MISHRA RAMA (US)
LUAN CHI-HAO (US)
WO2020247860A1 | 2020-12-10 |
US9725708B2 | 2017-08-08 |
LI ET AL.: "A patent review of the ubiquitin ligase system: 2015-2018", EXPERT OPIN THER PAT, vol. 28, no. 12, December 2018 (2018-12-01), pages 919 - 937, XP055644386, DOI: 10.1080/13543776.2018.1549229
CLAIMS We claim: 1. A method for inhibiting the activity of a HECT E3 ubiquitin ligase in a subject in need thereof, the method comprising: administering an effective amount of a compound of Formula I, a derivative, isomer, or a pharmaceutically acceptable salt thereof, or a combination thereof, wherein Formula I is: wherein R is selected from hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl optionally substituted with alkyl, and heteroaryl. 2. The method of claim 1, wherein R is selected from: 3. The method of claim 1 or 2 wherein the compound comprises Formula II: . 4. The method of claim 1 or 2, wherein the compound comprises Formula III: . 5. The method of any of the previous claims, wherein the subject in need thereof is diagnosed with or is suspected of having a disease or condition characterized by increased HECT E3 ubiquitin ligase activity. 6. The method of claim 5, wherein the HECT E3 ubiquitin ligase comprises UBE3A/E6AP. 7. The method of claim 6, wherein the disease or condition is cancer or a neurological disorder. 8. The method of claim 7, wherein the cancer is one or more of HPV associated cancer, HCV associated cancer, cancer characterized by PML downregulation, non-small cell lung cancer, and breast cancer. 9. The method of claim 8 wherein the cancer characterized by PML downregulation is one or more of Burkitt's lymphoma and prostate cancer. 10. The method of claim 7, wherein the neurological disorder is one or more of Angelman syndrome (AS), Autism Spectrum Disorder (ASD), and chromosome 15q11.2- q13.3 duplication syndrome (Dup15q). 11. The method of any of the preceding claims, wherein the compound is formulated as a pharmaceutical composition. 12. The method of any of the preceding claims, wherein the compound is administered to the subject orally, parenterally, topically, or locally. |
[0050] [0051] Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain. Non-conservative amino acids typically disrupt (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain. [0052] As used herein, the term “subject” may be used interchangeably with the term “patient” or “individual” and may include an “animal” and in particular a “mammal.” Mammalian subjects may include humans and other primates, domestic animals, farm animals, and companion animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like. [0053] As used herein, the phrase “effective amount” shall mean that drug dosage that provides the specific pharmacological response for which the drug is administered in a significant number of patients in need of such treatment. An effective amount of a drug that is administered to a particular patient in a particular instance will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art. [0054] As used herein, the terms “treat” or “treatment” encompass both “preventative” and “curative” treatment. “Preventative” treatment is meant to indicate a postponement of development of a disease, a symptom of a disease, or medical condition, suppressing symptoms that may appear, or reducing the risk of developing or recurrence of a disease or symptom. “Curative” treatment includes reducing the severity of or suppressing the worsening of an existing disease, symptom, or condition. Thus, treatment includes ameliorating or preventing the worsening of existing disease symptoms, preventing additional symptoms from occurring, ameliorating or preventing the underlying systemic causes of symptoms, inhibiting the disorder or disease, e.g., arresting the development of the disorder or disease, relieving the disorder or disease, causing regression of the disorder or disease, relieving a condition caused by the disease or disorder, or stopping the symptoms of the disease or disorder. [0055] As used herein "control," as in "control subject" or "control sample" has its ordinary meaning in the art, and refers to a sample, or a subject, that is appropriately matched to the test subject or test sample and is treated or not treated as appropriate. [0056] A "therapeutic agent" or "therapeutic molecule" includes a compound or molecule that, when present in an effective amount, produces a desired therapeutic effect, pharmacologic and/or physiologic effect on a subject in need thereof. It includes any compound, e.g., a small molecule drug, or a biologic (e.g., a polypeptide drug or a nucleic acid drug) that when administered to a subject has a measurable or conveyable effect on the subject, e.g., it alleviates or decreases a symptom of a disease, disorder or condition. [0057] As used herein the term "inhibit" or "inhibiting" with respect to the activity of a protein or enzyme (e.g., a HECT E3 ligase) refers to lessening, decreasing, or completely blocking or preventing a measurable activity. Inhibition may be permanent as to a specific molecule, or may be temporary, for example, inhibition may be reversible. [0058] Ubiquitin [0059] The ubiquitin protein is known in the art. (See, e.g., Herschko et al., The ubiquitin system. Annu. Rev. Biochem. 