Molecular Pharmacology最新文献

Antipsychotic activity of certain 5-HT_2A receptor (5-HT_2AR) antagonists has been attributed to inverse agonism. 5-HT_2AR hallucinogenic agonists exhibit biased agonism toward G_i/o proteins. Ketanserin and altanserin are 2 structurally similar orthosteric drugs used as 5-HT_2AR therapeutic and radiotracers tools. This study assessed the pharmacological properties of ketanserin and altanserin, previously described as neutral 5-HT_2AR antagonists, using postmortem human prefrontal cortex. Receptor radioligand binding experiments and functional [³⁵S]GTPγS (sulfur 35 labeled guanosine-5'-O-[gamma-thio]-triphosphate) binding assays followed by immunocapture with anti-Gα_q/11 and anti-Gα_i1 protein antibodies were performed. Molecular dynamics simulations were used to ascertain binding modes to the 5-HT_2AR. [³H]Ketanserin binding to 5-HT_2ARs was decreased by guanylyl-5'-imido-diphosphate, a GTP analog. Guanylyl-5'-imido-diphosphate transformed the biphasic inhibition of [³H]ketanserin binding by the agonist (±)DOI into a monophasic low-affinity curve. Ketanserin stimulated [³⁵S]GTPγS binding to Gα_q/11 but not Gα_i1 proteins. Conversely, altanserin inhibited [³⁵S]GTPγS binding to Gα_i1 but not Gα_q/11 proteins. These effects were blocked by the neutral antagonist MDL-11,939. Altanserin-induced inhibition was abolished by the Gα_i/o alkylating agent N-ethylmaleimide. Similar findings were observed in wild type but disappeared in 5-HT_2AR knockout mice. Ketanserin and altanserin showed distinct interactions within residues S2.61, S5.46, and N6.55 in the 5-HT_2AR binding pocket. Therefore, ketanserin and altanserin elicit biased signaling with different patterns of G protein modulation upon interacting with 5-HT_2ARs in brain. Ketanserin behaves as partial agonist on G_q/11, whereas altanserin acts as inverse agonist on G_i1 proteins. These findings should influence the design of antipsychotic drugs targeting this receptor and when used as antagonists and radiotracers for the 5-HT_2AR assessment. SIGNIFICANCE STATEMENT: Ketanserin and altanserin are categorized as neutral 5-HT_2A receptor antagonists. This study shows differential patterns of 5-HT_2A receptor coupling to Gα_q/11 and Gα_i1 proteins between ketanserin and altanserin and demonstrates biased signaling and inverse agonism in human brain.

In cardiomyocytes, Kv7.1 associates with the regulatory subunit KCNE1 to generate the delayed rectifier potassium current I_Ks, which plays a crucial role in cardiac repolarization at elevated heart rates. Gain-of-function mutations in either of these subunits are associated with short QT syndrome, a condition that increases the risk of cardiac arrhythmias including atrial fibrillation, syncope, and sudden death. Therefore, the study of pharmacological inhibitors of Kv7.1 and I_Ks is of significant therapeutic interest. In this work, we used whole-cell patch clamp recordings to characterize the electrophysiological effects of the Kv7.1 blocker 3-(triphenylmethylaminomethyl)pyridine (UCL2077) in both Kv7.1 and I_Ks (Kv7.1 + KCNE1) channels. We found that UCL2077 inhibited both Kv7.1 and I_Ks channels with high affinity (IC₅₀ in the picomolar range) and mild voltage dependence. The drug induced a biphasic time-dependent current decay and reduced current reactivation of Kv7.1, whereas the kinetics of I_Ks were unaffected. We examined state dependence using mutations that functionally stabilize Kv7.1/I_Ks in either the intermediate-open (E160R/R231E) or in the activated-open (E160R/R237E) state. In both channels, UCL2077 potency correlated with the strength of the electromechanical coupling. Our results are further supported by a kinetic Markov model simulating UCL2077 binding that closely resembles the experimental currents. Overall, our work provides an in-depth characterization of UCL2077's action on Kv7.1 and I_Ks channels, offering valuable insights for the development of Kv7.1/I_Ks inhibitors in the context of short QT syndrome and other cardiac arrhythmias. SIGNIFICANCE STATEMENT: This study characterizes UCL2077: a highly specific, high-affinity inhibitor of Kv7.1 and I_Ks channels with therapeutic implications for cardiac arrhythmia. Our work reveals a picomolar affinity, mild voltage dependence, and a kinetic modulation consistent with state-dependent open-channel block.

