest concentration tested ($CC_{50} > 20$ µM). Immunofluorescence and western blot analyses confirmed that **Biv5** inhibited SARS-CoV-2 infection in HEK-ACE2-TMPRSS2 cells, as evidenced

by decreased nucleoprotein detection (Fig. 4E and 4F, respectively).

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>
Fig. 4. Effect of 20 µM CK2 inhibitors on SARS-CoV-2 infection in (A) Vero cells and (B) HEK-ACE2/TMPRSS2 cells. Dose-dependent effect of **Biv5** in (C) Vero cells and (D) HEK-ACE2/TMPRSS2 cells. (E) Inhibition of SARS-CoV-2 infection in HEK-ACE2-TMPRSS2 cells by **Biv5**, visualized by immunofluorescence using an antibody against the SARS-CoV-2 nucleoprotein. Magnification: 10×; cell nuclei stained with DAPI. (F) Inhibition of nucleoprotein expression visualized by western blot using an antibody targeting the SARS-CoV-2 nucleoprotein.

We next investigated the time-dependent antiviral effects of **Biv5** in human nasal epithelial cells, an ex vivo physiologically relevant model. **Biv5** significantly reduced extracellular viral RNA production over time (Fig. 5A). By 96 hours post-treatment, intracellular viral RNA levels were also markedly reduced (Fig. 5B). Importantly, **Biv5** did not significantly alter transepithelial electrical resistance (TEER) which reflect the integrity of the epithelium (Fig. 5C). Finally, to evaluate the potential broad-spectrum antiviral activity of **Biv5**, we tested its effect on OC43, another β-CoV. Interestingly, **Biv5** dose-dependently reduced OC43 RNA production in HRT18 cells (Fig. 5E), suggesting that **Biv5** possesses a broad β-CoV spectrum.
A

<figure><img src="image_16.png" /></figure>

B

<figure><img src="image_17.png" /></figure>

C

<figure><img src="image_18.png" /></figure>

D

<figure><img src="image_19.png" /></figure>

Fig. 5. Kinetic evaluation of the antiviral effect of **Biv5** in human nasal epithelial cells. (A) Time-dependent changes in extracellular viral RNA production at the apical pole of HNECs, monitored by RT-qPCR. Results are shown for Biv5 (dashed line) and untreated cells (solid black line). (B) Relative intracellular viral RNA levels after 96 hours of Biv5 treatment in HNECs. (C) Evaluation of TEER over time in untreated (solid black line) and Biv5-treated (dashed line) HNECs. (D) Dose-dependent reduction of OC43 RNA production by Biv5 in HRT18 cells.

Finally, we evaluated the activity of **Biv5** against protein kinases targeted by CX-4945 [5] using a radiometric assay. ATP concentrations were adjusted to each kinase's Km value during the assay. Among the 18 protein kinases tested, **Biv5** exhibited the strongest activity against CK2α and CK2α' (Table 2, Fig. S4). Other kinases were either moderately inhibited (IC₅₀: 489-889 nM) or not inhibited (IC₅₀ > 1000 nM), except for DRAK2 which was slightly inhibited (IC₅₀ = 114 nM). These
results highlight the high selectivity of **Biv5** for CK2α and CK2α', most likely due to its binding to the αD pocket.

<table><caption>**Table 2.** Enzymatic assay (radiometric) of **Biv5** against 18 protein kinases, performed at ATP concentrations corresponding to each kinase's Km value</caption><thead><tr><th>Kinase</th><th>K<sub>m</sub> [ATP] (μM)</th><th>IC<sub>50</sub> (nM)</th></tr></thead><tbody><tr><td>CDK1/cyclinB(h)</td><td>45</td><td>&gt;1,000</td></tr><tr><td>CK2α1(h)</td><td>10</td><td>3</td></tr><tr><td>CK2α2(h)</td><td>10</td><td>3</td></tr><tr><td>CLK1(h)</td><td>15</td><td>666</td></tr><tr><td>CLK3(h)</td><td>90</td><td>&gt;1,000</td></tr><tr><td>DAPK1(h)</td><td>70</td><td>&gt;1,000</td></tr><tr><td>DRAK2(h)</td><td>15</td><td>114</td></tr><tr><td>DYRK1A(h)</td><td>45</td><td>713</td></tr><tr><td>DYRK1B(h)</td><td>45</td><td>&gt;1,000</td></tr><tr><td>DYRK2(h)</td><td>15</td><td>&gt;1,000</td></tr><tr><td>DYRK3(h)</td><td>10</td><td>549</td></tr><tr><td>Flt3(h)</td><td>200</td><td>&gt;1,000</td></tr><tr><td>HIPK1(h)</td><td>45</td><td>&gt;1,000</td></tr><tr><td>HIPK2(h)</td><td>10</td><td>489</td></tr><tr><td>HIPK3(h)</td><td>15</td><td>&gt;1,000</td></tr><tr><td>JAK3(h)</td><td>10</td><td>&gt;1,000</td></tr><tr><td>Pim-1(h)</td><td>90</td><td>899</td></tr><tr><td>TBK1(h)</td><td>155</td><td>&gt;1,000</td></tr></tbody></table>

### 3. Conclusion/Discussion

In this study, we have designed and synthesized bivalent CK2 inhibitors by modifying CX-4945, an ATP-competitive inhibitor, into compound **5**. Compound **5** showed reduced CK2 inhibition compared to CX-4945 but maintained a similar binding mode. Then, compound **5** was linked to two distinct αD pocket-binding moieties and two types of linkers. **Biv1** and **Biv2**, incorporating a dichlorophenethylamine moiety, demonstrated strong CK2 inhibition (IC<sub>50</sub>: 180 pM and 90 pM, respectively). Further modifications included linking compound **5** to a biphenyl-methanamine moiety, yielding inhibitors (**Biv3–Biv7**) with potent CK2 inhibition (IC<sub>50</sub>: 46–258 pM). Surprisingly, while the
polar linkers form hydrogen bonds with CK2 residues, the corresponding bivalent compounds do not exhibit improved activity compared to the bivalent inhibitors containing alkyl linkers.

<figure><img src="image_20.png" /><figcaption>Fig. 6. Chemical structures of CX-4945, **5** and **Biv5** and overlay of CX-4945 bound to CK2 with **5** and **Biv5** bound to CK2.</figcaption></figure>

Other bivalent CK2 inhibitors published in the literature include the compound CAM4066 ($K_D$ of 0.32 µM and $IC_{50}$ of 0.37 µM) [12], the compound KN2 ($K_i$ 6 nM for CK2 holoenzyme) [13], AB668 ($K_i$ of 41 nM for the holoenzyme) [16] and a series of bivalent inhibitors linking the biphenyl-methanamine moiety to ATP-binders (0,9-28 µM) [14]. To our knowledge, **Biv1-Biv7** are the most potent bivalent CK2 inhibitors targeting the αD pocket, the binding strength of the inhibitors appearing to be mostly dependent of the ATP-site binder. Other pM CK2 inhibitors have been published in the literature, corresponding to bivalent compounds that target the ATP site and the substrate site, ARC-3140 [17], where a peptide-like moiety is used to bind as the substrate site.
**Biv5**, the most promising inhibitor reported here, showed potent antiviral activity against SARS-CoV-2 in HEK-ACE2-TMPRSS2 and Vero cells, reducing viral RNA production. In human nasal epithelial cells, the CK2 inhibitor significantly diminished viral replication over time. In addition, **Biv5** exhibited excellent selectivity against 18 known CX-4945 targets. Consequently, **Biv5** emerges as a promising tool for further investigating the role of CK2 in the SARS-CoV-2 and β-CoV cell cycle. We anticipate that this compound will also serve as a powerful tool to deepen our understanding of CK2's role in other pathologies.

