Affinity maturation of the RLIP76 Ral binding domain to inform the design of stapled peptides targeting the Ral GTPases.

Ral GTPases have been implicated as critical drivers of cell growth and metastasis in numerous Ras-driven cancers. We have previously reported stapled peptides, based on the Ral effector RLIP76, that can disrupt Ral signaling. Stapled peptides are short peptides that are locked into their bioactive form using a synthetic brace. Here, using an affinity maturation of the RLIP76 Ral-binding domain, we identified several sequence substitutions that together improve binding to Ral proteins by more than 20-fold. Hits from the selection were rigorously analyzed to determine the contributions of individual residues and two 1.5 Å cocrystal structures of the tightest-binding mutants in complex with RalB revealed key interactions. Insights gained from this maturation were used to design second-generation stapled peptides based on RLIP76 that exhibited vastly improved selectivity for Ral GTPases when compared with the first-generation lead peptide. The binding of second-generation peptides to Ral proteins was quantified and the binding site of the lead peptide on RalB was determined by NMR. Stapled peptides successfully competed with multiple Ral-effector interactions in cellular lysates. Our findings demonstrate how manipulation of a native binding partner can assist in the rational design of stapled peptide inhibitors targeting a protein-protein interaction

The discovery and maturation of peptide biologics targeting the small G protein Cdc42: a bioblockade for Ras-driven signalling

Aberrant Ras signalling drives 30% of cancers and inhibition of Rho family small-GTPase signalling has been shown to combat Ras-driven cancers. Here we present the discovery of a 16mer cyclic peptide that binds to Cdc42 with nanomolar affinity. Affinity maturation of this sequence has produced a panel of derived candidates with increased affinity and modulated specificity for other closely related small-GTPases. The structure of the tightest binding peptide was solved by NMR and its binding site on Cdc42 determined. Addition of a cell penetrating sequence allowed the peptides to access the cell interior and engage with their target(s), modulating signalling pathways. In Ras-driven cancer cell models, the peptides have an inhibitory effect on proliferation and show suppression of both invasion and motility. As such they represent promising candidates for Rho-family small GTPase inhibitors and therapeutics targeting Ras-driven cancers. Our data adds to the growing literature demonstrating that peptides are establishing their place in the biologics arm of drug discovery.This research was supported by an MRC CASE Studentship (MR/K017101/1) to DO and RNC, a BBSRC DTP iCASE Studentship (BB/M011194/1) to DO and JR and a short-term Glover Research Fund Fellowship to GJNT

TREM2 shedding by cleavage at the H157-S158 bond is accelerated for the Alzheimer’s disease-associated H157Y variant

We have characterised the proteolytic cleavage events responsible for the shedding of Triggering Receptor Expressed on Myeloid cells 2 (TREM2) from primary cultures of human macrophages, murine microglia and TREM2-expressing human embryonic kidney (HEK293) cells. In all cell types, a soluble 17 kDa N-terminal cleavage fragment was shed into the conditioned media in a constitutive process that is inhibited by G1254023X and metalloprotease inhibitors and siRNA targeting ADAM10. Inhibitors of serine proteases and matrix metalloproteinases 2/9, and ADAM17 siRNA did not block TREM2 shedding. Peptidomimetic protease inhibitors highlighted a possible cleavage site and mass spectrometry confirmed that shedding occurred predominantly at the H157-S158 peptide bond for both wild type and H157Y human TREM2 and for the wild type murine orthologue. Crucially, we also show that the Alzheimer diseaseassociated H157Y TREM2 variant was shed more rapidly than wild type from HEK293 cells, possibly by a novel, batimastat- and ADAM10-siRNA-independent, sheddase activity. These insights offer new therapeutic targets for modulating the innate immune response in Alzheimer’s and other neurological diseases.Funding from the Wellcome Trust and the Canadian Institutes of Health Research contributed to the support of this study

The development of novel immobilised reagents and solid phase linkers

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PS-COD and PS-9-BBN: polymer-supported reagents for solution-phase parallel synthesist

1,5-Cyclooctadiene was deprotonated under LICKOR conditions and reacted with Merrifield resin to afford an immobilized cyclooctadiene in high yield. This polymer is effective as a halogen scavenger, while hydroboration leads to a supported 9-BBN analogue. The latter exhibits similar regioselectivity to 9-BBN in olefin hydroboration

Recombinant Expression and<i> In Vitro</i> Characterisation of Active Huwentoxin-IV

