Effects of Purinergic Receptor Deletion or Pharmacologic Modulation on Pulmonary Inflammation in Mice.

COVID-19 disease is associated with progressive accumulation of SARS-CoV-2-specific mRNA, which is recognized by innate immune receptors, such as TLR3. This in turn leads to dysregulated production of multiple cytokines, including IL-6, IFN-γ, CXCL1, and TNF-α. Excessive production of these cytokines leads to acute lung injury (ALI), which consequently compromises alveolar exchange of O2 and CO2. It is therefore of considerable interest to develop novel therapies that reduce pulmonary inflammation and stem production of pro-inflammatory cytokines, potentially for COVID-19 patients that are at high risk of developing severe disease. Purinergic signaling has a central role in fine-tuning the innate immune system, with P2 (nucleotide) receptor antagonists and adenosine receptor agonists having anti-inflammatory effects. Accordingly, we focused here on the potential role of purinergic receptors in driving neutrophilic inflammation and cytokine production in a mouse model of pulmonary inflammation. To mimic the effects of SARS-CoV-2-specific RNA accumulation in mice, we administered progressively increasing daily doses of a viral mimetic, polyinosinic:polycytidylic acid [poly(I:C)] into the airways of mice over the course of 1 week. Some mice also received increasing daily doses of ovalbumin to mimic virus-encoded protein accumulation. Animals receiving both poly(I:C) and ovalbumin displayed particularly high cytokine levels and neutrophilia, suggestive of both innate and antigen-specific, adaptive immune responses. The extent of these responses was diminished by genetic deletion (P2Y14R, P2X7R) or pharmacologic modulation (P2Y14R antagonists, A3AR agonists) of purinergic receptors. These results suggest that pharmacologic modulation of select purinergic receptors might be therapeutically useful in treating COVID-19 and other pulmonary infections.

Synthesis and pharmacological characterization of multiply substituted 2H-chromene derivatives as P2Y6 receptor antagonists.

P2Y6 receptor (P2Y6R) antagonists represent potential drugs for treating cancer, pain, neurodegeneration, asthma, diabetes, colitis and other disorders. However, there are few chemical classes of known competitive antagonists. We recently explored the structure activity relationship (SAR) of 2H-chromene derivatives as P2Y6R antagonists of moderate affinity. New analogues in this series modified at five positions were synthesized and shown to antagonize Ca2+ transients induced by the native agonist UDP in human (h) P2Y6R-expressing (but not turkey P2Y1R-, hP2Y2R- or hP2Y4R-expressing) astrocytoma cells. Alternatives to the reported 2-(trifluoromethyl)- and 3-nitro- substitutions of this scaffold were not identified. However, 6­fluoro 11 and 6­chloro 12 analogues displayed enhanced potency compared to other halogens, although still in the 1 - 2 µM range. Similar halogen substitution at 5, 7 or 8 positions reduced affinity. 5- or 8­Triethylsilylethynyl extension maintained hP2Y6R affinity, with IC50 0.46 µM for 26 (MRS4853). The 6,8­difluoro analogue 27 (IC50 2.99 µM) lacked off-target activities among 45 sites examined, unlike earlier analogues that bound to biogenic amine receptors. 11 displayed only one weak off-target activity (σ2). Mouse P2Y6R IC50s of 5, 25, 26 and 27 were 4.94, 17.6, 6.15 and 17.8 µM, respectively, but most other analogues had reduced affinity (>20 µM) compared to the hP2Y6R. These analogues are suitable for evaluation in in vivo inflammation and cancer models, which will be performed in the future studies.

Adipocyte P2Y14 receptors play a key role in regulating whole-body glucose and lipid homeostasis.

