A historical overview of protein kinases and their targeted small molecule inhibitors.

Protein kinases play a predominant regulatory role in nearly every aspect of cell biology and they can modify the function of a protein in almost every conceivable way. Protein phosphorylation can increase or decrease enzyme activity and it can alter other biological activities such as transcription and translation. Moreover, some phosphorylation sites on a given protein are stimulatory while others are inhibitory. The human protein kinase gene family consists of 518 members along with 106 pseudogenes. Furthermore, about 50 of the 518 gene products lack important catalytic residues and are called protein pseudokinases. The non-catalytic allosteric interaction of protein kinases and pseudokinases with other proteins has added an important regulatory feature to the biochemistry and cell biology of the protein kinase superfamily. With rare exceptions, a divalent cation such as Mg2+ is required for the reaction. All protein kinases exist in a basal state and are activated only as necessary by divergent regulatory stimuli. The mechanisms for switching between dormant and active protein kinases can be intricate. Phosphorylase kinase was the first protein kinase to be characterized biochemically and the mechanism of its regulation led to the discovery of cAMP-dependent protein kinase (protein kinase A, or PKA), which catalyzes the phosphorylation and activation of phosphorylase kinase. This was the first protein kinase cascade or signaling module to be elucidated. The epidermal growth factor receptor-Ras-Raf-MEK-ERK signaling module contains protein-tyrosine, protein-serine/threonine, and dual specificity protein kinases. PKA has served as a prototype of this enzyme family and more is known about this enzyme than any other protein kinase. The inactive PKA holoenzyme consists of two regulatory and two catalytic subunits. After binding four molecules of cAMP, the holoenzyme dissociates into a regulatory subunit dimer (each monomer binds two cAMP) and two free and active catalytic subunits. PKA and all other protein kinase domains have a small amino-terminal lobe and large carboxyterminal lobe as determined by X-ray crystallography. The N-lobe and C-lobe form a cleft that serves as a docking site for MgATP. Nearly all active protein kinases contain a K/E/D/D signature sequence that plays important structural and catalytic roles. Protein kinases contain hydrophobic catalytic and regulatory spines and collateral shell residues that are required to assemble the active enzyme. There are two general kinds of conformational changes associated with most protein kinases. The first conformational change involves the formation of an intact regulatory spine to form an active enzyme. The second conformational change occurs in active kinases as they toggle between open and closed conformations during their catalytic cycles. Because mutations and dysregulation of protein kinases play causal roles in human disease, this family of enzymes has become one of the most important drug targets over the past two decades. Imatinib was approved by the United States FDA for the treatment of chronic myelogenous leukemia in 2001; this small molecule inhibits the BCR-Abl protein kinase oncoprotein that results from the formation of the Philadelphia chromosome. More than two dozen other orally effective mechanism-based small molecule protein kinase inhibitors have been subsequently approved by the FDA. These drugs bind to the ATP-binding site of their target enzymes and extend into nearby hydrophobic pockets. Most of these protein kinase inhibitors prolong survival in cancer patients only weeks or months longer than standard cytotoxic therapies. In contrast, the clinical effectiveness of imatinib against chronic myelogenous leukemia is vastly superior to that of any other targeted protein kinase inhibitor with overall survival lasting a decade or more. However, the near universal and expected development of drug resistance in the treatment of neoplastic disorders requires new approaches to solve this therapeutic challenge. Cancer is the predominant indication for these drugs, but disease targets are increasing. For example, we can expect the approval of new drugs inhibiting other protein kinases in the treatment of illnesses such as hypertension, Parkinson's disease, and autoimmune diseases.

Roskoski R Jr 《-》

Properties of FDA-approved small molecule protein kinase inhibitors.

Because mutations, overexpression, and dysregulation of protein kinases play essential roles in the pathogenesis of many illnesses, this enzyme family has become one of the most important drug targets in the past 20 years. The US FDA has approved 48 small molecule protein kinase inhibitors, nearly all of which are orally effective with the exceptions of netarsudil (which is given as an eye drop) and temsirolimus (which is given intravenously). Of the 48 approved drugs, the majority (25) target receptor protein-tyrosine kinases, ten target non-receptor protein-tyrosine kinases, and 13 target protein-serine/threonine protein kinases. The data indicate that 43 of these drugs are used in the treatment of malignancies (36 against solid tumors including lymphomas and seven against non-solid tumors, e.g., leukemias). Seven drugs are used in the treatment of non-malignancies: baricitinib, rheumatoid arthritis; fostamatinib, chronic immune thrombocytopenia; ruxolitinib, myelofibrosis and polycythemia vera; nintedanib, idiopathic pulmonary fibrosis; sirolimus, renal graft vs. host disease; netarsudil, glaucoma; tofacitinib, rheumatoid arthritis, Crohn disease, and ulcerative colitis. Moreover, ibrutinib and sirolimus are used for the treatment of both malignant and non-malignant diseases. The most common drug targets include ALK, B-Raf, BCR-Abl, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor receptor (VEGFR). Most of the small molecule inhibitors (45) interact directly with the protein kinase domain. In contrast, sirolimus, temsirolimus, and everolimus are larger molecules (MW ≈ 1000) that bind to FKBP-12 to generate a complex that inhibits mTOR (mammalian target of rapamycin). This review presents the available drug-enzyme X-ray crystal structures for 27 of the approved drugs as well as the chemical structures and physicochemical properties of all of the FDA-approved small molecule protein kinase antagonists. Six of the drugs bind covalently and irreversibly to their target. Twenty of the 48 drugs have molecular weights greater than 500, exceeding a Lipinski rule of five criterion. Excluding the macrolides (everolimus, sirolimus, temsirolimus), the average molecular weight of drugs is 480 with a range of 306 (ruxolitinib) to 615 (trametinib). Nearly half of the antagonists (23) have a lipophilic efficiency with values of less than five while the recommended optima range from 5-10. One of the vexing problems is the near universal development of resistance that is associated with the use of small molecule protein kinase inhibitors for the treatment of cancer.

