Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Both human and bovine prothrombin fragment 2 (the second kringle) have been cocrystallized separately with human PPACK (D-Phe-Pro-Arg)-thrombin, and the structures of these noncovalent complexes have been determined and refined (R = 0.155 and 0.157, respectively) at 3.3-A resolution using X-ray crystallographic methods. The kringles interact with thrombin at a site that has previously been proposed to be the heparin binding region. The latter is a highly electropositive surface near the C-terminal helix of thrombin abundant in arginine and lysine residues. These form salt bridges with acidic side chains of kringle 2. Somewhat unexpectedly, the negative groups of the kringle correspond to an enlarged anionic center of the lysine binding site of lysine binding kringles such as plasminogens K1 and K4 and TPA K2. The anionic motif is DGDEE in prothrombin kringle 2. The corresponding cationic center of the lysine binding site region has an unfavorable Arg70Asp substitution, but Lys35 is conserved. However, the folding of fragment 2 is different from that of prothrombin kringle 1 and other kringles: the second outer loop possesses a distorted two-turn helix, and the hairpin beta-turn of the second inner loop pivots at Val64 and Asp70 by 60 degrees. Lys35 is located on a turn of the helix, which causes it to project into solvent space in the fragment 2-thrombin complex, thereby devastating any vestige of the cationic center of the lysine binding site. Since fragment 2 has not been reported to bind lysine, it most likely has a different inherent folding conformation for the second outer loop, as has also been observed to be the case with TPA K2 and the urokinase kringle. The movement of the Val64-Asp70 beta-turn is most likely a conformational change accompanying complexation, which reveals a new heretofore unsuspected flexibility in kringles. The fragment 2-thrombin complex is only the second cassette module-catalytic domain structure to be determined for a multidomain blood protein and only the third domain-domain interaction to be described among such proteins, the others being factor Xa without a Gla domain and Ca2+ prothrombin fragment 1 with a Gla domain and a kringle.
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PMID:Structures of the noncovalent complexes of human and bovine prothrombin fragment 2 with human PPACK-thrombin. 838 13

Trypsinogen is converted to trypsin by the removal of a peptide from the N terminus, which permits formation of a salt bridge between the new N-terminal Ile (residue 16) and Asp194. Formation of this salt bridge triggers a conformational change in the "activation domain" of trypsin, creating the S1 binding site and oxyanion hole. Thus, the activation of trypsinogen appears to represent an example of protein folding driven by electrostatic interactions. The following trypsin mutants have been constructed to explore this problem: Asp194Asn, Ile16Val, Ile16Ala, and Ile16Gly. The bovine pancreatic trypsin inhibitor (BPTI), benzamidine, and leupeptin affinities and activity and pH-rate profiles of these mutants have been measured. The changes in BPTI and benzamidine affinity measure destabilization of the activation domain. These experiments indicate that hydrophobic interactions of the Ile16 side chain provide 5 kcal/mol of stabilization energy to the activation domain while the salt bridge accounts for 3 kcal/mol. Thus, hydrophobic interactions provide the majority of stabilization energy for the trypsinogen to trypsin conversion. The pH-rate profiles of I16A and I16G are significantly different than the pH-rate profile of trypsin, further confirming that the activation domain has been destabilized. Moreover, these mutations decrease kcat/Km and leupeptin affinity in parallel with the decrease in stability of the activation domain. Acylation is selectively decreased, while substrate binding and deacylation are not affected. Together these observations indicate that the stability of protein structure is an important component of transition state stabilization in enzyme catalysis. These results also suggest that active zymogens can be created without providing a counterion for Asp194, and thus have important implications for the elucidation of the structural features which account for the zymogen activity of tissue plasminogen activator and urokinase.
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PMID:Hydrophobic interactions control zymogen activation in the trypsin family of serine proteases. 860 1

Many hypotheses have been proposed for renal stone formation. It has been argued that with infection-induced renal stones the hydrolysis of urea by bacterial urease increases urinary pH, with consequent stone formation. Unfortunately, this theory is not applicable to the micro-organisms that do not produce urease (e.g. Escherichia coli). It has been recently reported that E. coli reduces the urinary urokinase activity of male rats, but does not influence the urinary sialidase activity. This study has now been expanded to the urease-producing bacteria Proteus mirabilis, Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa and Micrococcus luteus. Subcutaneous injections with these bacteria were found to significantly (P < 0.003) reduce the UK activity of extrarenally obstructed kidneys. The urease-producing mammalian skin bacterium, M. luteus, was, however, the exception (P = 0.1079). In contrast to S. epidermidis, P. aeruginosa and M. luteus (P < 0.0213), P. mirabilis and S. aureus had no effect on renal sialidase activity (P < 0.4047). These results may explain why Proteus species are predominant in infection-induced renal stones. According to the urokinase-sialidase hypothesis, a decrease in urinary urokinase activity should increase the uromucoid levels, whilst no effect on the urinary sialidase activity should favour conversion of urinary uromucoid to mineralizable matrix. These conditions may lead to renal stone formation. An increase in urinary pH resulting from urease-producing micro-organisms will increase salt precipitation on the uromucoid. It is thus concluded that urease-producing bacteria may play a double role in renal stone formation.
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PMID:In vivo effects of urease-producing bacteria involved with the pathogenesis of infection-induced urolithiasis on renal urokinase and sialidase activity. 883 91

