Patent Application
20250163122
This application is accompanied by a sequence listing entitled .xml, created February ______, 2023, which is approximately ______ bytes in size. This sequence listing is incorporated herein by reference in its entirety. This sequence listing is submitted herewith via EFS-Web, and is in compliance with 37 C.F.R. § 1.824(a)(2)-(6) and (b).
Airway mucus plaques contribute to the pathogenesis of common lung diseases, including asthma, COPD, cystic fibrosis, and bronchiectasis. Plaques form because of increased mucin production with rapid mucin secretion (together, “mucin hypersecretion”) or inadequate surface liquid. Both result in the formation of mucus that is excessively concentrated, viscoelastic and adhesive.
Mucins are high molecular weight, highly glycosylated proteins that are packaged into secretory granules and secreted from airway epithelial cells. Mucin secretion occurs at both a low baseline rate and a high stimulated rate. Stimulated mucin secretion involves interactions between the Ca2+ sensitive synaptotagmin-2 (Syt2) protein with components of the SNARE (soluble NSF attachment protein receptor) complex. The SNARE complex is involved in packaging and delivering material to various parts of a cell via vesicles. In particular, there is a primary interface where the SNARE protein SNAP-23 binds to the C2B domain of Syt2 that is important for stimulated mucin secretion.
Available therapeutic drugs address mucin production and surface liquid production that occur when airway mucus plaques are formed, but do not target the stimulated hypersecretion of mucin mediated by the interaction between Syt2 and the SNAP-23.
Various examples are described for compositions and methods for treating mucus dysfunction.
Provided herein are polypeptide constructs comprising a first peptide comprising a portion of the SNARE protein SNAP-25A (a close homolog of SNAP-23) or a variant thereof attached to a cell penetrating peptide, wherein the first peptide is a hydrocarbon-stapled peptide comprising non-natural amino acids connected via at least one macrocyclic crosslink. In some embodiments, the polypeptide constructs are useful for specifically inhibiting mucin hypersecretion. In some embodiments, the polypeptide constructs are useful for specifically disrupting other Ca2+-triggered membrane fusion processes, such synaptic neurotransmitter release. In some embodiments, the cell penetrating domain is penetratin.
Also provided herein are various Syt2 expression inhibitors. Syt2 expression inhibitors include Syt2 targeting polynucleotides or polynucleotide complexes, such shRNAs, ASOs, siRNAs, or miRNAs, and one or more gene editing system components (e.g., at least one of a targeted nuclease or a guide RNA).
Also provided are pharmaceutical compositions comprising such a polypeptide construct and a pharmaceutically acceptable excipient. In addition, provided are pharmaceutical compositions comprising such a Syt2 expression inhibitor. Also provided are kits and devices containing such pharmaceutical compositions.
Also provided are methods for treating a subject with mucus hypersecretion-based airway obstruction and/or developed mucus occlusions. In some instances, the methods can include administering a therapeutically effective amount of a polypeptide construct as described herein to a subject in need thereof. The polypeptide constructs can be administered in conjunction with an inhibitor of at least one of Munc18, VAMP8, Munc13, or Stx3. These are homologs of neuronal synaptic proteins and are implicated in basal and/or stimulated mucin secretion in airway cells. In some instances, the methods can include administering a therapeutically effective amount of a Syt2 expression inhibitor as described herein to a subject in need thereof.
Also provided are methods of inhibiting mucin secretion in an airway epithelial cell and methods of inhibiting Syt2-mediated stimulated mucin secretion, triggered by Ca2+ release after ATP or methacholine bind to helpta-helical plasma membrane receptors coupled to Gq. In some instances, the methods include contacting a cell with an effective amount of a polypeptide construct as described herein. In some instances, the methods include contacting a cell with an effective amount of a Syt2 expression inhibitor as described herein.
These illustrative examples are mentioned not to limit or define the scope of this disclosure, but rather to provide examples to aid understanding thereof. Illustrative examples are discussed in the Detailed Description, which provides further description. Advantages offered by various examples may be further understood by examining this specification.
The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more certain examples and, together with the description of the example, serve to explain the principles and implementations of the certain examples.
In
Provided herein are compositions and methods for treating obstruction of airways due to mucin hypersecretion by airway epithelial cells, which contributes to the pathogenesis of a number of diseases, including asthma, COPD, cystic fibrosis, and bronchiectasis. Mucin hypersecretion includes elevated mucin production and stimulated mucin secretion. The invention includes compositions that disrupt stimulated mucin secretion and methods of using such compositions. In some instances, the compositions are polypeptide constructs that disrupt the interaction between soluble NSF attachment protein receptor (SNARE) vesicle fusion proteins and Ca2+ sensor proteins involved in stimulated mucin secretion. In some instances, the compositions are polynucleotides or polynucleotide complexes that result in reduced expression of Syt2 in an airway epithelial cell, such as an airway epithelial cell.
Secretion of proteins such as mucin relies on vesicle-mediated transport, which in turn relies on lipid bilayer fusion. Fusion proteins, such as SNARE proteins, play an essential role in secretion, supplying the energy to overcome the high kinetic barrier to mediate lipid bilayer fusion and mediating fusion of vesicles with a target membrane efficiently and specifically. Since membrane fusion is a key process within all living cells, SNARE proteins are highly conserved among different organisms and in different vesicle-mediated processes with a single organism.
The SNARE protein family consists of at least 60 membrane-associated proteins in mammalian cells. Among the best studied SNARE proteins are those involved in the targeting of synaptic vesicles, which carry neurotransmitters, in neurons (Gerald, K., 2002). SNARE proteins can be functionally distinguished as v-SNAREs, which are located on vesicles, and t-SNAREs which are located on the target membrane, which associate together to form the SNARE protein complex.
The underlying molecular mechanisms for membrane fusion are superficially similar for both class 1 viral fusion proteins and SNARE-mediated fusion, wherein formation of helical bundles drives membranes together, leading to membrane fusion. However, for stimulated mucin secretion, SNARE proteins must cooperate with other factors, in particular, the Ca2+ sensor synaptotagmin. Synaptotagmins are a family of proteins containing an N-terminal transmembrane region, a linker, and two C-terminal domains, called C2A and C2B. The C2 domain of a subset of synaptotagmins binds to Ca2+ (Mackler et al., 2002), an interacts with other SNARE components. For example, between 2 and 3 Ca2− ions bind to the C2B domain of synaptotagmin-1 (Syt1). Id.
The primary interface between the neuronal SNARE complex and the C2B domain of Syt1 is a specific interface that is conserved in all species and across the other fast isoforms for neurotransmitter release, synaptotagmin-2 (Syt2) and synaptotagmin-9 (Zhou et al., 2015). Many of the key residues involved in the primary interface are located in the t-SNARE SNAP-25A. Residues involved in the primary interface are critical for Ca2+-triggered fusion in a reconstituted system and in neuronal cultures (Zhou et al., 2015; Zhou et al., 2017).
Primary sequence conservation suggests that the primary interface also exists in other systems that utilize SNAREs and synaptotagmins, such as stimulated exocytosis in neurons, endocrine cells, and exocrine cells (Zhou et al., 2015). In particular, mucin exocytosis in airway secretory cells is mediated by SNAREs, Syt2, and other factors (Jaramillo et al., 2018; Davis and Dickey, 2008; Fahy et al., 2010). Syt2 is selectively expressed in airway secretory cells compared to ciliated cells, and it serves as a critical sensor for stimulated but not baseline mucin secretion (Tuvim et al., 2009). Syntaxin-3 (Stx3) and SNAP-23 are also highly expressed in airway epithelial cells (Riento et al., 1998; Ren et al. 2015). In stimulated mucin secretion, Ca2+ is released from the endoplasmic reticulum (ER) via the activated inositol triphosphate (IP3) receptor. IP3 is generated by phospholipase C (PLC) upon binding of agonists (such as ATP or methacholine) to hepta-helical receptors in the plasma membrane coupled to Gq. The released Ca2+ in turn binds to Syt2 on the granule vesicle and then triggers SNARE-mediated fusion of the granule with the plasma membrane, leading to mucin secretion (Jaramillo et al., 2018; Davis & Dickey, 2008; Tuvim et al., 2009).
Both baseline and stimulated mucin secretion are impaired in SNAP-23 heterozygous mutant mice (Ren et al., 2015), VAMP8 knock-out mice (Jones et al., 2012), and Munc13-2 knock-out mice (Zhu et al., 2008). In contrast, stimulated mucin secretion is selectively impaired in Munc18-2 mutant mice (Kim et al., 2012; Jaramillo et al., 2019). Stx3 binds and colocalizes with Munc18-2 (Riento et al., 1998) and overexpression of Munc18-2 reduces the level of Stx3/SNAP-23 binary complex (Kim et al., 2012). These finding suggested that, similar to neurotransmitter release, SNAREs (Stx3, SNAP-23, and VAMP8), Syt2, Munc13-2, and Munc18-2 are among the key components that drive membrane fusion between mucin-containing granules and the plasma membrane (Jaramillo et al., 2018; Davis & Dickey, 2008; Fahy et al., 2010).
The invention described in this disclosure is based in part on the discovery that hydrocarbon-stapled peptides can disrupts Ca2+-triggered membrane fusion by interfering with the primary interface between the neuronal SNARE complex and synaptotagmin-1. In reconstituted systems with these neuronal synaptic proteins or with their airway homologues syntaxin-3, SNAP-23, VAMP8, synaptotagmin-2, along with Munc13-2 and Munc18-2, SP9 strongly suppressed Ca2+-triggered fusion at physiological Ca2+ concentrations. Conjugation of cell penetrating peptides to SP9 resulted in efficient delivery into cultured human airway epithelial cells and mouse airway epithelium, where it markedly reduced stimulated mucin secretion in both systems, and substantially attenuated mucus occlusion of mouse airways. Based on this work, peptides that disrupt Ca2+-triggered membrane fusion may allow therapeutic modulation of mucin secretory pathways.
The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues linked by covalent peptide bonds. All three terms apply to naturally occurring amino acid polymers and non-natural amino acid polymers, as well as to amino acid polymers in which one (or more) amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid. Unless otherwise specified, the terms encompass amino acid chains of any length, including full-length proteins.
The term “amino acid” refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. Amino acids include naturally-occurring α-amino acids and their stereoisomers, as well as unnatural (non-naturally occurring) amino acids and their stereoisomers. “Stereoisomers” of a given amino acid refer to isomers having the same molecular formula and intramolecular bonds but different three-dimensional arrangements of bonds and atoms (e.g., an L-amino acid and the corresponding D-amino acid).
Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate and O-phosphoserine. Naturally-occurring α-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of a naturally-occurring α-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-Ile), 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-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.
Unnatural (non-naturally occurring) amino acids include, without limitation, amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids in either the L- or D-configuration that function in a manner similar to the naturally-occurring amino acids. For example, “amino acid analogs” can be unnatural amino acids that have the same basic chemical structure as naturally-occurring amino acids (i.e., a carbon that is bonded to a hydrogen, a carboxyl group, an amino group) but have modified side-chain groups or modified peptide backbones, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. “Amino acid mimetics” refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally-occurring amino acid.
As used herein, a “chemically modified amino acid” refers to an amino acid whose side chain has been chemically modified. For example, a side chain can be modified to comprise a new functional group, such as an olefin, a thiol, a carboxylic acid, or an amino group. A side chain can also be modified to comprise a signaling moiety, such as a fluorophore or a radiolabel. Post-translationally modified amino acids are also included in the definition of chemically modified amino acids.
Also contemplated are “conservative amino acid substitutions.” By way of example, a conservative amino acid substitution can be made in one or more of the amino acid residues, for example, in one or more lysine residues of any of the polypeptides provided herein. One of skill in the art would know that a conservative substitution is the replacement of one amino acid residue with another that is biologically and/or chemically similar. The following eight groups each contain amino acids that are conservative substitutions for one another:
By way of example, when an arginine to serine is mentioned, also contemplated is a conservative substitution for the serine (e.g., threonine). Nonconservative substitutions, for example, substituting a lysine with an asparagine, are also contemplated.
The terms “nucleic acid,” “nucleotide,” and “polynucleotide” refer to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers. The term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, and DNA-RNA hybrids, as well as other polymers comprising purine and/or pyrimidine bases or other natural, chemically modified, biochemically modified, non-natural, synthetic, or derivatized nucleotide bases. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), orthologs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
“Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence (e.g., a peptide of the invention) in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence that does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
“Identical” and “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Sequences are “substantially identical” to each other if they have a specified percentage of nucleotides or amino acid residues that are the same (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
“Similarity” and “percent similarity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of amino acid residues that are either the same or similar as defined by a conservative amino acid substitutions (e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% similar over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Sequences are “substantially similar” to each other if, for example, they are at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% similar to each other.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available at the National Center for Biotechnology Information website, ncbi.nlm.nih.gov. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=−2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA, 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, an amino acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test sequence to the reference sequence is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
When only two polypeptides are being compared, one sequence acts as the reference polypeptide sequence, the homologous regions are aligned, and the identical amino acids are counted and recorded as a percent of the total reference polypeptide sequence, which is the “percent identity.”
“Hydrocarbon-stapled peptides” are peptides whose structure has been stabilized or altered by, for example, incorporating at least one all-hydrocarbon macrocyclic crosslink.
A “macrocyclic crosslink” is a covalent bond formed when pairs of amino acids bearing two terminal alkenes, such as α,α-disubstituted non-natural amino acids bearing olefin tethers of various lengths, undergo ring closing metathesis. See, e.g., Schafmeister, C. E., et al., J. Am. Chem. Soc. 122(24): 5891-5892 (2000).
As used herein, the term “cell penetration peptide” refers to an amino acid sequence that, when linked to a second peptide, causes or enhances the ability of the second peptide to cross membranes of a cell when the cell is contacted by the cell penetration peptide linked to the second peptide.
An “airway epithelial cell” includes any one of a variety of cell types found in the respiratory epithelium including, for example, basal cells, club cells, ciliated cells, goblet cells, tuft cells, pulmonary ionocytes, pulmonary neuroendocrine cells, hillock cells, and microfold cells. An airway secretory cell is an airway epithelial cell that is variously termed a club, Clara, and goblet cell. While these cell types are of the same lineage, their appearance can change depending on the degree of mucin production.
