Inhibitors EPSP synthase

Dawson RMC, Elliott DC, Elliott WH, Jones KM. Data for Biochemical Research, 3rd edition. 1986. Clarendon Press, Oxford. Miller MJ, Braccolino DS, Cleary DG, Ream JE, Walker MC, Sikorski JA. EPSP synthase inhibitor design iv New aromatic substrate analogs and symmetrical inhibitors containing novel 3-phosphate mimics. Bioorg. Med. Chem. Lett. 1994 4 2605-2608. Boudreau MA, Vederas JC. Synthesis and biological evaluation of nucleoside dicarboxylates as potential mimics of nucleoside diphosphates. Org. Biomol. Chem. 2007 5 627-635. 

Shah, A., Font, J.L., Miller, M.J., Ream, J.E., Walker, M.C., and Sikorski, J.A., New aromatic inhibitors of EPSP synthase incorporating hydroxymalonates as novel 3-phosphate replacements. Bioorg. Med. Chem., 5, 323, 1997. 

The concept is not limited to analogs of simple linear organophosphates (e.g. phosphoenol pyruvate analog as inhibitor of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase [71]) but can be extended to cyclic phosphates (e.g. cyclic phosphoinositol analog as a phospholipase C inhibitor [72]) and transition state analogs (e. g. purine nucleoside phosphorylase (PNP) inhibitor [73]) (Scheme 4.30). 

The products of the phospho-aldol reaction, a-functionalised phosphonate esters such as a-hydroxy- and a-aminophosphonates, find widespread application directly or indirectly as precursors to enzyme inhibitors (such as the enzymes renin [6], thrombin [7],EPSP synthase [8], HIV protease [9] and various classes of PTK and PTPs [10]) and phosphate analogues [11], antibiotics [ 12], antiviral agents [ 13], and in nucleotide technology [ 14]. Within each application stereochemistry of the phosphonate is crucial to eliciting the required properties [15]. 

In this chapter, the discussion will concentrate on two inhibitors with a reasonable claim to selective action on enz3ones related to the shikimate pathway glyphosate, which inhibits 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase and L-a-aminooxy-3 phenylpropionic acid (L-AOPP), an inhibitor of phenylalanine ammonia-lyase (PAL) (Fig. 2). In addition to introducing a novel inhibitor of PAL, (R)-(l-amino-2-phenylethyl)phosphonic acid (APEP), previous and current efforts to design inhibitors of other shikimate pathway enzymes will be described. The treatment presented here will show that the deductions and predictions made on the basis of the abstract scheme in Figure 1 can be, and have been, tested on the basis of the real pathway presented in Figure 2. 

The information obtained from the application of glyphosate to complex systems strongly pointed to one of the following three enzymes as the target of the inhibitor in the shikimate pathway shikimate kinase (EC 2.7.1.71), 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19), and chorismate synthase (EC 4.6.1.4). Jointly, these three enzymes convert shikimic acid to chorismic acid in a series of interesting reactions >(Fig. 2). A defined system" had therefore to be found in which the conversion of shikimic acid to chorismic acid could be conveniently studied. 

Tissues and cultured cells accumulate predominantly shikimate, rather than S-3-P, in the presence of glyphosate, and in cultured buckwheat cells shikimate was shown to accumulate in the vacuole.As neither S-3-P nor shikimate compete with glyphosate for binding sites on EPSP synthase, and as PEP does not accumulate in glyphosate-treated cells (Amrhein, unpublished), the inhibition of EPSP synthase is not overcome. This example illustrates that in order to effectively inhibit an enzyme vivo the inhibitor need not necessarily be of the tight-binding or suicide (Kcat active-site-directed irreversible) type. 

Of the enzymes subsequent to EPSP synthase, only chorismate mutase has been the subject of inhibitor studies (see Chapter 5 by P. Bartlett in this volume). 

However, the monofluoro analog 12 has been made the enzyme itself will assemble it fiom Z-fluoro-PEP and S-3-P, as Mark Walker and the Monsanto group have recently shown (Figure 9).3 Actually, EPSP synthase induces formation of 12, it doesn t catalyze it This analog is a powerful inhibitor and the enzyme can t carry it further to fluoro-EPSP thus, turnover and true catalysis do not occur. We have tried to get to this monofluoro derivative fiom the other direction, namely from Z-fluoro-EPSP, but the enzyme will not oblige us. Enzymatic formation of 12 from S-3-P and Z-fluoro-PEP takes less than a minute, but even weeks of incubation of enzyme, phosphate, and Z-fluoro-EPSP give no evidence for formation of 12 or Z-fluoro-PEP.37... 

The synthesis of the amine, 62, a portion of which is the herbicide "glyphosate" has been reported as a novel inhibitor of EPSP synthase. ... 

A number of analogues that mimic the intermediate formed during the EPSP nthase conversion of shikimic-3-phosphate into EPSP (5-enolpyruvyl shikimate-3-phosphate) have been prepared and tested as inhibitors of the enzyme. The best was shown to be 63. (Z)-3-Fluoro-phosphoenolpyruvate functions as a pseudosubstrate for EPSP synthase producing in one step the same adduct 63, but as an isomeric mixture. ... 

The synthesis of compound (44) as a potential transition-state analogue inhibitor of isochorismate synthase (IS) has been reported/ Compounds (45) and (46) have been synthesized from the known 6-fluoroshikimic acids (J. Chem. Soc., Chem. Commun., 1989, 1386) by treatment first with shikimate kinase then 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase and shown to be competitive inhibitors of chorismate synthase/ ... 

Sikorski JA, Miller MJ, Braccolino DS et al (1993) EPSP synthase The design and synthesis of bisubstrate inhibitors incorporating novel 3-phosphate mimics. Phosphorus, Sulfur Silicon Relat Elem 76 115-118... 

The synthesis of an EPSP synthase inhibitor 91, vdiich relies on a malonate group to mimic a phosphate group, has been reported and the EPSP analogue 92 has been prepared to study inhibition of EPSP synthase. "... 

The study of inhibitors of photosynthetic carbon metabolism has, by contrast, been remarkably unsuccessful. Attempts to design herbicidal inhibitors on rational grounds on the basis of specific enzyme inhibition have not afforded herbicidal compounds that are effective on whole plants. It is possible that in many instances the chloroplast envelope proves to be an insuperable barrier. Nevertheless, recent work identifying specific enzymes such as 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, acetyl-CoA carboxylase, and acetohydroxy acid synthase has demonstrated the effectiveness of enzyme inhibitors, although none were developed on rational grounds. 

Glyphosate (Roundup), a phosphonomethyl derivative of the amino acid glycine (Figure 2.4), is a potent inhibitor of all the wild-type microbial and plant EPSP synthases that have been isolated (Table 2.1). The inhibition markedly increases over the pH range 6-8. While considerable differences in the inhibitor constants that have been measured are apparent (Table 2.1), the range of values is put into context by the report that glyphosate concentrations in the cytosol of treated plants can be in excess of 1 mM, with intrachloroplastic levels of >50... 

Inhibitors of 5-enolpyruvyl Shikimate 3-phosphase Synthase (EPSPS)... 

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