Small Molecules That Covalently Modify Transthyretin
AssigneeThe Scripps Research Institute
InventorsJeffery W. Kelly, Sungwook Choi
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Abstract
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.
Claims35
A compound corresponding in structure to Formula I
- wherein
- R1 is a halide, a methyl, monofluoro-, difluoro- or trifluoromethyl group;
- R2 is H, OH or NH2;
- n is zero or one;
- L is a linker that is Q=Q or CH2—CH2 when n is one and is absent when n is zero, so that the two depicted cyclic structures are bonded directly to each other, where Q=Q is N═N or HC═CH;
- the circle A is an aromatic or heteroaromatic ring structure containing one 5- or 6-membered ring or a fused ring system containing one 5- and one 6-membered ring or two 6-membered rings;
- X is a reactive substituent that reacts with an amine in an aqueous environment to bond the amine to the depicted compound;
- and
- Z is H, or a second linker, L2, that is an X group that is the same or different from the previously mentioned X group, NR3R4, where R3 and R4 are the same or different and are H, methyl or ethyl, a 1,3-diketo group or a metal ion chelating group.
The compound according to aspect 1, wherein X is a carboxylic ester or thioester, an azetidin-2-one, a Michael acceptor, an epoxide, aziridine or thienyl group or a β-haloacetyl group.
The compound according to aspect 2, wherein n is one.
The compound according to aspect 3, wherein circle A is an aromatic or heteroaromatic ring structure containing one 5- or 6-membered ring.
The compound according to aspect 4, wherein said heterocyclic ring contains one to three heteroatoms that are oxygen, nitrogen, sulfur and mixtures thereof.
The compound according to aspect 5, wherein said heterocyclic ring contains 6-members and the heteroatom is nitrogen.
The compound according to aspect 3, wherein circle A is an aromatic or heteroaromatic ring structure containing two fused rings having one 5- and one 6-membered ring or two 6-membered rings.
The compound according to aspect 7, wherein said heterocyclic ring contains one to three heteroatoms that are oxygen, nitrogen, sulfur and mixtures thereof.
The compound according to aspect 1, wherein L is Q=Q.
The compound according to aspect 1, wherein Z is L2.
The compound according to aspect 1, that corresponds in structure to Formula Ic, below,
- where L, n, X, Z, R1 and R2 are as defined before, W is N or C, preferably C, and the depicted structure A is an aromatic or heteroaromatic ring structure containing one 5- or 6-membered ring, and wherein L and X are in a 1,3-substitution relationship on the ring to which they are bonded.
The compound according to aspect 11, wherein structure A is a six-membered ring.
The compound according to aspect 12, wherein the six-membered ring is a hydrocarbyl ring.
The compound according to aspect 12, wherein Z is NR3R4.
A compound corresponding in structure to Formula Ia
- wherein
- R1 is a halide, a methyl, monofluoro-, difluoro- or trifluoromethyl group;
- R2 is H, OH or NH2;
- n is zero or one;
- L is a linker that is Q=Q or CH2—CH2 when n is one and is absent when n is zero, so that the two depicted cyclic structures are bonded directly to each other, where Q=Q is N═N or HC═CH;
- the circle A is an aromatic or heteroaromatic ring structure containing one 5- or 6-membered ring or a fused ring system containing one 5- and one 6-membered ring or two 6-membered rings;
- X is a reactive substituent that reacts with an amine in an aqueous environment to bond the amine to the depicted compound;
- and
- L2 is an X group that is the same or different from the previously mentioned X group, NR3R4, where R3 and R4 are the same or different and are H, methyl or ethyl, a 1,3-diketo group or a metal ion chelating group.
The compound according to aspect 15, wherein L2 is a metal ion chelating group.
The compound according to aspect 16, wherein said metal ion chelating group chelates a radionuclide.
The compound according to aspect 16, wherein said metal ion chelating group chelates a magnetic resonance imaging contrast ion.
The compound according to aspect 15, wherein L2 is NR3R4.
The compound according to aspect 19, wherein R3 and R4 are both methyl.
A conjugate compound corresponding in structure to Formula Ib
- wherein
- R1 is a halide, a methyl, monofluoro-, difluoro- or trifluoromethyl group;
- R2 is H, OH or NH2;
- n is zero or one;
- L is a linker that is Q=Q or CH2—CH2 when n is one and is absent when n is zero, so that the two depicted cyclic structures are bonded directly to each other, where Q=Q is N═N or HC═CH;
- the circle A is an aromatic or heteroaromatic ring structure containing one 5- or 6-membered ring or a fused ring system containing one 5- and one 6-membered ring or two 6-membered rings;
- X is a reactive substituent that reacts with an amine in an aqueous environment to bond the amine to the depicted compound;
- and
- L2′ is a reacted linker group that is an X group that is the same as or different from the previously mentioned X group, a 1,3-diketo group or a metal ion chelating group;
- and
- E is bonded to L2′ as a conjugated small molecule drug, a peptide, a peptide hormone or a chelator for a metal ion that can be a radionuclide or contrast agent, but only one of E and L2′ is a chelator for a metal ion.
The conjugate compound according to aspect 21, wherein E is a chelator for a radionuclide metal ion.
The conjugate compound according to aspect 21, wherein E is a chelator for a metal ion contrast agent.
The method according to aspect 24, wherein the sample to be assayed is blood plasma or serum.
The method according to aspect 24, wherein the presence of the protein-reacted compound is determined using mass spectral analysis.
The method according to aspect 24, wherein the presence of the protein-reacted compound is determined using fluorescence emitted by the protein-reacted compound.
The method according to aspect 24, wherein Q=Q is HC═CH.
A pharmaceutical composition that comprises a transthyretin (TTR) fibril formation-inhibiting amount of a compound of aspect 1 dissolved or dispersed in a pharmaceutically acceptable diluent.
A method of treating transthyretin (TTR) amyloidosis in a subject in need thereof that comprises administering to that subject a pharmaceutical composition according to aspect 29.
The method according to aspect 30 that comprises repeating said administration a plurality of times in one week.
A method of inhibiting transthyretin (TTR) fibril formation that comprises the steps of
- a) contacting an aqueous composition containing TTR with a stabilizing amount of a compound of aspect 1;
- and
- b) maintaining that contact for a time period sufficient for the compound to bind to, react with and thereby stabilize TTR.
A method for identifying a non-covalent transthyretin (TTR) kinetic stabilizer compound that comprises the steps of
- a) admixing a binding-sufficient amount of a candidate non-covalent transthyretin (TTR) kinetic stabilizer compound and an aqueous composition that contains a predetermined amount of TTR to form a first binding admixture;
- b) maintaining the first binding admixture for a time period sufficient for the candidate compound to bind to TTR;
- c) admixing a binding/reaction sufficient amount of a compound of aspect 1 wherein Q=Q is HC═CH with the first binding admixture to form a binding/reaction admixture;
- d) maintaining the binding/reaction admixture for a time period sufficient for the compound of step c) to bind to and react with TTR;
- and
- e) determining whether the reaction of the compound of step c) with TTR has been inhibited, wherein the presence of reaction inhibition identifies the candidate as a non-covalent TTR kinetic stabilizer compound.
The method according to aspect 33 wherein whether the reaction of the compound of step c) with TTR has been inhibited is determined using fluorescence data.
The method according to aspect 33 wherein whether the reaction of the compound of step c) with TTR has been inhibited is determined using liquid chromatography-mass spectral data.
Dependencies are inferred from the claim text.