CYCLIC PEPTIDE AS INHIBITORS OF FREE-LIVING AMOEBA
20260139006 · 2026-05-21
Assignee
Inventors
- Elizabeth Ivy Parkinson (West Lafayette, IN, US)
- Gabriela Coy (Bogotá, CO)
- Chenyang Lu (West Lafayette, IN, US)
- Samantha Nelson (West Lafayette, IN, US)
- Christopher Aaron Rice (West Lafayette, IN, US)
Cpc classification
C07K7/64
CHEMISTRY; METALLURGY
International classification
Abstract
Cyclic peptides that are inhibitors of a pathogenic free-living amoeba (e.g., Balamuthia mandrillaris, Naegleria fowleri, and Acanthamoeba castellanii), compositions comprising the same, and their use for inhibiting infections or diseases caused by the pathogenic free-living amoeba.
Claims
1. A cyclic peptide therapeutic agent selected from: ##STR00055## ##STR00056## ##STR00057## ##STR00058## ##STR00059## ##STR00060## ##STR00061## ##STR00062## ##STR00063## or a variant thereof.
2. The pharmaceutical composition comprising a cyclic peptide therapeutic agent of claim 1 and a pharmaceutically acceptable carrier, diluent, or excipient.
3. The pharmaceutical composition of claim 2, further comprising one or more additional therapeutic agents, one or more of which can be other than a cyclic peptide therapeutic agent.
4. A method of inhibiting an infection or a disease caused by a pathogenic free-living amoeba in a patient in need thereof, which method comprises administering an inhibitory effective amount of a cyclic peptide therapeutic agent selected from ##STR00064## ##STR00065## ##STR00066## ##STR00067## ##STR00068## ##STR00069## ##STR00070## or a variant thereof or a pharmaceutical composition comprising the same, whereupon the infection or disease caused by the pathogenic free-living amoeba in the patient is inhibited.
5. The method of claim 4, wherein the pathogenic free-living amoeba is Balamuthia mandrillaris.
6. The method of claim 4, wherein the pathogenic free-living amoeba is Naegleria fowleri.
7. The method of claim 4, wherein the pathogenic free-living amoeba is Acanthamoeba castellanii.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The present disclosure will be more readily understood from the detailed description of embodiments presented below considered in conjunction with the attached drawings of which:
[0016]
[0017]
[0018]
[0019]
[0020]
DETAILED DESCRIPTION
[0021] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the claimed invention is thereby intended.
[0022] The present disclosure is predicated, at least in part, on the discovery that the peptides can have activity against free-living amoebas (FLAs), for example, Naegleria fowleri, Acanthamoeba globifora, and Acanthamoeba castellanii (Journal of Trop Med Public Health, 2014, 45, 537-46; ACS Omega 2022, 7, 28797-28805; Int J Parasitol Drugs Drug Resist, 2021, 17, 67-79; Microorganisms 10, 2022; Acta Parasitol, 2022, 67, 511-517) but none have demonstrated activity against Balamuthia mandrillaris. Specifically, a tyrocidine-derived linear peptide exhibited IC.sub.50 of 112 g/mL (88 M) against Acanthamoeba castellanii and 82 g/mL (64 M) against Naegleria fowleri at 24 hours. Nisin, a ribosomally synthesized post-translationally modified peptide produced by Lactococcus lactis, appears to affect the growth of Acanthamoeba, resulting in smaller and rounder trophozoites. However, the trophozoites proliferated again after 72 hours. Overall, the IC.sub.50 of nisin was quite poor (4493.2 ILU/mL, 4.5 mg/mL at 24 h), suggesting that it is unlikely to be an effective treatment for Acanthamoeba.
[0023] In view of the above, provided is a pharmaceutical composition comprising: (a) a cyclic peptide therapeutic agent of formula (I):
##STR00018## [0024] wherein: [0025] each of AA1, AA2, AA3, AA4, AA5, and AA6 is independently an amino acid selected from phenylalanine (Phe), ornithine (Orn), threonine (Thr), arginine (Arg), valine (Val), alanine (Ala), 3-fluorophenylalanine (Phe, 3-F), lysine (Lys), histidine (His), serine (Ser), diaminobutyric acid (Dab), diaminopropionic acid (DAP), isoleucine (Ile), glycine (gly), and tryptophan (Trp), and (b) a pharmaceutically acceptable carrier, diluent, or excipient.
