Optimization and Anti-Cancer Properties of Fluoromethylketones as Covalent Inhibitors for Ubiquitin C-Terminal Hydrolase L1

Abstract

The deubiquitinating enzyme (DUB) UCHL1 is implicated in various disease states including neurodegenerative disease and cancer. However, there is a lack of quality probe molecules to gain a better understanding on UCHL1 biology. To this end a study was carried out to fully characterize and optimize the irreversible covalent UCHL1 inhibitor VAEFMK. Structure-activity relationship studies identified modifications to improve activity versus the target and a full cellular characterization was carried out for the first time with this scaffold. The studies produced a new inhibitor, 34, with an IC50 value of 7.7 µM against UCHL1 and no observable activity versus the closest related DUB UCHL3. The molecule was also capable of selectively inhibiting UCHL1 in cells and did not demonstrate any discernible off-target toxicity. Finally, the molecule was used for initial probe studies to assess the role of UCHL1 role in proliferation of myeloma cells and migration behavior in small cell lung cancer cells making 34 a new tool to be used in the biological evaluation of UCHL1.

Rational Development and Characterization of a Ubiquitin Variant with Selectivity for Ubiquitin C-Terminal Hydrolase L3

“…The recombinant UCHL3 and UCHL1 proteins were recombinantly expressed and purified according to our previously published protocols [ 20 , 29 , 30 ] and summarized below. A pET-15b plasmid construct was used for the expression of both hexa-histidine (His 6 )-tagged UCHL1 and His 6 -UCHL3 in bacterial culture.…”

“…The inhibition assays were carried out as previously described [ 20 , 29 , 30 ] and summarized below. UbVs were buffer exchanged into 50 mM Tris-HCl containing 0.5 mM EDTA pH 7.6 using 0.5 mL Zeba spin desalting columns (ThermoScientific, catalog number 89882).…”

Activity-Based Diagnostics: Recent Advances in the Development of Probes for Use with Diverse Detection Modalities

“…To address this limitation, fluorescein amidite (FAM) was conjugated to the iron oxide nanoparticles through an MMP9-specific substrate, resulting in accumulation of the FAM signal in the margins of the tumor in several xenograft models (Figure 5D). 87 This colocalization is further improved in vivo through additional conjugation of tumor-directing peptides onto the nanoparticles, such as an acidosis-mediated tumor targeting sequence. 88 Directing the in vivo localization of these nanoparticles improves the diagnostic ability of the platform by concentrating the ABNs to tumor tissue and thereby decreasing the chance of off-target processing in healthy tissues that also express MMP9.…”

“…By conjugating peptide sequences to these nanoparticles which couple MMP9-specific substrates with peptide sequences that direct hydrolyzed products toward renal clearance, a variety of xenograft and spontaneous tumor models have been sensitively detected by monitoring urine for cleaved MMP9 peptides via mass spectrometry or ELISA (Figure C). , Although this method enables noninvasive diagnosis of cancer, it provides no spatial location of where enzymatic processing of the MMP9 substrate occurred in vivo . To address this limitation, fluorescein amidite (FAM) was conjugated to the iron oxide nanoparticles through an MMP9-specific substrate, resulting in accumulation of the FAM signal in the margins of the tumor in several xenograft models (Figure D) . This colocalization is further improved in vivo through additional conjugation of tumor-directing peptides onto the nanoparticles, such as an acidosis-mediated tumor targeting sequence .…”

Covalent Fragment Screening and Optimization Identifies the Chloroacetohydrazide Scaffold as Inhibitors for Ubiquitin C-terminal Hydrolase L1

“…42 Our group performed SAR around this scaffold, showing that an alkyne-functionalized version of VAEFMK (Figure 1) had improved potency and selectively engaged UCHL1 in cells. 43 The majority of UCHL1 inhibitors are from the covalent cyanopyrrolidine-based scaffold, including IMP1710 38 and GK13S 44 (Figure 1). They demonstrated submicromolar potency, selectivity over other DUBs, and improvements in both off-target profile and toxicity compared to previous generations of cyanopyrrolidine UCHL1 inhibitors.…”