Showing papers by "Torrey Pines Institute for Molecular Studies published in 2001"
TL;DR: Improvements in flexible docking technology may lead to a bigger role for computational methods in lead discovery, and the docking and screening procedures can select small sets of likely lead candidates from large libraries of either commercially or synthetically available compounds.
342 citations
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TL;DR: The search for transformation-relevant targets of Jun is moving into a new stage with the application of DNA microarrays technology, and whether all genes essential for transformation reside within the overlap of the v-Jun and c-Jun target spectra remains to be determined.
Abstract: Cellular Jun (c-Jun) and viral Jun (v-Jun) can induce oncogenic transformation. For this activity, c-Jun requires an upstream signal, delivered by the Jun N-terminal kinase (JNK). v-Jun does not interact with JNK; it is autonomous and constitutively active. v-Jun and c-Jun address overlapping but not identical sets of genes. Whether all genes essential for transformation reside within the overlap of the v-Jun and c-Jun target spectra remains to be determined. The search for transformation-relevant targets of Jun is moving into a new stage with the application of DNA microarrays technology. Genetic screens and functional tests remain a necessity for the identification of genes that control the oncogenic phenotype.
294 citations
TL;DR: By using WOW-1, the selective recruitment of high-affinity integrins is identified as a mechanism by which lamellipodia promote formation of new adhesions at the leading edge in cell migration.
Abstract: Integrin alphavbeta3 has an important role in the proliferation, survival, invasion and migration of vascular endothelial cells. Like other integrins, alphavbeta3 can exist in different functional states with respect to ligand binding. These changes involve both affinity modulation, by which conformational changes in the integrin heterodimer govern affinity for individual extracellular matrix proteins, and avidity modulation, by which changes in lateral mobility and integrin clustering affect the binding of cells to multivalent matrices. Here we have used an engineered monoclonal antibody Fab (antigen-binding fragment) named WOW-1, which binds to activated integrins alphavbeta3 and alphavbeta5 from several species, to investigate the role of alphavbeta3 activation in endothelial cell behaviour. Because WOW-1 is monovalent, it is insensitive to changes in integrin clustering and therefore reports only changes in affinity. WOW-1 contains an RGD tract in its variable region and binds only to unoccupied, high-affinity integrins. By using WOW-1, we have identified the selective recruitment of high-affinity integrins as a mechanism by which lamellipodia promote formation of new adhesions at the leading edge in cell migration.
266 citations
TL;DR: T cells from prediabetic NOD mice respond spontaneously to these peptide analogs in culture; this finding validates them as being related to a critical autoantigen involved in the etiology of spontaneous diabetes and indicates that their further characterization is important for a better understanding of underlying disease mechanisms.
Abstract: Nonobese diabetic (NOD) mice spontaneously develop insulitis and destruction of pancreatic islet beta cells similar to type 1 diabetes mellitis in humans. Insulitis also occurs in the BDC2.5 TCR transgenic line of NOD mice that express the rearranged TCR alpha- and beta-chain genes of a diabetogenic NOD CD4 T cell clone. When activated with syngeneic islet cells in culture, BDC2.5 T cells adoptively transfer disease to NOD recipients, but the identity of the islet cell Ag responsible for pathogenicity is not known. To characterize the autoantigen(s) involved, BDC2.5 T cells were used to screen a combinatorial peptide library arranged in a positional scanning format. We identified more than 100 decapeptides that stimulate these T cells at nanomolar concentrations; they are then capable of transferring disease to NOD-scid mice. Surprisingly, some of the peptides include sequences similar (8 of 10 residues) to those found within the 528-539 fragment of glutamic acid decarboxylase 65. Although this 12-mer glutamic acid decarboxylase 65 fragment is only slightly stimulatory for BDC2.5 T cells (EC(50) > 100 microM), a larger 16-mer fragment, 526-541, shows activity in the low micromolar range (EC(50) = 2.3 microM). Finally, T cells from prediabetic NOD mice respond spontaneously to these peptide analogs in culture; this finding validates them as being related to a critical autoantigen involved in the etiology of spontaneous diabetes and indicates that their further characterization is important for a better understanding of underlying disease mechanisms.
