ABSTRACT
The flavin mononucleotide (FMN) riboswitch is an emerging target for the development of novel RNA-targeting antibiotics. We previously discovered an FMN derivative, 5FDQD, that protects mice against diarrhea-causing Clostridium difficile bacteria. Here, we present the structure-based drug design strategy that led to the discovery of this fluoro-phenyl derivative with antibacterial properties. This approach involved the following stages: (1) structural analysis of all available free and bound FMN riboswitch structures; (2) design, synthesis, and purification of derivatives; (3) in vitro testing for productive binding using two chemical probing methods; (4) in vitro transcription termination assays; and (5) resolution of the crystal structures of the FMN riboswitch in complex with the most mature candidates. In the process, we delineated principles for productive binding to this riboswitch, thereby demonstrating the effectiveness of a coordinated structure-guided approach to designing drugs against RNA.
Subject(s)
Anti-Bacterial Agents/pharmacology , Flavin Mononucleotide/pharmacology , Quinoxalines/pharmacology , RNA, Bacterial/antagonists & inhibitors , Riboswitch , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Bacteria/drug effects , Base Sequence , Binding Sites , Drug Design , Flavin Mononucleotide/chemical synthesis , Flavin Mononucleotide/chemistry , Ligands , Molecular Structure , Quinoxalines/chemical synthesis , Quinoxalines/chemistry , RNA, Bacterial/genetics , Structure-Activity RelationshipABSTRACT
An environmentally benign surfactant (TPGS-750-M), a diester composed of racemic α-tocopherol, MPEG-750, and succinic acid, has been designed and readily prepared as an effective nanomicelle-forming species for general use in metal-catalyzed cross-coupling reactions in water. Several "name" reactions, including Heck, Suzuki-Miyaura, Sonogashira, and Negishi-like couplings, have been studied using this technology, as have aminations, C-H activations, and olefin metathesis reactions. Physical data in the form of DLS and cryo-TEM measurements suggest that particle size and shape are key elements in achieving high levels of conversion and, hence, good isolated yields of products. This new amphiphile will soon be commercially available.
Subject(s)
Metals/chemistry , Succinates/chemistry , Temperature , Vitamin E/analogs & derivatives , Water/chemistry , Catalysis , Hydrophobic and Hydrophilic Interactions , Micelles , Polyethylene Glycols , Sulfonamides/chemistry , Surface-Active Agents/chemistry , Thiadiazoles/chemistry , Vitamin E/chemistryABSTRACT
Tumor protein 53 (p53) is a critical regulator of cell cycle and apoptosis that is frequently disabled in human tumors. In many tumor types, p53 is deleted or mutated, but in others p53 is inactivated by overexpression or amplification of its negative regulator mouse double minute 2 (MDM2). A high-throughput screening effort identified 6,7-bis(4-bromophenyl)-7,12-dihydro-6H-chromeno[4,3-d][1,2,4]triazolo[1,5-a]pyrimidine as a potent inhibitor of the MDM2-p53 protein-protein interaction. This screening hit was found to be chemically unstable and difficult to handle due to poor DMSO solubility. Co-crystallization with the target protein helped to direct further optimization and provided a tractable lead series of novel MDM2-p53 inhibitors. In cellular assays, these compounds were shown to upregulate p53 protein levels and p53 signaling and to cause p53-dependent inhibition of proliferation and apoptosis.
