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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C

Poster I3 - Investigation on the role of hemp extracts on the enzymatic activity of members of the ubiquitin-proteasome system (UPS)

Friday, October 25, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  The ubiquitin-proteasome system (UPS) is the primary pathway governing the degradation of misfolded or deregulated proteins that involves a cascade of three different enzymes which culminate in the conjugation of a polyubiquitin chain to a target protein for recognition by the proteasome, where it is then degraded. Protein ubiquitination is a reversible process where deubiquitinating enzymes (DUBs) cleave the polyubiquitin chain to rescue the protein from degradation. Recent studies have identified UPS-targeting drugs, including the proteasome inhibitors Bortezomib and Carfilzomib, can aid in the treatment of diseases that upregulate the UPS like multiple myeloma (MM) and lung cancer. While these inhibitors have shown clinical success, they are oftentimes coupled with severe side effects including nausea and peripheral neuropathy, motivating the search for new or alternative therapies for UPS inhibition. One area that has received limited attention is the role of hemp extracts, like cannabidiol (CBD), on the enzymatic activity on members of the UPS. Various studies have explored the antiproliferative effects of hemp and CBD through the observation of various biochemical pathways, and it has been shown to interfere with several intracellular signaling systems which can inhibit the expression of some genes or up- or down-regulate the function some proteins; however, its role on members of the UPS have not been explored. The goal of this work was to evaluate the effect of CBD and hemp extracts on UPS activity, as well as potential synergy effects when these compounds were co-dosed with established proteasome or DUB inhibitors.

Materials and

Methods:  To quantify enzyme activity, we utilized fluorometry using commercially available DUB and proteasome reporters in lysates obtained from model multiple myeloma (OPM2) and lung cancer (A549) cell lines. These peptide-based fluorescence reporters contain a C-terminal fluorophore (AMC, 7-amino-4-methylcoumarin) that only emits a fluorescence signal when cleaved by the enzyme. The DUB reporter used was Z-LRGG-AMC where the LRGG recognition sequence are the final four amino acids on ubiquitin and the proteasome reporter was Suc-LLVY-AMC where LLVY is the proteasome-specific recognition sequence. Hemp extracts were collected and purified at the LSU AgCenter while CBD, PR-619, and MG-132 were purchased from commercial sources. Drug experiments were performed at three concentrations of either CBD or hemp extracts (10, 100, and 250 µM) and co-dosed with three concentrations around the IC50 values of the known DUB inhibitor PR-619 and proteasome inhibitor MG-132. We utilized a 96-well plate to allow for high-throughput screening with each measurement performed in triplicate. The lysates were pre-incubated with the hemp-derived compounds and/or UPS inhibitors for 1 hour at 37°C followed by addition of the peptide reporter. Time-dependent fluorometric measurements were performed every 30 min over the course of ten hours. A DMSO vehicle control was also performed to obtained baseline enzymatic activity. The obtained fluorescent signals were background subtracted against the baseline signal of buffer and unreacted peptide reporter.

Results, Conclusions, and Discussions: We hypothesized that cells treated with CBD and/or hemp extracts would exhibit diminished DUB and proteasome activity similar to how they respond to commercial inhibitors PR-619 and MG-132. Preliminary fluorometric data was performed investigating how hemp extracts and pure CBD altered DUB and proteasome activity in OPM2 lysates. We pre-treated OPM2 lysates with increasing doses of either hemp extract (Fig. 1A, C) or CBD (Fig. 1B, D) and quantified enzyme activity using the two commercially available peptide reporters for the proteasome (Fig. 1A-B) or DUBs (Z-LRGG-AMC; Fig. 1C-D). We found that pre-treatment with hemp extracts resulted in a significant concentration-dependent decrease in both proteasome and DUB activity (Fig. 1A, C); however, the extent of inhibition was diminished in the lysates pre-treated with only CBD (Fig. 5B, D). This preliminary data supports both the potential for hemp extracts as inhibitors of members of the UPS and the concept of the “entourage effect” of using hemp extracts instead of pure CBD. Additionally, we evaluated the specificity of the two reporters for their enzymatic targets. Preliminary data examining enzyme activity of the DUB (Fig. 2A) and proteasome (Fig. 2B) reporters in the presence of MG-132, PR-619, or DMSO vehicle support the specificity of the two recognition sequences. A student’s t-test at the 360 minute time point showed no significant difference between DMSO and MG-132 for Z-LRGG-AMC (Fig. 2A) or DMSO and PR-619 for Z-LLVY-AMC (Fig. 2B), whereas there was a significant difference for the DUB and proteasome specific inhibitors and reporters (Fig. 2). Ongoing work is underway performing additional biological and technical replicates in addition to co-dosing experiments of lysates with CBD or hemp extracts and established UPS inhibitors. These findings provide new insight on the role of hemp extracts and CBD as potential modulators of the UPS and companion therapies to be used with more aggressive therapeutics.

Acknowledgements (Optional):