Inhibitors of phenylalanine aggregation and uses thereof in the treatment of metabolic disorders

Abstract

Compounds usable in treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) and methods of identifying same are provided. The compounds include morin hydrate and structural analogs thereof and / or are collectively represented by Formula with the variables being as defined in the specification and claims. The compounds alternatively include those presented in Table 1 and / or compounds capable of aligning with a pharmacophoric model as described in the specification and drawings herein. Methods of identifying compounds capable of interfering with phenylalanine aggregation are also provided.

Description

[0001] INHIBITORS OF PHENYLALANINE AGGREGATION AND USES THEREOF IN THE TREATMENT OF METABOLIC DISORDERS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of priority ofU.S. Provisional Application Nos. 63 / 557,638 and 63 / 557,632, co-filed on February 26, 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] FIELD AND BACKGROUND OF THE INVENTION

[0005] The present invention, in some embodiments thereof, relates to therapy and, more particularly, but not exclusively, to compounds, compositions and methods for inhibiting phenylalanine aggregation and to uses thereof in treating metabolic diseases and disorders associated with phenylalanine aggregation such as phenylketonuria.

[0006] Metabolic disorders, and, more specifically, inborn error of metabolism (IEM) disorders, are the result of inherited mutations in specific single genes encoding for metabolic enzymes. IEM disorders result in the accumulation of metabolites that could be toxic or interfere with normal functions of cells and tissues. Yet, little is known about the pathological mechanisms that cause the symptoms. Up to date, more than 50 metabolic disorders have been reported and described; most of them are rare, occurring in less than 1 per 250,000 persons in most populations. Collectively, however, these disorders constitute a very significant portion of pediatric genetic diseases, with an estimated incidence as high as 1 in 800 live births.

[0007] Phenylketonuria (PKU), one of the most common among the rare IEM disorders, is caused by a deficiency in the phenylalanine hydroxylase (PAH) enzyme. Consequently, due to the inability to metabolize phenylalanine, accumulation of phenylalanine occurs, leading to elevated phenylalanine levels in the tissues, sera, and urine of PKU patients. If not carefully monitored and treated from early infancy with a phenylalanine-restricted diet, the symptoms may include mental retardation, severe reduction in physical and communication abilities, seizures, hyperactivity, tremor, microcephaly, and failure to thrive. Untreated PKU patients typically show symptoms of profound mental retardation by the age of 1 year, and rarely achieve an IQ greater than 50. Importantly, thanks to the neonatal screening programs that allow for early diagnosis, these clinical manifestations are now rarely seen.

[0008] PKU is considered as the most common clinically encountered inborn error of amino acid metabolism disorder, with prevalence that exhibit considerable geographic variation. PKU incidence is 1 : 15,000 births in the United States and 1 : 10,000 births in Europe, while in Turkey, where the highest rate in the world is documented, the incidence is 1 :2,600 births. Currently, no disease-modifying course of treatment is available, with the mainstay of treatment being a very strict low-phenylalanine diet and special formula supplements. A survey of 625 people conducted by the National PKU Alliance in 2015 found that more than 60 % of adults and 25 % of children with PKU can’t keep their phenylalanine levels in check.

[0009] To date, there are only two proven, but imperfect, drugs that are approved for PKU treatment: Kuvan®, which is considered as the gold standard treatment for PKU, is an orally administered drug that can reduce blood levels of phenylalanine in only 30 % of patients, while most patients have little or no benefit; and Palynziq®, a subcutaneous injectable PEGylated recombinant phenylalanine ammonia lyase enzyme, which is approved only for adults and adolescents from 16 years of age and is prohibited for women who are pregnant or trying to conceive, and may cause severe immune reactions.

[0010] Overall, the development of a new therapeutic agent, which will affect the course of the disease, is certainly required.

[0011] Previous studies have indicated that phenylalanine, as a single amino acid, is capable of forming well-ordered fibrillar assemblies at the nano-scale, and that these assemblies have typical fibrillar morphology, characteristic birefringence, ThT fluorescence patterns and clear electron diffraction patterns, resembling properties of amyloid assemblies, which are related to numerous pathological disorders. The formed structures are ordered as amyloid fibrils and also have strong and clear cytotoxic activities, similarly to other amyloid assemblies. Importantly, the formation of phenylalanine aggregates could be detected in the brain of PKU model mice and individuals with PKU using Phe-specific antibodies, suggesting that PKU is closely related to the family of amyloid- related diseases and might have similar etiology. These studies further indicated that the phenylalanine fibrils represent a distinct immunological entity, like amyloid assemblies. See, for example, Adler-Abramovich et al., Nature Chemical B 2012, 8, 701-706. In further studies, biophysical characterization of these assemblies revealed that all assembled ultrastructures formed by the various metabolites present amyloidogenic properties, shown by electron microscopy and ThT and Congo red assays, self-assemble into supramolecular amyloid-like fibrillar structures, and demonstrate a clear apoptotic effect on neuronal model cells. See, Shaham-Niv et al. Sci. Adv. 2015, 1, el500137, 1-6.

[0012] The structural similarity between the protein and peptide-based amyloid and the metabolites amyloid-like assemblies, suggested that molecules found to halt the self-assembly of the proteinaceous amyloid can also inhibit the formation of the metabolite fibrils. This approach was proven to be efficient in vitro using several compounds that found to indeed inhibit both species [see, Porat et al. Chemical Biology and Drug Design 2006, 67, 27-37; Gazit, E. FEBS Journal 2005, 272, 5971-5978; Hard, T. & Lendel, C. Journal of Molecular Biology 421, 441-465 (2012); Shaham-Niv, S. et al. Commun. Chem. 2018, 1, 25],

[0013] Recently, two aromatic polyphenolic compounds, epigallocatechin gallate (EGCG) and Tannic acid (TA), which have been previously shown to generically inhibit the formation of protein and peptide amyloid structures, were shown to halt the formation of phenylalanine amyloid fibrils and reduce the resulting cytotoxicity of the assemblies, indicating their therapeutic potential. See, Shaham-Niv et al., Commun. Chem. 2018, 1, supra.

[0014] Additional studies of the metabolites amyloid-like assemblies are described, for example, in Shaham-Niv et al. Angew Chemie Int Ed. 2018, 57(38), 12444-7; Shaham-Niv et al. Sci Adv. 2015, l(August): 1-7; Shaham-Niv et al. Isr. J. Chem. 2016;57, 729-737; and Shaham-Niv et al. Chem Commun. 2018, 54, 4561-4.

[0015] Additional Background Art includes Gazit E. FASEB J. 2002, 16(1), 77-83; Shaham-Niv et al. Biophys. Chem., 2024, 208, 107215; a review by Rajput et al., Biomedicine & Pharmacotherapy, 2021, 138, 111511; and Noor et al. Protein Science, 21(3), 373-382.

[0016] SUMMARY OF THE INVENTION

[0017] The previously reported formation of amyloid-like toxic assemblies by aggregation of phenylalanine, and the showing that compounds that halt the formation of these assemblies also prevent a resulting toxicity that has been assumed to be associated with metabolic disorders such as phenylketonuria, have prompted the present inventors to search for additional compounds that can inhibit phenylalanine aggregation as potential candidates for treating these diseases.

[0018] The present inventors have performed high-throughput screening, and have thereby uncovered inhibitors of the phenylalanine aggregation. An exemplary such compound was tested in vivo, using phenylketonuria mice model, and was found to significantly improve the mice’ cognitive and motor functions, while maintaining high phenylalanine levels yet causing a reduction in the number of phenylalanine brain deposits. Additional exemplary compounds that were uncovered were tested in vitro, and derivatives of the best-performing compound, namely PCM1, were designed.

[0019] The present inventors have designed a pharmacophore model and have uncovered accordingly further candidates for treating metabolic diseases that are associated with phenylalanine aggregation.

[0020] Embodiments of the present invention relate to compounds and compositions usable in inhibiting phenylalanine aggregation, and to uses of such compounds, for example, in treating diseases associated with phenylalanine aggregation such as PKU. Embodiments of the present invention further relate to methods of identifying novel compounds that are capable of inhibiting phenylalanine aggregation, and of treating diseases associated with phenylalanine aggregation.

[0021] According to an aspect of some of any of the embodiments of the invention, there is provided a compound represented by Formula I:

[0022] Formula I wherein: the dashed line represents an optional double bond;

[0023] X is O, S or NRn; and

[0024] R1-R11 are each independently selected from hydrogen, alkyl, cycloalkyl, heteroalicyclic, hydroxy, thiol, alkoxy, thioalkoxy, amine, halo, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S -thiocarb amyl, C-amido, N- amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and likewise substituents, provided that at least one of R1-R4 is a hydrogen-bond donor substituent, preferably hydroxy; the compound being for use in treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) in a subject in need thereof.

[0025] According to some embodiments of any of the embodiments described herein, the dashed line represents a double bond.

[0026] According to some embodiments of any of the embodiments described herein, Ri is hydroxy.

[0027] According to some embodiments of any of the embodiments described herein, at least two of R1-R4 are each hydroxy. According to some embodiments of any of the embodiments described herein, Ri and R3 are each hydroxy.

[0028] According to some embodiments of any of the embodiments described herein, R2 and R4 are each hydrogen.

[0029] According to some embodiments of any of the embodiments described herein, R5 is hydroxy.

[0030] According to some embodiments of any of the embodiments described herein, at least one of Re to Rio is selected from hydroxy and alkoxy.

[0031] In some embodiments of any of the embodiments described herein, at least Rs is hydroxy or alkoxy.

[0032] In some embodiments of any of the embodiments described herein, Rs is hydroxy or alkoxy and Re, R7 and R9 are each hydrogen.

[0033] According to some embodiments of any of the embodiments described herein, at least two of Re to Rio are each independently selected from hydroxy and alkoxy.

[0034] In some embodiments of any of the embodiments described herein, Rs and Rio are each independently hydroxy or alkoxy.

[0035] According to some embodiments of any of the embodiments described herein, X is O.

[0036] According to some embodiments of any of the embodiments described herein, the compound is morin hydrate.

[0037] According to some embodiments of any of the embodiments described herein, the compound is Apigenin.

[0038] According to some embodiments of any of the embodiments described herein, the compound is selected from morin hydrate, Apigenin, Kaempferol, Kaempferide and Galangin.

[0039] According to some embodiments of any of the embodiments described herein, the compound is represented by Formula la:

[0040] Wherein R1-R10 are each independently as defined for Formula I. According to an aspect of some of any of the embodiments of the invention, there is provided a compound selected from:

[0041] the compound being for use in treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) in a subject in need thereof. According to some embodiments of any of the embodiments described herein, the compound is selected from:

[0042] According to some embodiments of any of the embodiments described herein, the compound is: or a structural analog thereof such as described herein in any of the respective embodiments. According to some embodiments of any of the embodiments described herein, the metabolic disorder is caused by an inborn error of metabolism.

[0043] According to some embodiments of any of the embodiments described herein, the metabolic disorder is phenylketonuria.

[0044] According to an aspect of some of any of the embodiments of the invention, there is provided a method of identifying a compound capable of interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation in a subject in need thereof, the method comprises screening in silico a library of compounds to thereby determine if a compound is capable of aligning with a pharmacophoric model featuring two aromatic motifs and a hydrogen-bond donor motif as depicted in FIG. 4A, wherein a compound capable of the aligning is identified as capable of inhibiting phenylalanine aggregation.

[0045] According to some embodiments of any of the embodiments described herein, once identifying the compound, the method is further effected by contacting the identified compound with a phenylalanine-containing medium (e.g., an aqueous solution and / or cellular medium), to thereby further determine if the identified compound is capable of inhibiting phenylalanine aggregation, wherein a compound that reduces formation of phenylalanine fibrils by at least 20 %, compared to a medium lacking the compound, is identified as capable of inhibiting phenylalanine aggregation.

[0046] According to some embodiments of any of the embodiments described herein, once identifying the compound and / or contacting the identified compound with a phenylalanine- containing medium, the method is further effected by in silico screening of compounds identified as capable of aligning with the pharmacophoric model to thereby determine a set of pharmacological parameters of each of these compounds, wherein compounds identified as featuring LogS higher than 1 and / or HIA, are identified as suitable for use capable of interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation in a subject in need thereof.

[0047] According to some embodiments of any of the embodiments described herein, once identifying the compound and / or contacting the identified compound with a phenylalanine- containing medium, the method is further effected by in silico screening of compounds identified as capable of aligning with the pharmacophoric model to thereby determine an oral CNS scoring profile of each of these compounds, wherein compounds featuring a scoring profile higher than 0.1 are identified as suitable for use in interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation in a subject in need thereof.

[0048] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0049] Implementation of the method and / or system of embodiments of the invention can involve performing or completing selected tasks manually, automatically, or a combination thereof. Moreover, according to actual instrumentation and equipment of embodiments of the method and / or system of the invention, several selected tasks could be implemented by hardware, by software or by firmware or by a combination thereof using an operating system.

[0050] For example, hardware for performing selected tasks according to embodiments of the invention could be implemented as a chip or a circuit. As software, selected tasks according to embodiments of the invention could be implemented as a plurality of software instructions being executed by a computer using any suitable operating system. In an exemplary embodiment of the invention, one or more tasks according to exemplary embodiments of method and / or system as described herein are performed by a data processor, such as a computing platform for executing a plurality of instructions. Optionally, the data processor includes a volatile memory for storing instructions and / or data and / or a non-volatile storage, for example, a magnetic hard-disk and / or removable media, for storing instructions and / or data. Optionally, a network connection is provided as well. A display and / or a user input device such as a keyboard or mouse are optionally provided as well.

[0051] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS )

[0052] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0053] In the drawings:

[0054] FIGs. 1 A-K (Background Art) present previously published phenylalanine amyloidogenic characteristics. FIG. 1A presents the molecular structure of phenylalanine. FIG. IB presents transmission electron microscopy (TEM) micrographs of elongated fibrils formed of phenylalanine. FIG. 1C presents confocal fluorescence microscopy images of assemblies stained with Thioflavin-T (ThT). FIG. ID shows ThT fluorescence emission spectra. FIG. IE presents confocal fluorescence microscopy images of assemblies stained with Congo Red (CR). FIG. IF is a graph obtained upon monitoring fibril formation using measurement of turbidity over time. FIG. 1G demonstrates the interaction of fibrils with inhibitor molecules using molecular dynamic simulation. FIG. 1H presents the inhibition of fibril formation by generic polyphenolic compounds as presented in the TEM micrographs. FIG. II shows intrinsic fluorescence properties of assemblies used for live cell imaging. FIGs. 1J and IK are bar graphs showing the cytotoxicity (FIG. 1J) and apoptotic effect (FIG. IK) of assemblies. FIG.s 1A-F, J-K are taken from Shaham- Niv, et al. Science advances 1 .7 (2015): el 500137. FIGs. 1G-H are taken from Shaham-Niv et al., Comm. Chem., 2018, 1 :25, pp. 1-11. FIGs. II is taken from Shaham-Niv, et al. Angewandte Chemie International Edition 57.38 (2018): 12444-12447.

[0055] FIG. 2 is a schematic illustration of metabolite amyloid properties.

[0056] FIGs. 3 A-D present a workflow scheme of the high-throughput screening for inhibitors of the phenylalanine self-assembly using computational and high-throughput screening (FIG. 3A); the molecular structure (left) and 2D chemical structure (right) of an exemplary inhibitor morin hydrate uncovered by the screening (FIG. 3B); and dose response curves and a resulting graph showing the inhibition of phenylalanine (40 mg / ml) aggregation by morin hydrate as examined using ThT fluorescence assay (FIG. 3C; emission was measured at 480 nm and excitation at 440 nm).

[0057] FIGs. 4A-C present showing a pharmacophoric model according to some embodiments of the present invention, AAR, featuring two aromatic motifs and 1 H donor motif and the distances between the motifs (FIG. 4A); and an alignment of the AAR model with morin hydrate (FIG. 4B) and with ECGC (FIG. 4C).

[0058] FIGs. 5 A-D present data obtained in efficacy studies of morin hydrate treatment at 10 and 50 mg / Kg (M10 and M50, respectively) in Pahenu2PKU mice model: Improvement in latency to platform in a Morris water maze long-term memory and spatial learning test (FIG. 5A); Probe test performed on the fifth day in a single swim (60 seconds) without platform (FIG. 5B); Learning trend in both frequency and duration in the novel arm of Y-maze short-term spatial learning test (FIG. 5C); and treatment effect on grip strength motor function (FIG. 5D).

[0059] FIGs. 6A-B are bar graphs showing that morin hydrate or Kuvan® treatment does not affect phenylalanine metabolism. After treatment, whole blood was extracted and sent for LC-MS analysis of phenylalanine (FIG. 6A) and phenylalanine / tyrosine ratio (FIG. 6B). Bars represent mean ± SD.; ***p < 0.001.

[0060] FIG. 7 is a bar graph showing that morin hydrate at both doses (M10 and M50) reduces plaque pathology. After treatment, mice were probed for plaque pathology. Right brain hemisphere was coronally sectioned into 25 pm sections and stained for Congo Red. Quantification analysis of sections stained with Congo Red was performed by image! software. The control group was set to 100 %. Bars represent mean±SEM.; *p< 0.05; **p< 0.01 FIGs. 8A-B are bar graphs showing Western blot analyses of brain homogenates of WT, vehicle, morin hydrate 50 mg / Kg, morin hydrate 10 mg / Kg and Kuvan® 50 mg / Kg (n=4) PKU treated mice. Densitometry quantification of the blots developed with anti-ChAT antibody, normalized to actin (FIG. 8A) and anti-NeuN antibody, normalized to actin (FIG. 8B). Bars represent mean ± SD.; *p < 0.05; **p < 0.01.

[0061] FIGs. 8C-D present images of the respective Western blots developed with anti-ChAT antibody (FIG. 8C) and anti-NeuN antibody (FIG. 8D).

[0062] FIGs. 9A-B are bar graphs showing Western blot analyses of brain homogenates of WT, vehicle, morin hydrate 50 mg / Kg, morin hydrate 10 mg / Kg and Kuvan® 50 mg / Kg (n=4) PKU treated mice. Densitometry quantification of the blots were developed with anti-GFAP antibody normalized to actin (FIG. 9A) and anti-Iba-1 antibody normalized to actin (FIG. 9B). Bars represent mean ± SD.; *p < 0.05.

[0063] FIGs. 9C-D present the respective Western blots developed with anti-GFAP antibody (FIG. 9C) and anti-Iba-1 antibody (FIG. 9D).

[0064] FIGs. 10A-G present chemical structures of derivatives and structural analogs of morin hydrate, according to some embodiments of the present invention.

[0065] FIGs. 11 A-D are bar graphs showing the effect of varying concentrations of apigenin (FIG. 11 A), Kaempferide (FIG. 11B), Galangin (FIG. 11C), and Kaempferol (FIG. 11D) on cell cytotoxicity in the presence of 10 mg / mL phenylalanine.

[0066] FIG. 12 is a scheme depicting a hit-to-lead process according to some of the present embodiments.

[0067] FIGs. 13A-B present the prioritization rules used in used in in silico analysis of inhibitors of the phenylalanine self-assembly (FIG. 13 A) and the scoring profile data obtained for five inhibitors according to some of the present embodiments (FIG. 13B).

[0068] FIGs. 14A-B present comparative plots showing the time-dependent fluorescence (FIG. 14A) and absorbance (FIG. 14B) obtained in ThT and turbidity assays of phenylalanine (Phe) in the absence and presence of the exemplary compounds PCM1, PCM2 and PCM3 in DMSO (. Measurements (time = 0 minutes (min)) began shortly after the addition of phenylalanine.

[0069] FIGs. 15A-E are transmission electron microscopy (TEM) images showing the formation of phenylalanine fibrils in a 20 mg / ml phenylalanine solution (FIG. 15 A); a 50 micromolar solution of PCM1 in the absence of phenylalanine; and the effect of 10, 50, and 100 micromolar PCM1, respectively, when added to a 20 mg / ml phenylalanine solution, on fibril formation.

[0070] FIG. 16 is a bar graph showing the effect of phenylalanine (6 mg / ml) and of different concentrations of PCM1 (0, 10, 50, and 100 micromolar) in the presence of phenylalanine (6 mg / ml) on cell viability. The data are presented as mean ± SD. The results represent three biological repeats.

[0071] FIG. 17A presents the chemical structure of the exemplary compound PCM1, highlighting its structural motifs.

