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PubMed2001

Selective cleavage of D-Ala-D-Lac by small molecules: re-sensitizing resistant bacteria to vancomycin.

Pathogenic enterococci are becoming resistant to currently available antibiotics, including vancomycin, the drug of last resort for Gram-positive infections. Enterococci pose a significant public health threat, not least because of the risk of transferring vancomycin resistance to the ubiquitous Staphylococcus aureus. Vancomycin resistance is manifested by cell wall peptidoglycan precursors with altered termini that cannot bind the antibiotic. Small molecules with well-oriented nucleophile-electrophile assembly and complementary chirality to the peptidoglycan termini were identified as catalytic and selective cleavers of the peptidoglycan precursor depsipeptide. These molecules were tested in combination with vancomycin and were found to re-sensitize vancomycin-resistant bacteria to the antibiotic.

Alanine↗
PubMed1989

Molecular cloning of a small prostate protein, known as beta-microsemenoprotein, PSP94 or beta-inhibin, and demonstration of transcripts in non-genital tissues.

In order to study the gene expression of the seminal plasma protein beta-microseminoprotein, also known as PSP94 and beta-inhibin, clones encoding this protein were isolated from a cDNA library constructed in lambda gt11. Nucleotide sequencing confirmed the structure of a previously cloned cDNA. By northern blot analysis identical sized transcripts were demonstrated in the prostate, the respiratory (tracheal, bronchial and lung) tissues and the antrum part of the gastric mucosa. Thus, the protein is not primarily associated with male reproductive function. Although probably of no physiological significance, a slight structural similarity to the ovarian inhibin beta-chains was identified in the C-terminal half of the molecule.

Amino Acid Sequence↗
PubMed2005

Design of ligand binding to an engineered protein cavity using virtual screening and thermal up-shift evaluation.

Proteins could be used to carry and deliver small compounds. As a tool for designing ligand binding sites in protein cores, a three-step virtual screening method is presented that has been optimised using existing data on T4 lysozyme complexes and tested in a newly engineered cavity in flavodoxin. The method can pinpoint, in large databases, ligands of specific protein cavities. In the first step, physico-chemical filters are used to screen the library and discard a majority of compounds. In the second step, a flexible, fast docking procedure is used to score and select a smaller number of compounds as potential binders. In the third step, a finer method is used to dock promising molecules of the hit list into the protein cavity, and an optimised free energy function allows discarding the few false positives by calculating the affinity of the modelled complexes. To demonstrate the portability of the method, several cavities have been designed and engineered in the flavodoxin from Anabaena PCC 7119, and the W66F/L44A double mutant has been selected as a suitable host protein. The NCI database has then been screened for potential binders, and the binding to the engineered cavity of five promising compounds and three tentative non-binders has been experimentally tested by thermal up-shift assays and spectroscopic titrations. The five tentative binders (some apolar and some polar), unlike the three tentative non-binders, are shown to bind to the host mutant and, importantly, not to bind to the wild type protein. The three-step virtual screening method developed can thus be used to identify ligands of buried protein cavities. We anticipate that the method could also be used, in a reverse manner, to identify natural or engineerable protein cavities for the hosting of ligands of interest.

Bacteriophage T4↗
PubMed1995

Generating a phage display antibody library against an identified neuron.

The generation of monoclonal antibodies by conventional hybridoma technology is limited by the diversity of the clones and the difficulties of screening the antibodies and of their large-scale production from isolated clones. As an alternative approach, we have investigated the suitability of phage display libraries for the production of recombinant antibodies against an identified neuron. Mice were immunized with isolated leech Retzius (R) neurons. The spleen poly A+ RNA was isolated and first-strand cDNA was prepared. The variable regions of light- and heavy-chain IgG molecules were amplified by the polymerase chain reaction (PCR) and separate libraries of each were constructed and combined in the pComb8 vector to yield a combinatorial library of approximately 10(7) transformants. Single R neurons that were plated in culture bound approximately 100 phages on a first screen and several thousand when the first batch was re-screened. Of these, 96 individual phage colonies were isolated and used for immunocytochemistry with leech CNS ganglia: 41 exhibited general staining, 20 showed no detectable staining and 30 stained selective subsets of neurons including (but not specific for) the R neuron. The phage display library approach thus simplifies the screening of large libraries with small numbers of (and even single) cells. However, the combinatorial antibodies with binding activity must still be tested individually by immunocytochemistry, which is further limited by their apparently low affinity.

