Colin W. G. Fishwick

Mutations in 15-hydroxyprostaglandin dehydrogenase cause primary hypertrophic osteoarthropathy

Digital clubbing, recognized by Hippocrates in the fifth century BC, is the outward hallmark of pulmonary hypertrophic osteoarthropathy, a clinical constellation that develops secondary to various acquired diseases, especially intrathoracic neoplasm. The pathogenesis of clubbing and hypertrophic osteoarthropathy has hitherto been poorly understood, but a clinically indistinguishable primary (idiopathic) form of hypertrophic osteoarthropathy (PHO) is recognized. This familial disorder can cause diagnostic confusion, as well as significant disability. By autozygosity methods, we mapped PHO to chromosome 4q33–q34 and identified mutations in HPGD, encoding 15-hydroxyprostaglandin dehydrogenase, the main enzyme of prostaglandin degradation. Homozygous individuals develop PHO secondary to chronically elevated prostaglandin E2 levels. Heterozygous relatives also show milder biochemical and clinical manifestations. These findings not only suggest therapies for PHO, but also imply that clubbing secondary to other pathologies may be prostaglandin mediated. Testing for HPGD mutations and biochemical testing for HPGD deficiency in patients with unexplained clubbing might help to obviate extensive searches for occult pathology.

Structure-based discovery of antibacterial drugs

The modern era of antibacterial chemotherapy began in the 1930s, and the next four decades saw the discovery of almost all the major classes of antibacterial agents that are currently in use. However, bacterial resistance to many of these drugs is becoming an increasing problem. As such, the discovery of drugs with novel modes of action will be vital to meet the threats created by the emergence of resistance. Success in discovering inhibitors using high-throughput screening of chemical libraries is rare. In this Review we explore the exciting opportunities for antibacterial-drug discovery arising from structure-based drug design.

Molecular Genetic and Structural Modeling Studies of Staphylococcus aureus RNA Polymerase and the Fitness of Rifampin Resistance Genotypes in Relation to Clinical Prevalence

ABSTRACT The adaptive and further evolutionary responses of Staphylococcus aureus to selection pressure with the antibiotic rifampin have not been explored in detail. We now present a detailed analysis of these systems. The use of rifampin for the chemotherapy of infections caused by S. aureus has resulted in the selection of mutants with alterations within the β subunit of the target enzyme, RNA polymerase. Using a new collection of strains, we have identified numerous novel mutations in the β subunits of both clinical and in vitro-derived resistant strains and established that additional, undefined mechanisms contribute to expression of rifampin resistance in clinical isolates of S. aureus. The fitness costs associated with rifampin resistance genotypes were found to have a significant influence on their clinical prevalence, with the most common clinical genotype (H481N, S529L) exhibiting no fitness cost in vitro. Intragenic mutations which compensate for the fitness costs associated with rifampin resistance in clinical strains of S. aureus were identified for the first time. Structural explanations for rifampin resistance and the loss of fitness were obtained by molecular modeling of mutated RNA polymerase enzymes.

Analysis of Mupirocin Resistance and Fitness in Staphylococcus aureus by Molecular Genetic and Structural Modeling Techniques

ABSTRACT Chromosomal resistance to mupirocin in clinical isolates of Staphylococcus aureus arises from V588F or V631F mutations in isoleucyl-tRNA synthetase (IRS). Whether these are the only IRS mutations that confer mupirocin resistance or simply those that survive in the clinic is unknown. Mupirocin-resistant mutants of S. aureus 8325-4 were therefore generated to examine their ileS genotypes and the in vitro and in vivo fitness costs associated with them before and after compensatory evolution. Most spontaneous first-step mupirocin-resistant mutants carried V588F or V631F mutations in IRS, but a new mutation (G593V) was also identified. Second-step mutants carried combinations of previously identified IRS mutations (e.g., V588F/V631F and G593V/V631F), but additional combinations also occurred involving novel mutations (R816C, H67Q, and F563L). First-step mupirocin-resistant mutants were not associated with substantial fitness costs, a finding that is consistent with the occurrence of V588F or V631F mutations in the IRS of clinical strains. Second-step mutants were unfit, but fitness could be restored by subculture in the absence of mupirocin. In most cases, this was the result of compensatory mutations that also suppressed mupirocin resistance (e.g., A196V, E190K, and E195K), despite retention of the original mutations conferring resistance. Structural explanations for mupirocin resistance and loss of fitness were obtained by molecular modeling of mutated IRS enzymes, which provided data on mupirocin binding and interaction with the isoleucyl-AMP reactive intermediate.

