TRPV1 analgesics disturb core body temperature via a biased allosteric mechanism involving conformations distinct from that for nociception.

Efforts on developing transient receptor potential vanilloid 1 (TRPV1) drugs for pain management have been hampered by deleterious hypo- or hyperthermia caused by TRPV1 agonists/antagonists. Here, we compared the effects of four antagonists on TRPV1 polymodal gating and core body temperature (CBT) in Trpv1+/+, Trpv1-/-, and Trpv1T634A/T634A. Neither the effect on proton gating nor drug administration route, hair coverage, CBT rhythmic fluctuations, or inflammation had any influence on the differential actions of TRPV1 drugs on CBT. We identified the S4-S5 linker region exposed to the vanilloid pocket of TRPV1 to be critical for hyperthermia associated with certain TRPV1 antagonists. PSFL2874, a TRPV1 antagonist we discovered, is effective against inflammatory pain but devoid of binding to the S4-S5 linker and inducing CBT changes. These findings implicate that biased allosteric mechanisms exist for TRPV1 coupling to nociception and CBT regulation, opening avenues for the development of non-opioid analgesics without affecting CBT.

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.

Vanilloid agonist-mediated activation of TRPV1 channels requires coordinated movement of the S1-S4 bundle rather than a quiescent state.

Transient receptor potential vanilloid1 (TRPV1) channel plays an important role in a wide range of physiological and pathological processes, and a comprehensive understanding of TRPV1 gating will create opportunities for therapeutic intervention. Recent incredible advances in cryo-electron microscopy (cryo-EM) have yielded high-resolution structures of all TRPV subtypes (TRPV1-6) and all of them share highly conserved six transmembrane (TM) domains (S1-S6). As revealed by the open structures of TRPV1 in the presence of a bound vanilloid agonist (capsaicin or resiniferatoxin), TM helicesS1 to S4 form a bundle that remains quiescent during channel activation, highlighting differences in the gating mechanism of TRPV1 and voltage-gated ion channels. Here, however, we argue that the structural dynamics rather than quiescence of S1-S4 domains is necessary for capsaicin-mediated activation of TRPV1. Using fluorescent unnatural amino acid (flUAA) incorporation and voltage-clamp fluorometry (VCF) analysis, we directly observed allostery of the S1-S4 bundle upon capsaicin binding. Covalent occupation of VCF-identified sites, single-channel recording, cell apoptosis analysis, and exploration of the role of PSFL828, a novel non-vanilloid agonist we identified, have collectively confirmed the essential role of this coordinated S1-S4 motility in capsaicin-mediated activation of TRPV1. This study concludes that, in contrast to cryo-EM structural studies, vanilloid agonists are also required for S1-S4 movement during TRPV1 activation. Redefining the gating process of vanilloid agonists and the discovery of new non-vanilloid agonists will allow the evaluation of new strategies aimed at the development of TRPV1 modulators.

Dynamic recognition of naloxone, morphine and endomorphin1 in the same pocket of µ-opioid receptors.

Morphine, the most widely used analgesic, relieves severe pain by activating the µ-opioid receptor (MOR), whereas naloxone, with only slight structural changes compared to morphine, exhibits inhibitory effect, and is used to treat opioid abuse. The mechanism by which the MOR distinguishes between the two is unclear. Molecular dynamics (MD) simulations on a 1-µs time scale and metadynamics-enhanced conformational sampling are used here to determine the different interactions of these two ligands with MOR: morphine adjusted its pose by continuously flipping deeper into the pocket, whereas naloxone failed to penetrate deeper because its allyl group conflicts with several residues of MOR. The endogenous peptide ligand endomorphin-1 (EM-1) underwent almost no significant conformational changes during the MD simulations. To validate these processes, we employed GIRK4S143T, a MOR-activated Gßγ-protein effector, in combination with mutagenesis and electrophysiological recordings. We verified the role of some key residues in the dynamic recognition of naloxone and morphine and identified the key residue I322, which leads to differential recognition of morphine and naloxone while assisting EM-1 in activating MOR. Reducing the side chain size of I322 (MORI322A) transformed naloxone from an inhibitor directly into an agonist of MOR, and I322A also significantly attenuated the potency of MOR on EM-1, confirming that binding deep in the pocket is critical for the agonistic effect of MOR. This finding reveals a dynamic mechanism for the response of MOR to different ligands and provides a basis for the discovery of new ligands for MOR at the atomic level.

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.