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.

Anticancer opportunities at every stage of chemokine function.

The chemokine system, comprising 48 chemokines and 23 receptors, is critically involved in several hallmarks of cancer. Yet, despite extensive efforts from the pharmaceutical sector, only two drugs aimed at this system are currently approved for clinical use against cancer. To date, numerous pharmacological approaches have been developed to successfully intervene at different stages of chemokine function: (i) chemokine availability; (ii) chemokine-glycosaminoglycan binding; and (iii) chemokine receptor binding. Many of these strategies have been tested in preclinical cancer models, and some have advanced to clinical trials as potential anticancer therapies. Here we will review the strategies and growing pharmacological toolbox for manipulating the chemokine system in cancer, and address novel methods poised for future (pre)clinical testing.

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.

Synthesis and Pharmacological Evaluation of Triazolopyrimidinone Derivatives as Noncompetitive, Intracellular Antagonists for CC Chemokine Receptors 2 and 5.

CC chemokine receptors 2 (CCR2) and 5 (CCR5) are involved in many inflammatory diseases; however, most CCR2 and CCR5 clinical candidates have been unsuccessful. (Pre)clinical evidence suggests that dual CCR2/CCR5 inhibition might be more effective in the treatment of such multifactorial diseases. In this regard, the highly conserved intracellular binding site in chemokine receptors provides a new avenue for the design of multitarget ligands. In this study, we synthesized and evaluated the biological activity of a series of triazolopyrimidinone derivatives in CCR2 and CCR5. Radioligand binding assays first showed that they bind to the intracellular site of CCR2, and in combination with functional assays on CCR5, we explored structure-affinity/activity relationships in both receptors. Although most compounds were CCR2-selective, 39 and 43 inhibited ß-arrestin recruitment in CCR5 with high potency. Moreover, these compounds displayed an insurmountable mechanism of inhibition in both receptors, which holds promise for improved efficacy in inflammatory diseases.

Fosfomycin as a potential therapy for the treatment of systemic infections: a population pharmacokinetic model to simulate multiple dosing regimens.

Fosfomycin has emerged as a potential therapy for multidrug-resistant bacterial infections. In most European countries, the oral formulation is only approved as a 3 g single dose for treatment of uncomplicated cystitis. However, for the treatment of complicated systemic infections, this dose regimen is unlikely to reach efficacious serum and tissue concentrations. This study aims to investigate different fosfomycin-dosing regimens to evaluate its rationale for treatment of systemic infections. Serum concentration-time profiles of fosfomycin were simulated using a population pharmacokinetic model based on published pharmacokinetic parameter values, their uncertainty, inter-individual variability and covariates. The model was validated on published data and used to simulate a wide range of dosing regimens for oral and intravenous administration of fosfomycin. Finally, based on the minimum inhibitory concentration for E. coli, surrogate pharmacodynamic indices were calculated for each dosing regimen. This is the first population pharmacokinetic model to describe the oral pharmacokinetics of fosfomycin using data from different literature sources. The model and surrogate pharmacodynamic indices provide quantitative evidence that a dosing regimen of 6-12 g per day divided in 3 doses is required to obtain efficacious exposure and may serve as a first step in the treatment of systemic multi-drug-resistant bacterial infections.

Synthesis and biological evaluation of chemokine receptor ligands with 2-benzazepine scaffold.

Targeting CCR2 and CCR5 receptors is considered as promising concept for the development of novel antiinflammatory drugs. Herein, we present the development of the first probe-dependent positive allosteric modulator (PAM) of CCR5 receptors with a 2-benzazepine scaffold. Compound 14 (2-isobutyl-N-({[N-methyl-N-(tetrahydro-2H-pyran-4-yl)amino]methyl}phenyl)-1-oxo-2,3-dihydro-1H-2-benzazepine-4-carboxamide) activates the CCR5 receptor in a CCL4-dependent manner, but does not compete with [3H]TAK-779 binding at the CCR5. Furthermore, introduction of a p-tolyl moiety at 7-position of the 2-benzazepine scaffold turns the CCR5 PAM 14 into the selective CCR2 receptor antagonist 26b. The structure affinity and activity relationships presented here offer new insights into ligand recognition by CCR2 and CCR5 receptors.

Sodium ion binding pocket mutations and adenosine A2A receptor function.

Recently we identified a sodium ion binding pocket in a high-resolution structure of the human adenosine A2A receptor. In the present study we explored this binding site through site-directed mutagenesis and molecular dynamics simulations. Amino acids in the pocket were mutated to alanine, and their influence on agonist and antagonist affinity, allosterism by sodium ions and amilorides, and receptor functionality was explored. Mutation of the polar residues in the Na(+) pocket were shown to either abrogate (D52A(2.50) and N284A(7.49)) or reduce (S91A(3.39), W246A(6.48), and N280A(7.45)) the negative allosteric effect of sodium ions on agonist binding. Mutations D52A(2.50) and N284A(7.49) completely abolished receptor signaling, whereas mutations S91A(3.39) and N280A(7.45) elevated basal activity and mutations S91A(3.39), W246A(6.48), and N280A(7.45) decreased agonist-stimulated receptor signaling. In molecular dynamics simulations D52A(2.50) directly affected the mobility of sodium ions, which readily migrated to another pocket formed by Glu13(1.39) and His278(7.43). The D52A(2.50) mutation also decreased the potency of amiloride with respect to ligand displacement but did not change orthosteric ligand affinity. In contrast, W246A(6.48) increased some of the allosteric effects of sodium ions and amiloride, whereas orthosteric ligand binding was decreased. These new findings suggest that the sodium ion in the allosteric binding pocket not only impacts ligand affinity but also plays a vital role in receptor signaling. Because the sodium ion binding pocket is highly conserved in other class A G protein-coupled receptors, our findings may have a general relevance for these receptors and may guide the design of novel synthetic allosteric modulators or bitopic ligands.