Biomedical subjects

V E Marquez

Publications and source records attributed to V E Marquez.

At least 19 recordsLinked to original sources

PubMed2004

Synthesis, stability, and protonation studies of a self-complementary dodecamer containing the modified nucleoside 2'-deoxyzebularine.

The nucleoside 2'-deoxyzebularine (K) was incorporated into the self-complementary dodecamer 5'-CGTACGKGTACG-3' by solid-phase 2-cyanoethylphosphoramidite chemistry using dimethoxytrityl (DMT) as the 5'-hydroxyl protecting group. Standard synthesis cycles using trichloroacetic acid and short ammonia treatment (50 degrees C for 30 min) were found to be the optimal conditions to obtain the desired dodecamer with minimum acid and basic degradation of the acid- and base-sensitive 2-pyrimidinone residue. The protonation equilibria of the K nucleoside and of the dodecamer at 37 degrees C were studied by means of spectroscopically monitored titrations. For the K nucleoside, a pK(a) value of 3.13 +/- 0.09 was obtained. For the dodecamer, four acid-base species were found in the pH range 2-12, with pK(a) values of 9.60 +/- 0.07, 4.46 +/- 0.16, and 2.87 +/- 0.19. Melting experiments were carried out to confirm the proposed acid-base concentration profiles. Finally, kinetic experiments were also carried out at several pH values to evaluate the stability of the K nucleoside and of the dodecamer. An increased stability was shown by the K nucleoside when incorporated into the dodecamer. Multivariate methods based on both hard- and soft-modeling were applied for the analysis of spectroscopic data, allowing the estimation of concentration profiles and pure spectra.

Chromatography, High Pressure Liquid↗

PubMed2001

Conformationally constrained analogues of diacylglycerol. 18. The incorporation of a hydroxamate moiety into diacylglycerol-lactones reduces lipophilicity and helps discriminate between sn-1 and sn-2 binding modes to protein kinase C (PK-C). Implications for isozyme specificity.

An approach to reduce the log P in a series of diacylglycerol (DAG)-lactones known for their high binding affinity for protein kinase C (PK-C) is presented. Branched alkyl groups with reduced lipophilicity were selected and combined with the replacement of the ester or lactone oxygens by NH or NOH groups. Compound 6a with an isosteric N-hydroxyl amide arm represents the most potent and least lipophilic DAG analogue known to date.

4-Butyrolactone↗

PubMed2001

Synthesis and purine receptor affinity of 6-oxopurine nucleosides and nucleotides containing (N)-methanocarba-pseudoribose rings.

6-Oxopurine derivatives containing a northern (N) methanocarba modification (i.e., fused cyclopropane and cyclopentane rings in place of the ribose) were synthesized and the adenosine receptor affinity measured. Guanine or hypoxanthine was coupled at the 7-position, or 1,3-dibutylxanthine was coupled at the 9-position. The pseudoribose ring was also substituted at the 5'-position with an N-methyluronamide or with phosphate groups.

Calcium↗

PubMed2001

An optimized protein kinase C activating diacylglycerol combining high binding affinity (Ki) with reduced lipophilicity (log P).

A small, focused combinatorial library encompassing all possible permutations of acyl branched alkyl chains-small and large, saturated and unsaturated-was generated from the active diacylglycerol enantiomer (S-DAG) to help identify the analogue with the highest binding affinity (lowest Ki) for protein kinase C (PK-C) combined with the minimum lipophilicity (log P). The selected ligand (3B) activated PK-C more effectively than sn-1,2-dioctanoylglycerol (diC8) despite being 1.4 log units more hydrophilic. Compound 3B indeed represents the most potent, hydrophilic DAG ligand to date. With the help of a green fluorescent protein (GFP)-tagged PK-Calpha, 3B was able to translocate the full length protein to the membrane with an optimal dose of 100 microM in CHO-K1 cells, while diC8 failed to achieve translocation even at doses 3-fold higher. Molecular modeling of 3B into an empty C1b domain of PK-Cdelta clearly showed the existence of a preferred binding orientation. In addition, molecular dynamic simulations suggest that binding discrimination could result from a favorable van der Waals (VDW) interaction between the large, branched sn-1 acyl group of 3B and the aromatic rings of Trp252 (PK-Cdelta) or Tyr252 (PK-Calpha). The DAG analogue of 3B in which the acyl groups are reversed (2C) showed a decrease in binding affinity reflecting the capacity of PK-C to effectively discriminate between alternative orientations of the acyl chains.