[0026] In some embodiments, the cyclic peptide therapeutic agent of formula (I) is BICyP1, a cyclic peptide of formula (II):
##STR00019## [0027] wherein, AA1 is Phe, AA2 is D-Orn, AA3 is Thr, AA4 is D-Arg, AA5 is Val, and AA6 is D-Phe. or a variant of the cyclic peptide of formula (II) selected from:
##STR00020## ##STR00021## ##STR00022## ##STR00023##
[0028] The BICyP1 and its variant can inhibit or treat infections or diseases caused by the pathogenic free-living amoeba. The pathogenic free-living amoeba can be a specific free-living amoeba, but not limited to Balamuthia mandrillaris (B. mandrillaris), Naegleria fowleri, and Acanthamoeba castellanii. Pathogenic free-living amoeba causes infection of the central nervous system and causes cutaneous and systemic diseases. Examples of the infections or diseases caused by these free-living amoeba include, but are not limited to, Balamuthia amoebic encephalitis (BAE), granulomatous amoebic encephalitis (GAE), primary amoebic meningoencephalitis (PAM), keratitis, lesions in skin and respiratory mucosa.
[0029] Provided is a cyclic peptide therapeutic agent selected from:
##STR00024## ##STR00025## ##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030## ##STR00031## ##STR00032## [0030] or a variant thereof.
[0031] Provided is a pharmaceutical composition comprising (a) a cyclic peptide therapeutic agent selected from BICyP5, BICyP18, BICyP19, BICyP20, BICyP21, BICyP26, pNP51a, GC-1-110, GC-1-112, GC-1-116, GC-1-120, GC-1-122, GC-1-124, GC-1-126, GC-1-128, TO-1-006, LX-1-001, LX-1-004, LX-1-006, LX-1-008, GC-1-132, GC-1-134, GC-1-136, and GC-1-138 and (b) a pharmaceutically acceptable carrier, diluent, or excipient.
[0032] In some embodiments, the pharmaceutical compositions can further comprise one or more additional therapeutic agents and one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, one or more additional therapeutic agents can be other than a cyclic peptide therapeutic agent. The carriers, excipients, or diluents can vary based on the particular route of administration (see, e.g., Remington's The Science and Practice of Pharmacy, 23.sup.rd ed. (2020)).
[0033] Provided is a method for inhibiting an infection or a disease caused by a pathogenic free-living amoeba in a patient in need thereof, which method comprises administering to the patient an inhibitory effective amount of a cyclic peptide therapeutic agent selected from
##STR00033## ##STR00034## ##STR00035## ##STR00036## ##STR00037## ##STR00038## ##STR00039## [0034] or a variant thereof or a pharmaceutical composition comprising the same, whereupon the infection or disease caused by the pathogenic free-living amoeba in the patient is inhibited.
[0035] In some embodiments, the patient has an infection of the central nervous system. In some embodiments, the patient has cutaneous or systemic disease. In some embodiments, the patient has an infection of the central nervous system, such as Balamuthia amoebic encephalitis (BAE). In some embodiments, the pathogenic free-living amoeba is Balamuthia mandrillaris. In some embodiments, the pathogenic free-living amoeba is Naegleria fowleri. In some embodiments, the pathogenic free-living amoeba is Acanthamoeba castellanii.
[0036] The other variants of BICyP1 of formula (II) screened for potency and toxicity by performing alanine scan are:
##STR00040## ##STR00041## ##STR00042##
[0037] The other variants of BICyP1 of formula (II) are: [0038] BICyP2, wherein AA4 in formula (II) is D-Orn; BICyP4, wherein AA4 in formula (II) is D-Dab; BICyP14, wherein AA1 in formula (II) is Phe (4F); BICyP15, wherein AA6 in formula (II) is D-Phe (4F); BICyP16, wherein AA1 in formula (II) is Phe (3, 4F); BICyP17, wherein AA6 in formula (II) is D-Phe (3, 4F); BICyP22, wherein AA2 in formula (II) is D-Dap; BICyP23, wherein AA2 in formula (II) is D-Dab; BICyP24, wherein AA4 in formula (II) is D-His; or BICyP27, wherein AA2 in formula (II) is D-Lys and AA4 is D-Trp; or BICyP28, wherein AA5 in formula (II) is Pra.
##STR00043## ##STR00044## ##STR00045## ##STR00046##
[0039] In some embodiments the cyclic peptide therapeutic agent pNP-51a comprises a formula
##STR00047## [0040] wherein AA1 is D-Orn, AA2 is Orn, AA3 is D-Orn, AA4 is Phe, AA5 is D-Phe (4F), and AA6 is Phe. The variants of pNP-51-a are: pNP-51b, wherein AA1 in pNP-51a formula is D-ala; pNP-51c, wherein AA1 in pNP-51a formula is D-lys; pNP-51d, wherein AA5 in pNP-51a formula is D-Phe (3, 4F); pNP-51e, wherein AA5 in pNP-51a formula is D-Phe (3F); or pNP-51f, wherein AA2 in pNP-51a formula is ala.