201 citations
TL;DR: The results suggest that PAI‐1 contributes to the development of the characteristic hyperglycemia and hyperinsulinemia associated with murine obesity, possibly by facilitating increased adipose tissue TNF‐α gene expression.
Abstract: SPECIFIC AIMElevated levels of plasma and adipose tissue plasminogen activator inhibitor 1 (PAI-1) are frequently observed in obese humans and rodents and correlate strongly with visceral fat mass ...
182 citations
TL;DR: This work proposes a fast yet accurate and robust implementation of the boundary element solution of the Poisson equation, the REBEL algorithm, and demonstrates for the first time that such a simulation can be performed in a realistic time frame.
Abstract: Solvation effects play a profound role in the energetics of protein folding. While a continuum dielectric model of solvation may provide a sufficiently accurate estimate of the solvation effects, until now this model was too computationally expensive and unstable for folding simulations. Here we proposed a fast yet accurate and robust implementation of the boundary element solution of the Poisson equation, the REBEL algorithm. Using our earlier double-energy scheme, we included for the first time the mathematically rigorous continuous REBEL solvation term in our Biased Probability Monte Carlo (BPMC) simulations of the peptide folding. The free energy of a 23-residue beta beta alpha-peptide was then globally optimized with and without the solvation electrostatics contribution. An ensemble of beta beta alpha conformations was found at and near the global minimum of the energy function with the REBEL electrostatic solvation term. Much poorer correspondence to the native solution structure was found in the "control" simulations with a traditional method to account for solvation via a distance-dependent dielectric constant. Each simulation took less than 40 h (21 h without electrostatic solvation calculation) on a single Alpha 677 MHz CPU and involved more than 40,000 solvation energy evaluations. This work demonstrates for the first time that such a simulation can be performed in a realistic time frame. The proposed procedure may eliminate the energy evaluation accuracy bottleneck in folding simulations.
137 citations
TL;DR: The predictive power of the novel strategy, combined with gene expression profiling by cDNA microarrays, leads to the identification of novel candidate autoantigens in the inflammatory autoimmune disease, multiple sclerosis.
Abstract: The interaction of TCRs with MHC peptide ligands can be highly flexible, so that many different peptides are recognized by the same TCR in the context of a single restriction element. We provide a quantitative description of such interactions, which allows the identification of T cell epitopes and molecular mimics. The response of T cell clones to positional scanning synthetic combinatorial libraries is analyzed with a mathematical approach that is based on a model of independent contribution of individual amino acids to peptide Ag recognition. This biometric analysis compares the information derived from these libraries composed of trillions of decapeptides with all the millions of decapeptides contained in a protein database to rank and predict the most stimulatory peptides for a given T cell clone. We demonstrate the predictive power of the novel strategy and show that, together with gene expression profiling by cDNA microarrays, it leads to the identification of novel candidate autoantigens in the inflammatory autoimmune disease, multiple sclerosis.
104 citations
TL;DR: The identity of the mammalian homolog to the yeast Sec12 guanine nucleotide exchange factor (18% identity) that promotes Sar1 activation is reported, and the dominant negative GDP‐restricted mutant Sar1[T39N] is shown to be a potent inhibitor of mSec12 activity.
