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D J Ecker

Publications and source records attributed to D J Ecker.

At least 19 recordsLinked to original sources
PubMed2001

RNAMotif, an RNA secondary structure definition and search algorithm.

RNA molecules fold into characteristic secondary and tertiary structures that account for their diverse functional activities. Many of these RNA structures are assembled from a collection of RNA structural motifs. These basic building blocks are used repeatedly, and in various combinations, to form different RNA types and define their unique structural and functional properties. Identification of recurring RNA structural motifs will therefore enhance our understanding of RNA structure and help associate elements of RNA structure with functional and regulatory elements. Our goal was to develop a computer program that can describe an RNA structural element of any complexity and then search any nucleotide sequence database, including the complete prokaryotic and eukaryotic genomes, for these structural elements. Here we describe in detail a new computational motif search algorithm, RNAMotif, and demonstrate its utility with some motif search examples. RNAMotif differs from other motif search tools in two important aspects: first, the structure definition language is more flexible and can specify any type of base-base interaction; second, RNAMotif provides a user controlled scoring section that can be used to add capabilities that patterns alone cannot provide.

3' Untranslated Regions↗
PubMed2001

De novo initiation of viral RNA-dependent RNA synthesis.

RNA viruses use several initiation strategies to ensure that their RNAs are synthesized in appropriate amounts, have correct termini, and can be translated efficiently. Many viruses with genomes of single-stranded positive-, negative-, and double-stranded RNA initiate RNA synthesis by a de novo (primer-independent) mechanism. This review summarizes biochemical features and variations of de novo initiation in viral RNA replication.

Flaviviridae↗
PubMed2001

Prediction of rho-independent transcriptional terminators in Escherichia coli.

A new algorithm called RNAMotif containing RNA structure and sequence constraints and a thermodynamic scoring system was used to search for intrinsic rho-independent terminators in the Escherichia coli K-12 genome. We identified all 135 reported terminators and 940 putative terminator sequences beginning no more than 60 nt away from the 3'-end of the annotated transcription units (TU). Putative and reported terminators with the scores above our chosen threshold were found for 37 of the 53 non-coding RNA TU and for almost 50% of the 2592 annotated protein-encoding TU, which correlates well with the number of TU expected to contain rho-independent terminators. We also identified 439 terminators that could function in a bi-directional fashion, servicing one gene on the positive strand and a different gene on the negative strand. Approximately 700 additional termination signals in non-coding regions (NCR) far away from the nearest annotated gene were predicted. This number correlates well with the excess number of predicted 'orphan' promoters in the NCR, and these promoters and terminators may be associated with as yet unidentified TU. The significant number of high scoring hits that occurred within the reading frame of annotated genes suggests that either an additional component of rho-independent terminators exists or that a suppressive mechanism to prevent unwanted termination remains to be discovered.

Algorithms↗
PubMed1999

Structure-activity relationships of novel 2-substituted quinazoline antibacterial agents.

High-throughput screening of in-house compound libraries led to the discovery of a novel antibacterial agent, compound 1 (MIC: 12-25 microM against S. pyogenes). In an effort to improve the activity of this active compound, a series of 2-substituted quinazolines was synthesized and evaluated in several antibacterial assays. One such compound (22) displayed improved broad-spectrum antibacterial activity against a variety of bacterial strains. This molecule also inhibited transcription/translation of bacterial RNA, suggesting a mechanism for its antibiotic effects. Structure-activity relationship studies of 22 led to the synthesis of another 24 compounds. Although some of these molecules were found to be active in bacterial growth assays, none were as potent as 22. Compound 22 was tested for its ability to cure a systemic K. pneumonia infection in the mouse and displayed moderate effects compared with a control antibiotic, gentamycin.

Animals↗
PubMed1999

Determinants of aminoglycoside-binding specificity for rRNA by using mass spectrometry.

