The substrate specificities of papain-like cysteine proteases (clan CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a completely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facilitated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences completely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids. A microtiter plate assay format permitted a rapid determination of the specificity profile of each enzyme. Individual peptide substrates were then synthesized and tested for a quantitative determination of the specificity of the human cathepsins. Despite the conserved three-dimensional structure and similar substrate specificity of the enzymes studied, distinct amino acid preferences that differentiate each enzyme were identified. The specificities of cathepsins K and S partially match the cleavage site sequences in their physiological substrates. Capitalizing on its unique preference for proline and glycine at the P2 and P3 positions, respectively, selective substrates and a substrate-based inhibitor were developed for cathepsin K. A cluster analysis of the proteases based on the complete specificity profile provided a functional characterization distinct from standard sequence analysis. This approach provides useful information for developing selective chemical probes to study protease-related pathologies and physiologies.Proteases hydrolyze amide bonds in proteins and peptides and represent one of the largest and most important protein families known. They comprise over 2% of the human genome and play diverse physiological roles (merops.sanger.ac.uk) (1). The substrate specificity of a protease enables the enzyme to preferentially cleave its substrates in the presence of other peptides or proteins. Therefore, specificity information can provide clues about the biological function of the protease and aid in the design of efficient substrates and potent, selective inhibitors. Various methods including both biological and chemicalbased approaches to study protease specificity have been developed and were recently reviewed (2).Positional scanning synthetic combinatorial libraries (PS-SCLs) 2 of fluorogenic substrates have emerged as useful reagents for the rapid and exhaustive determination of protease specificity (3). A peptide-based PS-SCL is composed of sublibraries in which one peptide position is fixed with an amino acid, whereas the remaining positions contain an equimolar mixture of amino acids. Assaying proteases with these sublibraries rapidly establishes the amino acid preferences at the defined position. Initially, the substrate specificities of caspases and granzyme B were profiled using PSSCLs with the P1 position fixed as an aspartic acid.The limitations of the original P1 fixed libraries were overcome through the development of a modified coumarin, 7-amino-...

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Voltage-gated sodium channels (Na V ) are functionally expressed in highly metastatic cancer cells derived from nonexcitable epithelial tissues (breast, prostate, lung, and cervix). MDA-MB-231 breast cancer cells express functional sodium channel complexes, consisting of Na V 1.5 and associated auxiliary ␤-subunits, that are responsible for a sustained inward sodium current at the membrane potential. Although these channels do not regulate cellular multiplication or migration, their inhibition by the specific blocker tetrodotoxin impairs both the extracellular gelatinolytic activity (monitored with DQ-gelatin) and cell invasiveness leading to the attenuation of colony growth and cell spreading in three-dimensional Matrigel-composed matrices. MDA-MB-231 cells express functional cysteine cathepsins, which we found play a predominant role (ϳ65%) in cancer invasiveness. Matrigel invasion is significantly decreased in the presence of specific inhibitors of cathepsins B and S (CA-074 and Z-FL-COCHO, respectively), and co-application of tetrodotoxin does not further reduce cell invasion. This suggests that cathepsins B and S are involved in invasiveness and that their proteolytic activity partly depends on Na V function. Inhibiting Na V has no consequence for cathepsins at the transcription, translation, and secretion levels. However, Na V activity leads to an intracellular alkalinization and a perimembrane acidification favorable for the extracellular activity of these acidic proteases. We propose that Na v enhance the invasiveness of cancer cells by favoring the pH-dependent activity of cysteine cathepsins. This general mechanism could lead to the identification of new targets allowing the therapeutic prevention of metastases.Breast cancer is the most common female cancer and the primary cause of death in women by cancer worldwide (1).Deaths occur primarily after the development of metastases. The invasive potential of malignant cells is mainly linked to their capacity to degrade basement membranes and extracellular matrices by various proteases. Studies have mostly focused on metalloproteases, including matrix metalloproteinases and the closely related ADAMs (a disintegrin and metalloproteinase) and ADAMTs (a disintegrin and metalloproteinase with thrombospondin motifs) (2), that are key factors in growth, invasion, and angiogenesis, and to a lesser extent on aspartyl and serine proteases. Pharmaceutical inhibitors of matrix metalloproteinases have been developed, but the results from clinical trials with these drugs have so far been disappointing (3,4