Enzyme-specific inhibitory activities

The fungal metabolites TAN-1496 A, C, and E (isolated from the culture broth of Microsphaeropsis sp. FL-16144) have been shown to have specific inhibitory activity against DNA topoisomerase 1. DNA topoisomerase 1 is an enzyme responsible for DNA metabolism and have been proposed as an intracellular target for cancer chemotherapy. ... 

Apart from these two Vertex compounds, only one other caspase inhibitor, BDN-6556, has been used in clinical trials. This compound belongs to the class of oxamyl dipeptides and was originally developed by Idun Pharmaceuticals (taken over by Pfizer). It is the only pan-caspase inhibitor that has been evaluated in humans. BDN-6556 displays inhibitory activity against all tested human caspases. It is also an irreversible, caspase-specific inhibitor that does not inhibit other major classes of proteases, or other enzymes or receptors. The therapeutic potential of BDN-6556 was first evaluated in several animal models of liver disease because numerous publications suggested that apoptosis contributes substantially to the development of some hepatic diseases, such as alcoholic hepatitis, hepatitis B and C (HBV, HCV), non-alcoholic steato-hepatitis (NASH), and ischemia/reperfusion injury associated with liver transplant. Accordingly, BDN-6556 was tested in a phase I study. The drug was safe and... 

Inhibitor assay A suitable amount of inhibitor was preincubated with 0.2 ml of polygalacturonase and buffer in a total volume of 1 ml for 10 minutes at 37°C. Control without inhibitor was run simultaneously. The enzyme reaction was initiated by the addition of 1 ml of substrate solution (1% polygalacturonic acid). The decrease in PG activity was a measure of the inhibitory activity. Proper controls containing only Dieffenbachia extract and no fungal PG in the assay mixture were also run to account for the inherent PG activity, if any, of Dieffenbachia extract. One unit of inhibitor activity is defined as the amount of inhibitor that reduces the polygalacturonase activity under the assay conditions by one unit. Specific activity of the inhibitor is expressed as units per mg protein. 

A series of carboxyl derivatized polyglucoses were studied as inhibitors of ribonuclease activity, in an attempt to relate charge density to inhibitory activity.202 In comparison with other factors, it was concluded that coulombic forces probably play a major role in complex-formation between enzyme and substrate, and between enzyme and inhibitor. However, other specific, nonelectrostatic forces were shown to participate in the binding of bovine pancreatic ribonuclease to ribonucleic acid.204... 

Since preliminary studies showed that 6-hydroxymellein-O-methyl-transferase activity was appreciably inhibited in the presence of the reaction products, the mode of product inhibition of the enzyme was studied in detail in order to understand the regulatory mechanism of in vivo methyltransfer. It is well known that S-adenosyl-Z.-homocysteine (SAH), which is a common product of many O-methyltransferases that use SAM as methyl donor, is usually a potent inhibitor of such enzymes. In the 6-hydroxymellein-Omethyltransferase catalyzing reaction another product of this enzyme, 6-methoxymellein, has pronounced inhibitory activity, in addition to SAH. Since the specific product of the transferase reaction, 6-methoxymellein, is capable of inhibiting transferase activity [88], this observation suggests that activity of the transferase is specifically regulated in response to increases in cellular concentrations of its reaction products in carrot cells. It has been also found that 6-methoxymellein inhibits transferase activity with respect not only to 6-hydroxymellein but also to SAM, competitively. This competitive inhibition was also found in SAH as a function of the co-substrates of the enzyme [89]. It follows that the reaction catalyzed by 6-hydroxymellein-O-methyltransferase proceeds by a sequential bireactant mechanism in which the entry of the co-substrates to form the enzyme-substrate complexes and the release of the co-products to generate free enzyme take place in random order [Fig. (7)]. This result also implies that 6-methoxymellein and SAH have to associate with the free transferase protein to exhibit their inhibitory activities, and cannot work as the inhibitors after the enzyme forms complexes with the the substrate. If, therefore, 6-hydroxymellein-O-methyltransferase activity is controlled in vivo by its specific product 6-methoxymellein, this compound should... 

