Azole-based inhibitors of AKT/PKB for the treatment of cancer

Through a combination of screening and structure-based rational design, we have discovered a series of N¹-(5-(heterocyclyl)-thiazol-2-yl)-3-(4-trifluoromethylphenyl)-1,2-propanediamines that were developed into potent ATP competitive inhibitors of AKT. Studies of linker strand-binding adenine isosteres identified SAR trends in potency and selectivity that were consistent with binding interactions observed in structures of the inhibitors bound to AKT1 and to the counter-screening target PKA. One compound was shown to have acceptable pharmacokinetic properties and to be a potent inhibitor of AKT signaling and of in vivo xenograft tumor growth in a preclinical model of glioblastoma.     

2,3,5-Trisubstituted pyridines as selective AKT inhibitors—Part I: Substitution at 2-position of the core pyridine for ROCK1 selectivity

2,3,5-Trisubstituted pyridines have been designed as potent AKT inhibitors that are selective against ROCK1 based on the comparison between AKT and ROCK1 structures. Substitution at the 2-position of the core pyridine is the key element to provide selectivity against ROCK1. An X-ray co-crystal structure of 9p in PKA supports the proposed rationale of ROCK1 selectivity.     

ROCK inhibitors 3: Design,synthesis and structure-activity relationships of 7-azaindole-based Rho kinase (ROCK) inhibitors

Rho kinase (ROCK) inhibitors are potential therapeutic agents for the treatment of a variety of disorders including hypertension, glaucoma and erectile dysfunction. Here we disclose a series of potent and selective ROCK inhibitors based on a substituted 7-azaindole scaffold. Substitution of the 3-position of 7-azaindole led to compounds such as 37, which possess excellent ROCK inhibitory potency and high selectivity against the closely related kinase PKA.     

Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway

The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling.     

Signaling pathways activated by PACAP in MCF-7 breast cancer cells

PACAP has opposing roles ranging from activation to inhibition of tumor growth and PACAP agonists/antagonists could be used in tumor therapy. In this study, the effect of PACAP stimulation on signaling pathways was investigated in MCF-7 human adenocarcinoma breast cancer cells. Results showed that MCF-7 cells express VPAC1 and VPAC2, but not PAC1, receptors. In addition, PACAP increased the phosphorylation levels of STAT1, Src and Raf within seconds, confirming their involvement in early stages of PACAP signaling whereas maximal phosphorylation of AKT, ERK and p38 was reached 10 to 20 min later. Moreover, selective inhibition of Src or PI3K resulted in a significant decrease in the phosphorylation of ERK and AKT, but not p38, demonstrating that PACAP signaling follows Src/Raf/ERK and PI3K/AKT pathways. On the other hand, selective inhibition of PLC or PKA resulted in a significant decrease in the phosphorylation of p38, but not AKT or ERK, indicating that PACAP signaling also follows the PLC and PKA/cAMP pathways. Furthermore, PACAP induced ROS through H₂O₂ production whereas pretreatment with NAC inhibitor decreased AKT and ERK phosphorylation, but not p38. Selective NOX2 inhibition affected Src/Raf/Erk and PI3K/Akt pathways, without affecting the p38/PLC/PKA pathway whereas other inhibitors (ML171, VAS2870) had no effect on PACAP induced ROS generation. On the other hand, PACAP induced calcium release, which was decreased by pretreatment with PLC inhibitor. Finally, PACAP stimulation promoted apoptosis by increasing Bax and decreasing Bcl2 expression. In conclusion, we demonstrated that PACAP signaling in MCF-7 cells follows the Src/Raf/ERK and PI3K/AKT pathways and is VPAC1 dependent in a ROS dependent manner, whereas it follows PLC and PKA/cAMP pathways and is VPAC2 dependent through p38 MAP kinase activation involving calcium.     

Discovery of chiral dihydropyridopyrimidinones as potent,selective and orally bioavailable inhibitors of AKT

During the course of our research efforts to develop potent and selective AKT inhibitors, we discovered enatiomerically pure substituted dihydropyridopyrimidinones (DHP) as potent inhibitors of protein kinase B/AKT with excellent selectivity against ROCK₂. A key challenge in this program was the poor physicochemical properties of the initial lead compound 5. Integration of structure-based drug design and physical properties-based design resulted in replacement of a highly hydrophobic poly fluorinated aryl ring by a simple trifluoromethyl that led to identification of compound 6 with much improved physicochemical properties. Subsequent SAR studies led to the synthesis of new pyran analog 7 with improved cell potency. Further optimization of pharmacokintetics properties by increasing permeability with appropriate fluorinated alkyl led to compound 8 as a potent, selective AKT inhibitors that blocks the phosphorylation of GSK3β in vivo and had robust, dose and concentration dependent efficacy in the U87MG tumor xenograft model.     

