Proximity-inducing modalities: the past, present, and future.

Living systems use proximity to regulate biochemical processes. Inspired by this phenomenon, bifunctional modalities that induce proximity have been developed to redirect cellular processes. An emerging example of this class is molecules that induce ubiquitin-dependent proteasomal degradation of a protein of interest, and their initial development sparked a flurry of discovery for other bifunctional modalities. Recent advances in this area include modalities that can change protein phosphorylation, glycosylation, and acetylation states, modulate gene expression, and recruit components of the immune system. In this review, we highlight bifunctional modalities that perform functions other than degradation and have great potential to revolutionize disease treatment, while also serving as important tools in basic research to explore new aspects of biology.

Novel Cannabinoid Receptor 2 (CB2) Low Lipophilicity Agonists Produce Distinct cAMP and Arrestin Signalling Kinetics without Bias.

Cannabinoid Receptor 2 (CB2) is a promising target for treating inflammatory diseases. We designed derivatives of 3-carbamoyl-2-pyridone and 1,8-naphthyridin-2(1H)-one-3-carboxamide CB2-selective agonists with reduced lipophilicity. The new compounds were measured for their affinity (radioligand binding) and ability to elicit cyclic adenosine monophosphate (cAMP) signalling and ß-arrestin-2 translocation with temporal resolution (BRET-based biosensors). For the 3-carbamoyl-2-pyridone derivatives, we found that modifying the previously reported compound UOSS77 (also known as S-777469) by appending a PEG2-alcohol via a 3-carbomylcyclohexyl carboxamide (UOSS75) lowered lipophilicity, and preserved binding affinity and signalling profile. The 1,8-naphthyridin-2(1H)-one-3-carboxamide UOMM18, containing a cis configuration at the 3-carboxamide cyclohexyl and with an alcohol on the 4-position of the cyclohexyl, had lower lipophilicity but similar CB2 affinity and biological activity to previously reported compounds of this class. Relative to CP55,940, the new compounds acted as partial agonists and did not exhibit signalling bias. Interestingly, while all compounds shared similar temporal trajectories for maximal efficacy, differing temporal trajectories for potency were observed. Consequently, when applied at sub-maximal concentrations, CP55,940 tended to elicit sustained (cAMP) or increasing (arrestin) responses, whereas responses to the new compounds tended to be transient (cAMP) or sustained (arrestin). In future studies, the compounds characterised here may be useful in elucidating the consequences of differential temporal signalling profiles on CB2-mediated physiological responses.

Chemical Tools for Studying Lipid-Binding Class A G Protein-Coupled Receptors.

Cannabinoid, free fatty acid, lysophosphatidic acid, sphingosine 1-phosphate, prostanoid, leukotriene, bile acid, and platelet-activating factor receptor families are class A G protein-coupled receptors with endogenous lipid ligands. Pharmacological tools are crucial for studying these receptors and addressing the many unanswered questions surrounding expression of these receptors in normal and diseased tissues. An inherent challenge for developing tools for these lipid receptors is balancing the often lipophilic requirements of the receptor-binding pharmacophore with favorable physicochemical properties to optimize highly specific binding. In this study, we review the radioligands, fluorescent ligands, covalent ligands, and antibodies that have been used to study these lipid-binding receptors. For each tool type, the characteristics and design rationale along with in vitro and in vivo applications are detailed.

Development and Applications of Chimera Platforms for Tyrosine Phosphorylation.

