Maria Jesus Perez-Perez

Anti-angiogenic activity of a novel multi-substrate analogue inhibitor of thymidine phosphorylase

7‐Deazaxanthine (7‐DX) was recently identified as the first purine derivative with pronounced inhibitory activity against Escherichia coli thymidine phosphorylase (TP) and angiogenesis. In order to ‘freeze’ the enzyme in an open, inactive conformation, a novel multi‐substrate analogue inhibitor of TP, containing an alkyl phosphonate moiety covalently linked to 7‐DX, was synthesized. The prototype compound TP65 (9‐(8‐phosphonooctyl)‐7‐deazaxanthine) (at 250 μM) completely inhibited TP‐induced formation of microvascular sprouts from endothelial cell aggregates in a three‐dimensional fibrin gel. In the chick chorioallantoic membrane assay, TP caused a dose‐dependent stimulation of angiogenesis, which was completely inhibited by 250 nmol TP65. This dose proved to be non‐toxic for the developing chick embryo. TP65 thus emerges as a potent and specific inhibitor of TP and TP‐induced angiogenesis, which opens new perspectives for multi‐substrate analogue inhibitors of TP as potential anti‐cancer agents and as inhibitors of angiogenesis and of diseases with enhanced expression of TP.

Exploring Acyclic Nucleoside Analogues as Inhibitors of Mycobacterium tuberculosis Thymidylate Kinase

In the search for novel inhibitors of the enzyme thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt), an attractive target for novel antituberculosis agents, we report herein the discovery of the first acyclic nucleoside analogues that potently and selectively inhibit TMPKmt. The most potent compounds in this series are (Z)‐butenylthymines carrying a naphtholactam or naphthosultam moiety at position 4, which display Ki values of 0.42 and 0.27 μM, respectively. Docking studies followed by molecular dynamics simulations performed to rationalize the interaction of this new family of inhibitors with the target enzyme revealed a key interaction between the distal substituent and Arg 95 in the target enzyme. The fact that these inhibitors are more easily synthesizable than previously identified TMPKmt inhibitors, together with their potency against the target enzyme, makes them attractive lead compounds for further optimization.

Hiv-1 Specific Reverse Transcriptase Inhibitors: why are Tsao-Nucleosides so Unique?

1. INTRODUCTION AIDS will still be one of the most important challenges for the Scientific Community in the approaching new century. Since the identification, in 1983-84,1,2 of human immunodeficiency virus (HIV) as the etiological agent of AIDS, significant progress has been made in the treatment of HIV-infected patients. This has been in part due to the discovery and clinical use of an increasing number of anti-HIV drugs. However, while highly active antiretroviral therapy (HAART)3 approaches have reduced the morbidity and mortality, the intertwined problems of drug induced viral resistance, poor compliance with complex regimens and therapy failure continue. Therefore, there remains a pressing need for the development of new antiviral agents that can be used not only as first line therapeutic candidates, but also in the antiretroviral-experienced patient population.

Antivascular and antitumor properties of the tubulin-binding chalcone TUB091.

We investigated the microtubule-destabilizing, vascular-targeting, anti-tumor and anti-metastatic activities of a new series of chalcones, whose prototype compound is (E)-3-(3’’-amino-4’’-methoxyphenyl)-1-(5’-methoxy-3’,4’-methylendioxyphenyl)-2-methylprop-2-en-1-one (TUB091). X-ray crystallography showed that these chalcones bind to the colchicine site of tubulin and therefore prevent the curved-to-straight structural transition of tubulin, which is required for microtubule formation. Accordingly, TUB091 inhibited cancer and endothelial cell growth, induced G2/M phase arrest and apoptosis at 1-10 nM. In addition, TUB091 displayed vascular disrupting effects in vitro and in the chicken chorioallantoic membrane (CAM) assay at low nanomolar concentrations. A water-soluble L-Lys-L-Pro derivative of TUB091 (i.e. TUB099) showed potent antitumor activity in melanoma and breast cancer xenograft models by causing rapid intratumoral vascular shutdown and massive tumor necrosis. TUB099 also displayed anti-metastatic activity similar to that of combretastatin A4-phosphate. Our data indicate that this novel class of chalcones represents interesting lead molecules for the design of vascular disrupting agents (VDAs). Moreover, we provide evidence that our prodrug approach may be valuable for the development of anti-cancer drugs.

