Protein Kinase Inhibitors

All individuals in Southern Sweden with a cancer diagnosis occurring between the ages of 0–18 years of age from 1970–01–01 -2016–12–31 were identified in the national cancer registry. The starting point of enrollment of patients into the registry was chosen due to the low survival rates of childhood cancer before 1970. Since its start in 1958, Swedish law mandates that all cancer cases be reported to the national registry which registers all cancer diagnoses (primary and subsequent) but not details on treatment. The vital status and personal details of the childhood cancer patients were retrieved from the Swedish population registry. Together this data formed the basis for the manual data collection of treatment history and details of the primary malignancy found in the medical records of each individual. In order for the treatment data to be collected and entered into BORISS, the medical records were manually gathered from the archives of the children’s clinic in Lund. Records were also found in the departments of Neurosurgery, Pediatric surgery, Medicine, Oncology and Pathology in Lund and in other hospitals in Southern Sweden.
The diagnosis of a malignancy was verified by an experienced pediatric oncologist (TW), by assessment of the pathology report. The diagnosis of malignancy is coded according to ICD-7, ICD-9, C24, ICD-O version 3 and ICD-10. The registry contains 219 different histological diagnoses, divided in 12 categories according to ICCC. Details of chemotherapy were extracted from prescription documents and later on from separate chemotherapy records. The dates and administration routes of all chemotherapy was recorded. All cytostatic agents (conventional drugs and more recently introduced drugs e.g. tyrosine kinase inhibitors, protein kinase inhibitors and antibodies) were recorded. The chemotherapy drugs (n = 91) are coded by a 3-digit number. Cumulative doses of each drug (mg/m² body surface area) were calculated. From the Radiation clinic, details of target organ(s), total irradiation dose (Gy) and fractionation was gathered. The target organ which received radiation therapy is coded by a 3-digit number and the target list covers 150 targets. When surgery was performed, details of the procedure, microscopic surgical margins, removal of organ(s) or an extremity, if any, were recorded. For stem cell treatment, details of conditioning therapy (with chemotherapy and/or total body irradiation), and dates of the procedure were recorded.
All entered data was cross-checked by two separate individuals. The registry contains personal data, diagnosis (in plain text and in code; ICD-7, ICD-9, C24, ICD-10 and ICD-0 version 3), and date of diagnosis. Phenotypic and cytogenetic data for leukemia, as well as cytogenetic aberrations with regards to solid tumors were gathered from medical records. Where available, data was recorded on other serious diseases, previous treatments, constitutional chromosomal aberrations, and other possible immunosuppressive treatments (pre-dating the cancer diagnosis), as was heredity for malignancies, and data on relapses. By annual updating of the database from the national cancer registry and the population registry, the development of secondary and subsequent primary neoplasms and vital status, are obtained.
Prospective collection of treatment data is carried out from 2016–01–01 and onwards under the governance of the Council of Skåne.

We previously developed a protocol [29 (link)] which associated domain families with drugs, by calculating the over-representation of drug targets within domain families. To identify druggable KinFams associated with human protein kinases, we adopted a similar approach: an FDA-approved kinase-inhibitor drug dataset was extracted from ChEMBL release 30 [41 (link)], https://www.ebi.ac.uk/chembl/, accessed on 2 November 2022). A drug was considered as a small molecule with therapeutic application, with direct binding to a single protein (ASSAY-TYPE = “B”), with a maximum phase of development = “4”, which indicates that the drug has been approved. Those with weak activity were filtered out by only considering a drug-target activity stronger than 1mM and a pChEMBL value of 6. The pChEMBL value is the measure of the half-maximal potency/affinity on a negative logarithmic scale. The anatomical therapeutic code (ATC-code) was used to select drugs that are protein kinase inhibitors. The ATC code classifies drugs into different groups at different levels (https://www.whocc.no/atc_ddd_index/, accessed on 24 January 2023). The code “L01E” corresponds to antineoplastic drugs which are protein kinase inhibitors.

CATH typically classifies the functional unit in protein kinases into two separate domains corresponding to the N- and C- lobes (or domains). These are represented as the CATH superfamilies 3.30.200.20 (N-domain) and 1.10.510.10 (C-domain), respectively (https://www.cathdb.info/, accessed on 24 January 2023). As the majority of the protein kinase inhibitors act at the hinge region between these two domains, we have created a new level within CATH to classify such ‘functional units’. This will clearly be valuable for enzymes and other proteins where the functional unit straddles more than one domain. In the context of the kinases, not only will it enable us to better understand the relationships between different kinases, but it will be essential for understanding kinase–drug interactions and enabling drug repurposing. The concept of a functional unit is illustrated in Figure 1 (illustrated using PDB ID: 1H8F).

Glutamate release from purified prefrontal cortical synaptosomes was monitored online with an assay that employed exogenous GDH and NADP⁺ to couple the oxidative deamination of the released glutamate to the generation of NADPH detected fluorometrically [49 (link)]. A 2 mL aliquot of synaptosomes (0.5 mg of protein) was transferred to a cuvette maintained at 37 °C and stirred with a magnetic bar. Synaptosomes were preincubated with PD 10 min before depolarization. Bafilomycin A1, dl-TBOA, ω-conotoxin GVIA, ω-agatoxin IVA, dantrolene or CGP37157 were added together with PD, and protein kinase inhibitors were added 30 min before the addition of PD. After 5 min, 2 mM NADP⁺ and 50 units/mL GDH were added and allowed to stir for 3 min, and then 1 mM CaCl₂ was added. After a further 2 min of incubation, 1.2 mM 4-AP or 15 mM KCl was added to depolarize the synaptosomes and stimulate glutamate release. The rate of increase in NADPH fluorescence at 460 nm emission (340 nm excitation) spectra was monitored over a 10 min time period using a PerkinElmer FL 6500 Fluorescence Spectrophotometer (PerkinElmer, Inc., Waltham, MA, USA). At the end of each assay, the traces were calibrated by adding 5 nmol of exogenous glutamate, and data were accumulated at 2 s intervals. The data were analyzed using GraphPad Prism 8.4.3 (GraphPad, San Diego, CA, USA), and the released glutamate was expressed as nanomoles of glutamate per milligram synaptosomal protein after 5 min of depolarization (nmol mg of protein⁻¹ 5 min⁻¹).