Structure-based design of selective and potent G quadruplex-mediated telomerase inhibitors

The telomerase enzyme is a potential therapeutic target in many human cancers. A series of potent inhibitors has been designed by computer modeling, which exploit the unique structural features of quadruplex DNA. These 3,6,9-trisubstituted acridine inhibitors are predicted to interact selectively with the human DNA quadruplex structure, as a means of specifically inhibiting the action of human telomerase in extending the length of single-stranded telomeric DNA. The anilino substituent at the 9-position of the acridine chromophore is predicted to lie in a third groove of the quadruplex. Calculated relative binding energies predict enhanced selectivity compared with earlier 3,6-disubstituted compounds, as a result of this substituent. The ranking order of energies is in accord with equilibrium binding constants for quadruplex measured by surface plasmon resonance techniques, which also show reduced duplex binding compared with the disubstituted compounds. The 3,6,9-trisubstututed acridines have potent in vitro inhibitory activity against human telomerase, with EC50 values of up to 60 nM.

Large vortex-like structure of dipole field in computer models of liquid water and dipole-bridge between biomolecules

We propose a framework to describe the cooperative orientational motions of water molecules in liquid water and around solute molecules in water solutions. From molecular dynamics (MD) simulation a new quantity “site-dipole field” is defined as the averaged orientation of water molecules that pass through each spatial position. In the site-dipole field of bulk water we found large vortex-like structures of more than 10 Å in size. Such coherent patterns persist more than 300 ps although the orientational memory of individual molecules is quickly lost. A 1-ns MD simulation of systems consisting of two amino acids shows that the fluctuations of site-dipole field of solvent are pinned around the amino acids, resulting in a stable dipole-bridge between side-chains of amino acids. The dipole-bridge is significantly formed even for the side-chain separation of 14 Å, which corresponds to five layers of water. The way that dipole-bridge forms sensitively depends on the side-chain orientations and thereby explains the specificity in the solvent-mediated interactions between biomolecules.

Structure and function of declarative and nondeclarative memory systems

This article reviews recent studies of memory systems in humans and nonhuman primates. Three major conclusions from recent work are that (i) the capacity for nondeclarative (nonconscious) learning can now be studied in a broad array of tasks that assess classification learning, perceptuomotor skill learning, artificial grammar learning, and prototype abstraction; (ii) cortical areas adjacent to the hippocampal formation, including entorhinal, perirhinal, and parahippocampal cortices, are an essential part of the medial temporal lobe memory system that supports declarative (conscious) memory; and (iii) in humans, bilateral damage limited to the hippocampal formation is nevertheless sufficient to produce severe anterograde amnesia and temporally graded retrograde amnesia covering as much as 25 years.

Molecular markers reveal that population structure of the human pathogen Candida albicans exhibits both clonality and recombination.

The life history of Candida albicans presents an enigma: this species is thought to be exclusively asexual, yet strains show extensive phenotypic variation. To address the population genetics of C. albicans, we developed a genetic typing method for codominant single-locus markers by screening randomly amplified DNA for single-strand conformation polymorphisms. DNA fragments amplified by arbitrary primers were initially screened for single-strand conformation polymorphisms and later sequenced using locus-specific primers. A total of 12 single base mutations and insertions were detected from six out of eight PCR fragments. Patterns of sequence-level polymorphism observed for individual strains detected considerable heterozygosity at the DNA sequence level, supporting the view that most C. albicans strains are diploid. Population genetic analyses of 52 natural isolates from Duke University Medical Center provide evidence for both clonality and recombination in C. albicans. Evidence for clonality is supported by the presence of several overrepresented genotypes, as well as by deviation of genotypic frequencies from random (Hardy-Weinberg) expectations. However, tests for nonrandom association of alleles across loci reveal less evidence for linkage disequilibrium than expected for strictly clonal populations. Although C. albicans populations are primarily clonal, evidence for recombination suggests that sexual reproduction or some other form of genetic exchange occurs in this species.

