An Fc-Small Molecule Conjugate for Targeted Inhibition of the Adenosine†2A Receptor.

The adenosine A2A receptor (A2A R) is expressed in immune cells, as well as brain and heart tissue, and has been intensively studied as a therapeutic target for multiple disease indications. Inhibitors of the A2A R have the potential for stimulating immune response, which could be valuable for cancer immune surveillance and mounting a response against pathogens. One well-established potent and selective small molecule A2A R antagonist, ZM-241385 (ZM), has a short pharmacokinetic half-life and the potential for systemic toxicity due to A2A R effects in the brain and the heart. In this study, we designed an analogue of ZM and tethered it to the Fc domain of the immunoglobulin IgG3 by using expressed protein ligation. The resulting protein-small molecule conjugate, Fc-ZM, retained high affinity for two Fc receptors: FcγRI and the neonatal Fc receptor, FcRn. In addition, Fc-ZM was a potent A2A R antagonist, as measured by a cell-based cAMP assay. Cell-based assays also revealed that Fc-ZM could stimulate interferon γ production in splenocytes in a fashion that was dependent on the presence of A2A R. We found that Fc-ZM, compared with the small molecule ZM, was a superior A2A R antagonist in mice, consistent with the possibility that Fc attachment can improve pharmacokinetic and/or pharmacodynamic properties of the small molecule.

Architectural organization of the metabolic regulatory enzyme ghrelin O-acyltransferase.

Ghrelin O-acyltransferase (GOAT) is a polytopic integral membrane protein required for activation of ghrelin, a secreted metabolism-regulating peptide hormone. Although GOAT is a potential therapeutic target for the treatment of obesity and diabetes and plays a key role in other physiologic processes, little is known about its structure or mechanism. GOAT is a member of the membrane-bound O-acyltransferase (MBOAT) family, a group of polytopic integral membrane proteins involved in lipid-biosynthetic and lipid-signaling reactions from prokaryotes to humans. Here we use phylogeny and a variety of bioinformatic tools to predict the topology of GOAT. Using selective permeabilization indirect immunofluorescence microscopy in combination with glycosylation shift immunoblotting, we demonstrate that GOAT contains 11 transmembrane helices and one reentrant loop. Development of the V5Glyc tag, a novel, small, and sensitive dual topology reporter, facilitated these experiments. The MBOAT family invariant residue His-338 is in the ER lumen, consistent with other family members, but conserved Asn-307 is cytosolic, making it unlikely that both are involved in catalysis. Photocross-linking of synthetic ghrelin analogs and inhibitors demonstrates binding to the C-terminal region of GOAT, consistent with a role of His-338 in the active site. This knowledge of GOAT architecture is important for a deeper understanding of the mechanism of GOAT and other MBOATs and could ultimately advance the discovery of selective inhibitors for these enzymes.

An Fc domain protein-small molecule conjugate as an enhanced immunomodulator.

Proteins as well as small molecules have demonstrated success as therapeutic agents, but their pharmacologic properties sometimes fall short against particular drug targets. Although the adenosine 2a receptor (A(2A)R) has been identified as a promising target for immunotherapy, small molecule A(2A)R agonists have suffered from short pharmacokinetic half-lives and the potential for toxicity by modulating nonimmune pathways. To overcome these limitations, we have tethered the A(2A)R agonist CGS-21680 to the immunoglobulin Fc domain using expressed protein ligation with Sf9 cell secreted protein. The protein small molecule conjugate Fc-CGS retained potent Fc receptor and A(2A)R interactions and showed superior properties as a therapeutic for the treatment of a mouse model of autoimmune pneumonitis. This approach may provide a general strategy for optimizing small molecule therapeutics.

Intersubject representational similarity analysis uncovers the impact of state anxiety on brain activation patterns in the human extrastriate cortex.

The current study used functional magnetic resonance imaging (fMRI) and showed that state anxiety modulated extrastriate cortex activity in response to emotionally-charged visual images. State anxiety and neuroimaging data from 53 individuals were subjected to an intersubject representational similarity analysis (ISRSA), wherein the geometries between neural and behavioral data were compared. This analysis identified the extrastriate cortex (fusiform gyrus and area MT) to be the sole regions whose activity patterns covaried with state anxiety. Importantly, we show that this brain-behavior association is revealed when treating state anxiety data as a multidimensional response pattern, rather than a single composite score. This suggests that ISRSA using multivariate distances may be more sensitive in identifying the shared geometries between self-report questionnaires and brain imaging data. Overall, our findings demonstrate that a transient state of anxiety may influence how visual information - especially those relevant to the valence dimension - is processed in the extrastriate cortex.

A selective phenelzine analogue inhibitor of histone demethylase LSD1.

Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme that oxidatively cleaves methyl groups from monomethyl and dimethyl Lys4 of histone H3 (H3K4Me1, H3K4Me2) and can contribute to gene silencing. This study describes the design and synthesis of analogues of a monoamine oxidase antidepressant, phenelzine, and their LSD1 inhibitory properties. A novel phenelzine analogue (bizine) containing a phenyl-butyrylamide appendage was shown to be a potent LSD1 inhibitor in vitro and was selective versus monoamine oxidases A/B and the LSD1 homologue, LSD2. Bizine was found to be effective at modulating bulk histone methylation in cancer cells, and ChIP-seq experiments revealed a statistically significant overlap in the H3K4 methylation pattern of genes affected by bizine and those altered in LSD1-/- cells. Treatment of two cancer cell lines, LNCaP and H460, with bizine conferred a reduction in proliferation rate, and bizine showed additive to synergistic effects on cell growth when used in combination with two out of five HDAC inhibitors tested. Moreover, neurons exposed to oxidative stress were protected by the presence of bizine, suggesting potential applications in neurodegenerative disease.

Ghrelin O-acyltransferase assays and inhibition.

Ghrelin O-acyltransferase (GOAT) is responsible for catalyzing the attachment of the eight-carbon fatty acid octanoyl to the Ser3 side chain of the peptide ghrelin to generate the active form of this metabolic hormone. As such, GOAT is viewed as a potential therapeutic target for the treatment of obesity and diabetes mellitus. Here, we review recent progress in the development of cell and in vitro assays to measure GOAT action and the identification of several synthetic GOAT inhibitors. In particular, we discuss the design, synthesis, and characterization of the bisubstrate analog GO-CoA-Tat and its ability to modulate weight and blood glucose in mice. We also highlight current challenges and future research directions in our biomedical understanding of this fascinating ghrelin processing enzyme.