Crystal structure of MET (gray) showing the design of the bioorthogonal MET imaging agents.

<p>(A) Foretinib-TCO (B) PF04217903-TCO. Note that the bioorthogonal transcyclooctene (TCO) is predicted to project outside from the target so that it is available for reaction with the fluorescent counter partner. (PDB ID: 3LQ8 and 3ZXZ) 3D models were rendered using PyMol.</p

Inhibitory effect of Foretinib based imaging agents.

<p>(A) The IC₅₀ values for Foretinib, Foretinib-TCO (<b>11</b>) and Foretinib-BODIPY-FL (<b>12</b>) against purified MET were determined using the z’-lyte kinase assay. Note the much lower affinity of the fluorochrome conjugated drug compared to the bioorthogonal version. (B) Representative western blot of MET phosphorylation inhibition by Foretinib, Foretinib-TCO (<b>11</b>) and Foretinib-BODIPY-FL(<b>12</b>) in OVCA429 cells. Following pre-treatment with increasing concentrations of inhibitors (0, 100, 500, or 1000 nM, respectively), MET phosphorylation was stimulated with HGF for 10 min, followed by cell lysis, SDS-PAGE, and Western blot with MET and phospho-MET antibodies. (C) Densiometric quantification of MET phosphorylation from the Western blot data using ImageJ. (D) The IC₅₀ values for Foretinib and Foretinib-TCO (<b>11</b>) against purified AXL, PDGFRα, RON and KDR were determined using the z’-lyte kinase assay. R² values for the dose-response curve fit (GraphPad, Prism) were 0.92 or greater.</p

Bioorthogonal labeling using the specific MET CID PF04217903-TCO (15) or the PCID Foretinib-TCO (11) in OVCA429 (MET positive) and SK-BR-3 (MET negative) cells.

<p>Cells were incubated for 1hr with 200 nM Foretinib-TCO (<b>11</b>) or 40 nM PF04217903-TCO (<b>15</b>), washed and incubated for 30 min with 1 μM Tz-CFDA (e-h) for bioorthogonal reaction inside living cells. After fixation with 2% paraformaldehyde, MET was labeled using a MET primary antibody and AlexaFluor 647 labeled secondary antibody (i-l). After nuclear staining with Hoechst 33342 (a-d) for 10 min, 40X images were collected using a DeltaVision microscope. Note the excellent co-localization between the MET antibody and affinity ligands on the membrane of the cells (m-p). Scale bar: 10 μm.</p

Synthetic scheme of Foretinib-TCO (11), Foretinib-BODIPY-FL (12) and PF04217903-TCO (15).

<p>Boc = <i>tert</i>-butyloxycarbonyl; Bn = Benzyl; BODIPY-FL = 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl; DMAP = 4-dimethylaminopyridine; DCM = dichloromethane; DIPEA = N,N-Diisopropylethylamine; DME = dimethoxyethane; DMF = dimethylformamide; Ms = methanesulfonyl; TCO = trans-cyclooctene; TEA = triethylamine; TFA = trifluoroacetic acid; THF = tetrahydrofuran; NHS = <i>N</i>-hydroxysuccinimide.</p

Optimized Near-IR Fluorescent Agents for in Vivo Imaging of Btk Expression

Bruton’s tyrosine kinase (Btk) is intricately involved in anti-apoptotic signaling pathways in cancer and in regulating innate immune response. A number of Btk inhibitors are in development for use in treating B-cell malignancies and certain immunologic diseases. To develop robust companion imaging diagnostics for in vivo use, we set out to explore the effects of red wavelength fluorochrome modifications of two highly potent irreversible Btk inhibitors, Ibrutinib and AVL-292. Surprisingly, we found that subtle chemical differences in the fluorochrome had considerable effects on target localization. Based on iterative designs, we developed a single optimized version with superb in vivo imaging characteristics enabling single cell Btk imaging in vivo. This agent (Ibrutinib-SiR-COOH) is expected to be a valuable chemical tool in deciphering Btk biology in cancer and host cells in vivo

Locomotion and anxiety-like behavior of GIRK2^WT and GIRK2^AgRP-KO mice.

