Hypomorphic CAMKK2 in EA.hy926 endothelial cells causes abnormal transferrin trafficking,iron homeostasis and glucose metabolism

We recently reported that loss of calcium/calmodulin-dependent protein kinase kinase-2 (CAMKK2), a serine/threonine kinase activated by intracellular calcium, in mice leads to tissue-specific aberrant turnover of transferrin (TF), a receptor-mediated iron-transporter that supplies iron to tissues. Iron dyshomeostasis is associated with the pathogenesis of several diseases, making TF transport relevant to health. In this study, we used hemizygous CAMKK2 hypomorphic human endothelial cell (EA.hy926) clones to demonstrate that cells with reduced CAMKK2 exhibit increased TF uptake and transcytosis, and decreased intracellular trafficking to subcellular organelles compared to wild-type. The abnormal TF trafficking in CAMKK2 hypomorphic cells correlated with a reduction in intracellular iron content and defective glucose metabolism including glycolysis and mitochondrial respiration. CAMKK2 deficiency also caused reduction in GAPDH and VDAC1 protein level which correlated to defective bioenergetics function. These findings have identified a novel mechanistic link between abnormal calcium signaling, iron dyshomeostasis and metabolic dysfunction involving CAMKK2.     

A complete map of the Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) signaling pathway

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine-protein kinase belonging to the Ca²⁺/calmodulin-dependent protein kinase subfamily. CAMKK2 has an autocatalytic site, which gets exposed when Ca²⁺/calmodulin (CAM) binds to it. This results in autophosphorylation and complete activation of CAMKK2. The three major known downstream targets of CAMKK2 are 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPKα), calcium/calmodulin-dependent protein kinase 1 (CAMK1) and calcium/calmodulin-dependent protein kinase 4 (CAMK4). Activation of these targets by CAMKK2 is important for the maintenance of different cellular and physiological processes within the cell. CAMKK2 is found to be important in neuronal development, bone remodeling, adipogenesis, and systemic glucose homeostasis, osteoclastgensis and postnatal myogensis. CAMKK2 is reported to be involved in pathologies like Duchenne muscular dystrophy, inflammation, osteoporosis and bone remodeling and is also reported to be overexpressed in prostate cancer, hepatic cancer, ovarian and gastric cancer. CAMKK2 is involved in increased cell proliferation and migration through CAMKK2/AMPK pathway in prostate cancer and activation of AKT in ovarian cancer. Although CAMKK2 is a molecule of great importance, a public resource of the CAMKK2 signaling pathway is currently lacking. Therefore, we carried out detailed data mining and documentation of the signaling events associated with CAMKK2 from published literature and developed an integrated reaction map of CAMKK2 signaling. This resulted in the cataloging of 285 reactions belonging to the CAMKK2 signaling pathway, which includes 33 protein–protein interactions, 74 post-translational modifications, 7 protein translocation events, and 22 activation/inhibition events. Besides, 124 gene regulation events and 25 activator/inhibitors involved in CAMKK2 activation were also cataloged. The CAMKK2 signaling pathway map data is made freely accessible through WikiPathway database (https://www.wikipathways.org/index.php/Pathway:WP4874). We expect that data on a signaling map of CAMKK2 will provide the scientific community with an improved platform to facilitate further molecular as well as biomedical investigations on CAMKK2 and its utility in the development of biomarkers and therapeutic targets.Electronic supplementary materialThe online version of this article (10.1007/s12079-020-00592-1) contains supplementary material, which is available to authorized users.     

Discovery of aryl ureas and aryl amides as potent and selective histamine H3 receptor antagonists for the treatment of obesity (Part II)

A novel series of histamine H₃ receptor (H₃R) antagonists was derived from an arylurea lead series (1) via bioisosteric replacement of the urea functionality by an amide linkage. The arylamide series was optimized through SAR studies by a broad variation of substituents in the left-hand side benzoyl residue (analogs 2a–2ag) or replacement of the benzoyl moiety by heteroarylcarbonyl residues (analogs 5a–5n). Compounds 2p and 2q were identified within the series as potent and selective H₃R antagonists/inverse agonists with acceptable overall profile. Compound 2q was orally active in food intake inhibition in diet-induced obese (DIO) mice. Compound 2q represents a novel H₃R antagonist template with improved in vitro potency and oral efficacy and has its merits as a new lead for further optimization.     

