Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rats received 0.1% lead acetate in their drinking water for 3 weeks or for 6 weeks, at which time renal brush border fractions were obtained for measurement of enzyme activity. Renal brush border preparations from Pb2+-exposed rats exhibited statistically significant decreases in the activity of gamma-glutamyl transpeptidase and alanine aminopeptidase after 3 or 6 weeks of treatment. There was an increase in the activity of alkaline phosphatase which was statistically significant after 3 weeks of Pb2+ exposure. The (Na+,K+) adenosine triphosphatase activity and urokinase activity, located in the basolateral membrane fractions, were unchanged by Pb2+ exposure, as were the protein and phospholipid contents of the brush border fractions. The results are compared to those following acute exposure to Pb2+ or Cd2+.
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PMID:Rat kidney brush border enzyme activity following subchronic oral lead exposure. 285 32

Urokinase activity was measured in kidney homogenate fractions obtained 2 days after injecting rats with 0.5 mg Pb2+/100 g body weight. The activity was higher in the membrane-containing fractions than in the soluble supernatant fractions and was markedly higher in the preparations derived from the lead-treated rats. The kidney poly(A)+ RNA was obtained from these animals and translated in a rabbit reticulocyte lysate system. In the preparations obtained from the lead-treated rats, there was an increased synthesis of a protein, believed to be urokinase, of pl 8.6 and molecular weight (Mr) 45,000.
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PMID:Kidney urokinase activity following acute exposure to lead. 670 45

The need to decrease the time scale for clinical compound discovery has led to innovations at several stages in the process, including genomics/proteomics for target identification, ultrahigh-throughput screening for lead identification, and structure-based drug design and combinatorial chemistry for lead optimization. A critical juncture in the process is the identification of a proper lead compound, because a poor choice may generate costly difficulties at later stages. Lead compounds are commonly identified from high-throughput screens of large compound libraries, derived from known substrates/inhibitors, or identified in computational prescreeusing X-ray crystal structures. Structural information is often consulted to efficiently optimize leads, but under the current paradigm, such data require preidentification and confirmation of compound binding. Here, we describe a new X-ray crystallography-driven screening technique that combines the steps of lead identification, structural assessment, and optimization. The method is rapid, efficient, and high-throughput, and it results in detailed crystallographic structure information. The utility of the method is demonstrated in the discovery and optimization of a new orally available class of urokinase inhibitors for the treatment of cancer.
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PMID:Discovering novel ligands for macromolecules using X-ray crystallographic screening. 1101 52

Thrombin inhibitors are potentially useful in medicine for their anticoagulant and antithrombotic effects. We synthesized and evaluated diverse heterocycle-activated ketones based on the d-Phe-Pro-Arg, and related thrombin active-site recognition motifs, as candidate inhibitors. The peptide-based alpha-ketoheterocycles were typically prepared by either an imidate or a Weinreb amide route (Schemes 1 and 2), the latter of which proved to be more general. Test compounds were generally assayed for inhibition of human alpha-thrombin and bovine trypsin. From a structure-based design standpoint, the heterocycle allows one to explore and adjust interactions within the S1' subsite of thrombin. The preferred alpha-ketoheterocycle is a pi-rich 2-substituted azole with at least two heteroatoms proximal to the carbon bearing the keto group, and a preferred thrombin inhibitor is 2-ketobenzothiazole 3, with a potent K(i) value of 0.2 nM and ca. 15-fold selectivity over trypsin. 2-Ketobenzothiazole 13 exhibited exceedingly potent thrombin inhibition (K(i) = 0.000 65 nM; slow tight binding). Several alpha-ketoheterocycles had thrombin K(i) values in the range 0.1-400 nM. The "Arg" unit in the alpha-ketoheterocycles can be sensitive to stereomutation under mildy basic conditions. For example, 2-ketothiazoles 4 and 59 readily epimerize at pH 7.4, although they are fairly stable stereochemically at pH 3-4; thus, suitable conditions had to be selected for the enzymatic assays. Lead d-Phe-Pro-Arg 2-benzothiazoles 3, 4, and 68 displayed good selectivity for thrombin over other key coagulation enzymes (e.g., factor Xa, plasmin, protein Ca, uPA, tPA, and streptokinase); however, their selectivity for thrombin over trypsin was modest (<25-fold). Compounds 3, 4, and 68 exhibited potent in vitro antithrombotic activity as measured by inhibition of gel-filtered platelet aggregation induced by alpha-thrombin (IC(50) = 30-40 nM). They also proved to be potent anticoagulant/antithrombotic agents in vivo on intravenous administration, as determined in the canine arteriovenous shunt (ED(50) = 0.45-0.65 mg/kg) and the rabbit deep vein thrombosis (ED(50) = 0.1-0.4 mg/kg) models. Intravenous administration of 3, and several analogues, to guinea pigs caused hypotension and electrocardiogram abnormalities. Such cardiovascular side effects were also observed with some nonguanidine inhibitors and inhibitors having recognition motifs other than d-Phe-Pro-Arg. 2-Benzothiazolecarboxylates 4 and 68 exhibited significantly diminished cardiovascular side effects, and benzothiazolecarboxylic acid 4 had the best profile with respect to therapeutic index. The X-ray crystal structures of the ternary complexes 3-thrombin-hirugen and 4-thrombin-hirugen depict novel interactions in the S(1)' region, with the benzothiazole ring forming a hydrogen bond with His-57 and an aromatic stacking interaction with Trp-60D of thrombin's insertion loop. The benzothiazole ring of 3 displaces the Lys-60F side chain into a U-shaped gauche conformation, whereas the benzothiazole carboxylate of 4 forms a salt bridge with the side chain of Lys-60F such that it adopts an extended anti conformation. Since 3 has a 10-fold greater affinity for thrombin than does 4, any increase in binding energy resulting from this salt bridge is apparently offset by perturbations across the enzyme (viz. Figure 4). The increased affinity and selectivity of 2-ketobenzothiazole inhibitors, such as 3, may be primarily due to the aromatic stacking interaction with Trp-60D. However, energy contour calculations with the computer program GRID also indicate a favorable interaction between the benzothiazole sulfur atom and a hydrophobic patch on the surface of thrombin.
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PMID:In-depth study of tripeptide-based alpha-ketoheterocycles as inhibitors of thrombin. Effective utilization of the S1' subsite and its implications to structure-based drug design. 1577 42