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A Micropatterning Assay for Measuring Cell Chirality

A Micropatterning Assay for Measuring Cell Chirality

Published on: March 11, 2022

Chirality and asymmetry increase the potency of candidate ADRM1/RPN13 inhibitors

Ravi K Anchoori^1,2, Logan George¹, Ssu-Hsueh Tseng¹

¹Department of Pathology, The Johns Hopkins University, Baltimore, Maryland, United States of America.

|September 10, 2021

Abstract:

Bortezomib and the other licensed 20S proteasome inhibitors show robust activity against liquid tumors like multiple myeloma, but have disappointed against solid tumors including ovarian cancer. Consequently, interest is mounting in alternative non-peptide based drugs targeting the proteasome's 19S regulatory particle subunit, including its ubiquitin receptor RPN13. RA183 and RA375 are more potent analogs of the prototypic inhibitor of RPN13 (iRPN13) called RA190, and they show promise for the treatment of ovarian cancer. Here we demonstrate that rendering these candidate RPN13 inhibitors chiral and asymmetric through the addition of a single methyl to the core piperidone moiety increases their potency against cancer cell lines, with the S-isomer being more active than the R-isomer. The enhanced cancer cell cytotoxicities of these compounds are associated with improved binding to RPN13 in cell lysates, ATP depletion by inhibition of glycolysis and mitochondrial electron chain transport, mitochondrial depolarization and perinuclear clustering, oxidative stress and glutathione depletion, and rapid accumulation of high molecular weight polyubiquitinated proteins with a consequent unresolved ubiquitin proteasome system (UPS) stress response. Cytotoxicity was associated with an early biomarker of apoptosis, increased surface annexin V binding. As for cisplatin, BRCA2 and ATM deficiency conferred increased sensitivity to these iRPN13s. Ubiquitination plays an important role in coordinating DNA damage repair and the iRPN13s may compromise this process by depletion of monomeric ubiquitin following its sequestration in high molecular weight polyubiquitinated protein aggregates. Indeed, a synergistic cytotoxic response was evident upon treatment of several ovarian cancer cell lines with either cisplatin or doxorubicin and our new candidate iRPN13s, suggesting that such a combination approach warrants further exploration for the treatment of ovarian cancer.

Insights

New chiral RPN13 inhibitors show enhanced potency against ovarian cancer cells by disrupting cellular energy and protein degradation pathways. These compounds, particularly the S-isomer, demonstrate promise for combination therapies with existing chemotherapeutics.

Area of Science:

Biochemistry
Molecular Biology
Oncology

Background:

20S proteasome inhibitors like bortezomib are effective against liquid tumors but not solid tumors such as ovarian cancer.
Research is shifting towards targeting the 19S proteasome regulatory subunit, specifically the ubiquitin receptor RPN13, with non-peptide drugs.
RA183 and RA375 are potent inhibitors of RPN13 (iRPN13) with potential for ovarian cancer treatment.

Purpose of the Study:

To investigate the impact of chirality on the potency of RPN13 inhibitors (RA183 and RA375) against cancer cell lines.
To elucidate the molecular mechanisms underlying the enhanced cytotoxicity of chiral RPN13 inhibitors.
To explore the potential of these novel inhibitors as a therapeutic strategy for ovarian cancer, including combination therapies.

Main Methods:

Synthesized chiral analogs of RA183 and RA375 by introducing a methyl group to the piperidone moiety.
Assessed cytotoxicity against ovarian cancer cell lines and measured binding affinity to RPN13.
Analyzed cellular effects including ATP levels, mitochondrial function, oxidative stress markers, and protein ubiquitination.
Investigated sensitivity in BRCA2 and ATM-deficient cells and evaluated synergistic effects with cisplatin and doxorubicin.

Main Results:

Chiral induction, specifically the S-isomer, significantly increased the potency of RPN13 inhibitors against cancer cell lines.
Enhanced cytotoxicity correlated with improved RPN13 binding, ATP depletion, mitochondrial dysfunction, oxidative stress, and accumulation of polyubiquitinated proteins.
Increased sensitivity was observed in BRCA2 and ATM-deficient cells, similar to cisplatin.
Combination treatment with cisplatin or doxorubicin resulted in synergistic cytotoxicity.

Conclusions:

Chiral RPN13 inhibitors represent a promising new class of anti-cancer agents, particularly for ovarian cancer.
The mechanism involves multifaceted cellular disruption, including proteasome inhibition and energy depletion.
These novel inhibitors may enhance efficacy when combined with DNA-damaging agents like cisplatin and doxorubicin, warranting further clinical investigation.

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