Potent inhibitors of malarial P. Falciparum protein kinase G: Improving the cell activity of a series of imidazopyridines

Development of a class of bicyclic inhibitors of the Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG), starting from known compounds with activity against a related parasite PKG orthologue, is reported. Examination of key sub-structural elements led to new compounds with good levels of inhibitory activity against the recombinant kinase and in vitro activity against the parasite. Key examples were shown to possess encouraging in vitro ADME properties, and computational analysis provided valuable insight into the origins of the observed activity profiles.     

Potent bicyclic inhibitors of malarial cGMP-dependent protein kinase: approaches to combining improvements in cell potency,selectivity and structural novelty

Focussed studies on imidazopyridine inhibitors of Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG) have significantly advanced the series towards desirable in vitro property space. LLE-based approaches towards combining improvements in cell potency, key physicochemical parameters and structural novelty are described, and a structure-based design hypothesis relating to substituent regiochemistry has directed efforts towards key examples with well-balanced potency, ADME and kinase selectivity profiles.     

Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signaling

The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca²⁺. Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca²⁺ indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca²⁺ signaling in the model apicomplexan Toxoplasma gondii. In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca²⁺. We define the pool of Ca²⁺ regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca²⁺ signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca²⁺. The enhancers identified are capable of releasing intracellular Ca²⁺ stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii. The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum. Inhibition of Ca²⁺-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca²⁺ stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca²⁺, underscoring the importance of these pathways and the therapeutic potential of their inhibition.     

Cyclic GMP-dependent Stimulation of Serotonin Transport Does Not Involve Direct Transporter Phosphorylation by cGMP-dependent Protein Kinase

The serotonin transporter (SERT) is responsible for reuptake of serotonin (5-hydroxytryptamine) after its exocytotic release from neurons. It is the primary target for antidepressants and stimulants, including “ecstasy” (3,4-methylenedioxymethamphetamine). SERT is regulated by several processes, including a cyclic GMP signaling pathway involving nitric oxide synthase, guanylyl cyclase, and cGMP-dependent protein kinase (PKG). Here, we show that SERT was phosphorylated in a PKG Iα-dependent manner in vitro, but that SERT was not a direct substrate of PKG. We generated an analog-sensitive gatekeeper residue mutant of PKG Iα (M438G) that efficiently used the ATP analog N⁶-benzyl-ATP. This mutant, but not the wild type (WT) kinase, used the ATP analog to phosphorylate both a model peptide substrate as well as an established protein substrate of PKG (vasodilator-stimulated phosphoprotein). PKG Iα M438G effectively substituted for the WT kinase in stimulating SERT-mediated 5-hydroxytryptamine transport in cultured cells. Addition of either WT or mutant PKG Iα M438G to membranes containing SERT in vitro led to radiolabel incorporation from [γ-³³P]ATP but not from similarly labeled N⁶-benzyl-ATP, indicating that SERT was phosphorylated by another kinase that could not utilize the ATP analog. These results are consistent with the proposed SERT phosphorylation site, Thr-276, being highly divergent from the consensus PKG phosphorylation site sequence, which we verified through peptide library screening. Another proposed SERT kinase, the p38 mitogen-activated protein kinase, could not substitute for PKG in this assay, and p38 inhibitors did not block PKG-dependent phosphorylation of SERT. The results suggest that PKG initiates a kinase cascade that leads to phosphorylation of SERT by an as yet unidentified protein kinase.     

Interactions between cAMP- and cGMP-dependent protein kinase inhibitors and phosphodiesterase IV inhibitors on arachidonate release from human monocytes

The effects of specific inhibitors of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) on the inhibitory activity of phosphodiesterase (PDE) type IV inhibitors and of the cell permeable analogue of cAMP, db-cAMP, were investigated on fMLP-induced arachidonate release from human monocytes. When monocytes were preincubated with the combined PKA/PKG inhibitor H8 (10⁻⁶ to 10⁻⁴ M) or the selective PKG inhibitor Rp-8-cpt-cGMPs (10⁻⁶ to 10⁻⁴ M) a concentration-dependent reduction of the inhibitory effect of db-cAMP (10⁻ M), rolipram (10⁻⁵ M) and Ro 20-1724 (10⁻⁵ M) was noted. When monocytes were preincubated with the selective PKA inhibitor H89 (10⁻⁶ to 10⁻⁴ M), only a small inhibition of the effect of db-cAMP and no inhibition of the effects of rolipram and Ro 20–1724 were observed. The present data indicate that db-cAMP and PDE IV inhibitors elicit an in vitro anti-inflammatory activity by a PKA-independent mechanism, which do not appear to be mainly mediated via the PKG activation.     

