BMK1 (Kd = 80 nM); BMK1 (Kd = 190 nM)

XMD8-92 significantly increases p21 expression in cells and inhibits the proliferation of cancer cells by inhibiting BMK1 activation.[1] In addition to blocking the down-regulation of MEF2C caused by hydroxysafflor yellow A (HSYA), XMD8-92 also significantly reduces the inhibitory effects of HSYA on the activation of hepatic stellate cells (HSCs).[2]

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BMK1 is activated by mitogens and oncogenic signals and, thus, is strongly implicated in tumorigenesis. We found that BMK1 interacted with promyelocytic leukemia protein (PML), and inhibited its tumor-suppressor function through phosphorylation. Furthermore, activated BMK1 notably inhibited PML-dependent activation of p21. To further investigate the BMK-mediated inhibition of the tumor suppressor activity of PML in tumor cells, we developed a small-molecule inhibitor of the kinase activity of BMK1, XMD8-92. Inhibition of BMK1 by XMD8-92 blocked tumor cell proliferation in vitro [1].

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HSYA suppresses the expression of Col I and type III alpha collagen collagen in culture-activated HSC [2]
HSC activation is characterized by expression of overproduction of ECM components, including Col I and type III alpha collagen (Col III) (Cho et al., Citation2004). To assess the effect of HSYA on ECM production in culture-activated HSC, real-time PCR analysis were performed for mRNA expression of Col I and Col III, enzyme-linked immunosorbent assay and Western blot analysis for content of Col I in culture-activated HSC or HSC-cultured media. As shown in Figure 3A, compared with control, HSYA caused significant decreases in contents of Col I in HSC-cultured media by 55%. No effect of 5 μM XMD8-92 on contents of Col I could be observed. However, XMD 8–92 significantly reversed the inhibitive effect of HSYA on Col I secretion. HSYA significantly repress Col I as assessed by Western blot analysis (Figure 3B and C). Similar trends were also observed in Col I mRNA transcript (Figure 3D). As shown in Figure 3E, compared with control, HSYA caused significant decreases in mRNA expression of Col III in HSC by 28%. XMD 8–92 tend to reverse the inhibitive effect of HSYA on mRNA expression of Col III. However, lack of Col III antibody prevented from conducting the experiments for detection of Col III content. These data suggest the possibility that the inhibitory effect of HSYA on Col I and Col III contents may involve ERK5 signaling.

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HSYA induce the suppression of HSC activation [2]
Alpha-SMA is the unique marker for activated HSC (Abergel et al., Citation2006). To further evaluate the effect of HSYA on activation of HSC, real-time PCR and Western blot analysis were performed for gene expression of α-SMA in culture-activated HSC. As shown in Figure 4A and B, compared with control, HSYA caused significant decreases in protein expression of α-SMA by 71%. XMD8-92 alone also led to similar decreases in protein expression of α-SMA by 34%. Compared with cells treated with HSYA, cells treated with both HSYA and XMD 8–92 partially abrogated the inhibitive effect of HSYA protein expression of α-SMA. Consistent with the results of protein levels, real-time PCR revealed that HSYA treatment significantly inhibit mRNA expression of α-SMA in culture-activated HSC (Figure 4C). These findings support the notion that the inhibition of HSC activation by HSYA might be at least partially mediated by ERK5 signaling.

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HSYA suppresses MEF2C in culture-activated HSC [2]
MEF2 as a key nuclear mediator participate in the pathologic process of fibrogenesis in vivo (Konno et al., Citation2010). Therefore, we examined the effect of HSYA on MEF2C gene expression in culture-activated HSC. As shown in Figure 5A and B, HSYA or XMD8-92 significantly inhibited protein expression of MEF2C in culture-activated HSC by 61 and 80%, respectively. The inhibitory effects of HSYA on MEF2C appeared to be enhanced by XMD 8–92. These observations were confirmed at the transcription level by real-time PCR analyses (Figure 5C).