Animals↗
PubMed2003

Determination of the HIV-1 gp41 fusogenic core conformation modeled by synthetic peptides: applicable for identification of HIV-1 fusion inhibitors.

Triggered by receptor binding of gp120, the human immunodeficiency virus type 1 (HIV-1) gp41 changes its conformation to a fusogenic six-helix bundle structure. In the present study, this core conformation modeled by the peptides derived from the gp41 N- and C-terminal heptad repeat regions was determined by fluorescence native polyacrylamide gel electrophoresis and size exclusion high-performance liquid chromatography (HPLC). Two previously described small molecule HIV-1 fusion inhibitors significantly blocked the six-helix bundle formation. It suggests that these biophysical techniques can be used in a novel way to study the conformational change of gp41 during virus entry into cells and to identify HIV-1 fusion inhibitors.

Amino Acid Sequence↗
PubMed2006

Molecular diversity of disintegrin-like domains within metalloproteinase precursors of Bothrops jararaca.

Disintegrins are small peptides isolated from the venom of several snake families which act as integrin-antagonists or agonists, interacting with a variety of biological processes mediated by integrins. In this work we describe five new disintegrin-like domains within metalloproteinase precursor sequences, obtained from a Bothrops jararaca venom gland cDNA library. Among the new disintegrin-like domains, four were contained in PIII metalloproteinase precursors, with three of them presenting ECD-motifs and one presenting a new KCD-motif. Moreover, we found three disintegrin-like domains within PII metalloproteinase precursors. Two of them are similar to the already described disintegrins jarastatin and jararacin. The third molecule is unusual, presenting some typical PIII metalloproteinase characteristics but lacking the cysteine-rich domain being, thus, classified as a PII metalloproteinase. Only few reports presented molecules with these characteristics. Sequence analysis suggests that these molecules are intermediate steps between the more ancient PIII and the more recent PII metalloproteinases. We also investigated disintegrin N-terminus diversity in B. jararaca crude venom by purifying jarastatin and jararacin and analyzing them by mass spectrometry.

Amino Acid Sequence↗
PubMed2006

[Aptamers in clinical diagnostics].

Aptamers are single-stranded DNA or RNA oligonucleotides selected in vitro from combinatorial libraries in a process called SELEX (Systematic Evolution of Ligands by EXponential Enrichment). Aptamers play a role of artificial nucleic acid ligands that can recognize and bind to various organic or inorganic target molecules with high specificity and affinity. They can discriminate even between closely related targets and can be easily chemically modified for radioactive, fluorescent and enzymatic labeling or biostability improvement. Aptamers can thus be considered as universal receptors that rival antibodies in diagnostics as a tool of molecular recognition. To date aptamers have been successively used instead of monoclonal antibodies in flow cytometry, immunochemical sandwich assays and in vivo imaging as well to detect wide range of small or large biomolecules.

Aptamers, Nucleotide↗
PubMed2006

Combining the rapid MTT formazan exocytosis assay and the MC65 protection assay led to the discovery of carbazole analogs as small molecule inhibitors of Abeta oligomer-induced cytotoxicity.

