Bisubstrate Analogues

If we are able to covalently link these two compounds together in a way that captures all their individual binding interactions, the free energy of binding for the bisubstrate inhibitor will be the sum of the free energies of the individual molecules that is, the free energy, assuming no loses of productive interactions, will be -14.4 kcal/mol. The Kt of this combined, bisubstrate inhibitor will therefore be 

A recent example of the success of the bisubstrate, transition state design approach comes from the work of Pope and coworkers (Pope et al., 1998a-c Brown et al., 2000) on the design of inhibitors of bacterial isoleucyl tRNA synthetase 

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Figure 8.18 Mechanism-based inactivators of steroid 5a-reductase. (A) Finasteride, (B) the bisubstrate analogue formed by reaction of NADP1 with finasteride catalyzed by the enzyme, and (C) dutasteride.

Binding measurements of the bisubstrate analogue N-(phosphoacetyl)-L-aspartate (PALA) in the presence and absence of ATP were reported by Newell, et al, (1989). Figure 8.21 shows the binding isotherm of ATCase, a plot of 0 = [PALA]t,Qy j/[ATCase] as a function of [PALA]f (at 23 °C and in a buffer solution). 

Example 17 Bertozzi and associates in their studies on sulfotransferases have performed synthesis of a bisubstrate analogue designed to inhibit estrogen sulfotransferase [45]. Synthesis of this diphosphate depends on the coupling of two phosphates prepared by phosphoroamidite methodology. The synthesis utilizes differently protected 3 -phosphoadenosine-5 -phos-phate allowing selective functionalization of the 5 -phosphate with the sulfate acceptor mimic. 

Osborne, T., Roska, R.L., Rajski, S.R. and Thompson, P.R. (2008) In Situ Generation of a Bisubstrate Analogue for Protein Arginine Methyltransferase 1. Journal of the American Chemical Society (in press). 

Three novel bisubstrate analogues (133a-c) acting as potent inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase have been synthesised and used in the co-crystallisation of the kinase. These compounds were synthesised by coupling adenosine nucleotides (AMP, ADP and ATP) to 6-hy-... 

Bull HG, Garcia-Calvo M, Andersson S, etal. Mechanism-based inhibition of human steroid 5a-reductase by finasteride enzyme-catalyzed formation of NADP-dihydrofinasteride, a potent bisubstrate analogue inhibitor. J Am Chem Soc 1996 118 2359-2365. 

Bisubstrate analogues are a variation on this approach. Covalent linkage of two of the substrates of a multisubstrate enzyme may lead to surprisingly potent inhibition, even exceeding what is expected on the basis of the enzyme s tinity for the two substrates individually. This approach requires that the two substrates are bound at the same time, i.e., a sequential rather than ping pong kinetic mechanism. 

Figure 6. Reaction intermediate analogue inhibitors of isopropylmalate isomeiase (top) and bisubstrate analogue inhibitors of ornithine transcarbamoylase (bottom).

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