# 4. Experimental procedures

## 4.1. Chemistry

All commercially available chemicals and solvents were purchased from Sigma-Aldrich, Acros Organics, Fischer scientific, Alfa Aesar, Enamine or BLDpharm. They were used without further purification unless otherwise stated, and the reaction solvents were of anhydrous grade (Acros ACROSEALTM). Reactions were carried out in oven-dried glassware under argon overpressure and monitored by HPLC-MS analysis or thin layer chromatography after UV visualizations (at 254 and 365 nm) or chemical staining (ninhydrin, p-anisaldehyde or phosphomolybdic acid solutions). Microwave synthesis was conducted using a CEM Discover 2017 Microwave Synthesis Instrument (CEM Corporation), monitored via CEM's Innovators in Microwave Technology Synergy system, under the following conditions: 17 bar pressure, 200 W power, air flush at 0.4 psi, high stirring, and 2 mL microwave vials. The products were purified on Macherey-Nagel silica gel 60 (particle size 0.040 - 0.063 mm) using flash technique and under a positive pressure. Flash column chromatography was performed on a Teledyneinterchim puriFlash XS 520plus using methanol and dichloromethane with silica gel.

The crude mixtures were adsorbed on Macherey-Nagel silica gel 60 (particle size 0.040 - 0.063 mm) prior to chromatographic purification. The purity of products and composition of the reaction mixtures were tested on a Waters HPLC-MS Agilent Technologies 1260 Infinity (flow rate 0.5 mL/min,
gradient 10-90 % MeCN/water, 0.1 % formic acid, over six minutes) with Agilent Poroshell 120 SB-C18 2.7 µm 2.1 x 50 mm column and Agilent Technologies 6120 Quadripole LC/MS using Agilent OpenLab ChemStation Chromatography Data System.

NMR spectra were recorded on Bruker Avance 400 (400 MHz) and Bruker Avance 500 (500 MHz) spectrometers. Chemical shifts (δ) were referenced to the solvent residual peak and reported in parts per million (ppm) to the nearest 0.01 ppm for ¹H and to the nearest 0.1 ppm for ¹³C. CDCl₃, d₆-DMSO and MeOD were used as deuterated solvents and the resonances were locked as internal standards (CDCl₃ ¹H δ = 7.26, ¹³C δ = 77.1, d₆-DMSO ¹H δ = 2.50, ¹³C δ = 39.5 and MeOD ¹H δ = 3.31, ¹³C δ = 49.00). Signal multiplicity is indicated by lower case letters (s singlet, d doublet, t triplet, q quadruplet, m multiplet or overlap of non-equivalent resonances or combination of letters) and coupling constants (J) are given in Hertz to the nearest 0.1 Hz. High-resolution mass spectra (HRMS) were recorded on QTOF Impact II – Bruker spectrometer using electrospray ionization (ESI).

The synthesis of the CX-4945 moiety was inspired by Pierre et al. [9].

**General method A: Boc deprotection**

The solid was dissolved in CH₂Cl₂ (0.5 mL) and trifluoroacetic acid was added dropwise to the solution (0.5:0.5) and stirred for 1h. At the end of the reaction, the volatile components were removed under reduced pressure. The crude product obtained was dissolved in methanol and heptane (1:2) or Et₂O and evaporated a several times under reduced pressure to yield the corresponding ammonium salt.

**General method B1: Pyridine-2-amine formation, microwave heating**

Compound 3 (1 eq.) was combined with the appropriate amine (1.2 eq.), K₂CO₃ (2.5 eq.) and N-methylpyrrolidinone in a 2 ml microwave tube and stirred for 15 min. The mixture was then heated to 150 °C for 5 min. The mixture was poured into saturated NaCl(aq) solution and extracted 5 times with
AcOEt. The volatile components were evaporated and the remaining solid was purified by flash chromatography (99.5/0.5 - 90/10 CH₂Cl₂/MeOH gradient) to yield the desired product.

**General method B2:** Pyridine-2-amine formation, conventional heating

The appropriate amine (1.1 eq), compound 3 (1 eq.), DIPEA (2.5 eq.) and NMP were dissolved in a microwave tube. The tube was argon flushed before sealing and placed at 90°C overnight. The mixture was poured into saturated NaCl(aq) solution and extracted 5 times with AcOEt. The volatile components were evaporated and the remaining solid was purified by flash chromatography (99.5/0.5 - 90/10 CH₂Cl₂/MeOH gradient) to yield the desired product.

**General method C:** Ester hydrolysis

In a microwave vial with a V-bottom, the ester was added in a 4:1:1 dioxane/THF/H₂O and lithium hydroxide (3.5 eq.) was added after sonication. The mixture was stirred at r.t. for 6 h. A solution of HCl 2M in Et₂O was slowly added to the mixture. The solution was concentrated under continuous nitrogen flushing and the excess salt was removed by washing with aqueous HCl 2.5%, the solid was filtered and dried under vacuum overnight to yield the corresponding carboxylic acid without further purification.

**General method D1:** Amide coupling, HATU agent

In a round bottom flask, the acid (1.1 eq.), DIPEA (2 eq.), HATU (1.2 eq.) and the amine (1 eq.) were dissolved in CH₂Cl₂ in this order. The mixture was stirred for 2 h and the solution was poured into water and extracted 3 times with CH₂Cl₂. The combined organic extract was evaporated and the remaining solid was purified by flash chromatography (99.5/0.5 - 90/10 CH₂Cl₂/MeOH gradient) to yield the desired product.

**General method D2:** Amide coupling, EDC/HOBt agents
In a round bottom flask, the amine (1 eq.), DIPEA (1.5 eq.), acid (1 eq.), EDC.HCl (1.2 eq.) and HOBt (1.2 eq.) were dissolved in anhydrous N,N-dimethylformamide at 0°C in the following order. The mixture was stirred at r.t. for 3 h and the solution was poured into water and extracted 3 times with AcOEt. The combined organic extract was evaporated and the remaining solid was purified by flash chromatography (99.5/0.5 - 90/10 CH₂Cl₂/MeOH gradient) to yield the desired product.

**General method E: reductive amination**

The corresponding amine (1 eq.) and aldehyde (1.2 eq.) were dissolved in MeOH/AcOH (9:1) and stirred at r.t. for 45 min. NaBH₃CN (1 eq.) was then added and stirred at r.t. for 1h30. The mixture was diluted with dichloromethane (100 ml), washed twice with NaHCO₃ sat. and the aqueous layer extracted with dichloromethane. The combined organic layers were directly evaporated under reduced pressure and purified by flash chromatography (99.5/0.5 - 90/10 CH₂Cl₂/MeOH gradient) to yield the desired product.

