Article

Peptide Length and Leaving-Group Sterics Influence Potency of Peptide Phosphonate Protease Inhibitors

Graduate Group in Biochemistry and Molecular Biology, University of California, San Francisco, CA 94158, USA.
Chemistry & biology (Impact Factor: 6.59). 01/2011; 18(1):48-57. DOI: 10.1016/j.chembiol.2010.11.007
Source: PubMed

ABSTRACT The ability to follow enzyme activity in a cellular context represents a challenging technological frontier that impacts fields ranging from disease pathogenesis to epigenetics. Activity-based probes (ABPs) label the active form of an enzyme via covalent modification of catalytic residues. Here we present an analysis of parameters influencing potency of peptide phosphonate ABPs for trypsin-fold S1A proteases, an abundant and important class of enzymes with similar substrate specificities. We find that peptide length and stability influence potency more than sequence composition and present structural evidence that steric interactions at the prime-side of the substrate-binding cleft affect potency in a protease-dependent manner. We introduce guidelines for the design of peptide phosphonate ABPs and demonstrate their utility in a live-cell labeling application that specifically targets active S1A proteases at the cell surface of cancer cells.

Download full-text

Full-text

Available from: Charles S Craik, Jul 04, 2015
0 Followers
 · 
135 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cercarial elastase is the major invasive larval protease in Schistosoma mansoni, a parasitic blood fluke, and is essential for host skin invasion. Genome sequence analysis reveals a greatly expanded family of cercarial elastase gene isoforms in Schistosoma mansoni. This expansion appears to be unique to S. mansoni, and it is unknown whether gene duplication has led to divergent protease function. Profiling of transcript and protein expression patterns reveals that cercarial elastase isoforms are similarly expressed throughout the S. mansoni life cycle. Computational modeling predicts key differences in the substrate-binding pockets of various cercarial elastase isoforms, suggesting a diversification of substrate preferences compared with the ancestral gene of the family. In addition, active site labeling of SmCE reveals that it is activated prior to exit of the parasite from its intermediate snail host. The expansion of the cercarial gene family in S. mansoni is likely to be an example of gene dosage. In addition to its critical role in human skin penetration, data presented here suggests a novel role for the protease in egress from the intermediate snail host. This study demonstrates how enzyme activity-based analysis complements genomic and proteomic studies, and is key in elucidating proteolytic function.
    PLoS Neglected Tropical Diseases 04/2012; 6(4):e1589. DOI:10.1371/journal.pntd.0001589 · 4.49 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Proteases responsible for the increased peritumoral proteolysis associated with cancer represent functional biomarkers for monitoring tumorigenesis. One attractive extracellular biomarker is the transmembrane serine protease matriptase. Found on the surface of epithelial cells, the activity of matriptase is regulated by its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). Quantitative mass spectrometry allowed us to show that, in selected cancers, HAI-1 expression decreases, leading to active matriptase. A preclinical probe specific for the measurement of emergent active matriptase was developed. Using an active-site-specific, recombinant human antibody for matriptase, we found that the selective targeting of active matriptase can be used to visualize the tumorigenic epithelium. Live-cell fluorescence imaging validated the selectivity of the antibody in vitro by showing that the probe localized only to cancer cell lines with active matriptase on the surface. Immunofluorescence with the antibody documented significant levels of active matriptase in 68% of primary and metastatic colon cancer sections from tissue microarrays. Labeling of the active form of matriptase in vivo was measured in human colon cancer xenografts and in a patient-derived xenograft model using near-infrared and single-photon emission computed tomography imaging. Tumor uptake of the radiolabeled antibody, (111)In-A11, by active matriptase was high in xenografts (28% injected dose per gram) and was blocked in vivo by the addition of a matriptase-specific variant of ecotin. These findings suggest, through a HAI-1-dependent mechanism, that emergent active matriptase is a functional biomarker of the transformed epithelium and that its proteolytic activity can be exploited to noninvasively evaluate tumorigenesis in vivo.
    Proceedings of the National Academy of Sciences 12/2012; 110(1). DOI:10.1073/pnas.1218694110 · 9.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Two novel synthetic methods for the preparation of bis(trifluoroethyl) esters of α-aminophosphonic acids are presented. Preliminary results on the application of the compounds synthesized as inhibitors of serine proteases are also reported. Structures originating from α-aminoalkylphosphonate diaryl esters represent a novel class of serine protease inactivators.
    Tetrahedron Letters 03/2013; 54(12):1566–1568. DOI:10.1016/j.tetlet.2013.01.039 · 2.39 Impact Factor