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WO-2026093979-A1 - PEPTIDE COACERVATES, METHODS, AND USES THEREOF

WO2026093979A1WO 2026093979 A1WO2026093979 A1WO 2026093979A1WO-2026093979-A1

Abstract

The present disclosure relates to innovative designer peptides for creating single peptide-based coacervates, also known as compartments, liquid-like droplets, or condensates. These designer peptide sequences are inspired by motifs found in disordered regions of intrinsically disordered proteins that undergo liquid-liquid phase separation (LLPS), also known as LLPS-promoting motifs.

Inventors

  • FIDALGO POMBO MENDES PINA CARTAXO, Ana Sofia
  • QUENEDIT PERALTA REIS, David Ruben
  • FERREIRA ANTUNES, Diogo
  • RAMOS DOS SANTOS CALVÁRIO, Joana
  • CARVALHO MORGADO, Maria Leonor
  • MATOS PEREIRA, Pedro

Assignees

  • UNIVERSIDADE NOVA DE LISBOA

Dates

Publication Date
20260507
Application Date
20251030
Priority Date
20241030

Claims (20)

  1. 1. Coacervate composition comprising a peptide and a suitable solvent, wherein the peptide is represented by the following formula: (A) n -(B)m-(A)p wherein, "A", "B" are independently selected of each other; "A" comprises a phase-separating peptide, wherein "A" is at least 90% identical to a sequence selected from a list consisting of SEQ ID No. 1-701; "B" comprises a peptide for molecular recognition or catalytic activity, wherein "B" is a peptide at least 90% identical to a sequence selected from a list consisting of SEQ ID No. 702-717, SED ID No. 764; wherein the peptide comprises up to 50 amino acids; wherein "n" is an integer from 0 to about 50, "m" is an integer from 0 to about 50, "p" is an integer from 0 to about 50; preferably wherein "n" is an integer from 1 to about 25, "m" is an integer from 1 to about 25, "p" is an integer from 1 to about 25; more preferably wherein n=m; at least one of n, m, p is different of 0.
  2. 2. Coacervate composition according to the previous claim, wherein the peptide comprises a sequence at least 90% identical to a sequence selected from a list consisting of SEQ ID No. 1-780, or combinations thereof.
  3. 3. Coacervate composition according to any of the previous claims, wherein the peptide comprises a sequence at least 95% - 98% identical to a sequence selected from a list consisting of SEQ. ID. 1-780, or combinations thereof; more preferably at least 98% - 99% identical; even more preferably identical.
  4. 4. Coacervate composition according to any of the previous claims comprising a peptide at least 90% identical to a sequence selected from SED ID No. 1, 7, 15, 30, 198, 381, 649, 655, 704, 706, 714, 717, 719, 724 preferably 95% - 98% identical; more preferably at least 98% - 99% identical; even more preferably identical.
  5. 5. Coacervate composition according to any of the previous claims, wherein the coacervate droplet size ranges from 1 pm - 50 pm; preferably 1 pm - 10 pm.
  6. 6. Coacervate composition according to any of the previous claims, wherein the zeta potential of the coacervates, measured by dynamic light scattering, ranges from +5mV to +15mV, preferably from +6 mV to +13 mV, at pH 8.0 and 25 °C. .
  7. 7. Coacervate composition according to any of the previous claims, comprising a structured domain wherein the peptide in the structured domain has a folded P-hairpin conformation.
  8. 8. Coacervate composition according to the previous claim, wherein the folded P-hairpin conformation exhibits a molar ellipticity of -0.25 X 10 5 to +0.25 X 10 5 deg.cm 2 .dmol -1 measured by circular dichroism spectroscopy in a wavelength ranging from 200 nm - 300 nm; preferably 210 nm - 230 nm; more preferably 215 - 225 nm.
  9. 9. Coacervate composition according to any of the previous claims, wherein the solvent is an aqueous solvent.
  10. 10. Coacervate composition according to any of the previous claims, wherein the volume ratio between the peptide and the solvent ranges from 1:20 (v/v) to 1:5 (v/v).
  11. 11. Coacervate composition according to any of the previous claims, wherein stock concentration of the peptide ranges from 1 mg mL 1 - 10 mg mL 1 ; preferably from 5 mg mL 1 - 10 mg mL 1 .
  12. 12. Coacervate composition according to any of the previous claims, wherein the solvent is a buffer selected from phosphate buffer, Tris Buffer, HEPES Buffer, MOPS Buffer, PIPES Buffer, MES Buffer, or mixtures thereof.
  13. 13. Coacervate composition according to the previous claim, wherein the buffer concentration ranges from 10 mM - 200 mM; preferably 50 mM - 150 mM; more preferably 100 mM.
  14. 14. Coacervate composition according to any of the previous claims 12-13, wherein the buffer further comprises a salt; preferably the salt is selected from sodium chloride (NaCI), potassium chloride (KCI), calcium chloride (CaCI 2 ), magnesium sulfate (MgS0 4 ), sodium phosphate (Na 2 HPO 4 or NaH 2 PO 4 ), ammonium sulfate ((NH 4 ) 2 SO 4 ), sodium bicarbonate (NaHCO 3 ), potassium nitrate (KNO 3 ), lithium chloride (LiCI), ammonium chloride (NH 4 CI), sodium acetate (CH 3 COONa), barium sulfate (BaS0 4 ), calcium carbonate (CaCO 3 ), calcium sulfate (CaS0 4 ), magnesium chloride (MgCI 2 ), sodium sulfate (Na 2 SO 4 ), potassium sulfate (K 2 SO 4 ), sodium citrate (Na 3 C 6 H 5 O 7 ), and sodium fluoride (NaF), or combinations thereof.
  15. 15. Coacervate composition according to the previous claim, wherein the salt concentration in the buffer ranges from 0.05 M - 2 M; preferably 0.5 M - 1.5 M; more preferably 0.5 M - 1 M.
  16. 16. Coacervate composition according to any of the previous claims, wherein the pH of the coacervate composition ranges from 1 - 10; preferably 7 - 9; more preferably 7 - 8.
  17. 17. Coacervate composition according to any of the previous claims, wherein the minimum coacervate droplet count per mL ranges from 10 - 100; preferably 50 - 1000; more preferably 100 - 100000.
  18. 18. Coacervate composition according to any of the previous claims further comprising nucleic acids, lipids or lipid-like molecules, carbohydrates, protein, peptide, organometallic molecules, organic molecules, inorganic molecules, a fluorophore, analyte-responsive molecule, or combinations thereof.
  19. 19. Coacervate composition according to the previous claim wherein the analyte-responsive molecule is a chemosensor comprising a pyrene fluorescent reporter and a Zn 2+ -chelate phosphate-binding group.
  20. 20. Coacervate composition according to any of the previous claims 19-20 wherein the nucleic acids, lipids or lipid-like molecules, carbohydrates, protein, peptide, organometallic molecules, organic molecules, inorganic molecules, fluorophore, analyte-responsive molecule, or combinations thereof has a partitioning efficiency into the coacervate phase between 5% and 90%.