[0072] FIGs. 17B-C presents the chemical structures of exemplary derivatives of PCM1 : SS18400- 1 and SSI 8400-7 (FIG. 17B) and SSI 8400-8, SSI 8400-9, SSI 8400- 10, and SSI 8400-11 (FIG. 17C), highlighting the structural motifs in each derivative.

[0073] FIG. 17D presents a general synthetic scheme the preparation, under microwave (MW) irradiation, of the PCM1 derivatives presented in FIGs. 17B-C.

[0074] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0075] The present invention, in some embodiments thereof, relates to therapy and, more particularly, but not exclusively, to compounds, compositions and methods for inhibiting phenylalanine aggregation and to uses thereof in treating metabolic diseases and disorders associated with phenylalanine aggregation such as phenylketonuria.

[0076] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0077] Previously reported findings have shown that phenylalanine can self-assemble to form amyloid-like fibrils possessing typical ultrastructural, biophysical and biochemical properties similar to those of protein and polypeptide amyloids, and that these phenylalanine assemblies are cytotoxic via apoptotic cell death mechanism and that antibodies raised against these species deplete fibril toxicity.

[0078] The present inventors have utilized this novel mechanistic approach as a novel therapeutic target for phenylketonuria (PKU), for designing disease-modifying agents for the treatment of PKU as an exemplary IEM disorder. PKU is one of the most common diseases among the rare inborn error of metabolism (IEM) disorders with a continuously expanding market, of which the mainstay of treatment being a very strict low-phenylalanine diet. Without proper treatment, most patients with PKU would develop a severe intellectual disability, mental retardation, behavioral abnormalities and seizures.

[0079] Currently available treatments of PKU focus around the reduction of phenylalanine concentration rather than avoiding its aggregation into toxic species. The present inventors have used high-throughput screening (HTS) to identify efficient phenylalanine self-assembly inhibitors, which resulted in the discovery of 14 hit compounds (see, Table 1 and FIG. 3 A).

[0080] One of the hit compounds, morin hydrate, which was found previously to inhibit the formation of proteinaceous amyloids, was chosen for further proof of concept in in-vivo studies. The ability of the treatment with morin hydrate to statistically improve cognitive function in PKU mice model was demonstrated. It was uncovered that while this treatment had no effect over phenylalanine levels, there is a significant reduction in its amyloid deposits (see, FIGs. 5 A-D, FIGs. 6A-D, FIG. 7, FIGs. 8A-D, and FIGs. 9A-D).

[0081] Structural derivatives of morin hydrate (see, FIGs. 10A-G) were also studied, and it was uncovered that morin hydrate derivatives, such as Apigenin, Kaempferol, Kaempferide, and Galangin, also exhibit inhibitory activity in the formation of phenylalanine fibrils (see, FIGs. 11 A- D).

[0082] Additional hit compounds of Table 1 (see, Table 2) were selected for further studies, three of which (namely PCM1, PCM2 and PCM3) were selected for in vitro validations. It was uncovered that all three compounds exhibited inhibitory activity, with PCM1 demonstrating full inhibition of the formation of fibrils (see, FIGs. 14A-B, FIGs. 15A-E and FIG. 16). Structural derivatives of PCM1 (FIGs. 17A-D) are also prepared and studied.

[0083] The uncovered 14 hit compounds allowed for the pharmacophore definition of active ingredients, which serves as a virtual screening tool in the identification and design of additional therapeutically active agents.

[0084] Referring to PKU as an amyloid disorder provides a new target and has led to the development of innovative therapeutic agent(s) for the disease-modifying treatment of PKU. The newly designed compounds are safe and potent and offer a new remedy for PKU patients, while circumventing the requirement of dietary restrictions.

[0085] Some embodiments of the present invention relate to the retrieval of compounds that are capable of inhibiting phenylalanine aggregation, which are capable of interacting with phenylalanine so as to inhibit its self-assembly into non-proteinaceous amyloid-like fibrils, per the pharmacophoric model depicted in FIG. 4A.

[0086] According to an aspect of some embodiments of the present invention there is provided a method of treating a metabolic disorder associated with formation of non-proteinaceous amyloid- like fibrils.

[0087] Herein, formation of non-proteinaceous amyloid-like fibrils describes the formation of aggregated fibrillar structures formed of non-proteinaceous materials such as carbohydrates, hydrocarbons, amino acids, etc. According to some of any of the embodiments described herein, the metabolic disorder is associated with formation of amyloid-like fibrils, which is also referred to herein as associated with phenylalanine aggregation (fibril formation), in a subject in need thereof.

[0088] As used herein throughout, the phrase "phenylalanine aggregation", which is also referred to herein interchangeably as “phenylalanine fibril formation” or simply as “fibril formation”, describes a self-assembly process of phenylalanine molecules into structured fibrillar aggregates, driven by, e.g., hydrophobic interactions and 7t-stacking. In subjects with phenylketonuria (PKU), phenylalanine aggregation leads to the formation of amyloid-like fibrils.

[0089] As used herein throughout, and as known in the art in the context of phenylalanine aggregation and / or phenylketonuria (PKU), the phrase "fibril formation" describes a process by which molecules of phenylalanine aggregate (self-assemble) into amyloid-like fibrils (e.g., elongated and insoluble fibers with a P-sheet-rich conformation).

[0090] According to some of any of the embodiments described herein, there is provided a method of treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in any of the respective embodiments in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound as described herein in any of the respective embodiments.

[0091] According to an aspect of some embodiments of the present invention there is provided a compound as described herein in any of the respective embodiments, for use in treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in any of the respective embodiments in a subject in need thereof.

[0092] According to an aspect of some embodiments of the present invention there is provided a use of a compound as described herein in any of the respective embodiments, in the manufacture of a medicament for treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in any of the respective embodiments in a subject in need thereof.

[0093] According to some of any of the embodiments described herein, there is provided a method of treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in any of the respective embodiments in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound selected from the compounds depicted in Table 1.

[0094] According to an aspect of some embodiments of the present invention there is provided a compound selected from the compounds depicted in Table 1, for use in treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in any of the respective embodiments in a subject in need thereof. According to an aspect of some embodiments of the present invention there is provided a use of a compound selected from the compounds depicted in Table 1, in the manufacture of a medicament for treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in any of the respective embodiments in a subject in need thereof.

[0095] According to some of any of the embodiments described herein, there is provided a method of treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in any of the respective embodiments in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of morin hydrate or a structural analog thereof as described herein in any of the respective embodiments.

[0096] According to an aspect of some embodiments of the present invention there is provided morin hydrate or a structural analog thereof as described herein in any of the respective embodiments, for use in treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in any of the respective embodiments in a subject in need thereof.

[0097] According to an aspect of some embodiments of the present invention there is provided a use of morin hydrate or a structural analog thereof as described herein in any of the respective embodiments, in the manufacture of a medicament for treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in any of the respective embodiments in a subject in need thereof.

[0098] Herein and in the art, a metabolic disorder describes a pathological condition characterized by an impairment in the biochemical pathways responsible for the synthesis, breakdown, or utilization of biomolecules, including carbohydrates, lipids, amino acids, peptides, proteins, and nucleic acids, resulting in abnormal accumulation or depletion of metabolic intermediates. These disorders may be congenital (genetic) or acquired and can lead to systemic dysfunction, including energy imbalances, organ failure, and structural abnormalities at the cellular level.

[0099] A subset of metabolic disorders is characterized by abnormal aggregation of proteins or peptides into insoluble fibrillar structures. These fibrils, typically composed of P-sheet-rich protein aggregates, can lead to cytotoxicity, tissue damage, and organ dysfunction. These disorders often involve disruptions in protein homeostasis, post-translational modifications, proteostasis mechanisms, or extracellular matrix regulation.

[0100] According to some of any of the embodiments described herein, the metabolic disorder is caused by an inborn error of metabolism.

[0101] Inborn errors of metabolism (IEM) refer to a group of genetic disorders that result from defects in the biochemical pathways responsible for metabolizing various substances within the body. Metabolism involves the complex set of chemical reactions that occur to maintain life, including the conversion of nutrients into energy and the synthesis of essential molecules. These disorders are typically caused by mutations in genes that encode enzymes involved in specific metabolic pathways. Enzymes play a crucial role in catalyzing the various chemical reactions required for the breakdown or synthesis of molecules. When there is a deficiency or dysfunction of a specific enzyme, it can lead to the accumulation of certain substances or a lack of essential products, disrupting normal metabolic processes.

[0102] The clinical manifestations of inborn errors of metabolism can vary widely depending on the specific enzyme and pathway affected. Symptoms may include developmental delays, intellectual disabilities, seizures, neurological problems, organ dysfunction, and metabolic crises. The age of onset and severity can also differ, with some conditions presenting in infancy, while others may become apparent later in childhood or adulthood.

[0103] Examples of inborn errors of metabolism include phenylketonuria (PKU), maple syrup urine disease, galactosemia, and lysosomal storage disorders such as Tay-Sachs disease.

[0104] According to some of any of the embodiments described herein, the metabolic disorder is an amino acid metabolism disorder.

[0105] As known in the art, the phrase “amino acid metabolism disorder” describes a group of inborn errors of metabolism characterized by defects in the enzymatic pathways responsible for the synthesis, breakdown, or transport of amino acids. The clinical manifestations of these disorders vary widely and may include neurological impairment, developmental delays, organ dysfunction, and metabolic crises. Non-limiting examples of amino acid metabolism disorders include phenylketonuria (PKU), maple syrup urine disease (MSUD), homocystinuria, tyrosinemia, and alkaptonuria.

[0106] According to some of any of the embodiments described herein, the metabolic disorder is phenylketonuria.

[0107] As used herein throughout, “phenylketonuria” (PKU) refers to an inherited metabolic disorder caused by a deficiency of the enzyme phenylalanine hydroxylase (PAH). This deficiency results in elevated levels of phenylalanine (Phe), which can aggregate into amyloid-like fibrils. The formation of these phenylalanine amyloid-like fibrils may further disrupt neurotransmitter balance, leading to severe intellectual disability, behavioral abnormalities, delayed speech, and seizures.

[0108] According to some of any of the embodiments described herein, compounds usable in any of the methods and uses described herein are capable of aligning or overlapping with a phenylalanine pharmacophoric model as depicted in FIG. 4A.

[0109] According to some embodiments, the compound features at least two aromatic moi eties and at least one hydrogen-bond donor moiety which are spatially arranged so as to form at least two aromatic interactions and at least one hydrogen bond with the phenylalanine pharmacophoric model, to thereby interfere with (e.g., inhibit, destabilize) phenylalanine aggregation (fibril formation), and / or treat the metabolic disorder.

[0110] As used herein, the phrase “aromatic moiety” or “aromatic group” describes a chemical moiety having one or more completely conjugated ring system, such as an aryl and / or a heteroaryl as defined herein, that follows Hiickel’s rule as known in the art, and possesses delocalized 71- electrons. Non-limiting examples include phenyl, polycyclic aromatic hydrocarbons (PAHs) (e.g., naphthalene), pyrrole, pyrazole, pyridine, thiophene, furans, imidazoles, triazoles, tetrazoles, oxazoles, thiazoles, purines, quinoline, isoquinoline, and pyrimidines.

[0111] As used herein and known in the art, a “hydrogen bond” is a non-covalent bond that forms a type of dipole-dipole attraction which occurs when a hydrogen atom bonded to a strongly electronegative atom exists in the vicinity of another electronegative atom with a lone pair of electrons.

[0112] The hydrogen atom in a hydrogen bond is partly shared between two relatively electronegative atoms.

[0113] Hydrogen bonds are typically formed between a hydrogen bond donor group and a hydrogen bond acceptor group.

[0114] As used herein, the phrase “hydrogen-bond donor” (substituent or moiety) describes a functional group that includes both the atom to which the hydrogen is more tightly linked and the hydrogen atom itself, and is capable of donating a hydrogen atom to participate in a hydrogen bond as defined herein. Non-limiting examples of hydrogen-bond donor substituents include amide, carboxylate, hydroxy, alkoxy, aryloxy, ether, amine, carbamate, urethane, hydrazine, a nitrogencontaining heteroalicyclic (e.g., piperidine, oxalidine), nitrile, an oxygen-containing heteroalicyclic (e.g., tetrahydrofuran, morpholine), and any other chemical moiety that comprises one or more nitrogen and / or oxygen atoms.

[0115] A hydrogen-bond acceptor is an electronegative atom capable of being linked to a hydrogen atom of another group. The relatively electronegative atom to which the hydrogen atom is covalently bonded pulls electron density away from the hydrogen atom so that it develops a partial positive charge (6+). Thus, it can interact with an atom having a partial negative charge (6‘) through an electrostatic interaction.

[0116] Atoms that typically participate in hydrogen bond interactions, as donors and / or acceptors, include oxygen, nitrogen and fluorine. These atoms typically form a part of a chemical group or moiety such as, for example, carbonyl, carboxylate, amide, hydroxyl, amine, imine, carbamate, alkyl fluoride, F2, and more. However, other electronegative atoms and chemical groups or moieties containing same may participate in hydrogen bonding. As used herein, the phrase “aromatic interactions” describes non-covalent interactions between aromatic moieties. Non-limiting aromatic interactions include 7t-7t stacking, CH-7t interactions, cation-7t interactions, and edge-to-face interactions.

[0117] As known in the art, the phrase “TC-TC stacking” describes a non-covalent interaction between two (or more) aromatic rings involving the alignment of their 7t-electron clouds in a parallel or offset configuration.

[0118] As known in the art, the phrase “CH-7t interaction” describes a non-covalent interaction between an aromatic 7t-system and a hydrogen atom chemically bound to a carbon.

[0119] As known in the art, the phrase “cation-7t interaction” describes an electrostatic interaction between a cation (a positively charged ion) and an aromatic 7t-system.

[0120] As known in the art, the phrase “edge-to-face interaction” describes a non-covalent interaction between two aromatic rings in which one ring’s edge interacts perpendicularly with the 7t-system of another ring.

[0121] As known in the art, the phrase “polycyclic system” describes a molecular structure composed of two or more interconnected or fused rings, which may be aromatic, partially aromatic, or non-aromatic. Non-limiting examples for polycyclic systems include carbon-based frameworks (e.g., naphthalene and anthracene), heteroaromatic polycyclic systems (e.g., quinoline and purine), and polycyclic non-aromatic systems (e.g., decalin).

[0122] As known in the art, the phrase “polycyclic aromatic hydrocarbon” (PAH) describes a hydrocarbon composed of multiple fused aromatic rings, as described herein, containing only carbon and hydrogen atoms.

[0123] According to some of any of the embodiments described herein, the compound (e.g., the compound that aligns or overlaps with the pharmacophoric model) is capable of spatially arranging such that a distance between two of the at least two aromatic moieties is in a range of from about 5.0 to about 7.5, or from about 5.5 to about 7.5, or from about 5.5 to about 6.5, or from about 6.0 to about 7.0, or from about 6.0 to about 6.5, or is about 6.3, angstroms, including any intermediate values and subranges therebetween; and a distance of the hydrogen-bond donor moiety from one aromatic moiety is in a range of from about 2.0 to about 4.0, or from about 2.5 to about 3.5, or from about 2.0 to about 3.0, or from about 3.5 to about 3.0, or is about 2.7, angstroms, including any intermediate values and subranges therebetween; and a distance of the hydrogen-bond donor moiety from a second (the other) aromatic moiety is in a range of from about 7.0 to about 9.0, or from about 7.5 to about 8.5, or from about 7.0 to about 8.5, or from about 7.5 to about 9.0, or from about 7.5 to about 8.0, or is about 7.9, angstroms, including any intermediate values and subranges therebetween. According to some of any of the embodiments described herein, the compound (e.g., the compound that aligns or overlaps with the pharmacophoric model) is capable of spatially arranging such that a distance between two of the at least two aromatic moieties is 6.3 angstroms ± 30 %, or ± 25 %, or ± 20 %, or ± 15 %, or ± 10 %, or ± 7.5 %, or ± 5 %, or ± 2 %, or ± 1 %, or even less (e.g., ± 0 %, “full alignment” or “complete overlap” with the pharmacophoric model as described herein), including any intermediate values and subranges therebetween; and / or a distance of the hydrogen-bond donor moiety from one aromatic moiety is 2.7 angstroms ± 30 %, or ± 25 %, or ± 20 %, or ± 15 %, or ± 10 %, or ± 7.5 %, or ± 5 %, or ± 2 %, or ± 1 %, or even less (e.g., ± 0 %, “full alignment” or “complete overlap” with the pharmacophoric model as described herein), including any intermediate values and subranges therebetween; and / or a distance of the hydrogen-bond donor moiety from a second (the other) aromatic moiety is 7.9 angstroms ± 30 %, or ± 25 %, or ± 20 %, or ± 15 %, or ± 10 %, or ± 7.5 %, or ± 5 %, or ± 2 %, or ± 1 %, or even less (e.g., ± 0 %, which means “full alignment” or “complete overlap” with the pharmacophoric model as described herein), including any intermediate values and subranges therebetween.

[0124] In some embodiments, determining if the compound is capable of spatially arranging in the distances as described herein (i.e., a distance between two of the at least two aromatic moieties; a distance of the hydrogen-bond donor moiety from one aromatic moiety; and / or a distance of the hydrogen-bond donor moiety from a second (the other) aromatic moiety) is affected using in silico tools as described herein (e.g., computational modeling, such that the tool enables measuring the distances between corresponding functional groups).

[0125] As used herein, the phrase “capable of spatially arranging” describes the ability of a compound to adopt a conformational state in which specific functional groups align with a pharmacophoric model within the defined spatial tolerances. This spatial arrangement may be influenced by molecular flexibility, steric hindrance, electronic interactions, and other structural properties that enable partial or substantial overlap (e.g., 60-100 %) with the pharmacophoric model.

[0126] As used herein, the phrase “compound that aligns or overlaps with the pharmacophoric model” describes a compound that adopts a spatial conformation in which its key functional groups (the hydrogen-bond donor and two aromatic moieties) position themselves within the defined spatial tolerances of the pharmacophoric model. This alignment or overlap is partial or substantial (e.g., 60-100 %).

[0127] According to some of any of the embodiments described herein, exemplary compounds usable in any of the methods and uses described herein are set forth in Table 1 herein. Compound 5 of Table 1 is morin hydrate. According to some of any of the embodiments described herein, the compound is morin hydrate or a structural analog thereof.

[0128] The phrase “structural analog thereof’ describes a compound that shares a core chemical scaffold with morin hydrate while containing modifications, substitutions, or functional group variations that retain or enhance its ability to interact with phenylalanine aggregates (fibril formation), and is optionally represented by Formula A, I or la.

[0129] The chemical structures of exemplary structural analogs of morin hydrate are presented in FIGs. 10A-G.

[0130] According to some of any of the embodiments described herein, compounds usable in any of the methods and uses described herein are collectively represented by Formula A: wherein: the dashed line represents an optional double bond;

[0131] X is O, S or NRn; and

[0132] R1-R5, R11 and Y are each independently selected from hydrogen, alkyl, cycloalkyl, heteroalicyclic, hydroxy, thiol, alkoxy, thioalkoxy, amine, aryl, heteroaryl, halo, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- thiocarbamyl, S -thiocarb amyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and likewise substituents.

[0133] According to some of any of the embodiments described herein, at least one of R1-R5 is a hydrogen-bond donor substituent, for example, hydroxy, amine, thiol, hydrazine, amide, oxime, and likewise substituents, and is preferably hydroxy.

[0134] In some embodiments, one or both of Y and Rs are each independently selected from hydrogen, an aryl, and an alkyl (preferably a methyl).

[0135] According to some of any of the embodiments described herein, one or both of Y and Rs are each independently hydrogen or an aryl, which can be unsubstituted or substituted. According to some of any of the embodiments described herein, Rs is an aryl, which can be unsubstituted or substituted. According to some of any of the embodiments described herein, Y is hydrogen and Rs is an aryl, which can be unsubstituted or substituted. When substituted, the substituent can be any of the substituents described herein. In some embodiments, the aryl is substituted by one or more of alkyl, cycloalkyl, heteroalicyclic, hydroxy, thiol, alkoxy, thioalkoxy, halo, and amine, and in some embodiments, the aryl is substituted by one or more of hydroxy and alkoxy (e.g., methoxy).

