Ruberto, R. A. | Search bioRxiv

bioRxiv2020

Modulation of ferroptosis sensitivity by TXNRD1 in pancreatic cancer cells

AO_SCPLOWBSTRACTC_SCPLOWThe selenoprotein thioredoxin reductase 1 (TXNRD1) plays a central role in ameliorating oxidative stress. Inhibition of TXNRD1 has been explored as a means of killing cancer cells that are thought to develop an enhanced reliance on such antioxidant proteins. In the context of ferroptosis, a non-apoptotic form of oxidative cell death, TXNRD1 has been proposed to cooperate with the phospholipid hydroperoxidase enzyme glutathione peroxidase 4 (GPX4) to protect cells from the lethal accumulation of lipid peroxides. Here, we report our unexpected finding that in pancreatic cancer cells, CRISPR-Cas9-mediated loss of TXNRD1 confers protection from ferroptosis induced by small-molecule inhibition of GPX4. Insights stemming from mechanistic interrogation of this phenomenon suggest that loss of TXNRD1 results in increased levels of GPX4 protein, potentially by influencing availability of selenocysteine, a scarce amino acid required by both proteins for proper synthesis and function. Increased abundance of GPX4 protein, in turn, protects cells from the effects of small-molecule GPX4 inhibition. These findings implicate selenoprotein regulation in governing ferroptosis sensitivity. Furthermore, by delineating a relationship between GPX4 and TXNRD1 contrary to that observed in numerous other settings, our discoveries underscore the context-specific nature of ferroptosis circuitry and its modulators.

cell biology↗

bioRxiv2018

Targeting a Therapy-Resistant Cancer Cell State Using Masked Electrophiles as GPX4 Inhibitors

We recently discovered that inhibition of the lipid peroxidase GPX4 can selectively kill cancer cells in a therapy-resistant state through induction of ferroptosis. Although GPX4 lacks a conventional druggable pocket, covalent small-molecule inhibitors are able to overcome this challenge by reacting with the GPX4 catalytic selenocysteine residue to eliminate enzymatic activity. Unfortunately, all currently-reported GPX4 inhibitors achieve their activity through reactive chloroacetamide groups. We demonstrate that such chloroacetamide-containing compounds are poor starting points for further advancement given their promiscuity, instability, and low bioavailability. Development of improved GPX4 inhibitors, including those with therapeutic potential, requires the identification of new electrophilic chemotypes and mechanisms of action that do not suffer these shortcomings. Here, we report our discovery that nitrile oxide electrophiles, and a set of remarkable chemical transformations that generates them in cells from masked precursors, provide an effective strategy for selective targeting of GPX4. Our results, which include structural insights, target engagement assays, and diverse GPX4-inhibitor tool compounds, provide critical insights that may galvanize development of improved compounds that illuminate the basic biology of GPX4 and therapeutic potential of ferroptosis induction. In addition, our discovery that nitrile oxide electrophiles engage in highly selective cellular interactions and are bioavailable in their masked forms may be relevant for targeting other currently undruggable proteins, such as those revealed by recent proteome-wide ligandability studies.

biochemistry↗