Chemoselective small molecules that covalently modify one lysine in a non-enzyme protein in plasma

Abstract

A small molecule that could bind selectively to and then react chemoselectively with a non-enzyme protein in a complex biological fluid, such as blood, could have numerous practical applications. Herein, we report a family of designed stilbenes that selectively and covalently modify the prominent plasma protein transthyretin in preference to more than 4,000 other human plasma proteins. They react chemoselectively with only one of eight lysine e-amino groups within transthyretin. The crystal structure confirms the expected binding orientation of the stilbene substructure and the anticipated conjugating amide bond. These covalent transthyretin kinetic stabilizers exhibit superior amyloid inhibition potency compared to their noncovalent counterparts, and they prevent cytotoxicity associated with amyloidogenesis. Though there are a few prodrugs that, upon metabolic activation, react with a cysteine residue inactivating a specific non-enzyme, we are unaware of designed small molecules that react with one lysine e-amine within a specific non-enzyme protein in a complex biological fluid.

Figure 1

Reverse phase (RP)-HPLC analysis of the chemoselectivity of compounds 1-4 in recombinant WT-TTR vs K15A-TTR homotetramer solutions and human plasma. (a) C₁₈-RP-HPLC analysis of WT-TTR (top) or K15A-TTR (bottom) pre-incubated (18 h) with candidate covalent kinetic stabilizer 4 (analogous data for 1-3 are described in Supplementary Fig. 1). (b) Rate of WT-TTR–(stilbene)_n conjugate formation analyzed by C₁₈-RP-HPLC. Samples were analyzed in triplicate and the error bars represent standard deviations. (c) C₁₈-RP-HPLC assessment of the modification of TTR in human blood plasma by compounds 3 and 4 (analogous data for 1 and 2 are shown in Supplementary Fig. 8).

Figure 2

A comparison of the potency of covalent kinetic stabilizers and their non-covalent counterparts and an assessment of WT-TTR tetramer dissociation kinetics in the presence of a covalent kinetic stabilizer. (a) Comparison of the potency of non-covalent kinetic stabilizers 8 and 9 (2.7 μM) with that of covalent kinetic stabilizers 2 and 4 (2.7 μM) assessed by their capacity to inhibit recombinant WT-TTR (3.6 μM) amyloidogenesis over a 120 h time course. (b) Concentration-dependent kinetic stabilization of recombinant WT-TTR by 1-4 and their non-covalent counterparts 8 and 9 assessed by % fibril formation after 72 h. (c) Kinetic stabilizer 4 prevents WT-TTR dissociation in a concentration-dependent fashion. Urea-mediated WT-TTR (1.8 μM) dissociation time course in the absence and presence of compound 4 as a function of the indicated concentrations, evaluated by linking slow tetramer dissociation to rapid and irreversible monomer denaturation in 6 M urea measured by far-UV circular dichroism at 215-218 nm¹⁹. Samples were analyzed in triplicate and the error bars represent standard deviations.

Figure 3

Inhibition of WT-TTR cytotoxicity in human IMR-32 neuroblastoma cells as a function of the dose of covalent and non-covalent TTR kinetic stabilizers. WT-TTR was pre-incubated at 37 °C in the absence (black bars) or presence of compounds for 18 h and then added to the cell culture media. The final concentration of WT-TTR was 8 μM and the final concentrations of compounds were 8, 6, 4 and 2 μM, as indicated. Cell viability was assessed using the resazurin reduction assay after 24 h. Cell viability results are reported relative to cells treated with vehicle only (100% cell viability). Columns represent the means of 2 independently performed experiments (n = 6) and the error bars represent standard errors.

Figure 4

Crystal structure of the WT-TTR–(benzoyl substructure of 4)₂ conjugate, showing the amide bond linkage to the Lys-15 ε-amino group (PDB 3HJ0). Individual TTR monomers are uniquely colored. (a) Three-dimensional ribbon diagram depiction. (b) Magnified image of the benzoyl substructure derived from 4 covalently bound in one of the T₄ binding sites. A Connolly analytical surface representation (green = hydrophobic, purple = polar, and blue = exposed) depicts the hydrophobicity of the binding site. The 3,5-methyl groups are placed in the halogen binding pockets 3 and 3′ and bridging hydrogen bonds are formed between the 4-OH of the benzoyl substructure of 4 and the Ser117 and 117′ hydroxyls from adjacent TTR monomers. Figure generated using MOE (2006.08), Chemical Computing Group, Montreal, Canada).