Rapid assembly and profiling of an anticoagulant sulfoprotein library

Abstract

Hematophagous organisms produce a suite of salivary proteins which interact with the host’s coagulation machinery to facilitate the acquisition and digestion of a bloodmeal. Many of these biomolecules inhibit the central blood-clotting serine proteinase thrombin that is also the target of several clinically approved anticoagulants. Here a bioinformatics approach is used to identify seven tick proteins with putative thrombin inhibitory activity that we predict to be posttranslationally sulfated at two conserved tyrosine residues. To corroborate the biological role of these molecules and investigate the effects of amino acid sequence and sulfation modifications on thrombin inhibition and anticoagulant activity, a library of 34 homogeneously sulfated protein variants were rapidly assembled using one-pot diselenide-selenoester ligation (DSL)-deselenization chemistry. Downstream functional characterization validated the thrombin-directed activity of all target molecules and revealed that posttranslational sulfation of specific tyrosine residues crucially modulates potency. Importantly, access to this homogeneously modified protein library not only enabled the determination of key structure–activity relationships and the identification of potent anticoagulant leads, but also revealed subtleties in the mechanism of thrombin inhibition, between and within the families, that would be impossible to predict from the amino acid sequence alone. The synthetic platform described here therefore serves as a highly valuable tool for the generation and thorough characterization of libraries of related peptide and/or protein molecules (with or without modifications) for the identification of lead candidates for medicinal chemistry programs.

TickSialoFam (TSFam): A Database That Helps to Classify Tick Salivary Proteins, a Review on Tick Salivary Protein Function and Evolution, With Considerations on the Tick Sialome Switching Phenomenon

“…Other previously identified enzymes that are associated with protein modification (Ribeiro et al, 2006 ; Francischetti et al, 2009 ) include those promoting proline hydroxylation and tyrosine sulfation. While tyrosine sulfation has been determined in tick salivary anticlotting peptides (Thompson et al, 2017 ; Watson et al, 2019 ), proline hydroxylation, a common modification in collagen (Bohmer, 1971 ), has not been confirmed in ticks despite the high abundance of salivary collagen-like cement proteins that display the motifs triggering proline hydroxylation (Rhoads and Udenfriend, 1969 ; de Jong et al, 1991 ; Shimizu et al, 2005 ). Proteomic studies should include these possible modifications in the databases used for identification of peptide fragments obtained by MS/MS.…”

Chemical synthesis of a haemathrin sulfoprotein library reveals enhanced thrombin inhibition following tyrosine sulfation

“…28 The final step to generate the target C-terminal diselenide fragments involved deprotection of the nP sulfate ester(s). While these esters are normally cleaved through the treatment of nucleophilic azide or acetate reagents, 17,29 we found that these could be efficiently and cleanly deprotected by simply incubating the fragments in Gnd buffer at 50 1C for 2 hours. Final purification by reverse-phase HPLC then provided the target C-terminal (sulfo)peptide fragments 20-27 as the corresponding diselenide dimers in 3-6% yield based on resin loading (Scheme 1).…”

“…While the archetypal leechderived hirudin anticoagulants have long been known to be sulfated, [11][12][13] it was not until very recently that Tyr O-sulfation was uncovered as a ubiquitous modification of other salivary anti-inflammatory and anticoagulant proteins from a range of blood feeding organisms. [13][14][15][16][17] Specifically, we have recently demonstrated that a number of thrombin inhibiting tick (from Haemaphysalis longicornis, 15,17 Hyalomma marginatum 17 and Dermacentor andersoni 17 ) and mosquito (from Anopheles albimanus and Anopheles gambiae) 16 salivary proteins are sulfated. Importantly, it has also been demonstrated that sulfation of these proteins uniformly enhances their thrombin inhibitory potency and anticoagulant activity.…”

“…Moreover, the final nP deprotection step prior to HPLC purification of the sulfoprotein targets proceeds sluggishly, representing the longest reaction step in the synthetic sequence (16 h). 17 In order to accelerate access to the haemathrin sulfoform library, we envisaged performing the ligation reactions on peptide fragments with unprotected sTyr residues in this work. Given that DSL reactions are performed at near neutral pH, we proposed that the unprotected sulfate esters would be stable under the reaction conditions and would avoid the aforementioned issues associated with the use of the nP protecting group.…”

“…Syntheses of the C-terminal fragments were achieved using Fmoc-strategy solid-phase peptide synthesis (SPPS) wherein sTyr residues were installed at positions Y31 and Y34 using the commercially available Fmoc-Tyr(SO 3 nP)-OH building block 9. Selenylated building blocks Boc-(g-SePMB)Glu-OH (10) and Boc-(b-SePMB)Asp-OH (11) were coupled as the N-terminal residues to afford resin-bound haemathrin-1 (12-15) and haemathrin-2 (16)(17)(18)(19) fragments, respectively. Each of the peptides were then deprotected and cleaved from resin using an acidic cocktail followed by PMB deprotection (see ESI, † for full synthetic details).…”