The chikungunya virus (CHIKV) has become a substantial global health threat due to its massive re-emergence, the considerable disease burden and the lack of vaccines or therapeutics. We discovered a novel class of small molecules ([1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones) with potent in vitro activity against CHIKV isolates from different geographical regions. Drug-resistant variants were selected and these carried a P34S substitution in non-structural protein 1 (nsP1), the main enzyme involved in alphavirus RNA capping. Biochemical assays using nsP1 of the related Venezuelan equine encephalitis virus revealed that the compounds specifically inhibit the guanylylation of nsP1. This is, to the best of our knowledge, the first report demonstrating that the alphavirus capping machinery is an excellent antiviral drug target. Considering the lack of options to treat CHIKV infections, this series of compounds with their unique (alphavirus-specific) target offers promise for the development of therapy for CHIKV infections.

Rationale Platelets contain abundant thymidine phosphorylase (TYMP), which is highly expressed in diseases with high risk of thrombosis, such as atherosclerosis and type II diabetes. Objective Test the hypothesis that TYMP participates in platelet signaling and promotes thrombosis. Methods and Results By using a ferric chloride (FeCl3) induced carotid artery injury thrombosis model, we found time to blood flow cessation was significantly prolonged in Tymp−/− and Tymp+/− mice compared to wild type (WT) mice. Bone marrow transplantation and platelet transfusion studies demonstrated that platelet TYMP was responsible for the antithrombotic phenomenon in the TYMP deficient mice. Collagen-, collagen-related peptide (CRP)-, adenosine diphosphate-and/or thrombin-induced platelet aggregation were significantly attenuated in Tymp+/− and Tymp−/− platelets, and in WT or human platelets pretreated with TYMP inhibitor KIN59. Tymp deficiency also significantly decreased agonist-induced P-select in expression. TYMP contains an N-terminal SH3 domain binding proline-rich motif and forms a complex with the tyrosine kinases Lyn, Fyn and Yes in platelets. TYMP-associated Lyn was inactive in resting platelets, and TYMP trapped and diminished active Lyn after collagen stimulation. Tymp/Lyn double haploinsufficiency diminished the antithrombotic phenotype of Tymp+/− mice. TYMP deletion or inhibition of TYMP with KIN59 dramatically increased PECAM-1 tyrosine phosphorylation and diminished CRP or collagen induced AKT phosphorylation. In vivo administration of KIN59 significantly inhibited FeCl3 induced carotid artery thrombosis without affecting hemostasis. Conclusion TYMP participates in multiple platelet signaling pathways and regulates platelet activation and thrombosis. Targeting TYMP might be a novel anti-platelet and anti-thrombosis therapy.