Patent Application
20240294570
The invention relates to isolated peptides and compositions, and their use in methods of causing cell death, specifically causing selective cell death of induced pluripotent stem cells even when present in mixed populations of cells.
Having unprecedented potential to generate a variety of cell types for cell therapy, pluripotent stem cells (PSCs), such as human embryonic stem cells (ESC) and human induced pluripotent stem cells (iPSCs), promise to revolutionize personalized medicine (Takahashi et al., “Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors,” Cell 131(5):861-872 (2007)). Increased research efforts have focused on the use of PSCs in clinical applications, such as PSC-derived dopamine (DA) neurons for treating Parkinson's disease (Kriks et al., “Dopamine Neurons Derived from Human ES Cells Efficiently Engraft in Animal Models of Parkinson's Disease,” Nature 480 (7378):547-51 (2011)), differentiating PSC into cardiomyocytes (Zhang et al., “Functional Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells,” Circ. Res. 104(4):e30-e41 (2009) or epicardium cells (Witty et al., “Generation of the Epicardial Lineage from Human Pluripotent Stem Cells,” Nat. Biotechnol. 32(10): 1026-35 (2014)) for treating heart disease, the generation of insulin-producing pancreatic beta cells from PSCs for treating diabetes (Pagliuca et al., “Generation of Functional Human Pancreatic Beta Cells in vitro,” Cell 159(2):428-39 (2014)), photoreceptor progenitors derived from PSCs for treating blindness (Barnea-Cramer et al., “Function of Human Pluripotent Stem Cell-derived Photoreceptor Progenitors in Blind Mice,” Sci. Rep. 6:29784 (2016)), and PSC-derived retinal pigment epithelium (RPE) for treating age-related macular degeneration (AMD) (Sharma et al., “Clinical-grade Stem Cell-derived Retinal Pigment Epithelium Patch Rescues Retinal Degeneration in Rodents and Pigs,” Sci. Transl. Med. 11(475):eaat5580 (2019)). Despite the rapid progresses of iPSC technology, considerable challenges remain to be met before safe clinical applications of PSCs (Yamanaka, “Pluripotent Stem Cell-Based Cell Therapy—Promise and Challenges,” Cell Stem Cell 27(4):523-531 (2020); Harding et al., “Preclinical Studies for Induced Pluripotent Stem Cell-based Therapeutics,” J. Biol. Chem 289(8):4585-4593 (2014)). Because a key feature of iPSCs is their potential for infinite proliferation, one major safety concern of iPSCs is their tumorigenicity (Knoepfler, “Deconstructing Stem Cell Tumorigenicity: A Roadmap to Safe Regenerative Medicine,” Stem Cells 27(5): 1050-1056 (2009)). For example, undifferentiated iPSCs exhibit comparable tumor producing potential with that of Hela cells in a rat model (Kanemura et al., “Tumorigenicity Studies of Induced Pluripotent Stem Cell (iPSC)-derived Retinal Pigment Epithelium (RPE) for the Treatment of Age-Related Macular Degeneration,” PLOS ONE 9(1):e85336 (2014)). Several studies even have shown a small number of residual iPSCs may produce teratomas in animals (Kawamata et al., “Design of a Tumorigenicity Test for Induced Pluripotent Stem Cell (iPSC)-Derived Cell Products,” J. Clin. Med. 4(1): 159-71 (2015); Liu et al., “The Tumourigenicity of iPS Cells and Their Differentiated Derivates,” J. Cell Mol. Med. 17(6): 782-91 (2013)). Moreover, inefficient differentiation protocols, variability of differentiation efficiency, or heterogeneity of iPSC clones all can lead to residual or large numbers of undifferentiated iPSCs after the differentiation procedure (Hu et al., “Neural Differentiation of Human Induced Pluripotent Stem Cells Follows Developmental Principles But With Variable Potency,” Proc. Natl. Acad. Sci. USA 107(9):4335-40 (2010)). Thus, it is necessary to eliminate the undifferentiated iPSCs without harming differentiated cells in a cell mixture prior to cell transplantation.
Considerable efforts have been spent on developing approaches to eliminate residual undifferentiated iPSCs (Schuldiner et al., “Selective Ablation of Human Embryonic Stem Cells Expressing a ‘Suicide’ Gene,” Stem Cells 21(3):257-265 (2003); Choo et al., “Selection Against Undifferentiated Human Embryonic Stem Cells By a Cytotoxic Antibody Recognizing Podocalyxin-like protein-1,” Stem Cells 26(6): 1454-1463 (2008); Tateno et al., “Elimination of Tumorigenic Human Pluripotent Stem Cells By a Recombinant Lectin-Toxin Fusion Protein,” Stem Cell Rep. 4(5):811-820 (2015); Tateno, “Development of Lectin-drug Conjugates for Elimination of Undifferentiated Cells and Cancer Therapy,” Trends Glycosci. Glycotechnol. 31(183):e121-E127 (2019); Fong et al., “Separation of SSEA-4 and TRA-1-60 Labelled Undifferentiated Human Embryonic Stem Cells from a Heterogeneous Cell Population Using Magnetic-Activated Cell Sorting (MACS) and Fluorescence-Activated Cell Sorting (FACS),” Stem Cell Rev. 5(1): 72-80 (2009); Menendez et al., “Increased Dosage of Tumor Suppressors Limits the Tumorigenicity of iPS Cells Without Affecting Their Pluripotency,” Aging Cell 11(1):41-50 (2012); Miki et al., “Efficient Detection and Purification of Cell Populations Using Synthetic MicroRNA Switches,” Cell Stem Cell 16(6): 699-711 (2015); Matsumoto et al., “Plasma-activated Medium Selectively Eliminates Undifferentiated Human Induced Pluripotent Stem Cells,” Regen. Ther. 5:55-63 (2016); Okada et al., “Selective Elimination of Undifferentiated Human Pluripotent Stem Cells Using Pluripotent State-specific Immunogenic Antigen Glypican-3,” Biochem. Biophys. Res. 511(3):711-717 (2019); Shiraki et al., “Methionine Metabolism Regulates Maintenance and Differentiation of Human Pluripotent Stem Cells,” Cell Metab. 19(5): 780-794 (2014); Nagashima et al., “Selective Elimination of Human Induced Pluripotent Stem Cells Using Medium with High Concentration of L-Alanine,” Sci. Rep. 8(1): 12427 (2018); Blum et al., “The Anti-apoptotic Gene Survivin Contributes to Teratoma Formation By Human Embryonic Stem Cells,” Nat. Biotechnol. 27(3):281-287 (2009); Ben-David et al., “Immunologic and Chemical Targeting of the Tight-junction Protein Claudin-6 Eliminates Tumorigenic Human Pluripotent Stem Cells,” Nat. Commun. 4:1992 (2013); Kuo et al., “Selective Elimination of Human Pluripotent Stem Cells by a Marine Natural Product Derivative,” J. Am. Chem. Soc. 136(28):9798-9801 (2014); Mao et al., “A Synthetic Hybrid Molecule for the Selective Removal of Human Pluripotent Stem Cells from Cell Mixtures,” Angew. Chem. Int. Ed. 56(7): 1765-1770 (2017); Mao et al., “Chemical Decontamination of iPS Cell-derived Neural Cell Mixtures,” Chem. Commun. 54(11): 1355-1358 (2018); Go et al., “Structure-activity Relationship Analysis of YM155 for Inducing Selective Cell Death of Human Pluripotent Stem Cells,” Front. Chem. 7:298 (2019); Kim et al., “Ethanol Extract of Magnoliae Cortex (EEMC) Limits Teratoma Formation of Pluripotent Stem Cells by Selective Elimination of Undifferentiated Cells Through the p53-dependent Mitochondrial Apoptotic Pathway,” Phytomedicine 69:153198 (2020); Kondo, “Selective Eradication of Pluripotent Stem Cells by Inhibiting DHODH Activity,” Stem Cells 39(1):33-42 (2021); Hermann et al., “Possible Applications of New Stem Cell Sources in Neurology,” Nervenarzt 84(8): 943-948 (2013); Rampoldi et al., “Targeted Elimination of Tumorigenic Human Pluripotent Stem Cells Using Suicide-Inducing Virus-like Particles,” ACS Chem. Biol. 13(8):2329-2338 (2018); Hong et al., “Suppression of Induced Pluripotent Stem Cell Generation By the p53-p21 Pathway,” Nature 460(7259): 1132-1135 (2009)) for the safe clinical applications of iPSC-based cell therapy, but current strategies still have many drawbacks. Therefore, there still exists a need to develop an innovative strategy that is rapid (≤2 hours), effective, and general for eliminating undifferentiated iPSCs in cell mixture.
