Prodrug-inspired probes selective to cathepsin B over other cysteine cathepsins.

Cathepsin B (CTB) is a cysteine protease believed to be an important therapeutic target or biomarker for several diseases including aggressive cancer, arthritis, and parasitic infections. The development of probes capable of assessing CTB activity in cell lysates, living cells, and animal models of disease are needed to understand its role in disease progression. However, discovering probes selective to cathepsin B over other cysteine cathepsins is a significant challenge due to overlap of preferred substrates and binding site homology in this family of proteases. Herein we report the synthesis and detailed evaluation of two prodrug-inspired fluorogenic peptides designed to be efficient and selective substrate-based probes for CTB. Through cell lysate and cell assays, a promising lead candidate was identified that is efficiently processed and has high specificity for CTB over other cysteine cathepsins. This work represents a key step toward the design of rapid release prodrugs or substrate-based molecular imaging probes specific to CTB.

[1]  J. Joyce,et al.  Proteolytic networks in cancer. , 2011, Trends in cell biology.

[2]  A. Barrett,et al.  L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. , 1982, The Biochemical journal.

[3]  C. D. Arnett,et al.  Mapping human brain monoamine oxidase A and B with 11C-labeled suicide inactivators and PET. , 1987, Science.

[4]  Olga Vasiljeva,et al.  Cysteine cathepsins: From structure, function and regulation to new frontiers☆ , 2011, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics.

[5]  A. D'Amico,et al.  An image contrast agent selectively activated by prostate specific antigen. , 2006, Bioorganic & medicinal chemistry.

[6]  P. Trail,et al.  Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. , 2002, Bioconjugate chemistry.

[7]  G. Dubowchik,et al.  Cathepsin B-sensitive dipeptide prodrugs. 1. A model study of structural requirements for efficient release of doxorubicin. , 1998, Bioorganic & medicinal chemistry letters.

[8]  B. Rzeszotarska,et al.  A Large Scale Synthesis of Mono- and Di-urethane Derivatives of Lysine , 1999 .

[9]  R. Weissleder,et al.  In vivo imaging of tumors with protease-activated near-infrared fluorescent probes , 1999, Nature Biotechnology.

[10]  F. Kratz,et al.  Development of protein-binding bifunctional linkers for a new generation of dual-acting prodrugs. , 2009, Bioconjugate chemistry.

[11]  J. Barrio,et al.  Positron emission tomography imaging of adenoviral-mediated transgene expression in liver cancer patients. , 2005, Gastroenterology.

[12]  Francesco Leonetti,et al.  Substrate Profiling of Cysteine Proteases Using a Combinatorial Peptide Library Identifies Functionally Unique Specificities* , 2006, Journal of Biological Chemistry.

[13]  G. Salvesen,et al.  Regulating cysteine protease activity: essential role of protease inhibitors as guardians and regulators. , 2002, Current pharmaceutical design.

[14]  Andrew E. Sloan,et al.  Topical Application of Activity-based Probes for Visualization of Brain Tumor Tissue , 2012, PloS one.

[15]  R. Jain,et al.  Challenges and key considerations of the enhanced permeability and retention effect for nanomedicine drug delivery in oncology. , 2013, Cancer research.

[16]  Daniel E Koshland.  The application and usefulness of the ratio k(cat)/K(M). , 2002, Bioorganic chemistry.

[17]  J. Katzenellenbogen,et al.  A novel connector linkage applicable in prodrug design. , 1981, Journal of medicinal chemistry.

[18]  D. Hicklin,et al.  Strategies for Delaying or Treating In vivo Acquired Resistance to Trastuzumab in Human Breast Cancer Xenografts , 2006, Clinical Cancer Research.

[19]  C. Olsen,et al.  Substrates for efficient fluorometric screening employing the NAD-dependent sirtuin 5 lysine deacylase (KDAC) enzyme. , 2012, Journal of medicinal chemistry.

[20]  E. Brogi,et al.  Macrophages and cathepsin proteases blunt chemotherapeutic response in breast cancer. , 2011, Genes & development.

[21]  I. Shin,et al.  Dual-targeting delivery system for selective cancer cell death and imaging , 2013 .

[22]  J. Kos,et al.  Intracellular and extracellular cathepsin B facilitate invasion of MCF-10A neoT cells through reconstituted extracellular matrix in vitro. , 2003, Experimental cell research.