We have developed methods for studying the interactions between small molecules and RNA and have applied them to characterize the binding of three classes of aminoglycoside antibiotics to ribosomal RNA subdomains. High-resolution MS was used to quantitatively identify the noncovalent binding interactions between mixtures of aminoglycosides and multiple RNA targets simultaneously. Signal overlap among RNA targets was avoided by the addition of neutral mass tags that direct each RNA target to a unique region of the spectrum. In addition to determining binding affinities, the locations of the binding sites on the RNAs were identified from a protection pattern generated by fragmenting the aminoglycoside/RNA complex. Specific complexes were observed for the prokaryotic rRNA A-site subdomain with ribostamycin, paromomycin, and lividomycin, whereas apramycin preferentially formed a complex with the eukaryotic subdomain. We show that differences in binding between paromomycin and ribostamycin can be probed by using an MS-MS protection assay. We have introduced specific base substitutions in the RNA models and have measured their impact on binding affinity and selectivity. The binding of apramycin to the prokaryotic subdomain strongly depends on the identity of position 1408, as evidenced by the selective increase in affinity for an A1408G mutant. An A1409-G1491 mismatch pair in the prokaryotic subdomain enhanced the binding of tobramycin and bekanamycin. These observations demonstrate the power of MS-based methods to provide molecular insights into small molecule/RNA interactions useful in the design of selective new antimicrobial drugs.

Aminoglycosides↗
PubMed1999

Multiplexed screening of neutral mass-tagged RNA targets against ligand libraries with electrospray ionization FTICR MS: a paradigm for high-throughput affinity screening.

We demonstrate that binding of mixtures of aminoglycosides can be measured simultaneously against multiple RNA targets of identical length and similar (or identical) molecular weight. Addition of a neutral mass tag to one of the RNA targets shifts the detected peaks to a higher mass/charge ratio, where complexes with small molecules can be identified unambiguously. An appropriately placed neutral mass tag does not alter RNA--ligand binding. The utility of this strategy is demonstrated with model RNAs corresponding to the decoding region of the prokaryotic and eukaryotic rRNAs and a mixture of five aminoglycosides. Complexes are observed between the aminoglycoside library and the prokaryotic rRNA model, while no aminoglycoside was observed to bind to the mass-tagged eukaryotic rRNA model. The differential binding data is consistent with the eukaryotic A-site rRNA having a different conformation compared with the prokaryotic A-site that prevents entry and binding of neomycin-class aminoglycosides. Mass spectrometric analysis of neutral mass-tagged macromolecular targets represents a new high-throughput screening paradigm in which the interaction of multiple targets against a collection of small molecules can be evaluated in parallel.

Escherichia coli↗
PubMed1998

Inhibition of human immunodeficiency virus type 1 infection in SCID-hu Thy/Liv mice by the G-quartet-forming oligonucleotide, ISIS 5320.

Viral replication was inhibited in a dose-dependent manner after administration of the phosphorothioate oligonucleotide TTGGGGTT (ISIS 5320) to human immunodeficiency virus type 1 (HIV-1)-infected SCID-hu Thy/Liv mice. Potent in vivo antiviral activity was observed against the T-cell-tropic molecular clone NL4-3; the agent was found to have weak activity against one primary HIV-1 isolate, and the agent was inactive against a second primary isolate.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Strategies for rapid deconvolution of combinational libraries: comparative evaluation using a model system.

Synthesis and testing of complex mixtures maximize the number of compounds that can be prepared and tested in a combinatorial library. When mixtures of compounds are screened, however, the identity of the compound(s) selected may depend on the deconvolution procedure employed. Previously, we developed a model system for evaluation of deconvolution procedures and used it to compare pooling strategies for iterative and noniterative deconvolution [Freier et al. J. Med. Chem. 1995, 38, 344-352]. We have now extended the model studies to include simulations of procedures with overlapping subsets such as subtractive pooling [Carell et al. Angew, Chem., Int. Ed. Engl. 1994, 33, 2061-2064], bogus coin pooling [Blake and Litzi-Davis. Bioconjugate Chem. 1992, 3, 510-513], and orthogonal pooling [D'Prez et al. J. Am. Chem. Soc. 1995, 117, 5405-5406]. These strategies required synthesis and testing of fewer subsets than did the more traditional nonoverlapping iterative strategies. The compounds identified using simulations of these strategies, however, were not the most active compounds in the library and were substantially less active than those identified by simulations of more traditional strategies.

