Structure-based design of selective, orally available salt-inducible kinase inhibitors that stimulate bone formation in mice

Abstract

Osteoporosis is a major public health problem. Currently, there are no orally available therapies that increase bone formation. Intermittent parathyroid hormone (PTH) stimulates bone formation through a signal transduction pathway that involves inhibition of salt-inducible kinase isoforms 2 and 3 (SIK2 and SIK3). Here, we further validate SIK2/SIK3 as osteoporosis drug targets by demonstrating that ubiquitous deletion of these genes in adult mice increases bone formation without extraskeletal toxicities. Previous efforts to target these kinases to stimulate bone formation have been limited by lack of pharmacologically acceptable, specific, orally available SIK2/SIK3 inhibitors. Here, we used structure-based drug design followed by iterative medicinal chemistry to identify SK-124 as a lead compound that potently inhibits SIK2 and SIK3. SK-124 inhibits SIK2 and SIK3 with single-digit nanomolar potency in vitro and in cell-based target engagement assays and shows acceptable kinome selectivity and oral bioavailability. SK-124 reduces SIK2/SIK3 substrate phosphorylation levels in human and mouse cultured bone cells and regulates gene expression patterns in a PTH-like manner. Once-daily oral SK-124 treatment for 3 wk in mice led to PTH-like effects on mineral metabolism including increased blood levels of calcium and 1,25-vitamin D and suppressed endogenous PTH levels. Furthermore, SK-124 treatment increased bone formation by osteoblasts and boosted trabecular bone mass without evidence of short-term toxicity. Taken together, these findings demonstrate PTH-like effects in bone and mineral metabolism upon in vivo treatment with orally available SIK2/SIK3 inhibitor SK-124.

Keywords: kinase inhibitor; osteoporosis; parathyroid hormone.

Fig. 1.

Whole-body Sik2/Sik3 ablation increases bone turnover markers and trabecular bone mass. Twelve-week-old control (Sik2^f/f; Sik3^f/f) and mutant (Sik2^f/f; Sik3^f/f; ubiquitin-Cre^ERt2) were treated with tamoxifen (1 mg IP Q48H ×3 doses). (A) Bone turnover markers (alkaline phosphatase (AlkP), P1NP), and CTX) were measured 2, 4, 8, and 16 wk after tamoxifen treatment. Serum analysis of mice after tamoxifen administration showed dramatic increases in turnover markers at each time point (n = 3 to 6 mice per group). (B) Tibia histology by hematoxylin and eosin (HE) stain. (C) Safranin O and (D) sclerostin immunohistochemistry were performed. Sclerostin levels were substantially down-regulated by Sik2/Sik3 gene deletion at each time point. (E) Distal femur micro-CT results showed significantly increased bone volume and mineral density 8 and 16 wk after tamoxifen treatment. Images are shown on the Left, and key trabecular (bone volume per tissue volume (BV/TV) and trabecular BMD) and cortical (cortical thickness) parameters are shown on the Right (n = 6 to 9 mice per group). See also Dataset S1 for full micro-CT data from 8- and 16-wk chase animals. P values versus control are shown in the figure. Two-sided t tests were used (A and E). Data are expressed as mean ± SEM.

Fig. 2.

Identification of SK-124 (compound 16) by in silico screening followed by iterative medicinal chemistry. (A) Homology model of SIK2 used for in silico docking. (B–E) Chemical structures of compounds. SIK2 kinase assay was assessed with a radioisotope kinase assay using recombinant full-length SIK2 and HDAC5 peptide substrate in a 10-point dose–response format. Respective IC₅₀ values are indicated below each compound.

Fig. 3.

Kinome selectivity and cellular activity of SK-124. (A) YKL-05-099 and SK-124 were tested at 0.5 µM on a panel of 300 human kinases. Dendrograms show kinases inhibited >80% by each compound in red. SIK isoforms are present at the 6-o’clock position. S(80) refers to the portion of kinases tested whose activity was inhibited >80% by 0.5 µM compounds. (B) Comparison of SK-124 and YKL-05-099 against 300 kinases as percentage of remaining activity. (C) NanoBRET assays using NanoLuc–SIK fusion proteins were performed in HEK293T cells using the indicated doses of SK-124 used for cell-based calculation of IC₅₀ values. (D) Cellular thermal shift assay was performed in Ocy454 cells. Top panels show the melting curve used to determine SIK2 thermostability. A melting temperature of 55 °C was selected for subsequent studies. Bottom panels show effects of SK-124 treatment (1 h) on SIK2 thermostability at 55 °C. (E) Ocy454 cells were treated with the indicated compounds/doses for 1 h followed by immunoblotting as indicated. (F) PathHunter^® U2OS CRTC2 (TORC2) nuclear translocation reporter cells were treated for 90 min with the indicated doses of SK-124. Data are reported as CRTC2 nuclear translocation relative to forskolin (10 µM, positive control). (G) Ocy454 cells were treated for 4 h with the indicated doses of SK-124 followed by RNA isolation for RT-qPCR, with β-actin used as a housekeeping gene.

Fig. 4.

SK-124 affects mineral metabolism in a PTH-like manner. (A) Mice were treated once daily with the indicated doses of SK-124 by oral gavage (n = 10/group). Two hours after dose #14 was administered, serum was collected to measure SK-124 levels by mass spectrometry. (B and C) 2 h after SK-124 dose #21 was administered, serum was collected to measure the indicated analyte. In all panels, data points represent individual mice. (D) Decalcified paraffin-embedded sections from the tibiae of mice after 3 wk of the indicated treatment were stained using hematoxylin and eosin (Top), safranin O (Middle), and TRAP (Bottom).

Fig. 5.

SK-124 affects bone remodeling in a PTH-like manner. (A) Representative micro-CT images from the femur of mice in the indicated treatment group. The red box denotes the PS region. Quantification of (B) PS bone volume fracture and (C) midshaft cortical TMD in the indicated treatment groups by micro-CT. See also Dataset S5 for complete micro-CT results at all sites analyzed. (D) Trichrome-stained sections from histomorphometry study demonstrating increased osteoblasts on trabecular bone surfaces of mice treated with high-dose SK-124 and PTH. Yellow arrowheads denote groups of active osteoblasts seen in response to high-dose SK-124 and PTH. (Scale bar, 20 µm.) Quantification of osteoblast (Ob.S/BS) and osteoclast (Oc.S/BS) measurements is shown on the Right. (E) Dual calcein (green) and demeclocycline (red) images are shown from mice in the indicated treatment groups. Both high-dose SK-124 and PTH increase MAR and BFR/BS but not the MS/BS. See also Dataset S6 for complete histomorphometry results. (F) Sclerostin immunohistochemistry (brown staining) was performed and quantified (Right). (G) Cortical bone RNA was isolated, and SOST (gene that encodes sclerostin) levels were determined by RT-qPCR. For all panels, one-way ANOVA was performed followed by Tukey’s post hoc analysis to compare treatment groups with vehicle. P values less than 0.05 are shown on the graphs.