Doubly constrained peptides as allosteric inhibitors of leucine rich repeat kinase 2

Abstract

This disclosure describes peptides useful in treating medical disorders, and more particularly to doubly constrained synthetic polypeptides capable of inhibiting or modulating the activity of Leucine Rich Repeat Kinase 2 (LRRK2).

Description

[0001] DOUBLY CONSTRAINED PEPTIDES AS ALLOSTERIC INHIBITORS OF LEUCINE RICH REPEAT KINASE 2

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to United States Provisional Application No. 63 / 466,533 filed May 15, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on May 15, 2024, is entitled “10067-072W01_ST26.xml”, and is 37,136 bytes in size.

[0006] TECHNICAL FIELD

[0007] This disclosure relates to peptides useful in treating medical disorders and, more particularly, to doubly constrained synthetic polypeptides capable of inhibiting or modulating the activity of Leucine Rich Repeat Kinase 2 (LRRK2).

[0008] BACKGROUND

[0009] The Leucine-Rich-Repeat-Kinase-2 (LRRK2) protein is a multi-domain protein consisting of seven domains comprising 2527 amino acid residues (see Berwick, D. C.; Heaton, G. R.; Azeggagh, S.; Harvey, K. LRRK2 Biology from structure to dysfunction: research progresses, but the themes remain the same. Mol Neurodegener 2019, 14 (1), 49). The different domains along the LRRK2 protein partake in both individual and overlapping functions. From the N-terminus to the C-terminus, LRRK2 contains Armadillo Repeats (ARM), Ankyrin Repeats (ANK), Leucine-Rich-Repeats (LRR), Ras of Complex (ROC) domain, C-terminal of Roc (COR) domain, Kinase domain, and the WD-40 domain. The different domains along LRRK2 partake in both individual and overlapping functions. LRRK2 is expressed in diverse tissues, including the brain, lungs, kidneys, and a subset of immune cells. In cells, LRRK2 is present as both a monomer and a dimer. In its monomeric form, the protein is largely distributed throughout the cytosol, whereas in its dimeric form, the protein is localized to specific cell organelles and membranes and performs discrete functions (see Civiero, L.; Russo, I.; Bubacco, L.; Greggio, E. Molecular Insights and Functional Implication of LRRK2 Dimerization. Adv Neurobiol 2017, 14, 107-121). Missense mutations along the different domains of LRRK2 are the most common cause of genetically associated Parkinson’s Disease (PD) (see Zimprich, A.; Muller-Myhsok, B.; Farrer, M.; Eeitner, P.; Sharma, M.; Hulihan, M.; Lockhart, P.; Strongosky, A.; Kachergus, J.; Caine, D. B.; et al. The PARK8 locus in autosomal dominant parkinsonism: confirmation of linkage and further delineation of the disease-containing interval. Am J Hum Genet 2004, 74 (1), 11-19). PD is caused by the loss of dopamine-producing nerve cells in the substantia nigra in the ventral midbrain (see Nguyen, A. P. T.; Tsika, E.; Kelly, K.; Levine, N.; Chen, X.; West, A. B.; Boularand, S.; Bameoud, P.; Moore, D. J. Dopaminergic neurodegeneration induced by Parkinson's disease-linked G2019S LRRK2 is dependent on kinase and GTPase activity. Proc Natl Acad Sci U S A 2020, 117 (29), 17296-17307). Pathogenic mis sense mutations in LRRK2 are linked with accumulation and extracellular propagation of a-synuclein, which in turn leads to microglia activation in the brain, causing neuroinflammation and nerve cell death (see Rivero-Rios, P.; Romo-Lozano, M.; Fasiczka, R.; Naaldijk, Y.; Hilfiker, S. LRRK2-Related Parkinson's Disease Due to Altered Endolysosomal Biology With Variable Lewy Body Pathology: A Hypothesis. Front Neurosci 2020, 14, 556). PD-related pathogenic mutations along LRRK2 are mostly localized to the ROC, COR, and Kinase domains (see Cookson, M. R. LRRK2 Pathways Leading to Neurodegeneration. Curr Neurol Neurosci Rep 2015, 75 (7), 42). Each of these mutations alters kinase and GTPase activity, which has downstream cell signaling effects, including altered lysosomal maintenance, cell apoptosis, disrupted mitochondrial function, and altered vesicular trafficking, all of which are pathologies of PD. Efforts to develop LRRK2 inhibitors have largely focused on small molecule ATP competitive-binding kinase inhibitors such as MLi-2 and DNL201 (see Azeggagh, S.; Berwick, D. C. The development of inhibitors of leucine-rich repeat kinase 2 (LRRK2) as a therapeutic strategy for Parkinson's disease: the current state of play. Br J Pharmacol 2022, 179 (8), 1478-1495; and Jennings, D.; Huntwork-Rodriguez, S.; Henry, A. G.; Sasaki, J. C.; Meisner, R.; Diaz, D.; Solanoy, H.; Wang, X.; Negrou, E.; Bondar, V. V.; et al. Preclinical and clinical evaluation of the LRRK2 inhibitor DNL201 for Parkinson's disease. Sci Transl Med 2022, 14 (648), eabj2658). While these small molecule inhibitors are successful at downregulating LRRK2 kinase activity, toxicities for many of these compounds include altered lysosomal function and vesicular trafficking, mitochondrial dysfunction, induced mislocalization of LRRK2, and lung and kidney pathologies (see Baptista, M. A. S.; Merchant, K.; Barrett, T.; Bhargava, S.; Bryce, D. K.; Ellis, J. M.; Estrada, A. A.; Fell, M. J.; Fiske, B. K.; Fuji, R. N.; et al. ERRK2 inhibitors induce reversible changes in nonhuman primate lungs without measurable pulmonary deficits. Sci Transl Med 2020, 12 (540)).

[0010] There is a clear need for further inhibitors of ERRK2, which are useful in treating neurological diseases, disorders, or conditions.

[0011] SUMMARY

[0012] The present disclosure provides doubly constrained peptides that mimic the first sheets in the “7+1” interface of the COR:COR dimer of ERRK2 which can inhibit or modulate the activity of ERRK2. These doubly constrained peptides are useful in treating neurological diseases, disorders, or conditions, for example, Parkinson’s disease.

[0013] In one aspect, a synthetic polypeptide is comprising a variant of an amino acid sequence of SEQ ID NO. 1, wherein the variant comprises at least two pairs of non-natural amino acids, wherein each pair of the at least two pairs of non-natural amino acids is crosslinked.

[0014] In another aspect, a pharmaceutical composition is provided comprising a synthetic polypeptide described herein and a pharmaceutically acceptable carrier.

[0015] In another aspect, a cell is provided comprising a synthetic polypeptide described herein.

[0016] In another aspect, a method is provided of treating a neurological disease, disorder, or condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a synthetic polypeptide or pharmaceutical composition thereof described herein. In some aspects, the neurological disease, disorder, or condition comprises Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, or amyotrophic lateral sclerosis (AES).

[0017] In another aspect, a method is provided of treating Crohn’s disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a synthetic polypeptide or a pharmaceutical composition thereof described herein.

[0018] In another aspect, a method is provided of treating a disorder or condition that is treated by inhibition or modulation of LRRK2 activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a synthetic polypeptide or pharmaceutical composition thereof described herein.

[0019] The details of one or more aspects of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims.

[0020] DESCRIPTION OF DRAWINGS

[0021] FIGs. 1A-1D depict the design and Synthesis of the Elongated COR (ECOR) Peptide Library. (FIG. 1A) Structure of the LRRK2 dimer interface highlighting the targeted COR:COR interface (shown in pink). The “7+1” stacked beta sheets are shown in teal. The ECOR peptide sequence is shown in lilac (SEQ ID NO.: 11). (FIG. IB) Schematic representation of the in silica alanine scan results for the ECOR peptide sequence residues (residues 1802-1828, n=10 models). Shown in navy blue is the hydrophobic triad that was identified as critical for binding (SEQ ID NOs. 1 and 12-15, respectively top to bottom). (FIG. 1C) The ECOR peptide library is shown. Red asterisks indicate the placement of the olefinic amino acid Ss. The glycine substitution is also shown in red (A12G). (FIG. ID) Schematic of synthetic strategy to generate singly and doubly constrained hydrocarbon stapled ECOR peptides.

[0022] FIGs. 2A-2B depict that doubly constrained peptides bind the RocCOR domain of LRRK2. (FIG. 2A) Fluorescence polarization (FP) assays using doubly constrained FAM- labeled peptides (ECOR and ECORA 12G) and the RocCOR domain of LRRK2 demonstrate that both peptides bind this construct with KD values ranging from 45-60 nM. (FIG. 2B) FP assays were performed using a RocCOR construct bearing the R1441C disease-associated mutation. Peptides bound this construct with higher affinities with KD values ranging between 25-35 nM. Data is representative of triplicate experiments.

[0023] FIGs. 3A-3B depict that stapled peptides are resistant to proteolytic degradation, but only doubly constrained peptides permeate cells. (FIG. 3A) Proteolytic stability was measured for each peptide in mouse serum over a 6-hour time course at 37 °C. The singly and doubly stapled peptides demonstrate considerable proteolytic stability with up to 30% degradation at the 6-hour time point, while the unstapled parent peptide underwent greater than 50% degradation within the first hour. Plots are representative of triplicate experiments. (FIG. 3B) HEK293 cells were treated with 2.5 pM of each FAM-labeled peptide for 6 h at 37 °C. Doubly stapled peptides were found to permeate cells while the singly stapled peptides and the unstapled parent control peptide did not.

[0024] FIGs. 4A-4D depict that doubly constrained peptides downregulate dimerization ad LRRK2 kinase activity and do not cause microtubule localization of LRRK2. (FIG. 4A) A proximity biotinylation assay was used to measure LRRK2 dimerization in cells. Both doubly constrained peptides downregulated dimerization by approximately 50%. Experiments were performed in triplicate, n.s. not significant, *p < 0.05, **p<0.005. (FIG. 4B) Western blot analysis of Rabl2 phosphorylation in cells expressing G2019S LRRK2 demonstrates that both doubly constrained peptides downregulate LRRK2 kinase activity as compared to the untreated control, # p < 0.05 (G2019S compared to WT), *p < 0.05, **p < 0.01. (FIG. 4C) HEK293 cells were transfected with GFP-LRRK2 for 24 hours and subsequently treated with DMSO, 1 pM MLi-2, 2.5 pM each of ECOR and ECOR A12G peptide for 6 hours at 37 °C. MLi-2 treatment shows skein-like filamentous structures as indicated with the arrows. DMSO and doubly constrained peptides treated cells show aggregation / homogenous expression of GFP-LRRK2. Scale bar 20pm. (FIG. 4D) A minimum of 500 transfected cells were quantified for skein-like structures for each condition as shown in (FIG. 4C). The graph represents the average percentage of cells and standard errors of the mean (SEM) for three independent experiments with at least two biological replicates are shown with p values: One-way ANOVA and Dunnett’s multiple comparisons test (DMSO as a control), **** p <0.0001. ns: not significant (p > 0.05).

[0025] FIGs. 5A-5B depict that doubly constrained peptides downregulate LRRK2- mediated neuronal apoptosis. (FIG. 5A) Representative images of primary cortical neurons expressing LRRK2 G2019S that are treated with 0.5 pM ECOR A12G. Nuclear condensation and fragmentation are reduced after peptide treatment. (FIG. 5B) Quantification of apoptotic primary neurons expressing LRRK2 with or without peptide treatments. Primary neurons were transiently transfected with Flag-LRRK2 (either WT or G2019S), followed by treatment with 0.5-1 pM of ECOR or ECOR A12G for 48 hours. Both doubly stapled peptides greatly reduced neuronal apoptosis with 0.5 pM peptide treatments. Neurons from three separate biological replicates were counted in a blinded manner.

[0026] Like reference symbols in the various drawings indicate like elements. DETAILED DESCRIPTION

[0027] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known aspects. Many modifications and other aspects disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain, benefiting from the teachings presented in the descriptions herein and the associated drawings. Therefore, it is understood that the disclosures are not limited to the specific aspects disclosed and that modifications and other aspects are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0028] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0029] As apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features that may be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the present disclosure.

[0030] Any recited method can be carried out in the order of events recited or any other order that is logically possible. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not explicitly state in the claims or descriptions that the steps are to be limited to a particular order, it is in no way intended that an order be inferred in any respect. This holds for any possible nonexpress basis for interpretation, including logic concerning arrangement of steps or operational flow, meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0031] All publications mentioned herein are incorporated by reference to disclose and describe the methods or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure before the filing date of the present application. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation. It is also to be understood that the terminology herein describes particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It can be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0032] Before describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.

[0033] Definitions

[0034] As used herein, “comprising” is interpreted as specifying the presence of the stated features, integers, steps, or components but does not preclude the presence or addition of one or more features, integers, steps, components, or groups thereof. Moreover, each of the terms “by,” “comprising,” “comprises,” “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, nonlimiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.”

[0035] As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context dictates otherwise.

[0036] Ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. Further, the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. There are many values disclosed herein, and each value is also disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value and to “about” another particular value. Similarly, when values are expressed as approximations, using the antecedent “about,” the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed. When a range is expressed, a further aspect includes from the one particular value and to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g., ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x,’ ‘about y,’ and ‘about z’ as well as the ranges of ‘less than x,’ ‘less than y.’ and ‘less than z.’ Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x,’ ‘about y,’ and ‘about z’ as well as the ranges of ‘greater than x,’ greater than y,’ and ‘greater than z.’ In addition, the phrase “about ‘x’ to ‘y’,” where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’.”

[0037] Such a range format is used for convenience and brevity and, thus, should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5% but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0038] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact but may be approximate, larger or smaller, as desired, reflecting tolerances, conversion factors, rounding, measurement error, and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, as used herein, “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter, or other quantity or characteristic is “about,” “approximate,” or “at or about,” whether or not expressly stated to be such. Where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself unless expressly stated otherwise.

