Methods of treating mitochondrial dysfunction

Abstract

Provided are compounds that covalently bind to Fis1. Also provided are methods of selectively inhibiting mitochondrial dysfunction. Aspects of the methods include covalently binding a compound to Fis1. Also provided are methods for treating mitochondrial dysfunction in a subject by administering an effective amount of a compound that covalently binds to Fis1. Aspects of the methods include treating a subject that has a neurodegenerative disease, an inflammatory disease or a cardiovascular disease.

Description

Atty Dkt No.: STAN-2169WO Client Ref.: S23-492 METHODS OF TREATING MITOCHONDRIAL DYSFUNCTION GOVERNMENT RIGHTS

[0001] This invention was made with Government support under contract W81XWH-22-1- 0203 awarded by the Department of Defense. The Government has certain rights in the invention. CROSS-REFERENCE

[0002] This application claims priority pursuant to 35 U.S.C. §119(e) to the filing date of U.S. Provisional Application No.63 / 604,688, filed on November 30, 2023, the disclosure of which application is incorporated herein by reference in its entirety. INTRODUCTION

[0003] Mitochondria are dynamic organelles, and fusion and fission maintain mitochondrial size, morphology and function. Fusion enables replenishment of damaged mitochondrial machinery and maintenance of the mitochondrial network whereas fission increases mitochondrial number and enables the removal of damaged mitochondria as a mitochondrial quality control measure. A tetratricopeptide repeat (TPR) motif containing mitochondrial outer membrane protein, mitochondrial fission protein 1 (Fis1), mediates mitochondrial fission by recruiting large cytosolic GTPase, Drp1, to the mitochondrial outer membrane. In mammals, the Fis1-Drp1 interaction mediates pathological mitochondrial fission. Fis1 interaction with Drp1 occurs during cellular stress, but not under physiological conditions, suggesting that cellular signals during stress activate Fis1. SUMMARY

[0004] Fis1-mediated mitochondrial anchoring of Drp1 and excessive mitochondrial fission occur in multiple diseases associated with oxidative stress. However, it was not known how Fis1 detects oxidative stress and what structural changes in Fis1 enable mitochondrial recruitment of Drp1. Inventors of the present invention found that the N-terminus of Fis1 is dynamic and that α1 helix unfolding exposes the only cysteine in Fis1. This hidden Cys41 is redox sensitive and forms a covalent homodimer via a disulfide bridge during oxidative stress,Atty Dkt No.: STAN-2169WO Client Ref.: S23-492 triggering Drp1 recruitment to mitochondria and excessive fission. These structural discoveries provide insight into compounds that covalently bind to Fis1 (e.g., Fis1 with exposed Cys41). Inhibitors that selectively bind to Fis1 (e.g., Fis1 with exposed Cys41) preserve mitochondrial integrity and function in cells during oxidative stress.

[0005] Provided are compounds that covalently bind to Fis1. Also provided are methods of selectively inhibiting mitochondrial dysfunction. Aspects of the methods include covalently binding a compound to Fis1. Also provided are methods for treating mitochondrial dysfunction in a subject by administering an effective amount of a compound that covalently binds to Fis1. Aspects of the methods include treating a subject that has a neurodegenerative disease, an inflammatory disease or a cardiovascular disease. BRIEF DESCRIPTION OF THE FIGURES

[0006] FIGS.1A-1C: Molecular Dynamic (MD) simulations predict dynamic nature of Fis1 N-terminus. (FIG.1A) 30 MD simulations of 1.2 µs of Fis1, phosphorylated pThr34 Fis1 and pTyr38 are shown. Plotted values are mass average root mean square fluctuations of backbone (Cα, C and N) atoms. RMSF of backbone atoms of first 35 residues are highlighted. (FIG. 1B) Relative positions and distances of known phosphorylation sites (Thr34 and Tyr38) and negatively charged residues (Glu18 and Glu24) are shown. (FIG. 1C) Average structural propensities of residues in the α1 helix residues 15-25 to form α helix are calculated from the MD simulations and shown as heat maps.

