Therapeutic Kinase Inhibitors
Kinases are required for cell cycle progression and cellular responses to growth factors. Given the regulatory role of kinases in cell growth and division, it is not surprising that mutations in kinase genes or misregulation of kinase expression occurs in many cancers. Indeed, nearly 75% of all human kinases are expressed in many breast cancer cell lines. Therefore, developing specific kinase inhibitors for the treatment of cancer and other diseases is an important goal for drug development.
Vichem Chemie, a Hungarian-German biotechnology company, specializes in the development of kinase inhibitors as therapeutics in collaboration with Axel Ullrich, Ph.D., director of molecular biology at the Max Planck Institute of Biochemistry. When Vichem screens for clinically relevant kinase inhibitors, it uses a knowledge-base approach, consulting its Nested Chemical Library, a collection of kinase inhibitory compounds of about 110 core structures and more than 600 scaffolds. The library encompasses both published and proprietary compounds.
According to Vichem’s CSO, György Kéri, Ph.D., D.Sc., the company’s knowledge-base approach is best suited for addressing the complex kinome changes in disease. “According to the Catalogue of Somatic Mutations in Cancer (COSMIC) database, there are 138 driver genes, of which 64 are oncogenic, and 74 are tumor suppressor genes,” notes Dr. Kéri. “Most of these driver genes are associated directly or indirectly with kinases of the 12 major cancer pathways.”
Relying on statistics generated by the BioSeeker Group, Dr. Kéri states that drug development efforts are focused on 224 different targets, 202 of which have been recorded with somatic mutations. Industry-wide, there are 563 protein kinase inhibitor drugs in development for 155 indications, of which 90 are cancer indications.
“At Vichem, we have developed reference compounds for 160 of these kinases, and we focus on compounds that can block the cancer driver pathways,” confides Dr. Kéri. “We try to develop multiple target compounds, which will kill cancer stem cells because these are the really bad guys, and they also happen to have more so-called survival factor kinases than other tumor cells.”
To facilitate the development of these inhibitors, Vichem and its collaborators culture tumor cells from cancer patients and identify mutations in the driver genes through genomic sequencing. According to Dr. Kéri, this approach “allows us to see if there are mutations in known oncogenes and tumor suppressor genes.” In addition, the approach makes it possible “to check for mutations in the downstream targets of these suppressors and to see if other pathways are activated in the patient.”
“Despite the heterogeneity of the patient samples, the current data indicate that we should expect at least two mutations in driver genes. but not more than eight,” explains Dr. Kéri. “This should give us a good chance for proof-of-concept studies for using our Nested Chemical Library to develop targeted inhibitors for kinases that are cancer drivers.”
Vichem is also developing a clonogenic assay to identify biomarkers for cancer stem cells. The company also intends to develop kinase inhibitors to specifically target and kill them. It wants to use these genomic or proteomic biomarkers for the survival cultures from the patient material to utilize so called “stemkill” compounds for killing the cancer stem cells from the patient.
Dr. Kéri believes that this approach—the use of the driver-hit combinations or stemkill compounds derived from phenotypical screening—offers the most promise. “I’ve been working on this for 30 years and can finally see light at the end of the tunnel,” he exclaims. “[The cancer driver and cancer stem cell approaches for developing targeted inhibitors] should provide a very dramatic and synergistic effect.”
Dr. Kéri also sees the development of kinase inhibitors as a community effort. Accordingly, he has made the Vichem resources available for various research collaborations.
Kinases assays are obviously important for validating the effectiveness of the kinase inhibitors that Vichem is developing. Dr. Kéri notes that the company uses a variety of approaches for kinase assays. The immobilized metal assay for phosphochemicals (IMAP) technology, the Transcreener kinase assay, and the fluorescence polarization assay are among the preferred assay systems.
Through partners, Vichem also uses binding assays for 400 kinases for selected inhibitors; high-throughput, homogeneous, time-resolved fluorescence (HTRF) assays for its allosteric kinase inhibitor library; and an assay that resolves fluorescence-resonance energy transfer over time (time-resolved FRET, or TR-FRET). Vichem outsources much of its in vivo work, including ADMET assays for drug development, but conducts in vitro kinases assay as well as various cellular assays in house. The company also uses classic Western Blotting and kits from R&D Systems in addition to functional assays.
“We need to determine the IC50 values for an inhibitor for a series of kinases,” Dr. Kéri states. “We also need to determine whether it is an ATP binding site inhibitor (Type I) or a non-ATP binding inhibitor (Type III or Type IV).” Vichem also measures the inhibitor’s residence time, which reflects the inhibitor’s effectiveness in vivo. To take such measurements, Vichem works with colleagues at Proteros biostructures in Martinsreid, Germany.