Selective disruption of protein aggregation by cyclodextrin dimers

Abstract

Beta-cyclodextrin (CD) dimers (n = 11) were synthesized and tested against eight enzymes, seven of which were dimeric or tetrameric, for inhibitor activity. Initial screening showed that only L-lactate dehydrogenase and citrate synthase were inhibited but only by two specific CD dimers in which two beta-CDs were linked on the secondary face by a pyridine-2,6-dicarboxylic group. Further investigation suggested that these CD dimers inhibit the activity of L-lactate dehydrogenase and citrate synthase at least in part by disruption of protein-protein aggregation.

Scheme 1

CD dimers studied.

Figure 1

Plot of initial rate of LDH versus concentration of CD dimer 1. Initial rate is expressed in units/liter; 1 unit of LDH is defined as the amount of LDH required to reduce 1.0 μmol pyruvate to l-lactate per min at pH 7.5 and at 37°C.

Figure 2

CD dimer 1 decreases BS cross-linking efficiency, whereas its components (β-CD, pyridine 2,6-dicarboxylic acid, and dimethyl-2,6-pyridine dicarboxylate) do not, as evidenced by SDS/PAGE of cross-linking experiments with 0.1 μM enzymes (LDH and CS): lane 1, enzyme + 100 μM BS; lane 2, enzyme + 100 μM BS; lane 3, enzyme + 100 μM BS + 77 μM CD dimer; lane 4, enzyme + 100 μM BS + 385 μM CD dimer; lane 5, enzyme + 100 μM BS + 1.5 mM CD dimer; lane 6, enzyme + 100 μM BS + 7.7 mM CD dimer; lane 7, enzyme + 100 μM BS + 15 mM β-CD; lane 8, enzyme + 100 μM BS + 7 mM pyridine 2,6-dicarboxylic acid; lane 9, enzyme + 100 μM BS + 7 mM dimethyl-2,6-pyridine dicarboxylate; lane 10, same as lane 1.

Figure 3

Both CD dimer 1 and dimer 5 decrease cross-linking efficiency, as evidenced by SDS/PAGE of cross-linking experiments with CS (Upper) and LDH (Lower). Lanes 1 and 10, molecular weight markers; lanes 2 and 3, enzyme alone; lanes 4 and 5, 1.0 μM enzyme + 7.0 mM BS; lanes 6 and 7, 1.0 μM enzyme + 0.7 mM dimer 5 + 7.0 mM BS; lanes 8 and 9, 1.0 μM enzyme + 0.7 mM dimer 1 + 7.0 mM BS.

Figure 4

Crystal structure of CS. Only one monomeric unit is shown for clarity, but the geometry is that seen in the dimer of CS. A computer model of CD dimer 1 is shown in close proximity to the protein–protein interfaces. Several residues of the proteins are highlighted for their potential to bind with CD dimers 1 and 5, which would disrupt protein aggregation.

Figure 5

Crystal structure of LDH. Only one monomeric unit is shown for clarity, but the geometry is that seen in the tetramer of LDH. A computer model of CD dimer 1 is shown in close proximity to the protein–protein interfaces. Several residues of the proteins are highlighted for their potential to bind with CD dimers 1 and 5, which would disrupt protein aggregation.