67, 425-479 (1998), the content of which is incorporated herein by reference in its entirety). In some embodiments, the wild-type UB comprises the amino acid sequence of SEQ ID NO:1 - 3, shown at https:// world wide web, at the NCBI website, National Library of Medicine at nih.gov /protein/NP_066289.3 (a product of the UBC gene, encoding multimer precursor of ubiquitin; SEQ ID NO: 1). Uniprot entry L8B196 (Uniprot entry about UBC, showing the multimer structure; SEQ ID NO: 2); and Uniprot blast, (/?about=L8B196[1-76]&key=Domain, a BLAST sequence of mature 76 amino acid ubiquitin; SEQ ID NO: 3). [0060] Ubiquitin-activating enzymes (E1) [0061] The ubiquitin-activating enzymes (E1) are known in the art. (See, e.g., Schulman et al., Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signaling pathways. Nat. Rev. Mol. Cell Biol.10, 319-331 (2009), the content of which is incorporated herein by reference in its entirety). In some embodiments, the wild-type E1 comprises the amino acid sequence of Uba1 (E1) SEQ ID NO:4 (see https://www.ncbi.nlm.nih.gov/protein/NP_003325.2). [0062] Ubiquitin-conjugating enzymes (E2) [0063] The ubiquitin-conjugating enzymes (E2) are known in the art. (See, e.g., Wenzel, et al., E2s: structurally economical and functional replete. Biochem. J.433, 31-42 (2011), the content of which is incorporated herein by reference in its entirety). In some embodiments the wild-type E2 comprises the amino acid sequence of UbcH7/UBE2L3 (E2) SEQ ID NO:5 (see https://www.ncbi.nlm.nih.gov/protein/NP_003338.1) [0064] In some embodiments, the E2 protein comprises UBE2A, UBE2B, UBE2C, UBE2D1, UBE2D2 (UBCH5B), UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2F, UBE2G1, UBE2G2, UBE2H, UBE2I, UBE2J1, UBE2J2, UBE2K, UBE2L3 (UBCH7), UBE2L6, UBE2M UBE2N, UBE2O, UBE2Q1, UBE2Q2, UBE2R1 (CDC34), UBE2R2, UBE2S, UBE2T, UBE2U, UBE2V1, UBE2V2, UBE2W, UBE2Z, ATG3, BIRC6, and UFC1. [0065] Ubiquitin ligase enzymes (E3) [0066] The ubiquitin ligase enzymes (E3) are known in the art. (See, e.g., Deshaies, et al., RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78, 399-434 (2009); and Jin et al., Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging. Nature. 447, 1135-1138 (2007); the contents of which are incorporated by reference in their entireties.). Several hundred E3 ligases have been identified in the human genome. (See, e.g., Medvar et al., Comprehensive database of human E3 ubiquitin ligases: application to aquaporin-2 regulation. Physiol Genomics 2016; 48(7)502-512, the content of which is incorporated herein by reference in its entirety). E3 ligases are predominantly of types referred to as HECT types, U-box types, RBR types, and/or Ring types and a comprehensive library of E3 ligases exists. (See id. citing to (hpcwebapps dot cit.nih.gov front slash ESBL/Database/E3-ligases/). Ubiquitination plays a pivotal role in several cellular processes and is critical for protein degradation and signaling. In the ubiquitination cascade, E3 ubiquitin ligases are responsible for substrate recognition. In order to achieve selectivity and specificity on their substrates, HECT E3 enzymes are tightly regulated and exert their function in a spatially and temporally controlled fashion in the cells. At their C-terminus, all HECT E3s present the catalytic HECT domain, composed of a bulkier N-terminal lobe (N-lobe) that contains the E2 binding domain, and a C-terminal lobe (C-lobe) carrying the catalytic cysteine (see e.g., Figure 1). The two lobes are connected by a flexible hinge region that allows the C-lobe to move around in order to facilitate the Ub transfer from the E2 to the E3. [0067] According to the domain organization present in the N-terminal part of the proteins, the HECT E3s can be subdivided into three main families. The best characterized family is the NEDD4 family, including nine human members: ITCH, SMURF1, SMURF2, WWP1, WWP2, NEDD4, NEDD4-2, HECW1, and HECW2. The NEDD4 members share similar domain structure and include a membrane/lipid-binding C2 domain, two to four WW domains for substrate recognition and a C-terminal HECT domain. The second class, the HERC family, is characterized by one or more regulators of chromatin condensation 1 (RCC)-like domains (RLD), which serve as a guanine nucleotide exchange factor (GEF) for the small GTPase in membrane trafficking processes. This family includes six members (HERC1-6) that can be subdivided into four ‘small’ and two ‘large’ HERCs, where the latter, HERC1 