Showing papers by "James P. Snyder published in 2005"

EF24, a novel synthetic curcumin analog, induces apoptosis in cancer cells via a redox-dependent mechanism.

Abstract: In this study, we show that the novel synthetic curcumin analog, EF24, induces cell cycle arrest and apoptosis by means of a redox-dependent mechanism in MDA-MB-231 human breast cancer cells and DU-145 human prostate cancer cells. Cell cycle analysis demonstrated that EF24 causes a G2/M arrest in both cell lines, and that this cell cycle arrest is followed by the induction of apoptosis as evidenced by caspase-3 activation, phosphatidylserine externalization and an increased number of cells with a sub-G1 DNA fraction. In addition, we demonstrate that EF24 induces a depolarization of the mitochondrial membrane potential, suggesting that the compound may also induce apoptosis by altering mitochondrial function. EF24, like curcumin, serves as a Michael acceptor reacting with glutathione (GSH) and thioredoxin 1. Reaction of EF24 with these agents in vivo significantly reduced intracellular GSH as well as oxidized GSH in both the wild-type and Bcl-xL overexpressing HT29 human colon cancer cells. We therefore propose that the anticancer effect of a novel curcumin analog, EF24, is mediated in part by redox-mediated induction of apoptosis.

Structural features of the glutamate binding site in recombinant NR1/NR2A N-methyl-D-aspartate receptors determined by site-directed mutagenesis and molecular modeling

Abstract: We have used site-directed mutagenesis of amino acids located within the S1 and S2 ligand binding domains of the NR2A N-methyl-D-aspartate (NMDA) receptor subunit to explore the nature of ligand binding. Wild-type or mutated NR1/NR2A NMDA receptors were expressed in Xenopus laevis oocytes and studied using two electrode voltage clamp. We investigated the effects of mutations in the S1 and S2 regions on the potencies of the agonists L-glutamate, L-aspartate, (R,S)-tetrazol-5yl-glycine, and NMDA. Mutation of each of the corresponding residues found in the NR2A receptor subunit, suggested to be contact residues in the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit, caused a rightward shift in the concentration-response curve for each agonist examined. None of the mutations examined altered the efficacy of glutamate as assessed by methanethiosulfonate ethylammonium potentiation of agonist-evoked currents. In addition, none of the mutations altered the potency of glycine. Homology modeling and molecular dynamics were used to evaluate molecular details of ligand binding of both wild-type and mutant receptors, as well as to explore potential explanations for agonist selectivity between glutamate receptor subtypes. The modeling studies support our interpretation of the mutagenesis data and indicate a similar binding strategy for L-glutamate and NMDA when they occupy the binding site in NMDA receptors, as has been proposed for glutamate binding to the GluR2 AMPA receptor subunit. Furthermore, we offer an explanation as to why "charge conserving" mutations of two residues in the binding pocket result in nonfunctional receptor channels and suggest a contributing molecular determinant for why NMDA is not an agonist at AMPA receptors.

3‐Fluoropiperidines and N‐Methyl‐3‐fluoropiperidinium Salts: The Persistence of Axial Fluorine

Abstract: It has previously been shown that the fluorine atom in N-protonated 3-fluoropiperidine salts in water strongly prefers the axial orientation in the six-membered ring chairs. In the present work we examine the proposition that the N-methyl salts are equally disposed to present axial fluorine. Initially, we explored this point by comparing the structures of the corresponding NH 2 + , NHMe + , and NMe 2 + salts by means of density functional theory (DFT), ab initio, and MMFF force field calculations with and without aqueous solvation models. The predictions unambiguously pointed to axial fluorine for all salts investigated, including those with simultaneous axial F and (N)Me. The calculations were followed by synthesis of the corresponding series of 4,4-diphenylpiperidinium salts. These were evaluated by one- and two-dimensional NMR spectroscopy in [D 6 ]DMSO to fully corroborate the axial disposition of the fluorine in each of the compounds. X-ray crystal structure determinations were likewise performed for the diphenyl-3-fluoro NH 2 + and NMe 2 + systems to substantiate axial-F. Comparison of the X-ray structures of the fluorinated and unfluorinated NMe 2 + salts reveals that the fluorine resides axial in spite of substantial steric compression. While the charge-dipole phenomenon responsible for the axial-F conformation in the parent protonated fluoropiperidinium compounds carries over to doubly alkylated salts, we show that it extends to molecular orientation in the packing of the unit cells in the solid state as well. Finally, using the computational methods that successfully motivated our synthesis and structural work, we have made predictions for a number of new structures and re-examined some parallel results reported by the Eliel group in the early 1970s. Although C-F...H-N hydrogen bonds are reported to be weak and few in number, the CF...HN charge-dipole orienting effect is a powerful directing force that matches the hydrogen-bond in both its energetic contribution and conformational consequences.

