Transport and inhibition mechanisms of the human noradrenaline transporter.

The noradrenaline transporter (also known as norepinephrine transporter) (NET) has a critical role in terminating noradrenergic transmission by utilizing sodium and chloride gradients to drive the reuptake of noradrenaline (also known as norepinephrine) into presynaptic neurons1-3. It is a pharmacological target for various antidepressants and analgesic drugs4,5. Despite decades of research, its structure and the molecular mechanisms underpinning noradrenaline transport, coupling to ion gradients and non-competitive inhibition remain unknown. Here we present high-resolution complex structures of NET in two fundamental conformations: in the apo state, and bound to the substrate noradrenaline, an analogue of the χ-conotoxin MrlA (χ-MrlAEM), bupropion or ziprasidone. The noradrenaline-bound structure clearly demonstrates the binding modes of noradrenaline. The coordination of Na+ and Cl- undergoes notable alterations during conformational changes. Analysis of the structure of NET bound to χ-MrlAEM provides insight into how conotoxin binds allosterically and inhibits NET. Additionally, bupropion and ziprasidone stabilize NET in its inward-facing state, but they have distinct binding pockets. These structures define the mechanisms governing neurotransmitter transport and non-competitive inhibition in NET, providing a blueprint for future drug design.

Synthesis and activity evaluation of selenazole-coupled CPI-1 irreversible bifunctional inhibitors for botulinum toxin A light chain.

A series of novel conjugates of benzoselenazole or selenazole and CPI-1 were designed, synthesized, and evaluated for inhibitory activities against the botulinum neurotoxin A (BoNT/A) light chain (LC) and BoNT/A in vivo. The results show that these compounds exhibit potent inhibitory activities to the LC with IC50 of 0.5-4.1 µM. The reaction kinetics and the mass spectra of the reaction products of LC with benzoselenazole- or selenazole- coupled CPI-1 demonstrate that the benzoselenazole group of most inhibitors is coupled to the LC of BoNT/A. These data indicate that the CPI-1 conjugates can inhibit both the active center of BoNT/A LC as well as Cys165, therefore functioning as irreversible bifunctional inhibitors. The detoxification activities in vivo show that one of the benzoselenazole-CPI-1 compounds prolongs the survival time of mice challenged by 2 × LD50 of BoNT/A. This work provides a new strategy to design potent antidotes of BoNT/A.

A Single Amino Acid Replacement Boosts the Analgesic Activity of α-Conotoxin AuIB through the Inhibition of the GABABR-Coupled N-Type Calcium Channel.

α-conotoxin AuIB is the only one of the 4/6 type α-conotoxins (α-CTxs) that inhibits the γ-aminobutyric acid receptor B (GABABR)-coupled N-type calcium channel (CaV2.2). To improve its inhibitory activity, a series of variants were synthesized and evaluated according to the structure-activity relationships of 4/7 type α-CTxs targeting GABABR-coupled CaV2.2. Surprisingly, only the substitution of Pro7 with Arg results in a 2-3-fold increase in the inhibition of GABABR-coupled CaV2.2 (IC50 is 0.74 nM); substitutions of position 9-12 with basic or hydrophobic amino acid and the addition of hydrophobic amino acid Leu or Ile at the second loop to mimic 4/7 type α-CTxs all failed to improve the inhibitory activity of AuIB against GABABR-coupled CaV2.2. Interestingly, the most potent form of AuIB[P7R] has disulfide bridges of "1-4, 2-3" (ribbon), which differs from the "1-3, 2-4" (globular) in the isoforms of wildtype AuIB. In addition, AuIB[P7R](globular) displays potent analgesic activity in the acetic acid writhing model and the partial sciatic nerve injury (PNL) model. Our study demonstrated that 4/6 type α-CTxs, with the disulfide bridge connectivity "1-4, 2-3," are also potent inhibitors for GABABR-coupled CaV2.2, exhibiting potent analgesic activity.

α-Conotoxin Bt1.8 from Conus betulinus selectively inhibits α6/α3ß2ß3 and α3ß2 nicotinic acetylcholine receptor subtypes.