Divergent allostery reveals critical differences between structurally homologous regulatory domains of Plasmodium falciparum and human protein kinase G

Malaria is a life-threatening infectious disease primarily caused by the Plasmodium falciparum parasite. The increasing resistance to current antimalarial drugs and their side effects has led to an urgent need for novel malaria drug targets, such as the P. falciparum cGMP-dependent protein kinase (pfPKG). However, PKG plays an essential regulatory role also in the human host. Human cGMP-dependent protein kinase (hPKG) and pfPKG are controlled by structurally homologous cGMP-binding domains (CBDs). Here, we show that despite the structural similarities between the essential CBDs in pfPKG and hPKG, their respective allosteric networks differ significantly. Through comparative analyses of chemical shift covariance analyses, molecular dynamics simulations, and backbone internal dynamics measurements, we found that conserved allosteric elements within the essential CBDs are wired differently in pfPKG and hPKG to implement cGMP-dependent kinase activation. Such pfPKG versus hPKG rewiring of allosteric networks was unexpected because of the structural similarity between the two essential CBDs. Yet, such finding provides crucial information on which elements to target for selective inhibition of pfPKG versus hPKG, which may potentially reduce undesired side effects in malaria treatments.     

(d)-Amino acid analogues of DT-2 as highly selective and superior inhibitors of cGMP-dependent protein kinase Iα

The cGMP-dependent protein kinase type I (PKG I) is an essential regulator of cellular function in blood vessels throughout the body. DT-2, a peptidic inhibitor of PKG, has played a central role in determining the molecular mechanisms of vascular control involving PKG and its signaling partners. Here, we report the development of (d)-amino acid DT-2 derivatives, namely the retro-inverso ri-(d)-DT-2 and the all (d)-amino acid analog, (d)-DT-2. Both peptide analogs were potent PKG Iα inhibitors with K_i values of 5.5 nM (ri-(d)-DT-2) and 0.8 nM ((d)-DT-2) as determined using a hyperbolic mixed-type inhibition model. Also, both analogs were proteolytically stable in vivo, showed elevated selectivity, and displayed enhanced membrane translocation properties. Studies on isolated arteries from the resistance vasculature demonstrated that intraluminally perfused (d)-DT-2 significantly inhibited vasodilation induced by 8-Br-cGMP. Furthermore, in vivo application of (d)-DT-2 established a uniform translocation pattern in the resistance vasculature, with exception of the brain. Thus, (d)-DT-2 caused significant increases in mean arterial blood pressure in unrestrained, awake mice. Further, mesenteric arteries isolated from (d)-DT-2 treated animals showed a markedly reduced dilator response to 8-Br-cGMP in vitro. Our results clearly demonstrate that (d)-DT-2 is a superior inhibitor of PKG Iα and its application in vivo leads to sustained inhibition of PKG in vascular smooth muscle cells. The discovery of (d)-DT-2 may help our understanding of how blood vessels constrict and dilate and may also aid the development of new strategies and therapeutic agents targeted to the prevention and treatment of vascular disorders such as hypertension, stroke and coronary artery disease.     