[0007] FIGS. 2A, 2B: N-terminus is disordered in activated Fis1. (FIG. 2A) X-ray structures showing electron density [composite omit map (σ = 1.0)] of structures of WT Fis1, Thr34Asp Fis1, Thr34Glu Fis1 and Tyr38Glu Fis1. All the mutant structures are missing electron density for α1 helix, which is shown as transparent cyan wire. (FIG.2B) Kratky plots of monomers of WT, Thr34Asp and Tyr38Glu Fis1 obtained from small angle X-ray scattering data are overlayed and porod volumes and AUC (up to the qRg = 4) calculated from the SAXS experimental data are modeled.

[0008] FIGS.3A-3D: N-terminus unfolding exposes Cys41. (FIG.3A) Fis1 protein shown with a surface and the Cys41 residue is highlighted. The surface is colored according to sequence conservation in vertebrates. Sequences aligned for this analysis are provided in Table 2. (FIG. 3B) Solvent accessible surface area of Cys41 (obtained from molecular dynamics simulations) for Fis1, phosphorylated Thr34 Fis1 (pThr34) and phosphorylated Tyr38 Fis1 (pTyr38) areAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 shown. Each box represents an independent 1.2 µs simulation. The average SASAs of Cys41 in WT, pThr34, and pTyr38 calculated from the 30 independent 1.2μs MD simulations each were 12.5±8.5 Å2, 13.9±10.0 Å2, and 29.2±14.7 Å2, respectively. (FIG. 3C) Schematic of the assay used to measure Cys41 exposure using maleimide based fluorescent probe, CPM. (FIG. 3D) CPM fluorescence-based binding assay is shown. 4 µM protein and 8 µM CPM were used. Plotted value represents mean of 3 independent experiments each with 3 technical replicates. Error bar represents standard deviation. One-way ANOVA with Dunnett’s multiple comparisons test (Thr34Asp and Tyr38Glu each against WT) was performed. p-value results indicated by stars (**** p < 0.0001).

[0009] FIGS. 4A-4G: Cys41 is oxidizable and mediates covalent dimerization. (FIG. 4A) X- ray structures showing electron density [composite omit map (σ = 1.0)] for Thr34Asp Fis1 covalent dimer. Missing N-terminal residues AA 1-32 is shown as grey transparent wire. Continuous electron density for disulfide bridge between the Cys41 of two monomers is highlighted. (FIG. 4B) Partial native SDS-PAGE gel showing covalent dimers for different constructs of Fis1 [pThr34 (T34D), pTyr38 (Y38E) and wildtype (WT)] under different conditions; equal amount of beads initially loaded with 20 µg protein (30% of total initial beads; about 6 µg protein) were loaded in each lane. Quantification of dimer / monomer band ratios from three independent experiments are shown. Error bars represent S.D. Uncropped images are provided in FIG. 22. (FIG. 4C) Thr34Asp Fis1 covalent dimers have large hydrophobic patches exposed (darker color). The structure is rotated by 180° to show the patches on both sides. (FIG. 4D) Crystal structure of WT Fis1 dimer has the concave hydrophobic patches buried between the interface of the dimer. (FIG. 4E) The theoretical Kratky plot of WT crystallographic dimer structure doesn’t fit the experimental Kratky plot of WT Fis1 dimer. (FIG. 4F) The theoretical Kratky plot of the WT Fis1 covalent dimer modeled based on Thr34Asp Fis1 dimer structure including unfolded N-terminal region fits the experimental Kratky plot of WT Fis1 dimer. The theoretical plots are shown in solid black line and experimental plot is shown in circles in FIGS. 4E, 4F. (FIG. 4G) The modeled WT Fis1 covalent dimer also fits both the P1 and P2 symmetry enforced the ab initio envelops (white envelope and dark blue mesh at 2 contour levels, 0.13 and 0.057) independently calculated from experimental SAXS curve.Atty Dkt No.: STAN-2169WO Client Ref.: S23-492