Description

D E S C R I P T I O N PEPTIDE COACERVATES, METHODS, AND USES THEREOF TECHN ICAL FIELD [0001] The present disclosure relates to coacervates comprising peptides that undergo liquid-liquid phase separation (LLPS). These peptide coacervates can be tailored for applications in drug delivery, biosensing, bioelectronics, and in a range of other biotechnological purposes. BACKGROUND [0002] Compartmentalization has played a critical role in the emergence of catalysis and in the development of more complex biological systems. For example, it is hypothesized that the first cells emerged from simple lipid vesicles, which provided a protective environment for the chemical reactions that gave rise to the first self-replicating molecules. Membrane confinement, however, is not the only compartmentalization strategy available in nature. Several organisms use liquid-liquid phase separation (LLPS) to concentrate protein and nucleic acid molecules into membrane-less compartments, which enables the organization of cellular functionalities and complex biochemical reactions. LLPS has been shown to promote the nucleation and assembly of actin filaments, and to enhance the enzymatic efficiency of p-carboxysomes and multi-enzyme complexes. These LLPS-derived biomolecular condensates, or coacervates, provide spatial organization and facilitate efficient substrate transfer between enzymes, leading to an overall increase in the efficiency of enzymatic reactions. [0003] The importance of LLPS in living systems has also fueled its use to create synthetic catalytic systems. Previous studies have used LLPS to confine enzymes in artificial cells via the use of polymers, or to confine substrates in peptide-based condensates. While the impact of phase transitions on enzyme activity is complex and not fully understood, LLPS can create microenvironments that concentrate substrates and enzymes, thus increasing the local substrate concentration and influencing enzyme activity and selectivity. [0004] Though LLPS has been explored as a strategy to increase the catalytic rates of enzymes in artificial cells, its use for potentiating the activity of catalytic peptides remains unexplored. These catalytic peptides, which are for example widely used as chiral catalysts in organic reactions, present substantial limitations when used in aqueous reactions as their conformational flexibility strongly limits catalytic efficiency. However, the compartmentalization of catalytically active peptides in coacervates presents an enticing avenue to bolster their efficiency by confining their conformational flexibility into more densely packed environments, which may lead to the formation of structured peptide domains with improved catalytic activity. Furthermore, catalytic peptides offer an exciting opportunity as core components of coacervates, owing to their remarkable programmability and potential to simultaneously provide structural organization and catalytic functionality. Despite this potential, the systematic investigation of coacervate designs incorporating catalytic peptides that inherently fuse condensate organization with functionality is still largely unexplored. Notably, the encapsulation of the L-dipeptide (Ser-His) within lipid- based vesicles represents the only example of compartmentalization involving catalytic peptides, and this system was mainly explored as a model for the evolutionary competition between protocells. [0005] Liquid-liquid phase separation (LLPS) in living cells provides innovative pathways for synthetic compartmentalized catalytic systems. While LLPS has been explored for enhancing enzyme catalysis, its potential application to catalytic peptides remains unexplored. [0006] Therefore, there is a need to improve the catalytic efficiency of peptides. Catalytic peptides are highly flexible molecules, which, while advantageous for adaptability, can suffer from reduced catalytic efficiency due to their conformational mobility, especially in aqueous solutions. This flexibility presents challenges in achieving consistent and efficient catalytic activity, as the lack of structure often hinders proper substrate alignment and lowers reaction rates. Thus, the technical challenge is to restrict the movement of peptides to effectively stabilize their conformations and form structured peptide domains, which will increase local reaction rates and improve overall catalytic efficiency. [0007] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure. GEN ERAL DESCRIPTION [0008] The present disclosure relates to a coacervate, namely peptide-based compartments, liquid-like droplets or condensates, comprising a single peptide with disordered regions that undergo liquid-liquid phase separation (LLPS). This peptide sequence forms biomolecular coacervates with structured peptide domains proficient in hydrolyzing phosphate ester molecules and selectively sequestering phosphorylated peptides, stabilizing p