Animals↗

PubMed2001

Ring-Constrained (N)-methanocarba nucleosides as adenosine receptor agonists: independent 5'-uronamide and 2'-deoxy modifications.

Novel methanocarba adenosine analogues, having the pseudo-ribose northern (N) conformation preferred at adenosine receptors (ARs), were synthesized and tested in binding assays. The 5'-uronamide modification preserved [N6-(3-iodobenzyl)] or enhanced (N6-methyl) affinity at A3ARs, while the 2'-deoxy modification reduced affinity and efficacy in a functional assay.

Adenine↗

PubMed2001

Role of hydrophobic residues in the C1b domain of protein kinase C delta on ligand and phospholipid interactions.

The C1 domains of conventional and novel protein kinase C (PKC) isoforms bind diacylglycerol and phorbol esters with high affinity. Highly conserved hydrophobic residues at or near the rim of the binding cleft in the second cysteine-rich domain of PKC-delta (PKC-deltaC1b) were mutated to probe their roles in ligand recognition and lipid interaction. [(3)H]Phorbol 12,13-dibutyrate (PDBu) binding was carried out both in the presence and absence of phospholipids to determine the contribution of lipid association to the ligand affinity. Lipid dependence was determined as a function of lipid concentration and composition. The binding properties of a high affinity branched diacylglycerol with lipophilicity similar to PDBu were compared with those of PDBu to identify residues important for ligand selectivity. As expected, Leu-20 and Leu-24 strongly influenced binding. Substitution of either by aspartic acid abolished binding in either the presence or absence of phosphatidylserine. Mutation of Leu-20 to Arg or of Leu-24 to Lys caused a dramatic (340- and 250-fold, respectively) reduction in PDBu binding in the presence of lipid but only a modest reduction in the weaker binding of PDBu observed in the absence of lipid, suggesting that the main effect was on C1 domain -phospholipid interactions. Mutation of Leu-20 to Lys or of Trp-22 to Lys had modest (3-fold) effects and mutation of Phe-13 to Tyr or Lys was without effect. Binding of the branched diacylglycerol was less dependent on phospholipid and was more sensitive to mutation of Trp-22 to Tyr or Lys, especially in the presence of phospholipid, than was PDBu. In terms of specific PKC isoforms, our results suggest that the presence of Arg-20 in PKC-zeta may contribute to its lack of phorbol ester binding activity. More generally, the results emphasize the interplay between the C1 domain, ligand, and phospholipid in the ternary binding complex.

Amino Acid Sequence↗

PubMed2001

5-acyloxy-5-hydroxymethyltetrahydro-2-furancarboxylate as a novel template for protein kinase C (PKC) binding.

A series of alkyl tetrahydrofuran-2-carboxylates (1-4) bearing a new set of three pharmacophoric groups were tested as protein kinase C (PKC) ligands. The compounds were synthesized from commercially available glycidyl 4-methoxyphenyl ether. The correlation between their binding affinities for PKC-alpha and a conformational fit to phorbol ester indicates they mimic a pharmacophore model comprising the C20-OH, C3-C=O and C9-OH rather than that including the C13-C=O moiety.

Binding, Competitive↗

PubMed2001

N-(3-Acyloxy-2-benzylpropyl)-N'-(4-hydroxy-3-methoxybenzyl) thiourea derivatives as potent vanilloid receptor agonists and analgesics.

A series of N-(3-acyloxy-2-benzylpropyl)-N'-(4-hydroxy-3-methoxybenzyl) thiourea derivatives were investigated as vanilloid receptor ligands in an effort to discover a novel class of analgesics. The proposed pharmacophore model of resiniferatoxin. which includes the C20 homovanillic moiety, the C3-carbonyl and the orthoester phenyl ring as key pharmacophoric groups, was utilized as a guide for drug design. The compounds were synthesized after several steps from diethylmalonate and evaluated in vitro in a receptor binding assay and in a capsaicin-activated channel assay. Additional evaluation of analgesic activity, anti-inflammatory activity and pungency was conducted in animal models by the writhing test, the ear edema assay, and the eye-wiping test, respectively. Among the new compounds, 23 and 28 were found to be the most potent receptor agonists of the series with Ki values of 19 nM and 11 nM, respectively. Their strong in vitro potencies were also reflected by an excellent analgesic profile in animal tests with ED50 values of 0.5 microg kg for 23 and 1.0 microg/kg for 28. Relative to capsaicin these compounds appear to be ca. 600 and 300 times more potent. Both 23 and 28 were found to be less pungent than capsaicin based on the eye-wiping test. However, the compounds did not show significant anti-inflammatory activity. A molecular modeling study comparing the energy-minimized structures of resiniferatoxin and 35 demonstrated a good correlation in the spatial disposition of the corresponding key pharmacophores. The thioureas described in this investigation, which were designed as simplified resiniferatoxin surrogates, represent a novel class of potent vanilloid receptor agonists endowed with potent analgesic activity and reduced pungency.

