An Isochroman Analog of CD3254 and Allyl-, Isochroman-Analogs of NEt-TMN Prove to Be More Potent Retinoid-X-Receptor (RXR) Selective Agonists Than Bexarotene.

Bexarotene is an FDA-approved drug for the treatment of cutaneous T-cell lymphoma (CTCL); however, its use provokes or disrupts other retinoid-X-receptor (RXR)-dependent nuclear receptor pathways and thereby incites side effects including hypothyroidism and raised triglycerides. Two novel bexarotene analogs, as well as three unique CD3254 analogs and thirteen novel NEt-TMN analogs, were synthesized and characterized for their ability to induce RXR agonism in comparison to bexarotene (). Several analogs in all three groups possessed an isochroman ring substitution for the bexarotene aliphatic group.

Modeling, Synthesis, and Biological Evaluation of Potential Retinoid-X-Receptor (RXR) Selective Agonists: Analogs of 4-[1-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahyro-2-naphthyl)ethynyl]benzoic Acid (Bexarotene) and 6-(Ethyl(4-isobutoxy-3-isopropylphenyl)amino)nicotinic Acid (NEt-4IB).

Five novel analogs of 6-(ethyl)(4-isobutoxy-3-isopropylphenyl)amino)nicotinic acid-or NEt-4IB-in addition to seven novel analogs of 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl]benzoic acid (bexarotene) were prepared and evaluated for selective retinoid-X-receptor (RXR) agonism alongside bexarotene (), a FDA-approved drug for cutaneous T-cell lymphoma (CTCL). Bexarotene treatment elicits side-effects by provoking or disrupting other RXR-dependent pathways. Analogs were assessed by the modeling of binding to RXR and then evaluated in a human cell-based RXR-RXR mammalian-2-hybrid (M2H) system as well as a RXRE-controlled transcriptional system.

Evidence that specific interactions play a role in the cholesterol sensitivity of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol's general effects on the membrane is not well-understood. We performed coarse-grained molecular dynamics (CGMD) simulations coupled with structural bioinformatics approaches on the β-adrenergic receptor (βAR) and the cholecystokinin (CCK) receptor subfamily. The βAR has been shown to be sensitive to membrane cholesterol and cholesterol molecules have been clearly resolved in numerous βAR crystal structures.

Harnessing the power of an X-ray laser for serial crystallography of membrane proteins crystallized in lipidic cubic phase.

Serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase; however, like most techniques, it has limitations. Here we attempt to address some of these limitations related to the use of a vacuum chamber and the need for attenuation of the XFEL beam, in order to further improve the efficiency of this method. Using an optimized SFX experimental setup in a helium atmosphere, the room-temperature structure of the adenosine A receptor (AAR) at 2.

Structural Basis of the Activation of Heterotrimeric Gs-Protein by Isoproterenol-Bound β-Adrenergic Receptor.

Cardiac disease remains the leading cause of morbidity and mortality worldwide. The β-adrenergic receptor (β-AR) is a major regulator of cardiac functions and is downregulated in the majority of heart failure cases. A key physiological process is the activation of heterotrimeric G-protein Gs by β-ARs, leading to increased heart rate and contractility.

Structure Determination from Lipidic Cubic Phase Embedded Microcrystals by MicroED.

The lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. However, the crystal size optimization process could be time and resource consuming, if it ever happens. Therefore, improved techniques for structure determination using these small crystals is an important strategy in diffraction technology development.

High-viscosity injector-based pink-beam serial crystallography of microcrystals at a synchrotron radiation source.

Since the first successful serial crystallography (SX) experiment at a synchrotron radiation source, the popularity of this approach has continued to grow showing that third-generation synchrotrons can be viable alternatives to scarce X-ray free-electron laser sources. Synchrotron radiation flux may be increased ∼100 times by a moderate increase in the bandwidth ('pink beam' conditions) at some cost to data analysis complexity. Here, we report the first high-viscosity injector-based pink-beam SX experiments.

Overview of Biochemical Assays in Lipidic Cubic Phase.

The development of novel biochemical methods to efficiently characterize membrane protein (MP) properties in lipidic cubic phase (LCP) is important for studying complicated MPs and their multimeric complexes. Here, we summarize recent LCP-related assays and provide an outlook on their applications in structure and function studies of MPs.

Structure of the Nanobody-Stabilized Active State of the Kappa Opioid Receptor.

The κ-opioid receptor (KOP) mediates the actions of opioids with hallucinogenic, dysphoric, and analgesic activities. The design of KOP analgesics devoid of hallucinatory and dysphoric effects has been hindered by an incomplete structural and mechanistic understanding of KOP agonist actions. Here, we provide a crystal structure of human KOP in complex with the potent epoxymorphinan opioid agonist MP1104 and an active-state-stabilizing nanobody.

High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions.

Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20-25°C) and under cryogenic conditions.

Allosteric Coupling of Drug Binding and Intracellular Signaling in the A Adenosine Receptor.

Signaling across cellular membranes, the 826 human G protein-coupled receptors (GPCRs) govern a wide range of vital physiological processes, making GPCRs prominent drug targets. X-ray crystallography provided GPCR molecular architectures, which also revealed the need for additional structural dynamics data to support drug development. Here, nuclear magnetic resonance (NMR) spectroscopy with the wild-type-like A adenosine receptor (AAR) in solution provides a comprehensive characterization of signaling-related structural dynamics.

Native phasing of x-ray free-electron laser data for a G protein-coupled receptor.

Serial femtosecond crystallography (SFX) takes advantage of extremely bright and ultrashort pulses produced by x-ray free-electron lasers (XFELs), allowing for the collection of high-resolution diffraction intensities from micrometer-sized crystals at room temperature with minimal radiation damage, using the principle of "diffraction-before-destruction." However, de novo structure factor phase determination using XFELs has been difficult so far. We demonstrate the ability to solve the crystallographic phase problem for SFX data collected with an XFEL using the anomalous signal from native sulfur atoms, leading to a bias-free room temperature structure of the human A adenosine receptor at 1.

Insight into the molecular basis of aromatic polyketide cyclization: crystal structure and in vitro characterization of WhiE-ORFVI.

Aromatic polyketides are biologically active natural products. Many important pharmaceuticals are derived from aromatic polyketides. Especially important in aromatic polyketide biosynthesis is the regiospecific cyclization of a linear, preassembled polyketide chain catalyzed by aromatase/cyclase (ARO/CYC), which serves as a key control point in aromatic ring formation.

Structural and biochemical characterization of ZhuI aromatase/cyclase from the R1128 polyketide pathway.

Aromatic polyketides comprise an important class of natural products that possess a wide range of biological activities. The cyclization of the polyketide chain is a critical control point in the biosynthesis of aromatic polyketides. The aromatase/cyclases (ARO/CYCs) are an important component of the type II polyketide synthase (PKS) and help fold the polyketide for regiospecific cyclizations of the first ring and/or aromatization, promoting two commonly observed first-ring cyclization patterns for the bacterial type II PKSs: C7-C12 and C9-C14.

Differential activation of a C/EBP beta isoform by a novel redox switch may confer the lipopolysaccharide-inducible expression of interleukin-6 gene.

C/EBP beta, a member of the CCAAT/enhancer binding protein (C/EBP) family, is one of the key transcription factors responsible for the induction of a wide array of genes, some of which play important roles in innate immunity, inflammatory response, adipocyte and myeloid cell differentiation, and the acute phase response. Three C/EBP beta isoforms (i.e.