The mode of action and the structure of a herbicide in complex with its target: binding of activated hydantocidin to the feedback regulation site of adenylosuccinate synthetase.

(+)-Hydantocidin, a recently discovered natural spironucleoside with potent herbicidal activity, is shown to be a proherbicide that, after phosphorylation at the 5' position, inhibits adenylosuccinate synthetase, an enzyme involved in de novo purine synthesis. The mode of binding of hydantocidin 5'-monophosphate to the target enzyme was analyzed by determining the crystal structure of the enzyme-inhibitor complex at 2.6-A resolution. It was found that adenylosuccinate synthetase binds the phosphorylated compound in the same fashion as it does adenosine 5'-monophosphate, the natural feedback regulator of this enzyme. This work provides the first crystal structure of a herbicide-target complex reported to date.

The solution structure of the Raf-1 cysteine-rich domain: a novel ras and phospholipid binding site.

The Raf-1 protein kinase is the best-characterized downstream effector of activated Ras. Interaction with Ras leads to Raf-1 activation and results in transduction of cell growth and differentiation signals. The details of Raf-1 activation are unclear, but our characterization of a second Ras-binding site in the cysteine-rich domain (CRD) and the involvement of both Ras-binding sites in effective Raf-1-mediated transformation provides insight into the molecular aspects and consequences of Ras-Raf interactions. The Raf-1 CRD is a member of an emerging family of domains, many of which are found within signal transducing proteins. Several contain binding sites for diacylglycerol (or phorbol esters) and phosphatidylserine and are believed to play a role in membrane translocation and enzyme activation. The CRD from Raf-1 does not bind diacylglycerol but interacts with Ras and phosphatidylserine. To investigate the ligand-binding specificities associated with CRDs, we have determined the solution structure of the Raf-1 CRD using heteronuclear multidimensional NMR. We show that there are differences between this structure and the structures of two related domains from protein kinase C (PKC). The differences are confined to regions of the CRDs involved in binding phorbol ester in the PKC domains. Since phosphatidylserine is a common ligand, we expect its binding site to be located in regions where the structures of the Raf-1 and PKC domains are similar. The structure of the Raf-1 CRD represents an example of this family of domains that does not bind diacylglycerol and provides a framework for investigating its interactions with other molecules.

Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene.

Nociceptin (orphanin FQ), the newly discovered natural agonist of opioid receptor-like (ORL1) receptor, is a neuropeptide that is endowed with pronociceptive activity in vivo. Nociceptin is derived from a larger precursor, prepronociceptin (PPNOC), whose human, mouse, and rat genes we have now isolated. The PPNOC gene is highly conserved in the three species and displays organizational features that are strikingly similar to those of the genes of preproenkephalin, preprodynorphin, and preproopiomelanocortin, the precursors to endogenous opioid peptides, suggesting the four genes belong to the same family-i.e., have a common evolutionary origin. The PPNOC gene encodes a single copy of nociceptin as well as of other peptides whose sequence is strictly conserved across murine and human species; hence it is likely to be neurophysiologically significant. Northern blot analysis shows that the PPNOC gene is predominantly transcribed in the central nervous system (brain and spinal cord) and, albeit weakly, in the ovary, the sole peripheral organ expressing the gene. By using a radiation hybrid cell line panel, the PPNOC gene was mapped to the short arm of human chromosome 8 (8p21), between sequence-tagged site markers WI-5833 and WI-1172, in close proximity of the locus encoding the neurofilament light chain NEFL. Analysis of yeast artificial chromosome clones belonging to the WC8.4 contig covering the 8p21 region did not allow to detect the presence of the gene on these yeast artificial chromosomes, suggesting a gap in the coverage within this contig.

Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation.