Nitrosyl-cobinamide (NO-Cbi), a new nitric oxide donor,improves wound healing through cGMP/cGMP-dependent protein kinase

Nitric oxide (NO) donors have been shown to improve wound healing, but the mechanism is not well defined. Here we show that the novel NO donor nitrosyl-cobinamide (NO-Cbi) improved in vitro wound healing in several cell types, including an established line of lung epithelial cells and primary human lung fibroblasts. On a molar basis, NO-Cbi was more effective than two other NO donors, with the effective NO-Cbi concentration ranging from 3 to 10 μM, depending on the cell type. Improved wound healing was secondary to increased cell migration and not cell proliferation. The wound healing effect of NO-Cbi was mediated by cGMP, mainly through cGMP-dependent protein kinase type I (PKGI), as determined using pharmacological inhibitors and activators, and siRNAs targeting PKG type I and II. Moreover, we found that Src and ERK were two downstream mediators of NO-Cbi's effect. We conclude that NO-Cbi is a potent inducer of cell migration and wound closure, acting via cGMP, PKG, Src, and extracellular signal regulated kinase (ERK).     

Discovery of a class of diheteroaromatic amines as orally bioavailable CDK1/4/6 inhibitors

The discovery of a class of diheteroaromatic amines based on LY2835219 as cyclin-dependent kinase (CDK1/4/6) inhibitors was described. The series was found to have much more improved CDK1 inhibition and potent in vitro anti-proliferative effects against cancer cell lines. The synthesis and structure–activity relationship studies of these compounds were reported. One promising compound was selected to evaluate as a novel lead compound after in vitro and in vivo profiling.     

cGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice

Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca²⁺ channel inositol 1,4,5-trisphosphate receptor 1 (IP₃R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP₃R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3^−/−/Nrl^−/− mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP₃R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca²⁺ channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency.     

Identification of new pyrrolo[2,3-d]pyrimidines as potent VEGFR-2 tyrosine kinase inhibitors: Design,synthesis, biological evaluation and molecular modeling

Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in cancer angiogenesis. In the current study, a series of novel pyrrolo[2,3-d]pyrimidine based-compounds was designed and synthesized as VEGFR-2 inhibitors, in accordance to the structure activity relationship (SAR) studies of known type II VEGFR-2 inhibitors. The newly synthesized compounds were evaluated for their ability to inhibit VEGFR-2 kinase enzyme in vitro. All the tested compounds demonstrated highly potent dose-related VEGFR-2 inhibition with IC₅₀ values in nanomolar range. Among these compounds, pyrrolo[2,3-d]pyrimidine derivatives carrying biaryl urea moieties (12d and 15c) exhibited IC₅₀ values of 11.9 and 13.6 nM respectively. Additionally, most of the newly synthesized final compounds were tested on 60 human cancer cell lines. Docking of these compounds into the inactive conformation of VEGFR-2 was performed which showed comparable binding modes to that of the FDA approved VEGFR-2 kinase inhibitors. These newly discovered potent kinase inhibitors could be considered as potential candidates for the development of new targeted anticancer agent.     

Discovery of new quinazoline derivatives as irreversible dual EGFR/HER2 inhibitors and their anticancer activities – Part 1

Overexpression of EGFR and HER2 are observed in many breast, ovarian, colon and prostate cancers. The second and third generation irreversible EGFR/HER2 dual kinase inhibitors became popular after the approval of Afatinib by FDA to overcome the mutation related problem. To find efficacious drug candidates, a series of novel quinazoline derivatives were designed, synthesized and evaluated as dual EGFR/HER2 tyrosine kinase (TK) inhibitors. Selected twenty four compounds were reported here with significant inhibitory activities against EGFR/HER2 tyrosine kinases. Several compounds showed nanomolar IC₅₀ values. In vitro studies of quinazoline derivatives were done on NCI-H1975, HCC827, A431, MDA MB-453 cell lines. The compounds 1a, 1d and 1v were found more potent compared to standard drug afatinib. In vivo efficacy study of 1d on nude mice NCI-H1975 tumour xenograft model was discussed.     

Non-hinge-binding pyrazolo[1,5-a]pyrimidines as potent B-Raf kinase inhibitors

As part of our research effort to discover B-Raf kinase inhibitors, we prepared a series of C-3 substituted N-(3-(pyrazolo[1,5-a]pyrimidin-7-yl)phenyl)-3-(trifluoromethyl)benzamides. X-ray crystallography studies revealed that one of the more potent inhibitors (10n) bound to B-Raf kinase without forming a hinge-binding hydrogen bond. With basic amine residues appended to C-3 aryl residues, cellular activity and solubility were enhanced over previously described compounds of this class.     

