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  • The effect of ET activity on

    2019-09-25

    The effect of ET-1 activity on the development of experimental pulmonary fibrosis has also been examined by other investigators. One study found that repeated administration of Bosentan, an ERA used to treat pulmonary arterial hypertension (PAH), significantly decreased bleomycin-induced pulmonary fibrosis [34]. Conversely, transgenic mice overexpressing human ET-1 developed rapidly progressive pulmonary fibrosis [35]. Whether other ERAs would have the same time-related effects on the BLM model as HJP272 may depend on their specific mode of action. HJP272 is a selective ERA, with a primary affinity for the ET-1 subtype A (ET-A) receptor, whereas mixed ERAs also bind to ET-1 subtype B (ET-B) receptors. However, the difference between selective and mixed antagonists is not well-defined pharmacologically [36]. Selective ERAs, when used at relatively high doses, may act on both receptors, while mixed ERAs usually demonstrate a greater affinity for ET-A receptors. Among the ERAs used to treat PAH, Ambrisentan is selective for ET-A receptors, whereas Bosentan and Macitentan are mixed antagonists. Clinical trials using each of these agents to treat pulmonary fibrosis have been largely unsuccessful [11], [12], [13], [14]. Although a subgroup of patients with less severe disease showed a trend toward improved survival with Bosentan, the result could not be confirmed in a subsequent trial [14].
    Introduction G-protein coupled receptors (GPCRs) are one of the most important membrane protein families as they are involved in a multitude of physiological processes and play key roles in prevalent human diseases and disorders [1]. Consequently, they represent a major target fraction for modern pharmaceutical drugs with 475 drugs targeting 108 unique GPCRs, corresponding to ∼34% of all drugs approved by the US Food and Drug Administration (FDA) by mid of 2017 [2]. Designing, screening and evaluation of new drugs requests fast and efficient platforms providing GPCRs in functionally folded and stable condition. Compared to classical detergent based approaches, lipid bilayers are more closely mimicking the native environment of membrane proteins and are therefore often superior for their characterization in vitro[3], [4], [5]. Most GPCRs are of low abundance in Sephin1 and their recombinant production is thus an almost mandatory prerequisite for their molecular analysis. Elaborated expression systems using insect or mammalian host cells are the current standard for GPCR production and they have considerably supported the progress in their structural evaluation [6]. With an optimized insect cell expression system, several mg of GPCR can be produced per L of cell culture. On the other hand, cell handling is relatively labor intense and the need to produce baculovirus for cell transfection is time consuming [7]. Further bottlenecks still exist in the efficient production and analysis of the vast majority of GPCRs as conventional cell-based expression systems are frequently faced with low synthesis rates, the need of potentially harmful membrane disruption procedures and altered expression efficiencies after genetical modification of the target protein. Cell-free protein synthesis offers an alternative approach to get access to a GPCR target in a defined environment. It allows straight-forward optimization procedures that are exclusively focused on the production of a single protein without the need of compromises to cell viability and has therefore emerged as an alternative platform for the production and functional or structural characterization of membrane proteins [8]. Structural studies by crystallization and NMR are possible with samples isolated from few mL of CF reaction. These include the lipid metabolizing kinase DgkA [9], the K+ channel KcsA [10] and the light-driven proton pump proteorhodopsin [11]. However, some types of membrane proteins such as the GPCRs are more difficult to address as their functional folding requires intensive screening of a variety of CF reaction conditions such as hydrophobic environments or redox conditions [12], [13]. Despite quantities of several nmol of the human endothelin B (ETB) receptor could be synthesized per mL CF reaction, only a minor fraction of few pmol/mL and representing less than 0.1% of the synthesized protein appeared to be functionally folded [12]. A similar low quality was obtained from ETB samples isolated from CF reactions based either on E. coli lysates or on Sf9 insect cell lysates [14]. We therefore intended to identify and to optimize parameters in the CF reaction still being unfavorable or limiting for ETB folding.