Extracellular calcium-sensing receptors (CaSRs) are required for whole-body calcium homeostasis and operate as homodimers, shown structurally to bind only one heterotrimeric G protein at a time. Whether the intracellular domain (ICD) of the other, uncoupled monomer is functionally redundant or still required for optimal CaSR signaling remains unknown. Cotransfection of human embryonic kidney 293 cells with receptors containing both extracellular (CaSR₁^S170A) and intracellular (CaSR₂^F801A) mutations, which are nonfunctional when transfected individually, partially rescues function via trans-activation in CaSR₁^S170A:CaSR₂^F801A heterodimers. Further, mutation of an inhibitory, intracellular protein kinase C site T888 permits gain-of-function in CaSR. Therefore, we disabled phosphorylation of this residue (CaSR^T888A) by mutating one, and then both monomers in the CaSR₁^S170A:CaSR₂^F801Atrans-activation-enabled heterodimers. The extracellular Ca²⁺-induced intracellular Ca²⁺ mobilization elicited by CaSR₁^S170A:CaSR₂^F801A was significantly enhanced by disabling one T888 inhibitory phosphorylation site in either CaSR₁^S170A/T888A:CaSR₂^F801A or CaSR₁^S170A:CaSR₂^F801A/T888A, and further enhanced in cells in which both T888 sites had been removed in CaSR₁^S170A/T888A:CaSR₂^F801A/T888A. The results demonstrate that both ICDs of CaSR dimers contribute independently to receptor signaling. Further, in the presence of N-(3-[2-chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine (NPS R-568; CaSR positive allosteric modulator), extracellular Ca²⁺-stimulated signaling from the nonfunctional CaSR^S170A mutant was rescued to wild-type levels by removal of the inhibitory T888 phosphorylation site in CaSR^S170A/T888A homodimers. Therefore, although CaSR^S170A destabilizes the closed (active) form of the Venus fly trap in wild-type homodimers, receptor function can be rescued by relieving intracellular C-terminal domain-dependent inhibition of signaling. SIGNIFICANCE STATEMENT: Despite the homodimeric calcium-sensing receptor coupling to only one G protein at a time, disinhibiting both intracellular domains elicits a greater increase in intracellular Ca²⁺ mobilization than disinhibiting only one, suggesting a positive functional contribution from both subunits.

The β1-adrenoceptor exists in at least 2 agonist-stabilized conformational ensembles: a "catecholamine" ensemble induced via the intrahelical binding site through which catecholamines and most agonists act and a "secondary" ensemble of conformations through which CGP12177 stimulates agonist responses. Several β-ligands stimulate agonist responses through both conformations, resulting in biphasic concentration responses, but little is known about the structure-activity relationship of such ligands. Using a structure-activity hypothesis built on the predicted poses CGP12177 and 3 biphasic agonists (alprenolol, oxprenolol, and bucindolol), predictions based on ligand similarity and structural compatibility reasoning were made about 11 other β1-ligands not yet tested for secondary conformation interaction and examined in radioligand binding and functional assays using human β1- and β2-adrenoceptors. Although the predictions matched with pharmacology in only 6/11 of cases, 3 novel compounds were found to induce an active-state secondary conformation. A CGP12177 derivative (methyl-pyrrole replacing the cyclic urea motif) retained catecholamine site antagonism with secondary site activation. Carteolol (related to CGP12177) and bunitrolol (similar to alprenolol) activated both conformations with biphasic concentration responses. Bunolol (CGP12177 derivative lacking nitrogen in the bicyclic system), as predicted, was a neutral antagonist with no secondary site activation. Moprolol and some bucindolol analogs appeared as conventional agonists, whereas other alprenolol and bucindolol analogs lost all receptor interaction. In a β1-adrenoceptor mutant (β1-V189T-L195Q-W199Y) where secondary site CGP12177 and pindolol interaction is lost, the 3 novel secondary-site compounds were also no longer able to stimulate secondary conformation responses, suggesting that there is a common TM4 secondary conformation-inducing interaction site. SIGNIFICANCE STATEMENT: The β1-adrenoceptor exists in 2 agonist-stabilized, pharmacologically distinguishable conformations. This study pinpointed the interaction site through which the alternative conformation is stabilized and suggested and evaluated additional ligands, thus providing possible molecular determinants.