As used herein, “treating” or “treatment” of any disease or disorder refers to preventing or ameliorating a disease or disorder in a subject or a symptom thereof. The term ameliorating refers to any therapeutically beneficial result in the treatment of a disease state, e.g., COPD or cystic fibrosis, lessening in the severity or progression, or curing thereof. Treating or treatment also encompass prophylactic treatments that reduce the incidence of a disease or disorder in a subject and/or reduce the incidence or reduce severity of a symptom thereof. Thus, treating or treatment includes ameliorating at least one physical parameter or symptom. Treating or treatment includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. Treating or treatment includes delaying, preventing increases in, or decreasing mucin hypersecretion. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, a method for treating airway obstruction in a subject by administering a fusion protein or modified protein as described in this disclosure is considered to be a treatment if there is at least a 10% reduction in one or more symptoms of airway obstruction in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. In some embodiments, formulations comprising a fusion protein or modified protein as described herein are administered to the subject until the subject exhibits amelioration of at least one symptom of airway obstruction and/or is demonstrated to have a sustained decrease in mucin secretion, e.g., as measured by immunoassay. In some instances, the formulation is administered to the subject until mucin hypersecretion is undetectable, i.e. below the level of detection, such that only basal mucin secretion can be detected after IL-13 and ATP treatment by the assay methodology employed. In some instances, the subject exhibits undetectable mucin hypersecretion 1-4 weeks, 2-4 weeks, 2-12 weeks, 4-12 weeks, or 12-24 weeks after last administration of the formulation. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
As used herein, the term “therapeutically effective amount” refers to an amount of polypeptide construct as provided herein that, when administered to a subject, is effective to achieve an intended purpose, e.g., to reduce airway obstruction, to reduce or ameliorate at least one symptom of an airway obstruction disease (e.g., COPD, cystic fibrosis), and/or otherwise reduce the length of time that a patient experiences a symptom of an airway obstruction disease, or extend the length of time before a symptom may recur. The term therapeutically effective amount may be referred to herein as effective amount, with the context depending on the subject who is receiving treatment or in reference to in vitro effects. An effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. In some instances, an effective amount is not a dosage so large as to cause adverse side effects, such as excessive coughing, increased propensity for pulmonary infections, and the like. An effective amount may vary with the subject's age, condition, and sex, the extent of the disease in the subject, frequency of treatment, the nature of concurrent therapy (if any), the method of administration, and the nature and scope of the desired effect(s) (Nies et ah, Chapter 3 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et ah, eds., McGraw-Hill, New York, NY, 1996). and can be determined by one of skill in the art. Other factors can include, e.g., other medical disorders concurrently or previously affecting the subject, the general health of the subject, the genetic disposition of the subject, diet, time of administration, rate of excretion, drug combination, and any other additional therapeutics or treatments that are administered to the subject. Although individual needs may vary, determination of optimal ranges for effective amounts of formulations is within the skill of the art. It should also be understood that a specific dosage and treatment regimen for any particular subject also depends upon the judgment of the treating medical practitioner (e.g., doctor or nurse).
As used herein, the term “subject” refers to animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like.
As used herein, the term “pharmaceutically acceptable excipient” refers to a substance that aids the administration of an active agent to a subject. By “pharmaceutically acceptable,” it is meant that the excipient is compatible with the other ingredients of the formulation and is not deleterious to the recipient thereof. Pharmaceutical excipients useful in the compositions include, but are not limited to, binders, fillers, disintegrants, lubricants, glidants, coatings, sweeteners, flavors and colors.
As used herein, the terms “about” and “around” indicate a close range around a numerical value when used to modify that specific value. If “X” were the value, for example, “about X” or “around X” would indicate a value from 0.9X to 1.1X, e.g., a value from 0.95X to 1.05X, or a value from 0.98X to 1.02X, or a value from 0.99X to 1.01X. Any reference to “about X” or “around X” specifically indicates at least the values X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and 1.1X, and values within this range.
Use herein of the word “or” is intended to cover inclusive and exclusive OR conditions. In other words, A or B or C includes any or all of the following alternative combinations as appropriate for a particular usage: A alone; B alone; C alone; A and B only; A and C only; B and C only; and A and B and C.
Provided herein are polypeptides comprising a hydrocarbon-stapled peptide attached to a cell penetrating peptide. Also provided herein are polypeptides comprising a hydrocarbon-stapled peptide attached to a non-biological material. The hydrocarbon-stapled peptide is derived from a portion of SNAP-25A that forms an α-helix in the full-length SNARE complex that is a part of the primary interface with synaptotagmin. The hydrocarbon-stapled peptide, also referred to as the first peptide in this disclosure, contains one or two macrocyclic crosslinks that stabilize the formation of an α-helical peptide conformation.
In some embodiments, the first peptide comprises one side that contains amino acid residues that interact with amino acid residues in a synaptotagmin protein. In the context of this disclosure, this side of the first peptide is the primary interface-facing side of the first peptide. In some embodiments, the first peptide also comprises one side that does not contain amino acids that interact with amino acid residues in a synaptotagmin protein. In the context of this disclosure, this side of the first peptide is the non-binding side of the first peptide. As discussed above, the primary interface of SNARE proteins with Syt2 and other synaptotagmin homologs is conserved across species. The crystal structure of the primary interface between the SNARE complex (VAMP-2), Stx1, SNAP-25A, and the C2B domain of Syt1 indicates which amino acid residues in the first peptide (e.g., SP9) likely face the primary interface (data not shown; also see EXTENDED DATA FIGS. 2B and 2C of U.S. Provisional Patent Application Ser. No. 63/311,001).
Primary sequence alignments between neuronal and airway systems (Stx1A vs Stx3, SNAP-25A vs SNAP-23, and Syt1 vs Syt2) indicate that the residues directly involved in interactions at the primary interface are identical for Syt1/Syt2 (see SEQ ID NO: 46, amino acid positions 268-407; SEQ ID NO:47, amino acid positions 269-408) and mostly identical for SNAP-25A/SNAP-23 (see SEQ ID NOs: 1-2 (amino acid positions 37-58) and SEQ ID NOs: 44-45 (amino acid positions 156-170); also see EXTENDED DATA FIGS. 2A and 2B of U.S. Provisional Patent Application Ser. No. 63/311,001). For example, residues that are important for the primary interface include R281, E295, Y338, R398, and R399 in Syt1 C2B (also corresponding to residues mutated in Syt1_QM); K40, D51, E52, E55, Q56, and D166 in SNAP-25A; and D231, E234, and E238 in Stx1A (see SEQ ID NOS: 42-47; also see EXTENDED DATA FIGS. 2A and 2B of U.S. Provisional Patent Application Ser. No. 63/311,001). These are among the highly conserved amino acid residues when comparing SEQ ID NO: 42 (Stx1A aa 222-247) to SEQ ID NO: 43 (Stx3 aa 222-247), when comparing SEQ ID NO: 44 (SNAP-25 aa 156-170) to SEQ ID NO: 45 (SNAP-23 aa 156-170), and when comparing SEQ ID NO: 46 (Syt1, aa 268-407) to SEQ ID NO: 47 (Syt2, aa 268-407) (also see EXTENDED DATA FIGS. 2A and 2B of U.S. Provisional Patent Application Ser. No. 63/311,001).
In some embodiments, the macrocyclic crosslinks are located on the first peptide so as to not interfere with the interaction of the first peptide with the region of a synaptotagmin homolog that forms part of the primary interface and binds to components of the SNARE complex. In some embodiments, the macrocyclic crosslink is located between non-natural amino acid pairs that are on the non-binding side of the first peptide. Said another way, in some embodiments, the macrocyclic crosslinks of the first peptide are positioned such that they oriented away from the primary interface-facing side of the first peptide.
In some embodiments, the first peptide comprises SEQ ID NO: 1 or a sequence having at least 64% identity (e.g., at least 64%, 65%, 70%, 75%, 80%, 85%, 90% 95%, or 99% identity) thereto. In some embodiments, the first peptide comprises SEQ ID NO: 2, or a sequence having at least 64% identity (e.g., at least 64%, 65%, 70%, 75%, 80%, 85%, 90% 95%, or 99% identity) thereto.
SEQ ID NO: 1 and SEQ ID NO: 2 correspond to residues 37-58 of human SNAP-25A and residues 32-53 of human SNAP-23, respectively. Other proteins within this family of SNARE proteins are identifiable by amino acid sequence homology using, for example, the BLAST algorithm. Since they may perform analogous functions in vivo, the corresponding peptide sequences from such sequence homologs may functionally substitute for SEQ ID NO: 1 and/or SEQ ID NO: 2.
In some embodiments, the first peptide comprises SEQ ID NO: 1 or SEQ ID NO: 2 or a portion or variant thereof (e.g., having at least 64% identity (e.g., at least 64%, 65%, 70%, 75%, 80%, 85%, 90% 95%, or 99% identity) thereto) and one or more pairs of non-natural amino acids in which each pair forms a macrocyclic crosslink. In some embodiments, the first peptide comprises a variant of SEQ ID NO: 1 or SEQ ID NO: 2 comprising a non-native amino acid residue in at least one amino acid position not predicted to interact with Syt1 C2B based on the crystal structure of the SP9-Syt2 C2B complex. In some embodiments, the first peptide comprises a variant of SEQ ID NO: 1 or SEQ ID NO: 2 with a non-native amino acid residue in a position corresponding to at least one of amino acid positions 2, 6, 9, 13, 17, or 18 of the SP9 sequence as set forth in SEQ ID NO:8. In some embodiments, the variant of SEQ ID NO: 1 or SEQ ID NO: 2 comprises a non-native amino acid residue at least one of positions 2, 6, 9, 13, 17, or 18 of SEQ ID NO: 8.
In some embodiments, the first peptide comprises one pair of non-natural amino acids that form a macrocyclic crosslink, and the non-natural amino acids flank six contiguous amino acid residues in the first peptide. In some embodiments, the one pair of non-natural amino acids corresponds to amino acids 6 and 13, 7 and 14, 10 and 17, or 11 and 18 of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the first peptide comprises SEQ ID NO: 3 or 4.
In some embodiments, the first peptide comprises two pairs of non-natural amino acids that each form a macrocyclic crosslink, and each pair of non-natural amino acids flanks three continguous amino acids. In some embodiments, the first pair of non-natural amino acids correspond to amino acids 3 and 7, 6 and 10, or 7 and 11 in SEQ ID NO: 1 or SEQ ID NO: 2, and the second pair of non-natural amino acids correspond to amino acids 10 and 14, 13 and 17, 14 and 18, or 17 and 21 in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the first peptide comprises SEQ ID NO: 5, 6, 7, 8, or 9.
In some embodiments, the polypeptide constructs comprise one or more cell penetration peptides linked to the first peptide (e.g., to the N-terminus of the first peptide or to the C-terminus of the first peptide). Cell penetration peptides are positively charged and 5-30 amino acids long, and characterized by their ability to penetrate into biological membranes, often taking their attached cargo with them. A number of cell penetration peptides can be linked to the first peptide so as to enhance delivery of the first peptide to target cells in vitro and/or in vivo (see, e.g., Guidotti 2017 and Derakhshankhah & Jafari 2018). The polypeptide construct can have the first peptide and one or more cell penetration peptides in any orientation. In some embodiments, the C-terminus of the first peptide is linked to the N-terminus of the cell penetration peptide. In some embodiments, the N-terminus of the first peptide is linked to the C-terminus of a first cell penetration peptide(s) and C-terminus of the first peptide is linked to the N-terminus of a second cell penetration peptide(s). In some embodiments, the cell penetration peptide is a cationic peptide having 5-25 total amino acid residues and at least 5 arginine residues, lysine residues, or a combination thereof. In some embodiments, the cell penetration peptide is a polyarginine ranging in length from 5 residues to 25 residues. In some embodiments, the cell penetration peptide comprises an amino acid sequence set forth in Table 1 or a sequence having at least 70% identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity) thereto. In some embodiments, the cell penetrating peptide is selected so that the polypeptide cannot stimulate IL-13-dependent release of mucin without stimulation by ATP or methacholine. IL-13 is the cytokine interleukin 13.
Table 1 shows stapled peptide sequences according to various embodiments of this disclosure. Shown are the staple types and location of linkages involved in macrocyclic crosslinks within each peptide. A single macrocyclic crosslink is typically formed by substituting the non-natural amino acid S-pentylalanine at i, i+4 positions, resulting in a three amino acid gap flanking the macrocyclic crosslink. Two macrocyclic crosslinks within a single polypeptide are typically formed by substituting R-octenylalanine (R8)/S-pentenylalanine (S5) or S-octenylalanine (S8)/R-pentenylalanine (R5) at i, i+7 positions. Also indicated are any N- or C-terminal modifications such as acetyl (Ac) or amide groups.
In some embodiments, the polypeptide constructs comprise the first peptide linked to a non-biological material. In some embodiments, the non-biological material is a small molecular compound, a metal chelate, a polymer, a hydrogel, or a nanoparticle. See, e.g., Shu, J. Y. et al., Peptide-polymer conjugates: from fundamental science to application. Annu. Rev. Phys. Chem. 64, 631-657 (2013) and Xiao, Y. et al., Diabetic wound regeneration using peptide-modified hydrogels to target re-epithelialization. Proc. Natl. Acad. Sci. USA. 113(40), E5792-E5801 (2016). Nanoparricles (NPs) have shown their potential to serve as conjugate scaffolds that not only improve the functionality of peptides but also implement abiotic characteristics, often resulting in synergistic effects. See, e.g., Jeong, Wj., et al. Peptide-nanoparticle conjugates: a next generation of diagnostic and therapeutic platforms?Nano Convergence 5, 38 (2018).
The polypeptide constructs as described herein may be synthesized by solid-phase peptide synthesis methods, during which N-α-protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminus to a solid support, e.g., polystyrene beads. Various chemistries, resins, protecting groups, protected amino acids and reagents can be employed as described, for example, by Barany and Merrifield, “Solid-Phase Peptide Synthesis,” in The Peptides: Analysis, Synthesis, Biology Gross and Meienhofer (eds.), Academic Press, N.Y., vol. 2, pp. 3-284 (1980); Atherton et al., SolidPhase Peptide Synthesis: A Practical Approach, IRL Press (1989); Bodanszky, Peptide Chemistry, A Practical Textbook, 2nd Ed., Springer-Verlag (1993)); and Chan et al. Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press (2000).
Non-limiting examples of support materials for solid-phase peptide synthesis include polystyrene (e.g., microporous polystyrene resin, mesoporous polystyrene resin, macroporous polystyrene resin; including commercially-available Wang resins, Rink amide resins, and trityl resins), glass, polysaccharides (e.g., cellulose, agarose), polyacrylamide resins, polyethylene glycol, or copolymer resins (e.g., comprising polyethylene glycol, polystyrene, etc.). The solid support may have any suitable form factor. For example, the solid support can be in the form of beads, particles, fibers, or in any other suitable form factor. Non-limiting examples of protecting groups (e.g., N-terminal protecting groups) include Fmoc, Boc, allyloxycarbonyl (Alloc), benzyloxycarbonyl (Z), and photolabile protecting groups. Sidechain protecting groups include, but are not limited to, Fmoc; Boc; cyclohexyloxycarbonyl (Hoc); allyloxycarbonyl (Alloc); mesityl-2-sulfonyl (Mts); 4-(N-methylamino)butanoyl (Nmbu); 2,4-dimethylpent-3-yloxycarbonyl (Doc); 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl (Dde); 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde); 4-methyltrityl (Mtt). Additional protecting groups and methods for their addition and removal from supported peptides are described, for example, by Isidro-Llobet et al. Chem. Rev. 2009, 19: 2455-2504.
A base may be used to activate or complete the activation of amino acids prior to exposing the amino acids to immobilized peptides. In some embodiments, the base is a non-nucleophilic bases, such as triisopropylethylamine, N,N-diisopropylethylamine, certain tertiary amines, or collidine, that are non-reactive to or react slowly with protected peptides to remove protecting groups. A coupling agent may be used to form a bond with the C-terminus of an amino acid to facilitate the coupling reaction and the formation of an amide bond. The coupling agent may be used to form activated amino acids prior to exposing the amino acids to immobilized peptides. Any suitable coupling agent may be used. In some embodiments, the coupling agent is a carbodiimide, a guanidinium salt, a phosphonium salt, or a uronium salt. Examples of carbodiimides include, but are not limited to, N,N′-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and the like. Examples of phosphonium salts include, but are not limited to, such as (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP); bromotris (dimethylamino) phosphonium hexafluorophosphate (BroP); and the like. Examples of guanidinium/uronium salts include, but are not limited to, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU); 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU); 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy) dimethylaminomorpholino)]uronium hexafluorophosphate (COMU); and the like.