Related to Fig 6. (A) Bar graphs and dots summarize ambulatory movement of GIRK2WT (n = 14) and GIRK2AgRP-KO (n = 16) mice (260.8 ± 48.5 counts, n = 14, for GIRK2WT and 256.9 ± 48.8 counts, n = 16, for GIRK2AgRP-KO, df = 28, t = 0.057, p = 0.955 in dark cycle; 58.0 ± 10.3 counts, n = 14, for GIRK2WT and 51.6 ± 8.1 counts, n = 16, for GIRK2AgRP-KO, df = 28, t = 0.4921, p = 0.627 in light cycle). (B) Bar graphs and dots summarize rearing activity of GIRK2WT (n = 14) and GIRK2AgRP-KO (n = 16) mice (148.3 ± 27.1 counts, n = 14, for GIRK2WT and 146.3 ± 32.0 counts, n = 16, for GIRK2AgRP-KO, df = 28, t = 0.049, p = 0.962 in dark cycle; 26.7 ± 9.6 counts, n = 14, for GIRK2WT and 18.7 ± 3.9 counts, n = 16, for GIRK2AgRP-KO, df = 28, t = 0.804, p = 0.428 in light cycle). (C) Trajectory of freely moving GIRK2WT (n = 8) and GIRK2AgRP-KO (n = 9) mice in the OFT chamber in dark and light cycles. (D) Bar graphs and dots summarize total moving distance of GIRK2WT (n = 8) and GIRK2AgRP-KO (n = 9) mice (95.1 ± 9.0 m, n = 8, for GIRK2WT and 108.1 ± 4.1 m, n = 9, for GIRK2AgRP-KO, df = 15, t = 1.370, p = 0.191 in dark cycle; 113.8 ± 6.6 m, n = 8, for GIRK2WT and 123.9 ± 7.8 m, n = 9, for GIRK2AgRP-KO, df = 15, t = 0.980, p = 0.343 in light cycle). (E) Image demonstrates a view of chamber by a camera that is installed on the ceiling of sound-proof booths. (F) Heat-maps demonstrate zone preference of GIRK2WT and GIRK2AgRP-KO mice in the chamber. (G) Bar graphs and dots summarize proportions of duration in center and outer zones of GIRK2WT (n = 8) and GIRK2AgRP-KO (n = 9) mice (10.6 ± 1.8%, n = 8, for GIRK2WT and 8.4 ± 0.6%, n = 9, for GIRK2AgRP-KO, df = 15, t = 1.224, p = 0.240 in dark cycle and center; 13.4 ± 1.4%, n = 8, for GIRK2WT and 12.3 ± 1.6%, n = 9, for GIRK2AgRP-KO, df = 15, t = 0.523, p = 0.609 in light cycle and center; 89.4 ± 1.8%, n = 8, for GIRK2WT and 91.6 ± 0.6%, n = 9, for GIRK2AgRP-KO, df = 15, t = 1.224, p = 0.240 in dark cycle and outer; 86.6 ± 1.4%, n = 8, for GIRK2WT and 87.8 ± 1.6%, n = 9, for GIRK2AgRP-KO, df = 15, t = 0.523, p = 0.609 in light cycle and outer). Data are presented as mean ± SEM. Unpaired t test was used for statistical analyses. ns = not significant. The numerical data for S9A, S9B, S9D, and S9G Fig can be found in S6 Data. (TIF)</p

GIRK channels stabilize RMP of NPY neurons.

(A) Brightfield illumination (Brightfield), fluorescent (FITC) illumination (Npy-hrGFP), fluorescent (TRITC) illumination (Alexa Fluor 594), and merged (Merge) images of targeted NPY neuron. Arrows indicate the cell targeted for whole-cell patch clamp recording. (B) Image demonstrates a depolarizing effect of tertiapin-Q. Dotted line indicates RMP. (C) Voltage deflections in response to small hyperpolarizing current steps (from −25 pA to 0 pA by 5 pA increments) before (control, black) and after (tertiapin-Q, red) the perfusion with tertiapin-Q as indicated by arrows in (B). (D) The voltage–current (V-I) relationship demonstrates increased input resistance by tertiapin-Q. Erev = reversal potential. (E) Lines and dots summarize effects of tertiapin-Q on RMP (from −47.7 ± 3.0 mV to −44.9 ± 2.1 mV, n = 11, df = 10, t = 2.787, p = 0.019). Red and black lines indicate changes of membrane potential in depolarized and nonresponsive neurons, respectively. (F) Lines and dots summarize effect of tertiapin-Q on input resistance (from 2.75 ± 0.27 GΩ to 3.03 ± 0.30 GΩ, n = 11, df = 10, t = 4.370, p = 0.001). Red and black lines indicate changes of input resistance in depolarized and nonresponsive neurons, respectively. (G, H) Lines and dots summarize effects of 100 nM tertiapin-Q (G) (from −41.2 ± 0.8 mV to −40.0 ± 1.1 mV, n = 11, df = 10, t = 2.040, p = 0.069) and 500 nM tertiapin-Q (H) (from −42.9 ± 1.2 mV to −40.5 ± 1.1 mV, n = 13, df = 12, t = 3.292, p = 0.006) on RMP. Red and black lines indicate changes of membrane potential in depolarized and nonresponsive neurons, respectively. (I) Histogram summarizes responses (no effects or depolarization) of NPY neurons to different concentrations of tertiapin-Q. (J) Bar graphs and dots summarize effects of K+ channel blockers. Each neuron was tested with only 1 K+ channel blocker. Data are presented as mean ± SEM. Paired t test was used for statistical analyses. *p p S1 Data. GIRK, G protein-gated inwardly rectifying K+; NPY, neuropeptide Y; RMP, resting membrane potential.</p