[0136] According to some of any of the embodiments described herein, Y is an aryl, which can be unsubstituted or substituted. According to some of any of the embodiments described herein, Rs is hydrogen and Y is an aryl, which can be unsubstituted or substituted. When substituted, the substituent can be any of the substituents described herein. In some embodiments, the aryl is substituted by one or more of alkyl, cycloalkyl, heteroalicyclic, hydroxy, thiol, alkoxy, thioalkoxy, halo, and amine, and in some embodiments, the aryl is substituted by one or more of hydroxy and alkoxy (e.g., methoxy).

[0137] According to some of any of the embodiments described herein, compounds usable in any of the methods and uses described herein are collectively represented by Formula I:

[0138] Formula I wherein: the dashed line represents an optional double bond;

[0139] X is O, S or NRn; and

[0140] R1-R11 are each independently selected from hydrogen, alkyl, cycloalkyl, heteroalicyclic, hydroxy, thiol, alkoxy, thioalkoxy, amine, halo, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S -thiocarb amyl, C-amido, N- amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and likewise substituents as described herein. According to some of any of the embodiments described herein for Formula I, at least one of R1-R4 is a hydrogen-bond donor substituent as described herein, preferably hydroxy.

[0141] According to some of any of the embodiments described herein for Formula I, two or more, or three or more, or four or more, or five or more, or all, of R1-R11 are each independently a hydrogen-bond donor substituent as described herein, preferably hydroxy.

[0142] According to some of any of the embodiments described herein for Formula I, two or more, or three or more, or four or more, or five or more, or all, of R1-R11 are each independently hydroxy or alkoxy as described herein.

[0143] According to an aspect of some embodiments of the present invention there is provided a compound represented by Formula I as described herein in any of the respective embodiments, for use in treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) in a subject in need thereof.

[0144] According to an aspect of some embodiments of the present invention there is provided a use of a compound represented by Formula I as described herein in any of the respective embodiments, in the manufacture of a medicament for treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) in a subject in need thereof.

[0145] According to some of any of the embodiments described herein, the dashed line represents a double bond.

[0146] According to some of any of the embodiments described herein for Formula I, X is O.

[0147] According to some of any of the embodiments described herein, the dashed line represents a double bond, and X is O.

[0148] According to some of any of the embodiments described herein for Formula I, Ri is hydroxy.

[0149] According to some of any of the embodiments described herein for Formula I, one or more, or two or more, or three or more, or all, of R1-R4 are each hydroxy.

[0150] According to some of any of the embodiments described herein for Formula I, R3 is hydroxy.

[0151] According to some of any of the embodiments described herein for Formula I, Ri and R3 are each hydroxy.

[0152] According to some of any of the embodiments described herein for Formula I, at least one of R2 and R4 is hydrogen.

[0153] According to some of any of the embodiments described herein for Formula I, R2 and R4 are each hydrogen.

[0154] According to some of any of the embodiments described herein for Formula I, Ri and R3 are each independently hydroxy, and R2 and R4 are each hydrogen. According to some of any of the embodiments described herein for Formula I, one or more, or two or more, or three or more, or four or more, or all, of Ri, R3, Rs, Re, Rs and Rio are each independently a hydrogen-bond donor substituent as described herein, preferably hydroxy.

[0155] According to some of any of the embodiments described herein for Formula I, one or more, or two or more, or three or more, or four or more, or all, of Ri, R3, Rs, Re, Rs and Rio are each independently hydroxy or alkoxy as described herein (e.g., a short alkoxy of 1-4 carbon atoms in length, for example, methoxy).

[0156] According to some of any of the embodiments described herein for Formula I, X is O, and one or more, or two or more, or three or more, or four or more, or all, of Ri, R3, Rs, Re, Rs and Rio are each independently hydroxy or alkoxy as described herein (e.g., a short alkoxy of 1-4 carbon atoms in length, for example, methoxy).

[0157] According to some of any of the embodiments described herein for Formula I, the dashed line represents a double bond, X is O, and one or more, or two or more, or three or more, or four or more, or all, of Ri, R3, Rs, Re, Rs and Rio are each independently hydroxy or alkoxy as described herein (e.g., a short alkoxy of 1-4 carbon atoms in length, for example, methoxy).

[0158] According to some of any of the embodiments described herein for Formula I, one or more, or two or more, or three or more, or all, of Ri, R3, Rs, and Rs are each independently a hydrogenbond donor substituent as described herein, preferably hydroxy.

[0159] According to some of any of the embodiments described herein for Formula I, one or more, or two or more, or three or more, or all, of Ri, R3, Rs, and Rs are each independently hydroxy or alkoxy as described herein.

[0160] According to some of any of the embodiments described herein for Formula I, X is O, and one or more, or two or more, or three or more, or all, of Ri, R3, Rs, and Rs are each independently hydroxy or alkoxy as described herein.

[0161] According to some of any of the embodiments described herein for Formula I, the dashed line represents a double bond, X is O, and one or more, or two or more, or three or more, or all, of Ri, R3, Rs, and Rs are each independently hydroxy or alkoxy as described herein.

[0162] According to some of any of the embodiments described herein for Formula I, Rs is selected from hydrogen, hydroxy and alkoxy.

[0163] According to some of any of the embodiments described herein for Formula I, X is O, and Rs is selected from hydrogen, hydroxy and alkoxy (e.g., a short alkoxy of 1-4 carbon atoms in length, for example, methoxy).

[0164] According to some of any of the embodiments described herein for Formula I, the dashed line represents a double bond, X is O, and Rs is selected from hydrogen, hydroxy and alkoxy. According to some of any of the embodiments described herein for Formula I, Rs is hydroxy.

[0165] According to some of any of the embodiments described herein for Formula I, X is O, and Rs is hydroxy.

[0166] According to some of any of the embodiments described herein for Formula I, the dashed line represents a double bond, X is O, and Rs is hydroxy.

[0167] According to some of any of the embodiments described herein for Formula I, Ri and Rs are each hydroxy, and optionally one or more, or two or more, or all, of R2-R4 is hydroxy or alkoxy (e.g., a short alkoxy of 1-4 carbon atoms in length, for example, methoxy).

[0168] According to some of any of the embodiments described herein for Formula I, Rs is hydroxy, and two or more, or three or more, or all, of Ri, R3, Rs, and Rs are each independently hydroxy or alkoxy as described herein.

[0169] According to some of any of the embodiments described herein for Formula I, X is O, Rs is hydroxy, and two or more, or three or more, or all, of Ri, R3, Rs, and Rs are each independently hydroxy or alkoxy as described herein.

[0170] According to some of any of the embodiments described herein for Formula I, the dashed line represents a double bond, X is O, Rs is hydroxy, and two or more, or three or more, or all, of Ri, R3, Rs, and Rs are each independently hydroxy or alkoxy as described herein.

[0171] According to some of any of the embodiments described herein for Formula I, R3 is selected from hydrogen, hydroxy and alkoxy as described herein.

[0172] According to some of any of the embodiments described herein for Formula I, R3 is hydroxy.

[0173] According to some of any of the embodiments described herein for Formula I, Ri, R3 and Rs are each independently hydroxy, and optionally R2 and R4 are each hydrogen.

[0174] According to some of any of the embodiments described herein for Formula I, X is O, Ri, R3 and Rs are each independently hydroxy, and optionally R2 and R4 are each hydrogen.

[0175] According to some of any of the embodiments described herein for Formula I, the dashed line represents a double bond, X is O, Ri, R3 and Rs are each independently hydroxy, and optionally R2 and R4 are each hydrogen.

[0176] According to some of any of the embodiments described herein for Formula I, at least one of Re-Rio is selected from hydroxy and alkoxy (e.g., a short alkoxy of 1-4 carbon atoms in length, for example, methoxy).

[0177] According to some of any of the embodiments described herein for Formula I, at least Rs is hydroxy or alkoxy as described herein. According to some of any of the embodiments described herein for Formula I, Rs is hydroxy or alkoxy as described herein and Re, R? and R9 are each hydrogen, and optionally Rio is also hydrogen.

[0178] According to some of any of the embodiments described herein for Formula I, Rs is hydroxy, Re, R7 and R9 are each hydrogen, and optionally Rio is also hydrogen.

[0179] According to some of any of the embodiments described herein for Formula I, X is O, and at least one of Re-Riois selected from hydroxy and alkoxy (e.g., a short alkoxy of 1-4 carbon atoms in length, for example, methoxy).

[0180] According to some of any of the embodiments described herein for Formula I, the dashed line represents a double bond, X is O, and at least one of Re-Rio is selected from hydroxy and alkoxy (e.g., a short alkoxy of 1-4 carbon atoms in length, for example, methoxy).

[0181] According to some of any of the embodiments described herein for Formula I, X is O, Ri, R3 and R5 are each independently hydroxy, and at least one of Re-Rio is selected from hydroxy and alkoxy (e.g., a short alkoxy of 1-4 carbon atoms in length, for example, methoxy). According to some of these embodiments, R2 and R4 are each hydrogen

[0182] According to some of any of the embodiments described herein for Formula I, the dashed line represents a double bond, X is O, Ri, R3 and Rs are each independently hydroxy, and optionally R2 and R4 are each hydrogen, and at least one of Re-Rio is selected from hydroxy and alkoxy (e.g., a short alkoxy of 1-4 carbon atoms in length, for example, methoxy). According to some of these embodiments, R2 and R4 are each hydrogen

[0183] According to some of any of the embodiments described herein for Formula I, X is O, Rs is hydroxy, Re, R7 and R9 are each hydrogen. According to some of these embodiments, Rio is hydrogen.

[0184] According to some of any of the embodiments described herein for Formula I, the dashed line represents a double bond, X is O, Rs is hydroxy, Re, R7 and R9 are each hydrogen. According to some of these embodiments, Rio is hydrogen. According to some of any of the embodiments described herein for Formula I, X is O, Ri, R3 and Rs are each independently hydroxy, Rs is hydroxy, Re, R7 and R9 are each hydrogen. According to some of these embodiments, R2 and R4 are each hydrogen. According to some of these embodiments, Rio is hydrogen. According to some of these embodiments, R2, R4 and Rio are each hydrogen.

[0185] According to some of any of the embodiments described herein for Formula I, the dashed line represents a double bond, X is O, Ri, R3 and Rs are each independently hydroxy, Rs is hydroxy, and Re, R7 and R9 are each hydrogen. According to some of these embodiments, R2 and R4 are each hydrogen. According to some of these embodiments, Rio is hydrogen. According to some of these embodiments, R2, R4 and Rio are each hydrogen. According to some of any of the embodiments described herein for Formula I, at least one of Re-Rio is selected from hydroxy and alkoxy (e.g., a short alkoxy of 1-4 carbon atoms in length, for example, methoxy).

[0186] According to some of any of the embodiments described herein for Formula I, one or more, or two or more, or three or more, or all, of Re-Rio are each independently selected from hydroxy and alkoxy as described herein.

[0187] According to some of any of the embodiments described herein for Formula I, X is O, and one or more, or two or more, or three or more, or all, of Re-Rio are each independently selected from hydroxy and alkoxy as described herein.

[0188] According to some of any of the embodiments described herein for Formula I, the dashed line represents a double bond, X is O, and one or more, or two or more, or three or more, or all, of Re-Rio are each independently selected from hydroxy and alkoxy as described herein.

[0189] According to some of any of the embodiments described herein for Formula I, one of Re- Rio is selected from hydroxy and alkoxy as described herein.

[0190] According to some of any of the embodiments described herein for Formula I, one of Re, Rs and Rio is selected from hydroxy and alkoxy as described herein.

[0191] According to some of any of the embodiments described herein for Formula I, X is O, and one of Re, Rs and Rio is selected from hydroxy and alkoxy as described herein.

[0192] According to some of any of the embodiments described herein for Formula I, the dashed line represents a double bond, X is O, and one of Re, Rs and Rio is selected from hydroxy and alkoxy as described herein.

[0193] According to some of any of the embodiments described herein for Formula I, X is O, one of Rs and Rio is selected from hydroxy and alkoxy as described herein, and Re, R? and R9 are each hydrogen.

[0194] According to some of any of the embodiments described herein for Formula I, the dashed line represents a double bond, X is O, one of Rs and Rio is selected from hydroxy and alkoxy as described herein, and Re, R7 and R9 are each hydrogen.

[0195] According to some of any of the embodiments described herein for Formula I, Rs and Rio are each independently hydroxy or alkoxy.

[0196] According to some of any of the embodiments described herein for Formula I, Ri, R3 and R5 are each independently hydroxy, and one or more of Re, Rs and Rio is hydroxy.

[0197] According to some of any of the embodiments described herein for Formula I, Ri, R3 and Rs are each independently hydroxy, and one of Re, Rs and Rio is hydroxy.

[0198] According to some of any of the embodiments described herein for Formula I, Ri, R3, Rs and Rio are each independently hydroxy. According to some of any of the embodiments described herein for Formula I, Ri and R3 are each independently hydroxy, and at least one of R2 and R4 is hydrogen.

[0199] According to some of any of the embodiments described herein for Formula I, Ri and R3 are each independently hydroxy, at least one of R2 and RHs hydrogen, and one or more of Re, Rs and Rio is hydroxy.

[0200] According to some of any of the embodiments described herein for Formula I, Ri and R3 are each independently hydroxy, at least one of R2 and R4 is hydrogen, and one of Re, Rs and Rio is hydroxy.

[0201] According to some of any of the embodiments described herein for Formula I, Ri and R3 are each independently hydroxy, and R2 and R4 are each independently hydrogen.

[0202] According to some of any of the embodiments described herein for Formula I, Ri and R3 are each independently hydroxy, R2 and R4 are each independently hydrogen, and one or more of Re, Rs and Rio is hydroxy.

[0203] According to some of any of the embodiments described herein for Formula I, Ri and R3 are each independently hydroxy, R2 and R4 are each independently hydrogen, and one of Re, Rs and Rio is hydroxy.

[0204] According to some of any of the embodiments described herein for Formula I, Ri, R3 and Rs are each independently hydroxy, R2 and R4 are each independently hydrogen, and Rio is hydroxy. According to some of these embodiments, X is O and the dashed line represents a double bond. An exemplary such compound is morin hydrate.

[0205] According to some of any of the embodiments described herein for Formula I, Ri, R3 and Rs are each independently hydroxy, R2 and R4 are each independently hydrogen, and Rs is hydroxy. According to some of these embodiments, X is O and the dashed line represents a double bond. An exemplary such compound is Kaempferol.

[0206] According to some of any of the embodiments described herein for Formula I, at least one of D6-D10 is alkoxy (e.g., a short alkoxy of 1-4 carbon atoms in length, for example, methoxy).

[0207] According to some of any of the embodiments described herein, at least Rs is alkoxy as described herein.

[0208] According to some of any of the embodiments described herein for Formula I, at least Rs is alkoxy as described herein, and two or more of Ri, R3 and Rs are independently hydroxy.

[0209] According to some of any of the embodiments described herein for Formula I, at least Rs is alkoxy as described herein, and Ri, R3 and Rs are each independently hydroxy.

[0210] According to some of any of the embodiments described herein for Formula I, at least Rs is alkoxy as described herein, Ri, R3 and Rs are each independently hydroxy, and R2 and R4 are each independently hydrogen. According to some of any of the embodiments described herein for Formula I, Rs is alkoxy as described herein, Ri, R3 and R5 are each independently hydroxy. According to some of these embodiments, R2 and R4 are each independently hydrogen. According to some of these embodiments, Re, R7, R9 and Rio are each hydrogen.

[0211] According to some of any of the embodiments described herein for Formula I, Ri, R3 and Rs are each independently hydroxy, R2 and R4 are each independently hydrogen, and Rs is alkoxy as described herein. According to some of these embodiments, Re, R7, R9 and Rio are each hydrogen. According to some of these embodiments, X is O and the dashed line represents a double bond. An exemplary such compound is Kaempferide.

[0212] According to some of any of the embodiments described herein for Formula I, at least two, or at least three, or at least four, or all, of De-Dio are each hydrogen.

[0213] According to some of any of the embodiments described herein for Formula I, at least two, or at least three, or at least four, or all, of De-Dio are each hydrogen, and Ri, R3 and Rs are each independently hydroxy or alkoxy as described herein.

[0214] According to some of any of the embodiments described herein for Formula I, De-Dio are each hydrogen, and Ri, R3 and Rs are each independently hydroxy or alkoxy as described herein.

[0215] According to some of any of the embodiments described herein for Formula I, De-Dio are each hydrogen, and Ri, R3 and Rs are each independently hydroxy. An exemplary such compound is Galangin.

[0216] According to some of any of the embodiments described herein for Formula I, Rs is hydrogen.

[0217] According to some of any of the embodiments described herein for Formula I, Rs is hydrogen, and Ri and R3 are each independently hydroxy.

[0218] According to some of any of the embodiments described herein for Formula I, Rs is hydrogen, at least one of Ri, R3 and Rs are each independently hydroxy, and optionally R2, R4 and Rs are each independently hydrogen.

[0219] According to some of any of the embodiments described herein for Formula I, Ri and R3 are each independently hydroxy, R2, R4 and Rs are each independently hydrogen, and Rs is hydroxy. An exemplary such compound is Apigenin.

[0220] According to some of any of the embodiments described herein, compounds usable in any of the methods and uses described herein are collectively represented by Formula la:

[0221] Formula la wherein R1-R10 are each independently as described for Formula I in any of the respective embodiments and any combination thereof.

[0222] According to some of any of the embodiments described herein for Formula la, R2 and R4 are each hydrogen. According to some of any of the embodiments described herein for Formula la, R7 and R9 are each hydrogen.

[0223] According to some of any of the embodiments described herein for Formula la, R2, R4 R7 and R9 are each hydrogen. According to some of these embodiments, one, two, three, four of all of Ri, R3 R5, Re, Rs and Rio is hydroxy, as described herein in any of the respective embodiments of Formula I. According to some of these embodiments, Ri and R3 are each hydroxy, and Rs is hydrogen. According to some of these embodiments, Ri and R3 are each hydroxy, and Rs is hydroxy. According to some of these embodiments, one or more of Re, Rs and Rio is hydroxy, as described herein in any of the respective embodiments of Formula I.

[0224] According to some of any of the embodiments described herein for Formula la, R2, R4 and R6-R9 are each hydrogen and Rio is hydroxy. According to some of these embodiments, Ri and R3 are each hydroxy, and Rs is hydroxy.

[0225] According to some of any of the embodiments described herein, the compound is morin hydrate.

[0226] Morin hydrate (3,5,7-trihydroxy-2-(2-hydroxyphenyl)-4H-chromen-4-one hydrate) (CAS No. 654055-01-3) is a plant-derived flavonoid that has the following chemical structure (which is also presented in FIG. 3B):

[0227] 3,5 ,7-trihydroxy-2-(2-hydroxyphenyl)-4H-chromen-4-one hydrate

[0228] According to some of any of the embodiments described herein for Formula la, R2, R4 and Re, R7, R9 and Rio are each hydrogen and Rs is hydroxy.

[0229] According to some of any of the embodiments described herein, the compound is Apigenin. Apigenin (5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one; also being referred to as 4',5,7-trihydroxyflavone) (CAS No. 520-36-5) is a plant-derived flavonoid that has the following chemical structure:

[0230] 5,7-dihydroxy-2-(4-hydroxyphenyl)-477-chromen-4-one

[0231] According to some of any of the embodiments described herein, the compound is Kaempferol.

[0232] Kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one; also being referred to as 3,4',5,7-tetrahydroxyflavone) (CAS No. 520-18-3) is a plant-derived flavonoid that has the following chemical structure:

[0233] 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4 / / -chromen-4-one According to some of any of the embodiments described herein, the compound is Kaempferide.

[0234] Kaempferide (3,5,7-trihydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one; also being referred to as 4'-methoxy-3,5,7-trihydroxyflavone) (CAS No. 491-54-3) is a plant-derived flavonoid that has the following chemical structure:

[0235] 3,5,7-trihydroxy-2-(4-methoxyphenyl)-477-chromen-4-one

[0236] According to some of any of the embodiments described herein, the compound is Galangin.

[0237] Galangin (3,5,7-trihydroxy-2-phenyl-4H-chromen-4-one; also being referred to as 3,5,7- trihydroxyflavone) (CAS No. 548-83-4) is a plant-derived flavonoid that has the following chemical structure:

[0238] 3 , 5 ,7-trihydroxy-2-phenyl-42 / -chromen-4-one

[0239] According to some of any of the embodiments described herein, the compound is selected from morin hydrate, Apigenin, Kaempferol, Kaempferide and Galangin.