[0041] In some embodiments, the variants of BICyP3 provided are:
##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052## ##STR00053##
[0042] In some embodiments, the BICyP3 and its variant can inhibit or treat infections or diseases caused by the pathogenic free-living amoeba. The pathogenic free-living amoeba can be a specific free-living amoeba, but not limited to Balamuthia mandrillaris (B. mandrillaris), Naegleria fowleri, and Acanthamoeba castellanii. Pathogenic free-living amoeba causes infection of the central nervous system and causes cutaneous and systemic diseases. Examples of the infections or diseases caused by these free-living amoeba include, but are not limited to, Balamuthia amoebic encephalitis (BAE), granulomatous amoebic encephalitis (GAE), primary amoebic meningoencephalitis (PAM), keratitis, lesions in skin and respiratory mucosa.
[0043] The term amino acid generally refers to an organic compound comprising both a carboxylic acid group and an amine group. The term amino acid includes both natural and unnatural or non-natural amino acids. Additionally, the term amino acid includes O-alkylated or N-alkylated amino acids, as well as amino acids having nitrogen or oxygen-containing side chains (such as Lys, Orn, or Ser) in which the nitrogen or oxygen atom has been acylated or alkylated. Amino acids may be pure L or D isomers or mixtures of L and D isomers, including racemic mixtures.
[0044] The term natural amino acid and equivalent expressions refer to L-amino acids commonly found in naturally occurring proteins. Examples of natural amino acids include, without limitation, alanine (Ala), cystein (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asp), proline (Pro), glutamine (Gin), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), -alanine (-ALA), and -aminobutyric acid (GABA).
[0045] The term unnatural amino acid refers to any derivative of a natural amino acid including D forms, and - and -amino acid derivatives. The terms unnatural amino acid and non-natural amino acid are used interchangeably herein and are meant to include the same moieties. It is noted that certain amino acids, e.g., hydroxyproline, that are classified as a non-natural amino acid herein, may be found in nature within a certain organism or a particular protein. Amino acids with many different protecting groups appropriate for immediate use in the solid-phase synthesis of peptides are commercially available.
[0046] The terms inhibit, inhibiting, inhibited, and inhibition with regard to inhibiting infection or disease caused by pathogenic free-living amoeba in a patient, such as a patient with BAE, refer to any degree of inhibition of one or more infections or diseases in the patient, wherein any degree of inhibition is beneficial to the patient. The term includes a method of inhibiting the onset of a disease or condition and/or its attendant symptoms or barring a subject from acquiring a disease. The term inhibitory effect or inhibition refers to the ability of a compound to reduce or block a specific biological or chemical activity, enzyme, or receptor, thereby preventing, suppressing, or altering its normal function.
[0047] The term inhibitory amount means any amount of a compound or a pharmaceutical composition comprising the compound that is sufficient to achieve an inhibitor effect or inhibition. The term effective amount or therapeutically effective amount means the amount of a compound that is effective to treat a disease or a disorder, such as metastatic cancer, at a reasonable benefit/risk ratio. The therapeutically effective amount of such compound will vary depending upon the patient and the disease or disorder being treated, the weight and age of the patient, the severity of the disease or disorder, the manner of administration, and the like, which can readily be determined by one of skill in the art.
[0048] The term therapeutically effective amount or effective amount refers to an amount of the active ingredient(s) that is(are) sufficient, when administered, to deliver efficaciously the active ingredient(s) for the treatment of a disease or condition of interest to a subject in need thereof. The therapeutically effective amount or effective amount of such composition will vary depending upon the patient and the disease or condition being treated, the weight and age of the patient, the severity of the disease or condition, the manner of administration, and the like, which can readily be determined by one of ordinary skill in the art.
[0049] For any compound, a therapeutically effective amount or effective amount can be initially determined from animal models. A therapeutically effective dose can also be determined from human data for compounds that have been tested in humans and for compounds that are known to exhibit similar pharmacological activities, such as other related active agents. Higher doses may be required for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.
[0050] The terms treat, treating, treatment, and the like refer to eliminating, reducing, or ameliorating a disease or condition, and/or symptoms associated therewith. Although not precluded, treating a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated. The term treat and synonyms contemplate administering a prophylactic or therapeutically effective amount of a combination or composition described herein to a subject in need of such treatment. The treatment can be orientated symptomatically, for example, to suppress symptoms. It can be effected over a short period, be oriented over a medium term, or can be a long-term treatment, for example within the context of maintenance therapy.
[0051] Generally, daily oral doses of a compound are from about 0.01 milligrams/kg per day to 1,000 milligrams/kg per day. Oral doses in the range of 0.5 to 50 milligrams/kg, in one or more administrations per day, can yield therapeutic results. Dosage can be adjusted appropriately to achieve the desired drug level, local or systemic, depending upon the mode of administration. For example, intravenous administration can vary from one order to several orders of magnitude lower dose per day. If the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) can be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of the compound.