[0010] FIGS. 5A-5D: Structural insights guided assay development and discovery of activated Fis1 inhibitor. (FIG. 5A) Schematic of CPM assay for the screening of covalent inhibitor of activated Fis1. (FIG. 5B) Proof of concept of CPM-based screening assay using maleimide. Unlabeled maleimide (0-50 µM) inhibited CPM fluorescence in Thr34Asp Fis1 and Tyr38Glu Fis1.4 µM protein and 8 µM CPM were used in the assay. Each data point represents mean of 3 independent experiments each with 3 technical replicates. Error bar represents standard deviation. (FIG.5C) Inhibition of CPM fluorescence by SP11 in Thr34Asp Fis1 but not in counter screen construct (Cys41Ser, Val56Cys Fis1 double mutant).4 µM protein and 8 µM CPM were used in the assay. Each data point represents mean of 3 independent experiments each with 3 technical replicates. Error bar represents standard deviation. (FIG.5D) Structure of SP11.

[0011] FIGS. 6A-6E: SP11 binds to Cys41 of activated Fis1. (FIG. 6A) MS-MS spectrum showing Fis1 peptides are modified at Cys41 by SP11. (FIG.6B) X-ray structures showing SP11 bound to Thr34Asp Fis1. Electron density [2Fo-Fc map (σ = 1.0)] for SP11 and pocket residues (shown as sticks) are highlighted. (FIG. 6C) Phenothiazine moiety sits on the hydrophobic patch formed by Tyr38. (FIG.6D) Two benzene rings of SP11 form proton-Pi interactions with Tyr38 residue. (FIG. 6E) CPM fluorescence inhibition assay for SP11. SP11 binds to Cys41 in Thr34Asp Fis1 mutant but does not bind to Cys41 when Tyr38 is mutated to Glutamic acid.4 µM protein and 8 µM CPM were used in the assay. Each data point represents mean of 3 independent experiments each with 3 technical replicates. Error bar represents standard deviation.

[0012] FIGS. 7A-7F: SP11 reduces H2O2-induced mitochondrial ROS production and fragmentation and increased Drp1 translocation to the mitochondria in HK-2 cells. (FIG.7A) HK-2 cells were co-treated with H2O2(50 µM) and SP11 (250 nM) for 24h. DMSO was used for vehicle control. Mitochondrial ROS was measured using MitoSOXTMmitochondrial superoxide indicator. MitoSOXTMsignal was normalized to Hoechst 33342. Each data point represents the mean of an independent experiment with 6 replicates each (n= 8). (FIG. 7B) Mitochondrial morphology in live HK-2 cells was analyzed using MitoTracker Deep Red FM (n= 39 cells for Veh; 27 cells for H2O2; 38 cells for H2O2+SP11) after co-treatment of H2O2 (100 µM) and SP11 (250 nM) for 24h. DMSO was used for vehicle control. (FIG.7C) Using the conditions described in FIG. 7B, the average mitochondrial area per cell was normalized to vehicle mean. (CenterAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 line, median; upper and lower dotted lines, quartiles) (FIG. 7D) Cells from FIG. 7C were categorized into three groups with two cut-off criteria, 100% and 60%. (FIG. 7E) HK-2 cells were pre-treated with SP11 (250 nM) or DMSO vehicle control for 30 min. Then the cells were treated with H2O2 (100 µM) in the presence of SP11 or DMSO, and incubated for additional 24h. Mitochondrial fraction was prepared using differential centrifugation method. Densitometry graph was obtained from three independent experiments. Uncropped blots are provided in FIG.22. (FIG.7F) Be(2)-M17 WT and Cys41Ser cells were co-treated with H2O2 (50 µM) and SP11 (250 nM). DMSO was used for vehicle control. Mitochondrial ROS was measured using MitoSOXTMmitochondrial superoxide indicator. MitoSOXTMsignal was normalized to Hoechst 33342. Each data point represents the mean of an independent experiment with 6 replicates each (n= 4). P-value results indicated by stars (ns p ≥ 0.05, * p < 0.05, ** p < 0.01, **** p < 0.0001). Error bar represents standard deviation.

