Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs

Abstract

Piperlongumine is a naturally occurring small molecule recently identified to be toxic selectively to cancer cells in vitro and in vivo. This compound was found to elevate cellular levels of reactive oxygen species (ROS) selectively in cancer cell lines. The synthesis of 80 piperlongumine analogs has revealed structural modifications that retain, enhance, and ablate key piperlongumine-associated effects on cells, including elevation of ROS, cancer cell death, and selectivity for cancer cells over nontransformed cell types. Structure/activity relationships suggest that the electrophilicity of the C2-C3 olefin is critical for the observed effects on cells. Furthermore, we show that analogs lacking a reactive C7-C8 olefin can elevate ROS to levels observed with piperlongumine but show markedly reduced cell death, suggesting that ROS-independent mechanisms, including cellular cross-linking events, may also contribute to piperlongumine's induction of apoptosis. In particular, we have identified irreversible protein glutathionylation as a process associated with cellular toxicity. We propose a mechanism of action for piperlongumine that may be relevant to other small molecules having two sites of reactivity, one with greater and the other with lesser electrophilicity.

Fig. 1.

(A) PL and its cellular phenotypes. (B) Convergent strategy for the synthesis of PL analogs. (C) PL reacts with small-molecule thiols at C3 under neutral conditions. Reaction time, 72 h.

Fig. 2.

Contribution of PL’s electrophilic functionalities to cellular phenotypes. (A) Measurement of cellular ATP as a surrogate of viability (CellTiter-Glo) and (B) cellular ROS levels (CM-H₂DCF-DA) in two cell lines. Data are expressed as mean ± SD for four (CTG) or three (ROS) independent experiments. (C) Representative fluorescence microscopy images of HeLa cells treated for 1 h with 20 μM PL, PL-H₂, or PL-2,3H₂.

Fig. 3.

Oligomerization of PL leads to greatly elevated potency for elevation of ROS and cell death in two cell lines. (A) Oligomers of PL: monomer (PL-MON), dimer (PL-DI), and trimer (PL-TRI). (B) ATP levels and (C) ROS levels in HeLa and H1703 cells after 48 h (ATP) or 1.5 h (ROS) treatment with the indicated concentrations of oligomer. Data are expressed as mean ± SD for four (CTG) or three (ROS) independent experiments.

Fig. 4.

PL analogs decouple ROS elevation and cellular viability. (A) ATP levels and (B) ROS levels in HeLa and H1703 cells after 48 h (ATP) or 1.5 h (ROS) treatment with the indicated concentrations of analog. Data are expressed as mean ± SD for four (CTG) or three (ROS) independent experiments.

Fig. 5.

PL’s C7-C8 olefin is unnecessary for depletion of cellular glutathione but essential for protein glutathionylation. (A) Levels of total cellular glutathione were measured after 3-h treatment of EJ (Left) or 6-h treatment of HeLa (Right) cells with the indicated concentrations of each analog. (B) Quantification of protein gluathionylation in HeLa cells after 6-h treatment, as detected by immunofluorescence using a monoclonal antibody against glutathione. (C) Protein glutathionylation induced by addition of GSSG is reversed by addition of dithiothreitol, but PL-induced glutathionylation is unchanged by DTT treatment. Data are expressed as mean ± SD for three (A and C) or four (B) independent experiments. (D) Representative fluorescence microscopy images showing protein glutathionylation following 6-h treatment with either DMSO, PL, or PL-H₂ (20 μM).

Fig. 6.

PL analogs show selective toxicity toward transformed human fibroblasts (BJ-ELR). Viability was measured by Crystal Violet staining after 48-h treatment with (A) PL-7, (B) PL-SO₂, (C) PL-DIM, or (D) PL-TRI. Data are expressed as mean ± SD for three independent experiments.

Fig. 7.

(A) Summary of the role of electrophilic functionalities of PL analogs on cellular assay performance. (B) Proposed model for PL-mediated protein glutathionylation of glutathione-binding proteins. Note that the proposed protein glutathionylation does not involve a direct reaction between the protein and glutathione. Rather, our model predicts that the glutathionylation involves a linking PL molecule between the protein and glutathione.