No abstract

Subtle structural differences in the S1 site of uPA compared with that of related proteases, which result in part from the presence of a serine residue at position 190, account for the selectivity of small thiophene-2-carboxamidines for uPA, and afford a framework for structure-based design of small, potent, selective uPA inhibitors.

Finasteride is employed in treatment of benign prostatic hyperplasia in man, where its target enzyme is steroid 5α-reductase. It is a novel, potent mechanism-based inhibitor of the human prostate (type 2) isozyme. Although it is accepted as an alternate substrate and is ultimately reduced to dihydrofinasteride, this proceeds through an enzyme-bound NADP−dihydrofinasteride adduct. Finasteride is processed with a second-order rate constant, k i/K i = 1 × 106 M-1 s-1, that approaches k cat/K m for reduction of testosterone, 3 × 106 M-1 s-1, and essentially every catalytic event is lethal (partition ratio ≤ 1.07). The membrane-bound enzyme−inhibitor complex formed from [3H]finasteride appears to release [3H]dihydrofinasteride with a half-life of 1 month at 37 °C (k = (2.57 ± 0.03) × 10-7 s-1), as identified by mass spectroscopy. The intermediate NADP−dihydrofinasteride adduct can be recovered intact by denaturation of the enzyme−inhibitor complex and has been purified. Free in solution, it likewise decomposes to dihydrofinasteride (half-life = 11 days). An extremely potent bisubstrate analog inhibitor, this NADP−dihydrofinasteride adduct binds to the free enzyme with a second-order rate constant equal to k cat/K m for turnover of testosterone and has a dissociation constant K i ≤ 1 × 10-13 M. Finasteride is also a mechanism-based inhibitor of the human skin (type 1) isozyme, but it is processed with a much smaller second-order rate constant, k i/K i = 3 × 103 M-1 s-1, which attenuates its activity against this isozyme in vivo. The mechanism explains the exceptional potency and specificity of finasteride in treatment of benign prostatic hyperplasia, and the concept may have application to other pyridine nucleotide-linked enzymes.

Hepsin is an integral membrane protein that may participate in cell growth and in maintaining proper cell morphology and is overexpressed in a number of primary tumors. We have determined the 1.75 A resolution structure of the extracellular component of human hepsin. This structure includes a 255-residue trypsin-like serine protease domain and a 109-residue region that forms a novel, poorly conserved, scavenger receptor cysteine-rich (SRCR) domain. The two domains are associated with each other through a single disulfide bond and an extensive network of noncovalent interactions. The structure suggests how the extracellular region of hepsin may be positioned with respect to the plasma membrane.

Thymopoietin (T7P) was orignlly sated as a 5-kDa 49-aa protein from bovine thymus in studies of the effects of thymic extracts on neuromuscular tAsi n and was subsequently observed to affect T-cell differentiation and function. We now report the Isolation of cDNA clones for three alternatively spliced mRNAs that encode three distinct human T-cell TPs. Proteins encoded by these mRNAs, whih we have Thymopoietin (TP) was originally isolated as a 5-kDa polypeptide from bovine thymus on the basis ofits ability to affect neuromuscular transmission when injected into mice (1). Subsequently, purified 5-kDa TP was found to affect certain immunological functions, inducing differentiation of prothymocytes to thymocytes, as measured by changes in expression of Thy-1 and other cell surface phenotypic markers (2), and enhancing allogeneic responses of peripheral T cells (3). The 5-kDa TP and a synthetic peptide corresponding to aa 32-36 of TP, Arg-Lys-Asp-Val-Tyr (thymopentin) gave similar results in both neurophysiological and immunological assays, suggesting that thymopentin may act as a TP mimetic (4, 5). Furthermore, in clinical studies with thymopentin being used as a putative immunoregulatory drug, promising findings have been obtained in double-blind, placebocontrolled studies of subjects with rheumatoid arthritis (6), recurrent herpes simplex (7), atopic dermatitis (8), and asymptomatic human immunodeficiency virus infection (9).To better define the cellular function(s) of TP and to provide a molecular basis for the mechanism of action of thymopentin, we isolated and characterized cDNAs encoding this protein. previously isolated a bovine cDNA that encodes the originally determined 49-aa bovine TP sequence (11, 12) at the 5' end of a larger open reading frame. Here we report the isolation, using a probe derived from the bovine sequence of Zevin-Sonkin et al. (10), of human TP cDNA clones that encode three distinct human TP proteins, derived from three alternatively spliced mRNAs, each ofwhich contains a sequence at the N terminus that is homologous to the originally determined bovine 49-aa TP sequence. TPa (75 kDa), TP(3 (51 kDa), and TPy(39 kDa), contain some domains in common and some unique domains,