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[PubMed] [Google Scholar] 16. the docking methodology and demonstrating its utility towards the design of substrate-mimetic inhibitors that can be used to explore PSAs role in the pathobiology of prostate cancer. INTRODUCTION PSA is a serine protease with chymotrypsin like specificity1 and a member of the human tissue kallikrein family comprising 12 tryspin-like and 3 chymotrypsin-like serine proteases2. Since the discovery of Prostate Specific Antigen (PSA) in seminal fluid in 19713, serological screening of PSA has become the most important tool to screen for prostate cancer, to detect recurrence and to follow response to local and systemic therapies. Consistent with its role as biomarker for prostate cancer, PSA levels are elevated in blood in both local adenocarcinoma of the prostate as well as metastatic disease, due to increased leakage of PSA into the circulation from the extracellular fluid surrounding invading prostate cancer cells. While a significant amount of research has been conducted towards developing and validating PSA as a biomarker, the physiological role of the serine protease activity of PSA in normal prostate as well as in prostate carcinogenesis MC-976 process remains ill defined4. One possible role of PSAs enzymatic activity of PSA is in the process of liquefaction of human semen wherein PSA cleaves Semenogelin I and Semenogelin II, the major gel-forming proteins in human semen5,6. This hydrolysis of semen results in the release of trapped spermatozoa, thereby aiding in the fertilization process. Another normal physiological pathway in which the proteolytic activity of PSA might be involved is the activation of the latent form of transforming growth factor (TGF-) present abundantly in semen7. Recent studies also implicate PSA mediated activation of the TGF- signaling cascade in the initiation and/or progression of prostate cancer.8. Prostate cancer cells express high levels of TGF- which is shown to be critical for prostate cancer growth and metastasis. Therefore, through activation of TGF- signaling in prostate cancer cells, PSAmight play an important role in stimulating angiogenesis and a variety of inflammatory processes. Similarly, PSA has been shown to directly cleave cytokine binding proteins (such as IGFBP) and release cytokines like IGF-1 that are involved in growth stimulation and inflammation9,10. Additional evidence suggests that PSA may play a significant role in osteoblastic bone metastasis11,12. In spite of growing evidence, a direct role for the enzymatic activity of PSA in prostate pathobiology has not been established in the normal prostate or prostate cancer. PSA contains the classic serine protease triad of serine, histidine and asparatic acid residues in its catalytic pocket that are juxtaposed to each other in correct orientation to drive catalysis of substrate hydrolysis13C15 (Figure 1). The hydrolysis reaction is initiated by early non-covalent interactions between the substrate and the PSA catalytic pocket (Figure 1A) that leads to the formation of an intermediate acyl-enzyme complex between the peptide fragment on the N-terminal side of the peptide bond being cleaved (Figure 1C) via a tetrahedral transition state (Figure 1B). The acyl-enzyme MC-976 complex is transient in nature and is quickly attacked by solvent water molecules resulting in the formation of a second tetrahedral transition state (Figure 1D) and subsequent release of the peptide product (Figure 1E) returning the enzyme to the ground state. The efficiency and Rabbit Polyclonal to GPR108 specificity of the hydrolysis reaction is dictated by the nature of the energetic interactions between the peptide backbone/sidechains and the protease residues in the specific binding sites. PSA exhibits a low protease activity (four orders of magnitude less than chymotrypsin)16 but it is unique among serine proteases in that it can use glutamine as a P1 residue during substrate hydrolysis17. Thus by incorporating glutamine in the P1 position, PSA-specific substrate and inhibitors MC-976 can be identified that lack activity against related homolog proteases such as chymotrypsin. Open in a separate window Figure 1 Mechanistic scheme for the substrate hydrolysis reaction catalyzed by PSA. Illustrated in the scheme are catalytic triad residues (HIS41, SER189 and.