[0052] The compounds can be typically administered in admixture with a pharmaceutical carrier to give a pharmaceutical composition selected with regard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and/or auxiliaries that facilitate the processing of the compound. The pharmaceutical compositions can be manufactured, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping, or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen. When a therapeutically effective amount of a compound described herein is administered orally, the composition typically is in the form of a tablet, capsule, powder, solution, or elixir. When administered in tablet form, the composition additionally can contain a solid carrier, such as a gelatin or an adjuvant. The tablet, capsule, and powder can contain about 0.01% to about 95%, and preferably from about 1% to about 50%, of the combination of compounds. When administered in liquid form, a liquid carrier can be added, such as water, petroleum, or oils of animal or plant origin. The liquid form of the composition can further contain the physiological saline solution, dextrose or other saccharide solutions, or glycols. When administered in liquid form, the composition contains about 0.1% to about 90%, and preferably about 1% to about 50%, by weight, of the combination of compounds.
[0053] For oral administration, the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers, excipients, or diluents well-known in the art. Such carriers, excipients, or diluents enable the compounds to be formulated as tablets, pills, powders, dragees, capsules, liquids, gels, syrups, slurries, suspensions, solutions, and the like for oral ingestion by a subject to be treated.
[0054] The exact formulation, route of administration, and dosage of a pharmaceutical composition comprising an effective amount of the compound are determined by an individual physician in view of the diagnosed condition or disease. The dosage amount and interval can be adjusted individually to provide levels of the compound that are sufficient to maintain a prophylactic or therapeutic effect.
[0055] Toxicity and therapeutic efficacy of the combination can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the maximum tolerated dose (MTD) of a compound, which is defined as the highest dose that causes no toxicity in animals. The therapeutic index is the dose ratio between the maximum tolerated dose and therapeutic effects (e.g., inhibition of tumor growth). The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The determination of a therapeutically effective amount is well within the capability of those ordinarily skilled in the art, especially in light of the detailed disclosure provided herein.
[0056] A combination can be administered in an amount of about 0.005 to about 500 milligrams per dose, about 0.05 to about 250 milligrams per dose, or about 0.5 to about 100 milligrams per dose. For example, the combination can be administered, per dose, in an amount of about 0.005, about 0.05, about 0.5, about 5, about 10, about 20, about 30, about 40, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 milligrams, including all doses between 0.005 and 500 milligrams.
[0057] As stated above, a combination or composition described herein can be administered with one or more other prophylactically or therapeutically active agents.
[0058] It will be appreciated by persons skilled in the art that the present disclosure is not limited by what has been particularly shown and described herein above. Rather, the scope of the present disclosure includes both combinations and sub-combinations of the various features described hereinabove as well as variations and modifications that would occur to persons skilled in the art upon reading the specification and which are not in the prior art.
Examples
[0059] The following examples serve to illustrate the present disclosure. The examples are not intended to limit the scope of the claimed invention in any way.
Methods
[0060] All the methods are carried out in accordance with a previously reported method in ACS Chem. Biol., 2021, 16, 2604-2611, which is hereby specifically incorporated by reference for its teaching regarding the same.
Maintenance of Balamuthia mandrillaris
[0061] The pathogenic Balamuthia mandrillaris (CDC V039; ATCC 50209), a BAE strain, was isolated from a pregnant baboon at the San Diego zoo, USA, in 1986. Balamuthia mandrillaris trophozoites were routinely grown axenically at 37 C. in Balamuthia mandrillaris ITSON (BMI) medium (bacto casitone, 20 g/liter; Hanks' balanced salts, 68 mL/liter and 912 mL of deionized water). After autoclaving, 10% fetal bovine serum and 1% penicillin/streptomycin antibiotics were added to produce the complete BMI. All experiments were performed using logarithmic-phase trophozoites. Before experimentation, 0.25% trypsin-EDTA was used to detach the cells from the vented 75 cm.sup.2 tissue culture flasks, and the cells were collected by centrifugation at 3214 RCF.
In Vitro Cell Titer-Glo Trophocidal Assay
[0062] The trophocidal activity of cyclic peptides was assessed using the CellTiter-Glo 2.0 luminescent viability assay (Rice et al., Antimicrobial Agents and Chemotherapy, 2020, 64, 9, 476). In brief, all cyclic peptides were diluted in BMI and serially diluted in 2-fold dilutions six times with a final concentration of 10% dimethyl sulfoxide (DMSO) in clear 96-well plates (Costar 3370) mother plates. After serial dilutions were performed, 10 L of peptides from the diluted stock plates were put into white 96-well plates, assay-ready plates. To combine parasites for screening, 90 L of Balamuthia mandrillaris trophozoites were seeded at 16,000 cells/well from a stock concentration of 1.77105/mL for a total time of 72 hours at 37 C. The peptide concentration was screened from 20 M to 0.625 M with a final DMSO (1%) screening concentration in the highest drug wells. DMSO (1%) and 90 M of atorvastatin were used as the negative and positive control, respectively. At the 72-hour time point, 25 L of CellTiter-Glo 2.0 Reagent was added to all wells of the white 96-well plates. The plates were shaken at 250 rpm in a dark environment for 2 minutes to induce cell lysis, then incubated at room temperature for 10 min to stabilize the luminescent signal. The adenosine triphosphate (ATP) luminescent signal (relative light units; RLUs) was measured at 490 nm with a SpectraMaxi3X plate reader. Each peptide was tested in a minimum of three biological replicates using different populations of cells. Half maximal inhibitory concentration (IC.sub.50) curves were generated using total ATP RLUs, where controls were calculated as the average of replicates using the Levenberg-Marquardt algorithm, using DMSO as the normalization control, as defined in CDD Vault.