Computer Simulation↗
PubMed1996

Deconvolution of combinatorial libraries for Drug discovery: theoretical comparison of pooling strategies.

Synthesis and testing of mixtures of compounds in a combinatorial library allow much greater throughput than synthesis and testing of individual compounds. When mixtures of compounds are screened, however, the possibility exists that the most active compound will not be identified. The specific strategies employed for pooling and deconvolution will affect the likelihood of success. We have used a nucleic acid hybridization example to develop a theoretical model of library deconvolution for a library of more than 250,000 compounds. This model was used to compare various strategies for pooling and deconvolution. Simulations were performed in the absence and presence of experimental error. We found iterative deconvolution to be most reliable when active molecules were assigned to the same subset in early rounds. Reliability was reduced only slightly when active molecules were assigned randomly to all subsets. Iterative deconvolution with as many as 65,536 compounds per subset did not drastically reduce the reliability compared to one-at-a-time testing. Pooling strategies compared using this theoretical model are compared experimentally in an accompanying paper.

Base Sequence↗
PubMed1996

Deconvolution of combinatorial libraries for drug discovery: experimental comparison of pooling strategies.

An experimental evaluation of several different pooling strategies for combinatorial libraries was conducted using a library of 810 compounds and an enzyme inhibition assay (phospholipase A2). The library contained compounds with varying degrees of activity as well as inactive compounds. The compounds were synthesized in groups of three and pooled together in various formats to realize different pooling strategies. With one exception, all iterative deconvolution strategies and position scanning resulted in identification of the same compound. The results are in good agreement with the predicted outcome from theoretical and computational methods. These data support the tenet that active compounds for pharmaceutically relevant targets can be successfully identified from combinatorial libraries organized in mixtures.

Drug Evaluation, Preclinical↗
PubMed1996

'Mutational SURF': a strategy for improving lead compounds identified from combinatorial libraries.

Synthesis and testing of mixtures of compounds in a combinatorial library offers the potential of much greater throughput than the 'one compound, one well' approach. When mixtures of compounds are screened, however, pooling and deconvolution strategies must be employed to identify the most active compound in the library. The possibility exists that the most active compound will not be identified. We have developed a theoretical model of library deconvolution using the well characterized properties of nucleic acid hybridization to calculate activities of individual molecules in libraries of more than 250,000 compounds. Calculations using this model have been employed to evaluate strategies for pooling and deconvolution. In the presence of errors in synthesis and testing, iterative deconvolution or position scanning sometimes identified a compound with sub-optimal activity. We describe a procedure called 'mutational SURF' in which 'mutants' of the selected compound are individually synthesized and tested. Simulations of mutational SURF using our model libraries suggest that mutational SURF provides an efficient method for improving the activity of lead compounds identified from combinatorial libraries.

Base Sequence↗
PubMed1995

Deconvolution of combinatorial libraries for drug discovery: a model system.

Iterative synthesis and screening strategies have recently been used to identify unique active molecules from complex synthetic combinatorial libraries. These techniques have many advantages over traditional screening methods, including the potential to screen large numbers of compounds to identify an active molecule while avoiding analytical separations and structural determination of unknown compounds. It is not clear, however, whether these techniques identify the most active molecular species in the mixtures and, if so, how often. Two key factors which may affect success of the selection process are the presence of many active compounds in the library with a range of activities and the chosen order of unrandomization. The importance of these factors has not been previously studied. Moreover, the impact of experimental errors in determination of subset activities or in randomization during library synthesis is not known. We describe here a model system based on oligonucleotide hybridization that addresses these questions using computer simulations. The results suggested that, within achievable experimental and library synthesis error, iterative deconvolution methods generally find either the best molecule or one with activity very close to the best. The presence of many active compounds in a library influenced the profile of subset activities, but did not preclude selection of a molecule with near optimal activity.

Base Sequence↗
PubMed1995

Combinatorial drug discovery: which methods will produce the greatest value?