Subject(s)
Drug Discovery , Proto-Oncogene Proteins c-mdm2/metabolism , Pyrimidines/chemistry , Pyrimidines/pharmacology , Tumor Suppressor Protein p53/metabolism , Apoptosis/drug effects , Cell Proliferation/drug effects , HCT116 Cells , Humans , Inhibitory Concentration 50 , Models, Molecular , Molecular Conformation , Protein Binding/drug effects , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Oxazolidinones possessing a C-5 carboxamide functionality (reverse amides) represent a new series of compounds that block bacterial protein synthesis. These reverse amides also exhibited less potency against monoamine oxidase (MAO) enzymes and thus possess less potential for the side effects associated with MAO inhibition. The title compound (14) showed reduced in vivo myelotoxicity compared to linezolid in a 14-day safety study in rats, potent in vivo efficacy in murine systemic infection models, and excellent pharmacokinetic properties.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Cyclic S-Oxides/chemical synthesis , Oxazolidinones/chemical synthesis , Acetamides/pharmacology , Administration, Oral , Animals , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/toxicity , Biological Availability , Cyclic S-Oxides/pharmacology , Cyclic S-Oxides/toxicity , Dogs , Drug Resistance, Bacterial , Female , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Injections, Intravenous , Linezolid , Male , Mice , Microbial Sensitivity Tests , Monoamine Oxidase Inhibitors/chemical synthesis , Monoamine Oxidase Inhibitors/pharmacology , Monoamine Oxidase Inhibitors/toxicity , Oxazolidinones/pharmacology , Oxazolidinones/toxicity , Rats , Rats, Sprague-Dawley , Staphylococcal Infections/drug therapy , Staphylococcus aureus , Streptococcal Infections/drug therapy , Streptococcus pyogenes , Structure-Activity RelationshipABSTRACT
The oxazolidinones are one of the newest classes of antibiotics. They inhibit bacterial growth by interfering with protein synthesis. The mechanism of oxazolidinone action and the precise location of the drug binding site in the ribosome are unknown. We used a panel of photoreactive derivatives to identify the site of action of oxazolidinones in the ribosomes of bacterial and human cells. The in vivo crosslinking data were used to model the position of the oxazolidinone molecule within its binding site in the peptidyl transferase center (PTC). Oxazolidinones interact with the A site of the bacterial ribosome where they should interfere with the placement of the aminoacyl-tRNA. In human cells, oxazolidinones were crosslinked to rRNA in the PTC of mitochondrial, but not cytoplasmic, ribosomes. Interaction of oxazolidinones with the mitochondrial ribosomes provides a structural basis for the inhibition of mitochondrial protein synthesis, which is linked to clinical side effects associated with oxazolidinone therapy.
Subject(s)
Mitochondria/drug effects , Oxazolidinones/pharmacology , Peptidyl Transferases/drug effects , Protein Synthesis Inhibitors/pharmacology , RNA, Ribosomal/drug effects , Software , Acetamides , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Binding Sites/drug effects , Cross-Linking Reagents/chemistry , Cross-Linking Reagents/pharmacology , Cytoplasm/drug effects , Cytoplasm/enzymology , Drug Resistance/genetics , Escherichia coli/drug effects , Escherichia coli/enzymology , Humans , Linezolid , Mitochondria/enzymology , Models, Molecular , Molecular Structure , Mutation/genetics , Oxazolidinones/chemistry , Peptidyl Transferases/metabolism , Protein Synthesis Inhibitors/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Ribosomal/metabolism , RNA, Ribosomal, 23S , RNA, Transfer, Amino Acyl/antagonists & inhibitors , RNA, Transfer, Amino Acyl/metabolism , Staining and Labeling , Staphylococcus aureus/drug effects , Staphylococcus aureus/enzymologyABSTRACT
Replacement of the morpholine C-ring of linezolid 1 with a 1,3,4-thiadiazolyl ring leads to oxazolidinone analogues 5 having potent antibacterial activity against both gram-positive and gram-negative organisms. Conversion of the C5 acetamide group to a thioacetamide further increases the potency of these compounds.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Gram-Positive Bacteria/drug effects , Oxazolidinones/chemical synthesis , Oxazolidinones/pharmacology , Phenols/chemistry , Thiadiazoles/chemistry , Acetamides/chemical synthesis , Acetamides/chemistry , Acetamides/pharmacology , Animals , Anti-Bacterial Agents/chemistry , Linezolid , Microbial Sensitivity Tests , Oxazolidinones/chemistry , Rats , Structure-Activity Relationship , Thioacetamide/chemical synthesis , Thioacetamide/chemistry , Thioacetamide/pharmacologyABSTRACT
Combinatorial libraries of N-acylated 5-(S)-aminomethyloxazolidinone derivatives of S-oxide and S,S-dioxide tetrahydro-4(2H)-thiopyranyl and thiomorpholine phenyloxazolidinone series have been synthesized on a solid phase and evaluated for antimicrobial activity. Several novel potent leads have been identified, including orally active oxazolidinones with enhanced activity against respiratory tract infection pathogens Haemophilus influenzae and Moraxella catarrhalis.