α-Conotoxins are small disulfide-rich peptides found in the venom of marine cone snails and are potent antagonists of nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential therapeutic applications for the treatment of chronic pain or neurological diseases and disorders. In the present study, we synthesized and functionally characterized a novel α-conotoxin Bt1.8, which was cloned from Conus betulinus. Bt1.8 selectively inhibited ACh-evoked currents in Xenopus oocytes expressing rat(r) α6/α3ß2ß3 and rα3ß2 nAChRs with an IC50 of 2.1 nM and 9.4 nM, respectively, and similar potency for human (h) α6/α3ß2ß3 and hα3ß2 nAChRs. Additionally, Bt1.8 had higher binding affinity with a slower dissociation rate for the rα6/α3ß2ß3 subtype compared to rα3ß2. The amino acid sequence of Bt1.8 is significantly different from other reported α-conotoxins targeting the two nAChR subtypes. Further Alanine scanning analyses demonstrated that residues Ile9, Leu10, Asn11, Asn12 and Asn14 are critical for its inhibitory activity at the α6/α3ß2ß3 and α3ß2 subtypes. Moreover, the NMR structure of Bt1.8 indicated the presence of a relatively larger hydrophobic zone than other α4/7-conotoxins which may explain its potent inhibition at α6/α3ß2ß3 nAChRs.

The 3/4- and 3/6-Subfamily Variants of α-Conotoxins GI and MI Exhibit Potent Inhibitory Activity against Muscular Nicotinic Acetylcholine Receptors.

α-Conotoxins GI and MI belong to the 3/5 subfamily of α-conotoxins and potently inhibit muscular nicotinic acetylcholine receptors (nAChRs). To date, no 3/4- or 3/6-subfamily α-conotoxins have been reported to inhibit muscular nAChRs. In the present study, a series of new 3/4-, 3/6-, and 3/7-subfamily GI and MI variants were synthesized and functionally characterized by modifications of loop2. The results show that the 3/4-subfamily GI variant GI[∆8G]-II and the 3/6-subfamily variants GI[+13A], GI[+13R], and GI[+13K] displayed potent inhibition of muscular nAChRs expressed in Xenopus oocytes, with an IC50 of 45.4-73.4 nM, similar to or slightly lower than that of wild-type GI (42.0 nM). The toxicity of these GI variants in mice appeared to be about a half to a quarter of that of wild-type GI. At the same time, the 3/7-subfamily GI variants showed significantly lower in vitro potency and toxicity. On the other hand, similar to the 3/6-subfamily GI variants, the 3/6-subfamily MI variants MI[+14R] and MI[+14K] were also active after the addition of a basic amino acid, Arg or Lys, in loop2, but the activity was not maintained for the 3/4-subfamily MI variant MI[∆9G]. Interestingly, the disulfide bond connectivity "C1-C4, C2-C3" in the 3/4-subfamily variant GI[∆8G]-II was significantly more potent than the "C1-C3, C2-C4" connectivity found in wild-type GI and MI, suggesting that disulfide bond connectivity is easily affected in the rigid 3/4-subfamily α-conotoxins and that the disulfide bonds significantly impact the variants' function. This work is the first to demonstrate that 3/4- and 3/6-subfamily α-conotoxins potently inhibit muscular nAChRs, expanding our knowledge of α-conotoxins and providing new motifs for their further modifications.

A Conantokin Peptide Con-T[M8Q] Inhibits Morphine Dependence with High Potency and Low Side Effects.

N-methyl-D-aspartate receptor (NMDAR) antagonists have been found to be effective to inhibit morphine dependence. However, the discovery of the selective antagonist for NMDAR GluN2B with low side-effects still remains challenging. In the present study, we report a selective NMDAR GluN2B antagonist con-T[M8Q](a conantokin-T variant) that potently inhibits the naloxone-induced jumping and conditioned place preference of morphine-dependent mice at nmol/kg level, 100-fold higher than ifenprodil, a classical NMDAR NR2B antagonist. Con-T[M8Q] displays no significant impacts on coordinated locomotion function, spontaneous locomotor activity, and spatial memory mice motor function at the dose used. Further molecular mechanism experiments demonstrate that con-T[M8Q] effectively inhibited the transcription and expression levels of signaling molecules related to NMDAR NR2B subunit in hippocampus, including NR2B, p-NR2B, CaMKII-α, CaMKII-ß, CaMKIV, pERK, and c-fos. The high efficacy and low side effects of con-T[M8Q] make it a good lead compound for the treatment of opiate dependence and for the reduction of morphine usage.

TAT-Modified ω-Conotoxin MVIIA for Crossing the Blood-Brain Barrier.