Discovery of a series of novel 5H-pyrrolo[2,3-b]pyrazine-2-phenyl ethers,as potent JAK3 kinase inhibitors

We report the discovery of a novel series of ATP-competitive Janus kinase 3 (JAK3) inhibitors based on the 5H-pyrrolo[2,3-b]pyrazine scaffold. The initial leads in this series, compounds 1a and 1h, showed promising potencies, but a lack of selectivity against other isoforms in the JAK family. Computational and crystallographic analysis suggested that the phenyl ether moiety possessed a favorable vector to achieve selectivity. Exploration of this vector resulted in the identification of 12b and 12d, as potent JAK3 inhibitors, demonstrating improved JAK family and kinase selectivity.     

Design and synthesis of benzofuro[3,2-b]pyridin-2(1H)-one derivatives as anti-leukemia agents by inhibiting Btk and PI3Kδ

Btk inhibitors and PI3Kδ inhibitors play crucial roles in the treatment of leukemia, and studies confirmed that the synergetic inhibition against Btk and PI3Kδ could gain an optimal response. Herein, a series of novel benzofuro[3,2-b]pyridin-2(1H)-one derivatives were designed and synthesized as dual Btk/PI3Kδ kinases inhibitors for the treatment of leukemia. Studies indicated that most compounds could suppress the proliferation of multiple leukemia or lymphoma cells (Raji, HL60 and K562 cells) at low micromolar concentrations in vitro. Further kinase assays identified several compounds could simultaneously inhibit Btk kinase and PI3Kδ kinase. Thereinto, compound 16b exhibited the best inhibitory activity (Btk: IC₅₀?=?139?nM; PI3Kδ: IC₅₀?=?275?nM) and showed some selectivity against PI3Kδ compared to PI3Kβ/γ. Finally, the SAR of target compounds was preliminarily discussed combined with docking results. In brief, 16b possessed of the potency for the further optimization as anti-leukemia drugs by inhibiting simultaneously Btk kinase and PI3Kδ kinase.     

Activation of cGMP-dependent protein kinase stimulates cardiac ATP-sensitive potassium channels via a ROS/calmodulin/CaMKII signaling cascade

Background

Cyclic GMP (cGMP)-dependent protein kinase (PKG) is recognized as an important signaling component in diverse cell types. PKG may influence the function of cardiac ATP-sensitive potassium (K_ATP) channels, an ion channel critical for stress adaptation in the heart; however, the underlying mechanism remains largely unknown. The present study was designed to address this issue.

Methods and Findings

Single-channel recordings of cardiac K_ATP channels were performed in both cell-attached and inside-out patch configurations using transfected human embryonic kidney (HEK)293 cells and rabbit ventricular cardiomyocytes. We found that Kir6.2/SUR2A (the cardiac-type K_ATP) channels were activated by cGMP-selective phosphodiesterase inhibitor zaprinast in a concentration-dependent manner in cell-attached patches obtained from HEK293 cells, an effect mimicked by the membrane-permeable cGMP analog 8-bromo-cGMP whereas abolished by selective PKG inhibitors. Intriguingly, direct application of PKG moderately reduced rather than augmented Kir6.2/SUR2A single-channel currents in excised, inside-out patches. Moreover, PKG stimulation of Kir6.2/SUR2A channels in intact cells was abrogated by ROS/H₂O₂ scavenging, antagonism of calmodulin, and blockade of calcium/calmodulin-dependent protein kinase II (CaMKII), respectively. Exogenous H₂O₂ also concentration-dependently stimulated Kir6.2/SUR2A channels in intact cells, and its effect was prevented by inhibition of calmodulin or CaMKII. PKG stimulation of K_ATP channels was confirmed in intact ventricular cardiomyocytes, which was ROS- and CaMKII-dependent. Kinetically, PKG appeared to stimulate these channels by destabilizing the longest closed state while stabilizing the long open state and facilitating opening transitions.