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The blockade of activation of the ERK5 by HSYA results in the reduction in culture-activated HSC proliferation [2]
Substantial evidence has indicated that the activation of ERK5 stimulates HSC proliferation (Rovida et al., Citation2008). As shown in Figure 6A and B, compared with control, HSYA caused significant decreases in p-ERK5 by 49%, and XMD 8–92 robustly inhibit phosphorylation of ERK5. The inhibitory effects of XMD8-92 on phosphorylation of ERK5 appeared not to be enhanced by with HSYA. This inhibition pattern of p-ERK5 correlated closely with the pattern of MEF2C presented above. Cell proliferation was determined by numbers of viable cells using MTS assays. As demonstrated in Figure 6C, HSYA significantly reduced viability of culture-activated HSC, as expected. XMD 8–92 itself had no detectable effect on viability of culture-activated HSC. Culture-activated HSC treated with both HSYA and XMD 8–92 apparently abrogated the inhibitory effect of HSYA on cell viability. Thus, ERK5 plays an important, but not exclusive role, in the inhibitive effects of HSYA on culture-activated HSC proliferation.

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XMD8-92 inhibits DCLK1, c-MYC, KRAS and NOTCH1 mRNA in AsPC-1 cells in vitro [3]
Based on the previously published report, XMD8-92 is known to inhibit BMK1 kinase activity and also bind to DCLK1. Taking this into consideration, we treated AsPC-1 human pancreatic cancer cells with doses upto 25 mM of XMD8-92 for 48 h. Proliferation of cancer cells was assessed using standard MTT assay. Total RNA isolated were subjected to quantitative real-time RTPCR analysis for p53 and p21, DCLK1, c-MYC, KRAS and NOTCH1. We observed a dose-dependent significant downregulation of AsPC-1 cancer cell proliferation (Fig. 1A). Following the RTPCR analysis, we did not observe increase in expression of tumor suppressor genes p21 or p53 mRNA following the treatment (Supplementary Fig. 1A and B). Subsequently, we observed significant dose-dependent downregulation of DCLK1 mRNA and protein (by Western blot analysis) following treatment with 10 and 15 μM of XMD8-92 (Fig. 1B and C). Furthermore, we also observed nearly 60% reduction in c-MYC, KRAS and NOTCH1 mRNA in AsPC-1 cells treated with XMD8-92 (Fig. 1D). These data taken together demonstrate that treatment AsPC-1 cells with XMD8-92 in vitro results in downregulation of DCLK1, c-MYC, KRAS and NOTCH1 mRNA.

XMD8-92 (50 mg/kg i.p.) significantly slows the growth of xenografted human or syngeneic mouse tumors by preventing tumor cell proliferation and tumor-associated angiogenesis.[1] XMD8-92 significantly downregulates DCLK1 and several of its downstream targets, which prevents pancreatic tumor xenograft growth.[3]

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BMK1 Is a Potential Drug Target for Treating Cancer [2]
To evaluate the effectiveness of XMD8-92 in inhibiting the activity of BMK1 in tumors, we tested the compound in mice xenografted with human tumors. XMD8-92 was found to effectively inhibit BMK1 activation as well as induce PML’s downstream effector, p21 (Figure 6A). More importantly, treatment of the mice with XMD8-92 one day, several days, or weeks after inoculation of the tumor cells all significantly inhibit the growth of the xenografted human or syngeneic mouse tumors (Figure 6B), without obvious side effects to the animals. Immuno-staining of tumor sections showed that XMD8-92 effectively inhibited the incorporation of BrdU during tumor cell division (Figure 6C) indicating XMD8-92 blocks tumor cell proliferation, one of the anti-cancer effects of PML. As both BMK1 and PML are involved in angiogenesis (Bernardi et al., 2006; Borden and Culjkovic, 2009; Hayashi et al., 2005; Hayashi et al., 2004), we tested whether XMD8-92 blocks angiogenesis in vivo and found that XMD8-92 significantly inhibits basic fibroblast growth factor (bFGF) induced angiogenesis in Matrigel plugs (Figure 6D). These results indicated that XMD8-92 exerts its anti-tumor effect by blocking tumor cell proliferation and tumor-associated angiogenesis, and, possibly, through other BMK1-dependent mechanisms yet to be tested or discovered.

