Discovery of Potent and Exquisitely Selective Inhibitors of Kinase CK1 with Tunable Isoform Selectivity

Casein kinases 1 (CK1) are key signaling molecules that have emerged recently as attractive therapeutic targets in particular for the treatment of hematological malignancies. Herein, we report the identification of a new class of potent and highly selective inhibitors of CK1α, δ and ϵ. Based on their optimal in vitro and in vivo profiles and their exclusive selectivity, MU1250, MU1500 and MU1742 were selected as quality chemical probes for those CK1 isoforms. At proper concentrations, MU1250 and MU1500 allow for specific targeting of CK1δ or dual inhibition of CK1δ/ϵ in cells. The compound MU1742 also efficiently inhibits CK1α and, to our knowledge, represents the first potent and highly selective inhibitor of this enzyme. In addition, we demonstrate that the central 1H-pyrrolo[2,3-b]pyridine-imidazole pharmacophore can be used as the basis of highly selective inhibitors of other therapeutically relevant protein kinases, e.g. p38α, as exemplified by the compound MU1299.     

Recent Advances in Pain Management: Relevant Protein Kinases and Their Inhibitors

The purpose of this review is to underline the protein kinases that have been established, either in fundamental approach or clinical trials, as potential biological targets in pain management. Protein kinases are presented according to their group in the human kinome: TK (Trk, RET, EGFR, JAK, VEGFR, SFK, BCR–Abl), CMGC (p38 MAPK, MEK, ERK, JNK, ASK1, CDK, CLK2, DYRK1A, GSK3, CK2), AGC (PKA, PKB, PKC, PKMζ, PKG, ROCK), CAMK, CK1 and atypical/other protein kinases (IKK, mTOR). Examples of small molecule inhibitors of these biological targets, demonstrating an analgesic effect, are described. Altogether, this review demonstrates the fundamental role that protein kinase inhibitors could play in the development of new pain treatments.     

Light-regulated sampling of protein tyrosine kinase activity

Protein tyrosine kinases serve as key mediators of signaling pathways, biochemical highways that control various aspects of cell behavior. Although fluorescent reporters of tyrosine kinases have been described, these species can suffer immediate phosphorylation upon exposure to the cellular milieu, thereby hindering a detailed analysis of kinase activity as a function of the cell cycle or exposure to environmental stimuli. The first example of a light-regulated tyrosine kinase reporter is described herein, which allows the investigator to control when kinase activity is sampled. In addition, the set of sensors created in this study contain different fluorophores, each with its own unique photophysical properties, thereby furnishing an array of choices that can be used in combination with other intracellular probes.     

Covalent‐Allosteric Inhibitors to Achieve Akt Isoform‐Selectivity

Isoforms of protein kinase Akt are involved in essential processes including cell proliferation, survival, and metabolism. However, their individual roles in health and disease have not been thoroughly evaluated. Thus, there is an urgent need for perturbation studies, preferably mediated by highly selective bioactive small molecules. Herein, we present a structure‐guided approach for the design of structurally diverse and pharmacologically beneficial covalent‐allosteric modifiers, which enabled an investigation of the isoform‐specific preferences and the important residues within the allosteric site of the different isoforms. The biochemical, cellular, and structural evaluations revealed interactions responsible for the selective binding profiles. The isoform‐selective covalent‐allosteric Akt inhibitors that emerged from this approach showed a conclusive structure–activity relationship and broke ground in the development of selective probes to delineate the isoform‐specific functions of Akt kinases.     

High-throughput kinase profiling: a more efficient approach toward the discovery of new kinase inhibitors

Selective protein kinase inhibitors have only been developed against a small number of kinase targets. Here we demonstrate that "high-throughput kinase profiling" is an efficient method for the discovery of lead compounds for established as well as unexplored kinase targets. We screened a library of 118 compounds constituting two distinct scaffolds (furan-thiazolidinediones and pyrimido-diazepines) against a panel of 353 kinases. A distinct kinase selectivity profile was observed for each scaffold. Selective inhibitors were identified with submicromolar cellular activity against PIM1, ERK5, ACK1, MPS1, PLK1-3, and Aurora A,B kinases. In addition, we identified potent inhibitors for so far unexplored kinases such as DRAK1, HIPK2, and DCAMKL1 that await further evaluation. This inhibitor-centric approach permits comprehensive assessment of a scaffold of interest and represents an efficient and general strategy for identifying new selective kinase inhibitors.     

Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example

Phosphorylation events catalyzed by protein kinases represent one of the most prevalent as well as important regulatory posttranslational modifications, and dysregulation of protein kinases is associated with the pathogenesis of different diseases. Therefore, interest in developing potent small molecule kinase inhibitors has increased enormously within the last two decades. A critical step in the development of new inhibitors is cell-free in vitro testing with the intention to determine comparable parameters like the commonly used IC₅₀ value. However, values described in the literature are often biased as experimental setups used for determination of kinase activity lack comparability due to different readout parameters, insufficient normalization or the sheer number of experimental approaches. Here, we would like to hold a brief for highly sensitive, radioactive-based in vitro kinase assays especially suitable for kinases exhibiting autophosphorylation activity. Therefore, we demonstrate a systematic workflow for complementing and validating results from high-throughput screening as well as increasing the comparability of enzyme-specific inhibitor parameters for radiometric as well as non-radiometric assays. Using members of the CK1 family of serine/threonine-specific protein kinases and established CK1-specific inhibitors as examples, we clearly demonstrate the power of our proposed workflow, which has the potential to support the generation of more comparable data for biological characterization of kinase inhibitors.     

Betulin,a Newly Characterized Compound in Acacia auriculiformis Bark,Is a Multi-Target Protein Kinase Inhibitor

The purpose of this work is to investigate the protein kinase inhibitory activity of constituents from Acacia auriculiformis stem bark. Column chromatography and NMR spectroscopy were used to purify and characterize betulin from an ethyl acetate soluble fraction of acacia bark. Betulin, a known inducer of apoptosis, was screened against a panel of 16 disease-related protein kinases. Betulin was shown to inhibit Abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase, casein kinase 1ε (CK1ε), glycogen synthase kinase 3α/β (GSK-3 α/β), Janus kinase 3 (JAK3), NIMA Related Kinase 6 (NEK6), and vascular endothelial growth factor receptor 2 kinase (VEGFR2) with activities in the micromolar range for each. The effect of betulin on the cell viability of doxorubicin-resistant K562R chronic myelogenous leukemia cells was then verified to investigate its putative use as an anti-cancer compound. Betulin was shown to modulate the mitogen-activated protein (MAP) kinase pathway, with activity similar to that of imatinib mesylate, a known ABL1 kinase inhibitor. The interaction of betulin and ABL1 was studied by molecular docking, revealing an interaction of the inhibitor with the ABL1 ATP binding pocket. Together, these data demonstrate that betulin is a multi-target inhibitor of protein kinases, an activity that can contribute to the anticancer properties of the natural compound and to potential treatments for leukemia.     

A small molecule discrimination map of the antibiotic resistance kinome

Kinase-mediated resistance to antibiotics is a significant clinical challenge. These enzymes share a common protein fold characteristic of Ser/Thr/Tyr protein kinases. We screened 14 antibiotic resistance kinases against 80 chemically diverse protein kinase inhibitors to map resistance kinase chemical space. The screens identified molecules with both broad and narrow inhibition profiles, proving that protein kinase inhibitors offer privileged chemical matter with the potential to block antibiotic resistance. One example is the flavonol quercetin, which inhibited a number of resistance kinases in vitro and in vivo. This activity was rationalized by determination of the crystal structure of the aminoglycoside kinase APH(2″)-IVa in complex with quercetin and its antibiotic substrate kanamycin. Our data demonstrate that protein kinase inhibitors offer chemical scaffolds that can block antibiotic resistance, providing leads for co-drug design.     

Crystal Structure-Guided Design of Bisubstrate Inhibitors and Photoluminescent Probes for Protein Kinases of the PIM Family

We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)₆ fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology.     

Structural basis for selective inhibition of Src family kinases by PP1.

