A CREB1-miR-181a-5p loop regulates the pathophysiologic features of bone marrow stromal cells in fibrous dysplasia of bone.

BACKGROUND: Fibrous dysplasia (FD) is a bone marrow stromal cell (BMSC) disease caused by activating mutations of guanine nucleotide-binding protein alpha-stimulating activity polypeptide (GNAS) and is characterized by increased proliferative activity and disrupted osteogenesis of BMSCs. However, the molecular mechanisms regulating the pathophysiologic features of BMSCs in FD remain unknown. This study aimed to identify and verify the roles of the CREB1-miR-181a-5p regulatory loop in FD pathophysiology. METHODS: MicroRNA (miRNA) sequencing analysis was used to identify the possible miRNAs implicated in FD. The proliferation, apoptosis, and osteogenic differentiation of BMSCs, as well as the osteoclast-induced phenotype, were measured and compared after exogenous miR-181a-5p transfection into FD BMSCs or miR-181a-5p inhibitor transfection into normal BMSCs. Chromatin immunoprecipitation and luciferase reporter assays were performed to verify the interactions between CREB1 and miR-181a-5p and their effects on the FD pathological phenotype. RESULTS: Compared to normal BMSCs, FD BMSCs showed decreased miR-181a-5p levels and exhibited increased proliferative activity, decreased apoptotic capacity, and impaired osteogenesis. FD BMSCs also showed a stronger osteoclast activation effect. miR-181a-5p overexpression reversed the pathophysiologic features of FD BMSCs, whereas miR-181a-5p suppression induced an FD-like phenotype in normal BMSCs. Mechanistically, miR-181a-5p was the downstream target of CREB1, and CREB1 was posttranscriptionally regulated by miR-181a-5p. CONCLUSIONS: Our study identifies that the interaction loop between CREB1 and miR-181a-5p plays a crucial role in regulating the pathophysiologic features of FD BMSCs. MiR-181a-5p may be a potential therapeutic target for the treatment of FD.

Blood and Salivary MicroRNAs for Diagnosis of Oral Squamous Cell Carcinoma: A Systematic Review and Meta-Analysis.

PURPOSE: This meta-analysis aimed to compare and evaluate the diagnostic accuracy of blood and salivary microRNAs (miRNAs) in discriminating oral squamous cell carcinoma (OSCC). METHODS: The PubMed, Embase, Web of Science, and Cochrane Library were searched (updated to February 2020) to identify all articles describing the diagnostic value of blood and salivary miRNAs for OSCC. The pooled parameters were calculated using Revman (v.5.3) and STATA (v.14.0). RESULTS: Twenty articles involving 1,106 patients and 732 controls were included in this meta-analysis. The pooled sensitivity and specificity of salivary miRNAs were 0.70 (95% CI: 0.63-0.77) and 0.82 (95% CI: 0.72-0.90). For blood miRNAs, they were 0.79 (95% CI: 0.73-0.84) and 0.82 (95% CI: 0.77-0.86). The areas under receiver operating characteristic curve in saliva, blood, and body fluid miRNAs were 0.80 (95% CI: 0.77-0.84), 0.88 (95% CI: 0.84-0.90), and 0.87 (95% CI: 0.84-0.90), respectively. CONCLUSIONS: The results of this meta-analysis indicate a moderate diagnostic accuracy of blood and salivary miRNAs presented for OSCC. These findings may provide less invasive and relatively reliable diagnostic tools for OSCC detection.

Investigating small molecules to inhibit germinal center kinase-like kinase (GLK/MAP4K3) upstream of PKCθ phosphorylation: Potential therapy to modulate T cell dependent immunity.

Germinal center kinase-like kinase (GLK, also known as MAP4K3) has been hypothesized to have an effect on key cellular activities, including inflammatory responses. GLK is required for activation of protein kinase C-θ (PKCθ) in T cells. Controlling the activity of T helper cell responses could be valuable for the treatment of autoimmune diseases. This approach circumvents previous unsuccessful approaches to target PKCθ directly. The use of structure based drug design, aided by the first crystal structure of GLK, led to the discovery of several inhibitors that demonstrate potent inhibition of GLK biochemically and in relevant cell lines.

Impaired autophagy-mediated macrophage polarization contributes to age-related hyposalivation.

