Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis

Definitions

• the dimers comprise (L,L,L,L,L)-(MVGGVV)k and (D,D,D,D,D)-(mvggvv)k, wherein k is an integer of 1 or greater.
• the dimers comprise (L,L,L,L,L,L)-(KLVFFAE)k and (D,D,D,D,D,D)-(klvffae)k, wherein k is an integer of 1 or greater.
• the dimers comprise (L,L,L,L,L)-(AILSS)k and (D,D,D,D,D)-(ailss)k, wherein k is an integer of 1 or greater.
• rippled p-sheet fibrils comprising a plurality of the rippled antiparallel cross-p dimers of the present disclosure.
• Materials comprising the rippled antiparallel cross-p dimers and rippled p-sheet fibrils of the present disclosure are also provided, as are compositions comprising such materials.
• methods of making the rippled antiparallel cross-p dimers and rippled p-sheet fibrils of the present disclosure are also provided.
• FIG. 1 Fully extended rippled p-sheet layers with FYYF:fyyf.
• FIG. 2 Rippled sheet with C-terminus of Ap40 MVGGVV.
• FIG. 3 Rippled sheet with central A04O cluster KLVFFAE.
• FIG. 4 The system forms fibers under suitable conditions.
• dimers, fibrils and methods of the present disclosure are described in greater detail, it is to be understood that the dimers, fibrils and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the dimers, fibrils and methods will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the dimers, fibrils and methods.
• dimers, fibrils and methods have the same meaning as commonly understood by one of ordinary skill in the art to which the dimers, fibrils and methods belong. Although any dimers, fibrils and methods similar or equivalent to those described herein can also be used in the practice or testing of the dimers, fibrils and methods, representative illustrative dimers, fibrils and methods are now described.
• amino acids include, but are not limited to, amino acids comprising photoactivatable cross-linkers, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, photocaged and/or photoisomerizable amino acids, radioactive amino acids, amino acids comprising biotin or a biotin analog, glycosylated amino acids, other carbohydrate modified amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, carbon-linked sugar-containing amino acids, redox- active amino acids, amino thioacid containing amino acids, and amino acids comprising one or more toxic moieties.
• amino acid includes, but is not limited to, naturally-occurring a-amino acids and their stereoisomers.
• “Stereoisomers” of amino acids refer to mirror image isomers of the amino acids, such as L-amino acids or D-amino acids.
• a stereoisomer of a naturally-occurring amino acid refers to the mirror image isomer of the naturally-occurring amino acid (/.e., the D-amino acid).
• Naturally-occurring a-amino acids are those encoded by the genetic code as well as those amino acids that are later modified (e.g., hydroxyproline, y-carboxyglutamate, and O- phosphoserine).
• Naturally-occurring a-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (lie), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), serine (Ser), threonine (Thr), valine (Vai), tryptophan (Trp), tyrosine (Tyr), and combinations thereof.
• Stereoisomers of a naturally- occurring a-amino acids include, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys), D- aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D- isoleucine (D-lle), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-GIn), D-serine (D-Ser), D-threonine (D- Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.
• D-alanine
• Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature.
• an L-amino acid may be represented herein by its commonly known three letter symbol (e.g., L-Met for L-methionine) or by an upper-case one- letter amino acid symbol (e.g., M for L-methionine).
• a D-amino acid may be represented herein by its commonly known three letter symbol (e.g., D-Met for D-methionine) or by a lower-case one- letter amino acid symbol (e.g., m for D-methionine).
• polypeptide refers to a polymeric form of amino acids of any length (e.g., connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues), which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
• rippled antiparallel cross-p dimers comprising (L,L,L,L,L)-(MVGGVV)k dimerized with (D,D,D,D,D,D)-(mvggvv)k, wherein k is an integer of 1 or greater.
• rippled antiparallel cross-p dimer comprising (L,L,L,L,L)-(AILSS)k dimerized with (D,D,D,D,D)-(ailss)k, wherein k is an integer of 1 or greater.
• k is an integer of from 1 to 1000, such as from 1 to 750, from 1 to 500, from 1 to 250, from 1 to 100, from 1 to 75, from 1 to 50, from 1 to 40, from 1 to 30, from 1 to 20, or from 1 to 10.
• rippled p-sheet fibrils comprise a plurality of the rippled antiparallel cross-p dimers of the present disclosure.
• compositions comprising the materials of the present disclosure.
• a composition includes a material of the present disclosure present in a liquid medium, e.g., an aqueous liquid medium.
• the liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like.
• One or more additives such as a salt (e.g., NaCI, MgCh, KOI, MgSO4), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine- N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N- Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a protease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions.
