Sulfonyl fluorides (SFs) have recently emerged as a promising warhead for the targeted covalent modification of proteins. Despite numerous examples of the successful deployment of SFs as covalent probe compounds, a detailed exploration of the factors influencing the stability and reactivity of SFs has not yet appeared. In this work we present an extensive study on the influence of steric and electronic factors on the reactivity and stability of the SF and related S-F groups. While SFs react rapidly with N-acetylcysteine, the resulting adducts were found to be unstable, rendering SFs inappropriate for the durable covalent inhibition of cysteine residues. In contrast, SFs afforded stable adducts with both N-acetyltyrosine and N-acetyllysine; furthermore, we show that the reactivity of arylsulfonyl fluorides towards these nucleophilic amino acids can be predictably modulated by adjusting the electronic properties of the warhead. These trends were largely conserved when the covalent reaction occurred within a protein binding pocket. We have also obtained a crystal structure depicting covalent modification of the catalytic lysine of a tyrosine kinase (FGFR1) by the ATP analog 5'-O-3-((fluorosulfonyl)benzoyl)adenosine (m-FSBA). Highly reactive warheads were demonstrated to be unstable with respect to hydrolysis in buffered aqueous solutions, indicating that warhead reactivity must be carefully tuned to provide optimal rates of protein modification. Our results demonstrate that the reactivity of SFs complements that of more commonly studied acrylamides, and we hope that this work spurs the rational design of novel SF-containing covalent probe compounds and inhibitors, particularly in cases where a suitably positioned cysteine residue is not present.

ABSTRACT:The Wnt pathway is an evolutionarily conserved and tightly regulated signaling network with important roles in embryonic development and adult tissue regeneration. Impaired Wnt pathway regulation, arising from mutations in Wnt signaling components, such as Axin, APC, and β-catenin, results in uncontrolled cell growth and triggers oncogenesis. To explore the reported link between CK2 kinase activity and Wnt pathway signaling, we sought to identify a potent, selective inhibitor of CK2 suitable for proof of concept studies in vivo. Starting from a pyrazolo[1,5-a]pyrimidine lead (2), we identified compound 7h, a potent CK2 inhibitor with picomolar affinity that is highly selectivity against other kinase family enzymes and inhibits Wnt pathway signaling (IC 50 = 50 nM) in DLD-1 cells. In addition, compound 7h has physicochemical properties that are suitable for formulation as an intravenous solution, has demonstrated good pharmacokinetics in preclinical species, and exhibits a high level of activity as a monotherapy in HCT-116 and SW-620 xenografts. KEYWORDS: CK2 kinase, pyrazolo[1,5-a]pyrimidine, Wnt, β-catenin T he serine/threonine protein kinase CK2 is a constitutively active heterotetrameric complex composed of two catalytic (α or α′) and two regulatory (β) subunits, 1 which has emerged as an attractive drug discovery target in oncology. 2 Researchers from Cylene have recently advanced CX-4945, a selective, orally available inhibitor of CK2 into the clinic for treatment of patients with solid tumors and hematological malignancies. 3 Among its diverse functions, CK2 interacts with and regulates multiple components of the Wnt pathway, an evolutionarily conserved signaling network that regulates embryonic development and the regeneration of intestinal epithelial cells. 4 Certain cancers, including colorectal carcinoma (CRC), arise due to gene mutations among constituents of the Wnt pathway, including the CK2 substrates dishevelled (Dvl), APC, and β-catenin. 5 Inhibition of CK2, either by RNA knockdown or with small molecules, decreases β-catenin−Tcfmediated transcription of Wnt target genes such as survivin and leads to cell death and apoptosis in a range of CRC lines. 6,7 In addition, elevated levels of CK2 activity have been reported in CRC tissue samples and expression levels correlate with poor prognosis in CRC patients. 8,9 Taken together, these data illustrate the potential utility of CK2 inhibitors in CRC and other cancers characterized by aberrant Wnt pathway activity.We sought to identify a potent, selective inhibitor of CK2 kinase for hypothesis testing in vivo using preclinical models of CRC. An early probe from our previously described series of ATP-competitive pyrazolo[1,5-a]pyrimidine-derived inhibitors of CK2 (1, 2; Figure 1) was used to assess the link between CK2 inhibition and Wnt signaling. 10 Treatment of DLD-1(APC mutant) cells with 2 inhibits β-catenin phosphorylation and decreases Wnt-mediated gene transcription as shown in a Luciferase reporter assay in APC

No abstract