CaMKK2 facilitates Golgi-associated vesicle trafficking to sustain cancer cell proliferation

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) regulates cell and whole-body metabolism and supports tumorigenesis. The cellular impacts of perturbing CAMKK2 expression are, however, not yet fully characterised. By knocking down CAMKK2 levels, we have identified a number of significant subcellular changes indicative of perturbations in vesicle trafficking within the endomembrane compartment. To determine how they might contribute to effects on cell proliferation, we have used proteomics to identify Gemin4 as a direct interactor, capable of binding CAMKK2 and COPI subunits. Prompted by this, we confirmed that CAMKK2 knockdown leads to concomitant and significant reductions in δ-COP protein. Using imaging, we show that CAMKK2 knockdown leads to Golgi expansion, the induction of ER stress, abortive autophagy and impaired lysosomal acidification. All are phenotypes of COPI depletion. Based on our findings, we hypothesise that CAMKK2 sustains cell proliferation in large part through effects on organelle integrity and membrane trafficking.Subject terms: Prostate cancer, Golgi     

Synthesis and structure-activity relationship of 4-alkoxy-thieno[2,3-b]pyridine derivatives as potent alkaline phosphatase enhancers for osteoporosis treatment

The synthesis and structure-activity relationships of a novel series of 3-aminothieno[2,3-b]pyridine-2-carboxamides were explored. Our efforts were focused on modifying the C-4 substituent of the thienopyridine ring to develop orally available bone anabolic agents. 4-Alkoxy derivatives were found to be novel ALPase enhancers without inhibitory effect on P450 activity. Among these derivatives, compound 6k was orally administered to ovariectomized rats, and it was found to significantly improve areal bone mineral density at a dose of 30 mg/kg/day.     

Arylphthalazines as potent,and orally bioavailable inhibitors of VEGFR-2

A series of arylphthalazine derivatives were synthesized and evaluated as antagonists of VEGF receptor II (VEGFR-2). IM-094482 57, which was prepared in two steps from commercially available starting materials, was found to be a potent inhibitor of VEGFR-2 in enzymatic, cellular and mitogenic assays (comparable activity to ZD-6474). Additionally, 57 inhibited the related receptor, VEGF receptor I (VEGFR-1), and showed excellent exposure when dosed orally to female CD-1 mice.     

Imidazole piperazines: SAR and development of a potent class of cyclin-dependent kinase inhibitors with a novel binding mode

A piperazine series of cyclin-dependent kinase (CDK) inhibitors have been identified. The compounds exhibit excellent physiochemical properties and a novel binding mode, whereby a bridging interaction via a water molecule with Asp 86 of CDK2, leads to selectivity for the CDK family of enzymes over other kinases. Piperazines 2e and 2i were subsequently shown to inhibit tumour growth when dosed orally in a nude mouse xenograft study. Additional chemical series that exploit this unexpected interaction with Asp 86 are also described.     

Discovery of a series of potent,orally active α,α-disubstituted piperidine NK1 antagonists

Modification of prototype NK₁ antagonist 2 resulted in the synthesis of a series of simple amides 6 and retroamides 9. These compounds were shown to be potent and orally active NK₁ antagonists.     

Novel 2-aminooctahydrocyclopentalene-3a-carboxamides as potent CCR2 antagonists

Novel CCR2 antagonists with a novel 2-aminooctahydrocyclopentalene-3a-carboxamide scaffold were designed. SAR studies led to a series of potent compounds. For example, compound 51 had a good PK profile in both dog and monkey, and exhibited excellent efficacy when dosed orally in an inflammation model in hCCR2 KI mice. In addition, an asymmetric synthesis to the core structures was developed.     

The discovery of an orally efficacious positive allosteric modulator of the calcium sensing receptor containing a dibenzylamine core

The discovery of a series of novel and orally efficacious type II calcimimetics, developed from the lead compound 1, is described herein. Compound 22 suppressed plasma PTH levels relative to vehicle when dosed orally in a rat pharmacodynamic model.     

Synthesis and in vitro biological evaluation of new pyrimidines as glucagon-like peptide-1 receptor agonists

The therapeutic success of peptide glucagon-like peptide-1 (GLP-1) receptor agonists for the treatment of type 2 diabetes mellitus has inspired discovery efforts aimed at developing orally available small-molecule GLP-1 receptor agonists. In this study, two series of new pyrimidine derivatives were designed and synthesized using an efficient route, and were evaluated in terms of GLP-1 receptor agonist activity. In the first series, novel pyrimidines substituted at positions 2 and 4 with groups varying in size and electronic properties were synthesized in a good yield (78–90%). In the second series, the designed pyrimidine templates included both urea and Schiff base linkers, and these compounds were successfully produced with yields of 77–84%. In vitro experiments with cultured cells showed that compounds 3a and 10a (10^?15–10^?9 M) significantly increased insulin secretion compared to that of the control cells in both the absence and presence of 2.8 mM glucose; compound 8b only demonstrated significance in the absence of glucose. These findings represent a valuable starting point for the design and discovery of small-molecule GLP-1 receptor agonists that can be administered orally.     

Orally bioavailable amine-linked macrocyclic inhibitors of factor XIa

The discovery of orally bioavailable FXIa inhibitors has been a challenge. Herein, we describe our efforts to address this challenge by optimization of our imidazole-based macrocyclic series. Our optimization strategy focused on modifications to the P2 prime, macrocyclic amide linker, and the imidazole scaffold. Replacing the amide of the macrocyclic linker with amide isosteres led to the discovery of substituted amine linkers which not only maintained FXIa binding affinity but also improved oral exposure in rats. Combining the optimized macrocyclic amine linker with a pyridine scaffold afforded compounds 23 and 24 that were orally bioavailable, single-digit nanomolar FXIa inhibitors with excellent selectivity against relevant blood coagulation enzymes.     

