Small molecules that covalently modify transthyretin
A family of covalent kinetic stabilizer compounds that selectively and covalently react with the prominent plasma protein transthyretin in preference to more than 4000 other human plasma proteins is disclosed. A contemplated compound corresponds in structure to Formula I, below, where the various substituents are defined within, and reacts chemoselectively with one or two of four Lys-15 ε-amino groups within the transthyretin tetramer. The crystal structure confirms the binding orientation of the compound substructure and the conjugating amide bond. A covalent transthyretin kinetic stabilizer exhibits superior amyloid inhibition potency, compared to a non-covalent counterpart, and inhibits cytotoxicity associated with amyloidogenesis.
1. A method for assaying for the presence of transthyretin (TTR) in a sample to be assayed that comprises the steps of:
a) admixing a binding/reaction sufficient amount of a covalent TTR kinetic stabilizer compound of Formula II with a sample to be assayed,
wherein
the depicted structure Ar is a hydrocarbyl aromatic ring structure,
R 1 is a halide, a methyl, monofluoro-, difluoro- or trifluoromethyl group;
R 2 is H, OH or NH 2 ;
n is one;
L is a linker that is HC═CH;
X is a reactive substituent that reacts with an amine in an aqueous environment to bond the amine to the depicted compound that is a carboxylic ester or thioester, an azetidin-2-one, a Michael acceptor, an epoxide, aziridine or thienyl group or a □-haloacetyl group, and L and X are in a 1,3-substitution relationship on the ring to which they are bonded; and
Z is H, or a second linker, L 2 , that is an X group that is the same as or different from the previously mentioned X group, a NR 3 R 4 group, where R 3 and R 4 are the same or different and are H, methyl or ethyl, a 1,3-diketo group or a metal ion chelating group;
b) maintaining that admixture for a time period sufficient for said compound to bind to TTR present in the sample, react therewith and form a covalently-bound reacted compound, and
c) determining the presence of said reacted compound.
2. The method according to claim 1 , wherein the sample to be assayed is blood plasma or serum.
3. The method according to claim 1 , wherein X substituent reactive group is a carboxylic acid ester or thioester prepared using an aromatic or heteroaromatic alcohol or thiol.
4. The method according to claim 1 , wherein Z is NR 3 R 4 .
5. The method according to claim 4 , wherein R 3 and R 4 are both methyl.
6. The method according to claim 1 , wherein compound of Formula II has the structural formula
7. The method according to claim 1 , wherein compound of Formula II has the structural formula