Novel penem molecules with heterocycle substitutions at the 6 position via a methylidene linkage were investigated for their activities and efficacy as beta-lactamase inhibitors. The concentrations of these molecules that resulted in 50% inhibition of enzyme activity were 0.4 to 3.1 nM for the TEM-1 enzyme, 7.8 to 72 nM for Imi-1, 1.5 to 4.8 nM for AmpC, and 14 to 260 nM for a CcrA metalloenzyme. All the inhibitors were more stable than imipenem against hydrolysis by hog and human dehydropeptidases. Piperacillin was combined with a constant 4-microg/ml concentration of each inhibitor for MIC determinations. The combinations reduced piperacillin MICs by 2- to 32-fold for extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae strains. The MICs for piperacillin-resistant (MIC of piperacillin, >64 microg/ml) strains of Enterobacter spp., Citrobacter spp., and Serratia spp. were reduced to the level of susceptibility (MIC of piperacillin, < or =16 microg/ml) when the drug was combined with 4, 2, or 1 microg of these penem inhibitors/ml. Protection against acute lethal bacterial infections with class A and C beta-lactamase- and ESBL-producing organisms in mice was also demonstrated with piperacillin plus inhibitor. Median effective doses were reduced by approximately two- to eightfold compared to those of piperacillin alone when the drug was combined with the various inhibitors at a 4:1 ratio. Pharmacokinetic analysis after intravenous administration of the various inhibitors showed mean residence times of 0.1 to 0.5 h, clearance rates of 15 to 81 ml/min/kg, and volumes of distribution between 0.4 and 2.5 liters/kg. The novel methylidene penem molecules inhibit both class A and class C enzymes and warrant further investigation for potential as therapeutic agents when used in combination with a beta-lactam antibiotic.

BACKGROUND:Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H), is based on the assumption that the loss of target genes is minimal, or at worst, managable. However, the expression of genes or gene fragments that are capable of interacting with E. coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost) in the amplified library.RESULTS:Analysis of candidate genes as Y2H fusion constructs has shown that, while stable in E. coli and yeast for genetic studies, they are rapidly lost in growth conditions for genomic libraries. This includes the rapid loss of a fragment of the E. coli cell division gene ftsZ which encodes the binding site for ZipA and FtsA. Expression of this clone causes slower growth in E. coli. This clone is also rapidly lost in yeast, when expressed from a GAL1 promoter, relative to a vector control, but is stable when the promoter is repressed. We have demonstrated in this report that the construction of libraries for the E. coli and B. subtilis genomes without passaging through E. coli is practical, but the number of transformants is less than for libraries cloned using E. coli as a host. Analysis of several clones in the libraries that are strongly growth inhibitory in E. coli include genes for many essential cellular processes, such as transcription, translation, cell division, and transport.CONCLUSION:Expression of Y2H clones capable of interacting with E. coli and yeast targets are rapidly lost, causing a loss of complexity. The strategy for preparing Y2H libraries described here allows the retention of genes that are toxic when inappropriately expressed in E. coli, or yeast, including many genes that represent potential antibacterial targets. While these methods are generally applicable to the generation of Y2H libraries from any source, including mammalian and plant genomes, the potential of functional clones interacting with host proteins to inhibit growth would make this approach most relevant for the study of prokaryotic genomes.

A screening system is described that can detect and confirm inhibitors of the late steps of cell wall biosynthesis. The primary high through-put screen monitors induction of beta-lactamase following exposure to samples, in an Escherichia coli envA- strain that carries the beta-lactamase gene from Citrobacter freundii on a plasmid. Positive samples were detected from compound libraries, from natural products libraries, and from fractions of natural products crude preparations. These samples were then subjected to in vitro assays that could detect the incorporation of soluble cell wall precursor into Lipid I, Lipid II, and polymerized cell wall, using a TLC system that was very accurate and unambiguous in detecting known cell wall inhibitors. One partially purified sample containing a novel antibacterial agent derived from natural products was found to inhibit the formation of Lipid I (50% inhibition at < or = 62.5 ng/ml), whereas another partially purified sample also derived from natural products inhibited transglycosylation into cell wall polymer (50% inhibition at < or = 10 microg/ml). This screening system proved to be especially useful because it was sufficiently sensitive and robust to detect inhibitors among samples of crude preparations or varying states of purity.