Obesity is the major driver of the worldwide epidemic in type 2 diabetes (T2D). In the obese state, chronically elevated plasma free fatty acid levels contribute to peripheral insulin resistance, which can ultimately lead to the development of T2D. For this reason, drugs that are able to regulate lipolytic processes in adipocytes are predicted to have considerable therapeutic potential. Gi-coupled P2Y14 receptor (P2Y14R; endogenous agonist, UDP-glucose) is abundantly expressed in both mouse and human adipocytes. Because activated Gi-type G proteins exert an antilipolytic effect, we explored the potential physiological relevance of adipocyte P2Y14Rs in regulating lipid and glucose homeostasis. Metabolic studies indicate that the lack of adipocyte P2Y14R enhanced lipolysis only in the fasting state, decreased body weight, and improved glucose tolerance and insulin sensitivity. Mechanistic studies suggested that adipocyte P2Y14R inhibits lipolysis by reducing lipolytic enzyme activity, including ATGL and HSL. In agreement with these findings, agonist treatment of control mice with a P2Y14R agonist decreased lipolysis, an effect that was sensitive to inhibition by a P2Y14R antagonist. In conclusion, we demonstrate that adipose P2Y14Rs were critical regulators of whole-body glucose and lipid homeostasis, suggesting that P2Y14R antagonists might be beneficial for the therapy of obesity and T2D.

Structure activity relationship of 3-nitro-2-(trifluoromethyl)-2H-chromene derivatives as P2Y6 receptor antagonists.

Various 6-alkynyl analogues of a known 3-nitro-2-(trifluoromethyl)-2H-chromene antagonist 3 of the Gq-coupled P2Y6 receptor (P2Y6R) were synthesized using a Sonogashira reaction to replace a 6-iodo group. The analogues were tested in a functional assay consisting of inhibition of calcium mobilization in P2Y6R-expressing astrocytoma cells elicited by native P2Y6R agonist UDP. 6-Ethynyl and 6-cyano groups were installed, and the alkynes were extended through both alkyl and aryl spacers. The most potent antagonists, with IC50 of ~1 µM, were found to be trialkylsilyl-ethynyl 7 and 8 (3-5 fold greater affinity than reference 3), t-butyl prop-2-yn-1-ylcarbamate 14 and p-carboxyphenyl-ethynyl 16 derivatives, and 3 and 8 displayed surmountable antagonism of UDP-induced production of inositol phosphates. Other chain-extended terminal carboxylate derivatives were less potent than the corresponding methyl ester derivatives. Thus, the 6 position in this chromene series is suitable for derivatization with flexibility of substitution, even with sterically extended chains, without losing P2Y6R affinity. However, a 3-carboxylic acid or 3-ester substitution did not serve as a nitro bioisostere, as the affinity was eliminated. These compounds provide additional ligand tools for the underexplored P2Y6R, which is a target for inflammatory, neurodegenerative and metabolic diseases.

Biological Evaluation of 5'-(N-Ethylcarboxamido)adenosine Analogues as Grp94-Selective Inhibitors.

The heat shock protein 90 kDa (Hsp90) family of chaperones is highly sought-after for the treatment of cancer and neurodegenerative diseases. Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum localized isoform that is responsible for the maturation of proteins involved in cell adhesion and the immune response, including Toll-like receptors, immunoglobulins, and integrins. Consequently, Grp94 has been implicated in many different diseases including cancer metastasis, glaucoma, and viral infection. 5'-(N-Ethylcarboxamido)adenosine (NECA) was identified from a high-throughput screen as one of the first molecules to exhibit isoform selectivity toward Grp94, with the ethyl group projecting into a unique pocket within the ATP binding site of Grp94. This pocket has since been exploited by several groups to develop Grp94 selective inhibitors. Despite success in the development of other classes of inhibitors, relatively little work has been done to further develop inhibitors with the NECA scaffold. Unfortunately, NECA is also a potent adenosine receptor agonist, which is likely to confound any biological activity. Therefore, structure-activity relationship studies were performed on the NECA scaffold leading to the discovery of several molecules that displayed similar selectivity and affinity as the parent compound.

