Inhibitor of cyclic GMP-amp synthase

Abstract

Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is an enzyme sensor of double-stranded DNA (dsDNA) that serves to trigger activation of the cGAS-stimulator of interferon genes (STING) pathway. The inhibition of cGAS with Cladophorol A is described herein.

Description

PCT / US25 / 53397 30 October 2025 (30.10.2025)SIREN.026WO PATENTINHIBITOR OF CYCLIC GMP-AMP SYNTHASECROSS-REFERNCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 715458, filed on November 1, 2024, the contents of which are hereby incorporated by reference in their entirety for all purposes.PARTIES OF JOINT RESEARCH AGREEMENT

[0002] The subject matter disclosed in this application was developed and the claimed invention was made by, or on behalf of, Sirenas LLC and / or The Scripps Research Institute or its affiliates. Sirenas LLC and the California Institution for Biomedical Research (now a division of The Scripps Research Institute) are parties to a joint research agreement which was in effect on or before the effective filing date of the claimed invention. The claimed invention was made as a result of activities undertaken within the scope of the joint research agreement.BACKGROUND

[0003] Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is an enzyme sensor of double-stranded DNA (dsDNA) that serves to trigger activation of the cGAS-stimulator of interferon genes (STING) pathway. Excessive activation of this pathway has been demonstrated to contribute to various forms of inflammatory disease. As such, cGAS has arisen as a potential therapeutic target with broad potential applications. Thus, there is a need for compounds capable of inhibiting cGAS.SUMMARY

[0004] Some embodiments described herein related to a pharmaceutical composition comprising Cladophorol A,PCT / US25 / 53397 30 October 2025 (30.10.2025)Cladophorol A or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0005] Some embodiments relate to a method of inhibiting cGAS, comprising contacting cGAS with Cladophorol A. Additional embodiments relate to method of treating a disease, disorder, or condition comprising administering an effective amount of Cladophorol A or a pharmaceutically acceptable salt thereof to a subject in need thereof. In some embodiments, the disease, disorder, or condition is cGAS-mediated.

[0006] In some embodiments, the disease, disorder, or condition is an inflammatory disease, disorder, or condition. In additional embodiments, the disease, disorder, or condition is selected from the group consisting of: STING-associated vasculopathy with onset in infancy (SAVI), Familial Chilblain Lupus, Aicardi-Goutieres Syndrome, COPA syndrome, Silica-Induced Fibrosis, and Sepsis.

[0007] In some embodiments, the disease, disorder, or condition is an autoimmune disease, disorder, or condition. In additional embodiments, the disease, disorder, or condition is selected from the group consisting of: Systemic Lupus Erythematosus, Lupus Nephritis, Sjogren’s Syndrome, Rheumatoid arthritis, Systemic Sclerosis (Scleroderma and Interstitial Lung Disease), Raynaud’s Syndrome, and Dermatomyositis / Polymyositis.

[0008] In some embodiments, the disease, disorder or condition is cancer. In additional embodiments, the disease, disorder, or condition is selected from the group consisting of: colorectal cancer, skin cancer, and metastases.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 depicts the results of in vitro cell assays for cGAS inhibition and cytotoxicity upon treatment with Cladophorol A.

[0010] FIG. 2 depicts the results of an enzyme assay for cGAS inhibition.PCT / US25 / 53397 30 October 2025 (30.10.2025)

[0011] FIG. 3 depicts the results of a study of 2’3’-cGAMP inhibition in Trexl knockout THP-1 cells upon incubation with Cladophorol A.

[0012] FIG. 4 depicts the results of a study of the impact of Cladophorol A on cGAS enzymatic activity in heart tissue after in vivo administration to Trexl - / - mice.DETAILED DESCRIPTION

[0013] The present embodiments are directed toward compositions of Cladophorol A and methods of treatment comprising administering an effective amount of Cladophorol A to a subject in need thereof.Definitions

[0014] Unless defined otherwise, technical, and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0015] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0016] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.25, or 0.1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.Preparation

[0017] Cladophorol A can be isolated from marine green macroalgae from the genus Cladophora using suitable extraction and purification techniques. For example, Cladophorol A can be extracted from the algae using MeOH and MeOH / CFECh and purified using reversed-phase chromatography.