We further examined whether the anti-tumor effect of XMD8-92 is specific to its anti-BMK1 capacity. The extent of tumor growth inhibition by XMD8-92 is identical to that elicited using dominant negative BMK1 administered through intratumoral injection of Ad-BMK1(AEF) (Figure 7A). Importantly, the additional treatment of Ad-BMK1(AEF) did not have a further inhibitory effect on the tumor-bearing mice treated with XMD8-92 alone (Figure 7A) indicating the anti-tumor capacity of XMD8-92 is, at least partly, through blocking the activity of BMK1.

We next evaluate the role of PML in XMD8-92-dependent inhibition of tumor growth. We found that depleting PML in tumor cells significantly lowered, but did not completely abrogate, the inhibitory effect of XMD8-92 on their growth in mice (Figure 7B). As XMD8-92 inhibits tumor cell proliferation and neovascularization (Figure 6C and 6D), the reason why PML depletion in tumor cells can only partly reverse the anti-tumor effect of XMD8-92 may be that PML in tumor cells is responsible only for mediating XMD8-92-mediated growth arrest of cancer cells and not for XMD8-92-dependent angiogenesis inhibition. Moreover, increasing the expression level of wild type PML in these PML-knockdown (KD) tumor cells restored their sensitivity to XMD8-92-mediated inhibition of tumor growth (Figure 7B). In contrast, expression of PML2D in the PML-KD cells did not re-establish their responsiveness to XMD8-92-mediated suppression of tumor proliferation (Figure 7B). These data indicate that PML plays a role in XMD8-92-mediated suppression of tumor growth.

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XMD8-92 inhibits pancreatic tumor xenograft growth [3]
Pancreatic tumor xenografts were generated by injecting AsPC-1 cells subcutaneously into the lower flanks of NOD/SCID mice. Tumors were allowed to develop for 30 days. When tumors were palpable, mice were treated with either XMD8-92 (50 mg/kg body weight) in DMSO and sterile corn oil (i.p.) or Control (injected with DMSO and corn oil) (n = 5 animals in each group). Treatments were given every day for 15 days and tumor volumes were measured every third day. Tumors were excised at day 45, and tumor volumes are represented in Fig. 2A. Control or vehicle-treated tumors grew exponentially throughout the experiment, whereas treatment with XMD8-92 not only arrested the tumor growth but resulted in decrease in the tumor volume compared to initial no treatment (day 0) (Fig. 2A). Treatment with XMD8-92 resulted in a significant (>80%) reduction (p < 0.01) in tumor volume compared to control tumors. We also observed more than 2-fold decrease in the tumor weight following treatment with XMD8-92 (Fig. 2B). mRNA analysis demonstrated a significant downregulation (p < 0.01) of DCLK1 mRNA in tumor treated with XMD8-92 compared to control tumors (Fig. 2C). Following immunohistochemical analysis, we observed significant downregulation of DCLK1 protein in tumors treated with XMD8-92 compared to control tumors (Fig. 2D and E). Similar to PDAC cell lines, in tumor xenografts following treatment with XMD8-92, we did not observe increase in expression of BMK1 downstream tumor suppressor genes p21 and p53 mRNA indicating that BMK1 related activity is no affected in pancreatic cancer (Supplementary Fig. 1C and D). These data taken together demonstrate that XMD8-92 inhibits AsPC-1 tumor xenograft growth and inhibits DCLK1 mRNA and protein.

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XMD8-92 treatment inhibits pluripotency in pancreatic tumor xenografts [3]
Pluripotency factors KLF4, OCT4, SOX2 and NANOG are upregulated in various aggressive cancers and in cancer stem cells. In our previous studies, following knockdown of DCLK1 using siRNA, we observed decreased expression of these pluripotency factors via miR-143/145 miRNA cluster-dependent mechanism. In this study, we wanted to determine whether treatment with XMD8-92 also resulted in regulation of the pluripotency factors via miR-145. In XMD8-92 treated tumors, we observed significant (p < 0.01) upregulation of miR-143/145 cluster compared to control tumors (Fig. 3A). Furthermore, we observed significant downregulation of pluripotency factors KLF4, OCT4, SOX2, and NANOG mRNA (Fig. 3B) and protein (Fig. 3C) in tumors treated with XMD8-92 compared to control tumors. Ras-responsive element binding protein 1 (RREB1) represses miR-143/145 promoter activity, which indicates that repression is an early event in pancreatic cancer initiation and progression. Additionally, KRAS and RREB1 are targets of miR-143/145, demonstrating a feed-forward mechanism that potentiates RAS signaling-mediated PDAC tumor progression. It has been recently demonstrated that ectopic expression of miR-143/145 results in repressed metastasis and increased adhesion of pancreatic cancer cells. Earlier, we have demonstrated that following knockdown of DCLK1 results in decreased expression of RREB1 via miR-143/145. Similarly, in this study, we observed >50% reduction in RREB1 mRNA following treatment with XMD8-92 compared to control tumors (Fig. 3B).

