SGC-CAMKK2-1: A Chemical Probe for CAMKK2

Abstract

The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2.

Keywords: CAMKK2; NanoBRET; chemical probe; kinase; kinase selectivity.

Figure 1

STO-609 and some reported (Price et al. numbering noted in parenthesis [47]) CAMKK2 inhibitors. Potencies are depicted as pIC₅₀ values. pIC₅₀ values are the negative log of the IC₅₀ value (in molar). Thus, a pIC₅₀ = 6 corresponds to an IC₅₀ of 1μM, a pIC₅₀ = 7 corresponds to an IC₅₀ = 100 nM, a pIC₅₀ = 8 corresponds to an IC₅₀ = 10 nM, and a pIC₅₀ = 9 corresponds to an IC₅₀ = 1 nM.

Scheme 1

Synthetic route to compounds 5 and 6. Reagents and conditions (a) 8, Pd(dppf)Cl₂ (0.05 eq.), Cs₂CO₃ (2 eq.), 1,4-dioxane/water (4:1), 120 °C microwave, 30 min; (b) ArB(OH)₂ (1 eq.), Pd(dppf)Cl₂ (0.05 eq.), Cs₂CO₃ (2 eq.), 1,4-dioxane/water (4:1), 120 °C microwave, 30 min; (c) 1 N NaOH, MeOH, 75 °C then aq. HCl, 1 h.

Figure 2

(a) The structure and enzyme inhibition IC₅₀ of the CAMKK2 probe molecule. (b) The selectivity of compound 5 (SGC-CAMKK2-1) depicted using a TREEspot diagram obtained from profiling SGC-CAMKK2-1 at 1 µM in the KinomeSCAN^® assay panel. PoC = percentage of control. A PoC of 100 indicates no binding and a PoC = 0 is reflective of complete binding. Thus, kinases with PoC values closer to zero are the assay hits and are the kinases to which our compound binds.

Figure 3

Structure and characterization of the negative control compound SGC-CAMKK2-1N (all data n = 1).

Scheme 2

Synthesis of negative control 7. Reagents and conditions: (a) PhB(OH)2, Cs₂CO₃, Pd(PPh₃)₄, DMF/H₂O, 16 h; (b) i. CHCl₃, Br₂, ii. 10% NaOH, MeOH; (c) 4-borono-2-chlorobenzoic acid, Pd(PPh₃)₄, Na₂CO₃, dioxane/H₂O, 90 °C, 16 h.

Figure 4

Predicted binding mode of probe SGC-CAMKK1 to CAMKK2 kinase domain. (a) Detailed view of the binding interactions between compound 6, a close furopyridine analog of probe SGC-CAMKK-1, and CAMKK2 ATP-binding pocket as seen in the complex co-crystal structure (PDB ID 5UY6). (b) Predicted binding mode of probe SGC-CAMKK1 to CAMKK2 ATP-binding pocket obtained by in silico docking (see Methods for details). In (a,b), dashed black lines indicate potential hydrogen bonds, water molecules are depicted as red spheres, carbon atoms are shown in green (ligand) or yellow (protein), the kinase domain α-helix C is shown in blue, and protein surface (for the bottom of the ATP-binding site) is shown in white.

Figure 5

NanoBRET in-cell target engagement of CAMKK2 probe (5) (n = 3) and STO-609 (n = 1) in HEK293 cells transiently transfected with NLuc-CAMKK2. Increasing doses of inhibitor displaces the fluorescent inhibitor ligand (called a tracer) used in the assay, leading to a reduction in the BRET signal.

Figure 6

Western blot analysis of endogenous CAMKK2 inhibition using p-AMPK(Thr172) levels as a marker of CAMKK2 cellular activity. Western blot images (top) and densitometry analyses (bottom) of C4-2 cells treated with increasing doses of (a) STO-609, (b) probe SGC-CAMKK2-1 (5), and (c) negative control SGC-CAMKK2-1N (7). IC₅₀ for STO-609 = 10.7 μM, SGC-CAMKK2-1 probe = 1.6 μM, and negative control SGC-CAMKK2-1N ≥10 μM. Results represent values from three individual experiments.

Figure 7

The probe compound SGC-CAMKK2-1 (5) inhibits CAMKK2 activity with higher efficacy than STO-609 in MDA-MB 231 triple-negative metastatic breast cancer cells. MDA-MB 231 cells were treated with increasing doses of STO-609 or 5 for 24 h as indicated. Compound 7 (10 μM) was used as the negative control for CAMKK2 inhibition (data not shown). Lysates were collected and immunoblotting was done to check for levels of p-AMPK(Thr172), a substrate of CAMKK2. (a) Representative image showing 5 inhibiting CAMKK2 downstream activity with improved efficacy compared to STO-609. (b) Quantification of immunoblot bands. Results represent values from three individual experiments. * p < 0.05.

Figure 8

Plasma concentration of SGC-CAMKK2-1 and STO-609 in a single dose IP mouse study. The compounds were administered at a dose of 10 mg/kg via IP administration to three animals for each compound. Blood was collected at t = 0.5, 1, 3, and 8 h and immediately spun for 3 min and the plasma was collected, and compound levels were quantified with HPLC/MS.