Iterative design of emetine-based prodrug targeting fibroblast activation protein (FAP) and dipeptidyl peptidase IV DPPIV using a tandem enzymatic activation strategy

Abstract

Background: There is an urgent need to develop new agents for treating metastatic prostate cancer to overcome multiple drug resistance to the current standard targeted cancer therapy. Emetine is a highly cytotoxic natural product protein synthesis inhibitor, which is toxic to all cell types. Its cytotoxicity can be blocked by derivatizing its N-2' position. Thus emetine can be selectively delivered to cancer cells in the region of metastatic cancer as a prodrug that will be activated by an enzyme selectively overexpressed within the metastatic tumor microenvironment. In this work, we convert emetine to a prodrug activatable by the fibroblast activation protein (FAP), a serine protease overexpressed by the carcinoma associated fibroblasts.

Method: By using an iterative structure-activity relationship strategy, several peptidyl emetine prodrug analogs (1-11) were synthesized by chemical derivatization of emetine at its N-2' position and tested for in-vitro activation by FAP. The lead prodrug 11 is made up of a DPPIV activatable prodrug precursor 10 (Ala-Pro-PABC-Emetine) coupled to FAP substrate (Ala-Ser-Gly-Pro-Ala-Gly-Pro). Activation assays of the prodrugs were performed in purified FAP, DPPIV, FBS, and human serum and were analyzed by LCMS. In vitro cytotoxicity assays of these prodrugs are carried out in prostate (LNCaP, PC3) and breast (MCF7 and MDA-MB-231) cancer cell lines. The prodrugs are also tested in normal immortalized human prostatic epithelial cell line (PrEC).

Results: The lead FAP activated emetine prodrug 11 is activated to emetine in tandem by FAP and DPPIV in about 70% conversion within 24 hr. In prostate and breast cancer cell lines treated with prodrug 11, it is found to be equipotent with emetine in the presence of FAP and DPPIV. However, in the PrEC cell line grown in serum free media, prodrug 11 is more than 200-fold less cytotoxic than emetine in the absence of FAP and DPPIV.

Conclusion: This FAP activated prodrug of cytotoxic agent emetine further shows the crucial role of the N-2' position of emetine in controlling its cytotoxicity. Significantly reduced toxicity observed in the PrEC cell line in the absence of FAP and DPPIV shows that prodrug 11 could be systemically delivered to regions of metastatic prostate cancer or other solid tumor for activation by cancer selective enzymes within the cancer microenvironment, such as FAP that is overexpressed by the carcinoma-associated fibroblasts. The two-step tandem enzymatic activation of prodrug 11 by FAP and DPPIV is a strategy for overcoming steric hindrance.

Keywords: dipeptidyl peptidase IV (DPPIV); emetine; fibroblast activation protein (FAP); linker; para-aminobenzyloxycarbonyl (PABC); prodrug.

Fig. 1.

(A) Structure of emetine showing the numbering of the atoms. (B) Schematic of emetine prodrug tandem protease activation strategy to release free emetine in the tumor microenvironment.

Fig. 2.

(A) General structure of the N-2′ derived emetine analogs and prodrugs. (B) Description of N-2′ substituents on the emetine analogs and prodrugs.

Fig. 3.

In vitro characterization of Ala-Pro-PABC-Emetine analog 10. (A) Conversion of compound 10 (200 μM) to emetine in the presence of serum free media (SFM), purified DPPIV (30 nM; activity ≥4,500units/μg protein), 10% fetal bovine serum (FBS), purified recombinant FAP (300 nM). (B) Inhibition of hydrolysis to free emetine by FAP inhibitor Ac-Gly-boroPro (200 μM) in 10% FBS. (C) Inhibition of growth of PrEC cell growing in FAP/DPPIV negative serum free media over 1–3 days exposure to emetine or 10 at 100 nM.

Fig. 4.

In vitro characterization of FAP/DPPIV activated emetine prodrug 11. (A) Schematic depicting tandem activation by FAP and DPPIV and spontaneous elimination of the self-cleaving linker PABC to release free emetine. Production of Ala-Pro-PABC-emetine (precursor 10) and free emetine following incubation with (B) purified FAP (300 nM); (C) mixture of purified FAP and DPPIV (FAP, 300 nM; DPPIV, 30 nM); (D) 10% FBS; (E) human plasma.

Fig. 5.

Cytoxicity of emetine prodrug 11 against human prostate and breast cancer cells. (A) IC50 values for emetine and prodrug 11 against human prostate cancer cells LNCaP and PC3 and human breast cancer cells MCF-7 and MDA-MB-231 following 3-day incubation. (B) Effect of emetine or prodrug 11 on growth of PrEC cells following 3-day exposure to 100 and 1,000 nM of emetine in serum free media or prodrug 11 in serum free media (no FAP or DPPIV) or following addition of purified FAP and DPPIV to the media (FAP, 300 nM; DPPIV, 30 nM).