and HERC2, are the largest HECT E3s with about 5000 residues. The remaining 13 HECTs do not share specific domains at the N-terminus and, for this reason, are classified as “other” HECT ligases (E6AP, HACE1, TRIP12, UBR5, UBE3B, UBE3C, HECTD1, HECTD2, HECTD4, HECTD3, G2D3, and AREL1). See e.g., Weber et al., Front. Physiol., 03 April 2019, incorporated herein by reference in its entirety). [0068] HECT-E3s ubiquitinate their specific substrate in a two-step process. First, an HECT-E3 binds to an E2 in complex with activated ubiquitin, leading to the formation of a thioester linkage between the C-terminus of ubiquitin and the catalytic cysteine residue in the HECT domain. This transient complex subsequently transfers ubiquitin to an interacting substrate with the formation of an isopeptide bond. [0069] In some embodiments, the wild-type E3 comprises a HECT E3 ligase, and is, for example, HECT-E3 ligase E6AP (also called, interchangeably UBE3A or E6AP/UBE3A), having the amino acid sequence of SEQ ID NO:6 (see https:// world wide web dot ncbi dot nlm dot nih dot gov front slash protein/NP_001341435.1). [0070] Structurally, E6AP possesses a Zn2+-binding N-terminal (amino-terminal Zn-finger of Ube3a Ligase (AZUL)) domain and a catalytic HECT domain of ~350 amino acids at the C terminus. A domain necessary for binding with the human papillomavirus (HPV) E6 oncoprotein is located between the AZUL and HECT domains. The AZUL domain is involved in substrate recruitment and also self-inhibitory regulation. [0071] Substrates [0072] In some embodiments, methods, compounds, and compositions of the present disclosure are provided that inhibit, prevent, or decrease, the level of ubiquitination of one or more substrates, thereby treating, ameliorating, or otherwise lessening disease symptoms, progression, and/or severity. By way of example but not by way of limitation, E6AP substrates involved in cancer are shown below in Table 1 (see e.g., Owais, et al., Ref. #2, incorporated herein by reference in its entirety). [0073] Compounds [0074] Disclosed herein are methods and processes for drug screening and drug discovery. Also disclosed herein are compounds identified in the drug screening methods that are useful for variety of medical and therapeutic applications. In some embodiments, the compounds are formulated into therapeutic compositions and are administered to subjects in need thereof. In some embodiments, the compound of Formula I, isomers, derivatives, or pharmaceutically salts thereof, is provided to a subject in need thereof. Formula I is shown below:
[0075] [0076] In some embodiments, R is selected from hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl optionally substituted with alkyl, and heteroaryl. [0077] In some embodiments, R is selected from: [0078] [0079] In some embodiments, the compound is one or more of Formula II or III, isomers, derivatives, or pharmaceutically acceptable salts thereof. [0080] Formula II. [0081] Formula II is the Zinc 23375107 compound, 1-(5-methoxy-2-{[(tetrahydro- 2H-pyran-4-ylmethyl)amino]methyl}phenoxy)-3-(4-methyl-1-pipe razinyl)-2-propanol, and is shown as compound #3 in Figure 3. [0082] [0083] Formula III. [0084] Formula III is #3-1 compound (1-(5-methoxy-2-{[(3- methylbenzyl)amino]methyl}phenoxy)-3-(4-methyl-1-piperazinyl )-2-propanol) and is shown as compound #3-1 in Figure 4. [0085] Formulations and modes of administration [0086] While the compositions disclosed herein may include pharmaceutical compositions comprising any of the compounds disclosed herein (e.g., Formula I, Formula II, Formula III, derivatives, isomers and pharmaceutically acceptable salts thereof), Formula I, derivatives, isomers and pharmaceutically acceptable salts thereof, will be used as an example throughout the discussion of the various embodiments. It is to be understood that any of the compounds disclosed herein can be formulated and administered as described in this section at a dosage effective to treat a subject in need thereof. [0087] Such compositions can be formulated and/or administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the route of administration. [0088] The compositions may include pharmaceutical solutions comprising carriers, diluents, excipients, and surfactants, as known in the art. Further, the compositions may include preservatives (e.g., anti-microbial or anti-bacterial agents such as benzalkonium chloride). The compositions also may include buffering agents (e.g., in order to maintain the pH of the composition between 6.5 and 7.5). [0089] The pharmaceutical compositions may be administered therapeutically. In therapeutic applications, the compositions are administered to a patient in an amount sufficient to elicit a therapeutic effect (e.g., a response which cures or at least partially arrests or slows symptoms and/or complications of disease (i.e., a “therapeutically