The catalytic, or third domain of Pseudomonas exotoxin A (PEIII) catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor-2 in eukaryotic cells, inhibiting protein synthesis. We have determined the structure of PEIII crystallized in the presence of NAD to define the site of binding and mechanism of activation. However, NAD undergoes a slow hydrolysis and the crystal structure revealed only the hydrolysis products, AMP and nicotinamide, bound to the enzyme. To better define the site of NAD binding, we have now crystallized PEIII in the presence of a less hydrolyzable NAD analog, beta-methylene-thiazole-4-carboxamide adenine dinucleotide (beta-TAD), and refined the complex structure at 2.3 angstroms resolution. There are two independent molecules of PEIII in the crystal, and the conformations of beta-TAD show some differences in the two binding sites. The beta-TAD attached to molecule 2 appears to have been hydrolyzed between the pyrophosphate and the nicotinamide ribose. However molecule 1 binds to an intact beta-TAD and has no crystal packing contacts in the vicinity of the binding site, so that the observed conformation and interaction with the PEIII most likely resembles that of NAD bound to PEIII in solution. We have compared this complex with the catalytic domains of diphtheria toxin, heat labile enterotoxin, and pertussis toxin, all three of which it closely resembles.

Crystal structure of the T state of allosteric yeast chorismate mutase and comparison with the R state.

The crystal structure of the tyrosine-bound T state of allosteric yeast Saccharomyces cerevisiae chorismate mutase was solved by molecular replacement at a resolution of 2.8 angstroms using a monomer of the R-state structure as the search model. The allosteric inhibitor tyrosine was found to bind in the T state at the same binding site as the allosteric activator tryptophan binds in the R state, thus defining one regulatory binding site for each monomer. Activation by tryptophan is caused by the larger steric size of its side chain, thereby pushing apart the allosteric domain of one monomer and helix H8 of the catalytic domain of the other monomer. Inhibition is caused by polar contacts of tyrosine with Arg-75 and Arg-76 of one monomer and with Gly-141, Ser-142, and Thr-145 of the other monomer, thereby bringing the allosteric and catalytic domains closer together. The allosteric transition includes an 8 degree rotation of each of the two catalytic domains relative to the allosteric domains of each monomer (domain closure). Alternatively, this transition can be described as a 15 degree rotation of the catalytic domains of the dimer relative to each other.

cDNA structure, tissue distribution, and chromosomal localization of rat PC7, a novel mammalian proprotein convertase closest to yeast kexin-like proteinases.

By using reverse transcription-coupled PCR on rat anterior pituitary RNA, we isolated a 285-bp cDNA coding for a novel subtilisin/kexin-like protein convertase (PC), called rat (r) PC7. By screening rat spleen and PC12 cell lambda gt11 cDNA libraries, we obtained a composite 3.5-kb full-length cDNA sequence of rPC7. The open reading frame codes for a prepro-PC with a 36-amino acid signal peptide, a 104-amino acid prosegment ending with a cleavable RAKR sequence, and a 747-amino acid type I membrane-bound glycoprotein, representing the mature form of this serine proteinase. Phylogenetic analysis suggests that PC7 represents the most divergent enzyme of the mammalian convertase family and that it is the closest member to the yeast convertases krp and kexin. Northern blot analyses demonstrated a widespread expression with the richest source of rPC7 mRNA being the colon and lymphoid-associated tissues. In situ hybridization revealed a distinctive tissue distribution that sometimes overlaps with that of furin, suggesting that PC7 has widespread proteolytic functions. The gene for PC7 (Pcsk7) was mapped to mouse chromosome 9 by linkage analysis of an interspecific backcross DNA panel.

An RNA structure involved in feedback regulation of splicing and of translation is critical for biological fitness.

