Central Core Structure Research Articles

Synthesis of Novel Nonpeptidic Thrombin Inhibitors

A novel class of nonpeptidic, active, and selective thrombin inhibitors has resulted from X-ray-structure-based design and subsequent improvement of the initial lead molecules. These inhibitors possess a bi- or tricyclic central core structure with attached side chains to reach the three binding pockets (selectivity S1 pocket, distal D pocket, and proximal P pocket) present in the active site of the enzyme. The key step in the preparation of these compounds is the 1,3-dipolar cycloaddition between an azomethine ylide, prepared in situ by the decarboxylative method from an aromatic aldehyde and an α-amino acid, with an N-substituted maleimide (e.g., see Schemes 1 and 2). All potent inhibitors contain an amidinium residue in the side chain for incorporation into the S1 pocket, which was introduced in the last step of the synthesis by a Pinner reaction. The compounds were tested in biological assays for activity against thrombin and the related serine protease trypsin. The first-generation lead compounds (±)-11 and (±)-19 (Scheme 1) with a bicyclic central scaffold showed Ki values for thrombin inhibition of 18 μM and 0.67 μM, respectively. Conformationally more restricted second-generation analogs (Scheme 2) were more active ((±)-22i: Ki=90 nM (Table 1)); yet the selectivity for thrombin over trypsin remained weak. In the third-generation compounds, a small lipophilic side chain for incorporation into the hydrophobic P pocket was introduced (Schemes 7 and 8). Since this pocket is present in thrombin but not in trypsin, an increase in binding affinity was accompanied by an increase in selectivity for thrombin over trypsin. The most selective inhibitor (Ki=13 nM, 760-fold selectivity for thrombin over trypsin; Table 2) was (±)-1 with an i-Pr group for incorporation into the P pocket. Optical resolution of (±)-1 (Scheme 9) provided (+)-1 with a Ki value of 7 nM and a 740-fold selectivity, whereas (−)-1 was 800-fold less active (Ki=5.6 μM, 21-fold selectivity). The absolute configuration of the stronger-binding enantiomer was assigned based on the X-ray crystal structure of the complex formed between thrombin and this inhibitor. Compound (+)-1 mimics the natural thrombin substrate, fibrinogen, which binds to the enzyme with the Ph group of a phenylalanine (piperonyl in (+)-1) in the distal D pocket, with the i-Pr group of a valine (i-Pr in (+)-1) in the proximal P pocket, and with a guanidinium side chain of an arginine residue (phenylamidinium group in (+)-1) in the selectivity S1 pocket of thrombin. A series of analogs of (±)-1 with the phenylamidinium group replaced by aromatic and aliphatic rings bearing OH or NH2 groups (Schemes 10 – 14) were not effectively bound by thrombin. A number of X-ray crystal-structure analyses of free inhibitors confirmed the high degree of preorganization of these compounds in the unbound state. Since all inhibitors prefer similar modes of association with thrombin, detailed information on the strength of individual intermolecular bonding interactions and their incremental contribution to the overall free energy of complexation was generated in correlative binding and X-ray studies. The present study demonstrates that defined mutations in highly preorganized inhibitors provide an attractive alternative to site-directed mutagenesis in exploring molecular-recognition phenomena at enzyme active sites.

X-ray crystal structure of glycinamide ribonucleotide synthetase from Escherichia coli.

Glycinamide ribonucleotide synthetase (GAR-syn) catalyzes the second step of the de novo purine biosynthetic pathway; the conversion of phosphoribosylamine, glycine, and ATP to glycinamide ribonucleotide (GAR), ADP, and Pi. GAR-syn containing an N-terminal polyhistidine tag was expressed as the SeMet incorporated protein for crystallographic studies. In addition, the protein as isolated contains a Pro294Leu mutation. This protein was crystallized, and the structure solved using multiple-wavelength anomalous diffraction (MAD) phase determination and refined to 1.6 A resolution. GAR-syn adopts an alpha/beta structure that consists of four domains labeled N, A, B, and C. The N, A, and C domains are clustered to form a large central core structure whereas the smaller B domain is extended outward. Two hinge regions, which might readily facilitate interdomain movement, connect the B domain and the main core. A search of structural databases showed that the structure of GAR-syn is similar to D-alanine:D-alanine ligase, biotin carboxylase, and glutathione synthetase, despite low sequence similarity. These four enzymes all utilize similar ATP-dependent catalytic mechanisms even though they catalyze different chemical reactions. Another ATP-binding enzyme with low sequence similarity but unknown function, synapsin Ia, was also found to share high structural similarity with GAR-syn. Interestingly, the GAR-syn N domain shows similarity to the N-terminal region of glycinamide ribonucleotide transformylase and several dinucleotide-dependent dehydrogenases. Models of ADP and GAR binding were generated based on structure and sequence homology. On the basis of these models, the active site lies in a cleft between the large domain and the extended B domain. Most of the residues that facilitate ATP binding belong to the A or B domains. The N and C domains appear to be largely responsible for substrate specificity. The structure of GAR-syn allows modeling studies of possible channeling complexes with PPRP amidotransferase.

