Inhibitor substrate analog

The three-dimensional structures of several ribonucleases have been determined by single-crystal X-ray diffraction methods and recently also by nuclear magnetic resonance (NMR) spectroscopy. For a number of RNases, several crystal structure analyses have been performed. In these crystal structures, different inhibitors, substrate analogs, or reaction products have been used, different protein variants generated by site-directed mutagenesis have been present, or different crystal forms of the same enzyme have been studied. 

Probably all adenylyl cyclases are inhibited competitively by substrate analogs, which bind at the site and to the enzyme configuration with which cation-ATP binds (cf Fig. 4). One of the best competitive inhibitors is (3-L-2, 3 -dideoxy adenosine-5 -triphosphate ( 3-L-2, 3 -dd-5 -ATP Table 4) [4], which allowed the identification of the two metal sites within the catalytic active site (cf Fig. 4) [3]. This ligand has also been labeled with 32P in the (3-phosphate and is a useful ligand for reversible, binding displacement assays of adenylyl cyclases [4]. The two inhibitors, 2, 5 -dd-3 -ATP and 3-L-2, 3 -dd-5 -ATP, are comparably potent... 

The carba-trehaloses are carbocyclic analogs of trehalose, in which one or both of the D-glucopyranose units is (are) replaced with D-carba-glucopy-ranose, and may be expected to act as a substrate analog or an inhibitor of... 

Inhibitors are usually classified according to their effect upon Vmax and Kn. Competitive inhibitors, such as substrate analogs, compete with the substrate for the same binding site on the enzyme, but do not interfere with the decomposition of the enzyme-substrate complex. Therefore, the primary effect of a competitive inhibitor is to increase the apparent value of Km. The effect of a competitive inhibitor can be reduced by increasing the substrate concentration relative to the concentration of the inhibitor. 

Fig. 7. Structures of various substrates and inhibitors for NOS. Only 1,2, and 10 are substrates for NOS. An important conclusion from studies on such substrate analogs is the requirement for the -amino group for catalysis.

Substrate analogs (2) have properties similar to those of one of the substrates of the target enzyme. They are bound by the enzyme, but cannot be converted further and therefore reversibly block some of the enzyme molecules present. A higher substrate concentration is therefore needed to achieve a halfmaximum rate the Michaelis constant increases (B). High concentrations of the substrate displace the inhibitor again. The maximum rate V ax is therefore not influenced by this type of inhibition. Because the substrate and the inhibitor compete with one another for the same binding site on the enzyme, this type of inhibition is referred to as competitive. Analogs of the transition state (3) usually also act competitively. 

The first reported selective inhibitors of the HATs p300 and PCAF were peptides representing structurally simple bi-substrate analogs of H3 and acetyl-CoA [10, 11]. Hence, p300 was inhibited by Lys-CoA 1 and the more specific PCAF by the 20-amino-acid peptide H3-CoA-20 2 (Figure 11.2). Unfortunately, these inhibitors showed low cell permeability and high metabolic instability, which decreases their suitability for investigations in vivo [12]. 

Pharmacology Saquinavir is an inhibitor of HIV protease which cleaves viral polyprotein precursors to generate functional proteins in HIV-infected cells. The cleavage of viral polyprotein precursors is essential for maturation of infectious virus. Saquinavir is a synthetic peptide-like substrate analog that inhibits the activity of HIV... 

Substrate Analog Inhibitors of Highly Specific Proteases... 

The use of substrate analogs has led to the rapid development of inhibitors for several highly specific proteases. Research from this laboratory on the development of renin Inhibitors, kalllkreln Inhibitors, and Inhibitors of IgA-) protease Is outlined below. 

Current research from this laboratory (Figure 2) shows that the blocked hexapeptlde Ac-Ser-Pro-Phe-Arg-Ser-Gln-NH2 Inhibits kalllkreln In vitro (Kj = 6l pH) and demonstrates that the substrate analog approach Is applicable to the design of Inhibitors of kalllkreln. 

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