Silent information regulatory protein modulating compounds comprising sirtuin activating compounds and uses thereof

Abstract

In one aspect, aryl carbodiimide compounds are described herein that comprise a carbodiimide molecule substructure and modulate mammalian silent information modulating proteases (SIRT1 to SIRT7), and in particular, to compounds for modulating SIRT3. A novel small molecule for regulating the activity of silent information regulatory protein is developed by utilizing a DNA coding library technology (ELT). Modulation of sirtuin activity includes sirtuin activation and / or sirtuin inhibition.

Description

[0001] Related application data

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Serial No. 63 / 353,248, filed on June 17, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present application relates to compounds that exhibit sirtuin regulatory function, and in particular to compounds for regulating SIRT3. Background Art

[0004] Sirtuin (sirtuin) enzymes catalyze NAD + Sirtuins are NAD-dependent post-translational modifications that have emerged as key regulators of many cellular pathways. In particular, these enzymes protect against age-related diseases and serve as key regulators of longevity in evolutionarily distant model organisms. + A lysine deacylase that requires the cofactor NAD + Cleaves acyl groups from lysine side chains of its substrate proteins.

[0005] A thorough understanding of sirtuin chemistry is not only of fundamental importance but also of considerable medical importance, as there is currently tremendous interest in developing novel mechanism-based sirtuin modulators. The overall catalytic process is proposed to proceed in two sequential stages. The initial stage involves the activation of NAD + Cleavage of the nicotinamide moiety and nucleophilic attack of the acetyl-Lys side chain of the protein substrate to form a positively charged O-alkyl imine ester intermediate. Nicotinamide-induced reversal of the intermediate (the so-called base exchange reaction) leads to NAD + and acetyl-Lys protein reformation. The energetics of this reversible reaction influence the potency of nicotinamide (NAM) to inhibit sirtuins and NAD + The Michaelis constant (K m,NAD+ The second phase of sirtuin catalysis consists of a rate-determining step involving four sequential steps that ultimately lead to the deacetylation of the Lys side chain of the protein substrate and the formation of O-acetyl ADP-ribose byproduct.

[0006] Recently, there has been a great deal of interest in the activation of seven mammalian sirtuin proteases (SIRT1 to SIRT7) in order to combat aging. Enzyme activators have considerable advantages over enzyme inhibitors, which constitute the vast majority of today's drugs. However, their design is much more difficult because enzyme catalysis has been optimized over billions of years of evolution. Existing research on sirtuin activation has focused solely on experimental screening, with a focus on allosteric activation of the SIRT1 enzyme. In fact, small molecule allosteric activators of SIRT1 have been shown to induce lifespan extension in model organisms, such as mice. Allosteric activation is one of the four known modes by which small molecules can activate enzymes. They do this by reducing the dissociation constant of the substrate (the acetylated protein dissociation constant K for sirtuins). d，Ac-Pr ) to play a role.

[0007] Almost all known sirtuin activators allosterically target SIRT1 and do not bind in the active site. However, allosteric activators only work with certain substrates of SIRT1. Other sirtuins, including SIRT2, SIRT3, and SIRT6, are now known to play important roles in regulating mammalian lifespan. Therefore, a general strategy for activation of any mammalian sirtuin, including activation of SIRT1 against other substrates, is critical but not yet understood. Typically, allosteric activation is performed to reduce substrate K d would not be an option for enzyme activation, making mechanism-based activation important.

[0008] The basis for the rational design of mechanism-based activators has been lacking. Several mechanism-based sirtuin inhibitors have recently been reported in the literature, including Ex-527. However, mechanism-based activation has proven much more challenging due to the difficulty in screening for the balance of properties required for active site binding and accelerated catalysis. While there are many ways to inhibit an enzyme’s mechanism, there are far fewer ways to activate it. Only a dozen different classes of small molecule enzyme activators are currently known, with only four known modes of activation across all enzyme families. None of these modes of activation exploit the unique catalytic reaction mechanism of the target enzyme. Summary of the invention

[0009] In one aspect, described herein are compounds that modulate sirtuin activity. Modulation of sirtuin activity includes sirtuin activation and / or sirtuin inhibition. In some embodiments, the sirtuin modulating compound and / or its salt has Formula I:

[0010]

[0011] Among them, Ar 1is aryl or heteroaryl, R1 to R7 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, -C(O)R8, alkoxy, halogen, nitro (-NO2) and hydroxy, wherein Ar 1 , alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10 )-alkyl, (C1-C 10 )-alkenyl, alkoxy, halogen, amine and hydroxy; and wherein R8 is selected from alkyl, alkenyl and NR9R 10 The group consists of R9 and R 10 is independently selected from the group consisting of hydrogen, alkyl and cycloalkyl; and wherein m and n are integers each having a value independently selected from 0 to 10. In some embodiments, the compound of Formula I and / or its salt is:

[0012]

[0013] In another aspect, the sirtuin-modulating compound and / or salt thereof has Formula II:

[0014]

[0015] wherein Ar is aryl or heteroaryl, R1 to R6 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, -C(O)R7, alkoxy, halogen, nitro (-NO2) and hydroxy, wherein Ar 1 , alkyl, alkenyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10 )-alkyl, (C1-C 10 )-alkenyl, alkoxy, halogen, amine, -alkoxy-amide and hydroxyl; and wherein R7 is selected from the group consisting of alkyl, alkenyl and NR8R9, wherein R8 and R9 are independently selected from the group consisting of hydrogen, alkyl and cycloalkyl; and wherein m and n are integers each having a value independently selected from 0 to 10. In some embodiments, for example, the compound of formula II and / or its salt is:

[0016]

[0017] In another embodiment, the compound of Formula II and / or its salt is:

[0018]

[0019] In another aspect, the sirtuin-modulating compound and / or salt thereof has Formula III:

[0020]

[0021] Among them, Ar 1 is aryl or heteroaryl, R1 is selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, amine and -C(O)NR3R4, wherein Ar 1 , alkyl, alkenyl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkyloxy, cycloalkenyl, alkylamine, acetamide, acetylamine, cycloalkyl, heterocycloalkyl, aryl, heteroaryl and amine are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10 )-alkyl, (C1-C 10 )-alkenyl, alkoxy, halogen, amine and hydroxyl; and wherein R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, aryl and heteroaryl, and wherein R2 is selected from the group consisting of hydrogen, alkyl, fluoroalkyl, alkenyl, aryl, heteroaryl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkoxy, cycloalkenyl, amine, alkylamine and O, and wherein Y is selected from the group consisting of N and CH, and wherein X is selected from the group consisting of NH, S and O. In some embodiments, compounds of Formula III and / or salts thereof include:

[0022]

[0023]

[0024] In another aspect, the sirtuin-modulating compound and / or salt thereof has Formula IV:

[0025]

[0026] Among them, Ar 1 is aryl or heteroaryl, R1 is selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, amine and -C(O)NR3R4, wherein Ar 1 , alkyl, alkenyl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkyloxy, cycloalkenyl, alkylamine, acetamide, acetylamine, cycloalkyl, heterocycloalkyl, aryl, heteroaryl and amine are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10 )-alkyl, (C1-C 10)-alkenyl, alkoxy, halogen, amine and hydroxyl; and wherein R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, aryl and heteroaryl, and wherein R2 is selected from the group consisting of hydrogen, alkyl, fluoroalkyl, alkenyl, aryl, heteroaryl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkoxy, cycloalkenyl, amine, alkylamine and O, and wherein Y is selected from the group consisting of N and CH, and wherein X is selected from the group consisting of NH, S and O. In some embodiments, compounds of Formula III and / or salts thereof include:

[0027]

[0028] In yet another aspect, the sirtuin-modulating compound and / or salt thereof has Formula V:

[0029]

[0030] wherein R1 is selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, alkenyl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkoxy, cycloalkenyl, and heterocycloalkyl; and wherein Ar 1 and Ar 2 are independently selected from aryl and heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10 )-alkyl, (C1-C 10 )-alkenyl, alkoxy, halogen, amine, sulfonyl-alkyl and hydroxy; and wherein X is selected from the group consisting of O, S and N; and wherein m and n are integers each having a value independently selected from 0 to 10.