Subject(s)
Anti-Bacterial Agents/pharmacokinetics , Haemophilus influenzae/drug effects , Moraxella catarrhalis/drug effects , Morpholines/chemistry , Oxazolidinones/pharmacokinetics , Oxides/chemistry , Oxygen Compounds/pharmacokinetics , Animals , Anti-Bacterial Agents/administration & dosage , Biological Availability , Combinatorial Chemistry Techniques , Haemophilus Infections/microbiology , Lipid Metabolism , Male , Microbial Sensitivity Tests , Moraxellaceae Infections/microbiology , Oxazolidinones/administration & dosage , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
Oxazolidinone antibiotics, an important new class of synthetic antibacterials, inhibit protein synthesis by interfering with ribosomal function. The exact site and mechanism of oxazolidinone action has not been elucidated. Although genetic data pointed to the ribosomal peptidyltransferase as the primary site of drug action, some biochemical studies conducted in vitro suggested interaction with different regions of the ribosome. These inconsistent observations obtained in vivo and in vitro have complicated the understanding of oxazolidinone action. To localize the site of oxazolidinone action in the living cell, we have cross-linked a photoactive drug analog to its target in intact, actively growing Staphylococcus aureus. The oxazolidinone cross-linked specifically to 23 S rRNA, tRNA, and two polypeptides. The site of cross-linking to 23 S rRNA was mapped to the universally conserved A-2602. Polypeptides cross-linked were the ribosomal protein L27, whose N terminus may reach the peptidyltransferase center, and LepA, a protein homologous to translation factors. Only ribosome-associated LepA, but not free protein, was cross-linked, indicating that LepA was cross-linked by the ribosome-bound antibiotic. The evidence suggests that a specific oxazolidinone binding site is formed in the translating ribosome in the immediate vicinity of the peptidyltransferase center.
Subject(s)
Anti-Bacterial Agents/pharmacology , Cross-Linking Reagents/pharmacology , Oxazolidinones/pharmacology , Protein Synthesis Inhibitors/pharmacology , Amino Acid Sequence , Binding Sites , Electrophoresis, Polyacrylamide Gel , Models, Chemical , Models, Genetic , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Peptides/chemistry , Protein Binding , Protein Conformation , Protein Structure, Tertiary , RNA/metabolism , RNA, Ribosomal, 23S/metabolism , RNA, Transfer/metabolism , Staphylococcus aureus/metabolism , Transcription Factors/chemistryABSTRACT
The oxazolidinones are a novel class of antimicrobial agents that target protein synthesis in a wide spectrum of gram-positive and anaerobic bacteria. The oxazolidinone PNU-100766 (linezolid) inhibits the binding of fMet-tRNA to 70S ribosomes. Mutations to oxazolidinone resistance in Halobacterium halobium, Staphylococcus aureus, and Escherichia coli map at or near domain V of the 23S rRNA, suggesting that the oxazolidinones may target the peptidyl transferase region responsible for binding fMet-tRNA. This study demonstrates that the potency of oxazolidinones corresponds to increased inhibition of fMet-tRNA binding. The inhibition of fMet-tRNA binding is competitive with respect to the fMet-tRNA concentration, suggesting that the P site is affected. The fMet-tRNA reacts with puromycin to form peptide bonds in the presence of elongation factor P (EF-P), which is needed for optimum specificity and efficiency of peptide bond synthesis. Oxazolidinone inhibition of the P site was evaluated by first binding fMet-tRNA to the A site, followed by translocation to the P site with EF-G. All three of the oxazolidinones used in this study inhibited translocation of fMet-tRNA. We propose that the oxazolidinones target the ribosomal P site and pleiotropically affect fMet-tRNA binding, EF-P stimulated synthesis of peptide bonds, and, most markedly, EF-G-mediated translocation of fMet-tRNA into the P site.