Age-related dysfunction of salivary glands (SGs) leading to xerostomia or dry mouth is typically associated with increased dental caries and difficulties in mastication, deglutition or speech. Inflammaging-induced hyposalivation plays a significant role in aged SGs; however, the mechanisms by which ageing shapes the inflammatory microenvironment of SGs remain unclear. Here, we show that reduced salivary secretion flow rate in aged human and mice SGs is associated with impaired autophagy and increased M1 polarization of macrophages. Our study reveals the crucial roles of SIRT6 in regulating macrophage autophagy and polarization through the PI3K/AKT/mTOR pathway, as demonstrated by generating two conditional knock out mice. Furthermore, triptolide (TP) effectively rejuvenates macrophage autophagy and polarization via targeting this pathway. We also design a local delivery of TP-loaded apoptotic extracellular vesicles (ApoEVs) to improve age-related SGs dysfunction therapeutically. Collectively, our findings uncover a previously unknown link between SIRT6-regulated autophagy and macrophage polarization in age-mediated hyposalivation, while our locally therapeutic strategy exhibits potential preventive effects for age-related hyposalivation.

A Tiny Pocket Packs a Punch: Leveraging Pyridones for the Discovery of CNS-Penetrant Aza-indazole IRAK4 Inhibitors.

We herein report the discovery, synthesis, and evolution of a series of indazoles and azaindazoles as CNS-penetrant IRAK4 inhibitors. Described is the use of structure-based and property-based drug design strategically leveraged to guide the property profile of a key series into a favorable property space while maintaining potency and selectivity. Our rationale that led toward functionalities with potency improvements, CNS-penetration, solubility, and favorable drug-like properties is portrayed. In vivo evaluation of an advanced analogue showed significant, dose-dependent modulation of inflammatory cytokines in a mouse model. In pursuit of incorporating a highly engineered bridged ether that was crucial to metabolic stability in this series, significant synthetic challenges were overcome to enable the preparation of the analogues.

Discovery of BIO-8169─A Highly Potent, Selective, and Brain-Penetrant IRAK4 Inhibitor for the Treatment of Neuroinflammation.

Interleukin receptor associated kinase 4 (IRAK4) plays an important role in innate immune signaling through Toll-like and interleukin-1 receptors and represents an attractive target for the treatment of inflammatory diseases and cancer. We previously reported the development of a potent, selective, and brain-penetrant imidazopyrimidine series of IRAK4 inhibitors. However, lead molecule BIO-7488 (1) suffered from low solubility which led to variable PK, compound accumulation, and poor in vivo tolerability. Herein, we describe the discovery of a series of pyridone analogs with improved solubility which are highly potent, selective and demonstrate desirable PK profiles including good oral bioavailability and excellent brain penetration. BIO-8169 (2) reduced the in vivo production of pro-inflammatory cytokines, was well tolerated in safety studies in rodents and dog at margins well above the predicted efficacious exposure and showed promising results in a mouse model for multiple sclerosis.

Catalytic asymmetric Petasis reactions of vinylboronates.

Binaphthol-catalyzed asymmetric Petasis reactions of salicylaldehydes with dibutyl vinylboronates and secondary amines in the presence of 4 Å molecular sieves (MS) afforded products with up to 99% ee in isolated yields of 39-94%. The 99% ee of the product indicated that the reaction by the binaphthol-catalyzed pathway was roughly 500 times faster than the uncatalyzed pathway. NMR experiments ((1)H and (11)B) showed that the amine component played a role in triggering the reaction between the binaphthol catalyst and the vinylboronate in the catalytic reaction sequence. The 4 Å MS enhanced both the rate and enantioselectivity by effective removal of water from the reaction system. A novel rearrangement reaction of the unconjugated allylic amine Petasis reaction product to a conjugated allylic amine was also observed.

SIRT6-regulated macrophage efferocytosis epigenetically controls inflammation resolution of diabetic periodontitis.