The discovery of small molecule inhibitors of cytotoxicity induced by amyloid-beta (Abeta) oligomers, either applied extracellularly or accumulated intraneuronally, is an important goal of drug development for Alzheimer's disease (AD), but has been limited by the lack of efficient screening methods. Here we describe our approach using two cell-based methods. The first method takes advantage of the unique ability of extracellularly applied Abeta oligomers to rapidly induce the exocytosis of formazan formed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). We employed a short protocol to quantify this toxicity, and quickly identified two novel inhibitors, code-named CP2 and A5, from two compound libraries. A second independent screen of the same libraries using our previously published MC65 protection assay, which identifies inhibitors of toxicity related to intracellular Abeta oligomers, also selected the same two leads, suggesting that both assays select for the same anti-Abeta oligomer properties displayed by these compounds. We further demonstrated that A5 attenuated the progressive aggregation of existing Abeta oligomers, reduced the level of intracellular Abeta oligomers, and prevented the Abeta oligomer-induced death of primary cortical neurons, effects similar to those demonstrated by CP2. Our results suggest that, when combined, the two methods would generate fewer false results and give a high likelihood of identifying leads that show promises in ameliorating Abeta oligomer-induced toxicities within both intraneuronal and extracellular sites. Both assays are simple, suitable for rapid screening of a large number of medicinal libraries, and amenable for automation.

Amyloid beta-Peptides↗
PubMed1996

Cloning of the mucosal addressin MAdCAM-1 from human brain: identification of novel alternatively spliced transcripts.

The mucosal addressin cell adhesion molecule-1 (MAdCAM-1), expressed selectively on high endothelial venules (HEV) and lamina propria venules, directs lymphocyte traffic by binding the lymphocyte Peyer's patch adhesion molecule-1 (LPAM-1, alpha 4 beta 7). Full-length DNA encoding human MAdCAM-1 was obtained by combining sequences from an expressed sequence tag (EST) identified in an early stage human brain cDNA library, a polymerase chain reaction-derived clone, and a MAdCAM-1 genomic clone. The deduced amino acid sequence revealed an 18 amino acid signal peptide, two N-terminal immunoglobulin (Ig)-like domains conserved (59-65%) in sequence with those of the mouse homologue, an 86 amino acid mucin-like region rich in serine-threonine residues, a 20 amino acid transmembrane domain and a 43 amino acid charged cytoplasmic domain. No counterpart to the third IgA-like domain of mouse MAdCAM-1 was present; however, the serine-threonine-rich mucin domain was extended as two distinguishable major and minor mucin regions unrelated to the mouse domain. The major domain is formed from six tandem repeats of an eight amino acid sequence having the MUC-2-related consensus DTTSPEP/SP. Human MAdCAM-1 mRNA transcripts were restricted to small intestine, colon, spleen, pancreas and brain. Alternatively spliced MAdCAM-1 variants were identified that lack parts of the second Ig domain and all or part of the major mucin domain, indicating that the function of this vascular addressin is regulated by extensive modifications to its multi-domain structure.

Alternative Splicing↗
PubMed1991

Molecular cloning and sequencing of cDNAs encoding the proteolipid subunit of the vacuolar H(+)-ATPase from a higher plant.

To understand the molecular structure of the vacuolar H(+)-translocating ATPase from plants, cDNAs encoding the N,N'-dicyclohexylcarbodiimide-binding 16-kDa proteolipid from oat (Avena sativa L. var. Lang) have been obtained. A synthetic oligonucleotide corresponding to a region of the bovine proteolipid cDNA (Mandel, M., Moriyama, Y., Hulmes, J.D., Pan, Y.-C.E., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5521-5524) was used to screen an oat cDNA library constructed in lambda gt11. The nucleotide sequences of several positive clones (VATP-P1, clones 12, 54, 93) demonstrated the presence of a small multigene family. The four clones showed extensive divergence in their codon usage and their 3'-untranslated regions; however, the deduced amino acid sequences of the proteins were 97-99% identical. These clones encoded the proteolipid subunit as one of them (clone 12) expressed a fusion protein that reacted with an antibody to the 16-kDa proteolipid. The open reading frame of one cDNA clone (VATP-P1) predicted a polypeptide of 165 amino acids with a molecular mass of 16,641. Based on hydropathy plots, a molecule with four membrane-spanning domains was predicted, in which domain IV was especially conserved among different species. This domain showed 80% identity in nucleotide or amino acid sequences between the oat and the bovine proteolipids and contained a glutamate residue that is the putative N,N'-dicyclohexylcarbodiimide-binding residue. The presence of a small multigene family of the 16-kDa proteolipid was confirmed by Southern blot analysis showing that several distinct restriction fragments of oat nuclear DNA hybridized with the VATP-P1 cDNA.