[0240] According to some of any of the embodiments described herein, compounds usable in any of the methods and uses described herein include those presented in Table 1 hereinbelow, or structural analogs thereof.

[0241] In some of any of the embodiments described herein, the compound as described herein in any of the respective embodiments is devoid of an electrophilic chemical moiety. Non-limiting examples of electrophilic chemical moieties include aldehydes, alkyl dihalides (an alkyl as defined herein, substituted by two halides), and quinones. As used herein and as known in the art, the phrase “electrophilic chemical moiety” describes a functional group or moiety capable of accepting an electron pair (e.g., by reacting with a nucleophilic chemical moiety to form a covalent bond).

[0242] According to some of any of the embodiments described herein, exemplary compounds usable in any of the methods and uses described herein include:

[0243] According to some of any of the embodiments described herein, exemplary compounds usable in any of the methods and uses described herein include: (N-[2-(lH-imidazol-4-yl)ethyl]-4-(5-methyl-2- furyl)benzamide, “PCM1”); (N-benzyl-l-(l-isopropyl-lH-pyrazol-4-yl)-N-(l,3-thiazol-2- ylmethyl)methanamine, “PCM2”); and (3-chloro-N-({2-[3-(trifluoromethyl)phenoxy]pyridin-3- yl}methyl)pyridin-2-amine, “PCM3”). According to some of any of the embodiments described herein, an exemplary compound usable in any of the methods and uses described herein is PCM1 as described herein, or structural analogs thereof.

[0244] According to some of any of the embodiments described herein, compounds usable in any of the methods and uses described herein are collectively represented by Formula II:

[0245] Formula II wherein L is a linking moiety or is absent,

[0246] Zi is an aryl or a heteroaryl as described herein, and

[0247] Z2 is a heteroaryl as described herein.

[0248] According to some of any of the embodiments described herein, the linking moiety L is or comprises an alkylene. In some of any of the embodiments described herein, the linking group is a saturated alkylene. In some of any of the embodiments described herein, the linking group is an alkylene of 1-4 carbon atoms. In some of any of the embodiments described herein, the linking group is a saturated alkylene of 1-4 carbon atoms. In some of any of the embodiments described herein, the linking group L is ethylene.

[0249] According to some of any of the embodiments described herein, the heteroaryl Z2 is a nitrogen-containing heteroaryl, which comprises one or more nitrogen atoms in the heteroaryl ring(s). Non-limiting examples for nitrogen-containing heteroaryls include diazoles, triazoles, pyridines, pyrimidines, pyrazines, pyridazines, imidazoles, tetrazoles, tetrazines, pyrazoles, indoles, quinolines, isoquinolines, purines, carbazoles, indazoles, benzimidazoles, pyrroles, and pyrrolidines. In some of any of the embodiments described herein, the heteroaryl Z2 is a fivemembered ring nitrogen-containing heteroaryl. Non-limiting examples for five-membered ring nitrogen-containing heteroaryls include pyrroles, imidazoles, pyrazoles, triazoles, tetrazoles, thiazoles, isothiazoles, oxazoles, isoxazoles, and thiadiazoles. In some of any of the embodiments described herein, the heteroaryl Z2 is a diazole.

[0250] In some of any of the embodiments described herein, the linking moiety L is an alkylene and the heteroaryl Z2 is a nitrogen-containing heteroaryl. In some of any of the embodiments described herein, the linking group L is an alkylene and the heteroaryl Z2 is a five-membered ring nitrogen-containing heteroaryl. In some of any of the embodiments described herein, the linking group L is an alkylene and the heteroaryl Z2 is a diazole. In some of any of the embodiments described herein, the linking group L is an alkylene of 1-4 carbon atoms and Z2 is a nitrogen- containing heteroaryl. In some of any of the embodiments described herein, the linking group L is an alkylene of 1-4 carbon atoms and Z2 is a diazole. In some of any of the embodiments described herein, the linking group L is ethylene and Z2 is a nitrogen-containing heteroaryl. In some of any of the embodiments described herein, the linking group L is ethylene and Z2 is a diazole.

[0251] In some of any of the embodiments described herein, Zi is an aryl, optionally a substituted aryl. According to some of any of the embodiments described herein, the aryl is phenyl, optionally a substituted phenyl. In some of any of the embodiments described herein, Zi is an unsubstituted phenyl. When substituted, the aryl can include one or more substituents independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S- thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein.

[0252] In some of any of the embodiments described herein, Zi is a phenyl substituted by a heteroaryl as described herein. In some of any of the embodiments described herein, aryl Zi is a phenyl substituted by a furan, optionally a substituted furan.

[0253] In some of any of the embodiments described herein, Zi is a phenyl substituted by a substituent selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl, halo, hydroxy, alkoxy, and aryloxy. In some of any of the embodiments described herein, Zi is a phenyl substituted by one or more, or two or more, halo, optionally one or more, or two or more, or all, are each independently chloride. In alternative embodiments, Zi is a phenyl substituted by an alkyl of 1-4 carbon atoms, optionally methyl.

[0254] In some of any of the embodiments described herein, Zi is a phenyl substituted at least at the para position, with respect to the amide substituent. In some of any of the embodiments described herein, Zi is a phenyl substituted at the para position with respect to the amide substituent.

[0255] In some of any of the embodiments described herein, the linking group L is an alkylene (optionally an alkylene of 1-4 carbon atoms, e.g., ethylene), the heteroaryl Z2 is a nitrogencontaining heteroaryl as described herein in any of the respective embodiments (e.g., a diazole), and Zi is a substituted aryl. In some of any of the embodiments described herein, the linking group L is an alkylene (optionally an alkylene of 1-4 carbon atoms, optionally ethylene), the heteroaryl Z2 is a nitrogen-containing heteroaryl as described herein in any of the respective embodiments (e.g., a diazole), and Zi is an unsubstituted phenyl. In some of any of the embodiments described herein, the linking group L is an alkylene (optionally an alkylene of 1-4 carbon atoms, e.g., ethylene), the heteroaryl Z2 is a nitrogen-containing heteroaryl as described herein in any of the respective embodiments (e.g., a diazole), and Zi is a phenyl substituted by a heteroaryl as described herein. In some of any of the embodiments described herein, the linking group L is an alkylene (optionally an alkylene of 1-4 carbon atoms, e.g., ethylene), the heteroaryl Z2 is a nitrogencontaining heteroaryl as described herein in any of the respective embodiments (e.g., a diazole), and Zi is a phenyl substituted by a furan, optionally by a substituted furan. In some of any of the embodiments described herein, the linking group L is an alkylene (optionally an alkylene of 1-4 carbon atoms, e.g., ethylene), the heteroaryl Z2 is a nitrogen-containing heteroaryl as described herein in any of the respective embodiments (optionally a diazole), and Zi is a substituted phenyl as described herein. In some of any of the embodiments described herein, the linking group L is an alkylene (optionally an alkylene of 1-4 carbon atoms, e.g., ethylene), the heteroaryl Z2 is a nitrogen-containing heteroaryl as described herein in any of the respective embodiments (e.g., a diazole), and Zi is a phenyl substituted by one or more, or two or more, halo, optionally one or more, or two or more, or all, are each chloro. In some of any of the embodiments described herein, the linking group L is an alkylene (optionally an alkylene of 1-4 carbon atoms, e.g., ethylene), the heteroaryl Z2 is a nitrogen-containing heteroaryl as described herein in any of the respective embodiments (e.g., a diazole), and Zi is a phenyl substituted by an alkyl of 1-4 carbon atoms, optionally methyl, the linking group L is an alkylene (optionally an alkylene of 1-4 carbon atoms, e.g., ethylene), the heteroaryl Z2 is a nitrogen-containing heteroaryl as described herein in any of the respective embodiments (e.g., a diazole), and Zi is a phenyl substituted at least at the para position. In some of any of the embodiments described herein, the linking group L is an alkylene (optionally an alkylene of 1-4 carbon atoms, e.g., ethylene), the heteroaryl Z2 is a nitrogencontaining heteroaryl as described herein in any of the respective embodiments (e.g., a diazole), and Zi is a phenyl substituted at the para position.

[0256] In some of any of the embodiments described herein for Formula II, Zi is a heteroaryl, optionally a substituted heteroaryl. In some of any of the embodiments described herein, the Zi is a furan, optionally a substituted furan. In some embodiments, Zi is a furan substituted by a substituent selected from alkyl, cycloalkyl, and aryl. In some such embodiments, Zi is a furan substituted by an alkyl, optionally an alkyl of 1-4 carbon atoms, optionally methyl.

[0257] In some of any of the embodiments described herein, the linking group L is an alkylene (optionally an alkylene of 1-4 carbon atoms, e.g., ethylene), the heteroaryl Z2 is a nitrogencontaining heteroaryl as described herein in any of the respective embodiments (e.g., a diazole),, and Zi is a heteroaryl, optionally a substituted heteroaryl. In some of any of the embodiments described herein, the linking group L is an alkylene (optionally an alkylene of 1-4 carbon atoms, e.g., ethylene), the heteroaryl Z2 is a nitrogen-containing heteroaryl as described herein in any of the respective embodiments (e.g., a diazole), and Zi is a furan, optionally a substituted furan. In some of any of the embodiments described herein, the linking group L is an alkylene (optionally an alkylene of 1-4 carbon atoms, e.g., ethylene), the heteroaryl Z2 is a nitrogen-containing heteroaryl as described herein in any of the respective embodiments (e.g., a diazole), and Zi is a furan substituted by a substituent selected from alkyl, cycloalkyl. In some of any of the embodiments described herein, the linking group L is an alkylene (optionally an alkylene of 1-4 carbon atoms, e.g., ethylene), the heteroaryl Z2 is a nitrogen-containing heteroaryl as described herein in any of the respective embodiments (e.g., a diazole), and Zi is a furan substituted by an alkyl (optionally an alkyl of 1-4 carbon atoms, optionally methyl).

[0258] According to some of any of the embodiments described herein, compounds usable in any of the methods and uses as described herein are collectively represented by Formula Ila:

[0259] Formula Ila wherein L and Zi are each independently as described herein in any of the respective embodiments of Formula II, and

[0260] R20 and R21 are each independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O- thiocarbamyl, N-thiocarbamyl, S -thiocarb amyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino.

[0261] According to some of any of the embodiments described herein for Formula Ila, R20 and / or R21 are each independently hydrogen.

[0262] According to some of any of the embodiments described herein, compounds usable in any of the methods and uses as described herein are collectively represented by Formula lib : wherein L and Z2 are each independently as described herein in any of the respective embodiments of Formula II, and

[0263] R22-R26 are each independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O- thiocarbamyl, N-thiocarbamyl, S -thiocarb amyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino.

[0264] According to some of any of the embodiments described herein for Formula lib, one or more, or two or more, or three or more, or all, of R22-R26 are each hydrogen. In some of any of the embodiments described herein, each of R22-R26 is hydrogen.

[0265] According to some of any of the embodiments described herein, one or more, or two or more, or three or more, or all, of R22-R26 are each independently an alkyl, preferably an alkyl of 1- 4 carbon atoms, more preferably methyl. In some of any of the embodiments described herein, at least R24 is an alkyl, preferably an alkyl of 1-4 carbon atoms, more preferably methyl.

[0266] According to some of any of the embodiments described herein, one or more, or two or more, or three or more, or all, of R22-R26 are each independently hydroxy or alkoxy. In some of any of the embodiments described herein, one or more, or two or more, or all of R23-R25 are each independently hydroxy or alkoxy. In some of any of the embodiments described herein, at least R24 is an alkoxy, preferably a methoxy. In some of any of the embodiments described herein, R24 is an alkoxy, preferably a methoxy.

[0267] According to some of any of the embodiments described herein, one or more, or two or more, or three or more, or all, of R22-R26 are each independently halo, preferably chloro. In some of any of the embodiments described herein, at least R24 is a halo, preferably chloro. In some of any of the embodiments described herein, R24 and R23 are each independently a halo, preferably chloro. According to some of any of the embodiments described herein, compounds usable in any of the methods and uses as described herein are collectively represented by Formula lie: wherein L is as described in any of the respective embodiments of Formula II, and R20-R26 are each independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O- thiocarbamyl, N-thiocarbamyl, S -thiocarb amyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino.

[0268] According to some embodiments of Formula lie, R20 and R21 are each independently as described herein for Formula Ila.

[0269] According to some embodiments of Formula lie, R22-R26 are each independently as described herein for Formula IIB.

[0270] Exemplary compound represented by Formula lie are as presented in FIGs. 17A-C. A nonlimiting process for the preparation thereof is as presented in FIG. 17D.

[0271] According to some of any of the embodiments described herein, R20 and / or R21 are each independently hydrogen, and one or more, or two or more, or three or more, or all, of R22-R26 are each hydrogen. According to some of any of the embodiments described herein, R20 and / or R21 are each independently hydrogen, and all R22-R26 are each hydrogen. An exemplary such compound is SS18400-7, as presented in FIGs. 17B-C.

[0272] According to some of any of the embodiments described herein, R20 and / or R21 are each independently hydrogen, and one or more, or two or more, or three or more, or all, of R22-R26 are each alkyl, preferably an alkyl of 1-4 carbon atoms, more preferably methyl. According to some of any of the embodiments described herein, R20 and / or R21 are each independently hydrogen, and at least R24 is an alkyl, preferably an alkyl of 1-4 carbon atoms, more preferably methyl. An exemplary such compound is SSI 8400-11, as presented in FIG. 17C. According to some of any of the embodiments described herein, R20 and / or R21 are each independently hydrogen, and one or more, or two or more, or three or more, or all, of R22-R26 are each independently hydroxy or alkoxy. According to some of any of the embodiments described herein, R20 and / or R21 are each independently hydrogen, and one or more, or two or more, or all of R22-R26 are each independently alkoxy. According to some of any of the embodiments described herein, R20 and / or R21 are each independently hydrogen, and at least R24 is an alkoxy, preferably methoxy. An exemplary such compound is SSI 8400- 10, as presented in FIG. 17C.

[0273] According to some of any of the embodiments described herein, R20 and / or R21 are each independently hydrogen, and one or more, or two or more, or three or more, or all, of R22-R26 are each independently halo, preferably chloro. According to some of any of the embodiments described herein, R20 and / or R21 are each independently hydrogen, and at least R24 is a halo, preferably chloro. An exemplary such compound is SS18400-9, as presented in FIG. 17C.

[0274] According to some of any of the embodiments described herein, R20 and / or R21 are each independently hydrogen, and R24 and R23 are each independently a halo, preferably chloro. An exemplary such compound is SSI 8400-8, as presented in FIG. 17C.

[0275] According to an aspect of some embodiments of the present invention, there is provided a method of treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound represented by Formula A.

[0276] According to an aspect of some embodiments of the present invention there is provided a compound represented by Formula A as described herein in any of the respective embodiments, for use in treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in a subject in need thereof.

[0277] According to an aspect of some embodiments of the present invention there is provided a use of a compound represented by Formula A as described herein in any of the respective embodiments, in the manufacture of a medicament for treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in a subject in need thereof.

[0278] According to an aspect of some embodiments of the present invention, there is provided a method of treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound represented by Formula I, as described herein in any of the respective embodiments.

[0279] According to an aspect of some embodiments of the present invention there is provided a compound represented by Formula I as described herein in any of the respective embodiments, for use in treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in a subject in need thereof.

[0280] According to an aspect of some embodiments of the present invention there is provided a use of a compound represented by Formula I as described herein in any of the respective embodiments, in the manufacture of a medicament for treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in a subject in need thereof.

[0281] According to an aspect of some embodiments of the present invention, there is provided a method of treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound represented by Formula la, as described herein in any of the respective embodiments.

[0282] According to an aspect of some embodiments of the present invention there is provided a compound represented by Formula la as described herein in any of the respective embodiments, for use in treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in a subject in need thereof.

[0283] According to an aspect of some embodiments of the present invention there is provided a use of a compound represented by Formula la as described herein in any of the respective embodiments, in the manufacture of a medicament for treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in a subject in need thereof.

[0284] According to an aspect of some embodiments of the present invention, there is provided a method of treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound represented by Formula II, Ila, lib or lie, as described herein in any of the respective embodiments.

[0285] According to an aspect of some embodiments of the present invention there is provided a compound represented by Formula II, Ila, lib or lie as described herein in any of the respective embodiments, for use in treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in a subject in need thereof.

[0286] According to an aspect of some embodiments of the present invention there is provided a use of a compound represented by Formula II, Ila, lib or lie as described herein in any of the respective embodiments, in the manufacture of a medicament for treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) as described herein in a subject in need thereof. According to an aspect of some embodiments of the present invention there is provided a compound selected from the compounds depicted in Table 1 as described herein in any of the respective embodiments, for use in treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) in a subject in need thereof.

[0287] According to an aspect of some embodiments of the present invention there is provided a use of a compound selected from a compound depicted in Table 1 as described herein in any of the respective embodiments, in the manufacture of a medicament for treating a metabolic disorder associated with phenylalanine aggregation (fibril formation) in a subject in need thereof.

[0288] According to some of any of the embodiments described herein, a compound as described herein in any of the aspect and embodiments described herein is usable in interfering with (e.g., inhibiting, destabilizing) formation of a non-proteinaceous amyloid-like fibril, and hence is usable in treating metabolic disorders that are associated with formation of non-proteinaceous amyloid- like fibrils (phenylalanine fibrils).

[0289] According to an aspect of some embodiments of the present invention there is provided a method of interfering with (e.g., inhibiting, destabilizing) a formation of a non-proteinaceous amyloid-like fibril, preferably a formation of phenylalanine fibrils, or of interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation, the method comprising contacting a phenylalanine-containing medium with a compound as described herein in any of the respective embodiments and any combination thereof. The phenylalanine-containing medium can be, for example, an aqueous solution or a cellular medium. According to some of these embodiments, the method is effected in vitro or ex- vivo.

[0290] As used herein, the phrase “phenylalanine-containing medium” describes any medium that includes phenylalanine molecules, either in solution or as part of a biological system. Non-limiting examples of phenylalanine-containing media include an aqueous solution containing dissolved phenylalanine, a buffered system (simulating physiological conditions) containing dissolved phenylalanine, and a biological fluid (e.g., blood, plasma, cerebrospinal fluid) suspected as containing, or containing, phenylalanine molecules and / or aggregates.

[0291] As used herein, the phrase “cellular medium” describes a biological environment that includes living cells or cellular components in which phenylalanine is present, metabolized, or capable of aggregating. Non-limiting examples for cellular media include cell cultures, tissue samples, extracellular matrices, intracellular environments, and physiological samples where phenylalanine metabolism, transport, or aggregation may take place.

[0292] According to some of any of the embodiments described herein there is provided a pharmaceutical composition comprising a compound as described herein in any of the respective embodiments, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is for use in any of the methods and uses described herein.

[0293] In any of the method and uses described herein, the compounds according to embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

[0294] As used herein a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0295] Herein the term “active ingredient” refers to the compound accountable for the biological effect (herein, interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation or fibril formation, as described herein).

[0296] Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier”, which may be interchangeably used, refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0297] Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0298] Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0299] Suitable routes of administration may, for example, include oral, rectal, topical, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0300] Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via inj ection of the pharmaceutical composition directly into a tissue region of a patient.

[0301] The term “tissue” refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.

[0302] Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0303] Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0304] For topical administration, an appropriate carrier may be selected and optionally other ingredients that can be included in the composition, as is detailed herein. Hence, the compositions can be, for example, in a form of a cream, an ointment, a paste, a gel, a lotion, and / or a soap.

[0305] Ointments are semisolid preparations, typically based on vegetable oil (e.g., shea butter and / or cocoa butter), petrolatum or petroleum derivatives. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and non-sensitizing.

[0306] Lotions are preparations that may to be applied to the skin without friction. Lotions are typically liquid or semiliquid preparations with a water or alcohol base, for example, an emulsion of the oil-in-water type. Lotions are typically preferred for treating large areas (e.g., as is frequently desirable for sunscreen compositions), due to the ease of applying a more fluid composition.

[0307] Creams are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases typically contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also called the “lipophilic” phase, optionally comprises petrolatum and / or a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase optionally contains a humectant. The emulsifier in a cream formulation is optionally a nonionic, anionic, cationic or amphoteric surfactant.