Rationale: Diabetes exacerbates the prevalence and severity of periodontitis, leading to severe periodontal destruction and ultimately tooth loss. Delayed resolution of inflammation is a major contributor to diabetic periodontitis (DP) pathogenesis, but the underlying mechanisms of this imbalanced immune homeostasis remain unclear. Methods: We collected periodontium from periodontitis with or without diabetes to confirm the dysfunctional neutrophils and macrophages in aggravated inflammatory damage and impaired inflammation resolution. Our in vitro experiments confirmed that SIRT6 inhibited macrophage efferocytosis by restraining miR-216a-5p-216b-5p-217 cluster maturation through ''non-canonical'' microprocessor complex (RNA pulldown, RIP, immunostaining, CHIP, Luciferase assays, and FISH). Moreover, we constructed m6SKO mice that underwent LIP-induced periodontitis to explore the in vitro and in vivo effect of SIRT6 on macrophage efferocytosis. Finally, antagomiR-217, a miRNA antagonism, was delivered into the periodontium to treat LIP-induced diabetic periodontitis. Results: We discovered that insufficient SIRT6 as a histone deacetylase in macrophages led to unresolved inflammation and aggravated periodontitis in both human and mouse DP with accumulated apoptotic neutrophil (AN) and higher generation of neutrophil extracellular traps. Mechanistically, we validated that macrophage underwent high glucose stimulation resulting in disturbance of the SIRT6-miR-216/217 axis that triggered impeded efferocytosis of AN through targeting the DEL-1/CD36 axis directly. Furthermore, we demonstrated the inhibitory role of SIRT6 for MIR217HG transcription and identified a non-canonical action of microprocessor that SIRT6 epigenetically hindered the splicing of the primary miR-216/217 via the complex of hnRNPA2B1, DGCR8, and Drosha. Notably, by constructing myeloid-specific deletion of SIRT6 mice and locally delivering antagomir-217 in DP models, we strengthened the in vivo effect of this axis in regulating macrophage efferocytosis and inflammation resolution in DP. Conclusions: Our findings delineated the emerging role of SIRT6 in mediating metabolic dysfunction-associated inflammation, and therapeutically targeting this regulatory axis might be a promising strategy for treating diabetes-associated inflammatory diseases.

Acceleration of Petasis reactions of salicylaldehyde derivatives with molecular sieves.

Mild reaction conditions for Petasis reactions of substituted salicylaldehydes with various amines and arylboronic acids in the presence of molecular sieves were developed. Molecular sieves (MS) significantly accelerated the reaction rates and drove the reactions to high conversions. The conditions were applied to the synthesis of the core structure of BIIB042, a γ-secretase modulator, without stereochemical erosion of a stereogenic center in the salicylaldehyde intermediate.

LncRNA NEAT1 controls the lineage fates of BMSCs during skeletal aging by impairing mitochondrial function and pluripotency maintenance.

Aged bone marrow mesenchymal stem cells (BMSCs) exhibit aberrant self-renewal and lineage specification, which contribute to imbalanced bone-fat and progressive bone loss. In addition to known master regulators of lineage commitment, it is crucial to identify pivotal switches governing the specific differentiation fate of aged BMSCs. Here, we profiled differences in epigenetic regulation between adipogenesis and osteogenesis and identified super-enhancer associated lncRNA nuclear-enriched abundant transcript 1 (NEAT1) as a key bone-fat switch in aged BMSCs. We validated that NEAT1 with high enhancer activity was transcriptionally activated by ATF2 and directed aged BMSCs to a greater propensity to differentiate toward adipocytes than osteoblasts by mediating mitochondrial function. Furthermore, we confirmed NEAT1 as a protein-binding scaffold in which phosphorylation modification of SOX2 Ser249/250 by CDK2 impaired SOX2/OCT4 complex stability and dysregulated downstream transcription networks of pluripotency maintenance. In addition, by sponging miR-27b-3p, NEAT1 upregulated BNIP3L, BMP2K, and PPARG expression to shape mitochondrial function and osteogenic/adipogenic differentiation commitment, respectively. In extracellular communication, NEAT1 promoted CSF1 secretion from aged BMSCs and then strengthened osteoclastic differentiation by extracellular vesicle delivery. Notably, Neat1 small interfering RNA delivery induced increased bone mass in aged mice and decreased fat accumulation in the bone marrow. These findings suggest that NEAT1 regulates the lineage fates of BMSCs by orchestrating mitochondrial function and pluripotency maintenance, and might be a potential therapeutic target for skeletal aging.

