Identification and Characterization of Dual Inhibitors of the USP25/ 28 Deubiquitinating Enzyme Subfamily

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

Identification and development of a subtype-selective allosteric AKT inhibitor suitable for clinical development

The serine/threonine protein kinase AKT plays a pivotal role within the PI3K pathway in regulating cellular proliferation and apoptotic cellular functions, and AKT hyper-activation via gene amplification and/or mutation has been implicated in multiple human malignancies. There are 3 AKT isoenzymes (AKT1-3) which mediate critical, non-redundant functions. We present the discovery and development of ALM301, a novel, allosteric, sub-type selective inhibitor of AKT1/2. ALM301 binds in an allosteric pocket created by the combined movement of the PH domain and the catalytic domain, resulting in a DFG out conformation. ALM301 was shown to be highly selective against a panel of over 450 kinases and potently inhibited cellular proliferation. These effects were particularly pronounced in MCF-7 cells containing a PI3KCA mutation. Subsequent cellular downstream pathway analysis in this sensitive cell line revealed potent inhibition of pAKT signalling up to 48 h post dosing. ALM301 treatment was well tolerated in an MCF-7 xenograft model and led to a dose-dependent reduction in tumour growth. Enhanced efficacy was observed in combination with tamoxifen. In summary, ALM301 is a highly specific AKT 1/2 inhibitor with an excellent pharmacological profile suitable for further clinical development

USP7 inhibitors suppress tumour neoangiogenesis and promote synergy with immune checkpoint inhibitors by downregulating fibroblast VEGF

BACKGROUND: Understanding how to modulate the microenvironment of tumors that are resistant to immune checkpoint inhibitors represents a major challenge in oncology.Here we investigate the ability of USP7 inhibitors to reprogram the tumor microenvironment (TME) by inhibiting secretion of vascular endothelial growth factor (VEGF) from fibroblasts.METHODS: To understand the role played by USP7 in the TME, we systematically evaluated the effects of potent, selective USP7 inhibitors on co-cultures comprising components of the TME, using human primary cells. We also evaluated the effects of USP7 inhibition on tumor growth inhibition in syngeneic models when dosed in combination with immune checkpoint inhibitors (ICIs).RESULTS: Abrogation of VEGF secretion from fibroblasts in response to USP7 inhibition resulted in inhibition of tumor neoangiogenesis and increased tumor recruitment of CD8-positive T-lymphocytes, leading to significantly improved sensitivity to immune checkpoint inhibitors. In syngeneic models, treatment with USP7 inhibitors led to striking tumor responses resulting in significantly improved survival.CONCLUSIONS: USP7-mediated reprograming of the TME is not linked to its previously characterized role in modulating MDM2 but does require p53 and UHRF1 in addition to the well-characterized VEGF transcription factor, HIF-1α. This represents a function of USP7 that is unique to fibroblasts, and which is not observed in cancer cells or other components of the TME. Given the potential for USP7 inhibitors to transform "immune desert" tumors into "immune responsive" tumors, this paves the way for a novel therapeutic strategy combining USP7 inhibitors with immune checkpoint inhibitors (ICIs).</p

Identification and Structure-Guided Development of Pyrimidinone Based USP7 Inhibitors

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 <i>in vivo</i> experiments. Overall, these discoveries will enable further research into the wider biological role of USP7