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Biomedical subjects

James P Snyder

Publications and source records attributed to James P Snyder.

At least 19 recordsLinked to original sources
PubMed2007

Rotational-echo double-resonance NMR distance measurements for the tubulin-bound Paclitaxel conformation.

The important anticancer drug Taxol (paclitaxel, PTX) owes its unique activity to its ability to bind to tubulin in a stoichiometric ratio and promote its assembly into microtubules. The conformation of the microtubule-bound drug has been the focus of numerous research efforts, since the inability of polymerized tubulin to form crystals precludes structure proof by X-ray crystallography. Likewise, although the alpha,beta-tubulin dimer structure has been solved by electron crystallography, the 3.7 A resolution is too low to permit direct determination of either ligand conformation or binding pose. In this article, we present experimental results from 2H{19F} REDOR NMR that provide direct confirmation that paclitaxel adopts a T-shaped conformation when it is bound to tubulin.

Animals↗
PubMed2006

Bridging converts a noncytotoxic nor-paclitaxel derivative to a cytotoxic analogue by constraining it to the T-Taxol conformation.

The synthesis of the bridged A-nor-paclitaxel 4 has been achieved from paclitaxel in a key test of the T-Taxol conformational hypothesis. Although the unbridged A-nor-paclitaxel 3 is essentially noncytotoxic, the bridged analogue 4 is strongly cytotoxic. This result provides strong evidence for the T-Taxol conformation as the bioactive tubulin-binding conformation of paclitaxel.

Animals↗
PubMed2006

Nonpeptide inhibitors of measles virus entry.

Measles virus (MV) is one of the most infectious pathogens known. Despite the existence of a vaccine, over 500,000 deaths/year result from MV or associated complications. Anti-measles compounds could conceivably reverse these statistics. Previously, we described a homology model of the MV fusion protein trimer and a putative binding site near the head-neck region. The resulting model permitted the identification of two nonpeptidic entry inhibitors. Here, we present the design, synthesis, and bioevaluation of several series of fusion inhibitors and describe their structure-activity relationships (SAR). Five simply substituted anilides show low-microM blockade of the MV, one of which (AS-48) exhibits IC50 = 0.6-3.0 microM across a panel of wild-type MV strains found in the field. Molecular field topology analysis (MFTA), a 2D QSAR approach based on local molecular properties (atomic charges, hydrogen-bonding capacity and local lipophilicity), applied to the anilide series suggests structural modifications to improve potency.

Anilides↗
PubMed2006

Synthesis of EF24-tripeptide chloromethyl ketone: a novel curcumin-related anticancer drug delivery system.

The blood coagulation cascade includes a step in which the soluble protein, factor VIIa (fVIIa), complexes with its transmembrane receptor, tissue factor (TF). The fVIIa/TF protein-protein complex is subsequently drawn into the cell by endocytosis. The observation that TF is aberrantly and abundantly expressed on many cancer cells offers an opportunity to specifically target those cells with an effective anticancer drug. Thus, we propose a new drug delivery system, drug-linker-Phe-Phe-Arg-mk-fVIIa, which can associate with TF on the surface of cancer cells, but release the cytotoxic agent in the cytoplasm. Synthetic procedures have been developed for the preparation of phenylalanine-phenylalanine-arginine chloromethyl ketone, (FFRck) followed by coupling with the cytotoxin EF24 and subsequently fVIIa to give EF-24-FFRmk-fVIIa. When breast cancer cells (MDA-MB-231) and human melanoma cells (RPMI-7951) are treated with the complex, the cells are arrested to a greater extent than EF24 alone by comparison with controls.

Amino Acid Chloromethyl Ketones↗
PubMed2006

The T-Taxol conformation.

T-Taxol is a proposal for the bioactive conformation of paclitaxel (PTX) derived from fitting ligand conformations to the electron crystallographic (EC) density. Although confirmed by a number of studies, some structural ambiguities based on the interpretation of two solid-state REDOR (13)C-(19)F distances in a fluorinated PTX derivative remain. An evaluation of the static and dynamic properties of the PTX-tubulin complex shows that small 6-12 degrees variations in calculated torsions and a justifiable increase of the REDOR distance error to > or = +/-0.7 A readily resolves key discrepancies around T-Taxol's service as the bioactive conformation. In addition, conformational analysis reveals a range of (13)C-(19)F separations compatible with the REDOR measurements suggesting that the present PTX REDOR distances may not provide a precise model for bioactive, tubulin-bound bridged taxanes. In addition, we show that New York-Taxol (PTX-NY), a recently proposed alternative to T-Taxol, is incompatible with both the EC density and the activity of a highly active series of bridged taxanes.

Binding Sites↗
PubMed2006

Diastereomers of dibromo-7-epi-10-deacetylcephalomannine: crowded and cytotoxic taxanes exhibit halogen bonds.