Abstract: The Sar1 GTPase is an essential component of COPII vesicle coats involved in export of cargo from the endoplasmic reticulum of mammalian cells. To begin to elucidate its mechanism of action, we now report the identity of the mammalian homolog to the yeast Sec12 guanine nucleotide exchange factor (18% identity) that promotes Sar1 activation. Mammalian Sec12 (mSec12) is a type II transmembrane protein with a large cytosolic domain, a fragment of which has previously been reported as the transcription factor prolactin regulatory element binding protein (PREB). mSec12 promotes efficient guanine nucleotide exchange on Sar1, but not Arf1 or Rab GTPases. mSec12 is localized to the endoplasmic reticulum and an antibody to the cytosolic domain of mSec12 potently inhibits Sar1 recruitment and the formation of COPII vesicles in vitro. The dominant negative GDP-restricted mutant Sar1[T39N] is shown to be a potent inhibitor of mSec12 activity, consistent with its role in preventing COPII vesicle formation in vitro and during transient expression in vivo. We propose that mSec12 is an evolutionarily distant guanine nucleotide exchange factor directing Sar1 GTPase activation in mammalian cells. Its divergence from yeast Sec12p may reflect the specialized needs of the mammalian endoplasmic reticulum involving the formation of Sar1-dependent transitional elements (Aridor M, et al. J Cell Biol 2001;152:213–229) and selection of cargo into prebudding complexes.
99 citations
TL;DR: Because grape extracts are a convenient alimentary source of salutary phytochemicals to supplement currently prevalent occidental food and resveratrol appears to be especially useful, it could conveniently be added in biosignificant amounts to the grape extracts provided that their extraction, contents, and quality controls are instituted.
Abstract: The recent and essential reports on the biological activity of the principal phytophenols of Vitis vinifera and wine, with special attention to resveratrol, are reviewed. The phytophenols are arbitrarily divisible into single-ring phenolic acids, bisphenols including stilbenes, tricyclic phenols (flavonoids) and their subclasses, and oligomeric and polymeric species, the proanthocyanidins and anthocyanidins. Their precursors and the stilbenes, including resveratrol with its analogs and conjugates, appear to be of preventative and possibly therapeutic value in atherosclerosis and certain neoplastic and inflammatory afflictions. The probable mechanisms are free radical scavenging and selective interference with a multitude of factors affecting the division cycle of rapidly and abnormally proliferating mammalian cells. Reviewed are studies of natural occurrence, extraction methods, bioavailability, analytical detection, and metabolism of resveratrol, as well as its effects on cancer and inflammation, atheros...
90 citations
TL;DR: In vitro stimulation of peripheral blood mononuclear cells with each of the peptide epitope analogs stimulated memory CTLs, which recognized CMV- infected targets among a high percentage of CMV+ individuals.
79 citations
TL;DR: The use of synthetic combinatorial peptide libraries in the positional scanning format together with novel biometric prediction approaches has allowed the recognition profiles of individual autoreactive T cell clones with unprecedented accuracy and provides firm evidence that the basic principles of cross-recognition and their pathogenetic significance are relevant in MS.
Journal Article•
TL;DR: Results indicate that, at least for some clones, most of the amino acids composing the native antigenic peptide can be identified through the use of PS-SCLs, and biometrical analysis of the data generated by PS- SCL screening allowed the identification of the native ligand in a public database.
Abstract: The recent identification of molecularly defined human tumor antigens recognized by autologous CTLs has opened new opportunities for the development of antigen-specific cancer vaccines. Despite extensive work, however, the number of CTL-defined tumor antigens that are suitable targets for generic vaccination of cancer patients is still limited, mostly because of the painstaking and lengthy nature of the procedures currently used for their identification. A novel approach is based on the combined use of combinatorial peptide libraries in positional scanning format (positional scanning synthetic combinatorial peptide libraries, PS-SCLs) and tumor-reactive CTL clones. To validate this approach, we herein analyzed in detail the recognition of PS-SCLs by Melan-A-specific CTL clones. Our results indicate that, at least for some clones, most of the amino acids composing the native antigenic peptide can be identified through the use of PS-SCLs. Interestingly, this analysis also allowed the identification of peptide analogues with increased antigenic activity as well as agonist peptides containing multiple amino-acid substitutions. In addition, biometrical analysis of the data generated by PS-SCL screening allowed the identification of the native ligand in a public database. Overall, these data demonstrate the successful use of PS-SCLs for the identification and optimization of tumor-associated CTL epitopes.