effective dose”). [0090] In some embodiments, compositions are formulated for systemic delivery, such as oral or parenteral delivery. In some embodiments, minimally invasive microneedles and/or iontophoresis may be used to administer the composition. In some embodiments, compositions are formulated for site-specific administration, such as by injection into a specific tissue or organ, or by topical administration (e.g., by patch applied to the target tissue or target organ, e.g., cancer tissue or brain/neuronal tissue, etc.). [0091] The therapeutic composition may include, in addition to a compound of Formula I, one or more additional active agents. By way of example, the one or more active agents may include an antibiotic, anti-inflammatory agent, a steroid, or a non- steroidal anti-inflammatory drug, and chemotherapeutics. [0092] According to various aspects, a compound of the present disclosure, and optionally the one or more active or inactive agents may be present in the composition as particles or may be soluble. By way of example, in some embodiments, micro particles or microspheres may be employed, and/or nanoparticles may also be employed, e.g., by utilizing biodegradable polymers and lipids to form liposomes, dendrimers, micelles, or nanowafers as carriers for targeted delivery of the compounds. In some embodiments, polymeric implants may be used. By way of example but not by way of limitation, in some embodiments, a therapeutic composition comprising any of the compounds disclosed herein is applied to a patch and placed in contact with the target tissue (e.g., a tumor). [0093] In some embodiments, the composition formulated for administration comprises between 500 mg/ml and 1000 mg/ml of the compound, e.g., a compound comprising Formula I. In some embodiments, the composition formulated for administration comprises between 0.1 ng and 500 mg/ml of the compound, e.g., a compound comprising Formula I. In some embodiments, the compositions if formulated such that between 0.1ng and 500 µg/ml of the compound (e.g., a compound comprising Formula I) is administered to a subject. [0094] In some embodiments, the methods include administration of the therapeutic compositions once per day; in some embodiments, the composition may be administered multiple times per day, e.g., at a frequency of one or two times per day, or at a frequency of three or four times per day or more. In some embodiments, the methods include administration of the composition once per week, once per month, or as symptoms dictate. [0095] In some embodiments, the composition is administered at between 500 mg/ml and 1000 mg/ml of HECT E3 ligase inhibitor; between 0.1 ng and 500 mg/ml of the inhibitor; or between about 0.1 ng and 500 µg/ml of the inhibitor. [0096] In some embodiments, the treatment reduces, alleviates, prevents, or otherwise lessens the symptoms of the disease or condition more quickly than if no treatment is provided to a subject suffering the same or similar disease, condition, or injury. By way of example, for a subject suffering from cancer, a treated subject would exhibit one or more of reduced tumor size, reduced tumor growth, reduced metastatic activity, reduced swelling near the tumor, and reduced pain, sooner or at a greater degree than a non-treated subject with the same or similar cancer. By way of example, for a subject suffering from a neurological disease or condition, a treated subject would exhibit an improvement in, or a reduced worsening of one or more of the following: sensory response, memory, judgement, speech, writing, general confusion, understanding verbal and/or written communication, eye contact, social interaction, motor coordination and epileptic seizures sooner or at a greater degree than a non-treated subject with the same or similar disease or condition. [0097] In some embodiments, improvements in the condition of the subject's condition is observed more quickly than if no treatment is provided for the same or similar condition or disease. [0098] By way of example, in some embodiments, improvements in the condition is observed within about 1 to about 3 days; within about 3 to about 5 days, or within about a week of the first administration. In some embodiments, improvements in the subject's condition is observed within about 10 days, about 14 days or within about 1 month of the first administration. In some embodiments, improvements in the subject's condition is observed within about 1-3 month, about 3-6 months or within about 1 year of the first administration. [0099] Disease and conditions [00100] Disclosed herein are compositions useful to treat a subject suffering from, or suspected of having a disease or condition characterized by an increased HECT E3 ligase activity, and/or ectopic HECT E3 ligase activity e.g., UBE3A/E6AP ligase activity. [00101] UBE3A plays important roles not