A rapid method is described to efficiently perform site-directed mutagenesis based on overlap extension polymerase chain reaction (OE-PCR). Two template DNA molecules in different orientations relative to only one universal primer were amplified in parallel. By choosing a high dilution of mutagenic primers it was possible to run an overlap extension PCR in only one reaction without purification of intermediate products. This method which we have named one-step overlap extension PCR (OOE-PCR) can in principle be applied to every DNA fragment which can be cloned into a multiple cloning site of any common cloning vector.

Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of <25 g/ml]). Our screening approach is exemplified by the discovery of classical and novel DNA synthesis and translation inhibitors. For instance, we show that the mechanistically underexplored antibiotic ferrimycin A1 selectively inhibits protein biosynthesis.

DsbA and DsbC proteins involved in the periplasmic formation of disulfide bonds in Pseudomonas aeruginosawere identified and shown to play an important role for the formation of extracellular enzymes. Mutants deficient in either dsbA or dsbC or both genes were constructed, and extracellular elastase, alkaline phosphatase, and lipase activities were determined. The dsbA mutant no longer produced these enzymes, whereas the lipase activity was doubled in the dsbC mutant. Also, extracellar lipase production was severely reduced in a P. aeruginosa dsbA mutant in which an inactive DsbA variant carrying the mutation C34S was expressed. Even when the lipase gene lipA was constitutively expressed in trans in a lipA dsbA double mutant, lipase activity in cell extracts and culture supernatants was still reduced to about 25%. Interestingly, the presence of dithiothreitol in the growth medium completely inhibited the formation of extracellular lipase whereas the addition of dithiothreitol to a cell-free culture supernatant did not affect lipase activity. We conclude that the correct formation of the disulfide bond catalyzed in vivo by DsbA is necessary to stabilize periplasmic lipase. Such a stabilization is the prerequisite for efficient secretion using the type II pathway.Disulfide bonds are important for the structure and stability of numerous proteins. For Escherichia coli it is now well established that the formation of disulfide bonds is an assisted process which occurs in the periplasm and is catalyzed by the thiol-disulfide oxidoreductase DsbA (7, 34). DsbA acts as a donor of disulfides to newly synthesized periplasmic proteins (23) and is reoxidized by DsbB, a second protein located in the inner membrane (5, 6). DsbA and DsbB are members of the Dsb system (the system for disulfide bond formation) which consists of at least six redox proteins belonging to the thioredoxin superfamily. These proteins contain a canonical C-X-X-C motif in the dithiol active site and seem to be conserved throughout the gram-negative bacteria (49). DsbC is another member which is suggested to act as a disulfide isomerase (56, 67). DsbD (39) keeps DsbC in a reduced state (57). More recently, DsbG was described as a novel member of the Dsb family in E. coli which oxidizes so far unknown substrates (4,8).Undoubtedly, the process of protein secretion in gram-negative bacteria is related to the function of the Dsb system. However, the results obtained so far are contradictory. Four major secretion pathways exist in gram-negative bacteria to direct proteins into the extracellular medium (37). In type I and type III pathways the secreted proteins directly pass both the inner and the outer membrane using a machinery formed by either three or more than 20 different proteins, respectively. In the type II pathway, which is also called the general secretory pathway, secretion occurs in two consecutive steps (48), with an intermediate state in the periplasm where the formation of disulfide bonds can take place. Since E. coli does not secrete exoprotein...