[0039] As used herein, the term “therapeutically effective amount” refers to an amount sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the particular compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts. In the case of treating a particular disease or condition, in some instances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to permanently halt the progression of the disease. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition can also be delaying the onset or even preventing the onset.

[0040] For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to increase the dosage gradually until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for administration. Consequently, single-dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The individual physician can adjust the dosage in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the disclosure (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. However, a patient may insist on a lower or tolerable dose for medical reasons, psychological reasons, or virtually any other reason.

[0041] A response to a therapeutically effective dose of a disclosed compound or composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following the administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. The amount of a treatment may be varied, for example, by increasing or decreasing the amount of a disclosed compound or pharmaceutical composition, changing the disclosed compound or pharmaceutical composition administered, changing the route of administration, changing the dosage timing, and so on. Dosage can vary and can be administered in one or more doses daily for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0042] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur. The description includes instances where said event or circumstance occurs and those where it does not.

[0043] As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g., human). “Subject” can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to a human and constituents thereof.

[0044] As used herein, “treating” and “treatment” generally refer to obtaining a desired pharmacological or physiological effect. The effect can be but does not necessarily have to be prophylactic in preventing or partially preventing a disease, symptom, or condition such as Parkinson’s disease. The effect can be therapeutic regarding a partial or complete cure of a disease, condition, symptom, or adverse effect attributed to the disease, disorder, or condition. The term “treatment” as used herein can include any treatment of a disorder in a subject, particularly a human. It can include any one or more of the following: (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease or its symptoms or conditions. The term “treatment,” as used herein, can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (i.e., subjects in need thereof) can include those already with the disorder or those in which the disorder is to be prevented. As used herein, the term “treating” can include inhibiting the disease, disorder, or condition, e.g., impeding its progress, and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder, or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0045] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0046] As used herein, “therapeutic” can refer to treating, healing, or ameliorating a disease, disorder, condition, or side effect or decreasing the rate of advancement of a disease, disorder, condition, or side effect.

[0047] “Amino acid,” as used herein, refers to a molecule containing both an amino group and a carboxyl group. Amino acids include a- amino acids and P-amino acids. In certain forms, an amino acid is an alpha amino acid. Amino acids can be natural or synthetic. Amino acids include but are not limited to, the twenty standard or canonical amino acids: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, L), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trk, W), Tyrosine (Tyr, Y), and Valine (Vai, V). Common non-standard or non-canonical amino acids include but are not limited to, selenocysteine, pyrrolysine, and N-formylmethionine. The term “synthetic amino acid” or “non-natural amino acid,” as used herein, refers to an organic compound that has a structure similar to a natural amino acid so that it mimics the structure and reactivity of a natural amino acid. The synthetic amino acid, as defined herein, generally increases or enhances the properties of a peptide (e.g., selectivity or stability) when the synthetic amino acid is either substituted for a natural amino acid or incorporated into a peptide.

[0048] The terms “peptide,” “protein,” “polypeptide,” or “polyamino acid” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the amino group of another. In addition, as used herein, the term “polypeptide” refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc., and may contain modified amino acids other than the 20 gene-encoded amino acids. The polypeptides can be modified by either natural processes, such as post- translation processing, or by chemical modification techniques, which are well-known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side chains, and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in the given polypeptide. Also, a given polypeptide can have many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleoside or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylating, iodination, methylation, myristoylation, oxidation, PEGylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to proteins such as arginylation. Also included in the term “polypeptides” are cis- and trans-isomers, R- and S-enantiomers, D-isomers, L-isomers, diastereomers, conformers, and mixtures thereof.

[0049] The term “residue,” as used herein, refers to an amino acid that is incorporated into a polypeptide. The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

[0050] A “variant,” as used herein, means a polypeptide comprising one or more modifications such as substitutions, deletions, and / or truncations of one or more specific amino acid residues in the corresponding wild-type peptide. A variant of a polypeptide may be naturally occurring or synthetic, and may have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the wild-type polypeptide.

[0051] Conservative substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic -hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Exemplary conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. Further exemplary conservative substitutions are provided in the table below, with others being known in the art:

[0052] Amino Acid Substitutions Original Exemplary Conservative

[0053] Residue Substitutions

[0054] Ala Ser

[0055] Arg Lys; Gin

[0056] Asn Gin; His

[0057] Asp Glu

[0058] Cys Ser

[0059] Gin Asn, Lys

[0060] Glu Asp

[0061] Gly Pro

[0062] His Asn;Gln

[0063] He Leu; Vai

[0064] Leu He; Vai

[0065] Lys Arg; Gin

[0066] Met Leu; lie

[0067] Phe Met; Leu; Tyr

[0068] Ser Thr

[0069] Thr Ser

[0070] Trp Tyr

[0071] Tyr Trp; Phe

[0072] The term “inhibit” refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90%, 100%, or any amount of reduction in between as compared to native or control levels.

[0073] Compounds are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. The compounds described herein include enantiomers, mixtures of enantiomers, diastereomers, tautomers, racemates, and other isomers, such as rotamers, as if each is specifically described unless otherwise indicated or otherwise excluded by context. It is to be understood that the compounds provided herein may contain chiral centers. Such chiral centers may be of either the (7?-) or (S-) configuration. The compounds provided herein may either be enantiomerically pure or be diastereo meric or enantiomeric mixtures. It is to be understood that the chiral centers of the compounds provided herein may undergo epimerization in vivo. As such, one of skill in the art will recognize that administration of a compound in its (7?-) form is equivalent, for compounds that undergo epimerization in vivo, to administration of the compound in its CS'-J form. Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer, diastereomer, and meso compound, and a mixture of isomers, such as a racemic or scalemic mixture.

[0074] Compounds described herein may contain one or more double bonds and, thus, potentially give rise to cis / trans (E / Z) isomers, as well as other conformational isomers. Unless stated to the contrary, all such possible isomers are contemplated, as well as mixtures of such isomers.

[0075] Compounds described herein may also present as an equilibrium of tautomers. For example, ketones with an a-hydrogen can exist in an equilibrium of the keto form and the enol form. Likewise, amides with an N-hydrogen can exist in an equilibrium of the amide form and the imidic acid form. Unless stated to the contrary, all possible tautomers of the compounds described herein are contemplated.

[0076] As used herein, the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compound. Exemplary derivatives include, but are not limited to, salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound.

[0077] Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers, such as Sigma-Aldrich (formally MilliporeSigma, Burlington, MA) or Thermo Fisher Scientific Inc. (Waltham, MA), or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis (John Wiley and Sons, 2007); Organic Reactions (John Wiley and Sons, 2004); March's Advanced Organic Chemistry, (John Wiley and Sons, 8thEdition); and Larock's Comprehensive Organic Transformations (John Wiley and Sons, 3rdedition, 2017).

[0078] Peptides

[0079] The present disclosure provides doubly constrained peptides that mimic the first sheets in the “7+1” interface of the COR:COR dimer of LRRK2 which can inhibit or modulate the activity of LRRK2. These doubly constrained peptides are useful in treating neurological diseases, disorders, or conditions, for example, Parkinson’s disease.

[0080] Protein-protein interactions (PPIs) are a major driving force for the activation of many cellular pathways and associated disease pathologies, making them an attractive target for drug discovery (see Wells, J. A.; McClendon, C. L. Reaching for high-hanging fruit in drug discovery at protein-protein interfaces. Nature 2007, 450 (7172), 1001-1009). It is a significant challenge to design small molecules that inhibit PPIs for multiple reasons, including their small inherent size relative to the large, hydrophobic surfaces that often encompass PPI interfaces (see Arkin, M. R.; Tang, Y.; Wells, J. A. Small-molecule inhibitors of protein-protein interactions: progressing toward the reality. Chem Biol 2014, 21 (9), 1102-1114). As an alternative approach, we previously developed constrained peptides derived from the Roc domain of LRRK2 to target and allosterically inhibit the dimer interface (see Helton, L. G.; Soliman, A.; von Zweydorf, F.; Kentros, M.; Manschwetus, J. T.; Hall, S.; Gilsbach, B.; Ho, F. Y.; Athanasopoulos, P. S.; Singh, R. K.; et al. Allosteric Inhibition of Parkinson's-Linked LRRK2 by Constrained Peptides. ACS Chem Biol 2021, 16 (11), 2326-2338). A second compound as described herein that targeted the COR domain showed limited cell uptake, weak binding affinity, and limited cellular activity as compared to the Roc-targeting peptide. However, earlier studies on Roco proteins demonstrated the significance of the COR domain on protein dimerization (see Terheyden, S.; Ho, F. Y.; Gilsbach, B. K.; Wittinghofer, A.; Kortholt, A. Revisiting the Roco G-protein cycle. Biochem J 2015, 465 (1), 139-147). In addition, a recently published full-length structure of the inactive LRRK2 dimer revealed that the COR domain comprises a significant portion of the LRRK2 dimer interface and may play a key role in mediating dimerization (see Myasnikov, A.; Zhu, H.; Hixson, P.; Xie, B.; Yu, K.; Pitre, A.; Peng, J.; Sun, J. Structural analysis of the full-length human LRRK2. Cell 2021, 184 (13), 3519-3527 e3510). As described herein, the LRRK2 COR:COR dimer interface was predominately composed of stacked beta-sheets that form a “7+1” structure with seven beta-sheets from one COR subunit stack and one beta- sheet from the second COR subunit through hydrophobic interactions. Further, the previous homology model of the cryo-EM structure of the C-terminal portion of LRRK2 containing the ROC- COR- kinase- WD40 domains (LRRK2RCKW) portion of LRRK2 also identified the COR domain as a key component for LRRK2 dimerization (see Deniston, C. K.; Salogiannis, J.; Mathea, S.; Snead, D. M.; Lahiri, I.; Matyszewski, M.; Donosa, O.; Watanabe, R.; Bohning, J.; Shiau, A. K.; et al. Structure of LRRK2 in Parkinson's disease and model for microtubule interaction. Nature 2020, 588 (7837), 344-349). Taken together, both structures highlight an important role for the COR domain on LRRK2 dimerization.

[0081] A strategic method to bestow drug-like properties onto a-helical peptides was developed called peptide “stapling” (see Schafmeister C. E., et al. J. Am. Chem. Soc. 2000 122(24):5891-2). This strategy involves the incorporation of two non-natural amino acids within the peptide sequence that are disubstituted to contain a-methyl and a-alkenyl groups. The peptide secondary structure is conformationally locked via, e.g., a Grubbs I catalyzed ring-closing metathesis reaction to form a macrocyclic ring using the a-alkenyl groups (see Mansuy D., et al. Med Sci (Paris). 2005 21(11):995-6). Further studies have shown that this chemical modification introduces an entropically favorable pre-ordered binding state that increases substrate binding affinity, causes resistance to proteolytic degradation, and greatly enhances cell permeability (see Manschwetus, J. T. et al. Molecules 2019, 24(8):E1567; Flaherty, B. R. et al. ACS Infect Dis 2019, 5(4):506-514; Fulton, M. D. et al. Bioorg Med Chem 2018, 26(6): 1167-1173; Teng, Y. et al. Cancer Res 2016, 76(17):5133-42; Wang, Y. et al. ACS Chem Biol 2015, 10(6): 1502-10; and Wang, Y. et al. ACS Chem Biol 2014, 9(3):635-642). By applying this chemical modification to a peptide -based scaffold, large binding areas on protein surfaces can be targeted with a high degree of specificity that would otherwise be elusive for targeting using a small molecule approach.

[0082] “Peptide stapling” is a term coined from a synthetic methodology wherein two olefin-containing side-chains present in a polypeptide chain are covalently joined (e.g., “stapled together”) using a ring-closing metathesis (RCM) reaction to form a cross-linked ring (see, the cover art for J. Org. Chem. 2001 66(16) describing metathesis-based crosslinking of alpha-helical peptides; Blackwell et al. Angew. Chem. Int. Ed. 1994 37:3281). However, the term “peptide stapling” as used herein encompasses the joining of two double bond-containing side chains, two triple bond-containing side chains, or one double bond-containing and one triple bond-containing side chain, which may be present in a polypeptide chain, using any number of reaction conditions and / or catalysts to facilitate such a reaction, to provide a single “staple” polypeptide. Additionally, the term “peptide stitching” as used herein refers to multiple and tandem “stapling” events in a single polypeptide chain to provide a “stitched” (multiply stapled) polypeptide. The disclosed polypeptides can contain a hydrocarbon staple to chemically stabilize an a-helical shape.

[0083] In some aspects, the disclosed peptides include a hydrocarbon staple. The genesis of the hydrocarbon stapling technique can be traced to the ruthenium-based Grubb’s catalyst used for ring-closing metathesis. The a-helix features 3.6 residues per complete turn, which places the i, i+4, i+7, and i+11 side chains on the same face of the folded structure. Therefore, stapling cross-links two a, a disubstituted amino acids bearing olefinic chains of variable length at positions “i" and “i+4” or “i+7” in the peptide sequence. In general, the first step in designing stapled peptides for macromolecular targets is the identification of appropriate sites for incorporating the non-natural amino acids used to from the hydrocarbon cross-link. Generally, residues which are not involved in the target recognition are chosen as potential sites for incorporation of olefin-bearing building blocks. These sites are subsequently used to incorporate various suitable stapling systems such as i and i+3; i and i+4; or i and i+7. The classical strategy to stabilize the a-helical conformation in peptides employs covalent bonds between the i and i+3, i and i+4, or i and i+7 side chain groups.