only in brain development but also in viral and non-viral carcinogenesis. Duplication or triplication of 15q11-13 (Dup15q syndrome) renders individuals highly susceptible to autism spectrum disorders (ASD). Indeed, Dup15q is one of the most common cytogenetic anomalies in ASD cohorts. Studies using mouse models of Dup15q suggest that overexpression of UBE3A in neurons accounts for most of the ASD phenotype. It is thought that neurons in developing brain requires proper control of the ubiquitin ligase activity of UBE3A, and excess UBE3A activity could perturb synaptic networks leading to autistic traits. [00102] Excess or ectopic activity of UBE3A could also drive cancer development. The E6 oncoprotein encoded by human papillomavirus (HPV) binds and facilitates UBE3A to ubiquitinate tumor suppressor proteins such as p53 and p27, thus leading to the development of cervical cancer and head/neck cancer. [00103] By way of example only, and not by way of limitation, diseases and conditions include cancer or a neurological disorder (see e.g., Owais et al., Ref. #2, incorporated herein by reference in its entirety). In some embodiments, the cancer is one or more of HPV associated cancer such as cervical, skin and head/neck cancers; HCV associated cancer; cancer characterized by PML downregulation, non-small cell lung cancer, and breast cancer. In some embodiments, the cancer characterized by PML downregulation is one or more of Burkitt's lymphoma and prostate cancer. In some embodiments, the neurological disorder is one or more of Angelman syndrome (AS), Autism Spectrum Disorders (ASD), and chromosome 15q11.2-q13.3 duplication syndrome (Dup15q). [00104] Applications and Advantages [00105] Compounds and compositions disclosed herein (HECT E3 inhibitors) are useful for a number of applications, and exhibit several advantages over known HECT E3 inhibitors. Non-limiting examples include the following. [00106] Use the UBE3A inhibitors as drugs to treat ASD patients, especially those with cytogenic parameters exhibiting Dup15q. [00107] Use the UBE3A inhibitors as drugs to treat patients with HPV-induced cervical, skin and head/neck cancers. [00108] Use the UBE3A inhibitors as drugs to treat patients with non-viral cancers, such as castration-resistant prostate cancers, some of which have been shown to depend on UBE3A activity for their malignant phenotypes. [00109] Use the UBE3A inhibitors as a research reagent to examine the biological functions of UBE3A in various experimental models. [00110] Advantages [00111] A previous report described macrocyclic N-methyl-peptides that bound to the HECT domain of UBE3A and inhibited UBE3A-mediated ubiquitination of p53 (20; PMID: 22195558). However, peptides are generally much more difficult to translate into clinical applications, mostly because of issues in drug delivery to neurons and tumors. [00112] Flavanoid compounds, Luteolin and CAF024, which mimic leucines in the conserved alpha helical motif of UBE3A have been found to inhibit the E6–UBE3A interaction (21, 22; PMID: 24376816, 30875378). Some zinc-ejecting compounds also have been shown to inhibit the E6 interaction with UBE3A (23; PMID: 10413422). However, these approaches are focused on the E6-UBE3A interaction, and will not inhibit the E3 activity of UBE3A in the absence of viral oncoproteins, which is important for treating ASD and non-viral cancers. [00113] N-acetyl phenylalanine has been shown to block UBE3A oligomerization by substituting Phe727 and inhibits its E3 activity at a very high concentration (Ki = 12 mM) (24; PMID: 24273172). This is a different way to inhibit the E3, but the efficacy is too low. [00114] Risperidone, a blocker of several neurotransmitter receptors such as dopamine type 2, serotonin type 2, and alpha-adrenergic receptors, is most widely used to treat children with ASD. Risperidone is somewhat effective to improve explosive and aggressive behaviors. However, not all patients with ASD respond to risperidone, and the drug has significant side effects such as weight gain, drowsiness, hormonal changes and involuntary movements. Aripiprazole, which is a serotonin 5-HT2A receptor antagonist and partial agonist of dopamine D2 receptor, is the only other drug approved by FDA to treat irritability of autistic children, but has similar side effects. Thus, there are only limited choices for treatment of ASD, which presents a major unmet need especially for drugs that directly target pathogenic proteins in brain. [00115] To prevent HPV-induced cancers, HPV vaccines have been demonstrated to be quite effective. However, a large number of HPV-infected individuals are still supposed to develop cancers in the coming 10-20 years, and additional targeted therapies to treat those individuals that are already infected is needed. This need is met by the disclosed compounds and compositions. [00116] The compounds and