Triazole derivatives: A series of Darapladib analogues as orally active Lp-PLA2 inhibitors

This Letter reports our efforts towards the optimization of our previously identified series of imidazole and triazole derivatives that lead to the discovery of a series of orally active Lp-PLA₂ inhibitors in C57 mice. These inhibitors are characterized by the presence of a diamine side chain in the molecules, such as 2c, 2f, and 4a. The introduction of the terminal-end amine succeeded in maintaining the in vitro activities at sub-nanomolar levels. The vivo activities could be greatly affected by variations in the two amines via modulating the metabolic stability and lipophilicity of the compounds.     

Discovery and SAR of PF-4693627, a potent,selective and orally bioavailable mPGES-1 inhibitor for the potential treatment of inflammation

Inhibition of mPGES-1, the terminal enzyme in the arachidonic acid/COX pathway to regulate the production of pro-inflammatory prostaglandin PGE2, is considered an attractive new therapeutic target for safe and effective anti-inflammatory drugs. The discovery of a novel series of orally active, selective benzoxazole piperidinecarboxamides as mPGES-1 inhibitors is described. Structure–activity optimization of lead 5 with cyclohexyl carbinols resulted in compound 12, which showed excellent in vitro potency and selectivity against COX-2, and reasonable pharmacokinetic properties. Further SAR studies of the benzoxazole ring substituents lead to a novel series of highly potent compounds with improved PK profile, including 23, 26, and 29, which were effective in a carrageenan-stimulated guinea pig air pouch model of inflammation. Based on its excellent in vitro and in vivo pharmacological, pharmacokinetic and safety profile and ease of synthesis, compound 26 (PF-4693627) was advanced to clinical studies.     

Identification of a novel 2-pyridyl-benzensulfonamide derivative,RQ-00203078, as a selective and orally active TRPM8 antagonist

A novel series of 2-pyridyl-benzensulfonamide derivatives have been identified as selective and orally active TRPM8 antagonists via high throughput screening (HTS). Exploration of the structure–activity relationships of compound 1 has led to the identification of RQ-00203078 (compound 36) as a highly selective, potent and orally available TRPM8 antagonist.RQ-00203078 demonstrated excellent in vivo activity in a dose dependent manner with an ED₅₀ value of 0.65 mg/kg in the icilin-induced wet-dog shakes model in rats after oral administration and may become an important pharmacological tool for fully assessing the potential therapeutic use of the targets activated by cold stimulation.     

Pyrrole-3-carboxamides as potent and selective JAK2 inhibitors

We report herein the discovery, structure guided design, synthesis and biological evaluation of a novel class of JAK2 inhibitors. Optimization of the series led to the identification of the potent and orally bioavailable JAK2 inhibitor 28 (NMS-P953). Compound 28 displayed significant tumour growth inhibition in SET-2 xenograft tumour model, with a mechanism of action confirmed in vivo by typical modulation of known biomarkers, and with a favourable pharmacokinetic and safety profile.     

Discovery of furan-2-carbohydrazides as orally active glucagon receptor antagonists

Furan-2-carbohydrazides were found as orally active glucagon receptor antagonists. Starting from the hit compound 5, we successfully determined the structure activity relationships of a series of derivatives obtained by modifying the acidity of the phenol. We identified the ortho-nitrophenol as a good scaffold for glucagon receptor inhibitory activity. Our efforts have led to the discovery of compound 7l as a potent glucagon receptor antagonist with good bioavailability and satisfactory long half-life.     

Spirocyclic ureas: Orally bioavailable 11β-HSD1 inhibitors identified by computer-aided drug design

Structure-guided drug design led to the identification of a class of spirocyclic ureas which potently inhibit human 11β-HSD1 in vitro. Lead compound 10j was shown to be orally bioavailable in three species, distributed into adipose tissue in the mouse, and its (R) isomer 10j2 was efficacious in a primate pharmacodynamic model.     

Lead optimization and structure-based design of potent and bioavailable deoxycytidine kinase inhibitors

A series of deoxycytidine kinase inhibitors was simultaneously optimized for potency and PK properties. A co-crystal structure then allowed merging this series with a high throughput screening hit to afford a highly potent, selective and orally bioavailable inhibitor, compound 10. This compound showed dose dependent inhibition of deoxycytidine kinase in vivo.     

Design,synthesis, and biological evaluation of a series of piperazine ureas as fatty acid amide hydrolase inhibitors

A series of piperazine ureas were designed, synthesized, and evaluated for their potential as novel orally efficacious fatty acid amide hydrolase (FAAH) inhibitors for the treatment of neuropathic and inflammatory pain. We carried out an optimization study of compound 5 to improve its in vitro FAAH inhibitory activity, and identified the 2-pyrimidinylpiperazine derivative 21d with potent inhibitory activity, favorable DMPK profile and brain permeability. Compound 21d showed robust and dose-dependent analgesic efficacy in animal models of both neuropathic and inflammatory pain.