A single macrocyclic crosslink is typically formed by substituting the non-natural amino acid S-pentylalanine at i, i+4 positions, resulting in a three amino acid gap flanking the macrocyclic crosslink. Two macrocyclic crosslinks within a single polypeptide are typically formed by substituting R-octenylalanine/S-pentenylalanine or S-octenylalanine/R-pentenylalanine at i, i−7 positions.
Ring closing metathesis is typically catalyzed by a ruthenium and/or molybdenum catalyst. Such a catalyst could be a first generation Grubb's catalyst (which consists of a ruthenium molecule substituted with two phosphine groups, two chlorine atoms, and a carbene compound), a second generation Grubb's catalyst (which is like the first generation Grubb's catalyst but consists of an N-Heterocyclic carbene in place of the phosphine groups), or a Schrock catalyst (which is a molybdenum-based catalyst).
Such polypeptides may exhibit increased stabilization of an α-helical conformation and protease resistance. Other means for stabilizing an α-helix include other ring-forming reactions such as: copper catalyzed azide alkyne cycloaddition (CuAAC), lactamization, cysteine-xylene stapling, cysteine-perfluorobenzene stapling, thiol-yne/-ene Click chemistry, selenocysteine stapling, tryptophan condensation, and C—H activation. Additionally, an α-helix might be stabilized by adding helical caps between terminal side-chains and the peptide backbone or via electrostatic or hydrogen bonding interactions at select positions.
In some instances, the polypeptide constructs described herein do not substantially aggregate, as determined by size exclusion chromatography as shown, for example, in
As described in
In some embodiments, the mRNA transcript of the SYT2 gene may be targeted for cleavage and degradation. Different portions of the mRNA transcript may be targeted to decrease or inhibit the expression of the SYT2 gene. In some embodiments, a DNA oligonucleotide may be used to target the mRNA transcript and form a DNA:RNA duplex with the mRNA transcript. The duplex may then be recognized and the mRNA cleaved by specific proteins in the cell. In other embodiments, an RNA oligonucleotide may be used to target the mRNA transcript of the SYT2 gene. In some embodiments, the oligonucleotide may be a shRNA, an ASOs, or an miRNA. In some embodiments, the oligonucleotide may mediate an RNase H-dependent cleavage of the mRNA transcript of the SYT2 gene.
A short hairpin RNA or small hairpin RNA (shRNA) is an artificial RNA molecule with a hairpin turn that can be used to silence target gene expression via the small interfering RNA (siRNA) it produced in cells. See, e.g., Fire et. al., Nature 391:806-811, 1998; Elbashir et. Al., Nature 411:494-498, 2001; Chakraborty et al., Mol Ther Nucleic Acids 8:132-143, 2017; Bouard et al., Br. J. Pharmacol. 157:153-165, 2009. Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. Suitable bacterial vectors include but not limited to adeno-associated viruses (AAVs), adenoviruses, and lentiviruses (as discussed further in Section IV). Once the vector has integrated into the host genome, the shRNA is then transcribed in the nucleus by polymerase II or polymerase III depending on the promoter choice. The resulting pre-shRNA is exported from the nucleus and then processed by Dicer and loaded into the RNA-induced silencing complex (RISC). The sense strand is degraded by RISC and the antisense strand directs RISC to an mRNA that has a complementary sequence. A protein called Ago2 in the RISC then cleaves the mRNA, or in some cases, represses translation of the mRNA, thus, leading to its destruction and an eventual reduction in the protein encoded by the mRNA. Thus, the shRNA leads to targeted gene silencing. shRNA is an advantageous mediator of siRNA in that it has relatively low rate of degradation and turnover.
In some embodiments, the methods described herein for treating airway obstruction in a subject comprise administering an shRNA to the subject. The methods may comprise administering to the subject a therapeutically effective amount of a vector, wherein the vector comprises a polynucleotide encoding an shRNA capable of hybridizing to a portion of an mRNA transcript of the SYT2 gene. In some embodiments, the vector may also include appropriate expression control elements known in the art, including, e.g., promoters (e.g., tissue specific promoters), enhancers, and transcription terminators. In particular, expression of the shRNA can be driven by a promoter solely or particularly active in airway epithelial cells such as, for example airway secretory cells. Such promoters include, for example, ScblA1 (also known as CCSP), cytokeratin 5 (CK5), Sox2, and Sonic Hedgehog (Shh). See, e.g., Li, H. et al. Cre-mediated recombination in mouse Clara cells. Genesis 46, 300-307, (2008).
Once the vector is delivered to the airway epithelial cell, the shRNA may be integrated into the cell's genome and undergo downstream processing by Dicer and RISC (described in detail further herein) to eventually hybridize to the mRNA transcript of the SYT2 gene, leading to mRNA cleavage and degradation.
The present disclosure also provides siRNA-based therapeutics for inhibiting expression of SYT2 in a subject. The double stranded RNAi therapeutic includes a sense strand complementary to an antisense strand. The sense or antisense strands of the siRNA may be about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The antisense strand of the siRNA-based therapeutic includes a region complementary to a part of an mRNA encoding SYT2. Additional methods to make therapeutic siRNA can be found in U.S. Pat. No. 9,399,775, which is incorporated by reference in its entirety for all purposes.
RNase H-dependent antisense oligonucleotides (ASOs) are single-stranded, chemically modified oligonucleotides that bind to complementary sequences in target mRNAs and reduce gene expression both by RNase H-mediated cleavage of the target RNA and by inhibition of translation by steric blockade of ribosomes. In some embodiments, to target the mRNA transcript of the SYT2 gene for degradation, a nucleic acid (e.g., DNA oligonucleotide) capable of hybridizing to a portion of the mRNA may be administered to the subject. Once inside the cell (e.g., an airway epithelial cell), the DNA oligonucleotide base pairs with its targeted mRNA transcript. RNase H may bind to the resulting duplex and cleave the mRNA transcript at one or more places. The DNA oligonucleotide may further bind to other mRNA transcripts to target them for RNase H degradation reducing the expression of the SYT2 gene.
A microRNA (miRNA) is a small non-coding RNA molecule that functions in RNA silencing and post-transcriptional regulation of gene expression. miRNAs base pair with complementary sequences within the mRNA transcript. As a result, the mRNA transcript may be silenced by one or more of the mechanisms such as cleavage of the mRNA strand, destabilization of the mRNA through shortening of its poly(A) tail, and decrease translation efficiency of the mRNA transcript into proteins by ribosomes. In some embodiments, miRNAs derive from regions of RNA transcripts that fold back on themselves to form short hairpins, which are also called pri-miRNA. Once transcribed as pri-miRNA, the hairpins are cleaved out of the primary transcript in the nucleus by an enzyme called Drosha. The hairpins, or pre-miRNA, are then exported from the nucleus into the cytosol. In the cytosol, the loop of the hairpin is cleaved off by an enzyme called Dicer. The resulting product is now a double strand RNA with overhangs at the 3′ end, which is then incorporated into RISC. Once in the RISC, the second strand is discarded and the miRNA that is now in the RISC is a mature miRNA, which binds to mRNAs that have complementary sequences. In some embodiments, an miRNA targeting the SYT2 gene may be used in methods described herein.
In some embodiments, the knocking out or knocking down of SYT2 gene expression is performed using a gene editing system such as, but not limited to, meganucleases designed against the Syt2 genomic sequence, the CRISPR/Cas system, TALENs, and other technologies for precise editing of genomes; Cre-lox site-specific recombination; FLP-FRT recombination; Bxbl-mediated integration; zinc-finger mediated integration; homologous recombination; prime editing and transposases; translocation; and inversion.
In some embodiments, the Syt2 expression inhibitor comprises one or more gene editing components such as a targeted nuclease and a guide RNA (gRNA). Four classes of CRISPR-based genome editing agents are nucleases, base editors, transposases/recombinases and prime editors. Components of any of these gene editing systems can be used to reduce or eliminate Syt2 expression. As used throughout, the term “targeted nuclease” refers to nuclease that is targeted to a specific DNA sequence in the genome of a cell to produce a strand break at that specific DNA sequence. The strand break can be single-stranded or double-stranded. Targeted nucleases include, but are not limited to, a Cas nuclease, a TAL-effector nuclease and a zinc finger nuclease. As used throughout, a guide RNA (gRNA) sequence is a sequence that interacts with a site-specific or targeted nuclease and specifically binds to or hybridizes to a target nucleic acid within the genome of a cell, such that the gRNA and the targeted nuclease co-localize to the target nucleic acid in the genome of the cell. Each gRNA includes a DNA targeting sequence or protospacer sequence of about 10 to 50 nucleotides in length that specifically binds to or hybridizes to a target DNA sequence in the genome. Any CRISPR/Cas system that is capable of altering a target polynucleotide sequence in a cell can be used in methods described here. See, for example, Sanders and Joung, Nature Biotechnol 32:347-355, 2014, Huang et al., J Cell Physiol 10:1-17, 2017 and Mitsunobu et al., Trends Biotechnol 17:30132-30134, 2017.
In some embodiments, inhibiting comprises contacting the polynucleotide encoding Syt2 with at least one gRNA and optionally a targeted nuclease, wherein the at least one gRNA comprises a sequence that specifically hybridizes to a genomic sequence of the SYT2 gene In some embodiments, inhibiting comprises mutating the polynucleotide encoding Syt2. In some embodiments, inhibiting comprises contacting the polynucleotide encoding Syt2 with a targeted nuclease. In some embodiments, inhibiting comprises performing clustered regularly interspaced short palindromic repeats (CRISPR)/Cas genome editing.
Also provided in the present disclosure are pharmaceutical compositions containing one or more of the polypeptide constructs as described herein and one or more pharmaceutically acceptable excipients. Also provided are pharmaceutical compositions comprising an inhibitor of Syt2 expression as described above in Section III, optionally in combination with one or more pharmaceutically acceptable excipients. The term “active ingredient” is used herein to refer individually and collectively to any of the polypeptide constructs or SYT2 expression inhibitors provided herein.
The pharmaceutical compositions can be prepared by any of the methods well known in the art of pharmacy and drug delivery. In general, methods of preparing the compositions include the step of bringing an active ingredient as described herein and any other additional active ingredients into association with a carrier containing one or more accessory ingredients. The pharmaceutical compositions are typically prepared by uniformly and intimately bringing the active ingredient(s) into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In addition to an active ingredients described herein in Sections II and III, pharmaceutical compositions provided herein may also contain additional active ingredients such as corticosteroids, anti-inflammatory drugs, agents to improve mucus clearance, and inhibitors of other SNARE complex proteins (e.g., Munc18, VAMP8, Munc13, or Stx3) as described below.
Pharmaceutical compositions containing an active ingredient as described herein can be in the form of aqueous or oleaginous solutions and suspensions (e.g., sterile solutions or suspensions for administration by oral inhalation or as a nasal spray or nasal drops). Such preparations can be formulated using non-toxic parenterally-acceptable vehicles including water, Ringer's solution, and isotonic sodium chloride solution, and acceptable solvents such as 1,3-butane diol. In addition, sterile, fixed oils can be used as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic monoglycerides, diglycerides, or triglycerides.
Aqueous suspensions can contain one or more of active ingredient as described herein in admixture with excipients including, but not limited to: suspending agents such as sodium carboxymethylcellulose, methylcellulose, oleagino-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin, polyoxyethylene stearate, and polyethylene sorbitan monooleate; and preservatives such as ethyl, n-propyl, and p-hydroxybenzoate. Dispersible powders and granules (suitable for preparation of an aqueous suspension by the addition of water) can contain an active ingredient as described herein in admixture with a dispersing agent, wetting agent, suspending agent, or combinations thereof. Oily suspensions can be formulated by suspending an an active ingredient as described herein in a vegetable oil (e.g., arachis oil, olive oil, sesame oil or coconut oil), or in a mineral oil (e.g., liquid paraffin). Oily suspensions can contain one or more thickening agents, for example beeswax, hard paraffin, or cetyl alcohol. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.
In certain embodiments, a pharmaceutical composition can be formulated for inhalation. The compositions described herein, either alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation (e.g., intranasally or intratracheally). Aerosol formulations can be placed into pressurized acceptable propellants, such as dichiorodifluoromethane, propane, nitrogen, and the like. In certain embodiments, the pharmaceutical composition can be nebulized. Methods for delivering compositions directly to the lungs via nasal aerosol sprays have been described, e.g., in U.S. Pat. No. 6,565,841. Likewise, the delivery of drugs using intranasal microparticle resins and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are also well-known in the pharmaceutical arts. Similarly, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045.
In some instances, the pharmaceutical composition can be formulated as a solid particulate. In some instances, the pharmaceutical composition can be can be formulated as a liquid. In some instances, the pharmaceutical composition can be formulated as a polymeric nanopartical or a lipid nanoparticle. A polymeric nanoparticle formed from natural or synthetic materials can comprise, for example, a polymersome, a dendrimer, a polymer micelle, or a nanosphere. A lipid-based nanoparticle can comprise, for example, a liposome or a lipid nanoparticle. Liposomes comprise phospholipids that form vesicular structures, whereas lipid nanoparticles comprise miceller structures within the particle core.
The active ingredients, and pharmaceutical compositions thereof, may be administered to a subject by any technique known in the art, including local or systemic delivery. Routes of administration include, but are not limited to, intrapulmonary inhalation, oral inhalation administration, and intranasal administration. Inhalation delivery can enhance targeting a pharmaceutical composition to airway epithelial cells. See, e.g., Manunta et al., Airway Deposition of Nebulized Gene Delivery Nanocomplexes Monitored by Radioimaging Agents, Am J Respir Cell Mol Biol 49(3): 471-480 (2013).
Depending on whether transient or stable expression is desired one can select an appropriate delivery vector for delivery of the polynucleotides provided herein for inhibition of Syt2 expression. Examples of delivery vectors that may be used with the present disclosure are viral vectors, plasmids, exosomes, liposomes, bacterial vectors, or nanoparticles. The present disclosure also provides for delivery by any means known in the art, some of which are described in Fumoto et al. (2021), which is hereby incorporated by reference in its entirety for all purposes.
For targeted delivery to epithelial cells, one skilled in the art would appreciate that some delivery vectors may be genetically modified to target a specific cell type or to tissue type. In addition, depending on the required therapeutic duration a viral delivery vector can be genetically modified to be continuously replicating, replication-defective, or conditionally replicating as described in, Sliva, K. and Schnierle, B. S., Selective gene silencing by viral delivery of short hairpin RNA Virology Journal (2010).
In some embodiments, the SYT2 shRNA or siRNA can be delivered by vector comprising an adenovirus vector, an adeno-associated viral vector, a retrovirus vector, a lentivirus vector, or a nanoparticle. Examples of nanoparticles that can be use with the present disclosure, include but are not limited to, exosomes, liposomes, organic nanoparticles, or inorganic nanoparticles. Other non-limiting examples of nanoparticles include, but are not limited to, e.g., those provided in Hong and Nam, Functional Nanostructures for Effective Delivery of Small Interfering RNA Therapeutics, Theranostics 4.12: 1211-1232 (2014), which is hereby incorporated by reference in its entirety for all purposes. In some embodiments, the siRNA active ingredient is administered in a solution. The siRNA may be administered in an unbuffered solution. In one embodiment, the siRNA is administered in water. In other embodiments, the siRNA is administered with a buffer solution, such as an acetate buffer, a citrate buffer, a prolamine buffer, a carbonate buffer, or a phosphate buffer or any combination thereof. In some embodiments, the buffer solution is phosphate buffered saline.