RNA Polymerase Inhibitors with Activity against Rifampin-Resistant Mutants of Staphylococcus aureus

ABSTRACT A collection of rifampin-resistant mutants of Staphylococcus aureus with characterized RNA polymerase β-subunit (rpoB) gene mutations was cross-screened against a number of other RNA polymerase inhibitors to correlate susceptibility with specific rpoB genotypes. The rpoB mutants were cross-resistant to streptolydigin and sorangicin A. In contrast, thiolutin, holomycin, corallopyronin A, and ripostatin A retained activity against the rpoB mutants. The second group of inhibitors may be of interest as drug development candidates.

Self-assembling β-Sheet Tape Forming Peptides

Biological proteins have intrinsically the ability to self-assemble, and this has been implicated in pathological situations called amyloid diseases. Conversely understanding protein self-assembly and how to control it can open up the route to new nanodevices and nanostructured materials for a wide range of applications in medicine, chemical industry and nanotechnology. Biological peptides and proteins have complex chemical structure and conformation. This makes it difficult to decipher the fundamental principles that drive their self-assembling behaviours. Here we review our work on the self-assembly of simple de novo peptides in solution. These peptides are designed so that: (i) the chemical complexity of the primary structure and (ii) the conformational complexity are both kept to a minimum. Each peptide adopts an extended β-strand conformation in solution and these β-strands self-assemble in one dimension to form elongated tapes as well as higher order aggregates with pure antiparallel β-sheet structure, without the presence of any other conformations such as turns, loops, α-helices or random coils. Experimental data of the self-assembling properties are fitted with an appropriate theoretical model to build a quantitative relationship between peptide primary structure and self-assembly. These simple systems provide us with the opportunity to reveal the generic properties of the pure β-sheet structures and expose the underlying physicochemical principles that drive the self-assembling behaviour of this biological motif.

Determinants of Hepatitis C Virus p7 Ion Channel Function and Drug Sensitivity Identified In Vitro

ABSTRACT Hepatitis C virus (HCV) chronically infects 170 million individuals, causing severe liver disease. Although antiviral chemotherapy exists, the current regimen is ineffective in 50% of cases due to high levels of innate virus resistance. New, virus-specific therapies are forthcoming although their development has been slow and they are few in number, driving the search for new drug targets. The HCV p7 protein forms an ion channel in vitro and is critical for the secretion of infectious virus. p7 displays sensitivity to several classes of compounds, making it an attractive drug target. We recently demonstrated that p7 compound sensitivity varies according to viral genotype, yet little is known of the residues within p7 responsible for channel activity or drug interactions. Here, we have employed a liposome-based assay for p7 channel function to investigate the genetic basis for compound sensitivity. We demonstrate using chimeric p7 proteins that neither the two trans-membrane helices nor the p7 basic loop individually determines compound sensitivity. Using point mutation analysis, we identify amino acids important for channel function and demonstrate that null mutants exert a dominant negative effect over wild-type protein. We show that, of the three hydrophilic regions within the amino-terminal trans-membrane helix, only the conserved histidine at position 17 is important for genotype 1b p7 channel activity. Mutations predicted to play a structural role affect both channel function and oligomerization kinetics. Lastly, we identify a region at the p7 carboxy terminus which may act as a specific sensitivity determinant for the drug amantadine.