A prominent difference between iPSCs and differentiated cells is that iPSCs overexpress (or upregulate) alkaline phosphatase (ALP) (Stefkova et al., “Alkaline Phosphatase in Stem Cells,” Stem Cells Int 2015:628368 (2015)), but the differentiated cells do not. Thus, it is possible to selectively kill iPSCs by using enzyme-instructed self-assembly (EISA) (Yang et al., “Enzymatic Formation of Supramolecular Hydrogels,” Adv. Mater. 16(16): 1440-1444 (2004); He et al., “Enzymatic Noncovalent Synthesis for Mitochondrial Genetic Engineering of Cancer Cells,” Cell Rep. Phys. Sci. 1(12): 100270 (2020); Feng et al., “Enzyme-Instructed Peptide Assemblies Selectively Inhibit Bone Tumors,” Chem 5(9):2442-2449 (2019); Wang et al., “Intercellular Instructed-Assembly Mimics Protein Dynamics To Induce Cell Spheroids,” J. Am. Chem. Soc. 141(18):7271-7274 (2019); Yan et al., “Activatable NIR Fluorescence/MRI Bimodal Probes for in Vivo Imaging by Enzyme-Mediated Fluorogenic Reaction and Self-Assembly,” J. Am. Chem. Soc. 141(26): 10331-10341 (2019); Yang et al., “Desuccinylation-Triggered Peptide Self-Assembly: Live Cell Imaging of SIRT5 Activity and Mitochondrial Activity Modulation,” J. Am. Chem. Soc. 142(42): 18150-18159 (2020); Cheng et al., “Autocatalytic Morphology Transformation Platform for Targeted Drug Accumulation,” J. Am. Chem. Soc. 141(10):4406-4411 (2019); Wang et al., “A Photoacoustic Probe for the Imaging of Tumor Apoptosis by Caspase-Mediated Macrocyclization and Self-Assembly,” Angew. Chem. Int. Ed. 58(15):4886-4890 (2019); Tanaka et al., “Cancer Cell Death Induced by the Intracellular Self-Assembly of an Enzyme-Responsive Supramolecular Gelator,” J. Am. Chem. Soc. 137(2):770-775 (2015); Chen et al., “Exploring the Condensation Reaction between Aromatic Nitriles and Amino Thiols To Optimize In Situ Nanoparticle Formation for the Imaging of Proteases and Glycosidases in Cells,” Angew. Chem. Int. Ed. 59(8):3272-3279 (2020); Shi et al., “De novo Design of Selective Membrane-Active Peptides by Enzymatic Control of Their Conformational Bias on the Cell Surface,” Angew. Chem. Int. Ed. 58(39): 13706-13710 (2019); Wang et al., “Tumour Sensitization Via the Extended Intratumoural Release of a STING Agonist and Camptothecin from a Self-assembled Hydrogel,” Nat. Biomed. Eng. 4(11): 1090-1101 (2020)), a molecular process that integrates enzyme reactions and self-assembly and is known to selectively kill cells based on overexpression of enzymes (Tanaka et al., “Cancer Cell Death Induced by the Intracellular Self-Assembly of an Enzyme-Responsive Supramolecular Gelator,” J. Am. Chem. Soc. 137(2): 770-775 (2015); Yang et al., “Intracellular Enzymatic Formation of Nanofibers Results in Hydrogelation and Regulated Cell Death,” Advanced Materials 19(20):3152-3156 (2007); Kuang et al., “Pericellular Hydrogel/Nanonets Inhibit Cancer Cells,” Angew. Chem. Int. Ed. 53(31): 8104-8107 (2014); Pires et al., “Controlling Cancer Cell Fate Using Localized Biocatalytic Self-Assembly of an Aromatic Carbohydrate Amphiphile,” J. Am. Chem. Soc. 137(2):576-579 (2015)). In fact, Saito et al. recently reported selectively eliminating iPSCs by EISA of D-phosphotetrapeptides (Kuang et al., “Efficient, Selective Removal of Human Pluripotent Stem Cells via Ecto-Alkaline Phosphatase-Mediated Aggregation of Synthetic Peptides,” Cell Chem. Biol. 24(6):685-694.e4 (2017)). Taking the advantage that ALP acts as an ectophosphatase to form pericellular nanofibers of D-peptides, Kuang et al. (“Pericellular Hydrogel/Nanonets Inhibit Cancer Cells,” Angew. Chem. Int. Ed. 53(31):8104-8107 (2014))) have shown that ALP overexpressed on iPSCs dephosphorylates the D-phosphotetrapeptides (e.g., 1, see
The present invention is directed to overcoming these and other deficiencies in the art.
A first aspect of the invention relates to an isolated peptide including the structure below:
where
A second aspect of the invention relates to supramolecular assembly of peptides according to the first aspect, where at least a portion of the peptides are dephosphorylated.
A third aspect of the invention relates to a pharmaceutical composition including one or more peptides according to the first aspect in an aqueous medium.
A fourth aspect of the invention relates to a method of causing cell death that includes the step of: contacting a cell that overexpresses a phosphatase with one or more peptides according to the first aspect, or a pharmaceutical composition according to the third aspect, which one or more peptides is phosphorylated, whereby the contacting step is effective to cause uptake of the one or more peptides and dephosphorylation of the phosphorylated amino acid residue(s) thereof by the phosphatase and thereby allow for intracellular self-assembly of the dephosphorylated one or more peptides.
