Journal: Nature Communications

Article Title: C5aR1 inhibition reprograms tumor associated macrophages and reverses PARP inhibitor resistance in breast cancer

doi: 10.1038/s41467-024-48637-y

Figure Lengend Snippet: A Design of the study in T22 and T127 models for olaparib treatment for 4 days or 8 days. 4d: 4 days, 8d: 8 days, B Growth curve (left) and weight of tumors at end of study (right) in T22 treated with olaparib (50 mg/kg, po, daily) for 4 days and 8 days. For vehicle treated, n = 6, and vehicle treated, n = 5. Data are presented as mean values±SD. p values are from one-way ANOVA. ** P < 0.01. C Growth curve (left) and weight of tumors at end of study (right) of T127 treated with olaparib for 4 days or 8 days. For vehicle treated, n = 6 and olaparib treated, n = 5. Data are presented as mean values±SD. p values are from one-way ANOVA. ** P < 0.01. D Stratification and cell-type identification of T22 and T127 tumors from mouse models indicated in A . Malignant cells from T127 (Malign_0, Malign_1) and T22 (Malign_2, Malign_3) show distinct cell clusters. Tcm: central memory T cells, Treg: regulator T cells, Tn: naive T cells, Tgd: γδT cells, Tprf: proliferative T cells, my_CAF: myofibroblastic cancer associated fibroblast cells, i_CAF: inflammatory cancer associated fibroblast cells, pDC: plasmacytoid dendritic cells. For vehicle treated, n = 2 and olaparib treated, n = 3. E Bar plot showing relative numbers of epithelial (high CNV cells), NK cells and i_CAF (in %) defined by cell lineage markers listed in (Fig. ) from the scRNAseq datasets in D . Data are presented as mean values ± SD. Statistical significance was not applicable due to the small size of each group. F Dot plot of summarized expression level of each pathway of indicated TAM clusters. Gene lists used for functional pathway scores are listed in Fig. . G Ligand-receptor interaction count changes estimated by CellChat between each TAM cluster and CD8 naive T cells (left) or NK cells (right and defined by cell lineage markers indicated in Fig. ) after olaparib treatment. No interactions were noted in T127 likely due to the low number of T cells in T127. Source data and exact p values are provided as a Source Data file. Source data are provided as a Source Data file.

Article Snippet: For CD8 depletion assays, mice were treated with vehicle, with PMX53C (3 mg/kg, subcutaneously, every second day, starting 7 days before olaparib treatment and continuing throughout the treatment protocol, 5697, Toris) or IgG (1 mg/kg, intra-peritoneal, every second day, starting 7 days before olaparib treatment and continuing throughout the treatment protocol, BE0089, BioXcell) or combination of olaparib (50 mg/kg, oral gavage, daily, Selleckchem, #S7110) and PMX53 (3 mg/kg, subcutaneously, every second day, starting 7 days before olaparib treatment and continuing throughout the treatment protocol, HY-106178, MedChemExpress) or anti-CD8 (5 mg/kg, intraperitoneal, every second day, starting 7 days before olaparib treatment and continuing throughout the treatment protocol, BE0004-1, BioXcell) for 13 days.

Techniques: Expressing, Functional Assay

Journal: Nature Communications

Article Title: C5aR1 inhibition reprograms tumor associated macrophages and reverses PARP inhibitor resistance in breast cancer