Discovery of 4-aminomethylphenylacetic acids as γ-secretase modulators via a scaffold design approach.

Starting from literature examples of nonsteroidal anti-inflammatory drugs (NSAIDs)-type carboxylic acid γ-secretase modulators (GSMs) and using a scaffold design approach, we identified 4-aminomethylphenylacetic acid 4 with a desirable γ-secretase modulation profile. Scaffold optimization led to the discovery of a novel chemical series, represented by 6b, having improved brain penetration. Further SAR studies provided analog 6q that exhibited a good pharmacological profile. Oral administration of 6q significantly reduced brain Aß42 levels in mice and rats.

Discovery of Potent Selective Nonzinc Binding Autotaxin Inhibitor BIO-32546.

Autotaxin (ATX) is a lysophospholipase D that is the main enzyme responsible for generating LPA in body fluids. Although ATX was isolated from a conditioned medium of melanoma cells, later it was discovered to play a critical role in vascular and neuronal development. ATX has also been implicated in primary brain tumor, fibrosis, and rheumatoid arthritis, as well as neurological diseases such as multiple sclerosis, Alzheimer's disease, and neuropathic pain. As ATX and LPA levels are increased upon neuronal injury, a selective ATX inhibitor could provide a new approach to treat neuropathic pain. Herein we describe the discovery of a novel series of nonzinc binding reversible ATX inhibitors, particularly a potent, selective, orally bioavailable, brain-penetrable tool compound BIO-32546, as well as its synthesis, X-ray cocrystal structure, pharmacokinetics, and in vivo efficacy.

Discovery of Potent, Selective, and Brain-Penetrant Apoptosis Signal-Regulating Kinase 1 (ASK1) Inhibitors that Modulate Brain Inflammation In Vivo.

Apoptosis signal-regulating kinase 1 (ASK1) is one of the key mediators of the cellular stress response that regulates inflammation and apoptosis. To probe the therapeutic value of modulating this pathway in preclinical models of neurological disease, we further optimized the profile of our previously reported inhibitor 3. This effort led to the discovery of 32, a potent (cell IC50 = 25 nM) and selective ASK1 inhibitor with suitable pharmacokinetic and brain penetration (rat Cl/Clu = 1.6/56 L/h/kg and Kp,uu = 0.46) for proof-of-pharmacology studies. Specifically, the ability of 32 to inhibit ASK1 in the central nervous system (CNS) was evaluated in a human tau transgenic (Tg4510) mouse model exhibiting elevated brain inflammation. In this study, transgenic animals treated with 32 (at 3, 10, and 30 mg/kg, BID/PO for 4 days) showed a robust reduction of inflammatory markers (e.g., IL-1ß) in the cortex, thus confirming inhibition of ASK1 in the CNS.

Discovery of CNS-Penetrant Apoptosis Signal-Regulating Kinase 1 (ASK1) Inhibitors.

Apoptosis signal-regulating kinase 1 (ASK1) is a key mediator in the apoptotic and inflammatory cellular stress response. To investigate the therapeutic value of modulating this pathway in neurological disease, we have completed medicinal chemistry studies to identify novel CNS-penetrant ASK1 inhibitors starting from peripherally restricted compounds reported in the literature. This effort led to the discovery of 21, a novel ASK1 inhibitor with good potency (cell IC50 = 138 nM), low clearance (rat Cl/Clu = 0.36/6.7 L h-1 kg-1) and good CNS penetration (rat K p,uu = 0.38).

Rational Design and Optimization of a Novel Class of Macrocyclic Apoptosis Signal-Regulating Kinase 1 Inhibitors.

Structural analysis of a known apoptosis signal-regulating kinase 1 (ASK1) inhibitor bound to its kinase domain led to the design and synthesis of the novel macrocyclic inhibitor 8 (cell IC50 = 1.2 µM). The profile of this compound was optimized for CNS penetration following two independent strategies: a rational design approach leading to 19 and a parallel synthesis approach leading to 26. Both analogs are potent ASK1 inhibitors in biochemical and cellular assays (19, cell IC50 = 95 nM; 26, cell IC50 = 123 nM) and have moderate to low efflux ratio (ER) in an MDR1-MDCK assay (19, ER = 5.2; 26, ER = 1.5). In vivo PK studies revealed that inhibitor 19 had moderate CNS penetration (Kpuu = 0.17) and analog 26 had high CNS penetration (Kpuu = 1.0).

