Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis

ACS Infect Dis. 2016 Nov 11;2(11):807-815. doi: 10.1021/acsinfecdis.6b00092. Epub 2016 Jul 15.


Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb "Hydrolase important for pathogenesis 1" (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections.

Keywords: Mycobacterium tuberculosis; activity-based probe; fluorogenic substrate; hydrolase important for pathogenesis 1 (Hip1); protease; substrate profiling.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antitubercular Agents / chemistry*
  • Antitubercular Agents / pharmacology
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Drug Design
  • Enzyme Inhibitors / chemistry*
  • Enzyme Inhibitors / pharmacology
  • Humans
  • Kinetics
  • Molecular Structure
  • Mycobacterium tuberculosis / drug effects
  • Mycobacterium tuberculosis / enzymology*
  • Mycobacterium tuberculosis / genetics
  • Serine Proteases / chemistry
  • Serine Proteases / genetics
  • Serine Proteases / metabolism*
  • Substrate Specificity
  • Tuberculosis / microbiology*


  • Antitubercular Agents
  • Bacterial Proteins
  • Enzyme Inhibitors
  • Hip1 protein, Mycobacterium tuberculosis
  • Serine Proteases