aa92593  (MedChemExpress)

Journal: Investigative Ophthalmology & Visual Science

Article Title: Blue Light Damages Retinal Ganglion Cells Via Endoplasmic Reticulum Stress and Autophagy in Chickens

doi: 10.1167/iovs.66.1.3

Figure Lengend Snippet: Both OPN4 and melatonin influence ER stress in chicken RGCs under BL in vitro. ( A ) Expression levels of genes associated with mitochondrial dynamics and damage in the retina following five weeks of monochromatic light treatment ( n = 5–9). ( B ) Fluorescent labeling of DNA fragmentation in chicken retinas. ( C ) Effects of light wavelength on the transcription levels of Opn4 orthologs in the chicken retina ( n = 5). ( D ) Protein levels of OPN4m in chicken retinas after five weeks of monochromatic light treatment ( n = 5). ( E ) BL exposure resulted in greater muscular pupil constriction in chickens ( n = 6). ( F , G ) BL exposure does not influence the kinetics of pupil contraction in chickens (one-phase association, n = 5). ( H ) Expression levels of melatonin-related proteins ( n = 5). ( I ) Localization of Mel1a in the chicken retina. ( J ) Localization of AANAT in the nuclei of RGCs ( n = 5). ( K ) Effects of the OPN4 inhibitor AA92593 on the viability of embryonic chicken retinal cells ( n = 4). The black arrows indicate the drug concentrations used in vitro. ( L ) Effects of melatonin on the viability of embryonic chicken retinal cells ( n = 4). The black arrows indicate the drug concentrations used in vitro. ( M , N ) Effects of the in vitro addition of AA92593 and melatonin on the intensity of p -PERK immune-positive signals in TUJ-1 + embryonic chicken retinal cells under BL ( n = 5). Data were presented as means ± SEM. The results were analyzed using the Kruskal-Wallis test with Dunn's multiple comparisons ( A ) or one-way ANOVA with Sidak's multiple comparisons tests ( C , D , E , H , and J ). Comparisons between drugs and their solvents were conducted using an unpaired two-tailed Student's t -test or Mann-Whitney U test ( K , L , and N ); * P < 0.05, ** P < 0.01, and **** P < 0.0001. Dnm1, dynamin 1; Opa1, optic atrophy 1; Mfn1, mitofusin-1; Mfn2, mitofusin-2; cgas, cyclic guanosine monophosphate (GMP)-AMP synthase; Sting, stimulator of interferon genes; Mel1b, melatonin receptor 2; NQO2, quinone dehydrogenase 2; Mel, melatonin.

Article Snippet: During the experiments, DMSO (0.1%), AA92593 (HY-125145; MedChemExpress, Monmouth Junction, NJ, USA), melatonin (HY-B0075; MedChemExpress), or a combination of AA92593 and melatonin was added 30 minutes before light treatment.

Techniques: In Vitro, Expressing, Labeling, Two Tailed Test, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis underlying the specificity of an antagonist AA92593 for mammalian melanopsins

doi: 10.1016/j.jbc.2025.108461

Figure Lengend Snippet: AA92593-induced suppression of G protein activation by mammalian melanopsins assessed by GsX Glo S ensor assay. A , light stimulus–response curves of GsX GloSensor assay for human melanopsin with Gsα/q11. Error bars indicate the SD values (n = 3). Fitting parameters: max , 113.43 ± 5.86; min , 2.71 ± 11.1; rate , 2.30 ± 1.85. The EC 50 value of the curve is ∼10 μW/cm 2 . B , chemical structure of AA92593 . C and D , AA92593 concentration-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of human ( C ) and mouse ( D ) melanopsins. Luminescence levels of cAMP biosensor (GloSensor) are normalized to the values at the starting point (time = 0 min). In ( C ) and ( D ), final AA92593 concentrations are 16.7 ( red ), 10 ( orange ), 5 ( brown ), 1 ( green ), 0.1 ( blue ), 0.01 ( purple ), and 0 μM ( black ), respectively. Light blue bars indicate white light illumination (10 s). Error bars indicate the SD values (n = 3). E and F , dose-dependent reduction in peak cAMP responses upon Gsα/q11 activation in human ( E ) and mouse ( F ) melanopsin-expressing COS-1 cells. Relative average peak values to the value in the absence of AA92593 are plotted against the final concentrations of AA92593. Error bars indicate the SD values (n = 3). Fitting parameters for human melanopsin: max , 102.59 ± 2.72; min , 13.65 ± 7.28; rate , −0.92 ± 0.21. Fitting parameters for mouse melanopsin: max , 98.86 ± 1.11; min , 46.53 ± 4.15; rate , −1.15 ± 0.18. IC 50 values for human and mouse melanopsins are 1.05 ± 0.28 μM and 2.98 ± 0.58 μM, respectively. G and H , AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα activation of human ( G ) and mouse ( H ) melanopsins. Error bars indicate the SD values (n = 3). I and J , AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/i11 activation of human ( I ) and mouse ( J ) melanopsins. Error bars indicate the SD values (n = 3). In ( G – J ), red and black curves indicate luminescence changes in the presence and absence of 16.7 μM AA92593, respectively. Light blue bars indicate white light illumination (10 s). Luminescence levels of cAMP biosensor (GloSensor) are normalized to the values at the starting point (time = 0 min).

