Discovery of PI-1840, a novel noncovalent and rapidly reversible proteasome inhibitor with anti-tumor activity

Abstract

The proteasome inhibitor bortezomib is effective in hematologic malignancies such as multiple myeloma but has little activity against solid tumors, acts covalently, and is associated with undesired side effects. Therefore, noncovalent inhibitors that are less toxic and more effective against solid tumors are desirable. Structure activity relationship studies led to the discovery of PI-1840, a potent and selective inhibitor for chymotrypsin-like (CT-L) (IC50 value = 27 ± 0.14 nm) over trypsin-like and peptidylglutamyl peptide hydrolyzing (IC50 values >100 μm) activities of the proteasome. Furthermore, PI-1840 is over 100-fold more selective for the constitutive proteasome over the immunoproteasome. Mass spectrometry and dialysis studies demonstrate that PI-1840 is a noncovalent and rapidly reversible CT-L inhibitor. In intact cancer cells, PI-1840 inhibits CT-L activity, induces the accumulation of proteasome substrates p27, Bax, and IκB-α, inhibits survival pathways and viability, and induces apoptosis. Furthermore, PI-1840 sensitizes human cancer cells to the mdm2/p53 disruptor, nutlin, and to the pan-Bcl-2 antagonist BH3-M6. Finally, in vivo, PI-1840 but not bortezomib suppresses the growth in nude mice of human breast tumor xenografts. These results warrant further evaluation of a noncovalent and rapidly reversible proteasome inhibitor as potential anticancer agents against solid tumors.

Keywords: Anticancer Drug; Apoptosis; Bcl-2 Family Proteins; CT-L; Cancer Therapy; Cell Proliferation; Noncovalent; PI-1840; Proteasome; p53.

FIGURE 1.

PI-1833 and its potent analog PI-1840 are selective inhibitors for CT-L activity in vitro. A, chemical structures of PI-1833 and PI-1840 with their IC₅₀ values against different proteasome activities. B, effects of PI-1840 and bortezomib on the CT-L activities of the 20 S constitutive rabbit proteasome and 20 S human immunoproteasome. Data are representative of at least two independent experiments.

FIGURE 2.

Lactacystin but not PI-1840 binds covalently to CT-L of the 20 S proteasome. A–C, LC-MS/MS analysis. Purified rabbit 20 S proteasome was incubated either with vehicle (A), PI-1840 (B), or lactacystin (C), and the tryptic digests were analyzed by LC-MS/MS as described under “Experimental Procedures.” The b ions (red) and y ions (blue) designate the N and C termini of the peptide, respectively. The number next to each ion represents the number of amino acids in that fragment (i.e. y4 = LAFK from C terminus of the peptide).

FIGURE 3.

A, unmodified TTTLAFK peptide; B, clasto-lactacystin-modified TTTLAFK peptide; C, dialysis. Purified rabbit 20 S proteasome was incubated with vehicle control, 1 μm PI-1840, or 2.5 μm lactacystin and was subjected to dialysis at 4 °C for different lengths of time as described under “Experimental Procedures.” Percentage of CT-L activity (relative to vehicle-treated control samples) was then determined at different time points. Data are representative of two (LC/MS-MS) and three (dialysis) independent experiments.

FIGURE 4.

Selective inhibition of the proteasomal CT-L activity in whole cells, accumulation of proteasome substrate proteins, inhibition of cell survival pathways, and induction of apoptosis. A, exponentially growing human breast cancer MDA-MB-468 cells were treated with 5 μm PI-1840 for the indicated time points, followed by measurement of CT-L, T-L, and PGPH activities in whole cell extracts as described under “Experimental Procedures.” B and C, human breast cancer MDA-MB-468 cells were treated with the indicated concentrations of PI-1833 and PI-1840 for 48 h, followed by Western blot assay using the indicated antibodies as described under “Experimental Procedures.” Data are representative of at least two independent experiments. PARP, poly(ADP-ribose) polymerase. Lane D, DMSO.

FIGURE 5.

Antitumor efficacy of PI-1840 and bortezomib against human breast cancer MDA-MB-231 xenografts in nude mice. Mice bearing MDA-MB-231 tumors were treated with vehicle (closed squares), PI-1840 (open circles), or bortezomib (filled circles) as described under “Experimental Procedures.” A, representative tumor growth curves form vehicle-, PI-1840-, and bortezomib-treated mice. B, average percent change in tumor volumes from mice treated with vehicle, PI-1840, and bortezomib. There were statistically significant differences between vehicle and PI-1840 in the average percent change in tumor volume on every day of measurement with p values of 0.006, 0.019, 0.009, 0.018, 0.039, and 0.045 on days 3, 5, 7, 10, 12, and 14 of treatment, respectively. In contrast, there were no statistically significant differences between vehicle and bortezomib in the average percent change in volume with p values of 0.243, 0.751, 0.951, 0.842, 0.709, and 0.786 on days 3, 5, 7, 10, 12, and 14 of treatment, respectively.