Mammalian Cytotoxicity Counter Screening
[0063] Cytotoxicity of the cyclic peptides was also determined by using the CellTiter-Glo 2.0 assay on A549 human lung carcinoma cells. A549 cells were seeded at a concentration of 1.610.sup.4 cells/mL in 96-well plates in the presence of serially diluted compounds, as described above. DMSO (1%) and 100 M of chlorhexidine were used as the negative and positive controls. A549 were grown in F12K medium supplemented with 10% FBS and 1% penicillin/streptomycin antibiotics. The peptide concentration was screened from 100 M to 3.125 M. The total volume of each well was 100 L, containing 10 L compounds and 90 L cells, and plates were incubated at 37 C., 5% CO.sub.2 for 72 hours. At the 72-hours time point, CellTiter-Glo 2.0 Reagent was added to all the wells and read by the SpectraMax i3X plate reader as described above. Curve fitting using non-linear regression was carried out using the average of replicates and the Levenberg-Marquardt algorithm, using DMSO as the normalization control, as defined in CDD Vault. The selectivity index (SI) was calculated by using the equation: SI=(IC.sub.50 of A549's)/(IC.sub.50 of Balamuthia mandrillaris) to determine the specificity of inhibitors. A drug with a SI value 10 was considered the minimum standard for further evaluation as a hit drug.
Statistical Analysis
[0064] The Z factor was used as a statistical measurement to assess the robustness of each drug plate. This factor uses the mean and standard deviation values of the positive and negative controls to assess data quality. The robustness of all of the plates screened had an excellent Z-score value of 0.75 or above.
Hemolysis Assay
[0065] 100 L of whole blood was combined with 500 L of sterile 0.9% NaCl in a 1.5 mL Eppendorf tube, followed by thorough mixing through inversion. The resulting mixture underwent centrifugation at 500 rcf for 7 minutes, after which the supernatant was removed. The pellet was subjected to two additional washes, and following the final wash, 800 L of red blood cell (RBC) buffer was added to resuspend the RBCs. Subsequently, 4 L of the compounds (ranging from 100 to 0.78 M in DMSO, final concentration), 76 L RBC buffer, and 40 L of resuspended RBC were added to a 96 U-well plate (VWR) and then incubated for 1 hour at 37 C. The plate underwent centrifugation at 500 g for 5 minutes, after which 75 L of the supernatant was transferred to a 96 flat-well plate for absorbance measurement at 540 nm using a SpectraMax iD3 plate reader. Percent hemolysis was determined based on the average absorbance of the positive (triton-X 100) and negative controls (DMSO), with a minimum of three biological triplicates conducted.
Resin Loading
[0066] Resin loading was performed using a protocol reported in ACS Chem Biol, 2021, 16 (11), 2604-2611. 2-chlorotrityl chloride (2-CTC) resin (1 g, with a loading capacity of 0.56 mmol/g, mesh size 100-200), underwent swelling in dimethylformamide (DMF) for 5 minutes. After the removal of DMF, the swollen 2-CTC resin was exposed to fluorenylmethyloxycarbonyl (Fmoc)-protected amino acid (3 equiv.), N, N-diisopropylethylamine (DIPEA) (4 equiv.), and DMF (0.07 mM) for a duration of 2 hours. Subsequently, the solution was drained, and a mixture comprising of dichloromethane (DCM):methanol (MeOH):DIPEA in a ratio of 17:2:1 (0.07 mM) was employed to treat the resin, followed by agitation for 30 minutes to cap any remaining unreacted resin. After draining the solution, the resin underwent filtration and was washed thrice with DCM (5 mL each) and MeOH (5 mL each) before being dried for 1 hour. Resin loading was determined by exposing a small sample (1-3 mg) of the resin to piperidine-DMF (1:4) and determining the absorbance of the piperidine-dibenzofulvene adduct.
Solid Phase Peptide Synthesis
[0067] Linear peptides were synthesized on a 0.05 mmol scale utilizing a PS3 peptide synthesizer (ACS Chem Biol, 2021, 16 (11), 2604-2611). Preloaded CTC resin, as detailed in the resin loading procedures above, was employed. Fmoc deprotection was carried out using a 20% piperidine/DMF solution (25 minutes). Dicyclohexylcarbodiimide (DIC), Oxyma Pure, and Fmoc-protected amino acids (each six equivalents) were utilized in a 1-hour coupling process. The coupling and deprotection steps were iterated until the desired linear peptide sequence was achieved.
