Evaluation of substituted 6-arylquinazolin-4-amines as potent and selective inhibitors of cdc2-like kinases (Clk)

A series of substituted 6-arylquinazolin-4-amines were prepared and analyzed as inhibitors of Clk4. Synthesis, structure–activity relationships and the selectivity of a potent analogue against a panel of 402 kinases are presented. Inhibition of Clk4 by these agents at varied concentrations of assay substrates (ATP and receptor peptide) highly suggests that this chemotype is an ATP competitive inhibitor. Molecular docking provides further evidence that inhibition is the result of binding at the kinase hinge region. Selected compounds represent novel tools capable of potent and selective inhibition of Clk1, Clk4, and Dyrk1A.     

Discovery of trans-3,4'-bispyridinylethylenes as potent and novel inhibitors of protein kinase B (PKB/Akt) for the treatment of cancer: Synthesis and biological evaluation

A novel series of Akt/PKB inhibitors derived from a screening lead (1) has been prepared. The novel trans-3,4'-bispyridinylethylenes described herein are potent inhibitors of Akt/PKB with IC(50) values in the low double-digit nanomolar range against Akt1. Compound 2q shows excellent selectivity against distinct families of kinases such as tyrosine kinases and CAMK, and displays poor to modest selectivity against closely related kinases in the AGC and CMGC families. The cellular activities including inhibition of cell growth and phosphorylation of downstream target GSK3 are also described. The X-ray structure of compound 2q complexed with PKA in the ATP binding site was determined.     

Discovery and SAR of oxindole-pyridine-based protein kinase B/Akt inhibitors for treating cancers

We describe a series of potent and selective oxindole-pyridine-based protein kinase B/Akt inhibitors. The most potent compound 11n in this series demonstrated an IC(50) of 0.17nM against Akt1 and more than 100-fold selectivity over other Akt isozymes. The selectivity against other protein kinases was highly dependent on the C-3 substitutions at the oxindole scaffold, with unsubstituted 9e or 3-furan-2-ylmethylene (11n) more selective and 3-(1H-pyrrol-2-yl)methylene (11f) or 3-(1H-imidazol-2-yl)methylene (11k) less selective. In a mouse xenograft model, 9d, 11f, and 11n inhibited tumor growth but with accompanying toxicity.     

Achieving selectivity between highly homologous tyrosine kinases: a novel selective erbB2 inhibitor

The discovery of small molecule kinase inhibitors for use as drugs is a promising approach for the treatment of cancer and other diseases, but the discovery of highly specific agents is challenging because over 850 kinases are expressed in mammalian cells. Systematic modification of the 4-anilino functionality of a selective quinazoline inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase can invert selectivity to favor inhibition of the highly homologous erbB2 tyrosine kinase. The selectivity pattern was demonstrated in assays of recombinant kinases and recapitulated in measures of kinase activity in intact cells. The most potent and selective erbB2 inhibitor of the analog series has anti-proliferative activity against an erbB2-overexpressing cell line that was lacking in the original EGFR-selective compound. Subtle changes to the molecular structure of ATP-competitive small molecule inhibitors of tyrosine kinases can yield dramatic changes in potency and selectivity. These results suggest that the discovery of highly selective small molecule inhibitors of very homologous kinases is achievable.     

Expression, purification, characterization and homology modeling of active Akt/PKB, a key enzyme involved in cell survival signaling.

Akt is a serine/threonine kinase that plays a critical role in cell survival signaling and its activation has been linked to tumorigenesis. Up-regulation of Akt as well as its upstream regulator phosphatidylinositol-3 kinase (PI3K) has been found in many tumors and the negative regulator of this pathway PTEN/MMAC is a tumor suppressor. As a target for drug discovery, we have expressed and purified an active Akt1 enzyme from a recombinant baculovirus-infected Sf9 cell culture. Coexpression of Akt1 with the catalytic subunit of PI3K or treatment with okadaic acid during expression was found to generate an active enzyme in the insect cell culture system. We have optimized the kinase activity and developed a simple quantitative kinase assay using biotinylated peptide substrates. Using the purified active enzyme, we have characterized its physical, catalytic and kinetic properties. Since Akt is closely related to protein kinase C (PKC) and protein kinase A, the issue of obtaining selective inhibitors of this enzyme was addressed by comparison of the structures of catalytic domains of Akt and PKC, derived by homology modeling methods. A number of amino acid differences in the ATP binding regions of these kinases were identified, suggesting that selective inhibitors of Akt can be discovered. However, the ATP binding regions are highly conserved in the three isoforms of Akt implying that the discovery of isoform-selective inhibitors would be very challenging.     