Roskoski R Jr 《-》

Properties of FDA-approved small molecule protein kinase inhibitors: A 2020 update.

Because genetic alterations including mutations, overexpression, translocations, and dysregulation of protein kinases are involved in the pathogenesis of many illnesses, this enzyme family is currently the subject of many drug discovery programs in the pharmaceutical industry. The US FDA approved four small molecule protein kinase antagonists in 2019; these include entrectinib, erdafitinib, pexidartinib, and fedratinib. Entrectinib binds to TRKA/B/C and ROS1 and is prescribed for the treatment of solid tumors with NTRK fusion proteins and for ROS1-postive non-small cell lung cancers. Erdafitinib inhibits fibroblast growth factor receptors 1-4 and is used in the treatment of urothelial bladder cancers. Pexidartinib is a CSF1R antagonist that is prescribed for the treatment of tenosynovial giant cell tumors. Fedratinib blocks JAK2 and is used in the treatment of myelofibrosis. Overall, the US FDA has approved 52 small molecule protein kinase inhibitors, nearly all of which are orally effective with the exceptions of temsirolimus (which is given intravenously) and netarsudil (an eye drop). Of the 52 approved drugs, eleven inhibit protein-serine/threonine protein kinases, two are directed against dual specificity protein kinases, eleven target non-receptor protein-tyrosine kinases, and 28 block receptor protein-tyrosine kinases. The data indicate that 46 of these drugs are used in the treatment of neoplastic diseases (eight against non-solid tumors such as leukemias and 41 against solid tumors including breast and lung cancers; some drugs are used against both tumor types). Eight drugs are employed in the treatment of non-malignancies: fedratinib, myelofibrosis; ruxolitinib, myelofibrosis and polycythemia vera; fostamatinib, chronic immune thrombocytopenia; baricitinib, rheumatoid arthritis; sirolimus, renal graft vs. host disease; nintedanib, idiopathic pulmonary fibrosis; netarsudil, glaucoma; and tofacitinib, rheumatoid arthritis, Crohn disease, and ulcerative colitis. Moreover, sirolimus and ibrutinib are used for the treatment of both neoplastic and non-neoplastic diseases. Entrectinib and larotrectinib are tissue-agnostic anti-cancer small molecule protein kinase inhibitors. These drugs are prescribed for the treatment of any solid cancer harboring NTRK1/2/3 fusion proteins regardless of the organ, tissue, anatomical location, or histology type. Of the 52 approved drugs, seventeen are used in the treatment of more than one disease. Imatinib, for example, is approved for the treatment of eight disparate disorders. The most common drug targets of the approved pharmaceuticals include BCR-Abl, B-Raf, vascular endothelial growth factor receptors (VEGFR), epidermal growth factor receptors (EGFR), and ALK. Most of the approved small molecule protein kinase antagonists (49) bind to the protein kinase domain and six of them bind covalently. In contrast, everolimus, temsirolimus, and sirolimus are larger molecules (MW ≈ 1000) that bind to FK506 binding protein-12 (FKBP-12) to generate a complex that inhibits the mammalian target of rapamycin (mTOR) protein kinase complex. This review presents the physicochemical properties of all of the FDA-approved small molecule protein kinase inhibitors. Twenty-two of the 52 drugs have molecular weights greater than 500, exceeding a Lipinski rule of five criterion. Excluding the macrolides (everolimus, sirolimus, temsirolimus), the average molecular weight of the approved drugs is 480 with a range of 306 (ruxolitinib) to 615 (trametinib). More than half of the antagonists (29) have lipophilic efficiency values of less than five while the recommended optima range from 5 to 10. One of the troublesome problems with both targeted and cytotoxic drugs in the treatment of malignant diseases is the near universal development of resistance to every therapeutic modality.

Roskoski R Jr 《-》

Targeting oncogenic Raf protein-serine/threonine kinases in human cancers.

