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Biomedical subjects

Federico Gago

Publications and source records attributed to Federico Gago.

At least 37 records · Page 2Linked to original sources
PubMed2005

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.

Animals↗
PubMed2004

Phosphorylation modulates the alpha-helical structure and polymerization of a peptide from the third tau microtubule-binding repeat.

Paired helical filaments (PHFs) isolated from patients with Alzheimer's disease (AD) mainly consist of the microtubule-associated protein tau in a hyperphosphorylated form. It has been found that PHFs are the first example of pathological protein aggregation associated with formation of alpha-helices [Biochemistry (2002) 41, 7150-5]. In an effort to investigate the interplay between phosphorylation and the putative role of short regions of alpha-helix in the polymerization of tau, we have focused on the region of tau encompassing residues 317 to 335. This region is able to form protein fibrils in vitro and has two serines that are often found phosphorylated in PHFs. Using trifluoroethanol as an indicator of the alpha-helix, we find that the stability of the alpha-helix conformation is enhanced by phosphorylation. Circular dichroism data show that the phosphorylated peptide in water presents a content in alpha-helix similar to the unphosphorylated peptide at 40% of trifluoroethanol. Phosphorylation also stimulates the effect of juglone in promoting the in vitro polymerization. Furthermore, Fourier transformed infrared spectroscopy of samples of phosphorylated peptide polymerized with juglone renders a spectrum with maxima at approximately 1665 and approximately 1675 cm(-1), which are suggestive of a mixture of turns and alpha-helix conformations. Our results provide a direct mechanistic connection between phosphorylation and polymerization in tau. The connection between phosphorylation and polymerization appears to involve formation of alpha-helix structure.

Amino Acid Sequence↗
PubMed2004

Structural basis for the binding of didemnins to human elongation factor eEF1A and rationale for the potent antitumor activity of these marine natural products.

Didemnins and tamandarins are closely related marine natural products with potent inhibitory effects on protein synthesis and cell viability. On the basis of available biochemical and structural evidence and results from molecular dynamics simulations, a model is proposed that accounts for the strong and selective binding of these compounds to human elongation factor eEF1A in the presence of GTP. We suggest that the p-methoxyphenyl ring of these cyclic depsipeptides is inserted into the same pocket in eEF1A that normally lodges either the 3' terminal adenine of aminoacylated tRNA, as inferred from two prokaryotic EF-Tu.GTP.tRNA complexes, or the aromatic side chain of Phe/Tyr-163 from the nucleotide exchange factor eEF1Balpha, as observed in several X-ray crystal structures of a yeast eEF1A:eEF1Balpha complex. This pocket, which has a strong hydrophobic character, is formed by two protruding loops on the surface of eEF1A domain 2. Further stabilization of the bound depsipeptide is brought about by additional crucial interactions involving eEF1A domain 1 in such a way that the molecule fits snugly at the interface between these two domains. In the GDP-bound form of eEF1A, this binding site exists only as two separate halves, which accounts for the much greater affinity of didemnins for the GTP-bound form of this elongation factor. This binding mode is entirely different from those seen in the complexes of the homologous prokaryotic EF-Tu with kirromycin-type antibiotics or the cyclic thiazolyl peptide antibiotic GE2270A. Interestingly, the set of interactions used by didemnins to bind to eEF1A is also distinct from that used by eEF1Balpha or eEF1Bbeta, thus establishing a competition for binding to a common site that goes beyond simple molecular mimicry. The model presented here is consistent with both available biochemical evidence and known structure-activity relationships for these two classes of natural compounds and synthetic analogues and provides fertile ground for future research.

Amino Acid Sequence↗
PubMed2004

Modulation of binding strength in several classes of active site inhibitors of acetylcholinesterase studied by comparative binding energy analysis.

