Eva-María Priego

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.

Structure, physiological role, and specific inhibitors of human thymidine kinase 2 (TK2): Present and future

Human mitochondrial thymidine kinase (TK2) is a pyrimidine deoxynucleoside kinase (dNK) that catalyzes the phosphorylation of pyrimidine deoxynucleosides to their corresponding deoxynucleoside 5′‐monophosphates by γ‐phosphoryl transfer from ATP. In resting cells, TK2 is suggested to play a key role in the mitochondrial salvage pathway to provide pyrimidine nucleotides for mitochondrial DNA (mtDNA) synthesis and maintenance. However, recently the physiological role of TK2turned out to have direct clinical relevance as well. Point mutations in the gene encoding TK2 have been correlated to mtDNA disorders in a heterogeneous group of patients suffering from the so‐called mtDNA depletion syndrome (MDS). TK2 activity could also be involved in mitochondrial toxicity associated to prolonged treatment with antiviral nucleoside analogues like AZT and FIAU. Therefore, TK2 inhibitors can be considered as valuable tools to unravel the role of TK2 in the maintenance and homeostasis of mitochondrial nucleotide pools and mtDNA, and to clarify the contribution of TK2 activity to mitochondrial toxicity of certain antivirals. Highly selective TK‐2 inhibitors having an acyclic nucleoside structure and efficiently discriminating between TK‐2 and the closely related TK‐1 have already been reported. It is actually unclear whether these agents efficiently reach the inner mitochondrial compartment. In the present review article,structural features of TK2, MDS‐related mutations observed in TK2 and their role in MDS will be discussed. Also, an update on novel and selective TK2 inhibitors will be provided.

5'-O-tritylated nucleoside derivatives: inhibition of thymidine phosphorylase and angiogenesis

Thymidine phosphorylase (TPase) is one of the key enzymes involved in the pyrimidine nucleoside salvage pathway. However, TPase also stimulates angiogenesis, and its expression correlates well with microvessel density and metastasis in a variety of human tumors. We have shown recently that 5′-O-trityl-inosine (KIN59) allosterically inhibits TPase enzymatic activity. KIN59 also inhibits TPase-induced angiogenesis in the chick chorioallantoic membrane (CAM) assay. The trityl group was found to be instrumental to preserve both the anti-TPase and antiangiogenic effect. We have now synthesized a variety of novel 5′-O-trityl nucleoside derivatives. Enzyme activity studies showed that the anti-TPase activity is significantly improved by replacement of the hypoxanthine base by thymine [3.5-fold; i.e., 5′-O-tritylthymidine (KIN6)] and the introduction of chloride on the trityl group [7-fold; i.e., 5′-O-(4-chlorotrityl)-inosine (TP136)], whereas removal of 2′-hydroxyl in the ribose did not significantly alter the anti-TPase activity. Enzyme kinetic studies also demonstrated that 1-(5′-O-trityl-β-d-ribofuranosyl)-thymine (TP124), like KIN59, inhibits TPase in a noncompetitive fashion both with respect to phosphate and thymidine. Most KIN59 analogs markedly inhibited TPase-induced angiogenesis in the CAM assay. In vitro studies showed that the antiangiogenic effect of these compounds is not attributed to endothelial cell toxicity. For several compounds, there was no stringent correlation between their anti-TPase and antiangiogenic activity, indicating that these compounds may also act on other angiogenesis mediators. The antiangiogenic 5′-O-trityl nucleoside analogs also caused degradation of pre-existing, immature vessels at the site of drug exposure. Thus, 5′-O-trityl nucleoside derivatives combine antiangiogenic and vascular-targeting activities, which opens perspectives for their potential use as anticancer agents.

Mitochondrial Thymidine Kinase Inhibitors

Mitochondrial thymidine kinase or TK-2 belongs to the family of mammalian deoxynucleoside kinases (dNKs) that catalyze the phosphorylation of deoxynucleosides to their corresponding deoxynucleoside monophosphates by gamma-phosphoryl transfer of ATP. These enzymes are instrumental in the activation of deoxynucleoside analogues with biological and therapeutic properties. Moreover, dNKs are fundamental to maintain dNTPs pools for DNA synthesis and repair. TK-2 has a mitochondrial localization and is the only thymidine kinase that is physiologically active in non-proliferating and resting cells. Several recent investigations point to an important role of TK-2 in the maintenance of mitochondrial dNTPs pools. Indeed, mutations in the gene encoding TK-2 have been associated with mitochondrial DNA (mtDNA) depletion that mostly affects skeletal muscle. Moreover, TK-2 has been suggested to be implicated in mitochondrial toxicity associated to prolonged treatments with nucleoside analogues (i.e AZT for the treatment of AIDS patients). In this scenario, TK-2 inhibitors could be a useful tool to further clarify both the physiological role of TK-2 in the maintenance of mitochondrial dNTP pools, and the possible contribution of TK-2 to the mitochondrial toxicity of pyrimidine nucleoside analogues. In the present article we review the most recent literature covering different aspects of TK-2 as well as published TK-2 inhibitors, with special emphasis on acyclic nucleoside analogues that have been described by our research groups and whose prototype compound is 1-[(Z)-4-(triphenylmethoxy)-2-butenyl]thymine.