[0018] As shown in Scheme 1, Cladophorol A can also be prepared synthetically.PCT / US25 / 53397 30 October 2025 (30.10.2025)Scheme 1

[0019] To prepare Cladophorol A, the hydroxyl group of compound 1 can be protected, and the resulting compound 2 can be brominated to give compound 3. Separately, the carboxy group of compound 4 can be esterified, and the resulting compound 5 can be converted to boronic ester-containing compound 6. Compounds 3 and 6 can then be coupled to afford compound 7, followed by deprotection of the hydroxyl groups resulting in the production of Cladophorol A.Administration and Pharmaceutical Compositions

[0020] In some embodiments, Cladophorol A may be in the form of a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts include acetate, adipate, besylate, bromide, camsylate, chloride, citrate, edisylate, estolate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hyclate, hydrobromide, hydrochloride, iodide, isethionate, lactate, lactobionate, maleate, mesylate, methylbromide, methylsulfate, napsylate, nitrate, oleate, palmoate, phosphate, polygalacturonate, stearate, succinate, sulfate, sulfosalicylate, tannate, tartrate, terphthalate, tosylate, and triethiodide. Alternatively, a pharmaceutically acceptable salt may be prepared by combining Cladophorol A with a counterion selected from: aluminum, arginine, benzathine, calcium, chloroprocaine, choline, diethanolamine, ethanolamine, ethylenediamine, lysine, magnesium, histidine, lithium, meglumine, potassium, procaine, sodium, triethylamine, and zinc.PCT / US25 / 53397 30 October 2025 (30.10.2025)

[0021] Compositions containing Cladophorol A may be in any form suitable for the intended method of administration. In some embodiments, the compounds of a method and / or composition described herein can be provided via oral administration, rectal administration, transmucosal administration, intestinal administration, enteral administration, topical administration, transdermal administration, intrathecal administration, intraventricular administration, intraperitoneal administration, intranasal administration, intraocular administration and / or parenteral administration.

[0022] When the compounds are administered via oral administration, for example, tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable. These excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

[0023] Formulations for oral use may be also presented as hard gelatin capsules where the active ingredient can be mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient can be mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

[0024] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain, for example, antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may includePCT / US25 / 53397 30 October 2025 (30.10.2025) suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0025] Pharmaceutical compositions comprising Cladophorol A may include one or more excipients from the following list: Magnesium Stearate, Microcrystalline Cellulose, Lactose, Starch (com), Silicone / Titanium Dioxide, Stearic Acid, Sodium Starch Glycolate, Gelatin, Talc, Sucrose, Calcium Stearate, Povidone, Pregelantinized Starch, HPMC, OPA products, Croscarmellose, Hydroxypropyl Cellulose, Ethycellulose, Calcium Phosphate, Crospovidone, and Shellac (and Glaze).

