Robert C. Gadwood

Oxazolidinone Antibiotics Target the P Site on Escherichia coli Ribosomes

ABSTRACT 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 Halobacteriumhalobium, Staphylococcusaureus, and Escherichiacoli 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.

Development of an in vitro primary screen for skin depigmentation and antimelanoma agents

An in vitro cell culture assay was developed to identify inhibitors of melanogenesis and agents which produce cytostatic or cytotoxic effects specifically in melanocytes. A total of 50 compounds related to tyrosine, dihydroxyphenylalanine, and hydroquinone (HQ) were tested in vitro in order to determine their effects upon a murine melanocyte cell line, Mel-Ab, that produces copious amounts of melanin in culture. The agents that demonstrated an inhibition of growth or pigment production by 50% (IC50) at < 100 micrograms/ml were considered active. The cytotoxicity of melanocyte-active compounds were also tested in vitro on a control nonmelanocyte cell line (HT 1080), using a simple crystal violet staining method to quantitate adherent cell number after treatment. The cell culture assay was validated with known potent melanocyte cytotoxic agents, including HQ and 4-S-cysteaminylphenol (4-S-CAP). Although most cytotoxic chemicals were nonspecific in this primary screen (i.e. killing both Mel-Ab and HT-1080 cells), several of the compounds tested exhibited high melanocyte-specific cytotoxicity, similar to HQ and 4-S-CAP. Potentially these compounds may be useful as either antimelanoma or skin depigmentation agents. All of the compounds identified as active in this primary screen were cytotoxic or cytostatic to melanocytes, except for the methyl ester of gentisic acid, which uniquely inhibited the de novo synthesis of melanin without cytotoxicity.

Design, Synthesis, and Evaluation of Novel Oxazolidinone Antibacterial Agents Active Against Multidrug-Resistant Bacteria

Throughout the human experience, diseases caused by pathogenic bacteria have exerted an enormous negative impact on society. Today, for example, approximately one third of the world’s population is infected with Mycobacterium tuberculosis and tuberculosis remains the leading cause of death in the world from infectious disease (Bloom, 1994). The emergence of effective antibacterial agents, from penicillin to more contemporary drugs, was initially thought to mark an end to the burden of microbial disease. However, subsequent events have illustrated the resiliency of bacteria to environmental pressures, including the threat of antibacterial agents.

Chapter 20. Pathogenesis and Treatment of Alopecias

Publisher Summary This chapter discusses the pathogenesis and treatment of alopecia areata and androgenetic alopecia; that are two common nonscarring alopecias because of fundamental changes in the functioning of the hair follicles. Both alopecia areata and androgenetic alopecia involve perturbation of the normal hair growth cycle. Alopecia areata and androgenetic alopecia are characterized by a higher percentage of telogen hair follicles and loss of hair from those follicles. Corticosteroids have frequently been used for the treatment of alopecia areata. Topical application and intralesional injection appear to be the safest and most efficacious routes of administration. Alopecia areata has also been treated with PUVA therapy that combines administration of topical or oral psoralens with UVA irradiation. Inosiplex and thymopentin are immunostimulants that have been used to treat alopecia areata. Androgenetic alopecia apparently involves undefined genetic factors that have been variously hypothesized to be levated androgen metabolism, decreased levels of sex hormone binding globulins, or increased androgen binding in target tissues. In females, oral spironolactone, cyproterone acetate, and cimetidine have been reported to result in some hair regrowth, presumably because of the antiandrogenic properties of these compounds. Other agents that are not involved with androgenic pathways have shown hair growth stimulating properties in androgenetic alopecia. Topical minoxidil has received by far the greatest amount of study for the treatment of androgenetic alopecia, and several recent reviews have appeared that summarize the results. Minoxidil has shown hair growth stimulating activity in both alopecia areata and androgenetic alopecia. Further development of hair growth promoters is critically dependent on the discovery of a rapid and accurate assay system that has some known relationship to alopecia in humans. Future areas of research should focus on the development of agents with greater efficacy and safety.

Chapter 12. Progress in the oxazolidinone antibacterials

Publisher Summary This chapter describes the progress in oxazolidinone antibacterials, which are a class of antibacterial agents with activity against a broad spectrum of gram-positive pathogens including staphylococci, streptococci, and enterococci. Because they are totally synthetic and mechanistically novel, the oxazolidinones lack cross-resistance to every clinically significant resistance mechanism tested to date. These antibacterial agents are effective against drug resistant gram-positive bacteria including methicillin resistant S. aureus (MRSA), vancomycin resistant enterococci (VRE), and penicillin resistant S. pneumoniae (PRSP). The oxazolidinone structure is relatively simple and allows for diverse synthetic modification. It inhibits protein synthesis in actively growing bacteria. Mechanism of action studies support oxazolidinone binding to the bacterial 50S ribosomal subunit and inhibition of formation of the 70S ribosomal initiation complex. Cell-free studies show that linezolid and eperezolid are potent inhibitors of the bacterial transcription/translation system. The chapter reviews some new oxazolidinone analogues. An overview of linezolid is presented and mechanism of action is discussed. Concepts related to A-ring, B-ring, and C-ring modifications are also explained.

Structure-Activity Relationship of Flavin Analogues That Target the Flavin Mononucleotide Riboswitch.

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.