**Methyl 5-chlorobenzo[c][2,6]naphthyridine-8-carboxylate (3)**

Pd(PPh₃)₄ (0.267 g, 0.231 mmol) was added to a pre-argon-degased solution containing methyl 3-bromoisonicotinate (2) (0.5 g, 2.31 mmol), (2-amino-4-(methoxycarbonyl)phenyl)boronic acid hydrochloride (1) (0.680 g, 3.470 mmol) and K₂CO₃ (0.959 g, 6.94 mmol) in 1,4-dioxane (9 mL) and water (1 mL) at r.t. under argon bubbling. The resulting mixture was stirred at 105 °C for 3 hours. The reaction mixture was poured into water (15 mL), and stirred at r.t. for 10 min. The precipitate was collected by filtration, washed with water (2 mL), EA:PE= 1:5 (5 mL twice) and dried under vacuum to give methyl 5-oxo-5,6-dihydrobenzo[c][2,6]naphthyridine-9-carboxylate (0.464 g, 79%) as a beige solid, which was used for the next step without further purification. The solid (0.464 mg, 1.83 mmol) was mixed with neat phosphorus oxychloride (2 mL, 21.84 mmol). The mixture was stirred at 130°C for 2 h. The reaction was evaporated, then diluted with a minimum of acetonitrile and poured onto ice water, and the resulting solid was filtered and dried. Methyl 5-chlorobenzo[c][2,6]naphthyridine-8-carboxylate (3) was isolated as a grey solid (449 mg, 90%).
### Methyl 5-(methylamino)benzo[c][2,6]naphthyridine-8-carboxylate (4)

Following **general method B1** with compound **3** (50 mg, 0.183 mmol), methylamine hydrochloride (14 mg, 0.440 mmol), K₂CO₃ (56 mg, 0.403) and N-methylpyrrolidinone (0.2 mL) to yield methyl 5-(methylamino)benzo[c][2,6]naphthyridine-8-carboxylate as a yellowish solid (29 mg, 59 %). LC-MS (ESI+): RT 1.814 min, 99 % pure at 254 nm. HRMS (ESI+) m/z: calcd. for C₁₅H₁₄N₃O₂⁺: 268.1081; [M+H]⁺ found: 268.1081. ¹H NMR (500 MHz, DMSO) δ 10.04 (s, 1H), 8.88 (d, *J* = 5.6 Hz, 1H), 8.72 (d, *J* = 8.5 Hz, 1H), 8.19 (d, *J* = 5.6 Hz, 1H), 8.16 (s, 1H), 8.06 (q, *J* = 4.5 Hz, 1H), 7.80 (dd, *J* = 8.4, 1.8 Hz, 1H), 3.91 (s, 3H), 3.08 (d, *J* = 4.5 Hz, 3H); ¹³C NMR (126 MHz, DMSO) δ 166.72, 153.83, 148.05, 148.01, 145.48, 130.62, 127.96, 126.98, 124.54, 123.17, 122.30, 122.07, 116.56, 52.72, 28.53. LC-MS (ESI+): RT 1.947 min, 99 % pure at 254 nm. HRMS (ESI+) m/z: calcd. for C₁₅H₁₄N₃O₂⁺: 268.1081; [M+H]⁺ found: 268.1081

### 5-(methylamino)benzo[c][2,6]naphthyridine-8-carboxylic (5)

Following **general method C**, compound **4** (4.9 mg, 18.33 µmol) was dissolved in 4/1/1 dioxane/THF/H₂O (1.2 mL), lithium hydroxide (1.62 mg, 67.83 µmol) and HCl 2M in Et₂O (33.9 µL), compound 5-(methylamino)benzo[c][2,6]naphthyridine-8-carboxylic acid (5) yielded as a yellow solid (3.9 mg, 84 %). ¹H NMR (500 MHz, DMSO) δ 9.99 (s, 1H), 8.79 (d, *J* = 5.5 Hz, 1H), 8.53 (d, *J* = 8.4 Hz, 1H), 8.21 – 8.18 (m, 2H), 7.90 (d, *J* = 4.7 Hz, 1H), 7.87 (dd, *J* = 8.4, 1.7 Hz, 1H), 3.08 (d, *J* = 4.4 Hz, 3H); ¹³C NMR (126 MHz, DMSO) δ 174.56, 169.48, 152.68, 147.17, 146.21, 144.91, 141.10, 127.33, 123.69, 123.29, 120.64, 118.30, 116.11, 28.11. LC-MS (ESI+): RT 1.348 min, 98 % pure at 254 nm. HRMS (ESI+) m/z: calcd. for C₁₄H₁₂N₃O₂⁺: 254.0924; [M+H]⁺ found: 254.0924.

### 3-((8-(methoxycarbonyl)benzo[c][2,6]naphthyridin-5-yl)amino)propan-1-aminium-2,2,2-trifluoroacetate (6)

Following **general method B2** with Boc-diaminopropane (127 mg, 0.605 mmol), compound **3** (150 mg, 0.550 mmol), DIPEA (0.238 mL) and NMP (3 mL) to yield methyl 5-((3-((tert-butoxycarbonyl)amino)propyl)amino)benzo[c][2,6]naphthyridine-9-carboxylate as a white solid (194
mg, 85%). Using **general method A** with CH₂Cl₂ (0.5 mL) and TFA (0.5 mL), yielded 3-((8-(methoxycarbonyl)benzo[c][2,6]naphthyridin-5-yl)amino)propan-1-aminium-2,2,2-trifluoroacetate (6) was obtained as a white solid (192 mg, 95%).

### 4-((8-(methoxycarbonyl)benzo[c][2,6]naphthyridin-5-yl)amino)butan-1-aminium-2,2,2-trifluoroacetate (7)

Following **general method B2** with Boc-diaminobutane (135 mg, 0.605 mmol), compound **3** (150 mg, 0.550 mmol), DIPEA (0.238 mL) and NMP (3 mL) to yield methyl 5-((4-((tert-butoxycarbonyl)amino)butyl)amino)benzo[c][2,6]naphthyridine-9-carboxylate as a white solid (193 mg, 82%). Using **general method A** with CH₂Cl₂ (0.5 mL) and TFA (0.5 mL), yielded 4-((8-(methoxycarbonyl)benzo[c][2,6]naphthyridin-5-yl)amino)butan-1-aminium-2,2,2-trifluoroacetate (7) as a white solid (189 mg, 97%).

### 4-((3,4-dichlorophenethyl)amino)-4-oxobutanoic acid (8)

Following **general method D1** with mono-terbutoxylester succinic acid (403 mg, 2.31 mmol), DIPEA (0.73 mL) µL), HATU (960 mg, 2.53 mmol), 2-(3,4-dichlorophenyl)ethanamine (400 mg, 2.10 mmol) and CH₂Cl₂ (5 mL), 5h r.t. stirring. After completion of the reaction, TIPS was added (0.432 mL, 2,10 mmol) and TFA (5 mL), and the mixture was stirred for 2h at r.t.. The volatile components were removed under reduced pressure and the solid was purified by flash chromatography (50/50 to 95/5) AcOEt/PE gradient) to yield compound **8** as white crystals (454 mg, 74%).

