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. 2024 Sep 2;29(17):4158.
doi: 10.3390/molecules29174158.

Strategic Fluorination to Achieve a Potent, Selective, Metabolically Stable, and Orally Bioavailable Inhibitor of CSNK2

Affiliations

Strategic Fluorination to Achieve a Potent, Selective, Metabolically Stable, and Orally Bioavailable Inhibitor of CSNK2

Han Wee Ong et al. Molecules. .

Abstract

The host kinase casein kinase 2 (CSNK2) has been proposed to be an antiviral target against β-coronaviral infection. To pharmacologically validate CSNK2 as a drug target in vivo, potent and selective CSNK2 inhibitors with good pharmacokinetic properties are required. Inhibitors based on the pyrazolo[1,5-a]pyrimidine scaffold possess outstanding potency and selectivity for CSNK2, but bioavailability and metabolic stability are often challenging. By strategically installing a fluorine atom on an electron-rich phenyl ring of a previously characterized inhibitor 1, we discovered compound 2 as a promising lead compound with improved in vivo metabolic stability. Compound 2 maintained excellent cellular potency against CSNK2, submicromolar antiviral potency, and favorable solubility, and was remarkably selective for CSNK2 when screened against 192 kinases across the human kinome. We additionally present a co-crystal structure to support its on-target binding mode. In vivo, compound 2 was orally bioavailable, and demonstrated modest and transient inhibition of CSNK2, although antiviral activity was not observed, possibly attributed to its lack of prolonged CSNK2 inhibition.

Keywords: CSNK2; SARS-CoV-2; antiviral; fluorination; pyrazolo[1,5-a]pyrimidine; β-coronavirus.

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Conflict of interest statement

Authors Jason W. Brown, Edcon Chang were employed by the company Takeda Development Center Americas, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Selectivity profile of compound 2 (10 μM) against the NanoBRET K192 panel. Only CSNK2A1 and CSNK2A2 had 97% occupancy (red). Kinases with 50–85% occupancy are shown in yellow. Kinases with <50% occupancy are shown in green. Kinases not in the NanoBRET K192 panel are shown in grey. Image generated using CORAL [31]. No kinases had occupancy > 85% except CSNK2A1 and CSNK2A2.
Figure 2
Figure 2
(A) Co-crystal structure of compound 2 (white) with CSNK2A1 (green) (PDB ID: 9FYF). (B) Overlay of the co-crystal structure of compound 2 (white) with CSNK2A1 (green) and of SGC-CK2-1 (rose) with CSNK2A1 (cyan) (PDB: 6Z83) [24]. Part of the P-loop is hidden for clarity. Water molecules are represented as red spheres. Intermolecular hydrogen bonds are shown as yellow dashed lines. Key residues in the ATP-binding pocket are labeled.
Figure 3
Figure 3
A 24 h PK study of compound 2 in CD-1 mice. The plasma concentrations of compound 2 at the 24 h time point when dosed at 3 mg/kg i.v. and 10 mg/kg p.o. are below the limit of quantification (1.02 nM). The lung concentration of compound 2 at the 24 h time point when dosed at 3 mg/kg i.v. is recorded for one mouse. The other two mice had lung concentrations below the limit of quantification.
Figure 4
Figure 4
Snapshot PK study of compound 2 (10 mg/kg) to CD-1 mice administered via i.p. injection, and effect of co-dosing with EA (30 mg/kg) and 1-ABT (100 mg/kg).
Figure 5
Figure 5
Multi-dose PK study of compound 2 in Balb/c mice. Compound 2 was administered at 30 mg/kg p.o. every 12 h. The free plasma concentration of compound 2 was calculated based on the total plasma concentration measured factored by the fraction unbound (fu) in mouse plasma. A comparison with the cellular SARS-CoV-2 IC50 and IC90 (yellow dashed lines) and the mean CSNK2A1 and CSNK2A2 IC50 and IC90 determined in the NanoBRET assay (gray dashed lines) are provided.
Figure 6
Figure 6
In vivo CSNK2 inhibition. (A) SARS-CoV-2 viral titer measured in mouse lungs 24 h post-inoculation when treated with vehicle or with compound 2 (30 mg/kg p.o. b.i.d.) (n = 5). (B) AKT (phospho-Ser129) levels normalized to total AKT levels in mouse lungs following treatment with compound 2 (30 mg/kg p.o. b.i.d.) for 3, 6, and 36 h (n = 3). (C) EIF2S2 (phospho-Ser2) levels normalized to total EIF2S2 levels in mouse lungs following treatment with compound 2 (30 mg/kg p.o. b.i.d.) for 3, 6, and 36 h (n = 3). **: p = 0.0028 using unpaired t-test with Welch’s correction.
Scheme 1
Scheme 1
Synthesis of compound 2. Reagents and conditions: (a) cyclopropylamine, EtOH, 25 °C, 2 h; (b) K2CO3, MeCN, 100 °C, 12 h; (c) CbzCl, K2CO3, THF, 0 °C; (d) Fe, NH4Cl, EtOH, H2O, 90 °C, 12 h; (e) propanoyl chloride, DIPEA, DCM, 0–25 °C, 12 h; (f) H2, Pd/C, MeOH, 25 °C, 12 h; (g) 4, Pd(OAc)2, BINAP, Cs2CO3, dioxane, μW, 130 °C, 2 h; (h) TFA, DCM, 25 °C, 2 h.

References

    1. WHO COVID-19 Dashboard. [(accessed on 22 April 2024)]; Available online: https://covid19.who.int/
    1. Payne S. Viruses. Elsevier; Amsterdam, The Netherlands: 2017. Family Coronaviridae; pp. 149–158.
    1. Shereen M.A., Khan S., Kazmi A., Bashir N., Siddique R. COVID-19 infection: Emergence, transmission, and characteristics of human coronaviruses. J. Adv. Res. 2020;24:91–98. doi: 10.1016/j.jare.2020.03.005. - DOI - PMC - PubMed
    1. Richman D.D., Nathanson N. Viral Pathogenesis. Elsevier; Amsterdam, The Netherlands: 2016. Antiviral Therapy; pp. 271–287.
    1. Ryu W.-S. Molecular Virology of Human Pathogenic Viruses. Elsevier; Amsterdam, The Netherlands: 2017. Antiviral Therapy; pp. 367–381.

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