Protein kinase A (PKA) isoform RIIbeta mediates the synergistic killing effect of cAMP and glucocorticoid in acute lymphoblastic leukemia cells

Protein kinase A (PKA) or cAMP-dependent protein kinase (cAPK) mediates the synergistic effects of cAMP- and glucocorticoid (GC)-induced apoptosis in lymphoid cells. Using two human acute lymphoblastic leukemia cell (CEM) clones with respective GC-sensitive and GC-resistant phenotypes, we discovered that the PKA regulatory subunit isoform RII(beta) is preferentially expressed in the GC-sensitive clone C7-14 cells, whereas other intracellular cAMP receptors, including the exchange proteins directly activated by cAMP (Epac), are expressed at similar levels in both GC-sensitive and GC-resistant clones. High RII(beta) expression level in C7-14 cells is associated with elevated total PKA cellular activity and cAMP sensitivity, which consequently lead to an increased basal PKA activity. cAMP analogs that selectively activate type II PKA recapitulate the effects of forskolin of promoting apoptosis and antagonizing AKT/PKB activity in both GC-sensitive and GC-resistant clones, whereas type I PKA-selective agonists do not. Furthermore, down-regulation of RII(beta) leads to increased AKT/PKB activation and enhanced GC resistance in C7-14 cells. These results demonstrate that PKA RII(beta) is responsible for increased GC sensitivity, critical for cAMP-mediated synergistic cell killing in CEM cells, and may represent a novel therapeutic target for GC-resistant lymphoid malignancy.     

Discovery of pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors: a template-based approach--part 2

Previous efforts by our group have established pyrazolo[1,5-a]pyrimidine as a viable core for the development of potent and selective CDK inhibitors. As part of an effort to utilize the pyrazolo[1,5-a]pyrimidine core as a template for the design and synthesis of potent and selective kinase inhibitors, we focused on a key regulator in the cell cycle progression, CHK1. Continued SAR development of the pyrazolo[1,5-a]pyrimidine core at the C5 and C6 positions, in conjunction with previously disclosed SAR at the C3 and C7 positions, led to the discovery of potent and selective CHK1 inhibitors.     

2-Aminothiadiazole inhibitors of AKT1 as potential cancer therapeutics

A series of 2-aminothiadiazole of inhibitors of AKT1 is described. SAR relationships are discussed, along with selectivity for protein kinase A (PKA) and cyclin-dependent kinase 2 (CDK2). Moderate selectivity observed in several compounds for AKT1 versus PKA is rationalized by X-ray crystallographic analysis. Key compounds showed activity in cellular assays measuring phosphorylation of two AKT substrates, PRAS40 and FKHRL1. Compound 30 was advanced to a mouse liver PD assay, where it showed dose-dependent inhibition of AKT activity, as measured by the inhibition of phospho-PRAS40.     

Inward rectification of the AKT2 channel abolished by voltage-dependent phosphorylation

The Arabidopsis K(+) channel AKT2 possesses the remarkable property that its voltage threshold for activation can be either within the physiological range (gating mode 1), or shifted towards considerably more positive voltages (gating mode 2). Gating mode 1 AKT2 channels behave as delayed K(+)-selective inward rectifiers; while gating mode 2 AKT2 channels are K(+)-selective 'open leaks' in the physiological range of membrane potential. In the present study we have investigated modulation of AKT2 current by effectors of phosphatases/kinases in COS cells and Xenopus oocytes. These experiments show that (i) dephosphorylation can result in AKT2 channel silencing; and (ii) phosphorylation by protein kinase A (PKA) favors both recruitment of silenced AKT2 channels and transition from gating mode 1 to gating mode 2. Interestingly, phosphorylation of AKT2 by PKA in COS cells and Xenopus oocytes is favored by hyperpolarization. Two PKA phosphorylation sites (S210 and S329) were pinpointed in the region of the pore inner mouth. The role of these phosphorylation sites in the switch between the two gating modes was assessed by electrophysiological characterization of mutant channels. The molecular aspects of AKT2 regulation by phosphorylation, and the possible physiological meaning of such regulation in the plant context, are discussed.     