Expression,purification, characterization and homology modeling of active Akt/PKB,a key enzyme involved in cell survival signaling

Akt is a serine/threonine kinase that plays a critical role in cell survival signaling and its activation has been linked to tumorigenesis. Up-regulation of Akt as well as its upstream regulator phosphatidylinositol-3 kinase (PI3K) has been found in many tumors and the negative regulator of this pathway PTEN/MMAC is a tumor suppressor. As a target for drug discovery, we have expressed and purified an active Akt1 enzyme from a recombinant baculovirus-infected Sf9 cell culture. Coexpression of Akt1 with the catalytic subunit of PI3K or treatment with okadaic acid during expression was found to generate an active enzyme in the insect cell culture system. We have optimized the kinase activity and developed a simple quantitative kinase assay using biotinylated peptide substrates. Using the purified active enzyme, we have characterized its physical, catalytic and kinetic properties. Since Akt is closely related to protein kinase C (PKC) and protein kinase A, the issue of obtaining selective inhibitors of this enzyme was addressed by comparison of the structures of catalytic domains of Akt and PKC, derived by homology modeling methods. A number of amino acid differences in the ATP binding regions of these kinases were identified, suggesting that selective inhibitors of Akt can be discovered. However, the ATP binding regions are highly conserved in the three isoforms of Akt implying that the discovery of isoform-selective inhibitors would be very challenging.     

Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC₅₀ < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.     

Pseudosubstrate peptides inhibit Akt and induce cell growth inhibition

We have designed peptide inhibitors that potently inhibit Akt both in vitro and inside cells. These peptide inhibitors are selective for Akt versus other closely related kinases. The peptides inhibit the in vitro phosphorylation of a biotinylated Bad peptide by Akt with potency up to 100 nM. We have shown that the binding between Akt1 and these peptide inhibitors requires MgATP. Mutating the two putative Akt phosphorylation sites to Ala (nonsubstrate) in these peptides increases the inhibitory potency while mutating the sites to aspartic acid (phosphorylation mimetic) reduces the potency. When delivered into cells, these peptide inhibitors can inhibit cellular Akt activity and cell growth. Thus, these Akt-specific peptide inhibitors provide prototypes for peptide mimetic drugs as well as very useful tools to dissect cellular functions of Akt.     

Targeting Conformational Activation of CDK2 Kinase

Cyclin‐dependent kinases constitute attractive pharmacological targets for cancer therapeutics, yet inhibitors in clinical trials target the ATP‐binding pocket of the CDK and therefore suffer from limited selectivity and emergence of resistance. The more recent development of allosteric inhibitors targeting conformational plasticity of protein kinases offers promising perspectives for therapeutics. In particular tampering with T‐loop dynamics of CDK2 kinase would provide a selective means of inhibiting this kinase, by preventing its conformational activation. To this aim we engineered a fluorescent biosensor that specifically reports on conformational changes of CDK2 activation loop and is insensitive to ATP or ATP‐competitive inhibitors, which constitutes a highly sensitive probe for identification of selective T‐loop modulators. This biosensor was successfully applied to screen a library of small chemical compounds leading to discovery of a family of quinacridine analogs, which potently inhibit cancer cell proliferation, and promote accumulation of cells in S phase and G2. These compounds bind CDK2/ Cyclin A, inhibit its kinase activity, compete with substrate binding, but not with ATP, and dock onto the T‐loop of CDK2. The best compound also binds CDK4 and CDK4/Cyclin D1, but not CDK1. The strategy we describe opens new doors for the discovery of a new class of allosteric CDK inhibitors for cancer therapeutics.     

Autoregulation of kinase dephosphorylation by ATP binding in AGC protein kinases

AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non- ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.     

Autoregulation of kinase dephosphorylation by ATP binding to AGC protein kinases

AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non-ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.Key words: inhibitors hijacking kinase activation, activation loop phosphorylation, dephosphorylation, phosphatase resistance, PKA, PKB, PKC     

Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate

Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis‐activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti‐cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down‐regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells.     

Analysis of water patterns in protein kinase binding sites

Deregulation of protein kinases is associated with numerous diseases, making them important targets for drug discovery. The majority of drugs target the catalytic site of these proteins, but due to the high level of similarity within the ATP binding sites of protein kinases, it is often difficult to achieve the required pharmacological selectivity. In this study, we describe the identification and subsequent analysis of water patterns in the ATP binding sites of 171 protein kinase structures, comprising 19 different kinases from various branches of the kinome, and demonstrate that structurally similar binding sites often have significantly different water patterns. We show that the observed variations in water patterns of different, but structurally similar kinases can be exploited in the structure-based design of potent and selective kinase inhibitors.     