A fifth aspect of the invention relates to a method for selectively causing cell death in a mixed population of cells that includes the steps of: providing a mixed population of cells including differentiated cells and one or more induced pluripotent stem cells; and contacting the mixed population of cells with one or more peptides according to the first aspect, or a pharmaceutical composition according to the third aspect, which one or more peptides is phosphorylated, whereby the contacting step is effective to cause uptake of the one or more peptides and dephosphorylation of the phosphorylated amino acid residue(s) thereof by a phosphatase overexpressed by the induced pluripotent stem cells, and thereby allow for intracellular self-assembly of the dephosphorylated one or more peptides in the induced pluripotent stem cells, but not differentiated cells, and selective induction of cell death in the induced pluripotent stem cells containing intracellular self-assemblies of the dephosphorylated one or more peptides.
As demonstrated in the accompanying Examples, an L-phosphopentapeptide (5), upon the dephosphorylation catalyzed by ALP, rapidly forms intranuclear peptide assemblies made of α-helix and aggregated strands that selectively kill iPSCs (see
One aspect of the invention relates to a peptide capable of induced self-assembly having the structure
where
The isolated peptide, when exposed to a suitable phosphatase, is dephosphorylated at the Z2 amino acid residue. This dephosphorylation of the Z2 amino acid residue allows the peptides to self-assemble when present at a suitable concentration, thereby forming nanofibers and nanoribbons, as well as supramolecular assembles thereof. As used herein, the term “nanofibril” is defined as a fiber of material having any shape wherein at least one dimension, e.g. the diameter, width, thickness, and the like, is about 100 nm or less. Nanofibril diameters may be about 50 nm or less, about 40 nm or less, about 30 nm or less, about 20 nm or less, about 10 nm or less, about 5 nm or less, about 4 nm or less, about 3 nm or less, about 2 nm or less, or about 1 nm or less in diameter. Nanoribbons possess a uniquely sheet-like cross section such that the ribbons are wider than their thickness and much longer than their thickness. Although the peptides of the present invention, upon self-assembly, as described herein, form nanofibrils or nanoribbons, persons of skill in the art should appreciate that such peptides may also form microfibrils or microribbons that are larger than 100 nm thick.
As used herein, the term “amino acid” is intended to embrace all compounds, whether natural or synthetic, which include both an amino functionality and an acid functionality, including amino acid analogues and derivatives. In certain embodiments, the amino acids contemplated in the present invention are those naturally occurring amino acids found in proteins, or the naturally occurring anabolic or catabolic products of such amino acids, which contain amino and carboxyl groups. Amino acids, as used herein, may include both non-naturally and naturally occurring amino acids. The peptides preferably contain all L-amino acids.
The α-helical amino acid sequence, whose length is defined above, is preferably formed of multiple L-amino acid residues that promote the formation of the α-helix. It is well established that formation of a single turn of an α-helix requires 3.6 amino acid residues, in which case multiples thereof will dictate how many turns are present in the α-helical amino acid sequence.
The amino acid residues present in the α-helical amino acid sequence preferably include those that promote the formation of the α-helical amino acid sequence, although a minority of amino acid residues that are neutral or slightly interfere in α-helix formation may be tolerated. Particularly preferred are one or more amino acid residues independently selected from the group of alanine, alpha-aminobutyric acid, norvaline, valine, norleucine, isoleucine, and leucine.
In one embodiment, the α-helical amino acid sequence is the tetrapeptide
wherein each of AA1, AA2, AA3, and AA4 is independently selected from the group of alanine, alpha-aminobutyric acid, norvaline, valine, norleucine, isoleucine, and leucine.
In one embodiment, the α-helical amino acid sequence is the pentapeptide
wherein each of AA1, AA2, AA3, AA4 and AA5 is independently selected from the group of alanine, alpha-aminobutyric acid, norvaline, valine, norleucine, isoleucine, and leucine.
In one embodiment, the α-helical amino acid sequence is the hexapeptide
wherein each of AA1, AA2, AA3, AA4, AA5, and AA6 is independently selected from the group of alanine, alpha-aminobutyric acid, norvaline, valine, norleucine, isoleucine, and leucine.
In one embodiment, the α-helical amino acid sequence is the heptapeptide
wherein each of AA1, AA2, AA3, AA4, AA5, AA6, and AA7 is independently selected from the group of alanine, alpha-aminobutyric acid, norvaline, valine, norleucine, isoleucine, and leucine.
In one embodiment, the α-helical amino acid sequence is the octapeptide
wherein each of AA1, AA2, AA3, AA4, AA5, AA6, AA7, and AA8 is independently selected from the group of alanine, alpha-aminobutyric acid, norvaline, valine, norleucine, isoleucine, and leucine.
In one embodiment, the α-helical amino acid sequence is the nonapeptide
wherein each of AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, and AA9 is independently selected from the group of alanine, alpha-aminobutyric acid, norvaline, valine, norleucine, isoleucine, and leucine.
In one embodiment, the α-helical amino acid sequence is the decapeptide
wherein each of AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, and AA10 is independently selected from the group of alanine, alpha-aminobutyric acid, norvaline, valine, norleucine, isoleucine, and leucine.
Aromatic amino acids may optionally be present in the α-helical amino acid sequence or as an amino acid residue present within the Z2 moiety. Such aromatic amino acids include phenylalanine, phenylalanine derivatives, napthylalanine, napthylalanine derivative, tyrosine, tyrosine derivatives, tryptophan, and tryptophan derivatives. In certain embodiments, aromatic residues of this type are present only in non-consecutive positions along the length of the α-helical amino acid sequence or along the length of the Z2 moiety (or along the length of the peptide as a whole). For example, if multiple phenylalanine residues are present, there is no instance where Phe-Phe appears within the α-helical amino acid sequence or the Z2 moiety. In alternative embodiments, the peptide contains not more than two consecutive aromatic amino acid residues positions along the length of the xx-helical amino acid sequence or along the length of the Z2 moiety (or along the length of the peptide as a whole). For example, if multiple phenylalanine residues are present, -Phe-Phe- may be present within the α-helical amino acid sequence or the Z2 moiety, but-Phe-Phe-Phe will not be present. In another embodiment, the peptide contains no more than three or, alternatively, no more than two aromatic amino acid residues along the length of the peptide; and preferably the peptide contains not more than two consecutive aromatic amino acid residues.
The N-terminal amino acid is covalently attached to Z1, which is a moiety comprising an aromatic group or a fluorophore. The aromatic group can be an aryl or heteroaryl, and may include single, multiple, or fused ring structures.
In certain embodiments, where Z1 comprises the aromatic group, the aromatic group is selected from the group consisting of phenylacetyl, naphthylacetyl, fluorenylacetyl, pyrenylacetyl, and cinnamoyl. Other aromatic groups can optionally be used to promote self-assembly.
In certain embodiments, where Z1 comprises the fluorophore, the fluorophore is 4-nitro-2,1,3-benzoxadiazolyl (“NBD”), 5-(dimethylamino)naphthalene-1-sulfonyl, 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazolyl, or 9-acridinyl. Other fluorophore groups can optionally be used to promote fluorescence and, optionally, self-assembly.
In certain embodiments, it may also be desirable to include a single amino acid residue within the α-helical amino acid sequence, or within the Z2 moiety, which allows for one or more agents to be conjugated to the peptide by coupling via side chains of amino acids, including the amino group of lysine, the guanidine group of arginine, the thiol group of cysteine, or the carboxylic acid group of glutamic acid or aspartic acid.
