Cell engineering with microfluidic squeezing preserves functionality of primary immune cells in vivo.

The translational potential of cell-based therapies is often limited by complications related to effectively engineering and manufacturing functional cells. While the use of electroporation is widespread, the impact of electroporation on cell state and function has yet to be fully characterized. Here, we use a genome-wide approach to study optimized electroporation treatment and identify striking disruptions in the expression profiles of key functional transcripts of human T cells.

From bacterial genomes to novel antibacterial agents: discovery, characterization, and antibacterial activity of compounds that bind to HI0065 (YjeE) from Haemophilus influenzae.

As part of a fully integrated and comprehensive strategy to discover novel antibacterial agents, NMR- and mass spectrometry-based affinity selection screens were performed to identify compounds that bind to protein targets uniquely found in bacteria and encoded by genes essential for microbial viability. A biphenyl acid lead series emerged from an NMR-based screen with the Haemophilus influenzae protein HI0065, a member of a family of probable ATP-binding proteins found exclusively in eubacteria. The structure-activity relationships developed around the NMR-derived biphenyl acid lead were consistent with on-target antibacterial activity as the Staphylococcus aureus antibacterial activity of the series correlated extremely well with binding affinity to HI0065, while the correlation of binding affinity with B-cell cytotoxicity was relatively poor.

N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1-methylprop-2-ynyl}carboxy derivatives as acetyl-coA carboxylase inhibitors--improvement of cardiovascular and neurological liabilities via structural modifications.

A preliminary safety evaluation of ACC2 inhibitor 1-(S) revealed serious neurological and cardiovascular liabilities of this chemotype. A systematic structure-toxicity relationship study identified the alkyne linker as the key motif responsible for these adverse effects. Toxicogenomic studies in rats showed that 1-(R) and 1-(S) induced gene expression patterns similar to that seen with several known cardiotoxic agents such as doxorubicin.

Phenoxy thiazole derivatives as potent and selective acetyl-CoA carboxylase 2 inhibitors: Modulation of isozyme selectivity by incorporation of phenyl ring substituents.

A phenyl ring substitution strategy was employed to optimize the ACC2 potency and selectivity profiles of a recently discovered phenoxy thiazolyl series of acetyl-CoA carboxylase inhibitors. Ring substituents were shown to dramatically affect isozyme selectivity. Modifications that generally impart high levels of ACC2 selectivity (>3000-fold) while maintaining excellent ACC2 potency (IC50s approximately 9-20 nM) were identified.

The synthesis and structure-activity relationship studies of selective acetyl-CoA carboxylase inhibitors containing 4-(thiazol-5-yl)but-3-yn-2-amino motif: polar region modifications.

The structure-activity relationship study focused on the polar region of the HTS hit A-80040 (1) producing several series of potent and selective ACC2 inhibitors. The SAR suggests a compact lipophilic pocket that does not tolerate polar and ionic groups. Replacement of the hydroxyurea group with isoxazoles improves ACC2 selectivity while maintaining potency.

An ultraefficient affinity-based high-throughout screening process: application to bacterial cell wall biosynthesis enzyme MurF.

The authors describe the discovery of a new class of inhibitors to an essential Streptococcus pneumoniae cell wall biosyn-thesis enzyme, MurF, by a novel affinity screening method. The strategy involved screening very large mixtures of diverse small organic molecules against the protein target on the basis of equilibrium binding, followed by iterative ultrafiltration steps and ligand identification by mass spectrometry. Hits from any affinity-based screening method often can be relatively nonselective ligands, sometimes referred to as "nuisance" or "promiscuous" compounds.

Structure-activity relationships for a novel series of thiazolyl phenyl ether derivatives exhibiting potent and selective acetyl-CoA carboxylase 2 inhibitory activity.

Structure-activity relationships for a recently discovered thiazolyl phenyl ether series of acetyl-CoA carboxylase (ACC) inhibitors were investigated. Preliminary efforts to optimize the series through modification of the distal aryl ether moiety of the lead scaffold resulted in the identification of compounds exhibiting low-nanomolar potency and isozyme-selective ACC2 activity.

Novel antibacterial class: a series of tetracyclic derivatives.

