Supplementary Figure 1 from Characterization of an Akt Kinase Inhibitor with Potent Pharmacodynamic and Antitumor Activity

Supplementary Figure 2 from Characterization of an Akt Kinase Inhibitor with Potent Pharmacodynamic and Antitumor Activity

Supplementary Fig. S1 from Pharmacokinetic-pharmacodynamic correlation from mouse to human with pazopanib, a multikinase angiogenesis inhibitor with potent antitumor and antiangiogenic activity

Supplementary Fig. S2 from Pharmacokinetic-pharmacodynamic correlation from mouse to human with pazopanib, a multikinase angiogenesis inhibitor with potent antitumor and antiangiogenic activity

Supplementary Methods and Figure Legends 1-2 from Characterization of an Akt Kinase Inhibitor with Potent Pharmacodynamic and Antitumor Activity

Supplementary Table S4 from Pharmacokinetic-pharmacodynamic correlation from mouse to human with pazopanib, a multikinase angiogenesis inhibitor with potent antitumor and antiangiogenic activity

Supplementary Fig. S1 from Pharmacokinetic-pharmacodynamic correlation from mouse to human with pazopanib, a multikinase angiogenesis inhibitor with potent antitumor and antiangiogenic activity

2,3,5-Trisubstituted pyridines have been designed as potent AKT inhibitors that are selective against ROCK1 based on the comparison between AKT and ROCK1 structures. Substitution at the 2-position of the core pyridine is the key element to provide selectivity against ROCK1. An X-ray co-crystal structure of 9p in PKA supports the proposed rationale of ROCK1 selectivity.

The discovery and development of a series of thiophenes as potent and selective inhibitors of PLK is described. Identification and characterization of 2, a useful in vitro PLK inhibitor tool compound, is also presented.

Inhibition of the vascular endothelial growth factor (VEGF) signaling pathway has emerged as one of the most promising new approaches for cancer therapy. We describe herein the key steps starting from an initial screening hit leading to the discovery of pazopanib, N(4)-(2,3-dimethyl-2H-indazol-6-yl)-N(4)-methyl-N(2)-(4-methyl-3-sulfonamidophenyl)-2,4-pyrimidinediamine, a potent pan-VEGF receptor (VEGFR) inhibitor under clinical development for renal-cell cancer and other solid tumors.

Overexpression of AKT has an antiapoptotic effect in many cell types, and expression of dominant negative AKT blocks the ability of a variety of growth factors to promote survival. Therefore, inhibitors of AKT kinase activity might be useful as monotherapy for the treatment of tumors with activated AKT. Herein, we describe our lead optimization studies culminating in the discovery of compound 3g (GSK690693). Compound 3g is a novel ATP competitive, pan-AKT kinase inhibitor with IC 50 values of 2, 13, and 9 nM against AKT1, 2, and 3, respectively. An X-ray cocrystal structure was solved with 3g and the kinase domain of AKT2, confirming that 3g bound in the ATP binding pocket. Compound 3g potently inhibits intracellular AKT activity as measured by the inhibition of the phosphorylation levels of GSK3beta. Intraperitoneal administration of 3g in immunocompromised mice results in the inhibition of GSK3beta phosphorylation and tumor growth in human breast carcinoma (BT474) xenografts.

A novel series of pyrazolo[1,5-b]pyridazines have been synthesized and identified as cyclin dependant kinase inhibitors potentially useful for the treatment of solid tumors. Modification of the hinge-binding amine or the C(2)- and C(6)-substitutions on the pyrazolopyridazine core provided potent inhibitors of CDK4 and demonstrated enzyme selectivity against VEGFR-2 and GSK3beta.

Abstract Akt kinases 1, 2, and 3 are important regulators of cell survival and have been shown to be constitutively active in a variety of human tumors. GSK690693 is a novel ATP-competitive, low-nanomolar pan-Akt kinase inhibitor. It is selective for the Akt isoforms versus the majority of kinases in other families; however, it does inhibit additional members of the AGC kinase family. It causes dose-dependent reductions in the phosphorylation state of multiple proteins downstream of Akt, including GSK3β, PRAS40, and Forkhead. GSK690693 inhibited proliferation and induced apoptosis in a subset of tumor cells with potency consistent with intracellular inhibition of Akt kinase activity. In immune-compromised mice implanted with human BT474 breast carcinoma xenografts, a single i.p. administration of GSK690693 inhibited GSK3β phosphorylation in a dose- and time-dependent manner. After a single dose of GSK690693, >3 μmol/L drug concentration in BT474 tumor xenografts correlated with a sustained decrease in GSK3β phosphorylation. Consistent with the role of Akt in insulin signaling, treatment with GSK690693 resulted in acute and transient increases in blood glucose level. Daily administration of GSK690693 produced significant antitumor activity in mice bearing established human SKOV-3 ovarian, LNCaP prostate, and BT474 and HCC-1954 breast carcinoma xenografts. Immunohistochemical analysis of tumor xenografts after repeat dosing with GSK690693 showed reductions in phosphorylated Akt substrates in vivo. These results support further evaluation of GSK690693 as an anticancer agent. [Cancer Res 2008;68(7):2366–74]

4171 Polo-like kinase 1 (Plk1) plays an important role in the entry to, progression through, and exit from mitosis. The multiple roles Plk1 plays in mitosis make it an intriguing target to inhibit to disrupt the cell cycle and treat cancer. This presentation will highlight the lead optimization studies and elucidation of the structure-activity relationship leading to the identification of a novel thiophene amide Plk1 inhibitor. It will include the synthesis of a Plk-selective inhibitor, in addition to optimization of potency and developability characteristics through several iterations of structural changes, culminating in the identification of the clinical candidate. These efforts led to the development of GSK461364, an extremely potent Plk1 inhibitor (IC 50 ~3 nM) with greater than 100-fold selectivity across a panel of 47 other kinases. It also potently inhibits the proliferation of many tumor cell lines in vitro , shows in vivo activity in tumor xenograft mouse models, and has appropriate physical and pharmacokinetic characteristics to support further evaluation as an anticancer agent.

