Cinnamic acid inhibitor

Moran, E. J. Sarshar, S. Cargill, J. F. Shahbaz, M. M. Lio, A. M. Mjalli, A. M. M. Armstrong, R. W. Radio Frequency Tag Encoded Combinatorial Library Method for the Discovery of Tripeptide-Substituted Cinnamic Acid Inhibitors of the Protein Tyrosine Phosphatase PTP1B, J. Am. Chem. Soc. 1995, 777,10787. 

Moran EJ, Sarshar S, Cargill JF, Shahbaz M, Lio A, Mjalli AMM, Armstrong RW, Radiofrequency tog encoded combinatorial library method for the discovery of tripeptide-substituted cinnamic acid inhibitors of the protein tyrosine phosphatase PTPSB J. Am. Chem. Soc., 117 10787-1788, 1995. 

EJ Moran, S Sarshar, JF Cargill, MM Shahbaz, A Lio, AMM Mjalli, RW Armstrong. Radio frequency tag encoded combinatorial library method for discovery of tri peptide-substituted cinnamic acid inhibitors of the protein tyrosine phosphatase PTP1B. J Am Chem Soc 117 10787-10788, 1995. 

Phenylalanine ammonia-lyase (PAL EC 4.3.1.5) is a pivotal enzyme in controlling flow of carbon from aromatic amino acids to secondary aromatic compounds (Figure 1) (28). PAL primarily deaminates phenylalanine to form t-cinnamic acid, however, in many species, it also less efficiently deaminates tyrosine to form -coumaric acid. Because PAL is restricted to plants and is an important enzyme in plant development, Jangaard (29) suggested that PAL inhibitors might make safe and effective herbicides, however, in his screen of several herbicides, he found no compound to have a specific effect on PAL. This was also the case in studies by Hoagland and Duke (30, 31.) in which 16 herbicides were screened. 

A large number of in vitro inhibitors of PAL are known, however, few of these effectively inhibit PAL activity in vivo without also causing phytotoxic effects unrelated to their effects on PAL. PAL inhibitors fall into four categories 1) PAL product-inhibitors such as t-cinnamic acid and p-coumaric acid (e.g. 35) ... 

Acrylic acid (AA) and methacrylic acid (MAA) (purchased from Merck) are freed from inhibitor on a neutral aluminium oxid column and distilled. Acrylamide (AM) from Kebo, Stockholm, is recrystallized once from chloroform solution before use. Other monomers of analytical grade were purchased from Merck and used as received crotonic acid (CA), tiglic acid (TA), 3-methyl crotonic acid (3-MCA), and a-methyl cinnamic acid (oi-MCia) (Table 1). Benzophenone (analytical grade, Kebo) and acetone (spectroscope grade, Merck) were used as supplied. 

In the experiments reported above, no strong indications were obtained that the allelochemlcals interfered directly with the synthesis of ATP by acting on the energy transfer pathway complex). However, some of the compounds did inhibit ATP hydrolysis, as measured with preparations in which the mitochondria had been ruptured by freeze-thawing (Table III). The flavones (except fl one itself) and cinnamic acids strongly inhibited the Mg -ATPase, whereas the benzoic acids and coumarlns were weak inhibitors, i.e., less than 15% inhibition at 10 mM concentrations. Results obtained with quercetin (Table III) agree with published reports in which the compound was shown not to affect ATP synthesis but to inhibit the hydrolysis of ATP by the mitochondrial Mg -ATPase (30). 

In mitochondria, the allelochemlcals acted primarily as electron transport inhibitors. Malate oxidation was more sensitive than either succinate or NADH oxidation. No evidence for interaction with a specific membrane complex was obtained. Instead, Inhibition of substrate oxidation seems to result from alterations and perturbations produced in the inner membrane as reflected in interference with the behavior of transport processes. The compounds did not act as uncouplers or directly inhibit ATP synthesis. However, naringenin, some of the flavones, and the cinnamic acids dj inhibit the hydrolysis of ATP catalyzed by mitochondrial Mg -ATPase. 

Tiburzy (22,31) obtained similar results by application of the PAL inhibitor aminooxyacetic acid (AOA). However, AOA does not specifically inhibit PAL (99), and PAL is not only involved in lignin biosynthesis (100). Thus, AOA and the related inhibitor aminooxyphenyl propionic acid (AOPP) (101,102) inhibit the biosynthesis of lignin (103,104), anthocyanins (105), other flavonoids (106), and conjugates of cinnamic acids (107) via PAL, as well as ethylene (108-110) via a pyridoxal phosphate dependent enzyme (110,111). In view of the possible function of phenolic compounds as phytoalexins (21,112,113) and the well documented role of ethylene in some resistance reactions (114-116), the above cited experiments with AOA (22,... 

Whether an inhibitor acts in a competitive or noncompetitive manner is deduced from a Lineweaver-Burk or direct linear plot using varying concentrations of inhibitor and substrate. In separate assays, two substances will be added to the dopa-tyrosinase reaction mixture, and the effect on enzyme activity will be quantified. The structures of the potential inhibitors, cinnamic acid and thiourea, are shown in Figure E5.9. The inhibition assays must be done immediately following the KM studies. To measure inhibition, reaction rates both with and without inhibitor must be used and the tyrosinase activity must not be significantly different. If it is necessary to do the inhibition studies later, the Ku assay for L-dopa must be repeated with freshly prepared tyrosinase solution. 

To set up the inhibition assay, prepare a table similar to Table E5. 1. Inhibitor should appear in the list of reagents before tyrosinase. Use the same level of tyrosinase and the same dopa stereoisomer as in part C. Vary the amount of dopa as in part C. A constant amount of inhibitor (cinnamic acid or thiourea) should be added to each cuvette. You will have to determine this level of inhibitor by trial and error. The desired inhibition rate with saturating substrate is about 50% of the uninhibited rate. Add all reagents except tyrosinase, mix well, and determine the blank rate, if any. Add tyrosinase, mix, and immediately record AA75 for 2 minutes. From recorder traces or graphs of A475 vs. time, calculate AA/min for each assay. 

Catecholases and laccases may be differentiated by the use of substrate specificity tests and selective specific inhibitors (Walker and McCallion, 1980 Ferrar and Walker, 1996 Table C4.1.1). Salicylhydroxamic acid (SHAM), PVP, and/or cinnamic acids (cinnamic, p-coumaric, or ferulic) are probably the best choice for catecholase inhibitors, whereas cetyltrimethylammonium bromide (CETAB) has been found to inhibit most laccases. 

In addition to this up-regulation, PAL is also under strict negative control. It has been postulated that the reaction product of PAL, cinnamic acid, is itself an inhibitor of PAL enzyme activity (Lawton et al., 1980). It has been demonstrated that after addition of exogenous cinnamic acid to suspension cultures, PAL enzyme activity is rapidly lost owing to an inactivation of the active site dehydroalanine of the enzyme (Bolwell et al., 1986). In addition, cinnamic acid has also been shown to inhibit PAL mRNA appearance (Bolwell et al., 1988) with a differential effect being exerted on the different members of the bean PAL family (Mavandad et al., 1990). 

---