[0308] Herein, the term “emulsion” refers to a composition comprising liquids in two or more distinct phases (e.g., a hydrophilic phase and a lipophilic phase). Non-liquid substances (e.g., dispersed solids and / or gas bubbles) may optionally also be present.

[0309] As used herein and in the art, a “water-in-oil emulsion” is an emulsion characterized by an aqueous phase which is dispersed within a lipophilic phase.

[0310] As used herein and in the art, an “oil-in-water emulsion” is an emulsion characterized by a lipophilic phase which is dispersed within an aqueous phase.

[0311] Pastes are semisolid dosage forms which, depending on the nature of the base, may be a fatty paste or a paste made from a single-phase aqueous gel. The base in a fatty paste is generally petrolatum, hydrophilic petrolatum, and the like. The pastes made from single-phase aqueous gels generally incorporate carboxymethylcellulose or the like as a base.

[0312] Gel formulations are semisolid, suspension-type systems. Single-phase gels optionally contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous; but also, preferably, contains a non-aqueous solvent, and optionally an oil. Preferred organic macromolecules (e.g., gelling agents) include crosslinked acrylic acid polymers such as the family of carbomer polymers, e.g., carboxypolyalkylenes, that may be obtained commercially under the trademark Carbopol®. Other types of preferred polymers in this context are hydrophilic polymers such as polyethylene oxides, polyoxy ethylene-polyoxypropylene copolymers and polyvinyl alcohol; cellulosic polymers such as hydroxypropyl cellulose, hydroxy ethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methyl cellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. In order to prepare a uniform gel, dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing or stirring, or combinations thereof.

[0313] A composition formulated for topical administration may optionally be present in a patch, a swab, a pledget, and / or a pad.

[0314] Dermal patches and the like may comprise some or all of the following components: a composition to be applied (e.g., as described herein); a liner for protecting the patch during storage, which is optionally removed prior to use; an adhesive for adhering different components together and / or adhering the patch to the skin; a backing which protects the patch from the outer environment; and / or a membrane which controls release of a drug to the skin.

[0315] Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.

[0316] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0317] For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0318] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0319] Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

[0320] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0321] The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0322] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0323] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

[0324] The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

[0325] Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of an active ingredient (e.g., a compound as described herein in any of the respective embodiments and any combination thereof) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., a metabolic disorder as described herein) or prolong the survival of the subject being treated.

[0326] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0327] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. l).

[0328] Dosage amount and interval may be adjusted individually to provide levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0329] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

[0330] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0331] Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed herein. As used herein, the term “subject” includes mammals, preferably human beings at any age which suffer from the pathology. Preferably, this term encompasses individuals who are at risk to develop the pathology.

[0332] In any of the methods and uses described herein, the compound can be used in combination with an additional therapeutically active agent that is usable in treating an indicated disease or disorder, as described herein. The additional active agent can be co-administered with the compound, or co-formulated with the compound in the same pharmaceutical composition.

[0333] Non-limiting examples of therapeutically active agents that can be beneficially used in some of any of the embodiments described herein include agonist agents (e.g., dopamine agonists such as pramipexole), amino acid agents (e.g., glutamine), analgesic agents (e.g., acetaminophen), antagonist agents (e.g., beta-blockers such as propranolol), antimicrobial agents (e.g., penicillin, amoxicillin), biological antibodies (e.g., rituximab), antidepressant agents (e.g., fluoxetine), antigen agents (e.g., hepatitis B antigen), anti-histamine agents (e.g., diphenhydramine), antihypertensive agents (e.g., losartan), anti-inflammatory drugs (e.g., ibuprofen), anti-metabolic agents (e.g., methotrexate), antioxidant agents (e.g., vitamin E), anti-proliferative drugs (e.g., sirolimus), nucleic acid therapeutics (e.g., antisense oligonucleotides such as mipomersen), chemotherapeutic drugs (e.g., doxorubicin), co-factors (e.g., coenzyme Q10), cytokines (e.g., interleukin-2), enzymes (e.g., trypsin), growth factors (e.g., epidermal growth factor), heparins (e.g., enoxaparin), hormones (e.g., insulin), immunoglobulins (e.g., IgG), inhibitors (e.g., protease inhibitors such as ritonavir), ligands (e.g., biotin), nucleic acids (e.g., RNA), peptides (e.g., oxytocin), phospholipids (e.g., phosphatidylcholine), prostaglandins (e.g., prostaglandin E2), proteins (e.g., albumin), toxins (e.g., botulinum toxin), vitamins (e.g., vitamin D), and any combination thereof.

[0334] According to some of any of the embodiments described herein, the therapeutically active agent is usable in the treatment of a metabolic disorder (associated with phenylalanine aggregation as described herein) and / or in interfering with (inhibiting, destabilizing) phenylalanine aggregation (fibril formation) in a subject in need thereof, as these are described herein. Non-limiting examples of such therapeutically active agents include enzyme replacement therapies (e.g., phenylalanine ammonia-lyase (PAL)), small-molecule inhibitors of phenylalanine aggregation (e.g., compounds as described herein in any of the respective embodiments), chaperone or chaperone-like molecules (e.g., sapropterin dihydrochloride (Kuvan®)), metabolic cofactors (e.g., tetrahydrobiopterin), other compounds targeting phenylalanine metabolism and / or clearance pathways, and any combination thereof

[0335] According to an aspect of some embodiments of the present invention, there is provided a method of identifying a compound capable of interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation (fibril formation) in a subject in need thereof. Identified compounds are also referred to herein as candidates or lead candidates for imparting the desired pharmacological effect (e.g., inhibiting phenylalanine aggregation and / or treating a metabolic disorder as described herein).

[0336] The method according to this aspect of the present embodiments is based on identifying the pharmacophoric model as depicted in FIG. 4A and on screening a library of compounds for identifying compounds that align (e.g., overlap) and / or are capable of aligning (overlapping) with the pharmacophoric model, as these are described herein in any of the respective embodiments. The identification can be performed using, for example, in silico tools. Compounds identified as having structural features that are aligned with or overlap the pharmacophoric model as described herein are determined as capable of interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation (fibril formation), and optionally as usable in treating a metabolic disorder in the subject, as described herein.

[0337] Herein throughout, and with respect to any of the aspects and embodiments described herein, interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation (fibril formation) encompasses any action, process, or effect that is caused by the presence of the compound as described herein in any of the respective embodiments and in any combination thereof and prevents, reduces or disrupts phenylalanine aggregation (fibril formation) as described herein, and / or alters the formation, stability, or structural integrity of phenylalanine fibrils, to thereby result in a reduced amount of phenylalanine fibrils compared to the amount in the absence of the compound. Comparing the amount of phenylalanine fibrils (following phenylalanine aggregation, fibril formation) in the presence and absence of the identified compound can be affected, for example, by measuring a fluorescence of a phenylalanine-containing medium (as described herein) in the presence of an agent that changes its fluorescence in response to phenylalanine aggregation, in the presence and absence of the compound as described herein in any of the respective embodiments and in any combination thereof, such that the fluorescence of the phenylalanine- containing medium as a whole in the presence of the compound is modified by at least 10, at least 20, or at least 25, or at least 30, or at least 40, or at least 50, or at least 70, or at least 80, or at least 90, %, or even more, and up to 100 %, including any intermediate values and subranges therebetween, compared to the fluorescence of the phenylalanine-containing medium in the absence of the compound.

[0338] As used herein, the “pharmacophoric model” (e.g., as depicted in FIG. 4A) describes a spatially optimized molecular model that defines key structural features required for interfering with (e.g., destabilizing) phenylalanine assemblies (aggregation). The model is designed to identify a compound capable of interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation (fibril formation), e.g., by interfering (e.g., chemically or spatially interfering) with interactions critical for phenylalanine aggregation, fibril formation and / or fibril stabilization.

[0339] According to some of any of the embodiments described herein, identifying a compound that aligns or overlaps with pharmacophoric model is effected in silico (using at least one in silico tool).

[0340] As used herein, the phrase “zn silico tools” describes computational methods and software used to model, analyze, and predict molecular structures and interactions. Such tools facilitate the spatial optimization of interactions between two or more molecules, enabling the determination of atomic distances, binding conformations, and molecular docking configurations. Non-limiting examples of in silico tools include molecular dynamics (MD) simulations (e.g., using AMBER® or CHARMM®), quantum mechanical calculations (e.g., using Gaussian®), molecular docking programs (e.g., using MOE®), force field-based energy minimization techniques (e.g., Chimera software), and any combination thereof. In the context of the present embodiments, an in silico tool is such that enables 3D simulation of the compound (spatially arranging the compound) and measuring distances (e.g., distances as described herein in any of the respective embodiments) between corresponding functional groups (e.g., hydrogen-bond donor and two aromatic moieties). A non-limiting example of an in silico tool is a combination of MD and force field-based energy minimization technique such as Chimera software.

[0341] Once candidate compounds are identified per the pharmacophoric model, the method may proceed to identify which of these candidate compounds interferes with (e.g., inhibits, destabilizes) phenylalanine aggregation (fibril formation) upon contacting each of the identified compounds with a phenylalanine-containing medium (e.g., an aqueous solution and / or cellular medium), to thereby identify candidate compounds that exhibit an activity of interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation (fibril formation).

[0342] In some of any of the embodiments described herein, once the compound is identified, the method is further effected by determining if the compound (the compound that aligns or overlaps with the pharmacophoric model as described herein) interferes with (e.g., inhibits, destabilizes) phenylalanine aggregation (fibril formation).

[0343] In some of any of the embodiments described herein, determining if the compound as described herein in any of the respective embodiments and in any combination thereof (e.g., identified compound) interferes with (e.g., inhibits, destabilizes) phenylalanine aggregation (fibril formation) is effected by contacting the compound with a phenylalanine-containing medium as described herein. In some of any of the embodiments described herein, determining if the compound as described herein in any of the respective embodiments and in any combination thereof (e.g., identified compound) interferes with (e.g., inhibits, destabilizes) phenylalanine aggregation (fibril formation) is effected by contacting the (identified) compound with a phenylalanine-containing medium (e.g., an aqueous solution and / or cellular medium as those are defined herein) (e.g., as demonstrated in the Examples section that follows), and determining a formation of phenylalanine aggregation, for example, by comparing the extent of phenylalanine aggregation in the presence and absence of a tested compound, to thereby determine if the (identified) compound is capable of interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation (fibril formation).

[0344] In some of any of the embodiments described herein, determining whether a compound interferes with (e.g., inhibits, destabilizes) phenylalanine aggregation (fibril formation) and / or reduces formation of phenylalanine fibrils compared to a phenylalanine-containing medium lacking the compound is effected by a fluorescence measurement (using, e.g., confocal fluorescence microscopy). Such a comparative examination of fibril formation (samples of phenylalanine-containing media in the absence and presence of the compound) can be performed in the presence of a fluorescent agent that produces a modified (e.g., enhanced or reduced) fluorescent signal in the presence of amyloid assemblies, for example, ThT. In some of any of the embodiments described herein, determining whether a compound inhibits phenylalanine aggregation (fibril formation) is effected by determining ThT fluorescence intensity of phenylalaine-containing medium as described herein, in the presence or absence of the compound, which allows assessment of inhibition (e.g., interference, destabilization). A decrease in ThT fluorescence intensity (compared to the untreated sample) is indicative of reduced fibril formation (i.e., inhibition (e.g., interference, destabilization) by the compound) whereas a similar or increased ThT fluorescence intensity is indicative of a lack of inhibition (lack of, e.g., interference, destabilization) by the compound. An exemplary ThT-based assay is described in Example 2 in the Examples section that follows.

[0345] As known in the art, the term “thioflavin-T” (ThT) describes an amyloid-specific fluorescent dye that binds to P-sheet-rich structures, such that it produces an enhanced fluorescent signal upon interaction with amyloid assemblies (phenylalanine aggregation, fibril formation).

[0346] As used herein, the phrase “ThT fluorescence intensity” describes a measurable emission signal produced by thioflavin-T (ThT) upon binding to amyloid-like fibrils. This intensity correlates with the extent of fibril formation, where higher fluorescence indicates a greater presence of P-sheet-rich structures, and lower fluorescence suggests reduced fibril formation or fibril disassembly. In some of any of the embodiments described herein, determining whether a compound interferes with (e.g., inhibits, destabilizes) phenylalanine aggregation (fibril formation) and / or reduces formation of phenylalanine fibrils compared to a phenylalanine-containing medium lacking the compound is effected by microscopy (e.g., transmission electron microscopy (TEM)). Such a comparative examination of fibril formation (samples of phenylalanine-containing media in the absence and presence of the compound) allows assessment of inhibition (e.g., interference, destabilization). A reduced presence or absence of fibrils in the treated sample (compared to a phenylalanine-containing medium lacking the compound (the untreated sample)) indicates inhibition (e.g., interference, destabilization) of phenylalanine aggregation (fibril formation) by the compound, whereas a similar extent of fibril formation in both samples indicates a lack of inhibition (e.g., interference, destabilization) by the compound.

[0347] In some of any of the embodiments described herein, the compound (e.g., identified compound) reduces formation of phenylalanine fibrils (phenylalanine fibrillar structures) by at least 10, at least 20, or at least 25, or at least 30, or at least 40, or at least 50, or at least 70, or at least 80, or at least 90, %, or even more, and up to 100 %, including any intermediate values and subranges therebetween, compared to a medium lacking the compound, thereby determining that the compound is capable of interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation (fibril formation). In some such embodiments, determining if the compound reduces formation of phenylalanine fibrils (phenylalanine fibrillar structures) compared to a medium lacking the compound is affected by a ThT fluorescence assay as described herein. In alternative embodiments, determining if the compound reduces formation of phenylalanine fibrils (phenylalanine fibrillar structures) compared to a medium lacking the compound is affected by analysing microscopic images, e.g., TEM images.

[0348] In some such embodiments, microscopy (e.g., TEM) images are assessed visually and / or by means of determining an area occupied by phenylalanine fibrils (aggregated fibrillar structures). In some embodiments, the means of determining whether an area of a microscopy image is occupied by phenylalanine fibrils is effected in silico (using, e.g., a custom-built model that identifies aggregated and non-aggregated phenylalanine structures in a microscopy image), thereby determining whether a compound interferes with (e.g., inhibits, destabilizes) phenylalanine aggregation (fibril formation) and / or reduces formation of phenylalanine fibrils compared to a phenylalanine-containing medium lacking the compound.

[0349] As known in the art, the phrase “microscopy image” describes an image obtained using a microscope, e.g., a confocal fluorescence microscope.

[0350] A compound determined as capable of interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation (fibril formation) in such studies can be subjected to further assays for identifying lead candidates for in vivo assays.

[0351] In some of any of the embodiments described herein, determining if a candidate compound (the compound identified as aligning or overlapping with the pharmacophoric model as described herein) is suitable for use in interfering with phenylalanine aggregation in a subject in need thereof is effected by in silico screening of compounds identified as capable of aligning with the pharmacophoric model to thereby determine a set of pharmacological parameters of each of these compounds.

[0352] In some such embodiments, compounds identified as featuring an aqueous solubility value (logS) higher than 1; and / or a human intestinal absorption (HIA) of at least 30 %, or at least 50 %, or at least 70 %; are identified as being suitable for use in interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation in a subject in need thereof.

[0353] In some such embodiments, compounds identified as featuring an aqueous solubility value (logS) higher than 1 ; and / or a human intestinal absorption (HIA) of at least 30 %; and / or a partition coefficient (logP) in a range of from 0 to 3.5; and / or a brain penetration prediction (BBB log([Brain]: [Blood])) in a range of from -0.2 to 1; and / or a positive blood-brain barrier permeability; and / or a negative P-gp category; and / or a cardiac toxicity value (hERG pICso) of up to 5; a CYP2C9 inhibition value (2C9 pKi) of up to 6; a low to medium CYP2D6 interaction value (2D6 affinity category); and / or a low plasma protein binding at 90 % (PPB90); are identified as being suitable for use in interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation in a subject in need thereof.

[0354] Exemplary parameters usable for identifying in silico lead candidates in silico are presented in FIG. 13 A.

[0355] In some embodiment, determining whether a compound (the compound that aligns or overlaps with the pharmacophoric model as described herein) is suitable for use in interfering with phenylalanine aggregation in a subject in need thereof is effected by applying oral central nervous system (CNS) score, as presented in FIGs. 13A.

[0356] As used herein, the phrase “oral central nervous system (CNS) score” describes how suitable a compound is for exhibiting central nervous system activity when administered orally, based on its pharmacokinetic and physicochemical properties. The score is calculated by: (i) assessing how well a pharmacological parameter of the compound aligns with a desired value of that property (as defined, e.g., in FIG. 13 A); (ii) assigning an importance score to each property (e.g., on a scale of from 0 to 1; e.g., as can be seen in FIG. 13A); multiplying (i) by (ii) to thereby obtain the oral central nervous system (CNS) score.

[0357] As used herein, the phrase “aqueous solubility value” (logS) describes the logarithm of a compound’s solubility in water, indicating its ability to dissolve in aqueous environments, such that compounds with a logS higher than 1 are considered highly soluble.

[0358] As used herein, the phrase “human intestinal absorption” (HIA) describes the percentage of a compound absorbed through the human intestine, reflecting its potential oral bioavailability, such that compounds with an HIA of at least 30 % have at least moderate absorption, increasing their likelihood of reaching systemic circulation and interfering with phenylalanine aggregation in vivo.

[0359] Hydrophilic, amphiphilic, lipophilic, and hydrophobic substances can be determined by the partition coefficient thereof. As known in the art, the phrase “partition coefficient" (logP) describes the ratio of concentrations of a compound in the two phases of a mixture of two immiscible liquids at equilibrium, typically at room temperature. Normally, one of the solvents chosen is water while the second is hydrophobic such as n-octanol. The logarithm of the ratio of the concentrations of the un-ionized solute in the solvents is called LogP.

[0360] As used herein, the phrase “brain penetration prediction” (BBB log([Brain]: [Blood])) describes the predicted ratio of a compound’s concentration in the brain compared to the blood, indicating its ability to cross the blood-brain barrier, such that compounds with a BBB log([Brain]: [Blood]) in the range of -0.2 to 1 are predicted to exhibit at least moderate brain penetration.

[0361] As used herein, the phrase “blood-brain barrier permeability describes a compound’s ability to cross the blood-brain barrier and enter the central nervous system, such that compounds with a positive BBB permeability can efficiently penetrate the brain.

[0362] As used herein, the phrase “P-gp category” describes whether a compound is a substrate or inhibitor of P-gly coprotein, such that a negative P-gp category describes a compound that is not actively transported out of cells by P-glycoprotein.

[0363] As used herein, the phrase “cardiac toxicity value” (hERG pICso) describes a compound’s potential to inhibit the hERG potassium channel, where lower values indicate a higher risk of cardiac toxicity, such that compounds with hERG pIC50 of up to 5 have a reduced likelihood of causing cardiac toxicity.

[0364] As used herein, the phrase “CYP2C9 inhibition value” (2C9 pKi) describes the binding affinity of a compound for the CYP2C9 enzyme, where lower values suggest weaker inhibition and reduced drug-drug interaction risk, such that compounds with a 2C9 pKi of up to 6 are expected to have low to moderate CYP2C9 inhibition, minimizing metabolic interactions and enhancing drug safety.

[0365] As used herein, the phrase ’’CYP2D6 interaction value” (2D6 affinity category) describes a compound’s likelihood of interacting with the CYP2D6 enzyme, affecting its metabolism and potential drug interactions, such that compounds with a low to medium CYP2D6 affinity category are preferable as they reduce the risk of metabolic complications.

[0366] As known in the art, the phrase “plasma protein binding at 90 %” (PPB90) describes the percentage of a compound bound to plasma proteins, where higher values indicate reduced free drug availability for therapeutic action, such that compounds with low plasma protein binding at 90% have a higher fraction of free drug available in circulation, potentially increasing their ability to effectively interact with, e.g., phenylalanine aggregates.

[0367] In some of any of the embodiments described herein, once compounds are identified as being capable of aligning (overlapping) with the pharmacophoric model and / or as being capable of interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation (fibril formation), the method is further effected by further screening the (identified) compounds for identifying lead candidates (using, e.g., in vitro studies and / or further in silico screening process), thereby identifying lead candidates (compounds).