### 5-((3-(4-((3,4-dichlorophenethyl)amino)-4-oxobutanamido)propyl)amino)benzo[c][2,6]naphthyridine-8-carboxylic acid (Biv1)

Following **general method D1** with compound **8** (60 mg, 0.207 mmol), DIPEA (52 µL), HATU (86 mg, 0.226 mmol), compound **6** (80 mg, 0.188 mmol) and CH₂Cl₂ (2 mL) afforded methyl 5-((3-(4-((3,4-dichlorophenethyl)amino)-4-oxobutanamido)propyl)amino)benzo[c][2,6]naphthyridine-8-
carboxylate as a pale yellow solid (94 mg, 86%). Using **general method C** with the corresponding ester (20 mg, 0.034 mmol) and lithium hydroxide (2.5 mg, 0.103 mmol) to give 5-((3-(4-((3,4-dichlorophenethyl)amino)-4-oxobutanamido)propyl)amino)benzo[c][2,6]naphthyridine-8-carboxylic acid (**Biv1**) was obtained as a yellow solid (17 mg, 87%). ¹H NMR (500 MHz, DMSO) δ 9.98 (s, 1H), 8.78 (d, J = 5.5 Hz, 1H), 8.49 (d, J = 8.3 Hz, 1H), 8.19 (d, J = 5.6 Hz, 1H), 8.14 (d, J = 1.6 Hz, 1H), 7.84 (dd, J = 8.3, 1.6 Hz, 1H), 7.53 – 7.43 (m, 2H), 7.18 (dd, J = 8.2, 2.0 Hz, 1H), 3.61 (t, J = 7.0 Hz, 2H), 3.25 (t, J = 7.1 Hz, 2H), 3.19 – 3.15 (m, 2H), 2.70 (t, J = 7.1 Hz, 2H), 2.33 (s, 4H), 1.83 (p, J = 6.8 Hz, 2H); ¹³C NMR (126 MHz, DMSO) δ 171.82, 169.12, 147.60, 146.49, 145.25, 144.60, 142.95, 141.43, 131.19, 131.13, 130.69, 129.69, 129.09, 127.62, 126.01, 124.48, 120.89, 118.40, 38.80, 37.00, 34.43, 31.64, 31.59, 29.35; LC-MS (ESI+): RT 2.658 min, 96.1 % pure at 254 nm; HRMS (ESI+) m/z: calcd. for C₂₈H₂₈Cl₂N₅O₄⁺: 568.1513; [M+H]⁺ found: 568.1512.

**5-((4-(4-((3,4-dichlorophenethyl)amino)-4-oxobutanamido)butyl)amino)benzo[c][2,6]naphthyridine-8-carboxylic acid (Biv2)**

Following **general method D1** with compound **8** (59 mg, 0.201 mmol), DIPEA (52 µL), HATU (84 mg, 0.220 mmol), compound **7** (80 mg, 0.183 mmol) and CH₂Cl₂ (2 mL) afforded methyl 5-((4-(4-((3,4-dichlorophenethyl)amino)-4-oxobutanamido)butyl)amino)benzo[c][2,6]naphthyridine-9-carboxylate as pale yellow solid (91 mg, 83%). Using **general method C** with the corresponding ester (20 mg, 33 mmol) and lithium hydroxide (2.6 mg, 0.107 mmol) was obtained 5-((4-(4-((3,4-dichlorophenethyl)amino)-4-oxobutanamido)butyl)amino)benzo[c][2,6]naphthyridine-8-carboxylic acid (**Biv2**) was obtained as a yellow solid (18 mg, 92%). ¹H NMR (500 MHz, DMSO) δ 9.97 (s, 1H), 8.76 (d, J = 5.6 Hz, 1H), 8.48 (d, J = 8.4 Hz, 1H), 8.22 (d, J = 5.6 Hz, 1H), 8.12 (d, J = 1.6 Hz, 1H), 7.83 (dd, J = 8.3, 1.6 Hz, 2H), 7.73 (s, 1H), 7.50 – 7.42 (m, 2H), 7.18 (dd, J = 8.2, 2.1 Hz, 1H), 3.58 (t, J = 7.0 Hz, 2H), 3.23 (t, J = 7.0 Hz, 2H), 3.17 (s, 2H), 3.11 (t, J = 6.9 Hz, 2H), 2.69 (t, J = 7.0 Hz, 2H), 2.27 (p, J = 2.6 Hz, 2H), 1.76 – 1.64 (m, 2H), 1.53 (p, J = 7.1 Hz, 2H); ¹³C NMR (126 MHz, DMSO) δ 171.84, 171.63, 169.22, 152.42, 147.56, 146.42, 145.28, 142.79, 141.43, 131.19, 131.14, 130.70, 129.70, 129.04, 127.97, 127.64, 124.22, 123.52, 120.82, 116.58, 41.02, 38.82, 34.44, 31.51,
27.42, 26.61; LC-MS (ESI+): RT 2.676 min, 95.3 % pure at 254 nm; HRMS (ESI+) m/z: calcd. for C29H30Cl2N5O4⁺: 582.1669; [M+H]⁺ found: 582.1667.

### 6-((8-(methoxycarbonyl)benzo[c][2,6]naphthyridin-5-yl)amino)hexan-1-aminium-2,2,2-trifluoroacetate (9)

Following **general method B1**, starting with compound **3** (200 mg, 0.733 mmol), the corresponding amine (190 mg, 0.880 mmol), K₂CO₃ (253 mg, 1.83 mmol) and NMP (2 mL) gave methyl 5-((6-((tert-butoxycarbonyl)amino)hexyl)amino)benzo[c][2,6]naphthyridine-8-carboxylate (332 mg, 99%). **General method A** with TFA (2 mL) was used to give compound **9** as a pale brown gel (176 mg, 68%).

### 7-((8-(methoxycarbonyl)benzo[c][2,6]naphthyridin-5-yl)amino)heptan-1-aminium-2,2,2-trifluoroacetate (10)

Following **general method B1**, starting with compound **3** (200 mg, 0.733 mmol), the corresponding amine (203 mg, 0.880 mmol) K₂CO₃ (253 mg, 1.87 mmol) and NMP (2 mL) yielded methyl 5-((7-((tert-butoxycarbonyl)amino)heptyl)amino)benzo[c][2,6]naphthyridine-8-carboxylate (341 mg, 99%). This was followed by **general method A** with TFA (2 mL) to give compound **10** as a pale brown gel (233 mg, 87%).

