Biology subjects

Page, C. P.

Publications and source records attributed to Page, C. P..

3 recordsLinked to original sources

bioRxiv2026

Discovery of a pathway-selective platelet P2Y1R inverse agonist that suppresses inflammation while preserving hemostasis

The platelet P2Y1 receptor (P2Y1R) is necessary for inflammation, signalling via Rho-GTPase pathways to elicit functions that are distinct from aggregation (PLC-dependent canonical signalling pathway). Whether these distinct platelet inflammatory functions can be selectively suppressed to preserve hemostasis through the rational design of P2Y1R antagonists has not been explored. In silico molecular docking analysis examined biased nucleotide interactions within the P2Y1R binding pocket. The identified possible key amino acid residues guided rational design to synthesize compounds for pathway selective inhibition, evolving from nucleotide to non-nucleotide structures. The nucleotide analogue KMR-82-13 was predicted to engage distinct regions of the binding pocket and selectively inhibited platelet chemotaxis while preserving aggregation. These findings informed the design of a non-nucleotide compound KSN-159-27, aiming to retain key KMR-82-13-like interactions while improving drug-like properties. Docking and molecular dynamics simulation supported a stable but dynamic binding mode for KSN-159-27 within the P2Y1R pocket, consistent with pathway-selective inhibition. KSN-159-27 displayed characteristics of a pathway selective inverse agonist at P2Y1R towards G12/13-mediated pathways, but not those associated by Gq activation in P2Y1R-transfected HEK293T cells. KSN-159-27 showed functionally selective inhibition for platelet P2Y1R-mediated functions. In vivo, KSN-159-27 suppressed inflammatory cell recruitment, whilst preserving bleeding time and ADP-induced thromboembolic responses, in contrast to the neutral P2Y1R antagonist MRS2500. This first demonstration for the rational design of a pathway selective inverse agonist at platelet P2Y1Rs has significant implications for novel therapeutic strategies developed to safely target platelet activation during inflammation, in contrast to current anti-platelet drugs used in the prevention of thrombosis. Key PointsO_LIBiased inverse platelet P2Y1R agonists selectively supress inflammation whilst preserving hemostasis and the ability of platelets to aggregate. C_LIO_LIBiased inverse agonism selectively inhibited P2Y1R G12/13 (Rho-GTPAse functions) but not Gq activities (PLC functions). C_LI

pharmacology and toxicology↗

bioRxiv2024

Platelet-specific P2Y1 receptor deficient mice have suppressed leukocyte recruitment in response to lipopolysaccharide.

A role for the P2Y1 receptor in inflammation has been established using a pharmacological approach over an acute 4 hour time span. However, nucleotide-structure P2Y1 receptor antagonists have limited experimental use due to inadequate pharmacokinetics, and inability to decipher global versus cell specific effects in vivo. The creation of a conditional knock out (platelet) P2Y1 transgenic mouse model was designed to overcome these restrictions. A homozygous P2Y1 LoxP mouse colony was created using CRISPR/Cas9 technology, and crossed with a hemizygous P2Y1 LoxP with PF4-cre to provide offspring that are homozygous for P2Y1 LoxP flanked allele, and hemizygous for the PF4cre (platelet P2Y1-/-) and offspring homozygous for P2Y1 LoxP flanked allele, but non-carriers for PF4cre (control mice). Animals were intranasally administered LPS to induce pulmonary inflammation to assess the influence of phenotype on leukocyte recruitment. 24 hours post intranasal LPS administration; pulmonary neutrophil and platelet recruitment were significantly suppressed, despite the fact that neutrophils retained the ability to migrate to fMLP ex vivo. Circulating platelet and leukocyte numbers were not different between control and platelet P2Y1-/- animals. Tail bleeding times revealed the platelet P2Y1-/- mice had a severe bleeding phenotype. This is the first demonstration of a platelet specific P2Y1-/- mouse model to confirm the importance of platelet P2Y1 receptors in the regulation of inflammatory responses with a 60-70% inhibition of leukocyte recruitment over an extended time period compared to previous pharmacological studies. platelet P2Y1-/- mice will help further elucidate the mechanisms by which P2Y1 receptors regulate platelet activation during inflammation. Key PointsO_LIMice selectively deficient in the platelet P2Y1 have suppressed leukocyte and platelet recruitment during inflammation. C_LIO_LIWe provide a methodology to determine mechanistic relevance that is otherwise limited by pharmacological approaches. C_LI

pharmacology and toxicology↗

bioRxiv2022

Stimulation of platelet P2Y1 receptors by differentendogenous nucleotides leads to functional selectivity via biased signalling.

Background and PurposePlatelet function during inflammation is dependent on activation by endogenous nucleotides. Non-canonical signalling via the P2Y1 receptor is important for these non-thrombotic functions of platelets. However, apart from ADP, the role of other endogenous nucleotides acting as agonists at P2Y1 receptors is unknown. This study compared the effects of ADP, Ap3A, NAD+, ADP-ribose, and Up4A on platelet functions contributing to inflammation or haemostasis. Experimental ApproachPlatelets obtained from healthy human volunteers were incubated with ADP, Ap3A, NAD+, ADP-ribose or Up4A, with aggregation and fibrinogen binding measured (examples of function during haemostasis) or before exposure to fMLP to measure platelet chemotaxis (an inflammatory function). In silico molecular docking of these nucleotides to the binding pocket of P2Y1 receptors was then assessed. Key ResultsPlatelet aggregation and binding to fibrinogen induced by ADP was not mimicked by NAD+, ADP-ribose, and Up4A. However, these endogenous nucleotides induced P2Y1-dependent platelet chemotaxis, an effect that required RhoA and Rac-1 activity, but not canonical PLC activity. Analysis of molecular docking of the P2Y1 receptor revealed distinct differences of amino acid interactions and depth of fit within the binding pocket by Ap3A, NAD+, ADP-ribose or Up4A compared to ADP. Conclusion and ImplicationsPlatelet function (aggregation vs motility) can be differentially modulated by biased-agonist activation of P2Y1 receptors. This may be due to the character of the ligand-binding pocket interaction. This has implications for future therapeutic strategies aimed to suppress platelet activation during inflammation without affecting haemostasis as is the requirement of current ant-platelet drugs.

pharmacology and toxicology↗