[0084] In some aspects, the polypeptide comprises two non-natural amino acids on the same side of the a-helix that are crosslinked to stabilize the a-helical shape. For example, two non-natural amino acids can be four (i and i+4) or seven (i and i+7) amino acids apart. In some cases, the non-natural amino acids can comprise olefinic side chains, such as: (S)- 2-(2’-propenyl)alanine (“S3”); (S)-2-(4’-pentenyl)alanine (“S5”); (S)-2-(5’-hexenyl)alanine (“S6”); (S)-2-(7’-octenyl)alanine (“S8”); (R)-2-(2’-propenyl)alanine (“R3”); (R)-2-(4’- pentenyl)alanine (“R5”); (R)-2-(5’-hexenyl)alanine (“R6”); and (S)-2-(7’-octenyl)alanine (“S8”). The disclosed peptides can be stapled in any suitable pairing, including, but not limited to, a pairing selected from the group consisting of an S5-S5 pairing (i.e., i and i+4), an S5-R8 pairing (i.e., i and i+7), an S8-R5 pairing (i.e., i and i+7), an R3-S6 pairing (i.e., i and i+3), an R6-S3 pairing (i.e., i and i+3), and R3-S5 pairing (i.e., i and i+3), an R5-S3 pairing (i.e., i and i+3), or combinations of pairings within the polypeptide sequence.

[0085] The hydrocarbon bridge can then be formed, for example, by a ring-closing metathesis reaction catalyzed by benzylidenebis(tricyclohexyl-phosphine)- dichlororuthenium (Grubb’s catalyst). In other aspects, the ring-closing metathesis reaction can be performed by any other suitable metathesis catalyst as would be available to a person of ordinary skill in the art.

[0086] Stapling a peptide using an all-hydrocarbon cross-link has been shown to help maintain its native conformation and / or secondary structure, particularly under physiologically relevant conditions. For example, stapling a polypeptide predisposed to having an a-helical secondary structure can constrain the polypeptide to its native a-helical conformation. The constrained secondary structure may, for example, increase the peptide’s resistance to proteolytic cleavage, may increase the peptide’s hydrophobicity, may allow for better penetration of the peptide into the target cell’s membrane (e.g., through an energydependent transport mechanism such a pinocytosis), and / or may lead to an improvement in the peptide’s biological activity relative to the corresponding uncrosslinked (e.g., “unstapled”) peptide.

[0087] A number of alternative stapling methods are known to those in the art, each using a different form of macrocyclization chemistry and giving rise to stapled peptides with different bioactive properties. For example, the stapling may be one-component stapling. One-component stapling involves a direct bond-forming reaction between the side-chains of two amino acids. In some aspects, the one-component stapling method may comprise formation of an amide bond between two side chains of amino acids in the peptide. In some aspects, the one-component stapling technique may comprise, for example, a ring-closing metathesis, a lactamization, a cycloaddition (such as the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC, “copper-catalyzed click reaction”) or ring-strained azide-alkyne cycloaddition), a reversible reaction (such as formation of a disulfide bride or an oxime linkage), or thioether formation. The stapling technique may alternatively be a two- component stapling. Two-component stapling involves a bifunctional linker compound which forms a staple by reacting with two complementary native or non-native amino acids in the peptide of interest. Two-component stapling may employ, for example, a photoswitchable linker or a functionalized “double click” linker. In some aspects, the precursors may independently comprise residues that are an amino acid analog having an alkyne group on the side chain or an amino acid having an azide group on the side chain, and these groups react with a precursor to the staple having complementary alkyne and / or azide groups to from a triazole. Additional examples of staples and stapling methods appropriate for use in the stapled peptides disclosed herein are described in Walensky, L.D. et al., J. Med. Chem. 2014, 57:6275-6288; Lau, Y. H. et al., Chem. Soc. Rev. 2014, 00:1-12; Joy, S.T., et al., Chem. Commun. 52(33):5738-5741; and Zhao, H. et al. Angew. Chem. Int. Ed. 2016, 55:12088-12093, each of which are incorporated herein by reference in their entireties.

[0088] Other forms of chemical stabilization may also be used in the disclosed peptides. For example, amino acids, and unstapled, partially stapled, and stapled peptides and proteins, and unstitched, partially stitched, and stitched peptides and proteins may exist in particular geometric or stereoisomeric forms. The disclosed peptides can include all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)- isomers, racemic mixtures thereof, and other mixtures thereof. Where an isomer-enantiomer is preferred, it may, in some aspects, be provided substantially free of the corresponding enantiomer, and may also be referred to as “optically enriched”. “Optically enriched”, as used herein, means that the compound is made up of a significantly greater proportion of one enantiomer. In certain aspects, the compound of the present disclosure is made up of at least about 90% by weight of a preferred enantiomer. In other aspects, the compound is made up of at least about 95%, 98%, or 99% or more by weight of a preferred enantiomer.

[0089] The polypeptide can be a synthetic peptide containing non-natural amino acids, or a peptidomimetic. As used herein, “peptidomimetic” means a mimetic of a peptide which includes some alteration of the normal peptide chemistry. Peptidomimetic s typically enhance some property of the original peptide, such as increased stability, increased efficacy, enhanced delivery, increased half-life, etc. Use of peptidomimetic s can involve the incorporation of a non-amino acid residue with non-amide linkages at a given position. One aspect of the present disclosure is a peptidomimetic wherein the compound has a bond, a peptide backbone, or an amino acid component replaced with a suitable mimic. Some nonlimiting examples of non-natural amino acids which may be suitable amino acid mimics include, but are not limited to, P-alanine, L-a-aminobutyric acid, L-y-aminobutyric acid, L- a-aminoisobutyric acid, L-s-aminocaproic acid, 7-aminoheptanoic acid, L-aspartic acid, L- glutamic acid, N-s-Boc-N-a-CBZ-L-lysine, N-s-Boc-N-a-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, N-a-Boc-N-5-Cbz-L-ornithine, N-5-Boc-N-a-Cbz-L- orinithine, Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, and Boc-L-thioproline.

[0090] The disclosed peptides may also be substituted with any number of substituents or functional moieties. In general, the term “substituted” refers to the replacement of a hydrogen group in a given structure with a specified substituent group. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. As used herein, the term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds, any of the substituents described herein (for example aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thioxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thio, halo, etc.), and any combination thereof (for example, aliphatic amino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, hetero aryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like) that results in the formation of a stable moiety. The disclosed peptides can contain any and all such combinations in order to arrive at a stable substituent / moiety. For the disclosed peptides, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.

[0091] Peptides and peptidomimetics can be prepared by any method, such as by synthesizing the peptide or peptidomimetic, or by expressing a nucleic acid encoding an appropriate amino acid sequence in a cell and harvesting the peptide from the cell. Of course, a combination of such methods also can be used.

[0092] Examples of chemical synthesis technologies are solid phase synthesis and liquid phase synthesis. Solid phase synthesis methods are largely classified by the tBoc method and the Fmoc method, depending on the type of protective group used. Typically used protective groups include tBoc (t-butoxycarbonyl), Cl— Z (2-chlorobenzyloxycarbonl), Br — Z (2-bromobenzyloxycarbonyl), Bzl (benzyl), Fmoc (9-fluorenylmethoxycarbonyl), Mbh (4,4’ -dimethoxy dibenzyhydryl), Mtr (4-methoxy-2,3,6-trimethylbenzenesulfonyl), Trt (trityl), Tos (tosyl), Z (Benzyloxycarbonyl), and Clz-Bzl (2,6-dichlrobenzyl) for the amino groups; NO2 (nitro) and Pmc (2,2,5,7,8-pentamethylchromane-6-sulfonyl) for the guanidino groups; and t-Bu (t-butyl) for the hydroxyl groups. After synthesis of the desired peptide, it is subjected to one or more deprotection reactions and cut out from the solid support. Such peptide cutting reactions may be carried out with hydrogen fluoride or trifluoromethane sulfonic acid for the Boc method, or with TFA for the Fmoc method. Methods of de novo synthesizing of peptides and peptidomimetics are described, for example, in Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2005; and Peptide and Protein Drug Analysis, ed. Redi., R., Marcel Dekker, Inc., 2000.

[0093] Alternatively, the peptide may be synthesized using recombinant techniques. In this case, a nucleic acid encoding the peptide is cloned into an expression vector under the control of expression control sequences (e.g., a promoter, a terminator and / or an enhancer) allowing its expression. The expression vector is then transfected into a host cell (e.g., a human, CHO, mouse, monkey, fungal or bacterial host cell), and the transfected host cell is cultivated under conditions suitable for the expression of the peptide. Standard recombinant DNA and molecular cloning techniques are described, for example, in: Sambrook and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Silhavy et al., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); and Ausubel et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc, and Wiley-Interscience (1987).

[0094] The method of producing the peptide may optionally comprise the steps of purifying said peptide, chemically modifying said peptide, and / or formulating said peptide into a pharmaceutical composition.

[0095] In some aspects, the stapled peptide includes a helical motif (i.e., a stapled helical peptide). Different amino acid residues have different propensities for forming different secondary structures. For example, methionine (M), alanine (A), leucine (L), glutamate (E), and lysine (K) all have especially high a-helix forming propensities. Thus in some aspects, the stapled polypeptide includes one or more amino acid residues selected from methionine (M), alanine (A), leucine (L), glutamate (E), and lysine (K). In contrast, proline (P) and glycine (G) are a-helix disruptors. Thus in some aspects, the stapled polypeptide does not include one or more proline (P) and glycine (G) amino acid residues. In one aspect, a synthetic polypeptide is provided comprising a variant of an amino acid sequence of SEQ ID NO. 1:

[0096] GEGETLLKKWALYSFNDGEEHQKILLDL (SEQ ID NO. 1) wherein the variant comprises at least two pairs of non-natural amino acids, wherein each pair of the at least two pairs of non-natural amino acids is cross-linked.

[0097] In some aspects, the polypeptide formed from a variant of an amino acid sequence of SEQ ID NO. 1 comprises at least four non-natural amino acids independently selected from (S)-2-(2’-propenyl)alanine, (S)-2-(4’-pentenyl)alanine, (S)-2-(5’-hexenyl)alanine, (S)- 2-(7’-octenyl)alanine, (R)-2-(2’-propenyl)alanine, (R)-2-(4’-pentenyl)alanine, (R)-2-(5’- hexenyl)alanine, and (S)-2-(7’-octenyl)alanine.

[0098] In some aspects, the synthetic polypeptide is formed from a peptide comprising an amino acid sequence having at least 80% sequence similarity to a sequence selected from SEQ ID NO. 2 to 3. In some aspects, the synthetic polypeptide is formed from a peptide comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to a sequence selected from SEQ ID NO. 2 to 3.

[0099] In some aspects, the polypeptide is formed from a peptide comprising an amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 3:

[0100] KGEGEXLLKXWALYSFNDGEKHXKKLXKL (SEQ ID NO. 2) or

[0101] KGEGEXLLKXWGLYSFNDGEKHXKKLXKL (SEQ ID NO. 3), wherein each X is independently a non-natural amino acid capable of being crosslinked. In particular aspects, X is (S)-2-(4’-pentenyl)alanine.

[0102] In some aspects, the polypeptide comprises a polypeptide selected from: wherein:

[0103] Ala at each occurrence comprises an alanine residue or a conservative substitution or derivative thereof;

[0104] Asn at each occurrence comprises an asparagine residue or a conservative substitution or derivative thereof;

[0105] Asp at each occurrence comprises an aspartate residue or a conservative substitution or derivative thereof;

[0106] Glu at each occurrence comprises a glutamate residue or a conservative substitution or derivative thereof;

[0107] Gly at each occurrence comprises a glycine residue or a conservative substitution or derivative thereof;

[0108] His at each occurrence comprises a histidine residue or a conservative substitution or derivative thereof;

[0109] Leu at each occurrence comprises a leucine residue or a conservative substitution or derivative thereof;

[0110] Lys at each occurrence comprises a lysine residue or a conservative substitution or derivative thereof;

[0111] Phe at each occurrence comprises a phenylalanine residue or a conservative substitution or derivative thereof;

[0112] Ser at each occurrence comprises a serine residue or a conservative substitution or derivative thereof;

[0113] Trp at each occurrence comprises a tryptophan residue or a conservative substitution or derivative thereof; and

[0114] Tyr at each occurrence comprises a tyrosine residue or a conservative substitution or derivative thereof. In some aspects, the peptide is about 5 to 100 amino acids in length, including about 5 to 50 amino acids in length. In some aspects, the peptide is less than 51 amino acids in length, including less than 50, 45, 40, 35, 30, 25, 20, 15, or 10 amino acids in length. Therefore, the provided polypeptide can further constitute a fusion protein or otherwise have additional N-terminal, C-terminal, or intermediate amino acid sequences.

