Given the importance of ubiquitin-specific protease 7 (USP7) in oncogenic pathways, identification of USP7 inhibitors has attracted considerable interest. Despite substantial efforts, however, the development of validated deubiquitinase (DUB) inhibitors that exhibit drug-like properties and a well-defined mechanism of action has proven particularly challenging. In this article, we describe the identification, optimization and detailed characterization of highly potent (IC < 10 nM), selective USP7 inhibitors together with their less active, enantiomeric counterparts. We also disclose, for the first time, co-crystal structures of a human DUB enzyme complexed with small-molecule inhibitors, which reveal a previously undisclosed allosteric binding site. Finally, we report the identification of cancer cell lines hypersensitive to USP7 inhibition (EC < 30 nM) and demonstrate equal or superior activity in these cell models compared to clinically relevant MDM2 antagonists. Overall, these findings demonstrate the tractability and druggability of DUBs, and provide important tools for additional target validation studies.

The ubiquitin proteasome system is widely postulated to be a new and important field of drug discovery for the future, with the ubiquitin specific proteases (USPs) representing one of the more attractive target classes within the area. Many USPs have been linked to critical axes for therapeutic intervention, and the finding that USP28 is required for c-Myc stability suggests that USP28 inhibition may represent a novel approach to targeting this so far undruggable oncogene. Here, we describe the discovery of the first reported inhibitors of USP28, which we demonstrate are able to bind to and inhibit USP28, and while displaying a dual activity against the closest homologue USP25, these inhibitors show a high degree of selectivity over other deubiquitinases (DUBs). The utility of these compounds as valuable probes to investigate and further explore cellular DUB biology is highlighted by the demonstration of target engagement against both USP25 and USP28 in cells. Furthermore, we demonstrate that these inhibitors are able to elicit modulation of both the total levels and the half-life of the c-Myc oncoprotein in cells and also induce apoptosis and loss of cell viability in a range of cancer cell lines. We however observed a narrow therapeutic index compared to a panel of tissue-matched normal cell lines. Thus, it is hoped that these probes and data presented herein will further advance our understanding of the biology and tractability of DUBs as potential future therapeutic targets.

Telomeres are essential for genomic stability and cell viability. Telomerase, the enzyme responsible for telomere maintenance, is composed of a reverse transcriptase protein subunit and an integral RNA component which contains the templating domain. In human telomerase, the template region consists of 11 nt (3'-rCAAUCCCAAUC-5') and comprises an alignment domain (italicised) plus a template sequence encoding the telomeric repeat d(GGT TAG). In this study, the alignment domain of human telomerase was systematically reduced from the 3' end and the resultant recombinant enzyme activity was evaluated in vitro. Deletion or substitution of one or two residues from the 3' end of the alignment domain caused only a slight reduction in overall catalytic activity and did not alter the processivity of the enzyme. Deletion or substitution of three or more residues from the 3' end of the alignment domain resulted in total loss of catalytic activity. These results suggest that the two most 3' terminal RNA residues are relevant but not essential for overall activity and that the minimal length requirement of the alignment domain is 3 nt. Furthermore, base pairing between the 3' end of the primer substrate and the first two residues of the alignment domain is also not an absolute requirement for processive synthesis.

Ubiquitin specific protease 7 (USP7, HAUSP) has become an attractive target in drug discovery due to the role it plays in modulating Mdm2 levels and consequently p53. Increasing interest in USP7 is emerging due to its potential involvement in oncogenic pathways as well as possible roles in both metabolic and immune disorders in addition to viral infections. Potent, novel, and selective inhibitors of USP7 have been developed using both rational and structure-guided design enabled by high-resolution cocrystallography. Initial hits were identified via fragment-based screening, scaffold-hopping, and hybridization exercises. Two distinct subseries are described along with associated structure-activity relationship trends, as are initial efforts aimed at developing compounds suitable for experiments. Overall, these discoveries will enable further research into the wider biological role of USP7.

The G-quadruplex nucleic acid structural motif is a target for designing molecules that could potentially modulate telomere length or have anticancer properties. We have recently described an engineered zinc finger protein (Gq1) that binds with specificity to the intramolecular Gquadruplex formed by the human telomeric sequence 5′-(GGTTAG) 5 -3′ (Isalan et al. (2001) Biochemistry 40, 830-836). Here, we report that Gq1 is able to arrest the action of a DNA polymerase on a template-containing telomeric sequence. Inhibition occurs in a concentrationdependent manner, probably by forming a stabilized G-quadruplex·protein complex. Furthermore, Gq1 inhibits the apparent activity of the enzyme telomerase in vitro, with an IC 50 value of 74.3 ± 11.1 nM. Possible molecular mechanisms of inhibition are discussed, together with the potential for using engineered zinc fingers to interfere with the cellular processes associated with telomere function.Telomeres are the termini of eukaryotic chromosomes and contain repetitive DNA sequences that reduce in length with each round of cell division and DNA replication (1, 2). Telomere shortening has been considered as a mitotic clock, which stops when the telomeres have become too short to allow further cell division (2). This process is believed to be one of the factors that limits the number of times normal human somatic cells are able to divide. In contrast, cancer cells are able to divide indefinitely and maintain their telomere lengths most likely by expressing the enzyme telomerase (3-5). Telomerase is a ribonucleoprotein that catalyses telomere elongation (6). It has been demonstrated that telomerase can allow apparently infinite cell proliferation that is not normally possible in human somatic cells (7). A significant number of studies suggest that cancer cells can be specifically prevented from dividing indefinitely if telomere maintenance by telomerase is inhibited (8, 9). There is therefore considerable interest in designing molecules that may block telomere maintenance and to understand the associated mechanisms because such approaches may ultimately lead to agents that inhibit cancer proliferation.Telomeric DNA sequences generally consist of short tandem repeats, where one strand is rich in guanine residues [for example, 5′-TTAGGG-3′ in humans and 5′-TTTTGGGG-3′ in Oxytrichia] (10). A single-stranded 3′ overhang of the G-rich telomeric strand is a general feature of all telomeres (11). The 3′ end is the primer terminus for telomere extension, and models for telomere regulation have been proposed based on the status of this † This work was supported by the BBSRC.