Analgesics↗

PubMed2001

Synthesis of oligonucleotide inhibitors of DNA (Cytosine-C5) methyltransferase containing 5-azacytosine residues at specific sites.

The incorporation of 5-azacytosine residues into DNA causes potent inhibition of DNA (Cytosine-C5) methyltransferases. The synthesis of oligodeoxyribonucleotides incorporating single or multiple 5-aza-2'-deoxycytidine residues at precise sites was undertaken to generate an array of sequences containing the reactive 5-azacytosine base as specific target sites for enzymatic methylation. Preparation of these modified oligonucleotides requires the use of 2-(p-nitrophenyl)ethyloxycarbonyl (NPEOC) groups for the protection of exocyclic amino functions. These groups are removed under mild conditions, thus avoiding conventional protocols that are detrimental to the integrity of the 5-azacytosine ring.

Base Sequence↗

PubMed2000

A remarkably simple chemicoenzymatic approach to structurally complex bicyclo[3.1.0]hexane carbocyclic nucleosides.

[reaction: see text] Intramolecular cyclopropanation of a carbene engendered from the corresponding diazo beta-ketoester produced the desired bicyclo[3.1. 0]hexane pseudosugar. Purine nucleosides obtained via Mitsunobu coupling were resolved with adenosine deaminase. The requisite beta-ketoester was assembled in one step from ethyl acetoacetate and acrolein.

Acetoacetates↗

PubMed2000

Interactions of conformationally biased north and south 2'-fluoro-2', 3'-dideoxynucleoside 5'-triphosphates with the active site of HIV-1 reverse transcriptase.

Molecular dynamics simulations of a ternary complex of HIV-1 reverse transcriptase (RT), double-stranded DNA, and bound dideoxynucleoside-5'-triphosphate (RT-DNA-ddNTP), utilizing the ddNTPs ddATP, betaFddATP, and alphaFddATP, explain the experimentally observed order of potency of these 5'-triphosphates as inhibitors of RT: ddATP > betaFddATP > alphaFddATP. On the basis of RT's known preference to bind the incoming dNTP (or ddNTP) with a north conformation at the polymerase site, alphaFddATP, which in solution prefers almost exclusively a north conformation, was predicted to be the most potent inhibitor. However, Tyr115, which appears to function as a steric gate to preclude the binding of ribonucleoside 5'-triphosphates, prevents the effective binding of alphaFddATP in its preferred north conformation. The south-biased betaFddATP, while able to bind to RT without hindrance by Tyr115, has to pay a high energy penalty to be flipped to the active north conformation at the polymerase site. Finally, the more flexible and less conformationally biased ddATP is able to switch to a north conformation at the RT site with a smaller energy penalty than betaFddATP. These results highlight the opposite conformational preferences of HIV-1 RT for alphaFddATP and betaFddATP and help establish conformational guidelines for optimal binding at the polymerase site of this enzyme.

Anti-HIV Agents↗

PubMed2000

Conformationally constrained analogues of diacylglycerol (DAG). 17. Contrast between sn-1 and sn-2 DAG lactones in binding to protein kinase C.

