The detection of microbial DNA but not cultured bacteria is associated with increased mortality in patients with suspected sepsis-a prospective multi-centre European observational study.

OBJECTIVES: Blood culture results inadequately stratify the mortality risk in critically ill patients with sepsis. We sought to establish the prognostic significance of the presence of microbial DNA in the bloodstream of patients hospitalized with suspected sepsis. METHODS: We analysed the data collected during the Rapid Diagnosis of Infections in the Critically Ill (RADICAL) study, which compared a novel culture-independent PCR/electrospray ionization-mass spectrometry (ESI-MS) assay with standard microbiological testing. Patients were eligible for the study if they had suspected sepsis and were either hospitalized or were referred to one of nine intensive care units from six European countries. The blood specimen for PCR/ESI-MS assay was taken along with initial blood culture taken for clinical indications. RESULTS: Of the 616 patients recruited to the RADICAL study, 439 patients had data on outcome, results of the blood culture and PCR/ESI-MS assay available for analysis. Positive blood culture and PCR/ESI-MSI result was found in 13% (56/439) and 40% (177/439) of patients, respectively. Either a positive blood culture (p 0.01) or a positive PCR/ESI-MS (p 0.005) was associated with higher SOFA scores on enrolment to the study. There was no difference in 28-day mortality observed in patients who had either positive or negative blood cultures (35% versus 32%, p 0.74). However, in patients with a positive PCR/ESI-MS assay, mortality was significantly higher in comparison to those with a negative result (42% versus 26%, p 0.001). CONCLUSIONS: Presence of microbial DNA in patients with suspected sepsis might define a patient group at higher risk of death.

Identification and typing of Francisella tularensis with a highly automated genotyping assay.

A PCR assay was developed to genotypically characterize Francisella tularensis and F. novicida. An integrated and partially redundant set of markers was selected to provide positive identification of these species, identify subspecies of F. tularensis and genotype 14 variable number tandem repeat (VNTR) markers. Assay performance was evaluated with 117 Francisella samples. Sample DNA was amplified, and the masses of the PCR products were determined with electrospray ionization/time of flight mass spectrometry (ESI-MS). The base compositions of the PCR amplicons were derived from these high-accuracy mass measurements and contrasted with databased information associated with each of the 25 assay markers. Species and subspecies determinations for all samples were fully concordant with results from established typing methods, and VNTR markers provided additional discrimination among samples. Sequence variants were observed with a number of assay markers, but these did not interfere with sample characterization, and served to increase the genetic diversity detected by the assay.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

'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.

Pharmacology of antisense therapeutic agents : cancer and inflamination.

Antisense oligonucleotides represent a new paradigm for drug discovery that holds great promise to deliver potent and specific drugs with fewer undesired side effects. The antisense paradigm offers the opportunity to identify rapidly lead compounds based on knowledge of the biology of a disease process, and a relevant target gene sequence. With this information, the practitioner of antisense drug discovery can rapidly design, synthesize, and test a series of compounds in cell culture and determine if the target gene is specifically inhibited. A compound thus identified can then be tested in an animal model, either to determine whether targeted gene expression can be inhibited in various animal tissues or to determine if there is activity in an animal model of a human disease. The length of time and the resources required to identify a lead compound by the antisense paradigm is much less than by any other drug discovery method.

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.

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.

Characterization of mammalian Gs-alpha proteins expressed in yeast.

The guanine nucleotide regulatory protein, GS, mediates transmembrane signaling by coupling membrane receptors to the stimulation of adenylyl cyclase activity. The full length coding sequences for the M(r) = 42-45,000, short form (S), and M(r) = 46-52,000, long form (L), of the alpha-subunits of rat GS were placed in yeast expression vectors under the regulatory control of the copper-inducible CUP1 promoter and transformed into Saccharomyces cerevisiae. In the presence of 100 microM CuSO4, the transformed yeast expressed GS-alpha mRNAs and proteins. In reconstitution experiments, rat GS-alpha(S and L), solubilized from yeast membranes with 1% cholate, conferred NaF-, (-)isoproterenol-, and guanine nucleotide-dependent sensitivity to adenylyl cyclase catalytic units in S49 lymphoma cyc- cell membranes, which are devoid of endogenous GS-alpha. GS-alpha (S) demonstrated twice the activity of GS-alpha(L) in reconstitution assays of fluoride-stimulated adenylyl cyclase activity. Comparison of GS-alpha (S) expressed in yeast with GS purified from rabbit liver or human erythrocytes showed that the crude recombinant protein was fully competent in reconstituting NaF-stimulated adenylyl cyclase activity, but was only 2-5% as potent as purified GS. Addition of bovine brain beta gamma subunits during reconstitution enhanced all parameters of adenylyl cyclase activity for GS-alpha(S and L) obtained from yeast. In contrast, transducin beta gamma only enhanced agonist-stimulated adenylyl cyclase activity for GS-alpha (S and L) following reconstitution. These results demonstrate that the expression of functional mammalian GS-alpha subunits in yeast may be useful for their biochemical characterization.

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.