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XMD8-92 inhibits EMT and Angiogenesis via miR-200 in tumor xenografts [3]
In this study, we observed significant upregulation (>1.5-fold) of miR-200a, miR-200b, and miR-200c in tumors treated with XMD8-92 compared to control tumors (Fig. 4A). Subsequently, we observed significant downregulation of EMT transcription factors ZEB1, ZEB2, SNAIL and SLUG (Fig. 4B) in XMD8-92 treated tumors. We also observed significant downregulation (>60%) VEGFR1 and VEGFR2 mRNA (Fig. 4C) and protein (Fig. 4D) in the tumors treated with XMD8-92 compared to control tumors. These data taken together demonstrate that similar to DCLK1 knockdown, treatment of XMD8-92 results in downregulation of EMT and angiogenesis via miR-200 in pancreatic tumor xenografts.

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XMD8-92 treatment results in inhibition of Let-7a downstream targets c-MYC, KRAS and LIN28B in pancreatic tumor xenografts [3]
It has been previously demonstrated that following siRNA-mediated knockdown of DCLK1 results in increased expression of tumor suppressor miRNA let-7a and downregulation of let-7 downstream targets c-MYC, KRAS and LIN28B. In this study, following treatment with XMD8-92, we observed significant (p < 0.01) upregulation of miRNA let-7a (Fig. 5A) and more than 60% reduction in c-MYC mRNA (Fig. 5B) and protein (Fig. 5B inset and 5C). We also observed significant downregulation of KRAS mRNA (>50%) (Fig. 5D – left panel) and LIN28B mRNA (>80%) (Fig. 5D – right panel). These data indicate that XMD8-92 treatment regulates oncogenes c-MYC, KRAS and LIN28B via let-7a. These data also indicate these actions are mediated via downregulation of DCLK1 in pancreatic tumor xenografts.

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XMD8-92 treated xenografts have less NOTCH1 [3]
Notch signaling is upregulated in various cancers, including that of pancreatic cancer. Previous reports have indicated that NOTCH1 is downstream of DCLK1, and DCLK1 regulates NOTCH1 via miR-144 miRNA. siRNA-mediated knockdown of DCLK1 results in upregulation of pri-miR-144 and subsequently downregulated NOTCH1 mRNA. Similar to the above observation, in this study, following treatment with XMD8-92 a significant upregulation (>2.0 folds) of pri-miR-144 (Fig. 6A) and more than 60% reduction in NOTCH1 mRNA (Fig. 6B) and protein (Fig. 6C). Furthermore, we also observed significant downregulation of activated NOTCH1 (cleaved NOTCH1) following treatment with XMD8-92 (Fig. 6D). These data indicate that XMD8-92 treatment results in decreased expression of NOTCH1 via downregulation of DCLK1 in pancreatic tumor xenografts. Additionally, all the data has been summarized and is represented in a graphical format (Fig. 6E).

The following modifications are made when performing KiNativ profiling on XMD8-92 using an ATP and ADP acylphosphate-desthiobiotin. Prior to adding the ATP or ADP acylphosphate probe (5 μM final probe concentration), HeLa cell lysates (total protein concentration: 5 mg/mL) are incubated with XMD8-92 at 50 μM, 10 μM, 2 μM, 0.8 μM, and 0 μM for 15 minutes. A duplicate of each reaction is carried out. Probe reactions continued for 10 minutes before being processed for MS analysis and stopping when urea was added. In order to collect MS/MS spectra from all kinase peptide-probe conjugates that can be found in HeLa cell lysates, samples are analyzed by LC-MS/MS on a linear ion trap mass spectrometer using a time-segmented "target list." This target list was created and verified through a thorough analysis of HeLa lysates in the past. A comparison of inhibitor-treated to control (untreated) lysates allows for the precise determination of% inhibition at each point. Up to four characteristic fragment ions for each kinase peptide-probe conjugate are used to extract signals for each kinase. The specifics of this targeted mass spectrometry strategy will be covered in a manuscript that is currently being written. [1]