In general, amino groups present in lysine side chains can be reacted with reagents possessing amine-reactive functional groups using known reaction schemes. Exemplary amine-reactive functional groups include, without limitation, activated esters, isothiocyanates, and carboxylic acids. Examples of conjugating a chemotherapeutic agent (e.g., doxorubicin, daunorubicin, taxol) to a Lys sidechain are described in DeFeo-Jones et al., Nature Med. 6(11): 1248-52 (2000), Schreier et al., PlosOne 9(4):e94041 (2014), Gao et al., J Am Chem Soc. 131:13576 (2009), each of which is hereby incorporated by reference in its entirety.
In general, guanidine groups present in arginine can be reacted with reagents possessing guanidine-reactive groups using known reaction schemes. Exemplary guanidine-reactive functional groups include, without limitation, NHS esters using gas phase synthesis (McGee et al., J. Am. Chem. Soc., 134 (28):11412-11414 (2012), which is hereby incorporated by reference in its entirety).
In general, thiol groups present in cysteine (or cysteine derivative) side chains can be reacted with reagents possessing thiol-reactive functional groups using known reaction schemes. Exemplary thiol-reactive functional groups include, without limitation, iodoacetamides, maleimides, and alkyl halides.
In general, carboxyl groups present in glutamic or aspartic acid side chains, or at the C-terminal amino acid residue, can be reacted with reagents possessing carboxyl-reactive functional groups using known reaction schemes. Exemplary carboxyl-reactive functional groups include, without limitation, amino groups, amines, bifunctional amino linkers.
In each of the types of modifications described above, it should be appreciated that the conjugate can be directly linked via the functional groups of the peptide and the reagent to be conjugated, or via a bifunctional linker that reacts with both the peptide functional groups and the functional groups on the reagent to be conjugated.
In certain embodiments, the peptides of the invention include naphthyl-(CH2)—C(O)— or NBD-(CH2)2—C(O)— (also referred to herein as NBD-βAla-) as the Z1 moiety, and phosphotyrosine or tyrosine as the Z2 moiety.
Exemplary peptides of the present invention include, without limitation:
A further aspect of the invention relates to a self-assembled product formed by exposing the peptide to a phosphatase that is suitable to cause dephosphorylation of the phosphorylated amino acid residue. In certain embodiments, the self-assembled product is in the form of an oligomerized product that includes two or more peptides of the invention in dephosphorylated form. The dephosphorylated peptides co-assemble during oligomerization and hydrogelation. Preferably, each of the two or more peptides have an alpha-helix structure.
In certain embodiments, the oligomerization and hydrogelation occurs in an aqueous environment, in which case the resulting product takes the form of a supramolecular hydrogel formed upon self-assembly of the activated peptide(s) of the invention in an aqueous medium. As described herein, the term “supramolecular hydrogel” refers to a network of nanofibers or nanoribbons formed by the self-assembly of peptides as the solid phase to encapsulate water (Du et al., Chem. Asian J. 9(6): 1446-1472 (2014), which is hereby incorporated by reference in its entirety).
A further aspect of the invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a peptide or oligomerized product of the invention.
According to one embodiment, two or more of the peptides are present.
In some embodiments, the carrier is an aqueous medium. In one embodiment, the aqueous medium is a sterile isotonic aqueous buffer, which is typically well tolerated for administration to an individual. Additional exemplary aqueous media include, without limitation, normal saline (about 0.9% NaCl), phosphate buffered saline (“PBS”), sterile water/distilled autoclaved water (“DAW”), as well as cell growth medium (e.g., MEM, with or without serum), aqueous solutions of dimethyl sulfoxide (“DMSO”), polyethylene glycol (“PEG”), and/or dextran (less than 6% per by weight.)
To improve patient tolerance to administration, the pharmaceutical composition may have a pH of about 5 to about 8. In one embodiment, the pharmaceutical composition has a pH of about 6.5 to about 7.4. In some embodiments, the sodium hydroxide or hydrochloric is added to the pharmaceutical composition to adjust the pH.
In other embodiments, the pharmaceutical composition includes a weak acid or salt as a buffering agent to maintain pH. Citric acid has the ability to chelate divalent cations and can thus also prevent oxidation, thereby serving two functions as both a buffering agent and an antioxidant stabilizing agent. Citric acid is typically used in the form of a sodium salt, typically 10-500 mM. Other weak acids or their salts can also be used.
The pharmaceutical composition may also include solubilizing agents, preservatives, stabilizers, emulsifiers, and the like. A local anesthetic (e.g., lidocaine, benzocaine, etc.) may also be included in the compositions, particularly for injectable forms, to ease pain at the site of the injection.
In some embodiments, the peptide or peptides may each be present at a concentration of about 1 μM to about 10 mM, about 10 μM to about 5 mM, about 50 M to about 2 mM, or about 100 μM to about 1 mM, such as from about 100 μM to about 500 μM. The volume of the composition administered, and thus, dosage of the peptide administered can be adjusted by one of skill in the art to achieve optimized results. In one embodiment, between 100 and about 800 μg can be administered per day, repeated daily or periodically (e.g., once every other day, once every third day, once weekly). This can be adjusted lower to identify the minimal effective dose, or tailored higher or lower according to the nature of the treatment being effected.
Additional aspects of the invention relate to administering one or more peptides or compositions or hydrogels of the invention to a subject to promote a desired effect. In these various embodiments, administering may be carried out topically, intraperitoneally, intralesionally, ocularly, intraocularly, intranasally, orally, rectally, transmucosally, intranasally, intradermally, intestinally, parenterally, intramuscularly, subcutaneously, intravenously, intraarterially, intramedullary by implantation, by intracavitary or intravesical instillation, intrathecally, as well as direct intraventricular, intraperitoneal, intrasynovially, by intraocular injection, or by introduction into one or more lymph nodes. Administration can be repeated periodically during the course of a treatment regimen, for example, one or more times per week, daily, or even one or more times per day.
In some embodiments, the subject is a mammal. Suitable mammals include, without limitation, rodents, rabbits, canines, felines, ruminants, and primates such as monkeys, apes, and humans. In one embodiment, the subject is a human.
The products and compositions of the present invention afford a number of uses.
In one aspect, the invention relates to a method of causing cell death that includes contacting a cell that overexpresses a phosphatase with one or more peptides of the invention, or a pharmaceutical composition containing the same, which one or more peptides is phosphorylated, whereby said contacting is effective to cause uptake of the one or more peptides and dephosphorylation of the phosphorylated amino acid residue thereof by the phosphatase and thereby allow for intracellular self-assembly of the dephosphorylated one or more peptides.
In certain embodiments, the cell that overexpresses the phosphatase is a cancer cell. The cancer cells to be treated in accordance with these aspects can be present in a solid tumor, present as a metastatic cell, or present in a heterogenous population of cells that includes both cancerous and noncancerous cells.
