Synthetic apolipoproteins, and related compositions methods and systems for nanolipoprotein particles formation

Patent metadata

Field	Value
Publication number	US-11279749-B2
Application number	US-201615744754-A
Country	US
Kind code	B2
Filing date	Sep 9, 2016
Priority date	Sep 11, 2015
Publication date	Mar 22, 2022
Grant date	Mar 22, 2022

Abstract

Synthetic apolipoproteins based on native/naturally occurring homolog proteins can be prepared using solid-phase peptide synthesis approaches combined with native chemical ligation methods to create analogs of full length apolipoproteins. The chemical synthesis is expected to allow introduction of non-natural amino acids, e.g., α,α′-dialkyl amino acids, with a periodicity that encourages both helix formation and amphipathicity. Such apolipoprotein analogs are expected to encourage, in some embodiments, facile and more complete NLP formation, enabling consideration of full spectrum of nanoparticle-based biotechnology applications ranging from therapeutic sequestration and delivery to energy/biofuel production to biopolymer production.

First claim

The invention claimed is:

1. A synthetic apolipoprotein configured to bind lipids to form lipoproteins comprising:
a plurality of helical segments each having a primary structure configured to form an alpha helix secondary structure,
wherein at least one helical segment of the plurality of helical segments comprises a plurality of hydrophobic amino acids and a plurality of hydrophilic amino acids positioned in the primary structure to provide an amphipathic alpha helix secondary structure,
wherein the plurality of hydrophobic amino acids form an hydrophobic amino acid cluster and the plurality hydrophilic amino acids form an hydrophilic amino acid cluster, and
wherein the at least one helical segment comprises one or more non-natural α,α′-dialkyl amino acids within the hydrophobic amino acid cluster,
the plurality of hydrophobic amino acids is positioned in the primary structure with a periodicity i o +x o where i o is a recurring position of a hydrophobic amino acid of plurality of hydrophobic amino acids in the primary structure and x o is a number of amino acids in the helical segment between a first occurrence and a second occurrence of the recurring position i o , and
the plurality of hydrophilic amino acids is positioned in the primary structure with a periodicity i i +x i where i i is a recurring position of a hydrophilic amino acid of the plurality of hydrophilic amino acids in the primary structure and x i is a number of amino acids in the helical segment between a first occurrence and a second occurrence of the recurring position i i and

wherein x o and x i are independently any integer number from 3 to 9.

2. The synthetic apolipoprotein of claim 1 , wherein a C1-C4 linear or branched alkyl side chain is independently presented in a′ position of the one or more of the one or more non-natural α,α′-dialkyl amino acids.

3. The synthetic apolipoprotein of claim 1 , wherein a methyl, ethyl propyl, or an isobutyric side chain is independently presented in α′ position of one or more of the one or more non-natural α,α′-dialkyl amino acids.

4. The synthetic apolipoprotein of claim 1 , wherein the synthetic apolipoprotein comprises one or more helical segments having a length of 6, 7, 10, 11, 13, 14, 17, 18, 21, 22, 24, 25, 28, 29 or 31 amino acid residues.

5. The synthetic apolipoprotein of claim 1 , wherein at least one helical segment comprises from 15 to 100 amino acids.

6. The synthetic apolipoprotein of claim 1 , wherein at least one helical segment comprises 20 to 50 amino acids.

7. The synthetic apolipoprotein of claim 1 , wherein x o and x i are independently 3 and/or 4.

8. The synthetic apolipoprotein of claim 1 , wherein the first occurrence i o is at position 1 of the alpha helical segment and the additional occurrence are at positions 4, 5, 8, 9, 11, 12, and 15.

9. The synthetic apolipoprotein of claim 1 , wherein the first occurrence i o is at position 1 of the alpha helical segment and the additional occurrence are at positions 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, and 49 of the alpha helical segment.

10. The synthetic apolipoprotein of claim 1 , wherein the first occurrence i i is at position 2 of the alpha helical segment and the additional occurrence are at positions 3, 6, 7, 9, 10, 13, and 14 of the alpha helical segment.

11. The synthetic apolipoprotein of claim 1 , wherein the plurality of helical segments is connected by a linker comprising 1 to 15 amino acids.

12. The synthetic apolipoprotein of claim 1 , wherein the plurality of helical segments is connected by a linker comprising 1 to 10 L-amino acids.

13. The synthetic apolipoprotein of claim 1 , wherein the number of helical segments ranges between three and twelve.

14. A nanolipoprotein particle (NLP), comprising a membrane forming lipid, and a scaffold protein, wherein the scaffold protein is the synthetic apolipoprotein of claim 1 .

15. A method to provide the synthetic apolipoprotein of claim 1 , the method comprising
synthesizing a plurality of helical segments
each helical segment comprising a plurality of hydrophobic amino acid and a plurality of hydrophilic amino acid,
each helical segment having a primary structure configured to form an amphipathic alpha helical secondary structure in which the plurality of hydrophobic amino acid form a hydrophobic cluster and the plurality of hydrophilic amino acid form a hydrophilic cluster,
each helical segment having an N-terminal end and a C-terminal end; and
ligating the plurality of alpha-helical segments through the N-terminal end or the C-terminal end to form a synthetic apolipoprotein via at least one synthetic chemical linkage,
wherein at least one helical segment of the plurality of helical segment comprises one or more α,α′-dialkyl amino acids within the hydrophobic and/or the hydrophilic cluster.

16. A method to form nanolipoprotein particles (NLPs) comprising
synthesizing a synthetic apolipoprotein according to the method of claim 15 ; and
combining the synthetic apolipoprotein with membrane forming lipids to form a nanolipoprotein particle.

Assignees

• L Livermore Nat Security Llc

Inventors

• Hoeprich Jr Paul D
• Camarero Julio A

Classifications

• C07K14/775 Primary

  Apolipopeptides · CPC title

• B82Y5/00

  Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery · CPC title

• C07K1/1077

  by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids · CPC title

• C07K1/113

  without change of the primary structure · CPC title

• G16C10/00

  Computational theoretical chemistry, i.e. ICT specially adapted for theoretical aspects of quantum chemistry, molecular mechanics, molecular dynamics or the like · CPC title