[0031] In some embodiments, the compound of formula V and / or its salt is selected from one of the following:

[0032]

[0033]

[0034] In another aspect, the sirtuin-modulating compound and / or salt thereof has Formula VI:

[0035]

[0036] wherein R1 is selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, alkenyl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkoxy, cycloalkenyl, and heterocycloalkyl; and wherein Ar 1 and Ar 2 are independently selected from aryl and heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10)-alkyl, (C1-C 10 )-alkenyl, alkoxy, halogen, amine, sulfonyl-alkyl, acyl, amide, acetylamine, alkylamine and hydroxyl. In some embodiments, the compound of formula VI and / or its salt is selected from one of the following:

[0037] In some embodiments, the compound of Formula VI and / or its salt is selected from one of the following:

[0038]

[0039]

[0040] In another aspect, the sirtuin-modulating compound and / or salt thereof has Formula VII:

[0041]

[0042] wherein R1 and R2 are independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl and heterocycloalkyl; and wherein Ar1 and Ar2 are independently selected from aryl and heteroaryl, wherein aryl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10 )-alkyl, (C1-C 10 )-alkenyl, alkoxy, halogen, amine, nitro, sulfonyl-alkyl, acyl, amide, acetylamine, alkylamine, alkynyl and hydroxyl. In some embodiments, the compound of formula VII and / or its salt is:

[0043] In some embodiments, the compound of Formula VII and / or its salt is:

[0044]

[0045] In another aspect, the sirtuin-modulating compound and / or salt thereof has Formula VIII:

[0046]

[0047] Wherein R1 to R3 are independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, alkenyl, alkynyl, acyl and heterocycloalkyl, wherein alkyl, heteroalkyl, cycloalkyl and heterocycloalkyl are optionally substituted with one or more substituents selected from the group consisting of alkoxy, halogen, amine, nitro and hydroxy. In some embodiments, the compound of formula VIII and / or its salt is:

[0048]

[0049] In another aspect, the sirtuin-modulating compound and / or salt thereof has Formula IX:

[0050]

[0051] Wherein R1 and R2 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, acyl, heteroalkyl, cycloalkyl, heterocycloalkyl, amide and keto, wherein alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, amide and keto are optionally substituted with one or more substituents selected from the group consisting of aryl, heteroaryl, alkoxy, halogen, amine, nitro and hydroxyl. In some embodiments, the compound of formula IX and / or its salt is:

[0052]

[0053] In another aspect, the sirtuin-modulating compound and / or salt thereof has Formula X:

[0054]

[0055] wherein R1 and R2 are independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, alkenyl, alkynyl, acyl, and heterocycloalkyl; and wherein Ar 1 is selected from aryl and heteroaryl, wherein cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10 )-alkyl, (C1-C 10 )-alkenyl, aryl, alkoxy, halogen, amine, nitro, sulfonyl-alkyl, acyl, amide, acetylamine, alkylamine, alkynyl and hydroxyl. In some embodiments, the compound of formula X and / or its salt is:

[0056]

[0057] In another aspect, the sirtuin-modulating compound and / or salt thereof has Formula XI:

[0058]

[0059] Among them, Ar 1 and Ar 2 are independently selected from aryl and heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10 )-alkyl, (C1-C 10 )-alkenyl, alkoxy, halogen, amine, sulfonyl-alkyl, acyl, amide, acetylamine, alkylamine, alkynyl and hydroxyl. In some embodiments, the compound of formula XI and / or its salt is:

[0060]

[0061] In another aspect, the sirtuin-modulating compound and / or salt thereof has Formula XII:

[0062]

[0063] Among them, Ar 1 and Ar 2 are independently selected from aryl and heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10 )-alkyl, (C1-C 10 )-alkenyl, alkoxy, halogen, amine, sulfonyl-alkyl, acyl, amide, acetylamine, alkylamine, alkynyl and hydroxyl. In some embodiments, the compound of Formula XII and / or its salt is:

[0064]

[0065] In another aspect, the sirtuin-modulating compound and / or salt thereof has Formula XIII:

[0066]

[0067] Among them, Ar 1 and Ar 2 are independently selected from aryl and heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10 )-alkyl, (C1-C 10 )-alkenyl, alkoxy, halogen, amine, sulfonyl-alkyl, acyl, amide, acetylamine, alkylamine, alkynyl and hydroxyl. In some embodiments, the compound of formula XIII and / or its salt is:

[0068]

[0069] In another aspect, the sirtuin-modulating compound and / or salt thereof has Formula XIV:

[0070]

[0071] Where R 1 is selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, alkenyl, alkynyl, acyl, heteroalkenyl, heteroalkynyl and heterocycloalkyl; and wherein Ar is selected from aryl and heteroaryl, wherein aryl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10 )-alkyl, (C1-C 10)-alkenyl, alkoxy, halogen, amine, sulfonyl-alkyl, acyl, amide, acetylamine, alkylamine, alkynyl and hydroxyl. In some embodiments, the compound of Formula XIV is:

[0072]

[0073] As further described herein, in some embodiments, compounds of Formula I to Formula XIV and / or salts thereof can modulate SIRT3. In addition, methods of treating patients are described herein. In some embodiments, the method comprises administering to the patient a therapeutically effective amount of a compound selected from Formula III to Formula XIV herein, wherein the compound increases the patient's NAD+. In some embodiments, administration of the compounds described herein enhances the patient's cellular metabolism and / or energy production. In some embodiments, compounds of Formula III to Formula XIV can restore the activity of the major mitochondrial sirtuin levels observed in youth by increasing the sensitivity of the sirtuin to NAD+, which provides effective treatment for age-related diseases (including mitochondrial diseases).

[0074] These and other embodiments are further described in the detailed description below. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 . Frequencies of all compounds in the natural library. All derivatives of the 20 amino acids show a similar distribution over their 194 carboxylic acid counterparts in the library.

[0076] Figure 2 SIRT3 selection in the presence of Carba-NAD and acetylated peptides. Derivatives of AC71 and AC92 showed the highest enrichment. Among them, a few derivatives of AC40 and AC56 also showed significant enrichment. The Y axis represents the enrichment calculated for each compound, which is the ratio of their frequency in the target selection to their frequency in the non-target selection.

[0077] Figure 3 SIRT3 selection in the presence of OAADPr and deacetylated peptides. Derivatives of AC71 and AC92 showed the highest enrichment. Among them, a few derivatives of AC40 and AC56 also showed significant enrichment. The Y axis represents the enrichment calculated for each compound, which is the ratio of their frequency in the target selection to their frequency in the non-target selection.

[0078] Figure 4 Effects of top hit compounds on SIRT3 deacetylase activity. Bar graph of % control for different compounds ([NAD +] = 1 mM, [FdL2 peptide] = 50 μM, [cpd] = 1, 10, 50 μM or maximum, [E]0 / [NAD]0 = 0.000245, time point = 30 min, n = 2).

[0079] Figure 5 . SIRT3 activation by top hit compounds. Bar graph of % control (HKL) for different compounds ([NAD + ]=50 μM, [MnSOD K122]=600 μM, [cpd]=10 μM or maximum, [E]0 / [NAD]0=0.1223, time point=2 min, n=3).