<div><p>Huwentoxin-IV (HwTx-IV) is a 35-residue neurotoxin peptide with potential application as a novel analgesic. It is a member of the inhibitory cystine knot (ICK) peptide family, characterised by a compact globular structure maintained by three intramolecular disulfide bonds. Here we describe a novel strategy for producing non-tagged, fully folded ICK-toxin in a bacterial system. HwTx-IV was expressed as a cleavable fusion to small ubiquitin-related modifier (SUMO) in the cytoplasm of the SHuffle T7 Express <i>lysY Escherichia coli</i> strain, which allows cytosolic disulfide bond formation. Purification by IMAC with selective elution of monomeric SUMO fusion followed by proteolytic cleavage and polishing chromatographic steps yielded pure homogeneous toxin. Recombinant HwTx-IV is produced with a C-terminal acid, whereas the native peptide is C-terminally amidated. HwTx-IV(acid) inhibited Na_v1.7 in a dose dependent manner (IC₅₀ = 463-727 nM). In comparison to HwTx-IV(amide) (IC₅₀ = 11 ± 3 nM), the carboxylate was ~50 fold less potent on Na_v1.7, which highlights the impact of the C-terminus. As the amide bond of an additional amino acid may mimic the carboxamide, we expressed the glycine-extended analogue HwTx-IV^G36(acid) in the SUMO/SHuffle system. The peptide was approximately three fold more potent on Na_v1.7 in comparison to HwTx-IV(acid) (IC₅₀ = 190 nM). In conclusion, we have established a novel system for expression and purification of fully folded and active HwTx-IV(acid) in bacteria, which could be applicable to other structurally complex and cysteine rich peptides. Furthermore, we discovered that glycine extension of HwTx-IV(acid) restores some of the potency of the native carboxamide. This finding may also apply to other C-terminally amidated peptides produced recombinantly.</p> </div

Separating stereoisomers of di-, tri-, and tetrapeptides using capillary electrophoresis with contactless conductivity detection

The separation and detection of small oligopeptides in CE with contactless conductivity detection were demonstrated. A strongly acidic separation buffer (0.5 M acetic acid) was employed in order to render the species cationic. Separation of the stereoisomers was achieved in typically 10-15 min by using either dimethyl-beta-CD (DM-beta-CD), (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C(6)H(4)), a combination of the two substances, or of histidine, as buffer additives. Calibration curves were determined for isomers of Gly-Asp and H-Pro-Asp-NH2, in the range of 0.05-0.5 mM and 0.1-1 mM, respectively, and were found to be linear. LODs were determined to be in the order of 1.0 mu M. The determination of isomeric impurities down to about 1 also be successfully determined on an electrophoretic lab-on-chip device, with analysis times of a few minutes

Characterisation of recombinant HwTx-IV(acid).

<p>A) Analytical RP-HPLC. The recombinant and synthetic HwTx-IV(acid) were analysed separately and combined (1:1). B) LC-MS analysis of the recombinant and synthetic HwTx-IV(acid). The peptides are identical. C) MALDI-TOF analysis of recombinant HwTx-IV(acid). The calculated and observed isotope distributions and m/z ions match. A minor cleaved form was detected: HwTx-IV^E1-V23. The theoretical m/z ions are: [M+H]⁺ = 4105.928 m/z, [M+2H]²⁺ = 2053.464 m/z, [M+3H]³⁺ = 1369.981 m/z and [M+4H]⁴⁺ = 1027.232 m/z. </p

Purification and characterisation of recombinant HwTx-IV^G36(acid).

<p>A) Preparative RP-HPLC. The main peak was collected and lyophilised. B) LC-MS of recombinant and synthetic HwTx-IV^G36(acid). Synthetic and recombinant HwTx-IV^G36(acid) are identical. E) MALDI-TOF analysis of recombinant HwTx-IV^G36(acid). The calculated and observed isotope distributions and m/z ions match. The minor cleaved form HwTx-IV^E1-V23 was detected again. The theoretical m/z ions are: [M+H]⁺ = 4162.950 m/z, [M+2H]²⁺ = 2081.975 m/z, [M+3H]³⁺ = 1388.317 m/z and [M+4H]⁴⁺ = 1041.488 m/z.</p

Electrophysiological characterisation of HwTx-IV(acid/amide) potency at hNa_v1.7.

<p>Na_v1.7 currents were measured from HEK cells stably expressing the hNa_v1.7 alpha subunit using whole cell voltage clamp. Peak sodium current in the presence of increasing doses of HwTx-IV (10 minutes incubation per dose) is plotted as a fraction of basal current (I/Ibasal). Statistics indicate significant difference in IC₅₀ between selected groups and synthetic HwTx-IV(amide), (one-way ANOVA with Tukey post-test; p<0.001, ***). No significant difference was observed between the recombinant and synthetic HwTx-IV(acid).</p