Mechanism of Partial Agonism at NMDA Receptors for a Conformationally Restricted Glutamate Analog

Abstract: The NMDA ionotropic glutamate receptor is ubiquitous in mammalian central neurons. Because partial agonists bind to the same site as glutamate but induce less channel activation, these compounds provide an opportunity to probe the mechanism of activation of NMDA-type glutamate receptors. Molecular dynamics simulations and site-directed mutagenesis demonstrate that the partial agonist homoquinolinate interacts differently with binding pocket residues than glutamate. Homoquinolinate and glutamate induce distinct changes in the binding pocket, and the binding pocket exhibits significantly more motion with homoquinolinate bound than with glutamate. Patch-clamp recording demonstrates that single-channel activity induced by glutamate or by homoquinolinate has identical single-channel current amplitude and mean open-channel duration but that homoquinolinate slows activation of channel opening relative to glutamate. We hypothesize that agonist-induced conformational changes in the binding pocket control the efficacy of a subunit-specific activation step that precedes the concerted global change in the receptor-channel complex associated with ion channel opening.

Design of a Small-Molecule Entry Inhibitor with Activity against Primary Measles Virus Strains

Abstract: The incidence of measles virus (MV) infection has been significantly reduced in many nations through extensive vaccination; however, the virus still causes significant morbidity and mortality in developing countries. Measles outbreaks also occur in some developed countries that have failed to maintain high vaccine coverage rates. While vaccination is essential in preventing the spread of measles, case management would greatly benefit from the use of therapeutic agents to lower morbidity. Thus, the development of new therapeutic strategies is desirable. We previously reported the generation of a panel of small-molecule MV entry inhibitors. Here we show that our initial lead compound, although providing proof of concept for our approach, has a short half-life (<16 h) under physiological conditions. In order to combine potent antiviral activity with increased compound stability, a targeted library of candidate molecules designed on the structural basis of the first lead has been synthesized and tested against MV. We have identified an improved lead with low toxicity and high stability (half-life 16 h) that prevents viral entry and hence infection. This compound shows high MV specificity and strong activity (50% inhibitory concentration 0.6 to 3.0 M, depending on the MV genotype) against a panel of wild-type MV strains representative of viruses that are currently endemic in the field. Paramyxoviruses are nonsegmented negative-stranded RNA viruses, most of which are highly contagious airborne pathogens that spread via the respiratory route. Members of this viral family constitute major human and animal pathogens such as measles virus (MV), human parainfluenza viruses (HPIV), mumps virus, rinderpest virus, and Newcastle disease virus (12). Despite the existence of an effective live-attenuated vaccine (6), MV remains a serious threat to human health globally, accounting for approximately 0.5 million deaths annually (1). While most of these cases occur in developing countries with limited access to vaccination, measles outbreaks still occur in some developed countries that have failed to maintain high vaccine coverage rates (4, 26). Recent outbreaks, in particular in the United Kingdom, have been attributed to declining herd immunity as a result of reduced vaccination coverage due to parental concerns about vaccination safety (8). Furthermore, vaccine-induced immunity is less robust than naturally acquired protection, which may, in fully vaccinated populations, result in a progressive loss of immunity in adults due to the absence of natural boosting through circulating virus (15, 16, 27). Taken together, these facts make desirable the development of novel therapeutics that could be produced cost-effectively and that could be used for the rapid control of local outbreaks and improved case management to limit severe outcomes of infection.

The taxol pharmacophore and the T-taxol bridging principle.

Abstract: Different approaches to developing an accurate model of the binding conformation of paclitaxel (Taxol, PTX) on beta-tubulin are discussed. Electron crystallography, molecular modeling, NMR and synthetic studies all point to the T-Taxol conformation as the bioactive form. The range of molecular designs represented by synthetic taxoids prepared to test the latter, with an emphasis on internally bridged analogs, is summarized. Key implications and conclusions derived from the retrospective are presented.

Conformations of Laulimalide in DMSO-d6

Abstract: Laulimalide is one of the newest naturally occurring macrolides known to act as a microtubule stabilizing agent with properties similar to Taxol. It also stands as being one of the most flexible with 18 rotatable bonds. This large number of rotatable bonds allows for approximately 318 potential conformers. To examine the conformational energy surface of laulimalide, we have performed an NAMFIS deconvolution analysis for laulimalide in DMSO-d6. The latter has been supplemented with a post-NAMFIS energy analysis at the Becke3LYP/6-31G* level that examines the opposing effects of internal hydrogen bonding and syn-pentane interactions. In this way, we have identified 15 laulimalide conformations that can be classified into 5 different families: Supine, Convex, Cobra, Stretch, and Concave motifs.