Conclusion

The present study provides novel evidence that PKG exerts dual regulation of cardiac K_ATP channels, including marked stimulation resulting from intracellular signaling mediated by ROS (H₂O₂ in particular), calmodulin and CaMKII, alongside of moderate channel suppression likely mediated by direct PKG phosphorylation of the channel or some closely associated proteins. The novel cGMP/PKG/ROS/calmodulin/CaMKII signaling pathway may regulate cardiomyocyte excitability by opening K_ATP channels and contribute to cardiac protection against ischemia-reperfusion injury.     

Design,synthesis and structure-activity relationship of diaryl-ureas with novel isoxazol[3,4-b]pyridine-3-amino-structure as multi-target inhibitors against receptor tyrosine kinase

Inspired by that the multi-target inhibitors against receptor tyrosine kinases (RTKs) have significantly improved the effect of clinical treatment for cancer, and based on the chemical structure of Linifanib (ABT-869, Abbott), two series of diaryl-ureas with novel isoxazol[3,4-b]pyridine-3-amino-structure were designed and synthesized as multi-target inhibitors against RTKs. The preliminary biological evaluation showed that several compounds exhibited comparable potency with Linifanib. Compound S21 was identified as the most potent inhibitor against Fms-like tyrosine kinase 3 (FLT-3), kinase insert domain containing receptor (KDR) and platelet-derived growth factor receptor β (PDGFR-β) with its IC₅₀ values were 4?nM, 3?nM and 8?nM respectively, it also showed potent inhibitory activities against several cancer cells.     

Isosteric replacement of the Z-enone with haloethyl ketone and E-enone in a resorcylic acid lactone series and biological evaluation

The synthesis of a small library of resorcylic acid lactones and evaluation of their biological properties as kinase inhibitors is described. Within the series E-enones were found more active than corresponding Z-enones as inhibitors of a subset of kinases containing a conserved cysteine. Replacement of the enone moiety with a β-haloketone group led to compounds with an interesting kinase selectivity profile and also antiproliferative activity against Jurkat cells. An E-enone derivative also showed activity against capillary tube formation based on a co-culture of primary human umbilical cord endothelial cells (HUVECs) and vascular smooth muscle cells (vSMCs).     

Adenosine analogue–oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIα)

Introduction

Type I cGMP-dependent protein kinase (PKGIα) belongs to the family of cyclic nucleotide-dependent protein kinases and is one of the main effectors of cGMP. PKGIα is involved in regulation of cardiac contractility, vasorelaxation, and blood pressure; hence, the development of potent modulators of PKGIα would lead to advances in the treatment of a variety of cardiovascular diseases. Aim: Representatives of ARC-type compounds previously characterized as potent inhibitors and high-affinity fluorescent probes of PKA catalytic subunit (PKAc) were tested towards PKGIα to determine that ARCs could serve as activity regulators and sensors for the latter protein kinase both in vitro and in complex biological systems. Results: Structure–activity profiling of ARCs with PKGIα in vitro demonstrated both similarities as well as differences to corresponding profiling with PKAc, whereas ARC-903 and ARC-668 revealed low nanomolar displacement constants and inhibition IC₅₀ values with both cyclic nucleotide-dependent kinases. The ability of ARC-based fluorescent probes to penetrate cell plasma membrane was demonstrated in the smooth muscle tissue of rat cerebellum isolated arteries, and the compound with the highest affinity in vitro (ARC-903) showed also potential for in vivo applications, fully abolishing the PKG1α-induced vasodilation.     

Identification of pyrrolo[2,3-g]indazoles as new Pim kinase inhibitors

The synthesis and Pim kinase inhibition potency of a new series of pyrrolo[2,3-g]indazole derivatives is described. The results obtained in this preliminary structure–activity relationship study pointed out that sub-micromolar Pim-1 and Pim-3 inhibitory potencies could be obtained in this series, more particularly for compounds 10 and 20, showing that pyrrolo[2,3-g]indazole scaffold could be used for the development of new potent Pim kinase inhibitors. Molecular modeling experiments were also performed to study the binding mode of these compounds in Pim-3 ATP-binding pocket.