RESUMEN
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 (IC50 < 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 (EC50 < 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.
Asunto(s)
Antineoplásicos/química , Descubrimiento de Drogas , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Sitio Alostérico , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Concentración 50 Inhibidora , Cinética , Oxidación-Reducción , Inhibidores de Proteasas/química , Unión Proteica , Conformación Proteica , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Especificidad por Sustrato , Proteína p53 Supresora de Tumor/químicaRESUMEN
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).
Asunto(s)
Neoplasias , Peptidasa Específica de Ubiquitina 7 , Factor A de Crecimiento Endotelial Vascular , Humanos , Proteínas Potenciadoras de Unión a CCAAT/farmacología , Fibroblastos/metabolismo , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neovascularización Patológica/tratamiento farmacológico , Microambiente Tumoral , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidoresRESUMEN
Direct electrophilic borylation using Y(2)BCl (Y(2) = Cl(2) or o-catecholato) with equimolar AlCl(3) and a tertiary amine has been applied to a wide range of arenes and heteroarenes. In situ functionalization of the ArBCl(2) products is possible with TMS(2)MIDA, to afford bench-stable and easily isolable MIDA-boronates in moderate to good yields. According to a combined experimental and computational study, the borylation of activated arenes at 20 °C proceeds through an S(E)Ar mechanism with borenium cations, [Y(2)B(amine)](+), the key electrophiles. For catecholato-borocations, two amine dependent reaction pathways were identified: (i) With [CatB(NEt(3))](+), an additional base is necessary to accomplish rapid borylation by deprotonation of the borylated arenium cation (σ complex), which otherwise would rather decompose to the starting materials than liberate the free amine to effect deprotonation. Apart from amines, the additional base may also be the arene itself when it is sufficiently basic (e.g., N-Me-indole). (ii) When the amine component of the borocation is less nucleophilic (e.g., 2,6-lutidine), no additional base is required due to more facile amine dissociation from the boron center in the borylated arenium cation intermediate. Borenium cations do not borylate poorly activated arenes (e.g., toluene) even at high temperatures; instead, the key electrophile in this case involves the product from interaction of AlCl(3) with Y(2)BCl. When an extremely bulky amine is used, borylation again does not proceed via a borenium cation; instead, a number of mechanisms are feasible including via a boron electrophile generated by coordination of AlCl(3) to Y(2)BCl, or by initial (heteroarene)AlCl(3) adduct formation followed by deprotonation and transmetalation.
Asunto(s)
Aminas/química , Compuestos de Boro/síntesis química , Hidrocarburos Aromáticos/química , Iminoácidos/síntesis química , Compuestos de Boro/química , Iminoácidos/química , Modelos Moleculares , Estructura MolecularRESUMEN
The first enantiospecific total synthesis of the antibacterial natural product (+)-pleuromutilin has been achieved. The approach includes the synthesis of a non-racemic cyclisation substrate from (+)-trans-dihydrocarvone, a highly selective SmI2-mediated cyclisation cascade, an electron transfer reduction of a hindered ester, and the first efficient conversion of (+)-mutilin to the target.
Asunto(s)
Antibacterianos/síntesis química , Productos Biológicos/síntesis química , Cetonas/química , Compuestos Policíclicos/química , Antibacterianos/química , Productos Biológicos/química , Ciclización , Monoterpenos Ciclohexánicos , Diterpenos/síntesis química , Diterpenos/química , Estructura Molecular , Monoterpenos/síntesis química , Monoterpenos/química , Estereoisomerismo , PleuromutilinasRESUMEN
The cis-hydrindane motif is found in a number of natural products that display important biological activity. A flexible, stereoselective approach to the framework has been developed that features highly diastereoselective, SmI(2)-mediated cyclisations. The strategy has been exploited in the first synthesis of the proposed structure of faurinone and an approach to the skeleton of the antibacterial natural product, pleuromutilin.
Asunto(s)
Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Indanos/química , Samario/química , Productos Biológicos/química , Ciclización , Diterpenos/síntesis química , Modelos Moleculares , Estructura Molecular , Oxidación-Reducción , Compuestos Policíclicos , Estereoisomerismo , PleuromutilinasRESUMEN
Readily prepared beta-silyl substituted crotylzinc reagents undergo highly selective allylation of carbonyl compounds leading to syn-homoallylic alcohols.
RESUMEN
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 in vivo experiments. Overall, these discoveries will enable further research into the wider biological role of USP7.
RESUMEN
Electrophilic direct borylation is facilitated, and arene substrate scope enhanced, by using electrophiles derived from inexpensive reagents; specifically an amine, BCl(3) and AlCl(3).
RESUMEN
The participation of alkynylboronates in [4 + 2] cycloadditions has been investigated using both kinetic and DFT studies. Kinetic studies of the cycloaddition of tetrazine 1 with alkynylboronate 2 strongly suggest that a concerted cycloaddition mechanism is in operation. This mechanism has been confirmed by DFT calculations; moreover, a highly synchronous transition state appears to operate in this process. The experimentally observed poor reactivity of electron-rich dienes with alkynylboronates has also been confirmed by theoretical studies by analyzing the transition states of the cycloadditions with bis-2,5-trimethylsilyloxyfuran. The surprising conclusion has been made that alkynylboronates are relatively electron rich and have a cycloaddition reactivity that resembles that of acetylene. In contrast, the related dichloroalkynylborane cycloaddition reactivity resembles that of dimethylacetylene dicarboxylate.
RESUMEN
A series of 3,6-dichloro-1H-pyridazin-4-ones have been prepared via the cycloaddition of 3,6-dichlorotetrazine with alkynylboronates, and their employment as useful synthetic intermediates was highlighted through a selection of highly regioselective C-O, C-S and C-C bond forming reactions.