In some embodiments, where the Syt2 expression inhibitor comprises one or more gene editing components including a targeted nuclease, the targeted nuclease can be introduced into a cell in polypeptide form. In certain embodiments, the targeted nuclease may be conjugated to a cell-penetrating polypeptide. Non-limiting examples of cell-penetrating peptides include, but are not limited to, e.g., those provided in Milletti et al., Drug Discov. Today 17: 850-860, 2012, the relevant disclosure of which is hereby incorporated by reference in its entirety. In some embodiments, the target motif in the SYT2 gene, to which the targeted nuclease is directed by a polynucleotide (e.g., a guide RNAs). The sgRNAs can be selected depending on the particular CRISPR/Cas system employed, and the sequence of the target polynucleotide, as will be appreciated by those skilled in the art. In some instances, the targeted nuclease and the sgRNA can be provide as separate the pharmaceutical compositions or can be provided together in a single pharmaceutical composition (e.g., a polynucleotide complex or ribonucleoprotein (RNP)). As used herein, the phrase “introducing” in the context of introducing a polynucleotide or a polynucleotide complex comprising a nucleic acid and a polypeptide refers to the translocation of the nucleic acid sequence or the polynucleotide complex from outside a cell to inside the cell. In some cases, introducing refers to translocation of the nucleic acid or the complex from outside the cell to inside the nucleus of the cell.
Pharmaceutical compositions of the present disclosure may be packaged as a single use “unit dose” container or as a multi-dose container. In some instances, a unit dose of the compositions described in this disclosure is provided. Examples of single use containers are blister packs or capsules. Examples of multi-dose containers are drop dispensers, or vials. Kits according to the present disclosure may include one or more unit doses of a composition and a device for administering the composition. Kits may include a single use “unit dose” container or a multi-dose container. Examples of single use containers are blister packs or capsules. Examples of multi-dose containers are drop dispensers, or vials. In some instances, the device for administering the composition may be an aerosolization device. For example, in some instances, the device may be an aerosolizer, an inhaler, or a nebulizer. Inhalers include metered dose inhalers (MDIs), dry powder inhalers (DPIs), and soft mist inhalers (SMIs). Other devices for aerosolization of liquid compositions are well-known in the art. In some instances, the kits may include a device for administrating the composition via injection. For example, the kits may include one or more syringes. In another example, the kits may include one or more needles. In another example, the kits may include one or more syringes and one or more needles. The kits may also include a pump or a pen device for administering the composition via injection. In some instances, the kit may include instructions describing use of the device to administer the composition.
Also provided herein are methods of inhibiting mucin secretion in an airway epithelial cell. In some embodiments, the airway epithelial cell is a airway secretory cell. Further provided herein are methods of inhibiting Syt2-mediated stimulated mucin secretion, triggered by Ca2+ release after ATP or methacholine bind to hepta-helical PM receptors coupled to Gq. In some instances, the methods include contacting the cell with an effective amount of at least one polypeptide construct as described above in Section II. In some instances, the methods include contacting the cell with an effective amount of an inhibitor of SYT2 expression as described above in Section III thereby reducing Syt2 expression in the cell. In some instances, the methods comprise contacting the cell with both at least one polypeptide construct and an inhibitor of SYT2 expression. “Contacting” cells may include addition of a polypeptide construct to a cell culture in vitro, or administering a polypeptide construct to a subject (e.g., in conjunction with a pharmaceutical composition as described above).
Also provided herein is a method of treating a subject having mucus hypersecretion-based airway obstruction and/or mucus occlusions by administering an effective amount of at least one polypeptide construct or pharmaceutical composition thereof as described above. Also provided herein are methods of treating a subject having mucus hypersecretion-based airway obstruction and/or mucus occlusions by administering an effective amount of an inhibitor of Syt2 expression or pharmaceutical composition thereof as described above. In some instances, the methods comprising administereing an effective amount of at least one polypeptide construct or pharmaceutical composition thereof and an effective amount of an inhibitor of Syt2 expression.
In some embodiments, the subject has one or more of a respiratory viral infection, asthma, chronic obstructive pulmonary disease (COPD), or cystic fibrosis. In some instances, treatment may further comprise administering a therapeutically effective amount of an inhibitor of at least one of Munc18, VAMP8, Munc13, or Stx3.
Mucus dysfunction is a very common cause of symptoms and disease progression in common diseases of the airways. Rapid mucin secretion is a major contributor to airway mucus dysfunction, yet there are no currently available therapies directed at this root cause. Patients with asthma (8% of Americans), COPD (6% of American adults), cystic fibrosis (1/3,000 white newborns), and bronchiectasis (139/100,000 adult Americans) could potentially benefit from this invention. In general, it could be anticipated that the greatest need would be among patients with persistent mucus plaques and plugs, and those with acute airflow obstruction due to mucus obstruction of the airways.
In some embodiments, the polypeptide constructs described herein reduce fractional secretion of intracellular mucin stimulated by methacholine by up to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% when administered to an airway epithelial cell.
In some embodiments, administration of the polypeptide constructs described herein reduces airway luminal mucus accumulation in the lung by at least about 10% (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, or 40%) when administered to an airway epithelial cell as described, for example, in Example 8.
Polypeptide constructs provided herein can be used acutely and chronically. In the acute setting, the rapid release of hyperproduced mucins is well-known to be the major cause of airflow obstruction and death in asthma, and plays a similar role in other airway diseases such as acute exacerbations of COPD, cystic fibrosis, and bronchiectasis. Administration of a polypeptide construct as provided herein could prevent further acute mucus occlusion, while other interventions such as corticosteroids can be administered to prevent further mucin production. Thus, in some embodiments, the polypeptide constructs provided herein are administered to the subject pro re nata (i.e., on an as needed basis). In the chronic setting, where persistent mucus plaques cause chronic airway obstruction and inflammation in asthma, COPD, cystic fibrosis, and bronchiectasis, the polypeptide construct as provided herein could be administered alongside anti-inflammatory drugs (to reduce mucin production) and agents such as hypertonic saline solution (to improve mucus clearance) to prevent further plaque formation. Thus, in some embodiments, the polypeptide constructs provided herein are administered to the subject on a regular dosing regimen (e.g., each at least once per day). Together, such a strategy might improve the therapy of these undertreated airway diseases. Examples of methods of administration of the formulation are generally described under “Pharmaceutical Compositions” above.
In some embodiments, the subject may also be administered a therapeutically effective amount of an inhibitor of at least one of Munc18, VAMP8, Munc13, or Stx3. Each of these proteins are implicated in basal and/or stimulated mucin secretion and are either a component of the SNARE complex that includes SNAP-23 or affects its function. For example, Stx3 and VAMP8 are components of the SNARE complex containing SNAP-23. Furthermore, Munc18-2 mutant mice are selectively impaired in stimulated mucin secretion. Stx2 binds and colocalizes with Munc18-2, and Munc18-2 overexpression reduces the amount of Stx3 associated with SNAP-23. Also, the importance of these proteins in mucin secretion can be inferred by the role of homologous SNARE protein complex components, synaptotagmins, and Munc proteins involved in neurotransmitter secretion. Example of such inhibitors are 2-amiinobenzothiazoles tht inhibit Munc13-4 (Bruinsma et al., 2018).
In certain embodiments, an active ingredient or a pharmaceutical composition as provided herein are administered to the lungs of the subject (i.e. pulmonary administration). The term “pulmonary administration” represents any method of administration in which an active agent can be administered through the pulmonary route by inhaling or otherwise administering into the lungs an aerosolized liquid or powder form (nasally or orally). Such aerosolized liquid or powder forms are traditionally intended to substantially release and or deliver the active agent to the epithelium of the lungs. In certain embodiments, the active agent is in liquid form. Pulmonary administration is further described in International Application Publication No. WO/1994/020069, which describes pulmonary delivery of chemically modified proteins.
The active ingredients, and pharmaceutical compositions thereof, may be administered in a single dose or in multiple doses (e.g., two, three, or more single doses per treatment) over a time period (e.g., hours or days). The active ingredients described herein, and pharmaceutical compositions thereof, will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat or prevent the particular disease being treated. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated and/or eradication or amelioration of one or more of the symptoms associated with the underlying disorder such that the patient reports an improvement in feeling or condition, notwithstanding that the patient may still be afflicted with the underlying disorder. Therapeutic benefit also generally includes halting or slowing the progression of the disease, regardless of whether improvement is realized.
The amount of active ingredient administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular active ingredient, the conversation rate and efficiency into active drug compound under the selected route of administration, etc.
Determination of an effective dosage of an active ingredient for a particular use and mode of administration is well within the capabilities of those skilled in the art. Effective dosages may be estimated initially from in vitro activity and metabolism assays. For example, an initial dosage of a polypeptide construct for use in animals may be formulated to achieve a circulating blood or serum concentration that is at or above an IC50 of the particular polypeptide construct as measured in an in vitro assay. The dosage can be calculated to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular polypeptide construct via the desired route of administration. Initial dosages of compound can also be estimated from in vivo data, such as animal models. For example, an average mouse weighs 0.025 kg. Administering 0.025, 0.05, 0.1 and 0.2 mg of a polypeptide constructs per day may therefore correspond to a dose range of 1, 2, 4, and 8 mg/kg/day. If an average human adult is assumed to have a weight of 70 kg, the corresponding human dosage would be 70, 140, 280, and 560 mg of the polypeptide construct per day. Dosages for other active agents may be determined in similar fashion. Animal models useful for testing the efficacy of the active metabolites to treat or prevent the various diseases described above are well-known in the art. Animal models suitable for testing the bioavailability and/or metabolism of compounds into active metabolites are also well-known. Ordinarily skilled artisans can routinely adapt such information to determine dosages suitable for human administration.
Dosage amounts will typically be in the range of from about 0.0001 mg/kg/day, 0.001 mg/kg/day or 0.01 mg/kg/day to about 100 mg/kg/day, but may be higher or lower, depending upon, among other factors, the activity of the polypeptide construct or other active compound, the bioavailability of the polypeptide construct or other active compound, its metabolism kinetics and other pharmacokinetic properties, the mode of administration and various other factors, discussed above. The dose of the polypeptide construct can be, for example, about 0.01-750 mg/kg, or about 0.01-500 mg/kg, or about 0.01-250 mg/kg, or about 0.01-100 mg/kg, or about 0.1-50 mg/kg, or about 1-25 mg/kg, or about 1-10 mg/kg, or about 5-10 mg/kg, or about 1-5 mg/kg. The dose of the polypeptide construct can be about 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg/kg.
Dosage amount and interval may be adjusted individually to provide levels of the an active ingredient that is sufficient to maintain therapeutic or prophylactic effect. For example, the compounds may be administered once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of teh an active ingredient may not be related to plasma concentration. The dosage may be adjusted by the individual physician in the event of any complication.
The compounds may be present in a therapeutically effective concentration. In certain embodiments, the concentration of said compound is about 0.1 nmol/L to about 1000 nmol/L at the time of administration; e.g., about 0.1 nmol/L to about 500 nmol/L, or about 0.1 nmol/L to about 250 nmol/L, or about 0.1 nmol/L to about 100 nmol/L, or about 0.1 nmol/L to about 50 nmol/L, or about 0.1 nmol/L to about 10 nmol/L, or about 0.1 nmol/L to about 1 nmol/L, or about 1 nmol/L to about 500 nmol/L, or about 1 nmol/L to about 250 nmol/L, or about 1 nmol/L to about 100 nmol/L, or about 1 nmol/L to about 50 nmol/L, or about 1 nmol/L to about 10 nmol/L, or about 10 nmol/L to about 500 nmol/L, or about 10 nmol/L to about 250 nmol/L, or about 10 nmol/L to about 100 nmol/L, or about 10 nmol/L to about 50 nmol/L, or about 100 nmol/L to about 500 nmol/L, or about 100 nmol/L to about 250 nmol/L. One of skill in the art will recognize that suitable volume of the dose may be selected based on the desired route of administration.
As used below, any reference to a series of embodiments is to be understood as a reference to each of those embodiments disjunctively (e.g., “Embodiments 1-4” is to be understood as “Embodiments 1, 2, 3, or 4”).
Embodiment 1 is a polypeptide comprising a SNAP-25A peptide, or homolog or variant thereof, attached to a cell penetrating peptide, the peptide comprising a first pair of non-natural amino acids comprising a macrocyclic crosslink.
Embodiment 2 is a polypeptide comprising a SNAP-25A peptide, or homolog or variant thereof, attached to a non-biological material, the peptide comprising a first pair of non-natural amino acids comprising a macrocyclic crosslink.
Embodiment 3 is a polypeptide comprising a first peptide having at least 64% identity to SEQ ID NO: 1 or SEQ ID NO: 2 attached to a cell penetrating peptide, the peptide comprising a first pair of non-natural amino acids comprising a macrocyclic crosslink.
Embodiment 4 is a polypeptide comprising a first peptide having at least 64% identity to SEQ ID NO: 1 or SEQ ID NO: 2 attached to a a non-biological material, the peptide comprising a first pair of non-natural amino acids comprising a macrocyclic crosslink.
Embodiment 5 is a polypeptide of any one of embodiments 1-4, wherein the first peptide comprises a second pair of non-natural amino acids comprising a macrocyclic crosslink.
Embodiment 6 is a polypeptide of any one of embodiments 1-4, wherein the first pair of non-natural amino acids flanks six contiguous amino acid residues in the peptide.
Embodiment 7 is a polypeptide of embodiment 5, wherein the first pair of non-natural amino acids and second pair of non-natural amino acids each flank three contiguous amino acid residues in the peptide, and wherein the three contiguous amino acid residues flanked by the first pair of non-natural amino acids are different than the three contiguous amino acid residues flanked by the second pair of non-natural amino acids.
Embodiment 8 is a polypeptide of any one of embodiments 1-4 or 6, wherein the first pair of non-natural amino acids correspond to amino acids 6 and 13, 7 and 14, 10 and 17, or 11 and 18 in SEQ ID NO: 1 or SEQ ID NO: 2.
Embodiment 9 is a polypeptide of any one of embodiments 1-4 or 7, wherein the first pair of non-natural amino acids correspond to amino acids 3 and 7, 6 and 10, or 7 and 11 in SEQ ID NO: 1 or SEQ ID NO: 2.
Embodiment 10 is a polypeptide according to any one of embodiments 1-4, 7, or 9, wherein the second pair of non-natural amino acids correspond to amino acids 10 and 14, 13 and 17, 14 and 18, or 17 and 21 in SEQ ID NO: 1 or SEQ ID NO: 2.
Embodiment 11 is a polypeptide according to any one of embodiments 1 to 10, wherein the macrocyclic crosslink is positioned on a non-binding side of the first peptide, wherein the non-binding side of the first peptide does not contain amino acid residues that interact with amino acid residues in the C2B domain of a synaptotagmin protein.
Embodiment 12 is a polypeptide according to any one of embodiments 1-4, 6, 8, or 11, wherein the first peptide comprises any one of SEQ ID NO: 3 or 4.
Embodiment 13 is a polypeptide according to any one of of embodiments 1-5, 7, 9, 10, or 11, wherein the first peptide comprises any one of SEQ ID NO: 5, 6, 7, 8, or 9.
Embodiment 14 is a polypeptide of embodiment 13, comprising SEQ ID NO: 8.
Embodiment 15 is a polypeptide according to any one of embodiments 1 to 14, wherein the cell penetrating peptide comprises at least one of the cell penetrating peptides listed in Table 1.
Embodiment 16 is a polypeptide according to any one of embodiments 1 to 15, wherein the cell penetrating peptide comprises penetratin.
Embodiment 17 is a polypeptide according to any one of embodiments 1 to 16, wherein the cell penetrating peptide is not an HIV-1 TAT peptide.
