A natural-product switch for a dynamic protein interface

Small ligands are a powerful way to control the function of protein complexes via dynamic binding interfaces. The classic example is found in gene transcription where small ligands regulate nuclear receptor binding to coactivator proteins via the dynamic activation function 2 (AF2) interface. Current ligands target the ligand-binding pocket side of the AF2. Few ligands are known, which selectively target the coactivator side of the AF2, or which can be selectively switched from one side of the interface to the other. We use NMR spectroscopy and modeling to identify a natural product, which targets the retinoid X receptor (RXR) at both sides of the AF2. We then use chemical synthesis, cellular screening and X-ray co-crystallography to split this dual activity, leading to a potent and molecularly efficient RXR agonist, and a first-of-kind inhibitor selective for the RXR/coactivator interaction. Our findings justify future exploration of natural products at dynamic protein interfaces

Identification and development of biphenyl substituted iminosugars as improved dual glucosylceramide synthase/neutral glucosylceramidase inhibitors

This work details the evaluation of a number of N-alkylated deoxynojirimycin derivatives on their merits as dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. Building on our previous work, we synthesized a series of D-gluco and L-ido-configured iminosugars N-modified with a variety of hydrophobic functional groups. We found that iminosugars featuring N-pentyloxymethylaryl substituents are considerably more potent inhibitors of glucosylceramide synthase than their aliphatic counterparts. In a next optimization round, we explored a series of biphenyl-substituted iminosugars of both configurations (D-gluco and L-ido) with the aim to introduce structural features known to confer metabolic stability to drug-like molecules. From these series, two sets of molecules emerge as lead series for further profiling. Biphenyl-substituted L-ido-configured deoxynojirimycin derivatives are selective for glucosylceramidase and the nonlysosomal glucosylceramidase, and we consider these as leads for the treatment of neuropathological lysosomal storage disorders. Their D-gluco-counterparts are also potent inhibitors of intestinal glycosidases, and because of this characteristic, we regard these as the prime candidates for type 2 diabetes therapeutic

Org 214007-0: a novel non-steroidal selective glucocorticoid receptor modulator with full anti-inflammatory properties and improved therapeutic index

Contains fulltext : 103595.pdf (publisher's version ) (Open Access)Glucocorticoids (GCs) such as prednisolone are potent immunosuppressive drugs but suffer from severe adverse effects, including the induction of insulin resistance. Therefore, development of so-called Selective Glucocorticoid Receptor Modulators (SGRM) is highly desirable. Here we describe a non-steroidal Glucocorticoid Receptor (GR)-selective compound (Org 214007-0) with a binding affinity to GR similar to that of prednisolone. Structural modelling of the GR-Org 214007-0 binding site shows disturbance of the loop between helix 11 and helix 12 of GR, confirmed by partial recruitment of the TIF2-3 peptide. Using various cell lines and primary human cells, we show here that Org 214007-0 acts as a partial GC agonist, since it repressed inflammatory genes and was less effective in induction of metabolic genes. More importantly, in vivo studies in mice indicated that Org 214007-0 retained full efficacy in acute inflammation models as well as in a chronic collagen-induced arthritis (CIA) model. Gene expression profiling of muscle tissue derived from arthritic mice showed a partial activity of Org 214007-0 at an equi-efficacious dosage of prednisolone, with an increased ratio in repression versus induction of genes. Finally, in mice Org 214007-0 did not induce elevated fasting glucose nor the shift in glucose/glycogen balance in the liver seen with an equi-efficacious dose of prednisolone. All together, our data demonstrate that Org 214007-0 is a novel SGRMs with an improved therapeutic index compared to prednisolone. This class of SGRMs can contribute to effective anti-inflammatory therapy with a lower risk for metabolic side effects

Identification and Development of Biphenyl Substituted Iminosugars as Improved Dual Glucosylceramide Synthase/Neutral Glucosylceramidase Inhibitors

