Supercharging Prions via Amyloid‐Selective Lysine Acetylation

Abstract

Repulsive electrostatic forces between prion‐like proteins are a barrier against aggregation. In neuropharmacology, however, a prion's net charge (Z) is not a targeted parameter. Compounds that selectively boost prion Z remain unreported. Here, we synthesized compounds that amplified the negative charge of misfolded superoxide dismutase‐1 (SOD1) by acetylating lysine‐NH3+ in amyloid‐SOD1, without acetylating native‐SOD1. Compounds resembled a “ball and chain” mace: a rigid amyloid‐binding “handle” (benzothiazole, stilbene, or styrylpyridine); an aryl ester “ball”; and a triethylene glycol chain connecting ball to handle. At stoichiometric excess, compounds acetylated up to 9 of 11 lysine per misfolded subunit (ΔZfibril=−8100 per 103 subunits). Acetylated amyloid‐SOD1 seeded aggregation more slowly than unacetylated amyloid‐SOD1 in vitro and organotypic spinal cord (these effects were partially due to compound binding). Compounds exhibited reactivity with other amyloid and non‐amyloid proteins (e.g., fibrillar α‐synuclein was peracetylated; serum albumin was partially acetylated; carbonic anhydrase was largely unacetylated).

A method for quantifying how the activity of an enzyme is affected by the net charge of its nearest crowded neighbor

“…The results of nonselective acylation are supported by previous studies showing that small molecule acylating agents (e.g., anhydrides, N‐hydroxysuccinimide [NHS]‐esters, and aryl esters) acylate lysine in proteins semi‐randomly 47,64–66 . Even partially buried lysine residues are reactive with small organic molecules, because these lysines can associate with amphiphilic/hydrophobic molecules (in some cases, more so than solvent accessible lysine residues) 67 .…”

“…The results of nonselective acylation are supported by previous studies showing that small molecule acylating agents (e.g., anhydrides, N-hydroxysuccinimide [NHS]esters, and aryl esters) acylate lysine in proteins semi-randomly. 47,[64][65][66] Even partially buried lysine residues are reactive with small organic molecules, because these lysines can associate with amphiphilic/hydrophobic molecules (in some cases, more so than solvent accessible lysine residues). 67 The heterodimers produced are likely to be a polydisperse mixture of multiple geometric dimers between RNase A and Mb, Cyt c, or S-Cyt c. We expect that after acylation of 9-15 lysine residues, the attachment of the linker to S-Cyt c would still produce a polydisperse mixture following attachment of phosphine linker.…”