Article Snippet: Under our experimental conditions, >50 μM AA92593 was not fully dissolved in the HBSS solution, and we set maximal concentration of AA92593 (Glixx Laboratories) at 33.3 μM (final concentration of 16.7 μM, see below).

Techniques: Activation Assay, Concentration Assay, Inhibition, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis underlying the specificity of an antagonist AA92593 for mammalian melanopsins

doi: 10.1016/j.jbc.2025.108461

Figure Lengend Snippet: Changes in the susceptibility of human melanopsin for AA92593 by substitutions of conserved amino acid residues in the putative retinal-binding site. A , amino acid residues at positions 94 2.61 , 188 ECL2 , 189 ECL2 , 207 5.42 , and 269 6.52 in various melanopsins and a non-melanopsin Gq-coupled opsin. “Mammalian melanopsin-type” amino acids are highlighted as bold. The amino acid residue numbering in this paper is based on the amino acid sequence of bovine rhodopsin. B , arrangement of the amino acid residues at positions 94 2.61 , 188 ECL2 , 189 ECL2 , 207 5.42 , and 269 6.52 in the AlphaFold2-predicted human melanopsin structure . The 11- cis -retinal molecule is adopted from the crystal structure of bovine rhodopsin (PDB ID: 1U19 ) . Note that the predicted structure of human melanopsin and the crystal structure of bovine rhodopsin with 11- cis -retinal were overlapped to obtain a structure that looks like retinal bound to human melanopsin. The structural models are prepared using PyMOL ( https://pymol.org/ ). C - F , AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of human melanopsin mutants F94 2.61 C ( C ), S188 ECL2 T ( D ), S269 6.52 A ( E ), and F94 2.61 C/S188 ECL2 T/S269 6.52 A ( F ). Red and black traces indicate luminescence changes in the presence and absence of 16.7 μM AA92593, respectively. Light blue bars indicate white light illumination (10 s). Luminescence levels of cAMP biosensor (GloSensor) are normalized to the values at the starting point (time = 0 min). Error bars indicate the SD values (n = 3). G , comparison of inhibition in peak cAMP responses by 16.7 μM AA92593 upon Gsα/q11 activation in human melanopsin WT and mutants. Error bars indicate the SD values of independent experiments (n = 46, 8, 4, 3, 7, 6, 7, and 4 for WT, F94 2.61 C, S188 ECL2 T, S188 ECL2 A, S269 6.52 A, F94 2.61 C/S269 6.52 A, F94 2.61 C/S188 ECL2 T/S269 6.52 A, and F94 2.61 C/S188 ECL2 A/S269 6.52 A, respectively). The statistical p values in differences from WT are 0.99, <0.00001∗, 1.00, <0.00001∗, <0.00001∗, <0.00001∗, and <0.00001∗ for F94 2.61 C, S188 ECL2 T, S188 ECL2 A, S269 6.52 A, F94 C/S269 6.52 A, F94 2.61 C/S188 ECL2 T/S269 6.52 A, and F94 2.61 C/S188 ECL2 A/S269 6.52 A, respectively (Dunnett's test following one-way ANOVA, F = 79, d. f. = 14, 91). H and I , NanoBiT Gq dissociation assay using Gqα/R183Q-LgBiT on human melanopsin WT ( H ) and F94 2.61 C/S188 ECL2 T/S269 6.52 A mutant ( I ). Red , blue , and black traces indicate luminescence changes in the presence of 16.7 μM ( red ), 1.67 μM ( blue ), and 0 μM ( black ) AA92593. Light blue bars indicate white light illumination (10 s). NanoLuc luminescence levels are normalized to the values at the starting point (time = 0 min). Error bars indicate the SD values (n = 3).

Article Snippet: Under our experimental conditions, >50 μM AA92593 was not fully dissolved in the HBSS solution, and we set maximal concentration of AA92593 (Glixx Laboratories) at 33.3 μM (final concentration of 16.7 μM, see below).

Techniques: Binding Assay, Residue, Sequencing, Inhibition, Activation Assay, Comparison, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis underlying the specificity of an antagonist AA92593 for mammalian melanopsins

doi: 10.1016/j.jbc.2025.108461

Figure Lengend Snippet: Changes in the susceptibility of mouse melanopsin for AA92593 by substitutions in the putative retinal-binding site. A – D , AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of mouse melanopsin mutants F94 2.61 C ( A ), S188 ECL2 T ( B ), S269 6.52 A ( C ), and F94 2.61 C/S188 ECL2 T/S269 6.52 A ( D ). Red and black traces indicate luminescence changes in the presence and absence of 16.7 μM AA92593, respectively. Light blue bars indicate white light illumination (10 s). Luminescence levels of cAMP biosensor (GloSensor) are normalized to the values at the starting point (time = 0 min). Error bars indicate the SD values (n = 3). E , comparison of inhibition in peak cAMP responses by 16.7 μM AA92593 upon Gsα/q11 activation in mouse melanopsin WT and mutants. Error bars indicate the SD values of independent experiments (n = 23, 5, 4, 3, 5, 6, 6, and 5 for WT, F94 2.61 C, S188 ECL2 T, S188 ECL2 A, S269 6.52 A, F94 2.61 C/S269 6.52 A, F94 2.61 C/S188 ECL2 T/S269 6.52 A and F94 2.61 C/S188 ECL2 A/S269 6.52 A, respectively). The statistical p values in differences from WT are 0.95, 0.88, 0.81, <0.00001∗, <0.00001∗, <0.00001∗, and <0.00001∗ for F94 2.61 C, S188 ECL2 T, S188 ECL2 A, S269 6.52 A, F94 2.61 C/S269 6.52 A, F94 2.61 C/S188 ECL2 T/S269 6.52 A, and F94 2.61 C/S188 ECL2 A/S269 6.52 A, respectively (Dunnett's test following one-way ANOVA, F = 33, d. f. = 9, 53). F and G , NanoBiT Gq dissociation assay on mouse melanopsin WT ( F ) and F94 2.61 C/S188 ECL2 T/S269 6.52 A mutant ( G ). Red , blue , and black traces indicate luminescence changes in the presence of 16.7 μM ( red ), 1.67 μM ( blue ), and 0 μM ( black ) AA92593. Light blue bars indicate white light illumination (10 s). NanoLuc luminescence levels are normalized to the values at the starting point (time = 0 min). Error bars indicate the SD values (n = 3).

