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. 2011 Jan 3;6(1):54-9.
doi: 10.1002/cmdc.201000478.

The legumain protease-activated auristatin prodrugs suppress tumor growth and metastasis without toxicity

Krishna Mohan Bajjuri  1 , Yuan Liu, Cheng Liu, Subhash C Sinha
Affiliations

The legumain protease-activated auristatin prodrugs suppress tumor growth and metastasis without toxicity

Krishna Mohan Bajjuri et al. ChemMedChem. .
No abstract available

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Figures

Figure 1
Figure 1
Structure of dolastatin 10, auristatins and auristatin prodrugs. Cleavage sites are indicated and labeled as follows: a) legumain-catalyzed proteolysis; b) autocleavage through 1,6-elimination; c) autocleavage through CO2 loss.
Figure 2
Figure 2
Cytotoxicity of cytotoxins and prodrugs to (a–c) MDA-MB-435 human and (d–e) 4T1 murine breast cancer cell lines. In a typical experiment, 5000 cells were plated in 96-well plates and allowed to grow overnight. Different concentration of prodrug and cytotoxin were added to each well, and cells were incubated for 72 h at 37 °C and processed using MTT reagent (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). f) Determination of the legumain expression in the cell lines. Cells were lysed in NP-40 buffer. Using clear lysate, an equal amount of protein was separated on an 8–16% SDS-Tris/glycine gel and transferred to nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% milk powder in Tris-buffered saline (TBS) plus Tween 20 before probing with antibody to legumain. g) Table showing EC50 values of cytotoxins and their prodrugs. h) Prodrug activation in the presence/absence of the cysteine protease, legumain. Prodrugs (10 mm in DMSO, 2 μL) were added to the legumain protease (0.1 mgmL−1, 100 μL in assay buffer containing 50 mm citric acid, 120 mm Na2HPO4, 1mm dithiothreitol (DTT), 1 mm ethylenediaminetetraacetic acid (EDTA), and 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1 propanesulfonate (CHAPS), pH 6.0) and the mixture was incubated at 37°C for 24 h and periodically analyzed using HPLC. Condition A: assay buffer containing 50 mm citric acid, 120 mm disodium hydrogenphosphate, 1 mm DTT, 1 mm EDTA, and 0.1% CHAPS, pH 6.0, and Condition B: legumain protease in assay buffer.
Figure 3
Figure 3
Comparison of the legumain activity in 4T1 tumor tissues (□) against 4T1 cells (■) grown in vitro. 4T1 tumor tissues and 4T1 cells were homogenized in n-octyl glucopyranoside (OG) buffer (50 mm OG, 50 mm Tris, 150 mm NaCl, 1 mm DTT, 1 mm EDTA pH 6.0) and normalized to equal protein concentrations. To determine the legumain activity, Z-Ala-Ala-Asn-NHMec substrate was added to these samples and incubated at room temperature for 1 h, and fluorescence was measured using Perkin–Elmer LS-50-B spectrofluorometer (λex: 360 nm; λem: 460 nm).
Figure 4
Figure 4
Tumoricidal effect of prodrug 6b as compared to cytotoxin 4 in mammary carcino a in vivo. a) In vivo effect of 4 and 6b on 4T1 mammary carcinoma. When the tumors averaged ≈4 mm in diameter (after around six days), the 4T1 tumor mice were treated with: saline alone (control), cytotoxin 4 (0.1 mgkg−1), or prodrug 6b (0.1 mgkg−1 or 0.5 mgkg−1), every fourth day starting on day 6; total five i.p. injections per mouse. b) Inhibition of 4T1 spontaneous lung metastasis by 6b (0.5 mgkg−1). c) H&E staining of tumor treated with saline alone, prodrug 4 (0.1 mgkg−1), or prodrug 6b (0.1 mgkg−1 or 0.5 mgkg−1). d) Weight loss of treatment groups with prodrug 4 (0.1 mgkg−1), or prodrug 6b (0.1 mgkg−1 or 0.5 mgkg−1) in 4T1 mammary carcinoma model. All experiments were conducted in compliance with all regulations relating to animal care and welfare.
Scheme 1
Scheme 1
Synthesis of monomethylauristatin E (MMAE) and di-desmethylauristatin E (DDAE) prodrugs. Reagents and conditions: a) 3, EDC, HOBt, DIPEA, CH2Cl2 ; b) 1. Piperidine, DMF; 2. EDC, HOBt, DIPEA, CH2Cl2; 3. TFA, CH2Cl2; c) 7, HATU, DIPEA, CH2Cl2, DMF; d) 1. Piperidine, DMF; 2. EDC, HOBt, DIPEA, CH2Cl2 ; e) Py, CHCl3; f) 1. 4, HOBt, DMF, Py, DIPEA; 2. TFA/CH2Cl2, 0°C.
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