[0368] In some embodiments, once determining that the (identified) compound is capable of inhibiting phenylalanine aggregation, the method is further effected by an (optionally, additional) in silico screening of compounds capable of aligning with the pharmacophoric model and are structural analogs of the (identified) compound.

[0369] In some embodiments, structural analogs of the (identified) compound are identified using a Topliss tree method.

[0370] As known in the art, the phrase “Topliss tree method” describes a stepwise process for optimizing phenyl groups based on a change in substituent that change hydrophobicity, electronics, and sterics of a compound. This method allows generating a preliminary SAR to identify a potentially more potent compounds which may be suitable for use, e.g., in interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation in a subject in need thereof.

[0371] In some of any of the embodiments described herein, once identifying the compound as capable of interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation (fibril formation), the method is further effected by in silico screening of compounds identified as capable of aligning with the pharmacophoric model as described herein to thereby determine an oral CNS score (oral CNS scoring profile) (as described herein) of each of these compounds. In some embodiments, compounds featuring a scoring profile higher than 0.1, or higher than 0.15, are identified as suitable for use in of interfering with (e.g., inhibiting, destabilizing) phenylalanine aggregation in a subject in need thereof. Compounds of Table 1 having an oral CNS scoring profile higher than 0.1 include PCM1, PCM2, PCM-0026104 and PCM-0085146.

[0372] As used herein the term “about” refers to ± 10 % or ± 5 %.

[0373] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0374] The term “consisting of’ means “including and limited to”.

[0375] The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0376] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0377] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0378] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0379] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0380] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0381] As used herein, the term “preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.

[0382] Herein, the phrase “linking group” describes a group (e.g., a substituent) that is attached to two or more moieties in the compound; whereas the phrase “end group” describes a group (e.g., a substituent) that is attached to a single moiety in the compound via one atom thereof. As used herein throughout, the term “alkyl” refers to any saturated aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 1 to 20 carbon atoms. Whenever a numerical range; e.g., “1 to 20”, is stated herein, it implies that the group, in this case the hydrocarbon, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. More preferably, the alkyl is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkyl is a lower alkyl having 1 to 4 carbon atoms. The alkyl group may be substituted or non- substituted. When substituted, the substituent group can be, for example, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S- thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein.

[0383] A “cycloalkyl” group refers to a saturated on unsaturated all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group wherein one of more of the rings does not have a completely conjugated pi-electron system. Examples, without limitation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane. A cycloalkyl group may be substituted or non- substituted. When substituted, the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C- amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein. When a cycloalkyl group is unsaturated, it may comprise at least one carbon-carbon double bond and / or at least one carboncarbon triple bond. The cycloalkyl group can be an end group, as this phrase is defined herein, wherein it is attached to a single adjacent atom, or a linking group, as this phrase is defined herein, connecting two or more moieties.

[0384] A “heteroalicyclic” group refers to a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. The heteroalicyclic may be substituted or non-substituted. When substituted, the substituted group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S- thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein. Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholine and the like. The heteroalicyclic group can be an end group, as this phrase is defined herein, wherein it is attached to a single adjacent atom, or a linking group, as this phrase is defined herein, connecting two or more moieties.

[0385] Herein, the terms “amine” and “amino” each refer to either a -NR’R” group or a - N+R’R”R’” group, wherein R’, R” and R’” are each hydrogen or a substituted or non- substituted alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic (linked to amine nitrogen via a ring carbon thereof), aryl, or heteroaryl (linked to amine nitrogen via a ring carbon thereof), as defined herein. Optionally, R’, R” and R’” are hydrogen or alkyl comprising 1 to 4 carbon atoms. Optionally, R’ and R” (and R’”, if present) are hydrogen. When substituted, the carbon atom of an R’, R” or R’” hydrocarbon moiety which is bound to the nitrogen atom of the amine is not substituted by oxo (unless explicitly indicated otherwise), such that R’, R” and R’” are not (for example) carbonyl, C-carboxy or amide, as these groups are defined herein.

[0386] An “alkoxy” group refers to any of an -O-alkyl, -O-alkenyl, -O-alkynyl, -O-cycloalkyl, and -O-heteroalicyclic end group, as defined herein, or to any of an -O-alkylene, -O-cycloalkyl- and - O-heteroalicyclic- linking group, as defined herein .

[0387] An “aryloxy” group refers to both an -O-aryl and an -O-heteroaryl group, as defined herein, or to an -O-arylene.

[0388] A “hydroxy” group refers to a -OH group.

[0389] A “thiohydroxy” or “thiol” group refers to a -SH group.

[0390] A “thioalkoxy” group refers to any of an -S-alkyl, -S-alkenyl, -S-alkynyl, -S-cycloalkyl, and -S-heteroalicyclic end group, as defined herein, or to any of an -S-alkylene-, -S-cycloalkyl- and -S-heteroalicyclic- linking group, as defined herein.

[0391] A “thioaryloxy” group refers to both an -S-aryl and an -S-heteroaryl group, as defined herein, or to an -S-arylene.

[0392] The term “alkylene” describes a saturated or unsaturated aliphatic hydrocarbon linking group, as this term is defined herein, which differs from an alkyl group (when saturated) or an alkenyl or alkynyl group (when unsaturated), as defined herein, only in that alkylene is a linking group rather than an end group.

[0393] Herein, the term “alkenyl” describes an unsaturated aliphatic hydrocarbon comprise at least one carbon-carbon double bond, including straight chain and branched chain groups. Preferably, the alkenyl group has 2 to 20 carbon atoms. More preferably, the alkenyl is a medium size alkenyl having 2 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkenyl is a lower alkenyl having 2 to 4 carbon atoms. The alkenyl group may be substituted or non-substituted. Substituted alkenyl may have one or more substituents, whereby each substituent group can independently be, for example, alkynyl, cycloalkyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- thiocarbamyl, S -thiocarb amyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino.

[0394] Herein, the term “alkynyl” describes an unsaturated aliphatic hydrocarbon comprise at least one carbon-carbon triple bond, including straight chain and branched chain groups. Preferably, the alkynyl group has 2 to 20 carbon atoms. More preferably, the alkynyl is a medium size alkynyl having 2 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkynyl is a lower alkynyl having 2 to 4 carbon atoms. The alkynyl group may be substituted or non-substituted. Substituted alkynyl may have one or more substituents, whereby each substituent group can independently be, for example, cycloalkyl, alkenyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S- thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino.

[0395] An “aryl” group refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) end groups having a completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl group may be substituted or non-substituted. When substituted, the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S- thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein.

[0396] A “heteroaryl” group refers to a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) end group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be substituted or non-substituted. When substituted, the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, imine, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- thiocarbamyl, S -thiocarb amyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as these terms are defined herein.

[0397] The term “arylene” describes a monocyclic or fused-ring polycyclic linking group, as this term is defined herein, and encompasses linking groups which differ from an aryl or heteroaryl group, as these groups are defined herein, only in that arylene is a linking group rather than an end group.

[0398] An “azide” group refers to a -N=N+=N" end group.

[0399] A “carbonyl” or “acyl” group refers to a -C(=O)-R’ end group, where R’ is defined as hereinabove, or to a -C(=O)- linking group.

[0400] A “thiocarbonyl” group refers to a -C(=S)-R’ end group, where R’ is as defined herein, or to a -C(=S)- linking group.

[0401] A “carboxy”, “carboxyl”, “carboxylic” or “carboxylate” group refers to both “C-carboxy” and “O-carboxy” end groups, as defined herein, as well as to a carboxy linking group, as defined herein.

[0402] A “C-carboxy” group refers to a -C(=O)-O-R’ group, where R’ is as defined herein.

[0403] An “O-carboxy” group refers to an R’C(=O)-O- group, where R’ is as defined herein.

[0404] A “carboxy linking group” refers to a -C(=O)-O- linking group.

[0405] An “imine” group refers to a =N-R’ end group, where R’ is as defined herein, or to an =N- linking group.

[0406] An “oxime” group refers to a =N-0H end group.

[0407] A “hydrazone” group refers to a =N-NR’R” end group, where each of R’ and R” is as defined herein, or to a =N-NR’- linking group where R’ is as defined herein.

[0408] A “halo” group refers to fluorine, chlorine, bromine or iodine.

[0409] A “sulfinyl” group refers to an -S(=O)-R’ end group, where R’ is as defined herein, or to an -S(=O)- linking group.

[0410] A “sulfonyl” group refers to an -S(=O)2-R’ end group, where R’ is as defined herein, or to an -S(=O)2- linking group. A “sulfonate” group refers to an -S(=O)2-O-R’ end group, where R’ is as defined herein, or to an -S(=O)2-O- linking group.

[0411] A “sulfate” group refers to an -O-S(=O)2-O-R’ end group, where R’ is as defined as herein, or to an -O-S(=O)2-O- linking group.

[0412] A “sulfonamide” or “sulfonamido” group encompasses both S-sulfonamido and N- sulfonamido end groups, as defined herein, as well as a sulfonamide linking group, as defined herein.

[0413] An “S-sulfonamido” group refers to a -S(=0)2-NR’R” end group, with each of R’ and R” as defined herein.

[0414] An “N-sulfonamido” group refers to an R’S(=0)2-NR”- end group, where each of R’ and R” is as defined herein.

[0415] A “sulfonamide linking group” refers to a -S(=0)2-NR’- linking group, where R’ is as defined herein.

[0416] A “carbamyl” group encompasses both O-carbamyl and N-carbamyl end groups, as defined herein, as well as a carbamyl linking group, as defined herein.

[0417] An “O-carbamyl” group refers to an -OC(=O)-NR’R” end group, where each of R’ and R” is as defined herein.

[0418] An “N-carbamyl” group refers to an R’OC(=O)-NR”- end group, where each of R’ and R” is as defined herein.

[0419] A “carbamyl linking group” refers to a -OC(=O)-NR’- linking group, where R’ is as defined herein.

[0420] A “thiocarbamyl” group encompasses O-thiocarbamyl, S-thiocarbamyl and N- thiocarbamyl end groups, as defined herein, as well as a thiocarbamyl linking group, as defined herein.

[0421] An “O-thiocarbamyl” group refers to an -OC(=S)-NR’R” end group, where each of R’ and R” is as defined herein.

[0422] An “N-thiocarbamyl” group refers to an R’OC(=S)NR”- end group, where each of R’ and R” is as defined herein.

[0423] An “S-thiocarbamyl” group refers to an -SC(=O)-NR’R” end group, where each of R’ and R” is as defined herein.

[0424] A “thiocarbamyl linking group” refers to a -OC(=S)-NR’- or -SC(=O)-NR’- linking group, where R’ is as defined herein.

[0425] An “amide” or “amido” group encompasses C-amido and N-amido end groups, as defined herein, as well as an amide linking group, as defined herein. A “C-amido” group refers to a -C(=O)-NR’R” end group, where each of R’ and R” is as defined herein.

[0426] An “N-amido” group refers to an R’C(=O)-NR”- end group, where each of R’ and R” is as defined herein.

[0427] An “amide linking group” refers to a -C(=O)-NR’- linking group, where R’ is as defined herein.

[0428] A “urea group” refers to an -N(R’)-C(=O)-NR”R”’ end group, where each of R’, R” and R” is as defined herein, or an -N(R’)-C(=O)-NR”- linking group, where each of R’ and R” is as defined herein .

[0429] A “thiourea group” refers to an -N(R’)-C(=S)-NR”R”’ end group, where each of R’, R” and R” is as defined herein, or an -N(R’)-C(=S)-NR”- linking group, where each of R’ and R” is as defined herein .

[0430] A “nitro” group refers to an -NO2 group.

[0431] A “cyano” group refers to a -C=N group.

[0432] The term “phosphonyl” or “phosphonate” describes a -P(=O)(OR’)(OR”) group, with R’ and R” as defined herein, or a -P(=O)(OR’)-O- linking group, with R’ as defined herein.

[0433] The term “phosphate” describes an -O-P(=O)(OR’)(OR”) end group, with each of R’ and R” as defined herein, or an -O-P(=O)(OR’)-O- linking group, with R’ as defined herein.

[0434] The term “phosphinyl” describes a -PR’R” end group, with each of R’ and R” as defined herein, or a -PR’ - linking group, with R’ as defined herein.

[0435] The term “hydrazine” describes a -NR’-NR”R”’ end group, where R’, R”, and R’” are as defined herein, or to a -NR’ -NR”- linking group, where R’ and R” are as defined herein.

[0436] As used herein, the term “hydrazide” describes a -C(=O)-NR’-NR”R’” end group, where R’, R” and R’” are as defined herein, or to a -C(=O)-NR’-NR”- linking group, where R’ and R” are as defined herein.

[0437] As used herein, the term “thiohydrazide” describes a -C(=S)-NR’-NR”R’” end group, where R’, R” and R’” are as defined herein, or to a -C(=S)-NR’-NR”- linking group, where R’ and R” are as defined herein.

[0438] A “guanidinyl” group refers to an -RaNC(=NRd)-NRbRc end group, where each of Ra, Rb, Rc and Rd can be as defined herein for R’ and R”, or to an -R’NC(=NR”)-NR”’- linking group, where R’, R” and R’” are as defined herein.

[0439] A “guanyl” or “guanine” group refers to an R’ ’ ’R”NC(=NR’)- end group, where R’, R” and

[0440] R’” are as defined herein, or to a -R”NC(=NR’)- linking group, where R’ and R” are as defined herein. For any of the embodiments described herein, the compound described herein may be in a form of a salt, for example, a pharmaceutically acceptable salt, and / or in a form of a prodrug.

[0441] As used herein, the phrase “pharmaceutically acceptable salt” refers to a charged species of the parent compound and its counter-ion, which is typically used to modify the solubility characteristics of the parent compound and / or to reduce any significant irritation to an organism by the parent compound, while not abrogating the biological activity and properties of the administered compound. A pharmaceutically acceptable salt of a compound as described herein can alternatively be formed during the synthesis of the compound, e.g., in the course of isolating the compound from a reaction mixture or re-crystallizing the compound.

[0442] In the context of some of the present embodiments, a pharmaceutically acceptable salt of the compounds described herein may optionally be an acid addition salt and / or a base addition salt.

[0443] An acid addition salt comprises at least one basic (e.g., amine and / or guanidinyl) group of the compound which is in a positively charged form (e.g., wherein the basic group is protonated), in combination with at least one counter-ion, derived from the selected acid, that forms a pharmaceutically acceptable salt. The acid addition salts of the compounds described herein may therefore be complexes formed between one or more basic groups of the compound and one or more equivalents of an acid.

[0444] A base addition salt comprises at least one acidic (e.g., carboxylic acid) group of the compound which is in a negatively charged form (e.g., wherein the acidic group is deprotonated), in combination with at least one counter-ion, derived from the selected base, that forms a pharmaceutically acceptable salt. The base addition salts of the compounds described herein may therefore be complexes formed between one or more acidic groups of the compound and one or more equivalents of a base.

[0445] Depending on the stoichiometric proportions between the charged group(s) in the compound and the counter-ion in the salt, the acid additions salts and / or base addition salts can be either mono-addition salts or poly-addition salts.

[0446] The phrase “mono-addition salt”, as used herein, refers to a salt in which the stoichiometric ratio between the counter-ion and charged form of the compound is 1 : 1, such that the addition salt includes one molar equivalent of the counter-ion per one molar equivalent of the compound.

[0447] The phrase “poly-addition salt”, as used herein, refers to a salt in which the stoichiometric ratio between the counter-ion and the charged form of the compound is greater than 1 : 1 and is, for example, 2: 1, 3 : 1, 4: 1 and so on, such that the addition salt includes two or more molar equivalents of the counter-ion per one molar equivalent of the compound. An example, without limitation, of a pharmaceutically acceptable salt would be an ammonium cation or guanidinium cation and an acid addition salt thereof, and / or a carboxylate anion and a base addition salt thereof.

[0448] The base addition salts may include a cation counter-ion such as sodium, potassium, ammonium, calcium, magnesium and the like, that forms a pharmaceutically acceptable salt.

[0449] The acid addition salts may include a variety of organic and inorganic acids, such as, but not limited to, hydrochloric acid which affords a hydrochloric acid addition salt, hydrobromic acid which affords a hydrobromic acid addition salt, acetic acid which affords an acetic acid addition salt, ascorbic acid which affords an ascorbic acid addition salt, benzenesulfonic acid which affords a besylate addition salt, camphorsulfonic acid which affords a camphorsulfonic acid addition salt, citric acid which affords a citric acid addition salt, maleic acid which affords a maleic acid addition salt, malic acid which affords a malic acid addition salt, methanesulfonic acid which affords a methanesulfonic acid (mesylate) addition salt, naphthalenesulfonic acid which affords a naphthalenesulfonic acid addition salt, oxalic acid which affords an oxalic acid addition salt, phosphoric acid which affords a phosphoric acid addition salt, toluenesulfonic acid which affords a p-toluenesulfonic acid addition salt, succinic acid which affords a succinic acid addition salt, sulfuric acid which affords a sulfuric acid addition salt, tartaric acid which affords a tartaric acid addition salt and trifluoroacetic acid which affords a trifluoroacetic acid addition salt. Each of these acid addition salts can be either a mono-addition salt or a poly-addition salt, as these terms are defined herein.

[0450] As used herein, the term “prodrug” refers to a compound which is converted in the body to an active compound (e.g., the compound of the formula described hereinabove). A prodrug is typically designed to facilitate administration, e.g., by enhancing absorption. A prodrug may comprise, for example, the active compound modified with ester groups, for example, wherein any one or more of the hydroxyl groups of a compound is modified by an acyl group, optionally (Ci- 4)-acyl (e.g., acetyl) group to form an ester group, and / or any one or more of the carboxylic acid groups of the compound is modified by an alkoxy or aryloxy group, optionally (Ci-4)-alkoxy (e.g., methyl, ethyl) group to form an ester group.

[0451] Further, each of the compounds described herein, including the salts thereof, can be in a form of a solvate or a hydrate thereof.

[0452] The term “solvate” refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-, penta- , hexa-, and so on), which is formed by a solute (the heterocyclic compounds described herein) and a solvent, whereby the solvent does not interfere with the biological activity of the solute.

[0453] The term “hydrate” refers to a solvate, as defined hereinabove, where the solvent is water. The compounds described herein can be used as polymorphs and the present embodiments further encompass any isomorph of the compounds and any combination thereof.

[0454] The compounds and structures described herein encompass any stereoisomer, including enantiomers and diastereomers, of the compounds described herein, unless a particular stereoisomer is specifically indicated.

[0455] As used herein, the term “enantiomer” refers to a stereoisomer of a compound that is superposable with respect to its counterpart only by a complete inversion / reflection (mirror image) of each other. Enantiomers are said to have “handedness” since they refer to each other like the right and left hand. Enantiomers have identical chemical and physical properties except when present in an environment which by itself has handedness, such as all living systems. In the context of the present embodiments, a compound may exhibit one or more chiral centers, each of which exhibiting an (R) or an (S) configuration and any combination, and compounds according to some embodiments of the present invention, can have any their chiral centers exhibit an (R) or an (S) configuration.

[0456] The term “diastereomers”, as used herein, refers to stereoisomers that are not enantiomers to one another. Diastereomerism occurs when two or more stereoisomers of a compound have different configurations at one or more, but not all of the equivalent (related) stereocenters and are not mirror images of each other. When two diastereoisomers differ from each other at only one stereocenter they are epimers. Each stereo-center (chiral center) gives rise to two different configurations and thus to two different stereoisomers. In the context of the present invention, embodiments of the present invention encompass compounds with multiple chiral centers that occur in any combination of stereo-configuration, namely any diastereomer.

[0457] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0458] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0459] EXAMPLES

[0460] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion. MATERIAL AND EXPERIMENTAL METHODS

[0461] Screening compounds: Compound collections from several chemical suppliers (Analyticon, Maybridge, Microsource, Selleck Chemicals) were maintained as DMSO stocks at 10 mM compound concentration. Prior to the execution of High Throughput Screening (HTS), assay-ready plates were produced by acoustic dispensing of library compounds with an Echo 555 liquid handler (Labcyte). Plates were sealed and stored at -30 °C until use.