Article Snippet: Under our experimental conditions, >50 μM AA92593 was not fully dissolved in the HBSS solution, and we set maximal concentration of AA92593 (Glixx Laboratories) at 33.3 μM (final concentration of 16.7 μM, see below).

Techniques: Binding Assay, Inhibition, Activation Assay, Comparison, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis underlying the specificity of an antagonist AA92593 for mammalian melanopsins

doi: 10.1016/j.jbc.2025.108461

Figure Lengend Snippet: Docking and MD simulations on human melanopsin with AA92593. A and B , structure of the human melanopsin–AA92593 complex predicted from DiffDock. Parenthesis show the shortest distances between AA92593 and selected ligands. The distances in ( A ) and ( B ) are between heavy atoms, whereas those in other panels are between all atoms including hydrogen. C , time evolution of the distances between AA92593 and Phe-94 2.61 , Ser-188 ECL2 , and Ser-269 6.52 over the 1 μs MD trajectory. D , per-residue decomposition of the binding energy calculated using MMPBSA. E and F , structure of the melanopsin–AA92593 complex before ( E ) and after ( F ) the 1 μs MD simulation.

Article Snippet: Under our experimental conditions, >50 μM AA92593 was not fully dissolved in the HBSS solution, and we set maximal concentration of AA92593 (Glixx Laboratories) at 33.3 μM (final concentration of 16.7 μM, see below).

Techniques: Residue, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis underlying the specificity of an antagonist AA92593 for mammalian melanopsins

doi: 10.1016/j.jbc.2025.108461

Figure Lengend Snippet: Changes in the susceptibility of human melanopsin for AA92593 by substitutions of amino acid residues interacting with AA92593 in simulations. A – G , AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of human melanopsin mutants W189 ECL2 F ( A ), W189 ECL2 I ( B ), W189 ECL2 V ( C ), L207 5.42 V ( D ), L207 5.42 A ( E ), L207 5.42 F ( F ), and F94 2.61 C/S188 ECL2 T/W189 ECL2 F/S269 6.52 A ( G ). Error bars indicate the SD values (n = 3). H , comparison of inhibition in peak cAMP responses by 16.7 μM AA92593 upon Gsα/q11 activation in human melanopsin WT and mutants. WT data is the same as Figure 2 G . Error bars indicate the SD values of independent experiments (n = 46, 3, 3, 3, 3, 3, 3, and 3 for WT, W189 ECL2 F, W189 ECL2 I, W189 ECL2 V, F94 2.61 C/S188 ECL2 T/W189 ECL2 F/S269 6.52 A, L207 5.42 V, L207 5.42 A, and L207 5.42 F, respectively). The statistical p values in differences from WT are <0.00001∗, <0.00001∗, 0.00013∗, <0.00001∗, 0.010∗, 0.072, and 0.59 for W189 ECL2 F, W189 ECL2 I, W189 ECL2 V, F94 2.61 C/S188 ECL2 T/W189 ECL2 F/S269 6.52 A, L207 5.42 V, L207 5.42 A, and L207 5.42 F, respectively (Dunnett's test following one-way ANOVA, F = 79, d. f. = 14, 91). I , dose-dependent reduction in peak cAMP responses upon Gsα/q11 activation by human melanopsin mutants F94 2.61 C/S188 ECL2 T/W189 ECL2 F/S269 6.52 A ( blue ) and L207 5.42 F ( red ). The curve of WT ( black ) is adopted from Figure 1 E . Relative average peak values to the value in the absence of AA92593 are plotted against the final concentrations of AA92593. Error bars indicate the SD values (n = 3). Fitting parameters for the L207 5.42 F mutant: max , 98.88 ± 4.88; min , 3.15 ± 9.9; rate , −0.88 ± 0.29. IC 50 values for the L207 5.42 F mutant is 0.35 ± 0.16 μM. J and K , absorption spectra of 9- cis -retinal bound human melanopsin WT ( J ) and F94 2.61 C/S188 ECL2 T/W189 ECL2 F/S269 6.52 A mutant ( K ). Respective absorption maximum (λmax) values are indicated. Our previous study reported that the 11- cis -retinal–bound human melanopsin WT shows the λmax at 468 nm . L , NanoBiT Gq dissociation assay using Gqα/R183Q-LgBiT on human melanopsin F94 2.61 C/S188 ECL2 T/W189 ECL2 F/S269 6.52 A mutant. Red , blue , and black traces indicate luminescence changes in the presence of 16.7 μM ( red ), 1.67 μM ( blue ), and 0 μM ( black ) AA92593. Light blue bars indicate white light illumination (10 s). NanoLuc luminescence levels are normalized to the values at the starting point (time = 0 min). Error bars indicate the SD values (n = 3). M , AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of mouse melanopsin L207 5.42 F mutant. Error bars indicate the SD values (n = 3). In ( A – G ), and ( M ), red and black traces indicate luminescence changes in the presence and absence of 16.7 μM AA92593, respectively. Light blue bars indicate white light illumination (10 s). Luminescence levels of cAMP biosensor (GloSensor) are normalized to the values at the starting point (time = 0 min). N , comparison of inhibition in peak cAMP responses by 16.7 μM AA92593 upon Gsα/q11 activation in mouse melanopsin WT and L207 5.42 F mutant. WT data is the same as Figure 3 E . Error bar indicates the SD value of independent experiments (n = 23 and 3 for WT and L207 5.42 F, respectively). The statistical p values of L207 5.42 F in difference from WT is 0.000036∗ (Dunnett's test following one-way ANOVA, F = 33, d. f. = 9, 53).