Exemplary cancer conditions include, without limitation, cancers or neoplastic disorders of the brain and CNS (glioma, malignant glioma, glioblastoma, astrocytoma, multiforme astrocytic gliomas, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma), pituitary gland, breast (Infiltrating, Pre-invasive, inflammatory cancers, Paget's Disease, Metastatic and Recurrent Breast Cancer), blood (Hodgkin's Disease, Leukemia, Multiple Myeloma, Lymphoma), lymph node cancer, lung (Adenocarcinoma, Oat Cell, Non-small Cell, Small Cell, Squamous Cell, Mesothelioma), skin (melanoma, basal cell, squamous cell, Kapsosi's Sarcoma), bone cancer (Ewing's Sarcoma, Osteosarcoma, Chondrosarcoma), head and neck (laryngeal, pharyngeal, and esophageal cancers), oral (jaw, salivary gland, throat, thyroid, tongue, and tonsil cancers), eye, gynecological (Cervical, Endrometrial, Fallopian, Ovarian, Uterine, Vaginal, and Vulvar), genitourinary (Adrenal, bladder, kidney, penile, prostate, testicular, and urinary cancers), and gastrointestinal (appendix, bile duct (extrahepatic bile duct), colon, gallbladder, gastric, intestinal, liver, pancreatic, rectal, and stomach cancers).
In this embodiment, the cancer cells to be treated can be either ex vivo or in vivo.
In certain embodiments, the cell that overexpresses the phosphatase is an induced pluripotent stem cell (iPSC). iPSCs, including human iPSCs, may serve as promising materials for regenerative therapy. However, as discussed above, their ability to undergo unlimited self-renewal and pluripotent differentiation makes iPSCs tumorigenic after transplantation. Therefore, complete differentiation or selective elimination of residual undifferentiated cells is essential for the clinical application of these derivatives.
The iPSC to be treated in accordance with these aspects can be present in a heterogenous population of cells that includes both the iPSCs and differentiated cells. This embodiment is particularly useful when carrying out an autologous transfer of cells that have been modified ex vivo, and then treated in accordance with the present invention prior to administration of such modified cells to the individual from whom they were initially obtained. In this manner, the contacting step is desirably carried out ex vivo.
A further aspect of the invention relates to a method for selectively causing cell death in a mixed population of cells. This method includes providing a mixed population of cells including differentiated cells and one or more iPSCs; and contacting the mixed population of cells with one or more peptides of the invention, or a pharmaceutical composition containing the same, which one or more peptides is phosphorylated, whereby said contacting is effective to cause uptake of the one or more peptides and dephosphorylation of the phosphorylated amino acid residue thereof by a phosphatase overexpressed by the induced pluripotent stem cells, and thereby allow for intracellular self-assembly of the dephosphorylated one or more peptides in the induced pluripotent stem cells, but not differentiated cells, and selective induction of cell death in the induced pluripotent stem cells containing intracellular self-assemblies of the dephosphorylated one or more peptides.
The ex vivo contacting step is preferably carried out for less than 2 hours, such as from about 30 minutes up to about 120 minutes, including about 30 to about 45 minutes, about 45 to about 60 minutes, about 60 to about 75 minutes, about 75 to about 90 minutes, about 90 to about 105 minutes, or about 105 minutes to about 120 minutes. Further, the contacting step is preferably carried out using a peptide present at a concentration defined above, preferably from about 100 μM to about 1 mM, such as from about 200 μM to about 800 μM. Persons of skill in the art will be able to optimize the peptide dose and duration of treatment depending on the level of ALPs expressed by the iPSCs. For example, where iPSCs express higher levels of ALPs, then a lower concentration of peptide may be used for shorter duration.
A further aspect relates to a population of differentiated cells recovered from the processes of the present invention, which population of differentiated cells is free of iPSCs and, thus, suitable for transplantation into a patient. Such a population of differentiated cells can be used for autologous transplant procedures or heterologous transplant procedures.
According to another aspect, the invention relates to a method of treating a patient for cancer or inhibiting cancer cell efflux of an antineoplastic agent, anticancer drug, or chemotherapeutic drug.
According to one embodiment, the method of treating cancer includes administering to the patient a peptide of the invention or a pharmaceutical composition containing the same. The administering the peptide allows cancer cells to take up the peptide, or an oligomerization product formed by the peptide, which selectively causes cell death of cancer cells that overexpress phosphatase enzymes. Numerous cancer types have been previously demonstrated to overexpress alkaline phosphatase, including those noted above.
While any class of antineoplastic agent, anticancer drug, or chemotherapeutic drug is contemplated for use in combination with the present invention, exemplary agents within these classes include alkylating agents, platinum drugs, antimetabolites, anthracycline and non-anthracycline antitumor antibiotics, topoisomerase inhibitors, and mitotic inhibitors, corticosteroids and targeted cancer therapies (such as imatinib, Gleevec®; gefitinib, Iressa®; sunitinib, Sutent®; and bortezomib, Velcade®).
According to one embodiment, the method of treating cancer includes administering to the patient an antineoplastic agent, an anticancer drug, or a chemotherapeutic drug; and administering to the patient a solution comprising a peptide of the invention. These steps of administering the agents/drugs and peptide allows cancer cells to take up the peptide, or an oligomerization product formed by the peptide, and the administered agents/drugs.
As a consequence of administering the agents/drugs and peptide, the peptide or oligomerization product inhibits efflux of the antineoplastic agent, anticancer drug, or chemotherapeutic drug from cancer cells, further enhancing cancer cell death as compared to the peptide alone.
The examples below are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof.
Materials and Instruments: 2-Cl-trityl chloride resin (1.0-1.2 mmol/g), HOBt, HBTU, Fmoc-OSu, and other Fmoc-amino acids were purchased from GL Biochem (Shanghai, China). Other chemical reagents and solvents were purchased from Fisher Scientific. Alkaline phosphatase was purchased from Biomatik (Cat. No. A1130, Alkaline Phosphatase [ALP], 30000 U/mL, in 50% Glycerol.), Fetal bovine serum (FBS) and penicillin-streptomycin were purchased from Gibco by Life Technologies. All precursors were purified with Agilent 1100 Series Liquid Chromatograph system, equipped with an XTerra C18 RP column and Variable Wavelength Detector. The LC-MS spectra were obtained with a Waters Acquity Ultra Performance LC with Waters MICROMASS detector, and 1HNMR spectra on Varian Unity Inova 400. Circular dichroism (CD) spectra were obtained with a Jasco J-810 Spectropolarimeter. UV-Vis spectra were obtained with a Varian Cary 50 Bio UV-Visible Spectrophotometer.
TEM Sample Preparation: After placing 5 μL samples on 400 mesh copper grids coated with continuous thick carbon film (˜35 nm) which is glowed discharged, we washed the grid with ddH2O and UA (uranyl acetate). The sample loaded grid was stained with the UA. The residual UA was removed by filter paper and then dried in air. TEM images were obtained with Morgagni 268 transmission electron microscope.
Critical Micelle Concentration ((MC) Measurement: The CMCs were determined using pyrene as the fluorescent probe. Different concentrations of compounds were prepared in pyrene-saturate solutions. The fluorescence spectra of pyrene solutions with different concentration compounds were obtained. The intensity ratio of 378 nm/393 nm (I378/I393) was determined by a Synergy H1 hybrid multi-mode microplate reader. Plot I378/I393 against the concentrations of compounds. The concentration at the turning point is the CMC.