BACKGROUND: Small-molecule inhibitors that can target individual kinases are powerful tools for use in signal transduction research. It is difficult to find such compounds because of the enormous number of protein kinases and the highly conserved nature of their catalytic domains. Recently, a novel, potent, Src family selective tyrosine kinase inhibitor was reported (PP1). Here, we study the structural basis for this inhibitor's specificity for Src family kinases. RESULTS: A single residue corresponding to Ile338 (v-Src numbering; Thr338 in c-Src) in Src family tyrosine kinases largely controls PP1's ability to inhibit protein kinases. Mutation of Ile338 to a larger residue such as methionine or phenylalanine in v-Src makes this inhibitor less potent. Conversely, mutation of Ile338 to alanine or glycine increases PP1's potency. PP1 can inhibit Ser/Thr kinases if the residue corresponding to Ile338 in v-Src is mutated to glycine. We have accurately predicted several non-Src family kinases that are moderately (IC(50) approximately 1 microM) inhibited by PP1, including c-Abl and the MAP kinase p38. CONCLUSIONS: Our mutagenesis studies of the ATP-binding site in both tyrosine kinases and Ser/Thr kinases explain why PP1 is a specific inhibitor of Src family tyrosine kinases. Determination of the structural basis of inhibitor specificity will aid in the design of more potent and more selective protein kinase inhibitors. The ability to desensitize a particular kinase to PP1 inhibition of residue 338 or conversely to sensitize a kinase to PP1 inhibition by mutation should provide a useful basis for chemical genetic studies of kinase signal transduction.     

Chemoproteomic profiling of kinases in live cells using electrophilic sulfonyl triazole probes

Sulfonyl-triazoles are a new class of electrophiles that mediate covalent reaction with tyrosine residues on proteins through sulfur-triazole exchange (SuTEx) chemistry. Recent studies demonstrate the broad utility and tunability of SuTEx chemistry for chemical proteomics and protein ligand discovery. Here, we present a strategy for mapping protein interaction networks of structurally complex binding elements using functionalized SuTEx probes. We show that the triazole leaving group (LG) can serve as a releasable linker for embedding hydrophobic fragments to direct molecular recognition while permitting efficient proteome-wide identification of binding sites in live cells. We synthesized a series of SuTEx probes functionalized with a lipid kinase fragment binder for discovery of ligandable tyrosines residing in catalytic and regulatory domains of protein and metabolic kinases in live cells. We performed competition studies with kinase inhibitors and substrates to demonstrate that probe binding is occurring in an activity-dependent manner. Our functional studies led to discovery of probe-modified sites within the C2 domain that were important for downregulation of protein kinase C-alpha in response to phorbol ester activation. Our proof of concept studies highlight the triazole LG of SuTEx probes as a traceless linker for locating protein binding sites targeted by complex recognition elements in live cells.

Sulfonyl-triazole probes modified with a kinase recognition element are developed for live cell activity-based profiling to identify tyrosine sites located in catalytic and regulatory domains that are important for kinase function.     

The structural basis for the specificity of pyridinylimidazole inhibitors of p38 MAP kinase

Background: The p38 mitogen-activated protein (MAP) kinase regulates signal transduction in response to environmental stress. Pyridinylimidazole compounds are specific inhibitors of p38 MAP kinase that block the production of the cytokines interleukin-1 β and tumor necrosis factor α, and they are effective in animal models of arthritis, bone resorption and endotoxin shock. These compounds have been useful probes for studying the physiological functions of the p38-mediated MAP kinase pathway.Results: We report the crystal structure of a novel pyridinylimidazole compound complexed with p38 MAP kinase, and we demonstrate that this compound binds to the same site on the kinase as does ATP. Mutagenesis showed that a single residue difference between p38 MAP kinase and other MAP kinases is sufficient to confer selectivity among pyridinylimidazole compounds.Conclusions: Our results reveal how pyridinylimidazole compounds are potent and selective inhibitors of p38 MAP kinase but not other MAP kinases. It should now be possible to design other specific inhibitors of activated p38 MAP kinase using the structure of the nonphosphorylated enzyme.     

Synthesis of Novel Halogenated Heterocycles Based on o-Phenylenediamine and Their Interactions with the Catalytic Subunit of Protein Kinase CK2

Protein kinase CK2 is a highly pleiotropic protein kinase capable of phosphorylating hundreds of protein substrates. It is involved in numerous cellular functions, including cell viability, apoptosis, cell proliferation and survival, angiogenesis, or ER-stress response. As CK2 activity is found perturbed in many pathological states, including cancers, it becomes an attractive target for the pharma. A large number of low-mass ATP-competitive inhibitors have already been developed, the majority of them halogenated. We tested the binding of six series of halogenated heterocyclic ligands derived from the commercially available 4,5-dihalo-benzene-1,2-diamines. These ligand series were selected to enable the separation of the scaffold effect from the hydrophobic interactions attributed directly to the presence of halogen atoms. In silico molecular docking was initially applied to test the capability of each ligand for binding at the ATP-binding site of CK2. HPLC-derived ligand hydrophobicity data are compared with the binding affinity assessed by low-volume differential scanning fluorimetry (nanoDSF). We identified three promising ligand scaffolds, two of which have not yet been described as CK2 inhibitors but may lead to potent CK2 kinase inhibitors. The inhibitory activity against CK2α and toxicity against four reference cell lines have been determined for eight compounds identified as the most promising in nanoDSF assay.     