[0080] Figure 6 DNA-tagged and C-terminally amidated forms of selected active and inactive ligands identified by affinity selection assay. (A) Structure of the DNA-tagged form of the active ligand AA12-AC71; (B) Structure of the C-terminally amidated form of AA12-AC71; (C) Structure of the DNA-tagged form of another active ligand AA20-AC92; (D) Structure of the C-terminally amidated form of AA20-AC92; (E) Structure of the DNA-tagged form of the inactive ligand AA5-AC117; (F) Structure of the C-terminally amidated form of AA5-CA117. The compounds shown in B, D, and F were used for all ligand docking studies instead of those in A, C, and E.

[0081] Figure 7 .Position of site 1 in 4BVG and top poses of docking hits. (A) The position of the first putative atypical site defined by pseudo-atoms in 4BVG is almost the exact opposite of the well-known internal sites. Surrounding residues are highlighted in cyan. (B) Close-up of the top pose of AA5 / 7 / 11 / 12-AC92 at the putative atypical site. (C) Superposition of the top poses of AA5 / 7 / 11 / 12-AC92 shows a high overlap of pharmacophores. (D) Superposition of the top poses of AA5 / 7 / 11 / 12-AC71 also shows a high overlap of pharmacophores at atypical site 1 in 4BVG.

[0082] Figure 8 .Hit compound 6875218( Fig.13 ) Docking postures in the open (4FVT) and closed (4BVG) conformations of SIRT3. The figure shows the ternary complex of SIRT3 bound to the Ac-ACS peptide and Carba-NAD. The protein is represented in cyan, the peptide in green, and NAD in blue +, and compounds are in pink. Protein residues that form interactions are shown as white sticks. Intermolecular hydrogen bonds are shown as yellow dashed lines with corresponding distance markers.

[0083] Fig. 9 . Modulatory effects of the top 70 selected compounds under steady-state conditions (N=3).

[0084] Fig.10 . Modulatory effects of the top 20 selected compounds under non-steady-state conditions (N=3).

[0085] Fig.11 .Common structural patterns in SIRT3 activator hit compounds. ChemBridge ID(A)6068318( Fig.12 ), (B)5749820, (C)5761865, (D)7965907( Fig.31 All of the compounds presented here appear to share certain structural features, particularly a backbone consisting of a large bicyclic aromatic ring connected to a six-membered aromatic group via an amide group.

[0086] Figure 12 to Figure 295 Various compounds of Formulas III, V-XI, and XIV described herein are illustrated.

[0087] Fig.296 is a schematic diagram illustrating the compound screening method used in some embodiments of the present application.

[0088] Figure 297 (A) to Figure 297 (D). Effects of VS hit compounds on SIRT3 deacetylase activity under steady-state conditions: dose-response curves and time series of enzyme activation. (A) In the presence of 1 mM NAD + and 50 μM MnSOD peptide, t = 30 min, N = 3, at [E]0 / [NAD + ]0=0.00157, the dose-response curves of the three VS hit compounds. (B) In which [NAD + ]=100μM, [MnSOD K122]=600μM, [E]0 / [NAD + ]0=0.00157, t=10 min, N=2, bar graph of % control of 2 hit compounds (20 μM 5329973 or 1 μM 5689785) against SIRT3 deacetylase activity. (*P<0.001). In the presence and absence of: (C) 20 μM 5329973, for [NAD + ]=100μM and 500μM, [MnSOD K122]=600μM, [E]0 / [NAD +]0=0.00157(N=2); (D) 1μM 5689785, for [NAD + ]=100μM and 500μM, [MnSOD K122]=600μM, [E]0 / [NAD + ]A plot of product formation versus time for case 0=0.00157 (N=2).

[0089] Figure 298(A) to Figure 298(E). Binding affinity measurements of complexes in the sirtuin response mechanism. (A, C, E) Pathways in the sirtuin response network; E is an enzyme; Ac-Pr is an acetylated peptide substrate; NAD is nicotinamide adenine dinucleotide; A is a modulator (HKL or hit compound 5329973); NAM is nicotinamide adenine mononucleotide. (B) Carba-NAD binding in the ternary complex: mechanism-based effect of the modulator HKL. Binding of carba NAD (c-NAD) to the Sirt3.Ac-MnSOD complex measured using MST in the presence and absence of 6.25 μM HKL. (D) HKL binding: effect of NAM. Binding of HKL to the Sirt3.Ac-MnSOD complex measured using MST in the presence and absence of NAM. (F) Hit compound 5329973 binds to the apo enzyme SIRT3.

[0090] Figure 299 (A) to Figure 299 (D). Non-steady-state activator HKL and steady-state activator hSIRT3 on MnSOD substrate 102 -399 Steady-state kinetic characterization of deacetylation. Double reciprocal plots of initial rate measurements of deacetylation in the presence and absence of HKL at (A) [NAM] = 0 μM, (B) [NAM] = 5 mM. Double reciprocal plots of initial rate measurements of SIRT3 deacetylation in the presence and absence of (C) 20 μM 5329973 and (D) 1 μM 5689785. + The initial rate was measured at a concentration of [MnSOD K122] = 600 μM (N = 2). DETAILED DESCRIPTION

[0091] By referring to the following detailed description and examples and their descriptions before and after, the embodiments described herein can be more easily understood. However, the elements, devices and methods described herein are not limited to the specific embodiments presented in the detailed description and examples. It should be appreciated that these embodiments are only illustrations of the principles of the present invention. Without departing from the spirit and scope of the present invention, many modifications and adaptations will be apparent to those skilled in the art.

[0092] definition

[0093] The term "alkyl" as used herein, alone or in combination, refers to a straight or branched chain saturated hydrocarbon group optionally substituted with one or more substituents. For example, an alkyl group can be C1-C 30 or C1-C 18 .

[0094] The term "alkenyl," as used herein, alone or in combination, refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon double bond and optionally substituted with one or more substituents.

[0095] The term "aryl" as used herein, alone or in combination, refers to an aromatic monocyclic or polycyclic ring system optionally substituted with one or more ring substituents. The term "aryl" includes fused ring systems and non-fused ring systems, wherein the non-fused ring system includes alkylene and sulfonamide linkers between the rings.

[0096] The term "heteroaryl" as used herein, alone or in combination, refers to an aromatic monocyclic or polycyclic ring system in which one or more of the ring atoms is an element other than carbon, such as nitrogen, oxygen and / or sulfur. The term "heteroaryl" includes fused ring systems and non-fused ring systems, wherein the non-fused ring systems include alkylene and sulfonamide linkers between the rings. For polycyclic ring systems, it is not required that each ring is aromatic.

[0097] The term "cycloalkyl," as used herein, alone or in combination, refers to a non-aromatic monocyclic or multicyclic ring system optionally substituted with one or more ring substituents.

[0098] The term "heterocycloalkyl," as used herein, alone or in combination, refers to a non-aromatic monocyclic or polycyclic ring system, wherein one or more atoms in the ring system, alone or in combination, is an element other than carbon, such as nitrogen, oxygen or sulfur, and wherein the ring system is optionally substituted with one or more ring substituents.

[0099] The term "heteroalkyl" as used herein, alone or in combination, refers to an alkyl moiety as defined above having one or more carbon atoms (e.g., one, two or three carbon atoms) in the chain replaced by one or more heteroatoms, which may be the same or different, wherein the point of attachment to the rest of the molecule is through the carbon atom of the heteroalkyl radical.

[0100] The term "alkoxy," as used herein, alone or in combination, refers to the group RO-, wherein R is alkyl or alkenyl as defined above.

[0101] The term "halogen" as used herein, alone or in combination, refers to an element of Group VIIA of the Periodic Table (halogen). Depending on the chemical environment, a halogen can be in a neutral state or an anionic state.