Staurosporine tethered peptide ligands that target cAMP-dependent protein kinase (PKA): Optimization and selectivity profiling

We have recently developed a fragment based selection strategy for targeting kinases, where a small molecule warhead can be non-covalently tethered to a phage-displayed library of peptides. This approach was applied to the conversion of the promiscuous kinase inhibitor, staurosporine, into a potent bivalent ligand for cAMP-dependent protein kinase (PKA). Herein we report a systematic evaluation of this new bivalent ligand (BL); (a) Lineweaver–Burke analysis revealed that the BL, unlike substrate-based bivalent kinase inhibitors, displayed non-competitive inhibition with respect to the peptide substrate, suggesting an allosteric mechanism of action; (b) linker optimization of the BL, afforded one of the most potent, sub-nanomolar, inhibitors of PKA reported to date; (c) the BL was found to be modular, where attachment of active site targeted small molecule warheads in lieu of staurosporine could achieve similar gains in affinity; and (d) profiling studies of both the staurosporine derivative and the BL (amide isostere) against a panel of 90 kinases revealed almost unique enhancement in selectivity against PKA (>5-fold) compared to the starting staurosporine derivative. These combined results provide new insights for BL discovery, which has the potential to provide guidance toward the development of kinase selective reagents while uncovering new allosteric sites on kinases for therapeutic targeting.     

Structure–activity relationship studies of 5-benzylaminoimidazo[1,2-c]pyrimidine-8-carboxamide derivatives as potent,highly selective ZAP-70 kinase inhibitors

Zeta-associated protein, 70 kDa (ZAP-70), a spleen tyrosine kinase (Syk) family kinase, is normally expressed on T cells and natural killer cells and plays a crucial role in activation of the T cell immunoresponse. Thus, selective ZAP-70 inhibitors might be useful not only for treating autoimmune diseases, but also for suppressing organ transplant rejection. In our recent study on the synthesis of Syk family kinase inhibitors, we discovered that novel imidazo[1,2-c]pyrimidine-8-carboxamide derivatives possessed potent ZAP-70 inhibitory activity with good selectivity for ZAP-70 over other kinases. In particular, compound 26 showed excellent ZAP-70 kinase inhibition and high selectivity for ZAP-70 over structurally related Syk. The discovery of a potent, highly selective ZAP-70 inhibitor would contribute a new therapeutic tool for autoimmune diseases and organ transplant medication.     

Galpha and Gbeta gamma require distinct Src-dependent pathways to activate Rap1 and Ras

The Src tyrosine kinase is necessary for activation of extracellular signal-regulated kinases (ERKs) by the beta-adrenergic receptor agonist, isoproterenol. In this study, we examined the role of Src in the stimulation of two small G proteins, Ras and Rap1, that have been implicated in isoproterenol's signaling to ERKs. We demonstrate that the activation of isoproterenol of both Rap1 and Ras requires Src. In HEK293 cells, isoproterenol activates Rap1, stimulates Rap1 association with B-Raf, and activates ERKs, all via PKA. In contrast, the activation by isoproterenol of Ras requires Gbetagamma subunits, is independent of PKA, and results in the phosphoinositol 3-kinase-dependent activation of AKT. Interestingly, beta-adrenergic stimulation of both Rap1 and ERKs, but not Ras and AKT, can be blocked by a Src mutant (SrcS17A) that is incapable of being phosphorylated and activated by PKA. Furthermore, a Src mutant (SrcS17D), which mimics PKA phosphorylation at serine 17, stimulates Rap1 activation, Rap1/B-Raf association, and ERK activation but does not stimulate Ras or AKT. These data suggest that Rap1 activation, but not that of Ras, is mediated through the direct phosphorylation of Src by PKA. We propose that the beta(2)-adrenergic receptor activates Src via two independent mechanisms to mediate distinct signaling pathways, one through Galpha(s) to Rap1 and ERKs and the other through Gbetagamma to Ras and AKT.     

Structural basis for producing selective MAP2K7 inhibitors

Mitogen-activated protein kinase kinase 7 (MAP2K7) in the c-Jun N-terminal kinase signal cascade is an attractive drug target for a variety of diseases. The selectivity of MAP2K7 inhibitors against off-target kinases is a major barrier in drug development. We report a crystal structure of MAP2K7 complexed with a potent covalent inhibitor bearing an acrylamide moiety as an electrophile, which discloses a structural basis for producing selective and potent MAP2K7 inhibitors.     

Exploration of 3-methylisoquinoline-4-carbonitriles as protein kinase A inhibitors of Plasmodium falciparum

A series of isoquinolines have been evaluated in a homology model of Plasmodium falciparum Protein Kinase A (PfPKA) using molecular dynamics. Synthesis of these compounds was then undertaken to investigate their structure–activity relationships. One compound was found to inhibit parasite growth in an in vitro assay and provides a lead to further develop 3-methylisoquinoline-4-carbonitriles as antimalarial compounds. Development of a potent and selective PfPKA inhibitor would provide a useful tool to shed further insight into the mechanisms enabling malaria parasites to establish infection.     