In previous work, we have obtained potent protein kinase C (PK-C) ligands with low-namomolar binding affinities by constructing diacylglycerol (DAG) mimetics in which the sn-2 carbonyl of DAG was constrained into a lactone ring. An additional structural element that helped achieve high binding affinity was the presence of branched acyl or alpha-alkylidene chains. In the present study, the effects of similarly branched chains on a different lactone system, where the lactone carbonyl is now equivalent to the sn-1 carbonyl of DAG, are investigated. In this new lactone template, the two chiral centers must have the S-configuration for enzyme recognition. As with the sn-2 DAG lactones, the branched chains were designed to optimize van der Waals contacts with a group of conserved hydrophobic amino acids located on the rim of the C1 domain of PK-C. The acyl and alpha-alkylidene chains were also designed to be lipophilically equivalent (8 carbons each). Eight new compounds (7-14) representing all possible combinations of linear and branched acyl and alpha-alkylidene were synthesized and evaluated. The sn-1 DAG lactones were less effective as PK-C ligands than the sn-2 DAG lactones despite having a similar array of linear or branched acyl and alpha-alkylidene chains

Diglycerides↗

PubMed2000

Kinetics and crystal structure of the wild-type and the engineered Y101F mutant of Herpes simplex virus type 1 thymidine kinase interacting with (North)-methanocarba-thymidine.

Kinetic and crystallographic analyses of wild-type Herpes simplex virus type 1 thymidine kinase (TK(HSV1)) and its Y101F-mutant [TK(HSV1)(Y101F)] acting on the potent antiviral drug 2'-exo-methanocarba-thymidine (MCT) have been performed. The kinetic study reveals a 12-fold K(M) increase for thymidine processed with Y101F as compared to the wild-type TK(HSV1). Furthermore, MCT is a substrate for both wild-type and mutant TK(HSV1). Its binding affinity for TK(HSV1) and TK(HSV1)(Y101F), expressed as K(i), is 11 microM and 51 microM, respectively, whereas the K(i) for human cytosolic thymidine kinase is as high as 1.6 mM, rendering TK(HSV1) a selectivity filter for antiviral activity. Moreover, TK(HSV1)(Y101F) shows a decrease in the quotient of the catalytic efficiency (k(cat)/K(M)) of dT over MCT corresponding to an increased specificity for MCT when compared to the wild-type enzyme. Crystal structures of wild-type and mutant TK(HSV1) in complex with MCT have been determined to resolutions of 1.7 and 2.4 A, respectively. The thymine moiety of MCT binds like the base of dT while the conformationally restricted bicyclo[3.1.0]hexane, mimicking the sugar moiety, assumes a 2'-exo envelope conformation that is flatter than the one observed for the free compound. The hydrogen bond pattern around the sugar-like moiety differs from that of thymidine, revealing the importance of the rigid conformation of MCT with respect to hydrogen bonds. These findings make MCT a lead compound in the design of resistance-repellent drugs for antiviral therapy, and mutant Y101F, in combination with MCT, opens new possibilities for gene therapy.

Amino Acid Substitution↗

PubMed2000

Methanocarba analogues of purine nucleosides as potent and selective adenosine receptor agonists.

Adenosine receptor agonists have cardioprotective, cerebroprotective, and antiinflammatory properties. We report that a carbocyclic modification of the ribose moiety incorporating ring constraints is a general approach for the design of A(1) and A(3) receptor agonists having favorable pharmacodynamic properties. While simple carbocyclic substitution of adenosine agonists greatly diminishes potency, methanocarba-adenosine analogues have now defined the role of sugar puckering in stabilizing the active adenosine receptor-bound conformation and thereby have allowed identification of a favored isomer. In such analogues a fused cyclopropane moiety constrains the pseudosugar ring of the nucleoside to either a Northern (N) or Southern (S) conformation, as defined in the pseudorotational cycle. In binding assays at A(1), A(2A), and A(3) receptors, (N)-methanocarba-adenosine was of higher affinity than the (S)-analogue, particularly at the human A(3) receptor (N/S affinity ratio of 150). (N)-Methanocarba analogues of various N(6)-substituted adenosine derivatives, including cyclopentyl and 3-iodobenzyl, in which the parent compounds are potent agonists at either A(1) or A(3) receptors, respectively, were synthesized. The N(6)-cyclopentyl derivatives were A(1) receptor-selective and maintained high efficacy at recombinant human but not rat brain A(1) receptors, as indicated by stimulation of binding of [(35)S]GTP-gamma-S. The (N)-methanocarba-N(6)-(3-iodobenzyl)adenosine and its 2-chloro derivative had K(i) values of 4.1 and 2.2 nM at A(3) receptors, respectively, and were highly selective partial agonists. Partial agonism combined with high functional potency at A(3) receptors (EC(50) < 1 nM) may produce tissue selectivity. In conclusion, as for P2Y(1) receptors, at least three adenosine receptors favor the ribose (N)-conformation.