[0102] I. DNA Encoded Library Selection (Formula I and Formula II)

[0103] To develop novel small molecules that modulate sirtuin activity, DNA-encoded library technology (ELT) was employed. This technology provides a robust hit identification method using a large and diverse library of DNA-encoded small molecules that are screened for their affinity to the desired protein target. This technology provides an efficient method for screening a broad chemical structure space. It is also an attractive strategy because it requires minute amounts of target protein to perform selection experiments and it can identify ligands regardless of their functional activity.

[0104] A 3880-member DNA-encoded library based on the design defined by Mannocci et al. and consisting of 20 amino acids coupled to 194 carboxylic acids was generated and screened. + Affinity-mediated selection for SIRT3 was performed in the presence of carba-NAD (carba-NAD analog) and OAADPR to identify compounds that differentially bind to SIRT3 under such conditions. Carba-NAD or OAADPr together with acetylated or deacetylated peptide substrates are required to display a functional SIRT3 conformation.

[0105] For each screening, a no-target screen was performed in parallel as a negative control. This allowed all compounds in the library that bound non-specifically to the nickel beads to be excluded. All screening experiments were performed at 4°C to preserve SIRT3 activity. During the selection process, special attention was paid to washing the beads after incubation of the protein and library to remove as many non-specific binders as possible. Only one round of selection was performed and the final eluate was amplified, clustered and sequenced by Illumina iSeq100. Prior to selection, we sequenced the natural library to ensure that all compounds were present at nearly similar concentrations so that the selection would not be biased towards any compound that might be present at a higher concentration than other compounds. Figure 1 A 3D plot of the natural library is shown, which shows the reads corresponding to the DNA barcodes for each compound. This represents the distribution of the compounds in the library. We observed that all 194 derivatives of each of the 20 amino acids showed similar distributions in terms of frequency after next generation sequencing. In the natural library, it seems that only the AA5 derivative is slightly over-represented. The initial frequencies of the compounds help us normalize the final output to eliminate any bias that may occur due to differences in the concentrations of individual compounds in the natural library.

[0106] Figure 2 and Figure 3A 3D plot of the enrichment of the compounds compared to the background (no target selection) is shown, which was calculated after normalizing their abundance relative to the total number of DNA sequence reads obtained for each selection. The top 10 highly enriched samples were selected for characterization, as shown in Table 1. All of these compounds showed 12 to 16-fold enrichment compared to the no target control selection.

[0107] Interestingly, for both selection conditions, the same derivatives of carboxylic acids AC71 and AC92 showed the highest enrichment in the presence of Carba-NAD and OAADPr. Among them, a few derivatives of AC40 and AC56 showed significant enrichment of 4 to 8 times compared with negative selection, and these derivatives are worth studying. These compounds did not over-appear in the natural library, further confirming the hypothesis that enrichment was observed only due to binding to SIRT3. When performed in duplicate for both selection conditions, these compounds showed similar enrichment. This highlights the role of AC71 and AC92 derivatives as putative binding partners of SIRT3, which is the rationale for our selection of them for further analysis. We observed that almost all compounds in the library did not show any affinity for nickel beads, indicating the accuracy of immobilized matrix selection. All of the top 10 hit compounds are derivatives of AC71 or AC92, and among their corresponding amino acids, four of them (i.e., AA5, AA8, AA11, and AA12) are common in the two derivatives.

[0108] Therefore, it was decided to synthesize 7 compounds, of which 6 (AA12AC92, AA12AC78, AA12AC71, AA8AC92, AA8AC71, AA8AC40) were the compounds showing the best enrichment (top 10 hit compounds), and one compound that showed no enrichment (AA12AC02Bis) was used as a negative control. These compounds were synthesized in the absence of DNA sequence (off-DNA) (the DNA strand had been substituted with a butyl group) in order to Their binding affinities were validated and their regulatory effects on SIRT3 activity were tested.

[0109] Table 1. Chemical structures of the top 10 hit compounds from SIRT3 selection experiments in the presence of Carba-NAD or OAADPr. These compounds were resynthesized in the absence of oligonucleotide conjugates for further characterization.

[0110]

[0111] SIRT3 Modulation Effects of Hit Compounds

[0112] The test compound is expected to undergo three phases (pre-steady state, steady state and post-steady state) during the enzymatic reaction. The pre-steady state and post-steady state can be regarded as non-steady state (condition II of activity test), in which the enzyme concentration is related to NAD + The substrate concentration ratio is high. Depending on the initial ratio of enzyme to limiting substrate in the system (and the ratio of enzyme to test compound concentration), the net effect of the test compound on the reaction can be inhibitory or activating. If the ratio is above a threshold, activation will occur.

[0113] The top six compounds from the DEL screen were tested for SIRT3 modulation using a fluorescence-based assay ( Figure 4 ). The selected compounds showed different degrees of inhibition under steady-state conditions (condition I of activity test). It was found that in the presence of 50 μM compound AA8AC40, SIRT3 deacetylase activity was inhibited by 66.3%. It was also found that the inhibition level increased with increasing compound dose. IC of the expected compound 50 Values ​​will fall in the μM range (Table 2). The negative control (AA12AC02BIS) did not contribute to SIRT3 modulation. The activity results are consistent with the DEL screening output.

[0114] Table 2. Potency of selected compounds on SIRT3 deacetylating activity.

[0115]

[0116] Allostatic activation of SIRT3

[0117] The deacetylase activity of SIRT3 was measured in the presence of 10 μM AA8AC40 compound under non-steady-state conditions using an HPLC-based assay ( Figure 5 ). In previous studies, HKL has been extensively studied as a model non-steady-state SIRT3 activator. Test compounds are expected to undergo three phases (pre-steady-state, steady-state, and post-steady-state) during the enzymatic reaction. The pre-steady-state and post-steady-state can be considered as non-steady-states, where the enzyme concentration is related to NAD + The substrate concentration ratio is high. Depending on the initial ratio of enzyme to limiting substrate in the system (and the ratio of enzyme to test compound concentration), the net effect of the test compound on the reaction can be inhibitory or activating. If the ratio is above a threshold, activation will occur.

[0118] In the current study, the top hit compound has a larger structural volume compared to HKL (structure below). Computational studies indicate that AA8AC40 binds to SIRT3 externally.

[0119] Protein-compound docking

[0120] As described in “Materials and methods”, ligand docking was performed on the optimal binding ligands from DEL, as identified by affinity-mediated selection assays. However, the large size and flexibility of each DNA-tagged and combinatorially generated ligand made it impossible to dock the entire complex to SIRT3. To address this issue, we decided to dock only the structures of the combinatorial ligands (especially their C-terminal amidated forms) to SIRT3. In addition to most of the active combinatorial ligands, we also selected some inactive ligands that were also identified by the same assay as negative controls to validate the choice of binding sites and docking procedures. The structures of some of the positive and negative controls used in our docking studies are shown in Figure 2. Figure 6 shown.

[0121] Despite the significant size difference between the DEL-derived ligands and compounds known to bind to internal sites on SIRT3 (e.g., Ex-527 and Honokiol), it was decided to first explore known internal sites in the protein. However, attempts to dock active and inactive hit compounds from the DEL library into internal sites of two SIRT3 structures, 4BVG and 4FVT, were unsuccessful. The results were not unexpected for two reasons: (1) the pharmacophore or other structural features of the hit compounds obtained from the DEL library were similar to those that could bind to the NAD peptide and SIRT3. + (2) even without their DNA tags, the hit compounds were significantly longer than either Ex-527 or honokiol, and in the presence of peptides and carba-NAD, the space available to accommodate compounds in the internal site was barely sufficient to accommodate these two known ligands. Although it is possible to accommodate larger compounds in this site, doing so would require displacement of NAD from it. + Or Carba-NAD, like some potent inhibitors of SIRT3.