[23]  V. Turk,et al.  Cysteine cathepsins are not critical for TRAIL- and CD95-induced apoptosis in several human cancer cell lines , 2012, Biological chemistry.

[24]  Bonnie F. Sloane,et al.  Inhibition of cathepsin B activity attenuates extracellular matrix degradation and inflammatory breast cancer invasion , 2011, Breast Cancer Research.

[25]  Raphael Dumeunier,et al.  Latent fluorophores based on a self-immolative linker strategy and suitable for protease sensing. , 2008, Bioconjugate chemistry.

[26]  Georges von Degenfeld,et al.  Noninvasive optical imaging of cysteine protease activity using fluorescently quenched activity-based probes. , 2007, Nature chemical biology.

[27]  R. Bjerkvig,et al.  The regulation of cysteine cathepsins and cystatins in human gliomas , 2012, International journal of cancer.

[28]  D. Turk,et al.  Revised Definition of Substrate Binding Sites of Papain-Like Cysteine Proteases , 1998, Biological chemistry.

[29]  M. Bogyo,et al.  Non-Invasive Imaging of Cysteine Cathepsin Activity in Solid Tumors Using a 64Cu-Labeled Activity-Based Probe , 2011, PloS one.

[30]  Bonnie F. Sloane,et al.  Increased gelatinase A (MMP-2) and cathepsin B activity in invasive tumor regions of human colon cancer samples. , 1994, The American journal of pathology.

[31]  Kwangmeyung Kim,et al.  Cathepsin B-sensitive nanoprobe for in vivo tumor diagnosis , 2011 .

[32]  Damon L. Meyer,et al.  Contribution of linker stability to the activities of anticancer immunoconjugates. , 2008, Bioconjugate chemistry.

[33]  Bonnie F. Sloane,et al.  Cathepsin B and tumor proteolysis: contribution of the tumor microenvironment. , 2005, Seminars in cancer biology.

[34]  Bonnie F. Sloane,et al.  Cysteine cathepsins: multifunctional enzymes in cancer , 2006, Nature Reviews Cancer.

[35]  Shuji Ogino,et al.  Cathepsin B Expression and Survival in Colon Cancer: Implications for Molecular Detection of Neoplasia , 2010, Cancer Epidemiology, Biomarkers & Prevention.

[36]  Chun-Wei Peng,et al.  Cathepsin B cleavable novel prodrug Ac‐Phe‐Lys‐PABC‐ADM enhances efficacy at reduced toxicity in treating gastric cancer peritoneal carcinomatosis , 2012, Cancer.

[37]  K. Jaskiewicz,et al.  Overexpression of cathepsin B correlates with angiogenesis in colon adenocarcinoma. , 2004, Neoplasma.

[38]  G. Taraboletti,et al.  Cathepsin B mediates the pH-dependent proinvasive activity of tumor-shed microvesicles. , 2008, Neoplasia.

[39]  H. Hausen,et al.  A new type of papillomavirus DNA, its presence in genital cancer biopsies and in cell lines derived from cervical cancer. , 1984, The EMBO journal.

[40]  W. Bode,et al.  HUMAN CATHEPSIN G , 1997 .

[41]  G. Lalmanach,et al.  CA-074, But Not Its Methyl Ester CA-074Me, Is a Selective Inhibitor of Cathepsin B within Living Cells , 2002, Biological chemistry.

[42]  T. Lah,et al.  Post-Translational Regulation of Cathepsin B, but not of Other Cysteine Cathepsins, Contributes to Increased Glioblastoma Cell Invasiveness In Vitro , 2009, Pathology & Oncology Research.

[43]  H. Chapman,et al.  Human Cathepsin F , 1998, The Journal of Biological Chemistry.

[44]  Matthew Bogyo,et al.  Activity-based probes that target diverse cysteine protease families , 2005, Nature chemical biology.

[45]  Bonnie F. Sloane,et al.  Cathepsin B: a potential prognostic marker for inflammatory breast cancer , 2011, Journal of Translational Medicine.

[46]  C. P. Huber,et al.  Characterization of cathepsin B specificity by site-directed mutagenesis. Importance of Glu245 in the S2-P2 specificity for arginine and its role in transition state stabilization. , 1993, The Journal of biological chemistry.