Application of base cleavable safety catch linkers to solid phase library production.

We have used sulfide "Safety Catch" linkers to anchor typical medicinal chemistry functional groups to amine resins. Compounds are loaded as the ester, carbamate, or amine. At the end of the synthesis, the linker is activated by peracid. The sulfone resins are then cleaved by beta-elimination in the gas phase or in solution by secondary amines to produce acids and primary, secondary, or tertiary amines. Comparison of cleavage rates to other sulfone resins including SEM showed significantly faster cleavage for this system with conditions similar to Fmoc deprotection. Application of this strategy to a medicinal chemistry library gives good yields and purities of the resulting compounds.

The nanometer-scale structure of amyloid-beta visualized by atomic force microscopy.

Amyloid-beta (A beta) is the major protein component of neuritic plaques found in Alzheimer's disease. Evidence suggests that the physical aggregation state of A beta directly influences neurotoxicity and specific cellular biochemical events. Atomic force microscopy (AFM) is used to investigate the three-dimensional structure of aggregated A beta and characterize aggregate/fibril size, structure, and distribution. Aggregates are characterized by fibril length and packing densities. The packing densities correspond to the differential thickness of fiber aggregates along a zeta axis (fiber height above the x-y imaging surface). Densely packed aggregates ( > or = 100 nm thick) were observed. At the edges of these densely packed regions and in dispersed regions, three types of A beta fibrils were observed. These were classified by fibril thickness into three size ranges: 2-3 nm thick, 4-6 nm thick, and 8-12 nm thick. Some of the two thicker classes of fibrils exhibited pronounced axial periodicity. Substructural features observed included fibril branching or annealing and a height periodicity which varied with fibril thickness. When identical samples were visualized with AFM and electron microscopy (EM) the thicker fibrils (4-6 nm and 8-12 nm thick) had similar morphology. In comparison, the densely packed regions of approximately > or = 100 nm thickness observed by AFM were difficult to resolve by EM. The small, 2- to 3-nm-thick, fibrils were not observed by EM even though they were routinely imaged by AFM. These studies demonstrate that AFM imaging of A beta fibrils can, for the first time, resolve nanometer-scale, zeta-axis, surface-height (thickness) fibril features. Concurrent x-y surface scans of fibrils reveal the surface submicrometer structure and organization of aggregated A beta. Thus, when AFM imaging of A beta is combined with, and correlated to, careful studies of cellular A beta toxicity it may be possible to relate certain A beta structural features to cellular neurotoxicity.

Binding affinities of tyrosine-phosphorylated peptides to the COOH-terminal SH2 and NH2-terminal phosphotyrosine binding domains of Shc.

The adaptor protein Shc has been implicated in Ras signaling via association with many tyrosine-phosphorylated receptors, including growth factor receptors, antigen receptors on T and B cells, and cytokine receptors. Shc could interact with the activated receptors through the carboxyl-terminal Src homology 2 (SH2) domain or the structurally unrelated amino-terminal phosphotyrosine binding (PTB) domain. Using NMR and surface plasmon resonance techniques, we have measured the binding affinities of the SH2 and the PTB domains of Shc to a series of phosphotyrosine-containing peptides derived from known Shc binding sites. Tyrosine-phosphorylated peptides derived from Trk (pY490), polyoma virus middle T-antigen (pY250), ErbB3 (pY1309), and epidermal growth factor receptor (pY1086, pY1148, and pY1114) that contain NPXpY sequences bind preferentially to the PTB domain of Shc with Kd values of 0.02-5.3 microM. The binding affinities of these peptides to the Shc SH2 domain were in the range of 220-1290 microM. In contrast, tyrosine-phosphorylated peptides from epidermal growth factor receptor (pY992, pY1173) and the zeta chain of the T-cell receptor bind preferentially to the SH2 domain (Kd = 50-130 microM) versus the PTB domain (Kd > 680 microM). From these studies, the relative contribution of the individual domains of Shc for binding to the phosphotyrosine-containing portions of these proteins was determined. In addition, our data indicate that the high affinity binding of the PTB domain to the NPXpY-containing peptides results from a very high association rate and a rapid dissociation rate, which is similar to previous results observed for the SH2-phosphopeptide complexes.