The μ-opioid receptor (MOR) is responsible for the analgesic actions of opioid drugs as well as their unwanted actions, including respiratory depression and addiction liability. Following agonist occupancy, MOR can signal via G-protein and/or β-arrestin. However, MOR agonists may show an imbalance between activating these 2 pathways. Evidence from studies of G-protein-coupled receptors suggests that allosteric modulators can influence agonist signaling profiles. However, no studies have examined this phenomenon at MOR. In this study, we compare the ability of various orthosteric MOR agonists to activate G-protein or recruit β-arrestin in the absence or presence of 2 structurally distinct MOR positive allosteric modulators, BMS-986187 (a xanthenedione) or BMS-986122 (a thiazolidine). We determined the potency and efficacy of 6 orthosteric agonists (DAMGO, fentanyl, methadone, morphine, Met-enkephalin, and SR17018) with and without BMS-986187 or BMS-986122, and determined bias factors for the agonists themselves and in the presence of a modulator. The 2 allosteric modulators enhanced the potency of agonists to different degrees showing probe dependence and differentially shifted the ability of agonists to activate G-protein as compared to recruitment of β-arrestin, indicating an effect on ligand bias. In both measures, in the presence of either modulator, the higher efficacy agonists showed a shift in potency. In contrast, lower efficacy compounds showed an increase in maximal effect with a smaller shift in potency. Overall, the studies provide evidence that positive allosteric modulators influence the degree and direction by which orthosteric agonists signal downstream of MOR. SIGNIFICANCE STATEMENT: Biased agonism and positive allosteric modulation at the μ-opioid receptor have been suggested as approaches to improve the therapeutic index of opioid agonists. This work shows how allosteric modulation alters the bias signaling profile of μ-opioid agonists.

Nav1.1 voltage-gated sodium (Nav) channels encoded by the SCN1A gene are critical to high frequency action potential generation in inhibitory interneurons and so play a crucial role in maintaining the excitatory-inhibitory balance in the brain. Rare, often loss-of-function, mutations in the SCN1A gene can lead to a spectrum of developmental and epileptic encephalopathies including Dravet syndrome with common SCN1A variants being risk factors for epilepsy, highlighting the potential for Nav1.1 as a therapeutic target for seizure disorders. Here, we describe a potent small molecule sodium channel potentiator ICA00600232 that exhibits >1000-fold selectivity for human Nav1.1 over all other Nav1.x family members except Nav1.3. Nav1.1 potentiation by ICA00600232 results from a slowing of channel inactivation. Employing both chimeras and single point mutations, we demonstrate that ICA00600232 interacts with the domain 4 voltage sensor region of Nav1.1. Three amino acid residues within the "extracellular" facing regions of the S2 and S3 transmembrane segments are major determinants of Nav1.1 potentiation and subtype selectivity. These same residues are also important for subtype selective inhibitor interactions with Nav1.1, albeit in a different structural conformation. Furthermore, we demonstrate that ICA00600232 at concentrations as low as 10 nM enhances firing frequency in parvalbumin positive fast-spiking interneurons recorded in brain slices from scn1a^+/- mice. The current study shows that a small molecule Nav1.1 selective potentiator targeting the fourth voltage sensor can slow inactivation of Nav1.1, increase excitability of parvalbumin positive interneurons and inhibitory tone that could be beneficial in treating developmental and epileptic encephalopathies and other seizure disorders. SIGNIFICANCE STATEMENT: Loss-of-function mutations of Nav1.1 sodium channel genes in human inhibitory interneurons are associated with a spectrum of epilepsies including developmental and epileptic encephalopathies. Potential treatments include enhancement of Nav1.1 activity via administration of small molecule potentiating agents. This study describes a potent subtype selective Nav1.1 channel potentiator that interacts with a region that controls voltage sensitivity and enhances action potential firing in inhibitory interneurons, supporting its potential utility in treating developmental and epileptic encephalopathy associated seizure disorders.