Amino Acid Sequence↗
PubMed1992

The computer program LUDI: a new method for the de novo design of enzyme inhibitors.

A new computer program is described, which positions small molecules into clefts of protein structures (e.g. an active site of an enzyme) in such a way that hydrogen bonds can be formed with the enzyme and hydrophobic pockets are filled with hydrophobic groups. The program works in three steps. First it calculates interaction sites, which are discrete positions in space suitable to form hydrogen bonds or to fill a hydrophobic pocket. The interaction sites are derived from distributions of nonbonded contacts generated by a search through the Cambridge Structural Database. An alternative route to generate the interaction sites is the use of rules. The second step is the fit of molecular fragments onto the interaction sites. Currently we use a library of 600 fragments for the fitting. The final step in the present program is the connection of some or all of the fitted fragments to a single molecule. This is done by bridge fragments. Applications are presented for the crystal packing of benzoic acid and the enzymes dihydrofolate reductase and trypsin.

Algorithms↗
PubMed2000

Neuroprotection by the inhibition of apoptosis.

Accumulating evidence strongly suggests that apoptosis contributes to neuronal cell death in a variety of neurodegenerative contexts. Activation of the cysteine protease caspase-3 appears to be a key event in the execution of apoptosis in the central nervous system (CNS). As a result, mice null for caspase-3 display considerable neuronal expansion usually resulting in death by the second week of life. At present, 14 caspase family members have been identified and subdivided into three subgroups on the basis of preference for specific tetrapeptide motifs using a positional scanning combinatorial substrate library. Caspase-3 is a group II member (2, 3, 7) categorized by an absolute substrate requirement for aspartic acid in the P4 position of the scissile bond. The preferred cleavage motif (DExD) for group II caspases is found in many structural, metabolic and repair proteins essential for cellular homeostasis. Consistent with the proposal that apoptosis plays a central in role human neurodegenerative disease, caspase-3 activation has recently been observed in stroke, spinal cord trauma, head injury and Alzheimer's disease. Indeed, peptide-based caspase inhibitors prevent neuronal loss in animal models of head injury and stroke suggesting that these compounds may be the forerunners of non-peptide small molecules that halt apoptosis processes implicated in these neurodegenerative disorders. A clear link between an hereditary neurodegenerative disorder and failed caspase inhibition has recently been proposed for spinal muscular atrophy (SMA). In severe SMA, the neuronal specific inhibitor of apoptosis (IAP) family member known as NAIP is often dysfunctional due to missense and truncation mutations. IAPs such as NAIP potently block the enzymatic activity of group II caspases (3 and 7) suggesting that NAIP mutations may permit unopposed developmental apoptosis to occur in sensory and motor systems resulting in lethal muscular atrophy. Conversely, adenovirally-mediated overexpression of NAIP or the X-linked IAP called XIAP reduces the loss of CA1 hippocampal neurons following transient forebrain ischemia. Taken together, these findings suggest that anti-apoptotic strategies may some day have utility in the treatment of neurodegenerative disease. The present review will summarize some of the recent evidence suggesting that apoptosis inhibitors may become a practical therapeutic approach for both acute and chronic neurodegenerative conditions.

