Crystals of the urokinase type plasminogen activator variant βc-uPA in complex with small molecule inhibitors open the way towards structure-based drug design 1 1Edited by A. Fersht

Żesławska, Ewa; Schweinitz, Andrea; Karcher, Annette; Sondermann, Peter; Sperl, S.; Stürzebecher, Jörg; Jacob, Uwe

doi:10.1006/jmbi.2000.3966

Cited by 74 publications

(90 citation statements)

References 29 publications

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“…Comparable K i values were determined; 195 lM for native uPA and 112 lM for recombinant uPA (Table 1). These values are similar to those reported previously for benzamidine inhibition of uPA (Renatus et al 1998;Katz et al 2000;Zeslawska et al 2000).…”

Section: Kinetic Comparison Of Recombinant and Native Human Upasupporting

confidence: 92%

Development of a Mammalian Suspension Culture for Expression of Active Recombinant Human Urokinase-type Plasminogen Activator

et al. 2005

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The development of specific catalytic inhibitors for the serine protease urokinase-type plasminogen activator (uPA) has been hindered due to difficulties in producing sufficient amounts of active recombinant uPA that is catalytically equivalent to native uPA. The purpose of this study was to develop an efficient system for the expression of recombinant human uPA that exhibits comparable proteolytic activity to that of the native protein. Since post-translational modifications (e.g. glycosylations) of uPA are necessary for efficient proteolytic activity, we have used a mammalian cell line [Chinese hamster ovary (CHO)-S] to express recombinant human uPA. CHO-S cells were selected to stably express full-length recombinant human uPA containing a hexahistidine tag at its C-terminus to permit purification by nickel-based affinity chromatography. Secretion of recombinant uPA into the culture media was confirmed by immunoblotting and the presence of an N-linked glycosylation was confirmed by PNGase sensitivity. Enzymatic activity of purified recombinant uPA was demonstrated using zymography and quantitatively compared to native uPA by kinetic analysis using an uPA-specific substrate. Native uPA and the recombinant uPA demonstrated comparable K m values (55.7 and 39 lM, respectively). Furthermore, inhibition studies using benzamidine resulted in a K i of 195 lM for native uPA, while recombinant uPA had a K i of 112 lM. These data indicate that recombinant human uPA expressed by CHO-S cells is functionally comparable to native uPA.Abbreviations: CHO-S -Chinese hamster ovary

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Section: Kinetic Comparison Of Recombinant and Native Human Upasupporting

confidence: 92%

Development of a Mammalian Suspension Culture for Expression of Active Recombinant Human Urokinase-type Plasminogen Activator

et al. 2005

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“…If amiloride were bound at more than one site, one would expect to be able to resolve some of the multiple affinities once affinity at one site had been altered. Given the structure of amiloride and its reported coordination in urokinase-type plasminogen activator, it likely interacts with multiple residues that collectively form a binding pocket (20). Based on dramatic changes in amiloride block caused by mutations at the putative selectivity filter, we proposed that the charged guanidinium moiety of amiloride interacts with the outer part of the filter (29).…”

Section: Discussionmentioning

confidence: 99%

“…Previously, mutations at ␣Ser-583 have been found to cause only modest changes in apparent amiloride binding affinity, in stark contrast to all mutations made at the analogous positions in the ␤ and ␥ subunits (␤Gly-525 and ␥Gly-542) where any mutation of the Gly residue greatly weakened amiloride block (8), suggesting that the backbone assumes and angles uniquely accessible to Gly at this position. In the only resolved structure of a protein with bound amiloride, that of urokinase-type plasminogen activator, several Ser and Gly residues were involved in the coordination of amiloride through either the backbone carbonyl or the Ser hydroxyl (20). Here, we have generated a series of point mutations at ␣Ser-583 to explore the role of this residue in amiloride block.…”

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confidence: 99%

On the Interaction between Amiloride and Its Putative α-Subunit Epithelial Na+ Channel Binding Site