The diastereomers of dibromo-7-epi-10-deacetylcephalomannine (6 and 7) have been isolated and characterized. Cytotoxicity and microtubule assembly assays demonstrate that cephalomannine analogue 6 possesses a potency profile very similar to that of Taxol, while isomer 7 is slightly less active. Solid state, solution, and tubulin-bound conformations of the two diastereomers were probed by using X-ray crystallography, 2-D NMR experiments in conjunction with the NAMFIS analysis, and the Glide docking protocol. In the crystal, isomer 7 exhibits an intermolecular halogen bond that may contribute to self-assembly. Neither crystal structure appears in the NAMFIS solution analysis, but both diastereomers are represented in solution by a T-shaped Taxol conformer. Glide docking demonstrates the latter to best fill the tubulin binding pocket, as has been shown for the parent Taxol drug. Each model of the bound complexes for 6 and 7 presents a single well-defined halogen bond from one of the ligand's bromines to Glu22 or Asp26 near the N-terminus of beta-tubulin, respectively. This first report of a halogen bond between taxanes and tubulin may prove useful in guiding the design and synthesis of other microtubule-stabilizing agents with a similar capacity.

Antineoplastic Agents↗
PubMed2006

Crossover point between dialkoxy disulfides (ROSSOR) and thionosulfites ((RO)2S=S): prediction, synthesis, and structure.

Isomeric preference between cyclic dialkoxy disulfides and thionosulfites is governed by the ring size of the heterocycle. Rings smaller than seven atoms prefer the thionosulfite connectivity, whereas larger rings or acyclic analogues favor the unbranched dialkoxy disulfide structure. Density functional calculations were employed to predict the crossover point at which both constitutional isomers are of comparable stability. Follow-up synthesis provides the previously unknown eight-membered ring dialkoxy disulfide 14 and seven-membered ring thionosulfite 15 from the same reaction. X-ray crystallography for all but one of the reaction products and complementary NMR analysis furnishes insights into both solid-state and solution conformations. A long-standing issue regarding the concerted vs catalyzed isomerization pathway between XSSX and X(2)S=S has been addressed for X = RO and shown to be acid dependent.

Journal Article↗
PubMed2006

Glutamate receptors: variation in structure-function coupling.

Fast excitatory synaptic transmission in the CNS relies almost entirely on the neurotransmitter glutamate and its family of ion channel receptors. An appreciation of the coupling between agonist binding and channel opening has advanced rapidly during the past five years, largely as a result of new structural information about the agonist-binding site. Recent studies suggest that despite many structural similarities different family members use different mechanisms to translate agonist binding into channel opening.

Animals↗
PubMed2006

Two domains that control prefusion stability and transport competence of the measles virus fusion protein.

Most viral glycoproteins mediating membrane fusion adopt a metastable native conformation and undergo major conformational changes during fusion. We previously described a panel of compounds that specifically prevent fusion induced by measles virus (MV), most likely by interfering with conformational rearrangements of the MV fusion (F) protein. To further elucidate the basis of inhibition and better understand the mechanism of MV glycoprotein-mediated fusion, we generated and characterized resistant MV variants. Spontaneous mutations conferring drug resistance were confirmed in transient assays and in the context of recombinant virions and were in all cases located in the fusion protein. Several mutations emerged independently at F position 462, which is located in the C-terminal heptad repeat (HR-B) domain. In peptide competition assays, all HR-B mutants at residue 462 revealed reduced affinity for binding to the HR-A core complex compared to unmodified HR-B. Combining mutations at residue 462 with mutations in the distal F head region, which we had previously identified as mediating drug resistance, causes intracellular retention of the mutant proteins. The transport competence and activity of the mutants can be restored, however, by incubation at reduced temperature or in the presence of the inhibitory compounds, indicating that the F escape mutants have a reduced conformational stability and that the inhibitors stabilize a transport-competent conformation of the F trimer. The data support the conclusion that residues located in the head domain of the F trimer and the HR-B region contribute jointly to controlling F conformational stability.

Amino Acid Sequence↗
PubMed2006

Paclitaxel-resistant cells have a mutation in the paclitaxel-binding region of beta-tubulin (Asp26Glu) and less stable microtubules.

