title: Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools.
abstract: BACKGROUND
  Analysis of global changes in gene transcription and translation by systems-based genomics and proteomics approaches provides only indirect information about protein function. In many cases, enzymatic activity fails to correlate with transcription or translation levels. Therefore, a direct method for broadly determining activities of an entire class of enzymes on a genome-wide scale would be of great utility.
  
  
  RESULTS
  We have engineered chemical probes that can be used to broadly track activity of cysteine proteases. The structure of the general cysteine protease inhibitor E-64 was used as a scaffold. Analogs were synthesized by varying the core peptide recognition portion while adding affinity tags (biotin and radio-iodine) at distal sites. The resulting probes containing a P2 leucine residue (DCG-03 and DCG-04) targeted the same broad set of cysteine proteases as E-64 and were used to profile these proteases during the progression of a normal skin cell to a carcinoma. A library of DCG-04 derivatives was constructed in which the leucine residue was replaced with all natural amino acids. This library was used to obtain inhibitor activity profiles for multiple protease targets in crude cellular extracts. Finally, the affinity tag of DCG-04 allowed purification of modified proteases and identification by mass spectrometry.
  
  
  CONCLUSIONS
  We have created a simple and flexible method for functionally identifying cysteine proteases while simultaneously tracking their relative activity levels in crude protein mixtures. These probes were used to determine relative activities of multiple proteases throughout a defined model system for cancer progression. Furthermore, information obtained from libraries of affinity probes provides a rapid method for obtaining detailed functional information without the need for prior purification/identification of targets.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco 94143, USA.
authors.forename: D
authors.initials: D
authors.lastname: Greenbaum
authors.forename: K F
authors.initials: KF
authors.lastname: Medzihradszky
authors.forename: A
authors.initials: A
authors.lastname: Burlingame
authors.forename: M
authors.initials: M
authors.lastname: Bogyo
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D003713:Dendritic Cells / Q000201:enzymology; D018450:Disease Progression; D004852:Epoxy Compounds / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D007668:Kidney / Q000737:chemistry / Q000201:enzymology; D007930:Leucine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000032:analysis / Q000378:metabolism; D009928:Organ Specificity; D019151:Peptide Library; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D051381:Rats; D012878:Skin Neoplasms / Q000201:enzymology; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
chemical_list: D000345:Affinity Labels; D015853:Cysteine Proteinase Inhibitors; C419227:DCG 03; C419228:DCG 04; D004852:Epoxy Compounds; C404948:JPM 565; D015335:Molecular Probes; D009363:Neoplasm Proteins; D019151:Peptide Library; D010455:Peptides; D003546:Cysteine Endopeptidases; D007930:Leucine; C024974:E 64