Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.

Histone–lysine acetylation is a vital chromatin post-translational modification involved in the epigenetic regulation of gene transcription. Bromodomains bind acetylated lysines, acting as readers of the histone-acetylation code. Competitive inhibitors of this interaction have antiproliferative and anti-inflammatory properties. With 57 distinct bromodomains known, the discovery of subtype-selective inhibitors of the histone–bromodomain interaction is of great importance. We have identified the 3,5-dimethylisoxazole moiety as a novel acetyl-lysine bioisostere, which displaces acetylated histone-mimicking peptides from bromodomains. Using X-ray crystallographic analysis, we have determined the interactions responsible for the activity and selectivity of 4-substituted 3,5-dimethylisoxazoles against a selection of phylogenetically diverse bromodomains. By exploiting these interactions, we have developed compound 4d, which has IC50 values of <5 μM for the bromodomain-containing proteins BRD2(1) and BRD4(1). These compounds are promising leads for the further development of selective probes for the bromodomain and extra C-terminal domain (BET) family and CREBBP bromodomains.

Since the discovery of qnrA in 1998, two additional qnr genes, qnrB and qnrS, have been described. These three plasmid-mediated genes contribute to quinolone resistance in gram-negative pathogens worldwide. A clinical strain of Proteus mirabilis was isolated from an outpatient with a urinary tract infection and was susceptible to most antimicrobials but resistant to ampicillin, sulfamethoxazole, and trimethoprim. Plasmid pHS10, harbored by this strain, was transferred to azide-resistant Escherichia coli J53 by conjugation. A transconjugant with pHS10 had low-level quinolone resistance but was negative by PCR for the known qnr genes, aac(6)-Ib-cr and qepA. The ciprofloxacin MIC for the clinical strain and a J53/pHS10 transconjugant was 0.25 g/ml, representing an increase of 32-fold relative to that for the recipient, J53. The plasmid was digested with HindIII, and a 4.4-kb DNA fragment containing the new gene was cloned into pUC18 and transformed into E. coli TOP10. Sequencing showed that the responsible 666-bp gene, designated qnrC, encoded a 221-amino-acid protein, QnrC, which shared 64%, 42%, 59%, and 43% amino acid identity with QnrA1, QnrB1, QnrS1, and QnrD, respectively. Upstream of qnrC there existed a new IS3 family insertion sequence, ISPmi1, which encoded a frameshifted transposase. qnrC could not be detected by PCR, however, in 2,020 strains of Enterobacteriaceae. A new quinolone resistance gene, qnrC, was thus characterized from plasmid pHS10 carried by a clinical isolate of P. mirabilis.Plasmid-mediated quinolone resistance was first described for a ciprofloxacin-resistant strain of Klebsiella pneumoniae in 1998 (15). The responsible gene, qnr (later named qnrA), was located on plasmid pMG252, which encodes multidrug resistance proteins. qnrB and qnrS were discovered in 2005 and 2006, respectively, and mediated similar levels of ciprofloxacin resistance (9, 11). Qnr proteins belong to the pentapeptide repeat protein (PRP) family and protect DNA gyrase and topoisomerase IV from quinolone inhibition (26,27,28). qnr genes show a high level of diversity; there are at least 6 qnrA, 20 qnrB, and 3 qnrS alleles reported, with one or more amino acid alterations within each family (12; http://www.lahey.org /qnrStudies). More recently, qnrD was found in Salmonella isolates (3). qnr genes are widely distributed in clinical Enterobacteriaceae isolates around the world and are usually associated with mobile elements (21). There were also qnr-like genes found on the chromosomes of Vibrio vulnificus, Vibrio parahaemolyticus, Photobacterium profundum, Stenotrophomonas maltophilia, and gram-positive genera such as Enterococcus, Listeria, Clostridium, and Bacillus (1,17,22,24). The wide distribution of qnr genes in different species of Enterobacteriaceae and their high degree of diversity raise the concern that there might be more qnr genes that have not yet been discovered. In this study, a new plasmid-mediated quinolone resistance gene, qnrC, was found on and cloned from a transferable plasmid, pHS10, in a clinical ...

The genes for multidrug efflux pump OqxAB, which is active on fluoroquinolones, were found in human clinical isolates on a plasmid in Escherichia coli and on the chromosome of Klebsiella pneumoniae. IS26-like sequences flanked the plasmid-mediated oqxAB genes, suggesting that they had been mobilized as part of a composite transposon.Plasmid-borne genes conferring quinolone resistance have been increasingly recognized (7,10). Recently a plasmid-encoded efflux pump, OqxAB, conferring resistance to the quinoxaline-di-N-oxide olaquindox, which has been used as a growth promoter in pigs, was discovered in Escherichia coli isolates of porcine origin in Denmark and Sweden (4-6). OqxAB was encoded by the genes oqxA and oqxB located on a 52-kb conjugative plasmid designated pOLA52 and conferred resistance to multiple agents, including fluoroquinolones (4, 9). We have investigated the prevalence of this plasmid-encoded multidrug efflux pump in clinical isolates of Enterobacteriaceae and have for the first time identified an oqxAB-encoding plasmid in an E. coli isolate of human origin.Isolates were from the collection of blood isolates from Seoul National University Hospital collected from 1998 to 2006. The same set of isolates was previously surveyed for other plasmid-mediated quinolone resistance (PMQR) genes (8). A total of 461 clinical isolates were screened by PCR for the oqxA gene. Isolates positive for oqxA were also tested for oqxB, and strains positive for both genes were confirmed by sequencing of the PCR products. The primers used are shown in Table 1.One (0.4%) of 261 E. coli isolates, 3 (4.6%) of 65 Enterobacter cloacae isolates, and 100 (74.1%) of 135 Klebsiella pneumoniae isolates were provisionally classified as positive for both oqxA and oqxB. The oqxAB-positive E. coli was isolated from the blood of a patient in 1999. A BLAST search of the nucleotide sequence similarity of the oqxB PCR products obtained from the three E. cloacae isolates gave identities of only 88% (399/453) with pOLA52 (GenBank accession number EU370913) and 86% (394/454) with the hydrophobe/amphiphile efflux-1 (HAE1) family transporter of Enterobacter sp. strain 638 (GenBank accession number CP000653). There was, however, substantial similarity between the complete nucleotide sequences of the tandem oqxA and oqxB genes from E. coli 1-12 (GenBank accession number GQ120634; 99.5% and 99.0%, respectively), K. pneumoniae 4-39 (GenBank accession number FJ975560; 98.2% and 99.0%, respectively), and K. pneumoniae 5-80 (GenBank accession number FJ975561; 99.4% and 98.9%, respectively) relative to those of pOLA52 and the chromosomal genes in K. pneumoniae MGH78578 (GenBank accession number NC009648). Since the oqxAB genes appear to be chromosomal in K. pneumoniae (3, 9), E. coli 1-12 was the most likely candidate to contain an oqxAB-encoding plasmid (Table 2).To test for the plasmid location of oqxAB, plasmid DNAs were obtained using a plasmid midi kit (Qiagen, Valencia, CA) and hybridized with a horseradish peroxidase-labeled oqxB probe as prev...