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Additional information to provide an insight into the full kinase selectivity profile of AT7519 and full details of its pharmakokinetic parameters

Cyclin-dependent kinases (CDK), and their regulatory cyclin partners, play a central role in eukaryotic cell growth, division, and death. This key role in cell cycle progression, as well as their deregulation in several human cancers, makes them attractive therapeutic targets in oncology. A series of CDK inhibitors was developed using Astex's fragment-based medicinal chemistry approach, linked to high-throughput X-ray crystallography. A compound from this series, designated AT7519, is currently in early-phase clinical development. We describe here the biological characterization of AT7519, a potent inhibitor of several CDK family members. AT7519 showed potent antiproliferative activity (40-940 nmol/L) in a panel of human tumor cell lines, and the mechanism of action was shown here to be consistent with the inhibition of CDK1 and CDK2 in solid tumor cell lines. AT7519 caused cell cycle arrest followed by apoptosis in human tumor cells and inhibited tumor growth in human tumor xenograft models. Tumor regression was observed following twice daily dosing of AT7519 in the HCT116 and HT29 colon cancer xenograft models. We show that these biological effects are linked to inhibition of CDKs in vivo and that AT7519 induces tumor cell apoptosis in these xenograft models. AT7519 has an attractive biological profile for development as a clinical candidate, and the tolerability and efficacy in animal models compare favorably with other CDK inhibitors in clinical development. Studies described here formed the biological rationale for investigating the potential therapeutic benefit of AT7519 in cancer patients. [Mol Cancer Ther 2009;8(2):324–32]

Supplementary Data from Biological characterization of AT7519, a small-molecule inhibitor of cyclin-dependent kinases, in human tumor cell lines

Cyclin-dependent kinases (CDK), and their regulatory cyclin partners, play a central role in eukaryotic cell growth, division, and death. This key role in cell cycle progression, as well as their deregulation in several human cancers, makes them attractive therapeutic targets in oncology. A series of CDK inhibitors was developed using Astex's fragment-based medicinal chemistry approach, linked to high-throughput X-ray crystallography. A compound from this series, designated AT7519, is currently in early-phase clinical development. We describe here the biological characterization of AT7519, a potent inhibitor of several CDK family members. AT7519 showed potent antiproliferative activity (40-940 nmol/L) in a panel of human tumor cell lines, and the mechanism of action was shown here to be consistent with the inhibition of CDK1 and CDK2 in solid tumor cell lines. AT7519 caused cell cycle arrest followed by apoptosis in human tumor cells and inhibited tumor growth in human tumor xenograft models. Tumor regression was observed following twice daily dosing of AT7519 in the HCT116 and HT29 colon cancer xenograft models. We show that these biological effects are linked to inhibition of CDKs in vivo and that AT7519 induces tumor cell apoptosis in these xenograft models. AT7519 has an attractive biological profile for development as a clinical candidate, and the tolerability and efficacy in animal models compare favorably with other CDK inhibitors in clinical development. Studies described here formed the biological rationale for investigating the potential therapeutic benefit of AT7519 in cancer patients. [Mol Cancer Ther 2009;8(2):324–32]

The application of fragment-based screening techniques to cyclin dependent kinase 2 (CDK2) identified multiple (>30) efficient, synthetically tractable small molecule hits for further optimization. Structure-based design approaches led to the identification of multiple lead series, which retained the key interactions of the initial binding fragments and additionally explored other areas of the ATP binding site. The majority of this paper details the structure-guided optimization of indazole (6) using information gained from multiple ligand−CDK2 cocrystal structures. Identification of key binding features for this class of compounds resulted in a series of molecules with low nM affinity for CDK2. Optimisation of cellular activity and characterization of pharmacokinetic properties led to the identification of 33 (AT7519), which is currently being evaluated in clinical trials for the treatment of human cancers.

AT7519 is a potent inhibitor of several cyclin-dependent kinases and is currently in early phase clinical development. Recently, cyclin-dependent kinases 7, 8, and 9 have been shown to regulate transcription through phosphorylation of RNA polymerase II. B-cell lymphoproliferative disorders, including chronic lymphocytic leukemia, rely on the expression of transcripts with a short half-life, such as Mcl-1, Bcl-2, and XIAP, for survival. Here, we describe the characterization of AT7519 in leukemia cell lines, and compare and contrast the response in cell lines derived from solid tumors. Finally, we use these mechanistic insights to show activity in peripheral blood mononuclear cells isolated from 16 chronic lymphocytic leukemia patients. AT7519 induced apoptosis at concentrations of 100 to 700 nmol/L and was equally effective regardless of Rai stage or known prognostic markers. Short-term treatments (4-6 hours) resulted in inhibition of phosphorylation of the transcriptional marker RNA polymerase II and downregulation of the antiapoptotic protein Mcl-1, with no effect on either XIAP or Bcl-2 levels. The reduction in Mcl-1 protein level was associated with an increase in cleaved poly(ADP-ribose) polymerase. Together the data suggest AT7519 offers a promising treatment for patients with advanced B-cell leukemia. Mol Cancer Ther; 9(4); 920–8. ©2010 AACR.

Degradation of cartilage is recognised as a key event in arthritis, yet this is a difficult process to accurately quantify in vivo. In the clinic, joint space narrowing is used as a measure of cartilage loss because cartilage itself cannot be visualised by radiography. There are limitations to this technique because of the precision of alignment of the joint needed to achieve reproducible quantification [1, 2]. High definition images of cartilage in the joint can be produced by magnetic resonance imaging [3]. Quantification of cartilage loss would then require MRI systems which capture slices from the entire joint to perform volumetric analysis of the total joint cartilage. At the present time this can be achieved, but it is expensive and requires long imaging runs of many hours which would be impossible with conscious patients.

Injections of labile copper complexes such as copper II bisglycinate [Cu(II)gly] induce marked inflammatory responses in rats in contrast to stable copper complexes like copper II bishistidinate which are nonirritant. The antirheumatic drugs D-penicillamine, mercaptopyridoxine, thiola and captopril, inhibit Cu(II)gly-induced cutaneous vascular permeability when given intravenously and show oral cupriuretic activity. Inhibition of Cu(II)gly inflammation alone or cupriuretic activity alone do not appear predictive of clinical antirheumatic activity since L-cysteine methylester and trien, which are active in the former and latter tests, respectively, are devoid of clinical antirheumatic activity. Indomethacin and aspirin are inactive in these tests, thus discounting the concept that such drugs act in part through their copper complexes. The mechanisms by which labile copper induces inflammation and by which D-penicillamine-like drugs modulate the response are discussed. It is suggested that copper can generate free radicals in vivo and that D-penicillamine may act by neutralization of labile copper complexes and/or by formation of copper complexes with the capacity to catalyse the dismutation of superoxide anion.