Selection of an ASIC1a-blocking combinatorial antibody that protects cells from ischemic death

Qiang, Min; Dong, Xue; Zha, Zhao; Zuo, Xiao-Kun; Song, Xing-Lei; Zhao, Lingjuan; Yuan, Chao; Huang, Chen; Tao, Pingdong; Hu, Qin; Li, Wei‐Guang; Hu, Wanhui; Li, Jie; Nie, Yan; Buratto, Damiano; Zonta, Francesco; Ma, Peixiang; Zheng, Yu; Liu, Huan; Zhang, Yi; Yang, Bei; Xie, Jia; Qu, Zhihu; Yang, Guang; Lerner, Richard A.

doi:10.1073/pnas.1807233115

Cited by 52 publications

(54 citation statements)

References 55 publications

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“…However, it is increasingly recognised that these highly basic peptides also modulate the activity of ionotropic receptors, such as tetrameric glutamate receptors (N-methyl-D-aspartate (NMDA) subtype) and trimeric acid-sensing ion channels (ASICs) (6)(7)(8)(9)(10). The latter interaction is of particular interest, as ASICs have emerged as mediators of both pain and stroke and thus represent potential targets in the treatment of these diseases (11)(12)(13)(14)(15)(16)(17). In fact, big dynorphin (BigDyn) is the most potent endogenous ASIC modulator described to date.…”

Section: Introductionmentioning

confidence: 99%

Mechanism and site of action of big dynorphin on ASIC1a

Borg

Braun

Heusser

et al. 2019

Preprint

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Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to synaptic plasticity, as well as initiation of pain and neuronal death following ischemic stroke.As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is upregulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs and non-canonical amino acidmediated photocrosslinking. We demonstrate that BigDyn binding induces ASIC1a conformational changes that are different from those induced by protonation and likely represent a distinct closed state. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is mediated through electrostatic interactions of basic amino acids in the BigDyn N-terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the non-canonical amino acid azidophenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn and thus highlight this cavity as an important site for the development of ASIC-targeting therapeutics.Note: This manuscript has not been peer-reviewed 3

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Section: Introductionmentioning

confidence: 99%

Mechanism and site of action of big dynorphin on ASIC1a

Borg

Braun

Heusser

et al. 2019

Preprint

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“…An MCAO stroke was induced in mice, and three hours after ischemia, 4 µL of PBS (vehicle solution) + 3 µg/µL ASC06-IgG1 was injected intracerebroventricularly. Compared to the control, the group treated with PBS + ASC06-IgG1 showed a 23% decrease in infarct volume [36]. Such results show that ASICs contribute to neuronal cell death.…”

Section: Mab Acts On Acid-sensing Ion Channelsmentioning

confidence: 60%

“…Such results show that ASICs contribute to neuronal cell death. Antibodies against ASICs are neuroprotective and might have therapeutic potential in stroke patient [36]. During an ischemic stroke, glutamate is released into the plasma and cerebrospinal fluid from damaged neuronal tissue; an increase in glutamate leads to activation of NMDARs [39].…”

Section: Mab Acts On Acid-sensing Ion Channelsmentioning

confidence: 99%

Monoclonal Antibody as an Emerging Therapies for Ischemic Stroke

Woods¹,

Jiang²,

Chu³

2019

Preprint

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Acute ischemic strokes are the third leading cause of death and the leading cause of neurological disability worldwide. The oxygen and glucose deprivation associated with ischemic strokes not only leads to neuronal cell death, but also increases the inflammatory response and decreases functional output of the brain. The only intervention approved by US Federal Drug and Food Administration for treatment of ischemic strokes is tissue plasminogen activator (tPA), however, such treatment can only be given within 4.5 hours of the onset of stroke-like symptoms. This narrow time-range limits its application, and it also might induce detrimental rather than beneficial effects to stroke patients by treatment of the tPA. In order to reduce the infarct volume of an acute ischemic stroke while increasing the time period for treatment, emerging therapies reveal great potential by targeting inflammation, growth factors, ion channels, and neurotransmitter receptors with monoclonal antibody (MAB). With successfully application in the treatment of cancer patient by MAB, in this review, we will focus on recent advances on stroke therapy by using MAB on the treatment of stroke by targeting inflammation, growth factors, ion channels, and neurotransmitter receptors. Therefore, developing specific MAB targeting the signaling pathway of stroke will contribute to stroke therapy.

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“…1 can be used to select for dual fluorescent yeast-mammalian cell complexes indicative of protein-target cell binding, we then used an antibody specific for the membrane protein hASIC1a (ASC06) to study directed affinity maturation and to determine whether the intensity of the dual fluorescent signals is positively correlated with affinity. This ASC06 antibody was previously reported to be a potent and specific antagonist that blocks ASIC1a (20). A mutagenic library was generated in the heavy chain CDR3 region of ASC06 by using a pool of oligonucleotide primers in which each single oligonucleotide in the pool encodes a CDR3 variant carrying no more than four mutated positions (21).…”

Section: Resultsmentioning

confidence: 99%

A cell–cell interaction format for selection of high-affinity antibodies to membrane proteins

Yang

Wan

Tao

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Generating and improving antibodies and peptides that bind specifically to membrane protein targets such as ion channels and G protein-coupled receptors (GPCRs) can be challenging using established selection methods. Current strategies are often limited by difficulties in the presentation of the antigen or the efficiency of the selection process. Here, we report a method for obtaining antibodies specific for whole cell membrane-associated antigens which combines a cell–cell interaction format based on yeast display technology with fluorescence-activated cell sorting of dual fluorescent complexes. Using this method, we were able to direct the affinity maturation of an antagonist antibody specific for the proton-gated ion channel ASIC1a and showed that both the affinity and potency were improved. We were also able to use this method to do kinetic selections to generate clones with better dissociation profiles. In addition, this method was employed successfully to handle the difficult problem of selecting antibodies specific to a GPCR target, the mu-opioid receptor.

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Selection of an ASIC1a-blocking combinatorial antibody that protects cells from ischemic death

Cited by 52 publications

References 55 publications

Mechanism and site of action of big dynorphin on ASIC1a

Mechanism and site of action of big dynorphin on ASIC1a

Monoclonal Antibody as an Emerging Therapies for Ischemic Stroke

A cell–cell interaction format for selection of high-affinity antibodies to membrane proteins

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