Embodiment 18 is a polypeptide according to any one of embodiments 1 to 17, wherein the C-terminus of the cell penetrating peptide is linked to the N-terminus of the peptide.
Embodiment 19 is a polypeptide construct as described herein comprising at least one chemically modified amino acid or at least one conservative amino acid substitution.
Embodiment 20 is a polypeptide construct as described herein that does not substantially aggregate, as determined by size exclusion chromatography.
Embodiment 21 is a polypeptide construct as described herein that binds to the C2B domain of Syt1 or Syt2 with a KD of around 24 μM to around 35 μM.
Embodiment 22 is a polypeptide construct as described herein that binds to the of a wild-type Syt1 or Syt2 C2B domain but not a mutant Syt1 C2B domain comprising mutations R281A, E295A, Y338W, R398A, and R399A.
Embodiment 23 is a polypeptide construct as described herein that blocks Ca2+-dependent binding of a Syt2 homolog to a SNARE peptide.
Embodiment 24 is a pharmaceutical composition comprising a polypeptide according to any one of the preceding embodiments and a pharmaceutically acceptable excipient.
Embodiment 25 is a method of treating a subject having mucus hypersecretion-based airway obstruction, the method comprising administering to the subject a therapeutically effective amount of a polypeptide or pharmaceutical composition according to any one of the preceding embodiments.
Embodiment 26 is a method according to embodiment 25, wherein the subject has a respiratory viral infection, asthma, chronic obstructive pulmonary disease (COPD), or cystic fibrosis.
Embodiment 27 is a method according to embodiments 25 or 26, wherein the subject has developed mucus occlusions.
Embodiment 28 is a method according to any one of embodiments 25 to 27, further comprising administering a therapeutically effective amount of an inhibitor of at least one of Munc18, VAMP8, Munc13, or Stx3.
Embodiment 29 is a method of inhibiting mucin secretion in an airway epithelial cell, the method comprising contacting the airway epithelial cell or a cell derived from an airway epithelial cell with a polypeptide or pharmaceutical composition according to any one of the preceding embodiments.
Embodiment 30 is a method of inhibiting Syt2-mediated stimulated mucin secretion in an airway epithelial cell or a cell derived from an airway epithelial cell, triggered by Ca21 release after ATP or methacholine bind to hepta-helical PM receptors coupled to Gq, the method comprising contacting the cell with a polypeptide or pharmaceutical composition according to any one of the preceding embodiments.
Embodiment 31 is a method according to any one of embodiments 25 to 30 wherein administration of the polypeptide or pharmaceutical composition reduces fractional secretion of intracellular mucin stimulated by methacholine by up to about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% when administered to an airway epithelial cell.
Embodiment 32 is a method according to any one of embodiments 25 to 31 wherein administration of the polypeptide or pharmaceutical composition as described herein reduces airway luminal mucus accumulation in the lung by at least about 10% (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, or 40%) when administered to an airway epithelial cell.
Embodiment 33 is a polynucleotide that inhibits expression of SYT2.
Embodiment 34 is a polynucleotide according to embodiment 30, wherein the polynucleotide is a SYT2 targeting siRNA, shRNA, ASO, miRNA, or CRISPR/Cas system guide RNA.
Embodiment 35 is a pharmaceutical composition comprising a polynucleotide according to embodiment 33 or 34 and a pharmaceutically acceptable excipient.
Embodiment 36 is a method of treating a subject having mucus hypersecretion-based airway obstruction, the method comprising administering to the subject a therapeutically effective amount of a Syt2 expression inhibitor according to embodiment 33 or 34 or a pharmaceutical composition thereof according to embodiment 35.
Embodiment 37 is a method of inhibiting mucin secretion in an airway epithelial cell, the method comprising contacting the airway epithelial cell or a cell derived from an airway epithelial cell with a Syt2 expression inhibitor according to embodiment 33 or 34 or a pharmaceutical composition thereof according to embodiment 35.
Embodiment 38 is a method of inhibiting Syt2-mediated stimulated mucin secretion in an airway epithelial cell or a cell derived from an airway epithelial cell, triggered by Ca2+ release after ATP or methacholine bind to hepta-helical PM receptors coupled to Gq, the method comprising contacting the cell with a Syt2 expression inhibitor according to embodiment 33 or 34 or a pharmaceutical composition thereof according to embodiment 35.
Embodiment 39 is a method according to any one of embodiments 36 to 38, wherein the Syt2 expression inhibitor is a polynucleotide or polynucleotide complex.
Embodiment 40 is a method according to any one of embodiments 36 to 39, wherein the Syt2 expression inhibitor is a shRNA, an siRNA, or an miRNA.
Embodiment 41 is a method according to any one of embodiments 36 to 39, wherein the Syt2 expression inhibitor is one or more gene editing system components.
Embodiment 42 is a method according to embodiment 41, wherein the one or more gene editing system components comprise at least one of a targeted nuclease or a guide RNA.
References referred to in this disclosure include:
The following examples are provided to illustrate, but not to limit, the claimed invention.
Mice. All experiments were approved by the Institutional Animal Care and Use Committee of MD Anderson Cancer Center. Syt2 conditional deletant mice with the second exon flanked by LoxP recombination sites (Syt2F/F) were obtained from Dr. Thomas C. Sudhof (Luo and Sudhof, 2017). These were generated on a mixed 129/Sv:C57BL/6 background and backcrossed by us for ten generations onto a C57BL/6J background. To delete Syt2 in airway epithelial cells, Syt2F/F mice were crossed with mice in which a Cre recombinase optimized for mammalian codon usage was knocked into the secretoglobin 1A1 locus (Scgb1a1Cre) (Liu et al., 2015; Li et al., 2008). Half the progeny from crossing Syt2F/F mice with Syt2F/F mice that were also heterozygous for the Scgb1a1cre allele were Syt2 deletant (Syt2D/D) mice, and the other half were Syt2F/F mice that served as littermate controls for the mucin secretion experiments. Genotyping was performed by PCR using the oligonucleotide primers for Syt2WT and mutant alleles described in Luo and Sudhof (2017). C57BL/6J mice were purchased from the Jackson Laboratory and used as controls to be certain the SytF/F allele was not hypomorphic in airway epithelium. Since Syt2F/F mice did not differ from Syt2WT at baseline (data not shown; see U.S. Provisional Appl. No. 63/311,001 at EXTENDED DATA
Mucin secretion and airway mucus occlusion in mice. The efficiency of stimulated mucin secretion and the extent of mucus accumulation in the airway lumen of Syt2 mutant mice were measured as described previously (Jaramillo et al, 2019). Briefly, to increase intracellular mucin content (i.e., induce mucous metaplasia), 3 μg IL-13 (BioLegend) in 40 μl PBS was instilled every other day×3 into the posterior pharynx of mice under isoflurane anesthesia to be aspirated during inhalation. Three days after the last instillation, mucin secretion was stimulated by exposing mice for 10 min to an aerosol of 100 mM ATP in 0.9% NaCl, then lungs were harvested 20 min later. Fractional mucin secretion was calculated as the percentage reduction in intracellular mucin content of individual mice after sequential treatment with IL-13 and ATP compared to the group mean mucin content of mice of the same genotype treated only with IL-13. To measure intracellular airway epithelial mucin content, lungs were inflated via the trachea with 10% neutral buffered formalin to 20 cm water pressure for 24 h at 4° C., then embedded in paraffin. A single transverse 5 μm section was taken through the axial bronchus of the left lung between lateral branches 1 and 2, deparaffinized, rehydrated and stained with periodic acid fluorescent Schiff (PAFS) reagent. Images were acquired using an upright microscope (Olympus BX 60) with a ×40 NA 0.75 objective lens, and intracellular mucin was measured around the circumferential section of the axial bronchus using ImagePro (Media Cybernetics). Images were analyzed by investigators blinded to mouse genotype and treatment, and data are presented as the epithelial mucin volume density, signifying the measured volume of mucin overlying a unit area of epithelial baseline lamina.
To measure airway lumenal mucus content, mucous metaplasia was induced as above, then mucin secretion and bronchoconstriction were induced by exposure for 10 min to an aerosol of 150 mM methacholine. Lungs were harvested and fixed by immersion for 48 h at 4° C. to avoid displacement of lumenal mucus and using methanol-based Carnoy's solution (methacarn) for fixation to minimize changes in mucus volume. A single transverse 5 μm section was taken through the axial bronchus of the left lung between lateral branches 1 and 2 and stained with PAFS as above to evaluate intracellular mucin to ensure secretion had been stimulated. Then, six 5 μm sections of the paraffin blocks caudal to the initial section were taken at 500 m intervals and stained with PAFS. Mucus in the lumens of airways was identified manually and the area summed for all twelve sections using ImagePro (Media Cybernetics).
To measure the efficiency of stimulated mucin secretion and the extent of mucus accumulation in the airway lumen of WT mice treated with peptides, the same procedures as those used for analysis of Syt2 mutant mice were followed, except that both outcomes were measured in the same mouse by fixation of the left lung by inflation with formalin to measure mucin secretion and fixation of the right lung by immersion in methacarn to measure mucus accumulation. Stimulation of mucin secretion with a single methacholine aerosol was used for both outcomes. In addition, a MicroSprayer Aerosolizer (Penn-Century) was used for peptide delivery to the airways, secretion was measured in the left axial bronchus at a site 3 mm distal to the site used in the Syt2 mutant mice, and mucus accumulation was measured in the right caudal lobe.
Protein expression and purification. The same constructs and protocols were used to purify cysteine-free Stx1A, SNAP-25A, VAMP2, and Syt1 as described in Lai et al., 2017. The same constructs and protocols were used to purify NSF, and α-SNAP as described in Choi et al., 2018. The protein sample concentrations were measured by UV absorption at 280 nm, aliquots were flash frozen in liquid nitrogen and stored at −80° C.
Stx3. Full-length human Stx3 was expressed in E. coli strain BL-21 (DE3) with an N-terminal, TEV protease-cleavable, hexa-histidine tag. The expression and purification protocols were mostly identical to that of Stx1A. The protein was expressed overnight at 30° C. in 8 l of autoinducing media. Cell pellets from 8 l of culture were suspended in 1× phosphate-buffered saline, 1 mM EDTA, 1 mM PMSF, and 8 EDTA free protease inhibitor tablets (Roche) supplemented with lysozyme and DNAse I (Sigma). The cells were lysed using a sonicator (Fisher Scientific) and an M-110EH microfluidizer (Microfluidics). Inclusion bodies were removed by a 30 min spin at 13,000 RPM in a JA-14 rotor (Beckman Coulter), and the supernatant was centrifuged at 43,000 RPM for 1.5 h in a Ti-45 rotor (Beckman Coulter) to pellet the membrane. Membranes were resuspended in 20 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, and centrifuged at 43,000 RPM for 1 h. The pellet was resuspended once more in the same buffer, dodecylmaltoside (Anatrace) was added to 2%, and stirred for 1.5 h at 4° C. The solubilized membrane was centrifuged at 40,000 RPM for 35 min, and the supernatant was loaded onto a 5 ml column of Nickel-NTA agarose (Qiagen). The column was washed with 20 mM HEPES pH 7.5, 20 mM imidazole, 300 mM NaCl, 110 mM octyl glucoside (Anatrace), 10% glycerol, and the protein was eluted with wash buffer supplemented with 450 mM imidazole and 1 M NaCl. The protein fractions were pooled, digested with 110 ug TEV protease, and dialyzed overnight against 20 mM HEPES pH 7.5, 50 mM NaCl, 110 mM OG, 10% glycerol. The fractions were loaded onto a MonoQ 4.6/100 PE column (GE Healthcare) previously equilibrated with dialysis buffer. The protein was eluted with a gradient of 50 mM to 1 M NaCl over 30 column volumes. Protein concentration was measured by absorption at 280 nm and aliquots were flash frozen in liquid nitrogen and stored at −80° C.
SNAP-23. The expression and purification protocols were mostly identical to those of SNAP-25A. Cysteine free SNAP-23, in which all cysteine residues were changed to serine, was expressed in BL21(DE3) cells using auto inducing media from a pGEX vector as an N-terminal GST tag with a thrombin protease cleavage site to remove the tag. Cells from 4.0 l of induced culture were resuspended in 250 ml of buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 4 mM DTT, 10% glycerol) containing 1 mM PMSF and 5 EDTA free protease inhibitor tablets. Cells were lysed by sonication. The lysate was clarified by centrifugation in a Ti45 rotor for 35 min at 40,000 RPM. The supernatant was bound to 10 ml of Glutathione Sepharose beads (GE Healthcare) for 1 h with stirring at 4° C. The beads were harvested by centrifugation, poured into a column, and washed with 100 ml of buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 4 mM DTT, 10% glycerol). 10 μl of 5 mg/ml thrombin was added to the washed beads along with 5 ml of buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 4 mM DTT, 10% glycerol) and the mixture was rocked overnight at 4° C. to remove the GST tag. The cleaved SNAP-23 sample was washed out of the column using buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 4 mM DTT, 10% glycerol) and concentrated to 5 ml. The sample was injected on to a Superdex 200 (16/60) column (GE Healthcare) equilibrated in 20 mM HEPES pH 7.5, 100 mM NaCl, 10% glycerol. Protein containing fractions were combined and concentrated to ˜100 μM SNAP23. The protein concentration was measured by absorption at 280 nm and aliquots were flash frozen in liquid nitrogen and stored at −80° C.
VAMP8. VAMP8 was expressed in E. coli strain BL-21 (DE3) with an N-terminal, TEV protease-cleavable, GST tag. The protein was expressed overnight at 25° C. in 8.0 l of autoinducing media. Cell pellets were suspended in 20 mM HEPES pH 7.5, 300 mM NaCl, 2 mM DTT, 1 mM EDTA, and 8 EDTA free protease inhibitor tablets supplemented with lysozyme and DNAse I. The cells were lysed using a sonicator (Fisher Scientific) and an M-110EH microfluidizer. Cell debris was removed by a 30 min spin at 13,000 RPM in a JA-14 rotor, and the supernatant was centrifuged at 43,000 RPM for 1 h in a Ti-45 rotor to pellet the membrane. The pellet was resuspended in 20 mM HEPES pH 7.5, 300 mM NaCl, 2 mM DTT, 1 mM EDTA and 1.5% DDM and solubilized at 4° C. with stirring for 2.5 h. The solubilized membrane was centrifuged at 43,000 RPM for 35 min, the supernatant was mixed with 5 ml of Glutathione Sepharose 4B and incubated overnight at 4° C. with end-over-end mixing. The beads were washed with 20 CV of 20 mM HEPES pH 7.5, 300 mM NaCl, 2 mM DTT, 1 mM EDTA, 110 mM OG. The protein was cleaved off the column by resuspending the beads in 2 ml of wash buffer supplemented with 2 mM DTT and 110 μg of TEV protease, and incubating at 4° C. for 1 hr. After digest the column was drained and the flow through (containing cleaved VAMP8) was injected on a Superdex 200 10/300 Increase column (GE Healthcare) equilibrated with 20 mM HEPES pH 7.5, 300 mM NaCl, 2 mM DTT, 110 mM OG. Fractions containing protein were pooled, and protein concentration was measured by absorption at 280 nm. Aliquots were flash frozen in liquid nitrogen and stored at −80° C.