• a salt e.g
• a tonicity agent may be included to modulate the tonicity of the formulation.
• Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof.
• the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable.
• the term "isotonic" denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum.
• Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.
• a surfactant may also be added to the formulation to reduce aggregation and/or minimize the formation of particulates in the formulation and/or reduce adsorption.
• Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS).
• suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20TM) and polysorbate 80 (sold under the trademark Tween 80TM).
• Suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188TM.
• suitable Polyoxyethylene alkyl ethers are those sold under the trademark BrijTM.
• Example concentrations of surfactant may range from about 0.001% to about 1% w/v.
• a lyoprotectant may also be added in order to protect the materials against destabilizing conditions during a lyophilization process.
• known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included, e.g., in an amount of about 10 mM to 500 nM.
• a composition of the present disclosure comprises the material and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m- cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof.
• a preservative is included in the composition, e.g., at concentrations ranging from about 0.001 to about 2% weight/volume (w/v).
• Also provided by the present disclosure are methods of making and using the rippled antiparallel cross-p dimers, rippled p-sheet fibrils, materials and compositions of the present disclosure.
• methods comprising producing a polypeptide comprising, consisting essentially of, or consisting of, (L,L,L,L,L,L)-(MVGGVV) k , and producing a polypeptide comprising, consisting essentially of, or consisting of, (D,D,D,D,D)-(mvggvv)k, wherein k is an integer of 1 or greater.
• methods comprising producing a polypeptide comprising, consisting essentially of, or consisting of, (L,L,L,L,L,L)-(KLVFFAE) k , and producing a polypeptide comprising, consisting essentially of, or consisting of, (D,D,D,D,D,D)-(klvffae)k, wherein k is an integer of 1 or greater.
• provided are methods comprising producing a polypeptide comprising, consisting essentially of, or consisting of, (L,L,L,L,L)-(AILSS) k , and producing a polypeptide comprising, consisting essentially of, or consisting of, (D,D,D,D,D)-(ailss)k, wherein k is an integer of 1 or greater.
• the polypeptides are produced by chemical synthesis.
• a non-limiting example of a chemical synthesis includes solid-phase polypeptide synthesis.
• Solidphase peptide synthesis involves the successive addition of protected amino acid derivatives to a growing peptide chain immobilized on a solid phase, including deprotection and washing steps to remove unreacted groups and also side products.
• Any number of solid supports may be employed, including resins such as polystyrene and polyamide based resins.
• the peptides may be covalently bound to a solid support, typically at their C-terminal end through linkers such as acid labile and photolabile linkers. In some embodiments the linker is an acid labile linker.
• the linker is a trityl linker such as a 2-ch lorotrity I linker.
• Peptide synthesis is typically performed by coupling a protected amino acid to the N-terminal end of the bound sample.
• the protected amino acid may contain N-terminal protecting groups such as a Boc (tert-butyloxycarbonyl) or Fmoc (9-fluorenylmethyloxycarbonyl) group as well as side chain protecting groups.
• the solid-phase synthesis is Fmoc-based solid-phase synthesis.
• the olypeptides may be synthesized using preloaded, Fmoc-[amino acid] 4-alkoxybenzyl alcohol Wang resin: Fmoc-L-[amino acid]- Wang (Sigma) or Fmoc-D-[amino acid]-Wang (Fisher). Syntheses may be performed manually at 0.2 mM scale relative to resin loading. An orbital shaker may be used for mixing in both the deprotection and coupling steps.
• the resin may be swelled in 3 mL of dimethylformamide (DMF) in a filter tube, housing 250 mg Fmoc-[amino acid] Wang resin (0.796 mmol/g loading) for 20 min.
• DMF dimethylformamide
• 30% piperidine (Spectrum) in DMF may be added to the resin, and allowed to shake on an orbital shaker for 20 min.
• the deprotection solution may be rinsed with DMF (3x) and dichloromethane (DCM, 2x) and the deprotection step may be repeated.
• Coupling reagents used may be 4 eq. A/,A/-diisopropylethylamine (Fisher), 3 eq.
• the aforementioned steps are repeated to produce the resin-bound polypeptides, NH 2 -L-polypeptide-COOH and NH 2 - D-polypeptide-COOH.
• the polypeptides may be cleaved and deprotected with a mixture consisting of trifluoroacetic acid (10 mL, Fisher), tri-isopropylsilane (1 mL, Fisher), and liquefied phenol (0.5 mL, Sigma).
• the polypeptide identities may be confirmed with mass spectrometry.