As the first in a new class of non-opioid drugs, ω-Conotoxin MVIIA was approved for the management of severe chronic pains in patients who are unresponsive to opioid therapy. Unfortunately, clinical application of MVIIA is severely limited due to its poor ability to penetrate the blood-brain barrier (BBB), reaching the central nervous system (CNS). In the present study, we have attempted to increase MVIIA's ability to cross the BBB via a fusion protein strategy. Our results showed that when the TAT-transducing domain was fused to the MVIIA C-terminal with a linker of varied numbers of glycine, the MVIIA-TAT fusion peptide exhibited remarkable ability to cross the bio-membranes. Most importantly, both intravenous and intranasal administrations of MVIIA-TAT in vivo showed therapeutic efficacy of analgesia. Compared to the analgesic effects of intracerebral administration of the nascent MVIIA, these systemic administrations of MVIIA-TAT require higher doses, but have much prolonged effects. Taken together, our results showed that TAT conjugation of MVIIA not only enables its peripheral administration, but also maintains its analgesic efficiency with a prolonged effective time window. Intranasal administration also rendered the MVIIA-TAT advantages of easy applications with potentially reduced side effects. Our results may present an alternative strategy to improve the CNS accessibility for neural active peptides.

Structure-Activity Analysis of N-Type Calcium Channel Inhibitor SO-3.

As an ω-conopeptide originally discovered from Conus striatus, SO-3 contains 25 amino acid residues and three disulfide bridges. Our previous study has shown that this peptide possesses potent analgesic activity in rodent pain models (mouse and rat), and it specifically inhibits an N-type calcium ion channel (Cav2.2). In the study presented here, we investigated the key amino acid residues for their inhibitory activity against Cav2.2 expressed in HEK 293 cells and analgesic activity in mice. To improve the inhibitory activity of SO-3, we also evaluated the effects of some amino acid residues derived from the corresponding residues of ω-peptide MVIIA, CVID, or GVIA. Our data reveal that Lys6, Ile11, and Asn14 are the important functional amino acid residues for SO-3. The replacement of some amino acid residues of SO-3 in loop 1 with the corresponding residues of CVID and GVIA improved the inhibitory activity of SO-3. The binding mode of Cav2.2 with SO-3 amino acids in loop 1 and loop 2 may be somewhat different from that of MVIIA. This study expanded our knowledge of the structure-activity relationship of ω-peptides and provided a new strategy for improving the potency of Cav2.2 inhibitors.

Cloning, Synthesis and Functional Characterization of a Novel α-Conotoxin Lt1.3.

α-Conotoxins (α-CTxs) are small peptides composed of 11 to 20 amino acid residues with two disulfide bridges. Most of them potently and selectively target nicotinic acetylcholine receptor (nAChR) subtypes, and a few were found to inhibit the GABAB receptor (GABABR)-coupled N-type calcium channels (Cav2.2). However, in all of α-CTxs targeting both receptors, the disulfide connectivity arrangement "C¹-C³, C²-C4" is present. In this work, a novel α4/7-CTx named Lt1.3 (GCCSHPACSGNNPYFC-NH2) was cloned from the venom ducts of Conus litteratus (C. litteratus) in the South China Sea. Lt1.3 was then chemically synthesized and two isomers with disulfide bridges "C¹-C³, C²-C4" and "C¹-C4, C²-C³" were found and functionally characterized. Electrophysiological experiments showed that Lt1.3 containing the common disulfide bridges "C¹-C³, C²-C4" potently and selectively inhibited α3ß2 nAChRs and not GABABR-coupled Cav2.2. Surprisingly, but the isomer with the disulfide bridges "C¹-C4, C²-C³" showed exactly the opposite inhibitory activity, inhibiting only GABABR-coupled Cav2.2 and not α3ß2 nAChRs. These findings expand the knowledge of the targets and selectivity of α-CTxs and provide a new structural motif to inhibit the GABABR-coupled Cav2.2.

Design, Synthesis, and Biological Evaluation of Axitinib Derivatives.

Axitinib is an approved kinase inhibitor for the therapy of advanced metastatic renal cell carcinoma (RCC). It prevents angiogenesis, cellular adhesion, and induces apoptosis of cancer cells. Here, nine axitinib derivatives were designed by replacing the C=C moiety with the N=N group, and the substituted benzene or pyrrole analogs were considered to replace the pyridine ring. Biological activity results showed that most of nascent derivatives exhibited favorable VEGFR-2 kinase inhibitory activities, and TM6, 7, 9, and 11 behaved more potent anti-proliferative activities than axitinib. This novel series of compounds shows a potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

Near-Infrared Photoswitching of Azobenzenes under Physiological Conditions.