compositions disclosed herein form the first generation of direct small molecule inhibitors of UBE3A. Given the extremely large cohort of ASD patients (1 in 59 US children), economic and impact of development of a new drug for ASD would be extremely high. Even on the assumption that the targeted population for a UBE3A inhibitor is restricted to Dup15q syndrome, its prevalence may be as high as 1 in 5,000 (25; PMID: 23992924). Dup15q is one of the most common cytogenetic alterations in ASD cohorts, and has been found at frequencies of 1:253-1:522 (26, 27, 28; PMID: 19278672, 22424231, 23044707). In addition, 44,000 HPV-associated cancers occur in the United States each year, according to the CDC statistics based on data from 2012-16. Accordingly, the methods, compounds, and compositions of the present disclosure fulfill an unmet need. [00117] To our knowledge, no other small molecule inhibitor against UBE3A has been reported. EXAMPLES [00118] The following Examples are illustrative and are not intended to limit the scope of the claimed subject matter. [00119] Example 1. In silico assessment of UBE3A structure for druggable hotspots. To identify small molecules that bind to the catalytic HECT domain of UBE3A, we analyzed the 3-dimensional (3-D) structure of UBE3A at residues 518-875, which includes the catalytic center C820 residue, according to the X-ray crystal structures published at the database (PDB ID: 1C4Z)(1,2)(Fig. 1). The druggability estimation of a ligand binding pocket has been based on the knowledge obtained from binding pockets for known drugs (3,4). The SiteMap program implemented in the Schrodinger suite (5) correctly identifies 86% of the known binding sites, and it also identifies > 98% of binding sites that bind sub-nanomolar ligands (6). Since the implementation of the SiteMap, many modifications have improved the predictive power of the method particularly for flexible and cryptic binding site identification (6). The SiteMap provides a quantitative score (D score) and graphical information which promote assessment of in silico hits, hit-to-lead chemistry and lead optimization of a compound. To enhance the predictive power of the score, Loving et al. (6) introduced Dscore+, which was validated with 19 F NMR data (7). To analyze the druggability of the HECT domain of UBE3A, we evaluated the Dscore+ as well as the volume of this flexible cryptic pocket and confirmed the druggability criteria (6). We then generated the Connolly surface areas of the two interacting proteins. Close examination of the interfaces of the two interacting partners revealed a putative small molecule binding groove (Fig.2). It was noted that the I804 residue, which is included in the region, is known to be susceptible for loss-of-function mutations in Angelman syndrome. [00120] Example 2. Virtual high throughput screening for small molecule that bind to hotspots in the UBE3A HECT domain. One of the key elements in any screening campaign is to ensure identified compounds are drug-like and chemically tractable. Often, hits identified through wet HTS campaigns possess non-drug like properties and are unsuitable for chemical modification. We created a curated small molecule database by multiple tiers of filters to the ZINC database (8), which contains approximately 45 million purchasable compounds. We used Lipinski (9), Veber (10) and 239 PAINs filters (11). This proprietary database has been used in all of our successful in silico screening campaigns (12-15). To identify potential UBE3A inhibitors, we screened this library at complexity of 1 million diverse compounds, using the three-tiered Glide (16) small molecule docking engine from Schrodinger. Small molecule hit sets obtained through Glide were cross-docked with Gold (17) and Surflex (18) docking engines, which are built upon orthogonal algorithms, and chose 46 compounds among the top hits (Glide XP Score < -6) for further validation. These hits showed good interactions with the catalytic C820 residue and the Ile-804 (I804) residue critical for the catalytic activity. [00121] Example 3. Validation of hits by an in vitro ubiquitination assay. We purchased 35 compounds among the 46 hits identified by the initial virtual screen, and tested their abilities to modulate the ubiquitin ligase activity of UBE3A. The in vitro ubiquitination assay was developed using purified ubiquitin (Ub), Uba1 (E1), UbcH7/UBE2L3 (E2), UBE3A (E3), and S5A/Angiocidin/PSMD4 (substrate), as a modification of our previously reported assay (19). The purchased compounds were dissolved in DMSO to generate a stock solution at 10 mM, and then their effects on UBE3A activity were tested by adding each compound at a final concentration of 100 µM. E1, E2 and E3 enzymes were first pre-incubated at 37°C for 30 min in a buffer containing MgCl2, ATP and each compound. Subsequently, ubiquitin and S5A were