Structure-activity relationships of GPX4 inhibitor warheads

Abstract

Direct inhibition of GPX4 requires covalent modification of the active-site selenocysteine. While phenotypic screening has revealed that activated alkyl chlorides and masked nitrile oxides can inhibit GPX4 covalently, a systematic assessment of potential electrophilic warheads with the capacity to inhibit cellular GPX4 has been lacking. Here, we survey more than 25 electrophilic warheads across several distinct GPX4-targeting scaffolds. We find that electrophiles with attenuated reactivity compared to chloroacetamides are unable to inhibit GPX4 despite the expected nucleophilicity of the selenocysteine residue. However, highly reactive propiolamides we uncover in this study can substitute for chloroacetamide and nitroisoxazole warheads in GPX4 inhibitors. Our observations suggest that electrophile masking strategies, including those we describe for propiolamide- and nitrile-oxide-based warheads, may be promising for the development of improved covalent GPX4 inhibitors.

Keywords: Covalent inhibitors; Ferroptosis; GPX4; Masked electrophiles.

Figure 1.

Synthesis and assessment of ML162 analogs. (A) Chemical structures of GPX4 inhibitors. (B) Preparation of ML162 analogs via Ugi 4-component reactions. i.) MeOH, 20 °C, 2–16 h, 39–87% yield. (C) Viability assessment and fer-1 (1.5 μM) rescue experiments in LOX-IMVI cells.

Figure 2.

Electrophilic warhead SAR with ML210 benzhydrylpiperazine scaffold. Cell viability measurements and fer-1 (1.5 μM) rescue experiments in LOX-IMVI cells.

Figure 3.

(A) Assessment of nitroisoxazole warheads on GPX4 inhibitor scaffolds. (B) Synthesis of nitrolic acid 40. (C) The LOX-IMVI cell-killing activity of 40 can be suppressed by fer-1 co-treatment (1.5 μM). Compound 39 does not affect cell viability at the concentrations tested.

Figure 4.

Characterization of low-MW nitroisoxazole GPX4 inhibitors. (A) Chemical structures of 43 and 44. (B) 43 and 44 induce ferroptosis in LOX-IMVI cells. Data are plotted as mean ± s.e.m. of n = 2 biological experiments performed in duplicate. See also Supplementary Table 1. (C) Assessment of 44 (100 μM, 16 h) proteome-wide reactivity reveals labeling of a major target protein at ~38 kDa. (D) Treatment of LOX-IMVI cells with 44 (100 μM, 16 h) enables pulldown of GPX4. (E) Compound 44 covalently binds GAPDH.