Expression of <i>Girk</i> mRNA by arcuate AgRP neurons.

Related to Fig 2. (A) Graph demonstrates percentage of Agrp (+) neurons that express mRNA of Girk1 and/or Girk2. Girk1 (green): Girk1-containing Agrp (+) neurons; Girk2 (magenta): Girk2-containing Agrp (+) neurons; Girk1 and Girk2 (gray): Agrp (+) neurons containing both Girk1 and Girk2. n = 3. (B) Graph demonstrates percentage of Agrp (+) neurons that express mRNA of Girk1 and/or Girk3. Girk1 (green): Girk1-containing Agrp (+) neurons; Girk3 (cyan): Girk3-containing Agrp (+) neurons; Girk1 and Girk3 (gray): Agrp (+) neurons containing both Girk1 and Girk3. n = 3. (C) Graph demonstrates percentage of Agrp (+) neurons that express mRNA of Girk1 and/or Girk4. Girk1 (green): Girk1-containing Agrp (+) neurons; Girk4 (orange): Girk4-containing Agrp (+) neurons; Girk1 and Girk4 (gray): Agrp (+) neurons containing both Girk1 and Girk4. n = 3. (D) Graph demonstrates percentage of Agrp (+) neurons that express mRNA of Girk2 and/or Girk3. Girk2 (magenta): Girk2-containing Agrp (+) neurons; Girk3 (cyan): Girk3-containing Agrp (+) neurons; and Girk2 and Girk3 (gray): Agrp (+) neurons containing both Girk2 and Girk3. n = 3. Data are presented as mean ± SEM. Twelve hypothalamic slices from each mouse (from bregma −1.58 mm to −2.02 mm) were included for analyses. See text for specific values. The numerical data for S3A–S3D Fig can be found in S2 Data. (TIF)</p

Original data for the graphs in Figs 4 and S7 and S8.

Each tab includes data for individual panels of Figs 4 and S7 and S8. (XLSX)</p

Effects of K⁺ channel blockers on RMP of NPY neurons.

Related to Fig 1. (A) Trace demonstrates depolarizing effects of linopirdine and XE991, M channels blockers. (B) Trace demonstrates no effects of PK-THPP, a TASK-3 channel blocker. (C) Trace demonstrates no effects of spadin, a TREK-1 channel blocker. (D) Trace demonstrates no effects of tolbutamide, a KATP channel blocker. (E–H) Bar graphs and dots summarize effects on RMP change of linopirdine and XE991 (from −40.4 ± 0.7 mV to −39.5 ± 0.7 mV, n = 12, df = 11, t = 1.650, p = 0.127) (E), PK-THPP (from −42.5 ± 1.0 mV to −42.1 ± 0.8 mV, n = 12, df = 11, t = 0.890, p = 0.393) (F), spadin (from −41.9 ± 1.1 mV to −42.3 ± 1.0 mV, n = 13, df = 12, t = 1.866, p = 0.087) (G), and tolbutamide (from −42.2 ± 0.7 mV to −41.7 ± 0.8 mV, n = 13,df = 12, t = 1.879, and p = 0.085) (H). Red and black lines indicate changes of membrane potential in depolarized and nonresponsive neurons, respectively. Data are presented as mean ± SEM. Paired t test was used for statistical analyses. ns = not significant. The numerical data for S2E–S2H Fig can be found in S1 Data. (TIF)</p