Peptide Cleavage
[0068] Following a reported method (ACS Chem Biol, 2021, 16 (11), 2604-2611), the peptide-linked resin obtained from solid phase peptide synthesis (SPPS) was swelled in DMF for 15 minutes. Following the draining of the DMF, the final Fmoc-protecting group was removed using a 20% piperidine/DMF solution for 15 minutes. The resin was then washed with DMF (32 mL) and DCM (32 mL) and dried. To confirm successful Fmoc deprotection, a Kaiser ninhydrin test was conducted. The deprotected peptide was cleaved from the resin using 25% HFIP/DCM for 30 minutes. The resultant solution was concentrated under vacuum, and the residue was resuspended in 50% H.sub.2O/acetonitrile (MeCN), frozen, and lyophilized. The crude compound obtained was used without additional purification.
Peptide Cyclization
[0069] Crude peptides (0.05 mmol), benzotriazolyloxy-tris[pyrrolidino]-phosphonium hexafluorophosphate (PyBop, 3 equivalents), DIPEA (6 equivalents), and DMF (1.25 mM) were subjected to overnight agitation (16-24 hours) and subsequently concentrated under vacuum. To the concentrated crude residue, 10 mL of 50% H.sub.2O/MeCN was added, vortexed, and centrifuged to isolate the precipitated peptide. The resulting precipitate underwent additional washing with 10 mL of 50% H.sub.2O/MeCN, followed by freezing and lyophilization. In case the residue did not precipitate, prep HPLC was employed to obtain the cyclized product.
Global Deprotection
[0070] The crude cyclized product underwent treatment with trifluoroacetic acid (TFA):DCM:triisopropylsilyl (TIPS) (50:45: 5, 20 mM) for 2 hours. The volatiles were removed using air, and the peptides were precipitated with 2 mL of methyl tert-butyl ether (TBME). The precipitate was vortexed, centrifuged, washed with additional TBME, dissolved in H.sub.2O:MeCN, frozen, and lyophilized.
HPLC
[0071] High-performance liquid chromatography (HPLC) analysis and purification were conducted using an Agilent Technologies 1260 Infinity II preparative HPLC system. Purity analysis utilized a Luna C18 reverse phase column (5 m, 1504.6 mm, Phenomenex), while purification was carried out employing a Luna C18 reverse phase column (5 m, 15021.2 mm, Phenomenex). Eluent A consisted of H.sub.2O with 0.1% formic acid (FA), while Eluent B comprised MeCN with 0.1% FA. The gradient for purity analysis was as follows: (A:B, flow rate 1 mL/min): 95:5, 0 min; 95:5, 1 min; 5:95, 20 min; 5:95, 25 min; 95:5, 30 min. For purification, the gradient was adjusted: (A:B, flow rate 20 mL/min): 95:5, 0 min; 95:5, 1 min; 5:95, 20 min; 5:95, 25 min; 95:5, 30 min.
Result and Discussion
Exhibition of Significant Trophocidal Activity Against Balamuthia mandrillaris
[0072] Balamuthia mandrillaris is a neglected infectious disease with a high mortality rate. Among the currently recommended drugs, only pentamidine has significant in vitro activity against Balamuthia mandrillaris, with a reported IC.sub.50 of between 9 and 18 M. To date, no one has shown curative in vivo activity due to the lack of robust translational pathobiological models. Recently, nitroxoline was screened against Balamuthia mandrillaris trophozoites in vitro and was identified to have an IC.sub.50 of 4.8 M (Laurie et al., mBio 2018, 9). Since the publication of these results, nitroxoline was successfully repurposed to treat a case of BAE disease in California, US (Spottiswoode et al., Emerg Infect Dis, 2023, 29, 197-201). The patient improved after nitroxoline administration and, excitingly, survived. However, kidney toxicity complications were observed. Given this dearth of effective, well-tolerated Balamuthia mandrillaris treatments and the great utility that cyclic peptides have shown as treatments for other diseases, forty-four cyclic peptides were screened from the SNaPP library at 16 g/mL for trophocidal activity against Balamuthia mandrillaris, Acanthamoeba castellanii, and Naegleria fowleri. Active molecules were defined as those with greater than 50% reduction in growth. Eight active peptides for Balamuthia mandrillaris, sixteen active peptides for Acanthamoeba castellanii, and one active peptide for Naegleria fowleri were identified and validated via dose-response. The dose-response studies identified seven promising hits (IC.sub.5010 g) for Balamuthia mandrillaris, one hit for Acanthamoeba castellanii, and one hit for Naegleria fowleri, giving us a hit rate of 22%, 3%, and 3%, respectively (
##STR00054##
TABLE-US-00001 TABLE 1 IC.sub.50 IC.sub.90 Name (g/mL) (g/mL) BICyP1 3.96 4.1 Balamuthia mandrillaris pNP-51a 5.98 >16.0 Naegleria fowleri pNP-12 5.27 >16.0 Acanthamoeba. castellanii
[0073] An alanine scan was further performed for each position of the cyclic hexapeptide to identify residues that are necessary for activity (Table 2).