added to the reaction and incubated for 60 more minutes. Samples were then analyzed by SDS-PAGE and immunoblotting, and polyubiquitination of the substrate S5A was determined as specific appearance of forms of anti-S5A immunoreactivity at higher molecular weights. Figure 3 represents the assay, demonstrating that one of the tested compounds (Zinc 23375107, labeled as #3 in Fig.3) significantly inhibits polyubiquitination of S5A. [00122] The Zinc 23375107 compound, 1-(5-methoxy-2-{[(tetrahydro-2H-pyran-4- ylmethyl)amino]methyl}phenoxy)-3-(4-methyl-1-piperazinyl)-2- propanol, can bind to the cleft near the catalytic center (Cys-820, C820) and one of the residues critical for the activity (Glu-550, E550) (Fig.2). [00123] We then examined effects of varying concentrations of Zinc 23375107, named as compound #3, on polyubiquitination of S5A (Figure 5A). This compound exhibited dosage-dependent inhibition of S5A polyubiquitination. We estimated the IC50 of the compound around 100 µM. While this compound consistently inhibits the ubiquitin ligase activity of UBE3A, its potency was weak, suggesting the necessity of identifying more potent compounds structurally analogous to it. [00124] Example 4. Search for UBE3A inhibitors with better efficacy. To identify more potent UBE3A inhibitor compounds than Zinc 23375107, we conducted in silico search for compounds that are structurally analogous to Zinc 23375107 (with similarity varying from 0.99-0.80) and could bind to the druggable hotspot of UBE3A. This second screen identified 140 compounds, of which 8 have been tested by the in vitro ubiquitination assay. We found that one of the eight compounds was capable of inhibiting the UBE3A-mediated polyubiquitination of S5A substrate at 100 µM (Fig.4), and named it as compound #3-1, which is 1-(5-methoxy-2-{[(3- methylbenzyl)amino]methyl}phenoxy)-3-(4-methyl-1-piperazinyl )-2-propanol (see Fig. 4B). Our experiments using titrating amounts of #3-1 suggested that this compound is a modestly more potent UBE3A inhibitor than the parental #3 compound, and preliminary data showed that the IC50 of this compound is still relatively high in the range of 10 µM (Figure 5B). This observation indicates that we need to look for more potent inhibitors that are structurally analogous to compounds #3 and #3-1. Therefore, we added more purchasable drug-like compounds to the library and repeated the screen for compounds analogous to #3 with potential binding to the druggable hotspot of UBE3A. This latest screen provided a list of 30 potential inhibitors, and we are currently testing them by the in vitro ubiquitination assay. [00125] Example 5. Fluorescence thermal shift assay to demonstrate physical interaction between the compound #3.1 and the HECT domain of UBE3A. To verify the physical interaction between the HECT domain of UBE3A and the identified compounds and also develop a high throughput assay for further screening, we have developed a fluorescence thermal shift analysis (FTS). we have prepared a truncated UBE3A protein (residues 495-852 of isoform 1) using E. coli and performed preliminary FTS test (Figure 6). The recombinant HECT domain of UBE3A showed a well-defined melting profile. Moreover, the melting profile was significantly modulated by compound #3-1. The FTS method has been widely used for measurement of protein-ligand interaction (Reference https:// world wide web, at ncbi dot nlm dot nih dot gov front slash pubmed front slash 24590724). FTS monitors protein thermal denaturation using Sypro- Orange which fluoresces when bound to hydrophobic surfaces, taking advantage of the changes in hydrophobic surface exposure in protein denaturation. Small molecule binding affects protein thermal stability, therefore can be detected through a shift in protein’s thermal denaturation (melting) temperature (Tm). These data suggest that the compound #3-1 physically binds to the HECT domain of UBE3A and inhibits its E3 activity, as hypothesized. [00126] Example 6. Cytotoxicity of the compound #3.1 in HPV-positive cancer cells. We have recently tested whether the compound #3.1 exert any cytotoxic action in HPV-positive cancer cells. Human cervical carcinoma HeLa cells are a widely used HPV18-positive cell lines. We incubated HeLa cells with the compound #3.1 at various concentrations for 72 hours and determined viability of the cells (Fig. 7). This study has shown that the compound #3.1 exerts cytotoxicity in a dose-dependent manner with EC50 at 30 µM. The data suggest that small molecule-based inhibition of UBE3A leads to cell death in the presence of HPV oncoproteins, and more potent UBE3A inhibitors may be used as a therapeutic agent against human HPV-positive cancers. [00127] References [00128] 1. Huang, L., Kinnucan, E., Wang, G., Beaudenon, S., Howley, P. M., Huibregtse, J. M., and Pavletich, N. P. (1999) Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade. Science 286, 1321-1326, https://www.ncbi.nlm.nih.gov/pubmed/10558980 [00129] 2. Owais, A., Mishra, R. 