In 1996, the World Health Organization warned of a global crisis, estimating that infectious diseases were responsible for the deaths of over 17 million people per year [1.The World Health Report (1996) Fighting disease, Fostering Development. World Health Organization, GenevaGoogle Scholar]. Since then, and despite significant progress in the battle to eradicate or eliminate poliomyelitis, leprosy, guinea-worm infection, neonatal tetanus, Chagas disease, measles and onchocerciasis, the spread of HIV, drug-resistant malaria and tuberculosis has served to highlight the continuing threat posed by infectious diseases. However, the situation is not totally bleak. Directly observed treatment, short course (DOTS), has had a major impact on tuberculosis, and insecticide treated bednets (ITN) have provided a much-needed boost to malaria control. The application of existing technologies (such as ITN for malaria) to greater effect has bought a little time – time in which it might be possible for us to exploit the many exciting new technologies in the search for new drugs, vaccines and microbicides. However, there is no room for complacency: ‘old’ diseases continue to pose problems, and ‘new’ diseases have emerged with alarming rapidity (See Table 1 in the review by Woolhouse). The predicted and predictable public health threat of widespread drug resistance to therapeutic antimicrobial and/or antiviral agents has become a clinical reality, and the varied strategies of pathogens to evade these anti-infectives are reviewed by McKeegan et al. There has therefore been a considerable effort, both academically and industrially, to not only identify anti-infective agents that are active against resistant pathogens, but also to develop and implement strategies to ameliorate the emergence and spread of resistant pathogens. Another, somewhat overlooked, force that is driving efforts to discover new anti-infective agents and approaches, is the rise of emerging and-re-emerging pathogens, as reviewed by Woolhouse. Indeed, many of these ‘emerging’ pathogens might always have been present at relatively low levels (e.g. Legionella pneumophila), while others are re-emerging specifically because of multidrug resistant strains (e.g. multidrug resistant Mycobacterium tuberculosis). However, further coloring the picture are perhaps the ‘genuinely’ novel diseases, such as AIDS and variant Creutzfeldt-Jacob disease (vCJD). Progress has been made in the rapid identification of pathogenic microbes, as reviewed by Versalovic and Lupski. Perhaps within the decade, we will have point-of-care diagnostics that will enable the detection of infectious organisms. To date, there is a reliance on broad-spectrum agents, often used in combination, for empiric therapy. Rapid diagnosis of the pathogen in question should enable the use of more specific agents that could serve to limit the emergence and spread of resistance. Market forces, however, are pulling in exactly the opposite direction. The considerable and growing expense in developing safe and effective antimicrobial agents and vaccines, demands for companies – both large and small – the promise of a considerable market for these novel products. Persing and colleagues illustrate new approaches to vaccine development and immunomodulation. A large amount of attention has been focused recently on the decline of antibacterial research at many large pharmaceutical companies (despite the continued growing need for new drugs). This is widely attributed to the perceived ‘failure’ of novel target drug discovery programs to identify new classes of broad-spectrum antibacterial agents, despite the new and powerful technologies that have been brought to bear. This is specifically discussed in the article by Schmid, which reviews proteomics approaches and the probable impact of high-throughput protein-structure determination. However, there have been some modest successes in finding novel agents, and some of these are progressing into development. In addition, our understanding of what is required of a new agent for it to be successful has also progressed, and this will facilitate the discovery process. An example of that improved understanding is the development of better and more predictive animal models for anti-infective development, as reviewed by Druilhe et al. Another important consideration is that, in many cases, anti-infectives research is not looking for agents that interact with a single target (e.g. the gyrA gene product of Staphylococcus aureus), but a family of more-or-less related targets (e.g. the type II topoisomerases of pathogenic bacteria). More often than not, one size really doesn't fit all. In many ways, we are entering the golden age of anti-infectives research. Many of the major pathogenic bacteria have been completely sequenced, and complete sequence information is available for most of the clinically important viruses and some pathogenic fungi. While the roles of many bacterial and viral genes have yet to be elucidated, a variety of methods, including proteomics, transcriptional profiling, in vivo expression technology and tightly regulatable in vivo gene expression, are providing new insights into the physiology of infections at the level of both the pathogen and the host. Parkinson describes the impact of these technologies in accelerating drug discovery and, more importantly, (pre)clinical development. As multitudes of potential targets have emerged from these and other studies, an increasing array of methods for finding substances that interact with these novel (and not so novel) targets has also been developed and are being improved upon daily. These technologies are making increasingly effective uses of: (1) automated approaches for screening and chemical syntheses; (2) structural biology approaches to rational drug design; and (3) ever-sophisticated animal models of human disease. Perhaps our greatest difficulty is being able to use effectively the vast amount of biological data being generated in the drug discovery process. It is now recognized that infectious diseases are a global socio-economic problem as well as a health problem, and that they have a significant impact on the development of many nations. In September 2000, the Millennium Summit of the United Nations adopted the Millennium Development Goals (Box 1), which demand a dramatic reduction in poverty, and improvements in the health of the poor. The indications are that there has been a much-needed change in political will. One-hundred-and-eighty-nine countries, including 147 Heads of State, signed the Millennium Declaration, but unless this intent is rapidly translated into a sustained effort, with vastly increased resources, the burden of infectious diseases will continue to weigh heavily. While most scientists working on infectious diseases in academia and industry shy away from the political arena, they have an important role in providing data that can lead to informed decision-making by politicians, thus helping to ensure that research into infectious diseases remains a high priority.Box 1. The Millennium Development Goals*Tabled 1Eradicate extreme poverty and hungerAchieve universal primary educationPromote gender equality and empower womenReduce child mortalityImprove maternal healthCombat HIV and AIDS, malaria, and other diseasesEnsure environmental sustainabilityDevelop a global partnership for development*For details visit: http://www.who.int/mdg/goals/en/ Open table in a new tab Tabled 1Eradicate extreme poverty and hungerAchieve universal primary educationPromote gender equality and empower womenReduce child mortalityImprove maternal healthCombat HIV and AIDS, malaria, and other diseasesEnsure environmental sustainabilityDevelop a global partnership for development*For details visit: http://www.who.int/mdg/goals/en/ Open table in a new tab A TRENDS Guide to Infectious Diseases highlights many of the difficulties that scientists face in their search to provide new tools for the diagnosis and control of infectious diseases. Importantly, the articles offer hope that, with adequate resources and the efforts of skilled personnel from academia and industry, the exploitation of recent scientific and technological advances will allow the threat of infectious diseases to be contained.