Naphthyl benzoates versus phenyl benzoates A molecular structure-liquid crystal and ferroelectric behaviour relationship study

Abstract A comparative study of the liquid crystal and ferroelectric properties of several achiral, racemic and non-racemic 2,6-naphthyl benzoates and their 1,4-phenyl benzoate analogues is reported. In terms of pure compounds, both central core structures give rise to similar mesophase sequences; however, the naphthalene derivatives exhibit broader and more thermally stable liquid crystalline states. On the other hand, both central core units give rise to equivalent electric and optoelectronic properties when the compounds are used as chiral dopants in ferroelectric liquid crystal mixtures.

Central core myopathy in a great dane

ABSTRACTAn eight‐month‐old female great dane was referred for investigation of exercise intolerance that had developed progressively over one month. Examination revealed poor muscle mass, elevated plasma levels of muscle‐associated enzymes and electromyographic abnormalities. The dog was euthanased at 15 months old because of severe exercise intolerance. Tissue obtained by biopsy and at post mortem examination revealed prominent well defined central core structures within most muscle fibres, but no other abnormalities. The condition was diagnosed as a central core myopathy.

Characterization of folding intermediates of human carbonic anhydrase II: probing substructure by chemical labeling of SH groups introduced by site-directed mutagenesis.

By measurement of UV absorbance, CD spectra, and enzyme activity, we have shown that human carbonic anhydrase II forms a stable and compact folding intermediate at a moderate concentration of guanidine hydrochloride. The major aim of this study was to map the intermediate structure. For that reason, site-directed mutagenesis was used to introduce cysteine residues in various parts of the central beta-structure to give in each case a single cysteine residue. Thereafter, the accessibility of the introduced SH group to specific chemical labeling was used to probe the stability and compactness of the area surrounding each cysteine residue. Our results indicate that the folding intermediate has an ordered native-like secondary structure in the central part of the beta-sheet, whereas the peripheral part of the beta-sheet seems to be less ordered. A large hydrophobic cluster situated between the central beta-sheet core and secondary structure elements on the surface appears to be intact in the intermediate and is remarkably stable even at high GuHCl concentrations (> 5 M). This unusually stable substructure might function as a "seed" during the initiation of the folding process.

Comparative study of crystal structures and thermal magnetic properties of a yttrium-copper Y2Cu8 and a neodymium-copper Nd2Cu8 complex

Two heteronuclear Y-Cu and Nd-Cu metal complexes with the formulas Y[sub 2]Cu[sub 8]([mu][sub 4]-O)[sub 2]([mu]-PyO)[sub 12]([mu]-Cl)[sub 2](NO[sub 3])[sub 4](H[sub 2]O)[sub 2] (1) and Nd[sub 2]Cu[sub 8]([mu][sub 4]-O)[sub 2]([mu]-PyO)[sub 12]([mu]-Cl)[sub 2](OCH[sub 3])[sub 4](H[sub 2]O)[sub 4] (2) have been synthesized by the reactions of 2-hydroxypyridine (PyOH) and Cu(OCH[sub 3])[sub 2] with Y(NO[sub 3])[sub 3][center dot]4H[sub 2]O and CuCl[sub 2] or NdCl[sub 3][center dot]6H[sub 2]O in methanol in good yields. The structural features displayed by both compounds are very similar. The central core structures of these two compounds contain a Y[sub 2]Cu[sub 4]O[sub 2] or Nd[sub 2]Cu[sub 4]O[sub 2] unit which resembles that of a YBa[sub 2]Cu[sub 3]O[sub 7[minus]x] superconductor. The thermal properties of both compounds were studied by TGA and mass spectrometry. At temperatures below 200[degrees]C, H[sub 2]O and methanol ligands were released as the major volatile compounents. At temperatures above 200[degrees]C, fragmentation of both compounds occurs. Volatile fragments of Cu(PyO)[sup +], Cu[sub 2](PyO)[sup +], and Cu[sub 2](PyO)[sub 2][sup +] along with PyOH were observed. Magnetic susceptibilities of both compounds were measured in the temperature range 5-300 K. Y[sub 2]Cu[sub 8] showed characteristic antiferromagnetic interaction of Cu(II) ions, while Nd[sub 2]Cu[sub 8] showed typical paramagnetic behavior dominated by the two Nd(III) ions.more » 18 refs., 7 figs., 5 tabs.« less

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe: The cytochrome b gene has an intron closely related to the first two introns in the Saccharomyces cerevisiae cox1 gene