Structure activity relationship of 2-arylalkynyl-adenine derivatives as human A3 adenosine receptor antagonists.

Recognition of nucleosides at adenosine receptors (ARs) is supported by multiple X-ray structures, but the structure of an adenine complex is unknown. We examined the selectivity of predicted A1AR and A3AR adenine antagonists that incorporated known agonist affinity-enhancing N 6 and C2 substituents. Adenines with A1AR-favoring N 6-alkyl, cycloalkyl and arylalkyl substitutions combined with an A3AR-favoring 2-((5-chlorothiophen-2-yl)ethynyl) group were human (h) A3AR-selective, e.g. MRS7497 17 (∼1000-fold over A1AR). In addition, binding selectivity over hA2AAR and hA2BAR and functional A3AR antagonism were demonstrated. 17 was subjected to computational docking and molecular dynamics simulation in a hA3AR homology model to predict interactions. The SAR of nucleoside AR agonists was not recapitulated in adenine AR antagonists, and modeling suggested an alternative, inverted binding mode with the key N2506.55 H-bonding to the adenine N 3 and N 9, instead of N 6 and N 7 as in adenosine agonists.

Discovery of Potent Antiallodynic Agents for Neuropathic Pain Targeting P2X3 Receptors.

Antagonism of the P2X3 receptor is one of the potential therapeutic strategies for the management of neuropathic pain because P2X3 receptors are predominantly localized on small to medium diameter C- and Aδ-fiber primary afferent neurons, which are related to the pain-sensing system. In this study, 5-hydroxy pyridine derivatives were designed, synthesized, and evaluated for their in vitro biological activities by two-electrode voltage clamp assay at hP2X3 receptors. Among the novel hP2X3 receptor antagonists, intrathecal treatment of compound 29 showed parallel efficacy with pregabalin (calcium channel modulator) and higher efficacy than AF353 (P2X3 receptor antagonist) in the evaluation of its antiallodynic effects in spinal nerve ligation rats. However, because compound 29 was inactive by intraperitoneal administration in neuropathic pain animal models due to low cell permeability, the corresponding methyl ester analogue, 28, which could be converted to compound 29 in vivo, was investigated as a prodrug concept. Intravenous injection of compound 28 resulted in potent antiallodynic effects, with ED50 values of 2.62 and 2.93 mg/kg in spinal nerve ligation and chemotherapy-induced peripheral neuropathy rats, respectively, indicating that new drug development targeting the P2X3 receptor could be promising for neuropathic pain, a disease with high unmet medical needs.

Prevalence rate and clinical characteristics of esophageal ectopic sebaceous glands in asymptomatic health screen examinees.

Ectopic sebaceous glands in the esophagus have rarely been reported and, thus, represent an obscure medical condition. The aim of this study is to identify the prevalence rate and clinical characteristics of this lesion in an asymptomatic population. We prospectively enrolled health screen examinees who underwent esophagogastroduodenoscopy for gastric cancer screening. An esophageal biopsy was performed in the cases in which esophageal ectopic sebaceous glands were suspected. The general characteristics of the examinees were analyzed based on their medical records. A total of 9989 examinees were enrolled, and five examinees were diagnosed with esophageal ectopic sebaceous glands between December 2012 and June 2014. The endoscopic findings of the esophageal ectopic sebaceous glands indicated multiple yellowish patches or papules, which varied in size. The histopathological findings indicated several lobulated sebaceous glands in the squamous epithelium with inflammatory infiltration. The follow-up endoscopic findings indicated that there was no grossly discernible change. In conclusion, esophageal ectopic sebaceous glands are present in 0.05% of asymptomatic subjects. This lesion is thought to be benign and is not related to clinical symptoms. Therefore, esophageal ectopic sebaceous glands do not require further treatment or follow-up, which makes endoscopists free from active efforts for differential diagnosis with other malignant diseases.