Discovery and characterization of CX-4945, a selective ...
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0 5 10 15 20 25 30 35 0 200 400 600 800 1000 Vehicle CX-4945 25 mg/kg CX-4945 75 mg/kg CX-4945 50 mg/kg Days Tumor Volume (mm3) Dose dependent reduction in biomarker Dose dependent efficacy •P21-T145 ℗ IHC •CX-4945 bid for 3 days •Tumors collected 2, 6, 24 hr after final dose • 4 tumors analyzed per group 0 5 10 15 20 25 30 35 15 20 25 30 Days Body Weights (g) Well tolerated PO, bid 0 5 10 15 20 25 30 35 0 200 400 600 800 1000 Vehicle CX-4945 25 mg/kg CX-4945 75 mg/kg CX-4945 50 mg/kg Days Tumor Volume (mm3) Dose dependent reduction in biomarker Dose dependent reduction in biomarker Dose dependent efficacy Dose dependent efficacy •P21-T145 ℗ IHC •CX-4945 bid for 3 days •Tumors collected 2, 6, 24 hr after final dose • 4 tumors analyzed per group •P21-T145 ℗ IHC •CX-4945 bid for 3 days •Tumors collected 2, 6, 24 hr after final dose • 4 tumors analyzed per group 0 5 10 15 20 25 30 35 15 20 25 30 Days Body Weights (g) Well tolerated Well tolerated 67% TGI PO, bid PO, bid CX-4945 Reduces Microvessel Density in Prostate Cancer PC3 Xenograft Model • Nude mice bearing PC3 xenografts were treated with CX-4945 75mg/kg BID for 3 days. Six hours after the last dose the mice were sacrificed, their tumor excised, bisected and fixed in formalin. The IHC analysis for murine CD31 was performed by Mosaic Laboratories (Lake Forest, CA). • CX-494 significantly reduced microvessel density in PC3 xenografts. Vehicle CX-4945 Analysis of CD31 IHC Data Vehicle 75 mpk CX-4945 0 50 100 150 P = 0.04 Microvessel Density (%) • Nude mice treated with CX-4945 for 3 days • Blood collected 4 hr after final dose P-p21 (T145) levels in the white blood cell fraction of CX-4945 treated mice 0 20 40 60 80 100 UTC 12.5 mg/Kg CX-4945 75 mg/Kg CX-4945 p21-T145 / p21 total AKT T308 AKT S473 Total AKT PBMC’s Purchased ALLCELLS 1. CX-4945 added to PBMC’s 2. Incubate over time Western Blot CK2 is a highly conserved, constitutively active serine/threonine protein kinase found in the cytoplasmic and nuclear compartments of multiple cell types. CK2 has been implicated in critical cellular processes such as cell cycle regulation, signal transduction, apoptosis and angiogenesis. Elevated CK2 activity has been associated with malignant transformation and aggressive tumor growth. Overexpression of CK2 has been documented in multiple cancer indications including breast tumors where CK2α protein levels and CK2 activity levels are elevated 10-fold when compared to normal tissue. CK2 represents a novel target for cancer therapy. Reduction of CK2 activity using siRNA directed toward CK2α caused a profound decrease in cell viability and induction of apoptosis, confirming the importance of CK2 in the regulation of cell survival. Here we present the discovery and biological characterization of CX-4945, a potent and selective inhibitor of CK2 with an IC 50 of 