Structure and ligand recognition of the phosphotyrosine binding domain of Shc.

The nuclear magnetic resonance structure of the phosphotyrosine binding (PTB) domain of Shc complexed to a phosphopeptide reveals an alternative means of recognizing tyrosine-phosphorylated proteins. Unlike in SH2 domains, the phosphopeptide forms an antiparallel beta-strand with a beta-sheet of the protein, interacts with a hydrophobic pocket through the (pY-5) residue, and adopts a beta-turn. The PTB domain is structurally similar to pleckstrin homology domains (a beta-sandwich capped by an alpha-helix) and binds to acidic phospholipids, suggesting a possible role in membrane localization.

Solution structure of the Shc SH2 domain complexed with a tyrosine-phosphorylated peptide from the T-cell receptor.

She is a widely expressed adapter protein that plays an important role in signaling via a variety of cell surface receptors and has been implicated in coupling the stimulation of growth factor, cytokine, and antigen receptors to the Ras signaling pathway. She interacts with several tyrosine-phosphorylated receptors through its C-terminal SH2 domain, and one of the mechanisms of T-cell receptor-mediated Ras activation involves the interaction of the Shc SH2 domain with the tyrosine-phosphorylated zeta chain of the T-cell receptor. Here we describe a high-resolution NMR structure of the Shc SH2 domain complexed to a phosphopeptide (GHDGLpYQGLSTATK) corresponding to a portion of the zeta chain of the T-cell receptor. Although the overall architecture of the protein is similar to other SH2 domains, distinct structural differences were observed in the smaller beta-sheet, BG loop, (pY + 3) phosphopeptide-binding site, and relative position of the bound phosphopeptide.

Focal adhesion kinase expressed by nerve cell lines shows increased tyrosine phosphorylation in response to Alzheimer's A beta peptide.

A beta is a 39-43-amino acid peptide that accumulates as extracellular aggregates in Alzheimer's disease-afflicted brain tissue. Contact between these aggregates and neurons is potentially pathogenic, although little is known about the cellular transduction mechanisms. We have investigated the impact of A beta aggregates on the neuronal control of protein tyrosine phosphorylation, which underlies signal transduction for multiple families of growth factor and adhesion receptors. Added to cultures of rat and human nerve cell lines, A beta aggregates evoked a non-desensitizing increase (1.3-3.6-fold) in tyrosine phosphorylation in a band at 118 kDa. The 118-kDa protein was determined by immunoprecipitation to be pp125FAK, not previously documented in cells of neuronal lineage. Immunoblots with anti-focal adhesion kinase (FAK) showed that A beta aggregates had no effect on FAK protein levels. The increase in FAK tyrosine phosphorylation occurred at doses of A beta aggregates that evoked lactate dehydrogenase release; evoked tyrosine phosphorylation preceded the first detectable lactate dehydrogenase release by 4 h. Like degeneration, the FAK response was dependent on A beta aggregation and neuronal differentiation. Since tyrosine phosphorylation of FAK is essential to its activity as a transduction component of integrin-, peptide-, and lysophosphatidic acid-mediated signaling, the data establish a link between A beta aggregates and signal transduction pathways implicated in diverse cell functions including neurite outgrowth, control of the cell cycle, and apoptosis.

Amyloid-beta aggregation: selective inhibition of aggregation in mixtures of amyloid with different chain lengths.

