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Subtle modifications to oxytocin produce ligands that retain potency and improved selectivity across species

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Sci. Signal.  05 Dec 2017:
Vol. 10, Issue 508, eaan3398
DOI: 10.1126/scisignal.aan3398
  • Fig. 1 Overview of the pharmacophore framework modifications to oxytocin and vasopressin.

    (A) NMR structure of oxytocin (OT), with framework residues marked in red that were the focus of this study. The table provides an overview of all synthesized peptides with details on their modifications. U, selenocysteine; d, deamino (N terminus); Δ, deletion of residue; bold, modification; P, position; term, terminus; AVP, arginine vasopressin. (B) Synthesis of selenocysteine building blocks for Boc-SPPS to enable sulfur to selenium replacements. (C and D) Functional screen of oxytocin analogs 1 to 10 (C) and vasopressin analogs 11 to 16 (D) at the human OT receptor (hOTR), hV1aR, and hV1bR using the fluorescent imaging plate reader (FLIPR) Ca2+ signaling assay and at the hV2R by measuring cyclic adenosine monophosphate (cAMP) accumulation. Taller bars in graphs indicate loss of function at that particular receptor. The last row of beige bars illustrates how modifications affected potency compared to oxytocin at the hOTR and vasopressin at the hV2R (black). (E) Functional screen of oxytocin analogs 1 to 7, vasopressin, and dAVP at the hOTR, hV1aR, and hV1bR as assessed by measuring second-messenger inositol 1-phosphate (IP1) accumulation and at the hV2R as assessed by measuring second-messenger cAMP accumulation. CNS (central nervous system) and PNS (peripheral nervous system) indicate where these receptors can be found in humans. Exact EC50 values are shown in tables S2 and S3.

  • Fig. 2 Pharmacological characterization of [Se-Se]-OT-OH at all four human oxytocin and vasopressin receptors.

    (A) Representative Ca2+ concentration–response curves of [Se-Se]-OT-OH at the hOTR, hV1aR, and hV1bR. (B) Representative cAMP concentration–response curves of oxytocin, vasopressin, and [Se-Se]-OT-OH at the hV2R. (C) Representative IP1 concentration–response curves of [Se-Se]-OT-OH at the hOTR, hV1aR, and hV1bR. (D) Representative radioligand concentration-displacement curves for [Se-Se]-OT-OH at the hOTR, hV1aR, hV1bR, and hV2R. All curves were normalized to percentage of response or displacement of the control ligand (oxytocin for OTR and vasopressin for AVPRs). Data in (A) to (D) are means ± SEM of results obtained from at least n = 3 separate experiments, each performed in triplicate.

  • Fig. 3 Comparison of selectivity, stability, and ability to augment myometrial strip contractions between [Se-Se]-OT-OH and oxytocin.

    (A) Functional selectivity profile of [Se-Se]-OT-OH and OT over all four human (h) and murine (m) oxytocin and vasopressin receptors. OTR, V1aR, and V1bR activity was measured by IP1 accumulation. V2R activity was measured by cAMP accumulation. (B) Metabolic stability of [Se-Se]-OT-OH (t1/2 = 25 hours) compared to oxytocin (t1/2 = 12 hours) in human serum. Data in (A) and (B) are means ± SEM of results obtained from at least n = 3 separate experiments, each performed in triplicate. (C and D) Representative contraction pattern for human myometrial strips exposed to increasing doses of oxytocin (C) or [Se-Se]-OT-OH (D). n = 5 women for [Se-Se]-OT-OH, and n = 8 women for oxytocin.

  • Fig. 4 Social fear conditioning (SFC) mouse study.

    (A and B) Unconditioned (SFC) and conditioned (SFC+) mice were intracerebroventricularly infused with either vehicle (2 μl of Ringer’s solution; n = 9 SFC mice; n = 10 SFC+ mice) or [Se-Se]-OT-OH (250 μM/2 μl; 500 pmol; n = 7 SFC mice; n = 7 SFC+ mice). Oxytocin (250 μM/2 μl; 500 pmol; n = 10 mice) and [Thr4,Gly7]-OT (250 μM/2 μl; 500 pmol; n = 4 mice) were only infused into SFC+ mice 10 min before extinction training. Percentage of mice that investigated three nonsocial stimuli (empty cage) and six social stimuli (cage with a conspecific) during social fear extinction (day 2; A) and six social stimuli during social fear extinction recall (day 3; B) is shown. Data are means ± SEM and were analyzed using two-way analysis of variance (ANOVA) for repeated measures. (A) #P < 0.05 SFC+/vehicle compared to SFC+/[Se-Se]-OT-OH; *P < 0.05 SFC+/vehicle compared to SFC+/OT, SFC+/[Thr4,Gly7]-OT, and SFC/vehicle; (B) $P < 0.05 SFC+/vehicle compared to SFC+/OT and SFC/vehicle.