doi: 10.1038/s41467-024-48637-y

Figure Lengend Snippet: A Growth curve of T127 (left) and images of representative T127 tumors (right) from co-transplanted T22 and T127 tumor models treated with control, olaparib, PMX53 or the combination for 14 days. n = 5. B Growth curve of T22 (left) and images of representative tumors of T22 tumors (right) from co-transplanted T22 and T127 tumor models treated with control, olaparib, PMX53 or the combination for 14 days. n = 5. (C) Fraction of pro-inflammatory (MHCII hi CD206 lo ) TAMs (left), pro-tumor (MHCII lo CD206 hi ) TAMs (center see gating strategy in Supplementary Fig. ) out of total macrophages (F4/80 + CD11b + ) and ratio of pro-inflammatory and pro-tumor TAMs (right) in each indicated treatment group assessed by flow cytometry. n = 5. D Fraction of CD3 + F4/80 − cells (upper left), CD3 − F4/80 − cells (upper right), CD3 + CD8 + cells (upper right) and CD3 − CD335 + cells (lower right) in each indicated group assessed by flow cytometry. NK cells were gated as CD3 − F4/80 − CD335 + population. n = 5. E Tumor growth curve of T127 (left) and T22 (right) of co-transplanted T22 and T127 tumor models treated with control, anti-CD8 (5 mg/kg, i.p., q.o.d.), PMX53 (3 mg/kg, s.c., q.o.d.) combined with anti-CD8 or the combination of anti-CD8, PMX53 and olaparib (50 mg/kg, o.g., q.d.) for 14 days. aCD8: anti-CD8. P + O: PMX53 combined with olaparib. n = 5. F Tumor growth curve of T22 in mice receiving (5×10 5 ) C5aR1 hi cells treated with vehicle or olaparib for 8 days. Treatment started on the day after transfer of C5aR1 hi cells. n = 5. G Fraction of MHC-II lo C5aR1 hi , CD3 + F4/80 − cells, CD3 − F4/80 − cells, CD3 + CD8 + cells and CD3 − CD335 + cells in each treatment group from E assessed by flow cytometry. n = 5. All data are presented as mean values± SD. p values are from one-way ANOVA. * p < 0.05, ** P < 0.01, *** p < 0.001, **** p < 0.0001. Source data and exact p values are provided as a Source Data file. Source data are provided as a Source Data file.

Article Snippet: For CD8 depletion assays, mice were treated with vehicle, with PMX53C (3 mg/kg, subcutaneously, every second day, starting 7 days before olaparib treatment and continuing throughout the treatment protocol, 5697, Toris) or IgG (1 mg/kg, intra-peritoneal, every second day, starting 7 days before olaparib treatment and continuing throughout the treatment protocol, BE0089, BioXcell) or combination of olaparib (50 mg/kg, oral gavage, daily, Selleckchem, #S7110) and PMX53 (3 mg/kg, subcutaneously, every second day, starting 7 days before olaparib treatment and continuing throughout the treatment protocol, HY-106178, MedChemExpress) or anti-CD8 (5 mg/kg, intraperitoneal, every second day, starting 7 days before olaparib treatment and continuing throughout the treatment protocol, BE0004-1, BioXcell) for 13 days.

Techniques: Control, Flow Cytometry

Journal: Nature Communications

Article Title: C5aR1 inhibition reprograms tumor associated macrophages and reverses PARP inhibitor resistance in breast cancer

doi: 10.1038/s41467-024-48637-y

Figure Lengend Snippet: A Work flow of T127 tumor cells purified from vehicle control mice as described in Fig. , CD11b + C5aR1 −/+ macrophages isolated from tumor naive mice and T cells from tumor naive mice ex vivo co-culture assay (see Methods for details). Briefly, on day 1, 1 × 10 6 T127 tumor cells from vehicle treated mice were co-cultured with 5 × 10 5 CD11b + C5aR1 −/+ macrophages with and without PMX53 (40 nM). On day 2, CD11b + C5aR1 −/+ macrophages were isolated and then co-cultured with 5 × 10 5 isolated CD8 T cells. CD11b + C5aR1 −/+ macrophages were isolated from CD11b + positive splenocytes as described in Methods. Fraction of GZMB + , PRF1 + , IFNγ + CD8 + T cells in each indicated group from left assessed by flow cytometry. n = 3. Data are presented as mean values±SD. Comparison was done by one-way ANNOVA. * p < 0.05, ** P < 0.01, *** p < 0.001, **** p < 0.0001. B Work flow of T127 tumor cells purified from mice treated with olaparib as described in Fig. , CD11b + C5aR1 −/+ macrophages isolated from tumor naive mice and T cells from tumor naïve mice ex vivo co-culture assay (see Methods for details). Briefly, on day 1, 1 × 10 6 T127 tumor cells from olaparib treated mice were co-cultured with 5 × 10 5 CD11b + C5aR1 −/+ macrophages. On day 2, CD11b + C5aR1 −/+ macrophages were isolated and then co-cultured with 5 × 10 5 isolated CD8 T cells. CD11b + C5aR1 −/+ macrophages were isolated from CD11b + positive splenocytes. Fraction of GZMB + , PRF1 + , IFNγ + cells of CD8 + T cells in each indicated group from left assessed by flow cytometry. n = 3. Data are presented as mean values ± SD. Comparison was done by one-way ANNOVA. * p < 0.05, ** P < 0.01, *** p < 0.001, **** p < 0.0001. Source data and exact p values are provided as a Source Data file. Source data are provided as a Source Data file.