Identification of polyoxometalates as nanomolar noncompetitive inhibitors of protein kinase CK2

Protein kinase CK2 is a multifunctional kinase of medical importance that is dysregulated in many cancers. In this study, polyoxometalates were identified as original CK2 inhibitors. [P2Mo18O62](6-) has the most potent activity. It inhibits the kinase in the nanomolar range by targeting key structural elements located outside the ATP- and peptide substrate-binding sites. Several polyoxometalate derivatives exhibit strong inhibitory efficiency, with IC50 values < or = 10 nM. Furthermore, these inorganic compounds show a striking specificity for CK2 when tested in a panel of 29 kinases. Therefore, polyoxometalates are effective CK2 inhibitors in terms of both efficiency and selectivity and represent nonclassical kinase inhibitors that interact with CK2 in a unique way. This binding mode may provide an exploitable mechanism for developing potent drugs with desirable properties, such as enhanced selectivity relative to ATP-mimetic inhibitors.     

Sensitive and versatile electrogenerated chemiluminescence biosensing platform for protein kinase based on Ru(bpy)3 functionalized gold nanoparticles mediated signal transduction

A novel, sensitive and versatile electrogenerated chemiluminescence biosensing platform is developed for monitoring activity and inhibition of protein kinase based on Ru(bpy)₃²⁺ functionalized gold nanoparticles (Ru(bpy)₃²⁺-AuNPs) mediated signal transduction. Ru(bpy)₃²⁺-AuNPs were formed by functionalizing AuNPs with Ru(bpy)₃²⁺ through electrostatic interactions and were used as thiol-versatile signal probe. Casein kinase II (CK2) and cAMP-dependent protein kinase (PKA), two classical protein kinase implicated in disease, were chosen as model protein kinases while a CK2-specific peptide (CRRRADDSDDDDD) and a PKA-specific peptide (CLRRASLG) were employed as molecular substrate for CK2 and PKA, respectively. The specific peptide was self-assembled onto the gold electrode via Au–S bond to form ECL biosensor. Upon thiophosphorylation of the peptide on the electrode in the presence of protein kinase and co-substrate adenosine-5’-(γ-thio)-triphosphate, Ru(bpy)₃²⁺-AuNPs was assembled onto the thiophosphorylated peptides via Au–S bond. The Ru(bpy)₃²⁺-AuNPs attached on electrode surface produce detectable ECL signal in the presence of coreactant tripropylamine. This strategy is promising for multiple protein kinase assay and kinase inhibitor profiling with high sensitivity, good selectivity and versatility. The ECL intensity is proportional to the activity of CK2 in the range of 0.01–0.5 unit/mL with a low detection limit of 0.008 unit/mL and to the activity of PKA in the range of 0.01–0.4 unit/mL with a detection limit of 0.005 unit/mL. Additionally, this assay was applied to the detection of CK2 in serum samples and the inhibition of CK2 and PKA. This work demonstrates that the developed ECL method can provide a sensitive and versatile platform for the detection of kinase activity and drug-screening.     

Structurally sophisticated octahedral metal complexes as highly selective protein kinase inhibitors

The generation of synthetic compounds with exclusive target specificity is an extraordinary challenge of molecular recognition and demands novel design strategies, in particular for large and homologous protein families such as protein kinases with more than 500 members. Simple organic molecules often do not reach the necessary sophistication to fulfill this task. Here, we present six carefully tailored, stable metal-containing compounds in which unique and defined molecular geometries with natural-product-like structural complexity are constructed around octahedral ruthenium(II) or iridium(III) metal centers. Each of the six reported metal compounds displays high selectivity for an individual protein kinase, namely GSK3α, PAK1, PIM1, DAPK1, MLCK, and FLT4. Although being conventional ATP-competitive inhibitors, the combination of the unusual globular shape and rigidity characteristics, of these compounds facilitates the design of highly selective protein kinase inhibitors. Unique structural features of the octahedral coordination geometry allow novel interactions with the glycine-rich loop, which contribute significantly to binding potencies and selectivities. The sensitive correlation between metal coordination sphere and inhibition properties suggests that in this design, the metal is located at a "hot spot" within the ATP binding pocket, not too close to the hinge region where globular space is unavailable, and at the same time not too far out toward the solvent where the octahedral coordination sphere would not have a significant impact on potency and selectivity. This study thus demonstrates that inert (stable) octahedral metal complexes are sophisticated structural scaffolds for the design of highly selective chemical probes.     

