Transduction of hedgehog signals across the plasma membrane is a key process during animal development. This is facilitated by the Class F G-protein-coupled-receptor (GPCR) Smoothened (SMO), a major drug target in the treatment of basal cell carcinomas. Recent studies have suggested that SMO is modulated via interactions of its transmembrane (TM) domain with cholesterol. Long time scale (>0.35 ms of simulation time) molecular dynamics simulations of SMO embedded in two different cholesterol containing lipid bilayers reveal direct interactions of cholesterol with the transmembrane domain at regions distinct from those observed in Class A GPCRs. In particular the extracellular tips of helices TM2 and TM3 form a well-defined cholesterol interaction site, robust to changes in membrane composition and in force field parameters. Potential of mean force calculations for cholesterol interactions yield a free energy landscape for cholesterol binding.Combined with analysis of equilibrium cholesterol occupancy these results reveal the existence of a dynamic 'greasy patch' interaction with the TM domain of SMO, which may be compared to previously identified lipid interaction sites on other membrane proteins. These predictions provide molecular level insights into cholesterol interactions with a biomedically relevant Class F GPCR, suggesting potential druggable sites.

No abstract

Introduction: Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that regulates several proteins involved in cell cycle, DNA repair, genomic stability, and epigenetics and has been implicated in cancer progression. A key substrate of USP7 is MDM2, the oncogenic E3 ubiquitin ligase that promotes degradation of the tumor suppressor p53. USP7-mediated stabilization of MDM2 leads to the degradation of p53, preventing cell cycle arrest and the induction of apoptosis and promoting tumor cell growth. In addition to the MDM2-p53 pathway, USP7 regulates a number of other substrates involved in cancer, including PIM2 kinase, MYCN, the DNA methyltransferase DNMT1, and the PTEN tumor suppressor. Consistent with its function promoting oncogenic signaling pathways, genetic and pharmacological inhibition of USP7 has been shown to inhibit growth of a number of tumor cell lines in vitro and in vivo, and this anti-tumor activity is significantly enriched among cells expressing wild type p53. Thus, inhibition of USP7 is a promising therapeutic strategy, especially in cancers carrying wild type p53 that can be reactivated by suppression of MDM2. Results: We have discovered a new class of potent and selective USP7 inhibitors. These compounds bind to USP7 and prevent deubiquitinase activity in biochemical activity assays with picomolar potency. Our compounds induce accumulation of p53 and exhibit IC50s below 50 nM in cell viability assays in Multiple Myeloma and Acute Myeloid Leukemia (AML) cell lines. In AML cell lines, our USP7 inhibitors strongly synergize with the approved Bcl-2 inhibitor venetoclax. Unlike MDM2 antagonists currently undergoing clinical trials in AML, these USP7 inhibitors do not lead to a significant increase in MDM2 levels, and our compounds result in a more modest induction of p53, both of which could provide significant safety benefits. We have demonstrated differential sensitivity to MDM2 antagonists and our USP7 inhibitors in select cell lines and show that this effect is dependent upon p53 induction. Our compounds are orally bioavailable with a desirable PK profile in mice and induce p53 in tumor cells in CDX mouse models of Multiple Myeloma. Conclusions: We have identified novel, potent, orally bioavailable USP7 inhibitors that lead to p53 accumulation and cytotoxicity in cancer cells. We demonstrate mechanistic differences between USP7 and MDM2-p53 antagonists, which may lead to a safety advantage. We observe strong synergy between USP7 inhibitors and an approved treatment. Our lead compounds have favorable drug-like properties, promising mouse PK profiles, and can induce p53 accumulation in mouse CDX tumor models. These data demonstrate the therapeutic potential of our USP7 inhibitors and reveal specific opportunities for their use in the treatment of Multiple Myeloma and AML. Citation Format: Alan Futran, Tao Lu, Katherine Amberg-Johnson, Xiaoxiao Yang, Saidi He, Jeffrey Bell, Sarah Boyce, Markus Dahlgren, Karen Dingley, Liping Fang, Heidi Koldsoe, Zef Konst, Fang-Yu Lin, Robert Pelleltier, Heng Qian, Mats Svensson, Michael Trzoss, Jie Xu, Shuping Xu, Zhaowu Xu, Engin Yapici, Yan Zhang, Li Xing, Takao Suzuki, Xianhai Huang, Jiayi Xu, Hamish Wright, Kristian Jensen, Wayne Tang, Tao Guo, Karen Akinsanya, David Madge. Discovery of novel, potent USP7 inhibitors that upregulate p53 leading to anti-proliferative effects in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1338.

The structural impact of high hydrostatic pressure (HHP) was studied with different types of LDL (native, oxidized and triglyceride rich) with SANS (PSI, Switzerland) and SAXS (Elettra, Italy). The HHP ranged from 50 to 3000 bar. Temperature points were chosen below, on and above T m. The pair distance distribution functions p(r) of the SAS curves revealed pressure dependent changes of the particle structure seen in the low q-region (~0.25 nm À1), also reflected by a decrease of Radii of Gyration (R g) with increasing pressure. Especially the p(r) functions from the SAXS data do not only show an overall particle change but also a highly pressure sensitive inner organization. A triplepeak feature indicating the lamellar lipid organization below T m was induced by raising the pressure up to 3000 bar. These pressure sensitive lipid layers were observed by scattering intensity changes in SAXS curves at q=1.7 nm À1. The shape alterations could be evidenced by fitting an ellipsoidal model to the SANS curves resulting in a decrease of the ellipsoidal radii under pressure. A new LDL model considering the cross-sectional electron density profile is developed and applied to the SAS data to get a more detailed insight into pressure dependent behavior.