[0115] In some aspects, introduction of a hydrocarbon staple results in poor water solubility and cell permeability. To increase cell permeability and solubility of these peptides, the disclosed polypeptide can be linked to a cell permeability moiety. A “cell permeability” or a “cell-penetration” moiety refers to any molecule known in the art which is able to facilitate or enhance penetration of molecules through membranes. Non-limiting examples include: hydrophobic moieties such as lipids, fatty acids, steroids, and bulky aromatic or aliphatic compounds; moieties which may cell-membrane receptors or carriers, such a steroids, vitamins and sugars, natural and non-natural amino acids and transporter peptides. Examples for lipidic moieties which may be used according to the present disclosure include: Lipofectamine, TransfectACE, Trasfectam, Cytofectic, DMRIE, DLRIE, GAP- DLRIE, DOTAP, DOPE, DMEAP, DODMP, DPOC, DDAB, DOSPA, EDLPC, EDMPC, DPH, TMADPH, CTAB, lysyl-PE, DC-Cho, -alanyl cholesterol, DCGS, DPPES, DCPE, DMAP, DMPE, DOGS, DOHME, DPEPC, Pluronic, Tween, BRIJ, plasmalogen, phosphatidylethanolamine, phosphatidylcholine, glycerol-3-ethylphosphatidylcholine, dimethyl ammonium propane, trimethyl ammonium propane, diethyl ammonium propane, triethylammonium propane, dimethyldioctadecylammonium bromide, a sphingolipid, sphingomyelin, a lysolipid, a glycolipid, a sulfatide, a glyco sphingolipid, cholesterol, cholesterol ester, cholesterol salt, N- succinyldioleoylphosphatidylethanoleamine. 1,2- dioleoyl-sn-glycerol, 1 ,3-dipalmitoyl-2-succinylglycerol, 1 ,2-dipalmatoyl-sn-3- succinylglycerol, l-hexadecyl-2-palmitoylglycerophosphatidylethanolamine, palmitoylhomocysteine, N,N’-bis(dodecylaminocarbonylmethylene)-N,N’-bis(N,N,N- trimethylammoniumethylaminocarbonylmethylene)ethylene diamine tetraiodide; N5,N”- bis(hexadecylaminocarbonylmethylene)-N,N’,N”-tris(N,N,N- trimethylammoniumetyhlaminocarbonylmetylene)diethyltriamine hexaiodide; N,N- bis(dodecylaminocarbonyhnethylene-N,N-bis(N,N,N- trimethylammoniumethylaminocarbonylmethylene)cyclohexene- 1 ,4-diamine tetraiodide; l,7,7-tetra(N,N,N,N-tetramethylammoniumethylaminocarbonylmethylene)-3- hexadecylaminocarbonylmethylene- 1 ,3,7-triazaheptane heptaiodide; N5,N5,N’ ,N’ - tetra(N,N,-trimethylammoniumethylaminocarbonylmethylene)-N’-(15,2-dioleoylglycero-3- phosphoethanolaminocarbonylmethylene)diethylene triamine tetraiodide; dioleolylphosphatidylethanolamine; a fatty acid, a lysolipid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, a sphingolipid, a glycolipid, a glucolipid, a sulfatide, a glyco sphingolipid, phosphatidic acid, palmitic acid, stearic acid, arachidonic acid, oleic acid, a lipid bearing a polymer, a lipid bearing a sulfonated saccharide, cholesterol, tocopherol hemisuccinate, a lipid with an ether-linked fatty acid, a lipid with an ester-linked fatty acid, a polymerized lipid, diacetyl phosphate, stearylamine, cardiolipin, a phospholipid with a fatty acid of 6 to 8 carbons in length, a phospholipid with asymmetric acyl chains, 6-(5-cholesten-3b-yloxy)-l-thio-b-D- galactopyranoside, digalactosylglyceride, 6-(5-cholesten-3b-yloxy)hexyl-6-amino-6-deoxy- 1-thio-b-D-galactopyranoside, 6-(5-cholesten-3b-yloxy)hexyl-6-amino-6-deoxyl-l-thio-a- D-mannopyranoside, 12(((7’-diethylamino-coumarin-3-yl)carbonyl)methylamino)- octadecanoic acid; N-[12-(((7’-diethylaminocoumarin-3- yl)carbonyl)methylamino)octadecanoyl]-2-aminopalmitic acid, (cholesteryl)-4’ - trimethylammonio)butanoate; N-succinyldioleoyl-phosphatidylethanoleamine; 1 ,2-dioleoyl- sn-glycerol; l’-dipalmitoyl-sn-S-succinyl-glycerol, l,3-dipalmitoyl-2-succinylglycerol, 1- hexadecyl-2-palmitoylglycero-phosphoethanolamine, and palmitoylhomocysteine.

[0116] In some aspects, the disclosed polypeptide can be linked to a protein transduction domain to effectively enter a cell. The protein transduction domain sequence can eb any internalization sequence known or newly discovered in the art, or conservative variants thereof. Non-limiting examples of cellular internalization transporters and sequences include polyarginine (e.g., R9), Antennapedia sequences, TAT, HIV-TAT, Pentratin, Antp-3A (Antp mutant), Buforin II, Transportan, MAP (model amphipathic peptide), K-FGF, Ku70, Prion, pVEC, Pep-1, SynBl, Pep-7, HN-1, BGSC (Bis-Guanidinium-Spermidine- Cholestero), and BGTG (Bis-Guanidinium-Tren-Cholesterol).

[0117] Addition of water soluble polymers or carbohydrates to polypeptide drugs has been shown to prevent their degradation and increase their half-life. For instance, “PEGylation” of polypeptide drugs protects them and improves their pharmacodynamic and pharmacokinetic profiles. The PEGylation process attaches repeating units of polyethylene glycol (PEG) to a polypeptide drug. PEGylation of molecules can lead to increased resistance of drugs to enzymatic degradation, increase half-life in vivo, reduced dosing frequency, decreased immunogenicity, increased physical and thermal stability, increased solubility, increase liquid stability, and reduced aggregation. Therefore, in some aspects the disclosed polypeptide is covalently linked to a water soluble polymer, such as polyethylene glycol.

[0118] The most common route for PEG conjugation of polypeptides has been to activate the PEG with functional groups suitable for reactions with lysine and N-terminal amino acid groups. The monofunctionality of methoxy PET makes it particularly suitable for protein and peptide modification because it yields reactive PEGs that do not produce cross-linked polypeptides, as long as diol PEG has been removed. Branched structures of PEG have also been proven to be useful for PEGylation of a protein or a peptide. For example, a branched PEG attached to a protein has properties of a much larger molecule than a corresponding linear mPEG of the same molecular weight. Branched PEGs also have the advantage of adding two PEG chains per attachment site on the protein, therefore reducing the chance of protein inactivation due to attachment. Furthermore, these structures are more effective in protecting proteins from proteolysis, in reducing antigenicity, and in reducing immunogenicity.

[0119] To increase cell permeability and solubility of these peptides, the peptides can be optimized to increase their amphipathic properties. In some cases, an overall net charge (neutral or positive) is needed for permeability. Any method that alters the overall net charge can affect permeability. In some cases, 1, 2, 3, 4, or more hydrophilic residues can be added on the solvent-exposed face of the helix. For example, the hydrophilic residue can be a lysine, aspartic acid, glutamic acid, arginine, histidine, serine, asparagine, or glutamine. In some cases, lysine and / or arginine is used since they have positive charges that help increase permeability. Non-natural amino acids bearing hydrophilic or charged properties can also be added.

[0120] Methods of Making

[0121] The polypeptides described herein can be prepared in a variety of ways known to one skilled in the art of organic synthesis or variations thereon as appreciated by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions can vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art.

[0122] Variations on the compounds described herein include the addition, subtraction, or movement of the various constituents as described for each compound. Similarly, when one or more chiral centers are present in a molecule, the chirality of the molecule can be changed. Additionally, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups, can be determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4thEd., Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.

[0123] The starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St. Louis, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck (Whitehouse Station, NJ), Johnson & Johnson (New Brunswick, NJ), Aventis (Bridgewater, NJ), AstraZeneca (Wilmington, DE), Novartis (Basel, Switzerland), Wyeth (Madison, NJ), Bristol-Myers-Squibb (New York, NY), Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott (Abbott Park, IL), Schering Plough (Kenilworth, NJ), or Boehringer Ingelheim (Ingelheim, Germany), or are prepared by methods known to those skilled in the art following procedures set forth in the references such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd’s Chemistry of Carbon Compounds, Volumes 1-5 and Suppiementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March’s Advanced Organic Chemistry (John Wiley and Sons, 4thEdition); and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Other materials, such as the pharmaceutical carriers disclosed herein, can be obtained from commercial sources.

[0124] Reactions to produce the compounds described herein can be carried out in solvents, which can be selected by one of skill in the art of organic synthesis. Solvents can be substantially reactive with the starting materials (reactants), the intermediates, or products under the conditions at which the reactions are carried out, i.e., temperature and pressure. Reactions can be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formulation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., or13C), infrared spectroscopy, spectrophotometry (e.g. UV-visible), or mass spectrometry, or by chromatography such as high-performance liquid chromatography (HPLC) or thin layer chromatography. The disclosed compounds can be prepared by solid phase peptide synthesis wherein the amino acid a-N-terminal is protected by an acid or base protecting group. Such protecting groups should have the properties of being stable to the conditions of peptide linkage formation while being readily removable without destruction of the growing peptide chin or racemization of any of the chiral centers contained therein. Suitable protecting groups are 9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, a,a-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o- nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, and the like. The 9- fluorenylmethyloxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of the disclosed compounds. Other preferred side chain protecting groups are: for side chain amino groups like lysine and arginine, 2,2,5,7,8-pentamethylchroman-6-sunfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzene- sulfonyl, Cbz, Boc, and adamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxycarbonyl, 2,6- dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopentyl, and acetyl (Ac); for serine, t-butyl, benzyl, and tetrahydropyranyl; for histidine, trityl, benzyl, Cbz, p- toluenesulfonyl and 2,4-dinitrophenyl; for tryptophan, formyl; for aspartic and glutamic acid, benzyl and t-butyl; and for cysteine, triphenylmethyl (trityl). In solid phase peptide synthesis methods, the a-C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports useful for the above synthesis are those materials which are inert to the reagents and reaction of the stepwise condensation-deprotection reactions, as well as being insoluble in the media use. Solid supports for synthesis of a-C-terminal carboxy peptides include 4-hydroxymethylphenoxymethyl-copoly(styrene-l% divinylbenzene) or 4-(2’,4’-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin available from Applied Biosystems (Foster City, CA). N,N’ -diisopropylcarbodiimide (DIC) or O-benzotriazol-l-yl-N,N,N’N’-tetramethyluronium hexafluorophosphate (HBTU), with or without 4-dimethylaminopyridine (DMAP), 1 -hydroxybenzotriazole (HOBt), benzotriazole- l-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCI), mediate coupling for from about 0.5 to about 24 hours at a temperature of between 10 °C and 50 °C in a solvent such as dichloromethane, DMF, or NMP When the solid support is 4-(2’,4’-dimethoxyphenyl- Fmoc-aminomethyl)phenoxy-acetamidoethyl resin, the Fmoc group is cleaved with a secondary amine, preferably piperidine, prior to coupling with the a-C-terminal amino acid as described above. One method for coupling to the deprotected (3’,4’-dimethoxyphenyl- Fmoc-aminomethyl)phenoxy-acetamidoethyl resin is O-benzotriazol-l-yl-N,N,N’,N’- tetramethyluronium hexafluorophosphate (HBTU, 1 equiv.) and 1 -hydroxybenzotriazole (HOBt, 1 equiv.) in DMF or O-(lH-6-chlorobenzotriazol-l-yl)-l , 1 ,3,3,-tctramcthyluronium hexafluorophosphate (HCTU, 1 equiv.) and N,N-diisopropylethylamine (DIEA, 1 equiv.) in NMP. The coupling of successive protected amino acids can be carried out in an automatic polypeptide synthesize. In one example, the a-N-terminal in the amino acids of the growing peptide chain are protected with Fmoc. The removal of the Fmoc protecting group from the a-N-terminal side of the growing peptide is accomplished by treatment with a secondary amine, preferably piperidine. Each protected amino acid is then introduced in about 3-fold molar excess, and the coupling is preferably carried out in DMF. The coupling agent can be O-benzotriazol-l-yl-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU, 1 equiv.) and 1 -hydroxybenzotriazole (HOBt, 1 equiv.). At the end of the solid phase synthesis, the polypeptide is removed from the resin and deprotected, either successively or in a single operation. Removal of the polypeptide and deprotection can be accomplished in a single operation by treating the resin-bound polypeptide with a cleave reagent comprising thianisole, water, ethanedithiol, and trifluoroacetic acid. In cases wherein the a-C-terminal of the polypeptide is an alkylamide, the resin is cleaved by aminolysis with an alkylamine. Alternatively, the peptide can be removed by transesterification, e.g. with methanol, followed by aminolysis or by direct transamidation. The protected peptide can be purified at this point or taken to the next step directly. The removal of the side chain protecting groups can be accomplished using the cleavage cocktail described above. The fully deprotected peptide can be purified by a sequence of chromatographic steps employing any or all of the following types: ion exchange on a weakly basic resin (acetate form); hydrophobic adsorption chromatography or underivatized polystyrene-divinylbenzene (for example, Amberlite XED); silica gel adsorption chromatography; ion exchange chromatography on carboxymethylcellulose; partition chromatography, e.g. on Sephadex G-25, LH-20 or countercurrent distribution; or high performance liquid chromatography (HPLC), especially reverse-phase HPLC on octyl- or octadecylsilyl-silica bonded column packing.

[0125] Pharmaceutical Compositions

[0126] Also disclosed are pharmaceutical composition comprising any of the polypeptides disclosed herein in a pharmaceutically acceptable carrier. The disclosed polypeptides can be incorporated in the formulations described below as neutral compounds, pharmaceutically acceptable salts, and / or prodrugs thereof. Pharmaceutical formulations can be designed for immediate release, sustained release, delayed release and / or a burst release of one or more of the disclosed polypeptides in a therapeutically effective amount.

[0127] The compounds described herein can be formulated for parenteral administration. Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Typically, such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for use in preparing solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and microemulsions thereof; liposomes, or emulsomes.

[0128] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.) and combinations thereof.

[0129] Solutions and dispersions of the polypeptides as neutral compounds or pharmaceutically acceptable salts thereof can be prepared in water or another solvent or dispersion medium suitable mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, or combinations thereof.

[0130] Suitable surfactants for use in the disclosed pharmaceutical compositions may be anionic, cationic, amphoteric, or nonionic surface active agents. Suitable anionic surfactants include, but are not limited, to, carboxylate, sulfonate, and sulfate ions. Examples of anionic surfactants include sodium, potassium, and / or ammonium salts of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate and sodium bis-(2- ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostereate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-P-alanine, sodium-N-lauryl-P- iminodipropionate, myristoamphoacetate, lauryl betaine, and lauryl sulfobetaine.

[0131] The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the one or more polypeptides.

[0132] The formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

[0133] Water soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.

[0134] Sterile injectable solutions can be prepared by incorporating the one or more polypeptides described herein in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required additional ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the one or more disclosed polypeptides plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are well known in the art.

