Identification and characterization of a novel integrin-linked kinase inhibitor

Abstract

Integrin-linked kinase (ILK) represents a relevant target for cancer therapy in light of its role in promoting oncogenesis and tumor progression. Through the screening of an in-house focused compound library, we identified N-methyl-3-(1-(4-(piperazin-1-yl)phenyl)-5-(4'-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide (22) as a novel ILK inhibitor (IC(50), 0.6 μM), which exhibited high in vitro potency against a panel of prostate and breast cancer cell lines (IC(50), 1-2.5 μM), while normal epithelial cells were unaffected. Compound 22 facilitated the dephosphorylation of Akt at Ser-473 and other ILK targets, including glycogen synthase kinase-3β and myosin light chain. Moreover, 22 suppressed the expression of the transcription/translation factor YB-1 and its targets HER2 and EGFR in PC-3 cells, which could be rescued by the stable expression of constitutively active ILK. Evidence indicates that 22 induced autophagy and apoptosis, both of which were integral to its antiproliferative activity. Together, this broad spectrum of mechanisms underlies the therapeutic potential of 22 in cancer treatment, which is manifested by its in vivo efficacy as a single oral agent in suppressing PC-3 xenograft tumor growth.

Figure 1

Chemical structures of 1–53 in the focused compound library used for the biomolecular screening of ILK inhibitors.

Figure 2

(A) Identification of putative PDK2 inhibitors by screening of an in-house focused compound library. Western blot analysis of the effects of 1 – 53 versus DMSO control on the phosphorylation of Akt on Ser-473 versus Thr-308 in PC-3 cells. Cells were exposed to individual test agents at 2.5 μM or DMSO vehicle in 5% FBS-supplemented RPMI 1640 medium for 24 h. (B) SAR analysis of 22/23 versus the structurally related but PDK2-inactive derivatives, 5, 14, 6, 21, 18, and 53. As shown, modifications in any of the three peripheral structural motifs, designated as a – c, of 22 led to loss of the Akt Ser-473 dephosphorylating activity, indicating a stringent structural requirement.

Figure 3

Effects of 22 on cell viability and ILK signaling. (A) Dose-dependent suppressive effects of 22 on the viability of PC-3 and LNCaP prostate cancer cells versus PrECs (left panel), and of MDA-MB-231, MDA-MB-468, SKBR3, and MCF-7 breast cancer cells versus MECs (right panel) in 5% FBS-supplemented medium after 24 h of treatment. Cell viability was determined by MTT assays. Points, means; bars, SD (n = 6). (B) Western blot analysis of the suppressive effects of shRNA-mediated knockdown of ILK and 22 on the phosphorylation of Akt at Ser-473 and Thr-308 and the downstream targets of ILK (GSK3β and MLC) versus those of mTORC2 (SGK and PKCα) in PC-3 and/or MDA-MB-231 cells. Cells were exposed to 22 at the indicated concentrations for 24 h in 5% FBS-supplemented medium. The immunoblots shown are representative of three independent experiments.

Figure 4

Evidence that 22 is an ILK inhibitor. (A) Dose-dependent suppressive effect of 22 on the kinase activity of immunoprecipitated ILK. Kinase activity was determined in the presence of 22 at the indicated concentrations by measuring ³²P-phosphorylation of the ILK substrate MBP as described in the Experimental Section. Data are presented as means ± SD (n = 3). (B) Effects of ectopic expression of GFP-tagged CA-ILK versus GFP alone on the phosphorylation of Akt, GSK3β, PKCα, and SGK in stable PC-3 transfectants (left panel) and on the viability of PC-3 cells after 24 h-treatment with 22 at different concentrations in 5% FBS-supplemented medium (right panel). Blot on the left confirms the presence of the CA-ILK protein (*) in transfected PC-3 cells. The immunoblots shown are representative of three independent experiments. Points, means; bars, SD (n = 6).

Figure 5

Dose-dependent effects of 22 on the protein (left panel) and mRNA (right panel) expression of YB-1 and its targets HER2 and EGFR in (A) PC-3 cells versus stable PC-3 transfectants expressing CA-ILK (PC-3/CA-ILK), and (B) SKBR3 cells versus transient SKBR3 transfectants expressing ectopic CA/ILK (SKBR3/CA-ILK). Cells were treated with 22 at the indicated concentrations in 5% FBS-supplemented medium for 24 h. These data are representative of three independent experiments. Blot on the left confirms the presence of the CA-ILK protein (*) in transfected SKBR3 cells.

Figure 6

Specificity of 22 in kinase inhibition. (A) Western blot analysis of the dose-dependent effect of 22 on the phosphorylation of p70S6K, S6, Tyr-397-FAK, ERKs, p38, and JNK in PC-3 cells. Cells were exposed to the indicated concentrations of 22 in 5% FBS-supplemented medium for 24 h. (B) Effects of the stable expression of CA-ILK, as depicted in Fig. 4B, on 22-mediated dephosphorylation of S6, ERKs, and p38 in PC-3 cells. The immunoblots shown are representative of three independent experiments.

Figure 7

Evidence that 22 induces cell death through both apoptosis and autophagy. (A) Histograms depicting the percentages of PC-3 cells at various phases of the cell cycle after exposure to the indicated concentrations 22 in 5% FBS-supplemented medium for 24 h. Each data point represents the mean ± SD (n = 3). (B) Dose-dependent effect of 22 on the extent of apoptosis induction, as detected by annexin V/PI staining. The values in the right upper and lower quadrants denote the average of three independent experiments. (C) Dose-dependent effects of 22 on PARP cleavage and LC3-II conversion in PC-3 cells after 24 h of treatment. (D) Protective effect of the stable transfection of CA-ILK (*), as depicted in Fig. 4B, on the induction of LC3-II conversion by 1 μM 22 in PC-3 cells. (E) siRNA-mediated knockdown of Atg5 inhibited 22-induced autophagy (left panel) as determined by LC3-II conversion, and provided partial protection against 22-mediated suppression of PC-3 cell viability (right panel). Points, means; bars, SD (n = 6). All immunoblots shown are representative of three independent experiments.

Figure 8

In vivoantitumor efficacy of 22. (A) Effect of oral 22 on PC-3 xenograft tumor growth in athymic nude mice. Mice with established s.c. PC-3 xenograft tumors were treated orally once daily with 22 at 25 and 50 mg/kg or vehicle for 35 days and tumor growth was monitored as described in the Experimental Section. Points, mean; bars, SEM (n = 6). (B) Western blot analysis of intratumoral biomarkers of drug activity in three representative PC-3 tumors from each group of mice treated for 35 days as described above in (A).

Scheme 1

General synthetic procedures for 5, 15–24, 27–30, and 53. Reaction conditions: a, 1-(4-bromophenyl)ethanone, Bu₄NBr, K₂CO₃, Pd(OAc)₂ (cat.), H₂O, 60 °C; b, diethyl oxalate, NaH, THF; c, ethyl 4-(1H-benzo[d][1,2,3]triazol-1-yl)-4-oxobutanoate, MgBr₂, CH₂Cl₂, DIPEA; (d) (4-nitrophenyl)hydrazine hydrochloride, TsOH, EtOH, microwave 130 °C, 10 min; e, NH₂R (R = CH₃ or C₂H₅) in EtOH, 120 °C; f, H₂(g) 70 psi, 10 % Pd/C, MeOH/EtOAc; g, bis(2-chloroethyl)amine hydrochloride, xylene, 170 °C; h, CH₃I, K₂CO₃, AcCN, 50 °C; i, 1-iodo-2-(2-iodoethoxy)ethane, xylene, 170 °C.