TY - JOUR
T1 - Targeting intrinsically disordered regions facilitates discovery of calcium channels 3.2 inhibitory peptides for adeno-associated virus-mediated peripheral analgesia
AU - Shin, Seung Min
AU - Lauzadis, Justas
AU - Itson-Zoske, Brandon
AU - Cai, Yongsong
AU - Fan, Fan
AU - Natarajan, Gayathri K.
AU - Kwok, Wai Meng
AU - Puopolo, Michelino
AU - Hogan, Quinn H.
AU - Yu, Hongwei
N1 - Funding Information:
The authors thank Dr. Markus Missler (Westfalische Wilhelms University, Germany) for providing HEK293 Cav2.2 stable cell line and Dr. Bin Pan (MCW) for his technical assistance for voltage-gated patch-clamp recording experiments. This research was supported by a grant from a National Institutes of Health grant R61NS116203 (to H.Y. and Q.H.), an MCW NRC grant FP00016291 (to H.Y.), and 2022 award from Dr. Ralph and Marian Falk Medical Research Trust, Bank of America, Private Bank (to H.Y. and Q.H.).
Publisher Copyright:
© 2022 Lippincott Williams and Wilkins. All rights reserved.
PY - 2022/12/1
Y1 - 2022/12/1
N2 - Ample data support a prominent role of peripheral T-type calcium channels 3.2 (CaV3.2) in generating pain states. Development of primary sensory neuron-specific inhibitors of CaV3.2 channels is an opportunity for achieving effective analgesic therapeutics, but success has been elusive. Small peptides, especially those derived from natural proteins as inhibitory peptide aptamers (iPAs), can produce highly effective and selective blockade of specific nociceptive molecular pathways to reduce pain with minimal off-target effects. In this study, we report the engineering of the potent and selective iPAs of CaV3.2 from the intrinsically disordered regions (IDRs) of CaV3.2 intracellular segments. Using established prediction algorithms, we localized the IDRs in CaV3.2 protein and identified several CaV3.2iPA candidates that significantly reduced CaV3.2 current in HEK293 cells stably expressing human wide-type CaV3.2. Two prototype CaV3.2iPAs (iPA1 and iPA2) derived from the IDRs of CaV3.2 intracellular loops 2 and 3, respectively, were expressed selectively in the primary sensory neurons of dorsal root ganglia in vivo using recombinant adeno-associated virus (AAV), which produced sustained inhibition of calcium current conducted by CaV3.2/T-type channels and significantly attenuated both evoked and spontaneous pain behavior in rats with neuropathic pain after tibial nerve injury. Recordings from dissociated sensory neurons showed that AAV-mediated CaV3.2iPA expression suppressed neuronal excitability, suggesting that CaV3.2iPA treatment attenuated pain by reversal of injury-induced neuronal hypersensitivity. Collectively, our results indicate that CaV3.2iPAs are promising analgesic leads that, combined with AAV-mediated delivery in anatomically targeted sensory ganglia, have the potential to be a selective peripheral CaV3.2-targeting strategy for clinical treatment of pain.
AB - Ample data support a prominent role of peripheral T-type calcium channels 3.2 (CaV3.2) in generating pain states. Development of primary sensory neuron-specific inhibitors of CaV3.2 channels is an opportunity for achieving effective analgesic therapeutics, but success has been elusive. Small peptides, especially those derived from natural proteins as inhibitory peptide aptamers (iPAs), can produce highly effective and selective blockade of specific nociceptive molecular pathways to reduce pain with minimal off-target effects. In this study, we report the engineering of the potent and selective iPAs of CaV3.2 from the intrinsically disordered regions (IDRs) of CaV3.2 intracellular segments. Using established prediction algorithms, we localized the IDRs in CaV3.2 protein and identified several CaV3.2iPA candidates that significantly reduced CaV3.2 current in HEK293 cells stably expressing human wide-type CaV3.2. Two prototype CaV3.2iPAs (iPA1 and iPA2) derived from the IDRs of CaV3.2 intracellular loops 2 and 3, respectively, were expressed selectively in the primary sensory neurons of dorsal root ganglia in vivo using recombinant adeno-associated virus (AAV), which produced sustained inhibition of calcium current conducted by CaV3.2/T-type channels and significantly attenuated both evoked and spontaneous pain behavior in rats with neuropathic pain after tibial nerve injury. Recordings from dissociated sensory neurons showed that AAV-mediated CaV3.2iPA expression suppressed neuronal excitability, suggesting that CaV3.2iPA treatment attenuated pain by reversal of injury-induced neuronal hypersensitivity. Collectively, our results indicate that CaV3.2iPAs are promising analgesic leads that, combined with AAV-mediated delivery in anatomically targeted sensory ganglia, have the potential to be a selective peripheral CaV3.2-targeting strategy for clinical treatment of pain.
KW - Adeno-associated virus
KW - Dorsal root ganglia
KW - Neuropathic pain
KW - Peptide aptamer
KW - Peripheral nervous system
KW - T-type/Cav3.2 channels
UR - http://www.scopus.com/inward/record.url?scp=85134128027&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85134128027&partnerID=8YFLogxK
U2 - 10.1097/j.pain.0000000000002650
DO - 10.1097/j.pain.0000000000002650
M3 - Article
C2 - 35420557
AN - SCOPUS:85134128027
SN - 0304-3959
VL - 163
SP - 2466
EP - 2484
JO - Pain
JF - Pain
IS - 12
ER -