We describe the synthesis and antibacterial activity of a series of tetracyclic naphthyridones. The members of this series act primarily via inhibition of bacterial translation and belong to the class of novel ribosome inhibitors (NRIs). In this paper we explore the structure-activity relationships (SAR) of these compounds to measure their ability both to inhibit bacterial translation and also to inhibit the growth of bacterial cells in culture.

Synthesis and structure-activity relationships of N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1- methylprop-2-ynyl}carboxy derivatives as selective acetyl-CoA carboxylase 2 inhibitors.

A structurally novel acetyl-CoA carboxylase (ACC) inhibitor is identified from high-throughput screening. A preliminary structure-activity relationship study led to the discovery of potent dual ACC1/ACC2 and ACC2 selective inhibitors against human recombinant ACC1 and ACC2. Selective ACC2 inhibitors exhibited IC50<20 nM and >1000-fold selectivity against ACC1.

Structure-based optimization of MurF inhibitors.

The D-Ala-D-Ala adding enzyme (MurF) from Streptococcus pneumoniae catalyzes the ATP-dependent formation of the UDP-MurNAc-pentapeptide, a critical component of the bacterial cell wall. MurF is a potential target for antibacterial design because it is unique to bacteria and performs an essential non-redundant function in the bacterial cell. The recent discovery and subsequent cocrystal structure determination of MurF in complex with a new class of inhibitors served as a catalyst to begin a medicinal chemistry program aimed at improving their potency.

Structure of MurF from Streptococcus pneumoniae co-crystallized with a small molecule inhibitor exhibits interdomain closure.

In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small-molecule compound collection identified several compounds for further validation as pharmaceutical leads.

Novel approach to mapping of resistance mutations in whole genomes by using restriction enzyme modulation of transformation efficiency.

Restriction enzyme modulation of transformation efficiencies (REMOTE) is a method that makes use of genome restriction maps and experimentally observed differences in transformation efficiencies of genomic DNA restriction digests to discover the location of mutations in genomes. The frequency with which digested genomic DNA from a resistant strain transforms a susceptible strain to resistance is primarily determined by the size of the fragment containing the resistance mutation and the distance of the mutation to the end of the fragment. The positions of restriction enzyme cleavage sites immediately flanking the resistance mutation define these parameters.

Synthesis and antibacterial activity of 5-methoxy- and 5-hydroxy-6-fluoro-1,8-naphthyridone-3-carboxylic acid derivatives.

A series of 5-methoxy- and 5-hydroxy-6-fluoro-1,8-naphthyridone-3-carboxylic acid derivatives were prepared and evaluated for cell-free bacterial protein synthesis inhibition and whole cell antibacterial activity. When compared to the analogous 5-hydrogen compounds, the presence of the 5-OH group negatively affects biochemical potency. However, a tolerance of the 5-methoxy group is indicated.

Mechanism of action of a novel series of naphthyridine-type ribosome inhibitors: enhancement of tRNA footprinting at the decoding site of 16S rRNA.

The novel ribosome inhibitors (NRIs) are a broad-spectrum naphthyridine class that selectively inhibits bacterial protein synthesis (P. J. Dandliker et al.

Novel inhibitors of bacterial protein synthesis: structure-activity relationships for 1,8-naphthyridine derivatives incorporating position 3 and 4 variants.

Structure-activity relationships for a recently discovered novel ribosome inhibitor (NRI) class of antibacterials were investigated. Preliminary efforts to optimize protein synthesis inhibitory activity of the series through modification of positions 3 and 4 of the naphthyridone lead template resulted in the identification of several biochemically potent analogues. A lack of corresponding whole cell antibacterial activity is thought to be a consequence of poor cellular penetration as evidenced by the enhancement of activity observed for a lead analogue tested in the presence of a cell permeabilizing agent.

Discovery of potent inhibitors of dihydroneopterin aldolase using CrystaLEAD high-throughput X-ray crystallographic screening and structure-directed lead optimization.

Potent inhibitors of 7,8-dihydroneopterin aldolase (DHNA; EC 4.1.2.

A strategy for discovery of novel broad-spectrum antibacterials using a high-throughput Streptococcus pneumoniae transcription/translation screen.

