SY-707 is a multi-kinase inhibitor against ALK, FAK and IFG1R. SY-707 was developed as an ALK inhibitor primitively. It showed very potent inhibition on ALK kinase with an IC50 value of 2.4 nM (Fig. 1B). To evaluate in vitro enzymatic potency and selectivity of SY-707, a kinase panel screening on 96 kinases was performed to determine inhibitory activities of SY-707 at concentration of 1 mM. Besides ALK, SY-707 was able to inhibit several other kinases, such as FAK, Pyk2, LTK, IRK and IGF1R markedly with IC50 values around 1–10 nM (Fig. 1B). Then, enzymatic kinetic analysis on ALK and FAK was executed to explore mechanisms of inhibition of SY-707. Activities of ALK or FAK were estimated at various concentration of ATP in presence of SY-707 or not. SY-707-mediated inhibition on ALK kinase activity can be significantly attenuated with increasing concentration of ATP (Fig. 1C). SY-707 had higher affinity to free ALK enzyme with a dissociation constant (Ki) value of 0.90 nM than the enzyme-ATP complex with a Ki’ value of 19.2 nM. Lineweaver-Burk plots (Double-reciprocal plots of 1/v versus 1/[s]) (inset of Fig. 1C) illustrated that the four plots were intersected on Y axes, indicating that SY-707 inhibited ALK kinase as an ATP-competitive inhibitor. Similarly, SY-707 also preferred to combine free FAK enzyme with a dissociation constant (Ki) value of 0.56 nM than to combine the enzyme-ATP complex with a Ki’ value of 16.1 nM. And the four Double-reciprocal plots were also intersected on Y axes, suggesting an ATP-competitive inhibitory behavior of SY-707 for FAK kinase (inset of Fig. 1D). Since there was sequence similarity between catalytic domains of multiple kinases, it was reasonable that SY-707 depressed multiple kinases in an ATP-dependent manner.
SY-707 inhibits proliferation of breast cancer cell lines. To evaluate the cellular anti-tumor activities of SY-707, the cell proliferation studies were executed to evaluate the inhibitory effects of SY-707 on a series of cell lines including breast cancer (MCF-7, MDA-MB-231, T47D), pancreas cancer (MIA Paca-2, PANC-1), glioblastoma (U87MG), colon cancer (SW620), melanoma carcinoma (HT29), and lung cancer (A549). SY-707 significantly suppressed the growth of the most of solid cancer cells with IC50 values of 0.5–10 µm (Fig. 2A). Late apoptosis induced by SY-707 was observed in MDA-MB-231 cells dose-dependently on concentration of SY-707 (Fig. 2B). The cleaved poly-ADP ribose polymerase (PARP) was markedly detected in these treated cells and the amounts of cleaved PARP were correlated with the concentration of compound, meaning that SY-707 induced cell apoptosis by the way of formation of active apoptotic executor PARP (Fig. 2C). Meanwhile, SY-707 was able to induce pronounced G2/M phase arrest and a decrease in the percentage of cells in S phase in a dose-dependent manner (Fig. 2D).
SY-707 suppresses FAK signaling pathway in breast cancer. To investigate the underlying mechanisms of SY-707 inhibition on cell proliferation, we evaluated the effects of SY-707 on FAK signaling pathways. SY-707 dose-dependently suppressed the phosphorylation of FAK at Tyrosine397 and the phosphorylation of downstream protein ERK in MDA-MB-231 and T47D cells (Fig. 3A, B). More importantly, SY-707 displayed higher potency than reference compounds PF-562271 on the inhibition of phosphorylation ERK.
SY-707 blocked cell migration and adhesion of breast cancer cells. As FAK played key roles in cancer migration and metastasis, the effects of SY-707 on cell invasion and cell adhesion was evaluated. It was found that migration distances were significantly decreased in SY-707 treatment group compared to that in control group in a dose-dependently manner. Meanwhile, the significant reduction of cell migration was also observed in PF-562271 treated group (Fig. 4A). In another hand, SY-707 characteristically repressed cell adhesion in a dose-dependent manner (Fig. 4B). Thus, SY-707 suppressed cell invasion and cell adhesion, and this also supplied evidences for further exploration of anti-metastasis potency in in vivo studies.
SY-707 has anti-tumor activity in vivo via FAK inhibition. Since SY-707 showed excellent in vitro potency in enzymatic and cellular assays, we further evaluated its pharmacokinetics profile in SD rats and beagle dogs by oral (peros, PO) before in vivo efficacy studies. SY-707 was eliminated slowly with a t1/2 of 12.1 and 9.72 hours in SD rat or beagle dog respectively, displayed high exposure in animal plasma with mean AUC0 − INF of 10964 hr*ng/mL for rat and 459 hr*ng/mL for beagle dog correspondingly (Table. 1). In addition, SY-707 also had appropriate clearance parameters and oral bioavailability in these two species.
