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. 2016 Nov 18;11(11):3052-3060.
doi: 10.1021/acschembio.6b00560. Epub 2016 Sep 19.

Antitumor Activity of Cytotoxic Cyclooxygenase-2 Inhibitors

Md Jashim Uddin  1 , Brenda C Crews  1 , Shu Xu  1 , Kebreab Ghebreselasie  1 , Cristina K Daniel  1 , Philip J Kingsley  1 , Surajit Banerjee  2   3 , Lawrence J Marnett  1
Affiliations

Antitumor Activity of Cytotoxic Cyclooxygenase-2 Inhibitors

Md Jashim Uddin et al. ACS Chem Biol. .

Abstract

Targeted delivery of chemotherapeutic agents to tumors has been explored as a means to increase the selectivity and potency of cytotoxicity. Most efforts in this area have exploited the molecular recognition of proteins highly expressed on the surface of cancer cells followed by internalization. A related approach that has received less attention is the targeting of intracellular proteins by ligands conjugated to anticancer drugs. An attractive target for this approach is the enzyme cyclooxygenase-2 (COX-2), which is highly expressed in a range of malignant tumors. Herein, we describe the synthesis and evaluation of a series of chemotherapeutic agents targeted to COX-2 by conjugation to indomethacin. Detailed characterization of compound 12, a conjugate of indomethacin with podophyllotoxin, revealed highly potent and selective COX-2 inhibition in vitro and in intact cells. Kinetics and X-ray crystallographic studies demonstrated that compound 12 is a slow, tight-binding inhibitor that likely binds to COX-2's allosteric site with its indomethacin moiety in a conformation similar to that of indomethacin. Compound 12 exhibited cytotoxicity in cell culture similar to that of podophyllotoxin with no evidence of COX-2-dependent selectivity. However, in vivo, compound 12 accumulated selectively in and more effectively inhibited the growth of a COX-2-expressing xenograft compared to a xenograft that did not express COX-2. Compound 12, which we have named chemocoxib A, provides proof-of-concept for the in vivo targeting of chemotherapeutic agents to COX-2 but suggests that COX-2-dependent selectivity may not be evident in cell culture-based assays.

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Figures

Figure 1
Figure 1
Kinetics of the time-dependent inhibition of COX-2 by compound 12. (A) Purified mCOX-2 was reconstituted with heme and pre-incubated with the indicated concentrations of inhibitor at 37 °C for the indicated times prior to the addition of the substrate (50 μM). Data are the mean ± range of duplicate determinations. (B) Secondary plot of kobs versus inhibitor concentration used to generate values for KI, k2, and k−2.
Figure 2
Figure 2
Structure of the mCOX-2:compound 12 complex (PDB ID: 4OTJ). Shown is a stereoview of Compound 12 bound to the active site and the lobby region of COX-2. Compound 12 is presented in orange, and COX-2 is shown as a ribbon in grey. Selected key residues are in green.
Figure 3
Figure 3
Effect of compound 12 on cell viability. Shown are the viabilities of 1483 HNSCC cells (A) or primary human mammary epithelial cells (B) in the presence of Compound 12 (dashed line, ●) or podophyllotoxin (solid line, □) after a 48 h treatment. Data are the mean ± standard deviation (n = 6).
Figure 4
Figure 4
Reversibility of compound 12-mediated cytotoxicity. (A) Viability of 1483 HNSCC cells following incubation in the presence of the vehicle dimethylsulfoxide (DMSO), 10 μM compound 12, or 10 μM podophyllotoxin for 72 h. (B) Viability of 1483 HNSCC cells following incubation in the presence of DMSO, 10 μM compound 12, or 10 μM podophyllotoxin for 5 h followed by washout and further incubation for 72 h. Data are the mean ± standard deviation (n = 6).
Figure 5
Figure 5
In vivo tumor growth inhibition by compound 12. (A) Female nude mice bearing 1483 HNSCC xenografts were treated with vehicle (solid line, ●), podophyllotoxin (dotted line, □) or compound 12 (dashed line, ▲) for the indicated number of days. Data are the mean ± standard deviation (n = 8). (B) Female nude mice bearing HCT116 cell xenografts were treated with vehicle (solid line, ●) or compound 12 (dashed line, ▲). Data are the mean ± standard deviation (n = 7). (C) Mass spectrometric analysis of extracts of a 1483 cell xenograft 3 h post treatment with compound 12. (D) Levels of compound 12 in 1483 and HCT116 cell xenografts 24 h after completion of a 14 day course of treatment. Data are the mean ± standard deviation (n = 5). (The asterisk denotes that the data are significantly different at p = 0.048).
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