Aldol reaction of nucleoside 5'-carboxaldehydes with acetone. Synthesis of 5'-C-chain extended thymidine derivatives

Abstract Reaction of 3′-0-(t-butyldimethylsilyl)-2′-deoxythymidine-5′-carboxaldehyde and 2′,3′-dideoxythymidine-5′-carboxaldehyde with acetone afforded a 3:2 mixture of the two (5′R)- and (5′S)-5′-acetonylthymidine derivatives.

Improving the selectivity of acyclic nucleoside analogues as inhibitors of human mitochondrial thymidine kinase: replacement of a triphenylmethoxy moiety with substituted amines and carboxamides.

Two series of analogues of the novel human mitochondrial thymidine kinase inhibitor 1-[(Z)-4-(triphenylmethoxy)-2-butenyl]thymine were synthesized by replacing the triphenylmethoxy moiety by a variety of substituted amines and carboxamides. In all the cases, the selectivity against the mitochondrial enzyme was either maintained or improved, and several derivatives were almost as potent as the parent compound. A molecular model was built that can account for the observed selectivities.

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.

Novel N-3 substituted TSAO-T derivatives: synthesis and anti-HIV-evaluation.

Novel derivatives of the anti-HIV-1 agent, TSAO-T, bearing at the N-3 position alkylating groups or photoaffinity labels were prepared and evaluated for their anti-HIV activity. All of these compounds demonstrated pronounced anti-HIV-1 activity and inhibited HIV-1 RT; however, we were unable to detect stable covalent linkages between inhibitor and enzyme. In addition, compounds with an alcohol functional group connected to the N-3 position through a cis or trans double bond have been prepared. These compounds have been useful to study how the conformational restriction of the linker affects in the interaction between the N-3 substituent and the HIV-1 RT enzyme.

Presence of 2' ,5'-Bis-O-(tert-butyldimethylsilyl)-3-spiro-5"(4"-amino-1",2"-oxathiole-2",2"-dioxide) (TSAO)-Resistant Virus Strains in TSAO-Inexperienced HIV Patients

HIV-1 samples from six patients undergoing diverse anti-HIV therapies possessed the E138A mutation in their reverse transcriptase (RT) genome. Patients were receiving the following therapies: TIBO monotherapy (one patient); zidovudine plus didanosine combination therapy (one); zidovudine monotherapy (one); sequential therapy with zidovudine, then stavudine and finally zalcitabine plus didanosine (one); and two were drug naive. E138K, not E138A, is a known TSAO-specific resistance mutation, emerging under selective pressure in vitro. Our phenotypic data on the patient isolates, confirmed by data on an E138A mutant acquired through in vitro mutagenesis, indicated that an alanine substitution for glutamate at codon 138 of the HIV-1 RT renders the virus TSAO resistant, confirming the importance of this amino acid residue in the activity of TSAO derivatives. In addition, we have demonstrated through phenotypic analysis of the E138A and A98S mutants (after in vitro mutagenesis) that the mutation A98S, found in one of these patients, could be partially responsible for the phenotypic reversal of TSAO resistance. This reversal could be explained by the restoration of a hydrogen bond between 98S and the main-chain residue L349, which compensates for the loss of the E138-G99 main-chain hydrogen bond. As TSAO derivatives have not been used in the clinical setting, the presence of the E138A mutation at a frequency of 6.7% in our study of 90 TSAO-inexperienced HIV-seropositive individuals implies that 138A of the RT must be a natural variant and that the mutant virus is replication competent. Our observations suggest that the E138A mutation may likely arise in patients under the selective pressure of TSAO or related compounds that show a decreased antiviral potency toward the E138A variant.

Anti-HIV-1 activity of a tripodal receptor that recognizes mannose oligomers

The glycoprotein gp120 of the HIV-1 viral envelope has a high content in mannose residues, particularly α-1,2-mannose oligomers. Compounds that interact with these high-mannose type glycans may disturb the interaction between gp120 and its (co)receptors and are considered potential anti-HIV agents. Previously, we demonstrated that a tripodal receptor (1), with a central scaffold of 1,3,5-triethylbenzene substituted with three 2,3,4-trihydroxybenzoyl groups, selectively recognizes α-1,2-mannose polysaccharides. Here we present additional studies to determine the anti-HIV-1 activity and the mechanism of antiviral activity of this compound. Our studies indicate that 1 shows anti-HIV-1 activity in the low micromolar range and has pronounced gp120 binding and HIV-1 integrase inhibitory capacity. However, gp120 binding rather than integrase inhibition seems to be the primary mechanism of antiviral activity of 1.