Discovery of piperidine-aryl urea-based stearoyl-CoA desaturase 1 inhibitors.

A series of structurally novel stearoyl-CoA desaturase1 (SCD1) inhibitors has been identified via molecular scaffold manipulation. Preliminary structure-activity relationship (SAR) studies led to the discovery of potent, and orally bioavailable piperidine-aryl urea-based SCD1 inhibitors. 4-(2-Chlorophenoxy)-N-[3-(methyl carbamoyl)phenyl]piperidine-1-carboxamide 4c exhibited robust in vivo activity with dose-dependent desaturation index lowering effects.

Discovery of potent, selective, orally bioavailable stearoyl-CoA desaturase 1 inhibitors.

Stearoyl-CoA desaturase 1 (SCD1) catalyzes the committed step in the biosynthesis of monounsaturated fatty acids from saturated, long-chain fatty acids. Studies with SCD1 knockout mice have established that these animals are lean and protected from leptin deficiency-induced and diet-induced obesity, with greater whole body insulin sensitivity than wild-type animals. In this work, we have discovered a series of potent, selective, orally bioavailable SCD1 inhibitors based on a known pyridazine carboxamide template. The representative lead inhibitor 28c also demonstrates excellent cellular activity in blocking the conversion of saturated long-chain fatty acid-CoAs (LCFA-CoAs) to monounsaturated LCFA-CoAs in HepG2 cells.

Germinal-center kinase-like kinase co-crystal structure reveals a swapped activation loop and C-terminal extension.

Germinal-center kinase-like kinase (GLK, Map4k3), a GCK-I family kinase, plays multiple roles in regulating apoptosis, amino acid sensing, and immune signaling. We describe here the crystal structure of an activation loop mutant of GLK kinase domain bound to an inhibitor. The structure reveals a weakly associated, activation-loop swapped dimer with more than 20 amino acids of ordered density at the carboxy-terminus. This C-terminal PEST region binds intermolecularly to the hydrophobic groove of the N-terminal domain of a neighboring molecule. Although the GLK activation loop mutant crystallized demonstrates reduced kinase activity, its structure demonstrates all the hallmarks of an "active" kinase, including the salt bridge between the C-helix glutamate and the catalytic lysine. Our compound displacement data suggests that the effect of the Ser170Ala mutation in reducing kinase activity is likely due to its effect in reducing substrate peptide binding affinity rather than reducing ATP binding or ATP turnover. This report details the first structure of GLK; comparison of its activation loop sequence and P-loop structure to that of Map4k4 suggests ideas for designing inhibitors that can distinguish between these family members to achieve selective pharmacological inhibitors.

Synthesis and structure-activity relationships of N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1- methylprop-2-ynyl}carboxy derivatives as selective acetyl-CoA carboxylase 2 inhibitors.

A structurally novel acetyl-CoA carboxylase (ACC) inhibitor is identified from high-throughput screening. A preliminary structure-activity relationship study led to the discovery of potent dual ACC1/ACC2 and ACC2 selective inhibitors against human recombinant ACC1 and ACC2. Selective ACC2 inhibitors exhibited IC50<20 nM and >1000-fold selectivity against ACC1. (S)-Enantiomer 9p exhibited high ACC2 activity and lowered muscle malonyl-CoA dose-dependently in acute rodent studies, whereas (R)-enantiomer 9o was weak and had no effect on the malonyl-CoA level.

2,4-diaminopyrimidine derivatives as potent growth hormone secretagogue receptor antagonists.

Ghrelin, a gut-derived orexigenic hormone, is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R). Centrally administered ghrelin has been shown to cause hunger and increase food intake in rodents. Inhibition of ghrelin actions with ghrelin antibody, peptidyl GHS-R antagonists, and antisense oligonucleosides resulted in weight loss and food intake decrease in rodents. Here we report the effects of GHS-R antagonists, some of which were potent, selective, and orally bioavailable. A structure-activity relationship study led to the discovery of 8a, which was effective in decreasing food intake and body weight in several acute rat studies.