Resistance to paclitaxel-based therapy is frequently encountered in the clinic. The mechanisms of intrinsic or acquired paclitaxel resistance are not well understood. We sought to characterize the resistance mechanisms that develop upon chronic exposure of a cancer cell line to paclitaxel in the presence of the P-glycoprotein reversal agent, CL-347099. The epidermoid tumor line KB-3-1 was exposed to increasing concentrations of paclitaxel and 5 micromol/L CL-347099 for up to 1 year. Cells grown in 15 nmol/L paclitaxel plus CL-347099 (KB-15-PTX/099) developed 18-fold resistance to paclitaxel and were dependent upon paclitaxel for maximal growth. They grew well and retained resistance to paclitaxel when grown in athymic mice. Cross-resistance (3- to 5-fold) was observed in tissue culture to docetaxel, the novel taxane MAC-321, and epothilone B. Collateral sensitivity (approximately 3-fold) was observed to the depolymerizing agents vinblastine, dolastatin-10, and HTI-286. KB-15-PTX/099-resistant cells did not overexpress P-glycoprotein nor did they have an alteration of [14C]paclitaxel accumulation compared with parental cells. However, a novel point mutation (T to A) resulting in Asp26 to glutamate substitution in class I (M40) beta-tubulin was found. Based on an electron crystallography structure of Zn-stabilized tubulin sheets, the phenyl ring of C-3' NHCO-C6H5 of paclitaxel makes contact with Asp26 of beta-tubulin, suggesting a ligand-induced mutation. Optimized model complexes of paclitaxel, docetaxel, and MAC-321 in beta-tubulin show a novel hydrogen bonding pattern for the glutamate mutant and rationalize the observed resistance profiles. However, a mutation in the paclitaxel binding pocket does not explain the phenotype completely. KB-15-PTX/099 cells have impaired microtubule stability as determined by a reduced percentage of tubulin in microtubules and reflected by less acetylated tubulin. These results suggest that a mutation in tubulin might affect microtubule stability as well as drug binding and contribute to the observed resistance profile.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

A stereoelectronic effect on turn formation due to proline substitution in elastin-mimetic polypeptides.

Stereoelectronic effects have been identified as contributing factors to the conformational stability of collagen-mimetic peptide sequences. To assess the relevance of these factors within other protein structural contexts, three polypeptide sequences were prepared in which the sequences were derived from the canonical repeat unit (Val-Pro-Gly-Val-Gly) of the protein material elastin. These elastin-mimetic polypeptides, elastin-1, elastin-2, and elastin-3, incorporate (2S)-proline, (2S,4S)-4-fluoroproline, and (2S,4R)-4-fluoroproline, respectively, at the second position of the elastin repeat. Calorimetric and spectroscopic investigations of these three polypeptides indicate that the incorporation of the substituted proline residues had a dramatic effect upon the self-assembly of the corresponding elastin peptide. The presence of (2S,4R)-4-fluoroproline in elastin-3 lowered the temperature of the phase transition and increased the type II beta-turn population with respect to the parent polypeptide, while the presence of (2S,4S)-4-fluoroproline in elastin-2 had the opposite effect. These results suggest that stereoelectronic effects could either enhance or hinder the self-assembly of elastin-mimetic polypeptides, depending on the influence of the proline analogue on the energetics of the beta-turn conformation that develops within the pentapeptide structural repeats above the phase transition. Density functional theory (DFT) was employed to model three possible turn types (betaI-, betaII-, and inverse gamma-turns) derived from model peptide segments (MeCO-Xaa-Gly-NHMe) (Xaa = Pro, 4S-F-Pro, or 4R-F-Pro) corresponding to the turn-forming residues of the elastin repeat unit (Val-Pro-Gly-Val-Gly). The results of the these calculations suggested a similar outcome to the experimental data for the elastin-mimetic polypeptides, in that type II beta-turn structures were stabilized for peptide segments containing (2S,4R)-fluoroproline and destabilized for segments containing (2S,4S)-fluoroproline relative to the canonical proline residue.

Biomimetic Materials↗
PubMed2005

T-Taxol and the electron crystallographic density in beta-tubulin.

[chemical structure: see text]. T-Taxol has been proposed as the bioactive conformation on beta-tubulin and subsequently utilized in the design of a series of highly active bridged taxane analogues. A modified T-form with a reversed C-13 side chain orientation has recently been proposed as an equally plausible bioactive shape. A comparison of the two spatial alternatives within the tubulin binding site electron crystallographic density suggests strongly that T-Taxol is the bioactive conformation.

Binding Sites↗
PubMed2005

Conformations of laulimalide in DMSO-d6.

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 3(18) 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.

Crystallography, X-Ray↗
PubMed2005

Mechanism of partial agonism at NMDA receptors for a conformationally restricted glutamate analog.

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.

Amino Acid Sequence↗
PubMed2005

The taxol pharmacophore and the T-taxol bridging principle.

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.

Animals↗
PubMed2005

3-Fluoropiperidines and N-methyl-3-fluoropiperidinium salts: the persistence of axial fluorine.

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 NH2+, NHMe+, and NMe2+ 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 [D6]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 NH2+ and NMe2+ systems to substantiate axial-F. Comparison of the X-ray structures of the fluorinated and unfluorinated NMe2+ 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.

Crystallography, X-Ray↗
PubMed2005

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

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.

Amino Acid Sequence↗