Article Snippet: For CD8 depletion assays, mice were treated with vehicle, with PMX53C (3 mg/kg, subcutaneously, every second day, starting 7 days before olaparib treatment and continuing throughout the treatment protocol, 5697, Toris) or IgG (1 mg/kg, intra-peritoneal, every second day, starting 7 days before olaparib treatment and continuing throughout the treatment protocol, BE0089, BioXcell) or combination of olaparib (50 mg/kg, oral gavage, daily, Selleckchem, #S7110) and PMX53 (3 mg/kg, subcutaneously, every second day, starting 7 days before olaparib treatment and continuing throughout the treatment protocol, HY-106178, MedChemExpress) or anti-CD8 (5 mg/kg, intraperitoneal, every second day, starting 7 days before olaparib treatment and continuing throughout the treatment protocol, BE0004-1, BioXcell) for 13 days.

Techniques: Purification, Control, Isolation, Ex Vivo, Co-culture Assay, Cell Culture, Flow Cytometry, Comparison

Journal: eLife

Article Title: AI-based discovery and cryoEM structural elucidation of a K ATP channel pharmacochaperone

doi: 10.7554/eLife.103159

Figure Lengend Snippet: ( A ) Representative western blots of SUR1 from COSm6 cells co-transfected with cDNAs of wild-type (WT) Kir6.2 and trafficking mutants of SUR1 TMD0 domain, A30T, A116P, or V187D, and treated with either 0.1% DMSO (0 μM) or 100 μM Aekatperone (AKP) for 16 hr. The core-glycosylated immature SUR1 and the complex-glycosylated mature SUR1 are indicated by the black and gray arrows, respectively. The tubulin blot below serves as a loading control. ( B ) Representative western blots of SUR1 from COSm6 cells co-transfected with cDNAs of WT Kir6.2 and a SUR1 trafficking mutant A30T, and treated with either 0.1% DMSO (0 μM) or various concentrations of Aekatperone (10, 50, 100, 150, 200 μM) for 16 hr. WT SUR1 from cells co-transfected with WT Kir6.2 and WT SUR1 without Aekatperone treatment served as a control (left lane). ( C ) Representative recording from COSm6 cells co-transfected with hamster SUR1 and rat Kir6.2. Channels were exposed to K-INT solution upon patch excision (arrow) and exposed to solutions containing MgATP, MgADP, or Aekatperone as indicated by the bars above the recordings and the labels on the right. The patch was exposed to 1 mM ATP periodically to ensure the baseline has not shifted (gray dashed line). ( D ) Quantification of currents (normalized to currents in K-INT/1 mM EDTA at the time of patch excision) in various solutions from recordings such as that shown in ( A ). Each bar represents the mean ± SEM of at least three patches, with circles showing individual patches. *p<0.05 by one-way ANOVA and Dunnet’s post hoc test. Figure 2—source data 1. PDF file containing original western blots for panels A and B, indicating the relevant bands and treatments. Figure 2—source data 2. Original files for western blot analysis displayed in panels A and B.

Article Snippet: Of note, to ensure Aekatperone is the active compound in our biochemical and functional assays, we tested Aekatperone synthesized from a different commercial source (Acme Bioscience, Palo Alto, CA, USA) and confirmed its effects on K ATP channels ( ).