TL;DR: The data indicated that although both ERK and p38 are required for PC12 cell differentiation, activation of p38 is not required when ERK is superactivated, and provided further evidence for the threshold theory that differentiation is determined by the duration of ERK activation.
Abstract: MAP kinases have important role in PC12 cell differentiation, since the activities of both extracellular regulated protein kinase (ERK) and p38 have been indicated as necessary signal for PC12 cell differentiation. Epidermal growth factor (EGF) and NGF both activate ERK and p38 in PC12 cells, but only NGF trigger differentiation. It has been proposed that the duration of ERK activation determines the switch from proliferation to differentiation, since EGF causes more transient activation of ERK than NGF in PC12 cells. Here we report that treatment of PC12 cells with EGF in the presence of SB203580, a widely used p38 inhibitor, caused differentiation. The pro-differentiation effect of SB203580 in EGF-treated PC12 cells was found to be independent of its function of p38 inhibition but was through an effect on the ERK pathway that has been recently reported (Kalmes et al. [1999] FEBS Lett. 444: 71–74; Hall-Jackson et al. [1999] Onc. 18: 2047–2054). We found that SB203580 by itself did not affect the activity of ERK1/2 but significantly extended EGF-induced ERK activation in PC12 cells, which resulted in early morphological differentiation. Our data indicated that although both ERK and p38 are required for PC12 cell differentiation, activation of p38 is not required when ERK is superactivated. Our data provided further evidence for the threshold theory that differentiation is determined by the duration of ERK activation. J. Cell. Biochem. 83: 585–596, 2001. © 2001 Wiley-Liss, Inc.
TL;DR: Protaxel's efficacy is substantially higher in human cancer line xenografts in athymic mice, especially in prostate PC-3, breast MDA-MB 468 and ovary OVCAR-1.
Abstract: 2' and 7 Polyol carbonates of paclitaxel were synthesized and screened as potential paclitaxel prodrugs. Paclitaxel is released from 7-(2",3"-dihydroxypropylcarbonato) paclitaxel (Protaxel) at rates inversely proportional to pH, by an intramolecular cyclization. Compared to paclitaxel, maximum tolerated i.v. or i.p. doses (MTD) of Protaxel are about 2.5- to 3-fold higher; its efficacy is substantially higher in human cancer line xenografts in athymic mice, especially in prostate PC-3, breast MDA-MB 468 and ovary OVCAR-1.
TL;DR: Efficient methods for the solid-phase synthesis of imidazoline-tethered 2,3-diketopiperazines, cyclic ureas, and cyclic thioureas are described and procedures have been extended to prepare mixture-based combinatorial libraries.
Abstract: Efficient methods for the solid-phase synthesis of imidazoline-tethered 2,3-diketopiperazines, cyclic ureas, and cyclic thioureas are described. Following the exhaustive reduction of resin-bound dipeptides derived from orthogonally protected diamino acids, the primary amine of the resulting tetraamines was selectively protected with Dde. The compounds were then selectively cyclized via their secondary amines with three different diimidazole derivatives ((COIm)2, COIm2, CSIm2). Upon Dde removal, the compounds were selectively N-acylated and dehydratively cyclized with POCl3 to afford the imidazoline-tethered analogues in moderate yield and high purity. These procedures have been extended to prepare mixture-based combinatorial libraries. Details of the selection of building blocks for preparation of the positional scanning libraries based on the “libraries from libraries” approach are discussed.
TL;DR: The data demonstrate that a homologous recombination event can be selected by positive negative selection in plants, and that the ADH locus was not changed.
TL;DR: An improved method for the solid-phase synthesis of bicyclic guanidines from reduced N-acylated dipeptides, and its use in the synthesis of urea-linked bicyclic Guanidine, is described.
