Development of peptide-based reversing agents for p-glycoprotein-mediated resistance to carfilzomib

Abstract

Carfilzomib is a novel class of peptidyl epoxyketone proteasome inhibitor and has demonstrated promising activity in multiple clinical trials to treat patients with multiple myeloma and other types of cancers. Here, we investigated molecular mechanisms underlying acquired resistance to carfilzomib and a potential strategy to restore cellular sensitivity to carfilzomib. H23 and DLD-1 cells (human lung and colon adenocarcinoma cell lines) with acquired resistance to carfilzomib displayed marked cross-resistance to YU-101, a closely related proteasome inhibitor, and paclitaxel, a known substrate of Pgp. However, carfilzomib-resistant cells remained sensitive to bortezomib, a clinically used dipeptide with boronic acid pharmacophore. In accordance with these observations, carfilzomib-resistant H23 and DLD-1 cells showed marked upregulation of P-glycoprotein (Pgp) as compared to their parental controls, and coincubation with verapamil, a Pgp inhibitor, led to an almost complete restoration of cellular sensitivity to carfilzomib. These results indicate that Pgp upregulation plays a major role in the development of carfilzomib resistance in these cell lines. In developing a potential strategy to overcome carfilzomib resistance, we as a proof of concept prepared a small library of peptide analogues derived from the peptide backbone of carfilzomib and screened these molecules for their activity to restore carfilzomib sensitivity when cotreated with carfilzomib. We found that compounds as small as dipeptides are sufficient in restoring carfilzomib sensitivity. Taken together, we found that Pgp upregulation plays a major role in the development of resistance to carfilzomib in lung and colon adenocarcinoma cell lines and that small peptide analogues lacking the pharmacophore can be used as agents to reverse acquired carfilzomib resistance. Our findings may provide important information in developing a potential strategy to overcome drug resistance.

Fig 1

Effects of carfilzomib, YU-101, bortezomib and paclitaxel on cell growth of carfilzomib-resistant cell lines, H23/Carf (A) and DLD-1/Carf (B) in comparison to their respective parental cell lines. The closed circles are for carfilzomib-resistant cells while the open circles are for their parental cell lines. Results are represented as means ± S.D.

Fig 2

Upregulation of P-glycoprotein (Pgp) in carfilzomib-resistant cell lines is the major mechanism for carfilzomib resistance. A: immunoblotting analyses showing a marked increase in of Pgp expression in H23/Carf and DLD-1/Carf cells in comparison to their respective parental controls. B: RT-PCR analyses showing the upregulation of MDR1 mRNA in H23/Carf and DLD-1/Carf cell lines in comparison to their respective parental controls. C & D: Inhibition of Pgp using verapamil (40 μM) restores sensitivity to carfilzomib in H23/Carf and DLD-1/Carf cells. Results are represented as means ± S.D.

Fig 3

Comparison of peptide analogs with differing lengths for their reversing effects on carfilzomib resistance A: Chemical structures of carfilzomib and structurally related peptide analogs, compounds 1 to 4. B: Co-incubation with peptide analogs leads to a partial reversal of carfilzomib resistance. H23/Carf and DLD-1/Carf cells were treated with 25 μM of compounds 1 to 4 in the presence or absence of carfilzomib for 72 hours. Relative cell viability was measured using an ATP-based assay. Results are represented as means ± S.D. *, p < 0.0001, compared to the groups treated with vehicle alone, carfilzomib or peptide analogs alone, by the one-way ANOVA, followed by Bonferroni post testing.

Fig 4

Comparison of peptide analogs with the pyridine substitution at the N-cap site for their reversing effects on carfilzomib resistance A: Chemical structures of di- and tri-peptide analogs, compounds 5 to 7. B: Co-incubation of di- or tri-peptide analogs with the pyridine group substitution (compounds 5 to 7) restores carfilzomib sensitivity to a greater extent than their counterparts with the morpholino group. H23/Carf and DLD-1/Carf cells were treated with 25 μM of compounds 5 to 7 in the presence or absence of carfilzomib for 72 hours. Relative cell viability was measured using an ATP-based assay. Results are represented as means ± S.D. *, p < 0.0001, compared to the groups treated with vehicle alone, carfilzomib or peptide analogs alone, by the one-way ANOVA, followed by Bonferroni post testing.

Fig 5

Comparison of peptide analogs with the Weinreb amide substitution at the C-terminus for their reversing effects on carfilzomib resistance A: Chemical structures of di- and tri-peptide analogs, compounds 8 to 10. B: Co-incubation of di- or tri-peptide analogs with the Weinreb amide substitution (compounds 8 to 10) restores carfilzomib sensitivity to a greater extent than their counterparts with the benzyl ester group. H23/Carf and DLD-1/Carf cells were treated with 25 μM of compounds 8 to 10 in the presence or absence of carfilzomib for 72 hours. Relative cell viability was measured using an ATP-based assay. Results are represented as means ± S.D. *, p < 0.0001, compared to the groups treated with vehicle alone, carfilzomib or peptide analogs alone by the ANOVA, followed by Bonferroni post testing.

Fig 6

Peptide analogs (compounds 8 to 10) show the ability to reverse acquired resistance to carfilzomib and paclitaxel and to inhibit Pgp-mediated extrusion of calcein. Results are represented as means ± S.D. A & B: Compounds 8 to 10 restore cellular sensitivity to carfilzomib in concentration-dependent manners in DLD-1/Carf (A) and H23/Carf cells (B). *, p < 0.001 compared to the groups treated with vehicle alone, carfilzomib alone or peptide analogs alone by the one-way ANOVA followed by Bonferroni post testing. C: Compounds 8 to 10 (12.5 μM) can also reverse cross-resistance to paclitaxel (2 μM) in DLD-1/Carf and H23/Carf cells. **, p<0.0001, compared to groups treated with vehicle alone, drug alone, or peptide analogs only by the one-way ANOVA followed by Bonferroni post testing. D: Compounds 8 to 10 (25 μM) lead to increased cellular retention of calcein, a P-glycoprotein substrate, in DLD-1/Carf and H23/Carf cells. *, p < 0.05 compared to the group treated with vehicle alone by Student’s t-test; †p < 0.0125 (Bonferroni-corrected p value threshold).

Fig 7

Peptide analogs (compounds 8 to 10) potentiate the sensitivity of the parental DLD-1 and H23 cells to carfilzomib. Results are represented as means ± S.D. A & B: Compounds 8 to 10 potentiate the effect of carfilzomib (15 nM) in the parental DLD-1 (A) and H23 (B) cells. *, p < 0.001 compared to the groups treated with vehicle alone, carfilzomib alone or peptide analogs alone by the one-way ANOVA followed by Bonferroni post testing.