### 8-((8-(methoxycarbonyl)benzo[c][2,6]naphthyridin-5-yl)amino)octan-1-aminium 2,2,2-trifluoroacetate (11)

Following **general method B1**, starting with compound **3** (100 mg, 0.366 mmol), the corresponding amine (107 mg, 0.440 mmol) K₂CO₃ (127 mg, 0.916 mmol) and NMP (1 mL) yielded methyl 5-((8-((tert-butoxycarbonyl)amino)octyl)amino)benzo[c][2,6]naphthyridine-8-carboxylate (168 mg, 95%). This was followed by **general method A** with TFA (1 mL) to give compound **11** as a slightly brown solid (100 mg, 75%).
### 5-((6-(((2-chloro-[1,1'-biphenyl]-4-yl)methyl)amino)hexyl)amino)benzo[c][2,6]naphthyridine-8-carboxylic acid (Biv3)

Following general method E with compound 9 (176 mg, 0.495 mmol), 2-chloro-[1,1'-biphenyl]-4-carbaldehyde (119 mg, 0.549 mmol) in MeOH/AcOH (2.7/0.3 mL) gave methyl 5-((6-(((2-chloro-[1,1'-biphenyl]-4-yl)methyl)amino)hexyl)amino)benzo[c][2,6]naphthyridine-8-carboxylate (140 mg, 51%). The ester was hydrolysed using general method C with ester (121 mg, 0.219 mmol), LiOH (11.5 mg, 0.481 mmol) to yield 5-((6-(((2-chloro-[1,1'-biphenyl]-4-yl)methyl)amino)hexyl)amino)benzo[c][2,6]naphthyridine-8-carboxylic acid (Biv 3) as a pale yellow solid (64 mg, 54%). ¹H NMR (400 MHz, DMSO) δ 10.05 (s, 1H), 8.87 (d, *J* = 5.5 Hz, 1H), 8.70 (d, *J* = 8.5 Hz, 1H), 8.29 (d, *J* = 5.6 Hz, 1H), 8.16 (d, *J* = 1.7 Hz, 1H), 7.92 (t, *J* = 5.4 Hz, 1H), 7.83 (dd, *J* = 8.4, 1.8 Hz, 1H), 7.58 (d, *J* = 1.6 Hz, 1H), 7.51 – 7.31 (m, 7H), 3.82 (s, 2H), 3.62 (q, *J* = 6.5 Hz, 2H), 2.61 (t, *J* = 7.1 Hz, 2H), 1.75 (q, *J* = 6.9 Hz, 2H), 1.59 – 1.39 (m, 6H); ¹³C NMR (201 MHz, DMSO) δ 169.55, 152.85, 147.72, 147.18, 145.39, 139.33, 138.84, 138.42, 136.51, 131.74, 131.55, 130.65, 129.60, 128.65, 128.56, 128.18, 127.94, 127.49, 124.03, 123.25, 122.03, 120.25, 116.65, 50.69, 47.84, 41.15, 28.89, 27.77, 26.90, 26.72; LC-MS (ESI+): RT 5.318 min, > 99% pure at 254 nm; HRMS (ESI+) m/z: calcd. for C₃₂H₃₂ClN₄O₂⁺: 539.2208; [M+H]⁺ found: 539.2210.

### 5-((7-(((2-chloro-[1,1'-biphenyl]-4-yl)methyl)amino)heptyl)amino)benzo[c][2,6]naphthyridine-8-carboxylic acid (Biv4)

Following general method E with compound 10 (233 mg, 0.636 mmol), 2-chloro-[1,1'-biphenyl]-4-carbaldehyde (152 mg, 0.697 mmol) in MeOH/AcOH (3.6/0.4 mL) gave methyl 5-((7-(((2-chloro-[1,1'-biphenyl]-4-yl)methyl)amino)heptyl)amino)benzo[c][2,6]naphthyridine-8-carboxylate (188 mg, 52%). The ester was hydrolysed using general method C with ester (170 mg, 0.300 mmol), LiOH (16 mg, 0.659 mmol) to give 5-((7-(((2-chloro-[1,1'-biphenyl]-4-yl)methyl)amino)heptyl)amino)benzo[c][2,6]naphthyridine-8-carboxylic acid (Biv 4) as a pale yellow solid (107 mg, 64%). ¹H NMR (400 MHz, DMSO) δ 10.06 (s, 1H), 8.87 (d, *J* = 5.5 Hz, 1H), 8.69 (d, *J* = 8.5 Hz, 1H), 8.28 (d, *J* = 5.5 Hz, 1H), 8.15 (d, *J* = 1.8 Hz, 1H), 7.94 (t, *J* = 5.5 Hz, 1H), 7.83 (dd, *J* = 8.4, 1.8 Hz, 1H), 7.62 (d, *J* = 1.6 Hz, 1H), 7.49 – 7.34 (m, 7H), 3.86 (s, 2H), 3.62 (q, *J* = 6.5 Hz, 2H), 2.63 (t, *J* = 7.3 Hz,
2H), 1.77 – 1.67 (m, 2H), 1.57 (p, *J* = 7.3 Hz, 2H), 1.49 – 1.32 (m, 7H); <sup>13</sup>C NMR (201 MHz, DMSO) δ 168.58, 152.51, 147.41, 146.94, 145.00, 138.86, 138.44, 138.29, 134.87, 131.32, 131.10, 130.16, 129.19, 128.22, 128.09, 127.75, 127.52, 127.00, 123.69, 122.61, 121.85, 120.20, 116.20, 50.39, 47.59, 40.57, 28.54, 28.43, 27.57, 26.55, 26.51; LC-MS (ESI+): RT 5.419 min, 96.6% pure at 254 nm; HRMS (ESI+) m/z: calcd. for C<sub>33</sub>H<sub>34</sub>ClN<sub>4</sub>O<sub>2</sub><sup>+</sup>: 553.2365; [M+H]<sup>+</sup> found: 553.2366.

### 5-((8-(((2-chloro-[1,1'-biphenyl]-4-yl)methyl)amino)octyl)amino)benzo[c][2,6]naphthyridine-8-carboxylic acid (Biv5)

Following **general method E** with compound **11** (100 mg, 0.263 mmol), 2-chloro-[1,1'-biphenyl]-4-carbaldehyde (63 mg, 0.287 mmol) in MeOH/AcOH (1.8/0.2 mL) gave methyl 5-((8-(((2-chloro-[1,1'-biphenyl]-4-yl)methyl)amino)octyl)amino)benzo[c][2,6]naphthyridine-8-carboxylate (71 mg, 46%). The ester was hydrolysed using **general method C** with ester (51 mg, 0.088 mmol), LiOH (4.6 mg, 0.193 mmol) to give 5-((8-(((2-chloro-[1,1'-biphenyl]-4-yl)methyl)amino)octyl)amino)benzo[c][2,6]naphthyridine-8-carboxylic acid (Biv 5) acid as a pale yellow solid (37 mg, 74%).<sup>1</sup>H NMR (400 MHz, DMSO) δ 10.09 (s, 1H), 8.90 (d, *J* = 5.6 Hz, 1H), 8.71 (d, *J* = 8.5 Hz, 1H), 8.30 (d, *J* = 5.6 Hz, 1H), 8.20 (d, *J* = 1.7 Hz, 1H), 7.96 (t, *J* = 5.5 Hz, 1H), 7.87 (dd, *J* = 8.3, 1.7 Hz, 1H), 7.68 (d, *J* = 1.6 Hz, 1H), 7.56 – 7.37 (m, 7H), 3.91 (s, 2H), 3.65 (q, *J* = 6.6 Hz, 2H), 2.67 (t, *J* = 7.3 Hz, 2H), 1.77 (p, *J* = 6.6 Hz, 2H), 1.61 (t, *J* = 7.2 Hz, 2H), 1.51 – 1.36 (m, 8H); <sup>13</sup>C NMR (201 MHz, DMSO) δ 169.11, 152.86, 147.82, 147.26, 145.43, 139.17, 138.93, 136.23, 131.74, 131.53, 130.48, 129.63, 128.65, 128.43, 128.16, 127.97, 127.51, 124.06, 123.14, 122.13, 120.35, 116.61, 51.00, 48.14, 40.94, 29.19, 28.96, 28.63, 28.23, 27.13, 26.85; LC-MS (ESI+): RT 5.641 min, 97.8% pure at 254 nm; HRMS (ESI+) m/z: calcd. for C<sub>34</sub>H<sub>36</sub>ClN<sub>4</sub>O<sub>2</sub><sup>+</sup>: 567.2521; [M+H]<sup>+</sup> found: 567.2524.