Animals↗

PubMed2000

The lipophilicity of phorbol esters as a critical factor in determining the pattern of translocation of protein kinase C delta fused to green fluorescent protein.

Our previous study showed differential subcellular localization of protein kinase C (PKC) delta by phorbol esters and related ligands, using a green fluorescent protein-tagged construct in living cells. Here we compared the abilities of a series of symmetrically substituted phorbol 12,13-diesters to translocate PKC delta. In vitro, the derivatives bound to PKC with similar potencies but differed in rate of equilibration. In vivo, the phorbol diesters with short, intermediate, and long chain fatty acids induced distinct patterns of translocation. Phorbol 12,13-dioctanoate and phorbol 12,13-nonanoate, the intermediate derivatives and most potent tumor promoters, showed patterns of translocation typical of phorbol 12-myristate 13-acetate, with plasma membrane and subsequent nuclear membrane translocation. The more hydrophilic compounds (phorbol 12,13-dibutyrate and phorbol 12,13-dihexanoate) induced a patchy distribution in the cytoplasm, more prominent nuclear membrane translocation, and little plasma membrane localization at all concentrations examined (100 nM to 10 microM). The highly lipophilic derivatives, phorbol 12,13-didecanoate and phorbol 12, 13-diundecanoate, at 1 microM caused either plasma membrane translocation only or no translocation at incubation times up to 60 min. Our results indicate that lipophilicity of phorbol esters is a critical factor contributing to differential PKC delta localization and thereby potentially to their different biological activities.

Animals↗

PubMed2000

Construction of the bicyclo[3.1.0]hexane template of a conformationally locked carbocyclic adenosine via an olefin keto-carbene cycloaddition.

An intramolecular olefin keto-carbene cycloaddition reaction created the bicyclo[3.1.0]hexane template 10 that was necessary for the synthesis of carbocyclic amine 15. This amine is a direct precursor to a family of rigid nucleosides that are conformationally locked in the Southern hemisphere of the pseudorotational cycle. The synthesis of the conformationally locked adenosine analogue is reported herein as an illustrative example of the methodology. The racemic (South)-methanocarba adenosine analogue (+/-)-4 is the first example of a conformationally locked ribonucleoside version in the Southern hemisphere.

Adenosine↗

PubMed2000

Diacylglycerols with lipophilically equivalent branched acyl chains display high affinity for protein kinase C (PK-C). A direct measure of the effect of constraining the glycerol backbone in DAG lactones.

New synthetic diacylglycerols (DAGs) with equivalent branched acyl chains were compared with commercially available DAGs as PK-C ligands. The results support the view that there is a minimal lipophilic requirement provided by the equivalent acyl groups that results in high binding affinity. Locking the glycerol backbone of the most potent DAG into a five-member lactone resulted in a 10-fold increase in potency.

Diglycerides↗

PubMed2000

Adenosine deaminase prefers a distinct sugar ring conformation for binding and catalysis: kinetic and structural studies.

Several recent X-ray crystal structures of adenosine deaminase (ADA) in complex with various adenosine surrogates have illustrated the preferred mode of substrate binding for this enzyme. To define more specific structural details of substrate preferences for binding and catalysis, we have studied the ADA binding efficiencies and deamination kinetics of several synthetic adenosine analogues in which the furanosyl ring is biased toward a particular conformation. NMR solution studies and pseudorotational analyses were used to ascertain the preferred furanose ring puckers (P, nu(MAX)) and rotamer distributions (chi and gamma) of the nucleoside analogues. It was shown that derivatives which are biased toward a "Northern" (3'-endo, N) sugar ring pucker were deaminated up to 65-fold faster and bound more tightly to the enzyme than those that preferred a "Southern" (2'-endo, S) conformation. This behavior, however, could be modulated by other structural factors. Similarly, purine riboside inhibitors of ADA that prefer the N hemisphere were more potent inhibitors than S analogues. These binding propensities were corroborated by detailed molecular modeling studies. Docking of both N- and S-type analogues into the ADA crystal structure coordinates showed that N-type substrates formed a stable complex with ADA, whereas for S-type substrates, it was necessary for the sugar pucker to adjust to a 3'-endo (N-type) conformation to remain in the ADA substrate binding site. These data outline the intricate structural details for optimum binding in the catalytic cleft of ADA.

Adenosine↗