Article Snippet: Under our experimental conditions, >50 μM AA92593 was not fully dissolved in the HBSS solution, and we set maximal concentration of AA92593 (Glixx Laboratories) at 33.3 μM (final concentration of 16.7 μM, see below).

Techniques: Inhibition, Activation Assay, Comparison, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis underlying the specificity of an antagonist AA92593 for mammalian melanopsins

doi: 10.1016/j.jbc.2025.108461

Figure Lengend Snippet: Changes in the susceptibility of non - mammalian-type melanopsins for AA92593 by amino acid substitutions. A – C , AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of Xenopus Opn4x WT ( A ) as well as mutants C94 2.61 F/T188 ECL2 S/A269 6.52 S ( B ) and L207 5.42 F ( C ). D – F , AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of chicken Opn4-1 WT ( D ) as well as mutants C94 2.61 F/T188 ECL2 S/A269 6.52 S ( E ) and L207 5.42 F ( F ). In ( A – F ), red and black traces indicate luminescence changes in the presence and absence of 16.7 μM AA92593, respectively. Light blue bars indicate white light illumination (10 s). Luminescence levels of cAMP biosensor (GloSensor) are normalized to the values at the starting point (time = 0 min). Error bars indicate the SD values (n = 3). G and H , comparison of inhibition in peak cAMP responses by 16.7 μM AA92593 upon Gsα/q11 activation in WT and mutant of Xenopus Opn4x ( G ) and chicken Opn4-1 ( H ). Error bars indicate the SD values of independent experiments (n = 12, 5, and 3 for Xenopus Opn4x WT, C94 2.61 F/T188 ECL2 S/A269 6.52 S, and L207 5.42 F, respectively, and 8, 3, and 3 for chicken Opn4-1 WT, C94 2.61 F/T188 ECL2 S/A269 6.52 S, and L207 5.42 F, respectively). The statistical p values in differences from WT are 0.000076∗ and 0.00025∗ for Xenopus Opn4x C94 2.61 F/T188 ECL2 S/A269 6.52 S and L207 5.42 F, respectively, and 0.0027∗ and 0.000041∗ for chicken Opn4-1 C94 2.61 F/T188 ECL2 S/A269 6.52 S and L207 5.42 F, respectively (Dunnett's test following one-way ANOVA, F = 22, d. f. = 2, 17 for Xenopus Opn4x, F = 28, d. f. = 2, 11for chicken Opn4-1). I , dose-dependent reduction in peak cAMP responses upon Gsα/q11 activation by Xenopus Opn4x WT ( red ) and C94 2.61 F/T188 ECL2 S/A269 6.52 S mutant ( blue ). The curve of human melanopsin WT ( black ) is adopted from Figure 1 E . Relative average peak values to the value in the absence of AA92593 are plotted against the final concentrations of AA92593. Error bars indicate the SD values (n = 3). J and K , absorption spectra of 9- cis -retinal bound Xenopus Opn4x WT ( J ) and C94 2.61 F/T188 ECL2 S/A269 6.52 S mutant ( K ). Respective λmax values are indicated.

Article Snippet: Under our experimental conditions, >50 μM AA92593 was not fully dissolved in the HBSS solution, and we set maximal concentration of AA92593 (Glixx Laboratories) at 33.3 μM (final concentration of 16.7 μM, see below).

Techniques: Inhibition, Activation Assay, Comparison, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Molecular basis underlying the specificity of an antagonist AA92593 for mammalian melanopsins