Syt2. Syt2 was expressed in BL21(DE3) cells using auto inducing medium from a pGEX vector as an N-terminal GST tag with a thrombin protease cleavage site to remove the tag. Cells from 4 l of induced culture were re-suspended in 200 ml of buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 1 mM EDTA, 2 mM DTT), containing 4 EDTA free protease inhibitor tablets. Cells were lysed by three passes through the Emulsiflex C5 homogenizer (Avestin) at 15000 psi. Unlysed cells and debris were removed by centrifugation in a JA-14 rotor for 10 min at 8000 RPM, the supernatant was centrifuged again in the same rotor for 10 min at 8000 RPM to remove any final debris. The supernatant from the second spin was then centrifuged in a Ti45 rotor for one h at 40,000 RPM to collect the membranes. Membranes were resuspended using a Dounce homogenizer in 100 ml of buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 1 mM EDTA, 2 mM DTT) and n-dodecylmaltoside was added to a final concentration of 2% (w/v) to solubilize the membranes overnight at 4° C. with stirring. The extract was clarified by centrifugation using the Ti45 rotor at 40000 RPM for 35 min. The extract was applied to a 5 ml bed of glutathione Sepharose by stirring at 4° C. for 2 h. The column was washed with buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 1 mM EDTA, 2 mM DTT) containing 110 mM 3-octyl-glucoside and eluted with buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 1 mM EDTA, 2 mM DTT) containing 110 mM 3-octyl-glucoside and 20 mM reduced glutathione. The GST tag was removed by cleavage with 10 μl of 5 mg/ml thrombin for 2 hours and the Syt2 sample was purified on a monoS column equilibrated in 20 mM HEPES pH 7.5, 100 mM NaCl, 110 mM 3-octyl-glucoside, 2 mM DTT (monoS buffer). After washing the column with monoS buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 110 mM 3-octyl-glucoside, 2 mM DTT), the protein was eluted using a linear gradient from 100 mM to 1 M NaCl. Protein containing fractions were combined, the protein concentration was measured by absorption at 280 nm and aliquots were flash frozen in liquid nitrogen and stored at −80° C.
Syt1 C2B and Syt1_QM. The Syt1 C2B domain and the Syt1_QM mutant (R281A, E295A, Y338W, R398A, R399A) were expressed as GST-tagged fusion proteins in E. coli BL21 (DE3) cells at 30° C. overnight. After harvesting the cells by centrifugation, the sample was resuspended in lysis buffer containing 50 mM HEPES-Na, pH 7.5, 300 mM NaCl, 2 mM DTT and EDTA-free protease inhibitor cocktail, and then subjected to sonication and centrifugation. The supernatant was incubated with Glutathione Sepharose beads. The resin was extensively washed with 50 ml of wash buffer I containing 50 mM HEPES-Na, pH 7.5, 300 mM NaCl, and 1 mM DDT, followed by 50 ml of wash buffer II containing 50 mM HEPES-Na, pH 7.5, 300 mM NaCl, 1 mM DTT, and 50 mM CaCl2). The GST tag was cleaved overnight at 4° C. with PreScission protease (GE Healthcare) in cleavage buffer containing 50 mM HEPES-Na, pH 7.5, 300 mM NaCl, 1 mM DTT, 2 mM EDTA. The cleaved proteins were purified by monoS column and gel filtration on Superdex 75 (GE Healthcare). The protein concentration was measured by absorption at 280 nm and aliquots were flash frozen in liquid nitrogen and stored at −80° C.
Munc13-2*. The Munc13-2* fragment of Munc13-2 (amino acid range 451-1407, that is, including the C1, C2B, and the C-terminally truncated MUN domains, but excluding residues 1326-1343) was cloned into a pFastBac HTB vector with a GST tag and a PreScission cleavage site. The deletion of residues 1326-1343 in this construct prevents aggregation (Li et al., 2011; Ma et al., 2013), and the C-terminal truncation improves solubility. Cells from 8 l of SF9 cell culture were re-suspended in 200 ml re-suspension buffer (RB) (50 mM Tris, pH 8.0, 500 mM NaCl, 1 mM EDTA, 0.5 mM TCEP, 10% glycerol) containing 6 EDTA-free protease inhibitor tablets. The cells were lysed via 3 passes through the Avestin C5 homogenizer at 15000 psi. The lysate was clarified by centrifugation for 35 min at 40,000 RPM in a Ti45 rotor. The supernatant was mixed with 15 ml Glutathione Sepharose beads at 4° C. stirring for 2 h. The beads were washed using an Akta Prime system (GE Healthcare) with 20 ml RB, 90 ml RB+1% triton X-100, then eluted with RB+50 mM reduced glutathione. Peak fractions were pooled and then 100 μl of 10 mg/ml PreScission protease was added and incubated overnight. The cleaved proteins were purified by gel filtration on Superdex 200. The protein concentration was measured by absorption at 280 nm and aliquots were flash frozen in liquid nitrogen and stored at −80° C.
Munc18-2. Munc18-2 (amino acid range 1-594) was cloned into a pFastBac HTB vector with an N-terminal hexa-histidine tag and a TEV cleavage site. Cells from 4.0 l of a SF9 cell culture were re-suspended in 100 ml re-suspension buffer (RB) (20 mM sodium phosphate, pH 8.0, 300 mM NaCl, 2 mM DTT, 10% glycerol with 1 mM PMSF) containing 6 EDTA-free protease inhibitor tablets. The cells were lysed via 3 passes through the Avestin C5 homogenizer at 15000 psi. The lysate was clarified by centrifugation for 35 min at 40,000 RPM in a Ti45 rotor. The supernatant was mixed with 3 ml Ni-NTA beads at 4° C. stirring for 1 h. The beads were washed using an Akta Prime system with 20 ml each of RB, then eluted with RB+300 mM imidazole. Peak fractions were pooled and then 100 μl of 11 mg/ml TEV protease was added. The mixture was dialyzed overnight against 1 l of 500 ml of 20 mM HEPES, pH 7.5, 300 mM NaCl, 2 mM DTT, 10% glycerol. The TEV cleaved protein was injected on a Superdex 200 column. Peak fractions were combined and the protein concentration was measured by UV absorption at 280 nm. Aliquots of 100 μl were flash frozen in liquid N2 and stored at −80° C.
Stapled peptides. All stapled peptides (SP1-SP12, Table 1), the non-stapled peptide P0 (
For binding of Biotin-SP9-Cy3 to bacterial toxins, C2 and CRM197 were conjugated to streptavidin. Biotin-SP9-Cy3 and streptavidin-conjugated toxins were mixed in a 10:1 ratio at 30° C. for 30 min before adding to cells.
CD spectroscopy. CD spectra were measured with an AVIV stop-flow CD spectropolarimeter at 190 to 250 nm using a cell with a 1 mm path-length. The sample containing 100 mM of synthesized peptides in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4) was measured at 20° C. For the correction of the baseline error, the signal from a blank run with PBS buffer was subtracted from all the experimental spectra. The α-helical content of each peptide was calculated by dividing the mean residue ellipticity [φ]222obs by the reported [φ]222obs for a model helical decapeptide (Yang et al., 1986).
Cryo-electron microscopy. PM and SG vesicles were separately vitrified on lacey carbon grids with a Vitrobot (Thermo Fisher Scientific), and imaged with a FEI Tecnai F20 transmission cryo-electron microscope with a field emission gun (FEI) operated at 200 kV. Images were recorded on a Gatan K2 Summit electron-counting direct detection camera (Gatan) in electron-counting mode (Li et al., 2013). Nominal magnifications of 5,000× and 9,600× (corresponding to pixel sizes of 7.4 Å and 3.8 Å) were used for airway PM and SG vesicles, respectively (
Bulk fluorescence anisotropy measurements. In the bulk fluorescence anisotropy experiments, the P0 and SP9 peptides were labeled with the fluorescent dye Cy3 at the C-terminus. Anisotropy was measured with a Tecan Infinite M1000/PRO fluorimeter using an excitation wavelength of 530±5 nm and emission wavelength of 580±5 nm at 27.2° C. The fluorescent dye labeled samples were diluted to 10 nM concentration in TBS (20 mM Tris, pH 7.5, 150 mM NaCl, 0.5 mM TCEP) for optimal read out.
Molecular dynamics simulations. The starting point for all molecular dynamics simulations was the crystal structure of the SNARE/Syt-1/complexin complex at 1.85 Å resolution (PDB ID 5W5C) (Zhou et al., 2017). Prior to the simulations, the Syt1 C2A domain, the crystallographic water molecules, Mg2+, and glycerol molecules were deleted from the crystal structure. Specifically, the following residues that were included in the simulations: Syt1 C2B [270-419], synaptobrevin-2[29-66], Stx1A[191-244], SNAP-25A[10-74 & 141-194]. Cpx1[51-75] was not included in the simulations. For all simulations the NAMD program was used (Phillips et al., 2020). For the primary interface (SNARE: Syt1_C2B) simulations, the Syt1 C2B molecule that produces the primary interface was used. For the simulations with SP9, P9, only residues 37-53 of SNAP-25_N were kept (sequence: EESKDAGIRTLVMLDEQ (SEQ ID NO:10)). The SP9, P9 peptides were simulated with an acetylated N-terminus, and an amidated C-terminus.
The staples for SP9 were created by using CHARMM topology and parameter files for S5 and the covalent bond between S5 residues (Speltz et al., 2016). Initial coordinates were generated by mutating the native residues into Lys using PyMol (Schrödinger, LLC.), and then using the VMD “mutate” command (Humphrey et al., 1996) to change Lys into S5.
For the primary interface (SNARE: Syt1 C2B) simulations, the starting models were placed in a 113×125×116 Å periodic boundary condition box. The empty space in the box was filled with 50,420 water molecules using the VMD solvate plugin. The system has a total of 157,833 atoms. The system was charge-neutralized and ionized by addition of 155 potassium and 138 chloride ions, corresponding to a salt concentration of ˜145 mM using the VMD autoionize plugin.
For the simulations with P9 and SP9, the starting models were placed in a 80×80×80 Å periodic boundary condition box. The empty space in the box was filled with ˜15,200 water molecules using the VMD “solvate” plugin. The system has a total of 48,486 atoms. The system was charge-neutralized and ionized by addition of 42 potassium and 44 chloride ions, corresponding to a salt concentration of ˜145 mM using the VMD “autoionize” plugin.
The CHARMM22 (P9 and SP9:Syt1 C2B simulations) or CHARMM36 (primary interface simulations) all-hydrogen force fields and parameters (Brooks et al., 2009) were used with a non-bonded cutoff of 11 Å. A constant pressure method was used by adjusting the size of the box. The Particle Mesh Ewald method was used to accelerate the calculation of long-range electrostatic nonbonded energy terms. Langevin dynamics (with a friction term and a random force term) was used to maintain the temperature of the simulation. All hydrogen-heavy-atom bonds were kept rigid using the Rattle method as implemented in NAMD.
For the simulations with stapled peptides, in the relaxation step, dihedral angle restraints were added to restrain the S5 CE-CE double bond in the cis conformation, the S5 aliphatic chains in the trans conformation, and all α-helices in the alpha-helical conformation (using the “ssrestraints” plugin for VMD. In all subsequent steps (heating steps, and production runs), all these dihedral angle restraints were turned off. For all other simulations with peptides without staples, in the relaxation step, α-helical (secondary structure) restraints were added for all α-helices (using the ssrestraints plugin for VMD). In all subsequent steps (heating steps, and production runs), all these dihedral angle restraints were turned off. The system was equilibrated by the following procedure: (1) relaxation step, ramping up the temperature from 0 to 50 K for 50 picoseconds with a 1 femtosecond time step; (2) first heating step, ramping up the temperature from 50 to 100 K for 50 picoseconds with a 1 femtosecond time step; (3) second heating step, ramping up the temperature from 100 to 250 K for 150 picoseconds with a 1 femtosecond time step. For all simulations, 1-nanosecond junks were run at a temperature of 300 K with a time step of 1 femtosecond. Five independent 1—μsec simulations were performed for each system (primary interface, SP9-Syt1 C2B, P9-Syt1 C2B) by using different initial random number seeds. All simulations were performed on the Stanford Sherlock Cluster using 4 nodes, each node consisting of dual 10-core CPU 2.4 Ghz Intel processors, i.e. a total of 80 CPUs were used for each simulation. The MPI-parallel NAMD2 2.14b1 executable was used (compiled by Kailu Yang). To visualize the results, only protein components are shown, and all structures were fitted to each other, and displayed with PyMOL.
Vesicle reconstitution. For the ensemble lipid mixing assay, the lipid composition of the SV vesicles was phosphatidylcholine (PC) (46%), phosphatidylethanolamine (PE) (20%), phosphatidylserine (PS) (12%), cholesterol (20%), and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) (Invitrogen) (2%); for the both neuronal and airway PM vesicles the lipid composition was Brain Total Lipid Extract supplemented 3.5 mol % PIP2, 0.1 mol % biotinylated phosphatidylethanolamine (PE) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) (Invitrogen). All the lipids are from Avanti Polar Lipids.
For single vesicle content mixing assay, the lipid composition of the SV, SG, VAMP2, or VAMP8 vesicles was phosphatidylcholine (PC) (48%), phosphatidylethanolamine (PE) (20%), phosphatidylserine (PS) (12%), and cholesterol (20%); for both the neuronal and airway PM vesicles the lipid composition was Brain Total Lipid Extract supplemented 3.5 mol % PIP2, and 0.1 mol % biotinylated phosphatidylethanolamine (PE).
The reconstitution method for neuronal PM and SV vesicles was described in detail previously (see Lai et al., 2017; Kyoung et al., 2011; and Lai et al., 2014). The same methods were used for airway PM, SG, VAMP2, and VAMP8 vesicles. Dried lipid films were dissolved in 110 mM OG buffer containing purified proteins at protein-to-lipid ratios of 1:200 for VAMP2 and Stx1A for SV and neuronal PM vesicles, respectively (or 1:200 for VAMP8 and Stx3 for SG and airway PM vesicles, respectively), and 1:800 for Syt1 for SV vesicles (or 1:1200 for Syt2 for SG vesicles).
A 3-5 fold excess of SNAP-25A or SNAP-23 (with respect to Stx1A or Stx3) and 3.5 mol % PIP2 were added to the protein-lipid mixture for neuronal or airway PM vesicles. Detergent-free buffer (20 mM HEPES, pH 7.4, 90 mM NaCl, and 0.1% 2-mercaptoethanol) was added to the protein-lipid mixture until the detergent concentration was at (but not lower than) the critical micelle concentration of 24.4 mM, i.e., vesicles did not yet form. For the preparation of SV, SG, VAMP2, or VAMP8 vesicles for single vesicle content mixing assay, 50 mM sulforhodamine B (Thermo Fisher Scientific) was added to the protein-lipid mixture. The vesicles subsequently formed during size exclusion chromatography using a Sepharose CL-4B column, packed under near constant pressure by gravity with a peristaltic pump (GE Healthcare) in a 5.5 ml column with a ˜5 ml bed volume that was equilibrated with buffer V (20 mM HEPES, pH 7.4, 90 mM NaCl) supplemented with 20 μM EGTA and 0.1% 2-mercaptoethanol. The eluent was subjected to dialysis into 21 of detergent-free buffer V supplemented with 20 μM EGTA, 0.1% 2-mercaptoethanol, 5 g of Bio-beads SM2 (Bio-Rad) and 0.8 g/L Chelex 100 resin (Bio-Rad). After 4 hours, the buffer was exchanged with 21 of fresh buffer V supplemented with 20 μM EGTA, 0.1% 2-mercaptoethanol and Bio-beads, and the dialysis continued for another 12 h (overnight). The chromatography equilibration and elution buffers did not contain sulforhodamine, so the effective sulforhodamine concentration inside SV, SG, VAMP2, or VAMP8 vesicles is considerably (up to ten-fold) lower than 50 mM. For ensemble lipid mixing assay, the reconstitution method is the same as that for the single vesicle content mixing assay, except that 50 mM sulforhodamine B was omitted for all the steps.
