Central neuropeptide Y infusion and melanocortin 4 receptor antagonism inhibit thyrotropic function by divergent pathways

Weight loss inhibits thyrotropic function and reduces metabolic rate, thereby contributing to weight regain. Under negative energy balance there is an increase in the hypothalamic expression of both neuropeptide Y (NPY) and agouti related peptide (AgRP), the endogenous antagonist of melanocortin 4 (MC4) receptors. Both NPY and MC4 receptor antagonism reduce thyrotropic function centrally, but it is not known whether these pathways operate by similar or distinct mechanisms. We compared the time-course of effects of acute or chronic intracerebroventricular (ICV) administration of NPY (1.2 nmol acute bolus, or 3.5 nmol/day for 6 days) or the MC4 receptor antagonist HS014 (1.5 nmol bolus, or 4.8 nmol/day) on plasma concentrations of thyroid stimulating hormone (TSH) or free thyroxine (T4) in male rats pair-fed with vehicle-infused controls. These doses equipotently induced hyperphagia in acute studies, reduced latency to feed, and increased white adipose tissue mass after 6 days of infusion. Acute central NPY but not HS014 administration significantly reduced plasma TSH concentrations within 30–60 min and plasma free T4 levels within 90–120 min. These inhibitory effects were sustained for up to 5–6 days of continuous NPY infusion. HS014 induced a transient decrease in plasma free T4 levels that was observed only after 1–2 days of continuous ICV infusion. While both NPY and HS014 significantly increased corticosteronemia within an hour after ICV injection, the effect of NPY was significantly more pronounced and was sustained for up to 4 days of administration. Both NPY and HS014 significantly decreased the brown adipose tissue protein levels of uncoupling protein-3. We conclude that central NPY and MC4 antagonism decrease thyrotropic function via partially distinct mechanisms with different time courses, possibly involving glucocorticoid effects of NPY. MC4 receptor antagonism increases adiposity via pathways independent of increased food intake or changes in circulating concentrations of TSH, free T4 or corticosterone.     

Non-Competitive Inhibition by Active Site Binders

Classical enzymology has been used for generations to understand the interactions of inhibitors with their enzyme targets. Enzymology tools enabled prediction of the biological impact of inhibitors as well as the development of novel, more potent, ones. Experiments designed to examine the competition between the tested inhibitor and the enzyme substrate(s) are the tool of choice to identify inhibitors that bind in the active site. Competition between an inhibitor and a substrate is considered a strong evidence for binding of the inhibitor in the active site, while the lack of competition suggests binding to an alternative site. Nevertheless, exceptions to this notion do exist. Active site–binding inhibitors can display non-competitive inhibition patterns. This unusual behavior has been observed with enzymes utilizing an exosite for substrate binding, isomechanism enzymes, enzymes with multiple substrates and/or products and two-step binding inhibitors. In many of these cases, the mechanisms underlying the lack of competition between the substrate and the inhibitor are well understood. Tools like alternative substrates, testing the enzyme reaction in the reverse direction and monitoring inhibition time dependence can be applied to enable distinction between ‘badly behaving’ active site binders and true exosite inhibitors.     

Evaluation of WO 2012/177618 A1 and US-2014/0179750 A1: novel small molecule antagonists of prostaglandin-E2 receptor EP2

Recent studies underscore that prostaglandin-E₂ exerts mostly proinflammatory effects in chronic CNS and peripheral disease models, mainly through a specific prostanoid receptor EP2. However, very few highly characterized EP2 receptor antagonists have been reported until recently, when Pfizer and Emory University published two distinct classes of EP2 antagonists with good potency, selectivity and pharmacokinetics. The purpose of this article is to evaluate recently published patents WO 2012/177618 A1 and US-2014/0179750 A1 from Emory, which describe a number of cinnamic amide- and amide-derivatives as a potent antagonists of EP2 receptor, and their neuroprotective effects in in vitro and in an in vivo model. A selected compound from this patent(s) also attenuates prostate cancer cell growth and invasion in vitro, suggesting these compounds should be developed for therapeutic use.     

Therapeutic potential of endothelin receptor antagonists in diabetes

Endothelins (ETs) are widely distributed in the body and perform several vascular and non-vascular functions. Experimental evidence indicates that abnormalities of the ET system occur in several organs affected in chronic diabetic complications. Furthermore, ET antagonists were found to prevent structural and functional changes in the target organs of chronic diabetic complications in animal models. Abnormalities of plasma ET levels have also been demonstrated in human diabetes. This review discusses the role of ET in the pathogenesis of chronic diabetic complications. The current experimental evidence suggests that ET antagonism may potentially represent an adjuvant therapeutic tool in the treatment of chronic diabetic complications.     

Icatibant,the bradykinin B2 receptor antagonist with target to the interconnected kinin systems

Introduction: HOE-140/Icatibant is a selective, competitive antagonist to bradykinin (BK) against its binding to the kinin B2 receptor. Substitution of five non-proteogeneic amino acid analogues makes icatibant resistant to degradation by metalloproteases of kinin catabolism. Icatibant has clinical applications in inflammatory and vascular leakage conditions caused by an acute (non-controlled) production of kinins and their accumulation at the endothelium B2 receptor. The clinical manifestation of vascular leakage, called angioedema (AE), is characterized by edematous attacks of subcutaneous and submucosal tissues, which can cause painful intestinal consequences, and life-threatening complications if affecting the larynx. Icatibant is registered for the treatment of acute attacks of the hereditary BK-mediated AE, i.e., AE due to C1 inhibitor deficiency.

Areas covered: This review discusses emerging knowledge on the kinin system: kinin pharmacological properties, biochemical characteristics of the contact phase and kinin catabolism proteases. It underlines the responsibility of the kinins in AE initiation and the potency of icatibant to inhibit AE formation by kinin–receptor interactions.

Expert opinion: Icatibant antagonist properties protect BK-mediated AE patients against severe attacks, and could be developed for use in inflammatory conditions. More studies are required to confirm whether or not prolonged and frequent applications of icatibant could result in the impairment of the cardioprotective effect of BK.     

Developmental shift in TcR-mediated rescue of thymocytes from glucocorticoid-induced apoptosis

Glucocorticoid hormone (GC) production by thymic epithelial cells influences TcR signalling in DP thymocytes and modifies their survival. In the present work, we focused on exploring details of GC effects on DP thymocyte apoptosis with or without parallel TcR activation in AND transgenic mice, carrying TcR specific for pigeon cytochrome C, in vivo. Here we show that the glucocorticoid receptor (GR) protein level was the lowest in DP thymocytes, and it was slightly down-regulated by GC analogue, anti-CD3, PCC and combined treatments as well. Exogenous GC analogue treatment or TcR stimulation alone lead to marked DP cell depletion, coupled with a significant increase of early apoptotic cell ratio (AnnexinV staining), marked abrogation of the mitochondrial function in DP cells (CMXRos staining), and significant decrease in the Bcl-2(high) DP thymocyte numbers, respectively. On the other hand, the simultaneous exposure to these two proapototic signals effectively reversed all the above-described changes. The parallel analysis of CD4 SP cell numbers, AnnexinV, CMXRos, Bcl-2 and GR stainings revealed, that the GR and TcR signals were not antagonistic on the mature thymocytes. These data provide experimental evidence in TcR transgenic mice, in vivo, that when TcR activation and GR signals are present simultaneously, they rescue double positive thymocytes from programmed cell death. The two separate signalling pathways merge in DP thymocytes at such important apoptosis regulating points as the Bcl-2 and GR, showing that their balanced interplay is essential in DP cell survival.     

A small-molecule inhibitor suppresses the tumor-associated mitochondrial NAD(P)+-dependent malic enzyme (ME2) and induces cellular senescence

Here, we found a natural compound, embonic acid (EA), that can specifically inhibit the enzymatic activity of mitochondrial NAD(P)⁺-dependent malic enzyme (m-NAD(P)-ME, ME2) either in vitro or in vivo. The in vitro IC₅₀ value of EA for m-NAD(P)-ME was 1.4 ± 0.4 μM. Mutagenesis and binding studies revealed that the putative binding site of EA on m-NAD(P)-ME is located at the fumarate binding site or at the dimer interface near the site. Inhibition studies reveal that EA displayed a non-competitive inhibition pattern, which demonstrated that the binding site of EA was distinct from the active site of the enzyme. Therefore, EA is thought to be an allosteric inhibitor of m-NAD(P)-ME. Both EA treatment and knockdown of m-NAD(P)-ME by shRNA inhibited the growth of H1299 cancer cells. The protein expression and mRNA synthesis of m-NAD(P)-ME in H1299 cells were not influenced by EA, suggesting that the EA-inhibited H1299 cell growth occurs through the suppression of in vivo m-NAD(P)-ME activity EA treatment further induced the cellular senescence of H1299 cells. However, down-regulation of the enzyme-induced cellular senescence was not through p53. Therefore, the EA-evoked senescence of H1299 cells may occur directly through the inhibition of ME2 or a p53-independent pathway.     

A rapid and sensitive non-competitive avidin-biotin assay for lactoferrin

We have developed an avidin-biotin assay for the detection of lactoferrin in human seminal plasma. We compared 5 modifications of this assay, and found the non-competitive avidin-biotin assay (NABA) to be the most sensitive. The detection limit of 3-step NABA was 0.2 or 0.1 ng lactoferrin/ml, depending whether avidin-biotin-peroxidase complex (ABC) or avidin-peroxidase was used. The intra-assay and inter-assay coefficients of variation for 3-step NABA were 6.2 and 8.5%, respectively. The lactoferrin levels of human seminal plasma samples measured by 3-step NABA and radioimmunoassay (RIA) showed good correlation (r = 0.96). The total performance time of 3-step NABA is flexible and the method can be modified for rapid (less than 1 h) or overnight assay according to need.     

几种常用药物联用计算方法的数学瑕疵和一带一线模型法的解决方案

多药联用在临床实践中很常见,药物间的相互作用会使药物效应发生改变,联用组合有可能使疗效增加和毒性减少,也有可能会带来药效降低和毒性增大的风险。已有多种计算方法用于定量评价药物联用产生的协同、相加和拮抗,但业内所用的许多计算方法均为共识性方法或经验公式,同一组数据用不同的方法处理,常会得出不同的、相互矛盾的结论。本文用数学方法分析了业内常用的几种计算方法的瑕疵,包括:Loewe等效线法、Bliss独立模型法、周氏中效法和金氏法等,并通过介绍本实验室发现的药物联用药效相加的数学规律,发展了具有自主知识产权的一带一线模型法,作为解决多药联用效应定量评价的通用方案,为充分体现多药联用带来的益处和规避多药联用带来的疗效和毒性风险提供可靠的计算方法。