Chimeric small molecules that induce post-translational modification (PTM) on a target protein by bringing it into proximity to a PTM-inducing enzyme are furnishing novel modalities to perturb protein function. Despite recent advances, such molecules are unavailable for a critical PTM, tyrosine phosphorylation. Furthermore, the contemporary design paradigm of chimeric molecules, formed by joining a noninhibitory binder of the PTM-inducing enzyme with the binder of the target protein, prohibits the recruitment of most PTM-inducing enzymes as their noninhibitory binders are unavailable. Here, we report two platforms to generate phosphorylation-inducing chimeric small molecules (PHICS) for tyrosine phosphorylation. We generate PHICS from both noninhibitory binders (scantily available, platform 1) and kinase inhibitors (abundantly available, platform 2) using cysteine-based group transfer chemistry. PHICS triggered phosphorylation on tyrosine residues in diverse sequence contexts and target proteins (e.g., membrane-associated, cytosolic) and displayed multiple bioactivities, including the initiation of a growth receptor signaling cascade and the death of drug-resistant cancer cells. These studies provide an approach to induce biologically relevant PTM and lay the foundation for pharmacologic PTM editing (i.e., induction or removal) of target proteins using abundantly available inhibitors of PTM-inducing or -erasing enzymes.

Chromenopyrazole-based High Affinity, Selective Fluorescent Ligands for Cannabinoid Type 2 Receptor.

Cannabinoid type 2 receptor (CB2R) is an attractive target for the treatment of pain and inflammatory disorders. Availability of a selective CB2R fluorescent ligand to study CB2R expression and localization in healthy and disease conditions would greatly contribute to improving our understanding of this receptor. Herein, we report a series of chromenopyrazole-based CB2R fluorescent ligands. The highest affinity fluorescent ligand was Cy5-containing 24 (hCB2R pK i = 7.38 ± 0.05), which had 131-fold selectivity over CB1R. In a cAMP BRET assay, 24 behaved as a potent CB2R inverse agonist. Widefield imaging experiments showed that 24 binds to CB2R in live cells with good selectivity and low levels of nonspecific fluorescence. The high affinity, selectivity, and suitable imaging properties of fluorescent ligand 24 make it a valuable tool for studying CB2R.

Synthesis of novel (benzimidazolyl)isoquinolinols and evaluation as adenosine A1 receptor tools.

G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane receptors in eukaryotes. The adenosine A1 receptor (A1AR) is a class A GPCR that is of interest as a therapeutic target particularly in the treatment of cardiovascular disease and neuropathic pain. Increased knowledge of the role A1AR plays in mediating these pathophysiological processes will help realise the therapeutic potential of this receptor. There is a lack of enabling tools such as selective fluorescent probes to study A1AR, therefore we designed a series of (benzimidazolyl)isoquinolinols conjugated to a fluorescent dye (31-35, 42-43). An improved procedure for the synthesis of isoquinolinols from tetrahydroisoquinolinols via oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and atmospheric oxygen is reported. This synthetic method offers advantages over previous metal-based methods for the preparation of isoquinolinols and isoquinolines, which are important scaffolds found in many biologically active compounds and natural products. We report the first synthesis of the (benzimidazolyl)isoquinolinol compound class, however the fluorescent conjugates were not successful as A1AR fluorescent ligands.

Development of selective, fluorescent cannabinoid type 2 receptor ligands based on a 1,8-naphthyridin-2-(1H)-one-3-carboxamide scaffold.

Cannabinoid type 2 (CB2) receptor has been implicated in several diseases and conditions, however no CB2 receptor selective drugs have made it to market. The aim of this study was to develop fluorescent ligands as CB2 receptor tools, to enable an increased understanding of CB2 receptor expression and signalling and thereby accelerate drug discovery. Fluorescent ligands have been successfully developed for other receptors, however none with adequate subtype selectivity or imaging properties have been reported for CB2 receptor. A series of 1,8-naphthyridin-2-(1H)-one-3-carboxamides with linkers and fluorophores appended in the N1 and C3-positions were developed. Molecular modelling indicated the C3 cis-cyclohexanol-linked compounds directed the linker out of the CB2 receptor between transmembrane helices 1 and 7. Herein we report fluorescent ligand 32 (hCB2 pK i = 6.33 ± 0.02) as one of the highest affinity, selective CB2 receptor fluorescent ligands reported. Despite 32 displaying poor specific labelling of CB2 receptor, the naphthyridine scaffold with this linker remains highly promising for future development of CB2 receptor tools.