Treatment of cells [2]
HSYA were dissolved in sterile pyrogen-free water at a concentration of 1 × 10–4 M. These solutions were subjected to autoclaving for 15 min. Semi-confluent HSC were serum-starved for 24 h in DMEM containing 0.4% FBS. Cells were maintained in DMEM with 10% FBS in the presence or absence of HSYA at the indicated concentrations for the indicated times. To determine whether the ERK5 signaling pathway is involved in the inhibitive effect of HSYA on HSC activation, XMD8-92 (5 μM), a selective inhibitor of ERK5, was used, which was added to cell cultures 30 min before treatment with HSYA (Yang et al., Citation2010). Control HSC was treated with the appropriate amount of sterile pyrogen-free water. At the end of treatment, cells were washed with cold phosphate buffered solution and scraped in the sterile pyrogen-free water.

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Cell proliferation assays [3]
Cells (104 cells per well) were seeded into a 96-well tissue culture plate in triplicate. The cells were cultured in the presence of XMD8-92 with DMSO as a vehicle at 0, 0.78, 1.56, 3.13, 6.25, 12.50 and 25 μM. 48 h post treatment, 10 μl of TACS MTT Reagent was added to each well and the cells were incubated at 37 °C until dark crystalline precipitate became visible in the cells. 100 μl of 266 mM NH4OH in DMSO was then added to the wells and placed on a plate shaker at low speed for 1 min. After shaking, the plate was allowed to incubate for 10 min protected from light and the OD550 for each well was read using a microplate reader. The results were averaged and calculated as a percentage of the DMSO (vehicle) control +/– the standard error of the mean.

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IC50s were calculated using the DRC Master Spreadsheet. Values obtained for cells treated with EGF and DMSO were defined as the maximum control, while values for cells treated with EGF and 10 μM of XMD8-92 (SN12C-MEF2-luc) or 1 μM Staurosporine (SN12C-CMV-luc) were defined as the minimum control (i.e., maximum inhibition) [5].

Nod/Scid mice bearing HeLa xenograft, C57Bl/6 mice bearing LL/2 xenograft
~50 mg/kg twice a day
i.p.

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HeLa Xenograft Model [1]
5 × 105 HeLa cells were resuspended in DMEM and injected subcutaneously into the right flank of 6-week-old Nod/Scid mice (day 0). On the second day (day 1) after tumor cell injection, mice were randomized into 2 groups {6 animals [XMD8-92 (1–28 day)] and 18 animals [control]}. The XMD8-92 (1–28 day) group was treated with XMD8-92 at the dose of 50 mg/kg twice a day intraperitoneally. The control group received daily injections of the carrier solution as control. On the day 7, the control group was randomized into 2 groups {6 animals [XMD8-92 (7–28 day)] and 12 animals [control]}. And on the day 14, the remaining control group was randomized into 2 groups {6 animals [XMD8-92 (14–28 day)] and 6 animals [control]}. Treatment with XMD8-92 in XMD8-92 (7–28 day) and XMD8-92 (14–28 day) groups was initiated on day 7 and day 14, respectively. Tumor size was measured using a caliper, and tumor volume was determined by using the formula: L × W2 × 0.52, where L is the longest diameter and W is the shortest diameter.

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LL/2 Xenograft Model [1]
1 × 106 LL/2 cells were resuspended in DMEM and injected subcutaneously into the right flank of 6-week-old C57Bl/6 mice (day 0). On the second day (day 1) after tumor cell injection, mice were randomized into 2 groups {6 animals [XMD8-92 (1–17 day)] and 18 animals [control]}. The XMD8-92 (1–17 day) group was treated with XMD8-92 at the dose of 50 mg/kg twice a day intraperitoneally. The control group received daily injections of the carrier solution as control. On the day 7, the control group was randomized into 2 groups {6 animals [XMD8-92 (7–17 day)] and 12 animals [control]}. And on the day 14, the remaining control group was randomized into 2 groups {6 animals [XMD8-92 (10–17 day)] and 6 animals [control]}. Treatment with XMD8-92 in XMD8-92 (7–17 day) and XMD8-92 (10–17 day) groups was initiated on day 7 and day 10, respectively.