[0026] In other embodiments, pharmaceutical compositions comprising Cladophorol A may include one or more excipients from the following list: Acacia, Acesulfame, Acesulfame potassium, Acetic acid, Acetone, Acetyltributyl citrate, Alcohol, Alginic acid, Alpha-tocopherol, Aluminum chloride, Aluminum chlorohydrex propylene glycol, Aluminum hydroxide, Aluminum lake dyes, Aluminum oxide, Aluminum silicate, Aluminum stearate, Aluminum sulfate, Amide resin, Aminobenzoate sodium, Ammonia, Ammonio methacrylate copolymer, Ammonio methacrylate copolymer type A, Ammonio methacrylate copolymer type B, Ammonio methacrylate copolymers, Ammonium chloride, Ammonium laureth-5 sulfate, Ammonium phosphate dibasic, Artificial flavor, Artificial grape flavor, Artificial mint flavor, Ascorbic acid, Ascorbyl palmitate, Aspartame. Banana, Barium sulfate, Benzalkonium chloride, Benzoic acid, Benzyl alcohol, Betadex, Black currant, Black currant flavor, Black ink, Black pigment, Blackberry, Blue dye, Butyl alcohol, Butylated hydroxyanisole, Butylated hydroxytoluene, Butylparaben, Calcium, Calcium carbonate, Calcium phosphate, Calcium phosphate dibasic anhydrous, Calcium phosphate dihydrate dibasic, Calcium silicate, Calcium stearate, Calcium sulfate, Calcium sulfate anhydrous, Calcium sulfate dihydrate, Candelilla wax, Carbomer, Carbomer 934, Carbomer homopolymer type A, Carbomer homopolymer type B, Carbomer homopolymer type C, Carboxymethylcellulose, Carboxymethylcellulose calcium, Carboxymethylcellulose sodium, Carmine, Carnauba wax, Carrageenan, Castor oil, Cellacefate, Cellulose acetate, Cellulose compounds, Cellulose powdered, Cellulosic polymers, Cetostearyl alcohol, Cetyl alcohol,PCT / US25 / 53397 30 October 2025 (30.10.2025)Cetylpyridinium chloride, Cherry, Citric acid anhydrous, Citric acid monohydrate, Cochineal, Coconut oil, Colophony, Colorants, Coloring agent, Compressible sucrose, Compressible sugar, Confectioners’ sugar, Copovidone, Corn, Com oil, Com starch, Corn syrup, Corn- derived proteins, Cottonseed oil, Cranberry, Croscarmellose sodium, Crospovidone, Cysteine hydrochloride, D&C Green No. 5, D&C Red No. 21, D&C Red No. 22, D&C Red No. 27, D&C Red No. 27 Aluminum Lake, D&C Red No. 28, D&C Red No. 30, D&C Red No. 33, D&C Red No. 6 Lake, D&C Red No. 7 Calcium Lake, D&C Yellow No. 10, D&C Yellow No. 10 Aluminium Lake, Dextrates, Dextrose, Dextrose monohydrate, Dibutyl phthalate, Dibutyl sebacate, Diethyl phthalate, Dihydroxyaluminum sodium carbonate, Dimethicone, Dimethylaminoethyl methacrylate - butyl methacrylate - methyl methacrylate copolymer, Docusate sodium, Edetate calcium disodium, Edetate disodium, Edible black ink, Egg phospholipids, Ethanolamine, Ethyl acrylate - methyl methacrylate copolymer, Ethyl butyrate, Ethyl isovalerate, Ethylcellulose, Ethylcellulose, Ethylene glycol monoethyl ether, Ethylvanillin, Eudragit, FD&C Blue No. 1, FD&C Blue No. 1 Aluminium Lake, FD&C Blue No. 2, FD&C Blue No. 2 Aluminium Lake, FD&C Green No. 3, FD&C Red No. 3, FD&C Red No. 4, FD&C Red No. 40, FD&C Yellow No. 5, FD&C Yellow No. 5 Aluminum Lake, FD&C Yellow No. 6, Ferric oxide brown, Ferric oxide red, Ferric oxide yellow, Ferric oxides, Ferrosoferric oxide, Ferrous fumarate, Ferrous oxide, Flavor, Flavors, Fragrances, Fumaric acid, Gelatin, Glucosamine, Glucosamine hydrochloride, Glutamic acid hydrochloride, Glycerin, Glycerol monooleate, Glyceryl behenate, Glyceryl di stearate, Glyceryl monostearate, Glycine, Glycolate, Glycyrrhizin ammoniated, Guar gum, Hard gelatin capsule, High fructose corn syrup, Hydrochloric acid, Hydrogen peroxide, Hydrogenated castor oil, Hydrogenated cottonseed oil, Hydrogenated soybean oil, Hydrogenated vegetable oil, Hydroxyethyl cellulose, Hydroxypropyl cellulose, Hydroxypropyl methylcellulose phthalate, Hypromellose, Hypromellose phthalate, Hypromelloses, Indigotindi sulfonate