[0122] Attempts to dock hit compounds and controls at internal sites within both SIRT structures (4FVT and 4BVG) revealed more problems. First, at internal sites within both structures, the optimal poses for hit compounds from active ligands clustered poorly, which was not helped by the fact that a large proportion of them were partially located outside the cavity (not shown). In addition, the directionality of the optimal docking poses for many hit compounds was incompatible with the location where the DNA tag was attached. When docked to internal sites in both structures, inactive control compounds from the library also exhibited significantly better clustering than the hit compounds (not shown). Therefore, the hit compounds from the DEL library are unlikely to bind to internal sites of SIRT3, which leads us to believe that they may bind to more exposed sites on (or near) the protein surface.

[0123] A search was then conducted for atypical or external putative binding sites in SIRT3. Two such sites were easily identified in 5H4D and 408Z, one in each. Both sites were defined by a co-crystallized but weak SIRT ligand called amiodarone. However, it was again found that when docked at external sites in either protein structure, the optimal docking poses of the hit compounds derived from the DEL library did not show any significant superposition with each other (not shown). In addition, the optimal docking poses of the positive compounds had little or no hydrogen bonding interactions with the residues around the sites in 5H4D and 4O8Z.

[0124] Next, 4FVT and 4BVG were scanned using a geometry-based approach to locate potential binding sites. We were able to identify an atypical binding site in 4FVT that was large enough to partially accommodate positive library hit compounds (in addition to known internal sites). Similar scans of 4BVG revealed two more atypical potential binding sites that were large enough to accommodate these compounds. Attempts to dock positive hit compounds with the atypical binding sites in 4FVT revealed a degree of overlap between the optimal docking poses of some positive hit compounds, but the pharmacophore overlap of the optimal poses of different ligands was still poor, and no conservative pattern of interaction with the receptor was observed.

[0125] Docking of the first putative atypical binding site in 4BVG revealed a high overlap of the optimal docking poses for most of the positive hit compounds. Figure 7As shown, only 2 / 10 positive hit compounds (AA8-AC71, AA8-AC92) did not consistently overlap with the optimal docking poses of the other 8 / 10 positive hit compounds. The optimal poses of most positive hit compounds also showed a high degree of pharmacophore overlap and conservative interactions with residues around the site. It is worth noting that the inactive control also docked well at this site, but showed fewer interactions (hydrophobic and hydrophilic) with surrounding residues.

[0126] The second putative atypical site in 4BVG is similar to the atypical sites defined by amiodarone in 5H4D and 4O8Z. The optimal poses of the hit compounds do show some pharmacophore overlap with each other (not shown), but the extent of this overlap is nowhere near that seen at atypical site 1 in 4BVG, which is therefore the most likely binding site for the hit compounds in the DEL library. However, it should be noted that 2 / 10 of the hit compounds tested did not dock correctly at atypical site 1. In addition, developing ligands for atypical site 1 in 4BVG may be more difficult than usual because this site is shallower and more easily exposed to solvent than typical sites for small molecule sites.

[0127] II. Virtual screening was used to identify hit compounds that activated sirtuin-3 based on mechanism (Formula III to XIV)

[0128] AWS cloud server is used to use AutoDock Vina to virtually screen the 1.2 million compound database from ChemBridge. Two QM-MM geometry-optimized SIRT3 complexes (4FVT and 4BVG) are used as receptors in this docking exercise. Hit compounds are identified based on the docking scores of these two receptors (in AutoDock Vina and MOE) and their hydrogen bond interactions with specific residues around the binding site (including those residues in the cofactor binding loop). Honokiol is included in the library as a positive control and is subsequently identified as a hit compound according to these criteria. The top 20 compounds are selected from the top 1000 compounds based on their docking scores (as shown in Table 3, between -11.7 and -9.5). The AutoDock Vina score of the selected compound is significantly better than the average of the entire data set (median of -5.5), indicating a good fit to the binding site, proving that compounds with higher scores show better overall interactions with SIRT3 than compounds with lower values. Residues with hydrogen bonds to the compound and the degree of receptor burial of each compound are also reported in Table 3.

[0129] Table 3. Docking scores and in vitro SIRT3 modulation of the top 20 compounds selected by virtual screening (hit compounds*).

[0130]

[0131]

[0132] Comparison of top hit compounds with HKL binding mode

[0133] The top hit compounds were analyzed for the interactions of residues in the binding site involved in docking (especially the flexible loops) (see Table 4). The top hit compounds had better AutoDock scores than the reference ligand (HKL). Pro155, Arg158, and Ile230 interacted with the top hit compounds through hydrogen bonds, and their pattern was similar to HKL. Our previous studies have shown that HKL forms hydrogen bonds with Pro155 in the open-ring conformation of SIRT3, and with Arg158 in the closed-ring conformation of SIRT3. Interestingly, hit compounds 6068318 and 5761865 have the same binding mode ( Figure 8 ).

[0134] Validation of SIRT3-modulating effects of selected hit compounds

[0135] Respectively Fig. 9 and Fig.10 The hit compounds were validated in vitro using a commercial Fluor de Lys enzyme assay and HPLC assay under steady-state conditions (top 70) and non-steady-state conditions (top 20).

[0136] Under steady-state conditions, the compounds showed inhibitory potency against SIRT3 in the range of 0-58.4% at a concentration of 10 μM modulator (Table 3). Two hit compounds showed good inhibitory potency (>50% inhibition) at a concentration of 10 μM modulator. Under non-steady-state conditions, 9 of the 20 compounds were found to activate SIRT3 (3.1%-14.0%) at a concentration of 10 μM modulator. The hit rate of VS (the number of hit compounds divided by the number of compounds tested) was 45%, indicating that the current VS workflow is successful.

[0137] Comparison of top hits regarding HKL binding mode

[0138] The residues involved in docking (especially the flexible loop region) were analyzed for the top hit compounds (Table 4). The top hit compounds had better AutoDock scores than the reference ligand (HKL). Pro155, Arg158 and Ile230 were found to interact with the top hit compounds through hydrogen bonds, which is similar to the role of HKL. Our previous studies have shown that HKL forms hydrogen bonds with Pro155 in the open-loop conformation of SIRT3, and with Arg158 in the closed-loop conformation of SIRT3. Interestingly, hit compounds 6068318 and 5761865 present the same binding characteristics. The definition of docking sites, docking templates and ligand queries plays an important role in ensuring the high hit rate of the current VS (45% for activators and 55% for inhibitors and activators).

[0139] Table 4. Summary of ligand-protein interactions observed during docking. Color coding: blue, after docking minimization (hydrogen bonding); pink, conventional hydrogen bonding.

[0140]

[0141] Structural similarity of top hits

[0142] When searching for analogs of the top nine assay-validated hits with two SIRT3 receptor structures (4BVG and 4FVT) from our 1.2 million compound docking study, it was found that some of the hits shared common structural features. These common features were not sufficient to construct an appropriate SAR model, but were sufficient to influence the selection of combinatorial library building blocks. The most obvious recurring motif was a bicyclic ring (aromatic, partially aromatic, or with partial delocalization) connected to another single aromatic ring via an amide (or urea) linker ( Fig.11 ). These criteria can be applied when designing the backbones of future ligands and combinatorial libraries.

