Artificial self-sufficient cytochrome P450s

Patent metadata

Field	Value
Publication number	US-10138205-B2
Application number	US-201615552081-A
Country	US
Kind code	B2
Filing date	Feb 18, 2016
Priority date	Feb 19, 2015
Publication date	Nov 27, 2018
Grant date	Nov 27, 2018

Abstract

The disclosure relates to the field of fusion proteins. In some aspects, the invention relates to artificial fusion proteins comprising cytochrome P450 enzymes linked to reductase enzymes and uses thereof. In some aspects, the disclosure relates to compounds produced by artificial cytochrome P450 enzymes.

First claim

What is claimed is:

1. A fusion protein comprising:
(i) a cytochrome P450 enzyme which catalyzes transfer of a nitro functional group to provide a compound represented by Formula V:

wherein:
X 1 is halogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted C 2-6 alkenyl, substituted or unsubstituted C 2-6 alkynyl, substituted or unsubstituted, monocyclic, 3- to 6-membered carbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6- membered heterocyclyl, substituted or unsubstituted phenyl, substituted or unsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR A1a , —N(R A1a ) 2 , or —SR A1a ,
wherein R A1a is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two instances of R A1a are joined to form a substituted or unsubstituted, heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;
each of X 2 and X 3 is, independently, hydrogen, halogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted C 2-6 alkenyl, substituted or unsubstituted C 2-6 alkynyl, substituted or unsubstituted, monocyclic, 3- to 6-membered carbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstituted phenyl, substituted or unsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR A1a , —N(R A1a ) 2 , or —SR A1a ,
wherein R A1a is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two instances of R A1a are jointed to form a substituted or unsubstituted, heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;
R 1 is H or optionally substituted alkyl;
R 2 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, or optionally substituted heteroaryl; and
Y is NO 2 ;

or a pharmaceutically acceptable salt thereof;
(ii) an amino acid linker; and,
(iii) a catalytic domain of a reductase enzyme;

wherein the linker joins (iii) to a terminus of (i).

2. The fusion protein of claim 1 , wherein the P450 enzyme occurs naturally in Streptomyces.

3. The fusion protein of claim 1 , wherein the P450 enzyme is a TxtE enzyme, wherein a TxtE enzyme is defined as:
(i) TxtE;
(ii) a portion of TxtE which catalyzes transfer of a nitro functional group to provide a compound of represented by Formula V:

wherein
X 1 is halogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted C 2-6 alkenyl, substituted or unsubstituted C 2-6 alkynyl, substituted or unsubstituted, monocyclic, 3- to 6-membered carbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6- membered heterocyclyl, substituted or unsubstituted phenyl, substituted or unsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR A1a , —N(R A1a ) 2 , or —SR A1a ,
wherein R A1a is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two instances of R A1a are joined to form a substituted or unsubstituted, heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;
each of X 2 and X 3 is, independently, hydrogen, halogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted C 2-6 alkenyl, substituted or unsubstituted C 2-6 alkynyl, substituted or unsubstituted, monocyclic, 3- to 6-membered carbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstituted phenyl, substituted or unsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR A1a , —N(R A1a ) 2 , or —SR A1a ,
wherein R A1a is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two instances of R A1a are jointed to form a substituted or unsubstituted, heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;
R 1 is H or optionally substituted alkyl;
R 2 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, or optionally substituted heteroaryl; and
Y is NO 2 ;

or a pharmaceutically acceptable salt thereof;
(iii) an enzyme which is at least 95% homologous to the amino acid sequence of TxtE and which catalyzes transfer of a nitro functional group to provide a compound represented by formula V:

wherein
X 1 is halogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted C 2-6 alkenyl, substituted or unsubstituted C 2-6 alkynyl, substituted or unsubstituted, monocyclic, 3- to 6-membered carbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6- membered heterocyclyl, substituted or unsubstituted phenyl, substituted or unsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR A1a , —N(R A1a ) 2 , or —SR A1a ,
wherein R A1a is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two instances of R A1a are joined to form a substituted or unsubstituted, heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;
each of X 2 and X 3 is, independently, hydrogen, halogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted C 2-6 alkenyl, substituted or unsubstituted C 2-6 alkynyl, substituted or unsubstituted, monocyclic, 3- to 6-membered carbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstituted phenyl, substituted or unsubstituted,

Assignees

• Univ Florida

Inventors

• Ding Yousong

Classifications

• C07D209/16 Primary

  Tryptamines · CPC title

• C12N5/00

  Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; (plant reproduction by tissue culture techniques A01H4/00) · CPC title

• C12N15/00

  Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) · CPC title

• A61K31/4045

  Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin · CPC title

• C07D401/04

  directly linked by a ring-member-to-ring-member bond · CPC title