sodium, Iron, Isobutylparaben, Isopropyl alcohol, Lactitol, Lactitol monohydrate, Lactose, Lactose anhydrous, Lactose monohydrate, Lemon oil, Leucine, Light mineral oil, Low substituted hydroxypropyl cellulose, Magnesium, Magnesium aluminum silicate, Magnesium carbonate, Magnesium hydroxide, Magnesium oxide, Magnesium silicate, Magnesium stearate, Magnesium trisilicate, Maleic acid, Malic acid, Maltodextrin, Mannitol, Medium-chain triglycerides, Meglumine, Menthol, Methacrylic acid, Methacrylic acid - ethyl acrylatePCT / US25 / 53397 30 October 2025 (30.10.2025) copolymer (1 :1) type a, Methacrylic acid - methyl methacrylate copolymer (1: 1), Methacrylic acid - methyl methacrylate copolymer (1 :2), Methacrylic acid copolymer, Methyl alcohol, Methyl cinnamate, Methyl methacrylate, Methylcellulose, Methylene chloride, Methylparaben, Methylparaben sodium, Microcrystalline cellulose, Microcrystalline wax, Mineral oil, Mint, Mint cream flavor, Mint menthol, Modified corn starch, Monosodium citrate, Natural and artificial orange flavor, Natural flavor, Natural mint flavor, Natural peppermint flavor, Natural resin, Nonoxynol-100, Oleic acid, Olive oil, Opacode black, Orange cream flavor, Orange juice, Orange oil, Orange-pineapple flavor, Other ingredients, Palm kernel oil, Paraffin, Partially hydrogenated soybean and palm oils, Peanut oil, Peppermint, Peppermint flavor, Peppermint oil, Phenylalanine, Phosphoric acid, Piperazine, Polacrilin potassium, Polacrilin sodium, Pol oxamer, Poloxamer 188, Pol oxamer 407, Polyacrylate dispersion 30%, Polycarbophil, Polydextrose, Polyethylene glycol, Polyethylene glycol 1450, Polyethylene glycol 300, Polyethylene glycol 3000, Polyethylene glycol 3350, Polyethylene glycol 400, Polyethylene glycol 4000, Polyethylene glycol 600, Polyethylene glycol 6000, Polyethylene glycol 800, Polyethylene glycol 8000, Polygalacturonic acid, Polysorbate, Polysorbate 20, Polysorbate 80, Polyvinyl acetate, Polyvinyl alcohol, Polyvinylpyrrolidone, Potassium, Potassium bicarbonate, Potassium bitartrate, Potassium carbonate, Potassium chloride, Potassium gluconate, Potassium hydroxide, Potassium sorbate, Potato starch, Povidone, Povidone kl2, Povidone k25, Povidone k30, Povidone k90, Propyl gallate, Propylene glycol, Propylene glycol alginate, Propylparaben, Propylparaben sodium, Raspberry, Raw sugar, Riboflavin, Rice starch, Saccharin, Saccharin sodium, Sd-45 alcohol, Sda-3a alcohol, Sesame oil, Shellac, Silicified microcrystalline cellulose, Silicon dioxide, Silicone, Sodium, Sodium alginate, Sodium ascorbate, Sodium benzoate, Sodium bicarbonate, Sodium carbonate, Sodium carbonate monohydrate, Sodium caseinate, Sodium chloride, Sodium citrate, Sodium glycolate, Sodium hydroxide, Sodium laureth sulfate, Sodium lauryl sulfate, Sodium metabisulfite, Sodium monolaurate, Sodium phosphate, Sodium phosphate dibasic, Sodium propionate, Sodium starch glycolate, Sodium starch glycolate type A potato, Sodium stearate, Sodium stearyl fumarate, Sodium thioglycolate, Sodium tripolyphosphate, Sorbic acid, Sorbitan, Sorbitan monolaurate, Sorbitan monooleate, Sorbitol, Sorbitol special, Soya lecithin, Soybean oil, Spearmint, Starch, Stearic acid, Stearyl alcohol, Strawberry, Strawberry guarana flavor, Succinic acid, Sucralose, Sucrose, Sucrose stearate, Sugar 6xPCT / US25 / 53397 30 October 2025 (30.10.2025) powder, Sugar spheres, Sunflower oil, Synthetic ferric oxide, Synthetic ferric oxide red, Synthetic ferric oxide yellow, Synthetic ferric oxides, Tapioca starch, Tartaric acid, Taurine, TIMERx-N, Titanium dioxide, Tragacanth, Triacetin, Tribehenin, Tricalcium phosphate, Triethyl citrate, Trimyristin, Trisodium citrate anhydrous, Trisodium citrate dihydrate, Tromethamine, Tropical blend flavor, Vanilla, Vanilla flavor, Vanillin, Water, Wheat starch, White wax, Xanthan gum, Xylitol, Yellow wax, Zinc gluconate, and Zinc stearate.

