ABSTRACT
The discovery of potent, peptide site directed, tyrosine kinase inhibitors has remained an elusive goal. Herein we describe the discovery of two such clinical candidates that inhibit the tyrosine kinase Src. Compound 1 is a phase 3 clinical trial candidate that is likely to provide a first in class topical treatment for actinic keratosis (AK) with good efficacy and dramatically less toxicity compared to existing standard therapy. Compound 2 is a phase 1 clinical trial candidate that is likely to provide a first in class treatment of malignant glioblastoma and induces 30% long-term complete tumor remission in animal models. The discovery strategy for these compounds iteratively utilized molecular modeling, along with the synthesis and testing of increasingly elaborated proof of concept compounds, until the final clinical candidates were arrived at. This was followed with mechanism of action (MOA) studies that revealed tubulin polymerization inhibition as the second MOA.
Subject(s)
Acetamides/pharmacology , Drug Discovery , Morpholines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Tubulin Modulators/pharmacology , src-Family Kinases/antagonists & inhibitors , Acetamides/metabolism , Amino Acid Sequence , Catalytic Domain , Cell Line, Tumor , Humans , Molecular Docking Simulation , Morpholines/metabolism , Protein Kinase Inhibitors/metabolism , Pyridines/metabolism , Signal Transduction/drug effects , Tubulin Modulators/metabolism , src-Family Kinases/chemistry , src-Family Kinases/metabolismABSTRACT
OBJECTIVES: The aim of this study is to determine whether amitotic division or nuclear proliferation is involved in the formation of giant cells (GCs) in giant cell hepatitis (GCH). PATIENTS AND METHODS: Liver sections from 18 pediatric patients with idiopathic infantile GCH and 12 patients with postinfantile GCH were evaluated for the expression of proliferating cell nuclear antigen (PCNA) and human histone 3 (H3) mRNA, transforming growth factor-alpha (TGF-α), TGF-ß1, hepatocyte growth factor (HGF), and epidermal growth factor receptor (EGFR). RESULTS: Proliferation markers were detected in 1% to 80% in the nuclei of GC and non-GC hepatocytes in 10 of 18 (56%) infantile GCH biopsies and 11 of 12 (92%) postinfantile GCH biopsies, but not in normal liver. The expression of proliferation markers in GCs paralleled that in non-GC hepatocytes (P < 0.05 for both markers). TGF-α and EGFR were detected in both GCs (9/29 and 4/30 patients with infantile or postinfantile GCH, respectively) and non-GC hepatocytes (15/29 and 11/30 patients with infantile or postinfantile GCH, respectively). TGF-ß1 and HGF were detected mainly in sinusoidal cells in 20 of 29 and 10 of 30 patients with infantile or postinfantile GCH, respectively; the expression of HGF was positively correlated with PCNA and H3 mRNA in non-GC hepatocytes and with H3 mRNA in GCs (P < 0.01). CONCLUSIONS: Hepatic expressions of nuclear proliferation markers and growth factors were similar in infantile and postinfantile GCH, nuclear proliferation markers were detected in both GCs and non-GC hepatocytes in a high proportion of patients, and expression of HGF correlated positively with the proliferation markers. These data indicate that nuclear proliferation may contribute to the pathogenesis of GCs in at least a proportion of patients with GCH. A model for the pathogenesis of GCH is proposed.
Subject(s)
Cell Proliferation , Giant Cells/metabolism , Hepatitis/metabolism , Hepatitis/pathology , Hepatocytes/metabolism , Adolescent , Adult , Age Factors , Aged , Biomarkers/metabolism , Biopsy , Child , ErbB Receptors/metabolism , Female , Giant Cells/pathology , Hepatocyte Growth Factor/metabolism , Hepatocytes/pathology , Histones/genetics , Histones/metabolism , Humans , Infant , Male , Middle Aged , Proliferating Cell Nuclear Antigen/metabolism , RNA, Messenger/metabolism , Serologic Tests , Statistics, Nonparametric , Transforming Growth Factor alpha/metabolism , Transforming Growth Factor beta/metabolismABSTRACT
The expressions of c-Src and focal adhesion kinase (FAK) were studied in 65 Chinese patients with hepatocellular carcinoma (HCC) by immunohistochemistry using rabbit monoclonal antibodies. Expressions of total Src, an active form of Src, and FAK were found in 44/65 (67.7%), 36/45 (55.4%), and 33/56 (58.9%) HCC cases, respectively. There was a good correlation between the expression of total Src, active form of Src, and FAK in these HCC cases (P < 0.001). Expression of Src was not correlated to any clinical parameters, cancer cell phenotypic markers, and pathologic features apart from a positive correlation with alpha-fetoprotein (P < 0.01). The expression of FAK was correlated with earlier onset and the expression of Ki-67 but not proliferating cell nuclear antigen (PCNA) in these HCC cases. Four liver-cancer-derived cell lines (three derived from HCC and one from hepatoblastoma) were then tested with inhibitors against Src. A small molecule, KX2-391, designed to target the substrate binding pocket of Src, was found to have more broad-spectrum activity and better potency than Dasatinib, an adenosine triphosphate (ATP)-competitive inhibitor in vitro. Our data indicates that Src and FAK expression are both elevated and active in Chinese patients with HCC and that Src may play a key role in supporting HCC progression. Src antagonism with specific inhibitors may be an attractive treatment paradigm for patients with HCC.