The concentrations of matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-8 (MMP-8), matrix metalloproteinase-9 (MMP-9), lactoferrin and urokinase plasminogen activator (uPA), tissue-type plasminogen activator (tPA) and the inhibitors, tissue inhibitor of metalloproteinase-1 (TIMP-1), plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor (PAI-2), and alpha2-macroglobulin in the synovial fluids of patients with rheumatoid arthritis was determined before and during chemical synoviorthesis with a sodium salt of the fatty acids from cod-liver oil (Varicocid). Synovial fluids were obtained before treatment from 37 patients with rheumatoid arthritis and, in most cases, at 8 and 24 h after injection of the agent. Well-established ELISAs were used to determine the amounts of all proteins. All patients with rheumatoid arthritis revealed very high levels of metalloproteinases (about 1-15 mu g/ml) in their synovial fluids. During the inflammation inducing treatment the granulocyte enzymes increased. In contrast to this, the level of MMP-1 decreased. All granulocyte-derived enzymes were strongly correlated with each other, whereas their dependence on the granulocyte count was only weak. uPA and PAI-2 showed good correlations with the granulocytes-derived enzymes, but were also only weakly correlating with the cell counts. t-PA was not detected by the ELISA used. The proteases, MMP-8, MMP-9 and uPA were increased 8 h after the treatment, whereas the specific inhibitors TIMP-1, PAI-1 and PAI-2 showed significant changes only 24 h after the injection. Matrix metalloproteinases are important factors in the pathogenesis of rheumatoid arthritis. The inflammatory activity in the joint could be better correlated to the granulocyte enzymes than to the granulocyte counts. The levels of uPA and PAI-2 are also parallel to the granulocyte enzyme levels and might underly the same regulatory mechanism.
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PMID:Determination of metalloproteinases, plasminogen-activators and their inhibitors in the synovial fluids of patients with rheumatoid arthritis during chemical synoviorthesis. 891 99

Tissue type plasminogen activator (tPA) is the physiological initiator of fibrinolysis, activating plasminogen via highly specific proteolysis; plasmin then degrades fibrin with relatively broad specificity. Unlike other chymotrypsin family serine proteinases, tPA is proteolytically active in a single-chain form. This form is also preferred for therapeutic administration of tPA in cases of acute myocardial infarction. The proteolytic cleavage which activates most other chymotrypsin family serine proteinases increases the catalytic efficiency of tPA only 5- to 10-fold. The X-ray crystal structure of the catalytic domain of recombinant human single-chain tPA shows that Lys156 forms a salt bridge with Asp194, promoting an active conformation in the single-chain form. Comparisons with the structures of other serine proteinases that also possess Lys156, such as trypsin, factor Xa and human urokinase plasminogen activator (uPA), identify a set of secondary interactions which are required for Lys156 to fulfil this activating role. These findings help explain the anomalous single-chain activity of tPA and may suggest strategies for design of new therapeutic plasminogen activators.
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PMID:Lysine 156 promotes the anomalous proenzyme activity of tPA: X-ray crystal structure of single-chain human tPA. 930 22

Regardless of the primary cause, progressive renal deterioration with sclerosis is a hallmark of many renal diseases. Several studies have shown the superiority of angiotensin-converting enzyme inhibitors compared with other antihypertensive agents in providing protection from progressive renal deterioration. Furthermore, animal studies have shown that angiotensin II antagonists in excess of antihypertensive doses can also ameliorate or reverse glomerulosclerosis, leading to the hypothesis that angiotensin II has nonhemodynamic effects that mediate the renoprotective effects shown in these investigations. Although historically angiotensin II has been associated with salt and fluid homeostasis, recent data show that angiotensin II induces cell growth and matrix accumulation in glomerular cells. Plasminogen activator inhibitor-1 has been shown to be the major inhibitor of tissue plasminogen activator and urokinase-like plasminogen activator, with potentially important effects not only on thrombosis/fibrinolysis, but also on matrix degradation because of the proteolytic actions of these substances. Angiotensin II has been shown to influence the actions of plasminogen activator inhibitor-1 and, consequently, its thrombotic and sclerotic effects. Various studies, both in vitro and in vivo, have shown that direct hemodynamic actions, modulation of endothelial injury, and growth factor actions also may be important in the development of sclerosis. These factors can be directly modulated by angiotensin II inhibition. Sclerosis may even be reversed when therapies augment matrix degradation processes, both by directly increasing proteolytic activity and by downregulating inhibitors of matrix degradation. These observations indicate that angiotensin II is important in fibrotic as well as thrombotic renal injuries that lead to progressive renal disease and also in the development of therapies such as specific angiotensin receptor antagonists to prevent or reverse these conditions.
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PMID:The role of angiotensin II and plasminogen activator inhibitor-1 in progressive glomerulosclerosis. 1067 14

