# Chemical Synthesis and Structure–Activity Relationship Studies of the Coagulation Factor Xa Inhibitor Tick Anticoagulant Peptide from the Hematophagous Parasite Ornithodoros moubata

## TL;DR

Scientists synthesized a tick protein that stops blood clotting and found it behaves differently from a similar protein in terms of structure and function.

## Contribution

A new method for synthesizing TAP and comparing its structure and dynamics to BPTI is presented.

## Key findings

- TAP was synthesized with high yield and characterized in detail.
- TAP shows higher conformational flexibility than BPTI.
- TAP's unique properties suggest potential for improved drug design.

## Abstract

Tick Anticoagulant Peptide (TAP), a 60-amino acid protein from the soft tick Ornithodoros moubata, inhibits activated coagulation factor X (fXa) with almost absolute specificity. Despite TAP and Bovine Pancreatic Trypsin Inhibitor (BPTI) (i.e., the prototype of the Kunitz-type protease inhibitors) sharing a similar 3D fold and disulphide bond topology, they have remarkably different amino acid sequence (only ~24% sequence identity), thermal stability, folding pathways, protease specificity, and even mechanism of protease inhibition. Here, fully active and correctly folded TAP was produced in reasonably high yields (~20%) by solid-phase peptide chemical synthesis and thoroughly characterised with respect to its chemical identity, disulphide pairing, folding kinetics, conformational dynamics, and fXa inhibition. The versatility of the chemical synthesis was exploited to perform structure–activity relationship studies on TAP by incorporating non-coded amino acids at positions 1 and 3 of the inhibitor. Using Hydrogen–Deuterium Exchange Mass Spectrometry, we found that TAP has a remarkably higher conformational flexibility compared to BPTI, and propose that these different dynamics could impact the different folding pathway and inhibition mechanisms of TAP and BPTI. Hence, the TAP/BPTI pair represents a nice example of divergent evolution, while the relative facility of TAP synthesis could represent a good starting point to design novel synthetic analogues with improved pharmacological profiles.