[0462] High-throughput screening (HTS): 55,000 compounds from various compound libraries, i.e. Analyticon 2014 collection, MayBridge- HitFinderTM Collection, MicroSource, and Selleck Chemicals, were screened at 88 pM using turbidity assay. Phe (phenylalanine) at a final concentration of 240 mM was heated to 90 °C using a 3 -neck flask with a thermocouple and a condenser set to 6 °C. Heated Phe flowed to a stainless steel trough on a heating station occupying a deck position of a liquid handler (Agilent Bravo with 384 tip ST head). 22 pl of Phe was transferred to assay plates containing the library compounds. Plates were shaken continuously, and the turbidity at three wavelengths: 405, 450, and 600 nm was measured at time point zero, after two minutes of shaking, and after two hours (endpoint measurement). 503 hit compounds that completely blocked assembly / structure formation were selected for further investigation at the database level. After the elimination of potentially promiscuous, covalent, or limited availability compounds, 385 candidates were selected for re-examination in technical duplicates at 80 pM and 10 pM. Compounds with confirmed activity were tested for >70 % purity by LC-MS.

[0463] IC50 experiments - ThT fluorescence intensity: Phenylalanine (Phe) was dissolved at 90 °C in PBS to a final concentration of 40 mg / mL until a monomeric solution was obtained. The obtained solution was mixed with Morin hydrate at several concentrations ranging from 1-100 pM, in a black 96-well, clear, and flat bottom microplate (Greiner). As a control, Phe was diluted with DMSO alone to the same final concentrations. ThT in PBS was added to a final concentration of 40 pM. Following excitation at 450 nm, ThT emission data at 480 nm were measured over time and recorded using a TEC AN Infinite 200 PRO plate reader. Data processing was performed using OriginLab software.

[0464] In vivo efficacy studies: All animal experiments were approved and conducted in accordance with the Guidelines of the Institutional Animal Care and Use Committee (license no. 04-18-009). Every effort was made to relieve animal stress and minimize animal usage. Eight weeks old, male PKU Pahemu mice were purchased from The Jackson Laboratory and kept in a 12 hours light-dark cycle with free access to water and food pellets. After an acclimation period of two weeks, the 10 weeks old mice were treated for six weeks with daily IP injections of vehicle (PBS with 10% carboxymethyl cellulose), 50 mg / kg Kuvan®, 50 mg / kg Morin hydrate, or 10 mg / kg Morin hydrate (suspended in PBS with 10 % carboxymethyl cellulose) (n=7 per group). Control mice of the background strain (WT) were also analyzed. Mice (n=7 per group) were randomly assigned to each group.

[0465] After 42 days, treated mice were subjected to cognitive tests, as follows.

[0466] Morris water-maze: Mice were introduced to a round pool arena (1.5 m in diameter, 30 cm deep, 22-24 °C) with external and internal clues for orientation. A 5x5 cm hidden platform submerged under 1 cm of water was located inside the arena. The mice were subjected to three swims per day for 4 days (60 seconds duration with an inter-trial time interval of 60 minutes) to find the platform. The probe test was performed on the fifth day in a single swim (60 seconds) without the platform. Live tracking of the mice was reordered using GigE color 1 / 2" Basler acA1300- 60gc camera and the results were analyzed using the Ethovision ver. 13 XT (Noldus, The Netherlands) software. The time to reach the platform (latency to first) was recorded.

[0467] Y-maze: The test was performed in a symmetrical black Plexiglas Y-maze with three arms (30 cm long* 10 cm wide* 15 cm high) at 120° angles, designated old and novel. The mice were placed in the distal end of the old arm and allowed to explore the maze for 5 minutes with only the old arm available to explore. After a 5-minute waiting period, mice were reintroduced to the maze with both arms (old and new) available to explore and documented for 5 minutes. The percentage of time spent / frequency of visits to the novel arm was calculated as time / frequency in the novel arm divided by the sum of time / frequency in both novel and old arms.

[0468] Grip strength: Mice were allowed to grip a metal grid using their forelimb. Then, a steadily increasing force was applied to pull the mice back from the tail until their grip was broken. The force at which the grip was broken was recorded using a Grip-Strength Meter model 47200 (Ugo Basile, Gemonio, ITALY) measuring device connected to the metal grid.

[0469] Congo-red staining: The right hemisphere of the brain was fixed in 4 % formaldehyde in PBS for 48 hours. After fixation, brains were immersed in 30 % sucrose-PBS overnight. Brains were frozen with dry ice and kept at -70 °C. 25 pm free-floating coronal sections were obtained using a cryostat (LEICA CM 1900, Germany). The coronal brain sections were prepared and fixed in 70 % (v / v) ethanol for 1 minute, washed in double distilled water (ddEEO) for 2 minutes, and stained with filtered Congo red solution for 10 minutes. Following staining, the samples were washed in ddEEO for 2 minutes and washed 8-10 times in NaOH-ethanol solution (0.5 ml 1 % (w / v) NaOH, 49.5 ml 50 % (v / v) ethanol) until the excess red color disappeared. Finally, the samples were washed in ddEEO. The samples were viewed with a Nikon Eclipse TI fluorescent microscope (excitation at 498 nm, emission at 614 nm). Digitized images were obtained using a Nikon DS Ril digital camera. Brain lysate preparation: Following cognitive tests, mice were anesthetized IP with ketamine / xylazine and perfused with saline. Brain tissues were cut sagittally and the left hemisphere was frozen in liquid nitrogen and stored at -70 °C until homogenized on ice in 5 volumes (w / v) of T-per extraction buffer (Pierce, USA) supplemented with protease inhibitor (Complete Mini Protease Inhibitor Tablets, Roche) and phosphatase inhibitor cocktail (phosSTOP, Roche). After sonication, homogenates were centrifuged at 100,000 g for one hour at 4 °C. The resulting supernatants represent the soluble enriched fraction which was used for the analysis presented. The pellets were re-suspended in T-per extraction buffer supplemented with protease and phosphatase inhibitors, 0.5 % Triton X-100, 1 % sodium deoxycholate, and 3 % SDS. After sonication, homogenates were centrifuged at 100,000 g for 1 hour at 4 °C. The resulting supernatants represent the membrane-enriched fraction. The pellets were re-suspended in 6M guanidine-HCl overnight at 4 °C and centrifuged at 20,000 g for 1 hour at 4 °C. The resulting supernatants represent the insoluble fraction. Protein concentration was determined using a BCA protein assay kit (Thermo, USA) according to the manufacturer’s instructions.

[0470] Western immunoblot analysis: Equal amounts of mice brain homogenate protein (50 mg) were resolved on SDS-PAGE, transferred to a nitrocellulose membrane, and blocked overnight with 5 % skim milk in TBS-T (0.1 % Tween® 20). Blots were probed with primary antibodies and the corresponding peroxidase-conjugated secondary antibodies.

[0471] Table A below presents the antibodies used in this study.

[0472] Table A

[0473] Immunoblots were developed using the EZ-ECL detection kit (Biological Industries, Israel), and quantitative densitometric analysis (densitometry quantification) was performed using the Image Studio™ Lite (version 3.1) software.

[0474] Tandem Mass Spectrometry: Eleven amino acids (including Phe and Tyr), 30 acylcarnitines, free carnitine, and succinylacetone were tested using tandem mass spectrometry. In brief, 100-pl extract liquor containing internal standards was added into U bottom plates. After incubating for 45 minutes at 45 °C, 75-ul extract liquor was transferred into V bottom plates. After 2 hours standing at ambient temperature, 25-pl liquor was injected into tandem mass spectrometry for metabolite analyses. Three levels of internal quality controls including blank, low, and high were used for quality control.

[0475] EXAMPLE 1

[0476] Background Art

[0477] As discussed hereinabove, it has been demonstrated previously that phenylalanine (see, FIG. 1 A), as a single amino acid, can self-assemble to form amyloid-like fibrils possessing typical ultrastructural, biophysical, biochemical and intrinsic fluorescence properties similar to those of protein and polypeptide amyloids, that these phenylalanine assemblies are cytotoxic and that antibodies raised against these species deplete fibril toxicity (Background Art FIGs. 1B-K, taken from Shaham-Niv, et al. Science advances 1.7 (2015), supra, Shaham-Niv et al. Commun Chem. 2018 Dec 3;1(1):25 supra,' Shaham-Niv, et al. Angewandte Chemie International Edition 57.38 (2018)).

[0478] To study whether these observations represent a general amyloid-like mechanism prevalent in other IEM disorders, metabolites that accumulate in IEM disorders were screened. It was revealed that several other metabolites could self-assemble to form ordered amyloid-like ultrastructures in solution with molecular dimensions, dye-binding specificity and intrinsic fluorescence properties, similar to canonical amyloid fibrils [Shaham-Niv et al. Angew Chemie Int Ed. 2018, supra], It has been shown that these fibrillar self-assemblies are cytotoxic via induction of apoptotic programmed cell death, as observed for many amyloid disorders [Shaham-Niv et al. Sci Adv. 2015, supra,' Shaham-Niv et al. Isr. J. Chem. 2016, supra], A strong and differential mechanism of interaction of the metabolite assemblies with model membranes was detected, as was previously established for protein and polypeptide amyloids [Shaham-Niv et al. Chem Commun. 2018, 54, supra]. Molecular dynamic simulations provided mechanistic insights to further deepen the knowledge on the mechanism of action of the assemblies.

[0479] The discrete immunological entities of metabolite assemblies was also demonstrated, and the presence of antibodies, specific to the phenylalanine amyloid-like assemblies, in the serum of PKU patients was also demonstrated (unpublished data).

[0480] Overall, these studies indicated that exploring the formation of structures by IEM disorders- associated metabolites may lead to deciphering the pathological course in these maladies with no disease-modifying treatment and can attribute to the discovery of new routes for future therapy.

[0481] As described hereinabove, two aromatic polyphenolic compounds, epigallocatechin gallate (EGCG) and Tannic acid (TA), which have been previously shown to generically inhibit the formation of protein- and peptide-based amyloid structures, were shown to halt the formation of metabolite amyloid fibrils and inhibit the resulting cytotoxicity of the metabolite assemblies, indicating their therapeutic potential [Shaham-Niv et al. Commun Chem. 2018 Dec 3; 1(1):25], See, Background Art FIG. 1H.

[0482] Overall, it was demonstrated that the general phenomenon of amyloid formation is not limited to proteins and peptides, and is actually extended to the formation of amyloid-like assemblies by metabolites, which are also called “metabolite amyloid”. See, FIG. 2.

[0483] EXAMPLE 2

[0484] Screening Assay and Drug Design

[0485] The screening system has been up-scaled towards finding potential inhibitors of the selfassembly of phenylalanine amyloid-like structures, utilizing diverse compounds library and high- throughput screening (HTS) platform. The inhibition of phenylalanine structure formation was determined using an in-vitro turbidity assay. Briefly, phenylalanine was heated to 90 °C and inserted into 384-well plates containing the potential inhibitors (to a final concentration of 240 mM). The turbidity was measured at time point zero, after two minutes shaking, and after two hours (which served as an endpoint). TA (tannic acid) was used as a positive control and PBS with DMSO as a negative control.

[0486] As shown in FIG. 3A, 55,000 compounds were screened from diverse compound libraries, including, inter alia, Analyticon 2014 collection, MayB ridge- HitFinderTM Collection, MicroSource and Selleck Chemicals. 503 hits demonstrated more than 90 % inhibition in the turbidity assay and were further screened using known HTS physical property and structural filters, PAINS and Tier-1 [Baell J and Walters MA, Nature, 2014 Sep 24;513(7519):481-3], The final 385 candidates were re-examined in a validation round (the in-vitro turbidity assay as described herein) and were subjected to further quality control investigations using LC-MS. The LC contains three ACQUITY UPLC I-class separation systems (Waters Corporation), Each LC part augmented with ACQUITY UPLC Photodiode Array (PDA) e&lambda detector for UV-VIS absorption, which allows to detect and quantify low concentrations of compounds containing chromophores from 190 to 800 nm in a broad linear dynamic range. The MS contains two triple quadrupole (TQ-S) and one time-of-flight (G2-S) machines (Waters Corporation). Innovative StepWave technology enables high sensitivity and robustness at low specimen levels and in the complex samples. The re-examination and LC-MC quality control investigation resulted in 14 hit molecules, as presented in Table 1. Table 1

[0487] (Table 1; Cont.)

[0488] One of the uncovered compounds, morin hydrate (Table 1, No. 5; FIG. 3B), was already shown to inhibit amyloid formation by human Islet Amyloid Polypeptide (IAPP) and to disaggregate pre-formed IAPP amyloid fibrils [Noor et al. Protein Sci. 2012, supra].

[0489] The retrieval of morin hydrate, which was found previously to halt the formation of other, proteinaceous, amyloidal structures, served as a further reinforcement to the amyloidgenic nature of the phenylalanine structures. Thus, this compound was further investigated for its pharmacological properties utilizing other orthogonal assays and as a proof of concept in in-vivo studies.

[0490] To this end, it was examined whether this compound could inhibit phenylalanine fibril formation using the thioflavin-T (ThT) fluorescence assay, a dye indicative of the formation of amyloid assemblies [Levine H. Protein Sci. 2008, 2(3), 404-1012; Biancalana M. and Koide S.

[0491] Biochim Biophys Acta - Proteins Proteomics 2010, 1804(7), 1405-12],

[0492] As shown in FIGs. 3C-D, morin hydrate demonstrated a remarkable inverted dose- dependent inhibition effect upon phenylalanine fibril formation, presenting ICso of about 6 pM.

[0493] These results suggest that materials that are known to inhibit amyloid structure formation may also inhibit other amyloid-like structures.

[0494] Analysis of very short functional fragments from unrelated amyloid forming proteinaceous entities presented a remarkably high occurrence of aromatic residues [Gazit E. FASEB J. 2002, supra}. The aromatic residues may play an important role in the amyloidogenic process by very short fragments and in the stabilization of amyloidal structures by geometrically-restricted interactions between planar aromatic chemical entities [Makin O.S. and Serpell L.C. FEBS J. 2005, 272(23), 5950-61], These proteins and poly-peptides are interconnected by an extensive network of hydrogen bonds aligned in parallel to the fibril axis, which further stabilizes the amyloid fibrils and is considered as the origin of their remarkable properties [Pinotsi et al. ChemBioChem. 2013, 14(7), 846-50],

[0495] Notably, all the 14 compounds identified in the screening assay were aromatic, suggesting they might have a similar motif or structural resemblance.

[0496] Thus, a pharmacophore model for the 14 hit compounds was sought for.

[0497] To this end, a training set was prepared by including the 14 hits and their isomers. As a negative control, a compound that does not inhibit the phenylalanine self-assembly, acetylsalicylic acid [Shaham-Niv et al., 2018, supra], was used. The best pharmacophore model, which gave the best fitting results and was selected for further analysis, found to contain two aromatic motifs with a distance of 6.3 angstroms therebetween and one H+(proton) donor motif (ARR) as shown in FIG. 4A.

[0498] As mentioned above, it has been reported that aromatic and hydrogen bonds interactions are needed for stabilization of amyloid beta sheet structures. A pharmacophore presenting these two motifs should specifically target the destabilization of such phenylalanine amyloid assemblies. Alignments of the known inhibitor of amyloid fibril formation, ECGC, and of the uncovered morin hydrate, on the pharmacophore model are depicted in FIGs. 4C and 4B, respectively.

[0499] EXAMPLE 3 In vivo Studies

[0500] Pahenu2PKU mice model:

[0501] Efficacy studies were conducted in order to validate the new paradigm-shifting hypothesis that inhibition of phenylalanine aggregation serves as a valid therapeutic target to PKU. Preliminary studies were conducted with one of the hit compounds uncovered in the screening, morin hydrate (see, FIG. 3B), in Pahenu2PKU mice model. As a control, one group received Kuvan® (gold standard treatment of PKU). Two concentrations of morin hydrate were examined: 10 mg / Kg and 50 mg / Kg, denoted M10 and M50, respectively.

[0502] The efficacy study using PKU Pahenu2mice model was blinded and well controlled.

[0503] Animal studies experimental design:

[0504] PKU Homozygous Pahenu2mice were treated (injected IP) for six weeks daily with:

[0505] 1. vehicle (n=7)

[0506] 2. 50 mg / kg Kuvan® (n=7) 3. 50 mg / kg morin hydrate (n=6)

[0507] 4. 10 mg / kg morin hydrate (n=6)

[0508] Kuvan® concentration was chosen based on previous usage [Winn et al. Mol. Genet. Metab. 117, 5-11 (2016)], while considering the administration route and duration of the treatment.

[0509] The treatment groups were monitored and evaluated for disease progression by: weight loss, motor functions assessment (grip strength) and behavioral studies (Water maze and Y-Maze) compared to vehicle and Kuvan® controls.

[0510] Since cognitive functions are impaired in an age-dependent manner in the Pahenu2mice model, in order to assess the cognitive functions following treatment, mice were subjected to Morris water and Y maze spatial learning tests. The obtained data is presented in FIGs. 5A-D.

[0511] As can be seen, in water maze long-term memory and spatial learning test, all treated mice, with both doses of morin hydrate and Kuvan®, demonstrated a learning curve throughout the four days (FIG. 5A).

[0512] The probe test on the fifth day is presented in FIG. 5B, and show statistical significant improvement in all treated mice compared to the control. Statistical significance was achieved for the higher dose of the morin hydrate compared to the Kuvan®.

[0513] To further support morin hydrate effect on PKU mice model cognitive deficiency, mice were subjected to Y maze short-term spatial learning test, and the obtained data are presented in FIG. 5C. Vehicle treated mice did not show any preference to the novel arm, while treated mice spend a longer time and exhibit a higher frequency of visits to the novel arm, which indicates an improvement in short-term memory. These results show the same trend and thus further support the results reported for the water maze test.

[0514] The effect of morin hydrate on motor function via grip strength test was also tested, and the obtained data are shown in FIG. 5D. Improvement in grip strength was achieved for the higher dose of morin hydrate compared to vehicle treated group.

[0515] Phenylalanine metabolism:

[0516] Pathophysiological evaluation was studied by further analyzing control mice of the background strain (WT).

[0517] Mice (n=4 per group) were randomly assigned for each group. Mice were sacrificed by perfusion and harvested at treatment endpoint. Biochemical and histological analysis of brain tissue and plasma were used to assess treatment effect on disease pathology.

[0518] To explore the effect of treatment on phenylalanine metabolism whole blood was collected via cardiac puncture, spotted on a Guthrie card for a dried blood spot testing and sent to MS analysis. The bloodspots were digested using proteases and analyzed using Xevo G2-S QTOF MS (Waters Corporation) set for MS and non-targeted analysis provides high mass resolution, high mass accuracy and broad dynamic range.

[0519] As shown in FIGs. 6A-B, phenylalanine concentration and phenylalanine / tyrosine ratio were 14-folds and 28-folds, respectively, higher in PKU mice model compared to WT mice.

[0520] To examine the pathohistological effect of morin hydrate treatment on phenylalanine amyloid deposits, brain sections of mice from each group were histologically analyzed using staining with Congo Red, a commonly used amyloid indicative fluorescent dye. After treatment, mice were probed for plaque pathology. Right brain hemisphere was coronal sectioned into 25 pm sections and stained for Congo Red. The stained brain sections were imaged using brightfield and fluorescence microscopy. Quantification analysis of sections stained with Congo Red by imaged software.

[0521] The obtained data is presented in FIG. 7 and shows that the treatment with morin hydrate reduces the number of amyloid plaques as well as the plaques’ total area.

[0522] The fact that morin hydrate treatment had no effect over phenylalanine levels, while reducing its amyloid deposits and showing improvement in cognitive function goes to further supports the underlying basis of the present embodiments, that phenylalanine amyloid-like assemblies are the toxic species that play an important role in PKU.

[0523] The method described herein emphasizes the relevance of the observed metabolite amyloid fibrils in diseases such as PKU and the ability to target the disease pharmacologically without the need to reduce the levels of the metabolites, demonstrating the role of inhibitors of metabolite amyloid fibrils in treating also other disorders associated with metabolite imbalance.

[0524] ChAT and NeuN expression:

[0525] To go further into the molecular mechanism by which morin hydrate exerts its beneficial effect, brain homogenates of treated mice were analyzed by Western Blot (WB). More specifically, brain homogenates of WT, vehicle, morin hydrate 50 mg / Kg (M50), morin hydrate 10 mg / Kg (MIO) and Kuvan® 50 mg / Kg (Kuv) (n=4) PKU treated mice were analyzed by WB (FIGs. 8C-D). Densitometry quantification of the blots were developed with anti-ChAT antibody, normalized to actin, or with anti-NeuN antibody, normalized to actin.