The enzyme was purified from Candida utilis in 1965 by Rosen et al. (8Q). Dried yeast was allowed to autolyze in phosphate buffer at pH 7.5 for 48 hr, and the enzyme was isolated in crystalline form from these autolysates by a procedure which included heating to 55° at pH 5.0, fractionation with ammonium sulfate, and purification on phospho-cellulose columns from which the enzyme was specifically eluted with malonate buffer containing 2.0 mM FDP. Crystallization was carried out by addition of ammonium sulfate in the presence of mM magnesium chloride. The Candida enzyme was more active than the mammalian FDPases at room temperature and pH 9.5 the crystalline protein catalyzed the hydrolysis of 83 /nnoles of FDP per minute per milligram of protein. The enzyme was completely inactive with other phosphate esters, including sedoheptulose diphosphate, ribulose diphosphate, and fructose 1- or fructose 6-phosphates. Nor was the activity of the enzyme inhibited by any of these compounds. Optimum activity was observed at concentrations of FDP between 0.05 and 0.5 mM higher concentrations of FDP (5 mM) were inhibitory. 

Fraenkel et al. (17), who isolated mutant strains which had lost the ability to grow on glycerol, succinate, or acetate but grew normally on hexoses or pentoses. These organisms were shown to be deficient in a specific FDPase, which could be distinguished from the nonspecific acid hexosephosphatase present in both mutant or wild-type strains by the fact that the latter was present in the periplasmic space (86) and did not require a divalent metal cation. The properties of the specific FDPase were confirmed with a partially purified preparation (87) the E. coli enzyme was shown to be highly specific for FDP and to be active with very low concentrations of this substance. The requirement for a divalent cation was satisfied by Mg2+, which was far more effective than Mn2+ other divalent cations were either inactive or inhibitory. The partially purified enzyme showed optimum activity at pH 7.8, with very little activity below pH 7 or above pH 9. The enzyme resembled mammalian and Candida FDPases in its sensitivity to low concentrations of AMP it was approximately 50% inhibited at an AMP concentration of 2.5 X 10-° M. 

Some inhibitory activity of gossypol against mouse mammary adenocarcinoma 755 and borderline activity against leukaemia P388 were reported [323, 324], The effective dose range of gossypol is rather narrow because of toxicity [324]. Gossypol has recently been found to be a specific inhibitor of DNA polymerase a [325], a major enzyme involved in DNA replication. This compound may also interfere with the DNA repair process [325]. 

This geminal type of functionality occurs when the sugar moiety is in specific stereo orientation, and with acetamido functionality. Additionally, the basic functional group (-NHAc) may act as a binding site with receptors. Such disaccharides should be valuable tools to probe any enzyme inhibitory activity of synthesized (1 -2)-S-thiodisaccharides. 

As for deaminase, the kinetic analysis suggests a partial mixed-type inhibition mechanism. Both the Ki value of the inhibitor and the breakdown rate of the enzyme-substrate-inhibitor complex are dependent on the chain length of the PolyP, thus suggesting that the breakdown rate of the enzyme-substrate-inhibitor complex is regulated by the binding of Polyphosphate to a specific inhibitory site (Yoshino and Murakami, 1988). More complicated interactions were observed between PolyP and two oxidases, i.e. spermidine oxidase of soybeen seedling and bovine serum amine oxidase. PolyP competitively inhibits the activities of both enzymes, but may serve as an regulator because the amino oxydases are also active with the polyamine-PolyP complexes (Di Paolo et al., 1995). 

Rifampicin was first shown by Hartmann et al. 54 to have a specific inhibitory effect on RNA polymerase from E. coli. Later, other active ansamycins were found and RNA polymerases from a large variety of bacteria other than E. coli proved to be sensitive to the drug. More recently, an RNA polymerase from E. coli containing only one subunit and probably involved in the initiation of DNA replication (dna G gene product) has been shown to be resistant to rifampicin5 s This holds true also for the various mammalian RNA polymerases. In contrast to non-specific inhibitors of transcription such as actinomycin and mitomycin, rifampicin interacts specifically with the bacterial enzyme itself. With the aid of 14C-labelled rifampicin it could be shown that the drug forms a very stable complex with the enzyme in a molar ratio of 1 1S6> 57 The dissociation constant of this complex is 10-9 M at 37 °C and... 

After the challenge of the dynamic thiolester system (CDS-1 A) with acetylcholinesterase, the optimal constituents were immediately identified and hydrolyzed to acid and thiol products. The thiol product formed was simultaneously incorporated in the dynamic system to regenerate the optimal thiolester. During the acetylcholinesterase resolution process, two acid products, acetic and propionic acid, respectively, were mainly detected, with the acetyl ester hydrolyzed more rapidly (tl/2 = 210 min) than the propionyl ester (t1/2 = 270 min) as shown in Fig. 2. Only after significant hydrolysis of these two acyl species did the enzyme start to hydrolyze slowly the butyrate thiolester (t1/2 = 1,100 min), a lag phase possibly caused by inhibitory activities of the present thiolesters [8]. All the other acyl groups remained untouched by enzyme, a result which is in accordance with the known specificity of acetylcholinesterase. 

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