Effect of peptide-based caspase inhibitors on SKNAS cells as determined using the Caspase-Glo® 6 assay.

<p>SKNAS cells were treated with Ac-VEID-CHO (•) or Ac-DEVD-CHO (▪) prior to addition of 3 µM staurosporine for 6 hours and detection of VEID-ase activity as described in Experimental Procedures. Concentration inhibition curves were performed in duplicate and represent 1 of at least 3 experiments with similar results. Concentration-response curves for each inhibitor were normalized to zero and 100% based on no staurosporine or DMSO, respectively. The mean and standard error of the mean are reported.</p

Comparison of potency of peptide-derived caspase inhibitors in cellular lamin cleavage assay, enzymatic activity and cell permeability.

<p>nd = not determined</p><p>a = z-VEID-TFPM enzymatic IC₅₀ is less than 0.0017 due to limit of enzymatic assay detection.</p><p>*Enzymatic IC₅₀ values were determined after a 15 minute enzyme/inhibitor preincubation and 40 minute enzyme reaction.</p

Effect of peptide-based caspase inhibitors on the generation of cleaved lamin A/C in SKNAS cells.

<p>(A) SKNAS cells were treated with z-VEID-FMK (♦), z-DEVD-FMK (▪), Q-VD-OPh (•), Ac-VEID-CHO (▴) or Ac-DEVD-CHO (○) prior to addition of 3 µM staurosporine for 6 hours. (B) SKNAS cells were treated with z-ID-TFPM (•), z-EID-TFPM (▴) or z-VEID-TFPM (▪) prior to addition of 3 µM staurosporine for 6 hours. Detection of the small lamin A/C cleavage product was performed as described in Experimental Procedures. Concentration inhibition curves were performed in duplicate and represent 1 of at least 3 experiments with similar results. Concentration-response curves for each inhibitor were normalized to zero and 100% based on no staurosporine or DMSO, respectively. The mean and standard error of the mean are reported.</p

Apoptosis-mediated cleavage of lamin A/C is elevated in wild-type relative to caspase-6 KO fibroblasts.

<p>Fibroblasts derived from caspase-6 KO (▪) or wild type (•) mice were treated with the indicated concentration of staurosporine for 6 hours prior to detection of the small lamin A/C cleavage product. The assay was performed in quadruplicate two times with similar results; mean and standard error of the mean are reported.</p

Potency of peptide-derived caspase inhibitors possessing aldehyde (CHO) and fluoromethyl ketone (FMK) warheads against executioner caspases.

<p>*IC₅₀ values were determined after a 15 minute enzyme/inhibitor preincubation and 40 minute enzyme reaction. Due to the capacity for time-dependent inhibition with irreversible inhibitors the values reported can be considered “apparent IC₅₀”.</p

Effect of staurosporine on the generation of cleaved lamin A/C in SKNAS cells.

<p>SKNAS cells were treated with the indicated concentration of staurosporine for 6 hours prior to detection of the small lamin A/C cleavage product as described in Experimental Procedures. The assay was performed in triplicate one time. The mean and standard error of the mean are reported.</p

Western blot detection of lamin A/C from SKNAS neuroblastoma cells upon staurosporine treatment.

<p>(A) Schematic of the N- and C-terminal globular domains of Lamin A/C with VEID-containing central α-helical region as the site of caspase-6 proteolysis. (B) SKNAS cells were treated with DMSO control or staurosporine for 6 hours prior to cell lysis. Lysates were probed for small lamin A/C subunit (Lanes 1–2), large lamin A/C subunit (Lanes 3–4) or total lamin A/C (Lanes 5–6). (C) SKNAS cells were treated with DMSO or staurosporine for the indicated time prior to cell lysis. Lysates were probed for large lamin A/C subunit (red) or β-Actin (green).</p

Western blot detection of recombinant GST-lamin A processing by purified caspases.

<p>(A) The indicated concentration of caspase-6 was incubated with GST-lamin A for two hours. (B) 300 Units of caspases 1–9 were incubated with GST-lamin A for two hours. Intact and cleaved lamin A were detected via western blotting using anti-GST antibody.</p

Effect of staurosporine on the release of Lactate Dehydrogenase in SKNAS cells.

<p>SKNAS cells were treated with the indicated concentration of staurosporine for 2 (•), 4 (▪), 6 (▴) or 8 (♦) hours prior to detection of LDH release to the cell supernatant. The assay was performed in triplicate and represents 1 of at least 2 experiments with similar results. The data was normalized to fold increase over DMSO treatment. The mean and standard error of the mean are reported.</p

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

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