Cadazolid is a new oxazolidinone-type antibiotic currently in clinical development for the treatment of Clostridium difficile-associated diarrhea. Here, we report investigations on the mode of action and the propensity for spontaneous resistance development in C. difficile strains. Macromolecular labeling experiments indicated that cadazolid acts as a potent inhibitor of protein synthesis, while inhibition of DNA synthesis was also observed, albeit only at substantially higher concentrations of the drug. Strong inhibition of protein synthesis was also obtained in strains resistant to linezolid, in agreement with low MICs against such strains. Inhibition of protein synthesis was confirmed in coupled transcription/translation assays using extracts from different C. difficile strains, including strains resistant to linezolid, while inhibitory effects in DNA topoisomerase assays were weak or not detectable under the assay conditions. Spontaneous resistance frequencies of cadazolid were low in all strains tested (generally <10(-10) at 2× to 4× the MIC), and in multiple-passage experiments (up to 13 passages) MICs did not significantly increase. Furthermore, no cross-resistance was observed, as cadazolid retained potent activity against strains resistant or nonsusceptible to linezolid, fluoroquinolones, and the new antibiotic fidaxomicin. In conclusion, the data presented here indicate that cadazolid acts primarily by inhibition of protein synthesis, with weak inhibition of DNA synthesis as a potential second mode of action, and suggest a low potential for spontaneous resistance development.
BAL0030543, BAL0030544, and BAL0030545 are dihydrophthalazine inhibitors with in vitro potency against gram-positive pathogens. The MIC(50)s for methicillin (meticillin)-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, hetero-vancomycin-resistant Staphylococcus aureus, and vancomycin-resistant Staphylococcus aureus (VISA) range from 0.015 to 0.25 microg/ml (MIC(90)s < or = 0.5 microg/ml). MIC(50)s for beta-hemolytic streptococci range from 0.03 to 0.06 microg/ml, MIC(50)s for Streptococcus pneumoniae range from 0.06 to 0.12 microg/ml, MIC(50)s for Listeria monocytogenes range from 0.015 to 0.06 microg/ml, and MIC(50)s for Streptococcus mitis are < or = 0.015 microg/ml. These three dihydrophthalazine antifolates have improved potency compared to that of trimethoprim and activity against gram-positive pathogens resistant to other drug classes. (This work was presented in part at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2008.).
The gene for the insertion sequence (IS) 30 transposase is placed under the control of the tac promoter, and large quantities of transposase are expressed upon induction. The resulting protein precipitates inside the Escherichia coli cells in the form of inclusion bodies which, upon cell lysis, cannot be dissolved under nondenaturing conditions. In contrast, the N-terminal third of the transposase, a 17-kDa protein produced by a truncated gene, can be purified and is able to interact site specifically with the ends of the IS30 element. In DNase I footprint experiments, regions of 26 nucleotides on one DNA strand and 19 nucleotides on the other strand at either end of the element are protected from nuclease digestion. It is concluded that a functional DNA-binding domain can be formed by expression of only one-third of the complete IS30 transposase. Sequence comparison shows a homology of the IS30 ends to the ends of IS4351 and to the L1 end of bacteriophage Mu.
Technological advances in the field of mass spectrometry (MS) are providing powerful analytical means for the investigation of proteins and peptides. In the present work we have used pneumatically assisted electrospray (ion‐spray) MS for the biochemical characterization of recombinant human catechol O ‐methyltransferase (rhCOMT). hCOMT could be expressed in Escherichia coli in large quantities but in two forms of different size, both enzymically active. Electrospray MS analysis showed that the smaller rhCOMT protein had a molecular mass of 24352±2Da, corresponding to the calculated value for native hCOMT (without the initiating methionine), whereas that mass of the larger protein was of 25775±4Da. To investigate the molecular differences between the two proteins, they were digested with trypsin and the peptides produced analysed by electrospray MS. Neither protein apparently contained disulfide bridges and the observed molecular masses of the tryptic peptides corresponded to the calculated values. It was possible to determine, however, that the larger protein contained an extended C‐terminus with the correct sequence GPGSEAGP plus an additional stretch, EDLR. This C‐terminal extension resulted from ribosomal frameshift at the codon of the last proline (CCC, rare codon in prokaryotes). In fact, rightward frameshifting would produce a hCOMT form with an additional stretch of 11 amino acid (EDLRSHHHHHH) and the calculated molecular mass of this protein (25773.5Da) is in good agreement with our experimental result. The differential reactivity of the cysteine residues of the correct rhCOMT enzyme, in the presence and in the absence of S ‐adenosyl‐L‐methionine (AdoMet) and MgCl 2 , was also studied. 5‐Iodoacetamido fluorescein (5‐IAF) was used as thiol‐modifying reagent. Under the conditions used, 5‐IAF rapidly inactivated rhCOMT but the presence of AdoMet and MgCl 2 partially protected it from inactivation. The 5‐IAF‐labeled tryptic peptides were separated by HPLC and then submitted to electrospray MS and tandem MS. Several cysteine residues appeared to be readily available to chemical modification by 5‐IAF. Incorporation of 5‐IAF occurred to a larger extent into Cys32, Cys68, Cys94 and Cys172. AdoMet and MgCl 2 markedly reduced the label incorporation into Cys68 and Cys94, therefore suggesting that these residues belong to a region at or near the binding site of AdoMet.
Ribosomal protein synthesis is an important target in antibacterial drug discovery. Numerous natural products have served as starting points for the development of antibiotics. We report here the total synthesis of xenocoumacin 1, a natural product that binds to 16S ribosomal RNA at a highly conserved region, as well as analogues thereof. Preliminary structure-activity relationship studies were aimed at understanding and modulating the selectivity between eukaryotic and prokaryotic ribosomes. Modifications were mainly tolerated in the aromatic region. Whole-cell activity against Gram-negative bacteria is limited by efflux and penetration, as demonstrated in genetically modified strains of E. coli. Analogues with high selectivity for eukaryotic ribosomes were identified, but it was not possible to obtain inhibitors selective for bacterial protein synthesis. Achieving high selectivity (albeit not the desired one) was thus possible despite the high homology between eukaryotic and prokaryotic ribosomes in the binding region.
We have evaluated 3 molecularly defined polypeptides encoded by encloned Plasmodium falciparum genes for their ability to serve as antigens for detecting antimalaria antibodies. The recombinant proteins correspond to (i) a conserved part of 190–200 kDa schizont merozoite surface component, (ii) the carboxy terminal part of the P. falciparum aldolase, and (iii) the 5·1 antigen. Antibodies were detected using enzyme-linked immunosorbent assays (ELISA) in a high percentage of sera from individuals from a malaria endemic area in The Gambia (up to 99% for some adult groups). These results were further improved, especially for detection of antimalaria antibodies in children, when a pool of all 3 polypeptides (ELISA MIXT) was used as antigen. This ELISA MIXT improves presently available assays for the detection of antimalaria antibodies directed against asexual blood stages in respect of standardization, sensitivity and specificity.
