Highly Selective Drug-Derived Fluorescent Probes for the Cannabinoid Receptor Type 1 (CB1R).

The cannabinoid receptor type 1 (CB1R) is pivotal within the endocannabinoid system regulating various signaling cascades with effects in appetite regulation, pain perception, memory formation, and thermoregulation. Still, understanding of CB1R's cellular signaling, distribution, and expression dynamics is very fragmentary. Real-time visualization of CB1R is crucial for addressing these questions. Selective drug-like CB1R ligands with a defined pharmacological profile were investigated for the construction of CB1R fluorescent probes using a reverse design-approach. A modular design concept with a diethyl glycine-based building block as the centerpiece allowed for the straightforward synthesis of novel probe candidates. Validated by computational docking studies, radioligand binding, and cAMP assay, this systematic approach allowed for the identification of novel pyrrole-based CB1R fluorescent probes. Application in fluorescence-based target-engagement studies and live cell imaging exemplify the great versatility of the tailored CB1R probes for investigating CB1R localization, trafficking, pharmacology, and its pathological implications.

Dual allosteric and orthosteric pharmacology of synthetic analog cannabidiol-dimethylheptyl, but not cannabidiol, on the cannabinoid CB2 receptor.

Cannabinoid CB2 receptor (CB2R) is a class A G protein-coupled receptor (GPCR) involved in a broad spectrum of physiological processes and pathological conditions. For that reason, targeting CB2R might provide therapeutic opportunities in neurodegenerative disorders, neuropathic pain, inflammatory diseases, and cancer. The main components from Cannabis sativa, such as Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), have been therapeutically exploited and synthetically-derived analogs have been generated. One example is cannabidiol-dimethylheptyl (CBD-DMH), which exhibits anti-inflammatory effects. Nevertheless, its pharmacological mechanism of action is not yet fully understood and is hypothesized for multiple targets, including CB2R. The aim of this study was to further investigate the molecular pharmacology of CBD-DMH on CB2R while CBD was taken along as control. These compounds were screened in equilibrium and kinetic radioligand binding studies and various functional assays, including G protein activation, inhibition of cAMP production and ß-arrestin-2 recruitment. In dissociation studies, CBD-DMH allosterically modulated the radioligand binding. Furthermore, CBD-DMH negatively modulated the G protein activation of reference agonists CP55,940, AEA and 2-AG, but not the agonist-induced ß-arrestin-2 recruitment. Nevertheless, CBD-DMH also displayed competitive binding to CB2R and partial agonism on G protein activation, inhibition of cAMP production and ß-arrestin-2 recruitment. CBD did not exhibit such allosteric behavior and only very weakly bound CB2R without activation. This study shows a dual binding mode of CBD-DMH, but not CBD, to CB2R with the suggestion of two different binding sites. Altogether, it encourages further research into this dual mechanism which might provide a new class of molecules targeting CB2R.

Piperazine squaric acid diamides, a novel class of allosteric P2X7 receptor antagonists.

The P2X7 receptor (P2X7R) stands out among the purinergic receptors due to its strong involvement in the regulation of tumor growth and metastasis formation as well as in innate immune responses and afferent signal transmission. Numerous studies have pointed out the beneficial effects of P2X7R antagonism for the treatment of a variety of cancer types, inflammatory diseases, and chronic pain. Herein we describe the development of novel P2X7R antagonists, incorporating piperazine squaric diamides as a central element. Besides improving the antagonists' potency from pIC50 values of 5.7-7.6, ADME properties (logD7.4 value, plasma protein binding, in vitro metabolic stability) of the generated compounds were investigated and optimized to provide novel P2X7R antagonists with drug-like properties. Furthermore, docking studies revealed the antagonists binding to the allosteric binding pocket in two distinct binding poses, depending on the substitution of the central piperazine moiety.

Design and Characterization of an Intracellular Covalent Ligand for CC Chemokine Receptor 2.

Covalently acting inhibitors constitute a large and growing fraction of approved small-molecule therapeutics as well as useful tools for a variety of in vitro and in vivo applications. Here, we aimed to develop a covalent antagonist of CC chemokine receptor 2 (CCR2), a class A GPCR that has been pursued as a therapeutic target in inflammation and immuno-oncology. Based on a known intracellularly binding CCR2 antagonist, several covalent derivatives were synthesized and characterized by radioligand binding and functional assays. These studies revealed compound 14 as an intracellular covalent ligand for CCR2. In silico modeling followed by site-directed mutagenesis confirmed that 14 forms a covalent bond with one of three proximal cysteine residues, which can be engaged interchangeably. To our knowledge, compound 14 represents the first covalent ligand reported for CCR2. Due to its unique properties, it may represent a promising tool for ongoing and future studies of CCR2 pharmacology.

High-titer production of lathyrane diterpenoids from sugar by engineered Saccharomyces cerevisiae.

Euphorbiaceae are an important source of medically important diterpenoids, such as the anticancer drug ingenol-3-angelate and the antiretroviral drug prostratin. However, extraction from the genetically intractable natural producers is often limited by the small quantities produced, while the organic synthesis of terpene-derived drugs is challenging and similarly low-yielding. While transplanting the biosynthetic pathway into a heterologous host has proven successful for some drugs, it has been largely unsuccessful for diterpenoids due to their elaborate biosynthetic pathways and lack of genetic resources and tools for gene discovery. We engineered casbene precursor production in S. cerevisiae, verified the ability of six Euphorbia lathyris and Jatropha curcas cytochrome P450s to oxidize casbene, and optimized the expression of these P450s and an alcohol dehydrogenase to generate jolkinol C, achieving ~800mg/L of jolkinol C and over 1g/L total oxidized casbanes in millititer plates, the highest titer of oxidized diterpenes in yeast reported to date. This strain enables the semisynthesis of biologically active jolkinol C derivatives and will be an important tool in the elucidation of the biosynthetic pathways for ingenanes, tiglianes, and lathyranes. These findings demonstrate the ability of S. cerevisiae to produce oxidized drug precursors in quantities that are sufficient for drug development and pathway discovery.