[0526] The obtained data is shown in FIG. 8A (blots developed with anti-ChAT antibody, normalized to actin) and FIG. 8B (blots developed with anti-NeuN antibody, normalized to actin), and show that Choline acetyl transferase (ChAT) expression is increased in PKU mice model compared to WT mice (Veh vs. WT, FIG. 8A).

[0527] ChAT is a transferase enzyme responsible for the synthesis of the neurotransmitter acetylcholine. Both the dopaminergic and adrenergic neurotransmitter systems are compromised in PKU mice, as they are the products of downstream metabolism of Phenylalanine. The obtained results suggest that the cholinergic neurotransmitter system attempts to compensate over the defective systems via increasing ChAT expression. Treatment with either concentration of morin hydrate reverts this overcompensation to similar levels as WT, while treatment with Kuvan® did not affect this system (FIG. 8A).

[0528] To further support this hypothesis, the expression levels of the neuronal marker Neuronal Nuclei (NeuN), a protein localized to the nuclei and perinuclear cytoplasm of most neurons in the CNS, was explored. NeuN is used as a reliable marker for assessment of neuronal status studies in both norm and pathology. As shown in FIG. 8B, NeuN expression is decreased in PKU mice model compared to WT mice (Vehicle vs. WT, FIG. 8B) indicating neuronal stress. Treatment with either concentration of morin hydrate or Kuvan® reverts expression level to similar levels as WT. The presynaptic marker, Synapthophysin, expression did not change between WT and any of the PKU model groups, suggesting that synaptic transmission does not play a role in this model (data not shown). Altogether, these results suggest that treatment with morin hydrate reverses the overcompensation of the cholinergic system, which in turn results in reduction in neuronal stress.

[0529] Neuroin flammation :

[0530] Neuro-inflammation by both astrocytes and microglia in PKU patients was also tested. Glial fibrillary acidic protein (GFAP) is a protein involved in the shape, movement and function modulation of astroglial cells and is commonly used as a marker for astrocytes activation. Ionized calcium binding adaptor molecule 1 (Iba-1) is a calcium-binding protein expressed in microglia cells in the brain. It plays a significant role in modulating the function of microglia, which act as a first line of defense against altered autologous antigens.

[0531] Brain homogenates of WT, vehicle, morin hydrate 50 mg / Kg (M50), morin hydrate 10 mg / Kg (MIO) and Kuvan® 50 mg / Kg (Kuv) (n=4) PKU treated mice were analyzed by WB (FIGs. 9C-D), with densitometry quantification of the blots developed with anti-GFAP antibody normalized to actin and anti -Iba-1 antibody normalized to actin.

[0532] The obtained data is presented in FIGs. 9A-B, and show that GFAP expression is increased in PKU mice model as compared to WT mice (Vehicle vs. WT, FIG. 9A), indicating the involvement of astrocyte originated inflammation in PKU. Treatment with either concentration of morin hydrate reduces GFAP expression levels, while treatment with Kuvan® had a less pronounced effect.

[0533] Iba-1 expression is increased in PKU mice model compared to WT mice (Vehicle vs. WT,

[0534] FIG. 9B), indicating the involvement of microglia originated inflammation in PKU. Treatment with either concentration of morin hydrate reduces Iba-1 expression levels, while treatment with Kuvan® had a more pronounced effect. Without being bound by any particular theory, the biochemical analyses of mice brain lysate suggest a mechanism of action by which morin hydrate treatment reverses the overcompensation of the cholinergic neurotransmitter system due to the defective dopaminergic and adrenergic neurotransmitter systems. This may lead to the observed reduction in neuronal stress by NeuN marker (FIG. 8B). Reduction of neuro-inflammation is presumably effected by the treatment via reducing both astrocyte and microglia markers (FIGs. 9A-B).

[0535] EXAMPLE 4

[0536] Structural Analogs of Morin Hydrate

[0537] Exemplary compounds having structural features similar to those of morin hydrate are presented in FIG. 10A-G.

[0538] FIG. 10A presents compounds encompassed by Formula I as described herein, in which variables X and R1-R5 are the same as in morin hydrate, and some of variables Re-Rio are different.

[0539] FIG. 10B presents compounds encompassed by Formula I as described herein, in which X and Rs are the same as in morin hydrate and some of variables R1-R4 and Re-Rio are different, yet are selected from hydrogen, hydroxy and alkoxy (e.g., methoxy), and include at least one hydroxy or alkoxy.

[0540] FIG. IOC presents compounds encompassed by Formula I as described herein, in which X and Rs are the same as in morin hydrate and some of variables R1-R4 and Re-Rio are different, and are selected from hydrogen, hydroxy, alkyl and alkoxy (e.g., methoxy).

[0541] FIG. 10D presents compounds encompassed by Formula I as described herein, in which X is the same as in morin hydrate, Rs is hydrogen and not hydroxy as in morin hydrate, and some of variables R1-R4 and Re-Rio are different, yet are selected from hydrogen, hydroxy and alkoxy (e.g., methoxy), and include at least one hydroxy or alkoxy.

[0542] FIG. 10E presents compounds featuring higher variation from the structure of morin hydrate, some of which are not encompassed by Formula I as described herein.

[0543] FIG. 10F presents compounds featuring a flavanone skeleton, some of which are encompassed by Formula I as described herein, and all share the following structural features in Formula I: X is oxygen and the bond ring fused to the phenyl is saturated.

[0544] FIG. 10G presents compounds encompassed by Formula I as described herein, in which X is NH.

[0545] Selected compounds were tested for their activity in a ThT assay as described herein (data not shown), as follows: Kaempferol (FIG. 10A), Quercetin (FIG. 10A), Apigenin (FIG. 10D), Luteolin (FIG. 10D), Narigenin (FIG. 10F), Kaempferide (FIG. 10 A), Chrysin (FIG. 10D), Galangin, Flavanone (FIG. 10F) and Flavone (FIG. 10D).

[0546] Of these, Apigenin, Kaempferol, Kaempferide and Galangin, which exhibited the most pronounced inhibitory activity, were further tested in an MTT assay as described herein, using SH- SY5Y cells, in the presence of 10 mg / mL Phe, at varying concentrations, and the obtained data are shown in FIGs. 11 A-D.

[0547] As can be seen, all compounds were found active also in the cytotoxicity assays, with apigenin exhibiting the best performance.

[0548] EXAMPLE 5

[0549] Hit to Lead Optimization

[0550] A general depiction of further stages of the drug design is depicted in FIG. 12.

[0551] In silico screening and computational guided medicinal chemistry were used in order to discover and develop new therapeutic agents based on the uncovered pharmacophore model described in Example 1 hereinabove (see, FIGs. 4A-C). The Schrodinger virtual eMolecules database, containing about 14 million individual compounds, was screened against the best pharmacophore model, which contains the required properties needed to inhibit the formation of the phenylalanine assemblies. 11,000 molecular probes were identified and clustered according to their structural resemblance (data not shown).

[0552] In parallel, the uncovered 14 hit compounds shown in Table 1 were triaged using Multi Parameters Optimization (MPO) (MPO Explorer™ module on StarDrop software, Optibrium) in order to prioritize them by chemical matter. The potential intractable and 'risky' compounds with unfavorable properties were marked by applying structural rules and physicochemical properties using in silico tools (see, FIG. 12 and FIG. 13 A). Generally, the list was filtered by removing molecules possessing biologically reactive electrophilic moieties, such as aldehydes or alkyl dihalides. Quinones, quinone-like structures, and compounds possessing four or more electronrich aromatic systems, were also filtered at this stage.

[0553] Five compounds out of the 14 hits, presented in Table 2 below, were selected for further structure and in vitro validation. Table 2

[0554] (Table 2; Cont.)

[0555] The calculated oral Central Nervous System (CNS) scoring profile of the selected top five molecules is presented in FIG. 13B, as was calculated while considering biologically relevant physicochemical and biochemical properties of an orally-administered and CNS-directed drug, as presented in FIG. 13 A. Three synthesized hit compounds, namely PCM1, PCM2 and PCM3, successfully passed the quality control for further in vitro validation (data not shown).

[0556] The structure and in vitro validation were performed by pharmacophore based analog-to- catalog. For this process, structurally resembling compounds, answering to the pharmacophore model, were tested in order to validate the structural specificity and importance of the compound to the phenylalanine aggregates’ inhibition.

[0557] In vitro validation assays included the following:

[0558] ThT fluorescence and turbidity kinetic assays: The in vitro efficacy was determined using time-dependent monitoring of ThT fluorescence intensity and turbidity in the presence of increasing concentrations of each of the tested compounds, as described herein, for determining the IC50 of each compound.

[0559] Monomeric solutions of phenylalanine (38 mg / ml) were obtained as described above. These solutions were mixed with 100 micromolar (pM) of each of the tested compounds PCM1, PCM2 or PCM3 in DMSO (50 pM). As a control, phenylalanine monomeric solution was diluted with DMSO alone to the same final concentration.

[0560] For the ThT experiments, the solutions were plated in a black 96-well, clear, and flat bottom microplate and ThT in PBS was added to a final concentration of 40 pM. Following excitation at 450 nm, ThT emission data at 480 nm was measured over time and recorded using a BMG CLARIOstar plate reader.

[0561] The data are presented in FIG. 14 A. A decrease in fluorescence signal can be seen following the addition of each of the exemplary compounds PCM1, PCM2 and PCM3, indicating their fibril formation inhibitory activity.

[0562] For the turbidity experiments, the solutions were plated in similar experimental settings as the ThT measurements and the absorbance was monitored at 405 nm.

[0563] The data showing the effect of the inhibitors on the turbidity values are presented in FIG. 14B, and demonstrate that full inhibition of the turbidity alongside the ThT signal was obtained in the presence of PCM1. PCM2 also inhibited the fibrils formation to some extent, as the time to reach a plateau was approximately 10 times longer than Phe without inhibitor (FIG. 14B).

[0564] Based on these results, the exemplary compound PCM1 was chosen as a candidate for additional optimization studies.

[0565] Transmission electron microscopy: The effect of the compounds on the formation of ordered phenylalanine fibrillary assemblies was tested. Phenylalanine (20 mg / ml) was dissolved in phosphate-buffered saline (PBS) to mimic physiological conditions and reflect physiological pH and ionic strength. The solution was heated to 90 °C to obtain a homogenous monomeric solution. Next, the monomeric phenylalanine solution was mixed with different concentrations of the exemplary compound PCM1 (10, 50, and 100 micromolar). As controls, phenylalanine monomeric solution was diluted with PBS alone to the same final concentrations and the effect of the tested compound alone, without the phenylalanine monomeric solution, was also tested. Samples were examined using a JEOL 1200EX electron microscope operating in order to gain insight into the morphological changes of phenylalanine fibrillar assemblies when incubated with the exemplary compound PCM1.

[0566] The results are presented in FIGs. 15A-E. As can be seen, in the absence of PCM1, phenylalanine presented a typical fibrillar morphology of amyloid-like assemblies (FIG. 15 A), whereas in the presence of various concentration of PCM1, inhibition of fibrils formation was observed in a concentration-dependent manner (FIGs. 15C-E). PCM1 alone was imaged as well for comparison (FIG. 15B).

[0567] These results are consistent with the relative inhibitory effect of several polyphenolic and flavonoid compounds towards the formation of amyloids by protein, peptide and metabolite building blocks. In addition, the lack of structure formation observed in TEM, correlates well with the results of the ThT and turbidity assays.

[0568] Cell cytotoxicity experiments: The correlation between the inhibition of phenylalanine assemblies’ formation and the resulting cytotoxicity was determined. The effect on cell viability was investigated by performing an MTT cell viability assay on SH-SY5Y neuronal cell model. Cell viability was evaluated using the MTT assay. Briefly, to obtain monomeric solution, phenylalanine was dissolved at 90 °C in DMEM / Nutrient Mixture F 12 (Ham’s) (1 : 1) without fetal bovine serum (FBS), to a final concentration of 6 mg / ml. Once fully dissolved, phenylalanine solution was mixed with the exemplary compound PCM1 at different concentrations ranging from 5 to 50 micromolar (dissolved in DMSO), or with DMSO only to a final concentration of 1 % solvent. As a control, medium with DMSO and no phenylalanine, which was treated in the same manner, was examined. Cells were incubated for 24 hours with the different treatments in the absence or presence of PCM1, followed by addition of MTT reagents. Following a 4 hour-period of incubation, extraction buffer was added for an additional 30 minute-long incubation, and absorbance was determined at 570 nm and 680 nm, and the results are presented in FIG. 16.

[0569] The results show cell viability restored up to about 90 %, indicating inhibition of the cytotoxic effect of phenylalanine fibrils by the exemplary compound PCM1.

[0570] Hit to Lead studies: PCM1 was re-designed and studied, in order to assess the effect of each of its structural motifs: the presence and nature of the aromatic substitution, the presence and nature of the linker and the presence and nature of the heteroaromatic ring, which surround its amide core as presented in FIG. 17 A. The effect of the structural features of both parts of the molecule, including the amide, is studied in order to establish the complete Structure-Activity Relationship (SAR).

[0571] Optimizations and SAR studies of PCM1 began with the phenyl group in the aromatic substitution motif, and are presented in FIGs. 17B-C. For this purpose, the Topliss methodology was applied [Topliss, J. G. J. Med. Chem. 1977, 20 (4), 463-469; Topliss, J. G. J. Med. Chem. 1972],

[0572] The Topliss tree is a simple stepwise process for phenyl group optimization, based on the fundamental assumption that there are three broad properties that a substituent might change about a compound: hydrophobicity, electronics, and sterics. Thus, design, synthesis, and activity measurement of several compounds sequentially following the Topliss tree allow to generate a preliminary SAR and identify a more potent study-compound.

[0573] To rationally evaluate the SAR through the Topliss methodology, the molecular scaffold of PCM1 was first simplified by synthesizing two new molecules: (1) SS18400-1, consisting of methyl-furan only, and SSI 8400-7, consisting of the phenyl ring only, as depicted in FIG. 17B [Bemis, G. W. and Murcko, M. A. J. Med. Chem. 1996; Schuffenhauer et al. J. Chem. Inf. Model. 2006, 47 (1), 47-58], The activity measured for these two molecules indicates the importance of the methyl-furan substitution (data not shown).

[0574] Derivatives of PCM1, presented in FIGs. 17B-C, each lacking one structural motif, were synthesized. A general synthesis for the preparation of PCM1 derivatives in which the nature of the aromatic substitution was modified is as depicted in FIG. 17D. The process utilized simple and traditional coupling conditions, while applying microwave irradiation to accelerate the reaction.

[0575] The physiochemical properties (such as logP; MW; number of hydrogen bond acceptors; number of hydrogen bond donors; total polar surface area; flexibility; number of rotatable bonds etc.) are evaluated in silica. Potency and toxicity of the designed molecules is tested in vitro and the correlation between the structure; physiochemical properties; potency, and toxicity is studied in order to optimize the drug design to provide the most potent and safe molecule (See, e.g., FIGs. 17B-C). The SPR / SAR (Structure Property Relationship / Structure Activity Relationship) process is conducted in cycles toward a drug-like compound possessing proper properties and efficacy. Higher-level properties such as solubility, permeability, metabolic stability, P-glycoprotein (Pgp) efflux and plasma protein binding are also effected by the structural modification and are analyzed.

[0576] In-vitro and in vivo validations: Absorption; distribution; metabolism, and excretion (ADME) of the lead compounds is studied by in vitro and complementary in vivo assays. Drugmetabolism pharmacokinetics (DMPK) is improved by adding / removing / changing relevant chemical motifs while conserving the structural template. Each synthetic cycle includes design and synthesis 3-5 new molecules, their bioavailability evaluation, including the physio- and biochemical properties listed above and the correlation between all the parameters.

[0577] The identified leads are subjected to efficacy studies using PKU Pahenu2mouse model, similarly to the assays described hereinabove (Example 3). Toxicity, pharmacodynamics and pharmacokinetics evaluation of the most potent compound(s) is thereafter effected, including Maximum Tolerated Dose (MTD) studies, Dose Range Finding (DRF) studies, 14-day toxicity studies, in vivo drug metabolism and pharmacokinetic (DMPK), biotransformation, and metabolism studies.

[0578] PKU cell line model toxicity ofPCMl derivatives: Two cell lines, each featuring a single mutation in the human phenylalanine hydroxylase enzyme (I65T mutation in the regulatory domain and R261Q mutation in the catalytic domain), are used. Both mutations cause loss of function of the enzyme, thus resulting in high concentrations of phenylalanine and serve as suitable PKU cell model system. The cell lines are treated with increasing concentrations of phenylalanine, and the concentration that triggers 50 % cell cytotoxicity is determining using the MTT assay. Next, the determined phenylalanine assemblies’ ECso is used with increasing concentrations of the tested compounds, to determine the inhibition of the phenylalanine-associated cytotoxicity. All solutions and treatments are prepared as described herein.

[0579] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0580] It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

WHAT IS CLAIMED IS:

1. A compound represented by Formula I:Formula I wherein: the dashed line represents an optional double bond;X is O, S or NRn; andR1-R11 are each independently selected from hydrogen, alkyl, cycloalkyl, heteroalicyclic, hydroxy, thiol, alkoxy, thioalkoxy, amine, halo, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxime, hydrazone, carbonyl, thiocarbonyl, a urea group, a thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S -thiocarb amyl, C-amido, N- amido, C-carboxy, O-carboxy, sulfonamido, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and likewise substituents, provided that at least one of R1-R4 is a hydrogen-bond donor substituent; the compound being for use in treating a metabolic disorder associated with phenylalanine aggregation in a subject in need thereof.

2. The compound for use of claim 1 , wherein the dashed line represents a double bond.

3. The compound for use of claim 1 or 2, wherein Ri is hydroxy.

4. The compound for use of any one of claims 1 to 3, wherein at least two of R1-R4 are each hydroxy.

5. The compound for use of any one of claims 1 to 4, wherein Ri and Ra are each hydroxy.

6. The compound for use of claim 5, wherein R2 and R4 are each hydrogen.

7. The compound for use of any one of claims 1 to 6, wherein Rs is hydroxy.

8. The compound for use of any one of claims 1 to 7, wherein at least one of Re to Rio is selected from hydroxy and alkoxy.

9. The compound for use of claim 8, wherein at least Rs is hydroxy or alkoxy.

10. The compound for use of claim 9, wherein Rs is hydroxy or alkoxy and Re, R7 andR9 are each hydrogen.

11. The compound for use of any one of claims 1 to 9, wherein at least two of Re to Rio are each independently selected from hydroxy and alkoxy.

12. The compound for use of claim 11, wherein Rs and Rio are each independently hydroxy or alkoxy.

13. The compound for use of any one of claims 1 to 12, wherein X is O.

14. The compound for use of any one of claims 1 to 13, wherein the compound is morin hydrate.

15. The compound for use of any one of claims 1 to 13, wherein the compound is Apigenin.

16. The compound for use of any one of claims 1 to 13, wherein the compound is selected from morin hydrate, Apigenin, Kaempferol, Kaempferide and Galangin.

17. The compound for use of any one of claims 1 to 16, wherein the compound is represented by Formula la:Wherein R1-R10 are each independently as defined for Formula I.

18. A compound selected from:the compound being for use in treating a metabolic disorder associated with phenylalanine aggregation in a subject in need thereof.

19. The compound for use of claim 18, wherein the compound is selected from:

20. The compound for use of claim 18 or 19, wherein the compound is:or a structural analog thereof.

21. The compound for use of any one of claims 1 to 20, wherein said metabolic disorder is caused by an inborn error of metabolism.

22. The compound for use of any one of claims 1 to 21, wherein said metabolic disorder is phenylketonuria.

23. A method of identifying a compound capable of interfering with phenylalanine aggregation in a subject in need thereof, the method comprising screening in silico a library of compounds to thereby determine if a compound is capable of aligning with a pharmacophoric model featuring two aromatic motifs and a hydrogen-bond donor motif as depicted in FIG. 4A, wherein a compound capable of said aligning is identified as capable of inhibiting phenylalanine aggregation.

24. The method of claim 23, further comprising contacting the identified compound with a phenylalanine-containing medium, to thereby further determine if the identified compound is capable of inhibiting phenylalanine aggregation, wherein a compound that reduces formation of phenylalanine fibrils by at least 20 %, compared to a medium lacking the compound, is identified as capable of inhibiting phenylalanine aggregation.

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