Abstract: The general concept of tethered combinatorial libraries of compounds in which two pharmacophores are found is described. In particular, an improved method for the solid-phase synthesis of bicyclic guanidines from reduced N-acylated dipeptides, and its use in the synthesis of urea-linked bicyclic guanidines, is described. The exhaustive reduction of glutamine-containing resin-bound N-acylated dipeptides, using borane-THF, generated compounds containing three secondary amines and one primary amine. Following selective trityl protection of the primary amine, treatment of the three secondary amines with thiocarbonyldiimidazole (CSIm2) and mercuric acetate (Hg(OAc)2) generated the resin-bound bicyclic guanidines. Following trityl deprotection, an Fmoc-amino acid was coupled. Upon removal of the Fmoc protecting group, the resulting primary amine was treated with hexyl isocyanate to generate the urea-linked bicyclic guanidines. The desired products were cleaved from the resin using hydrogen fluoride. The selection of building blocks and characterization of controls for the synthesis of a combinatorial library is discussed.
TL;DR: An efficient method for the solid-phase synthesis of bis-heterocyclic compounds from resin-bound orthogonally protected lysine in excellent yield and high purity is presented.
Abstract: An efficient method for the solid-phase synthesis of bis-heterocyclic compounds from resin-bound orthogonally protected lysine is presented. The initial reaction step involves the exhaustive reduction of resin-bound tetra-amides using borane-THF, followed by cyclization of the resulting tetra-amine with either carbonyldiimidazole, thiocarbonyldiimidaziole, or oxalyldiimidazole to generate resin-bound bis-cyclic ureas, bis-cyclic thioureas, and bis-cyclic diketopiperazines, respectively. Cleavage from the solid support using hydrogen fluoride, followed by extraction and lyophilization, yields the desired bis-heterocyclic compounds in excellent yield and high purity.
TL;DR: Findings suggest that the lysophospholipid LPC--generated during the breakdown of low density lipoproteins and plasma membranes--is a master inhibitor of T cell activation, which could lower the threshold for T cellactivation leading to SLE and other multiorgan autoimmune diseases.
Abstract: What causes the aberrant immune responses that result in chronic multiorgan autoimmune diseases such as systemic lupus erythematosus (SLE)? As
Carson and Lo
explain in their Perspective, new findings suggest that the lysophospholipid LPC--generated during the breakdown of low density lipoproteins and plasma membranes--is a master inhibitor of T cell activation (
Kabarowski
et al.). Defects in the LPC pathway could lower the threshold for T cell activation leading to SLE and other multiorgan autoimmune diseases.
Patent•
07 Mar 2001TL;DR: Novel FBPase inhibitors of formula (I) are useful in the treatment of diabetes and other conditions associated with elevated blood glucose as discussed by the authors. But their effectiveness depends on the formulation of the formula.
Abstract: Novel FBPase inhibitors of formula (I) are useful in the treatment of diabetes and other conditions associated with elevated blood glucose.
TL;DR: Experiments are conducted on four 10-dimensional benchmark functions where the number of strategy parameter vectors is varied over 1, 2, 3, 4, 5, 10, and 20, and the results indicate advantages for using multiple strategy parameters vectors.
Abstract: Self-adaptation is a common method for learning online control parameters in an evolutionary algorithm. In one common implementation, each individual in the population is represented as a pair of vectors ( x , σ ), where x is the candidate solution to an optimization problem scored in terms of f ( x ), and σ is the so-called strategy parameter vector that influences how offspring will be created from the individual. Experimental evidence suggests that the elements of σ can sometimes become too small to explore the given response surface adequately. The evolutionary search then stagnates, until the elements of σ grow sufficiently large as a result of random variation. A potential solution to this deficiency associates multiple strategy parameter vectors with a single individual. A single strategy vector is active at any time and dictates how offspring will be generated. Experiments are conducted on four 10-dimensional benchmark functions where the number of strategy parameter vectors is varied over 1, 2, 3, 4, 5, 10, and 20. The results indicate advantages for using multiple strategy parameter vectors. Furthermore, the relationship between the mean best result after a fixed number of generations and the number of strategy parameter vectors can be determined reliably in each case.