Dephosphorylation Rate Measurement: To 100 μL solution of 5, 7 or 9 in PBS, ALP was added, and the mixtures were shaken at 37° C. At different time point, 900 μL methanol was added to quench the enzyme reaction. The reaction mixtures were analyzed with LC-MS.
Cell Culture: Human induced pluripotent stem cell line A21 was generated from human normal dermal fibroblasts by using the StemRNA™-NM Reprogramming kit (Stemgent, Cat #00-0076). hiPSCs were routinely cultured and passaged on 6-well plates coated with 0.25 μg/cm2 iMatrix-511 (Recombinant Laminin-511) (ReproCell) with NutriStem XF/FFTM medium (Biological Industries). HS-5 cell line and HEK293 cell line were purchased from American Type Culture Collection (ATCC, USA). HS-5 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. HEK293 cells were cultured in Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. All cells were maintained at 37° C. in a humidified atmosphere of 5% CO2.
Differentiation of Human iPSC to iPS-derived Hematopoietic Progenitor Cells (HPC's): iPSCs were differentiated into hematopoietic progenitor cells (HPCs) by using a 3D-bioreactor platform1. HPCs released from iPSC-spheroids after 9-10 days' differentiation were collected and characterized. Hematopoietic lineage specific marker expression of harvested HPCs were analyzed by flow cytometry. About 97.6% of these HPCs were CD31+CD43+ double positive, but only about 13% are OCT4+, indicative of commitment to hematopoietic lineage. These iPS-derived HPCs were used for 5 (400 μM, 2 hr) cytotoxicity assay.
Cell Viability of iPSC's: iPSCs were plated in 6-well plates and incubated for 24 to 48 hours, then media were replaced with fresh one (2 ml) supplemented with PBS (control) 5 (200 μM, 300 μM, and 400 μM), 7 (400 μM) or 9 (400 μM), and incubated for 2 hours. Media were removed and cells were rinsed with PBS once, fresh normal cultural media were added and incubated for 30 min. All cells were collected and stained with trypan blue, live cells were counted using Cellometer Auto 2000 (Nexcelom Bioscience). Data were obtained by from three independent wells (n=3).
Cell Viability of iPS-derived HPC's: iPS-derived HPCs were plated in 12-well plates and incubated overnight, then media were replaced with fresh one (2 ml) supplemented with PBS (control) 5 (400 μM) incubated for 2 hours. All cells were collected and stained with trypan blue, live cells were counted using Cellometer Auto 2000 (Nexcelom Bioscience). Data were obtained by from three independent wells (n=3).
Cell Viability of Differentiated Cells: Cytotoxicity against HS-5 cells and HEK293 cells was determined by using MTT assay. Cells were seeded in 96-well plates at 1×105 cells/well for 24 hours followed by culture medium removal and subsequently addition of culture medium containing different concentration of 5 (immediately diluted from fresh prepared 10 mM stock solution). After 1/2/3 hours, the culture medium with 5 was replaced by fresh culture medium and 10 μL MTT solution (5 mg/mL) was added to each well and incubated at 37° C. for another 4 h. Then 100 μL of SDS-HCl solution was added to stop the reduction reaction and dissolve the formazan. The absorbance of each well at 595 nm was measured by a DTX880 Multimode Detector. The results were calculated as cell viability percentage relative to untreated cells. Data were obtained by from three independent wells (n=3).
Confocal Laser Scanning Microscopy (CLSM) Imaging: For live cell imaging, cells in exponential growth phase were seeded in a confocal dish (3.5 cm) at 1.0×105 cells per dish and then incubated in incubator for 24 h. We removed culture medium, and added fresh medium containing precursors for different time points. After removing the medium and washing the cells with live cell imaging solution (2 mL×2), the cells were used for CLSM imaging. For time-dependent live cell imaging, cells in exponential growth phase were seeded in a confocal dish (3.5 cm) at 1.0×105 cells per dish and then incubated in incubator for 24 h. After removing culture medium, we treated the cells with 1 mL of Hoechst 33342 (1 μg mL−1) for 10 minutes. After being washed with culture medium (2 mL×2), the cells were incubated with fresh medium containing precursor 5 in a Tokai Hit stage top incubator (STXF-WSKMX-SET) to be used for CLSM imaging. All the CLSM images were obtained using Zeiss LSM 880 confocal microscopy at the lens of 63× with oil. The lasers used are 405 nm and 488 nm.
Degradation: 5 million HS-5 cells were made into 1 mL lysate by freeze-thaw lysis method. To 100 μL lysate, 5 (200 μM) and rhodamine 6G (inner standard) were added. The mixtures were shaken at 37° C. At different time point, 900 μL methanol was added to quench the reaction. The reaction mixtures were analyzed with LC-MS.
Two precursors, NBD-β-Alanine and Fmoc-L-phospho-Tyrosine, were prepared for the subsequent synthesis of peptides 3, 5, 7, and 9. The synthesis of NBD-β-Alanine and Fmoc-L-phospho-Tyrosine are illustrated in Scheme 1 below.
Synthesis of NBD-β-Alanine: To the solution of β-Alanine (5 mmol, 1 g) and K2CO3 (15 mmol, 2 g) in H2O (15 mL), the solution of NBD-Cl in MeOH (30 mL) was added dropwise under the protection of N2. After reaction at room temperature for 3 h, the MeOH was removed by evaporation. After adding 70 mL H2O, the pH was the solution was adjusted by 1 M HCl to ˜3. The solution was extracted by diethyl ether (200 mL×3), and the organic part was dried by Na2SO4, filtered and concentrated by evaporation. The structure was confirmed by 1H NMR and MS. 1H NMR: (400 MHZ, CD3OD-d4) δ (ppm): 8.55 (m, 1H), 6.40 (d, 1H), 3.82 (s, 2H), 2.79 (t, 2H). MS: calc. [M-H]−=251.04, obsvd. ESI-MS: M/Z=250.95.
Synthesis of Fmoc-L-Tyr(PO3H2)—OH: The mixture of P2O5 (35 mmol, 10 g), H3PO4 (133 mmol, 13 g) and H-L-Tyr-OH (18 mmol, 3.22 g) was stirred for 24 h at 80° C. in N2 atmosphere. After adding 30 mL H2O and stirred for 30 min at 80° C., the reaction mixture was cool to room temperature. The reaction mixture was added to butanol (650 mL) dropwise and recrystallized at 4° C. overnight, filtration provided H-L-Tyr(PO3H2)—OH as white power. To the solution of H-L-Tyr(PO3H2)—OH (2 mmol, 522 mg) in H2O (20 mL), the solution of Fmoc-OSu (2.4 mmol, 808 mg) in MeCN (20 mL) was added. After adjusting pH to ˜8 by triethylamine (TEA), the solution was stirred at room temperature for 2 h. After removal of MeCN by evaporation, 60 mL H2O was added and the pH of the solution was adjusted to ˜3 by 1 M HCl. After extraction by ethyl acetate (100 mL×3), the organic part was washed by 1 M HCl (100 mL×2) and brine (100 mL×1). After being dried by Na2SO4, filtered and concentrated by evaporation, Fmoc-L-Tyr(PO3H2)—OH was provided as white powder.