doi: 10.1016/j.jbc.2025.108461

Figure Lengend Snippet: Changes in the susceptibility of invertebrate melanopsins and a non - melanopsin Gq-coupled opsin for AA92593 by amino acid substitutions. A , AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of belcheri melanopsin WT. Error bars indicate the SD values (n = 3). B – G , AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of lanceolatum melanopsin WT ( B ) as well as mutants F189 ECL2 W ( C ), I207 5.42 L ( D ), I207 5.42 F ( E ), A269 6.52 S ( F ), and F189 ECL2 W/I207 5.42 F ( G ). Error bars indicate the SD values (n = 3). H , comparison of inhibition in peak cAMP responses by 16.7 μM AA92593 upon Gsα/q11 activation in belcheri melanopsin WT and lanceolatum melanopsin WT and mutants. Error bars indicate the SD values of independent experiments (n = 3, 8, 4, 3, 3, 3, and 3 for belcheri WT, lanceolatum WT, F189 ECL2 W, I207 5.42 L, I207 5.42 F, A269 6.52 S, F189 ECL2 W/I207 5.42 F, respectively). The statistical p values in differences from WT are <0.001∗, 0.23, <0.001∗, 1.00, and <0.001∗ for F189 ECL2 W, I207 5.42 L, I207 5.42 F, A269 6.52 S, F189 ECL2 W/I207 5.42 F, respectively (Dunnett's test following one-way ANOVA, F = 77, d. f. = 5, 18). I , dose-dependent reduction in peak cAMP responses upon Gsα/q11 activation by lanceolatum melanopsin WT ( red ) and F189 ECL2 W/I207 5.42 F mutant ( blue ). The curve of human melanopsin WT ( black ) is adopted from Figure 1 E . Relative average peak values to the value in the absence of AA92593 are plotted against the final concentrations of AA92593. Error bars indicate the SD values (n = 3). Fitting parameters for the F189 ECL2 W/I207 5.42 F mutant: max , 107.65 ± 12.6; min , −5.22 ± 4.53; rate , −0.84 ± 0.75. IC 50 values for the F189 ECL2 W/I207 5.42 F mutant is 1.40 ± 0.88 μM. J and K , absorption spectra of 9- cis -retinal bound lanceolatum melanopsin WT ( J ) and F189 ECL2 W/I207 5.42 F mutant ( K ). λmax value of WT is indicated. Note that the sharp abruption peak at ∼420 nm in ( K ) ( blue arrowhead) is caused by contamination with cytochrome, not by retinal bound to melanopsin ( purple arrow). L – O , AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of jumping spider rhodopsin-1 WT ( L ), M94 2.61 F/T188 ECL2 S/L269 6.52 S ( M ), I189 ECL2 W ( N ), and Y207 5.42 F ( O ). Error bars indicate the SD values (n = 3). In ( A – G ) and ( L – O ), red and black traces indicate luminescence changes in the presence and absence of 16.7 μM AA92593, respectively. Light blue bars indicate white light illumination (10 s). Luminescence levels of cAMP biosensor (GloSensor) are normalized to the values at the starting point (time = 0 min).

Article Snippet: Under our experimental conditions, >50 μM AA92593 was not fully dissolved in the HBSS solution, and we set maximal concentration of AA92593 (Glixx Laboratories) at 33.3 μM (final concentration of 16.7 μM, see below).

Techniques: Inhibition, Activation Assay, Comparison, Mutagenesis

Journal: bioRxiv

Article Title: Molecular basis underlying specific interaction of mammalian melanopsins with an antagonist AA92593

doi: 10.1101/2024.11.01.621458

Figure Lengend Snippet: ( A and B ) AA92593 concentration-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of human ( A ) and mouse ( B ) melanopsins. Luminescence levels of cAMP biosensor (GloSensor) are normalized to the values at the starting point (time = 0 min). In panel A , final AA92593 concentrations are 16.7 μM (red), 5 μM (orange), 1 μM (purple), 0.1 μM (green), 0.01 μM (blue), and 0 (black), respectively. In panel B , final AA92593 concentrations are 16.7 μM (red), 10 μM (orange), 5 μM (purple), 1 μM (green), 0.1 μM (blue), and 0 (black), respectively. Light blue bars indicate white light illumination (10 sec). Error bars indicate the SD values (n = 3). ( C and D ) Dose-dependent reduction in peak cAMP responses upon Gsα/q11 activation in human ( C ) and mouse ( D ) melanopsin-expressing COS-1 cells. Relative average peak values to the value in the absence of AA92593 are plotted against the final concentrations of AA92593. Error bars indicate the SD values (n = 3). EC 50 values for-human and mouse melanopsins are 1.05±0.28 μM and 2.98±0.58 μM, respectively. ( E and F ) AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα activation of human ( E ) and mouse ( F ) melanopsins. Error bars indicate the SD values (n = 3). ( G and H ) AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/i11 activation of human ( G ) and mouse ( H ) melanopsins. Error bars indicate the SD values (n = 3). In panels E - H , Red and black curves indicate luminescence changes in the presence and absence of 16.7 μM AA92593, respectively. Light blue bars indicate white light illumination (10 sec). Luminescence levels of cAMP biosensor (GloSensor) are normalized to the values at the starting point (time = 0 min).

Article Snippet: The transfected COS-1 cells were incubated at 37°C, 5% CO 2 for 1 day, and the medium was aspirated, followed by addition of 50 μL of HBSS containing 33.3 μM or 3.33 μM AA92593, 0.5% DMSO, and 10 μM coelenterazine h (Wako, Japan).