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Recombinant Adenovirus Treatment [1]
1 × 106 LL/2 cells were injected subcutaneously into C57Bl/6 mice (day 0). Recombinant adenoviral particles were generated as described in our previous study (Hayashi et al., 2001). On the day 7, mice were randomized into 4 groups [Ad-EV, Ad-BMK1(AEF), Ad-EV+XMD8-92 and Ad-BMK1(AEF) +XMD8-92] (6 animals per group). Mice were injected intratumorally with either empty adenovirus (Ad-EV) or recombinant adenovirus encoding BMK1(AEF) [Ad-BMK1(AEF)] on day 7, day 11, day 15, day 19, using the procedure previously described (Kim et al., 2004). Ad-EV+XMD8-92 and Ad-BMK1 (AEF)+XMD8-92 groups were treated with XMD8-92 from day 8 to day 20 using the procedure described in the LL/2 xenograft model. The other two groups [Ad-EV and Ad-BMK1(AEF)] received injections of the carrier solution instead as control.

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PML Reconstitution [1]
PML shRNAi knockdown and control cell lines were built using pGIPZ-shRNAmir-PML (Lentiviral) and pGIPZ (Lentiviral) plasmids from Openbiosystems. 1 × 106 knockdown (48 animals) or control (12 animals) LL/2 cells were injected subcutaneously into C57Bl/6 mice (day 0). On the second day (day 1) after tumor cell injection, mice were randomized into 10 groups (6 animals per group) [KD, Ctrl, KD+XMD8-92 and Ctrl+XMD8-92; KD+Ad-EV, KD+Ad-PML, KD+Ad-PML2D(S403D/T409D), KD+Ad-EV+XMD8-92, KD+Ad-PML+XMD8-92 and KD+Ad-PML2D(S403D/T409D)+XMD8-92]. The KD+XMD8-92 and Ctrl+XMD8-92 groups were treated with XMD8-92 at the dose of 50 mg/kg twice a day intraperitoneally for 16 days. The KD and Ctrl groups received daily injections of the carrier solution as control for 16 days. The KD+Ad-EV, KD+Ad-PML, KD+Ad-PML2D, KD+Ad-EV+XMD8-92, KD+Ad-PML+XMD8-92 and KD+Ad-PML2D+XMD8-92 groups were injected intratumorally with either empty adenovirus (Ad-EV), Ad-PML or Ad-PML2D recombinant adenovirus encoding PML or PML2D on day 7, day 11, day 15, day 19, using the procedure previously described (Kim et al., 2004). KD+Ad-EV+XMD8-92, KD+Ad-PML+XMD8-92 and KD+Ad-PML2D+XMD8-92 groups were treated with XMD8-92 from day 8 to day 20 using the procedure described in the LL/2 xenograft model. The KD+Ad-EV and KD+Ad-PML and KD+Ad-PML2D groups received injections of the carrier solution instead as control.

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A549 Xenograft Model [1]
To ascertain that XMD8-92 can block BMK1 in vivo, 1 × 106 A549 cells, whose endogenous BMK1 autophosphorylation was detectable by western blotting, were resuspended in DMEM and injected subcutaneously into the right flank of 6-week-old Nod/Scid mice. On the 21st day after the injection, mice were randomized into 2 groups (2 animals per group). One group was treated with XMD8-92 at the dose of 50 mg/kg twice a day. The other group was treated with the carrier solution as control. After 2 days, the A549 tumor was homogenized in E1A buffer followed by western blot analysis.

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Xenograft tumor model [3]
NOD/SCID mice were housed in pathogen-free conditions. AsPC-1 cells (1 × 107) were injected subcutaneously into the flanks of 4- to 6-wk-old mice (n = 5). Tumors were measured using a caliper and the volume was calculated as (length × width2) × 0.5. The tumors were palpable 30 days after injection of cells. XMD8-92was reconstituted in sterile corn oil and injected intraperitoneally (50 mg/kg body weight). Each animal bearing the tumor was injected with XMD8-92 or corn oil (vehicle control) on days 30–44 (15 doses, 1 dose/day). All mice were killed on day 45.