Techniques: Western Blot, Transfection, Control, Mutagenesis

Journal: eLife

Article Title: AI-based discovery and cryoEM structural elucidation of a K ATP channel pharmacochaperone

doi: 10.7554/eLife.103159

Figure Lengend Snippet: Top : Surface staining of COSm6 cells transiently co-transfected with Kir6.2 WT and various FLAG-tagged (extracellular N-terminus) SUR1 constructs (WT, F27S, or A30T) treated with or without Aekatperone using M2 anti-FLAG mouse monoclonal antibody followed by Alexa Fluor 546-conjugated anti-mouse secondary antibody. Bottom : Total cellular staining of FLAG-tagged SUR1. Cells were fixed and permeabilized prior to incubation with primary and secondary antibodies. Images shown are stacked Olympic Fluoview confocal sections, with nuclei counterstained using DAPI. Scale bar , 10 μm.

Article Snippet: Of note, to ensure Aekatperone is the active compound in our biochemical and functional assays, we tested Aekatperone synthesized from a different commercial source (Acme Bioscience, Palo Alto, CA, USA) and confirmed its effects on K ATP channels ( ).

Techniques: Staining, Transfection, Construct, Incubation

Journal: eLife

Article Title: AI-based discovery and cryoEM structural elucidation of a K ATP channel pharmacochaperone

doi: 10.7554/eLife.103159

Figure Lengend Snippet: ( A ) Representative western blot of SUR1 from COSm6 cells co-transfected with cDNAs of wild-type (WT) human Kir6.2 and human SUR1 A30T trafficking mutant and treated with either 0.1% DMSO (0 μM) or various concentrations of Aekatperone (AKP) for 16 hr. ( B ) Representative recording from COSm6 cells co-transfected with human SUR1 and Kir6.2. Channels were exposed to K-INT solution upon patch excision (arrow) and exposed to solutions containing MgATP, MgADP, or Aekatperone as indicated by the bars above the recordings and the labels on the right. The patch was exposed to 1 mM ATP periodically to ensure the baseline has not shifted (gray dashed line). ( C ) Rb + efflux assay results showing Aekatperone dose-dependently inhibits human WT K ATP channels expressed in COSm6 cells and opened by metabolic inhibition (see Methods). The fractional Rb + efflux was calculated by subtracting efflux in untransfected cells and normalizing to efflux in cells treated with 0.1% DMSO. ( D ) Aekatperone inhibition dose-response curve from data shown in ( C ) fitted with a Hill equation with variable slope using GraphPad Prism 10. Figure 2—figure supplement 2—source data 1. PDF file containing original western blots for panel A, indicating the relevant bands and treatments. Figure 2—figure supplement 2—source data 2. Original files for western blot analysis displayed in panel A.

Article Snippet: Of note, to ensure Aekatperone is the active compound in our biochemical and functional assays, we tested Aekatperone synthesized from a different commercial source (Acme Bioscience, Palo Alto, CA, USA) and confirmed its effects on K ATP channels ( ).

Techniques: Western Blot, Transfection, Mutagenesis, Inhibition

Journal: eLife

Article Title: AI-based discovery and cryoEM structural elucidation of a K ATP channel pharmacochaperone