The comparative binding energy (COMBINE) methodology has been used to identify the key residues that modulate the inhibitory potencies of three structurally different classes of acetylcholinesterase inhibitors (tacrines, huprines, and dihydroquinazolines) targeting the catalytic active site of this enzyme. The extended set of energy descriptors and the partial least-squares methodology used by COMBINE analysis on a unique training set containing all the compounds yielded an interpretable model that was able to fit and predict the activities of the whole series of inhibitors reasonably well (r2 = 0.91 and q2 = 0.76, 4 principal components). A more robust model (q2 = 0.81 and SDEP = 0.25, 3 principal components) was obtained when the same chemometric analysis was applied to the huprines set alone, but the method was unable to provide predictive models for the other two families when they were treated separately from the rest. This finding appears to indicate that the enrichment in chemical information brought about by the inclusion of different classes of compounds into a single training set can be beneficial when an internally consistent set of pharmacological data can be derived. The COMBINE model was externally validated when it was shown to predict the activity of an additional set of compounds that were not employed in model construction. Remarkably, the differences in inhibitory potency within the whole series were found to be finely tuned by the electrostatic contribution to the desolvation of the binding site and a network of secondary interactions established between the inhibitor and several protein residues that are distinct from those directly involved in the anchoring of the ligand. This information can now be used to advantage in the design of more potent inhibitors.

Algorithms↗
PubMed2004

Identification of the minimal conserved structure of HIV-1 protease in the presence and absence of drug pressure.

OBJECTIVE: To define the extent of amino acid protease (PR) conservation in vivo in the absence and presence of pharmacological pressure in a large patient cohort. METHODS: Plasma-derived complete protein PR sequences from a well-defined cohort of 1096 HIV-1 infected individuals (457 drug-naive and 639 under antiretroviral therapy including PR-inhibitors) were obtained and analysed, and are discussed in a structural context. RESULTS: In naive patients, the PR sequence showed conservation (< 1% variability) in 68 out of 99 (69%) residues. Five large conserved regions were observed, one (P1-P9) at the N-terminal site, another (E21-V32) comprised the catalytic active-site, a third (P44-V56) contained the flap, a fourth contained the region G78-N88, and another (G94-F99) contained the C-terminal site. In PR-inhibitor treated patients, the appearance of mutations primarily associated with drug resistance determined a decrease of amino acid invariance to 45 out of 99 residues (45% conservation). The overall degree of enzyme conservation, when compared to the PR sequences in drug-naive patients, was preserved at the N- and C-terminal regions, whereas the other large conserved areas decreased to smaller domains containing, respectively, the active-site residues D25-D29, the tip of the flap G49-G52, and the G78-P81 and G86-R87 turns. CONCLUSIONS: Amino acid conservation in HIV PR can be minimally present in 45 residues out of 99. Identification of these invariable residues, with crucial roles in dimer stability, protein flexibility and catalytic activity, and their mapping on the three-dimensional structure of the enzyme will help guide the design of novel resistance-evading drugs.

Amino Acid Sequence↗
PubMed2004

Benzo[f]azino[2,1-a]phthalazinium cations: novel DNA intercalating chromophores with antiproliferative activity.

New azaquinolizinium-type cations have been obtained from isochromane. The synthesis was completed over seven steps and included as the key feature an intramolecular Westphal condensation. This first example of the intramolecular process allowed the preparation of benzo[f]pyrido[2,1-a]phthalazinium and benzo[f]quino[2,1-a]phthalazinium salts, which were evaluated as DNA intercalators, DNA topoisomerase I inhibitors, and antiproliferative compounds. Both cationic systems behave as DNA intercalators and exhibit antiproliferative activity. The pentacyclic benzo[f]quino[2,1-a]phthalazinium cations also have an inhibitory effect on the catalytic activity of DNA topoisomerase I, without trapping of cleavage complexes. Structural characterization using density functional theory indicates that the fused ring systems are slightly nonplanar, and additional molecular modeling studies suggest a preferred orientation for the intercalating chromophores within a typical CpG or TpG intercalation site.

Antineoplastic Agents↗
PubMed2004

Role of histidine-85 in the catalytic mechanism of thymidine phosphorylase as assessed by targeted molecular dynamics simulations and quantum mechanical calculations.