TABLE-US-00002 TABLE 2 Balamuthia. mandrillaris A549 Hemolysis Name IC.sub.50 (M) IC.sub.50 (M) SI at 100 M BICyP1 5.0 0.1 >100 >20 <10% BICyP3 3.8 0.4 >100 >26 <10% BICyP5 4.1 0.6 70.5 17 <10% BICyP7 >20 ND ND ND BICyP8 11.9 1.8 47.7 4.0 <10% BICyP9 8.5 0.7 >100 >12 <10% BICyP10 >20 ND ND ND BICyP11 >20 ND ND ND BICyP12 >20 ND ND ND BICyP13 >20 ND ND <10% BICyP18 4.7 1.0 >100 >21 <10% BICyP19 4.2 0.8 64.4 15 <10% BICyP21 4.6 0.2 >100 >22 ND BICyP26 3.9 0.5 97.8 25 <10%
[0074] While all amino acids were found to contribute to the Balamuthia mandrillaris inhibitory activity, the D-orthinine (position 2 of BICyP1) and the threonine (position 3 of BICyP1) were found not to be essential (BICyP8 and BICyP9, respectively). A linear version of BICyP1 (BICyP13) was also tested, but it demonstrated a significant decrease in activity, confirming that the cyclic conformation is essential for activity. Following the alanine scan, further derivatization of BICyP1 was performed to identify more potent analogues. Modifying either of the phenylalanines (position 1 or 6 of BICyP1) with either a 4-flurophenylalanine (BICyP14 or BICyP15) or 3,4-flurophenylalanine (BICyP16 or BICyP17) resulted in a decrease in activity (Table 3). However, when a 3-flurophenylalanine (BICyP18 or BICyP19) was substituted at this position, the activity was maintained, suggesting that an electron-withdrawing group is tolerated in the meta position, but not the para position. Tyrosine substitution at the one position and six positions (BICyP20 and BICyP21) maintained activity.
TABLE-US-00003 TABLE 3 IC.sub.50 of cyclic peptides against trophozoites of Balamuthia mandrillaris in vitro. B. mandrillaris B. mandrillaris A549 Compound Mean IC.sub.50 Mean IC.sub.90 Mean IC.sub.50 Selectivity Name (M) SD (M) SD (M) Index (SI) BICyP1 5.03 0.06 5.73 0.11 >100 >22 BICyP3 3.79 0.37 5.83 0.33 >100 >26.39 BICyP5 4.11 0.56 5.66 0.04 70.50 17.15 BICyP18 4.68 0.99 7.64 1.94 >100 >21.37 BICyP19 4.22 0.77 5.89 0.45 64.40 15.26 BICyP20 5.41 0.99 8.85 1.62 >100 >18.48 BICyP21 4.62 0.17 7.38 1.12 >100 >21.65 BICyP25 6.25 1.27 8.06 5.28 49.00 7.84 BICyP26 3.89 0.54 6.65 1.00 97.80 25.14 pNP-51a 3.50 0.05 5.08 0.12 ND ND NDdata not determined.
[0075] Explored by varying the chain length of nucleophilic amino acids at positions 2 and 4. First, D-arginine at position 4 was exchanged for an amino acid with a shorter chain length, such as D-lysine (BICyP3), D-ornithine (BICyP2), and D-diaminobutyric acid (BICyP4). While D-lysine (BICyP3) was well tolerated, the other modifications were not, suggesting that a side chain with 4 or more carbons at position 4 is important for activity. When the D-ornithine at position 2 was modified with D-lysine (BICyP5), resulting in a longer chain length, the inhibitory activity was maintained. However, shortening the chain with a D-diaminopropionic acid (BICyP22) and D-diaminobutyric acid (BICyP23) resulted in decreased activity. This suggests that a chain length of at least 3 carbons between the alpha-carbon and the basic residue is important for inhibitory activity. Interestingly, these results are similar to those previously found in the study of cationic quarternary ammonium compounds and alkylphosphocholines (Mooney et al., Sci Rep 2020, 10, 6420). While these molecules are quite structurally different from the cyclic peptides studied here, they also showed increased activity with increasing alkyl chain length. This points to the activity of these molecules being due, at least in part, to their ability to interact with the protist plasma membrane. This is consistent with one of the major mechanisms of antimicrobial peptides, interaction with the cell wall through hydrophobic or electrostatic interactions, leading to membrane damage or lysis, cytoplasmic leakage, and cell death.