[0027] In some embodiments unit dosage formulations contain a daily dose or unit, daily sub-dose, or an appropriate fraction thereof, of a drug. It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the individual being treated; the time and route of administration; the rate of excretion; other drugs which have previously been administered; and the severity of the particular disease undergoing therapy, as is well understood by those skilled in the art.

[0028] The actual dose of the compounds described herein depends on the specific compound, and on the condition to be treated; the selection of the appropriate dose is well within the knowledge of the skilled artisan. In some embodiments, a daily dose may be from about 0.1 mg / kg to about 100 mg / kg or more of body weight, from about 0.25 mg / kg or less to about 50 mg / kg from about 0.5 mg / kg or less to about 25 mg / kg, from about 1.0 mg / kg to about 10 mg / kg of body weight. Thus, for administration to a 70 kg person, the dosage range would be from about 7 mg per day to about 7000 mg per day, from about 35 mg per day or less to about 2000 mg per day or more, from about 70 mg per day to about 1000 mg per day.

[0029] In some embodiments, Cladophorol A comprises between 0.0001% to 90%, 0.0001% to 10%, 0.001% to 10%, 0.01% to 10%, 0.1% to 10%, 0.1% to 1%, 1% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 99.9% of the composition by weight.

[0030] In some embodiments, the pharmaceutical composition comprising Cladophorol A does not comprise any other phenolic compound. In other embodiments, the pharmaceutical composition comprising Cladophorol A does not comprise any chlorophylls. In further embodiments, the pharmaceutical composition comprising Cladophorol A does not comprise any carotenoids.PCT / US25 / 53397 30 October 2025 (30.10.2025)Methods of Treatment

[0031] Some embodiments include methods of treating an inflammatory disease, disorder, or condition selected from the group consisting of: STING-associated vasculopathy with onset in infancy (SAVI), Familial Chilblain Lupus, Aicardi-Goutieres Syndrome, COPA syndrome, Silica-Induced Fibrosis, and Sepsis with Cladophorol A or a pharmaceutically acceptable salt thereof.

[0032] Some embodiments include methods of treating an autoimmune disease, disorder, or condition selected from the group consisting of: Systemic Lupus Erythematosus, Lupus Nephritis, Sjogren’s Syndrome, Rheumatoid arthritis, Systemic Sclerosis (Scleroderma and Interstitial Lung Disease), Raynaud’s Syndrome, and Dermatomyositis / Polymyositis with Cladophorol A or a pharmaceutically acceptable salt thereof.

[0033] Some embodiments include methods of treating a neurological disease, disorder, or condition selected from the group consisting of: Ischaemic brain injury, Parkinson’s disease, general neurodegeneration, Huntington disease, amyotropic lateral sclerosis and frontotemporal dementia, age-dependent macular degeneration, and traumatic brain injury with Cladophorol A or a pharmaceutically acceptable salt thereof.