2 nM against recombinant CK2. CX-4945 was found to be remarkably selective for CK2 compared to a panel of over 100 protein kinases. CX-4945 showed a broad range of antiproliferative activity against various tumor cell lines including inflammatory breast cancer cells. CX-4945 potently inhibited endogenous CK2 activity in Jurkat cells with an IC 50 of 100 nM. CX-4945 induced cell line dependent G 1 or G 2 cell cycle arrest. The protein p21 plays a critical role in cell cycle regulation and has been identified as a CK2 substrate. CX-4945 showed dose and time dependent dephosphorylation of p21 at threonine 145 in multiple cell lines. CX-4945 induced apoptosis via activation of caspase 3/7. CX-4945 showed favorable pharmacokinetics and oral bioavailability in multiple species. CX-4945 demonstrated robust antitumor activity in multiple xenografts, including 70% tumor free survival, in BxPC3 xenografts. Preclinical evaluation of inhibition of human CYP450 enzymes, genotoxicity and hERG activity suggest a favorable safety profile. CK2 has emerged as an attractive anticancer target and selective inhibitors of CK2 represent a potential therapeutic strategy to target aberrant CK2 activity perpetuating many cancers. A phase 1 clinical trial of CX-4945 in patients with advanced solid tumors, Castleman’s disease or multiple myeloma has been initiated. A Chronicle of CK2 Discovery Abstract CX-4945 Induces Cell Cycle Arrest Summary CX-4945 shows potent inhibition of CK2 enzymatic activity. CX-4945 kinase profile is highly selective for CK2 vs other kinases. CX-4945 selectively inhibits phosphorylation of ser/thr substrates. CX-4945 shows broad spectrum antiproliferative activity in cancer cell lines. CX-4945 inhibits phosphorylation of the cell cycle regulatory protein p21 at T145. CX-4945 inhibits phosphorylation of AKT at S129, T308 and S473. CX-4945 induces cell cycle arrest and apoptosis. CX-4945 shows potent antitumor activity in BxPC3 and PC3 xenografts. *Multi-center phase 1 trial ongoing. KINASE IC 50 (nM) KINASE IC 50 (nM) CK2 α’ 1 HIPK3 45 CK2 α 3 PIM1 46 DAPK3 17 CDK1/cyclin B 56 FLT3 35 DYRK2 91 CX-4945 Blocks PI3K/AKT Signaling BxPC3 Cells G2/M Arrest SUM 149PT G1 Arrest BxPC3 Cell line Tumor Type Phase Untreated (% of cells) CX-4945 (10μM) (% of cells) MDA-MB-468 Breast G1 44 28 S 36 33 G2/M 20 39 MDA-MB-231 Breast G1 53 64 S 35 26 G2/M 11 10 *SUM 149PT IBC G1 39 39 S 44 19 G2/M 16 42 SUM 190PT IBC G1 57 44 S 24 20 G2/M 20 36 BxPC3 Pancreas G1 51 71 S 36 15 G2/M 13 13 PC3 Prostate G1 33 24 S 39 10 G2/M 28 66 CX-4945 Blocks PI3K/AKT Signaling SUM149 Cells 5uM G0/G1 39% S 19% G2+M 42% UTC G0/G1 56% S 31% G2+M 13% 10uM CX-4945 G0/G1 71% S 15% G2+M 13% UTC G0/G1 39% S 44% G2+M 16% CX-4945 Modulates P-p21-T145 Biomarker in PBMC’s CX-4945 Reduction in P-p21-T145 is Reversible CX-4945 Selectively Blocks Phosphorylation of Ser/Thr Substrates BxPC3 cells were treated with or without CX-4945 for 2.5 hrs. Whole cell lysate