Kinase-kernel models: accurate in silico screening of 4 million compounds across the entire human kinome

Reliable in silico prediction methods promise many advantages over experimental high-throughput screening (HTS): vastly lower time and cost, affinity magnitude estimates, no requirement for a physical sample, and a knowledge-driven exploration of chemical space. For the specific case of kinases, given several hundred experimental IC(50) training measurements, the empirically parametrized profile-quantitative structure-activity relationship (profile-QSAR) and surrogate AutoShim methods developed at Novartis can predict IC(50) with a reliability approaching experimental HTS. However, in the absence of training data, prediction is much harder. The most common a priori prediction method is docking, which suffers from many limitations: It requires a protein structure, is slow, and cannot predict affinity. (1) Highly accurate profile-QSAR (2) models have now been built for roughly 100 kinases covering most of the kinome. Analyzing correlations among neighboring kinases shows that near neighbors share a high degree of SAR similarity. The novel chemogenomic kinase-kernel method reported here predicts activity for new kinases as a weighted average of predicted activities from profile-QSAR models for nearby neighbor kinases. Three different factors for weighting the neighbors were evaluated: binding site sequence identity to the kinase neighbors, similarity of the training set for each neighbor model to the compound being predicted, and accuracy of each neighbor model. Binding site sequence identity was by far most important, followed by chemical similarity. Model quality had almost no relevance. The median R(2) = 0.55 for kinase-kernel interpolations on 25% of the data of each set held out from method optimization for 51 kinase assays, approached the accuracy of median R(2) = 0.61 for the trained profile-QSAR predictions on the same held out 25% data of each set, far faster and far more accurate than docking. Validation on the full data sets from 18 additional kinase assays not part of method optimization studies also showed strong performance with median R(2) = 0.48. Genetic algorithm optimization of the binding site residues used to compute binding site sequence identity identified 16 privileged residues from a larger set of 46. These 16 are consistent with the kinase selectivity literature and structural biology, further supporting the scientific validity of the approach. A priori kinase-kernel predictions for 4 million compounds were interpolated from 51 existing profile-QSAR models for the remaining >400 novel kinases, totaling 2 billion activity predictions covering the entire kinome. The method has been successfully applied in two therapeutic projects to generate predictions and select compounds for activity testing.     

Rational inhibitor design and iterative screening in the identification of selective plasmodial cyclin dependent kinase inhibitors

New chemical classes of compounds must be introduced into the malaria drug development pipeline in an effort to develop new chemotherapy options for the fight against malaria. In this review we describe an iterative approach designed to identify potent inhibitors of a kinase family that collectively functions as key regulators of the cell cycle. Cyclin-dependent protein kinases (CDKs) are attractive drug targets in numerous diseases and, most recently, they have become the focus of rational drug design programs for the development of new antimalarial agents. Our approach uses experimental and virtual screening methodologies to identify and refine chemical inhibitors and increase the success rate of discovering potent and selective inhibitors. The active pockets of the plasmodial CDKs are unique in terms of size, shape and amino acid composition compared with those of the mammalian orthologues. These differences exemplified through the use of screening assays, molecular modeling, and crystallography can be exploited for inhibitor design. To date, several classes of compounds including quinolines and oxindoles have been identified as selective inhibitors of the plasmodial CDK7 homologue, Pfmrk. From these initial studies and through the iterative rational drug design process, more potent, selective, and most importantly, chemically unique compound classes have been identified as effective inhibitors of the plasmodial CDKs and the malarial parasite.     

Discovery of EGFR selective 4,6-disubstituted pyrimidines from a combinatorial kinase-directed heterocycle library

The epidermal growth factor receptor (EGFR) tyrosine kinase was one of the first receptor tyrosine kinases to be targeted for drug development by the pharmaceutical industry due to its ubiquitous overexpression in a variety of tumors. Despite the validation of several quinazoline-based scaffolds in the clinic, there is a dearth of alternative chemical structure classes that are capable of inhibiting EGFR kinase activity selectively. Here we describe the discovery of potent and highly selective 4,6-disubstituted pyrimidine inhibitors of enzymatic and cellular EGFR activity and provide an explanation for their exceptional degree of kinase selectivity.