[0143] Compounds of formula III, V, VI, VII, VIII, IX, X, XI and XIV were included in the SIRT3 docking study as follows (Table 6-32). Initial 3D structures of the compounds were generated from SMILES strings using RDKit (2019 version) or, if that failed, OpenBabel version 3.0 was used. In ambiguous cases (e.g. axial versus equatorial substitution), all conformers were generated. In addition, all stereoisomers were generated for racemates. For structures with non-standard annular folds, the structures were minimized using DFT to ensure viable geometries for docking. This was performed using ORCA version 3.0.1. All compounds were docked into SIRT3 using GOLD v 5.2 using 3 PDB files (4BVG and 4FVT and an in-house generated X-ray structure of human SIRT3 (called Xtal)). In each case, the binding pocket was constrained to the ligand in the original PDB file. 30 docking partners were required for each ligand for any substance within 1000. All other options were set to default options. An internal algorithm was used to identify and evaluate the quality of all hydrogen bonds (H bonds) formed between the ligand and the protein, and this information was used together with the GOLD docking score to select the best solution. Two docking partners were selected for each compound, the highest scoring docking partner containing the most hydrogen bonds and the highest scoring remaining docking partners. These docking partners were manually evaluated and compared with the parent reference compounds used to determine the seed design.

[0144] Among the docking results in the table below, the docking partners with the highest scores were selected regardless of the number of hydrogen bonds. Compounds with docking scores at least 90% higher than the reference compound docking score (GoldScore) were considered potential SIRT3 modulators.

[0145] The reference compounds on which the docking studies were based are marked in Tables 6 to 32. In fact, these are the compounds from the top 20 of the virtual screening that have been involved in laboratory activity tests and have been shown to modulate Sirtuin. The results of the activity tests of the reference compounds are summarized in Table 5, and the values ​​given are based on tests performed in triplicate.

[0146] Table 5. Activity test results of reference compounds (from top 20 VS).

[0147]

[0148] The results of docking studies using compounds of Formulae III, V to XI, and XIV are provided in Tables 6 to 32 below.

[0149] Table 6. SIRT3 docking results of compounds of formula III

[0150]

[0151]

[0152] Table 7. SIRT3 docking results of compounds of formula III

[0153]

[0154] Table 8. SIRT3 docking results of compounds of formula III

[0155]

[0156]

[0157] Table 9. SIRT3 docking results of compounds of formula VI

[0158]

[0159] Table 10. SIRT3 docking results of compounds of formula VI

[0160]

[0161]

[0162] Table 11. SIRT3 docking results of compounds of formula VI

[0163]

[0164] Table 12. SIRT3 docking results of compounds of Formula V

[0165]

[0166]

[0167] Table 13. SIRT3 docking results of compounds of Formula V

[0168]

[0169]

[0170] Table 14. SIRT3 docking results of compounds of Formula V

[0171]

[0172] Table 15. SIRT3 docking results of compounds of formula VII

[0173]

[0174]

[0175] Table 16. SIRT3 docking results of compounds of formula VII

[0176]

[0177]

[0178] Table 17. SIRT3 docking results of compounds of formula VII

[0179]

[0180]

[0181] Table 18. SIRT3 docking results of compounds of formula VIII

[0182]

[0183] Table 19. SIRT3 docking results of compounds of formula VIII

[0184]

[0185]

[0186] Table 20. SIRT3 docking results of compounds of formula VIII

[0187]

[0188]

[0189] Table 21. SIRT3 docking results of compounds of Formula IX

[0190]

[0191] Table 22. SIRT3 docking results of compounds of Formula IX

[0192]

[0193]

[0194] Table 23. SIRT3 docking results of compounds of Formula IX

[0195]

[0196]

[0197] Table 24. SIRT3 docking results of compounds of formula X

[0198]

[0199]

[0200] Table 25. SIRT3 docking results of compounds of formula X

[0201]

[0202]

[0203] Table 26. SIRT3 docking results of compounds of formula X

[0204]

[0205] Table 27. SIRT3 docking results of compounds of formula XI

[0206]

[0207] Table 28. SIRT3 docking results of compounds of formula XI

[0208]

[0209]

[0210] Table 29. SIRT3 docking results of compounds of formula XI

[0211]

[0212]

[0213] Table 30. SIRT3 docking results of compounds of Formula XIV

[0214]

[0215] Table 31. SIRT3 docking results of compounds of Formula XIV

[0216]

[0217]

[0218] Table 32. SIRT3 docking results of compounds of Formula XIV

[0219]

[0220]

[0221] The docking results showed that the docking scores of the analogs were very close to or lower than those of the reference compounds (whose regulation (activation or inhibition) of SIRT3 had been identified by activity tests performed in the laboratory). This suggests that these analogs also interact with the SIRT3 protein in a similar manner and regulate its activity.

[0222] The activity of additional compounds belonging to formula III, V, VII and XI was tested under condition I (steady state). These activity tests showed that these compounds modulated SIRT3 activity (activation or inhibition), confirming the results obtained from the docking study. Table 33 summarizes the results of these activity tests.

[0223] Table 33. Sirtuin-modulating compounds identified by activity assays

[0224]

[0225]

[0226] Homeostatic SIRT3 Activating Compounds

[0227] Importantly, our drug discovery workflow ( Fig.296 ) identified the first reported steady-state activators of SIRT3 (Table 5), which qualitatively improved the properties of honokiol and are ideal candidates for SIRT activator drug development. The steady-state activators 5329973 (Formula VII) and 5689785 (Formula XI) were characterized and found to be physiologically relevant [NAD + ]=100μM, SIRT3 activity increased by 184.5% (at 20μM) and 194.4% (at 1μM) respectively (Figure 297). These two compounds increased the catalytic efficiency (k cat / K m ) was almost doubled (Table 34). The MST binding affinity results of compound 5329973 fell into the nM range, indicating that compound 5329973 is a strong binder of SIRT3 ( Figure 298F ). Steady-state parameter estimates (Table 34) suggest that the catalytic efficiency is mainly increased by decreasing K, as originally proposed in our previous theoretical study. mTo improve, the theoretical study predicts that this may be achieved by regulating the conformation of the flexible ring induced by the active site binding of appropriately designed small molecules. The observations in Table 34 and Figure 299 (Michaelis-Menten diagram) are very consistent with the predicted properties of silent information regulatory protein activators based on the steady-state mechanism. The dose-response curves of compounds 5689785 and 6068318 (Formula III) (Figure 397A) also show that these compounds achieve more than 50% of their maximum activation effect (AC50) at concentrations of 100nM and <1uM, respectively, which is promising to further develop these hit compounds into drug-like leads.

[0228] Table 34. SIRT3 model parameter estimates based on Michaelis-Menten global nonlinear fitting in the presence and absence of 1 μM 5689785 and 20 μM 5329973. [E0] = 1.568 μM.

[0229]

[0230] In saturated NAD + ( Figure 297A ) and saturated peptide substrates ( Figure 297B-Figure 297D ) conditions, all of which demonstrated activation of SIRT3 by these steady-state activators. + (This is consistent with the NAD + concentrations are similar because mitochondrial NAD + The deacetylase rate of SIRT3 is approximately doubled at 200 μM in youth and may decrease by 50% or more in old age, and the levels of SIRT3 activation by these compounds are sufficient to largely restore the mitochondrial deacetylase activity of SIRT3 characteristic of youth. In addition, compared with honokiol, steady-state activators such as these compounds are less affected by changes in physiological conditions related to SIRT3 activity (e.g., enzyme expression levels).

[0231] Materials and methods

[0232] Activity assay - Activity test of reference compounds Table 5

[0233] Chemicals and reagents

[0234] MnSOD (KGELLEAI-(KAc)-RDFGSFDKF) was synthesized by GenScript (Piscataway, NJ). Human SIRT3 (recombinant) (His-tag), NAD +(SIRT substrate), FdL2 (QPKKAC-AMC) peptide (also known as p53-AMC peptide), nicotinamide, and HDAC buffer were purchased from Enzo Life Sciences (Farmingdale, NY). Dimethyl sulfoxide and trifluoroacetic acid were purchased from Sigma-Aldrich (St. Louis, MO). Test compounds were purchased from ChemBridge, Inc. (San Diego, CA). Carba-NAD was synthesized by Dalton Pharma (Toronto, ON).