[0013] FIG.8: Secondary structural propensities of residues in α1 helix (aa 15-25) from 30 independent 1.2μs MD simulations for WT, pT34, and pY38.

[0014] FIG.9: Overlay of WT Fis1 and Tyr38Glu Fis1. N-terminus of Tyr38Glu is intact but shifted.

[0015] FIG. 10: Overlay of Thr34Asp covalent dimer and WT Fis1 structure. WT Fis1with intact N-terminus overlaid with Thr34Asp Fis1 covalent dimer and Cys41 shown in green. α1 helix of the WT Fis1 occupies same space as the α2 helix of another monomer suggesting unlikelihood of Cys41 covalent dimerization in WT Fis1 with intact α1 helix.

[0016] FIGS. 11A, 11B: Crystal structures of Thr34Glu and Tyr38Glu Fis1 covalent dimer. Covalent dimer structures of Thr34Glu Fis1 (FIG. 11A) and Tyr38Glu Fis1 (FIG. 11B). Continuous electron density (Composite omit map (σ = 1.0)) of the disulfide bonds between two monomers is highlighted.

[0017] FIG. 12: Partial native SDS-PAGE showing monomers and dimers of different constructs of Fis1 immobilized to HisPurTMNi-NTA magnetic beads at different time points. Equal amounts of beads initially loaded with about 6 µg protein were loaded in each lane. Uncropped images are provided in FIG.23.

[0018] FIG. 13: Partial native SDS-PAGE showing inhibition of dimerization by SP11 treatment and quantification of dimer bands. Equal amounts of beads initially loaded with 20 µg protein (53% of total initial beads; about 10.6 µg protein) were loaded in each lane (left panel). Plotted value represents mean of 3 independent experiments each with 3 technicalAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 replicates (right panel). Error bar represents standard deviation. Two tailed unpaired t-test was performed. P-value results indicated by stars (** p < 0.01).

[0019] FIGS.14A-14D: Inhibitory properties of SP11 and SP22. (FIG.14A) Structure of SP11 and SP22. (FIG. 14B) Inhibition of CPM fluorescence by SP22 in Thr34Asp Fis1 but not in counter screen construct (Cys41Ser, Val56Cys Fis1 double mutant). (FIG. 14C) SP22 inhibits Thr34Asp Fis1 with slightly lower IC50 compared to SP11. (FIG.14D) SP22 binds to Cys41 in Thr34Asp Fis1 mutant but doesn’t bind to Cys41 when Tyr38 is mutated.4 µM protein and 8 µM CPM were used in the assays. Each data point represents mean of 3 independent experiments each with 3 technical replicates. Error bar represents standard deviation.

[0020] FIG. 15: Effect of SP11 on non-stimulated cells in MitoSOX assay. Be(2)-M17 WT and Cys41Ser cells were treated with DMSO or SP11 (250 nM). Mitochondrial ROS was measured using MitoSOXTMmitochondrial superoxide indicator. MitoSOXTMsignal was normalized to Hoechst 33342. Each data point represents the mean of an independent experiment with 6 replicates each (n= 3).

[0021] FIGS.16A-16C: (FIG.16A) Porod Volumes with different Dmax values (+ / - 5Å) using Primus. The q-range used for this analysis was fixed (0.016 < q < 0.45) for comparison. (FIG. 16B) Image number vs Rg and I(0) plots for WT and phosphorylation mimic mutants of Fis1. (FIG.16C) P(r) plots for WT and phosphorylation mimic mutants of Fis1.

[0022] FIG.17: Inhibition of CPM fluorescence by SP11 in Thr34Asp Fis1 at different time points.4 µM protein and 8 µM CPM were used in the assay. Each data point represents mean and error bar represents standard deviation of 4 technical replicates.

[0023] FIG. 18: Intact mass spectra from the same sample used in peptide mapping. Thr34Asp Fis1 + SP11 sample shows a new peak shifted by about 310 Da (weight of SP11 adduct) compared to untreated Thr34Asp Fis1.