A liquid chromatography/mass spectrometry-based method for the selection of ATP competitive kinase inhibitors

The currently approved kinase inhibitors for therapeutic uses and a number of kinase inhibitors that are undergoing clinical trials are directed toward the adenosine triphosphate (ATP) binding site of protein kinases. The 5'-fluorosulfonylbenzoyl 5'-adenosine (FSBA) is an ATP-affinity reagent that covalently modifies a conserved lysine present in the nucleotide-binding site of most kinases. The authors have developed a liquid chromatography/mass spectrometry-based method to monitor binding of ATP competitive protein kinase inhibitors using FSBA as a nonselective activity-based probe for protein kinases. Their method provides a general, rapid, and reproducible means to screen and validate selective ATP competitive inhibitors of protein kinases.     

In vitro protein kinase activity measurement by flow cytometry

Protein kinases are important drug targets, and a wide variety of methods have been developed for assessing their activity. A key element in developing selective kinase inhibitors is the ability to rapidly compare the effects of an inhibitor on several related or unrelated kinases. We describe a simple, nonradioactive, bead-based method for detecting kinase activity in vitro. Biotinylated peptide substrates are immobilized on beads and phosphorylation is detected with anti-phosphopeptide antibodies with no separation steps required. Phosphorylation is dependent on the amount of kinase in the assay and can be inhibited by known kinase inhibitors in a concentration-dependent manner. Using Luminex technology, we measured the activity of three kinases (PKA, PKC-μ, and Akt) on multiple substrates simultaneously. We also discuss conditions necessary to optimize measurement of the activity of several kinases in a single sample.     

Kinase selectivity profiling by inhibitor affinity chromatography

As new drugs rapidly advance into clinical trials, comprehensive identification of their intracellular targets becomes fundamental for the full understanding of the molecular basis of their efficacy and toxicity. This is particularly important when the targets belong to a large family and the inhibitors recognize a conserved site among different members of the class. A typical example is the kinase family, where efforts are aimed at the development of inhibitors of distinct kinases for therapeutic applications in oncology, inflammation and other disease areas. In this case, inhibitors targeting the ATP pocket may cross react with different kinases, as well as with other proteins that bind ATP. This review critically discusses the available approaches for kinase selectivity profiling. It also reviews some examples of inhibitor affinity chromatography applied to inhibitors of kinases and other protein families as a tool to identify and characterize their intracellular targets.     

Kinase selectivity profiling by inhibitor affinity chromatography

As new drugs rapidly advance into clinical trials, comprehensive identification of their intracellular targets becomes fundamental for the full understanding of the molecular basis of their efficacy and toxicity. This is particularly important when the targets belong to a large family and the inhibitors recognize a conserved site among different members of the class. A typical example is the kinase family, where efforts are aimed at the development of inhibitors of distinct kinases for therapeutic applications in oncology, inflammation and other disease areas. In this case, inhibitors targeting the ATP pocket may cross react with different kinases, as well as with other proteins that bind ATP. This review critically discusses the available approaches for kinase selectivity profiling. It also reviews some examples of inhibitor affinity chromatography applied to inhibitors of kinases and other protein families as a tool to identify and characterize their intracellular targets.     

Erasure of kinase phosphorylation in astrocytes during oxygen-glucose deprivation is controlled by ATP levels and activation of phosphatases

We have examined the relationship between adenosine triphosphate (ATP) concentration and loss of maintenance of kinase-signalling cascades in primary cortical astrocytes during oxygen-glucose deprivation (OGD) as this may constitute an irreversible step that commits astrocytes to cell death. We report that the phosphorylation of Akt, ERK, JNK and p38 kinases, whose activities depend on serine, threonine and tyrosine phosphorylation, were all increased during OGD. All these phosphorylations were reduced to below detection limits when ATP levels were less than 10% of normal levels. Using ERK and Akt as representative examples, we show that this erasure is not irreversible as both ERK and Akt phosphorylations can be partially restored by addition of glucose under anoxic conditions. We further investigated whether OGD caused any change in phosphatase activity. The PP1/PP2A phosphatase inhibitors okadaic acid and caliculyn A, but not cyclosporine A, delayed the removal of ERK and Akt phosphorylation under OGD. By comparing the extent of phosphorylation increase under OGD and normoxic conditions, we calculate that phosphatase activity was increased by approximately 3.6-fold during OGD. These data show that ATP levels control an important checkpoint in kinase function, and that ATP levels may need to be considered when studies of kinase function in relation to OGD are conducted.     

Rapid assembly of diverse and potent allosteric Akt inhibitors

This paper describes the rapid assembly of four different classes of potent Akt inhibitors from a common intermediate. Among them, a pyridopyrimidine series displayed the best intrinsic and cell potency against Akt1 and Akt2. This series also showed a promising pharmacokinetic profile and excellent selectivity over other closely related kinases.