Negating coordinative cysteine and methionine residues during metathesis of unprotected peptides.

Ru-Alkylidene catalysed olefin metathesis generates metabolically stable cystine bridge peptidomimetics with defined geometry. Deleterious coordinative bonding to the catalyst by sulfur-containing functionality found in cysteine and methionine residues can be negated by in situ and reversible oxidation of thiol and thioether functionality, as disulfides and S-oxides respectively, to facilitate high yielding ring-closing and cross metathesis of bioorthogonally protected peptides.

Inhibition of human N- and T-type calcium channels by an ortho-phenoxyanilide derivative, MONIRO-1.

BACKGROUND AND PURPOSE: Voltage-gated calcium channels are involved in nociception in the CNS and in the periphery. N-type (Cav 2.2) and T-type (Cav 3.1, Cav 3.2 and Cav 3.3) voltage-gated calcium channels are particularly important in studying and treating pain and epilepsy. EXPERIMENTAL APPROACH: In this study, whole-cell patch clamp electrophysiology was used to assess the potency and mechanism of action of a novel ortho-phenoxylanilide derivative, MONIRO-1, against a panel of voltage-gated calcium channels including Cav 1.2, Cav 1.3, Cav 2.1, Cav 2.2, Cav 2.3, Cav 3.1, Cav 3.2 and Cav 3.3. KEY RESULTS: MONIRO-1 was 5- to 20-fold more potent at inhibiting human T-type calcium channels, hCav 3.1, hCav 3.2 and hCav 3.3 (IC50 : 3.3 ± 0.3, 1.7 ± 0.1 and 7.2 ± 0.3 µM, respectively) than N-type calcium channel, hCav 2.2 (IC50 : 34.0 ± 3.6 µM). It interacted with L-type calcium channels Cav 1.2 and Cav 1.3 with significantly lower potency (IC50  > 100 µM) and did not inhibit hCav 2.1 or hCav 2.3 channels at concentrations as high as 100 µM. State- and use-dependent inhibition of hCav 2.2 channels was observed, whereas stronger inhibition occurred at high stimulation frequencies for hCav 3.1 channels suggesting a different mode of action between these two channels. CONCLUSIONS AND IMPLICATIONS: Selectivity, potency, reversibility and multi-modal effects distinguish MONIRO-1 from other low MW inhibitors acting on Cav channels involved in pain and/or epilepsy pathways. High-frequency firing increased the affinity for MONIRO-1 for both hCav 2.2 and hCav 3.1 channels. Such Cav channel modulators have potential clinical use in the treatment of epilepsies, neuropathic pain and other nociceptive pathophysiologies. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Ring closing metathesis of unprotected peptides.

An efficient and expedient route to the synthesis of dicarba peptides from protecting group-free sequences is reported using Ru-alkylidene catalysed olefin metathesis. A range of cyclic peptides was prepared from linear peptides containing two Z-crotyl glycine residues. Free amine groups were masked as salts with Brønsted acids preventing in situ catalyst decomposition. Excellent RCM conversion was obtained in both DMF and methanol.

Stereoselective synthesis and structural elucidation of dicarba peptides.

A facile stereoselective synthesis of cis and trans unsaturated dicarba peptides has been established using preformed diaminosuberic acid derivatives as bridging units. In addition, characteristic spectral differences in the (13)C-NMR spectra of the cis- and trans-isomers show that the chemical shift of carbons in the Δ4,5-diaminosuberic acid residue can be used to assign stereochemistry in unsaturated dicarba peptides formed from ring closing metathesis of linear peptide sequences.

Divergent Approach to a Family of Tyrosine-Derived Ru-Alkylidene Olefin Metathesis Catalysts.

A simple and generic approach to access a new family of Ru-alkylidene olefin metathesis catalysts with specialized properties is reported. This strategy utilizes a late stage, utilitarian Hoveyda-type ligand derived from tyrosine, which can be accessed via a multigram-scale synthesis. Further functionalization allows the catalyst properties to be tuned, giving access to modified second-generation Hoveyda-Grubbs-type catalysts. This divergent synthetic approach can be used to access solid-supported catalysts and catalysts that function under solvent-free and aqueous conditions.

Inhibition of N-type calcium channels by fluorophenoxyanilide derivatives.

A set of fluorophenoxyanilides, designed to be simplified analogues of previously reported ω-conotoxin GVIA mimetics, were prepared and tested for N-type calcium channel inhibition in a SH-SY5Y neuroblastoma FLIPR assay. N-type or Cav2.2 channel is a validated target for the treatment of refractory chronic pain. Despite being significantly less complex than the originally designed mimetics, up to a seven-fold improvement in activity was observed.

Application of fragment-based screening to the design of inhibitors of Escherichia coli DsbA.

The thiol-disulfide oxidoreductase enzyme DsbA catalyzes the formation of disulfide bonds in the periplasm of Gram-negative bacteria. DsbA substrates include proteins involved in bacterial virulence. In the absence of DsbA, many of these proteins do not fold correctly, which renders the bacteria avirulent. Thus DsbA is a critical mediator of virulence and inhibitors may act as antivirulence agents. Biophysical screening has been employed to identify fragments that bind to DsbA from Escherichia coli. Elaboration of one of these fragments produced compounds that inhibit DsbA activity in vitro. In cell-based assays, the compounds inhibit bacterial motility, but have no effect on growth in liquid culture, which is consistent with selective inhibition of DsbA. Crystal structures of inhibitors bound to DsbA indicate that they bind adjacent to the active site. Together, the data suggest that DsbA may be amenable to the development of novel antibacterial compounds that act by inhibiting bacterial virulence.