Novel charged sodium and calcium channel inhibitor active against neurogenic inflammation
- Boston Children's Hospital, United States
- Harvard Medical School, United States
- Université de Montréal, Canada
- Renaissance School of Medicine at Stony Brook University, United States
- Sage Partner International, United States
Figures
Figure 1
State dependent inhibition of Cav2.2 channels by the neutral compound NNCB-2.
(A) Time course of inhibition of Cav2.2 current by external NNCB-2 applied at concentrations from 3 to 100 µM. Current carried by 5 mM Ba2+ was evoked by 50-millisecond steps from −70 to +10 mV …
Figure 2
State dependent inhibition of Cav2.2 channels by the charged compound CNCB-2.
(A) Time course of inhibition of Cav2.2 current by external CNCB-2 applied at concentrations from 10 to 300 µM. Current carried by 5 mM Ba2+ current was evoked by 50-millisecond steps from −70 to …
Figure 3
Test of whether Cav2.2 inhibition by NNCB-2 and CNCB-2 requires channel opening and comparison of internal and external application of CNCB-2 .
(A) Calcium channel inhibition by NNCB-2 or CNCB-2 does not require channel opening. Ba2+ current was elicited in a whole-cell recording by 50-millisecond steps from −70 to + 10 mV delivered every …
Figure 4
Dose-dependent inhibition of native N-type Ca2+ channels in mouse sympathetic neurons by CNCB-2 applied extracellularly.
(A) Time course of inhibition by 10 µM CNCB-2. Current carried by 5 mM Ba2+ was evoked by 50 ms steps from −70 to +10 mV delivered every 3 s. The external solution contained 5 μM nimodipine to block …
Figure 5
CNCB-2 applied extracellularly inhibits Nav1.7 sodium channels.
(A) Time course of inhibition by 3 µM CNCB-2. Current was evoked by 10 ms steps from −70 to 0 mV delivered every 5 s. Mean ± SEM, n = 5. (B) Inhibition by 3 µM CNCB-2 at different holding …
Figure 6
Use-dependent inhibition of Nav1.7 sodium channels by externally-applied CNCB-2 compared to lidocaine and bupivacaine.
(A) Inhibition of Nav1.7 current by 3 µM CNCB-2 applied with stimulation at 0.1 Hz (20-msec depolarizations from −100 mV to 0 mV) for two minutes followed by two minutes of stimulation at 10 Hz, a …
Figure 7
Inhibition of action potential generation in mouse DRG neurons by CNCB-2.
(A) Action potential firing evoked by injections of 40 pA, 150 pA, and 200 pA in a small-diameter mouse DRG neuron before and after 5 min exposure to 3 µM CNCB-2. Resting potential in CNCB-2 was …
Figure 8
Inhibition of TTX-resistant sodium current in mouse DRG neurons by CNCB-2.
(A) Time-course of inhibition of TTX-resistant sodium current in a small mouse DRG neuron by 3 µM CNCB-2. TTX-resistant sodium current was isolated by solutions containing 300 nM TTX. Current was …
Figure 9
Effect of CNCB-2 in mouse pain models.
(A) Inhibition of paw withdrawal in paw incision model of post-operative pain. Threshold for response to von Frey filaments tested 1, 4 and 8 hr after injection of CNCB-2 into an injured paw 24 hr …
Figure 10
Inhibition of CGRP release and neurogenic lung inflammation by NNCB-2 and CNCB-2.
(A) Effect of 30 µM NNCB-2 or 30 µM CNCB-2 on release of CGRP induced by application of 50 mM KCl to DRG cultures (p=0.15 for 30 µM NNCB-2 (n = 4) compared to 50 mM KCl with no drug (n = 4), p=0.028 …
Tables
Key resources table
| Reagent type | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain (Mus musculus) | Swiss Webster CFW | Charles River | Cat#024 | |
| Strain (Mus musculus) | C57BL6/J | Jackson Lab | 000664 | |
| Strain (Mus musculus) | BALB/c | Jackson Lab | 001026 | |
| Cell line | rat Cav2.2 tsA201 cell line | PMID: 15201306 | Line #201719 | Dr. Diane Lipscombe, Brown University |
| Cell line | human Nav1.7 HEK cell line | PMID: 22442564 | Dr. Sooyeon Jo, Harvard Medical School | |
| Antibody | FITC anti-mouse CD45 | BD Biosciences | Cat 553079 RRID:AB_394609 | |
| Antibody | PR anti-mouse Siglec-F | BD Biosciences | Cat 552126 RRID:AB_394341 | |
| Antibody | APC anti-mouse GR-1 | Thermo Fisher | Cat 17-5931-81 RRID:AB_469475 | |
| Antibody | PE-Cy7 anti-mouse CD3e | Thermo Fisher | Cat 25-0031-81 RRID:AB_469571 | |
| Antibody | PerCP anti-mouse F4/80 | Biolegend | Cat 123125 RRID:AB_893495 | |
| Commercial assay or kit | MycoAlert PLUS Mycoplasma Detection Kit | Lonza | LT07-703 | |
| Commercial assay or kit | Rat CGRP Enzyme Immunoassay Kit | Bertin Pharma/Cayman Chemical | #589001 | |
| Chemical compound | NNCB-2 (3-Cyclopentylmethylsulfanyl-2-(3,3-dimethyl-butylamino)-N-(4-methoxy-benzyl)-propionamide) | This paper | Synthesized by Sundia MediTech, structure verified by NMR and mass spectrometry. | |
| Chemical compound | CNCB-2 ([2-Cyclopentylmethylsulfanyl-1-(4-methoxy-benzylcarbamoyl)-ethyl]−(3,3-dimethyl-butyl)-dimethyl-ammonium; chloride salt) | This paper | Synthesized by Sundia MediTech, structure verified by NMR and mass spectrometry. | |
| Chemical compound | Papain | Worthington Biochemical | LS003126 | |
| Chemical compound | Collagenase Type I | Sigma | 10103578001 | |
| Chemical compound | Dispase Type II | Sigma | 4942078001 | |
| Chemical compound | L-15 | Invitrogen | 11415–064 | |
| Chemical compound | Nerve growth factor | Invitrogen | 13290–010 | |
| Chemical compound | Neurobasal A Medium | Invitrogen | 10888–022 | |
| Chemical compound | B-27 | Invitrogen | 17504–010 | |
| Chemical compound | Penicillin-Streptomycin | Invitrogen | 15140–122 | |
| Chemical compound | Fetal calf serum | Invitrogen | 10082–147 | |
| Chemical compound | Minimal Essential Medium | American Tissue Type Collection | EMEM 30–2003 | |
| Chemical compound | Hank's Balanced Salt Solution | Gibco | 14170–112 | |
| Chemical compound | DMEM/F12 | Gibco | 11330–032 | |
| Chemical compound | Tetrodotoxin w/citrate | Abcam | Ab120055 | |
| Chemical compound | Zymosan | Sigma | Z4250 | |
| Software | Clampex | Molecular Devices | Versions 9.2, 10.3.1.5 RRID:SCR_011323 | https://www.moleculardevices.com |
| Software | Igor Pro | Wavemetrics | Version 6.12A RRID:SCR_000325 | https://www.wavemetrics.com |
| Software | DataAccess | Bruxton Corporation | http://www.bruxton.com/DataAccess/index.html | |
| Software | GraphPad Prism | Graph Pad | RRID:SCR_002798 | Version 5 |
Additional files
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Source data 1
- https://cdn.elifesciences.org/articles/48118/elife-48118-data1-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/48118/elife-48118-transrepform-v1.docx