doi: 10.7554/eLife.103159

Figure Lengend Snippet: ( A ) Rb + efflux assay results showing Aekatperone (AKP) dose-dependently inhibited wild-type (WT) pancreatic K ATP channels (hamster SUR1 and rat Kir6.2) expressed in COSm6 cells and opened by metabolic inhibition (see Methods). The fractional Rb + efflux was calculated by subtracting efflux in untransfected cells and normalizing to efflux in cells treated with 0.1% DMSO. ( B ) Dose-response curve of Aekatperone inhibition from data shown in ( A ) fitted with a Hill equation with variable slope using GraphPad Prism 10. The IC 50 is 9.23 µM±0.36 µM. Error bars represent the SEM. ( C, D ) Bar graphs showing dose-response enhancement in K ATP channel activity as assessed by Rb + efflux assay in COSm6 cells expressing two different trafficking mutations, SUR1 F27S ( A ) and SUR1 A30T ( B ). The cells were treated with varying concentrations of Aekatperone (10, 30, 50, 100, or 200 μM), glibenclamide (GBC) at 10 μM, or 0.1% DMSO as a vehicle control (0 μM Aekatperone) for 16 hr. Aekatperone and GBC was excluded from the efflux solutions during the efflux assay. Untransfected (UT) cells were included to quantify background Rb + efflux, which was subtracted from other experimental readings. The data were normalized to the fractional Rb + efflux of cells expressing WT channels. Error bars represent the SEM of at least three independent experiments (circles are individual data points from three to six different experiments). Statistical significance was performed using one-way ANOVA followed by Dunnett’s post hoc multiple comparison test, alpha = 0.05. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. A red dashed line is shown to indicate the basal efflux of the mutant channels under vehicle control conditions (in the absence of Aekatperone). ( E ) Schematic of experimental design for ( F ). COSm6 cells transfected with WT or various mutant channels were treated with 0.1% DMSO (vehicle control) or 100 Aekatperone (AKP) overnight in the presence of Rb + . Before efflux measurements, cells were washed in an RbCl containing buffer lacking AKP for 30 min. Efflux was then measured for 30 min in a Ringer’s solution±diazoxide (Diaz) at 200 μM. Note, diazoxide was included in Ringer’s solution during the efflux assay but not during the overnight incubation. ( F ) Rb + efflux experiments showing overnight treatment with AKP enhances acute Diaz response in COSm6 cells expressing trafficking mutants. Each bar represents the mean ± SEM of at least three different biological repeats, with circles indicating individual data points, alpha = 0.05. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: Of note, to ensure Aekatperone is the active compound in our biochemical and functional assays, we tested Aekatperone synthesized from a different commercial source (Acme Bioscience, Palo Alto, CA, USA) and confirmed its effects on K ATP channels ( ).

Techniques: Inhibition, Activity Assay, Expressing, Control, Comparison, Mutagenesis, Transfection, Incubation

Journal: eLife

Article Title: AI-based discovery and cryoEM structural elucidation of a K ATP channel pharmacochaperone

doi: 10.7554/eLife.103159

Figure Lengend Snippet: Note the exact concentrations of Aekatperone may not precisely match those shown in due to the very small quantity received from Acme Bioscience, which made reconstitution less accurate.

Article Snippet: Of note, to ensure Aekatperone is the active compound in our biochemical and functional assays, we tested Aekatperone synthesized from a different commercial source (Acme Bioscience, Palo Alto, CA, USA) and confirmed its effects on K ATP channels ( ).

Techniques:

Journal: eLife

Article Title: AI-based discovery and cryoEM structural elucidation of a K ATP channel pharmacochaperone

doi: 10.7554/eLife.103159

Figure Lengend Snippet: ( A ) Representative recordings from COSm6 cells expressing SUR2A/Kir6.2 (top) or SUR2B/Kir6.2 channels. Channels were exposed to K-INT solution upon patch excision (gray arrow) and exposed to solutions containing MgATP, MgADP, or Aekatperone as indicated by the bars above the recordings and the labels on the right. The patch was exposed to 1 mM ATP periodically to ensure the baseline has not shifted (gray dashed line). ( B ) Half-maximal inhibitory concentration (IC 50 ) of Aekatperone on SUR2A/Kir6.2 or SUR2B/Kir6.2 channels transiently expressed in COSm6 cells Kir6.2 using Rb + efflux assay. Data were fit with a Hill equation with variable slope using GraphPad Prism 10. The Aekatperone IC 50 is 41.22 µM±2.22 µM (SEM) for SUR2A/Kir6.2 channels and 41.85 µM±2.08 µM (SEM) for SUR2B/Kir6.2 channels. The error bar for each data point represents the SEM. Red dotted line represents the dose-response curve of Aekatperone on SUR1/Kir6.2 channels from for comparison.

Article Snippet: Of note, to ensure Aekatperone is the active compound in our biochemical and functional assays, we tested Aekatperone synthesized from a different commercial source (Acme Bioscience, Palo Alto, CA, USA) and confirmed its effects on K ATP channels ( ).