Table.1 Pharmacokinetics parameters of SY-707
Species | Rat 5 mg/kg (PO) | | Beagle Dog 5 mg/kg (PO) |
Male | Female | All | | Male | Femal | All |
t1/2 (hr) | 13 | 11.2 | 12.1 | | 9.33 | 10.1 | 9.72 |
Tmax (hr) | 3.33 | 4.33 | 3.83 | | 3 | 2.67 | 2.83 |
Cmax (ng/mL) | 909 | 639 | 774 | | 37.8 | 34.2 | 36 |
AUC0 − INF (hr*ng/mL) | 13335 | 8593 | 10964 | | 479 | 438 | 459 |
Cl_F_obs (mL/hr/kg) | 378 | 586 | 482 | | 11257 | 12131 | 11694 |
MRTINF_obs (hr) | 15.5 | 13.8 | 14.7 | | 13.6 | 13.7 | 13.6 |
F (%) | 38.3 | 33.2 | 36.2 | | 23.8 | 18.8 | 21.1 |
Due to strong cell growth inhibition potency of SY-707, we firstly evaluate its in vivo anti-cancer activities in mouse xenograft model bearing T47D cells. In this model, mice were orally administered with vehicle or SY-707 at dose of 100, 50 or 25 mg/kg twice a day (BID) after tumor size reached to 150–300 mm3. The results showed that SY-707 suppressed T47D tumor growth in a dose-dependent manner (Fig. 5A). After treatment with SY-707 for 21 days, tumor growth inhibition was estimated at 61.0% (p < 0.01), 39% (p < 0.05), 31% (p < 0.05) in 100, 50, and 25 mg/kg administrational groups respectively, while only reached 20% in reference compound PF-562217 dosing group.
To explore the relationship between the in vivo anti-tumor growth activities and the inhibition on FAK signaling transduction, pharmacokinetics /pharmacodynamics studies were performed in T47D xenograft model. Phosphorylation level of FAK at Tyrosine397 in SY-707 treatment groups were significantly decreased (Fig. 5B). Meanwhile, higher concentration of SY-707 in tumor samples were also detected compared to that in plasma, suggesting that SY-707 was able to access to tumor tissue with high exposure.
Next step, we evaluated the combination effects of SY-707 and paclitaxel (an approved drug for breast cancer therapy) in T47D xenograft model. Mice bearing T47D breast cancer were divided randomly into four groups and received vehicle, SY-707 (100 mg/kg, once a day (QD)), paclitaxel (10 mg/kg, once a week (QW)) or SY-707 plus paclitaxel respectively. After dosing for 24 days, tumor growth inhibition of xenograft mice in SY-707 (100 mg/kg, QD) and paclitaxel (10 mg/kg, QW) treatment groups was 14% and 24% (p < 0.05) respectively, while that in combinational group achieved to 66% (p < 0.01) (Fig. 5C).
In 4T1 xenograft model, SY-707 was administered with or without paclitaxel at indicated concentration, and then anti-tumor activities were evaluated three times a week. Only slight suppression on 4T1 tumor growth was observed in all of monotherapy groups, while significant inhibition on tumor growth was detected in combined group (100 mg/kg SY-707 plus 20 mg/kg paclitaxel) with a tumor growth inhibition value of 51.5% (p < 0.05) (Fig. 5D).
SY-707 repressed spontaneous metastasis of breast cancer to the lung. Cancer metastasis from primary area to other organs is a major cause of morbidity and mortality for breast cancer, since SY-707 displayed notable anti-migration activities in cellular assays, in vivo anti-metastasis effects were consequently evaluated in 4T1 xenograft models, in which the tumor metastasized to the lung spontaneously[18]. Alone treatment of SY-707 (100 mg/kg, PO, QD), paclitaxel, or paclitaxel (HS-15) (20 mg/kg or 10 mg/kg, IV (intravenous injection), QW) reduced the number of metastatic nodules in lungs but not statistically significantly (Fig. 6C, D, G, H). In contrast, combination of SY-707 and paclitaxel was able to eliminate the number of metastatic nodules in nude mice bearing 4T1 breast cancer significantly. Metastatic nodules in 100 mg/kg SY-707 (PO, QD) plus 20 mg/kg paclitaxel (IV, QW) group decreased for more than 40% compared to that in vehicle group (p < 0.01), and the number in 100 mg/kg SY-707 (PO, QD) plus 10 mg/kg paclitaxel (IV, QW) group also decreased for 31.1% compared to vehicle group (p < 0.05) (Fig. 6E, F). In conclusion, SY-707 plus paclitaxel notably depressed cancer metastasis to lungs from primary area.