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[00156] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. [00157] Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification. SEQUENCES SEQ ID NO: 1 This is a product of UBC gene, encoding multimer precursor of ubiquitin LOCUS NP_066289 685 aa linear PRI 12-DEC-2020 DEFINITION polyubiquitin-C [Homo sapiens]. ACCESSION NP_066289 XP_947802 XP_951381-XP_951420 VERSION NP_066289.3 1 mqifvktltg ktitleveps dtienvkaki qdkegippdq qrlifagkql edgrtlsdyn 61 iqkestlhlv lrlrggmqif vktltgktit levepsdtie nvkakiqdke gippdqqrli 121 fagkqledgr tlsdyniqke stlhlvlrlr ggmqifvktl tgktitleve psdtienvka 181 kiqdkegipp dqqrlifagk qledgrtlsd yniqkestlh lvlrlrggmq ifvktltgkt 241 itlevepsdt ienvkakiqd kegippdqqr lifagkqled grtlsdyniq kestlhlvlr 301 lrggmqifvk tltgktitle vepsdtienv kakiqdkegi ppdqqrlifa gkqledgrtl 361 sdyniqkest lhlvlrlrgg mqifvktltg ktitleveps dtienvkaki qdkegippdq 421 qrlifagkql edgrtlsdyn iqkestlhlv lrlrggmqif vktltgktit levepsdtie 481 nvkakiqdke gippdqqrli fagkqledgr tlsdyniqke stlhlvlrlr ggmqifvktl 541 tgktitleve psdtienvka kiqdkegipp dqqrlifagk qledgrtlsd yniqkestlh 601 lvlrlrggmq ifvktltgkt itlevepsdt ienvkakiqd kegippdqqr lifagkqled 661 grtlsdyniq kestlhlvlr lrggv
SEQ ID NO: 2 UniProtKB - L8B196 (L8B196_HUMAN) This is a Uniprot entry about UBC, showing the multimer structure. SEQ ID NO: 3 This shows BLAST sequence of mature 76 aa ubiquitin >tr|L8B196|1-76 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN IQKESTLHLVLRLRGG
SEQ ID NO: 4 LOCUS NP_0033251058 aa linear PRI 10-DEC-2020 DEFINITION ubiquitin-like modifier-activating enzyme 1 [Homo sapiens]. ACCESSION NP_003325 VERSION NP_003325.2 DBSOURCE REFSEQ: accession NM_003334.4 KEYWORDS RefSeq; MANE Select. SOURCE Homo sapiens (human) 1 msssplskkr rvsgpdpkpg sncspaqsvl sevpsvptng makngseadi deglysrqly 61 vlgheamkrl qtssvlvsgl rglgveiakn iilggvkavt lhdqgtaqwa dlssqfylre 121 edigknraev sqprlaelns yvpvtaytgp lvedflsgfq vvvltntple dqlrvgefch 181 nrgiklvvad trglfgqlfc dfgeemiltd sngeqplsam vsmvtkdnpg vvtcldearh 241 gfesgdfvsf sevqgmveln gnqpmeikvl gpytfsicdt snfsdyirgg ivsqvkvpkk 301 isfkslvasl aepdfvvtdf akfsrpaqlh igfqalhqfc aqhgrpprpr needaaelva 361 laqavnaral pavqqnnlde dlirklayva agdlapinaf igglaaqevm kacsgkfmpi 421 mqwlyfdale clpedkevlt edkclqrqnr ydgqvavfgs dlqeklgkqk yflvgagaig 481 cellknfami glgcgeggei ivtdmdtiek snlnrqflfr pwdvtklksd taaaavrqmn 541 phirvtshqn rvgpdteriy dddffqnldg vanaldnvda rmymdrrcvy yrkpllesgt 601 lgtkgnvqvv ipfltesyss sqdppeksip ictlknfpna iehtlqward efeglfkqpa 661 envnqyltdp kfvertlrla gtqplevlea vqrslvlqrp qtwadcvtwa chhwhtqysn 721 nirqllhnfp pdqltssgap fwsgpkrcph pltfdvnnpl hldyvmaaan lfaqtygltg 781 sqdraavatf lqsvqvpeft pksgvkihvs dqelqsanas vddsrleelk atlpspdklp 841 gfkmypidfe kdddsnfhmd fivaasnlra enydipsadr hkskliagki ipaiatttaa 901 vvglvclely kvvqghrqld sykngflnla lpffgfsepl aaprhqyynq ewtlwdrfev 961 qglqpngeem tlkqfldyfk tehkleitml sqgvsmlysf fmpaaklker ldqpmteivs 1021 rvskrklgrh vralvlelcc ndesgedvev pyvrytir
SEQ ID NO: 5 LOCUS NP_003338 154 aa linear PRI 20-DEC-2020 DEFINITION ubiquitin-conjugating enzyme E2 L3 isoform 1 [Homo sapiens]. ACCESSION NP_003338 VERSION NP_003338.1 DBSOURCE REFSEQ: accession NM_003347.4 KEYWORDS RefSeq; MANE Select. SOURCE Homo sapiens (human) 1 maasrrlmke leeirkcgmk nfrniqvdea nlltwqgliv pdnppydkga frieinfpae 61 ypfkppkitf ktkiyhpnid ekgqvclpvi saenwkpatk tdqviqslia lvndpqpehp 121 lradlaeeys kdrkkfckna eeftkkygek rpvd SEQ ID NO: 6 LOCUS NP_001341435 852 aa linear PRI 13-DEC-2020 DEFINITION ubiquitin-protein ligase E3A isoform 1 [Homo sapiens]. ACCESSION NP_001341435 XP_005268326 VERSION NP_001341435.1 DBSOURCE REFSEQ: accession NM_001354506.2 KEYWORDS RefSeq. SOURCE Homo sapiens (human) 1 mkraaakhli eryyhqlteg cgneactnef cascptflrm dnnaaaikal elykinaklc 61 dphpskkgas saylenskga pnnscseikm nkkgaridfk dvtylteekv yeilelcrer 121 edysplirvi grvfssaeal vqsfrkvkqh tkeelkslqa kdedkdedek ekaacsaaam 181 eedseasssr igdssqgdnn lqklgpddvs vdidairrvy trllsnekie taflnalvyl 241 spnvecdlty hnvysrdpny lnlfiivmen rnlhspeyle malplfckam sklplaaqgk 301 lirlwskyna dqirrmmetf qqlitykvis nefnsrnlvn dddaivaask clkmvyyanv 361 vggevdtnhn eeddeepipe sseltlqell geerrnkkgp rvdpletelg vktldcrkpl 421 ipfeefinep lnevlemdkd ytffkveten kfsfmtcpfi lnavtknlgl yydnrirmys 481 erritvlysl vqgqqlnpyl rlkvrrdhii ddalvrlemi amenpadlkk qlyvefegeq 541 gvdeggvske ffqlvveeif npdigmftyd estklfwfnp ssfetegqft ligivlglai 601 ynncildvhf pmvvyrklmg kkgtfrdlgd shpvlyqslk dlleyegnve ddmmitfqis 661 qtdlfgnpmm ydlkengdki pitnenrkef vnlysdyiln ksvekqfkaf rrgfhmvtne 721 splkylfrpe eiellicgsr nldfqaleet teydggytrd svlirefwei vhsftdeqkr 781 lflqfttgtd rapvgglgkl kmiiakngpd terlptshtc fnvlllpeys skeklkerll 841 kaityakgfg ml
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