Combinatorial strategies are important new approaches to drug discovery, and it seems quite likely that they will result in the discovery of interesting potential pharmaceuticals. However, it is less clear whether combinatorial approaches will result in quantum advances in therapeutics. Nor is there general agreement about the factors most important in defining how combinatorial strategies will provide value to the discovery of lead and therapeutic compounds. In this review, we propose criteria that define the value of combinatorial strategies and categorize the various approaches by: (a) the type of chemical space to be searched, (b) the tactics employed to synthesize and screen libraries, and (c) the structures of individual molecules in libraries. We evaluate the strengths and weaknesses of the various strategies and suggest milestones that can help to track their success.

Biotechnology↗
PubMed1994

Combinatorially selected guanosine-quartet structure is a potent inhibitor of human immunodeficiency virus envelope-mediated cell fusion.

The phosphorothioate oligonucleotide T2G4T2 was identified as an inhibitor of HIV infection in vitro by combinatorial screening of a library of phosphorothioate oligonucleotides that contained all possible octanucleotide sequences. The oligonucleotide forms a parallel-stranded tetrameric guanosine-quartet structure. Tetramer formation and the phosphorothioate backbone are essential for antiviral activity. The tetramer binds to the human immunodeficiency virus envelope protein gp120 at the V3 loop and inhibits both cell-to-cell and virus-to-cell infection.

Antiviral Agents↗
PubMed1994

Potent and specific inhibition of HIV envelope-mediated cell fusion and virus binding by G quartet-forming oligonucleotide (ISIS 5320).

We have previously reported identification of a phosphorothioate oligonucleotide TTGGGGTT (ISIS 5320) as a potent inhibitor of HIV infection in vitro. The oligonucleotide forms a parallel-stranded, tetrameric guanosine quartet (G-quartet) structure that specifically binds to the HIV envelope glycoprotein (gp120) and inhibits both cell-to-cell and virus-to-cell infection at submicromolar concentrations. In the current study we demonstrate that the tetramer inhibits the infection of laboratory-derived isolates of HIV-1 and HIV-2 in a variety of phenotypically distinct, established human cell lines and a panel of biologically diverse clinical isolates in fresh human peripheral blood lymphocytes and macrophages. The compound was also active against all drug-resistant virus isolates tested. In combination with AZT, ISIS 5320 exhibits additive to slightly synergistic anti-HIV activity. Cell-based mechanism of action studies demonstrate that the compound inhibits the binding of infectious virus and virus-infected cells to uninfected target cells by binding to the cationic V3 loop of the envelope glycoprotein. The G-quartet structure is a potential candidate for use in anti-HIV chemotherapy.

Antiviral Agents↗
PubMed1994

Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant.

The degradation of many proteins requires their prior attachment to ubiquitin. Proteolytic substrates are characteristically multiubiquitinated through the formation of ubiquitin-ubiquitin linkages. Lys-48 of ubiquitin can serve as a linkage site in the formation of such chains and is required for the degradation of some substrates of this pathway in vitro. We have characterized the recessive and dominant effects of a Lys-48-to-Arg mutant of ubiquitin (UbK48R) in Saccharomyces cerevisiae. Although UbK48R is expected to terminate the growth of Lys-48 multiubiquitin chains and thus to exert a dominant negative effect on protein turnover, overproduction of UbK48R in wild-type cells results in only a weak inhibition of protein turnover, apparently because the mutant ubiquitin can be removed from multiubiquitin chains. Surprisingly, expression of UbK48R complements several phenotypes of polyubiquitin gene (UB14) deletion mutants. However, UbK48R cannot serve as a sole source of ubiquitin in S. cerevisiae, as evidenced by its inability to rescue the growth of ubi1 ubi2 ubi3 ubi4 quadruple mutants. When provided solely with UbK48R, cells undergo cell cycle arrest with a terminal phenotype characterized by replicated DNA, mitotic spindles, and two-lobed nuclei. Under these conditions, degradation of amino acid analog-containing proteins is severely inhibited. Thus, multiubiquitin chains containing Lys-48 linkages play a critical role in protein degradation in vivo.

Cell Cycle↗