As described previously (Kyoung et al., 2011), the presence and purity of reconstituted proteins in the airway system was confirmed by SDS-PAGE of the vesicle preparations (
Bulk lipid mixing experiments with neuronal synaptic proteins. Protein-reconstituted neuronal PM and SV vesicles were mixed at a molar ratio of 1:1, both at 0.1 mM lipid concentration. Lipid mixing is measured by monitoring the fluorescent emission (670 nm) of the DiD dyes in the SV vesicles by FRET using 530 nm laser light excitation of the DiI dyes in neuronal PM vesicles. The fluorescence intensity was monitored in two channels at 570 nm and 670 nm, respectively. Fluorescence changes were recorded with a Varian Cary Eclipse model fluorescence spectrophotometer using a quartz cell of 100 μl with a 5 mm path length. All measurements were performed at 35° C.
Single vesicle content mixing experiments. All single vesicle fusion and single molecule experiments were performed on a prism-type total internal reflection fluorescence (TIRF) microscope using 532 nm (green) laser (CrystaLaser) excitation. Two observation channels were created by a 640 nm single-edge dichroic beamsplitter (FF640-FDi01-25x36, Shemrock): one channel was used for the fluorescence emission intensity of the content dyes and the other channel for that of the Cy5 dye that are part of the injected Ca2+-solution. The two channels were recorded on two adjacent rectangular areas (45×90 m2) of a charge-coupled device (CCD) camera (iXon+DV 897E, Andor Technology). The imaging data were recorded with the smCamera software developed by Taekjip Ha, Johns Hopkins University, Baltimore.
Flow chambers were assembled by creating a “sandwich” consisting of a quartz slide and a glass coverslip that were both coated with polyethylene glycol (PEG) molecules consisting 0.1% (w/v) biotinylated-PEG except when stated otherwise, and using double-sided tape to create up to five flow-chambers.
The single vesicle content mixing assay described in Lai et al. (2014) was used to monitor vesicle-vesicle association, Ca2+-independent, and Ca2+-triggered fusion. The surface of the quartz slides was passivated by coating the surface with polyethylene glycol (PEG) molecules which alleviated non-specific binding of vesicles. The same protocol and quality controls (surface coverage and non-specific binding) were used as described previously (Kyoung et al., 2011; Kyoung et al., 2012) except that PEG-SVA (Laysan Bio) instead of mPEG-SCM (Laysan Bio) was used since it has a longer half-life. The surface was functionalized by inclusion of biotin-PEG (Laysan Bio) during pegylation. A quartz slide was assembled into a flow chamber and incubated with neutravidin for 30 min (0.1 mg/ml).
For the fusion experiments described in
Multiple acquisition rounds and repeats for the single vesicle content mixing experiments. In order to increase the throughput of the assay and make better use of the vesicle samples, after intensive washing (3×120 μL) with buffer V (which includes 20 μM EGTA to remove Ca2+ from the sample chamber), the entire acquisition sequence (SV, SG, VAMP2, or VAMP8 vesicle loading, counting the number of freshly associated vesicle-vesicle pairs, monitoring of Ca2+-independent fusion, Ca2+-injection, and monitoring of Ca2+-triggered fusion) was repeated in a different imaging area within the same flow chamber. Five such acquisition rounds were performed with the same sample chamber. SV, SG, VAMP2, or VAMP8 vesicles were diluted 1000× for the first and second acquisition rounds, 200× for the third and fourth acquisition rounds, and 100× for the fifth acquisition round in order to offset the slightly increasing saturation of the surface with SG, SV, VAMP2 or VAMP8 vesicles. The entire experiment (each with five acquisition rounds) was then repeated several times (TABLE 4) (referred to as repeat experiment). Among the specified number of repeats there are at least three different protein preps and vesicle reconstitutions, so the variations observed in the bar charts reflect sample variations as well as variations among different flow chambers.
Cell culture. Primary human airway epithelial cells (HAECs) from several donors were obtained from Promocell (Heidelberg, Germany) at passage 2. HAECs from individual donors were thawed and expanded in a T75 flask (Sarstedt) in Airway Epithelial Cell Basal Medium supplemented with Airway Epithelial Cell Growth Medium SupplementPack (both Promocell). Growth medium was replaced every two days. Upon reaching 90 percent confluence, HAECs were detached using DetachKIT (Promocell) and seeded into 6.5 mm Transwell filters (Corning Costar). Filters were precoated with Collagen Solution (StemCell Technologies) overnight and irradiated with UV light for 30 min before cell seeding for collagen crosslinking and sterilization. 3.5×104 cells in 200 μl growth medium were added to the apical side of each filter, and an additional 600 μl of growth medium was added basolaterally. The apical medium was replaced after 48 h. After 72-96 h, when cells reached confluence, the apical medium was removed and basolateral medium was switched to differentiation medium+/−10 ng/ml IL-13 (IL012; Merck Millipore). Differentiation medium consisted of a 50:50 mixture of DMEM-H and LHC Basal (Thermo Fisher) supplemented with Airway Epithelial Cell Growth Medium SupplementPack and was replaced every 2 days. Air-lifting (removal of apical medium) defined day 0 of air-liquid interface (ALI) culture, and cells were grown at ALI conditions until experiments were performed at day 25 to 28. To avoid mucus accumulation on the apical side, HAEC cultures were washed apically with Dulbecco's phosphate buffered solution (DPBS) for 30 min every three days from day 14 onwards.
Mucin secretion assay in HAECs. Mucin secretion experiments under static, i.e., non-perfused, conditions were conducted as detailed previously (Winkelmann et al., 2019; Zhu et al., 2015; Abdullah et al., 2012) with modifications for the peptide treatments. In brief, for the 24 h peptide treatment, 20 μl of DMEM+/−100 μM peptides was added to the apical surface 24 h before stimulation. On the day of the assay, cells were washed 5 times with 100 μl DMEM for 1 h for each wash on the apical side. Apical supernatants were collected after every wash (wash 15), then 100 μl of DMEM+/−100 μM peptides was added to the apical surface and HAECs incubated for 15 min before collecting the supernatant (baseline wash). HAECs were then incubated for an additional 15 min with 100 μl DMEM+/−100 μM ATP (Sigma) before collection of supernatants (experimental washes). Following sample collection, cells were lysed in 100 μl of lysis buffer (lysate) containing 50 mM Tris-HCl pH 7.2, 1 mM EDTA, 1 mM EGTA, 1% Triton-X (Sigma), protease inhibitor cOmplete mini EDTA-free and phosphatase inhibitor PhosSTOP (Roche, Germany). The protocol was adapted for 30 min peptide treatment as follows. After wash 5, 100 μl of DMEM+/−10 μM or 100 μM of peptides was added to the apical surface and HAECs incubated for 30 min (baseline wash). HAECs were then incubated for 30 min with 100 μl DMEM+/−100 μM ATP to collect experimental washes.
All samples were diluted 1:10 in PBS (washes and cell lysates) and 50 μl of each sample were vacuum-aspirated onto a 0.45 μm pore nitrocellulose membrane using the Bio-Dot® MicrofiltrationApparatus (Bio-Rad). Subsequently, membranes were incubated with Intercept blocking buffer (Li-Cor®, USA) for 1 h before probing with anti-MUC5AC (MA1-21907, Invitrogen) added at 1:250 in Intercept blocking buffer for 1 h. Membranes were then washed four times for 10 min in PBS-Tween 20 (PBST) before incubation with the IRDye secondary antibody (#926-33212 or #926-68072; Li-Cor®) for 1 h. All steps were performed at room temperature. Fluorescent signals were acquired using the Odyssey® Fc Imaging System (Li-Cor®, USA) and quantified using ImageJ (v.2.0.0; NIH, USA). Equal volumes of samples were loaded on the gels for control and peptide treatments. Differences in total Muc5ac signal result from differences in IL-13 induced metaplasia between individual filters. Stimulated secretion is therefore normalized to baseline secretion within individual filters to account for filter to filter heterogeneities.
Immunofluorescence staining in HAECs. HAECs grown on Transwell filters were incubated with 20 μl of DMEM+/−100 μM specified peptides on day 28 of establishing ALI. 24 h later, cells were fixed for 20 min in 2% paraformaldehyde in DPBS (data not shown; also see FIG. 4A of U.S. Provisional Patent Application Ser. No. 63/311,001). Cells were then permeabilized for 10 min with 0.2% saponin and 10% FBS (Thermo Scientific) in DPBS. Cells were washed 2 times with DPBS and stained with anti-MUC5AC (clone 45M1; MA1-21907, Thermo Scientific) diluted 1:100 in DPBS, 0.2% saponin and 10% FBS over night at 4° C. Subsequently cells were washed 2 times with DPBS and incubated for 1 h at room temperature in DPBS, 0.2% saponin and 10% FBS containing AlexaFluor 488-labelled anti-mouse secondary antibody (1:500; Thermo Scientific) and DAPI (1: 5000; Thermo Scientific). Images were taken on an inverted confocal microscope (Leica TCS SP5) using a 40× lens (Leica HC PL APO CS2 40×1.30 OIL). Images for the blue (DAPI), green (AlexaFluor 488), red (Cy3) channels were taken in sequential mode using appropriate excitation and emission settings.
Immunofluorescence staining of CALU-3 cells. CALU-3 cells (cells derived from a lung adenocarcinoma patient) were cultivated on a 18-well p-slide (IBIDI) for 2 days. For uptake experiments, cells were left untreated (control), incubated for 30 min with dynasore (80 μM) or chloroquine (100 μM) or put on ice before adding peptides. Cells were incubated for 15, 30, or 60 min with either SP9-Cy3 or PEN-SP9-Cy3, washed, and fixed (15 min 4% PFA in PBS). Cells were counterstained with DAPI. Image acquisition involved acquiring Z-stacks covering the height of the cell (1 m intervals) in 3 different areas per well for each experiment. Z-stacks were collapsed into a single plane and individual cells were identified using “Cellpose 2.0” segmentation algorithm in the nuclear channel. Mean fluorescence intensity in the Cy3 channel was measured for each individual cells identified by the segmentation algorithm.
Image analysis for analysis of CPP uptake in HAECs. Serial sections of images along the basolateral to apical cell axis (z-axis) were acquired with 0.28 μm distance between individual z-sections to analyze distribution of intracellular Cy3 fluorescence (
Quantification and Statistical Analysis. Origin, Matlab, and Prism were used for the generation of all curves and graphs. The fusion experiments were conducted at least three times with different protein preps and vesicle reconstitutions and properties were calculated as mean±SEM. Student's t-test was used to test statistical significance in
Animal statement. All the mice work was conducted in accordance with the UT MD Anderson Cancer Center IACUC guidelines, and under the IACUC supervision; protocol No 00001214-RN02.
Stimulated secretion of mucins hyperproduced in response to inflammation is a major cause of airway obstruction in the pathophysiology of respiratory viral infection, asthma, COPD and cystic fibrosis (Goldblatt et al., 2020; Evans et al., 2015; Bossé et al., 2010; Hays and Fahy, 2003; Boucher et al., 2019). To validate Syt2 as a target of pharmacologic inhibition, whether deletion of Syt2 in airway epithelial cells might protect against mucus occlusion was tested. To accomplish this, Syt2F/F mice (Luo et al., 2017) were crossed with Scgb1a1cre knock-in mice (Liu et al, 2015; Li et al., 2008) as Syt2 knock-out mice die from complications of ataxia by postnatal day 24 (Pang et al., 2006), precluding study of the pathophysiologic role of airway mucin secretion in adult Syt2 knock-out mice. The deletant progeny of this cross (Syt2D/D) were born in a Mendelian ratio and appeared healthy, and the efficiency of deletion was essentially complete (data not shown; also see EXTENDED DATA FIG. 1 of U.S. Provisional Patent Application Ser. No. 63/311,001). There was no spontaneous mucin accumulation in SytD/D mice that would indicate impairment of baseline mucin secretion (Jaramillo et al., 2018; Zhu et al., 2008). To test impairment of stimulated mucin secretion in these mice, mucous metaplasia was induced by intrapharyngeal instillation of IL-13, then secretion was stimulated with an ATP aerosol. Fractional secretion of intracellular mucin in response to ATP by WT and Syt2F/F mice was 71% and 65%, respectively, whereas it was only 30% in Syt2D/D mice (
When bronchial airways of Syt2WT and Syt2D/D mice were stained with antibodies to Syt2 (Abcam #113545, rabbit polyclonal), there was less intense linear staining of tufted ciliated cells in the region of ciliary basal bodies, which often stain non-specifically (data not shown; also see EXTENDED DATA FIG. 1 of U.S. Provisional Patent Application Ser. No. 63/311,001). Secretory cells in Syt2WT mice have a domed appearance with their apical poles staining intensely for Syt2. Secretory cells in Syt2D/D mice did not stain for Syt2, but linear staining of ciliated cells was similar to Syt2WT (data not shown; also see EXTENDED DATA FIG. 1 of U.S. Provisional Patent Application Ser. No. 63/311,001). We enumerated secretory cells in 3 mice of each genotype and found 46% in Syt2WT and 48D/D in Syt2D/D±4%, which did not differ significantly, indicating there was no loss of viability of secretory cells in Syt2D/D mice (data not shown; also see EXTENDED DATA FIG. 1 of U.S. Provisional Patent Application Ser. No. 63/311,001). In Syt2WTmice, 91% of secretory cells stained for Syt2, whereas in Syt2D/Dmice, only 7% stained for Syt2, indicating a deletion efficiency ˜92%. Results for Syt2F/Fmice were indistinguishable from those for Syt2WT (data not shown). Together these data validated Syt2 as a therapeutic target in muco-obstructive airway disease.
Theoretically, helical peptides, such as a fragment of SNAP-25A involved in the primary interface), could be used to selectively interfere with this synaptotagmin-SNARE interaction and thereby disrupt the process of Ca2+-triggered membrane fusion (data not shown; also see FIG. 2A and EXTENDED DATA FIG. 2 of U.S. Provisional Patent Application Ser. No. 63/311,001). From a therapeutic perspective, peptide-based strategies have been successfully applied to inhibit virus-host membrane fusion (Xia et al., 2020; Kilby et al., 1998; Russell et al., 2001; Watanabe et al., 2000; Lu et al., 2014). However, the molecular mechanisms are quite different since viral membrane fusion is mediated by formation of a six-helix bundle (Harrison, 2015) whereas Ca2+-triggered membrane fusion is mediated by formation of four-helix SNARE bundles in cooperation with synaptotagmins and other factors (Zhou et al., 2015; Sutton et al., 1998). Moreover, peptide-based viral inhibitors function extracellularly whereas peptide-based secretory/synaptic vesicle inhibitors need to act intracellularly. Thus, the question if a peptide inhibitor strategy can be applied to disrupt Ca2+-triggered membrane fusion was investigated.