In the previously reported study (Kuang et al., “Efficient, Selective Removal of Human Pluripotent Stem Cells via Ecto-Alkaline Phosphatase-Mediated Aggregation of Synthetic Peptides,” Cell Chem. Biol. 24(6):685-694.e4 (2017), which is hereby incorporated by reference in its entirety), the substrate for EISA was a D-phosphotetrapeptide (1,
To minimize the formation of β-sheets and to maintain hydrophobicity for self-assembly in water, L-leucine (Leu)—which is known to have high helix propensity (Lyu et al., “Alpha-helix Stabilization by Natural and Unnatural Amino Acids with Alkyl Side Chains,” Proc. Natl. Acad. Sci. U.S.A. 88(12):5317-5320 (1991), which is hereby incorporated by reference in its entirety)—was instead selected as the amino acid for constructing the peptide backbone. To visualize the location of the peptide assemblies in cellular environment after EISA, the 2-naphthylacetyl group was replaced with NBD (4-nitro-2,1,3-benzoxadiazole, an environment-sensitive fluorophore) that is particularly useful for revealing peptide assemblies in cells (Feng et al., “Artificial Intracellular Filaments,” Cell Rep Phys Sci 1(7): 100085 (2020); Gao et al., “Imaging Enzyme-triggered Self-assembly of Small Molecules Inside Live Cells,” Nat Commun 3:1033 (2012), each of which is hereby incorporated by reference in its entirety). It was not known a priori, however, whether the Leu-rich peptides would self-assemble and selectively kill iPSCs.
Based on the foregoing, the phosphopeptide, NBD-βA-LLLpY (3) (see SEQ ID NO: 1), was designed and synthesized in the manner described above. Although 3 is able to turn into 4 upon dephosphorylation catalyzed by ALP (see
Based on the molecular design shown in
The structures of peptides 3, 5, 6, 7, and 9 were confirmed by 1H NMR and LC-MS as follows:
MS of 3: calc. [M-H]−=833.32, obsvd. ESI-MS: M/Z=833.61.
1H NMR of 5 (400 MHZ, DMSO-d6) δ (ppm): 7.77 (d, 1H, J=12 Hz), 7.14 (d, 2H, J=8 Hz), 7.04 (d, 2H, J=8 Hz), 6.45 (d, 1H, J=8 Hz), 4.36 (m, 1H), 4.28 (m, 4H), 2.98 (m, 2H), 2.86 (m, 2H), 2.61 (m, 2H), 1.54 (m, 3H), 1.41 (m, 8H), 1.25 (m, 1H), 0.79 (m, 24H).
MS of 5: calc. [M-H]−=946.41, obsvd. ESI-MS: M/Z=946.63.
MS of 6: calc. [M-H]−==866.44, obsvd. ESI-MS: M/Z=866.52.
1H NMR of 7 (400 MHZ, DMSO-d6) δ (ppm): 7.72 (d, 1H, J=12 Hz), 7.08 (d, 2H, J=8 Hz), 7.02 (d, 2H, J=8 Hz), 6.44 (d, 1H, J=12 Hz), 4.36 (m, 1H), 4.28 (m, 4H), 2.98 (m, 2H), 2.80 (m, 2H), 2.62 (m, 2H), 1.52 (m, 2H), 1.39 (m, 8H), 1.24 (m, 2H), 0.79 (m, 24H). MS of 7: calc. [M-H]−=946.41, obsvd. ESI-MS: M/Z=946.60.
1H NMR of 9 (400 MHZ, DMSO-d6) δ (ppm): 7.75 (m, 1H), 7.12 (d, 2H, J=8 Hz), 7.00 (d, 2H, J=8 Hz), 6.44 (d, 1H, J=8 Hz), 4.46 (m, 1H), 4.23 (m, 4H), 2.98 (m, 2H), 2.75 (m, 2H), 2.61 (m, 2H), 1.48 (m, 12H), 0.81 (m, 24H).
MS of 9: calc. [M-H]−=946.41, obsvd. ESI-MS: M/Z=946.53.
After obtaining all the precursors, their behaviors for EISA were evaluated in vitro using transmission electron microscopes (TEM) to examine the nanostructures formed before and after the ALP catalyzed dephosphorylation of precursors 5, 7, and 9. At 400 μM and in phosphate buffered saline (PBS), 5 self-assembles to form short nanofibers with the diameter of 9±2 nm and a few nanoparticles (
A key requirement for selectively eliminating iPSCs by EISA is that the L-peptide nanoribbons only form on and in iPSCs, which overexpress ALP, but not on and in the differentiated cells that express normal level of ALP. That is, L-peptide nanoribbons should only rapidly form at high level of ALP, but not at normal level of ALP. Considering that the normal level of ALP in serum is about 0.1 U/mL (Burtis et al., Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, Elsevier Saunders (2006), which is hereby incorporated by reference in its entirety) and abnormally high level ALP can be 0.6-0.8 U/mL (Abdallah et al., “Serial Serum Alkaline Phosphatase as an Early Biomarker for Osteopenia of Prematurity,” Medicine (Baltimore) 95(37):e4837 (2016); Gibson et al., “Clinical Problem-solving; Out of the Blue,” N Engl J Med 370(18): 1742-8 (2014), each of which is hereby incorporated by reference in its entirety), EISA of 5 was tested in the presence of different concentrations ALP (from 0.1 U/mL to 0.8 U/mL) for 1 hour and 2 hour (
The morphological differences shown in
When incubated with 0.5 U/mL of ALP, 7 has a slower dephosphorylation rate than 5; the half-lives of 7 are 127.0, 112.0, and 125.5 minutes at 100, 200, and 400 μM, respectively (
To understand the secondary structures of the L-phosphopentapeptide (5) in the assemblies without dephosphorylation and L-pentapeptide (6, NBD-βA-LLLLY, SEQ ID NO: 2) in the assemblies formed by the dephosphorylation of 5, the circular dichroism (CD) spectra of 5 was measured at different concentrations before and after the addition of ALP. The CD spectra of 5 at the concentrations ranging from 100 to 800 μM show slight negative trough at 225-250 nm (
As shown in
Peptides 5, 7 or 9 were incubated with iPSCs and cells counts were obtained using trypan blue staining. Incubation of 5 at the concentrations of 200, 300, and 400 μM with iPSCs (Takahashi et al., “Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors,” Cell 126(4):663-676 (2006), which is hereby incorporated by reference in its entirety) for 2 hours results in the cell viabilities of 31.05±3.20%, 18.50±1.50%, and 6.96±1.71%, respectively, confirming that 5 potently kills iPSCs (
The cytotoxicity of 5 was tested against iPS-derived HPCs. After 9-10 days' differentiation, the HPCs were released from iPSC-spheroids and collected, and then the hematopoietic lineage specific marker expression of harvested HPCs was analyzed by flow cytometry. About 97.6% of these HPCs were CD31+CD43+ double positive, indicating that HPCs has high purity. After incubation with 5 (400 μM) for 2 h, the viability of the HPC cells is 96.8% (
Because the fluorescence of NBD increases drastically from unassembled to assembled state, confocal laser scanning microscopy (CLSM) was suitable to reveal the cellular location of the peptide assemblies formed after dephosphorylation catalyzed by ALP. After incubation with 5 at 400 μM for 2 hours, the iPSCs exhibit strong NBD fluorescence in nuclei, and much weaker fluorescence in cytoplasm and on membrane except a few puncta (