Techniques: Concentration Assay, Inhibition, Activation Assay, Expressing

Journal: bioRxiv

Article Title: Molecular basis underlying specific interaction of mammalian melanopsins with an antagonist AA92593

doi: 10.1101/2024.11.01.621458

Figure Lengend Snippet: ( A ) Amino acid residues at positions 94, 188, 189, 207, and 269 in various melanopsins and a non-melanopsin Gq-coupled opsin. “Mammalian melanopsin-type” amino acids are highlighted as bold. The amino acid residue numbering in this paper is based on the amino acid sequence of bovine rhodopsin. ( B ) Arrangement of the amino acid residues at positions 94, 188, 189, 207, and 269 in the AlphaFold2-predicted human melanopsin structure . The 11- cis -retinal molecule is adopted from the crystal structure of bovine rhodopsin (PDB ID: 1U19) . Note that the predicted structure of human melanopsin and the crystal structure of bovine rhodopsin with 11- cis -retinal were overlapped to obtain a structure that looks like retinal bound to human melanopsin. The structural models are prepared using PyMOL ( https://pymol.org/ ). ( C - F ) AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of human melanopsin mutants F94C ( C ), S188T ( D ), S269A ( E ), and F94C/S188T/S269A ( F ). Red and black traces indicate luminescence changes in the presence and absence of 16.7 μM AA92593, respectively. Light blue bars indicate white light illumination (10 sec). Luminescence levels of cAMP biosensor (GloSensor) are normalized to the values at the starting point (time = 0 min). Error bars indicate the SD values (n = 3). ( G ) Comparison of inhibition in peak cAMP responses by 16.7 μM AA92593 upon Gsα/q11 activation in human melanopsin WT and mutants. Error bars indicate the SD values (n = 46, 8, 4, 3, 7, 6, 7, and 4 for WT, F94C, S188T, S188A, S269A, F94C/S269A, F94C/S188T/S269A and F94C/S188A/S269A, respectively). The statistical P values in differences from WT are 0.99, <0.001***, 1.00, <0.001***, <0.001***, <0.001***, and <0.001*** for F94C, S188T, S188A, S269A, F94C/S269A, F94C/S188T/S269A, and F94C/S188A/S269A, respectively (Dunnett’s test following one-way ANOVA). ( H and I ) NanoBiT Gq dissociation assay using Gqα/R183Q-LgBiT on human melanopsin WT ( H ) and F94C/S188T/S269A mutant ( I ). Red, blue, and black traces indicate luminescence changes in the presence of 16.7 μM (red), 1.67 μM (blue), and no (black) AA92593. Light blue bars indicate white light illumination (10 sec). NanoLuc luminescence levels are normalized to the values at the starting point (time = 0 min). Error bars indicate the SD values (n = 3).

Article Snippet: The transfected COS-1 cells were incubated at 37°C, 5% CO 2 for 1 day, and the medium was aspirated, followed by addition of 50 μL of HBSS containing 33.3 μM or 3.33 μM AA92593, 0.5% DMSO, and 10 μM coelenterazine h (Wako, Japan).

Techniques: Residue, Sequencing, Inhibition, Activation Assay, Comparison, Mutagenesis

Journal: bioRxiv

Article Title: Molecular basis underlying specific interaction of mammalian melanopsins with an antagonist AA92593

doi: 10.1101/2024.11.01.621458

Figure Lengend Snippet: Note that in each experiment, F94C showed a less AA92593-induced suppression, although the difference was not statistically significant (see main text).

Article Snippet: The transfected COS-1 cells were incubated at 37°C, 5% CO 2 for 1 day, and the medium was aspirated, followed by addition of 50 μL of HBSS containing 33.3 μM or 3.33 μM AA92593, 0.5% DMSO, and 10 μM coelenterazine h (Wako, Japan).

Techniques:

Journal: bioRxiv

Article Title: Molecular basis underlying specific interaction of mammalian melanopsins with an antagonist AA92593

doi: 10.1101/2024.11.01.621458

Figure Lengend Snippet: ( A - D ) AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of mouse melanopsin mutants F94C ( A ), S188T ( B ), S269A ( C ), and F94C/S188T/S269A ( D ). Red and black traces indicate luminescence changes in the presence and absence of 16.7 μM AA92593, respectively. Light blue bars indicate white light illumination (10 sec). Luminescence levels of cAMP biosensor (GloSensor) are normalized to the values at the starting point (time = 0 min). Error bars indicate the SD values (n = 3). (E) Comparison of inhibition in peak cAMP responses by 16.7 μM AA92593 upon Gsα/q11 activation in mouse melanopsin WT and mutants. Error bars indicate the SD values (n = 23, 5, 4, 3, 5, 6, 6, and 5 for WT, F94C, S188T, S188A, S269A, F94C/S269A, F94C/S188T/S269A and F94C/S188A/S269A, respectively). The statistical P values in differences from WT are 0.95, 0.88, 0.81, <0.001***, <0.001***, <0.001***, and <0.001*** for F94C, S188T, S188A, S269A, F94C/S269A, F94C/S188T/S269A and F94C/S188A/S269A, respectively (Dunnett’s test following one-way ANOVA). ( F and G ) NanoBiT Gq dissociation assay on mouse melanopsin WT ( F ) and F94C/S188T/S269A mutant ( G ). Red, blue, and black traces indicate luminescence changes in the presence of 16.7 μM (red), 1.67 μM (blue), and no (black) AA92593. Light blue bars indicate white light illumination (10 sec). NanoLuc luminescence levels are normalized to the values at the starting point (time = 0 min). Error bars indicate the SD values (n = 3).

Article Snippet: The transfected COS-1 cells were incubated at 37°C, 5% CO 2 for 1 day, and the medium was aspirated, followed by addition of 50 μL of HBSS containing 33.3 μM or 3.33 μM AA92593, 0.5% DMSO, and 10 μM coelenterazine h (Wako, Japan).