The structural changes taking place in the enzyme thymidine phosphorylase (TPase, also known as PD-ECGF) that are required to achieve catalytic competence upon binding thymidine and phosphate have been simulated by means of targeted molecular dynamics (tMD). The hinge regions were characterized by structural homology comparisons with pyrimidine nucleoside phosphorylase, whose X-ray structure has been solved both in a closed and in an open form. The rearrangement of residues around the substrate that was observed during the tMD trajectory suggested that His-85 could be playing an important role in the catalytic mechanism. A quantum mechanical study of the reaction in the presence of the most relevant active site residues was then performed at the semiempirical level. The results revealed that His-85 could be involved in the protonation of the pyrimidine base at the O2 position to yield the enol tautomer of the base. To establish the role of this oxygen atom in the reaction, ground states, transition states, and final products were studied using higher level ab initio methods starting from both thymidine and 2-thiothymidine as alternative substrates. Comparison of both transition states showed that replacing the oxygen at position 2 of the pyrimidine base by sulfur should accelerate the reaction rate. Consistent with this result, 2-thiothymidine was shown to be a better substrate for TPase than the natural substrate, thymidine. For simulating the final step of the reaction, tMD simulations were used to study domain opening upon product formation considering both the enol and keto tautomers of thymine. Product release from the enzyme was easiest in the simulation that incorporated the keto tautomer of thymine, suggesting that the enol intermediate spontaneously tautomerizes back to the more energetically stable keto form. These results highlight a previously unreported role for His-85 in the catalytic mechanism of TPase and can have important implications for the design of novel TPase inhibitors.

Amino Acid Sequence↗
PubMed2004

In silico activation of Src tyrosine kinase reveals the molecular basis for intramolecular autophosphorylation.

Structural data suggest that important hinge-bending motions of the two lobes that shape the catalytic domain of Src tyrosine kinase, together with reorganization of an alpha helix (helix C), are needed for the activation loop to adopt the catalytically competent conformation. The phosphorylation of a Tyr residue (Tyr-416) in this loop also seems to be essential for enzyme activation. However, no information is available about the dynamics of this activation process. By comparing the inactive and active forms of the catalytic domains of Src and Lck, another member of the Src family, we first identified a short stretch that can act as a hinge for the interlobe motion. The opening of the lobes was then simulated using a targeted molecular dynamics approach. The results obtained suggested that pulling the two lobes apart is not enough to induce the required conformational change in the activation loop. Rather unexpectedly, however, swinging of the lobes situated Tyr-416 in a suitable position for intramolecular autophosphorylation, and further simulation of Tyr-416-phosphorylated Src in the presence of ADP did then result in a conformational change that placed the activation loop in a position similar to that found in the active open conformation of Lck. Taken together, our results establish a physical link between intramolecular autophosphorylation and loop activation.

Adenosine Diphosphate↗
PubMed2004

ET-18-OCH3 (edelfosine): a selective antitumour lipid targeting apoptosis through intracellular activation of Fas/CD95 death receptor.

Synthetic ether-linked analogues of phosphatidylcholine and lysophosphatidylcholine, collectively named as antitumour lipids (ATLs), were initially synthesized in the late 60s, but have attracted a renewed interest since the finding that the ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3, edelfosine), a synthetic analogue of 2-lysophosphatidylcholine considered the ATL prototype, induces a selective apoptotic response in tumour cells, sparing normal cells. Unlike most chemotherapeutic agents currently used, ET-18-OCH3 does not interact with DNA, but act at the cell membrane, and thereby its effects seem to be independent of the proliferative state of target cells. Each part of the molecular structure of ET-18-OCH3 is important for its optimal proapoptotic activity. Recent progress has unveiled the molecular mechanism underlying the apoptotic action of ET-18-OCH3, involving membrane rafts and Fas/CD95 death receptor, and has led to the proposal of a two-step model for the ET-18-OCH3 selective action on cancer cells, namely: a) ET-18-OCH3 uptake into the tumour cell, but not in normal cells; b) intracellular activation of Fas/CD95 through its translocation and capping into membrane rafts. ET-18-OCH3 constitutes the first antitumour drug acting through the intracellular activation of the Fas/CD95 death receptor. Computational docking studies have allowed us to propose a molecular model for the putative interaction of ET-18-OCH3 with the intracellular Fas/CD95 death domain. This novel mechanism of action represents a new way to target tumour cells in cancer chemotherapy and can be of interest as a new framework in designing novel and more selective proapoptotic antitumour drugs.

Animals↗
PubMed2004

Modelling and simulation: a computational perspective in anticancer drug discovery.

The availability of high-quality molecular graphics tools in the public domain is changing the way macromolecular structure is perceived by researchers, educators and students alike. Computational methods have become increasingly important in a number of areas such as comparative or homology modelling, functional site location, characterization of ligand-binding sites in proteins, docking of small molecules into protein binding sites, protein-protein docking, and molecular dynamics simulations. The results obtained yield information that sometimes is beyond current experimental possibilities and can be used to guide and improve a vast array of experiments. On the basis of our improved level of understanding of molecular recognition and the widespread availability of target structures, it is reasonable to assume that computational methods will continue aiding not only in the design and interpretation of hypothesis-driven experiments in the field of cancer research but also in the rapid generation of new hypotheses.

Amino Acid Sequence↗
PubMed2004

HIV protease inhibition: limited recent progress and advances in understanding current pitfalls.

The identification of HIV-1 protease (HIVp) as a target for therapeutic intervention against AIDS was soon followed by major efforts to understand its substrate specificity, reaction kinetics and three-dimensional structure, both in the free state and in complex with a number of ligands including substrate mimics, products, and inhibitors. On the whole these studies have been extremely successful and have had a major impact on our understanding of ligand-receptor interactions and enzyme inhibition mechanisms. HIVp has also become a paradigm for the development and testing of new drug-design methodologies both in vitro and in silico. Even though thousands of potential HIVp inhibitors exhibiting amazing chemical diversity have been synthesized or identified from natural sources, only a few have turned out to be useful for human therapy. Although the alternative goal of preventing enzyme dimerization has been achieved as a proof of concept, this approach has not yet yielded a clinical candidate. The review covers the general strategies that led to some of the most useful inhibitors, the reasons for our limited success in effectively inhibiting this retroviral target in a clinical setting, current progress with second-generation inhibitors, and new avenues for research.

Anti-HIV Agents↗
PubMed2004

TSAO compounds: the comprehensive story of a unique family of HIV-1 specific inhibitors of reverse transcriptase.

Emergence of drug-resistant viral strains is one of the major milestones and the main cause for the failure of antiretroviral therapy. Combination of different anti-HIV agents has become the standard clinical practice to keep the viral load at low or even undetectable levels and to prevent emergence of virus-drug resistance. Among the human immunodeficiency virus (HIV) reverse transcriptase (RT) inhibitors, the so called nonnucleoside RT inhibitors (NNRTIs) have gained a definitive place in the treatment of HIV infections in combination with nucleoside analogue RT inhibitors (NRTIs) and HIV protease inhibitors (PIs). The virus can be markedly suppressed for a relatively long period of time when exposed to multiple drug combination therapy (highly active antiretroviral therapy, HAART). TSAO derivatives are a peculiar group of highly functionalized nucleosides that belong to the so-called nonnucleoside RT inhibitors (NNRTIs). They exert their unique selectivity for HIV-1 through a specific interaction with the p51 subunit of HIV-1 RT. They are the first small molecules that seem to interfere with the dimerization process of the enzyme. This review covers the work carried out with this unique class of specific inhibitors of HIV-1 reverse transcriptase, including structure activity relationship studies (SAR), its mechanism of action, resistance studies, model of interaction with the enzyme, etc.