While studies of the regulation of gene expression have generally concerned qualitative changes in the selection or the level of expression of a gene, much of the regulation that occurs within a cell involves the continuous subtle optimization of the levels of proteins used in macromolecular complexes. An example is the biosynthesis of the ribosome, in which equimolar amounts of nearly 80 ribosomal proteins must be supplied by the cytoplasm to the nucleolus. We have found that the transcript of one of the ribosomal protein genes of Saccharomyces cerevisiae, RPL32, participates in such fine tuning. Sequences from exon I of the RPL32 transcript interact with nucleotides from the intron to form a structure that binds L32 to regulate splicing. In the spliced transcript, the same sequences interact with nucleotides from exon II to form a structure that binds L32 to regulate translation, thus providing two levels of autoregulation. We now show, by using a sensitive cocultivation assay, that these RNA structures and their interaction with L32 play a role in the fitness of the cell. The change of a single nucleotide within the 5' leader of the RPL32 transcript, which abolishes the site for L32 binding, leads to detectably slower growth and to eventual loss of the mutant strain from the culture. Experiments designed to assess independently the regulation of splicing and the regulation of translation are presented. These observations demonstrate that, in evolutionary terms, subtle regulatory compensations can be critical. The change in structure of an RNA, due to alteration of just one noncoding nucleotide, can spell the difference between biological success and failure.

Structure-activity relationships derived by machine learning: the use of atoms and their bond connectivities to predict mutagenicity by inductive logic programming.

We present a general approach to forming structure-activity relationships (SARs). This approach is based on representing chemical structure by atoms and their bond connectivities in combination with the inductive logic programming (ILP) algorithm PROGOL. Existing SAR methods describe chemical structure by using attributes which are general properties of an object. It is not possible to map chemical structure directly to attribute-based descriptions, as such descriptions have no internal organization. A more natural and general way to describe chemical structure is to use a relational description, where the internal construction of the description maps that of the object described. Our atom and bond connectivities representation is a relational description. ILP algorithms can form SARs with relational descriptions. We have tested the relational approach by investigating the SARs of 230 aromatic and heteroaromatic nitro compounds. These compounds had been split previously into two subsets, 188 compounds that were amenable to regression and 42 that were not. For the 188 compounds, a SAR was found that was as accurate as the best statistical or neural network-generated SARs. The PROGOL SAR has the advantages that it did not need the use of any indicator variables handcrafted by an expert, and the generated rules were easily comprehensible. For the 42 compounds, PROGOL formed a SAR that was significantly (P < 0.025) more accurate than linear regression, quadratic regression, and back-propagation. This SAR is based on an automatically generated structural alert for mutagenicity.

Intron phase correlations and the evolution of the intron/exon structure of genes.

Two issues in the evolution of the intron/exon structure of genes are the role of exon shuffling and the origin of introns. Using a large data base of eukaryotic intron-containing genes, we have found that there are correlations between intron phases leading to an excess of symmetric exons and symmetric exon sets. We interpret these excesses as manifestations of exon shuffling and make a conservative estimate that at least 19% of the exons in the data base were involved in exon shuffling, suggesting an important role for exon shuffling in evolution. Furthermore, these excesses of symmetric exons appear also in those regions of eukaryotic genes that are homologous to prokaryotic genes: the ancient conserved regions. This last fact cannot be explained in terms of the insertional theory of introns but rather supports the concept that some of the introns were ancient, the exon theory of genes.

Genomic structure of human microtubule-associated protein 2 (MAP-2) and characterization of additional MAP-2 isoforms.

We have determined that the gene for human microtubule-associated protein 2 (MAP-2) spans 19 exons, including 6 exons identified in this study, 1-4, 8, and 13; all six of these exons are transcribed. The alternative splicing of coding exons generates a greater diversity of MAP-2 transcripts and isoforms. The first three exons encode alternate 5' untranslated regions that can be spliced to additional untranslated sequences contained in exons 4 and 5. Exons 8 and 13 are transcribed in human fetal spinal cord, adult brain, MSN cells, and rat brain, and each exon maintains an open reading frame with both high and low molecular weight MAP-2 isoforms. Antibodies generated to synthetic peptides of exons 8 and 13 demonstrate that these exons are translated and MAP-2 isoforms containing these exons are generated.