Biological tissue exhibits an absorbance minimum in the near-infrared between 700 and 900 nm that permits deep penetration of light. Molecules that undergo photoisomerization in this bio-optical window are highly desirable as core structures for the development of photopharmaceuticals and as components of chemical-biological tools. We report the systematic design, synthesis, and testing of an azobenzene derivative tailored to undergo single-photon photoswitching with near-infrared light under physiological conditions. A fused dioxane ring and a methoxy substituent were used to place oxygen atoms in all four ortho positions, as well as two meta positions, relative to the azobenzene N═N double bond. This substitution pattern, together with a para pyrrolidine group, raises the pKa of the molecule so that it is protonated at physiological pH and absorbs at wavelengths >700 nm. This azobenzene derivative, termed DOM-azo, is stable for months in neutral aqueous solutions, undergoes trans-to-cis photoswitching with 720 nm light, and thermally reverts to the stable trans isomer with a half-life near 1 s.

A novel 4/6-type alpha-conotoxin ViIA selectively inhibits nAchR α3ß2 subtype.

Conotoxins (CTxs) are typically small peptides composed of 12-50 amino acid residues with 2-5 disulfide bridges. Most of them potently and selectively target a wide variety of ion channels and membrane receptors. They are highly valued as neuropharmacological probes and in pharmaceutical development. In this work, a novel α4/6-CTx named ViIA (RDCCSNPPCAHNNPDC-NH2) was identified from a cDNA library of the venom ducts of Conus virgo (C. virgo). ViIA was then synthesized chemically and its disulfide connectivity was identified as 'C(1)-C(3), C(2)-C(4)'. Its molecular targets were further assessed using two-electrode voltage clamping. The results indicated that ViIA selectively inhibited nicotinic acetylcholine receptor (nAChR) α3ß2 subtype with an IC50 of 845.5 nM, but did not target dorsal root ganglion sodium (Na(+))-, potassium (K(+))- or calcium (Ca(2+))-ion channels. Further structure-activity relationship analysis demonstrated that Arg(1) and His(11) but not Asp(2) were the functional residues. To the best of our knowledge, ViIA is the first 4/6 α-CTx that selectively inhibits nAChR α3ß2 subtype. This finding expands the knowledge of targets of α4/6-family CTxs.

Structural and Functional Characterization of a Novel α-Conotoxin Mr1.7 from Conus marmoreus Targeting Neuronal nAChR α3ß2, α9α10 and α6/α3ß2ß3 Subtypes.

In the present study, we synthesized and, structurally and functionally characterized a novel α4/7-conotoxin Mr1.7 (PECCTHPACHVSHPELC-NH2), which was previously identified by cDNA libraries from Conus marmoreus in our lab. The NMR solution structure showed that Mr1.7 contained a 310-helix from residues Pro7 to His10 and a type I ß-turn from residues Pro14 to Cys17. Electrophysiological results showed that Mr1.7 selectively inhibited the α3ß2, α9α10 and α6/α3ß2ß3 neuronal nicotinic acetylcholine receptors (nAChRs) with an IC50 of 53.1 nM, 185.7 nM and 284.2 nM, respectively, but showed no inhibitory activity on other nAChR subtypes. Further structure-activity studies of Mr1.7 demonstrated that the PE residues at the N-terminal sequence of Mr1.7 were important for modulating its selectivity, and the replacement of Glu2 by Ala resulted in a significant increase in potency and selectivity to the α3ß2 nAChR. Furthermore, the substitution of Ser12 with Asn in the loop2 significantly increased the binding of Mr1.7 to α3ß2, α3ß4, α2ß4 and α7 nAChR subtypes. Taken together, this work expanded our knowledge of selectivity and provided a new way to improve the potency and selectivity of inhibitors for nAChR subtypes.

Characterization of a T-superfamily conotoxin TxVC from Conus textile that selectively targets neuronal nAChR subtypes.

