Structure-based discovery of opioid analgesics with reduced side effects.

Morphine is an alkaloid from the opium poppy used to treat pain. The potentially lethal side effects of morphine and related opioids-which include fatal respiratory depression-are thought to be mediated by µ-opioid-receptor (µOR) signalling through the ß-arrestin pathway or by actions at other receptors. Conversely, G-protein µOR signalling is thought to confer analgesia. Here we computationally dock over 3 million molecules against the µOR structure and identify new scaffolds unrelated to known opioids. Structure-based optimization yields PZM21-a potent Gi activator with exceptional selectivity for µOR and minimal ß-arrestin-2 recruitment. Unlike morphine, PZM21 is more efficacious for the affective component of analgesia versus the reflexive component and is devoid of both respiratory depression and morphine-like reinforcing activity in mice at equi-analgesic doses. PZM21 thus serves as both a probe to disentangle µOR signalling and a therapeutic lead that is devoid of many of the side effects of current opioids.

The Hairpin Form of r(G4C2)exp in c9ALS/FTD Is Repeat-Associated Non-ATG Translated and a Target for Bioactive Small Molecules.

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an expanded G4C2 repeat [(G4C2)exp] in C9ORF72. ALS/FTD-associated toxicity has been traced to the RNA transcribed from the repeat expansion [r(G4C2)exp], which sequesters RNA-binding proteins (RBPs) and undergoes repeat-associated non-ATG (RAN) translation to generate toxic dipeptide repeats. Using in vitro and cell-based assays, we identified a small molecule (4) that selectively bound r(G4C2)exp, prevented sequestration of an RBP, and inhibited RAN translation. Indeed, biophysical characterization showed that 4 selectively bound the hairpin form of r(G4C2)exp, and nuclear magnetic resonance spectroscopy studies and molecular dynamics simulations defined this molecular recognition event. Cellular imaging revealed that 4 localized to r(G4C2)exp cytoplasmic foci, the putative sites of RAN translation. Collectively, these studies highlight that the hairpin structure of r(G4C2)exp is a therapeutically relevant target and small molecules that bind it can ameliorate c9ALS/FTD-associated toxicity.

Development of Photoactivatable Allosteric Modulators for the Chemokine Receptor CXCR3.

The CXCR3 receptor, a class A G protein-coupled receptor (GPCR), is involved in the regulation and trafficking of various immune cells. CXCR3 antagonists have been proposed to be beneficial for the treatment of a wide range of disorders including but not limited to inflammatory and autoimmune diseases. The structure-based design of CXCR3 ligands remains, however, hampered by a lack of structural information describing in detail the interactions between an allosteric ligand and the receptor. We designed and synthesized photoactivatable probes for the structural and functional characterization, using photoaffinity labeling followed by mass spectrometry, of the CXCR3 allosteric binding pocket of AMG 487 and RAMX3, two potent and selective CXCR3 negative allosteric modulators. Photoaffinity labeling is a common approach to elucidate binding modes of small-molecule ligands of GPCRs through the aid of photoactivatable probes that convert to extremely reactive intermediates upon photolysis. The photolabile probe N-[({1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-{1-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl}piperidin-4-yl)methyl]acetamide (10) showed significant labeling of the CXCR3 receptor (80%) in a [(3) H]RAMX3 radioligand displacement assay. Compound 10 will serve as an important tool compound for the detailed investigation of the binding pocket of CXCR3 by mass spectrometry.

RNA Structures as Mediators of Neurological Diseases and as Drug Targets.

RNAs adopt diverse folded structures that are essential for function and thus play critical roles in cellular biology. A striking example of this is the ribosome, a complex, three-dimensionally folded macromolecular machine that orchestrates protein synthesis. Advances in RNA biochemistry, structural and molecular biology, and bioinformatics have revealed other non-coding RNAs whose functions are dictated by their structure. It is not surprising that aberrantly folded RNA structures contribute to disease. In this Review, we provide a brief introduction into RNA structural biology and then describe how RNA structures function in cells and cause or contribute to neurological disease. Finally, we highlight successful applications of rational design principles to provide chemical probes and lead compounds targeting structured RNAs. Based on several examples of well-characterized RNA-driven neurological disorders, we demonstrate how designed small molecules can facilitate the study of RNA dysfunction, elucidating previously unknown roles for RNA in disease, and provide lead therapeutics.

Identifying modulators of CXC receptors 3 and 4 with tailored selectivity using multi-target docking.