Ceramide-activated protein kinases A and C zeta inhibit kidney proximal tubule cell Na-ATPase

The basolateral membranes of kidney proximal tubule cells have (Na⁺+K⁺)-ATPase and Na⁺-ATPase activities, involved in Na⁺ reabsorption. We showed that ceramide (Cer) modulates protein kinase A (PKA) and protein kinase C (PKC), which are involved in regulating ion transporters. Here we show that ceramide, promotes 60% inhibition of Na⁺-ATPase activity (I₅₀ ≈ 100 nM). This effect was completely reversed by inhibiting PKA but did not involve the classic PKC signaling pathway. In these membranes we found the Cer-activated atypical PKC zeta (PKCζ) isoform. When PKCζ is inhibited, Cer ceases to inhibit the Na⁺-ATPase, allowing the cAMP/PKA signaling pathway to recover its stimulatory effect on the pump. There were no effects on the (Na⁺+K⁺)-ATPase. These results reveal Cer as a potent physiological modulator of the Na⁺-ATPase, participating in a regulatory network in kidney cells and counteracting the stimulatory effect of PKA via PKCζ.     

Energy metabolism regulates clathrin adaptors at the trans-Golgi network and endosomes

Glucose is a master regulator of cell behavior in the yeast Saccharomyces cerevisiae. It acts as both a metabolic substrate and a potent regulator of intracellular signaling cascades. Glucose starvation induces the transient delocalization and then partial relocalization of clathrin adaptors at the trans-Golgi network and endosomes. Although these localization responses are known to depend on the protein kinase A (PKA) signaling pathway, the molecular mechanism of this regulation is unknown. Here we demonstrate that PKA and the AMP-regulated kinase regulate adaptor localization through changes in energy metabolism. We show that genetic and chemical manipulation of intracellular ATP levels cause corresponding changes in adaptor localization. In permeabilized cells, exogenous ATP is sufficient to induce adaptor localization. Furthermore, we reveal distinct energy-dependent steps in adaptor localization: a step that requires the ADP-ribosylation factor ARF, an ATP-dependent step that requires the phosphatidyl-inositol-4 kinase Pik1, and third ATP-dependent step for which we provide evidence but for which the mechanism is unknown. We propose that these energy-dependent mechanisms precisely synchronize membrane traffic with overall proliferation rates and contribute a crucial aspect of energy conservation during acute glucose starvation.     

Evidence for a regulatory role of Cullin-RING E3 ubiquitin ligase 7 in insulin signaling

Dysfunctional regulation of signaling pathways downstream of the insulin receptor plays a pivotal role in the pathogenesis of insulin resistance and type 2 diabetes. In this study we report both in vitro and in vivo experimental evidence for a role of Cullin-RING E3 ubiquitin ligase 7 (CRL7) in the regulation of insulin signaling and glucose homeostasis. We show that Cul7^−/− mouse embryonic fibroblasts displayed enhanced AKT and Erk MAP kinase phosphorylation upon insulin stimulation. Depletion of CUL7 by RNA interference in C2C12 myotubes led to increased activation of insulin signaling pathways and cellular glucose uptake, as well as a reduced capacity of these cells to execute insulin-induced degradation of insulin receptor substrate 1 (IRS1). In vivo, heterozygosity of either Cul7 or Fbxw8, both key components of CRL7, resulted in elevated PI3 kinase/AKT activation in skeletal muscle tissue upon insulin stimulation when compared to wild-type controls. Finally, Cul7^+/− or Fbxw8^+/− mice exhibited enhanced insulin sensitivity and plasma glucose clearance. Collectively, our findings point to a yet unrecognized role of CRL7 in insulin-mediated control of glucose homeostasis by restraining PI3 kinase/AKT activities in skeletal muscle cells.     

Regulation of actin function by protein kinase A‐mediated phosphorylation of Limk1

Proper regulation of the cAMP-dependent protein kinase (protein kinase A, PKA) is necessary for cellular homeostasis, and dysregulation of this kinase is crucial in human disease. Mouse embryonic fibroblasts (MEFs) lacking the PKA regulatory subunit Prkar1a show altered cell morphology and enhanced migration. At the molecular level, these cells showed increased phosphorylation of cofilin, a crucial modulator of actin dynamics, and these changes could be mimicked by stimulating the activity of PKA. Previous studies of cofilin have shown that it is phosphorylated primarily by the LIM domain kinases Limk1 and Limk2, which are under the control of the Rho GTPases and their downstream effectors. In Prkar1a^−/− MEFs, neither Rho nor Rac was activated; rather, we showed that PKA could directly phosphorylate Limk1 and thus enhance the phosphorylation of cofilin. These data indicate that PKA is crucial in cell morphology and migration through its ability to modulate directly the activity of LIM kinase.