Effect of Akkermansia muciniphila on pancreatic islet ß-cell function in rats with prediabetes mellitus induced by a high-fat diet.

Prediabetes is an important stage in the development of diabetes. It is necessary to find a safe, effective and sustainable way to delay and reverse the progression of prediabetes. Akkermansia muciniphila (A. muciniphila) is one of the key bacteria associated with glucose metabolism. Recent studies mainly focus on the effect of A. muciniphila on obesity and insulin resistance, but there is no research on the effect of A. muciniphila on pancreatic ß-cell function and its mechanism in prediabetes. In this study, we investigated the effects of A. muciniphila on ß-cell function, apoptosis and differentiation, as well as its effects on the gut microbiome, intestinal barrier, metaflammation and the expression of Toll-like receptors (TLRs) in a high-fat diet (HFD)-induced prediabetic rat model. The effect of A. muciniphila was compared with dietary intervention. The results showed both A. muciniphila treatment and dietary intervention can reduce metaflammation by repairing the intestinal barrier in rats with prediabetes induced by an HFD and improve ß-cell secretory function, apoptosis and differentiation through signaling pathways mediated by TLR2 and TLR4. Additionally, A. muciniphila can further elevate ß-cell secretion, attenuate apoptosis and improve differentiation and the TLR signaling pathway on the basis of diet.

Deciphering the Exact Sequence of Endogenous Soluble B Cell Maturation Antigen and Unbiased Quantitation in Multiple Myeloma Patient Samples by LC-MS.

BACKGROUND: B-cell maturation antigen is a pivotal therapeutic target for multiple myeloma (MM). Membrane-bound BCMA can be cleaved by γ-secretase and shed as soluble BCMA (sBCMA). sBCMA can act as a neutralizing sink to compete with drug, as well as serve as a diagnostic/prognostic biomarker for MM. Antibody-capture based methods, such as enzyme-linked immunosorbent assay (ELISA) and immunoaffinity-liquid chromatography-multiple reaction monitoring (IA-LC-MRM), have been reported and well adopted to measure sBCMA in clinical samples. However, both methods are biased by capturing antibodies. METHODS: We have used various LC-MS workflows to characterize and quantify endogenous sBCMA in MM patient samples, including bottom-up peptide mapping, intact analysis, IA-based, and reagent-free (RF)-LC-MRM quantitation. RESULTS: We have confirmed that sBCMA contains a variable N-terminus and a C-terminus that extends to the transmembrane domain, ending at amino acid 61. Leveraging an in-house synthesized G-1-61 sBCMA recombinant standard, we developed a RF-LC-MRM method for unbiased sBCMA quantitation in MM patient samples. By comparing the results from RF-LC-MRM with ELISA and IA-LC-MRM, we demonstrated that RF-LC-MRM measures a more complete pool of endogenous sBCMA compared to the antibody-based methods. CONCLUSIONS: This work fills the knowledge gap of the exact sequence of endogenous sBCMA for the first time, which differs from the current commercially available standard. Additionally, this work highlights the necessity of identifying the actual sequence of an endogenous soluble target such as sBCMA, both for bioanalytical purposes and to underpin pharmacodynamic measurements.

Analytical comparability demonstrated for an IgG4 molecule, inclacumab, following transfer of manufacturing responsibility from Roche to Global Blood Therapeutics.

BACKGROUND: Inclacumab is a recombinant, fully human, immunoglobulin IgG4 monoclonal antibody that selectively binds to P-selectin. Initially discovered and developed by Roche through phase 2 clinical studies in peripheral arterial disease and coronary artery disease, inclacumab has been in-licensed by Global Blood Therapeutics (GBT) as a potential treatment to reduce the frequency of vaso-occlusive crises in individuals with sickle cell disease. RESEARCH DESIGN AND METHODS: GBT sought to demonstrate the analytical comparability between material produced by Roche and material produced by GBT to ensure that no meaningful differences in identity, safety, purity, potency, or bioavailability exist between the GBT and Roche lots. RESULTS: Inclacumab samples produced by GBT were found to be comparable to the Roche v0.2 inclacumab samples based on (1) comparable primary and higher-order structures; (2) comparable purity profiles; (3) comparable potency, in vitro functional activities, and in vivo plasma exposures and pharmacokinetic profiles; and (4) comparable degradation patterns and kinetics under forced degradation conditions. CONCLUSIONS: Based on the design of this comparability study and the results obtained, the US Food and Drug Administration approved the changes to the manufacturing process and gave clearance for GBT to proceed with phase 3 clinical trials.

The gut microbiome is associated with bone turnover markers in postmenopausal women.

OBJECTIVE: The association of the gut microbiome with bone turnover markers (BTMs) in postmenopausal women is poorly understood. METHODS: Fecal samples were collected from 97 Chinese postmenopausal women, and the serum CTX and P1NP were determined. Individuals with serum CTX lower or higher than the median value were divided into LCTX and P1NP groups; and individuals with serum P1NP lower or higher than the median value were grouped into LP1NP and HP1NP groups. Microbiota profiles were determined by high-throughput 16S rRNA gene sequencing. RESULTS: In postmenopausal women, only Faecalibacterium showed significant alteration in the HCTX group compared with the LCTX group (P=0.004, q=0.143). Linear discriminant analysis effect size (LEfSe) analysis revealed that Clostridiaceae (P=0.015, LDA=2.89), Faecalibacterium (P=0.017, LDA=4.60), Prevotella (P=0.040, LDA=3.61) and Clostridium (P=0.007, LDA=2.79) were abundant in the LCTX group, and Facklamia (P=0.044, LDA=3.10) was enriched in the HCTX group. Peptostreptococcaceae (P=0.048, LDA=2.83) and the SMB53 (P=0.028, LDA=2.05) genus were enriched in the LPINP group, and Veillonellaceae (P=0.025, LDA=4.43) and the S24_7 (P=0.023, LDA=3.08) family were enriched in the HPINP group. Six taxa correlated with BTMs in all subjects, including Clostridium (Clostridiaceae) that was negatively correlated with serum CTX amounts significantly (r=-0.34, P<0.001). CONCLUSION: This study identified taxa-specific differences in the intestinal microflora associated with BTMs, notably CTX. These findings may help in uncovering the roles of gut microbiota on bone metabolism.

Glucagon­like peptide­1 protects mouse podocytes against high glucose­induced apoptosis, and suppresses reactive oxygen species production and proinflammatory cytokine secretion, through sirtuin 1 activation in vitro.

Glucagon­like peptide­1 (GLP­1) is a gut incretin hormone that is considered to be a promising target for the treatment of patients with type 2 diabetes. However, the mechanisms underlying the protective effects of GLP­1 on diabetic nephropathy are yet to be fully elucidated. Sirtuin (SIRT)1 encodes a member of the SIRT family of proteins that serves an important role in mitochondrial function and is reported to be associated with the pathogenesis of chronic kidney disease. The present study treated mouse podocytes with various concentrations of D­glucose to establish a high glucose (HG)­induced model of renal injury. The results of a 2',7'­dichlorodihydrofluorescein diacetate assay, Annexin V/propidium iodide staining and ELISA demonstrated that treatment of podocytes with HG significantly enhanced the production of reactive oxygen species (ROS), promoted cell apoptosis and increased the secretion of proinflammatory cytokines, respectively. The cytokines increased following HG treatment included tumor necrosis factor­α, interleukin (IL)­1ß and IL­6. Notably, treatment with GLP­1 attenuated HG­induced increases in ROS production and podocyte apoptosis, which may occur via downregulation of the expression of caspase­3 and caspase­9, and increased expression of nephrin, podocin and SIRT1, as determined by reverse transcription­quantitative polymerase chain reaction and western blot analysis. Treatment with GLP­1 led to protective effects in podocytes that were similar to those of resveratrol. Furthermore, SIRT1 knockdown using short hairpin RNA significantly enhanced the expression of caspase­3 and caspase­9 in mouse podocytes, compared with normal mouse podocytes. SIRT1 knockdown with or without GLP­1 administration significantly decreased the expression of caspase­3 and caspase­9 in mouse podocytes, compared with SIRT1 knockdown mouse podocytes. In conclusion, the results of the present study indicated that GLP­1 may be a promising target for the development of novel therapeutic strategies for HG­induced nephropathy, and may function through the activation of SIRT1.

