Molecular basis of USP7 inhibition by selective small-molecule inhibitors.

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.

Discovery and optimization of novel piperazines as potent inhibitors of fatty acid synthase (FASN).

The discovery, structure-activity relationships, and optimization of a novel class of fatty acid synthase (FASN) inhibitors is reported. High throughput screening identified a series of substituted piperazines with structural features that enable interactions with many of the potency-driving regions of the FASN KR domain binding site. Derived from this series was FT113, a compound with potent biochemical and cellular activity, which translated into excellent activity in in vivo models.

Discovery of Potent Isoindolinone Inhibitors that Target an Active Conformation of PARP1 Using DNA-Encoded Libraries.

Inhibition of poly (ADP-ribose) polymerase-1 (PARP1), a DNA repair enzyme, has proven to be a successful strategy for the treatment of various cancers. With the appropriate selection conditions and protein design, DNA-encoded library (DEL) technology provides a powerful avenue to identify small molecules with the desired mechanism of action towards a target of interest. However, DNA-binding proteins, such as PARP1, can be challenging targets for DEL screening due to non-specific protein-DNA interactions. To overcome this, we designed and screened a PARP1 catalytic domain construct without the autoinhibitory helical domain. This allowed us to interrogate an active, functionally-relevant form of the protein resulting in the discovery of novel isoindolinone PARP1 inhibitors with single-digit nanomolar potency. These inhibitors also demonstrated little to no PARP1-DNA trapping, a property that could be advantageous in the clinic.

The deubiquitylase USP9X controls ribosomal stalling.

When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases, respectively, through ubiquitylation of the eS10 and uS10 subunits of the ribosome. We have developed a specific small-molecule inhibitor of the deubiquitylase USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome-associated protein stability but also reveals a loss of Makorin 2 and ZNF598. We show that USP9X interacts with both these ubiquitin E3 ligases, regulating their abundance through the control of protein stability. In the absence of USP9X or following chemical inhibition of its catalytic activity, levels of Makorins and ZNF598 are diminished, and the ribosomal quality control pathway is impaired.

USP28 deletion and small-molecule inhibition destabilizes c-MYC and elicits regression of squamous cell lung carcinoma.

Lung squamous cell carcinoma (LSCC) is a considerable global health burden, with an incidence of over 600,000 cases per year. Treatment options are limited, and patient's 5-year survival rate is less than 5%. The ubiquitin-specific protease 28 (USP28) has been implicated in tumourigenesis through its stabilization of the oncoproteins c-MYC, c-JUN, and Δp63. Here, we show that genetic inactivation of Usp28-induced regression of established murine LSCC lung tumours. We developed a small molecule that inhibits USP28 activity in the low nanomole range. While displaying cross-reactivity against the closest homologue USP25, this inhibitor showed a high degree of selectivity over other deubiquitinases. USP28 inhibitor treatment resulted in a dramatic decrease in c-MYC, c-JUN, and Δp63 proteins levels and consequently induced substantial regression of autochthonous murine LSCC tumours and human LSCC xenografts, thereby phenocopying the effect observed by genetic deletion. Thus, USP28 may represent a promising therapeutic target for the treatment of squamous cell lung carcinoma.

Design and Optimization of Benzopiperazines as Potent Inhibitors of BET Bromodomains.

A protein structure-guided drug design approach was employed to develop small molecule inhibitors of the BET family of bromodomains that were distinct from the known (+)-JQ1 scaffold class. These efforts led to the identification of a series of substituted benzopiperazines with structural features that enable interactions with many of the affinity-driving regions of the bromodomain binding site. Lipophilic efficiency was a guiding principle in improving binding affinity alongside drug-like physicochemical properties that are commensurate with oral bioavailability. Derived from this series was tool compound FT001, which displayed potent biochemical and cellular activity, translating to excellent in vivo activity in a mouse xenograft model (MV-4-11).