ND-2 Over-expression of AKT has an anti-apoptotic effect in many cell types and expression of dominant negative AKT blocks the ability of a variety of growth factors to promote survival. In addition, PTEN, a critical negative regulator of AKT, is lost in many cancers, including breast and prostate carcinomas, glioblastomas, and several cancer syndromes including Bannayan-Zonana syndrome, Cowden disease, and Lhermitte-Duclos disease. Therefore inhibitors of AKT kinase activity might be useful as monotherapy for the treatment of tumors with activated AKT. Herein, we will describe our lead optimization studies culminating in the discovery of GSK690693. GSK690693 is a novel ATP competitive, pan-AKT kinase inhibitor with IC 50 s of 2, 13 and 9 nM against AKT-1, 2 and 3, respectively. This compound binds in the ATP binding site as shown by an x-ray co-crystal structure with AKT2. >Treatment of tumor cells with GSK690693 causes dose dependent reductions in the phosphorylation state of multiple proteins downstream of AKT, including GSK3β, PRAS40 and Forkhead (FOXO1/FOXO3a). GSK690693 inhibits proliferation and induces apoptosis in a subset of tumor cells with potency that is consistent with intracellular inhibition of AKT kinase activity. >A single intraperitoneal (IP) administration of GSK690693 inhibits GSK3β phosphorylation in SCID mice bearing BT474 breast tumor xenografts in a dose and time-dependent manner. GSK690693 treatment (once daily for 21 days) produces significant anti-tumor activity in mice bearing established human SK-OV-3 ovarian, LNCaP prostate, and BT474 and HCC-1954 breast carcinoma xenografts. Immunohistochemical analysis of tumor xenografts after repeat dosing with GSK690693 demonstrates reductions in phosphorylated AKT substrates. The pharmacodynamic and antitumor effects of GSK690693 support its evaluation as an anticancer agent. >A summary of the pharmacokinetic properties and the description of a scalable synthetic route to GSK690693 will also be presented.

Polo-like kinase 1 (PLK1) plays key roles in the regulation of mitotic progression, including mitotic entry, spindle formation, chromosome segregation, and cytokinesis. PLK1 expression and activity are strongly linked to proliferating cells. Many studies have shown that PLK1 expression is elevated in a variety of tumors, and high expression often correlates with poor prognosis. Using a variety of methods, including small-molecule inhibition of PLK1 function and/or activity, apoptosis in cancer cell lines, cell cycle arrest in normal cell lines, and antitumor activity in vivo have been observed. In the present study, we have examined the in vitro biological activity of a novel and selective thiophene benzimidazole ATP-competitive inhibitor of PLK1 and PLK3 (5-(5,6-dimethoxy-1H-benzimidazol-1-yl)-3-{[2-(trifluoromethyl)-benzyl]oxy}-thiophene-2-carboxamide, called compound 1). Compound 1 has low nanomolar activity against the PLK1 and PLK3 enzymes and potently inhibits the proliferation of a wide variety of tumor cell lines. In the lung adenocarcinoma cell line NCI-H460, compound 1 induces a transient G(2)-M arrest, mitotic spindle defects, and a multinucleate phenotype resulting in apoptosis, whereas normal human diploid fibroblasts arrest in G2-M and show little apoptosis. We also describe a cellular mechanistic assay that was developed to identify potent intracellular inhibitors of PLK1. In addition to its potential as a therapeutic agent for treating cancer, compound 1 is also a useful tool molecule for further investigation of the biological functions of PLK1 and PLK3.

Abstract With the development of targeted therapeutics, especially for small-molecule inhibitors, it is important to understand whether the observed in vivo efficacy correlates with the modulation of desired/intended target in vivo. We have developed a small-molecule inhibitor of all three vascular endothelial growth factor (VEGF) receptors (VEGFR), platelet-derived growth factor receptor, and c-Kit tyrosine kinases, pazopanib (GW786034), which selectively inhibits VEGF-induced endothelial cell proliferation. It has good oral exposure and inhibits angiogenesis and tumor growth in mice. Because bolus administration of the compound results in large differences in Cmax and Ctrough, we investigated the effect of continuous infusion of a VEGFR inhibitor on tumor growth and angiogenesis. GW771806, which has similar enzyme and cellular profiles to GW786034, was used for these studies due to higher solubility requirements for infusion studies. Comparing the pharmacokinetics by two different routes of administration (bolus p.o. dosing and continuous infusion), we showed that the antitumor and antiangiogenic activity of VEGFR inhibitors is dependent on steady-state concentration of the compound above a threshold. The steady-state concentration required for these effects is consistent with the concentration required for the inhibition of VEGF-induced VEGFR2 phosphorylation in mouse lungs. Furthermore, the steady-state concentration of pazopanib determined from preclinical activity showed a strong correlation with the pharmacodynamic effects and antitumor activity in the phase I clinical trial. [Mol Cancer Ther 2007;6(7):2012–21]

A series of dianilinopyrimidineureas demonstrate potency as VEGFR2 kinase inhibitors.