T-superfamily conotoxins have a typical cysteine pattern of "CC-CC", and are known to mainly target calcium or sodium ion channels. Recently, we screened the targets of a series of T-superfamily conotoxins and found that a new T-superfamily conotoxin TxVC (KPCCSIHDNSCCGL-NH2) from the venom of Conus textile. It selectively targeted the neuronal nicotinic acetylcholine receptor (nAChR) subtypes α4ß2 and α3ß2, with IC50 values of 343.4 and 1047.2nM, respectively, but did not exhibit obvious pharmacological effects on voltage-gated potassium, sodium or calcium channel in DRG cells, the BK channels expressed in HEK293 cells, or the Kv channels in LßT2 cells. The changes in the inhibitory activities of its Ala mutants, the NMR structure, and molecular simulation results based on other conotoxins targeting nAChR α4ß2, all demonstrated that the residues Ile(6) and Leu(14) were the main hydrophobic pharmacophores. To our best knowledge, this is the first T-superfamily conotoxin that inhibits neuronal nAChRs and possesses high binding affinity to α4ß2. This finding will expand the knowledge of the targets of T-superfamily conotoxins and the motif information could help the design of new nAChR inhibitors.

Design, synthesis, and biological activity of novel 1,4-disubstituted piperidine/piperazine derivatives as CCR5 antagonist-based HIV-1 entry inhibitors.

A series of novel 1,4-disubstituted piperidine/piperazine derivatives were designed, synthesized and evaluated for their in vitro activities against HIV-1 Bal (R5) infection in CEMX174 5.25M7 cells. A majority of these compounds showed potent anti-HIV-1 activities with IC(50) at nanomolar levels. N-(4-Fluoro-benzyl)piperazine analog B07 hydrochloride exhibited potency against HIV-1 activity similar to that of TAK-220 hydrochloride, but it had much better water solubility (25 mg/ml in phosphate sodium buffer at 25 °C) and oral bioavailability (56%) than TAK-220 hydrochloride (a solubility of 2 mg/ml and oral bioavailability of 1.4%). These results suggest that B07 hydrochloride may serve as a better lead for the development of new anti-HIV-1 therapies or microbicides for treatment and prevent of HIV-1 infection.

Discovery and Structural and Functional Characterization of a Novel A-Superfamily Conotoxin Targeting α9α10 Nicotinic Acetylcholine Receptor.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels widely distributed in the central peripheral nervous system and muscles which participate in rapid synaptic transmission. The α9α10 nAChR is an acetylcholine receptor subtype and is involved in chronic pain. In the present study, a new A-superfamily conotoxin Bt14.12 cloned from Conus betulinus was found to selectively inhibit α9α10 nAChRs with an IC50 of 62.3 nM. Unlike α-conotoxins and other A-superfamily conotoxins, Bt14.12 contains a four Cys (C-C-C-C) framework with a unique disulfide bond connection "C1-C4, C2-C3". The structure-activity studies of Bt14.12 demonstrate that all amino acid residues contribute to its potency. Interestingly, mutation experiments show that the deletion of Asp2 or the addition of three Arg residues at the N-terminus of Bt14.12 significantly enhances its inhibitory activity (IC50 is 21.9 nM or 12.7 nM, respectively) by 2- or 4-fold compared to the wild-type Bt14.12. The NMR structure of Bt14.12 shows that it contains α-helix- and ß-turn-like elements, and further computational modelings of the interaction between Bt14.12 and the α9α10 nAChR demonstrate that Bt14.12 possesses a distinctive mode of action and displays a different structure-activity relationship from known α9α10 nAChR targeting α-conotoxins. Our findings provide a novel conotoxin that potently targets α9α10 nAChRs and a new motif for designing potent inhibitors against α9α10 nAChRs.

A new protein-coupled antigen of α-conotoxin MI displays high immunogenicity and can produce antiserum with high detoxification activity.

α-conotoxin (α-CTX) MI is a small peptide toxin with 14 amino acids and two disulfide bonds. It potently inhibits muscle-type nicotinic acetylcholine receptors (nAChRs), and poses a threat as a toxin to tropical fishermen. However, there are currently no effective drugs for the treatment of MI envenomation due to the toxin's low immunogenicity. In this report, we generated neutralizing antiserum and F(ab')2 to MI by synthesizing a new MI antigen through the coupling of alkynyl-modified MI and azide-modified bovine serum albumin (BSA), followed by immunization into mouse and horse. The new MI-BSA antigen generated high titers of mouse and horse antiserum (1:204,800 and 1:51,200, respectively), and both the antiserum as well as the horse F(ab')2 displayed highly potent neutralization and detoxification efficacy. 12.5 µL of mouse or horse antiserum preincubated with MI could completely neutralize a lethal dose of the MI (0.4 µg, 1.7 × LD50), while 6.25 µL (mouse) or 10.41 µL (horse) of the antiserum could exert complete detoxification of mice injected with 1.7 × LD50 of MI. Moreover, the mouse and horse antiserum exhibited medium cross-reactivity for highly toxic α-CTX GI. These results demonstrate that the integrity of MI's antigen epitope and carrier effect of BSA can improve MI's immunogenicity, and provides an effective detoxification treatment for highly toxic α-conotoxins as well as an effective method for the preparation of antiserum of small peptide toxins.