[0235] Hit compounds against hSIRT3 102-399 Effect of deacetylation activity - HPLC determination using native peptides

[0236] The enzymatic reaction included 1000 μM NAD + and 50 μM MnSOD peptide (Condition I) or 50 μM NAD + and 600 μM peptide substrate (condition II) in the presence of hit compound (50 μM (condition I) or 10 μM (condition II)) in dimethyl sulfoxide solution 95 / 5 in HDAC buffer (50 mM TRIS-HCl, pH 8.0, containing 137 mM sodium chloride, 2.7 mM potassium chloride and 1 mM magnesium chloride). 102-399 The reaction was initiated and incubated at 37°C for 30 minutes. The reaction was terminated by adding stop solution (final concentration 2% TFA, 5mM nicotinamide). The peptide product and substrate were separated using HPLC (reverse phase C18 HPLC column). Solvent A consisted of 90% HPLC grade water and 10% acetonitrile with 0.05% (v / v) TFA. Solvent B consisted of acetonitrile with 0.02% (v / v) TFA. A linear gradient was performed from 0% B to 51% (v / v) B for 20 minutes. The solvent B percentage was increased to 100% in 5 minutes. Then it was returned to the starting condition (0% B) in 5 minutes. The solvent A percentage (100%) was maintained at 100% for another 5 minutes (36 minutes total run time).

[0237] Hit compounds against hSIRT3 102-399 Effect of deacetylation activity - fluorescence-based assay using fluorine-labeled peptides

[0238] Using FdL2 peptide to determine the effects of hit compounds on SIRT3 102-399The enzymatic reaction was carried out similarly to the above. The reaction was terminated by adding 1X developer 2mM NAM solution and the fluorescence was measured on a TECAN microplate reader. The raw data were fitted to the defined model equation using GraphPad Prism (GraphPad Software, Inc, California).

[0239] Activity determination - Activity test table 33

[0240] Chemicals and reagents

[0241] MnSOD (KGELLEAI-(KAc)-RDFGSFDKF) was synthesized by GenScript (Piscataway, NJ). Human SIRT3 (recombinant) (His-tag), NAD + (SIRT substrate), nicotinamide, and HDAC buffer were purchased from Enzo Life Sciences (Farmingdale, NY). Dimethyl sulfoxide and trifluoroacetic acid were purchased from Sigma-Aldrich (St. Louis, MO). Test compounds were purchased from ChemBridge, Inc. (San Diego, CA).

[0242] HPLC assay

[0243] The enzymatic reaction included 1000 μM NAD + and 50 μM MnSOD peptide in the presence of hit compound (50 μM) (Condition I) in dimethyl sulfoxide solution 95 / 5 in HDAC buffer (50 mM TRIS-HCl, pH 8.0, containing 137 mM sodium chloride, 2.7 mM potassium chloride and 1 mM magnesium chloride). 102-399 The reaction was initiated and incubated at 37°C for 30 minutes. The reaction was terminated by adding stop solution (12% TFA, 30 mM nicotinamide). The peptide product and substrate were separated using HPLC (reverse phase C18 HPLC column). Solvent A consisted of 90% HPLC grade water and 10% acetonitrile with 0.05% (v / v) TFA. Solvent B consisted of acetonitrile with 0.02% (v / v) TFA. A linear gradient was performed from 0% B to 51% (v / v) B for 20 minutes. The solvent B percentage was increased to 100% in 5 minutes. Then it was returned to the starting condition (0% B) in 10 minutes. The solvent A percentage (100%) was maintained at 100% for another 10 minutes (41 minutes total run time).

[0244] Virtual screening of millions of compounds (Table 3 and Table 4)

[0245] High-throughput batch docking

[0246] A database (Chembridge) containing 1.2 million compounds was screened against two SIRT3 structures (PDB: 4FVT and 4BVG). Our existing knowledge of the bioactive conformation of HKL and its binding site for SIRT3 was applied to define the docking site and make the selection of protein conformations. Structure-based docking was used in a sequential process for achieving VS (Figure 299). In brief, the 1.2 million compounds were first imported into the database and converted from 2D→3D using the relevant modules in MOE. Next, additional modules (iMOE) were used to add hydrogens as needed, calculate partial charges and perform a fast round of energy minimization using the MMFF94x force field. When appropriate, the most likely tautomeric form was generated and automatically selected.

[0247] Figure 299 is a schematic diagram illustrating the compound screening method used in the present application. A series of operations were performed on a database containing 1.2 million compounds, starting with a 2D to 3D conversion and ending with a fast energy minimization of each compound. These compounds were then docked independently with two SIRT3 receptor structures (4BVG and 4FVT). The best poses (by calculated binding energy) of the top 1000 compounds in each docking run were selected for further post-docking analysis-implemented using MOE as described in "Post-docking Analysis". The final step involved carefully examining and selecting 20 compounds from two docking runs, which provided us with skeleton diversity, optimal coverage without duplication, and the maximum number of favorable interactions with residues in the putative binding site.

[0248] These prepared ligand structures were converted back to the input file format preferred by AutoDock Vina. Our existing studies on the bioactive conformations of HKL and its binding site in SIRT3 were applied to define the docking site and make the selection of protein conformations. Docking was performed using AutoDock Vina on AWS cloud server. Grid centers and volumes were defined as: for 4FVT, centers (x, y, z) = 23.19, 31.19, -14.31, volume = 29.6x34.8x33.8; for 4BVG, centers (x, y, z) = 22.24, 30.39, -7.98, volume = 25.0x25.0x25.0. 20 possible conformations were collected for each ligand.

[0249] Post-docking analysis

[0250] The docked ligands were then ranked according to the docking score (predicted affinity in kcal / mol) in AutoDock Vina. The top 1000 compounds were then screened for: (1) non-covalent interactions with the 4FVT and 4BVG binding cavities; (2) hydrogen bonding with residues located around the binding site; (3) interactions with specific residues within the SIRT3 cofactor binding loop; (4) predicted ADME properties such as LogP, LogD and (5) predictions regarding synthetic feasibility. Only the poses of each compound from the top 1000 best ranked compounds were included in the list.

[0251] The list of the top 1000 compounds was then subjected to further post-docking analysis using MOE. The ligands were imported into the database and converted to the native form used by MOE after adding hydrogen atoms and recalculating the partial charges of each structure. A small portion of these structures was carefully examined to determine that the treatment did not introduce any undesirable artifacts therein. Next, a truncated post-docking energy minimization of the ligands and hydrogen atoms from adjacent residues was performed using the MOE script named "analysis_dock.svl" to further optimize the interactions between the docking pose and site residues. The energy optimization step provides a new value for the approximate calculated binding energy (MOE) of the compound. The optimized structure is then used for all subsequent post-docking analysis steps.

[0252] We screened the optimized poses for the presence of hydrogen bonds between residues around the binding site. Based on the previous docked structure of honokiol, the following residues were considered to be part of this site: 4FVT (G145, S149, G153, G163, L168, Y171, F180, K195, E198, Y204, Q228 and H248). 4BVG (D156, P176, I179, F294, S321, E323 (and NLE7 from the co-crystallized Ac-ACS peptide chain)). The docked pose of honokiol was also used to measure the relative “receptor burial” of the optimized poses in the two receptor structures. The BHB (buried, hydrogen bonding and binding energy) score for each compound was calculated using the general formula first described by Feher M. et al. Hit compounds for hSIRT3 118-399 Effect of deacetylation activity - fluorescence-based assay using fluorine-labeled peptides

[0253] Using FdL2 peptide to determine the effects of hit compounds on SIRT3 118-399The enzymatic reaction was carried out similarly to the above. The reaction was terminated by adding 1X developer 2mM NAM solution and the fluorescence was measured on a TECAN microplate reader. The raw data were fitted to the defined model equation using GraphPad Prism (GraphPad Software, Inc, California).

