Discovery and SAR Study of Boronic Acid-Based Selective PDE3B Inhibitors from a Novel DNA-Encoded Library.

Human genetic evidence shows that PDE3B is associated with metabolic and dyslipidemia phenotypes. A number of PDE3 family selective inhibitors have been approved by the FDA for various indications; however, given the undesirable proarrhythmic effects in the heart, selectivity for PDE3B inhibition over closely related family members (such as PDE3A; 48% identity) is a critical consideration for development of PDE3B therapeutics. Selectivity for PDE3B over PDE3A may be achieved in a variety of ways, including properties intrinsic to the compound or tissue-selective targeting. The high (>95%) active site homology between PDE3A and B represents a massive obstacle for obtaining selectivity at the active site; however, utilization of libraries with high molecular diversity in high throughput screens may uncover selective chemical matter. Herein, we employed a DNA-encoded library screen to identify PDE3B-selective inhibitors and identified potent and selective boronic acid compounds bound at the active site.

Angiotensin II promotes an osteoblast-like phenotype in porcine aortic valve myofibroblasts.

OBJECTIVES: The mechanisms for pathogenesis of cardiac valve calcification were explored by studying the regulation of the Wnt signaling pathway during the transformation from cardiac valvular myofibroblasts to osteoblast-like phenotype. METHODS: Studies were carried on primary cultured porcine aortic valvular myofibroblasts. The cells were randomly divided into four groups and treated with angiotensin II (Ang II) according to the following: Ang II (10(-6) mol/l), Valsartan (Val) (10(-5) mol/l), Ang II plus Val (Ang II 10(-6) mol/l + Val 10(-5) mol/l) or mock treated as the control. Protein expression of Bone morphogenetic protein 2 (BMP2), Alkaline phosphatase (ALP), and Wnt pathway components, Wnt3a and ß-catenin, was investigated to assess the activation of the Wnt signaling pathway and determine whether cells undergo the transformation to osteoblast-like phenotype. RESULT: Ang II treatment of myofibroblasts led to significant up-regulation of α-SMA expression and activation of the cells. Neither the BMP2 or ALP proteins, nor the mRNA was detectable in the control group or the Val-treated group; however, there was a significant increase in Ang II-treated group (P < 0.01). The Wnt/ß-catenin signaling ligand, Wnt3a, was not expressed in the control or Val-treated groups, whereas in Ang II-treated cells, both Wnt3a and ß-catenin gene expression were enhanced (P < 0.01).The effect of Ang II can be inhibited by the addition of Val (P < 0.05). CONCLUSION: Ang II might act on the Ang II receptor on valvular interstitial cells (VICs) and lead to activation of the Wnt/ß-catenin pathway and hence cause the activation, differentiation and proliferation of myofibroblasts, and finally, osteoblast-like phenotype transformation, leading to calcification of heart valves.

[Mechanism of tanshinone II A in inhibiting transformation of aortic valvular myofibroblast to osteoblast-like phenotype].

Aortic valve calcification (AVC) is a pathological process correlated with multiple disease causes and actively regulated by cardiac valve cells. In this study, porcine aortic valve myofibroblasts cultured in vitro were treated with 50 µg z L(-1) of pathological factor tumor necrosis factor α (TNF-α). Tanshinone II A (TSN) with the concentration of 50 mg x L(-1) and TNF-α were combined in incubating cells for 72 h (3 d) and 120 h (5 d). The Western blotting and Real-time PCR were adopted to detect the changes in smooth muscle α actin (α-SMA), bone morphogenetic protein 2 ( BMP2), alkaline phosphatase (ALP) in cells, and expressions of key effect proteins GSK-3ß and ß-catenin on Wnt/ß-catenin signal pathway. According to the findings, TNF-α can significantly increase the expression of myofibroblasts α-SMA and add the transformation activity to them, with nearly no expression of BMP2, ALP and mRNA in the control group and the TSN group but significant increase in their expressions in the TNF-α group (P < 0.01), which showed osteoblast-like phenotype. Moreover, TNF-α down-regulated the expression of up-streaming regulator GSK-3ß and mRNA expression (P < 0. 01) , notably increased the expression of key effect protein ß-catenin, but with no significant difference in mRNA with the control group and the TSN group. The result demonstrated that TSN showed a certain inhibitory effect on TNF-α's pathological impact (P < 0.05) in a time-dependent manner. Inflammatory factor TNF-α may promote the transformation of aortic valvular myofibroblasts to osteoblast-like phenotype by activating Wnt/ß-catenin signal pathway in aortic valvular myofibroblasts, so as to cause AVC. Tanshinone II A can have a preventive effect in AVC by activating GSK-3ß proteins and regulating signal transduction of Wnt/ß-catenin signal pathway.

Dolutegravir (S/GSK1349572) exhibits significantly slower dissociation than raltegravir and elvitegravir from wild-type and integrase inhibitor-resistant HIV-1 integrase-DNA complexes.

The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10(-6) s(-1) and a dissociative half-life (t(1/2)) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding (t(1/2), at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the single substitution. For all of the IN substitutions tested, the off-rate of DTG from IN-DNA complexes was significantly slower (from 5 to 40 times slower) than the off-rate of RAL or ELV. These data are consistent with the potential for DTG to have a higher genetic barrier to resistance, provide evidence that the INI off-rate may be an important component of the mechanism of INI resistance, and suggest that the slow dissociation of DTG may contribute to its distinctive resistance profile.

Limited role for SREBP-1c in defective glucose-induced insulin secretion from Zucker diabetic fatty rat islets: a functional and gene profiling analysis.

Accumulation of intracellular lipid may contribute to defective insulin secretion in type 2 diabetes. Although Zucker diabetic fatty (ZDF; fa/fa) rat islets are fat-laden and overexpress the lipogenic master gene, sterol regulatory element binding protein 1c (SREBP-1c), the contribution of SREBP-1c to the secretory defects observed in this model remains unclear. Here we compare the gene expression profile of lean control (fa/+) and ZDF rat islets in the absence or presence of dominant-negative SREBP-1c (SREBP-1c DN). ZDF islets displayed elevated basal insulin secretion at 3 mmol/l glucose but a severely depressed response to 17 mmol/l glucose. While SREBP-1c DN reduced basal insulin secretion from ZDF islets, glucose-stimulated insulin secretion was not improved. Of 57 genes differentially regulated in ZDF islets and implicated in glucose metabolism, vesicle trafficking, ion fluxes, and/or exocytosis, 21 were upregulated and 5 were suppressed by SREBP-1c DN. Genes underrepresented in ZDF islets were either unaffected (Glut-2, Kir6.2, Rab3), stimulated (voltage-dependent Ca(2+) channel subunit alpha1D, CPT2, SUR2, rab9, syt13), or inhibited (syntaxin 7, secretogranin-2) by SREBP-1c inhibition. Correspondingly, SREBP-1c DN largely corrected decreases in the expression of the transcription factors Pdx-1 and MafA but did not affect the abnormalities in Pax6, Arx, hepatic nuclear factor-1alpha (HNF1alpha), HNF3beta/Forkhead box-a2 (Foxa2), inducible cyclic AMP early repressor (ICER), or transcription factor 7-like 2 (TCF7L2) expression observed in ZDF islets. We conclude that upregulation of SREBP-1c and mild increases in triglyceride content do not explain defective glucose-stimulated insulin secretion from ZDF rats. However, overexpression of SREBP-1c may contribute to enhanced basal insulin secretion in this model.

Suppression of the cell-mediated immune response by a Fas-immunoglobulin fusion protein.

INTRODUCTION: Immunosuppressive agents must not only be effective in impairing the host's allo-immune response, but should also be selective in targeting only those elements of the immune system activated by the allograft. The fact that allo-activated T cells express surface protein molecules that are not typically present on resting T cells can be exploited to specifically target this population. Fas ligand is one such molecule whose cell surface expression on T cells is dramatically up-regulated upon activation. METHODS: We constructed a murine fusion protein by linking the extracellular domain of Fas to the Fc region of IgG2a. The rationale being to selectively target activated T cells via binding of its Fas moiety to cell surface Fas ligand, and then to allow the Fc moiety to invoke its usual effector mechanisms resulting in the destruction of the allo-activated T cell. Here, we describe the design and expression of Fas-IgG2a and characterize several key in vitro and in vivo properties of this fusion protein including its ability to impact on both cell-mediated immune responses and cellular apoptotic activity using a murine model of delayed-type hypersensitivity. RESULTS: In vitro, our Fas-IgG2a construct bound activated T cells via FasL and invoked cytotoxicity. In vivo, it demonstrated a prolonged half-life characteristic of an immunoglobulin-like molecule. Most significantly, it suppressed the cell-mediated immune response and diminished cellular apoptotic activity in lymphoid tissue in our murine model. CONCLUSION: Fas-IgG2a is a novel agent for delivering target-specific immunosuppression with potential applicability in the transplant setting.

Decrease in brain POMC mRNA expression and onset of obesity in guinea pigs exposed to 2-chloroethyl ethyl sulfide, a mustard analogue.

The full spectrum of physiological effects resulting from exposure to sulfur mustard and its analogs is currently unknown. In a guinea pig model, initially selected to study the role of an inflammatory cytokine cascade in mustard gas induced lung injury, we observed significant body weight gain in guinea pigs exposed to an intratracheally injected single dose of 2-chloroethyl ethyl sulfide, a mustard analog. The body weight gain was not associated with any apparent change in appetite. To further elucidate a molecular basis for the observed weight gain, we evaluated candidate genes for the obese phenotype by quantitative RT-PCR. We observed a time- and dose-dependent decrease in guinea pig pro-opiomelanocortin (POMC) message following treatment with mustard gas. This reduction in POMC message is consistent with the onset of obesity in the animals. We hypothesize that the POMC melanocortin pathway provides a mechanistic basis for the observed effects of sulfur mustard on body weight.

Quantification of donor microchimerism in sex-mismatched porcine allotransplantation by competitive PCR.

The persistence of donor cells in recipient circulation and peripheral tissues post-transplantation has been demonstrated in solid organ allotransplantation and xenotransplantation models. Although this state of microchimerism has been postulated as the basis for graft acceptance, chimerism has not been directly linked to the maintenance of peripheral tolerance or prevention of rejection. Studies have demonstrated that the qualitative presence or absence of donor microchimerism bears no association with graft acceptance. Our preliminary work suggests that there is a threshold chimerism necessary for the induction of donor-specific hyporesponsiveness. Because the kinetics of donor cell accumulation and distribution in allograft recipients are largely unknown, quantitative analyses are needed to evaluate chimerism's significance to donor-specific tolerance. We developed a quantitative, competitive PCR assay to precisely measure the amount of chimerism in male to female transplant pairs by targeting the sex-determining region of the Y chromosome (SRY gene). Traditionally, this technique requires that serial known amounts of an SRY-specific competitive template (CT) be coamplified with a constant amount of sample DNA to determine the equivalence point of the relative band intensities of the PCR products. However running a panel of PCRs with CT amounts above and below the equivalence point to generate a standard curve for ever' sample is laborious. Here we describe the generation of a single standard curve that permits the rapid and reliable quantification of microchimerism after coamplification of sample DNA with a single amount of CT.