The pharmacokinetics of XMD8-92 was evaluated in Sprague-Dawley rats given a single intravenous or oral dose. XMD8-92 was found to have a 2.0 hr half life clearance of 26 mL/min/kg. The compound had moderate tissue distribution with a calculated volume of distribution of 3.4 L/kg. XMD8-92 had high oral bioavailability with 69% of the dose absorbed. After a single oral dose of 2 mg/kg, maximal plasma concentrations of approximately 500 nM were observed by 30 minutes, with 34 nM remaining 8 hr post dose. (Figure S1). In tolerability experiments (Table S3, S4 and S5), high plasma concentrations of drug (approximately 10 μM following IP dosing of 50 mg/kg) were maintained throughout the 14 days.[1]

XMD8-92 appeared to be well tolerated and the mice appeared healthy with no sign of distress. No vasculature instability was observed in the XMD8-92-treated mice (Table S4, S5 and Figure 1F). Together, these results demonstrated that XMD8-92 is an effective and specific inhibitor of BMK1 in vitro and in vivo. [1]

[1]. Cancer Cell . 2010 Sep 14;18(3):258-67.

[2]. Pharm Biol . 2013 Nov 5.

[3]. Cancer Lett . 2014 Aug 28;351(1):151-61.

[4]. Proc Natl Acad Sci U S A . 2016 Oct 18;113(42):11865-11870.

[5]. J Med Chem . 2019 Jan 24;62(2):928-940.

XMD8-92 is a dimethylpyrimido[4,5-b][1,4]benzodiazepin-6-one carrying at C-2 on the pyrimidine ring a [2-ethoxy-4-(4-hydroxypiperidin-1-yl)phenyl]amino substituent. It is an inhibitor of the BMK1 kinase pathway. It has a role as a protein kinase inhibitor.

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Abstract Context: Hepatic fibrosis ultimately leads to cirrhosis if not treated effectively. Hepatic stellate cells (HSC) are a main mediator of hepatic fibrosis through the accumulation of extracellular matrix proteins. Suppression activation of passaged HSC has been proposed as therapeutic strategies for the treatment and prevention of hepatic fibrosis. Objective: To evaluate the effect of hydroxysafflor yellow A (HSYA), an active chemical compound derived from the flowers of Carthamus tinctorius L. (Compositae), on HSC inhibition, and to begin elucidating underlying mechanisms. Materials and methods: Primary HSCs were isolated from rats by in situ pronase/collagenase perfusion. Culture-activated HSCs were treated with or without HSYA at 30 μM in the presence or absence of PD98059 for 48 h, and then cell proliferation was measured by MTS assays. Messenger RNA (mRNA) expression was quantified by polymerase chain reaction, and protein was quantified by Western blots or enzyme-linked immunosorbent assays. Results: HSYA significantly inhibits culture-activated HSC proliferation in a dose-dependent and time-dependent manner with an IC50 value of 112.79 μM. HSYA (30 μM) induce the suppression of HSC activation, as indicated by decreases in contents of type I alpha collagen in HSC-cultured media and expression of α-smooth muscle actin protein in culture-activated HSC by 55 and 71%, respectively. HSYA (30 μM) also caused significant decreases in mRNA expression of type III alpha collagen in HSC by 28%. HSYA (30 μM) suppresses myocyte enhancer factor 2 C (MEF2C) expression both at its mRNA and protein levels by 60 and 61%, respectively. Further study demonstrated that HSYA (30 μM) caused significant decreases in p-ERK5 by 49%. Blocking extracellular signal-regulated protein kinase 5 (ERK5) activity by XMD 8--92, an ERK5 inhibitor, markedly abrogated the inhibitive effects of HSYA on HSC activation, and blocked the HSYA-mediated MEF2C down-regulation. Conclusions: HSYA suppress HSC activation by ERK5-mediated MEF2C down-regulation and makes it a potential candidate for prevention and treatment of hepatic fibrogenesis.[2]

