Mechanistic insights into the inhibition of prostate specific antigen by β-lactam class compounds

Pratap Singh, Simon A. Williams, Meha H. Shah, Thomas Lectka, Gareth J. Pritchard, John T. Isaacs, Samuel R. Denmeade

Research output: Contribution to journalArticlepeer-review

Abstract

Prostate Specific Antigen (PSA) is a biomarker used in the diagnosis of prostate cancer and to monitor therapeutic response. However, its precise role in prostate carcinogenesis and metastasis remains largely unknown. A number of studies arguing in the favor of an active role of PSA in prostate cancer development and progression have implicated this serine protease in the release and activation of growth factors such as insulin-like growth factor 1 (IGF1) through cleavage of insulin like growth factor binding protein 3 and Transforming Growth Factor β (TGF-β) through cleavage of Latent TGF-β. In contrast, other studies suggest that PSA activity might hinder tumor development and progression. In light of these contradictory findings, efficient inhibitors of PSA are needed for exploring its biological role in tumor development and metastasis. Towards the goal of developing potent inhibitors of PSA, we have explored the molecular mechanism of a series of β-lactam based compounds on binding to PSA using activity assays, matrix assisted laser desorption ionization with a time-of-flight mass spectrometry, and GOLD docking methodology. The mass spectrometry experiments and the activity assays confirmed the time-dependent and covalent nature of β-lactam binding. To gain insights on the reaction intermediates at the molecular level, we docked β-lactam inhibitors to a homology modeled PSA using the GOLD docking program in noncovalent and covalent binding modes. The docking studies elucidated the molecular details of the early noncovalent Michaelis complex, the acyl-enzyme covalent complex, and the nature of conformational reorganization required for the long term stability of the covalent complex. Additionally, the molecular basis for the effect of stereochemistry of the lactam ring on the inhibitory potency was elucidated through docking of β-lactam enantiomers. As a validation of our docking methodology, two novel enantiomers were synthesized and evaluated for their inhibitory potency using fluorogenic substrate based activity assays. Additionally, cis enantiomers of eight β-lactam compounds reported in a previous study were docked and their GOLD scores and binding modes were analyzed in order to assess the general applicability of our docking results. The close agreement of our docking results with the experimental data validates the mechanistic insights revealed through the docking studies and paves the way for the design and development of potent and specific inhibitors of PSA.

Original languageEnglish (US)
Pages (from-to)1416-1428
Number of pages13
JournalProteins: Structure, Function and Genetics
Volume70
Issue number4
DOIs
StatePublished - Mar 2008

Keywords

  • Covalent inhibitors
  • Docking
  • GOLD
  • Homology model
  • MALDI-TOF
  • Serine proteases

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology

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