De Novo Design

Most early efforts in proving the rational design technology was based upon dye structures. To date all dyes considered have been anionic, presumably because the charged chromophores of these ligands mimic the binding of naturally occurring anionic het-erocycles such as NAD +, NAPD +, ATP, coenzyme A, folate, pyridoxal phosphate, oligonucleotides and polynucleotides. However, some proteins, particularly proteolytic enzymes, interact with cationic substrates. The trypsin-like family of enzymes forms

Figure 8 Putative binding pocket for the terminal-ring analogue (m-COO~) of Cibacron Blue F3G-A in the coenzyme binding site of horse liver alcohol dehydrogenase (ADH). The site lies lateral to the main coenzyme binding site and comprises the side chains of two juxtaposed cationic residues Arg47 and His51.

Figure 8 Putative binding pocket for the terminal-ring analogue (m-COO~) of Cibacron Blue F3G-A in the coenzyme binding site of horse liver alcohol dehydrogenase (ADH). The site lies lateral to the main coenzyme binding site and comprises the side chains of two juxtaposed cationic residues Arg47 and His51.

Table 4 Apparent affinities of terminal-ring analogues of an-thraquinone dyes for horse liver alcohol dehydrogenase (ADH)

Table 4 Apparent affinities of terminal-ring analogues of an-thraquinone dyes for horse liver alcohol dehydrogenase (ADH)

by insertion of a hexamethylene spacer arm between the designed ligand and the matrix. After synthesis of this medium it was demonstrated that purified pancreatic kallikrein was strongly bound, with over 90% of activity being recovered on elution with 4-aminobenzamidine, whereas trypsin appeared largely in the void of the column. This medium was able to purify kallikrein 110-fold from a crude pancreatic acetone powder in a single step.

There is an alternative to the rational design approach - the use of combinatorial libraries.

R ApparentKd (^mol L 1)

m-COO-

0.06

H

0.2

o-COO-

0.2

o-SO--

0.4

m-SO--

1.6

m-CH2OH

4.5

m-CONH2

5.7

p-COO-

5.9

P-SO-

9.3

P-PO3-

10.5

p-N#(CH3)3

172.0

one of the largest groups of enzymes requiring cationic substrates and includes enzymes involved in digestion (trypsin); blood clotting (kallikrein, thrombin, Factor Xa); fibrinolysis (urokinase, tissue plasminogen activator) and complement fixation. These enzymes possess similar catalytic mechanisms and bind the side chains of lysine or arginine in a primary pocket proximal to the reactive serine (Ser195), with specificity being determined partly by the side chain of Asp189 lying at the bottom of the pocket, and partly by the ability of the individual enzymes to form secondary interactions with the side chains of other nearnearby substrate amino acids. For example, tissue kallikrein differs from pancreatic trypsin in that it displays a marked preference for phenylalanine in the secondary site, probably because the phenyl ring on the phenylalanine residue neatly slips into a hydrophobic wedge-shaped pocket between the aromatic side chains of residues Trp215 and Tyr99 (Figure 10). Specificity for the secondary amino acid residue is less stringent in trypsin since Tyr99 is replaced by Ala99 and the hydrophobic pocket cannot be formed. By designing a mimic for the Ph-Arg dipeptide should result in a specificity for kallikrein. Figure 11 uses p-aminobenzamidine and phene-thylamine functions substituted on a monochloro-triazine moiety. However, the active site of pancreatic kallikrein lies in a depression in the surface of the enzyme. The expected steric hindrance is eliminated

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