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XMD8-92 is a kinase inhibitor with anti-cancer activity against lung and cervical cancers, but its effect on pancreatic ductal adenocarcinoma (PDAC) remains unknown. Doublecortin-like kinase1 (DCLK1) is upregulated in various cancers including PDAC. In this study, we showed that XMD8-92 inhibits AsPC-1 cancer cell proliferation and tumor xenograft growth. XMD8-92 treated tumors demonstrated significant downregulation of DCLK1 and several of its downstream targets (including c-MYC, KRAS, NOTCH1, ZEB1, ZEB2, SNAIL, SLUG, OCT4, SOX2, NANOG, KLF4, LIN28, VEGFR1, and VEGFR2) via upregulation of tumor suppressor miRNAs let-7a, miR-144, miR-200a-c, and miR-143/145; it did not however affect BMK1 downstream genes p21 and p53. These data taken together suggest that XMD8-92 treatment results in inhibition of DCLK1 and downstream oncogenic pathways (EMT, pluripotency, angiogenesis and anti-apoptotic), and is a promising chemotherapeutic agent against PDAC.[3]

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Unlike other members of the MAPK family, ERK5 contains a large C-terminal domain with transcriptional activation capability in addition to an N-terminal canonical kinase domain. Genetic deletion of ERK5 is embryonic lethal, and tissue-restricted deletions have profound effects on erythroid development, cardiac function, and neurogenesis. In addition, depletion of ERK5 is antiinflammatory and antitumorigenic. Small molecule inhibition of ERK5 has been shown to have promising activity in cell and animal models of inflammation and oncology. Here we report the synthesis and biological characterization of potent, selective ERK5 inhibitors. In contrast to both genetic depletion/deletion of ERK5 and inhibition with previously reported compounds, inhibition of the kinase with the most selective of the new inhibitors had no antiinflammatory or antiproliferative activity. The source of efficacy in previously reported ERK5 inhibitors is shown to be off-target activity on bromodomains, conserved protein modules involved in recognition of acetyl-lysine residues during transcriptional processes. It is likely that phenotypes reported from genetic deletion or depletion of ERK5 arise from removal of a noncatalytic function of ERK5. The newly reported inhibitors should be useful in determining which of the many reported phenotypes are due to kinase activity and delineate which can be pharmacologically targeted.[4]

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The availability of a chemical probe to study the role of a specific domain of a protein in a concentration- and time-dependent manner is of high value. Herein, we report the identification of a highly potent and selective ERK5 inhibitor BAY-885 by high-throughput screening and subsequent structure-based optimization. ERK5 is a key integrator of cellular signal transduction, and it has been shown to play a role in various cellular processes such as proliferation, differentiation, apoptosis, and cell survival. We could demonstrate that inhibition of ERK5 kinase and transcriptional activity with a small molecule did not translate into antiproliferative activity in different relevant cell models, which is in contrast to the results obtained by RNAi technology.[5]

C26H30N6O3

474.55

474.237

C, 65.80; H, 6.37; N, 17.71; O, 10.11

1234480-50-2

46843772

White to beige solid powder.

1.3±0.1 g/cm3

741.8±70.0 °C at 760 mmHg

402.4±35.7 °C

0.0±2.6 mmHg at 25°C

1.655

1.14

2

8

5

35

719

0

O([H])C1([H])C([H])([H])C([H])([H])N(C2C([H])=C([H])C(=C(C=2[H])OC([H])([H])C([H])([H])[H])N([H])C2=NC([H])=C3C(=N2)N(C([H])([H])[H])C2=C([H])C([H])=C([H])C([H])=C2C(N3C([H])([H])[H])=O)C([H])([H])C1([H])[H]

QAPAJIZPZGWAND-UHFFFAOYSA-N

InChI=1S/C26H30N6O3/c1-4-35-23-15-17(32-13-11-18(33)12-14-32)9-10-20(23)28-26-27-16-22-24(29-26)30(2)21-8-6-5-7-19(21)25(34)31(22)3/h5-10,15-16,18,33H,4,11-14H2,1-3H3,(H,27,28,29)

2-[2-ethoxy-4-(4-hydroxypiperidin-1-yl)anilino]-5,11-dimethylpyrimido[4,5-b][1,4]benzodiazepin-6-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.27 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.27 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)