To trace the dynamics of the formation and distribution of 5 inside iPSC cells, time-lapse CLSM was also used to image the changes of the fluorescence in the iPSCs incubated by 5 (400 μM). Using one cell as a representative case, after 6 minutes incubation fluorescent puncta appear on the membrane, likely originating from the aggregates of assemblies of 5 and those of 6 generated by the dephosphorylation catalyzed by ALP. This observation indicates that, as a surfactant-like peptide (Vauthey et al., “Molecular Self-assembly of Surfactant-like Peptides to Form Nanotubes and Nanovesicles,” Proc. Natl. Acad. Sci. USA 99(8):5355-5360 (2002), which is hereby incorporated by reference in its entirety), 5 firstly adheres to the cell membrane, and then is hydrolyzed by ALP to form 6. After 24 minutes incubation, fluorescence starts to grow in the cytoplasm. Considering the LLLLY motif in SEQ ID NO: 2 constitutes the transmembrane domains of 18 human membrane proteins, it is possible that the affinity of 5 or 6 to membrane allows the assemblies of the mixture of 5 and 6 to interact with cellular membranes, which then facilitates nuclear localization. At about 28 minutes of incubation, fluorescence appears in the nucleus. Moreover, the nuclei shrink after the assemblies emerge in the nuclei. In addition, the nuclei also show nuclear blebbing (Stephens et al., “Chromatin Histone Modifications and Rigidity Affect Nuclear Morphology Independent of Lamins,” Mol. Biol. Cell 29(2):220-233 (2018), which is hereby incorporated by reference in its entirety). The shrinkage of nuclei and nuclear blebbing likely associate with iPSC death. To further examine the dynamics of EISA-formed peptide assemblies in iPSCs over different time, the increase of fluorescence in two cells over 2 hours was monitored; their nuclei, A and B, are shown in
As a L-peptide, 5 should be proteolytic susceptible to proteases, especially after it is converted to 6. Thus, the stability of 5 was tested in the lysate of HS-5. Cell lysate was prepared from five million HS-5 cells and resuspended in 1 mL, then incubated with 200 μM of 5. All of 5 disappears (transform to 6) and only about 20% of 6 remains after 2 h of incubation. After 4 hours incubation, only 6.01% of 6 remains. This result agrees with the results demonstrating that 5 hardly inhibits HS-5 cells, and confirms that:
The preceding Examples demonstrate that a L-leucine-rich phosphopentapeptide (5) rapidly and selectively kills iPSCs by generation of intranuclear peptide assemblies via ALP catalyzed enzymatic self-assembly. Because the morphology of the peptide assemblies is controlled by the level of ALP and concentration of the precursors, EISA of 5 is able to control cell fates according to both the levels of enzyme expression and precursor concentrations. Unlike molecules that localize in nuclei by positive charge (Cai et al., “Supramolecular “Trojan Horse” for Nuclear Delivery of Dual Anticancer Drugs,” J. Am. Chem. Soc. 139(8): 2876-2879 (2017), which is hereby incorporated by reference in its entirety), the L-leucine-rich phosphopeptide bears negative charges. Although the exact pathway for 5 enters the nuclei of iPSCs remains to be elucidated, it is believed that the assemblies of 5 likely cluster ALP on cell surface to facilitate cellular uptake. Then, further dephosphorylation by ALP leads to their endosomal escape before entering the nuclei of iPSCs. Such an unconventional mode of cellular uptake of phosphopeptide assemblies is recently demonstrated by overexpressing ALP on HEK293 cells (He et al., “Dynamic Continuum of Nanoscale Peptide Assemblies Facilitates Endocytosis and Endosomal Escape,” Nano Lett 21(9):4078-4085 (2021), which is hereby incorporated by reference in its entirety). Moreover, the shrinkage of nuclei and nuclear blebbing suggest that the rapid formation of nuclear assemblies of 6 may generate local oncotic pressure to contribute to the iPSC death. In addition, it is believed that the nuclear accumulation of 6, without involving canonical nuclear location sequences (Dingwall et al., “Nuclear Targeting Sequences—A Consensus?” Trends in Biochemical Sciences 16:478-481 (1991), which is hereby incorporated by reference in its entirety), implies a possible new mechanism for nucleocytoplamic transport. The results from the peptides 7 and 9 indicate that both the rate of the enzymatic reaction and the molecule structures (e.g., sequence and stereochemistry) control the morphology of the resulted peptide assemblies. Although the effect of 5 on function of normal cells and iPSC-derived cells remains to be determined, the rapid degradation of 5 or 6 as unassembled L-peptide by HS-5 cells indicate that the long-term effects of the 5 or 6 likely would be minimal. While EISA has frequently resulted in the peptide assemblies made of β-sheets, the exploration of α-helical peptides for EISA has received less attention. This work illustrates the potential of enzymatic noncovalent synthesis for generating peptide assemblies of α-helices, because considerable amount of studies has already established a useful pool of peptides for generating helical assemblies of peptides (Castelletto et al., “Alpha Helical Surfactant-like Peptides Self-assemble into pH-dependent Nanostructures,” Soft Matter 17(11):3096-3104 (2021); Rhys et al., “Navigating the Structural Landscape of De Novo α-Helical Bundles,” J. Am. Chem. Soc. 141(22):8787-8797 (2019); Wang et al., “Structural Analysis of Cross α-helical Nanotubes Provides Insight into the Designability of Filamentous Peptide Nanomaterials,” Nat. Commun. 12(1):407 (2021), each of which is hereby incorporated by reference in its entirety) and there is rich information of the transmembrane domains of proteins.
Submitted with this application is a Sequence Listing in the form of an ASCII text (.txt) file, which is hereby incorporated by reference into the specification of the application. The ASCII text file (6 KB) was created on Jun. 22, 2022 and has the file name 147376_000691.txt.
Having thus described the basic concept of the invention, it will be rather apparent to those skilled in the art that the foregoing detailed disclosure is intended to be presented by way of example only, and is not limiting. Various alterations, improvements, and modifications will occur and are intended to those skilled in the art, though not expressly stated herein. All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. Additionally, the recited order of processing elements or sequences, or the use of numbers, letters, or other designations therefore, is not intended to limit the claimed processes to any order except as may be specified in the claims. These alterations, improvements, and modifications are intended to be suggested hereby, and are within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims and equivalents thereto.
This application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63/213,484 filed Jun. 22, 2021, which is hereby incorporated by reference in its entirety.
This invention was made with government support under grant number CA142746 awarded by the National Institutes of Health and grant number DMR-2011846 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/034482 | 6/22/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63213484 | Jun 2021 | US |
