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

Abstract

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.

Cytochrome P450 oxidoreductase contributes to phospholipid peroxidation in ferroptosis

“…We employed two approaches to rule out potential POR-dependent ML210/RSL3 processing events. First, we synthesized alkyne analogs of ML210 and RSL3 -namely ML210-yne (3) and RSL3-yne (4) 15 , both of which preserved their ferroptosis-inducing activities in a POR-dependent manner (Figs. 3a-b, Supplementary Fig.…”

“…Distinct from other GPXs, GPX4 is the only enzyme identified thus far that acts on phospholipid hydroperoxides 6,13 . Hence, inhibitors of GPX4, including ML210, ML162 and 1S,3R-RSL3 (RSL3), are used as specific inducers of ferroptosis in cells with high levels of polyunsaturated lipids -lipids that are highly susceptible to oxidation 1,14,15 .…”

Structure–activity Relationships of Glutathione Peroxidase 4 Inhibitor Warheads

“…We found that the chloroacetamide group cannot be replaced with other less-reactive electrophiles (2-9). These include α-chlorofluoroacetamide (2), other haloacetamides (3)(4)(5)(6), and acrylamide (7). However, we observed that the propiolamide analog 8 exhibited potent ferroptosis induction with improved selectivity compared to ML162.…”

“…[1][2][3] Induction of ferroptosis via inhibition of GPX4 has promising therapeutic potential, especially for targeting cancer cells that are otherwise therapy resistant. [4][5][6] However, GPX4 represents a difficult-to-drug target because it has a shallow active site that is not amenable to interacting with small molecules. 7,8 This topology constitutes a unique feature among the broader family of glutathione peroxidases, enabling reduction of structurally-diverse lipid hydroperoxide substrates.…”

Targeting Scavenger Receptor Type B1 In Cholesterol-Addicted Lymphomas Abolishes Glutathione Peroxidase 4 and Results in Ferroptosis

“…This has driven significant interest in the development of treatments to induce ferroptosis in cancer cells. Small molecule inhibitors of GPX4 have been developed and tested, but they are toxic and lack specificity (15), which limits in vivo use and clinical relevance.…”