[0135] The parenteral formulations described herein can be formulated for controlled release including immediate release, delayed release, extended release, pulsatile release, and combinations thereof. For parenteral administration, the polypeptides described herein and optionally one or more additional active agents can be incorporated into microparticles, nanoparticles, or combinations thereof that provide controlled release. For example, the compounds and / or one or more additional active agents can be incorporated into polymeric microparticles which provide controlled release of the polypeptide(s). Release of the polypeptide(s) is controlled by diffusion of the polypeptide(s) out of the microparticles and / or degradation of polymeric particles by hydrolysis and / or enzymatic degradation. Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives.

[0136] Polymers which are slowly soluble and form a gel in an aqueous environment, such as hydroxypropyl methylcellulose or polyethylene oxide may also be suitable as material for drug-containing microparticles. Other polymers include, but are not limited to, poly anhydrides, poly(ester anhydrides), polyhydroxyacids such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3 -hydroxybutyrate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof.

[0137] The polypeptide can also be formulated for depot injection. In a depot injection, the active agent is formulated with one or more pharmaceutically acceptable carriers that provide for the gradual release of the polypeptide over a period of hours or days after injection. The depot formulation can be administered by any suitable means; however, the depot formulation is typically administered via subcutaneous or intramuscular injection. A variety of carriers may be incorporated into the depot to provide for the controlled release of the active agent. In some cases, depot formulations contain one or more biodegradable polymeric or oligomeric carriers. Suitable polymeric carriers include, but are not limited to, poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid)- polyethylene glycol (PLA-PEG) block copolymers, poly anhydrides, poly(ester anhydrides), poly(glycolic acid) (PGA), poly-3-hydroxybutyrate (PHB) and copolymers thereof, poly-4- hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone, cellulose, hydroxypropyl methylcellulose, ethylcellulose, as well as blends, derivatives, copolymers, and combinations thereof. In depot formulations containing a polymeric or oligomeric carrier, the carrier and the polypeptide can be formulated as a solution, an emulsion, or a suspension. One or more polypeptides, and optionally one or more additional active agents, can also be incorporated into polymeric or oligomeric microparticles, nanoparticles, or combinations thereof.

[0138] Formulations may also be in the form of an organogel (assuming the polypeptide is relatively water insoluble) or a hydrogel. Numerous gel formulations are known. See, for example, U.S. Patent No. 5,411,737. Hydrogels, especially those further including nanoparticles or microparticles for sustained, immediate and / or delayed release, can also be used.

[0139] Oral pharmaceutical dosage forms are either solid, gel or liquid. The solid dosage forms are tablets, capsules, granules, and bulk powders. Types of oral tablets include compressed, chewable lozenges and tablets which may be enteric-coated, sugar-coated or film-coated. Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form with the combination of other ingredients known to those skilled in the art.

[0140] The polypeptides may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the polypeptides alone or in combination with other pharmaceutically acceptable excipients can also be administered. These solutions, particularly those intended for ophthalmic use, may be formulated as 0.01% - 10% isotonic solutions, having a pH of about 5-7, with appropriate salts.

[0141] Other routes of administration, such as transdermal patches, including iontophoretic and electrophoretic devices, vaginal and rectal administration, are also contemplated herein. Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those of skill in the art. For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect. Rectal suppositories as used herein mean solid bodies for insertion into the rectum which melt and soften at body temperature releasing one or more pharmacologically or therapeutically active ingredients. Pharmaceutically acceptable substances utilized in rectal suppositories are bases or vehicles and agents to raise the melting point. Examples of bases include cocoa butter (Theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) and appropriate mixtures of mono-, di-, and triglycerides of fatty acids. Combinations of the various bases may be used. Agents to raise the melting point of suppositories include spermaceti and wax. Rectal suppositories may be prepared either by the compressed method or by molding. The weight of a rectal suppository, in one aspect, is about 2 to 3 g.

[0142] Methods of Treatment The polypeptides described herein can be used to treat or prevent a disease, disorder, or condition in a patient in need thereof. In some aspects, treatment refers to partial or complete alleviation, amelioration, relief, inhibition, delaying onset, reducing severity, and / or incidence of the disease, disorder, or condition in the patient.

[0143] The terms “improve”, “increase”, “reduce”, “decrease”, and the like, as used herein, indicate values that are relative to a control. In some aspects, a suitable control is a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein.

[0144] In some aspects, the patient is an individual who has recently been diagnosed with a disease, disorder, or condition. Typically, early treatment (treatment commencing as soon as possible after diagnosis) is important to minimize the effects of the disease, disorder or condition and to maximize the benefits of treatment.

[0145] In some aspects, the polypeptides described herein can be used to treat or prevent a neurological disease or condition. In some aspects, the disease, disorder, or condition is a neurodegenerative disease. In some aspects, the neurological or neurogenerative disease or condition that may be treated includes, for example, Alzheimer’s disease, cerebral edema, cerebral ischemia, multiple sclerosis, neuropathies, Parkinson’s disease, Huntington’s disease, blunt or surgical trauma (including postsurgical cognitive dysfunction and spinal cord or brain stem injury), as well as neurological aspects of disorders such as degenerative disc disease and sciatica.

[0146] Further examples of neurodegenerative disorders include, but are not limited to, Alexander’s disease, Alper’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, ataxia telangiectasia, Batten disease, bovine spongiform encephalopathy, Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt- Jakob disease, Huntington’s disease, HIV- associated dementia, Kennedy’s disease, Krabbe’s disease, lewy body dementia, Machado -Joseph disease, multiple sclerosis, multiple system atrophy, narcolepsy, neuroborreliosis, Parkinson’s disease, Pelizaeus-Merzbacher disease, Pick’s disease, primary lateral sclerosis, prion diseases, Refsum’s disease, Sandhoff’s disease, Schilder’s disease, subacute combined degeneration of spinal cord secondary to pernicious anemia, schizophrenia, spinocerebellar ataxia, spinal muscular atrophy, Steele-Richardson- Olszewski disease, and tabes dorsalis. In some aspects, a neurodegenerative disease includes any pathological state involving neuronal degeneration, including Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis. Polyglutamine disease, including Huntington’s disease, are neurodegenerative diseases caused by an abnormally expanded polyglutamine tract in the causative gene products.

[0147] Thus in one aspect, a method of treating a neurodegenerative disease in a subject is provided, comprising administering to the subject a therapeutically effective amount of a polypeptide described herein. In some aspects, the subject is an individual suffering from or susceptible to a neurodegenerative disease. In some aspects, the subject is a human.

[0148] In some aspects, a method of treating Parkinson’s disease in a subject is provided comprising administering to the subject a therapeutically effective amount of a polypeptide described herein.

[0149] In some aspects, a method of treating Huntington’s disease in a subject is provided comprising administering to the subject a therapeutically effective amount of a polypeptide described herein.

[0150] In some aspects, a method of treating Alzheimer’s disease in a subject is provided comprising administering to the subject a therapeutically effective amount of a polypeptide described herein.

[0151] In some aspects, a method of treating amyotrophic lateral sclerosis (ALS) in a subject is provided comprising administering to the subject a therapeutically effective amount of a polypeptide described herein.

[0152] In another aspect, a method is provided for treating a disorder or condition that is treated by inhibiting LRRK2 activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide described herein.

[0153] In another aspect, a method is provided for treating or preventing nerve cell degeneration, the method comprising administering to a subject suffering or susceptible to nerve cell degeneration a therapeutically effective amount of a polypeptide described herein.

[0154] In particular aspects, the disorder or condition comprises Parkinson’s disease or a Parkinson-plus syndrome. Parkinson-plus syndromes include multiple system atrophy and progressive supranuclear party (PSP). In certain aspects, the polypeptides described herein are used to treat Parkinson’s disease that presents in one or more forms, including, but not limited to sporadic Parkinson’s disease, a familial form of Parkinson’s disease, autosomal recessive early-onset Parkinson’s disease, or post-encephalitic Parkinson’s disease. In some aspects, a therapeutically effective amount of the polypeptides described herein, when administered to a subject having Parkinson’s disease or a Parkinson-plus syndrome, ameliorates or lessens the severity of one or more of the symptoms of the disease, including but not limited to tremor, rigidity of the limbs and trunk, akinesia, bradykinesia, and postural abnormalities.

[0155] Further provided herein are methods to treat, prevent, delay the onset or progression of, or alleviate the symptoms of a disorder or condition that can be treated by inhibiting or diminishing LRRK2 activity in a subject in need of such treatment, the method comprising administering a therapeutically effective amount of a polypeptide described herein.

[0156] The polypeptides provided herein can treat the above-described diseases, disorders, or conditions, for instance, by disrupting native protein-protein interactions with LRRK2. In some particular aspects, the polypeptides provided herein may prevent dimerization of LRRK2.

[0157] In some aspects, the polypeptides described herein may be used in a method of treating a disorder or condition selected from: Parkinson’s disease; migraine; epilepsy; Alzheimer’s disease; brain injury; stroke; cerebrovascular disease (including cerebral arteriosclerosis, cerebral amyloid angiopathy, hereditary cerebral hemorrhage, and brain hypoxia-ischemia); cognitive disorders (including amnesia, senile dementia, HIV-associated dementia, Alzheimer’s disease, Huntington’s disease, Lewy body dementia, vascular dementia, drug-related dementia, tardive dyskinesia, myoclonus, dystonia, delirium, Pick’s disease, Creutzfeldt-Jacob disease, HIV disease, Gilles de la Tourette’s syndrome, epilepsy, muscular spasms and disorders associated with muscular spasticity or weakness including tremors, and mild cognitive impairment); mental deficiency (including spasticity, Down syndrome and fragile X syndrome); sleep disorders (including hypersomnia, circadian rhythm sleep disorder, insomnia, parasomnia, and sleep deprivation); psychiatric disorders such as anxiety (including acute stress disorder, generalized anxiety disorder, social anxiety disorder, panic disorder, post-traumatic stress disorder, agoraphobia, and obsessive compulsive disorder); factitious disorder (including acute hallucinatory mania); impulse control disorders (including compulsive gambling and intermittent explosive disorder); mood disorders (including bipolar I disorder, bipolar II disorder, mania, mixed affective state, major depression, chronic depression, seasonal depression, psychotic depression, premenstrual syndrome (PMS), premenstrual dysphoric disorder (PDD), and postpartum depression); psychomotor disorder; psychotic disorders (including schizophrenia, schizoaffective disorder, schizophreniform, and delusional disorder); drug dependence (including narcotic dependence, alcoholism, amphetamine dependence, cocaine addiction, nicotine dependence, and drug withdrawal syndrome); eating disorders (including anorexia, bulimia, binge eating disorder, hyperphagia, obesity, compulsive eating disorders and pagophagia); sexual dysfunction disorders; urinary incontinence; neuronal damage disorders (including ocular damage, retinopathy or macular degeneration of the eye, tinnitus, hearing impairment and loss, and brain edema) and pediatric psychiatric disorders (including attention deficit disorder, attention deficit / hyperactivity disorder, conduct disorder, and autism) in a subject, preferably a human, wherein the method comprises administering to a subject a therapeutically effective amount of the polypeptides described herein.

[0158] Other disorder which may be treated with the compounds described herein include, but are not limited to, lysosomal disorders (for example Niemann-Pick Type C disease, Gaucher disease), Crohn’s disease, thyroid, renal (including papillary renal), breast, lung, and prostate cancers, leukemias (including acute myelogenous leukemia), lymphomas, multiple sclerosis, rheumatoid arthritis, system lupus erythematosus, autoimmune hemolytic anemia, pure red cell aplasia, idiopathic thrombocytopenic purpura (ITP), Evans syndrome, vasculitis, bullous skin disorders, type 1 diabetes mellitus, Sjogren’s syndrome, Devic’s disease and inflammatory myopathies.

[0159] In some aspects, a method of treating Crohn’s disease in a subject in need thereof is provided comprising administering a therapeutically effective amount of a synthetic stapled peptide to the subject.

[0160] In view of the described compounds, compositions, and methods, hereinbelow are described certain more particular aspects of the disclosure. These particularly recited aspects should not, however, be interpreted to have any limiting effect on any different claims containing different or more general teachings described herein, or that the “particular” aspects are somehow limited in some way other than the inherent meanings of the language and formulae literally used therein. Aspect 1. A synthetic polypeptide comprising a variant of an amino acid sequence of SEQ ID NO. 1, wherein the variant comprises at least two pairs of non-natural amino acids, wherein each pair of the at least two pairs of non-natural amino acids is cross-linked.

[0161] Aspect 2. The synthetic polypeptide of aspect 1, wherein the synthetic polypeptide is formed from a variant of an amino acid sequence of SEQ ID NO. 1 comprising at least four non-natural amino acids independently selected from (S)-2-(2’-propenyl)alanine, (S)-2-(4’- pentenyl)alanine, (S)-2-(5’-hexenyl)alanine, (S)-2-(7’-octenyl)alanine, (R)-2-(2’- propenyl)alanine, (R)-2-(4’-pentenyl)alanine, (R)-2-(5’-hexenyl)alanine, and (S)-2-(7’- octenyl)alanine.

[0162] Aspect 3. The synthetic polypeptide of aspect 1 or aspect 2, wherein each of the at least two pairs of non-natural amino acids are three, four, or seven amino acids apart.

[0163] Aspect 4. The synthetic polypeptide of aspect 1, wherein the synthetic polypeptide is formed from a peptide comprising an amino acid sequence having at least 80% sequence similarity to a sequence selected from SEQ ID NO. 2 to 3.

[0164] Aspect 5. The synthetic polypeptide of aspect 1, wherein the synthetic polypeptide is formed from a peptide comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to a sequence selected from SEQ ID NO. 2 to 3.

[0165] Aspect 6. The synthetic polypeptide of aspect 1, wherein the synthetic polypeptide is formed from a peptide comprising an amino acid sequence selected from SEQ ID NO. 2 to 3.