The G protein-coupled receptors of the C-X-C subfamily form a group among the chemokine receptors whose endogenous ligands are peptides with a common Cys-X-Cys motif. The CXC chemokine receptors 3 and 4 (CXCR3, CXCR4), which are investigated in this study, are linked to severe diseases such as cancer, multiple sclerosis, and HIV infections. Of particular interest, this receptor pair potentially forms a target for a polypharmacological drug treatment. Considering known ligands from public databases, such dual binders have not been identified yet. We therefore applied large-scale docking to the structure of CXCR4 and a homology model of CXCR3 with the goal to predict such dual binders, as well as compounds selective for either one of the receptors. Using signaling and biochemical assays, we showed that more than 50% of these predictions were correct in each category, yielding ligands with excellent binding efficiencies. These results highlight that docking is a suitable tool for the identification of ligands with tailored binding profiles to GPCRs, even when using homology models. More importantly, we present novel CXCR3-CXCR4 dual modulators that might pave the road to understanding the mechanisms of polypharmacological inhibition of these receptors.

Ligand-biased and probe-dependent modulation of chemokine receptor CXCR3 signaling by negative allosteric modulators.

Over the last decade, functional selectivity (or ligand bias) has evolved from being a peculiar phenomenon to being recognized as an essential feature of synthetic ligands that target G protein-coupled receptors (GPCRs). The CXC chemokine receptor 3 (CXCR3) is an outstanding platform to study various aspects of biased signaling, because nature itself uses functional selectivity to manipulate receptor signaling. At the same time, CXCR3 is an attractive therapeutic target in the treatment of autoimmune diseases and cancer. Herein we report the discovery of an 8-azaquinazolinone derivative (N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)methyl]butanamide, 1 b) that can inhibit CXC chemokine 11 (CXCL11)-dependent G protein activation over ß-arrestin recruitment with 187-fold selectivity. This compound also demonstrates probe-dependent activity, that is, it inhibits CXCL11- over CXCL10-mediated G protein activation with 12-fold selectivity. Together with a previously reported biased negative allosteric modulator from our group, the present study provides additional information on the molecular requirements for allosteric modulation of CXCR3.

Boronic acids as probes for investigation of allosteric modulation of the chemokine receptor CXCR3.

The chemokine receptor CXCR3 is a G protein-coupled receptor, which conveys extracellular signals into cells by changing its conformation upon agonist binding. To facilitate the mechanistic understanding of allosteric modulation of CXCR3, we combined computational modeling with the synthesis of novel chemical tools containing boronic acid moiety, site-directed mutagenesis, and detailed functional characterization. The design of boronic acid derivatives was based on the predictions from homology modeling and docking. The choice of the boronic acid moiety was dictated by its unique ability to interact with proteins in a reversible covalent way, thereby influencing conformational dynamics of target biomolecules. During the synthesis of the library we have developed a novel approach for the purification of drug-like boronic acids. To validate the predicted binding mode and to identify amino acid residues responsible for the transduction of signal through CXCR3, we conducted a site-directed mutagenesis study. With the use of allosteric radioligand RAMX3 we were able to establish the existence of a second allosteric binding pocket in CXCR3, which enables different binding modes of structurally closely related allosteric modulators of CXCR3. We have also identified residues Trp109(2.60) and Lys300(7.35) inside the transmembrane bundle of the receptor as crucial for the regulation of the G protein activation. Furthermore, we report the boronic acid 14 as the first biased negative allosteric modulator of the receptor. Overall, our data demonstrate that boronic acid derivatives represent an outstanding tool for determination of key receptor-ligand interactions and induction of ligand-biased signaling.

Synthesis and application of the first radioligand targeting the allosteric binding pocket of chemokine receptor CXCR3.

Strategies for the identification of allosteric modulators of chemokine receptors largely rely on various cell-based functional assays. Radioligand binding assays are typically not available for allosteric binding sites. We synthesized, purified, and applied the first tritium-labeled allosteric modulator of the human chemokine receptor CXCR3 (RAMX3, [(3) H]N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-[(1-methylpiperidin-4-yl)methyl]acetamide). RAMX3 is chemically derived from 8-azaquinazolinone-type allosteric modulators and binds to the CXCR3 receptor with a K(d) value of 1.08 nM (specific activity: 80.4 Ci mmol(-1) ). Radioligand displacement assays showed potent negative cooperativity between RAMX3 and chemokine CXCL11, providing a basis for the use of RAMX3 to investigate other potential allosteric modulators. Additionally, the synthesis and characterization of a number of other full and truncated 8-azaquinazoline analogues were used to validate the binding properties of RAMX3. We demonstrate that RAMX3 can be efficiently used to facilitate the discovery and characterization of small molecules as allosteric modulators of the CXCR3 receptor.