Animals↗
PubMed1988

Partial molecular genetic map of the rabbit VH chromosomal region.

Thirty VH-containing cosmid clones, isolated from rabbit germ-line DNA libraries, were restriction mapped and shown to contain approximately 100 VH genes in 765-kb of DNA. Twenty-two of the cosmid clones were grouped into seven distinct clusters. The VH genes were separated by an average of 8 kb, although some were separated by less than 3 kb. Comparison of the nucleotide sequences of two of these VH genes with the sequences of another 11 VH genes showed that they were all generally more than 80% homologous suggesting that rabbit VH genes are members of one highly homologous gene family. Most rabbit Ig molecules have the VH allotypic specificities a1, a2, or a3 and are designated VHa-positive. A small number (less than 30%) of Ig molecules lack these VHa allotypic specificities and are designated VHa-negative. The VH containing cosmid clones were hybridized with synthetic oligomer probes designed to be specific for genes encoding VHa-positive or VHa-negative molecules. At least 50% of the germ-line VH genes hybridized with the VHa-negative oligomer and thus presumably encode VHa-negative molecules; as few as 15% of the genes could be identified as encoding VHa-positive molecules based on hybridization with the VHa-positive oligomer. Approximately 35% of the VH genes did not hybridize with either oligomer and could not be classified as VHa-negative or VHa-positive. We propose that the predominance of serum VHa-positive molecules, in contrast to the predominance of VHa-negative encoding germ-line genes, may reflect preferential usage of a few germline VH genes. The implications of this idea toward explaining the allelic inheritance of VHa allotypes are discussed.

Amino Acid Sequence↗
PubMed1995

Characterisation of two tomato fruit-expressed cDNAs encoding xyloglucan endo-transglycosylase.

Xyloglucan endo-transglycosylase (XET) catalyses the cleavage and concomitant transfer of one xyloglucan molecule to another. It is thought to be an important component of cell wall metabolism, particularly in expanding tissue and ripening fruits. The recently reported cloning of a cDNA encoding a seed-expressed XET from nasturtium [9] has enabled two XET-encoding cDNAs to be isolated from a tomato fruit (breaker stage) cDNA library. Their deduced amino acid sequences exhibit ca. 40% identity to nasturtium XET. One of the tomato cDNA clones (tXET-B2) was over-expressed in Escherichia coli; following purification and refolding, the recombinant protein was shown to have XET activity, with no detectable hydrolytic activity. Southern hybridisation analysis suggests that these clones are members of a small multi-gene family encoding tomato XET. Ribonuclease protection assays show that transcripts protected by one of the clones (tXET-B1) are most abundant in pink fruit pericarp and were also detected in stems.

Amino Acid Sequence↗
PubMed2006

Receptor-selective coactivators as tools to define the biology of specific receptor-coactivator pairs.

In the absence of specific high-affinity agonists and antagonists, it has been difficult to define the target genes and biological responses attributable to many of the orphan nuclear receptors (ONRs). Indeed, it appears that many members of this receptor superfamily are not regulated by classical small molecules but rather their activity is controlled by interacting cofactors. Motivated by this finding, we have developed an approach to genetically isolate specific receptor-cofactor pairs in cells, allowing us to define the biological responses attributable to each complex. This is accomplished by using combinatorial peptide phage display to engineer the receptor interacting domain of each cofactor such that it interacts selectively with one nuclear receptor. In this study, we describe the customization of PGC-1alpha and its use to study the biology of the estrogen-related receptor alpha (ERRalpha) in cultured liver cells.

Cell Line, Tumor↗
PubMed2005

High affinity targets of protein kinase inhibitors have similar residues at the positions energetically important for binding.

