Role of Asn2 and Glu7 residues in the oxidative folding and on the conformation of the N‐terminal loop of apamin

Le‐Nguyen, Dung; Chiche, Laurent; Hoh, François; Martin-Eauclaire, M.F.; Dumas, Christian; Nishi, Yoshinori; Kobayashi, Yuji; Aumelas, André

doi:10.1002/bip.20755

Cited by 11 publications

(17 citation statements)

References 45 publications

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“…Data integration and scaling, and structure solution and refinement were performed as described [3]. For stingin-5, the crystal structure of wild type apamin [8] was used as a starting model to determine initial phase information. For syn MDM2-stingin-1, syn MDM2 coordinates extracted from the syn MDM2-PMI complex structure [3] were used as a search model for molecular replacement.…”

Section: Methodsmentioning

confidence: 99%

“…We and others have also found that Tyr22 is superior to Leu22 in contributing to MDM2/MDMX binding [3,7]. Apamin consists of an N-terminal loop and a C-terminal α-helix globally stabilized by two disulfide bridges (Cys1–Cys11 and Cys3–Cys15) [8], providing an attractive structural template for de novo design of new functionalities [9]. To convert apamin to an inhibitor that emulates the activity of 18–26 p53, we grafted Phe19, Tyr22, Trp23, and Leu26 to the topologically equivalent positions of apamin in its α-helical region, generating, via additional C-terminal truncation, stingins 1–5, ranging from 16 to 18 amino acid residues (Table 1).…”

mentioning

confidence: 99%

“…Replacement of the former resulted in stingins 1–3, while substitutions of the latter yielded stingins 4–5. Notably, as residues Arg13, Arg14, and Gln17 are functional determinants of apamin [8], conversion to stingins is expected to conveniently decimate its neurotoxin activity. All five stingin peptides along with apamin were chemically synthesized, spontaneously and quantitatively folded within 2 hours under thiol-disulfide shuffling conditions, and purified to homogeneity by reversed-phase HPLC.…”

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confidence: 99%

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Apamin as a Template for Structure‐Based Rational Design of Potent Peptide Activators of p53

Pazgier

Liu

et al. 2009

Angew Chem Int Ed

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Apamin as a Template for Structure‐Based Rational Design of Potent Peptide Activators of p53

Pazgier

Liu

et al. 2009

Angew Chem Int Ed

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“…7,10 By grafting the four underlined hydrophobic residues (Phe9, Tyr12, Trp13, and Leu16) to apamin, we succeeded in converting the neurotoxin into a potent inhibitor of the interaction of the p53 tumor suppressor with its negative regulators MDM2 and MDMX. 7 …”

Section: Introductionmentioning

confidence: 99%

Functional consequences of retro-inverso isomerization of a miniature protein inhibitor of the p53–MDM2 interaction

Zhan

Zhao

et al. 2013

Bioorganic & Medicinal Chemistry

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Peptide retro-inverso isomerization is thought to be functionally neutral and has been widely used as a tool for designing proteolytically stable D-isomers to recapitulate biological activities of their parent L-peptides. Despite success in a wide range of applications, exceptions amply exist that clearly defy this rule of thumb when parent L-peptides adopt an α-helical conformation in their bound state. The detrimental energetic effect of retro-inverso isomerization of an α-helical L-peptide on its target protein binding has been estimated to be 3.0-3.4 kcal/mol. To better understand how the retro-inverso isomer of a structured protein works at the molecular level, we chemically synthesized and functionally characterized the retro-inverso isomer of a rationally designed miniature protein termed stingin of 18 amino acid residues, which adopts an N-terminal loop and a C-terminal α-helix stabilized by two intra-molecular disulfide bridges. Stingin emulated the transactivation peptide of the p53 tumor suppressor protein and bound with high affinity and via its C-terminal α-helix to MDM2 and MDMX – the two negative regulators of p53. We also prepared the retro isomer and D-enantiomer of stingin for comparative functional studies using fluorescence polarization and surface plasmon resonance techniques. We found that retro-inverso isomerization of L-stingin weakened its MDM2 binding by 720 fold (3.9 kcal/mol); while enantiomerization of L-stingin drastically reduced its binding to MDM2 by three orders of magnitude, sequence reversal completely abolished it. Our findings demonstrate the limitation of peptide retro-inverso isomerization in molecular mimicry and reinforce the notion that the strategy works poorly with biologically active α-helical peptides due to inherent differences at the secondary and tertiary structural levels between an L-peptide and its retro-inverso isomer despite their similar side chain topologies at the primary structural levelaCollege of Pharmaceutical SciencesSouthwest UniversityChongqing 400715China.

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“…The apamin-PM 400 -DiR, apamin-PM 200 -DiR, apamin-PM 100 -DiR, and apamin-PM 50 -DiR refer to fluorophore encapsulated micelles with different particle sizes (400, 200, 100, 50 nm, respectively), which were evaluated to investigate the influence of particle size on in vivo distribution of micelles. 50 50.03 ± 1.07 0.177 ± 0.03 a PDI, polydispersity index. From our investigation, approximately 80% of CUR and 75% of DiR can be successfully loaded into polymeric micelles (Table 1).…”

Section: Resultsmentioning

confidence: 99%

Apamin-Mediated Actively Targeted Drug Delivery for Treatment of Spinal Cord Injury: More Than Just a Concept

Jiang

et al. 2014

Mol. Pharmaceutics

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Faced with the complex medical challenge presented by spinal cord injuries (SCI) and considering the lack of any available curative therapy, the development of a novel method of delivering existing drugs or candidate agents can be perceived to be as important as the development of new therapeutic molecules. By combining three ingredients currently in clinical use or undergoing testing, we have designed a central nervous system targeted delivery system based on apamin-modified polymeric micelles (APM). Apamin, one of the major components of honey bee venom, serves as the targeting moiety, poly(ethylene glycol) (PEG) distearoylphosphatidylethanolamine (DSPE) serves as the drug-loaded material, and curcumin is used as the therapeutic agent. Apamin was conjugated with NHS (N-hydroxysuccinimide)-PEG-DSPE in a site-specific manner, and APM were prepared by a thin-film hydration method. A formulation comprising 0.5 mol % targeting ligand with 50 nm particle size showed strong targeting efficiency in vivo and was evaluated in pharmacodynamic assays. A 7-day treatment by daily intravenous administration of low doses of APM (corresponding to 5 mg/kg of curcumin) was performed. Significantly enhanced recovery and prolonged survival was found in the SCI mouse model, as compared to sham-treated groups, with no apparent toxicity. A single dose of apamin-conjugated polymers was about 700-fold lower than the LD50 amount, suggesting that APM and apamin have potential for clinical applications as spinal cord targeting ligand for delivery of agents in treatment of diseases of the central nervous system.

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Role of Asn² and Glu⁷ residues in the oxidative folding and on the conformation of the N‐terminal loop of apamin

Abstract: ABSTRACT:The X-ray structure of [N-acetyl]-apamin has been solved at 0.95 Å resolution. It consists of an 1-7 N-terminal loop stabilized by an Asn-b-turn motif (2-5 residues) and a helical structure spanning the 9-18 residues tightly linked together by two disulfide bonds.

Cited by 11 publications

References 45 publications

Apamin as a Template for Structure‐Based Rational Design of Potent Peptide Activators of p53

Apamin as a Template for Structure‐Based Rational Design of Potent Peptide Activators of p53

Functional consequences of retro-inverso isomerization of a miniature protein inhibitor of the p53–MDM2 interaction

Apamin-Mediated Actively Targeted Drug Delivery for Treatment of Spinal Cord Injury: More Than Just a Concept

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