A 4/8 Subtype α-Conotoxin Vt1.27 Inhibits N-Type Calcium Channels With Potent Anti-Allodynic Effect.

A novel 4/8 subtype α-conotoxin, Vt1.27 (NCCMFHTCPIDYSRFNC-NH2), was identified from Conus vitulinus in the South China Sea by RACE methods. The peptide was synthesized and structurally characterized. Similar to other α-conotoxins that target neuronal nicotinic acetylcholine receptor (nAChR) subtypes, Vt1.27 inhibited the rat α3ß2 nAChR subtype (IC50 = 1160 nM) and was inactive at voltage-gated sodium and potassium channels in rat sensory neurons. However, Vt1.27 inhibited high voltage-activated N-type (CaV2.2) calcium channels expressed in HEK293T cells with an IC50 of 398 nM. An alanine scan of the peptide showed that residues Phe5, Pro9, Ile10, and Ser13 contribute significantly to the inhibitory activity of Vt1.27. The molecular dockings indicate that Vt1.27 inhibits the transmembrane region of CaV2.2, which is different from that of ω-conotoxins. Furthermore, Vt1.27 exhibited potent anti-allodynic effect in rat partial sciatic nerve injury (PNL) and chronic constriction injury (CCI) pain models at 10 nmol/kg level with the intramuscular injection. The pain threshold elevation of Vt1.27 groups was higher than that of α-conotoxin Vc1.1 in CCI rat models. These findings expand our knowledge of targets of α-conotoxins and potentially provide a potent, anti-allodynic peptide for the treatment of neuropathic pain.

Potent HIV fusion inhibitors against Enfuvirtide-resistant HIV-1 strains.

T20 (generic name: Enfuvirtide, brand name: Fuzeon) is the only FDA-approved HIV fusion inhibitor that is being used for treatment of HIV/AIDS patients who have failed to respond to current antiretroviral drugs. However, it rapidly induces drug resistance in vitro and in vivo. On the basis of the structural and functional information of anti-HIV peptides from a previous study, we designed an HIV fusion inhibitor named CP32M, a 32-mer synthetic peptide that is highly effective in inhibiting infection by a wide range of primary HIV-1 isolates from multiple genotypes with R5- or dual-tropic (R5X4) phenotype, including a group O virus (BCF02) that is resistant to T20 and C34 (another anti-HIV peptide). Strikingly, CP32M is exceptionally potent (at low picomolar level) against infection by a panel of HIV-1 mutants highly resistant to T20 and C34. These findings suggest that CP32M can be further developed as an antiviral therapeutic against multidrug resistant HIV-1.

A novel ω-conotoxin Bu8 inhibiting N-type voltage-gated calcium channels displays potent analgesic activity.

ω-Conotoxins inhibit N-type voltage-gated calcium (CaV2.2) channels and exhibit efficacy in attenuating neuropathic pain but have a low therapeutic index. Here, we synthesized and characterized a novel ω-conotoxin, Bu8 from Conus bullatus, which consists of 25 amino acid residues and three disulfide bridges. Bu8 selectively and potently inhibits depolarization-activated Ba2+ currents mediated by rat CaV2.2 expressed in HEK293T cells (IC50 = 89 nmol/L). Bu8 is two-fold more potent than ω-conotoxin MVIIA, a ω-conotoxin currently used for the treatment of severe chronic pain. It also displays potent analgesic activity in animal pain models of hot plate and acetic acid writhing but has fewer side effects on mouse motor function and lower toxicity in goldfish. Its lower side effects may be attributed to its faster binding rate and higher recovery ratios. The NMR structure demonstrates that Bu8 contains a small irregular triple ß-strand. The structure-activity relationships of Bu8 Ala mutants and Bu8/MVIIA hybrid mutants demonstrate that the binding mode of CaV2.2 with the amino acid residues in loop 1 and loop 2 of Bu8 is different from that of MVIIA. This study characterizes a novel, more potent ω-conotoxin and provides new insights for designing CaV2.2 antagonists.