Techniques: Expressing, Concentration Assay, Comparison

Journal: eLife

Article Title: AI-based discovery and cryoEM structural elucidation of a K ATP channel pharmacochaperone

doi: 10.7554/eLife.103159

Figure Lengend Snippet: ( A ) Structural model of the pancreatic K ATP channel in complex with Aekatperone showing SUR1 in NBD-separated conformation and the Kir6.2 pore (red) constricted at the helical bundle crossing (HBC) and the selectivity filter (SF) gates. Aekatperone is shown as purple mesh (0.08 V contour) and N-terminal domain of Kir6.2 (KNtp) as blue mesh (0.08 V contour). Only one SUR1 subunit attached to Kir6.2 core is shown based on the focused refinement cryo-electron microscopy (cryoEM) map of the Kir6.2 tetramer and a single SUR1 subunit. SUR1 transmembrane domain (TMD) 1 and 2 are colored in dark green and light green, respectively. NBD, nucleotide binding domain; L0, L0 loop of SUR1. ( B, C ) Close-up side view ( B ) and top-down view ( C ) of the Aekatperone binding site. Aekatperone cryoEM density and model are shown in magenta and SUR1 residues that interact with Aekatperone are shown as sticks. KNtp is shown as a blue main chain peptide. Red numbers indicate the numbers of helices of SUR1. ( D ) Aekatperone cryoEM density map (0.08 V contour) and model fitting in two different views. Note at the contour shown, some surrounding density from interacting SUR1 and Kir6.2 is included.

Article Snippet: Of note, to ensure Aekatperone is the active compound in our biochemical and functional assays, we tested Aekatperone synthesized from a different commercial source (Acme Bioscience, Palo Alto, CA, USA) and confirmed its effects on K ATP channels ( ).

Techniques: Cryo-Electron Microscopy, Binding Assay

Journal: eLife

Article Title: AI-based discovery and cryoEM structural elucidation of a K ATP channel pharmacochaperone

doi: 10.7554/eLife.103159

Figure Lengend Snippet: ( A ) Aekatperone (AKP)-bound map (EMD-46820) superimposed with the structural model (PDB ID 9DFX) and ( B ) an AKP-free apo map (EMD-26320), also superimposed with the protein structural model (PDB ID 9DFX) for comparison.The cryo-electron microscopy (cryoEM) density in both maps was filtered to 4.5 Å, aligned in ChimeraX, normalized and displayed using PyMOL at 4.6 RMSD (equivalent to 0.08 V contour). The cryoEM density corresponding to AKP colored in magenta in ( A ) is absent in ( B ). The Kir6.2 N-terminal peptide (KNtp) density (modeled as a polyalanine chain) colored in blue is stronger in AKP-bound map than in the apo map. The cryoEM density corresponding to SUR1 is colored in green in both panels.

Article Snippet: Of note, to ensure Aekatperone is the active compound in our biochemical and functional assays, we tested Aekatperone synthesized from a different commercial source (Acme Bioscience, Palo Alto, CA, USA) and confirmed its effects on K ATP channels ( ).

Techniques: Comparison, Cryo-Electron Microscopy

Journal: eLife

Article Title: AI-based discovery and cryoEM structural elucidation of a K ATP channel pharmacochaperone

doi: 10.7554/eLife.103159

Figure Lengend Snippet: Calculated fluctuations in membrane thickness ( A ) and area per lipid indicating membrane density (black and cyan colors correspond to the upper and lower leaflet of the membrane, respectively) ( B ) suggest sufficient equilibration of the system with regard to the membrane. An additional 500 ns of simulation (5 repetitions) and the calculated RMSD values for the Kir6.2 tetramer and SUR1 protein ( C , black and cyan respectively) indicate fairly good conformational stability of our system. Root mean square fluctuations for the N-terminal residues of Kir6.2 ( D ) located in the SUR1 pocket adjacent to the docked Aekatperone show that the first three amino acids of KNtp buried in the SUR1 pocket retained some conformational freedom and freely sampled the available space.

Article Snippet: Of note, to ensure Aekatperone is the active compound in our biochemical and functional assays, we tested Aekatperone synthesized from a different commercial source (Acme Bioscience, Palo Alto, CA, USA) and confirmed its effects on K ATP channels ( ).