AIC246 (letermovir) is a potent anticytomegalovirus drug in clinical development. Here, we report a consistent antiviral efficacy of AIC246 against human cytomegalovirus laboratory strains, clinical isolates, and virus variants resistant to approved drugs. Furthermore, we describe a remarkable selectivity of AIC246 for human cytomegaloviruses compared to that of other alpha-, beta-, or gammaherpesviruses or nonrelated pathogenic viruses, including adeno-, hepadna-, retro-, orthomyxo-, and flaviviruses. Our data confirm and support an excellent and selective anticytomegaloviral activity of AIC246. Human cytomegalovirus (HCMV) is one of eight human herpesviruses with worldwide distribution and a high clinical importance. Despite diagnostic and therapeutic advances, HCMV infection has remained a significant complication during pregnancy and in clinical situations associated with inefficient immunocompetence, such as organ or bone marrow transplantation, cancer, and AIDS. Currently approved anticytomegaloviral drugs include ganciclovir (GCV), its prodrug valganciclovir (VGCV), foscarnet (FOS), and cidofovir (CDV), which all uniformly target the viral DNA polymerase. Although efficacious, the use of these drugs is limited because of severe toxic side effects, low oral bioavailability (with the exception of VGCV), and the occurrence of drug resistance (10). Thus, there is an urgent need for novel safe and tolerable anticytomegalovirus drugs.The 3,4-dihydroquinazoline AIC246 (letermovir) has shown a potent anticytomegaloviral activity in vitro and in vivo, a favorable safety and pharmacokinetic profile in phase I clinical trials, and proof of concept in a phase IIa trial (8,9,23). Recently, a clinical phase IIb dose-finding trial has been completed (clinicaltrials .gov). Analyses of the mode of action of AIC246 revealed that the drug interferes with DNA concatemer maturation and exerts its antiviral effect mainly through targeting the HCMV terminase complex, a heterodimeric enzyme which has no counterpart in mammalian cells (4). Based on this novel mode of action, AIC246 should provide new treatment options even for HCMV infections with virus variants that are resistant to current drugs. In fact, AIC246 recently proved to be highly efficacious in the treatment of a lung transplant recipient suffering from multidrug-resistant HCMV disease (6). Here, we extend our in vitro characterization of the AIC246 activities against (i) several cytomegalovirus variants and isolates; (ii) other alpha-, beta-, or gammaherpesviruses; and (iii) a panel of important human pathogenic viruses belonging to various families.First we analyzed the efficacy of AIC246 against a panel of 17 different clinical HCMV isolates. Virus isolates WT1 to WT17 (Table 1) were collected from various sources, including reference laboratories and medical centers regularly conducting HCMV diagnostic assays and therapy. The susceptibilities of the virus isolates to AIC246 were determined by standard plaque reduction assays essentially as described by Pepin et a...

One of the most promising viral targets in current hepatitis B virus (HBV) drug development is the core protein due to its multiple roles in the viral life cycle. Here we investigated the differences in the mode of action and antiviral activity of representatives of six different capsid assembly modifier (CAM) scaffolds: three from the well-characterized scaffolds heteroarylpyrimidine (HAP), sulfamoylbenzamide (SBA), and phenylpropenamide (PPA), and three from novel scaffolds glyoxamide-pyrrolamide (GPA), pyrazolyl-thiazole (PT), and dibenzo-thiazepin-2-one (DBT). The target activity and antiviral efficacy of the different CAMs were tested in biochemical and cellular assays. Analytical size exclusion chromatography and transmission electron microscopy showed that only the HAP compound induced formation of aberrant non-capsid structures (class II mode of action), while the remaining CAMs did not affect capsid gross morphology (class I mode of action). Intracellular lysates from the HepAD38 cell line, inducibly replicating HBV, showed no reduction in the quantities of intracellular core protein or capsid after treatment with SBA, PPA, GPA, PT, or DBT compounds; however HAP-treatment led to a profound decrease in both. Additionally, immunofluorescence staining of compound-treated HepAD38 cells showed that all non-HAP CAMs led to a shift in the equilibrium of HBV core antigen (HBcAg) towards complete cytoplasmic staining, while the HAP induced accumulation of HBcAg aggregates in the nucleus. Our study demonstrates that the novel scaffolds GPA, PT, and DBT exhibit class I modes of action, alike SBA and PPA, whereas HAP remains the only scaffold belonging to class II inhibitors.