This work details the evaluation of a number of N-alkylated deoxynojirimycin derivatives on their merits as dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. Building on our previous work, we synthesized a series of d-<i>gluco</i> and l-<i>ido</i>-configured iminosugars N-modified with a variety of hydrophobic functional groups. We found that iminosugars featuring <i>N</i>-pentyloxy­methylaryl substituents are considerably more potent inhibitors of glucosylceramide synthase than their aliphatic counterparts. In a next optimization round, we explored a series of biphenyl-substituted iminosugars of both configurations (d-<i>gluco</i> and l-<i>ido</i>) with the aim to introduce structural features known to confer metabolic stability to drug-like molecules. From these series, two sets of molecules emerge as lead series for further profiling. Biphenyl-substituted l-<i>ido</i>-configured deoxynojirimycin derivatives are selective for glucosylceramidase and the nonlysosomal glucosylceramidase, and we consider these as leads for the treatment of neuropathological lysosomal storage disorders. Their d-<i>gluco</i>-counterparts are also potent inhibitors of intestinal glycosidases, and because of this characteristic, we regard these as the prime candidates for type 2 diabetes therapeutics

Org 214007-0 does not effect rates of hepatic enzyme fluxes.

<p>Mass Isotopomer Distribution Analysis (MIDA), as described in detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048385#s4" target="_blank">Materials and Methods</a>, was performed in mice treated p.o., once daily, for 7 days with either vehicle, prednisolone (10 mg/kg) or Org 214007-0 (1.5 mg/kg). These doses of each compound are equi-efficacious in suppression of CIA. Neither the glucose-6-phosphatase flux (A) nor the glycogen phosphorylase flux (B) were affected by treatment with prednisolone or Org 214007-0. The glucokinase flux rate (C) was not changed by Org 214007-0, but significantly differed from the effect by prednisolone (##: p = 0.01 <i>vs</i> prednisolone). The glycogen synthase flux rate (D) was significantly decreased by prednisolone (**: p = 0.005 <i>vs</i> vehicle), whereas Org 214007-0 had no significant effect on this flux, but differed significantly from prednisolone (##: p = 0.002 <i>vs</i> prednisolone). Neither Org 214007-0 nor prednisolone, at equi-efficacious dosages, effects the gluconeogenic flux (<i>de novo</i> synthesis of glucose-6-phophate) (E).</p

Org 214007-0 is equally effective as prednisolone in the mouse CIA model.

<p>A) Inhibition of arthritis in the CIA model. Mean clinical score of each group (n = 12), shown as area under the curve (AUC) of the arthritis score monitored every other day during 3 weeks, corrected for baseline, is indicated (± SEM). **  =  significantly different from placebo (p<0.01; ANOVA-test). B) Reduction of bone destruction in the CIA model as measured by X-ray. Mean radiological score (sum of the X-ray scores of left and right hindpaws and knees) of each group of mice (n = 12) at the end of the CIA experiment is indicated (± SEM). **  =  significantly different from placebo (p<0.01; ANOVA-test).</p

Structure and predicted binding mode of Org 214007-0.

<p>A) The structure of ORG 214007-0. This compound, [(-)-N-(2S,10S,14bS)]-N-(8-cyano-1,2,3,4,10,14b-hexahydro-10-methyl dibenzo<i>[c,f]</i>pyrido[1,2-<i>a</i>]azepin-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide] has a molecular weight of 430 g/mole (C₂₄H₂₃N₅OS) B) The predicted binding mode of Org 214007-0 modeled in complex with the glucocorticoid receptor and demonstrating conservation of interactions typical to steroidal glucocorticoids (Gln564, Asn570, Arg611 and Gln642).</p

Org 214007-0 behaves as a partial agonist in vitro.

<p>A) In a co-factor recruitment assay, Org 214007-0, in comparison to prednisolone, shows potent but partial recruitment of a 0.1 μM peptide presenting TIF2 -3. On the Y-axis average fluorescence counts (+/− SD) are shown. EC50 values (%CV) and percentages maximal efficacy (%CV) for Org 214007-0 versus prednisolone were 10 (7.4) nM versus 48 (3.2) nM and 67 (7.1) % vs 100% respectively. B) In THP1 cells Org 214007-0 shows partial induction of FKBP51 protein expression and C) under inflammatory conditions represses the IL-6 protein expression almost as good as prednisolone does.</p