were prepared and subjected to immunoprecipitation using antibodies against phospho-Tyr, phospho-Ser/thr( ArgXXS/T), and phospho-Thr proteins. The antibodies/beads-bound proteins were then eluted, and applied to 4-12% SDS-PAGE. The SDS-PAGE gel was silver-stained. P-Tyr P-Ser/Thr P-Thr Beads only - CX-4945 - CX-4945 - CX-4945 - CX-4945 M M 250 150 100 75 50 37 25 20 15 KDa CX-4945 Shows Antiproliferative Activity in Alamar Blue Assay 2 hrs 4 hrs 16 hrs 24 hrs p70S6 p70S6–T389 p21 total p21-T145 AKT AKT-T308 AKT-S473 Tubulin 0 0.1 1.0 5.0 10 0.1 1.0 5.0 10 μΜ CX-4945 0 0.1 1.0 5.0 10 0.1 1.0 5.0 10 No CDK Inhibition with CX-4945 CX-4945 was found to inhibit CDK1 activity (in nM range) in the molecular screen, but does not inhibit CDK1 activity in the cell. This further demonstrates selectivity for CK2 CX-4945 Displays Antitumor Activity and Inhibits P-p21-T145 in BxPC3 Pancreatic Xenograft Model Cell Line Cancer Type Cell Viability EC 50 (μM) Cell Line Cancer Type Cell Viability EC 50 (μM) A375 Melanoma 3.9 MCF7 Breast 6.0 A549 Lung 3.0 MDA-MB-231 Breast 1.3 BxPC3 Pancreas 4.4 MDA-MB-468 Breast 5.1 Colo205 Colon 2.0 MiaPaCa 2 Pancreas 1.1 H1299 Lung 2.4 PANC1 Pancreas 15.3 H460 Lung 10.3 PC3 Prostate 2.1 HCT-116 Colon 2.2 LNCaP Prostate 4.7 HL60 Leukemia 1.1 SK-MES-1 Lung 6.8 HT29 Colon 1.6 SK-OV-3 Ovarian 9.0 Jurkat Leukemia 4.2 SUM 149PT Breast (IBC) 1.3 K-562 Leukemia 3.0 SUM 190PT Breast (IBC) 1.7 RPMI 8226 Myeloma 4.0 Hs578T Breast 0.67 Discovery and characterization of CX-4945, a selective orally bioavailable small molecule inhibitor of protein kinase CK2: Phase 1 initiated K. Anderes , A. Siddiqui-Jain, N. Streiner, P. Chua, F. Pierre, M. Omori, L. Darjarnia, Ryan Stansfield, Jennifer Phung, J. Bliesath, C. Ho, D. Drygin, S. O’Brien, W. Rice Cylene Pharmaceuticals Inc., San Diego CA, USA CX-4945 Blocks PI3K/AKT Signaling PC3 Cells CX-4945 Displays Antitumor Activity and Inhibits P-p21-T145 in Prostate Cancer PC3 Xenograft Model RTK IRS PI3K PIP 3 PTEN PTEN P P S370 S380 CK2 PDK1 AKT CK2 CK2 P21 P P P P Active Inactive P27 Bad FRAP/ mTOR GβL Rictor FRAP/ mTOR GβL Raptor Translation/ Cell growth Survival Cell cycle/ proliferation P P P P = CK2 Phospho-site RTK IRS PI3K PIP 3 PTEN PTEN P P S370 S380 CK2 PDK1 AKT CK2 CK2 P21 P P P P Active Inactive P27 Bad FRAP/ mTOR GβL Rictor FRAP/ mTOR GβL Raptor Translation/ Cell growth Survival Cell cycle/ proliferation P P P P = CK2 Phospho-site p70S6K CK2 is “Multi-Tasking” Kinase Regulation of signal transduction Continuous identification of substrates Role in oncogenic transformation CK2α knock-ins, knock-outs High levels associated with poor prognosis in multiple indications Regulation of oncogenic signaling pathways ie; PI3K/AKT/mTOR CK2 Kinase discovered Crystal structure solved CX-4945 1 st in class, Only in class CK2 inhibitor enters Phase 1 clinical trial Role in cell cycle regulation 1954 1998 2004 2005 2009 2001 1995 N HN N OH O Cl CX-4945 3D Model of CX-4945 