The authors report the development of a high-throughput screen for inhibitors of Streptococcus pneumoniae transcription and translation (TT) using a luciferase reporter, and the secondary assays used to determine the biochemical spectrum of activity and bacterial specificity. More than 220,000 compounds were screened in mixtures of 10 compounds per well, with 10,000 picks selected for further study. False-positive hits from inhibition of luciferase activity were an extremely common artifact.

SOS-NMR: a saturation transfer NMR-based method for determining the structures of protein-ligand complexes.

An NMR-based alternative to traditional X-ray crystallography and NMR methods for structure-based drug design is described that enables the structure determination of ligands complexed to virtually any biomolecular target regardless of size, composition, or oligomeric state. The method utilizes saturation transfer difference (STD) NMR spectroscopy performed on a ligand complexed to a series of target samples that have been deuterated everywhere except for specific amino acid types. In this way, the amino acid composition of the ligand-binding site can be defined, and, given the three-dimensional structure of the protein target, the three-dimensional structure of the protein-ligand complex can be determined.

Structure-activity relationships of novel potent MurF inhibitors.

A novel class of MurF inhibitors was discovered and structure-activity relationship studies have led to several potent compounds with IC(50)=22 approximately 70 nM. Unfortunately, none of these potent MurF inhibitors exhibited significant antibacterial activity even in the presence of bacterial cell permeabilizers.

Novel antibacterial class.

We report the discovery and characterization of a novel ribosome inhibitor (NRI) class that exhibits selective and broad-spectrum antibacterial activity. Compounds in this class inhibit growth of many gram-positive and gram-negative bacteria, including the common respiratory pathogens Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Moraxella catarrhalis, and are nontoxic to human cell lines. The first NRI was discovered in a high-throughput screen designed to identify inhibitors of cell-free translation in extracts from S.

A strategy for high-throughput assay development using leads derived from nuclear magnetic resonance-based screening.

A strategy is described for the development of high-throughput screening assays against targets of unknown function that involves the use of nuclear magnetic resonance (NMR) spectroscopy. Using this approach, molecules that bind to the protein target are identified from an NMR-based screen of a library of substrates, cofactors, and other compounds that are known to bind to many proteins and enzymes. Once a ligand has been discovered, a fluorescent or radiolabeled analog of the ligand is synthesized that can be used in a high-throughput screen.

Synthesis and SAR evaluation of oxadiazolopyrazines as selective Haemophilus influenzae antibacterial agents.

The parallel synthesis and antibacterial activity of 5-hydroxy[1,2,5] oxadiazolo[3,4-b]pyrazines is reported. The compounds were synthesized by condensing diaminofurazan with alpha-keto acids to give a variety of aryl-substituted analogues. Halogenated phenyl groups at C-6 give rise to the greatest Haemophilus influenzae antibacterial activity.

Antibacterial drug discovery in the post-genomics era.

Antibacterial research has evolved dramatically over the past five decades. Early work relied on serendipity of finding drug-like molecules, usually natural products that had desirable antibacterial and nontoxic properties without regard to mechanism of action. In the past decade, however, significant technological advances in the fields of genomics, molecular biology, high-throughput screening, and structural biochemistry have led to a fundamentally new paradigm in the pursuit of novel antibacterial agents.

Complex gel permeation assays for screening combinatorial libraries.

Gel permeation methods have been commonly used to screen combinatorial libraries synthesized on a solid support. We report here three screens of combinatorial libraries using gel permeation assays. These include a simple enzymatic assay to identify inhibitors of the influenza enzyme neuraminidase, and two more complex assays designed to screen for inhibitors of the interleukin-8 (IL-8)-IL-8 receptor and the urokinase-urokinase receptor interactions, respectively.

Development of a p38 Kinase Binding Assay for High Throughput Screening.

p38 is a member of the mitogen-activated protein kinase (MAPK) family of serine/threonine kinases, which is activated by cellular stressors and has been shown to be a critical enzyme in the synthesis and action of proinflammatory cytokines, tumor necrosis factor-a (TNF-alpha) and interleukin-1beta (IL-1beta). A group of pyridinyl imidazole compounds such as SB202190 have been identified as selective inhibitors of p38 that bind directly to the ATP pocket of the enzyme. These compounds inhibit the p38 kinase activity, block TNF-alpha and IL-1beta secretion both in vivo and in vitro and are found to be effective in animal models of arthritis, bone resorption, and endotoxin shock.