Compositions and uses of motor protein-binding moieties
The invention provides methods and reagents for efficient transport of macromolecules (such as drug therapeutics including nucleic acids and polypeptides) linked to motor protein-binding moieties (MPBM) intracellularly to a specific subcellular localization. One exemplary MPBP is dynein binding moiety (DBM). Drug therapeutics linked to DBM can be selectively, specifically, and actively transported intracellularly to the nucleus or peri-nuclear region, thus enhancing the uptake of such drug therapeutics into the target subcellular localization.
1 . A motor protein therapeutic represented by one of the general formulas:
A-L-B (I)
or
A::B (II)
wherein
A represents a moiety which binds to a motor protein;
B represents a drug moiety which produces a change in the growth state or phenotype of a cell, or a change in concentrations or rates of protein internalization, processing, or excretion, in a manner dependent on or enhanced by a particular subcellular localization of B;
L represents a bond or a linker group which covalently attaches A and B, and
:: represents a non-covalent interaction between A and B.
2 . The motor protein therapeutic of claim 1 , wherein said motor protein is a conventional kinesin (kinesin I), a heterotrimeric kinesin II, a homodimeric kinesin II, an Unc104/KIF1 protein, or a myosin V.
3 . The motor protein therapeutic of claim 1 , wherein said motor protein is a dynein.
4 . The motor protein therapeutic of claim 3 , wherein said motor protein is a cytoplasmic dynein, and wherein said moiety A binds to a subunit of said cytoplasmic dynein.
5 . The motor protein therapeutic of claim 4 , wherein said subunit is a dynein light chain (DLC).
6 . The motor protein therapeutic of claim 5 , wherein said dynein light chain (DLC) is DLC8.
7 . The motor protein therapeutic of claim 6 , wherein said moiety A comprises a polypeptide or peptidomimetic with an amino acid sequence consisting essentially of SEQ ID NO: 1, or SEQ ID NO: 1.
8 . The motor protein therapeutic of claim 6 , wherein said moiety A comprises a polypeptide or peptidomimetic with an amino acid sequence consisting essentially of SEQ ID NOs: 2, 4, 5, 6, 7, or 8, or SEQ ID NOs: 2, 4, 5, 6, 7, or 8.
9 . The motor protein therapeutic of claim 6 , wherein said moiety A comprises a polypeptide or peptidomimetic with an amino acid sequence consisting essentially of Xaa1-Xaa2-Thr-Gln-Thr (SEQ ID NO: 3) or SEQ ID NO: 3,
wherein,
Xaa1 represents an amino acid residue with a positively charged sidechain,
Xaa2 represents an amino acid residue with a polar or a neutral sidechain.
10 . The motor protein therapeutic of claim 9 , wherein said Xaa1 is Lys, His or Arg.
11 . The motor protein therapeutic of claim 9 , wherein said Xaa2 is a polar sidechain amino acid selected from Arg, Asn, Asp, Cys, Glu, Gln, His, Lys, Ser or Thr.
12 . The motor protein therapeutic of claim 9 , wherein said Xaa2 is a neutral sidechain amino acid selected from Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val.
13 . The motor protein therapeutic of claim 12 , wherein said Xaa2 is a small neutral sidechain amino acid selected from Gly, Ala or Ser.
14 . The motor protein therapeutic of claim 11 , wherein said Xaa2 is a negative polar sidechain amino acid selected from Asp or Glu.
15 . The motor protein therapeutic of claim 9 , wherein said Xaa2 is Ala, Gly, Glu or Ser.
16 . The motor protein therapeutic of claim 6 , wherein said moiety A is a DLC8-binding domain peptide sequence from neuronal nitric-oxide synthase (nNOS), proapoptotic Bcl-2 family protein Bim, Drosophilia mRNA localization protein Swallow, transcriptional regulator IκB, or postsynaptic scaffold protein GKAP.
17 . The motor protein therapeutic of claim 16 , wherein said moiety A is a DLC8-binding domain of nNOS (neuronal nitric-oxide synthase) represented by SEQ ID NO: 9.
18 . The motor protein therapeutic of claim 6 , wherein said moiety A is a small organic molecule which selectively binds to DLC8.
19 . The motor protein therapeutic of claim 4 , wherein said moiety A binds to cytoplasmic dynein with a dissociation constant K d of no more than 10 −4 M.
