Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic virus that causes severe respiratory illness accompanied by multiorgan dysfunction, resulting in a case fatality rate of approximately 40%. As found in other coronaviruses, the majority of the positive-stranded RNA MERS-CoV genome is translated into two polyproteins, one created by a ribosomal frameshift, that are cleaved at three sites by a papain-like protease and at 11 sites by a 3C-like protease (3CL pro ). Since 3CL pro is essential for viral replication, it is a leading candidate for therapeutic intervention. To accelerate the development of 3CL pro inhibitors, three crystal structures of a catalytically inactive variant (C148A) of the MERS-CoV 3CL pro enzyme were determined. The aim was to co-crystallize the inactive enzyme with a peptide substrate. Fortuitously, however, in two of the structures the C-terminus of one protomer is bound in the active site of a neighboring molecule, providing a snapshot of an enzyme-product complex. In the third structure, two of the three protomers in the asymmetric unit form a homodimer similar to that of SARS-CoV 3CL pro ; however, the third protomer adopts a radically different conformation that is likely to correspond to a crystallographic monomer, indicative of substantial structural plasticity in the enzyme. The results presented here provide a foundation for the structure-based design of small-molecule inhibitors of the MERS-CoV 3CL pro enzyme.

The DNA sequence of the 15,155-bp O-antigen gene cluster of Escherichia coli O121 was determined, and 14 open reading frames were identified (all had the same transcriptional direction). Analyses of results indicated that the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes were E. coli O121 specific, so regions in these two genes were chosen for development of PCR assays. The PCR assays using DNA from 99 E. coli O121 strains, strains representative of non-O121 E. coli serogroups, and strains of other bacterial genera and PCR assays using DNA from seven enrichments of swine fecal samples naturally contaminated with E. coli O121 showed specificity for E. coli O121. Thus, the PCR assay can be employed to reliably identify E. coli O121 and to potentially detect the organism in food, fecal, and environmental samples. More than 200Escherichia coli serotypes isolated from humans have been identified as Shiga toxin-producing E. coli (STEC), and more than 100 of these serotypes have been associated with human illness (24). E. coli O157:H7 is the most common STEC. In the United States, E. coli O157:H7 is more often associated with hemorrhagic colitis and hemolytic-uremic syndrome (HUS) than any of the other STEC serogroups. However, in other countries, such as Argentina, Germany, and Australia, non-O157 STEC strains have become an important public health problem (2,5,7,9). Unlike E. coli O157:H7, which generally does not ferment sorbitol or have ␤-glucuronidase activity, the non-O157 STEC strains do not have identifiable biochemical markers to facilitate screening for and identification of these pathogens. Detection of non-O157 STEC requires testing for the Shiga toxins or for genes which encode Shiga toxins, followed by serotyping using antisera produced against the ca. 179 different E. coli serogroups. Thus, due to the lack of simple and rapid methods for detection and identification of non-O157 STEC, the incidence of disease caused by these organisms is likely underestimated.Shiga toxin-producing E. coli O121 strains are classified as enterohemorrhagic E. coli (EHEC), since they have been isolated from patients with hemorrhagic colitis or HUS (3,11,12,18,24,25). Additionally, strains of E. coli O121 serogroup, possessing virulence characteristics similar to those of Shigella and enteroinvasive E. coli, have caused shigellosis-like illnesses (8, 10). In 1999, E. coli O121:H19 was associated with an outbreak of HUS at a lake in Connecticut (11). Due to the public health concern over E. coli O121 infection, assays specific for this serogroup are needed to rapidly and reliably detect this pathogen and to further define its role in causing human illness. Tarr et al. (19) characterized 24 isolates of E. coli O121:H19 and nonmotile variants using multilocus enzyme electrophoresis and multilocus sequencing and found that the isolates represented a single bacterial clone. The isolates possessed a virulence gene profile typical of EHEC clones; however, the results of sequencing analyses showed that the O121: H19 cl...

Tyrosyl DNA-phosphodiesterase I (TDP1) repairs type IB topoisomerase (TOP1) cleavage complexes generated by TOP1 inhibitors commonly used as anticancer agents. TDP1 also removes DNA 3′ end blocking lesions generated by chain-terminating nucleosides and alkylating agents, and base oxidation both in the nuclear and mitochondrial genomes. Combination therapy with TDP1 inhibitors is proposed to synergize with topoisomerase targeting drugs to enhance selectivity against cancer cells exhibiting deficiencies in parallel DNA repair pathways. A crystallographic fragment screening campaign against the catalytic domain of TDP1 was conducted to identify new lead compounds. Crystal structures revealed two fragments that bind to the TDP1 active site and exhibit inhibitory activity against TDP1. These fragments occupy a similar position in the TDP1 active site as seen in prior crystal structures of TDP1 with bound vanadate, a transition state mimic. Using structural insights into fragment binding, several fragment derivatives have been prepared and evaluated in biochemical assays. These results demonstrate that fragment-based methods can be a highly feasible approach toward the discovery of small-molecule chemical scaffolds to target TDP1, and for the first time, we provide co-crystal structures of small molecule inhibitors bound to TDP1, which could serve for the rational development of medicinal TDP1 inhibitors.