Dataset Information

Quadruplex-Duplex Junction: A High-Affinity Binding Site for Indoloquinoline Ligands.

ABSTRACT: A parallel quadruplex derived from the Myc promoter sequence was extended by a stem-loop duplex at either its 5'- or 3'-terminus to mimic a quadruplex-duplex (Q-D) junction as a potential genomic target. High-resolution structures of the hybrids demonstrate continuous stacking of the duplex on the quadruplex core without significant perturbations. An indoloquinoline ligand carrying an aminoalkyl side chain was shown to bind the Q-D hybrids with a very high affinity in the order K_a ?10⁷ ?m^-1 irrespective of the duplex location at the quadruplex 3'- or 5'-end. NMR chemical shift perturbations identified the tetrad face of the Q-D junction as specific binding site for the ligand. However, calorimetric analyses revealed significant differences in the thermodynamic profiles upon binding to hybrids with either a duplex extension at the quadruplex 3'- or 5'-terminus. A large enthalpic gain and considerable hydrophobic effects are accompanied by the binding of one ligand to the 3'-Q-D junction, whereas non-hydrophobic entropic contributions favor binding with formation of a 2:1 ligand-quadruplex complex in case of the 5'-Q-D hybrid.

SUBMITTER: Vianney YM

PROVIDER: S-EPMC7756412 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Quadruplex-Duplex Junction: A High-Affinity Binding Site for Indoloquinoline Ligands.

Vianney Yoanes Maria YM Preckwinkel Pit P Mohr Swantje S Weisz Klaus K

Chemistry (Weinheim an der Bergstrasse, Germany) 20201116 70

A parallel quadruplex derived from the Myc promoter sequence was extended by a stem-loop duplex at either its 5'- or 3'-terminus to mimic a quadruplex-duplex (Q-D) junction as a potential genomic target. High-resolution structures of the hybrids demonstrate continuous stacking of the duplex on the quadruplex core without significant perturbations. An indoloquinoline ligand carrying an aminoalkyl side chain was shown to bind the Q-D hybrids with a very high affinity in the order K<sub>a</sub> ≈10 ...[more]

PMID: 32975874

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Project description:BackgroundPregnane X receptor (PXR) agonists inhibit liver fibrosis. However, the rodent PXR activator pregnenolone 16alpha carbonitrile (PCN) blocks, in vitro, hepatic stellate cell-to-myofibroblast trans-differentiation and proliferation in cells from mice with a disrupted PXR gene, suggesting there is an additional anti-fibrogenic drug target for PCN. The role of the low affinity glucocorticoid binding site (LAGS) - which may be identical or associated with the progesterone receptor membrane component 1 (PGRMC1) - in mediating this anti-fibrogenic effect has been examined, since binding of dexamethasone to the LAGS in liver microsomal membranes has previously been shown to be inhibited by PCN.ResultsQuiescent rat and human hepatic stellate cells (HSC) were isolated from livers and cultured to generate liver myofibroblasts. HSC and myofibroblasts expressed PGRMC1 as determined by RT-PCR and Western blotting. Quiescent rat HSC also expressed the truncated HC5 variant of rPGRMC1. Rat PGRMC1 was cloned and expression in COS-7 cells gave rise to specific binding of radiolabelled dexamethasone in cell extracts that was inhibited by PCN, suggesting that PGRMC1 may be identical to LAGS or activates LAGS binding activity. Liver microsomes were used to screen a range of structurally related compounds for their ability to inhibit radiolabelled dexamethasone binding to rat LAGS. These compounds were also screened for their ability to activate rat and human PXR and to inhibit rat HSC-to-myofibroblast trans-differentiation/proliferation. A compound (4 androstene-3-one 17beta-carboxylic acid methyl ester) was identified which bound rat LAGS with high affinity and inhibited both rat and human HSC trans-differentiation/proliferation to fibrogenic myofibroblasts without showing evidence of rat or human PXR agonism. However, despite potent anti-fibrogenic effects in vitro, this compound did not modulate liver fibrosis severity in a rat model of liver fibrosis. Immunohistochemical analysis showed that rat liver myofibroblasts in vivo did not express rPGRMC1.ConclusionLAGS ligands inhibit HSC trans-differentiation and proliferation in vitro but show little efficacy in inhibiting liver fibrosis, in vivo. The reason(s) for this disparity is/are likely associated with an altered myofibroblast phenotype, in vitro, with expression of rPGMRC1 in vitro but not in vivo. These data emphasize the limitations of in vitro-derived myofibroblasts for predicting their activity in vivo, in studies of fibrogenesis. The data also demonstrate that the anti-fibrogenic effects of PCN in vivo are likely mediated entirely via the PXR.

Dataset Information

Quadruplex-Duplex Junction: A High-Affinity Binding Site for Indoloquinoline Ligands.

Publications

Quadruplex-Duplex Junction: A High-Affinity Binding Site for Indoloquinoline Ligands.

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