Anti-HIV Agents↗
PubMed2003

Development of a new family of conformationally restricted peptides as potent nucleators of beta-turns. Design, synthesis, structure, and biological evaluation of a beta-lactam peptide analogue of melanostatin.

Novel enantiopure (i)-(beta-lactam)-(Gly)-(i+3) peptide models, defined by the presence of a central alpha-alkyl-alpha-amino-beta-lactam ring placed as the (i+1) residue, have been synthesized in a totally stereocontrolled way by alpha-alkylation of suitable N-[bis(trimethylsilyl)methyl]-beta-lactams. The structural properties of these beta-lactam pseudopeptides have been studied by X-ray crystallography, Molecular Dynamics simulation, and NOESY-restrained NMR simulated annealing techniques, showing a strong tendency to form stable type II or type II' beta-turns either in the solid state or in highly coordinating DMSO solutions. Tetrapeptide models containing syn- or anti-alpha,beta-dialkyl-alpha-amino-beta-lactam rings have also been synthesized and their conformations analyzed, revealing that alpha-alkyl substitution is essential for beta-turn stabilization. A beta-lactam analogue of melanostatin (PLG amide) has also been prepared, characterized as a type-II beta-turn in DMSO-d6 solution, and tested by competitive binding assay as a dopaminergic D2 modulator in rat neuron cultured cells, displaying moderate agonist activity in the micromolar concentration range. On the basis of these results, a novel peptidomimetic design concept, based on the separation of constraint and recognition elements, is proposed.

Animals↗
PubMed2003

Synthesis, activity, and molecular modeling studies of novel human aldose reductase inhibitors based on a marine natural product.

Aldose reductase (ALR2) has been implicated in the etiology of diabetic complications, including blindness. Because of the limited number of currently available drugs for the prevention of these long-term complications, the discovery of new ALR2 inhibitors appears highly desirable. In this study, a polybrominated diphenyl ether (1) naturally occurring in a marine sponge was found to inhibit recombinant human ALR2 with an IC(50) of 6.4 microM. A series of polyhalogenated analogues that were synthesized and tested in vitro to explore the structure-activity relationships displayed various degrees of inhibitory activity. The most active compounds were also capable of preventing sorbitol accumulation inside human retinal cells. In this cell-based assay, the most potent synthesized analogue (16) showed a 17-fold increase in inhibitory activity compared to that of sorbinil (IC(50) = 0.24 vs 4 microM). A molecular representation of human ALR2 in complex with the natural product was built using homology modeling, automated docking, and energy refinement methods. AMBER parameters for the halogen atoms were derived and calibrated using condensed phase molecular dynamics simulations of fluorobenzene, chlorobenzene, and bromobenzene. Inhibitor binding is proposed to cause a conformational change similar to that recently reported for zenarestat. A free energy perturbation thermodynamic cycle allowed us to assess the importance of a crucial bromine atom that distinguishes the active lead compound from a much less active close natural analogue. Remarkably, the spatial location of this bromine atom is equivalent to that occupied by the only bromine atom present in zenarestat.

Aldehyde Reductase↗
PubMed2003

Solution structure and stability of a disulfide cross-linked nucleopeptide duplex.

NMR methods are used to study the structure and stability of the duplex formed by the nucleopeptide [Ac-Cys-Gly-Ala-Hse(p3'dGCATGC)-Ala-OH]2[S-S], in which the oligonucleotide is self-complementary and the cysteine residues of the two peptide chains form a disulfide bridge; thermal transitions and NMR-derived structural calculations are consistent with a 3-D structure in which the oligonucleotide forms a standard B-DNA helix without significant distortions; the peptide chains are relatively disordered in solution and lie in the minor groove of the DNA helix; this nucleopeptide duplex exhibits a high melting temperature, indicating that peptide-oligonucleotide conjugates containing cysteines are suitable molecules to establish cross-links between DNA strands and stabilize the duplex.

Amino Acid Sequence↗
PubMed2003

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.

Enzyme Inhibitors↗