Discovery of Small-Molecule Inhibitors of Ubiquitin Specific Protease 7 (USP7) Using Integrated NMR and in Silico Techniques

Abstract

USP7 is a deubiquitinase implicated in destabilizing the tumor suppressor p53, and for this reason it has gained increasing attention as a potential oncology target for small molecule inhibitors. Herein we describe the biophysical, biochemical, and computational approaches that led to the identification of 4-(2-aminopyridin-3-yl)phenol compounds described by Kategaya ( Nature 2017 , 550 , 534 - 538 ) as specific inhibitors of USP7. Fragment based lead discovery (FBLD) by NMR combined with virtual screening and re-mining of biochemical high-throughput screening (HTS) hits led to the discovery of a series of ligands that bind in the "palm" region of the catalytic domain of USP7 and inhibit its catalytic activity. These ligands were then optimized by structure-based design to yield cell-active molecules with reasonable physical properties. This discovery process not only involved multiple techniques working in concert but also illustrated a unique way in which hits from orthogonal screening approaches complemented each other for lead identification.

Targeting the overexpressed USP7 inhibits esophageal squamous cell carcinoma cell growth by inducing NOXA‐mediated apoptosis

“…Several inhibitors targeting USP7/HAUSP were screened and exhibited anti‐tumor effect . Chauhan et al discovered that P5091 exhibited specific and selective deubiquitylating activity against USP7 and did not inhibit other DUBs.…”

“…Several inhibitors targeting USP7/HAUSP were screened and exhibited anti-tumor effect. 16,25,[28][29][30][31][32] Chauhan et al 25 discovered that P5091 exhibited specific and selective deubiquitylating activity against USP7 and did not inhibit other DUBs. P5091 significantly inhibited HCT116 (wt) cells growth; conversely, a slight effect on HCT116 USP7 −/− cells, which suggested that P5091 specifically targeted USP7.…”

Small molecules that target the ubiquitin system

“…Medicinal chemistry optimization of a fragment yielded the small molecules GNE6640 (20) and GNE6776 (21) shown in Figure 2. This class of allosteric inhibitors has been demonstrated to bind to the 'palm' region of the USP7 catalytic domain thereby impeding ubiquitin binding (orange, Figure 2B) [99,100]. NMR-based saturation transfer difference (STD) experiments showed that these compounds bind to a novel functional site 12 Å away from the catalytic cysteine residue [100].…”

“…This class of allosteric inhibitors has been demonstrated to bind to the 'palm' region of the USP7 catalytic domain thereby impeding ubiquitin binding (orange, Figure 2B) [99,100]. NMR-based saturation transfer difference (STD) experiments showed that these compounds bind to a novel functional site 12 Å away from the catalytic cysteine residue [100]. These molecules potently inhibited the activity of USP7 (IC50 values of 1.34 μM and 0.75 μM, respectively) and did not inhibit the activity of a panel of 36 DUBs at concentrations of 100 μM, which is convincing evidence of high target selectivity [99].…”

Targeting ubiquitin specific protease 7 in cancer: A deubiquitinase with great prospects

“…For example, nuclear magnetic resonance‐based screening has directed to the development of GNE‐6640 and GNE‐6776. Both compounds can non‐covalently target and selectively inhibit USP7 relative to 36 other deubiquitinases and induce cell death with significant impact on cellular USP7, MDM2, and p53 signalling pathways . Recently a new class of USP7 inhibitors has been identified based on P5091 and P22077 as the lead compounds.…”