A virtual screen identified C96 as a novel inhibitor of phosphatidylinositol 3-kinase that displays potent preclinical activity against multiple myeloma in vitro and in vivo

Abstract

The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is emerging as a promising therapeutic target for multiple myeloma (MM). In the present study, we performed a virtual screen against 800,000 of small molecule compounds by targeting PI3Kγ. C96, one of such compounds, inhibited PI3K activated by insulin-like growth factor-1 (IGF-1), but did not suppress IGF-1R activation. The cell-free assay revealed that C96 preferred to inhibit PI3Kα and δ, but was not active against AKT1, 2, 3 or mTOR. C96 inhibited PI3K activation in a time- and concentration-dependent manner. Consistent with its inhibition on PI3K/AKT, C96 downregulated the activation of mTOR, p70S6K, 4E-BP1, but did not suppress other kinases such as ERK and c-Src. Inhibition of the PI3K/AKT signaling pathway by C96 led to MM cell apoptosis which was demonstrated by Annexin V staining and activation of the pro-apoptotic signals. Furthermore, C96 displayed potent anti-myeloma activity in a MM xenograft model in nude mice. Oral administration of 100 mg/kg bodyweight almost fully suppressed tumor growth within 16 days, but without gross toxicity. Importantly, AKT activation was suppressed in tumor tissues from C96-treated mice, which was consistent with delayed tumor growth. Thus, we identified a novel PI3K inhibitor with a great potential for MM therapy.

Figure 1. The virtual screening workflow

C96 was generated from a virtual screen using PI3Kγ as the subject against 800,000 compounds in total from Specs and ChemBridge Chemicals. The molecular docking and scoring were accomplished by using the Schrodinger (Glide), HTVS, SP, and XP modes, followed by Sybyl clustering. Top hits were then verified at the cell-based experiments and singled out for further studies.

Figure 2. C96 inhibits AKT and mTOR signaling

(A) LP1 and OPM2 were starved overnight, then treated with C96 at the indicated concentrations, or 100 μM of S14161 for 2 hrs, followed by IGF-1 (100 ng/mL) for 15 min. Cells were collected for the analysis of the expression of p-AKT and total AKT (T-AKT) by immunoblotting. (B) LP1 and OPM2 cells were starved overnight, then treated with C96 (50 μM) for different time periods, or S14161 (100 (μM) for 2 hrs, followed by IGF-1 (100 ng/mL) for 15 min. Cells were for the analysis of the expression of p-AKT and T-AKT by immunoblotting. (C) LP1, OPM2, and JJN3 cells were treated with C96 at the indicated concentrations for 24 hrs. Cell lysates were prepared and subjected to immunoblotting assay against p-AKT, AKT, p-mTOR, mTOR, p-4E-BP1, and 4E-BP1. GAPDH was used as a loading control.

Figure 3. C96 inhibits AKT but not IGF-1R, ERK or c-Src kinase activation

(A) LP1 and JJN3 cells were starved overnight, then treated with 100 (μM of C96 or S14161 for 2 hrs, followed by IGF-1 (100 ng/mL) for 15 min. After incubation, cells were harvested and the expression of p-AKT, p-IGF-1R, total AKT (T-AKT), and IGF-1R proteins were detected by immunoblotting. (B) LP1 and JJN3 were treated with C96 at indicated concentrations for 24 hrs followed by immunoblotting assay for the expression of p-Src, c-Src, p-ERK, and ERK. GAPDH was used as a loading control.

Figure 4. C96 inhibits PI3K activity

PI3K activity analyses in an in vitro cell-free system. Increasing concentrations of S14161 were incubated with the PI3K isoforms α, β, δ, and γ, respectively. Activity of each kinase was determined with HotSpot technology as described in “Kinase activity in cell-free assay”.

Figure 5. C96 induces MM cell apoptosis

MM cell lines LP1, OPM2, OCI-My5 (My5), KMS11, RPMI-8226 (8226), and JJN3 were treated with C96 at 0 or 10 μM for 24 hrs, followed by propidium iodide and Annexin V-FITC staining and flow cytometric analysis.

Figure 6. C96 activates apoptotic signaling in MM cells

(A) OPM2, U266, KMS11, RPMI-8226, OCI-My5, and LP1 cells were treated with C96 (20 μM) for 24 hrs. Cell lysates were then prepared and subjected to immunoblotting assay against apoptosis-associated proteins PARP, and apoptotic executive enzyme pro-caspase-3 (Pro-Casp3). GAPDH was used as a loading control. (B) LP1, OPM2, and JJN3 were treated with C96 at the indicated concentrations for 12 hrs, followed by immunoblotting assay for pro-caspase-3 (Pro-Casp3), cleaved caspase-3 (Cle-Casp3), and PARP. GAPDH was used as a loading control. (C) LP1, OPM2, and JJN3 were treated with C96 at 10 μM for different time points, followed by immunoblotting assay for caspase-3 and PARP. GAPDH was used as a loading control.

Figure 7. C96 delays myeloma tumor growth in a xenograft mouse model

(A) JJN3 cells were injected subcutaneously into nude mice with a density of 30 million cells/site/mouse. When tumors were palpable, mice (n=5/group) were orally given C96 (100 mg/kg body weight) in PBS containing 10% Tween 80 and 10% DMSO daily for continuous 16 days. Tumor volumes were monitored every other day. (B) Mouse body weight was monitored every other day. (C) At the end of the experiment, tumor samples from each group were subjected to immunoblotting analysis for the expression levels of p-AKT, T-AKT, p-mTOR and mTOR with specific antibodies. GAPDH was used as a loading control.