Binding to Catalytic Site of CK2α Subunit Yellow Arrow points at CX-4945 (purple) CX-4945 μM 0 0.1 1 5 10 0.1 1 5 10 0 0.1 1 5 10 0.1 1 5 10 PTEN P-PTEN (S370) P-PTEN (S380) P-AKT (S129) AKT p21 p21-T145 P-p70S6 P-p70S6 (T389) 2 hrs 4 hrs 16 hrs 24 hrs P-AKT (S473) CX-4945 μM 0 0.1 1 5 10 0.1 1 5 10 0 0.1 1 5 10 0.1 1 5 10 PTEN P-PTEN (S370) P-PTEN (S380) P-AKT (S129) AKT p21 p21-T145 P-p70S6 P-p70S6 (T389) 2 hrs 4 hrs 16 hrs 24 hrs P-AKT (S473) CX-4945 μM 0 0.1 1 5 10 0.1 1 5 10 0 0.1 1 5 10 0.1 1 5 10 P-AKT (S129) AKT p21 p21-T145 P-p70S6 P-p70S6 (T389) 2 hrs 4 hrs 16 hrs 24 hrs CX-4945 μM 0 0.1 1 5 10 0.1 1 5 10 0 0.1 1 5 10 0.1 1 5 10 P-AKT (S129) AKT p21 p21-T145 P-p70S6 P-p70S6 (T389) 2 hrs 4 hrs 16 hrs 24 hrs Effect of CX-4945 on AKT phosphorylation in PC3 cells by ELISA, 24 hr 0 20 40 60 80 100 UTC 0.1uM CX4945 1uM CX4945 5uM CX4945 10uM CX4945 Relative Intensity AKT Total AKT-S473 AKT-T308 Effect of CX-4945 on AKT phosphorylation in PC3 cells by ELISA, 24 hr 0 20 40 60 80 100 UTC 0.1uM CX4945 1uM CX4945 5uM CX4945 10uM CX4945 Relative Intensity AKT Total AKT-S473 AKT-T308 0 .01 0.1 1.0 0 25 50 75 100 CX-4945 ( μM) Endogenous CK2 Activity in Jurkat Cells 0 5 10 15 20 25 30 35 0 200 400 600 800 1000 Control Gem 120 mg/kg CX4945 12.5 mg/kg CX4945 25 mg/kg CX4945 50 mg/kg CX4945 75 mg/kg Days Tumor Volume (mm3) Well tolerated UTC Gemzar CX-4945 CX-4945 0 5 10 15 20 % Staining 12.5 mg/Kg 75 mg/Kg •P21-T145 ℗ IHC •CX-4945 bid for 5 days; tumors collected 4 hr after final dose • 3 tumors analyzed per group; 4 random images per tumor 0 10 20 30 15 20 25 30 Days Post Treatment Body Weight (g) Dose dependent efficacy Dose dependent reduction in biomarker PO, bid 93% TGI 4/9 regressions 0 5 10 15 20 25 30 35 0 200 400 600 800 1000 Control Gem 120 mg/kg CX4945 12.5 mg/kg CX4945 25 mg/kg CX4945 50 mg/kg CX4945 75 mg/kg Days Tumor Volume (mm3) Well tolerated Well tolerated Vehicle CX-4945 (75 mg/kg ) UTC Gemzar CX-4945 CX-4945 0 5 10 15 20 % Staining 12.5 mg/Kg 75 mg/Kg UTC Gemzar CX-4945 CX-4945 0 5 10 15 20 % Staining 12.5 mg/Kg 75 mg/Kg •P21-T145 ℗ IHC •CX-4945 bid for 5 days; tumors collected 4 hr after final dose • 3 tumors analyzed per group; 4 random images per tumor •P21-T145 ℗ IHC •CX-4945 bid for 5 days; tumors collected 4 hr after final dose • 3 tumors analyzed per group; 4 random images per tumor 0 10 20 30 15 20 25 30 Days Post Treatment Body Weight (g) Dose dependent efficacy Dose dependent efficacy Dose dependent reduction in biomarker Dose dependent reduction in biomarker PO, bid PO, bid CX-4945 Suppresses Endogenous CK2 Activity IC50s were determined for 8 most sensitive kinases Concentration and Time Dependent MDA-MB-468 PC3 CX-4945 Activates Caspase 3/7 8 20 32 44 56 72 0 100 200 300 400 Time (h) % ↑ Caspase 3/7 Activity 8 20 32 44 56 72 0 100 200 300 400 Time (h) % ↑ Caspase 3/7 Activity • Human blood treated ex-vivo with CX-4945, 2hr 0 20 40 60 80 100 UTC 1 uM CX-4945 10 uM CX-4945 p21-T145 / p21 total P-p21 (T145) levels in ex-vivo CX-4945 treated human PBMCs Effects of CX-4945 on Translation in PC3 Cells, 24 hr 0 20 40 60 80 100 0 0.1 1 5 10 [CX-4945] uM 35S-Met incorporation (%) Inhibition of S35-Met Incorporation in PC3 Cells at 24 hrs 0 20 40 60 80 100 0 0.1 1 5 10 35D-Met incorporation (%) [CX-4945] uM Effects of CX-4945 on Transaltion in BxPC3 Cells, 24 hr Inhibition of S35-Met Incorporation in BxPC3 Cells at 24 hrs CX-4945 Inhibits S35-Methionine Incorporation CX-4945 Molecular Structure and Kinase Selectivity CX-4945 Pharmacokinetics Species CL s (L/kg/hr) Vd ss (L/kg) Terminal T 1/2 (hr) %F 0-inf Mouse 2.7 19.5 7.1 21.6 Rat 0.1 2.0 12.3 47.8 Dog 0.7 8.9 5.2 47.3 Cellular CK2 kinase activity assessed by preparing cell lysates and exposing to a CK2-specific peptide fragment in the presence of radio-labeled ATP 145 Kinases < 50% inhibition at 500nM OR K d > 500nM > 50% inhibition at 500nM OR K d < 500nM > 90% inhibition at 500nM OR K d < 100nM
Transcript of Discovery and characterization of CX-4945, a selective ...
0 5 10 15 20 25 30 350
200
400
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1000 VehicleCX-4945 25 mg/kg
CX-4945 75 mg/kgCX-4945 50 mg/kg
Days
Tum
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olum
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m3)
Dose dependent reduction in biomarkerDose dependent efficacy
•P21-T145 ℗ IHC•CX-4945 bid for 3 days•Tumors collected 2, 6, 24 hr after final dose• 4 tumors analyzed per group
0 5 10 15 20 25 30 3515
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Bod
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eigh
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Well tolerated
67% TGI
PO, bid
0 5 10 15 20 25 30 350
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1000 VehicleCX-4945 25 mg/kg
CX-4945 75 mg/kgCX-4945 50 mg/kg
Days
Tum
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olum
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m3)
Dose dependent reduction in biomarkerDose dependent reduction in biomarkerDose dependent efficacyDose dependent efficacy
•P21-T145 ℗ IHC•CX-4945 bid for 3 days•Tumors collected 2, 6, 24 hr after final dose• 4 tumors analyzed per group
•P21-T145 ℗ IHC•CX-4945 bid for 3 days•Tumors collected 2, 6, 24 hr after final dose• 4 tumors analyzed per group
0 5 10 15 20 25 30 3515
20
25
30
Days
Bod
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eigh
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Well toleratedWell tolerated
67% TGI
PO, bidPO, bid
CX-4945 Reduces Microvessel Density in Prostate Cancer PC3 Xenograft Model
• Nude mice bearing PC3 xenografts were treated with CX-4945 75mg/kg BID for 3 days. Six hours after the last dose the mice were sacrificed, their tumor excised, bisected and fixed in formalin. The IHC analysis for murine CD31 was performed by Mosaic Laboratories (Lake Forest, CA).
• CX-494 significantly reduced microvessel density in PC3 xenografts.