[0254] Docking studies of hit compounds

[0255] MD simulation

[0256] Computational studies were performed using MOE (Molecular Operating Environment from Chemical Computing Group Inc., Montreal, Quebec) version MOE 2020.09 on a computer with a quad-core Intel x86-64 (Intel Core i7-8th Gen) processor, 16 GB RAM, and running Windows 10 Pro. The MMFF94x force field and solvation implemented in MOE were used for all computational studies.

[0257] Ligand docking program

[0258] Two methods were used to define the ligand binding site in the receptor protein structure. The first was used for 4BVG, which defined the site using a co-crystallized ligand (EX-527). The second was used for 4FVT and 4BVG, which used the "Site Finder module" in MOE to create virtual atoms and place them into the potential ligand binding sites of the two structures. This step was performed after removing molecules of all minor compounds present in the receptor structure (e.g., sulfate ions, glycerol, 1,2-ethylene glycol, etc.). Unexpectedly, the main site of 4BVG identified by the second method was almost identical to the site defined by the co-crystallized ligand. Therefore, it is assumed that the main putative (and internal) ligand binding site identified in 4FVT is the best candidate site for docking studies in the receptor structure.

[0259] Energy-optimized models of selected compounds were sequentially docked to these sites using the “Dock” application in MOE; all docking studies were performed with default settings of parameters using the “rigid receptor” scheme. Reproducibility was demonstrated in three independent docking simulations for a given site. The best pose is the best pose for that site. The main settings for ligand docking were as follows: Docking Scheme = Rigid Receptor; Placement = Triangle Matcher; Rescoring = London dG; Keep = 100, Refinement = Force Field; Rescoring = GBVI / WSA dG; Keep = 100 best poses. The default settings of docking parameters using the "Rigid Receptor" scheme were found to be satisfactory for the docking of honokiol and EX-527 used as a test ligand. The highest scoring pose of the ligand for each docking run was used to calculate the approximate binding energy and other inputs to calculate the final docking score.

[0260] Calculation of ligand binding energy

[0261] The binding energy of the optimal docking pose for each ligand at each site was calculated using an updated version of a published method. It involves: calculating the energy of the docked receptor-ligand complex (Ecpx); the unbound ligand in solution (Elig) and the unbound solvated rigid receptor from the complex (Eprot), for the equation: Ebind = Ecpx-(Elig+Eprot). The best selected docking pose for each ligand obtained from three independent docking runs was used to calculate the approximate ligand binding energy. The optimization of the semi-rigid (fixed heteroatom) receptor-ligand complex was performed under distance-dependent dielectric conditions, and the final step was performed under solvation, using a scheme where only the ligand and hydrogens in the cavity could be subjected to further structural optimization.

[0262] Binding energies calculated using molecular mechanics should always be considered as approximations of the actual binding energies, since any semi-rigid receptor approximation will almost always underestimate the effect of the induced fit, while a fully flexible receptor approximation will usually overestimate it. The actual binding energies are therefore expected to lie somewhere between the rigid and flexible receptor values. The methods used to calculate receptor burials for optimally docked (and energy-optimized) ligand poses and their counts of hydrogen-bonding interactions with residues in the binding site have been described previously; and the code is available upon request.

[0263] In order to achieve various purposes of the present invention, various embodiments of the present invention have been described. It should be recognized that these embodiments are only illustrations of the principles of the present invention. Without departing from the spirit and scope of the present invention, many modifications and adaptations will be apparent to those skilled in the art.

Claims

1. A sirtuin-modulating compound and / or a salt thereof having Formula VII: wherein R1 and R2 are independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl and heterocycloalkyl; and wherein Ar1 and Ar2 are independently selected from aryl and heteroaryl, wherein aryl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10 )-alkyl, (C1-C 10 )-alkenyl, alkoxy, halogen, amine, nitro, sulfonyl-alkyl, acyl, amide, acetylamide, alkylamine, alkynyl, and hydroxy.

2. The sirtuin-modulating compound of claim 1, wherein R1 and R2 are independently selected from the group consisting of hydrogen and alkyl, and Ar2 is nitrobenzene.

3. The sirtuin-modulating compound of claim 2, wherein Formula VII and / or its salt is:

4. The sirtuin-modulating compound of claim 1, wherein Formula VII and / or its salt is: wherein R3 is selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, halogen and heterocycloalkyl.

5. The sirtuin-modulating compound of claim 1, wherein the compound is a sirtuin activator.

6. The sirtuin-modulating compound of claim 1, wherein the compound is a sirtuin inhibitor.

7. The sirtuin-modulating compound of claim 1, wherein the compound modulates SIRT3 activity.

8. A sirtuin-modulating compound and / or a salt thereof having Formula III: Among them, Ar 1 is aryl or heteroaryl, R1 is selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, amine and -C(O)NR3R4, wherein Ar 1 , alkyl, alkenyl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkyloxy, cycloalkenyl, alkylamine, acetamide, acetylamine, cycloalkyl, heterocycloalkyl, aryl, heteroaryl and amine are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10 )-alkyl, (C1-C 10 )-alkenyl, alkoxy, halogen, amine and hydroxy; and wherein R3 and R4 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, aryl and heteroaryl, and wherein R2 is selected from the group consisting of hydrogen, alkyl, fluoroalkyl, alkenyl, aryl, heteroaryl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkoxy, cycloalkenyl, amine, alkylamine and O, and wherein Y is selected from the group consisting of N and CH, and wherein X is selected from the group consisting of NH, S and O.

9. The sirtuin-modulating compound of claim 8, wherein R1 is selected from the group consisting of aryl and -C(O)NR3R4.

10. The sirtuin-modulating compound of claim 8, wherein Formula III and / or its salt is:

11. The sirtuin-modulating compound of claim 8, wherein Formula III and / or its salt is:

12. The sirtuin-modulating compound of claim 8, wherein the compound is a sirtuin activator.

13. The sirtuin-modulating compound of claim 8, wherein the compound is a sirtuin inhibitor.

14. The sirtuin-modulating compound of claim 8, wherein the compound modulates SIRT3 activity.

15. A sirtuin-modulating compound and / or a salt thereof having Formula XI: Among them, Ar 1 and Ar 2 are independently selected from aryl and heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10 )-alkyl, (C1-C 10 )-alkenyl, alkoxy, halogen, amine, sulfonyl-alkyl, acyl, amide, acetylamine, alkylamine, alkynyl, and hydroxy.

16. A sirtuin-modulating compound and / or a salt thereof having Formula V: wherein R1 is selected from the group consisting of hydrogen, alkyl, heteroalkyl, cycloalkyl, alkenyl, alkynyl, acyl, alkoxy, alkenyloxy, cycloalkoxy, cycloalkenyl, and heterocycloalkyl; and wherein Ar 1 and Ar 2 are independently selected from aryl and heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of: (C1-C 10 )-alkyl, (C1-C 10 )-alkenyl, alkoxy, halogen, amine, sulfonyl-alkyl and hydroxy; and wherein X is selected from the group consisting of O, S and N; and wherein m and n are integers each having a value independently selected from 0 to 10.

17. The sirtuin-modulating compound of claim 16, wherein Formula V and / or its salt is:

18. The sirtuin-modulating compound of claim 16, wherein Formula V and / or its salt is:

19. The sirtuin-modulating compound of claim 16, wherein Formula V and / or its salt is:

20. The sirtuin-modulating compound of claim 16, wherein the compound is a sirtuin activator.

21. The sirtuin-modulating compound of claim 16, wherein the compound is a sirtuin inhibitor.

22. The sirtuin-modulating compound of claim 16, wherein the compound modulates SIRT3 activity.

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