Efficient synthesis and anti-enteroviral activity of 9-arylpurines

To further explore the anti-enteroviral activity of 9-aryl-6-chloropurines, three different series of compounds with a dialkylamino, (alkyl)amido, or oxazolidinone substituent at the aryl ring have been synthesized, in most cases with the aid of microwave-assisted synthesis. The resulting compounds efficiently inhibit Coxsackie virus type B3 (CVB3) replication with EC(50) values varying from 3 to 15 μM, and with no significant toxicity in Vero cells. The most potent compounds also selectively inhibit the replication of other enteroviruses including Coxsackie virus B4 and Echo virus 11. The cross-resistance studies performed with different 9-aryl-6-chloropurines indicate that they all belong to the same pharmacological family and differ from other CVB3 drugs such as enviroxime.

CRDOCK: An Ultrafast Multipurpose Protein–Ligand Docking Tool

An ultrafast docking and virtual screening program, CRDOCK, is presented that contains (1) a search engine that can use a variety of sampling methods and an initial energy evaluation function, (2) several energy minimization algorithms for fine tuning the binding poses, and (3) different scoring functions. This modularity ensures the easy configuration of custom-made protocols that can be optimized depending on the problem in hand. CRDOCK employs a precomputed library of ligand conformations that are initially generated from one-dimensional SMILES strings. Testing CRDOCK on two widely used benchmarks, the ASTEX diverse set and the Directory of Useful Decoys, yielded a success rate of ~75% in pose prediction and an average AUC of 0.66. A typical ligand can be docked, on average, in just ~13 s. Extension to a representative group of pharmacologically relevant G protein-coupled receptors that have been recently cocrystallized with some selective ligands allowed us to demonstrate the utility of this tool and also highlight some current limitations. CRDOCK is now included within VSDMIP, our integrated platform for drug discovery.

Selective Human Adenosine A3 Antagonists based on Pyrido[2,1-f]purine-2,4-diones: Novel Features of hA3 Antagonist Binding

Based on our previous results on the potent antagonist effect of 1H,3H‐pyrido[2,1‐f]purine‐2,4‐diones at the human A3 adenosine receptor, new series of this family of compounds have been synthesized and evaluated in radioligand binding studies against the human A1, A2A, A2B, and A3 receptors. A remarkable improvement in potency, and most noticeable, in selectivity has been achieved, as exemplified by the 3‐cyclopropylmethyl‐8‐methoxy‐1‐(4‐methylbenzyl)‐1H,3H‐pyrido[2,1‐f]purine‐2,4‐dione (10) that combines a very high affinity at hA3 (Ki=2.24 nM), with lack of affinity for the A1, A2A, and A2B receptors. On the basis of the published hA3 receptor model (PDB 1OEA), molecular modeling studies, including molecular dynamics (MD) simulations, have been performed to depict the binding mode of the 1 H,3H‐pyrido[2,1‐f]purine‐2,4‐diones and to justify the selectivity against the other adenosine receptors. These studies have led to novel features of the cavity where our antagonists are bound so that the cavity is lined by the hydrogen‐bonded Gln 167‐Asn 250 pair and by the highly conserved Phe 168.

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.

Thymidine Phosphorylase is Noncompetitively Inhibited by 5′-O-Trityl-Inosine (KIN59) and Related Compounds

We found that 5′-O-trityl-inosine (KIN59) inhibits recombinant bacterial (E. coli) and human thymidine phosphorylase (TPase) with an IC50 of 44 μM and 67 μM, respectively. In contrast to previously described TPase inhibitors, KIN59 does not compete with thymidine (dThd) at the pyrimidine nucleoside-binding site or with inorganic phosphate (Pi) at the phosphate-binding site of the enzyme. These findings are strongly suggestive for the presence of an allosteric binding site at the enzyme. TPase is identical to the angiogenic protein platelet-derived endothelial cell growth factor (PD-ECGF). As such, PD-ECGF stimulates angiogenesis in the chick chorioallantoic membrane (CAM) assay. This angiogenic response was completely inhibited by KIN59. Inosine did not inhibit the enzyme or the angiogenic effect of TPase, confirming that the 5′-O-trityl group in KIN59 is essential for the observed effect. Our observations indicate that allosteric sites in TPase may regulate its biological activity.