Peptides typically have little secondary structure in solution when taken out of context of the intact system. Thus, their efficacy as in vivo reagents may be limited by their loss of secondary structure. In analogy to the use of stapled peptides to inhibit HIV virus infection (Bird et al., 2010), non-natural amino acids containing olefin-bearing groups were used to generate hydrocarbon-stapled peptides by a Grubbs catalyst (Schafmeister et al., 2000) to interfere with the primary interface. A close-up view of the primary interface indicates the locations of residues that are important for the primary interface include R281, E295, Y338, R398, R399 in Syt1 C2B; which correspond to residues mutated in Syt1_QM) and K40, D51, E52, E55, Q56, D166 in SNAP-25A and D231, E234, E238 in Stx1A (data not shown; also see EXTENDED DATA FIG. 2C of U.S. Provisional Patent Application Ser. No. 63/311,001). The residues directly involved in the primary interface are identical for Syt1/Syt2 and mostly identical for SNAP-25A/SNAP-23, as discussed above in this disclosure (see SEQ ID NOS: 1, 2, 444-47; also see EXTENDED DATA FIGS. 2A and 2B of U.S. Provisional Patent Application Ser. No. 63/311,001). Since the crystal structure is known for the neuronal system, it was first used for the design of stapled peptides that disrupt this interaction, and the peptides in the airway epithelial system were subsequently tested. A series of hydrocarbon-stapled peptides consisting of SNAP-25A fragments that included many of the key residues involved in the primary interface (described previously in Section II; also see FIG. 2A and EXTENDED DATA FIGS. 2A-2C of U.S. Provisional Patent Application Ser. No. 63/311,001)) were designed. Within the peptides, α,α-distributed non-natural amino acids containing varying length of olefinic side chains were synthesized (see Example 1). The hydrocarbon staple was made to flank three (substitution position i and i+4) or six (substitution position i and i+7) amino acids within the SNAP-25A fragments (data not shown; also see FIGS. 2B and 2C of U.S. Provisional Patent Application Ser. No. 63/311,001). The positions of these substitutions were chosen to be away from the primary interface. P0, a non-stapled SNAP-25A fragment, that displays only 5% helicity in solution thus indicating that it is largely a random coil, (
To determine whether the stapled peptides specifically interact with the C2B domains of Syt1 and Syt2, the most helical peptide, SP9, and was chosen and labeled with the fluorescent dye Cy3 at the C-terminus and nothing, Biotin, PEN, or TAT at the N-terminus (Table 3). A TAT-P9-Cy3 peptide was used as a control. In Table 3, Ac is acetyl; LCBiot is a biotinylation at the N terminus by cross-linking biotin through the carbon spacer 6-aminohexaonic; and Cy3Mal-amide is Cy3 maleimide, a mono-reactive dye containing maleimide group, which can selectively and efficiently attach Cyanine3 fluorophore.
Bulk fluorescence anisotropy was recorded after mixing Cy3 labeled SP9 with varying concentrations of Syt1 C2B, Syt2 C2B, or a quintuple mutant of Syt1 C2B (C2B_QM) (SEQ ID NOS: 49-51; also see FIG. 2B of U.S. Provisional Patent Application Ser. No. 63/311,001). SP9 binds to Syt1 C2B and Syt2 C2B with a dissociation constant (Kd) of 24 μM and 35 μM, respectively, which is comparable to the Kd between Syt1 and the SNARE complex (˜20 μM) (Zhou et al., 2015). As a control and as expected, SP9 does not bind to the quintuple mutant of Syt1 C2B_QM since that mutation disrupts the primary interface. Id. Moreover, no binding of the non-stapled peptide PO to the C2B domain of either Syt1 or Syt2 in the conditions of these experiments was observed.
These binding experiments suggest that the stabilization of SP9 peptide by staples is important for binding. To corroborate this finding, the stability of the SP9 peptide interactions with the C2B domain of Syt1 by five 1-μsec molecular dynamics simulations was assessed, starting with a conformation derived from the crystal structure of the primary complex (data not shown; also see EXTENDED DATA FIGS. 2F and 2G of U.S. Provisional Patent Application Ser. No. 63/311,001). The simulations were performed in a periodic box of water molecules, and potassium and chloride ions at physiological concentrations. Both P9 and SP9 changed conformations during these simulations. Four of the five simulations of SP9 adopt binding poses at the end of the 1 sec simulations that would interfere with the formation of the primary interface (
Next, whether the stapled peptides disrupt membrane fusion with reconstituted neuronal SNAREs and nearly full-length Syt1 was tested. A simple lipid mixing ensemble assay (Methods) was used, allowing rapid screening of many peptides. Among the peptides tested, the stapled peptides (SP1, SP4, SP9, SP10) substantially inhibited ensemble lipid mixing in a peptide concentration-dependent fashion with or without Ca2+ (
After secretory granule—airway PM vesicle association, vesicle pairs either undergo Ca2+-independent fusion or remain associated until fusion is triggered by Ca2+ addition. To test if the stapled peptides have an inhibitory effect on the process of mucin secretion, two types of vesicles were reconstituted to mimic mucin secretion: vesicles with reconstituted Stx3 and SNAP-23 that mimic the plasma membrane of epithelial cells (airway PMvesicles), and vesicles with reconstituted VAMP8 and Syt2 that mimic mucin-containing secretory granules (SG vesicles) (Methods) (
TABLE 4 shows a summary of data from the single vesicle fusion experiments as indicated. Among each repeat experiment there are at least three different protein preparations and vesicle reconstitutions, so the variations observed in the bar charts reflect sample variations as well as variations among different flow chambers. For the definition of the repeat experiments see Methods. The effects of 10 μM of each of the specified peptides on the vesicle association, the corresponding Ca2+-independent fusion probabilities, the corresponding average probabilities of Ca2+-independent fusion events per second, the effects of 10 μM of each of the specified peptides on vesicle association, the corresponding Ca2+-independent fusion probabilities, the corresponding average probabilities of Ca2+-independent fusion events per second, the corresponding Ca2+-triggered fusion probabilities, the corresponding Ca2+-triggered fusion amplitude of the first 1-sec time bin upon 500 μM Ca2+-injection, the cumulative Ca2+-triggered fusion probability within 1 min, and the decay rate (1/T) of the Ca2+-triggered fusion histogram were also measured (data not shown; also see EXTENDED DATA FIGS. 5I-5R of U.S. Provisional Patent Application Ser. No. 63/311,001).
As the physiological functions of Munc13 are to catalyze the transition of syntaxin from the syntaxin/Munc18 complex into the ternary SNARE complex (Basu et al., 2005; Ma et al., 2013; Yang et al., 2015) and to promote proper SNARE complex formation (Lai et al., 2017), the effect of the SP9 stapled peptide was tested in a more complete reconstitution that includes airway epithelial SNAREs, Syt2, the C1C2B_MUN2 fragment of Munc13-2 (referred to as Munc13-2*) (Zhu et al., 2008), and Munc18-2 (
In contrast, in the presence of Munc13-2*, robust Ca2+-triggered fusion was observed at Ca2+ concentrations of both 50 and 500 μM (
When 10 μM SP9 was added in the more complete reconstitution assay, both the 50 μM and 500 μM Ca2−-triggered fusion amplitude, the cumulative fusion probability, and the synchronization were strongly inhibited (
Whether the selected stapled peptides could also inhibit mucin secretion in fully differentiated primary human airway epithelial cells (HAECs) was tested. To facilitate cellular entry of the stapled peptides, the N-terminus of SP9 was conjugated with cell penetrating peptides (CPPs) (Guidotti et al., 2017). Time traces of FRET efficiency upon mixing neuronal PM- and SV-vesicles shows that the non-stapled peptide (PO) had little effect on Ca2+-independent ensemble lipid mixing measurements of vesicle-vesicle fusion (data not shown; also see EXTENDED DATA FIG. 4A of U.S. Provisional Patent Application Ser. No. 63/311,001). The two groups of vesicles were mixed at the same molar ratio with a final lipid concentration of 0.1 mM with 10 μM and 100 μM P0 or SP9, respectively. For assessment of cellular entry, these peptides were conjugated with the Cy3 fluorescent dye at the C-terminus. In addition, SP9-Cy3 was conjugated with biotin and bound it to streptavidin-conjugated bacterial toxins (non-toxic mutants of clostridial C2 toxin or diphtheria toxin (CRM197)) as a possible alternative for intracellular delivery. These bacterial toxins can deliver biotin-conjugated peptides into mammalian cells via endocytosis (Fellerman et al., 2020; Fahrer et al., 2013).
Confocal imaging of fixed HAECs treated with SP9-Cy3, conjugated to either bacterial toxins or CPPs, indicated that only CPP-modified SP9-Cy3 penetrated into the cell interior (
To investigate the effect of CPP-conjugated SP9-Cy3 on baseline and stimulated secretion under control and mucous metaplastic conditions, HAECs were cultured in the absence or presence of 10 ng/ml IL-13, respectively. IL-13 treatment induces goblet cell hyperplasia and metaplasia in vitro (Turner et al., 2011; Winkelmann et al., 2019) mimicking IL-13 induced mucous metaplasia in vivo (Zhu et al., 2015; Wills-Karp et al., 1998). Consistently, Muc5ac expression was upregulated in IL-13 treated cells (data not shown; also see EXTENDED DATA FIG. 8A of U.S. Provisional Patent Application Ser. No. 63/311,001). Next, experiments were performed in metaplastic conditions with a 30 min peptide pre-incubation at a peptide concentration of 10 μM (data not shown; also see EXTENDED DATA FIG. 4E of U.S. Provisional Patent Application Ser. No. 63/311,001). Baseline secretion was low under metaplastic conditions and not affected by peptide treatment (
Since the inhibitory effect of TAT-P9-Cy3 was statistically significant while PEN-SP9-Cy3 was just below being statistically significant, the effect of the CPP-conjugated SP9-Cy3 peptides at a higher concentration and longer duration were tested. HAECs were incubated with 100 μM CPP-conjugated SP9-Cy3 for 24 h before stimulation (
To investigate whether SP9 could have therapeutic benefit in vivo, SP9 conjugated to CPPs and fluorophores was introduced into mouse airways using a microsprayer inserted into the distal trachea under direct visualization with a laryngoscope. Initial pilot experiments using TAT-SP9-Cy3 and PEN-SP9-Cy3 showed fluorescent labeling of scattered airway epithelial cells, but the forcefully injected peptide solution mostly bypassed the left proximal axial bronchus between lateral branches L1 and L2. All subsequent studies of airway mucin secretion were therefore performed in the distal axial bronchus. Unexpectedly, when TAT-SP9-Cy3 was introduced into the airways of mice with mucous metaplasia, serial sections showed that Cy3-labeled cells had secreted their mucin stores without stimulation by a secretagogue such as ATP or methacholine (data not shown; also see EXTENDED DATA FIGS. 9A and 9B of U.S. Provisional Patent Application Ser. No. 63/311,001). This effect was not observed with the injection of buffer alone. Hence, secretion was not induced by the shear force of the microsprayer, but it appears to be a side-effect of the TAT-SP9-Cy3 compound in this system. Fortunately, PEN-SP9-Cy3 did not show this problem (
The peptide solution was injected into the airway under pressure to generate a droplet aerosol. A high concentration of peptides was used in these experiments in part because the solution is expected to be in contact with the airway epithelium only briefly and to mostly end up in the alveolar region. Serial lung sections after reintroduction of fluorescent labeled peptide confirmed this expectation (data not shown; also see EXTENDED DATA FIG. 9A-9D of U.S. Provisional Patent Application Ser. No. 63/311,001). In serial transverse sections of the left axial bronchus of a mouse with mucous metaplasia induced by prior instillation of IL-13, then treated with 300 μM aerosolized TAT-SP9-Cy3, staining with PAFS demonstrates intracellular mucin, and shows some cells with high mucin content and other cells with low mucin content, presumably due to induced mucin secretion (data not shown; also see EXTENDED DATA FIG. 9A of U.S. Provisional Patent Application Ser. No. 63/311,001). Cells that internalize TAT-SP9-Cy3 tend to have low intracellular mucin content (data not shown; also see EXTENDED DATA FIG. 9A of U.S. Provisional Patent Application Ser. No. 63/311,001).
Sections of the left axial bronchus of mice with mucous metaplasia induced by prior instillation of IL-13subsequently treated with aerosolized 100 mM ATP or 1 mM TAT-SP9-Cy3 show high intracellular mucin content in the mice not treated with an aerosolized drug, extensive secretion of intracellular mucin in the mice treated with ATP, and extensive apocrine mucin secretion in the mice treated with TAT-SP9-Cy3 (data not shown; also see EXTENDED DATA FIG. 9B of U.S. Provisional Patent Application Ser. No. 63/311,001) Transverse section of the left axial bronchus of a mouse taken 30 min after treatment with aerosolized 20 μM PEN-SP9-Cy3 and immunofluorescent staining for CCSP show that secretory cells are observed to not take up PEN-SP9-Cy3, which is presumed to be internalized into ciliated cells, the other major airway epithelial cell type (data not shown; also see EXTENDED DATA FIG. 9C of U.S. Provisional Patent Application Ser. No. 63/311,001). Ciliary tufts were clearly visible by differential interference microscopy, but no staining of intervening secretory cells (data not shown; also see EXTENDED DATA FIG. 9C of U.S. Provisional Patent Application Ser. No. 63/311,001).
Approximate concentrations of PEN-SP9 required for jet nebulizer can be inferred based upon the concentration of PEN-SP9 required to achieve a therapeutic effect in cultured epithelial cells (
For allometric scaling for human drug delivery, it is generally believed that concentrations of drug in a jet nebulizer can be kept constant since the lung size of mammals, and hence the inspiratory volume, scales with size. This is certainly true with aerosol drug delivery to human children and adults using the same device and formulation. Consistent with this, here precisely the same concentration of drug in a jet nebulizer in mice administered for a defined period of time that fell on the upper inflection point for a pharmacodynamic marker (Alfaro V Y, et al., 2014) also fell on the upper inflection point of a biomarker in a human dose-escalation study (NCT 02124278). Substantially lower concentrations and/or dosages could be administered if the inhaler used can deliver drug to the conducting airways with high efficiency.
Aerosol administration of 200 μM PEN-SP9-Cy3 labeled 76% of epithelial cells in the distal left axial bronchus, while 200 μM of the control peptide PEN-P9-Cy3 labeled 77% of epithelial cells (
As shown in
A listing of the sequences by SEQ ID NO is provided below in Table 5.
The foregoing description of some examples has been presented only for the purpose of illustration and description and is not intended to be exhaustive or to limit the disclosure to the precise forms disclosed. It is recognized that those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.
It is noted that the description and/or claims may be drafted to exclude any optional element. As such, this statement is intended to serve as an antecedent basis for use of such exclusive terminology as “solely,” “only,” and the like in connection with the recitation of claim elements or use of a “negative” limitation. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order that is logically possible.
Reference herein to an example or implementation means that a particular feature, structure, operation, or other characteristic described in connection with the example may be included in at least one implementation of the disclosure. The disclosure is not restricted to the particular examples or implementations described as such. The appearance of the phrases “in one example,” “in an example,” “in one implementation,” or “in an implementation,” or variations of the same in various places in the specification does not necessarily refer to the same example or implementation. Where applicable or not specifically disclaimed, any one of the embodiments described herein is contemplated to be able to combine with any other one or more embodiments, even though the embodiments are described, under different aspects of the invention. As such, the preceding general areas of utility are given by way of example only and are not intended to be limiting on the scope of the present disclosure and appended claims. Additional objects and advantages associated with the compounds, compositions, methods, and processes of the present invention will be appreciated by one of ordinary skill in the art in light of the instant claims, description, and examples. For example, the various aspects and embodiments of the invention may be utilized in numerous combinations, all of which are expressly contemplated by the present description. These additional advantages, objects, and embodiments are expressly included within the scope of the present invention.
This application claims priority to and the benefit of U.S. Provisional Patent Application Ser. No. 63/311,001, filed Feb. 16, 2022, the contents of which is incorporated herein in its entirety by this reference as if fully set forth herein.
This invention was made with Government support under grant numbers R01 HL129795, RO1 A1137319, and MH063105 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/062758 | 2/16/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63311001 | Feb 2022 | US |