Reagent-free LC-MS/MS-based pharmacokinetic quantification of polyhistidine-tagged therapeutic proteins.

AIM: Immobilized metal ion affinity chromatography is widely employed for purifying polyhistidine-tagged recombinant proteins from cell lysates. The technique can be applied for quantification of therapeutic proteins in biological matrices by LC-MS/MS. RESULTS: A protein reagent-free workflow was developed for quantifying polyhistidine-tagged proteins by LC-MS/MS. The workflow includes target protein enrichment by immobilized metal ion affinity chromatography, on-bead trypsin digestion and quantification of signature peptides by LC-MS/MS. It was applied to quantify a 6×His-tagged protein in a mouse pharmacokinetic study with assay sensitivity of 10.0 ng/ml and linearity up to 10,000 ng/ml. CONCLUSION: The protein reagent-free workflow developed herein can overcome reagent limitation and serve as a viable approach for quantifying polyhistidine-tagged therapeutic proteins to support discovery pharmacokinetic and pharmacodynamic studies.

SIRT4 overexpression protects against diabetic nephropathy by inhibiting podocyte apoptosis.

Diabetic nephropathy is a diabetic complication associated with capillary damage and increased mortality. Sirtuin 4 (SIRT4) plays an important role in mitochondrial function and the pathogenesis of metabolic diseases, including aging kidneys. The aim of the present study was to investigate the association between SIRT4 and diabetic nephropathy in a glucose-induced mouse podocyte model. A CCK-8 assay showed that glucose simulation significantly inhibited podocyte proliferation in a time- and concentration-dependent manner. Reverse transcription-quantitative polymerase chain reaction and western blot analysis showed that the mRNA and protein levels of SIRT4 were notably decreased in a concentration-dependent manner in glucose-simulated podocytes. However, SIRT4 overexpression increased proliferation and suppressed apoptosis, which was accompanied by increases in mitochondrial membrane potential and reduced production of reactive oxygen species (ROS). Notably, SIRT4 overexpression downregulated the expression of apoptosis-related proteins NOX1, Bax and phosphorylated p38 and upregulated the expression of Bcl-2 in glucose-simulated podocytes. In addition, SIRT4 overexpression significantly attenuated the inflammatory response, indicated by reductions in the levels of TNF-α, IL-1ß and IL-6. These results demonstrate for the first time that the overexpression of SIRT4 prevents glucose-induced podocyte apoptosis and ROS production and suggest that podocyte apoptosis represents an early pathological mechanism leading to diabetic nephropathy.

Discovery of Phosphodiesterase 10A (PDE10A) PET Tracer AMG 580 to Support Clinical Studies.

We report the discovery of PDE10A PET tracer AMG 580 developed to support proof of concept studies with PDE10A inhibitors in the clinic. To find a tracer with higher binding potential (BPND) in NHP than our previously reported tracer 1, we implemented a surface plasmon resonance assay to measure the binding off-rate to identify candidates with slower washout rate in vivo. Five candidates (2-6) from two structurally distinct scaffolds were identified that possessed both the in vitro characteristics that would favor central penetration and the structural features necessary for PET isotope radiolabeling. Two cinnolines (2, 3) and one keto-benzimidazole (5) exhibited PDE10A target specificity and brain uptake comparable to or better than 1 in the in vivo LC-MS/MS kinetics distribution study in SD rats. In NHP PET imaging study, [(18)F]-5 produced a significantly improved BPND of 3.1 and was nominated as PDE10A PET tracer clinical candidate for further studies.

Silencing of USP22 suppresses high glucose-induced apoptosis, ROS production and inflammation in podocytes.

