α,β-Unsaturated carbonyl system of chalcone-based derivatives is responsible for broad inhibition of proteasomal activity and preferential killing of human papilloma virus (HPV) positive cervical cancer cells

Abstract

Proteasome inhibitors have potential for the treatment of cervical cancer. We describe the synthesis and biological characterization of a new series of 1,3-diphenylpropen-1-one (chalcone) based derivatives lacking the boronic acid moieties of the previously reported chalcone-based proteasome inhibitor 3,5-bis(4-boronic acid benzylidene)-1-methylpiperidin-4-one and bearing a variety of amino acid substitutions on the amino group of the 4-piperidone. Our lead compound 2 (RA-1) inhibits proteasomal activity and has improved dose-dependent antiproliferative and proapoptotic properties in cervical cancer cells containing human papillomavirus. Further, it induces synergistic killing of cervical cancer cell lines when tested in combination with an FDA approved proteasome inhibitor. Exploration of the potential mechanism of proteasomal inhibition by our lead compound using in silico docking studies suggests that the carbonyl group of its oxopiperidine moiety is susceptible to nucleophilic attack by the γ-hydroxythreonine side chain within the catalytic sites of the proteasome.

Figure 1. Effect of compounds 2–5 treatment on the levels of poly-ubiquitinated proteins in HeLa cervical cancer cells

Immunoblot analysis of ubiquitinated proteins in HeLa cervical cancer cells eight hours after treatment with or without compounds 2–5 at the indicated concentrations. Bortezomib was used as positive control. Equal protein loading in each lane, was verified by using and antibody against β-actin.

Figure 2. Inhibition of the 20S proteasome activity by compounds 2–4

20S purified proteasomes were treated for 30 min. with of without compounds 2–4 and with positive control at the indicated concentrations following addition of the specific fluorogenic substrates for chymotrypsin-like, trypsin-like and peptidylglutamyl peptide hydrolyzing-like hydrolytic proteasome capacities. Representative examples of two independent experiments.

Figure 3. Effect of compound 2 treatment upon cervical cancer cell lines versus normal cells

Cultures of HPV-transformed cervical cancer cells (SiHa and ME180) or primary human Keratinocytes were treated with the indicated concentrations of compound 2 for 48 hours. Cell viability was determined by XTT assay and plotted as a fraction of the untreated control cultures.

Figure 4. Sub-optimal doses of Bortezomib and lead compound 2 results in an increased bioluminescence from Ub-FL as compared to single agent alone

Transiently transfected firefly luciferase (CMV-FL) and Ub-FL HeLa cervical cancer cells were either mock treated or treated with Bortezomib, lead compound 2 or combination at the indicated concentrations for 8 hours. Luciferase activity on cell lysate was quantified in Relative Luminescence Units (RLU) and expressed as % of control. Error bars are Standard Errors (SE) for three independent experiments.

Figure 5. Bortezomib and compound 2 synergistically kill HPV-transformed HeLa cervical cancer cells

HeLa cells were treated with checker board dilution series of Bortezomib and lead compound 2. Cell viability was measured by XTT assay and calculated as percent of control untreated cultures. Synergy is shown by plotting the interaction between drugs in isobologram. The dotted diagonal corresponds to an additive effect while the points below indicate synergy. CI=combination index.

Figure 6. Morphological changes in HeLa cervical cancer cells treated with combination of Bortezomib and lead compound 2

Phase contrast microscopy of HeLa cultures exposed to Bortezomib, compound 2 or combination at the indicated concentration for 48 hours.

Figure 7. Docking simulation of lead compound 2 to 20S proteasome

A, LUMO (in blue) is localized around the carbonyl group of the oxo-piperidine of lead compound 2. B, Schematic view lead compound 2 docked to the crystal structure of 20S proteasome. C, lead compound 2 depicted in the 20S proteasome active site. D, superposition of the three inhibitors, compound 2, homobelactosin C and 2-spiro-lactacystin.

Scheme I

Reagents and Conditions: (a) dry HCl gas, glacial AcOH, 24 h, (b) Triethyl amine/Dichloromethane

Scheme II

Reagents and Conditions: (a) DIEA/THF, 0°C-rt, 45 min, (b) NMM/THF, 0°C-rt, (c) 20 % Piperidine-DMF, rt, 30 min