Techniques: Inhibition, Activation Assay, Comparison, Mutagenesis

Journal: bioRxiv

Article Title: Molecular basis underlying specific interaction of mammalian melanopsins with an antagonist AA92593

doi: 10.1101/2024.11.01.621458

Figure Lengend Snippet: Red and blue traces indicate luminescence changes using Gqα-LgBiT and Gqα/R183Q-LgBiT, respectively, in the absence of AA92593. Light blue bars indicate white light illumination (10 sec). NanoLuc luminescence levels are normalized to the values at the starting point (time = 0 min). Error bars indicate the SD values (n = 3).

Article Snippet: The transfected COS-1 cells were incubated at 37°C, 5% CO 2 for 1 day, and the medium was aspirated, followed by addition of 50 μL of HBSS containing 33.3 μM or 3.33 μM AA92593, 0.5% DMSO, and 10 μM coelenterazine h (Wako, Japan).

Techniques:

Journal: bioRxiv

Article Title: Molecular basis underlying specific interaction of mammalian melanopsins with an antagonist AA92593

doi: 10.1101/2024.11.01.621458

Figure Lengend Snippet: Location of predicted binding positions of AA92593 from AutoDock. Colors indicate different predicted positions.

Article Snippet: The transfected COS-1 cells were incubated at 37°C, 5% CO 2 for 1 day, and the medium was aspirated, followed by addition of 50 μL of HBSS containing 33.3 μM or 3.33 μM AA92593, 0.5% DMSO, and 10 μM coelenterazine h (Wako, Japan).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Molecular basis underlying specific interaction of mammalian melanopsins with an antagonist AA92593

doi: 10.1101/2024.11.01.621458

Figure Lengend Snippet: ( A , B ) Structure of the human melanopsin-AA92593 complex predicted from DiffDock. Parenthesis in panel B show the shortest distances between AA92593 and selected ligands. ( C ) Time evolution of the distances between AA92593 and Phe-94 (blue), Ser-188 (red), and Ser-269 (green) over the 1 µs MD trajectory. ( D ) Per-residue decomposition of the binding energy calculated using MMPBSA.

Article Snippet: The transfected COS-1 cells were incubated at 37°C, 5% CO 2 for 1 day, and the medium was aspirated, followed by addition of 50 μL of HBSS containing 33.3 μM or 3.33 μM AA92593, 0.5% DMSO, and 10 μM coelenterazine h (Wako, Japan).

Techniques: Residue, Binding Assay

Journal: bioRxiv

Article Title: Molecular basis underlying specific interaction of mammalian melanopsins with an antagonist AA92593

doi: 10.1101/2024.11.01.621458

Figure Lengend Snippet: Time evolution of the distances between AA92593 and selected residues in human melanopsin along the 1 µs MD trajectory of the complex. The results for Ile-122 (green), Leu-207 (red) and Trp-189 (blue) (in panel A ), and Val-211 (red) and Trp-265 (blue) (in panel B ) are shown.

Article Snippet: The transfected COS-1 cells were incubated at 37°C, 5% CO 2 for 1 day, and the medium was aspirated, followed by addition of 50 μL of HBSS containing 33.3 μM or 3.33 μM AA92593, 0.5% DMSO, and 10 μM coelenterazine h (Wako, Japan).

Techniques:

Journal: bioRxiv

Article Title: Molecular basis underlying specific interaction of mammalian melanopsins with an antagonist AA92593

doi: 10.1101/2024.11.01.621458

Figure Lengend Snippet: ( A - C ) AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of Xenopus Opn4x WT ( A ) as well as mutants C94F/T188S/A269S ( B ) and L207F ( C ). ( D - F ) AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of chicken Opn4-1 WT ( D ) as well as mutants C94F/T188S/A269S ( E ) and L207F ( F ). In panels A - F , red and black traces indicate luminescence changes in the presence and absence of 16.7 μM AA92593, respectively. Light blue bars indicate white light illumination (10 sec). Luminescence levels of cAMP biosensor (GloSensor) are normalized to the values at the starting point (time = 0 min). Error bars indicate the SD values (n = 3). ( G and H ) Comparison of inhibition in peak cAMP responses by 16.7 μM AA92593 upon Gsα/q11 activation in WT and mutant of Xenopus Opn4x ( G ) and chicken Opn4-1 ( H ). Error bars indicate the SD values (n = 8, 4, and 3 for Xenopus Opn4x WT, C94F/T188S/A269S, and L207F, respectively, and 8, 3, and 3 for chicken Opn4-1 WT, C94F/T188S/A269S, and L207F, respectively). The statistical P values in differences from WT are <0.001*** and <0.001*** for Xenopus Opn4x C94F/T188S/A269S and L207F, respectively, and 0.003** and <0.001*** for chicken Opn4-1 C94F/T188S/A269S and L207F, respectively (Dunnett’s test following one-way ANOVA).

Article Snippet: The transfected COS-1 cells were incubated at 37°C, 5% CO 2 for 1 day, and the medium was aspirated, followed by addition of 50 μL of HBSS containing 33.3 μM or 3.33 μM AA92593, 0.5% DMSO, and 10 μM coelenterazine h (Wako, Japan).