Ubiquitin-specific protease 22 (USP22) has been reported to mediate various cellular processes, including cell proliferation and apoptosis. However, its role in high glucose-induced podocytes and diabetic rats remains unknown. In the current study, podocytes were treated with different concentrations of d-glucose to establish a high glucose-induced injury model. Additionally, intravenous tail injection of rats with 65 mg kg(-1) of streptozotocin (STZ) was performed to establish a diabetic rat model. Our findings showed that the treatment of podocytes with high d-glucose significantly increased the USP22 expression level. Silencing of USP22 in podocytes attenuated high d-glucose-induced apoptosis and inflammatory responses, evidenced by increases in proliferation and MMP levels and decreases in the apoptotic rate, ROS production, the Bax/Bcl-2 ratio, caspase-3 expression and secretion of TNF-α, IL-1ß, IL-6 and TGF-ß1. In addition, podocytes with USP22 overexpression significantly enhanced the effect of high d-glucose-induced apoptosis and inflammatory responses. Similar to the protective effect of USP22 knockdown, resveratrol (RSV) depressed not only high d-glucose- and USP22 overexpression-induced cytotoxicity, but also the secretion of TNF-α, IL-1ß, IL-6 and TGF-ß1. Notably, silencing of USP22 in diabetic rats conferred a similar protective effect against high glucose-induced apoptosis and inflammation. Taken together, the findings of the present study have demonstrated for the first time that USP22 inhibition attenuates high glucose-induced podocyte injuries and inflammation.

AMG 580: a novel small molecule phosphodiesterase 10A (PDE10A) positron emission tomography tracer.

Phosphodiesterase 10A (PDE10A) inhibitors have therapeutic potential for the treatment of psychiatric and neurologic disorders, such as schizophrenia and Huntington's disease. One of the key requirements for successful central nervous system drug development is to demonstrate target coverage of therapeutic candidates in brain for lead optimization in the drug discovery phase and for assisting dose selection in clinical development. Therefore, we identified AMG 580 [1-(4-(3-(4-(1H-benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)-2-fluoropropan-1-one], a novel, selective small-molecule antagonist with subnanomolar affinity for rat, primate, and human PDE10A. We showed that AMG 580 is suitable as a tracer for lead optimization to determine target coverage by novel PDE10A inhibitors using triple-stage quadrupole liquid chromatography-tandem mass spectrometry technology. [(3)H]AMG 580 bound with high affinity in a specific and saturable manner to both striatal homogenates and brain slices from rats, baboons, and human in vitro. Moreover, [(18)F]AMG 580 demonstrated prominent uptake by positron emission tomography in rats, suggesting that radiolabeled AMG 580 may be suitable for further development as a noninvasive radiotracer for target coverage measurements in clinical studies. These results indicate that AMG 580 is a potential imaging biomarker for mapping PDE10A distribution and ensuring target coverage by therapeutic PDE10A inhibitors in clinical studies.

Discovery of clinical candidate 1-(4-(3-(4-(1H-benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)ethanone (AMG 579), a potent, selective, and efficacious inhibitor of phosphodiesterase 10A (PDE10A).

We report the identification of a PDE10A clinical candidate by optimizing potency and in vivo efficacy of promising keto-benzimidazole leads 1 and 2. Significant increase in biochemical potency was observed when the saturated rings on morpholine 1 and N-acetyl piperazine 2 were changed by a single atom to tetrahydropyran 3 and N-acetyl piperidine 5. A second single atom modification from pyrazines 3 and 5 to pyridines 4 and 6 improved the inhibitory activity of 4 but not 6. In the in vivo LC-MS/MS target occupancy (TO) study at 10 mg/kg, 3, 5, and 6 achieved 86-91% occupancy of PDE10A in the brain. Furthermore, both CNS TO and efficacy in PCP-LMA behavioral model were observed in a dose dependent manner. With superior in vivo TO, in vivo efficacy and in vivo PK profiles in multiple preclinical species, compound 5 (AMG 579) was advanced as our PDE10A clinical candidate.

Discovery of Novel Imidazo[4,5-b]pyridines as Potent and Selective Inhibitors of Phosphodiesterase 10A (PDE10A).

We report the discovery of novel imidazo[4,5-b]pyridines as potent and selective inhibitors of PDE10A. The investigation began with our recently disclosed ketobenzimidazole 1, which exhibited single digit nanomolar PDE10A activity but poor oral bioavailability. To improve oral bioavailability, we turned to novel scaffold imidazo[4,5-b]pyridine 2, which not only retained nanomolar PDE10A activity but was also devoid of the morpholine metabolic liability. Structure-activity relationship studies were conducted systematically to examine how various regions of the molecule impacted potency. X-ray cocrystal structures of compounds 7 and 24 in human PDE10A helped to elucidate the key bonding interactions. Five of the most potent and structurally diverse imidazo[4,5-b]pyridines (4, 7, 12b, 24a, and 24b) with PDE10A IC50 values ranging from 0.8 to 6.7 nM were advanced into receptor occupancy studies. Four of them (4, 12b, 24a, and 24b) achieved 55-74% RO at 10 mg/kg po.

Design, optimization, and biological evaluation of novel keto-benzimidazoles as potent and selective inhibitors of phosphodiesterase 10A (PDE10A).

Our development of PDE10A inhibitors began with an HTS screening hit (1) that exhibited both high p-glycoprotein (P-gp) efflux ratios in rat and human and poor metabolic stability. On the basis of cocrystal structure of 1 in human PDE10A enzyme, we designed a novel keto-benzimidazole 26 with comparable PDE10A potency devoid of efflux liabilities. On target in vivo coverage of PDE10A in rat brain was assessed using our previously reported LC-MS/MS receptor occupancy (RO) technology. Compound 26 achieved 55% RO of PDE10A at 30 mg/kg po and covered PDE10A receptors in rat brain in a dose-dependent manner. Cocrystal structure of 26 in PDE10A confirmed the binding mode of the novel scaffold. Further optimization resulted in the identification of keto-benzimidazole 34, which showed an increased in vivo efficacy of 57% RO in rats at 10 mg/kg po and an improved in vivo rat clearance and oral bioavailability.

Rapid identification of a novel small molecule phosphodiesterase 10A (PDE10A) tracer.

A radiolabeled tracer for imaging therapeutic targets in the brain is a valuable tool for lead optimization in CNS drug discovery and for dose selection in clinical development. We report the rapid identification of a novel phosphodiesterase 10A (PDE10A) tracer candidate using a LC-MS/MS technology. This structurally distinct PDE10A tracer, AMG-7980 (5), has been shown to have good uptake in the striatum (1.2% ID/g tissue), high specificity (striatum/thalamus ratio of 10), and saturable binding in vivo. The PDE10A affinity (K(D)) and PDE10A target density (B(max)) were determined to be 0.94 nM and 2.3 pmol/mg protein, respectively, using [(3)H]5 on rat striatum homogenate. Autoradiography on rat brain sections indicated that the tracer signal was consistent with known PDE10A expression pattern. The specific binding of [(3)H]5 to rat brain was blocked by another structurally distinct, published PDE10A inhibitor, MP-10. Lastly, our tracer was used to measure in vivo PDE10A target occupancy of a PDE10A inhibitor in rats using LC-MS/MS technology.

Development of a robust and sensitive LC-MS/MS method for the determination of adenine in plasma of different species and its application to in vivo studies.

A simple, robust, and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the measurement of endogenous adenine in mouse, rat, cynomolgus monkey, and human plasma. A "surrogate analyte" strategy was adopted by employing [(13)C(U)]-adenine as the surrogate analyte. The plasma samples were processed by protein precipitation, and the extracted supernatant samples were subjected directly to LC-MS/MS analysis. The analysis was carried out in the negative ion detection mode using selected-reaction monitoring (SRM). The method achieved a lower limit of quantification (LLOQ) of 5.0nM with a signal-to-noise ratio of 10. The intra- and inter-day assay coefficients of variation (CV) were ≤6.67% in rat plasma, and the mean recoveries and matrix effects across species and at various concentrations ranged from 88.8% to 104.2% and 86.0% to 110.8%, respectively. Using this methodology, the endogenous concentration of adenine in plasma of four species was found to range from 8.7nM in human to 93.1nM in cynomolgus monkey plasma. The assay was further applied to both an adenine pharmacokinetic study and a pivotal pharmacodynamic study evaluating the plasma concentration of adenine after a dose of 5'-deoxy-5'-methylthioadenosine (MTA).

A fully automated plasma protein precipitation sample preparation method for LC-MS/MS bioanalysis.

This report describes the development and validation of a robust robotic system that fully integrates all peripheral devices needed for the automated preparation of plasma samples by protein precipitation. The liquid handling system consisted of a Tecan Freedom EVO 200 liquid handling platform equipped with an 8-channel liquid handling arm, two robotic plate-handling arms, and two plate shakers. Important additional components integrated into the platform were a robotic temperature-controlled centrifuge, a plate sealer, and a plate seal piercing station. These enabled unattended operation starting from a stock solution of the test compound, a set of test plasma samples and associated reagents. The stock solution of the test compound was used to prepare plasma calibration and quality control samples. Once calibration and quality control samples were prepared, precipitation of plasma proteins was achieved by addition of three volumes of acetonitrile. Integration of the peripheral devices allowed automated sequential completion of the centrifugation, plate sealing, piercing and supernatant transferral steps. The method produced a sealed, injection-ready 96-well plate of plasma extracts. Accuracy and precision of the automated system were satisfactory for the intended use: intra-day and the inter-day precision were excellent (C.V.<5%), while the intra-day and inter-day accuracies were acceptable (relative error<8%). The flexibility of the platform was sufficient to accommodate pharmacokinetic studies of different numbers of animals and time points. To the best of our knowledge, this represents the first complete automation of the protein precipitation method for plasma sample analysis.

Allosteric inhibition of the protein-protein interaction between the leukemia-associated proteins Runx1 and CBFbeta.

The two subunits of core binding factor (Runx1 and CBFbeta) play critical roles in hematopoiesis and are frequent targets of chromosomal translocations found in leukemia. The binding of the CBFbeta-smooth muscle myosin heavy chain (SMMHC) fusion protein to Runx1 is essential for leukemogenesis, making this a viable target for treatment. We have developed inhibitors with low micromolar affinity which effectively block binding of Runx1 to CBFbeta. NMR-based docking shows that these compounds bind to CBFbeta at a site displaced from the binding interface for Runx1, that is, these compounds function as allosteric inhibitors of this protein-protein interaction, a potentially generalizable approach. Treatment of the human leukemia cell line ME-1 with these compounds shows decreased proliferation, indicating these are good candidates for further development.

[Thrombin and angiogenesis of cancer--review].

Thrombin is the most important factor in hemostasis. In recent years, it has been found that thrombin is a potent mitogen capable of inducing cellular functions. Therefore, it is proved to be of importance in promoting the growth, metastasis and angiogenesis of cancer. Anticoagulant therapy not only reduce the characteristic hypercoagulability of cancer, but also inhibits growth and metastasis of cancer, and alters the fundamental biology of cancer. In this paper thrombin and its receptor, relationship of thrombin and its receptor with cancer growth, metastasis and angiogenesis, the mechanisms of thrombin influence on cancer angiogenesis, as well as application prospects on anti-angiogenesis and anti-coagulation therapy were reviewed.

Protein signal assignments using specific labeling and cell-free synthesis.

The goal of structural genomics initiatives is to determine complete sets of protein structures that represent recently sequenced genomes. The development of new high throughput methods is an essential aspect of this enterprise. Residue type and sequential assignments obtained from specifically labeled samples, when combined with 3D heteronuclear data, can significantly increase the efficiency and accuracy of the assignment process, the first step in structure determination by NMR. A protocol for the design of specifically labeled samples with high information content is presented along with a description of the experiments used to extract essential information using 2D versions of 3D heteronuclear experiments. In vitro protein synthesis methods were used to produce four specifically labeled samples of the 23.5 kDa protein phosphoserine phosphatase (PSP) from Methanoccous jannaschii (MJ1594). Each sample contained two (13)C/(15)N-labeled amino acids and one (15)N-labeled amino acid. The 135 type and 14 sequential assignments obtained from these samples were used in conjunction with 3D data obtained from uniformly (13)C/(15)N-labeled and (2)H/(13)C/(15)N-labeled protein to manually assign the backbone (1)H(N), (15)N, (13)CO, (13)C(alpha), and (13)C(beta) signals. Using an automated assignment algorithm, 30% more assignments were obtained when the type and sequential assignments were used in the calculations.