• the above-described methods may further comprise purifying the produced polypeptides. Any suitable approach for purifying the polypeptides may be employed.
• the polypeptides are purified by chromatography, a non-limiting example of which is High Performance Liquid Chromatography (HPLC).
• HPLC High Performance Liquid Chromatography
• An example HPLC-based approach suitable for purifying the polypeptides is described in Warner et al. (2017) JoVE, 2017, e55482.
• polypeptides may be purified by reverse-phase high-performance liquid chromatography (HPLC) with PLRP-S columns (Agilent), yielding polypeptides with purities exceeding 95%.
• HPLC may be conducted under basic conditions (0.1 % NH 4 OH), to reduce aggregation and/or precipitation. Samples may be lyophilized and stored as solid powders at - 40 °C.
• the above-described methods may further comprise combining the produced polypeptides into a racemic mixture.
• the combining is under conditions suitable for formation of the rippled antiparallel cross-p dimers of the present disclosure.
• the combining is under conditions suitable for formation of a rippled p-sheet fibril of the present disclosure.
• Approaches for dimerizing polypeptides in a manner suitable for producing the dimers of the present disclosure are known and described in, e.g., Hazari et al. (2022) Chem. Sci.
• a rippled antiparallel cross-p dimer comprising (L,L,L,L,L)-(MVGGVV)k dimerized with (D,D,D,D,D,D)-(mvggvv)k, wherein k is an integer of 1 or greater.
• a rippled antiparallel cross-p dimer comprising (L,L,L,L,L,L,L)-(KLVFFAE)k dimerized with (D,D,D,D,D,D,D)-(klvffae)k, wherein k is an integer of 1 or greater.
• a rippled antiparallel cross-p dimer comprising (L,L,L,L,L)-(AILSS)k dimerized with (D,D,D,D,D)-(ailss)k, wherein k is an integer of 1 or greater.
• a rippled p-sheet fibril comprising a plurality of the rippled antiparallel cross-p dimers of any one of embodiments 1 to 3.
• a material comprising a plurality of the rippled antiparallel cross-p dimers of any one of embodiments 1 to 3.
• a composition comprising the rippled antiparallel cross-P dimers of any one of embodiments 1 to 3, the rippled p-sheet fibril of embodiment 4, or the material of any one of embodiments 5 to 7.
• composition of embodiment 8, wherein the dimer or material is present in a liquid medium is present.
• composition of embodiment 9, wherein the liquid medium is an aqueous liquid medium.
• a method comprising: producing a polypeptide comprising, consisting essentially of, or consisting of, (L,L,L,L,L)-(MVGGVV) k ; and producing a polypeptide comprising, consisting essentially of, or consisting of, (D,D,D,D,D)-(mvggvv)k, wherein k is an integer of 1 or greater.
• a method comprising: producing a polypeptide comprising, consisting essentially of, or consisting of, (L,L,L,L,L,L)-(KLVFFAE) k ; and producing a polypeptide comprising, consisting essentially of, or consisting of, (D,D,D,D,D,D)-(klvffae)k, wherein k is an integer of 1 or greater.
• a method comprising: producing a polypeptide comprising, consisting essentially of, or consisting of, (L,L,L,L,L)-(AILSS) k ; and producing a polypeptide comprising, consisting essentially of, or consisting of, (D,D,D,D,D)-(ailss) k , wherein k is an integer of 1 or greater.
• a method comprising: combining (L,L,L,L,L,L)-(MVGGVV)k and (D,D,D,D,D,D)-(mvggvv)k in a mixture under conditions in which rippled antiparallel cross-p dimers comprising (L,L,L,L,L)-(MVGGVV)k and (D,D,D,D,D)-(mvggvv) k are formed, wherein k is an integer of 1 or greater.
• a method comprising: combining (L,L,L,L,L,L,L)-(KLVFFAE) k and (D,D,D,D,D,D)-(klvffae)k in a mixture under conditions in which rippled antiparallel cross-p dimers comprising (L,L,L,L,L,L)-(KLVFFAE)k and (D,D,D,D,D,D)-(klvffae)k are formed, wherein k is an integer of 1 or greater.
• a method comprising: combining (L,L,L,L,L)-(AILSS) k and (D,D,D,D,D)-(ailss)k in a mixture under conditions in which rippled antiparallel cross-P dimers comprising (L,L,L,L)-(AILSS)k and (D,D,D,D,D)- (ailss)k are formed, wherein k is an integer of 1 or greater. 22. The method according to any one of embodiments 19 to 21 , wherein a rippled p-sheet fibril comprising the rippled antiparallel cross-P dimers is formed.

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