The Ras-Raf-MEK-ERK signal transduction cascade is arguably the most important oncogenic pathway in human cancers. Ras-GTP promotes the formation of active homodimers or heterodimers of A-Raf, B-Raf, and C-Raf by an intricate process. These enzymes are protein-serine/threonine kinases that catalyze the phosphorylation and activation of MEK1 and MEK2 which, in turn, catalyze the phosphorylation and activation of ERK1 and ERK2. The latter catalyze the regulatory phosphorylation of dozens of cytosolic and nuclear proteins. The X-ray crystal structure of B-Raf-MEK1 depicts a face-to-face dimer with interacting activation segments; B-Raf is in an active conformation and MEK1 is in an inactive conformation. Besides the four traditional components in the Ras-Raf-MEK-ERK signaling module, scaffolding proteins such as Kinase Suppressor of Ras (KSR1/2) play an important role in this signaling cascade by functioning as a scaffold protein. RAS mutations occur in about 30% of all human cancers. Moreover, BRAFV600E mutations occur in about 8% of all cancers making this the most prevalent oncogenic protein kinase. Vemurafenib and dabrafenib are B-RafV600E inhibitors that were approved for the treatment of melanomas bearing the V600E mutation. Coupling MEK1/2 inhibitors with B-Raf inhibitors is more effective in treating such melanomas and dual therapy is now the standard of care. Vemurafenib and cobimetanib, dabrafenib and trametinib, and encorafenib plus binimetinib are the FDA-approved combinations for the treatment of BRAFV600E melanomas. Although such mutations occur in other neoplasms including thyroid, colorectal, and non-small cell lung cancers, these agents are not as effective in treating these non-melanoma neoplasms. Vemurafenib and dabrafenib produce the paradoxical activation of the MAP kinase pathway in wild type BRAF cells. The precise mechanism for this activation is unclear, but drug-induced Raf activating side-to-side dimerization appears to be an essential step. Although 63%-76% of all people with advanced melanoma with the BRAF V600E mutation derive clinical benefit from combination therapy, median progression-free survival lasts only about nine months and 90% of patients develop resistance within one year. The various secondary resistance mechanisms include NRAS or KRAS mutations (20%), BRAF splice variants (16%), BRAFV600E/K amplifications (13%), MEK1/2 mutations (7%), and non-MAP kinase pathway alterations (11%). Vemurafenib and dabrafenib bind to an inactive form of B-Raf (αC-helixout and DFG-Din) and are classified as type I½ inhibitors. LY3009120 and lifirafenib, which are in the early drug-development stage, bind to a different inactive form of B-Raf (DFG-Dout) and are classified as type II inhibitors. Besides targeting B-Raf and MEK protein kinases, immunotherapies that include ipilimumab, pembrolizumab, and nivolumab have been FDA-approved for the treatment of melanomas. Current clinical trials are underway to determine the optimal usage of targeted and immunotherapies.

Roskoski R Jr 《-》

Src protein-tyrosine kinase structure, mechanism, and small molecule inhibitors.

The physiological Src proto-oncogene is a protein-tyrosine kinase that plays key roles in cell growth, division, migration, and survival signaling pathways. From the N- to C-terminus, Src contains a unique domain, an SH3 domain, an SH2 domain, a protein-tyrosine kinase domain, and a regulatory tail. The chief phosphorylation sites of human Src include an activating pTyr419 that results from phosphorylation in the kinase domain by an adjacent Src molecule and an inhibitory pTyr530 in the regulatory tail that results from phosphorylation by C-terminal Src kinase (Csk) or Chk (Csk homologous kinase). The oncogenic Rous sarcoma viral protein lacks the equivalent of Tyr530 and is constitutively activated. Inactive Src is stabilized by SH2 and SH3 domains on the rear of the kinase domain where they form an immobilizing and inhibitory clamp. Protein kinases including Src contain hydrophobic regulatory and catalytic spines and collateral shell residues that are required to assemble the active enzyme. In the inactive enzyme, the regulatory spine contains a kink or a discontinuity with a structure that is incompatible with catalysis. The conversion of inactive to active Src is accompanied by electrostatic exchanges involving the breaking and making of distinct sets of kinase domain salt bridges and hydrogen bonds. Src-catalyzed protein phosphorylation requires the participation of two Mg(2+) ions. Although nearly all protein kinases possess a common K/E/D/D signature, each enzyme exhibits its unique variations of the protein-kinase reaction template. Bosutinib, dasatinib, and ponatinib are Src/multikinase inhibitors that are approved by the FDA for the treatment of chronic myelogenous leukemia and vandetanib is approved for the treatment of medullary thyroid cancer. The Src and BCR-Abl inhibitors saracatinib and AZD0424, along with the previous four drugs, are in clinical trials for a variety of solid tumors including breast and lung cancers. Both ATP and targeted therapeutic Src protein kinase inhibitors such as dasatinib and ponatinib make hydrophobic contacts with catalytic spine residues and form hydrogen bonds with hinge residues connecting the small and large kinase lobes.

Roskoski R Jr 《-》