Inhibition of protein kinase activity is a focus of intense drug discovery efforts in several therapeutic areas. Major challenges facing the field include understanding of the factors determining the selectivity of kinase inhibitors and the development of compounds with the desired selectivity profile. Here, we report the analysis of sequence variability among high and low affinity targets of eight different small molecule kinase inhibitors (BIRB796, Tarceva, NU6102, Gleevec, SB203580, balanol, H89, PP1). It is observed that all high affinity targets of each inhibitor are found among a relatively small number of kinases, which have similar residues at the specific positions important for binding. The findings are highly statistically significant, and allow one to exclude the majority of kinases in a genome from a list of likely targets for an inhibitor. The findings have implications for the design of novel inhibitors with a desired selectivity profile (e.g. targeted at multiple kinases), the discovery of new targets for kinase inhibitor drugs, comparative analysis of different in vivo models, and the design of "a-la-carte" chemical libraries tailored for individual kinases.

Amino Acid Sequence↗
PubMed1988

Tissue-specific expression and cDNA cloning of small nuclear ribonucleoprotein-associated polypeptide N.

Sera from some patients with systemic lupus erythematosus and other autoimmune diseases have antibodies against nuclear antigens. An example is anti-Sm sera, which recognize proteins associated with small nuclear RNA molecules [small nuclear ribonucleoprotein (snRNP) particles]. In this paper anti-Sm sera were used to probe immunoblots of various rat tissues. A previously unidentified Mr 28,000 polypeptide was recognized by these anti-Sm sera. This polypeptide, referred to as "N," is expressed in a tissue-specific manner, being most abundant in rat brain, less so in heart, and undetectable in the other tissues examined. Immunoprecipitation experiments using antibodies directed against the cap structure of small nuclear RNAs have demonstrated that N is a snRNP-associated polypeptide. Anti-Sm serum was also used to isolate a partial cDNA clone (lambda rb91) from a rat brain phage lambda gt11 cDNA expression library. On RNA blots, the 450-base-pair cDNA insert of this clone hybridized to a 1600-nucleotide mRNA species with an identical tissue distribution to N, suggesting that lambda rb91 encodes at least part of N. A longer cDNA clone was obtained by rescreening the library with lambda rb91. In vitro transcription and subsequent translation of this subcloned, longer insert (pGMA2) resulted in a protein product with the same electrophoretic and immunological properties as N, confirming that pGMA2 encodes N. The tissue distribution of N and the involvement of snRNP particles in nuclear pre-mRNA processing may imply a role for N in tissue-specific pre-mRNA splicing.

Animals↗
PubMed2006

Allosteric activation of the follicle-stimulating hormone (FSH) receptor by selective, nonpeptide agonists.

The pituitary glycoprotein hormones, luteinizing hormone and follicle-stimulating hormone (FSH), act through their cognate receptors to initiate a series of coordinated physiological events that results in germ cell maturation. Given the importance of FSH in regulating folliculogenesis and fertility, the development of FSH mimetics has been sought to treat infertility. Currently, purified and recombinant human FSH are the only FSH receptor (FSH-R) agonists available for infertility treatment. By screening unbiased combinatorial chemistry libraries, using a cAMP-responsive luciferase reporter assay, we discovered thiazolidinone agonists (EC50's = 20 microm) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC50 = 2 nm) with full efficacy compared with FSH that was FSH-R-selective and -dependent. The compound mediated progesterone production in Y1 cells transfected with the human FSH-R (EC50 = 980 nm) and estradiol production from primary rat ovarian granulosa cells (EC50 = 10.5 nm). This and related compounds did not compete with FSH for binding to the FSH-R. Use of human FSH/thyroid-stimulating hormone (TSH) receptor chimeras suggested a novel mechanism for receptor activation through a binding site independent of the natural hormone binding site. This study is the first report of a high affinity small molecule agonist that activates a glycoprotein hormone receptor through an allosteric mechanism. The small molecule FSH receptor agonists described here could lead to an oral alternative to the current parenteral FSH treatments used clinically to induce ovarian stimulation for both in vivo and in vitro fertilization therapy.

Allosteric Regulation↗