[0034] Some embodiments include methods of treating a metabolic disease, disorder, or condition selected from the group consisting of: nonalcoholic steatohepatitis, alcoholic liver disease, and acute pancreatitis with Cladophorol A or a pharmaceutically acceptable salt thereof.

[0035] Some embodiments include methods of treating a cardiovascular disease, disorder, or condition selected from the group consisting of: myocardial infarction and chronic heart failure with Cladophorol A or a pharmaceutically acceptable salt thereof.

[0036] Some embodiments include treating cancerous disease, disorder, or condition selected from the group consisting of: colorectal cancer, skin cancer, and metastases with Cladophorol A or a pharmaceutically acceptable salt thereof.

[0037] Some embodiments include methods of treating a senescence or ageing disease, disorder, or condition selected from the group consisting of: senescence and ageing with Cladophorol A or a pharmaceutically acceptable salt thereof.

[0038] Some methods include administering a compound, composition, pharmaceutical composition described herein to a subject in need thereof. As used herein, aPCT / US25 / 53397 30 October 2025 (30.10.2025)“subject” or a “patient” refers to an animal that is the object of treatment. “Animal” includes, but is not limited to, mammals. “Mammal” comprises, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys (e.g., rhesus and macaque), chimpanzees, and apes, and, in particular, humans. In some alternatives, the subject is human.

[0039] Some embodiments disclosed herein relate to selecting a subject or patient in need. In some embodiments, a patient is selected who is in need of treatment, amelioration, inhibition, progression, prophylaxis, or improvement in disease symptoms or who is in need of curative therapy.

[0040] As used herein, the term “treatment” refers to an intervention made in response to a disease, disorder, or physiological condition manifested by a subject. The terms treating, treatment, therapeutic, or therapy do not necessarily mean total cure or abolition of the disease or condition. For example, in some embodiments, treatments reduce, alleviate, ameliorate, or eradicate the symptom(s) of the disease and / or provide curative therapy of the disease.

[0041] Further embodiments include administering a combination of compounds to a subject in need thereof. A combination can include a compound, composition, pharmaceutical composition described herein with an additional medicament.

[0042] Some embodiments include co-administering a compound, composition, and / or pharmaceutical composition described herein, with an additional medicament or additional therapeutic agent(s). By “co-administration,” it is meant that the two or more agents may be found in the patient’s bloodstream at the same time, regardless of when or how they are actually administered. In one embodiment, the agents are administered simultaneously. In one such embodiment, administration in combination is accomplished by combining the agents in a single dosage form. In another embodiment, the agents are administered sequentially. In one embodiment, the agents are administered through the same route, such as orally. In another embodiment, the agents are administered through different routes, such as one being administered orally and another being administered i.v.

[0043] The disclosed compounds may be used alone or in combination with other treatments. These compounds, when used in combination with other agents, may be administered as a daily dose or an appropriate fraction of the daily dose (e.g., bid). ThePCT / US25 / 53397 30 October 2025 (30.10.2025) compounds may be administered after a course of treatment by another agent, during a course of therapy with another agent, administered as part of a therapeutic regimen, or may be administered prior to therapy with another agent in a treatment program.

[0044] The term “therapeutically effective amount” is used to indicate an amount of a composition that elicits the biological or medicinal response indicated. Determination of a therapeutically effective amount is within the capability of those skilled in the art, in view of the disclosure provided herein. The therapeutically effective amount of the composition disclosed herein required as a dose will depend on the route of administration, the type of animal, including human, being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication, and other factors which those skilled in the medical arts will recognize.EXAMPLESExample 1 : Natural Product Isolation