[0076] When position 4 is substituted with a D-histidine (BICyP24), activity decreases. However, when this modification is combined with position 2 being changed to a D-lysine (BICyP25) activity increases. For this reason, additional substitutions of position 4 were explored while keeping position 2 a D-lysine. Changing position 4 to D-lysine (BICyP26) maintains activity, while D-tryptophan (BICyP27) completely abolishes activity. This further confirms that a chain length greater than 4 is needed at position 4 and suggests that a locked conformation is not tolerated at that position. Given this information, a structure-activity relationship was determined (
[0077] The ability of these molecules to inhibit the growth of other FLA was screened. Interestingly, neither our initial hit (BICyP1) nor the derivatives tested showed strong activity (IC.sub.50<5 M). The selectivity of these cyclic peptides for Balamuthia mandrillaris over Acanthamoeba and Naegleria is quite interesting and points to a unique mechanism of action. It is possible that this selectivity may be due to differences in the composition of plasma membranes, given that many cyclic peptides act at the plasma membrane.
[0078] Table 4 illustrates IC.sub.50 values of variants of BICyP3.
TABLE-US-00004 TABLE 4 IC.sub.50 of variants of BICyP3 against trophozoites of Balamuthia mandrillaris in vitro Compound B. mandrillaris Name IC.sub.50 (M) BICyP3 3.79 0.37 GC-1-120 4.45 TO-1-006 5.48 GC-1-116 4.69 GC-1-128 3.34 LX-1-001 6.84 GC-1-110 5.66 LX-1-004 4.76
Exhibition of Minimal Toxicity Against Human Cells
[0079] Many of the currently used medicines for Balamuthia mandrillaris have severe toxicities. For example, a 57-year-old cutaneous Balamuthia mandrillaris infected patient died with severe liver damage, potentially due to complications of the diminazene aceturate treatment they received (Wang et al., Clin Infect Dis, 2022, 75, 1637-1640). Another patient with Balamuthia mandrillaris GAE was treated for more than 2 months with the recommended multiple-drug regimen (flucytosine, pentamidine, fluconazole, sulfadiazine, azithromycin, miltefosine, and albendazole) and developed adverse effects, including acute kidney injury and myelosuppression (Haston et al., Curr Opin Infect Dis, 2023, 36, 186-191). These examples indicate that new treatments with lower toxicities are needed for Balamuthia mandrillaris. Cyclic peptide natural products are crucial sources of drug candidates because of their wide range of activities. Compared with linear peptides, the circular structures enable higher rigidity of cyclic peptides to reduce the entropic cost of the receptor binding that provides higher affinity, specificity, and selectivity to the target, which results in lower to minimal cytotoxicity. Given the promising activity of several cyclic peptides against Balamuthia mandrillaris, evaluated the most promising compounds (IC.sub.50<5 M) for their selectivity. Specifically, their cytotoxicity against a human cell line (A549) and their hemolytic activity (Table 2,
[0080] Overall, inhibitors of Balamuthia mandrillaris were identified. The initial screen revealed 8 cyclic peptides with an IC.sub.50 less 16 g/mL. Derivatives were then synthesized, enabling the development of a robust structural activity relationship. The multiple derivatives were identified to have an IC.sub.50 of less than 5 M and an IC.sub.90 of less than 10 M, with minimal to no toxicity to mammalian cells. Given that pentamidine, one of the current gold standards of Balamuthia mandrillaris treatment, has a lower efficacy against Balamuthia mandrillaris with an IC.sub.50 of 7.2 M, higher cytotoxicity, and poor selectivity, these cyclic peptides show great promise as leads for challenging to-treat disease.
[0081] While specific embodiments of the subject invention have been disclosed herein, the above specification is illustrative and not restrictive. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. Many variations of the invention will become apparent to those of skilled art upon review of this specification. Unless otherwise indicated, all numbers expressing reaction conditions, quantities of ingredients, and so forth, as used in this specification, are to be understood as being modified in all instances by the term about Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that can vary depending upon the desired properties sought to be obtained by the present disclosure.
[0082] As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art.
[0083] The term about can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range.
[0084] The term substantially can allow for a degree of variability in a value or range, for example, within 90%, within 95%, or within 99% of a stated value or of a stated limit of a range.
[0085] The terms a, an, or the are used to include one or more than one unless the context clearly dictates otherwise. The term or is used to refer to a nonexclusive or unless otherwise indicated. In addition, the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid the reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section heading may occur within or outside of that particular section. The terms including and having are defined as comprising (i.e., open language).
[0086] All patents, patent application publications, journal articles, textbooks, and other publications mentioned in the specification are indicative of the level of skill of those in the art to which the disclosure pertains. All such publications are incorporated herein by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference.