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Surprisingly, there was also a slight decrease of basal activity by another SMO agonist, SAG (Chen et?al


Nov 5, 2021

Surprisingly, there was also a slight decrease of basal activity by another SMO agonist, SAG (Chen et?al. of drugs is assay dependent. Furthermore, we explored topical efficacies of SMO antagonists on depilated mice using Gli1 and Ptch1 mRNA quantification in skin as biomarkers of the HH signaling inhibition. This topical model rapidly discriminated drugs in terms of efficacies and potencies for inhibition of both biomarkers. SMO antagonists showed also a large variation CL2-SN-38 in their blood and skin partition, suggesting that some drugs are more favorable for topical application. Overall, our data suggested that in?vitro and in?vivo efficacious drugs such as LEQ\506 and TAK\441 may be of interest for topical treatment of less invasive BCC with minimal side effects. test with o em P /em ? ?0.05; * em P /em ? ?0.001; # em P /em ? ?0.0001. Quantitative determination of compound concentration in plasma samples and skin homogenates The punch of the skin was put in a lysis tube (Bertin Technologies, France) containing 500? em /em L of water and homogenized with the Fastprep device (MP Biomedicals, Illkirch, France). The plasma samples or skin homogenates were processed using acetonitrile (AcN) precipitation and analyzed by HPLC\MS/MS (Supplementary Materials and Methods). Animal handling Animals were handled and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal SAT1 Resources on Life Sciences, U.S. National Research Council, 2011) and the European Directive 2010/63/EU, and the protocols were carried out in compliance with French regulations and the local ethical committee guidelines for animal research, in an AAALAC International accredited facility (compare Supplementary Materials and Methods, for further details). Data analysis All data are expressed as mean??SEM. Isotherms were analyzed by nonlinear regression, using Prism software (GraphPad Software, La Jolla, CA, USA) to yield IC50 values. Drugs Compound sources are given in the Supplementary Material and Methods. Results Determination of G\protein activation in CHO cells by [35S]GTP em /em S binding SMO\mediated G\protein activation was assessed in a [35S]GTP em /em S binding assay (Riobo et?al. 2006; Shen et?al. 2013) using a CHO cell line stably expressing the human SMO receptor isoform. The reference SMO agonist purmorphamine activated [35S]GTP em /em S incorporation in these cells, while the reference antagonist cyclopamine massively decreased basal [35S]GTP em /em S beyond basal levels (Fig.?S1). Cyclopamine thus acted as an inverse agonist at the G\protein level, inhibiting constitutively active SMO (Riobo et?al. 2006; Shen et?al. 2013). Cyclopamine was defined as reference inverse agonist and included in each experiment (10? em /em M). Surprisingly, there was also a slight decrease of basal activity by another SMO agonist, SAG (Chen et?al. 2002), which thus seems to act as a protean agonist at SMO (Fig.?S1). In the pharmacological comparison, all tested SMO antagonists yielded CL2-SN-38 reductions in SMO constitutive activity (Table?1 and Fig.?1). Inhibitor pIC50 values of the compounds were comprised between 8.06 (MRT\83) and 6.08 (CUR\61414). In terms of efficacy, most antagonists decreased basal signaling similar to cyclopamine and can thus be considered as equally efficacious inverse agonists. The notable exceptions are PF\5274857 and the antifungal itraconazole (Table?1). It should be noted that inhibition concentrationCresponse curves of most compounds appeared biphasic and yielded slopes that were less than unity, indicating the possible implication of a two\site process (Fig.?1). This was however not observed for IPI\926 (Fig.?1), cyclopamine, CUR\61414, itraconazole and PF\5274857 (not shown). Open in a separate window Figure 1 Comparison of eight CL2-SN-38 selected smoothened antagonists in three in?vitro assays for smoothened activity. Figures show concentrationCresponse data of the indicated compounds in a [35S]GTP em /em S incorporation assay using SMO\CHO cell membranes (squares, [35S]GTP em /em S), a GLI1 mRNA quantification test using DAOY cells CL2-SN-38 (triangles, GLI1), and rat CGNP cell proliferation experiments (circles, cell proliferation). All figures show representative duplicate or quadruplicate (CGNP cell proliferation) experimental determinations, each repeated at least three times. Data were fitted by nonlinear regression, using GraphPad Prism software. Please note the different scaling for inverse agonist activity ([35S]GTP em /em S binding, right em y /em \axis) and antagonism against SHH\induced effects (left em y /em \axis, both other tests). The average pIC 50 data of all compounds tested are given in Table?1. Table 1 Activity of SMO inhibitors in different in?vitro assays thead valign=”top” th align=”left” rowspan=”2″ style=”border-bottom:solid 1px #000000″ valign=”top” colspan=”1″ SMO wt inhibition /th th align=”left” valign=”top” rowspan=”1″ colspan=”1″ [3H]Thymidine incorporation /th CL2-SN-38 th align=”left” valign=”top” rowspan=”1″ colspan=”1″ GLI1 mRNA qPCR /th th.