Techniques: Inhibition, Activation Assay, Comparison, Mutagenesis

Journal: bioRxiv

Article Title: Molecular basis underlying specific interaction of mammalian melanopsins with an antagonist AA92593

doi: 10.1101/2024.11.01.621458

Figure Lengend Snippet: ( A - G ) AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of human melanopsin mutants W189F ( A ), W189I ( B ), W189V ( C ), L207V ( D ), L207A ( E ), L207F ( F ), and F94C/S188T/W189F/S269A ( G ). Error bars indicate the SD values (n = 3). ( H ) Comparison of inhibition in peak cAMP responses by 16.7 μM AA92593 upon Gsα/q11 activation in human melanopsin WT and mutants. WT data is the same as . Error bars indicate the SD values (n = 46, 3, 3, 3, 3, 3, 3, and 3 for WT, W189F, W189I, W189V, F94C/S188T/W189F/S269A, L207V, L207A, and L207F, respectively). The statistical P values in differences from WT are <0.001***, <0.001***, <0.001***, <0.001***, 0.010**, 0.07, and 0.59 for W189F, W189I, W189V, F94C/S188T/W189F/S269A, L207V, L207A, and L207F, respectively (Dunnett’s test following one-way ANOVA). ( I ) NanoBiT Gq dissociation assay using Gqα/R183Q-LgBiT on human melanopsin F94C/S188T/W189F/S269A mutant. Red, blue, and black traces indicate luminescence changes in the presence of 16.7 μM (red), 1.67 μM (blue), and no (black) AA92593. Light blue bars indicate white light illumination (10 sec). NanoLuc luminescence levels are normalized to the values at the starting point (time = 0 min). Error bars indicate the SD values (n = 3). ( J ) AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of mouse melanopsin L207F mutant. Error bars indicate the SD values (n = 3). In panels A - G , and J , Red and black traces indicate luminescence changes in the presence and absence of 16.7 μM AA92593, respectively. Light blue bars indicate white light illumination (10 sec). Luminescence levels of cAMP biosensor (GloSensor) are normalized to the values at the starting point (time = 0 min). ( K ) Comparison of inhibition in peak cAMP responses by 16.7 μM AA92593 upon Gsα/q11 activation in mouse melanopsin WT and L207F mutant. WT data is the same as . Error bar indicates the SD value (n = 23 and 3 for WT and L207F, respectively). The statistical P values of L207F in difference from WT is <0.001*** (Dunnett’s test following one-way ANOVA).

Article Snippet: The transfected COS-1 cells were incubated at 37°C, 5% CO 2 for 1 day, and the medium was aspirated, followed by addition of 50 μL of HBSS containing 33.3 μM or 3.33 μM AA92593, 0.5% DMSO, and 10 μM coelenterazine h (Wako, Japan).

Techniques: Inhibition, Activation Assay, Comparison, Mutagenesis

Journal: bioRxiv

Article Title: Molecular basis underlying specific interaction of mammalian melanopsins with an antagonist AA92593

doi: 10.1101/2024.11.01.621458

Figure Lengend Snippet: (A) AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of belcheri melanopsin WT. Error bars indicate the SD values (n = 3). ( B - G ) AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of lanceolatum melanopsin WT ( B ) as well as mutants F189W ( C ), I207L ( D ), I207F ( E ), A269S ( F ), and F189W/I207F ( G ). Error bars indicate the SD values (n = 3). ( H ) Comparison of inhibition in peak cAMP responses by 16.7 μM AA92593 upon Gsα/q11 activation in belcheri melanopsin WT and lanceolatum melanopsin WT and mutants. Error bars indicate the SD values (n=3, 8, 4, 3, 3, 3, and 3 for belcheri WT, lanceolatum WT, F189W, I207L, I207F, A269S, F189W/I207F, respectively). The statistical P values in differences from WT are <0.001***, 0.23, <0.001***, 1.00, and <0.001*** for F189W, I207L, I207F, A269S, F189W/I207F, respectively (Dunnett’s test following one-way ANOVA). ( I - L ) AA92593-dependent inhibition of intracellular cAMP elevation in COS-1 cells upon Gsα/q11 activation of jumping spider rhodopsin-1 WT ( I ), M94F/T188S/L269S ( J ), I189W ( K ), and Y207F ( L ). Error bars indicate the SD values (n = 3). In panels A - G and I - L , Red and black traces indicate luminescence changes in the presence and absence of 16.7 μM AA92593, respectively. Light blue bars indicate white light illumination (10 sec). Luminescence levels of cAMP biosensor (GloSensor) are normalized to the values at the starting point (time = 0 min).

Article Snippet: The transfected COS-1 cells were incubated at 37°C, 5% CO 2 for 1 day, and the medium was aspirated, followed by addition of 50 μL of HBSS containing 33.3 μM or 3.33 μM AA92593, 0.5% DMSO, and 10 μM coelenterazine h (Wako, Japan).

Techniques: Inhibition, Activation Assay, Comparison

Journal: bioRxiv

Article Title: Molecular basis underlying specific interaction of mammalian melanopsins with an antagonist AA92593

doi: 10.1101/2024.11.01.621458

Figure Lengend Snippet: Red and black traces indicate luminescence changes in the presence and absence of 16.7 μM AA92593, respectively. Light blue bars indicate white light illumination (10 sec). Luminescence levels of cAMP biosensor (GloSensor) are normalized to the values at the starting point (time = 0 min). Error bars indicate the SD values (n = 3).

Article Snippet: The transfected COS-1 cells were incubated at 37°C, 5% CO 2 for 1 day, and the medium was aspirated, followed by addition of 50 μL of HBSS containing 33.3 μM or 3.33 μM AA92593, 0.5% DMSO, and 10 μM coelenterazine h (Wako, Japan).

Techniques: