Selective covalent targeting of GPX4 using masked nitrile-oxide electrophiles

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.

Modulation of ferroptosis sensitivity by TXNRD1 in pancreatic cancer cells

“…In concurrence with results from the initial genome-scale screen, knockout of TXNRD1 in these cells conferred decreased sensitivity to the ferroptosis-inducing small molecules ML210, (1S,3R)-RSL3 (hereafter referred to as RSL3), and erastin (Figures 1A,C and S1-S3). 1,2,22 This decreased sensitivity was specific to ferroptosis inducers, as TXNRD1 loss did not impact cell killing by a range of other compounds representing targeted therapies, general ROS inducers, and other cytotoxic substances (Figures 1D and S1-S3). These results were recapitulated in another PDAC cell line, PANC-1, in which ablation of TXNRD1 yielded similar protection against ferroptosis, thereby suggesting that this phenomenon may be more broadly extensible to other pancreatic cancer cells ( Figures S4 and S5).…”

“…ML210 and (1S,3R)-RSL3 were prepared in accordance with previously published procedures. 2,22,41 Doxorubicin, erastin, paclitaxel, and piperlongumine were purchased from SelleckChem. Auranofin, ferrostatin-1 (fer-1), and sodium selenite were purchased from Sigma-Aldrich.…”

“…23 Such transformations can be critical for activation of prodrug molecules; for example, the covalent GPX4 inhibitor ML210 requires conversion to its active form in live cells. 22 We assessed direct interaction between GPX4 and ML210 through label-free cellular thermal shift assay (CETSA) 24,25 within intact KP-4 WT and TXNRD1 -/cells. We observed thermal stabilization of GPX4 in the ML210-treated condition irrespective of TXNRD1 knockout (Figure 2A,B).…”

Role of GPX4-Mediated Ferroptosis in the Sensitivity of Triple Negative Breast Cancer Cells to Gefitinib

“…Previous study has identi ed a potential therapeutic bene t of blocking EGFR with ge tinib in basal-like subtypes of TNBC in vitro [21]). GPX4 is a promising target for killing therapy-resistant cancer cells via ferroptosis [33]. However, the role of GPX4-mediated ferroptosis in ge tinib sensitivity in TNBC cells is largely unknown.…”

Targeting the Redox Landscape in Cancer Therapy

“…A number of published studies show the involvement of GPx proteins in tumorigenesis and chemotherapy resistance, which is summarized in these reviews [195,196]. Currently, there is no selective inhibitor for GPx proteins for therapeutic application; however, recent developments in medicinal chemistry show promising advancements for targeting GPx1 [197] and GPx4 [198].…”