Characterization of Apo-Form Selective Inhibition of Indoleamine 2,3-Dioxygenase*.

Indoleamine-2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that catalyzes the rate-limiting step in the kynurenine pathway of tryptophan (TRP) metabolism. As it is an inflammation-induced immunoregulatory enzyme, pharmacological inhibition of IDO1 activity is currently being pursued as a potential therapeutic tool for the treatment of cancer and other disease states. As such, a detailed understanding of the mechanism of action of IDO1 inhibitors with various mechanisms of inhibition is of great interest. Comparison of an apo-form-binding IDO1 inhibitor (GSK5628) to the heme-coordinating compound, epacadostat (Incyte), allows us to explore the details of the apo-binding inhibition of IDO1. Herein, we demonstrate that GSK5628 inhibits IDO1 by competing with heme for binding to a heme-free conformation of the enzyme (apo-IDO1), whereas epacadostat coordinates its binding with the iron atom of the IDO1 heme cofactor. Comparison of these two compounds in cellular systems reveals a long-lasting inhibitory effect of GSK5628, previously undescribed for other known IDO1 inhibitors. Detailed characterization of this apo-binding mechanism for IDO1 inhibition might help design superior inhibitors or could confer a unique competitive advantage over other IDO1 inhibitors vis-à-vis specificity and pharmacokinetic parameters.

The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twist.

Inhibition of acetyl-CoA carboxylase (ACC) may prevent lipid-induced insulin resistance and type 2 diabetes, making the enzyme an attractive pharmaceutical target. Although the enzyme is highly conserved amongst animals, only the yeast enzyme structure is available for rational drug design. The use of biophysical assays has permitted the identification of a specific C-terminal truncation of the 826-residue human ACC2 carboxyl transferase (CT) domain that is both functionally competent to bind inhibitors and crystallizes in their presence. This C-terminal truncation led to the determination of the human ACC2 CT domain-CP-640186 complex crystal structure, which revealed distinctions from the yeast-enzyme complex. The human ACC2 CT-domain C-terminus is comprised of three intertwined alpha-helices that extend outwards from the enzyme on the opposite side to the ligand-binding site. Differences in the observed inhibitor conformation between the yeast and human structures are caused by differing residues in the binding pocket.

Helix 1/8 interactions influence the activity of nuclear receptor ligand-binding domains.

The ligand-binding domain (LBD) of apo-nuclear receptors in solution is thought to be a very dynamic structure with many possible conformations. Upon ligand binding, the structure is stabilized to a more rigid conformation. The dynamic stabilization assay is a LBD reassembly assay that takes advantage of the high specificity of the intramolecular interactions that comprise the ligand-bound LBD. Here, we demonstrate dynamic stabilization for the nuclear receptors peroxisome proliferator-activated receptor (PPAR)gamma and nerve growth factor inducible (NGFIB)beta and identify residues important for stabilization of the intramolecular interactions induced by PPARgamma ligands. Site-directed mutagenesis studies identified residues in helices 1 and 8 required for LBD reassembly. Further, disrupting the helix 1/8 interaction in the context of the holo-LBD alters the response of the receptor in a compound-specific manner, suggesting that residues far from the ligand-binding pocket can influence the stability of the ligand-bound receptor. Thus, these results support and extend models of the apo-LBD of PPARgamma as a dynamic structure.

Correlation between in vitro peptide binding profiles and cellular activities for estrogen receptor-modulating compounds.

Numerous biochemical and structural studies have shown that the conformation of the estrogen receptor alpha (ERalpha) can be influenced by ligand binding. In turn, the conformational state of ERalpha affects the ability of the receptor to interact with a wide variety of protein accessory factors. To globally investigate ligand-based cofactor recruitment activities of ERalpha, we have applied a flow cytometric multiplexed binding assay to determine the simultaneous binding of ERalpha to over 50 different peptides derived from both known cofactor proteins and random peptide phage display. Using over 400 ERalpha-binding compounds, we have observed that the multiplexed in vitro peptide-binding profiles are distinct for a number of compounds and that these profiles can predict the effect that ERalpha ligands have on various cellular activities. These cell-based activities include transcriptional regulation at an estrogen response element, MCF-7 cell proliferation, and Ishikawa endometrial cell stimulation. The majority of the compound-induced diversity in the peptide profiling assay is provided by the unique phage display peptides. Importantly, some of these peptides show a sequence relationship with the corepressor motif, suggesting that peptides identified via phage display might represent natural binding partners of ERalpha. These in vitro:cellular correlations may in part explain tissue-specific activities of ERalpha-modulating compounds.

A new series of estrogen receptor modulators that display selectivity for estrogen receptor beta.

A series of 1,3,5-triazine-based estrogen receptor (ER) modulators that are modestly selective for the ERbeta subtype are reported. Compound 1, which displayed modest potency and selectivity for ERbeta vs ERalpha, was identified via high-throughput screening utilizing an ERbeta SPA-based binding assay. Subsequent analogue preparation resulted in the identification of compounds such as 21 and 43 that display 25- to 30-fold selectivity for ERbeta with potencies in the 10-30 nM range. These compounds profile as full antagonists at ERbeta and weak partial agonists at ERalpha in a cell-based reporter gene assay. In addition, the X-ray crystal structure of compound 15 complexed with the ligand binding domain of ERbeta has been solved and was utilized in the design of more conformationally restrained analogues such as 31 in an attempt to increase selectivity for the ERbeta subtype.

Effect of microsphere binding site density on the apparent affinity of an interaction partner.