### 3-((4-((8-(methoxycarbonyl)benzo[c][2,6]naphthyridin-5-yl)amino)butyl)amino)-3-oxopropan-1-aminium-2,2,2-trifluoroacetate (12)

According to **general method D2** with compound **7** (71 mg, 0.219 mmol), Boc-β-alanine (41 mg, 0.219 mmol), DIPEA (57 µL, 0.328 mmol), EDC.HCl (50 mg, 0.263 mmol), HOBt (44 mg, 0.263 mmol), DMF (1 mL) to give methyl 5-((4-((tert-
butoxycarbonyl)amino)propanamido)butyl)amino)benzo[c][2,6]naphthyridine-8-carboxylate (107 mg, 99%). **General method A** with the corresponding protected amine, $CH_2Cl_2$ (0.5 mL) and TFA (0.5 mL) afforded compound **12** as a white solid (101 mg, 91%).

### 5-((4-(3-(((2-chloro-[1,1'-biphenyl]-4-yl)methyl)amino)propanamido)butyl)amino)benzo[c][2,6]naphthyridine-8-carboxylic acid (Biv6)

Following general method E with compound **12** (137 mg, 0.346 mmol), 2-chloro-[1,1'-biphenyl]-4-carbaldehyde (83 mg, 0.381 mmol) in MeOH/AcOH (2.7/0.3 mL) gave methyl 5-((4-(3-(((2-chloro-[1,1'-biphenyl]-4-yl)methyl)amino)propanamido)butyl)amino)benzo[c][2,6]naphthyridine-8-carboxylate (161 mg, 78%). The ester was hydrolysed by **general method C** with ester (120 mg, 0.201 mmol), LiOH (17 mg, 0.705 mmol) to give 5-((4-(3-(((2-chloro-[1,1'-biphenyl]-4-yl)methyl)amino)propanamido)butyl)amino)benzo[c][2,6]naphthyridine-8-carboxylic acid (Biv 6) acid as a pale yellow solid (117 mg, 99%). ¹H NMR (400 MHz, DMSO) δ 10.06 (s, 1H), 9.33 (s, 1H), 8.88 (d, $J = 5.5$ Hz, 1H), 8.73 (d, $J = 8.5$ Hz, 1H), 8.34 (d, $J = 5.6$ Hz, 1H), 8.19 (q, $J = 9.8, 7.7$ Hz, 1H), 8.13 (d, $J = 1.8$ Hz, 1H), 8.05 (t, $J = 5.3$ Hz, 1H), 7.81 (dd, $J = 7.8, 1.7$ Hz, 2H), 7.59 (dd, $J = 7.9, 1.8$ Hz, 1H), 7.51 – 7.38 (m, 7H), 4.18 (s, 2H), 3.68 – 3.58 (m, 3H), 3.13 (dt, $J = 24.9, 7.0$ Hz, 5H), 2.60 (t, $J = 7.3$ Hz, 3H), 1.80 – 1.68 (m, 2H), 1.56 (q, $J = 7.2$ Hz, 2H); ¹³C NMR (201 MHz, DMSO) δ 168.90, 167.43, 152.85, 147.55, 147.41, 145.04, 140.14, 138.19, 133.40, 131.62, 131.48, 131.44, 131.28, 129.30, 129.21, 128.36, 128.02, 127.70, 126.79, 124.03, 122.52, 122.21, 121.30, 116.49, 48.88, 42.81, 40.49, 38.50, 31.03, 26.81, 26.06; LC-MS (ESI+): RT 5.056 min, >99 % pure at 254 nm; HRMS (ESI+) m/z: calcd. for $C_{33}H_{33}ClN_5O_3^+$: 582.2266; [M+H]+ found: 582.2268.

### 5-((4-(3-(((2'-methoxy-[1,1'-biphenyl]-4-yl)methyl)amino)propanamido)butyl)amino)benzo[c][2,6]naphthyridine-8-carboxylic acid (Biv7)

Following general method E with compound **12** (141 mg, 0.346 mmol), 2'-methoxy-[1,1'-biphenyl]-4-carbaldehyde (83 mg, 0.382 mmol) in MeOH/AcOH (2.7/0.3 mL) gave methyl 5-((4-(3-(((2'-methoxy-[1,1'-biphenyl]-4-yl)methyl)amino)propanamido)butyl)amino)benzo[c][2,6]naphthyridine-8-carboxylate (174 mg, 82%). The ester was hydrolysed according to general method C with ester (134
mg, 0.226 mmol), LiOH (19 mg, 0.793 mmol) to give 5-((4-(3(((2'-methoxy-[1,1'-biphenyl]-4-yl)methyl)amino)propanamido)butyl)amino)benzo[c][2,6]naphthyridine-8-carboxylic acid (Biv 7) as pale yellow solid (105.5 mg, 81%). ¹H NMR (400 MHz, DMSO) δ 10.05 (s, 1H), 8.78 (dd, *J* = 64.7, 7.0 Hz, 2H), 8.31 – 7.75 (m, 5H), 7.46 – 6.96 (m, 8H), 3.89 (s, 2H), 3.74 (s, 4H), 3.61 (d, *J* = 6.2 Hz, 3H), 3.14 (q, *J* = 6.2 Hz, 3H), 2.89 (t, *J* = 7.1 Hz, 2H), 2.41 (t, *J* = 7.1 Hz, 3H), 1.74 (p, *J* = 7.2 Hz, 2H), 1.54 (p, *J* = 7.1 Hz, 2H); ¹³C NMR (201 MHz, DMSO) δ 169.79, 168.22, 156.12, 152.65, 147.49, 147.13, 145.00, 137.96, 133.79, 133.57, 130.36, 129.35, 129.31, 129.04, 127.64, 126.95, 123.82, 122.55, 122.10, 120.81, 120.60, 116.30, 111.73, 55.47, 50.63, 43.53, 40.57, 38.39, 32.70, 26.85, 26.03; LC-MS (ESI+): RT 4.874 min, >99 % pure at 254 nm; HRMS (ESI+) m/z: calcd. for C₃₄H₃₆N₅O₄⁺: 578.2762; [M+H]⁺ found: 578.2761.