[0045] Cladophorol A may be extracted from marine green macroalgae by methods known in the art. For example, a sample containing the target chemistry may be extracted with an organic (e.g., methanol, methylene chloride, chloroform, ethyl acetate), wherein extraction may involve partitioning the biological sample between aqueous and organic solvent layers, collecting fractions from the organic layer, and then analyzing the organic fractions for the presence of natural products by conventional methods (e.g., LCMS, HPLC). The collected fractions containing a mixture of natural products (e.g., including Cladophorol A) may be resolved by common chromatographic methods in the art (e.g. size-exclusion chromatography, gel-electrophoresis, ion exchange chromatography, flash chromatography, reversed- and normal -phase HPLC) to provide the compound (e.g. , Cladophorol A) as an isolated compound. Further purification methods known in the art may be employed. The natural product (e.g., Cladophorol A) may then be analyzed and characterized by standard methods known to the skilled artisan (e.g. IR, ID and 2D NMR, HRMS, LCMS).Procurement of biological material for the isolation of Cladophorol APCT / US25 / 53397 30 October 2025 (30.10.2025)

[0046] A filamentous green macroalgae was collected from the US Virgin Islands. A green algae assemblage predominantly composed of a branched filamentous chiorophyte structure, was collected from John’s Folly Bay on St. John Island (Global Positioning System coordinates 18.318341, -64.700365). The sample was collected by hand, vacuumed sealed, and frozen. Specimens were preserved in RNAlater or frozen directly. The biomass was frozen and stored at -20°C until extraction.Extraction and isolation of Cladophorol A

[0047] A frozen mixture of the green algae as described above, was thawed and exhaustively extracted with MeOH and1 :2). The organic layers were combined, concentrated under reduced pressure, and then loaded onto a HP20ss size-exclusion chromatography resin. The resin was then washed stepwise with 1) H2O, 2) 9: 1 FEO / MeOH, 3) MeOH, and 4) CH2CI2. The 9: 1 H20 / Me0H fraction was further separated using reversed- phase chromatography (Phenomenex Luna 5pm C18(2), 150 x 30 mm, 15 mL min1) with a MeCN / FEO and 0.5% trifluoroacetic acid (TFA) linear gradient (20% MeCN to 50% MeCN over 15 min), generating 21 fractions. Final purification with fractions containing the target mass were then separated using reversed-phase HPLC (Luna Phenyl hexyl, 250 x 4.6 mm, 5 pm; 3 mL min'1) with a linear gradient from 20-70% aqueous MeCN and 0.1% formic acid (FA) over 25 min to yield SMD-3218 (3 mg).Table 1. NMR Chemical Shifts for Cladophorol A.PCT / US25 / 53397 30 October 2025 (30.10.2025)Mult = MultiplicityHMBC = Heteronuclear multiple bond correlationS = Singlet qC = Quaternary carbonCH = Methine carbonExample 2: In Vitro Inhibition of cGASCell Culture

[0048] THPl-Lucia™ ISG cells (cat. no. thpl-isg) were purchased from InvivoGen and maintained in growth media consisting of RPMI 1640 (ATCC modification), 2 mM L- glutamine, 25 mM HEPES, 10% (v / v) Gibco™ Heat-inactivated FBS, 1000 units / ml penicillin, 1000 pg / ml streptomycin, 0.25 pg / ml Amphotericin B, and 100 pg / ml zeocin.ISRE Luciferase Assay

[0049] A high throughput phenotypic screen was conducted to identify active compounds. 100 nL of compound or vehicle (DMSO) were transferred to 384-well white greiner plates using the Echo Liquid Handler. THP1 -Lucia™ ISG cells (cat. no. thpl-isg) were resuspended in low-serum growth media (2% FBS) at a density of 2 x 105 cells / ml. Precomplexed VacV70 / LyoVec (Invivogen) was resuspended in 500 uL LAL water and added to the cells at a final concentration of 2 pg / mL. 50 uL of cells with and without VacV70 were plated on top of the transferred compounds and incubated for 48 hours at 37 °C, 5% CCh. To evaluate expression of the luciferase reporter, 30 pl of Quanti-luc (cat. rep-qlc41gl, InvivoGen) detection reagent was added to each well and the plates were shaken for 5 seconds before luminescence was immediately read using an Envision plate reader (PerkinElmer) set with an integration time of 0.1 seconds. For each cell type, luminescence signals for test article samples were normalized to VacV70-treated samples and reported as relative light units (RLU).PCT / US25 / 53397 30 October 2025 (30.10.2025)Cytotoxicity Assay