[0166] Aspect 7. The synthetic polypeptide of aspect 1, wherein the polypeptide is selected from: wherein:

[0167] Ala at each occurrence comprises an alanine residue or a conservative substitution or derivative thereof;

[0168] Asn at each occurrence comprises an asparagine residue or a conservative substitution or derivative thereof;

[0169] Asp at each occurrence comprises an aspartate residue or a conservative substitution or derivative thereof;

[0170] Glu at each occurrence comprises a glutamate residue or a conservative substitution or derivative thereof;

[0171] Gly at each occurrence comprises a glycine residue or a conservative substitution or derivative thereof;

[0172] His at each occurrence comprises a histidine residue or a conservative substitution or derivative thereof;

[0173] Leu at each occurrence comprises a leucine residue or a conservative substitution or derivative thereof;

[0174] Lys at each occurrence comprises a lysine residue or a conservative substitution or derivative thereof;

[0175] Phe at each occurrence comprises a phenylalanine residue or a conservative substitution or derivative thereof;

[0176] Ser at each occurrence comprises a serine residue or a conservative substitution or derivative thereof;

[0177] Trp at each occurrence comprises a tryptophan residue or a conservative substitution or derivative thereof; and

[0178] Tyr at each occurrence comprises a tyrosine residue or a conservative substitution or derivative thereof. Aspect 8. A pharmaceutical composition comprising a synthetic polypeptide of any one of aspects 1-7 and a pharmaceutically acceptable carrier.

[0179] Aspect 9. A cell comprising a synthetic polypeptide of any one of aspects 1-7.

[0180] Aspect 10. A method of treating a neurological disease, disorder, or condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a synthetic polypeptide of any one of aspects 1-7 or a pharmaceutical composition of aspect 8.

[0181] Aspect 11. The method of aspect 10, wherein the neurological disease, disorder, or condition comprises Parkinson’s disease.

[0182] Aspect 12. The method of aspect 10, wherein the neurological disease, disorder, or condition comprises Huntington’s disease.

[0183] Aspect 13. The method of aspect 10, wherein the neurological disease, disorder, or condition comprises Alzheimer’s disease.

[0184] Aspect 14. The method of aspect 10, wherein the neurological disease, disorder, or condition comprises amyotrophic lateral sclerosis (ALS).

[0185] Aspect 15. A method of treating Crohn’s disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a synthetic polypeptide of any one of aspects 1-7 or a pharmaceutical composition of aspect 8.

[0186] Aspect 16. A method of treating a disorder or condition that is treated by inhibition or modulation of LRRK2 activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a synthetic polypeptide of any one of aspects 1-7 or a pharmaceutical composition of aspect 8.

[0187] A number of aspects of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other aspects are within the scope of the following claims.

[0188] By way of non-limiting illustration, examples of certain aspects of the present disclosure are given below. EXAMPLES

[0189] The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, and methods claimed herein are made and evaluated and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy concerning numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in degrees Celsius or is at ambient temperature, and pressure is at or near atmospheric pressure.

[0190] Example 1. Doubly Constrained COR-Derived Peptides Inhibit LRRK2 Dimerization

[0191] Mis sense mutations along the Leucine-Rich Repeat Kinase-2 (LRRK2) protein are a major contributor to Parkinson’s Disease (PD), the second most commonly occurring neurodegenerative disorder worldwide. We recently reported the development of allosteric constrained peptide inhibitors that target and downregulate LRRK2 activity through disruption of LRRK2 dimerization. In this example, we designed doubly constrained peptides with the objective of inhibiting COR-COR mediated dimerization at the LRRK2 dimer interface. We show that the doubly constrained peptides are cell-permeant, bind wildtype and pathogenic LRRK2, inhibit LRRK2 dimerization and kinase activity, inhibit LRRK2-mediated neuronal apoptosis, and in contrast to ATP-competitive LRRK2 kinase inhibitors, they do not induce the mislocalization of LRRK2 to skein-like structures in cells. This example highlights the significance of COR-mediated dimerization on LRRK2 activity while also highlighting the use of doubly constrained peptides to stabilize discrete secondary structural folds within a peptide sequence.

[0192] In this example, we describe the design and synthesis of doubly constrained peptides that mimic the first sheets in the “7+1” interface of the COR:COR dimer. These doubly constrained peptides permeate cells, bind to both wild- type and PD-associated pathogenic forms of LRRK2, inhibit LRRK2 dimerization, downregulate LRRK2-mediated kinase activity, inhibit LRRK2-mediated neuronal apoptosis and do not induce mislocalization of LRRK2 to skein-like structures in cells. This example supports the hypothesis that the COR:COR dimer interface is critical for LRRK2 dimerization and provides an alternative strategy to LRRK2-targeted PD therapy. Further, we demonstrate that the addition of a single staple was not sufficient for cell permeation and the second staple was necessary for cell-based experiments, highlighting an additive impact of a second staple for promoting cell permeation.

[0193] METHODS

[0194] Peptide Synthesis

[0195] All solvents used in these peptide syntheses were HPLC grade. N-a-Fmoc protected amino acids and rink amide MB HA resin were purchased from Novabiochem. Fmoc- 11- amino-3,6,9-trioxaundecanoic acid (PEG3) was purchased from ChemPep. Olefinic amino acid, S5 ((S)-N-Fmoc-2-(4-pentenyl) alanine)), labeling reagent 5,6- carboxyfluorescein (FAM), and Grubbs 1stgeneration catalyst were all purchased from Sigma-Aldrich. Labeling reagent D-biotin was purchased from GoldBio. All other synthesis reagents and organic solvents were purchased from Fisher Scientific unless stated otherwise.

[0196] Peptides were synthesized on Rink amide MBHA resin using standard N-a-Fmoc amino acids and following the standard Fmoc solid phase peptide synthesis. First, MBHA resin was calibrated in N-methylpyrrolidinone (NMP) with agitation for 10 minutes. Fmoc- group deprotection was carried out using 25% (v / v) piperidine in 75% (v / v) NMP solution for 25 minutes with agitation. After each deprotection, peptides were washed three times for 30 seconds in NMP prior to amino acid coupling.

[0197] During each coupling reaction, 10 equivalents of standard amino acid, 9.9 equivalents of 2-(6-chloro- IH-benzo triazole- 1-yl)- 1 , 1 ,3, 3 -tetramethy laminium hexafluorophosphate (HCTU) in NMP, and 20 equivalents of N, N-diisopropyl ethylamine (DIEA) were added to the resin with agitation for 45 minutes followed by three 30-second washes with NMP. To incorporate the olefinic amino acid, 4 equivalents of S5 ((S)-N- Fmoc-2-(4-pentenyl) alanine)), 3.9 equivalents of HCTU, and 20 equivalents of DIEA were added to the resin with agitation for 45 minutes. Following the addition of all amino acids, two separate cycles of ring closing metathesis (RCM) were performed using 0.4 equivalents of first- generation Grubbs in 1,2-dichloroethane (DCE) for 1 hour each.

[0198] Modifications were made to the N-terminus of each sequence to improve solubility and label peptides based on experimental need. First, Fmoc- 11- amino-3,6,9- trioxaundecanoic acid (PEG3) was added to each peptide using 4 equivalents under standard coupling conditions with agitation. For fluorescently labeled peptides, 2 equivalents of 5,6- carboxyfluorescein in N,N-Dimethylformamide (DMF), 1.8 equivalents of HCTU, and 4.6 equivalents of DIEA were added to the (5 / 6FAM)-labeled versions of the sequences overnight with agitation. For biotin-labeled peptides, 10 equivalents of D-biotin, 9.9 equivalents of HCTU, and 20 equivalents of DIEA were added to the resin with a 1:1 mixture of dimethyl sulfoxide (DMSO) and DMF overnight with agitation.

[0199] Following labeling, all peptides were separately cleaved from the resin using a solution of 95% (v / v) trifluoroacetic acid (TFA), 2.5% (v / v) triisopropylsilane (TIS), and 2.5% (v / v) distilled water. This solution was incubated for 5 hours at room temperature under constant rotation. Peptide products were precipitated in methyl-tert-butyl ether (MTBE) and air-dried overnight prior to purification and characterization.

[0200] Sequences of each peptide used in this example are as follows. Red asterisks (*) represent Ss residues:

[0201] FAM ECOR parent: (5 / 6 FAM)-PEG3- GEGETLLKKWALYSFNDGEEHQKILLDL

[0202] (SEQ ID NO. 4)

[0203] FAM ECOR: (5 / 6 FAM)- PEG3- KGEGE*LLK*WALYSFNDGEKH*KKL*KL

[0204] (SEQ ID NO. 5)

[0205] FAM ECOR SS: (5 / 6 FAM)- PEG3- KGEGE*LLK*WALYSFNDGEKHQKKLLKL

[0206] (SEQ ID NO. 6)

[0207] FAM ECOR A12G: (5 / 6 FAM)- PEG3- KGEGE*LLK*WGLYSFNDGEKH*KKL*KL

[0208] (SEQ ID NO. 7)

[0209] FAM ECOR A12G SS: (5 / 6 FAM)- PEG3- KGEGE*LLK*WGLYSFNDGEKHQKKLLKL

[0210] (SEQ ID NO. 8)

[0211] BIO ECOR: (D-Biotin)- PEG3- KGEGE*LLK*WALYSFNDGEKH*KKL*KL

[0212] (SEQ ID NO. 9)

[0213] BIO ECOR A12G: (D-Biotin)- PEG3- KGEGE*LLK*WGLYSFNDGEKH*KKL*KL

[0214] (SEQ ID NO. 10)

[0215] Molecular weight of the purified peptides used in this example are as follows:

[0216] FAM ECOR parent: 3948.8 (Expected mass = 3949.5)

[0217] FAM ECOR: 3978.9 (Expected mass = 3979.6) FAM ECOR SS: 4099.2 (Expected mass = 4099.7)

[0218] FAM ECOR A12G: 3965.4 (Expected mass = 3965.6)

[0219] FAM ECOR A12G SS: 4085.2 (Expected mass = 4085.7)

[0220] BIO ECOR: 3847.1 (Expected mass = 3847.63)

[0221] BIO ECOR A12G: 3833.0 (Expected mass = 3833.6)

[0222] Peptide Characterization

[0223] Following cleavage and drying, peptides were redissolved in ImL methanol and filtered using a 45um syringe filter. Peptides were then separated using an Agilent 1200 Reversed Phase-High Performance Liquid Chromatographer (RP-HPLC) with a Zorbax SB- C18 column. The RP-HPLC mobile phase linear gradient contained 0.1% TFA in 10-100% water to acetonitrile with a flow rate of 0.5mL / minute. Molecular weights were used to characterize each peptide on an Agilent 6120 Single Quadrupole ESI-Mass Spectrometer. Following confirmation of the peptide presence with ESI-MS, peptides were purified over a semi-preparatory column at a flow rate of 4mL / min. Final peptide characterizations were performed by ESI-MS.

[0224] All peptides were quantified using a Synergy 2 Microplate Reader (Bio-Tek). For the FAM labeled peptides, a 495 nm absorbance was used with an extinction coefficient of 69000 M'1A small volume of each peptide was redissolved in Tris Buffer (pH 8, lOmM) to quantify the FAM-labeled peptide concentration. To quantify the biotin-labeled peptides, an absorbance of 500nm was utilized. A small volume of each peptide was redissolved in a 2-hydroxyazobenzen-4’ -carboxylic acid avidin cocktail (HABA-avidin) to perform these quantifications. Following quantification, peptides were all dried and redissolved in an appropriate volume of DMSO.

[0225] Cell Culture

[0226] HEK293 cells (CRL-1573) and HEK293T (CRL-3216) were purchased from ATCC. Dulbecco’s Modified Eagle’s medium (DMEM) was purchased from Gibco (11960044). Fetal Bovine Serum (FBS) was purchased from HyClone (SH30910.03), trypsin (25-043- Cl) was purchased from Corning and penicillin- streptomycin was purchased from Gibco (10378016). Cells were cultured and maintained in media supplemented with 10% FBS and 1% penicillin- streptomycin at 37 °C with 5% CO2. Cells were passaged using 0.25% trypsin solution containing 2.21 mM EDTA ,1X, with respective neutralizing media at least twice prior to each cell assay. All experiments were carried out in triplicates at different passage numbers.

[0227] Peptide Uptake Assay

[0228] 40,000 HEK293 cells were plated into 8-well Ibidi slide with Poly-L-Lysine coating and polymer coverslip (80824). The cells were incubated in complete DMEM (10% FBS, 1% Penicillin Streptomycin) overnight and subsequently treated with 2.5 mM of each FAM- labeled peptide at 37 °C for 6 hours. Following peptide incubation, cells were fixed using 4% paraformaldehyde solution (PFA) for 20 minutes. IX PBS containing DAPI was added to the cells after aspirating 4% PFA. Cells were then stored at 4 °C overnight in dark prior to imaging. Uptake of the FAM-labeled peptides was visualized using 63x oil-immersion objective of a Zeiss ESM800 confocal laser scanning microscope.

[0229] Proteolytic Stability Assay

[0230] 0.2 mM of each peptide was incubated in a proteolytic cocktail containing 50% mouse serum, 0.4% benzoic acid, and 15% DMSO in PBS at 37 °C with agitation. At each indicated time point, aliquots of the solution were drawn, and serum was precipitated using 0.1% Trifluoroacetic acid (TFA) in acetonitrile. Each precipitate was collected and centrifuged at 14,000 rpm for five minutes and the supernatants were retained for analysis. Proteolytic degradation was monitored by EC-MS as a ratio of peptide to control relative to peptide at t=0 at 280 nm using a Zorbax Eclipse XDB-C18 column. A 0-100% water to acetonitrile gradient containing 0.1% TFA was used at a flow rate of 1.0 mE / min at 45 °C. This process was repeated in triplicate for each time point for each peptide.

[0231] Fluorescence Polarization (FP) Assay

[0232] Fluorescence polarization assays were conducted on the lead doubly constrained compounds (ECOR and ECOR A12G) with two separate LRRK2 protein constructs: MBP- tagged RocCOR LRRK2 and MBP-tagged R1441C RocCOR LRRK2, both in the presence of 2 mM GTP and 10 mM MgCh. Protein / peptide interactions were measured in an FP buffer (20 mM MOPS pH 7, 150 mM NaCl, and 0.005% CHAPS) at room temperature. FAM-labeled peptides were plated in 384- well microtiter plates at a final concentration of 10 nM. Proteins were added over concentrations ranging from 5 mM to 1 nM. Each protein / peptide interaction experiment was carried out at 10 different protein concentrations in triplicate. Each protein / peptide mixture was incubated at room temperature for 2 hours and the final reading was performed at the end of this 2-hour period.

[0233] Rabl2 Immunoblotting

[0234] LRRK2 auto-phosphorylation and Rab phosphorylation were measured by western immunoblotting. Cells were transiently transfected with WT or G2019S LRRK2 (Flag- tagged), and the following day were treated with increasing concentrations of the candidate peptides (as indicated in the figure legend). Extracts of HEK293T cells were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in nonfat milk and probed with: total LRRK2 (clone UDD3, Abeam); total Rab 12 (ab86349), pS106-Rabl2 (rabbit clone MJF-R25-9; Abeam), overnight at 4°C followed by incubation with HRP anti-mouse or rabbit secondary antibodies and developed using ECL.

[0235] Proximity Biotinylation of Dimeric LRRK2 Assay

[0236] LRRK2 dimerization was assessed using either wild- type LRRK2 homodimers or LRRK2 G2019S homodimers. Experiments were carried out using various concentrations (0.5, mM, 1 mM or 2.5 mM) of FAM-labeled ECOR or ECO AR12G. The LRRK2 dimers used were purified as previously described19. To generate these dimers, two cDNAs encoding LRRK2 fusions with biotin ligase (BirA; N-term, Flag-tagged) or an acceptor peptide (AP, N-term; c-Myc tagged) were created. These constructs were expressed in HEK293T cells grown in biotin-depleted medium, (OptiMEM+2% FBS). FAM-labeled ECOR12A and ECOR12G were diluted in serum- free medium and added 24 hours after transfection. 48 hours following the initial treatment (after 72 hours of total expression), cells were washed in PBS, given a brief biotin pulse (50 mM, 5 min, 37 °C), washed three more times in PBS and centrifuged. The cell pellet was snap frozen in a dry ice / methanol bath and stored at -80°C until analysis. Cells were lysed and extracts were diluted in a TBST / BSA solution (10 mM Tris HC1, pH 7.6; 100 mM NaCl; 0.1% Triton X-100; 1% BSA) with 0.5 mg protein loaded in parallel ELISA plates coated with streptavidin (SA; to capture biotinylated LRRK2 dimers) and anti-LRRK2 (to quantify LRRK2 expression). Protein-bound SA coated plates were incubated with HRP-conjugated anti-Flag antibodies for 1 hour at room temperature to quantify dimeric LRRK2. Total LRRK2 overexpression was quantified using HRP-conjugated LRRK2 antibody (clone N241) on the parallel anti- LRRK2 coated plates (pre-coated with anti-LRRK2, clone c41-2) and was used to normalize dimeric LRRK2 content. Proximity biotinylation experiments were performed in biological triplicates with 3-4 technical replicates for each condition in the ELISA.

[0237] LRRK2 Microtubule Localization Assay in Live Cells

[0238] HEK293 cells (40,000) were plated onto an 8-well Ibidi slide with glass bottom (80827) in complete DMEM (10% FBS, 1% penicillin- streptomycin) and incubated overnight for cells to adhere to the slide. Cells were transfected with pcDNA 3.0 GFP tagged-LRRK2 using jetPEI (101-10N) from Polyplus according to the manufacturer’s protocol for a 48-well plate and incubated for 24 hrs at 37 °C. The next day, the media was replaced and cells were incubated with 2.5 mM biotin-labeled peptides, 1 mM MLi-2 and DMSO as controls for 6 hrs at 37 °C. Live cells were imaged using 63x oil-immersion objective of a Zeiss LSM800 confocal laser scanning microscope. The Zeiss microscope software ZEN was used to generate channel overlays and quantify the data.

[0239] Neuronal Apoptosis

[0240] Quantification of apoptotic neuronal profiles was performed using the approach previously described (see Antoniou, N.; Vlachakis, D.; Memou, A.; Leandrou, E.; Valkimadi, P. E.; Melachroinou, K.; Re, D. B.; Przedborski, S.; Dauer, W. T.; Stefanis, L.; et al. A motif within the armadillo repeat of Parkinson's-linked LRRK2 interacts with FADD to hijack the extrinsic death pathway. Sci Rep 2018, 8 (1), 3455). Cortices were removed from embryonic day 16 (E16) pregnant C57BL mice and cut into small pieces for enzymatic digestion (trypsin 0.05% and lOOpg / mL DNase) and mechanical dissociation. Cells were collected, centrifuged, and grown at 150000 / cm2cell density in BrainPhys neuronal culture medium (containing SMI Neuronal Supplement, L-glutamine (0.5 mM, and penicillin / streptavidin). Neurons were then transfected using Lipofectamine 2000 with Flag-tagged WT LRRK2 or G2019S LRRK2. The following day, neurons were treated with 0.5 pM and 1 pM of both ECOR12A and ECOR12G for 48 hours. Cells were washed in PBS and fixed in 3.7% paraformaldehyde for 20 min at 4 °C prior to antibody treatments. Images were analyzed and quantifications were performed as previously described30. For the determination of apoptotic neuronal death, fixed and stained neurons were visualized under a 40X magnification objective (dry). On each coverslip, a minimum of 100 Flagpositive neurons were identified and the percentage of those with apoptotic profiles was determined. Apoptotic nuclei were defined as neurons having condensed chromatin, fragmented into at least 2 or more “apoptotic bodies”. Statistical Analysis

[0241] GraphPad Prism was utilized to perform statistical analysis. Additionally, FIJI / ImageJ, ZEN software and Image Studio (Li-COR) were used to quantify cell uptake images, microtubule localization images and immunoblotting images, respectively. Oneway ANOVA and Dunnett’s multiple comparisons test were used for the analysis of western blots. For the analysis of the neuronal apoptosis and in vitro dimerization assays, one-way ANOVA with Tukey posthoc tests were performed. All experiments were performed in triplicate unless stated otherwise. Graphical data are presented as means ± SEM.

[0242] RESULTS AND DISCUSSION

[0243] Design of Doubly Constrained Peptide Inhibitors Destabilizing COR:COR-mediated Dimerization of LRRK2

[0244] Some differences between the recently solved structures of full-length LRRK2 and the truncated LRRK2RCKWstructure highlight the potentially dynamic nature of the LRRK2 dimerization interface as it transitions between monomeric and dimeric states. In addition, structural changes may occur as the kinase domain transitions from an inactive to an active conformer. The full-length structure of LRRK2 was used as a starting point for PPI inhibitor design where key protein interactions in both the monomeric and a dimeric state were revealed. Further, the dimer structure suggested a potentially important interaction within the terminal beta sheets of the “7+1” COR dimer hydrophobic interface. Based on these interactions, we sought to design constrained peptides that mimic a portion of this interface as a strategy to disrupt the packing interactions at the PPI, thereby resulting in inhibition of COR:COR-mediated LRRK2 dimerization. We designed a series of peptides that mimicked the C-terminal portion of the COR domain consisting of a portion of the “7+1” sheet stacking interface (residues 1802-1828) (FIG 1A). Using this sequence, we performed an in silica Alanine scan (BudeAlaScan) to identify residues that appear to be critical for binding the targeted PPI (see Wood, C. W.; Ibarra, A. A.; Bartlett, G. J.; Wilson, A. J.; Woolfson, D. N.; Sessions, R. B. BAlaS: fast, interactive and accessible computational alanine- scanning using BudeAlaScan. Bioinformatics 2020, 36 (9), 2917-2919; and Ibarra, A. A.; Bartlett, G. J.; Hegedus, Z.; Dutt, S.; Hobor, F.; Homer, K. A.; Hetherington, K.; Spence, K.; Nelson, A.; Edwards, T. A.; et al. Predicting and Experimentally Validating Hot-Spot Residues at Protein-Protein Interfaces. ACS Chem Biol 2019, 14 (10), 2252-2263). A hydrophobic triad (1811W, 1814Y, and 1816F) was identified from this scan that was predicted to serve as high energetic contributors to binding (FIG. IB). Further, while many of the “strand” residues were predicted to contribute to binding, several of the “turn” residues were found to be less critical for binding. Olefinic amino acids (pentenyl alanine, S5) were introduced in positions that were predicted to play minor roles in binding. In addition, since this sequence contained two non-continuous secondary structural elements, we designed a variant sequence bearing an alanine to glycine substitution at position 1812 (ECOR A12G). The aim of this substitution was to provide more flexibility at the end of the first secondary structural element to reinforce flexibility at the “loop” region. Moreover, since the overall sequence was 28 residues in length, it was unclear whether one staple would be sufficient to promote cell permeation, so two versions were generated that are singly or doubly stapled (“SS denotes single staple) (FIG. 1C). Peptides were synthesized via Fmoc solid phase peptide synthesis on rink amide MBHA resin. Non-natural amino acids were introduced at i, i+4 positions. In the instance of the double- stapled variants, the sequence was synthesized up to the point of the incorporation of the first pair of olefinic amino acids and the ring-closing metathesis (RCM) reaction was performed while the last incorporated amino acid was still Fmoc-protected. The remaining sequence was then completed prior to performing an additional RCM reaction to form the second macrocycle to eliminate the possibility of cross-metathesis products being formed (FIG. ID). For all sequences, select, non-critical residues within the sequence were substituted with Lysine residues and an N-terminal PEG3 linker to improve hydrophilicity and overall net charge. Upon cleavage from resin, peptides were purified using RP-HPLC and products were confirmed by ESI-MS.

[0245] Doubly Constrained Peptides Directly Bind to LRRK2

[0246] We first sought to determine whether the peptides could bind to the LRRK2 RocCOR domain. We performed fluorescence polarization (FP) assays using either the wild-type RocCOR domain or a pathogenic mutant form (R1441C) in the presence of fluorescently labeled, doubly stapled versions of ECOR (FIGs. 2A-2B). Both ECOR and ECOR A12G exhibited binding with dissociation constants between 45-60 nM for wild-type RocCOR. Further, we observed that both ECOR and ECOR A12G displayed slightly stronger binding towards pathogenic LRRK2 as compared to the wild-type binding with KD values ranging from 25-35 nM. Together, this demonstrates that the doubly constrained ECOR peptides can effectively target and bind the RocCOR domain of LRRK2. Peptides are Resistant to Proteolytic Degradation

[0247] Since these peptide sequences are relatively long, we sought to determine whether they may be vulnerable to proteolytic degradation. To assess stability, the peptide library was incubated with mouse serum to measure their resistance to proteolytic degradation over time by mass spectrometry (see Hanold, L. E.; Oruganty, K.; Ton, N. T.; Beedle, A. M.; Kannan, N.; Kennedy, E. J. Inhibiting EGFR dimerization using triazolyl-bridged dimerization arm mimics. PLoS One 2015, 10 (3), eOl 18796). Stability was assessed over a 6-hour time course, whereby the amount of peptide remaining over time was analyzed by ESI-MS using benzoic acid as an internal control. As expected, the non-constrained parent peptide was readily degraded with less than 50% detected by one hour and nearly completely degraded at the 4-hour timepoint (FIG. 3A). On the other hand, both doubly stapled peptides (ECOR and ECOR A12G) were shown to be highly stable with over 70% remaining after 6 hours. In addition, the singly stapled peptides were also stable under the conditions tested with comparable levels of mild degradation as compared to the doubly stapled versions.

[0248] ECOR and ECOR A12G Permeate Cells

[0249] LRRK2 is localized within the intracellular space, therefore we monitored whether the constrained peptides could permeate cells. HEK293 cells were incubated in the presence of peptides at 37 °C for 6 hours prior to imaging by confocal microscopy (FIG. 3B). ECOR and ECOR A12G exhibited significant uptake compared to the parent peptide and the singly stapled versions. While the single stapled peptides demonstrate low levels of cellular uptake compared to the native peptide, the uptake of doubly constrained peptides is substantially higher as determined by fluorescence intensity. These findings demonstrate the significance of the second hydrocarbon staple in improving cell permeation for this peptide sequence.

[0250] ECOR and ECOR A12G Inhibit LRRK2 Dimerization in Cells

[0251] Since the peptides were derived from the COR:COR interface of the LRRK2 dimer, we sought to assess whether these peptides could effectively inhibit LRRK2 dimer formation in cells. We used the previously published in situ LRRK2 proximity biotinylation approach where LRRK2 constructs bearing either BirA or an acceptor peptide are cotransfected, and the level of biotinylated LRRK2 is measured (see Leandrou, E.; Markidi, E.; Memou, A.; Melachroinou, K.; Greggio, E.; Rideout, H. J. Kinase activity of mutant LRRK2 manifests differently in hetero-dimeric vs. homo-dimeric complexes. Biochem J 2019, 476 (3), 559-579). Both ECOR and ECOR A12G were found to downregulate dimerization of a PD-related pathogenic form of LRRK2 (G2019S) that exists primarily in the dimeric form (FIG. 4A). Doubly constrained peptides were tested over a concentration range of 0.5- 2.5 pM and were found to downregulate intracellular LRRK2 G2019S dimerization by approximately 50-60%, demonstrating that these COR interface-derived peptides can indeed prevent LRRK2 dimerization.

[0252] ECOR and ECOR A12G Downregulate LRRK2 Kinase Activity but do not Induce Mislocalization of LRRK2

[0253] Since LRRK2 kinase activity is linked to LRRK2 dimerization (see Sen, S.; Webber, P. J.; West, A. B. Dependence of leucine-rich repeat kinase 2 (LRRK2) kinase activity on dimerization. J Biol Chem 2009, 284 (52), 36346-36356; and Greggio, E.; Zambrano, I.; Kaganovich, A.; Beilina, A.; Taymans, J. M.; Daniels, V.; Lewis, P.; Jain, S.; Ding, J.; Syed, A.; et al. The Parkinson disease-associated leucine-rich repeat kinase 2 (LRRK2) is a dimer that undergoes intramolecular autophosphorylation. J Biol Chem 2008, 283 (24), 16906-16914), we aimed to determine whether the COR-derived peptides downregulate this kinase activity as a function of disrupted dimerization. HEK293T cells were transfected with a pathogenic mutant form of LRRK2 with high kinase activity, G2019S. Cells were treated with doses of the doubly stapled peptides ranging from 0.5- 2.5 pM for 8 hours before lysis. Lysates were analyzed by western blotting to probe for phosphorylation of a LRRK2 substrate, Rabi 2 (FIG. 4B). Both ECOR and ECOR A12G were found to downregulate Rabi 2 phosphorylation by nearly 50% at all concentrations tested. When compared to the small molecule ATP-competitive kinase inhibitor MLi-2, which nearly completely inhibited Rabi 2 phosphorylation, neither of the doubly constrained peptides was as potent, however, using an allosteric approach, they both demonstrate that they can significantly downshift the kinase activity of LRRK2.

[0254] Classical ATP-competitive LRRK2 kinase inhibitors induce cellular recruitment of LRRK2 to skein-like structures on microtubules and block kinesin and dynein- 1 -mediated transport in vitro, which might partly induce the side-effect reported for these compounds (FIGs. 4C-4D) (see Kett, L. R.; Boassa, D.; Ho, C. C.; Rideout, H. J.; Hu, J.; Terada, M.; Ellisman, M.; Dauer, W. T. LRRK2 Parkinson disease mutations enhance its microtubule association. Hum Mol Genet 2012, 21 (4), 890-899). To test whether peptides would have a similar effect, GFP-tagged LRRK2 was overexpressed in HEK293 cells for 24 hours. The cells were then incubated at 37°C for 6 hours with either DMSO, MLi-2, ECOR or ECOR A12G prior to live imaging. Although it was previously reported that cells overexpressing LRRK2 show some aggregation22, in the presence of the ECOR and ECOR A12G peptides, no significant localization of LRRK2 to microtubules as filamentous skein-like structures could be observed (FIGs. 4C-4D).

[0255] ECOR and ECOR A12G Inhibit LRRK2-induced Neuronal Apoptosis

[0256] We next sought to determine if our lead compounds could downregulate a downstream pathology related to pathogenic LRRK2, namely neuronal apoptosis (see Melachroinou, K.; Leandrou, E.; Valkimadi, P. E.; Memou, A.; Hadjigeorgiou, G.; Stefanis, L.; Rideout, H. J. Activation of FADD-Dependent Neuronal Death Pathways as a Predictor of Pathogenicity for LRRK2 Mutations. PLoS One 2016, 11 (11), e0166053; Fuji, R. N.; Flagella, M.; Baca, M.; Baptista, M. A.; Brodbeck, J.; Chan, B. K.; Fiske, B. K.; Honigberg, L.; Jubb, A. M.; Katavolos, P.; et al. Effect of selective LRRK2 kinase inhibition on nonhuman primate lung. Sci Transl Med 2015, 7 (273), 273ra215; and Rideout, H. J. Neuronal death signaling pathways triggered by mutant LRRK2. Biochem Soc Trans 2017, 45 (1), 123-129). To test whether these COR-derived peptides could provide a neuroprotective effect, we measured whether they could inhibit neuronal apoptosis in isolated primary cortical neurons (FIG. 5 ). Cortical neurons expressing LRRK2 G2019S demonstrated hallmarks of apoptosis including nuclear condensation and fragmentation as well as activation of caspase-3 (not shown). Both doubly constrained peptides (ECOR and ECOR A12G) were found to reduce cortical neuronal apoptosis by over 50% (FIG. 5B). This reduction in neuronal cell death was most pronounced using 500 nM concentrations of either peptide. This effect may be due to insolubility or aggregation of the peptides at higher concentrations. However, it is apparent that the doubly constrained peptides can reverse the neuronal apoptotic effects that are otherwise driven by pathogenic LRRK2.

[0257] CONCLUSION

[0258] Recently, we reported on the development of constrained peptides, each containing a single hydrocarbon staple, targeting the proposed dimer interface in the ROC-COR domains of LRRK2 which were able to disrupt dimerization and kinase activity of LRRK2. We demonstrated that constrained peptides derived from the Roc domain of LRRK2, termed LRIP, could effectively target and allosterically inhibit dimerization, thereby downregulating kinase activity. We additionally developed a second constrained peptide that was designed to target the COR domain, termed LOP, but this peptide had a weak binding affinity for the RocCOR domain, showed limited cell uptake and had inferior cellular activity as compared to the Roc-targeting peptide. This finding was unexpected since recent structural studies demonstrate that the COR domain is a critical component of the LRRK2 dimer interface and thus is likely crucial for dimerization.

[0259] The goal of this example was to increase the potency of newly developed COR- derived peptides that had improved cellular uptake and efficacy while utilizing the recently characterized cryo-EM structures of full-length LRRK2 as a guide for peptide design. These structural studies confirmed and clarified the importance of the COR domain in facilitating LRRK2 dimerization. The peptides in this example overlap with the originally designed COR-targeting peptides. While initial characterization studies of the newly designed library yielded no discernable differences in proteolytic stability or binding affinities towards the RocCOR domain, the cell permeation studies revealed unexpected results. Multiple studies have demonstrated that hydrocarbon stapling of a peptide sequence can increase cell permeability and thus we expected either singly or doubly constrained peptides to both permeate cells (see Walensky, L. D.; Bird, G. H. Hydrocarbon-stapled peptides: principles, practice, and progress. J Med Chem 2014, 57 (15), 6275-6288; Limaye, A. J.; Whittaker, M. K.; Bendzunas, G. N.; Cowell, J. K.; Kennedy, E. J. Targeting the WASF3 complex to suppress metastasis. Pharmacol Res 2022, 182, 106302; and Hanold, L. E.; Fulton, M. D.; Kennedy, E. J. Targeting kinase signaling pathways with constrained peptide scaffolds. Pharmacol Ther 2017, 173, 159-170). The fact that two hydrocarbon staples were required for this particular sequence demonstrates a limitation of hydrocarbon staples on cell permeation, at least in this particular instance. The ability of only the doubly stapled peptides to permeate cells could be due to a multitude of factors including the flexibility, conformation and overall molecular weight of these peptides.

[0260] There are several key advantages of using constrained peptides to disrupt PPIs. First, since they are designed to occupy a relatively flat and large binding surface that is mediated by side chain specificity, there is considerable opportunity to yield highly selective targeting agents with reduced non-specific, off-target effects. Secondly, the addition of synthetic conformational constraints on the peptide sequence allows for the peptide sequence to maintain a secondary structure that may mimic the pre-ordered binding state and may thereby reduce or eliminate an energetic penalty from undergoing a disorder-to-order transition upon binding. While the potential therapeutic use of constrained peptides represents an attractive alternative to ATP-competitive LRRK2 kinase inhibitors, there are several obstacles still to overcome including the propensity to form aggregates, challenges of generating an orally bioavailable peptide-based therapeutic and their delivery into the brain which is complicated by their inability to cross the BBB. However, peptide-based inhibitors are also valuable tools for target validation and may therefore uncover new strategies for inhibitor development.

[0261] LRRK2 kinase activity is tightly linked to its dimerization (see Berger, Z.; Smith, K. A.; Lavoie, M. J. Membrane localization of LRRK2 is associated with increased formation of the highly active LRRK2 dimer and changes in its phosphorylation. Biochemistry 2010, 49 (26), 5511-5523) and inhibition of dimerization may serve as an effective strategy to downregulate mutant LRRK2-induced neurodegeneration in cellular and in vivo models. While the consequences are not yet clear, there are indications of unexpected, but on-target, effects of small molecule LRRK2 kinase inhibitors such as ATP-competitive inhibitors. For example, multiple structurally distinct LRRK2 inhibitors lead to a re-distribution of LRRK2 in the cell to microtubule (MT)-associated cytoplasmic filaments, and can elicit mild pathology in pulmonary lamellar cells in rodents and non-human primates. Our recent description of peptide-based, Roc-derived LRRK2 dimer disruptors showed that this redistribution of LRRK2 to cytoplasmic filaments is not universally observed following inhibition of kinase activity. Similarly, in this example, we did not observe the relocalization of LRRK2 to cytoplasmic microtubule -bound filaments. Interestingly, however, our data reveals that loss of phosphorylated Ser 935 levels, which correlates with pharmacological kinase inhibition-induced filament formation, is not sufficient to induce this re-distribution of LRRK2. Genetic inhibition of LRRK2 kinase activity, such as the K1906M / R mutation, also does not lead to re-distribution of LRRK2 into microtubule- associated filaments and pS935-LRRK2 loss, further highlighting the complexity of this relationship.

[0262] Together, these results indicate for the first time that COR-derived doubly constrained peptides inhibit LRRK2 dimerization, downregulate its kinase activity, and reduce cortical neuronal apoptosis, even in the presence of a pathogenic form of LRRK2. Further, it was found that adding a second hydrocarbon staple to this sequence greatly improved cell uptake and overall LRRK2 inhibition. This work demonstrates that doubly constrained peptides targeting LRRK2 PPIs provide an alternative approach for LRRK2- targeted therapeutics by acting as an allosteric inhibitor of LRRK2 activity, via disruption of LRRK2 dimerization. Critically, we showed that this approach could successfully rescue neuronal death induced by the expression of mutant (G2019S) LRRK2. This work thus also highlights the significance of the COR domain for regulating LRRK2 dimerization and kinase activity and may also demonstrate new strategic approaches for downregulating aberrant kinase activity in disease states. The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compositions and method steps disclosed herein are specifically described, other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein; however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

Claims

WHAT IS CLAIMED IS:

1. A synthetic polypeptide comprising a variant of an amino acid sequence of SEQ ID NO. 1, wherein the variant comprises at least two pairs of non-natural amino acids, wherein each pair of the at least two pairs of non-natural amino acids is cross-linked.

2. The synthetic polypeptide of claim 1, wherein the synthetic polypeptide is formed from a variant of an amino acid sequence of SEQ ID NO. 1 comprising at least four non- natural amino acids independently selected from (S)-2-(2’-propenyl)alanine, (S)-2-(4’- pentenyl)alanine, (S)-2-(5’-hexenyl)alanine, (S)-2-(7’-octenyl)alanine, (R)-2-(2’- propenyl)alanine, (R)-2-(4’-pentenyl)alanine, (R)-2-(5’-hexenyl)alanine, and (S)-2-(7’- octenyl)alanine.

3. The synthetic polypeptide of claim 1 or claim 2, wherein each of the at least two pairs of non-natural amino acids are three, four, or seven amino acids apart.

4. The synthetic polypeptide of claim 1, wherein the synthetic polypeptide is formed from a peptide comprising an amino acid sequence having at least 80% sequence similarity to a sequence selected from SEQ ID NO. 2 to 3.

5. The synthetic polypeptide of claim 1, wherein the synthetic polypeptide is formed from a peptide comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to a sequence selected from SEQ ID NO. 2 to 3.

6. The synthetic polypeptide of claim 1, wherein the synthetic polypeptide is formed from a peptide comprising an amino acid sequence selected from SEQ ID NO. 2 to 3.

7. The synthetic polypeptide of claim 1, wherein the polypeptide is selected from:wherein:Ala at each occurrence comprises an alanine residue or a conservative substitution or derivative thereof;Asn at each occurrence comprises an asparagine residue or a conservative substitution or derivative thereof;Asp at each occurrence comprises an aspartate residue or a conservative substitution or derivative thereof;Glu at each occurrence comprises a glutamate residue or a conservative substitution or derivative thereof;Gly at each occurrence comprises a glycine residue or a conservative substitution or derivative thereof;His at each occurrence comprises a histidine residue or a conservative substitution or derivative thereof;Leu at each occurrence comprises a leucine residue or a conservative substitution or derivative thereof;Lys at each occurrence comprises a lysine residue or a conservative substitution or derivative thereof;Phe at each occurrence comprises a phenylalanine residue or a conservative substitution or derivative thereof;Ser at each occurrence comprises a serine residue or a conservative substitution or derivative thereof;Trp at each occurrence comprises a tryptophan residue or a conservative substitution or derivative thereof; andTyr at each occurrence comprises a tyrosine residue or a conservative substitution or derivative thereof.

8. A pharmaceutical composition comprising a synthetic polypeptide of any one of claims 1-7 and a pharmaceutically acceptable carrier.

9. A cell comprising a synthetic polypeptide of any one of claims 1-7.

10. A method of treating a neurological disease, disorder, or condition in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a synthetic polypeptide of any one of claims 1-7 or a pharmaceutical composition of claim 8.

11. The method of claim 10, wherein the neurological disease, disorder, or condition comprises Parkinson’s disease.

12. The method of claim 10, wherein the neurological disease, disorder, or condition comprises Huntington’s disease.

13. The method of claim 10, wherein the neurological disease, disorder, or condition comprises Alzheimer’s disease.

14. The method of claim 10, wherein the neurological disease, disorder, or condition comprises amyotrophic lateral sclerosis (ALS).

15. A method of treating Crohn’s disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a synthetic polypeptide of any one of claims 1-7 or a pharmaceutical composition of claim 8.

16. A method of treating a disorder or condition that is treated by inhibition or modulation of LRRK2 activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a synthetic polypeptide of any one of claims 1-7 or a pharmaceutical composition of claim 8.

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