BACKGROUND: Flow cytometric microsphere-based binding assays can be used to measure molecular interactions with high sensitivity. We have used multiplexed microsphere technology to explore the effect that binding site density has on the apparent affinity of a soluble interaction partner. METHODS: The interaction of a nuclear receptor, peroxisome proliferator-activated receptor gamma ligand binding domain (PPARgamma LBD), with a synthetic peptide derived from a nuclear receptor coactivator protein, PPARgamma coactivator-1 alpha (PGC-1alpha), is the interacting system being studied. The density of this peptide coupled to fluorescently unique microsphere populations is varied by co-incubating the biotinylated peptide and avidin-coated microsphere populations with increasing the amounts of free D-biotin. The discrete-density peptide-coupled microsphere populations are combined to conduct a multiplexed binding experiment with Alexa 532-labeled PPARgamma LBD, in the absence or presence of a small molecule ligand. RESULTS: As the immobilized binding site density of PGC-1alpha peptide on fluorescent microspheres is increased the measured apparent affinity for PPARgamma LBD is increased. CONCLUSIONS: The density of binding sites immobilized to a surface has a pronounced effect on the apparent affinity for soluble binding partners. By controlling and varying the binding site density it is possible to increase the sensitivity of an interaction assay. In multiplexed assay formats it should be possible to normalize intrinsically unequal binding interactions by individually optimizing the binding site density of the immobilized interaction partner. However, to quantitatively measure intrinsic affinities of molecular interactions, low binding site densities are required and multivalent reagents must be avoided.

X-ray crystal structures of the estrogen-related receptor-gamma ligand binding domain in three functional states reveal the molecular basis of small molecule regulation.

X-ray crystal structures of the ligand binding domain (LBD) of the estrogen-related receptor-gamma (ERRgamma) were determined that describe this receptor in three distinct states: unliganded, inverse agonist bound, and agonist bound. Two structures were solved for the unliganded state, the ERRgamma LBD alone, and in complex with a coregulator peptide representing a portion of receptor interacting protein 140 (RIP140). No significant differences were seen between these structures that both exhibited the conformation of ERRgamma seen in studies with other coactivators. Two structures were obtained describing the inverse agonist-bound state, the ERRgamma LBD with 4-hydroxytamoxifen (4-OHT), and the ERRgamma LBD with 4-OHT and a peptide representing a portion of the silencing mediator of retinoid and thyroid hormone action protein (SMRT). The 4-OHT structure was similar to other reported inverse agonist bound structures, showing reorientation of phenylalanine 435 and a displacement of the AF-2 helix relative to the unliganded structures with little other rearrangement occurring. No significant changes to the LBD appear to be induced by peptide binding with the addition of the SMRT peptide to the ERRgamma plus 4-OHT complex. The observed agonist-bound state contains the ERRgamma LBD, a ligand (GSK4716), and the RIP140 peptide and reveals an unexpected rearrangement of the phenol-binding residues. Thermal stability studies show that agonist binding leads to global stabilization of the ligand binding domain. In contrast to the conventional mechanism of nuclear receptor ligand activation, activation of ERRgamma by GSK4716 does not appear to involve a major rearrangement or significant stabilization of the C-terminal helix.

Functional consequences of cysteine modification in the ligand binding sites of peroxisome proliferator activated receptors by GW9662.

In the course of a high throughput screen to search for ligands of peroxisome proliferator activated receptor-gamma (PPARgamma), we identified GW9662 using a competition binding assay against the human ligand binding domain. GW9662 had nanomolar IC(50) versus PPARgamma and was 10- and 600-fold less potent in binding experiments using PPARalpha and PPARdelta, respectively. Pretreatment of all three PPARs with GW9662 resulted in the irreversible loss of ligand binding as assessed by scintillation proximity assay. Incubation of PPAR with GW9662 resulted in a change in the absorbance spectra of the receptors consistent with covalent modification. Mass spectrometric analysis of the PPARgamma ligand binding domain treated with GW9662 established Cys(285) as the site of covalent modification. This cysteine is conserved among all three PPARs. In cell-based reporter assays, GW9662 was a potent and selective antagonist of full-length PPARgamma. The functional activity of GW9662 as an antagonist of PPARgamma was confirmed in an assay of adipocyte differentiation. GW9662 showed essentially no effect on transcription when tested using both full-length PPARdelta and PPARalpha. Time-resolved fluorescence assays of ligand-modulated receptor heterodimerization, coactivator binding, and corepressor binding were consistent with the effects observed in the reporter gene assays. Control activators increased PPAR:RXR heterodimer formation and coactivator binding to both PPARgamma and PPARdelta. Corepressor binding was decreased. In the case of PPARalpha, GW9662 treatment did not significantly increase heterodimerization and coactivator binding or decrease corepressor binding. The experimental data indicate that GW9662 modification of each of the three PPARs results in different functional consequences. The selective and irreversible nature of GW9662 treatment, and the observation that activity is maintained in cell culture experiments, suggests that this compound may be a useful tool for elucidation of the role of PPARgamma in biological processes.

Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition.

Transcriptional regulation by the glucocorticoid receptor (GR) is mediated by hormone binding, receptor dimerization, and coactivator recruitment. Here, we report the crystal structure of the human GR ligand binding domain (LBD) bound to dexamethasone and a coactivator motif derived from the transcriptional intermediary factor 2. Despite structural similarity to other steroid receptors, the GR LBD adopts a surprising dimer configuration involving formation of an intermolecular beta sheet. Functional studies demonstrate that the novel dimer interface is important for GR-mediated activation. The structure also reveals an additional charge clamp that determines the binding selectivity of a coactivator and a distinct ligand binding pocket that explains its selectivity for endogenous steroid hormones. These results establish a framework for understanding the roles of protein-hormone and protein-protein interactions in GR signaling pathways.