Vehicle
CX-4945
Analysis of CD31 IHC Data
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• Blood collected 4 hr after final dose
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PBMC’s PurchasedALLCELLS
1. CX-4945 added to PBMC’s2. Incubate over time
Western Blot
CK2 is a highly conserved, constitutively active serine/threonine protein kinase found in the cytoplasmic and nuclear compartments of multiple cell types. CK2 has been implicated in critical cellular processes such as cell cycle regulation, signal transduction, apoptosis and angiogenesis. Elevated CK2 activity has been associated with malignant transformation and aggressive tumor growth. Overexpression of CK2 has been documented in multiple cancer indications including breast tumors where CK2α protein levels and CK2 activity levels are elevated 10-fold when compared to normal tissue. CK2 represents a novel target for cancer therapy. Reduction of CK2 activity using siRNAdirected toward CK2α caused a profound decrease in cell viability and induction of apoptosis, confirming the importance of CK2 in the regulation of cell survival. Here we present the discovery and biological characterization of CX-4945, a potent and selective inhibitor of CK2 with an IC50 of 2 nM against recombinant CK2. CX-4945 was found to be remarkably selective for CK2 compared to a panel of over 100 protein kinases. CX-4945 showed a broad range of antiproliferative activity against various tumor cell lines including inflammatory breast cancer cells. CX-4945 potently inhibited endogenous CK2 activity in Jurkat cells with an IC50 of 100 nM. CX-4945 induced cell line dependent G1 or G2 cell cycle arrest. The protein p21 plays a critical role in cell cycle regulation and has been identified as a CK2 substrate. CX-4945 showed dose and time dependent dephosphorylation of p21 at threonine 145 in multiple cell lines. CX-4945 induced apoptosis via activation of caspase 3/7. CX-4945 showed favorable pharmacokinetics and oral bioavailability in multiple species. CX-4945 demonstrated robust antitumor activity in multiple xenografts, including 70% tumor free survival, in BxPC3 xenografts. Preclinical evaluation of inhibition of human CYP450 enzymes, genotoxicity and hERG activity suggest a favorable safety profile. CK2 has emerged as an attractive anticancer target and selective inhibitors of CK2 represent a potential therapeutic strategy to target aberrant CK2 activity perpetuating many cancers. A phase 1 clinical trial of CX-4945 in patients with advanced solid tumors, Castleman’s disease or multiple myeloma has been initiated.
A Chronicle of CK2 Discovery
Abstract CX-4945 Induces Cell Cycle Arrest
Summary
CX-4945 shows potent inhibition of CK2 enzymatic activity.CX-4945 kinase profile is highly selective for CK2 vs other kinases.CX-4945 selectively inhibits phosphorylation of ser/thr substrates. CX-4945 shows broad spectrum antiproliferative activity in cancer cell lines.CX-4945 inhibits phosphorylation of the cell cycle regulatory protein p21 at T145.CX-4945 inhibits phosphorylation of AKT at S129, T308 and S473.CX-4945 induces cell cycle arrest and apoptosis.CX-4945 shows potent antitumor activity in BxPC3 and PC3 xenografts.*Multi-center phase 1 trial ongoing.
KINASE IC50 (nM) KINASE IC50 (nM)
CK2 α’ 1 HIPK3 45
CK2 α 3 PIM1 46
DAPK3 17 CDK1/cyclin B 56
FLT3 35 DYRK2 91
CX-4945 Blocks PI3K/AKT Signaling BxPC3 Cells
G2/M Arrest SUM 149PTG1 Arrest BxPC3
Cell line Tumor Type Phase Untreated (% of cells)
CX-4945 (10μM)(% of cells)
MDA-MB-468 BreastG1 44 28S 36 33G2/M 20 39
MDA-MB-231 BreastG1 53 64S 35 26G2/M 11 10
*SUM 149PT IBCG1 39 39S 44 19G2/M 16 42
SUM 190PT IBCG1 57 44S 24 20G2/M 20 36
BxPC3 PancreasG1 51 71S 36 15G2/M 13 13
PC3 ProstateG1 33 24S 39 10G2/M 28 66
CX-4945 Blocks PI3K/AKT Signaling SUM149 Cells
5uM
G0/G1 39%
S 19%
G2+M 42%
UTC
G0/G1 56%S 31%
G2+M 13%
10uM CX-4945
G0/G1 71%S 15%
G2+M13%
UTC
G0/G1 39%
S 44%
G2+M 16%
CX-4945 Modulates P-p21-T145 Biomarker in PBMC’s
CX-4945 Reduction in P-p21-T145 is Reversible
CX-4945 Selectively Blocks Phosphorylation of Ser/Thr Substrates
BxPC3 cells were treated with or without CX-4945 for 2.5 hrs.
Whole cell lysate were prepared and subjected to immunoprecipitation using antibodies against phospho-Tyr, phospho-Ser/thr( ArgXXS/T), and phospho-Thr proteins.
The antibodies/beads-bound proteins were then eluted, and applied to 4-12% SDS-PAGE.