Kashlan¹,

Sheng²,

Kleyman

2005

Journal of Biological Chemistry

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The epithelial Na ؉ channel (ENaC) belongs to the structurally conserved ENaC/Degenerin superfamily. These channels are blocked by amiloride and its analogues. Several amino acid residues have been implicated in amiloride binding. Primary among these are ␣Ser-583, ␤Gly-525, and ␥Gly-542, which are present at a homologous site within the three subunits of ENaC. Mutations of the ␤ and ␥ glycines greatly weakened amiloride block, but, surprisingly, mutation of the serine of the ␣ subunit resulted in moderate (<5-fold) weakening of amiloride K i . We investigated the role of ␣Ser-583 in amiloride binding by systematically mutating ␣Ser-583 and analyzing the mutant channels with two-electrode voltage clamp. We observed that most mutations had moderate effects on amiloride block, whereas those introducing rings showed dramatic effects on amiloride block. In addition, mutations introducing a ␤-methyl group at this site altered the electric field of ENaC, affecting both amiloride binding and the voltage dependence of channel gating. We also found that the His mutation, in addition to greatly weakening amiloride binding, appends a voltage-sensitive gate within the pore of ENaC at low pH. Because diverse residues at ␣583, such as Asn, Gln, Ser, Gly, Thr, and Ala, have similar amiloride binding affinities, our results suggest that the wild type Ser side chain is not important for amiloride binding. However, given that some ␣Ser-583 mutations affect the electrical properties of the channel whereas those introducing rings greatly weaken amiloride block, we conclude that amiloride binds at or near this site and that ␣Ser-583 may have a role in ion permeation through ENaC.The epithelial sodium channel (ENaC) 1 is the primary target of the potassium-sparing diuretic, amiloride. ENaC is expressed in the apical membrane of sodium-absorptive epithelia such as the distal nephron, lung airway and alveoli, and descending colon (1). As such, ENaC plays a critical role in maintaining Na ϩ homeostasis, controlling blood pressure and airway fluid volume. ENaC is a member of the ENaC/Degenerin superfamily, all of which conduct Na ϩ , are inhibited by amiloride, and have some structural features in common. They each have two hydrophobic membrane-spanning domains (M1 and M2) with intracellular N and C termini and a large extracellular loop containing two or three cysteine-rich domains (2). To date, ␣, ␤, ␥, and ␦ ENaC subunits, which share 30 -40% sequence identity, have been identified in mammals. ENaC is largely found in the kidney, lung, and colon as comprised of ␣, ␤, and ␥ subunits, although the subunit stoichiometry of functional channels remains controversial. Several groups have proposed a tetrameric ␣ 2 ␤␥ quaternary structure, although a nonameric ␣ 3 ␤ 3 ␥ 3 arrangement has also been proposed (3-6). The ␦ subunit may substitute for ␣ in some tissues.Understanding the mode of amiloride binding and the mechanism by which it inhibits ENaC has been the objective of numerous studies (2). Apparent amiloride binding affinity was found...

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“…The lack of measurable binding to DFP-treated uPA show that covalent modification of the serine hydroxyl by DFP precludes binding of the inhibitor to the S 1 pocket, most likely through steric hindrance by the diisopropyl moiety on the modified ␤-hydroxyl of Ser 356/195 , because mutation of this residue to an alanine still gives measurable binding to upain-1 phage and upain-1-D1D2 construct (see below). Additional phage ELISA experiments showed that 80 M PAB or 2 mM amiloride, two small molecule compounds known to bind in the S 1 specificity pocket of uPA (33,34), showed an effective competition for the binding of upain-1 phage particles, further suggesting a direct interaction with the S 1 pocket and the active site of uPA (data not shown).…”

Section: Determination Of the Equilibrium Binding Constant (K D ) Formentioning

confidence: 96%

A Urokinase-type Plasminogen Activator-inhibiting Cyclic Peptide with an Unusual P2 Residue and an Extended Protease Binding Surface Demonstrates New Modalities for Enzyme Inhibition

Hansen

Wind

Blouse

et al. 2005

Journal of Biological Chemistry

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To find new principles for inhibiting serine proteases, we screened phage-displayed random peptide repertoires with urokinase-type plasminogen activator (uPA) as the target. The most frequent of the isolated phage clones contained the disulfide bridgeconstrained sequence CSWRGLENHRMC, which we designated upain-1. When expressed recombinantly with a protein fusion partner, upain-1 inhibited the enzymatic activity of uPA competitively with a temperature and pH-dependent K i , which at 25°C and pH 7.4 was ϳ500 nM. At the same conditions, the equilibrium dissociation constant K D , monitored by displacement of p-aminobenzamidine from the specificity pocket of uPA, was ϳ400 nM. By an inhibitory screen against other serine proteases, including trypsin, upain-1 was found to be highly selective for uPA. The cyclical structure of upain-1 was indispensable for uPA binding. Alanine-scanning mutagenesis identified Arg 4 of upain-1 as the P 1 residue and indicated an extended binding interaction including the specificity pocket and the 37-, 60-, and 97-loops of uPA and the P 1 , P 2 , P 3 , P 4 , and the P 5 residues of upain-1. Substitution with alanine of the P 2 residue, Trp 3 , converted upain-1 into a distinct, although poor, uPA substrate. Upain-1 represents a new type of uPA inhibitor that achieves selectivity by targeting uPA-specific surface loops. Most likely, the inhibitory activity depends on its cyclical structure and the unusual P 2 residue preventing the scissile bond from assuming a tetrahedral geometry and thus from undergoing hydrolysis. Peptide-derived inhibitors such as upain-1 may provide novel mechanistic information about enzyme-inhibitor interactions and alternative methodologies for designing effective protease inhibitors.Serine proteases of the trypsin family (clan SA) have many physiological and pathophysiological functions. There is therefore extensive interest in generating specific inhibitors to be used for pharmacological interference with their enzymatic activity. Moreover, serine proteases are classical subjects for studies of catalytic and inhibitory mechanisms.Serine protease-catalyzed peptide bond hydrolysis proceeds through a tetrahedral transition state formed by a nucleophilic attack on the carbonyl group of the substrate P 1 amino acid by the hydroxyl group of Ser 195 (using the chymotrypsin template numbering (1)), with His 57 and Asp 102 acting as a charge relay system. The protonated His 57 functions as a general acid to facilitate collapse of the tetrahedral intermediate that is stabilized through interactions at the oxyanion hole and main chain ␤-strand-type hydrogen bonds between the P 1 -P 3 and P 2 Ј amino acids of the substrate and residues within the polypeptide binding cleft, as well as specific contacts within the S 1 , S 2 , S 3 , S 1 Ј, and S 2 Ј pockets, which bind respective side chains of the P 1 , P 2 , P 3 , P 1 Ј, and P 2 Ј residues (for reviews, see Refs. 2 and 3). Substrate specificity is governed by the fit of the P residues into their corresponding S-pockets as ...

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Crystals of the urokinase type plasminogen activator variant βc-uPA in complex with small molecule inhibitors open the way towards structure-based drug design 1 1Edited by A. Fersht

Cited by 74 publications

References 29 publications

Development of a Mammalian Suspension Culture for Expression of Active Recombinant Human Urokinase-type Plasminogen Activator

Development of a Mammalian Suspension Culture for Expression of Active Recombinant Human Urokinase-type Plasminogen Activator

On the Interaction between Amiloride and Its Putative α-Subunit Epithelial Na+ Channel Binding Site

A Urokinase-type Plasminogen Activator-inhibiting Cyclic Peptide with an Unusual P2 Residue and an Extended Protease Binding Surface Demonstrates New Modalities for Enzyme Inhibition

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