TL;DR: A new strategy was described for development of bifunctional inhibitors, which combine the protease inhibitor and another enzyme inhibitor in one molecule, and it contains Phe at P3, but no P3' residue.
TL;DR: The exhaustive reduction of solid-support bound amides with borane afforded the requisite secondary amines, which following treatment with phenyl isocyanatoformate and cleavage provided the corresponding triazepane-2,4-diones.
TL;DR: In this paper, solid phase synthesis of [3,5,7]-1 H -imidazo[1,5-a ]imidazol-2(3 H )-ones from acylated dipeptides via cyclization using Bischler-Napieralski conditions is described.
TL;DR: Analysis of glycine-substitution analogues demonstrated that all the amino acids that make up the peptide sequence are important for the stabilization of thealpha-helical conformation, suggesting that a well-defined set of interactions is indispensable to allow alpha-helix formation in this short hexapeptide.
Abstract: The conformational properties of two hexapeptides, Ac-LWRILW-NH(2) and its D-amino acid counterpart Ac-lwrilw-NH(2), identified as calmodulin inhibitors using mixture-based synthetic combinatorial library approaches, have been characterised by NMR and CD spectroscopy. The peptides fold into an alpha-helical conformation in aqueous solution. The observed short- and medium-range nuclear Overhauser effects were consistent with the formation of an alpha-helical structure and a reasonably well-defined set of structures was obtained by using restraints from the NMR data in simulated annealing calculations. Analysis of glycine-substitution analogues demonstrated that all the amino acids that make up the peptide sequence are important for the stabilization of the alpha-helical conformation. The results suggest that a well-defined set of interactions is indispensable to allow alpha-helix formation in this short hexapeptide.
TL;DR: On the basis of their high yield and purity, bis cyclic guanidines derived from N(alpha)-Fmoc-N(epsilon)-Boc-lysine and N-acylated guanidsines were chosen for preparation of mixture-based combinatorial libraries.
Abstract: An efficient method for the solid-phase synthesis of cyclic guanidines from N-acylated amino acid amides, bis cyclic guanidines from N-acylated dipeptides derived from orthogonally protected diamino acids, and N-acylated guanidines from disubstituted cyclic guanidines is described. The exhaustive reduction of N-acylated amino acid amides yields diamines that on treatment with cyanogen bromide lead to the formation of cyclic guanidines. Resin-bound orthogonally protected diamino acids (i.e., Nα-Fmoc-Nx-(Boc)-diamino acid, x = β, γ, δ, e) were N-acylated following removal of the Fmoc group. Removal of the Boc functionality from the side chain then generated a primary amine. Subsequent coupling of Boc amino acids, followed by removal of the Boc group, generated dipeptides that were N-acylated. Exhaustive reduction of amide bonds of the N-acylated dipeptides generated tetraamines having four secondary amines, which upon cyclization with cyanogen bromide afforded the resin-bound trisubstituted bis cyclic guani...
TL;DR: The development of soluble mixture-based heterocyclic combinatorial libraries derived from amino acids and peptides is described, each composed of tens of thousands of low molecular weight acyclic and heterocyClic compounds.
Abstract: The development of soluble mixture-based heterocyclic combinatorial libraries derived from amino acids and peptides is described. Starting with a "toolbox" of various chemical transformations, including alkylations, reductions, acylations, and the use of a variety of bifunctional reagents, the "libraries from libraries" concept has been expanded to encompass the development of more than fifty positional scanning combinatorial libraries each composed of tens of thousands of low molecular weight acyclic and heterocyclic compounds.