Structure-based design, synthesis, and characterization of inhibitors of human and Plasmodium falciparum dihydroorotate dehydrogenases

Pyrimidine biosynthesis is an attractive drug target in a variety of organisms, including humans and the malaria parasite Plasmodium falciparum. Dihydroorotate dehydrogenase, an enzyme catalyzing the only redox reaction of the pyrimidine biosynthesis pathway, is a well-characterized target for chemotherapeutical intervention. In this study, we have applied SPROUT-LeadOpt, a software package for structure-based drug discovery and lead optimization, to improve the binding of the active metabolite of the anti-inflammatory drug leflunomide to the target cavities of the P. falciparum and human dihydroorotate dehydrogenases. Following synthesis of a library of compounds based upon the SPROUT-optimized molecular scaffolds, a series of inhibitors generally showing good inhibitory activity was obtained, in keeping with the SPROUT-LeadOpt predictions. Furthermore, cocrystal structures of five of these SPROUT-designed inhibitors bound in the ubiquinone binding cavity of the human dihydroorotate dehydrogenase are also analyzed.

Linezolid and Tiamulin Cross-Resistance in Staphylococcus aureus Mediated by Point Mutations in the Peptidyl Transferase Center

ABSTRACT Oxazolidinone and pleuromutilin antibiotics are currently used in the treatment of staphylococcal infections. Although both antibiotics inhibit protein synthesis and have overlapping binding regions on 23S rRNA, the potential for cross-resistance between the two classes through target site mutations has not been thoroughly examined. Mutants of Staphylococcus aureus resistant to linezolid were selected and found to exhibit cross-resistance to tiamulin, a member of the pleuromutilin class of antibiotics. However, resistance was unidirectional because mutants of S. aureus selected for resistance to tiamulin did not exhibit cross-resistance to linezolid. This contrasts with the recently described PhLOPSA phenotype, which confers resistance to both oxazolidinones and pleuromutilins. The genotypes responsible for the phenotypes we observed were examined. Selection with tiamulin resulted in recovery of mutants with changes in the single-copy rplC gene (Gly155Arg, Ser158Leu, or Arg149Ser), whereas selection with linezolid led to recovery of mutants with changes (G2576U in 23S rRNA) in all five copies of the multicopy operon rrn. In contrast, cross-resistance to linezolid was exhibited by tiamulin-resistant mutants generated in a single-copy rrn knockout strains of Escherichia coli, illustrating that the copy number of 23S rRNA is the limiting factor in the selection of 23S rRNA tiamulin-resistant mutants. The interactions of linezolid and tiamulin with the ribosome were modeled to seek explanations for resistance to both classes in the 23S rRNA mutants and the lack of cross-resistance between tiamulin and linezolid following mutation in rplC.

High-Risk Human Papillomavirus E5 Oncoprotein Displays Channel-Forming Activity Sensitive to Small-Molecule Inhibitors

ABSTRACT High-risk human papillomavirus type 16 (HPV16) is the primary causative agent of cervical cancer and therefore is responsible for significant morbidity and mortality worldwide. Cellular transformation is mediated directly by the expression of viral oncogenes, the least characterized of which, E5, subverts cellular proliferation and immune recognition processes. Despite a growing catalogue of E5-specific host interactions, little is understood regarding the molecular basis of its function. Here we describe a novel function for HPV16 E5 as an oligomeric channel-forming protein, placing it within the virus-encoded “viroporin” family. The development of a novel recombinant E5 expression system showed that E5 formed oligomeric assemblies of a defined luminal diameter and stoichiometry in membranous environments and that such channels mediated fluorescent dye release from liposomes. Hexameric E5 channel stoichiometry was suggested by native PAGE studies. In lieu of high-resolution structural information, established de novo molecular modeling and design methods permitted the development of the first specific small-molecule E5 inhibitor, capable of both abrogating channel activity in vitro and reducing E5-mediated effects on cell signaling pathways. The identification of channel activity should enhance the future understanding of the physiological function of E5 and could represent an important target for antiviral intervention.