## 4.2 CK2 purification

CK2α (1-335 AA) sample was prepared for TSA and ADP-Glo assays. The pGEX-2T-CK2α plasmid was kindly given by Odile Filhol. CK2α as a glutathione S-transferase (GST) fusion protein expression was expressed in E. coli BL21 (Invitrogen). Bacteria were grown in LB-Broth medium (Thermofischer) containing 100 mg/L ampicillin (Euromedex). When the absorbance at 600 nm of the cultures reached 0.6, induction was performed by the addition of 0.1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside, Euromedex) for a period of 20 hours at 25°C. The bacterial pellet was recovered by centrifugation at 4,000 g at 4°C for 20 min and lysed with cold lysis buffer (40 mM Tris-HCl, pH 7.5, 0.3 % Triton X-100, 750mM NaCl, 2mM EDTA, 2mM EGTA, 2mM DTT, 1 mg/mL lysozyme (prepared extemporaneously), 0.1U/µL benzonase (Sigma), and protease inhibitor cocktail with EDTA (1 tablet/10 mL, Sigma). The lysis process was continued on a rotating wheel for 30 min at 4°C. The lysate was passed through a 21" syringe and subjected to centrifugation for one hour at 10,000 g. The resulting supernatant was filtered through a 0.45 µm filter. CK2α-GST was purified by affinity chromatography on a GSTrapTM FF column (Cytiva) according to the manufacturer's recommendations. GST tag cleavage was performed on the affinity column with 80 U of thrombin (Dutscher) overnight at 4°C. CK2α was eluted in PBS (10 mM Na₂HPO₄, 140 mM NaCl, 2.7 mM KCl, 1.8 mM KH2PO4, pH 7.4) containing 2 % glycerol. Protein was quantified spectrophotometrically at 280 nM and concentrated to 4 mg/mL on centrifugal filter with a cut off of
15 kDa (Amicon Ultra 15, Merck Millipore). Protein purity was verified by 12 % SDS polyacrylamide
gel electrophoresis.

For the structural studies, the plasmid pNIC28-Bsa4 containing the cDNA encoding amino acids 1-337
of CK2α1A was used. The plasmid was kindly provided by Stephan Knapp's team. E.coli BL21
bacteria were transformed with this plasmid. expression was performed in LB medium and
purification of CK2α was carried out by the SFR biosciences Protein Science Facility (Lyon) as
described previously [18].

**4.3 Thermal shift assay**

Thermal shift assays were performed in PBS-0.9% glycerol. Briefly, recombinant CK2α (10 µM) was incubated with DMSO (vehicle control), compounds X-Y, with a final drug concentration of 10 µM, and a final DMSO concentration of 5% (v/v). Sypro-Orange dye (Invitrogen) was added to each drug treatment (5x final concentration). Reagent buffer was PBS-0.9% glycerol. Thermal scanning (20-80 °C at 0.01°C/s) was performed in 96 well plate (Armadillo, Thermo Scientific) using a Real-Time PCR System (Roche Lighcycler 480) and fluorescence intensity was measured (50 acquisitions per °C). Subsequent analysis and Tm determination were performed using the TSA-CRAFT software [19].

**4.4. Kinase inhibition using the ADP-glo assay**

The ADP-Glo™ (Promega) was used to measure ADP formed during the phosphorylation of a CK2α-
peptide substrate, RRREDEESDDEE (80 µg/mL), by recombinant CK2α (50 nM) in presence of 25
µM ATP. The kinase assays were performed in 40 mM Tris-HCl pH7.5, 200 mM NaCl, 20 mM
MgCl2, 0.1 mg/mL BSA containing various test compounds at a constant concentration of DMSO (5
%). After 40 min at 25°C, ADP-Glo reagent was added to stop the kinetic reaction and degrade
residual ATP. After 60 min, the Kinase Detection Reagent is added to simultaneously convert ADP to
ATP and allow the newly synthesized ATP to be measured using a luciferase/luciferin reaction. After
30 min, luminescence was measured using a TECAN Infinite 200Pro plate reader.

**4.5 Crystallization and structure refinement**
Wild-Type protein concentration was 9 mg/ml. Crystals of human CK2α were grown at 20 °C using the hanging-drop vapor-diffusion method with a reservoir solution containing 33 % polyethylene glycol methyl ether 5000, 0.2 M ammonium sulfate, 0.1 MES pH 6.5. The drops contained 1µl of the reservoir solution and 1 µl of the protein. The crystals were soaked by adding 0.2 µl of ligand solution at 50 to 100 mM in DMSO. The crystals were cryo-protected with reservoir solution supplemented with 20 % glycerol and then flash-cooled in liquid nitrogen. X-ray diffraction data were collected at the ESRF Synchrotron in Grenoble, France, on beamlines ID30-A1 and ID23-2 (https://doi.org/10.15151/ESRF-DC-2123184537). Data were processed and scaled with the program suites installed at the ESRF[20]. The crystals belong to the space group P43212 with two monomers in the asymmetric unit. The structures were solved by molecular replacement using PDB entry 6Z84 as the search model. Bound ligands were manually identified and fitted into Fo–Fc electron density using Coot [21] and the phenix ligand pipeline from Phenix suite (https://phenix-online.org/documentation/reference/ligand_pipeline.html). The generation of geometry restraint information for refinement of novel ligands was done using Grade Server [22] or eLBOW software [23]. The structures were refined by rounds of rebuilding in Coot and refinement using Phenix [24].

**4.6. Radiometric kinase assay**

The kinase assays were performed at Eurofins Discovery. Protein kinase was incubated with 20 mM HEPES pH 7.4, 0.15 M NaCl, 0.1 mM EDTA, 5 mM DTT, 0.1% Triton X-100, 165 µM RRRDDDSDDD, 10 mM Magnesium acetate and [γ-33P]-ATP (at concentration as indicated in Table 2). The reaction was initiated by the addition of the Mg/ATP mix. After incubation for 40 minutes at room temperature, the reaction was stopped by the addition of phosphoric acid to a concentration of 0.5%. An aliquot of the reaction was then spotted onto a filter and washed four times for 4 minutes in 0.425% phosphoric acid and once in ethanol prior to drying and scintillation counting.

**4.7. Cell culture**
Vero, Huh7, HEK-ACE2-TMPRSS2, and HRT-18 cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco™, Thermo Fisher Scientific®, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 50 IU/mL penicillin, 100 µg/mL streptomycin, and 0.1 µg/mL amphotericin B. For HRT-18 cells, the medium was further enriched with 10% sodium bicarbonate (Gibco™) and 1% non-essential amino acids. The HEK-ACE2-TMPRSS2 cells were maintained with additional supplements, including 100 µg/mL hygromycin, 1.5 µg/mL puromycin, and 2 µg/mL poly-D-lysine.

## 4.8. Viruses

SARS-CoV-2 strain B.117.18 was isolated from nasopharyngeal swabs of patient at Henri Mondor University Hospital in Créteil, France and propagated in Vero cells. HCoV-OC43 (VR-1558 virus, batch number 58087760, ATCC) were propagated in and HRT-18 cells.

