Enzyme-Instructed Assembly and Disassembly Processes for Targeting Downregulation in Cancer Cells

Cancer cells differ from normal cells in both gain of functions (i.e., upregulation) and loss of functions (i.e., downregulation). While it is common to suppress gain of function for chemotherapy, it remains challenging to target downregulation in cancer cells. Here we show the combination of enzyme-instructed assembly and disassembly to target downregulation in cancer cells by designing peptidic precursors as the substrates of both carboxylesterases (CESs) and alkaline phosphatases (ALPs). The precursors turn into self-assembling molecules to form nanofibrils upon dephosphorylation by ALP, but CES-catalyzed cleavage of the ester bond on the molecules results in disassembly of the nanofibrils. The precursors selectively inhibit the cancer cells that downregulate CES (e.g., OVSAHO) but are innocuous to a hepatocyte that overexpresses CES (HepG2), while the two cell lines exhibit comparable ALP activities. This work illustrates a potential approach for the development of chemotherapy via targeting downregulation (or loss of functions) in cancer cells.


S1. Experiment materials and instruments
All chemical reagents and solvents were used as receiving from commercial sources without further purification. 2-Cl-trityl chloride resin (1.0-1.2 mmol/g), Fmoc-OSu and other Fmoc-amino acids were obtained from GL Biochem (Shanghai, China). Other chemical reagents and solvents were obtained from Fisher Scientific; alkaline phosphatase was purchased from Biomatik. Minimal Essential Medium (MEM), RPMI 1640 Medium were purchased from ATCC and fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Gibco by life technologies.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from ACROS Organics. All precursors were purified with Water Delta600 HPLC system, equipped with an XTerra C18 RP column and an in-line diode array UV detector. We obtained LC-MS spectrum on Waters Acquity Ultra Performance LC with Waters MICROMASS detector, ultraviolet-visible (UV) spectra on JASCO J-810 spectrophotometer, and 1 H-NMR spectra on Varian Unity Inova 400, and TEM images on Morgagni 268 transmission electron microscope. MTT assay for cell toxicity test on DTX880 Multimode Detector.

S2. Synthesis and characterization of the precursors
We synthesized Fmoc-Tyr(PO3H2)-OH based on previous work for directly use of solid phase peptide synthesis (SPPS). With SPPS, we synthesized 1-OH-OP based on protocols in published paper. 2 The following scheme demonstrate the synthetic route for 1-OH-OP and 1-OMe-OP. The synthetic route of other compounds are the same with 1-OH-OP or 1-OMe-OP. Then, all compounds were purified by reverse phase HPLC using acetonitrile (0.1% TFA) and double-distilled water (0.1% TFA) as the eluents.

S3. CMC measurements
A series of precursor/derivative solutions from the concentration of 8 mM to 0.4 µM was prepared in pH 7.4 PBS buffer. After incubating with Rhodamine 6G (5 µM), the λmax was determined by measuring the absorbance from 520 to 540 nm using a Biotek Synergy 4 hybrid multi-mode microplate reader.

S4. TEM sample preparation
We first place 5 µL samples (preparation procedure described in S3) on 400 mesh copper grids coated with continuous thick carbon film (~ 35 nm) which is glowed discharged. Then we washed the grid with ddH2O twice. Finally we stained the sample loaded grid with a large drop of the UA (uranyl acetate) and allow to dry in air.

S5. Static light scattering measurement
We performed static light scattering on using an ALV (Langen, Germany) goniometer and correlator system with a 22 mW HeNe (λ = 633 nm) laser and an avalanche photodiode detector.
We prepared the samples of 1-OMe-OP at the concentration of 20 µM, in pH 7.4 PBS buffer. After addition of 1 U/mL ALP or 1 U/mL CES or both, we incubated the tubes for 24 hours and test the static light scattering at degree of 30°. The resulting intensity ratios are proportional to the amount of aggregates in the samples.

S6. Cell culture and MTT assay
Cell culture: HepG2 cells were purchased from American-type Culture Collection (ATCC, USA).