The Role of Disulfide Bond Replacements in Analogues of the Tarantula Toxin ProTx-II and Their Effects on Inhibition of the Voltage-Gated Sodium Ion Channel Nav1.7 - PubMed (original) (raw)
. 2017 Sep 20;139(37):13063-13075.
doi: 10.1021/jacs.7b06506. Epub 2017 Sep 7.
Stephen McCarthy 1, Rachael Dickman 1, Francis E Reyes 2, Silvia Sanchez-Martinez 2, Adam Cryar 3 4, Ian Kilford 5, Adrian Hall 5, Andrew K Takle 5, Maya Topf 4, Tamir Gonen 2, Konstantinos Thalassinos 3 4, Alethea B Tabor 1
Affiliations
- PMID: 28880078
- PMCID: PMC5618157
- DOI: 10.1021/jacs.7b06506
The Role of Disulfide Bond Replacements in Analogues of the Tarantula Toxin ProTx-II and Their Effects on Inhibition of the Voltage-Gated Sodium Ion Channel Nav1.7
Zoë V F Wright et al. J Am Chem Soc. 2017.
Abstract
Spider venom toxins, such as Protoxin-II (ProTx-II), have recently received much attention as selective Nav1.7 channel blockers, with potential to be developed as leads for the treatment of chronic nocioceptive pain. ProTx-II is a 30-amino acid peptide with three disulfide bonds that has been reported to adopt a well-defined inhibitory cystine knot (ICK) scaffold structure. Potential drawbacks with such peptides include poor pharmacodynamics and potential scrambling of the disulfide bonds in vivo. In order to address these issues, in the present study we report the solid-phase synthesis of lanthionine-bridged analogues of ProTx-II, in which one of the three disulfide bridges is replaced with a thioether linkage, and evaluate the biological properties of these analogues. We have also investigated the folding and disulfide bridging patterns arising from different methods of oxidation of the linear peptide precursor. Finally, we report the X-ray crystal structure of ProTx-II to atomic resolution; to our knowledge this is the first crystal structure of an ICK spider venom peptide not bound to a substrate.
Conflict of interest statement
The authors declare no competing financial interest.
Figures
Figure 1
Amino acid sequences and disulfide bond connectivities of the ICK scaffold peptides ProTx-II, HwTx-IV, PaTx-1, GpTx-I, CcoTx-I, and Prialt.
Figure 2
Single ring thioether and disulfide analogues of ProTx-II. The positions of the Cys groups, and their Met replacements, are marked according to the numbering from the full-length wt ProTx-II sequence.
Scheme 1. Synthesis of Lanthionine-Bridged Peptide 2a
Reagents and conditions: (i) (allyl, Aloc/Fmoc)-lanthionine 1, PyAOP, HOAt, DIPEA, DMF, μwave, 5 min, 60 °C; (ii) incorporation of standard protected amino acids with HBTU, DIPEA, DMF, followed by deprotection with piperidine; (iii) Pd(PPh3)4, 1,3-dimethylbarbituric acid, DMF, CH2Cl2, then 40% piperidine/DMF; (iv) PyAOP, HOAt, DIPEA, DMF, μwave, 5 min, 60 °C; (v) Fmoc-Lys(Boc)-OH, HBTU, DIPEA, DMF, then 20% piperidine/DMF; (vi) TFA, ethanedithiol, iPr3SiH, H2O.
Figure 3
Lanthionine-bridged analogues of ProTx-II.
Scheme 2. Synthesis of Lanthionine-Bridged ProTx-II Analogue 12
Standard protecting groups were used for the amino acids, with additional Hmb protection as indicated; see Experimental Section. Reagents and conditions: (i) 1, PyAOP, HOAt, DIPEA, DMF, μwave, 5 min, 60 °C; (ii) incorporation of standard protected amino acids with HBTU, DIPEA, DMF, followed by deprotection with piperidine; (iii) 40% piperidine/DMF, then Pd(PPh3)4, 1,3-dimethylbarbituric acid, DMF, CH2Cl2; (iv) PyAOP, HOAt, DIPEA, DMF, μwave, 5 min, 60 °C; (v) incorporation of standard protected amino acids with HBTU, DIPEA, DMF, followed by deprotection with piperidine; (vi) TFA, iPr3SiH, H2O.
Scheme 3. Synthesis of Lanthionine-Bridged ProTx-II Analogue 13
Standard protecting groups were used for the amino acids, with additional Hmb protection as indicated; see Experimental Section. Reagents and conditions: (i) 1, PyAOP, HOAt, DIPEA, DMF, μwave, 5 min, 60 °C; (ii) incorporation of standard protected amino acids with HBTU, DIPEA, DMF, followed by deprotection with piperidine; (iii) 40% piperidine/DMF, then Pd(PPh3)4, 1,3-dimethylbarbituric acid, DMF, CH2Cl2; (iv) PyAOP, HOAt, DIPEA, DMF, μwave, 5 min, 60 °C; (v) incorporation of standard protected amino acids with HBTU, DIPEA, DMF, followed by deprotection with piperidine; (vi) 5% DTT in 0.1 M NMM, DMF; (vii) NCS (2 equiv), DMF; (viii) TFA, iPr3SiH, H2O.
Figure 4
HPLC of ProTx-II/24h and ProTx-II/7d peptides. All experiments were run using analytical HPLC Method B.
Figure 5
NanoESI spectra of (A) ProTx-II/24h and (B) ProTx-II/7d peptides. The predominant charge states observed are +4 and +5 for the ProTx-II/24h and ProTx-II/7d, respectively. Peaks annotated with * correspond to contaminant species.
Figure 6
Zoom-in of the +4 charge states of (A) ProTx-II/24h and (B) ProTx-II/7d peptides and theoretical isotope distribution corresponding to a ProTx-II with (C) all cysteines oxidized and (D) all cysteines reduced.
Figure 7
Arrival time distribution (ATD) from the IM-MS analysis of the +4 ion for (A) ProTx-II/24h and (B) ProTx-II/7d peptides. (C) The corresponding mass spectrum for ProTx-II/24h and (D–F) mass spectra corresponding to the different colored ATD regions for ProTx-II/7d.
Figure 8
(A) 2Fo-Fc Electron density map calculated using experimental phases, showing traceable peptide backbone and well-resolved side chains. (B) Fragment of ProTx-II crystal structure showing hydrogen bonding between R13 and D10. (C) Comparison of the X-ray crystal structure (left) with previously published NMR ensemble (PDB ID: 2N9T) showing similar overall folding and structure.
References
- King G. F.Venoms to drugs: venom as a source for the development of human therapeutics; Royal Society of Chemistry: Cambridge, 2015.
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- WT_/Wellcome Trust/United Kingdom
- HHMI/Howard Hughes Medical Institute/United States
- 109073/Z/15/Z/WT_/Wellcome Trust/United Kingdom
- 104913/Z/14/ZBM/WT_/Wellcome Trust/United Kingdom
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