The SDS-PAGE gel was silver-stained.
P-Tyr P-Ser/Thr P-ThrBeads only
- CX-4945 - CX-4945 - CX-4945 - CX-4945M M
25015010075
5037
252015
KDa
CX-4945 Shows Antiproliferative Activity in Alamar Blue Assay
2 hrs 4 hrs 16 hrs 24 hrs
p70S6
p70S6–T389
p21 total
p21-T145
AKT
AKT-T308
AKT-S473
Tubulin
0 0.1 1.0 5.0 10 0.1 1.0 5.0 10 μΜ CX-4945 0 0.1 1.0 5.0 10 0.1 1.0 5.0 10
No CDK Inhibition with CX-4945
CX-4945 was found to inhibit CDK1 activity (in nM range) in the molecular screen, but does not inhibit CDK1 activity in the cell. This further demonstrates selectivity for CK2
CX-4945 Displays Antitumor Activity and Inhibits P-p21-T145 in BxPC3 Pancreatic Xenograft Model
Cell Line Cancer Type Cell ViabilityEC50 (μM)
Cell Line Cancer Type Cell ViabilityEC50 (μM)
A375 Melanoma 3.9 MCF7 Breast 6.0A549 Lung 3.0 MDA-MB-231 Breast 1.3BxPC3 Pancreas 4.4 MDA-MB-468 Breast 5.1Colo205 Colon 2.0 MiaPaCa 2 Pancreas 1.1H1299 Lung 2.4 PANC1 Pancreas 15.3H460 Lung 10.3 PC3 Prostate 2.1HCT-116 Colon 2.2 LNCaP Prostate 4.7HL60 Leukemia 1.1 SK-MES-1 Lung 6.8HT29 Colon 1.6 SK-OV-3 Ovarian 9.0Jurkat Leukemia 4.2 SUM 149PT Breast (IBC) 1.3K-562 Leukemia 3.0 SUM 190PT Breast (IBC) 1.7RPMI 8226 Myeloma 4.0 Hs578T Breast 0.67
Discovery and characterization of CX-4945, a selective orally bioavailable small molecule inhibitor of protein kinase CK2: Phase 1 initiatedK. Anderes, A. Siddiqui-Jain, N. Streiner, P. Chua, F. Pierre, M. Omori, L. Darjarnia, Ryan Stansfield, Jennifer Phung, J. Bliesath, C. Ho, D. Drygin, S. O’Brien, W. RiceCylene Pharmaceuticals Inc., San Diego CA, USA
CX-4945 Blocks PI3K/AKT Signaling PC3 Cells
CX-4945 Displays Antitumor Activity and Inhibits P-p21-T145 in Prostate Cancer PC3 Xenograft Model
RTK
IRSPI3K PIP3
PTEN
PTEN
PP
S370S380
CK2
PDK1
AKT
CK2
CK2
P21
P
PP P
Active
Inactive
P27Bad
FRAP/mTOR
GβL Rictor
FRAP/mTOR
GβL Raptor
Translation/Cell growth
SurvivalCell cycle/proliferation
PP
P
P = CK2 Phospho-siteRTK
IRSPI3K PIP3
PTEN
PTEN
PP
S370S380
CK2
PDK1
AKT
CK2
CK2
P21
P
PP P
Active
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P27Bad
FRAP/mTOR
GβL Rictor
FRAP/mTOR
GβL Raptor
Translation/Cell growth
SurvivalCell cycle/proliferation
PP
P
P = CK2 Phospho-site
p70S6K
CK2 is “Multi-Tasking” Kinase
Regulation of signal transduction
Continuous identification of substrates
Role in oncogenictransformation
CK2α knock-ins, knock-outs
High levels associated with poor prognosis in
multiple indications
Regulation of oncogenic signaling
pathways ie; PI3K/AKT/mTOR
CK2 Kinasediscovered
Crystal structure
solved
CX-49451st in class, Only in class
CK2 inhibitor enters Phase 1 clinical trial
Role in cell cycle regulation
1954 1998 2004 2005 200920011995
N
HN
N
OH
O
Cl
CX-4945
3D Model of CX-4945 Binding to Catalytic Site of CK2α Subunit Yellow Arrow points at CX-4945 (purple)