[0050] THP1 -Lucia™ ISG cells (cat. no. thpl-isg) were resuspended in low-serum growth media (2% FBS) at a density of 2 x 105 cells / ml. Precomplexed VacV70 / LyoVec (Invivogen) was resuspended in 500 uL LAL water and added to the cells at a final concentration of 2 pg / mL. 50 uL of cells with and without VacV70 were plated on top of the transferred compounds and incubated for 48 hours at 37 °C, 5% CO2. To evaluate cytoxicity, plates were incubated with 20 uL of CellTiter-Glo reagent (Promega), shaken for 5 seconds, and incubated for 10 minutes in the dark at room temperature before being read.Results

[0051] Based on concentration-dependent inhibition of VacV70-dependent luciferase activation using authentic resynthesized material, Cladophorol A was identified as a non-cytotoxic (CellTiter-Glo IC50 > 30 pM) putative cGAS-STING pathway inhibitor (IRF- Luc IC50 = 370 nM). Figure 1 shows the inhibition of the ISRE-luciferase reporter signal and corresponding impact on cell viability in the VacV70-stimulated Cladophorol A-treated THP1- Lucia ISG™ cells.

[0052] To directly evaluate enzyme inhibition, an endpoint mass spectrometrybased enzyme assay was employed using recombinant human cGAS protein that is based on detection of formed 2’,3’-cGAMP. When evaluated at concentrations corresponding to observed Km values for dsDNA, ATP and GTP, Cladophorol A was found to inhibit recombinant human cGAS at physiologically relevant concentrations (<5 pM, Figure 2).Example 3: In vivo measurement of cGAMP in tissue

[0053] 2’3’ cGAMP levels in Trexl knockout TPH-1 cells after treatment withCladophorol A or DMSO. The time-dependent results are depicted in Figure 3 and demonstrate that Cladophorol A inhibited cGAMP levels

[0054] Trexl - / - or wildtype mice were administered five 30 mg / kg doses of Cladophorol A or vehicle twice daily, IP. Heart tissue was harvested four hours after the last dose and 2’3’-cGAMP levels were evaluated using ELISA. The results are depicted in Figure 4 and demonstrate in vivo inhibition of cGAMP upon administration of Cladophorol A.

Claims

WHAT TS CLAIMED IS:

1. A pharmaceutical composition comprising Cladophorol A,Cladophorol A or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

2. A method of inhibiting cGAS, comprising contacting cGAS with Cladophorol A.

3. A method of treating a disease, disorder, or condition comprising administering an effective amount of Cladophorol A or a pharmaceutically acceptable salt thereof to a subj ect in need thereof.

4. The method of claim 3, wherein the disease, disorder, or condition is cGAS- mediated.

5. The method of claim 3, wherein the disease, disorder, or condition is an inflammatory disease, disorder, or condition.

6. The method of claim 5, wherein the disease, disorder, or condition is selected from the group consisting of: STING-associated vasculopathy with onset in infancy (SA VI), Familial Chilblain Lupus, Aicardi-Goutieres Syndrome, COP A syndrome, Silica-Induced Fibrosis, and Sepsis.

7. The method of claim 3, wherein the disease, disorder, or condition is an autoimmune disease, disorder, or condition.

8. The method of claim 7, wherein the disease, disorder, or condition is selected from the group consisting of: Systemic Lupus Erythematosus, Lupus Nephritis, Sjogren’s Syndrome, Rheumatoid arthritis, Systemic Sclerosis (Scleroderma and Interstitial Lung Disease), Raynaud’s Syndrome, and Dermatomyositis / Polymyositis.

9. The method of claim 3, wherein the disease, disorder or condition is cancer.

10. The method of claim 9, wherein the disease, disorder, or condition is selected from the group consisting of: colorectal cancer, skin cancer, and metastases.

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