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The dynamically active and inactive conformations of kinases play a crucial role in the activation of intracellular downstream signaling pathways. The all-atom molecular dynamics (MD) simulations at microsecond (µs) timescale and longer provide robust insights into the structural details of conformational alterations in kinases that contribute to their cellular metabolic activities and signaling pathways. Tyro3, Axl and Mer (TAM) receptor tyrosine kinases (RTKs) are overexpressed in several types of human cancers. Cabozantinib, a small molecule inhibitor constrains the activity of TAM kinases at nanomolar concentrations. The apo, complexes of ATP (active state) and cabozantinib (active and inactive states) with TAM RTKs were studied by 1 µs MD simulations followed by trajectory analyses. The dynamic mechanistic pathways intrinsic to the kinase activity and protein conformational landscape in the cabozantinib bound TAM kinases are revealed due to the alterations in the P-loop, α-helix and activation loop that result in breaking the regulatory (R) and catalytic (C) spines, while the active states of ATP bound TAM kinases are retained. The co-existence of dynamical states when bound to cabozantinib was observed and the long-lived kinetic transition states of distinct active and inactive structural models were deciphered from MD simulation trajectories that have not been revealed so far.Communicated by Ramaswamy H. Sarma.

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In recent years, protein kinases have been one of the most pursued drug targets. These determined efforts have resulted in ever increasing numbers of small-molecule kinase inhibitors reaching to the market, offering novel treatment options for patients with distinct diseases. One essential component related to the activation and normal functionality of a protein kinase is the regulatory spine (R-spine). The R-spine is formed of four conserved residues named as RS1-RS4. One of these residues, RS3, located in the C-terminal part of αC-helix, is usually accessible for the inhibitors from the ATP-binding cavity as its side chain is lining the hydrophobic back pocket in many protein kinases. Although the role of RS3 has been well acknowledged in protein kinase function, this residue has not been actively considered in inhibitor design, even though many small-molecule kinase inhibitors display interactions to this residue. In this minireview, we will cover the current knowledge of RS3, its relationship with the gatekeeper, and the role of RS3 in kinase inhibitor interactions. Finally, we comment on the future perspectives how this residue could be utilized in the kinase inhibitor design.

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Owing to the dysregulation of protein kinase activity in many diseases including cancer, this enzyme family has become one of the most important drug targets in the 21st century. There are 68 FDA-approved therapeutic agents that target about two dozen different protein kinases and six of these drugs were approved in 2021. Of the approved drugs, twelve target protein-serine/threonine protein kinases, four are directed against dual specificity protein kinases (MEK1/2), thirteen block nonreceptor protein-tyrosine kinases, and 39 target receptor protein-tyrosine kinases. The data indicate that 58 of these drugs are prescribed for the treatment of neoplasms (49 against solid tumors including breast, lung, and colon, five against nonsolid tumors such as leukemias, and four against both solid and nonsolid tumors: acalabrutinib, ibrutinib, imatinib, and midostaurin). Three drugs (baricitinib, tofacitinib, upadacitinib) are used for the treatment of inflammatory diseases including rheumatoid arthritis. Of the 68 approved drugs, eighteen are used in the treatment of multiple diseases. The following six drugs received FDA approval in 2021 for the treatment of these specified diseases: belumosudil (graft vs. host disease), infigratinib (cholangiocarcinomas), mobocertinib and tepotinib (specific forms of non-small cell lung cancer), tivozanib (renal cell carcinoma), and trilaciclib (to decrease chemotherapy-induced myelosuppression). All of the FDA-approved drugs are orally effective with the exception of netarsudil, temsirolimus, and the newly approved trilaciclib. This review summarizes the physicochemical properties of all 68 FDA-approved small molecule protein kinase inhibitors including lipophilic efficiency and ligand efficiency.

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Dysregulation and mutations of protein kinases play causal roles in many diseases including cancer. The KLIFS (kinase-ligand interaction fingerprint and structure) catalog includes 85 ligand binding-site residues occurring in both the small and large protein kinase lobes. Except for allosteric inhibitors, all FDA-approved drug-target enzyme complexes display hydrophobic interactions involving catalytic spine residue-6 (KLIFS-77), catalytic spine residue-7 (KLIFS-11), and catalytic spine residue-8 (KLIFS-15) within the small lobe and residues within the hinge-linker region (KLIFS-46-52). Except for allosteric antagonists, the approved drugs form hydrogen bonds with the third hinge residue (KLIFS-48) of their target. Most of the approved drugs, including the allosteric inhibitors, interact with the small lobe gatekeeper residue (KLIFS-45). The type IIA inhibitors have the most hydrophobic interactions with their target enzymes. These include interactions with KLIFS-27/31/35/61/66 residues of the back pocket within both the small and large lobes. There is also interaction with KLIFS-68 (regulatory spine residue-1), the conserved histidine of the catalytic loop that is found in the back pocket of type II antagonists, but within the front pocket of the other types of inhibitors. Owing to the participation of protein kinase signaling cascades in a wide variety of physiological and pathological processes, one can foresee the increasing use of targeted inhibitors both as primary and secondary treatments for many illnesses. Further studies of protein kinase signal transduction pathways promise to yield new and actionable information that will serve as a basis for fundamental and applied biomedical breakthroughs.

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Owing to the dysregulation of protein kinase activity in many diseases including cancer, the protein kinase enzyme family has become one of the most important drug targets in the 21st century. There are 62 FDA-approved therapeutic agents that target about two dozen different protein kinases and eight of these were approved in 2020. All of the FDA-approved drugs are orally effective with the exception of netarsudil (a ROCK1/2 non-receptor protein-serine/threonine kinase antagonist given as an eye drop for the treatment of glaucoma) and temsirolimus (an indirect mTOR inhibitor given intravenously for the treatment of renal cell carcinoma). Of the approved drugs, ten target protein-serine/threonine protein kinases, four are directed against dual specificity protein kinases (MEK1/2), thirteen block non-receptor protein-tyrosine kinases, and 35 target receptor protein-tyrosine kinases. The data indicate that 55 of these drugs are prescribed for the treatment of neoplasms (52 against solid tumors including breast, lung, and colon, nine against non-solid tumors such as leukemias, and four against both solid and non-solid tumors: acalabrutinib, ibrutinib, imatinib, and midostaurin). A total of three drugs (baricitinib, tofacitinib, upadacitinib) is used for the treatment of inflammatory diseases including rheumatoid arthritis. Seven of the approved drugs form covalent bonds with their target enzymes and are classified as TCIs (targeted covalent inhibitors). Of the 62 approved drugs, eighteen are used in the treatment of multiple diseases. Imatinib, for example, is approved for the treatment of eight different 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. The following eight drugs received FDA approval in 2020 for the treatment of the specified diseases: avapritinib and ripretinib (gastrointestinal stromal tumors), capmatinib (non-small cell lung cancer), pemigatinib (cholangiocarcinoma), pralsetinib and selpercatinib (non-small cell lung cancer, medullary thyroid cancer, differentiated thyroid cancer), selumetinib (neurofibromatosis type I), and tucatinib (HER2-positive breast cancer). All of the eight drugs approved in 2020 fulfill Lipinski's rule of five criteria for an orally effective medicine (MW of 500 Da or less, five or fewer hydrogen bond donors, 10 or fewer hydrogen bond acceptors, calculated log10 of the partition coefficient of five or less) with the exception of three drugs with a molecular weight greater that 500 Da: pralsetinib (534), selpercatinib (526) and ripretinib (510). This review summarizes the physicochemical properties of all 62 FDA-approved small molecule protein kinase inhibitors.

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Because dysregulation of protein kinases owing to mutations or overexpression plays causal roles in human diseases, this family of enzymes has become one of the most important drug targets of the 21st century. Of the 62 protein kinases inhibitors that are approved by the FDA, seven of them form irreversible covalent adducts with their target enzymes. The clinical success of ibrutinib, an inhibitor of Bruton tyrosine kinase, in the treatment of mantle cell lymphomas following its approval in 2013 helped to overcome a general bias against the development of irreversible drug inhibitors. The other approved covalent drugs include acalabrutinib and zanubrutinib, which also inhibit Bruton tyrosine kinase. Furthermore afatinib, dacomitinib, and osimertinib, inhibitors of members of the epidermal growth factor receptor family (ErbB1/2/3/4), are used in the treatment of non-small cell lung cancers. Neratinib is an inhibitor of ErbB2 and is used in the treatment of ErbB2/HER2-positive breast cancer. The seven drugs considered in this review have a common mechanism of action; this process involves the addition of a protein cysteine thiolate anion (protein‒S:-) to an acrylamide derivative (CH2=CHC(=O)N(H)R) where R represents the pharmacophore. Such reactions are commonly referred to as Michael additions and each reaction results in the formation of a covalent bond between carbon and sulfur; the final product is a thioether. This process consists of two discrete steps; the first step involves the reversible association of the drug with its target enzyme so that a weakly electrophilic functionality, a warhead, is bound near an appropriately positioned nucleophilic cysteine. In the second step, a reaction occurs between the warhead and the target enzyme cysteine to form a covalently modified and inactive protein. For this process to work, the warhead must be appropriately juxtaposed in relationship to the cysteinyl thiolate so that the covalent addition can occur. Covalent inhibitors have emerged from the ranks of drugs to be avoided to become an emerging paradigm. Much of this recent success can be attributed to the clinical efficacy of ibrutinib as well as the other antagonists covered in this review. Moreover, the covalent inhibitor methodology is swiftly gaining acceptance as a valuable component of the medicinal chemist's toolbox and is primed to make a significant impact on the development of enzyme antagonists and receptor modulators.

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Hyperactive RAS/RAF/MEK/ERK signaling has a well-defined role in cancer biology. Targeting this pathway results in complete or partial regression of most cancers. In recent years, cancer genomic studies have revealed that genetic alterations that aberrantly activate the RAS/RAF/MEK/ERK signaling mainly occur on RAF or upstream, which motivated the extensive development of RAF inhibitors for cancer therapy. Currently, the first-generation RAF inhibitors have been approved for treating late-stage cancers with BRAF(V600E) mutations. Although these inhibitors have achieved promising outcomes in clinical treatments, their efficacy is abolished by quick-rising drug resistance. Moreover, cancers with hyperactive RAS exhibit intrinsic resistance to these drugs. To resolve these problems, the second-generation RAF inhibitors have been designed and are undergoing clinical evaluations. Here, we summarize the recent findings from mechanistic studies on RAF inhibitor resistance and discuss the critical issues in the development of next-generation RAF inhibitors with better therapeutic index, which may provide insights for improving targeted cancer therapy with RAF inhibitors.

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Protein Kinase-Like Non-Kinases (PKLNKs), commonly known as "pseudokinases", are homologous to eukaryotic Ser/Thr/Tyr protein kinases (PKs) but lack the crucial aspartate residue in the catalytic loop, indispensable for phosphotransferase activity. Therefore, they are predicted to be "catalytically inactive" enzyme homologs. Analysis of protein-kinase like sequences from Arabidopsis thaliana led to the identification of more than 120 pseudokinases lacking catalytic aspartate, majority of which are closely related to the plant-specific receptor-like kinase family. These pseudokinases engage in different biological processes, enabled by their diverse domain architectures and specific subcellular localizations. Structural comparison of pseudokinases with active and inactive conformations of canonical PKs, belonging to both plant and animal origin, revealed unique structural differences. The currently available crystal structures of pseudokinases show that the loop topologically equivalent to activation segment of PKs adopts a distinct-folded conformation, packing against the pseudoenzyme core, in contrast to the extended and inhibitory geometries observed for active and inactive states, respectively, of catalytic PKs. Salt-bridge between ATP-binding Lys and DFG-Asp as well as hydrophobic interactions between the conserved nonpolar residue C-terminal to the equivalent DFG motif and nonpolar residues in C-helix mediate such a conformation in pseudokinases. This results in enhanced solvent accessibility of the pseudocatalytic loop in pseudokinases that can possibly serve as an interacting surface while associating with other proteins. Specifically, our analysis identified several residues that may be involved in pseudokinase regulation and hints at the repurposing of pseudocatalytic residues to achieve mechanistic control over noncatalytic functions of pseudoenzymes.

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Flt3 is expressed by early myeloid and lymphoid progenitor cells and it regulates the proliferation and differentiation of hematopoietic cells. Flt3 is activated by the Flt3 ligand, the monomeric form of which is a polypeptide of about 200 amino acid residues. Both membrane-associated and soluble Flt3 ligands, which are a product of the same gene, function as noncovalent dimers. FLT3 mutations occur in about one-third of newly diagnosed acute myelogenous leukemia (AML) patients. This disease is a malignancy of hematopoietic progenitor cells with a variable clinical course; the incidence of this disorder is more than twice that of patients with chronic myelogenous leukemias (20,000 vs. 8500 new patients per year, respectively, in the United States). FLT3 internal tandem duplication (ITD) results from the head-to-tail duplication of from one to more than 100 amino acids within the juxtamembrane domain and such duplication occurs in about 20-25 % of patients with acute myelogenous leukemias. FLT3 tyrosine kinase (FLT3 TK) mutations, usually within the activation segment, occur in 5-10 % of these patients. The mainstay for the care of acute myelogenous leukemias include daunorubicin or idarubicin and cytarabine. Older patients who are not candidates for such traditional therapy are usually given 5-azacitidine, decitabine, or clofarabine. The addition of orally effective small molecule Flt3 inhibitors to these therapies may prolong event-free and overall survival, a subject of ongoing clinical studies. Midostaurin is US FDA-approved in combination with standard cytarabine and daunorubicin for first-line induction chemotherapy and in combination with cytarabine for second-line consolidation chemotherapy in the treatment of acute myelogenous leukemias with FLT3-postive mutations. Moreover, gilteritinib is a Flt3 multikinase inhibitor that is also FDA approved for the care of adult patients with relapsed or refractory acute myelogenous leukemias with FLT3 mutations. Quizartinib is a Flt3 multikinase inhibitor that was approved by the Ministry of Health, Labor and Welfare (MHLW) of Japan for the treatment of adult patients with relapsed/refractory Flt3-positive acute myelogenous leukemias. Gilteritinib and quizartinib bind to Flt3 with the inactive DFG-Dout structure and are classified as type II inhibitors. Furthermore, ponatinib is a multikinase inhibitor that is approved as therapy for Philadelphia chromosome-positive acute lymphoblastic and chronic myelogenous leukemias; it is used off label for the treatment of patients with acute myelogenous leukemias. Moreover, sorafenib is FDA-approved for the treatment of hepatocellular, renal cell, and differentiated thyroid cancers and it is used off label as maintenance therapy following allogeneic hematopoietic stem cell transplantation in the treatment of acute myelogenous leukemias. Other drugs that are in clinical trials for the treatment of this disorder include sunitinib, crenolanib, FF10101, and lestaurtinib. Unlike chronic myelogenous leukemias, which result solely from the formation of the BCR-Abl chimeric protein kinase, acute myelogenous leukemias result from multi-factorial causes and are prone to be resistant to both cytotoxic and targeted therapies. Consequently, there is a pressing need for better understanding the etiologies of acute myelogenous leukemias and for the development of more effective therapies.

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The human fibroblast growth factor family consists of 22 factors and five transmembrane receptors. Of the 22 factors, eighteen are secreted while four of them function exclusively within the cell. Four of the fibroblast growth factor receptors (FGFRs) possess intracellular protein-tyrosine kinase activity while the fifth (FGFRL1) has a short 105-residue intracellular non-enzymatic component. The FGFR protein kinase domain consists of a bi-lobed structure that is similar to that of all other protein kinases. FGFR gene alterations occur in a wide variety of cancers including those of the urinary bladder, breast, ovary, prostate, endometrium, lung, and stomach. The majority (66 %) of FGFR gene alterations involve gene amplifications, followed by mutations (26 %), and rearrangements that produce fusion proteins (8 %). Erdafitinib was the first orally effective FGFR antagonist approved by the FDA (2019) for the treatment of advanced cancer, that of the urinary bladder. FGF23 suppresses phosphate reabsorption in the proximal tubules of the kidney; FGF23 blockade allows phosphate reabsorption to occur and leads to elevated serum phosphate levels. Erdafitinib and several other, but not all, FGFR antagonists produce hyperphosphatemia. Erdafitinib binds to an inactive DGF-Din conformation of FGFR1 and is classified as a type I½ inhibitor. Similarly, dovitinib, AZD4547, CH5183284, infigratinib, lenvatinib, LY2874455, and lucitanib are type I½ inhibitors. The inactive conformations contain an autoinhibitory brake that is made up of three main residues: an asparagine (N) within the αC-ß4 back loop, a glutamate (E) corresponding to the second hinge residue, and a lysine (K) in the ß8-strand (the NEK triad). PDGFRα/ß, Kit, CSF1R, VEGFR1/2/3, Flt3, Tek, and Tie protein kinases are also regulated by a similar autoinhibitory brake mechanism. Ponatinib binds to FGFR4 in a DFG-Dout conformation and is classified as a type II inhibitor. Futibatinib, roblitinib, H3B-6527, fisogatinib, and PRN1371 bind covalently to their FGFR target and are classified as type VI inhibitors. Nintedanib, pazopanib, pemigatinib, rogaratinib, fisogatinib, and PRN1371 are FGFR inhibitors lacking drug-enzyme crystal structures. All of the aforementioned FGFR antagonists are orally effective. The development of FGFR inhibitors has lagged behind those of other receptor protein-tyrosine kinases. However, the FDA approval of erdafitinib for the treatment of urinary bladder cancers may stimulate additional work targeting the many other FGFR-driven neoplasms.

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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.

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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.

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ERK1 and ERK2 are key protein kinases that contribute to the Ras-Raf-MEK-ERK MAP kinase signalling module. This pathway participates in the control of numerous processes including apoptosis, cell proliferation, the immune response, nervous system function, and RNA synthesis and processing. MEK1/2 activate human ERK1/2 by first catalyzing the phosphorylation of Y204/187 and then T202/185, both residues of which occur within the activation segment. The phosphorylation of both residues is required for enzyme activation. The only Raf substrates are MEK1/2 and the only MEK1/2 substrates are ERK1/2. In contrast, ERK1/2 catalyze the phosphorylation of many cytoplasmic and nuclear substrates including transcription factors and regulatory molecules. The linear MAP kinase pathway branches extensively at the ERK1/2 node. ERK1/2 are proline-directed kinases that preferentially catalyze the phosphorylation of substrates containing a PxS/TP sequence. The dephosphorylation and inactivation of ERK1/2 is catalyzed by dual specificity phosphatases, protein-tyrosine specific phosphatases, and protein-serine/threonine phosphatases. The combined functions of kinases and phosphatases make the overall process reversible. To provide an idea of the complexities involved in these reactions, somatic cell cycle progression involves the strict timing of more than 32,000 phosphorylation and dephosphorylation events as determined by mass spectrometry. The MAP kinase cascade is perhaps the most important oncogenic driver of human cancers and the blockade of this signalling module by targeted inhibitors is an important anti-tumor strategy. Although numerous cancers are driven by MAP kinase pathway activation, thus far the only orally effective approved drugs that target this signaling module are used for the treatment of BRAF-mutant melanomas. The best treatments include the combination of B-Raf and MEK inhibitors (dabrafenib and trametinib, encorafenib and binimetinib, vemurafenib and cobimetanib). However, resistance to these antagonists occurs within one year and additional treatment options are necessary. Owing to the large variety of malignancies that are driven by dysregulation of the MAP kinase pathway, additional tumor types should be amenable to MAP kinase pathway inhibitor therapy. In addition to new B-Raf and MEK inhibitors, the addition of ERK inhibitors should prove helpful. Ulixertinib, MK-8353, and GDC-0994 are orally effective, potent, and specific inhibitors of ERK1/2 that are in early clinical trials for the treatment of various advanced/metastatic solid tumors. These agents are effective against cell lines that are resistant to B-Raf and MEK1/2 inhibitor therapy. Although MK-8353 does not directly inhibit MEK1/2, it decreases the phosphorylation of ERK1/2 as well as the phosphorylation of RSK, an ERK1/2 substrate. The decrease in RSK phosphorylation appears to be a result of ERK inhibition and the decrease in ERK1/2 phosphorylation is related to the inability of MEK to catalyze the phosphorylation of the ERK-MK-8353 complex; these decreases characterize the ERK dual mechanism inhibition paradigm. Additional work will be required to determine whether ERK inhibitors will be successful in the clinic and are able to forestall the development of drug resistance of the MAP kinase pathway.

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Cyclins and cyclin-dependent protein kinases (CDKs) are important proteins that are required for the regulation and expression of the large number of components necessary for the passage through the cell cycle. The concentrations of the CDKs are generally constant, but their activities are controlled by the oscillation of the cyclin levels during each cell cycle. Additional CDK family members play significant roles in a wide range of activities including gene transcription, metabolism, and neuronal function. In response to mitogenic stimuli, cells in the G1-phase of the cell cycle produce D type cyclins that activate CDK4/6. These activated enzymes catalyze the monophosphorylation of the retinoblastoma protein. Subsequently, CDK2-cyclin E catalyzes the hyperphosphorylation of Rb that promotes the release and activation of the E2F transcription factor, which in turn lead to the biosynthesis of dozens of proteins required for cell cycle progression. Consequently, cells pass the G1-restriction point and are committed to complete cell division in the absence of mitogenic stimulation. CDK2-cyclin A, CDK1-cyclin A, and CDK1-cyclin B are required for S-, G2-, and M-phase progression. A crucial mechanism in controlling cell cycle progression is the precise timing of more than 32,000 phosphorylation and dephosphorylation reactions catalyzed by a network of protein kinases and phosphoprotein phosphatases as determined by mass spectrometry. Increased cyclin or CDK expression or decreased levels of endogenous CDK modulators/inhibitors such as INK4 or CIP/KIP have been observed in a wide variety of carcinomas, hematological malignancies, and sarcomas. The pathogenesis of neoplasms because of mutations in the CDKs are rare. Owing to their role in cell proliferation, CDKs represent natural targets for anticancer therapies. Palbociclib, ribociclib, and abemaciclib are FDA-approved CDK4/6 inhibitors used in the treatment of breast cancer. These drugs have IC50 values for CKD4/6 in the low nanomolar range. These inhibitors bind in the cleft between the N-terminal and C-terminal lobes of the CDKs and they inhibit ATP binding. Like ATP, these agents form hydrogen bonds with hinge residues that connect the small and large lobes of protein kinases. Like the adenine base of ATP, these antagonists interact with catalytic spine residues CS6, CS7, and CS8. These and other CDK antagonists are in clinical trials for the treatment of a wide variety of malignancies. As inhibitors of the cell cycle, it is not surprising that one of their most common toxicities is myelosuppression with decreased neutrophil production.

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Platelet-derived growth factor (PDGF) was discovered as a serum-derived component necessary for the growth of smooth muscle cells, fibroblasts, and glial cells. The PDGF family is a product of four gene products and consists of five dimeric isoforms: PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, and the PDGF-AB heterodimer. This growth factor family plays an essential role in embryonic development and in wound healing in the adult. These growth factors mediate their effects by binding to and activating their receptor protein-tyrosine kinases, which are encoded by two genes: PDGFRA and PDGFRB. The functional receptors consist of the PDGFRα/α and PDGFRß/ß homodimers and the PDGFRα/ß heterodimer. Although PDGF signaling is most closely associated with mesenchymal cells, PDGFs and PDGF receptors are widely expressed in the mammalian central nervous system. The PDGF receptors contain an extracellular domain that is made up of five immunoglobulin-like domains (Ig-d1/2/3/4/5), a transmembrane segment, a juxtamembrane segment, a protein-tyrosine kinase domain that contains an insert of about 100 amino acid residues, and a carboxyterminal tail. Although uncommon, activating mutations in the genes for PDGF or PDGF receptors have been documented in various neoplasms including dermatofibrosarcoma protuberans (DFSP) and gastrointestinal stromal tumors (GIST). In most neoplastic diseases, PDGF expression and action appear to involve the tumor stroma. Moreover, this family is pro-angiogenic. More than ten PDGFRα/ß multikinase antagonists have been approved by the FDA for the treatment of several neoplastic disorders and interstitial pulmonary fibrosis (www.brimr.org/PKI/PKIs.htm). Type I protein kinase inhibitors interact with the active enzyme form with DFG-D of the proximal activation segment directed inward toward the active site (DFG-Din). In contrast, type II inhibitors bind to their target with the DFG-D pointing away from the active site (DFG-Dout). We used the Schrödinger induced-fit docking protocol to model the interaction of several antagonists with PDGFRα including imatinib, sorafenib, and sunitinib. The results indicate that these antagonists are able to bind to the DFG-Dout conformation of the receptor and are thus classified as type II inhibitors. Owing to the multiplicity of less active protein kinase conformations when compared with the canonical more active conformation, it was hypothesized that type II drugs would be less promiscuous than type I drugs which bind to the typical active conformation. Although type II inhibitors may be more selective, most - if not all - inhibit more than one target protein kinase and the differences are a matter of degree only.

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RET is a transmembrane receptor protein-tyrosine kinase that is required for the development of the nervous system and several other tissues. The mechanism of activation of RET by its glial-cell derived neurotrophic factor (GDNF) ligands differs from that of all other receptor protein-tyrosine kinases owing to the requirement for additional GDNF family receptor-α (GFRα) co-receptors (GFRα1/2/3/4). RET point mutations have been reported in multiple endocrine neoplasia (MEN2A, MEN2B) and medullary thyroid carcinoma. In contrast, RET fusion proteins have been reported in papillary thyroid and non-small cell lung adenocarcinomas. More than a dozen fusion partners of RET have been described in papillary thyroid carcinomas, most frequently CCDC6-RET and NCOA4-RET. RET-fusion proteins, commonly KIF5B-RET, have also been found in non-small cell lung cancer (NSCLC). Several drugs targeting RET have been approved by the FDA for the treatment of cancer: (i) cabozantinib and vandetanib for medullary thyroid carcinomas and (ii) lenvatinib and sorafenib for differentiated thyroid cancers. In addition, alectinib and sunitinib are approved for the treatment of other neoplasms. Each of these drugs is a multikinase inhibitor that has activity against RET. Previous X-ray studies indicated that vandetanib binds within the ATP-binding pocket and forms a hydrogen bond with A807 within the RET hinge and it makes hydrophobic contact with L881 of the catalytic spine which occurs in the floor of the adenine-binding pocket. Our molecular modeling studies indicate that the other antagonists bind in a similar fashion. All of these antagonists bind to the active conformation of RET and are therefore classified as type I inhibitors. The drugs also make variable contacts with other residues of the regulatory and catalytic spines. None of these drugs was designed to bind preferentially to RET and it is hypothesized that RET-specific antagonists might produce even better clinical outcomes. Currently the number of new cases of neoplasms bearing RET mutations or RET-fusion proteins is estimated to be about 10,000 per year in the United States. This is about the same as the incidence of chronic myelogenous leukemia for which imatinib and second and third generation BCR-Abl non-receptor protein-tyrosine kinase antagonists have proven clinically efficacious and which are commercially successful. These findings warrant the continued development of specific antagonists targeting RET-driven neoplasms.

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The Ras-Raf-MEK-ERK (Map kinase) cellular pathway is a highly conserved eukaryotic signaling module that transduces extracellular signals from growth factors and cytokines into intracellular regulatory events that are involved in cell growth and proliferation or the contrary pathway of cell differentiation. Dysregulation of this pathway occurs in more than one-third of all malignancies, a process that has fostered the development of targeted Map kinase pathway inhibitors. Cutaneous melanomas, which arise from skin melanocytes, are the most aggressive form of skin cancer. Mutations that activate the Map kinase pathway occur in more than 90% of these melanomas. This has led to the development of the combination of dabrafenib and trametinib or vemurafenib and cobimetanib for the treatment of BRAF V600E mutant melanomas. Dabrafenib and vemurafenib target V600E/K BRAF mutants while trametinib and cobimetanib target MEK1/2. The latter two agents bind to MEK1/2 at a site that is adjacent to, but separate from, the ATP-binding site and are therefore classified as type III allosteric protein kinase inhibitors. These agents form a hydrogen bond with a conserved ß3-lysine and they make numerous hydrophobic contacts with residues within the αC-helix, the ß5 strand, and within the activation segment, regions of the protein kinase domain that exhibit greater diversity than those found within the ATP-binding site. One advantage of such allosteric inhibitors is that they do not have to compete with millimolar concentrations of cellular ATP, which most FDA-approved small molecule competitive inhibitors such as imatinib must do. Owing to the wide spread activation of this pathway in numerous neoplasms, trametinib and cobimetinib are being studied in combination with other targeted and cytotoxic drugs in a variety of clinical situations. Except for BRAF and NRAS mutations, there are no other biomarkers correlated with treatment responses following MEK1/2 inhibition and the discovery of such biomarkers would represent an important therapeutic breakthrough.

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The Janus kinase (JAK) family of non-receptor protein-tyrosine kinases consists of JAK1, JAK2, JAK3, and TYK2 (tyrosine kinase-2). Each of these proteins contains a JAK homology pseudokinase (JH2) domain that regulates the adjacent protein kinase domain (JH1). JAK1/2 and TYK2 are ubiquitously expressed whereas JAK3 is found predominantly in hematopoietic cells. The Janus kinase family is regulated by numerous cytokines including interleukins, interferons, and hormones such as erythropoietin, thrombopoietin, and growth hormone. Ligand binding to cytokine and hormone receptors leads to the activation of associated Janus kinases, which then mediate the phosphorylation of the receptors. The SH2 domain of STATs (signal transducers and activators of transcription) binds to the receptor phosphotyrosines thereby promoting STAT phosphorylation by the Janus kinases and consequent activation. STAT dimers are translocated to the nucleus where they participate in the regulation of the expression of thousands of proteins. JAK-STAT dysregulation results in autoimmune disorders such as rheumatoid arthritis, ulcerative colitis, and Crohn disease. JAK-STAT dysregulation also plays a role in the pathogenesis of myelofibrosis, polycythemia vera, and other myeloproliferative illnesses. An activating JAK2 V617F mutation occurs in 95% of people with polycythemia vera and in a lower percentage of people with other neoplasms. JAK1/3 signaling participates in the pathogenesis of inflammatory afflictions while JAK1/2 signaling participates in the development of several malignancies including leukemias and lymphomas as well as myeloproliferative neoplasms. Tofacitinib is a pan-JAK inhibitor that is approved by the FDA for the treatment of rheumatoid arthritis and ruxolitinib is a JAK1/2 inhibitor that is approved for the treatment of polycythemia vera and myelofibrosis.

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Cyclins and cyclin-dependent protein kinases (CDKs) are important regulatory components that are required for cell cycle progression. The levels of the cell cycle CDKs are generally constant and their activities are controlled by cyclins, proteins whose levels oscillate during each cell cycle. Additional CDK family members were subsequently discovered that play significant roles in a wide range of activities including the control of gene transcription, metabolism, and neuronal function. In response to mitogenic stimuli, cells in the G1 phase of the cell cycle produce cyclins of the D type that activate CDK4/6. These activated enzymes catalyze the monophosphorylation of the retinoblastoma protein. Then CDK2-cyclin E catalyzes the hyperphosphorylation of Rb that promotes the release and activation of the E2F transcription factors, which in turn lead to the generation of several proteins required for cell cycle progression. As a result, cells pass through the G1-restriction point and are committed to complete cell division. CDK2-cyclin A, CDK1-cyclin A, and CDK1-cyclin B are required for S, G2, and M-phase progression. Increased cyclin or CDK expression or decreased levels of endogenous CDK inhibitors such as INK4 or CIP/KIP have been observed in various cancers. In contrast to the mutational activation of EGFR, Kit, or B-Raf in the pathogenesis of malignancies, mutations in the CDKs that cause cancers are rare. Owing to their role in cell proliferation, CDKs represent natural targets for anticancer therapies. Abemaciclib (LY2835219), ribociclib (Lee011), and palbociclib (Ibrance(®) or PD0332991) target CDK4/6 with IC50 values in the low nanomolar range. Palbociclib and other CDK inhibitors bind in the cleft between the small and large lobes of the CDKs and inhibit the binding of ATP. Like ATP, palbociclib forms hydrogen bonds with residues in the hinge segment of the cleft. Like the adenine base of ATP, palbociclib interacts with catalytic spine residues CS6 and CS7. CDK antagonists are in clinical trials for the treatment of a variety of malignancies. Significantly, palbociclib has been approved by the FDA for the treatment of hormone-receptor positive/human epidermal growth factor receptor-2 negative breast cancer in conjunction with letrozole as a first-line therapy and with fulvestrant as a second-line treatment. As inhibitors of the cell cycle, it is not surprising that one of their most common toxicities is myelosuppression with decreased neutrophil production.

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Because dysregulation and mutations 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. The X-ray crystal structures of 21 of the 27 FDA-approved small molecule inhibitors bound to their target protein kinases are depicted in this paper. The structure of the enzyme-bound antagonist complex is used in the classification of these inhibitors. Type I inhibitors bind to the active protein kinase conformation (DFG-Asp in, αC-helix in). Type I½ inhibitors bind to a DFG-Asp in inactive conformation while Type II inhibitors bind to a DFG-Asp out inactive conformation. Type I, I½, and type II inhibitors occupy part of the adenine binding pocket and form hydrogen bonds with the hinge region connecting the small and large lobes of the enzyme. Type III inhibitors bind next to the ATP-binding pocket and type IV inhibitors do not bind to the ATP or peptide substrate binding sites. Type III and IV inhibitors are allosteric in nature. Type V inhibitors bind to two different regions of the protein kinase domain and are therefore bivalent inhibitors. The type I-V inhibitors are reversible. In contrast, type VI inhibitors bind covalently to their target enzyme. Type I, I½, and II inhibitors are divided into A and B subtypes. The type A inhibitors bind in the front cleft, the back cleft, and near the gatekeeper residue, all of which occur within the region separating the small and large lobes of the protein kinase. The type B inhibitors bind in the front cleft and gate area but do not extend into the back cleft. An analysis of the limited available data indicates that type A inhibitors have a long residence time (minutes to hours) while the type B inhibitors have a short residence time (seconds to minutes). The catalytic spine includes residues from the small and large lobes and interacts with the adenine ring of ATP. Nearly all of the approved protein kinase inhibitors occupy the adenine-binding pocket; thus it is not surprising that these inhibitors interact with nearby catalytic spine (CS) residues. Moreover, a significant number of approved drugs also interact with regulatory spine (RS) residues.

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