In pulmonary epithelial cells, baculoviral inhibitor of apoptosis repeat-containing (BIRC) gene, BIRC3, and to a lesser extent, BIRC2, mRNAs were upregulated by interleukin (IL)-1β and tumor necrosis factor-α. Glucocorticoids also induced BIRC3 mRNA and the glucocorticoid receptor (GR) was recruited to GR-binding regions (GBRs) proximal to, and within, BIRC3 in A549 and BEAS-2B cells. Four such GBRs drove glucocorticoid-inducible, Organon 34517-antagonized, transcription in A549 cells. IL-1β and tumor necrosis factor-α recruited the nuclear factor (NF)-κB transactivating subunit, v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), to RELA-binding regions (RBRs) (R4 and R7) upstream of the BIRC3 and BIRC2 transcription start sites. These RBRs drove IL-1β-inducible transcription that involved NF-κB. Thus, direct regulation of BIRC3 by GR and BIRC3/BIRC2 by NF-κB is indicated. IL-1β-plus-budesonide also recruited RELA to multiple GBRs, whereas GR was recruited to the main IL-1β-induced RBR (R4), effects that correlated with positive IL-1β/glucocorticoid transcriptional cooperativity or additivity. At the R5 GBR, RELA was not recruited and both GR binding and glucocorticoid-dependent transcription reduced (infra-additivity) on cotreatment. Similarly, the R7 RBR barely recruited GR and IL-1β-induced transcription showed infra-additivity with IL-1β-plus-glucocorticoid. R8 recruited GR and RELA primarily with IL-1β-plus-glucocorticoid and revealed transcriptional synergy. Thus, GR/RELA-corecruitment yielded positive cooperative and additive transcriptional effects, whereas recruiting one factor alone associated with infra-additivity. Furthermore, DNA looping revealed how multiple RBRs/GBRs may integrate to control transcription. Because IL-1β- and glucocorticoid-dependent coupregulation of apoptotic/antiapoptotic genes was widespread, the combinatorial recruitment of RELA/GR to regulatory genes, including BIRC3, CFLAR plus others in the NF-κB pathway, may be critical to cell fate determination in inflammation. SIGNIFICANCE STATEMENT: Identification of functional GR- and RELA-binding regions at the BIRC3/BIRC2 locus explains the upregulation of BIRC3 expression by glucocorticoids and inflammatory cytokines. Cytokine-plus-glucocorticoid cotreatment revealed positive cooperative and additive interactions between GR and RELA, whereas DNA regions binding only one factor showed reduced effects on binding and transcription. These region-specific outcomes, combined with DNA looping between regulatory regions, provides insight as to how factors at multiple DNA regions may integrate their outputs to produce combinatorial regulation of apoptotic/antiapoptotic genes.

Membrane cholesterol modulates the ability of cholecystokinin to act at the type 1 cholecystokinin receptor (CCK1R), with elevated levels of this lipid that can be present in obesity, disrupting stimulus-activity coupling. This occurs via direct effect on a cholesterol association (cholesterol recognition/interaction amino acid consensus) motif within CCK1R, whereas an analogous motif is present in the type 2 cholecystokinin receptor (CCK2R) with no functional impact of cholesterol on that receptor. We recently demonstrated that surface residues surrounding this motif in these 2 G protein-coupled receptors could be exchanged as a group to convert their cholesterol sensitivity. We now evaluate each of these residues individually and use molecular dynamics to study cholesterol interactions with this site. These studies show that cholesterol occupies this motif in both CCK1R and CCK2R with similar dominant poses in both receptors in normal, as well as in elevated cholesterol environments. The dominant pose of cholesterol changes in both receptors with elevated cholesterol, shifting proximity from transmembrane segment (TM) 3 toward transmembrane segment 5. Of note, cholesterol residence time at this motif is longer for CCK2R than CCK1R and longer in high cholesterol conditions at both receptors. This suggests that the cholesterol sensitivity of CCK1R is likely a dynamic event mediated by short, transient cholesterol interaction with CCK1R under normal conditions. One residue in position 5.52 was critical for this cholesterol effect on CCK1R, despite the prediction that this is not due to direct interaction with cholesterol. Instead, the packing of hydrophobic residues around 5.52 is tighter in CCK1R than in CCK2R, likely affecting the position and movement of transmembrane segment 5 that are important for stimulus-activity coupling. SIGNIFICANCE STATEMENT: Membrane cholesterol can affect the function of select G protein-coupled receptors, such as the type 1 cholecystokinin receptor, that are targets for drugs in disease states in which membrane cholesterol can be elevated. This article studied the molecular basis for high cholesterol disruption of stimulus-activity coupling at this receptor using site-directed mutagenesis and molecular dynamics. The findings demonstrate that cholesterol residence time at a key cholesterol association motif within CCK1R is responsible for this effect.