The DNA sequence of the cob region of the Schizosaccharomyces pombe mitochondrial DNA has been determined. The cytochrome b structural gene is interrupted by an intron of 2526 base-pairs, which has an open reading frame of 2421 base-pairs in phase with the upstream exon. The position of the intron differs from those found in the cob genes of Saccharomyces cerevisiae, Aspergillus nidulans or Neurospora crassa. The Sch. pombe cob intron has the potential of assuming an RNA secondary structure almost identical to that proposed for the first two cox1 introns (group II) in S. cerevisiae and the p1- cox1 intron in Podospora anserina. It has most of the consensus nucleotides in the central core structure described for this group of introns and its comparison with other group II introns allows the identification of an additional conserved nucleotide stretch. A comparison of the predicted protein sequences of group II intronic coding regions reveals three highly conserved blocks showing pairwise amino acid identities of 34 to 53%. These regions comprise over 50% of the coding length of the intron but do not include the 5′ region, which has strong secondary structural features. In addition to the potential intron folding, long helical structures involving repetitive sequences can be formed in the flanking cob exon regions. A comparison of the Sch. pombe cytochrome b sequence with those available from other organisms indicates that Sch. pombe is evolutionarily distant from both budding yeasts and filamentous fungi. As was seen for the Sch. pombe cox1 gene (Lang, 1984), the cob exons are translated using the universal genetic code and this distinguishes Sch. pombe mitochondria from all other fungal and animal mitochondrial systems.

Core Structure of a Bay of Bengal Monsoon Depression

Summer MONEX aircraft flight level and dropwindsonde data have been used to examine the central core structure of a mature Bay of Bengal monsoon depression on 7 July 1979. Continuous aircraft data including cloud photographs were obtained at three flight levels. The depression sloped toward the southwest with height, with buoyant cloudy ascent within cold air to the south and west of the axis and warm subsidence of clear air to the northeast of the axis. Marked departures from gradient balance were found. In the northeast subgradient subsiding flow was found throughout the lower troposphere. In the southeast, new cloud base, supergradient flow accompanied areas of convergence; near 700 hPa, the overlying flow was subgradient and subsident; near 400 hPa, marked variations in flow were related to clouds above flight level. Near cloud base, convergence reached magnitudes of approximately -2 × 10-4 s-l in areas of ∼300 km2 to the south and west of the center (ascent reaching roughly -1 Pa s-1) with divergence and subsidence of similar magnitude in and to the northeast of the center. These two regimes were separated by cloud arcs in the boundary layer below a subsidence inversion. Northward out of the cold region of cloudy ascent in the westerlies south of the depression center, flow at 700 hPa subsided at ∼0.1 Pa s-1 around the cast side of the depression. A cumulonimbus anvil spread at 150 hPa over the area of convergence and ascent west of the centers of circulation at low levels. Airborne radar showed ∼1% area coverage by echoes of reflectivity ≳ 35 dB(Z) (rainfall rate ≳ 6 mm h-1), with average rainfall ∼0.5 mm h-1. The flow configuration was consistent with ascent in cumulus convection, coupled with compensating dry adiabatic subsidence.

Freeze-fracture confirmation of the presence of a core in the specialized tip structure of Mycoplasma pneumoniae.

The presence of a specialized terminal region in Mycoplasma pneumoniae was seen in thin sections viewed in an electron microscope. Actively growing cells were examined by the freeze-fracture technique in the absence of fixation to further establish the core as a significant structural entity. Cross fractures revealed a cytoplasmic matrix surrounding a central core structure of about 54 nm. This structure disappeared rapidly in aging cells. The convex protoplastic faces of the membrane around the core had characteristic 5- to 10-nm intramembrane particles evenly distributed across the cell surface, with no apparent difference in the region of the specialized tip. A periodicity previously noted in negatively stained preparations was clearly defined here in thin sections. Attachment of actively growing cells to sheep erythrocytes was seen primarily as a side attachment rather than attachment at the tip alone. This association between the mycoplasma and the sheep erythrocytes seriously deformed the sheep erythrocytes, but no membrane fusion could be detected.

Chromosome core structure revealed by silver staining.

Chromosomes were subjected to either prolonged hypotonic solution pretreatment or aging. Both conditions greatly loosened and dispersed the overlying epichromatin from the central chromosome core structure. This was followed by silver staining and examination with bright-field microscopy. The chromosome core selectively reduced the silver and stained black while the surrounding epichromatin stained yellow. A single core was seen extending the length of each chromatid. Nucleolus organizer regions appeared to be attached to the core, while kinetochores seemed to be specialized regions of the core itself. Cytochemical tests indicated that the core component(s) responsible for silver staining was non-histone protein(s).

Some facts concerning the nature and formation of axial core structures in spermatids of Gryllus domesticus

Examination of living spermatid nuclei of Gryllus domesticus has revealed the presence of the same structures, the X chromosome, the round body and the axial core structures, which have been described from electron microscopic observations. The outer ribbons of the axial core structures and the round body are composed of 100 A fibres indiscernible from and often continuous with the fibres composing the X chromosome. That the outer ribbons of the axial core structures and the round body are chromosomal is further substantiated by the results of cytochemical examinations of formaldehyde fixed material which show that the axial core structures and the round body contain RNA, DNA and basic protein. Neither acetic acid-ethanol nor cold ethanol fixation preserve the round body and the axial core structures suggesting that a protein may be responsible for maintenance of the central core structure. The central core structures are always found in close association with condensed chromatin in regions where the chromosome elements are about 1000 A apart, suggesting that the relative state of condensation of the chromatin and the spacial relationship between condensed regions may be two of the chief factors concerned in central core formation. Maintainance of the condensed state of the chromatin, however, may in turn depend upon central core integrity.