ABSTRACT
In recent years, the phenomenon of allostery has witnessed growing attention driven by a fundamental interest in new ways to regulate the functional properties of proteins, as well as the prospects of using allosteric sites as targets to design novel drugs with lower toxicity due to a higher selectivity of binding and specificity of the mechanism of action. The currently available bioinformatic methods can sometimes correctly detect previously unknown ligand binding sites in protein structures. However, the development of universal and more efficient approaches requires a deeper understanding of the common and distinctive features of the structural organization of both functional (catalytic) and allosteric sites, the evolution of their amino acid sequences in respective protein families, and allosteric communication pathways. The CASBench benchmark set contains 91 entries related to enzymes with both catalytic and allosteric sites within their structures annotated based on the experimental information from the Allosteric Database, Catalytic Site Atlas, and Protein Data Bank. The obtained dataset can be used to benchmark the performance of existing computational approaches and develop/train perspective algorithms to search for new catalytic and regulatory sites, as well as to study the mechanisms of protein regulation on a large collection of allosteric enzymes. Establishing a relationship between the structure, function, and regulation is expected to improve our understanding of the mechanisms of action of enzymes and open up new prospects for discovering new drugs and designing more efficient biocatalysts. The CASBench can be operated offline on a local computer or online using built-in interactive tools at https://biokinet.belozersky.msu.ru/casbench.
ABSTRACT
BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a major regulator of clotting initiation and a promising target for pro- and anticoagulation therapy. The aptamer BAX499 (formerly ARC19499) is a high-affinity specific TFPI antagonist designed to improve hemostasis. However, it is not clear how stimulation of coagulation onset by inactivating TFPI will affect spatial and temporal clot propagation. OBJECTIVE: To examine the BAX499 effect on clotting in a spatial, reaction-diffusion experimental system in comparison with that of recombinant activated factor VII (rVIIa). METHODS: Clotting in plasma activated by immobilized tissue factor (TF) was monitored by videomicroscopy. RESULTS: BAX499 dose-dependently improved coagulation in normal and hemophilia A plasma activated with TF at 2 pmole m(-2) by shortening lag time and increasing clot size by up to ~2-fold. The effect was TFPI specific as confirmed by experiments in TFPI-depleted plasma with or without TFPI supplementation. Clotting improvement was half-maximal at 0.7 nm of BAX499 and reached a plateau at 10 nm, remaining there at concentrations up to 1000 nm. The BAX499 effect decreased with TF surface density increase. RVIIa improved clotting in hemophilia A plasma activated with TF at 2 or 20 pmole m(-2) , both by shortening lag time and increasing spatial velocity of clot propagation; its effects were strongly concentration dependent. CONCLUSIONS: BAX499 significantly improves spatial coagulation by inhibiting TFPI in a spatially localized manner that is different to that observed with rVIIa.