CX-4945 μM 0 0.1 1 5 10 0.1 1 5 10 0 0.1 1 5 10 0.1 1 5 10
PTEN
P-PTEN (S370)
P-PTEN (S380)
P-AKT (S129)
AKT
p21
p21-T145
P-p70S6
P-p70S6 (T389)
2 hrs 4 hrs 16 hrs 24 hrs
P-AKT (S473)
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P-PTEN (S370)
P-PTEN (S380)
P-AKT (S129)
AKT
p21
p21-T145
P-p70S6
P-p70S6 (T389)
2 hrs 4 hrs 16 hrs 24 hrs
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P-AKT (S129)
AKT
p21
p21-T145
P-p70S6
P-p70S6 (T389)
2 hrs 4 hrs 16 hrs 24 hrs
CX-4945 μM 0 0.1 1 5 10 0.1 1 5 10 0 0.1 1 5 10 0.1 1 5 10
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AKT
p21
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P-p70S6 (T389)
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Effect of CX-4945 on AKT phosphorylation in PC3 cells by ELISA, 24 hr
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% S
tain
ing
12.5 mg/Kg 75 mg/Kg
•P21-T145 ℗ IHC•CX-4945 bid for 5 days; tumors collected 4 hr after final dose• 3 tumors analyzed per group; 4 random images per tumor
•P21-T145 ℗ IHC•CX-4945 bid for 5 days; tumors collected 4 hr after final dose• 3 tumors analyzed per group; 4 random images per tumor
0 10 20 3015
20
25
30
Days Post Treatment
Body
Weig
ht (g
)
Dose dependent efficacyDose dependent efficacy Dose dependent reduction in biomarkerDose dependent reduction in biomarker
PO, bidPO, bid
CX-4945 Suppresses Endogenous CK2 Activity
IC50s were determined for 8 most sensitive kinases
Concentration and Time Dependent
MDA-MB-468 PC3
CX-4945 Activates Caspase 3/7
8 20 32 44 56 72
0
100
200
300
400
Time (h)
%↑
Casp
ase
3/7
Activ
ity
8 20 32 44 56 72
0
100
200
300
400
Time (h)
%↑
Cas
pase
3/7
Act
ivity
• Human blood treated ex-vivo with CX-4945, 2hr
0
20
40
60
80
100
UTC 1 uM CX-4945 10 uM CX-4945
p21-
T145
/ p2
1 to
tal
P-p21 (T145) levels in ex-vivo CX-4945 treated human PBMCs
Effects of CX-4945 on Translation in PC3 Cells, 24 hr
0
20
40
60
80
100
0 0.1 1 5 10
[CX-4945] uM
35S-
Met
inco
rpor
atio
n (%
)
Inhibition of S35-Met Incorporation in PC3 Cells at 24 hrs
0
20
40
60
80
100
0 0.1 1 5 10
35D
-Met
inco
rpor
atio
n (%
)
[CX-4945] uM
Effects of CX-4945 on Transaltion in BxPC3 Cells, 24 hrInhibition of S35-Met Incorporation in
BxPC3 Cells at 24 hrs
CX-4945 Inhibits S35-Methionine Incorporation
CX-4945 Molecular Structure and Kinase Selectivity
CX-4945 Pharmacokinetics
Species CLs (L/kg/hr) Vdss
(L/kg)Terminal T1/2 (hr)
%F0-inf
Mouse 2.7 19.5 7.1 21.6
Rat 0.1 2.0 12.3 47.8
Dog 0.7 8.9 5.2 47.3
Cellular CK2 kinase activity assessed by preparing cell lysates and exposing to a CK2-specific peptide fragment in the presence of radio-labeled ATP
145 Kinases
< 50% inhibition at 500nM OR Kd > 500nM
> 50% inhibition at 500nM OR Kd < 500nM
> 90% inhibition at 500nM OR Kd < 100nM