Techniques: Membrane

Journal: eLife

Article Title: AI-based discovery and cryoEM structural elucidation of a K ATP channel pharmacochaperone

doi: 10.7554/eLife.103159

Figure Lengend Snippet: ( A ) Scatter of the positions of the centers of mass of individual fragments of Aekatperone during the simulation. The initial position is marked as sticks. Green helices are TMD1/2 of SUR1, and the blue fragment is KNtp. ( B ) Standard deviation of the positions of the centers of mass of individual fragments during the simulation. The fragments are color-coded throughout the figure. ( C ) Frequency of staying in close contact with SUR1 and KNtp residues during the simulation, divided by individual fragments. ( D, E ) An example snapshot from the simulation of Aekatperone in the pocket showing its interactions with SUR1 and KNtp residues - side view ( D ) and top view ( E ).

Article Snippet: Of note, to ensure Aekatperone is the active compound in our biochemical and functional assays, we tested Aekatperone synthesized from a different commercial source (Acme Bioscience, Palo Alto, CA, USA) and confirmed its effects on K ATP channels ( ).

Techniques: Standard Deviation

Journal: eLife

Article Title: AI-based discovery and cryoEM structural elucidation of a K ATP channel pharmacochaperone

doi: 10.7554/eLife.103159

Figure Lengend Snippet: Close contact frequency between first six residues of KNtp (labeled as KNt followed by residue number) and its surroundings in the Aekatperone (AKP) binding pocket.

Article Snippet: Of note, to ensure Aekatperone is the active compound in our biochemical and functional assays, we tested Aekatperone synthesized from a different commercial source (Acme Bioscience, Palo Alto, CA, USA) and confirmed its effects on K ATP channels ( ).

Techniques: Labeling, Residue, Binding Assay

Journal: eLife

Article Title: AI-based discovery and cryoEM structural elucidation of a K ATP channel pharmacochaperone

doi: 10.7554/eLife.103159

Figure Lengend Snippet: ( A ) 2D Aekatperone binding site model showing chemical interactions between the compound and surrounding SUR1 residues and KNtp. ( B ) Rb + efflux experiments testing the effect of mutating select binding site residues on channel response to Aekatperone. Each bar is the mean and error bars represent the SEM of at least three independent experiments (individual data points shown as circles). Statistical significance is based on one-way ANOVA with Dunnett’s post hoc multiple comparisons test, with alpha = 0.05. **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Of note, to ensure Aekatperone is the active compound in our biochemical and functional assays, we tested Aekatperone synthesized from a different commercial source (Acme Bioscience, Palo Alto, CA, USA) and confirmed its effects on K ATP channels ( ).

Techniques: Binding Assay

Journal: eLife

Article Title: AI-based discovery and cryoEM structural elucidation of a K ATP channel pharmacochaperone

doi: 10.7554/eLife.103159

Figure Lengend Snippet: ( A ) Cryo-electron microscopy (cryoEM) density map of the pancreatic K ATP channel in complex with Aekatperone (AKP), with the SUR1 subunit in the front shown in vertical sliced view. KNtp is highlighted in blue and Aekaperone in magenta. ( B ) Close-up view of the AKP (magenta) and KNtp (blue) structures overlayed with corresponding cryoEM densities (at 0.08 V) showing the close proximity of the imidazole group of Aekatperone to KNtp. ( C ) Comparison of the effects of Aekatperone on COSm6 cells expressing either wild-type (WT) K ATP channels or channels with KNtp deletions of either 5 or 10 amino acids (Δ5 or Δ10 KNtp) using the Rb + efflux assay. Rb + efflux assays were performed in the presence of metabolic inhibitors with various concentrations of Aekatperone as indicated on the x-axis. The data were normalized against the fractional Rb + efflux of untreated cells expressing either WT or KNtp mutant channels with metabolic inhibition but without Aekatperone. Each bar is the mean and error bars represent the SEM of five to six independent experiments (individual data points shown as light black circles). Statistical significance is based on one-way ANOVA and Tukey’s post hoc multiple comparisons test. Alpha = 0.05. **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Of note, to ensure Aekatperone is the active compound in our biochemical and functional assays, we tested Aekatperone synthesized from a different commercial source (Acme Bioscience, Palo Alto, CA, USA) and confirmed its effects on K ATP channels ( ).

Techniques: Cryo-Electron Microscopy, Comparison, Expressing, Mutagenesis, Inhibition