Sepharose 4B-based affinity adsorbent was prepared using cefazoline as a ligand, and epichlorohydrin as an activator. The density of the ligand in the affinity adsorbents was about 43 &mgr;mol/g wet gel. The optimal adsorption pH for urokinase was 6.0, and the optimal adsorption salt concentration was 1.0 mol/L NaCl. The optimal conditions of elution were 0.1 mol/L glycine buffer, pH 9.0, containing 0.5 mol/L NaCl. A crude urokinase with specific activity 500 u/mg protein was purified on an affinity chromatography column (1 cm x 12 cm), yielding a product of specific activity of 49 300 u/mg with 78% recovery and 98.6-fold purification.
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PMID:Affinity Chromatography of Urokinase Using Cefazoline as a Ligand. 1216 2

The present study characterized the persistent changes (ie, off-treatment) resulting from short-term antihypertensive treatments on mean arterial pressure (MAP) and structurally based vascular resistance. Rats were treated for 14 days with enalapril (30 mg x kg(-1) x d(-1)) with regular (ENAL, 0.4%) or low salt (ELS, 0.04%) diets, or a triple therapy (Triple: hydralazine 45 mg x kg(-1) x d(-1), hydrochlorothiazide 100 mg/L, and nifedipine 200 mg/d). MAP was continuously recorded via radiotelemetry. Structurally based hindlimb vascular resistance properties (resistance at maximum dilation [Max Dil]; resistance at maximum constriction [Max Con]) were assessed after 14-day enalapril treatment and 2 to 3 weeks after all drugs were withdrawn. Aortic urokinase plasminogen activator (uPA) activity was measured by zymography after 14 days of ELS. All treatments induced a significant, persistent decrease in the off-treatment MAP (ENAL downward arrow 12+/-4.6%, ELS downward arrow 16+/-2.6%, Triple downward arrow 5+/-4.17%). During treatment (14 days) the enalapril group had significant changes in the index of medial bulk (Max Con downward arrow 15+/-2.6%), but only minimal changes in lumen properties (Max Dil downward arrow 3+/-6.5%, NS). After stopping therapy, vascular properties at Max Dil were significantly decreased only in the 2 enalapril groups (ENAL downward arrow 15+/-7.9%, P<0.05; ELS downward arrow 9+/-6.0%, P<0.05; Triple downward arrow 2+/-9.8%, NS), whereas Max Con was significantly decreased in all groups (ENAL downward arrow 12+/-8.0%, ELS downward arrow 16+/-6.1%, Triple downward arrow 7+/-5.4%). At 14 days of ELS treatment, there was increased aortic uPA activity (1.6-fold). The findings reveal that various short-term antihypertensive treatments can produce persistent long-term changes in MAP and vascular structure. Further, the magnitude of the depressor response may be as important in inducing persistent changes as is the removal of angiotensin II.
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PMID:Time course of vascular structural changes during and after short-term antihypertensive treatment. 1281 Jul 56

The developments and trends of hemostatic and antithrombotic drugs in Japan were investigated chronologically for the last 50 years after the 2nd World War. 1. Hemostatic drugs are classified into three groups ; capillary stabilizers, blood coagulants and antifibrinolytics. l) As to capillary stabilizers, flavonoid (rutin, 1949), adrenochrome derivative (carbazochrome, 1954) and conjugated estrogen (Premarin, 1964) were introduced therapeutically. Especially, the soluble types of adrenochrome compounds (Adona 1956, S-Adchnon, 1962) were devised and used widely in Japan. 2) Drugs concerning blood coagulation, thrombin, introduced in 1953, and hemocoagulase, a snake venom introduced in 1966, were used clinically. V.K. groups producing various coagulation factors were introduced as V.K1 (Phytonadione, 1962) and V.K2 (rnenatetrenone,1972), and they were admitted in "The Japanese Pharmacopoeia"editions 8 and 14, respectively). 3) Regarding antifibrinolytic drugs, Japanese researchers have made remarkable contributions. e-Aminocapronic acid (Ipsilon, 1962) and tranexamic acid (Transamin, 1965) were developed and used for various abnormal bleedings or hemorrhage associated with plasmin over-activation. tranexamic acid also proved to suppress inflammations of the throat such as tonsillitis, pharyngitis or laryngitis. 2. Antithrombotic drugs are also divided into three groups; anticoagulants, antiplatelet drugs and fibrinolytics.1) The anticoagulants used therapeutically by injection are heparins (Na-salt, 1951; Ca-salt, 1962) and low-molecular-weight heparins such as dalteparin (1992), parnaparin (1994) and reviparin (1999). The low molecule compounds are superior to the original heparins in reducing the risk of bleeding. As oral anticoagulants, coumarin derivatives, dicumarol (1950), ethylbiscoumacetate (1954), phenylindandione (1956) and warfarin (1962) are known. Warfarin potassium is the main drug for oral therapy of thromboembolism lately. Gabexate mesilate (1989) and nafamostat mesilate (1989) were developed in Japan and used for DIC and acute pancreatitis to inhibit protease enzymes. Argatroban is a unique antithrombin product developed by Japanese researchers in 1990, and is used for vascular or cerebral thrombosis. After noticing in 1968 that aspirin inhibits platelet aggregation and prevents myocardial infraction, projects for developing antiplatelet drugs were initiated worldwide. Ticlopidine, originally developed in France, was introduced in 1981 and prevailed widely in Japan for reducing the risk of thrombotic stroke. Aspirin itself was recognized by the FDA (USA) as an antithrombotic drug in 1988, and was also approved by Japanese authorities in 2000. PGE1 clathrate compounds have also been developed as antiplatelet drugs; alprostadil alfadex for injection (1979), and limaprost alfadex for oral use (1988). The PGI2 product, beraprost sodium, for oral use followed them in 1992. Other antiplatelet drugs with unique mechanisms explored in Japan: Ozagrel (1988), which inhibits TXA2 synthetase, cilostazol (1988), which inhibits cAMP phosphodiesterase, and sarpogrelate (1993), which blocks 5HT in platelets, are the notable drugs in this field. Ethyl icosapentate, from fish oil, is available for antiplatelet therapy. Concerning the fibrinolytic system, plasminogen activators are useful for thromboembolism. The streptokinase from bacterial origin developed in the USA and Europe was not introduced, and urokinase (1965) was the first plasminogen activator developed in Japan. Then tissue plasminogen activators (t-PA) tisokinase (cell culture, 1991), alteplase (genetical recombination, 1991), nateplase (genetical recombination, 1996), monteplase (1998) and pamiteplase (1998) were developed and approved for acute myocardial infarction. Nasaruplase (prourokinase, cell culture,1991) was also approved for the same indication. While the development of the hemostatic drugs ceased in the 1960s, avid project studies for antithrombotic drugs including fibrinolytics began in the 1980s and are progressing now towards new molecular targets. This may be due to the increasing tendency of cardiovascular thromboembolic diathesis in Japan. (The figures in parentheses are the years approved by the Japanese Ministry of Health, Labor and Welfare.)
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PMID:[A 50-year history of new drugs in Japan-the development and trends of hemostatics and antithrombotic drugs]. 1457 69

The preparation and assessment of biological activity of 6-substituted 2-naphthamidine inhibitors of the serine protease urokinase plasminogen activator (uPA, or urokinase) is described. 2-Naphthamidine was chosen as a starting point based on synthetic considerations and on modeling of substituent vectors. Phenyl amides at the 6-position were found to improve binding; replacement of the amide with other two-atom linkers proved ineffective. The phenyl group itself is situated near the S1' subsite; substitutions off of the phenyl group accessed S1' and other distant binding regions. Three new points of interaction were defined and explored through ring substitution. A solvent-exposed salt bridge with the Asp60A carboxylate was formed using a 4-alkylamino group, improving affinity to K(i) = 40 nM. Inhibitors also accessed two hydrophobic regions. One interaction is characterized by a tight hydrophobic fit made with a small dimple largely defined by His57 and His99; a weaker, less specific interaction involves alkyl groups reaching into the broad prime-side protein binding region near Val41 and the Cys42-Cys58 disulfide, displacing water molecules and leading to small gains in activity. Many inhibitors accessed two of these three regions. Affinities range as low as K(i) = 6 nM, and many compounds had K(i) < 100 nM, while moderate to excellent selectivity was gained versus four of five members of a panel of relevant serine proteases. Also, some selectivity against trypsin was generated via the interaction with Asp60A. X-ray structures of many of these compounds were used to inform our inhibitor design and to increase our understanding of key interactions. In combination with our exploration of 8-substitution patterns, we have identified a number of novel binding interactions for uPA inhibitors.
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PMID:Identification of novel binding interactions in the development of potent, selective 2-naphthamidine inhibitors of urokinase. Synthesis, structural analysis, and SAR of N-phenyl amide 6-substitution. 1471 4


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