20 . The motor protein therapeutic of claim 1 , wherein said moiety A comprises two or more repeats of a polypeptide which binds to said motor protein.
21 . The motor protein therapeutic of claim 1 , wherein said moiety A comprises a peptidomimetic of a polypeptide which binds to said motor protein.
22 . The motor protein therapeutic of claim 1 , wherein said moiety A comprises a small organic molecule.
23 . The motor protein therapeutic of claim 1 , wherein said drug moiety B is a nucleic acid.
24 . The motor protein therapeutic of claim 23 , wherein said nucleic acid is an oligonucleotide, an anti-sense oligonucleotide, an siRNA, or a plasmid.
25 . The motor protein therapeutic of claim 1 , wherein said drug moiety B is a polypeptide or a peptidomimetic thereof.
26 . The motor protein therapeutic of claim 25 , wherein said polypeptide is a transcriptional regulator, an inducer or inhibitor of programmed cell death, or an intrabody (functional antibody) with intracellular targets.
27 . The motor protein therapeutic of claim 1 , wherein said drug moiety B is a microsphere, a liposome, a small organic molecule, or a large synthetic molecule.
28 . The motor protein therapeutic of claim 1 , wherein said drug moiety B interacts with a nuclear target.
29 . The motor protein therapeutic of claim 28 , wherein said nuclear target is a transcription factor, a histone, or a protein or protein complex which interacts with DNA and regulate gene expression or chromatin structure, a nuclear hormone or steroid receptor, a histone acetylase or deacetylase, a DNA methyltransferase, an enzyme which covalently modifies DNA, a kinase, a phosphatase, a protease, a lipase, an RNA polymerase, a DNA polymerase, a DNA primase, a DNA topoisomerase, a DNA helicase, a nuclease, or an ATPase.
30 . The motor protein therapeutic of claim 29 , wherein said drug moiety B is an inhibitor or activator of said nuclear target.
31 . The motor protein therapeutic of claim 1 , wherein either said A or said B or both include functionalities for enhancing cellular uptake and/or transmembrane movement.
32 . The motor protein therapeutic of claim 1 , wherein either said A or said B or both includes groups which can be cleaved to form an active drug moiety B not linked to A.
33 . The motor protein therapeutic of claim 1 , wherein said subcellular localization is nucleus.
34 . The motor protein therapeutic of claim 1 , wherein said subcellular localization is lysosome, mitochondria, ER (Endoplasmic Reticulum), Golgi complex, or a membrane fraction.
35 . The motor protein therapeutic of claim 1 , wherein said A and said B are covalently linked by said L through chemical cross-linking.
36 . The motor protein therapeutic of claim 1 , wherein said L is an amino acid or a polypeptide.
37 . The motor protein therapeutic of claim 35 , wherein said L includes a bond that can be cleaved or enzymatically degraded under physiological condition once at said subcellular localization.
38 . The motor protein therapeutic of claim 1 , wherein said non-covalent interaction :: is ionic interaction, hydrogen bond, van der Waals interaction, hydrophobic interaction, or simultaneous binding of said A and B to a third molecule.
39 . The motor protein therapeutic of claim 38 , wherein said non-covalent interaction :: is direct binding between said A and said B.
40 . The motor protein therapeutic of claim 39 , wherein said direct binding between said A and said B is mediated by a pair of heterologous interacting polypeptides.
41 . The motor protein therapeutic of claim 40 , wherein said pair of heterologous interacting polypeptides are biotin and streptavidin or avidin.
42 . The motor protein therapeutic of claim 38 , wherein said non-covalent interaction becomes unstable under physiological condition once at said subcellular localization.
43 . The motor protein therapeutic of claim 36 , wherein said amino acid is Cysteine.
44 . The motor protein therapeutic of claim 36 , wherein said polypeptide comprises a terminal Cysteine and a spacer polypeptide.
45 . The motor protein therapeutic of claim 44 , wherein said spacer polypeptide comprises one or more repeats of Gly-Gly-Gly-Ser (SEQ ID NO: 15).
46 . A nucleic acid encoding a proteinaceous motor protein therapeutic of claim 1 , or the complement nucleic acid thereof.
47 . A vector comprising the nucleic acid of claim 46 .
48 . A host cell comprising the vector of claim 47 or the nucleic acid of claim 46 .
49 . A method of delivering a drug moiety B to a particular subcellular localization of a cell, comprising contacting the cell with the motor protein therapeutic of claim 1.