One of the clinical manifestations of Alzheimer's disease is the deposition of the 39-43 residue amyloid-beta (A beta) peptide in aggregated fibrils in senile plaques. Characterization of the aggregation behavior of A beta is one of the critical issues in understanding the role of A beta in the disease process. Using solution hydrodynamics, A beta was observed to form three types of species in phosphate-buffered saline: insoluble aggregates with sedimentation coefficients of approximately 50,000 S and molecular masses of approximately 10(9) Da, "soluble aggregates" with sedimentation coefficients of approximately 30 S and masses of approximately 10(6) Da, and monomer. When starting from monomer, the aggregation kinetics of A beta 1-40 (A beta 40) and A beta 1-42 (A beta 42), alone and in combination, reveal large differences in the tendency of these peptides to aggregate as a function of pH and other solution conditions. At pH 4.1 and 7.0-7.4, aggregation is significantly slower than at pH 5 and 6. Under all conditions, aggregation of the longer A beta 42 was more rapid than A beta 40. Oxidation of Met-35 to the sulfoxide in A beta 40 enhances the aggregation rate over that of the nonoxidized peptide. Aggregation was found to be dependent upon temperature and to be strongly dependent on peptide concentration and ionic strength, indicating that aggregation is driven by a hydrophobic effect. When A beta 40 and A beta 42 are mixed together, A beta 40 retards the aggregation of A beta 42 in a concentration-dependent manner. Shorter fragments have a decreasing ability to interfere with A beta 42 aggregation. Conversely, the rate of aggregation of A beta 40 can be significantly enhanced by seeding slow aggregating solutions with preformed aggregates of A beta 42. Taken together, the inhibition of A beta 42 aggregation by A beta 40, the seeding of A beta 40 aggregation by A beta 42 aggregates, and the chemical oxidation of A beta 40 suggest that the relative abundance and rates of production of different-length A beta and its exposure to radical damage may be factors in the accumulation of A beta in plaques in vivo.

Binding affinities of synthetic peptides, pyridine-2-carboxamidonetropsin and 1-methylimidazole-2-carboxamidonetropsin, that form 2:1 complexes in the minor groove of double-helical DNA.

The designed peptides pyridine-2-carboxamidonetropsin (2-PyN) and 1-methylimidazole-2-carboxamidonetropsin (2-ImN) are crescent-shaped analogs of the natural products netropsin and distamycin A. 2-PyN and 2-ImN bind the 5'-TGTCA-3' sequence as antiparallel side-by-side dimers in the minor groove of DNA. The binding affinities of 2-PyN and 2-ImN to four different 5-bp sites on DNA were determined by quantitative MPE-Fe(II) footprint titration and compared with the tripeptide D from distamycin. The binding affinities of D to the sites 5'-TTTTT-3' and 5'-TGTCA-3' are 2.6 x 10(7) and < 1 x 10(5) M-1, respectively (pH 7.0, 100 mM NaCl). 2-PyN binds these sites with similar affinities, 2.3 x 10(5) and 2.7 x 10(5) M-1, respectively. The affinities of 2-ImN to the same two sites are < 5 x 10(4) and 1.4 x 10(5) M-1, respectively. Substitution of an N-methylpyrrole-2-carboxamide of the distamycin tripeptide by 1-methylimidazole-2-carboxamide has changed the specificities for the two binding sites by a factor of 10(3). The data for 2-PyN and 2-ImN binding the 5'-TGTCA-3' site are best fit by a cooperative binding curve consistent with 2:1 peptide-DNA complexes.

Antiparallel side-by-side dimeric motif for sequence-specific recognition in the minor groove of DNA by the designed peptide 1-methylimidazole-2-carboxamide netropsin.

The designed peptide 1-methylimidazole-2-carboxamide netropsin (2-ImN) binds specifically to the sequence 5'-TGACT-3'. Direct evidence from NMR spectroscopy is presented that this synthetic ligand binds DNA as a 2:1 complex, which reveals that the structure is an antiparallel dimer in the minor groove of DNA. This is in contrast to the 1:1 complexes usually seen with most crescent-shaped minor groove binding molecules targeted toward A+T-rich tracts but reminiscent of a dimeric motif found for distamycin at high concentrations. These results suggest that sequence-dependent groove width may play an important role in allowing an expanded set of DNA binding motifs for synthetic peptides.