Alba Gigante

Identification of [1,2,3]Triazolo[4,5-d]pyrimidin-7(6H)-ones as Novel Inhibitors of Chikungunya Virus Replication

Chikungunya virus (CHIKV) is a re-emerging Alphavirus that is transmitted to humans by Aedes mosquitoes. Currently, there are still no drugs or vaccines available for the treatment or prevention of this disease. Although traditionally restricted to Africa and Asia, the adaptation of the virus to Aedes albopictus, a mosquito species with an almost worldwide distribution, has contributed to the geographical spread of this virus in the past decade. Here, we report on a new family of compounds named [1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones that inhibit CHIKV replication in the low micromolar range with no toxicity to the host (Vero) cells. The most potent compound in this series has an EC50 value below 1 μM with no cytotoxicity detected up to 668 μM, therefore affording a selectivity index greater than 600. Interestingly, the compounds have little or no antiviral activity on the replication of other members of the Togaviridae family. The exploration and study of this class of selective inhibitors of CHIKV replication will contribute to deeper insights into the CHIKV life cycle and may be a first step toward the development of a clinical drug candidate.

The viral capping enzyme nsP1: a novel target for the inhibition of chikungunya virus infection.

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.

The thymidine phosphorylase inhibitor 5′-O-tritylinosine (KIN59) is an antiangiogenic multitarget fibroblast growth factor-2 antagonist

5′-O-Tritylinosine (KIN59) is an allosteric inhibitor of the angiogenic enzyme thymidine phosphorylase. Previous observations showed the capacity of KIN59 to abrogate thymidine phosphorylase–induced as well as developmental angiogenesis in the chicken chorioallantoic membrane (CAM) assay. Here, we show that KIN59 also inhibits the angiogenic response triggered by fibroblast growth factor-2 (FGF2) but not by VEGF in the CAM assay. Immunohistochemical and reverse transcriptase PCR analyses revealed that the expression of laminin, the major proteoglycan of the basement membrane of blood vessels, is downregulated by KIN59 administration in control as well as in thymidine phosphorylase- or FGF2-treated CAMs, but not in CAMs treated with VEGF. Also, KIN59 abrogated FGF2-induced endothelial cell proliferation, FGF receptor activation, and Akt signaling in vitro with no effect on VEGF-stimulated biologic responses. Accordingly, KIN59 inhibited the binding of FGF2 to FGF receptor-1 (FGFR1), thus preventing the formation of productive heparan sulphate proteoglycan/FGF2/FGFR1 ternary complexes, without affecting heparin interaction. In keeping with these observations, systemic administration of KIN59 inhibited the growth and neovascularization of subcutaneous tumors induced by FGF2-transformed endothelial cells injected in immunodeficient nude mice. Taken together, the data indicate that the thymidine phosphorylase inhibitor KIN59 is endowed with a significant FGF2 antagonist activity, thus representing a promising lead compound for the design of multitargeted antiangiogenic cancer drugs. Mol Cancer Ther; 11(4); 817–29. ©2012 AACR.

Thymidine Phosphorylase Participates in Platelet Signaling and Promotes Thrombosis

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 mellitus. Objective: To 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 with wild-type 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–, adenosine diphosphate-, or thrombin-induced platelet aggregation were significantly attenuated in Tymp+/− and Tymp−/− platelets, and in wild type or human platelets pretreated with TYMP inhibitor KIN59. Tymp deficiency also significantly decreased agonist-induced P-selectin 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 platelet-endothelial cell adhesion molecule 1 tyrosine phosphorylation and diminished collagen-related peptide– or collagen-induced AKT phosphorylation. In vivo administration of KIN59 significantly inhibited FeCl3-induced carotid artery thrombosis without affecting hemostasis. Conclusions: TYMP participates in multiple platelet signaling pathways and regulates platelet activation and thrombosis. Targeting TYMP might be a novel antiplatelet and antithrombosis therapy.