## 4.9. Cellular Viability

HEK-ACE2-TMPRSS2 cells were seeded at a density of 15,000 cells per well in a 96-well plate and incubated for 24 hours. Upon reaching approximately 75% confluency, the cells were treated with bivalent CK2 inhibitors at concentrations ranging from 0.1 µM to 20 µM in complete culture medium. After 24 hours of treatment, cell viability was assessed using the MTT assay. Briefly, 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to the culture medium, and the cells were incubated further. The absorbance was measured at 550 nm, with background absorbance from the culture medium and lysis solution subtracted.

## 4.10. Virus Inhibition Assay

Vero cells (20,000 cells per well in a 96-well plate), HEK-293T ACE2/TMPRSS2 cells (150,000 cells per well in a 24-well plate), Huh7 cells (10,000 cells per well in a 96-well plate), and HRT-18 cells (30,000 cells per well in a 96-well plate) were infected with the indicated virus at a multiplicity of infection (MOI) of 1 in DMEM supplemented with 2% FBS. The cells were incubated at 37°C with 5% CO₂ for 2 hours in the presence of the indicated concentrations of the bivalent CK2
inhibitor. Following the 2-hour infection period, the cells were washed with PBS and fresh medium containing the bivalent CK2 inhibitors was added. Supernatants were collected 24 hours post-infection, while the cells were washed once with PBS and harvested for RNA extraction and RT-qPCR analysis.

## 4.11. Human Nasal Epithelial Cells

Human nasal epithelial cells (HNECs) derived from healthy donors were commercially obtained from Epithelix (Sarl, Geneva, Switzerland) and cultured according to the manufacturer's instructions in Mucil Air™ medium. SARS-CoV-2 infection was conducted at the apical surface by applying 20 µL of viral inoculum for 4 hours at 37°C in the presence of bivalent CK2 inhibitors at a final concentration of 20 µM. At 4, 24, 48, 72, and 96 hours post-infection, the viral inoculum was collected, and the cells were washed twice with 200 µL of PBS. Supernatants were analyzed using RT-qPCR to assess the dynamics of viral replication. At 96 hours p.i., the HNECs were lysed and harvested for intracellular viral RNA quantification via RT-qPCR. Cell viability was evaluated daily using optical microscopy to monitor cellular morphology and overall health. Epithelial barrier integrity was assessed by measuring transepithelial electrical resistance (TEER) with an EVOM volt-ohmmeter (World Precision Instruments, Sarasota, FL, USA).

## 4.12. Immunofluorescence

Vero-E6 and HEK-T ACE2/TMPRSS2 cells were seeded at a density of 500,000 cells per well in 8-well Labtek Permanox plates (Sigma-Aldrich, Saint-Louis, USA) and incubated at 37°C with 5% CO₂ for 24 hours. Following incubation, cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 1 in the presence of varying concentrations 20 µM of the indicated compounds. After 24 hours of infection, cells were fixed using 4% paraformaldehyde (Sigma-Aldrich) and permeabilized in a saturation buffer containing 0.3% Triton X-114 and 10 mg/ml bovine serum albumin (BSA). For immunofluorescence, cells were incubated overnight at 4°C with a primary antibody targeting the SARS-CoV-2 nucleoprotein (Genetex GTX1357357, 1:1000 dilution). Then for
one hour at room temperature with the goat anti-rabbit Alexa Fluor secondary antibodies (Thermo
Fisher Scientific, A11037, 1:1000 dilution).

4.13. *Viral RNA Extraction and RT-qPCR*

SARS-CoV-2 RNA was extracted from the cell supernatant using the QIAmp Viral RNA Mini Kit (Qiagen, Hilden, Germany; Reference: 52906) for HNCEs and the 96 Virus Core Kit from Macherey-Nagel for all other infection experiments. Intracellular viral RNA was extracted utilizing the RNeasy® Mini Kit (Qiagen, Hilden, Germany) for the HNCEs, and the SV96 Total RNA Isolation System from Promega for the remaining infection experiments. Intracellular RNA samples were reverse-transcribed using The High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific®). For intracellular RNA quantification, the TaqMan Gene Expression Master Mix (Applied Biosystems by Thermo Fisher Scientific) was employed, whereas the GoTaq® Probe One-Step RT-qPCR System (Promega; Reference) was utilized for extracellular RNA. Relative quantification of SARS-CoV-2, HCoV-OC43, and HCoV-229E RNA levels was performed using the 2-ΔΔCT method. All samples were normalized to endogenous eukaryotic controls, specifically glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA or 18S rRNA (VIC™/TAMRA™ probe, Life Technologies), depending on the experimental setup.

4.14. Western Blot

Cells were lysed using EZ Lysis Buffer (20 mM Tris-HCl, pH 6.8; 100 mM NaCl; 1 mM EDTA; 0.5% NP40; and 10% glycerol) and total protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) following the manufacturer's protocol. Denatured protein samples were resolved on a Criterion™ XT Precast Gel (4-12% Bis-Tris polyacrylamide, BioRad) via SDS-PAGE and transferred to nitrocellulose membrane (Thermo Fisher Scientific®) in transfer buffer (2.5 mM Tris base, 19.2 mM glycine, 2% methanol, pH 8.3). The membrane was blocked for 1 hour at room temperature in TBS buffer containing 0.05% Tween-20 and supplemented with 5% non-fat dry milk. Blocked membranes were incubated overnight at 4 °C with a primary anti-rabbit antibody targeting SARS-CoV-2 Nucleoprotein (GeneTex,) at a 1:1000 dilution.
Next, membranes were incubated with an HRP-conjugated secondary antibody (Thermo Fisher Scientific®, Carlsbad, CA, USA) for 1 hour at room temperature. Protein bands were visualized using ECL™ Prime Western Blotting Detection Reagent (Cytiva, Amersham,). Chemiluminescent signals were detected and analyzed using ImageQuant LAS4000 software for quantification.

## 5. Author information

Manuscript was drafted by IK and edited by all authors. DG, JFG and IK designed compounds which were synthesized by DG. JGD performed TSA and ADP-Glo measurements with CM. AM provided the protein for the TSA and ADP-Glo assay. MG performed the X-ray crystallography and MG, JFG and IK performed the structural analyses; YY recorded the ¹³C NMR experiments; DSLM and NA performed the virus inhibition assay in HNEC, HEK-AT, and VERO cells, western blot, viral RNA extraction, RT-qPCR, and immunofluorescence. DSLM performed the viral production and cellular viability assay. Scientific supervision was performed by AAB and IK.

## Declaration of competing interest

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.

## Acknowledgement

We thank the Institut Convergence PLAsCAN (ANR-17-CONV-002) as well as ANR-21-CE18-0014-01 for project ANR-CK2COV. We acknowledge the contribution of SFR Biosciences (UAR3444/CNRS, US8/Inserm, ENS de Lyon, UCBL): Protein Science Facilities. We are grateful to Dr David Knapp and Dr Andreas Krämer for the gift of the CK2α plasmid and their advice for CK2α crystallization. We would like to thank the staff of the ESRF and EMBL Grenoble for assistance and support in using beamline(s) ID30-A1 and ID23-2 under proposal number mx2600. The CBS is a member of the French Infrastructure for Integrated Structural Biology (FRISBI), a national infrastructure supported by the French National Research Agency (ANR-10-INBS-05).
## Data availability

Data will be made available on request.

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