Chronic cough relief by allosteric modulation of P2X3 without taste disturbance.

P2X receptors are cation channels that sense extracellular ATP. Many therapeutic candidates targeting P2X receptors have begun clinical trials or acquired approval for the treatment of refractory chronic cough (RCC) and other disorders. However, the present negative allosteric modulation of P2X receptors is primarily limited to the central pocket or the site below the left flipper domain. Here, we uncover a mechanism of allosteric regulation of P2X3 in the inner pocket of the head domain (IP-HD), and show that the antitussive effects of quercetin and PSFL2915 (our nM-affinity P2X3 inhibitor optimized based on quercetin) on male mice and guinea pigs were achieved by preventing allosteric changes of IP-HD in P2X3. While being therapeutically comparable to the newly licensed P2X3 RCC drug gefapixant, quercetin and PSFL2915 do not have an adverse effect on taste as gefapixant does. Thus, allosteric modulation of P2X3 via IP-HD may be a druggable strategy to alleviate RCC.

Diagnostic value of lncRNA HOTAIR as a biomarker for detecting and staging of non-small cell lung cancer.

BackgroundSince the role of long non-coding RNA (lncRNA) HOTAIR is yet to be established in non-small cell lung cancer (NSCLC), we tried to explore the expression of lncRNA HOTAIR in NSCLC and evaluate the correlation between the combined detection of lncRNA HOTAIR and routine tumour markers and the pathological staging of lung cancer.MethodsThis study prospectively included 148 patients with NSCLC selected from our hospital from January 2017 to September 2020 as the lung cancer group, and 148 healthy volunteers who referred for physical examination were selected as the control group. Fluorescence in situ hybridisation was used to detect the expression of lncRNA HOTAIR in the cancerous tissues and adjacent tissues of lung cancer patients; the immunofluorescence method was used to detect the serum NSE, CEA and CYFRA21-1 levels of the two groups of testers. Correlation analysis was used to evaluate any relation between cancer staging and markers. In addition, ROC curve analysis was used to estimate sensitivity, specificity, positive predictive value, and negative predictive value.ResultsThe expression of lncRNA HOTAIR in lung cancer tissues was higher than control or surrounding tissue (p < 0.05). Also, high levels of NSE, CEA and CYFRA21-1 were observed in lung cancer group (p < 0.05). In both N and T stage, the expression of lncRNA HOTAIR combined with NSE, CEA and CYFRA21-1 levels increased with the increase in the number of stages (p < 0.05). The results of single factor analysis showed that NSE, CEA, CYFRA21-1 and lncRNA HOTAIR all have appropriate diagnostic value for detecting lung cancer (specificity of 92.6, 91.5, 90.6, 86.9%, respectively and the sensitivity of 61.3, 62.9, 55.4, 52.3%, respectively).ConclusionLncRNA HOTAIR is a novel diagnostic test with high diagnostic value for detecting of pathological staging of NSCLC; however, the diagnostic accuracy of lncRNA HOTAIR is not higher than other tumour biomarkers.

A Highly Conserved Salt Bridge Stabilizes the Kinked Conformation of ß2,3-Sheet Essential for Channel Function of P2X4 Receptors.

Significant progress has been made in understanding the roles of crucial residues/motifs in the channel function of P2X receptors during the pre-structure era. The recent structural determination of P2X receptors allows us to reevaluate the role of those residues/motifs. Residues Arg-309 and Asp-85 (rat P2X4 numbering) are highly conserved throughout the P2X family and were involved in loss-of-function polymorphism in human P2X receptors. Previous studies proposed that they participated in direct ATP binding. However, the crystal structure of P2X demonstrated that those two residues form an intersubunit salt bridge located far away from the ATP-binding site. Therefore, it is necessary to reevaluate the role of this salt bridge in P2X receptors. Here, we suggest the crucial role of this structural element both in protein stability and in channel gating rather than direct ATP interaction and channel assembly. Combining mutagenesis, charge swap, and disulfide cross-linking, we revealed the stringent requirement of this salt bridge in normal P2X4 channel function. This salt bridge may contribute to stabilizing the bending conformation of the ß2,3-sheet that is structurally coupled with this salt bridge and the α2-helix. Strongly kinked ß2,3 is essential for domain-domain interactions between head domain, dorsal fin domain, right flipper domain, and loop ß7,8 in P2X4 receptors. Disulfide cross-linking with directions opposing or along the bending angle of the ß2,3-sheet toward the α2-helix led to loss-of-function and gain-of-function of P2X4 receptors, respectively. Further insertion of amino acids with bulky side chains into the linker between the ß2,3-sheet or the conformational change of the α2-helix, interfering with the kinked conformation of ß2,3, led to loss-of-function of P2X4 receptors. All these findings provided new insights in understanding the contribution of the salt bridge between Asp-85 and Arg-309 and its structurally coupled ß2,3-sheet to the function of P2X receptors.