  • Fig. 5 Effects of vasopressin, oxytocin, and [Se-Se]-OT-OH on maximal intracellular Ca2+ in human cardiomyocytes.

    FLIPR assays showing the effect of vasopressin, oxytocin, and [Se-Se]-OT-OH on the maximal intracellular Ca2+ concentrations. Data are means ± SEM, n = 4 wells per data point.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/508/eaan3398/DC1

    Materials and Methods

    Fig. S1. Representative raw FLIPR data.

    Fig. S2. Schild plot analysis.

    Fig. S3. Functional study of [Se-Se]-OT-OH and d[Se-Se]-OT-OH at the mOTR, mV1aR, mV1bR, and mV2R.

    Fig. S4. Binding study of [Se-Se]-OT-OH and d[Se-Se]-OT-OH at the mOTR, mV1aR, mV1bR, and mV2R.

    Fig. S5. Human myometrial cell contractility assay.

    Fig. S6. Human myometrial strip contractility assay.

    Table S1. Overview of the synthesized peptides including details on their modifications.

    Table S2. Functional potencies for oxytocin and vasopressin analogs at the hOTR, hV1aR, hV1bR, and hV2R.

    Table S3. Functional potencies for oxytocin and vasopressin analogs at the human and murine OTR, V1aR, V1bR, and V2R.

    Table S4. Comparison of functional data from the FLIPR and HTRF-IP1 assays.

    Table S5. Radioligand displacement data for oxytocin and vasopressin analogs at the human and murine OTR, V1aR, V1bR, and V2R.

    Table S6. Overview of the number of electric foot shocks per mouse.

    Reference (110)

  • Supplementary Materials for:

    Subtle modifications to oxytocin produce ligands that retain potency and improved selectivity across species

    Markus Muttenthaler,* Åsa Andersson, Irina Vetter, Rohit Menon, Marta Busnelli, Lotten Ragnarsson, Christian Bergmayr, Sarah Arrowsmith, Jennifer R. Deuis, Han Sheng Chiu, Nathan J. Palpant, Margaret O'Brien, Terry J. Smith, Susan Wray, Inga D. Neumann, Christian W. Gruber, Richard J. Lewis, Paul F. Alewood*

    *Corresponding author. Email: markus.muttenthaler{at}univie.ac.at, markus.muttenthaler{at}univie.ac.at (M.M.);
    p.alewood{at}uq.edu.au (P.F.A.)

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Representative raw FLIPR data.
    • Fig. S2. Schild plot analysis.
    • Fig. S3. Functional study of [Se-Se]-OT-OH and d[Se-Se]-OT-OH at the mOTR, mV1aR, mV1bR, and mV2R.
    • Fig. S4. Binding study of [Se-Se]-OT-OH and d[Se-Se]-OT-OH at the mOTR, mV1aR, mV1bR, and mV2R.
    • Fig. S5. Human myometrial cell contractility assay.
    • Fig. S6. Human myometrial strip contractility assay.
    • Table S1. Overview of the synthesized peptides including details on their modifications.
    • Table S2. Functional potencies for oxytocin and vasopressin analogs at the hOTR, hV1aR, hV1bR, and hV2R.
    • Table S3. Functional potencies for oxytocin and vasopressin analogs at the human and murine OTR, V1aR, V1bR, and V2R.
    • Table S4. Comparison of functional data from the FLIPR and HTRF-IP1 assays.
    • Table S5. Radioligand displacement data for oxytocin and vasopressin analogs at the human and murine OTR, V1aR, V1bR, and V2R.
    • Table S6. Overview of the number of electric foot shocks per mouse.
    • Reference (110)

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    Citation: M. Muttenthaler, Å. Andersson, I. Vetter, R. Menon, M. Busnelli, L. Ragnarsson, C. Bergmayr, S. Arrowsmith, J. R. Deuis, H. S. Chiu, N. J. Palpant, M. O’Brien, T. J. Smith, S. Wray, I. D. Neumann, C. W. Gruber, R. J. Lewis, P. F. Alewood, Subtle modifications to oxytocin produce ligands that retain potency and improved selectivity across species. Sci. Signal. 10, eaan3398 (2017).

    © 2017 American Association for the Advancement of Science

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