Antiviral activity of [1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones against chikungunya virus targeting the viral capping nsP1

Abstract Chikungunya virus (CHIKV) is a re‐emerging alphavirus transmitted to humans by Aedes mosquitoes. Since 2005, CHIKV has been spreading worldwide resulting in epidemics in Africa, the Indian Ocean islands, Asia and more recently in the Americas. CHIKV is thus considered as a global health concern. There is no specific vaccine or drug available for the treatment of this incapacitating viral infection. We previously identified 3‐aryl‐[1,2,3]triazolo[4,5‐d]pyrimidin‐7(6H)‐ones as selective inhibitors of CHIKV replication and proposed the viral capping enzyme nsP1 as a target. This work describes the synthesis of novel series of related compounds carrying at the aryl moiety a methylketone and related oximes combined with an ethyl or an ethyl‐mimic at 5‐position of the triazolopyrimidinone. These compounds have shown antiviral activity against different CHIKV isolates in the very low &mgr;M range based on both virus yield reduction and virus‐induced cell‐killing inhibition assays. Moreover, these antivirals inhibit the in vitro guanylylation of alphavirus nsP1, as determined by Western blot using an anti‐cap antibody. Thus, the data obtained seem to indicate that the anti‐CHIKV activity might be related to the inhibition of this crucial step in the viral RNA capping machinery. Highlights3‐Aryl‐[1,2,3]triazolo[4,5‐d]pyrimidin‐7(6H)‐ones are selective inhibitors of CHIKV replication.EC50 values of the most potent compounds are around 1 &mgr;M against several clinical isolates.These compounds inhibit the in vitro guanylyl‐transfer activity of alphavirus nsP1.They represent the first compounds inhibiting the guanylyl‐transfer in the mRNA capping of VEEV.

Microwave-assisted synthesis of C-8 aryl and heteroaryl inosines and determination of their inhibitory activities against Plasmodium falciparum purine nucleoside phosphorylase

8-Arylinosines have been scarcely studied for therapeutic purposes, probably due to difficulties in their synthesis. The recently described direct arylation reaction at position 8 of purine nucleosides has been employed to synthesize a series of 8-aryl and 8-pyridylinosines. These compounds have been studied for hydrolytic stability and subjected to biological evaluation. Three compounds have shown a pronounced specific inhibition of Plasmodium falciparum-encoded purine nucleoside phosphorylase, an important target for antimalarial chemotherapy.

5′-Trityl-Substituted Thymidine Derivatives as a Novel Class of Antileishmanial Agents: Leishmania infantum EndoG as a Potential Target

Two series of 5′‐triphenylmethyl (trityl)‐substituted thymidine derivatives were synthesized and tested against Leishmania infantum axenic promastigotes and amastigotes. Several of these compounds show significant antileishmanial activity, with IC50 values in the low micromolar range. Among these, 3′‐O‐(isoleucylisoleucyl)‐5′‐O‐(3,3,3‐triphenylpropanoyl)thymidine displays particularly good activity against intracellular amastigotes. Assays performed to characterize the nature of parasite cell death in the presence of the tritylthymidines indicated significant alterations in mitochondrial transmembrane potential, an increase in superoxide concentrations, and also significant decreases in DNA degradation during the cell death process. Results point to the mitochondrial nuclease LiEndoG as a target for the action of this family of compounds.

Leishmania infantum EndoG Is an Endo/Exo-Nuclease Essential for Parasite Survival

EndoG, a member of the DNA/RNA non-specific ββα-metal family of nucleases, has been demonstrated to be present in many organisms, including Trypanosomatids. This nuclease participates in the apoptotic program in these parasites by migrating from the mitochondrion to the nucleus, where it takes part in the degradation of genomic DNA that characterizes this process. We now demonstrate that Leishmania infantum EndoG (LiEndoG) is an endo-exonuclease that has a preferential 5′ exonuclease activity on linear DNA. Regardless of its role during apoptotic cell death, this enzyme seems to be necessary during normal development of the parasites as indicated by the reduced growth rates observed in LiEndoG hemi-knockouts and their poor infectivity in differentiated THP-1 cells. The pro-life role of this protein is also corroborated by the higher survival rates of parasites that over-express this protein after treatment with the LiEndoG inhibitor Lei49. Taken together, our results demonstrate that this enzyme plays essential roles in both survival and death of Leishmania parasites.