[0024] FIGS. 19A-19C: (FIG. 19A) SP1113C NMR spectrum. (FIG. 19B) SP111H NMR spectrum. (FIG.19C) SP11 HRMS quality control data.

[0025] FIGS. 20A-20C: (FIG. 20A) SP2213C NMR spectrum. (FIG. 20B) SP221H NMR spectrum. (FIG.20C) SP22 HRMS quality control data.

[0026] FIG.21: Uncropped SDS-PAGE for FIG.4B.

[0027] FIG.22: Uncropped blot for FIG.7E.

[0028] FIG.23: Uncropped SDS-PAGE for FIG.12.Atty Dkt No.: STAN-2169WO Client Ref.: S23-492

[0029] FIG.24: Sequencing results for WT and Fis1 C41S Be(2)-M17 cells. DETAILED DESCRIPTION

[0030] Provided are compounds that covalently bind to Fis1. Also provided are methods of selectively inhibiting mitochondrial dysfunction. Aspects of the methods include covalently binding a compound to Fis1. Also provided are methods for treating mitochondrial dysfunction in a subject by administering an effective amount of a compound that covalently binds to Fis1. Aspects of the methods include treating a subject that has a neurodegenerative disease, an inflammatory disease or a cardiovascular disease.

[0031] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0032] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and exemplary methods and materials may now be described. Any and all publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It isAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0034] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a droplet" includes a plurality of such droplets and reference to "the discrete entity" includes reference to one or more discrete entities, and so forth. It is further noted that the claims may be drafted to exclude any element, e.g., any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only” and the like in connection with the recitation of claim elements, or the use of a “negative” limitation.

[0035] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. To the extent the definition or usage of any term herein conflicts with a definition or usage of a term in an application or reference incorporated by reference herein, the instant application shall control.

[0036] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. DEFINITIONS

[0037] “Alkyl” refers to a branched or linear, non-cyclic, saturated hydrocarbon group. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, cyclopentyl, and cyclohexyl. In some cases, the alkyl group has 1 to 24 carbon atoms, e.g.1 to 12, 1 to 6, or 1 to 3.

[0038] “Alkenyl” refers to a branched or linear, non-cyclic hydrocarbonyl group that comprises a carbon-carbon double bond. Exemplary alkenyl groups include ethenyl, n- propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, and tetracosenyl.Atty Dkt No.: STAN-2169WO Client Ref.: S23-492

[0039] “Alkynyl” refers to a branched or linear, non-cyclic hydrocarbonyl group that comprises a carbon-carbon triple bond. Exemplary alkynyl groups include ethynyl and n- propynyl.

[0040] “Cycloalkyl” refers to a cyclic, saturated hydrocarbon group. Similarly, “cycloalkenyl” refers to a cyclic group having carbon-carbon double bond whereas “cycloalkynyl” refers to a cyclic group having carbon-carbon triple bond.

[0041] “Heterocyclyl” refers to a cyclic group that contains a heteroatom (e.g., O, S, N) as a ring atom and that is not aromatic (i.e., distinguishing heterocyclyl groups from heteroaryl groups). Exemplary heterocyclyl groups include piperidinyl, tetrahydrofuranyl, dihydrofuranyl, and thiocanyl.

[0042] “Aryl” refers to an aromatic group containing at least one aromatic ring, wherein each of the atoms in the ring are carbon atoms, i.e., none of the ring atoms are heteroatoms (e.g., O, S, N). In some cases, the aryl group has a second aromatic ring, e.g., that is fused to the first aromatic ring. Exemplary aryl groups are phenyl, naphthyl, biphenyl, diphenylether, diphenylamine, and benzophenone.

[0043] “Heteroaryl” refers to an aromatic group containing at least one aromatic ring, wherein at least one of the atoms in the aromatic ring is a heteroatom (e.g., O, S, N). Exemplary heteroaryl groups include those obtained from removing a hydrogen atom from pyridine, pyrimidine, furan, thiophene, or benzothiophene.

[0044] The term “substituted” refers the removal of one or more hydrogens from an atom (e.g., from a C or N atom) and their replacement with a different group. For instance, a hydrogen atom on a phenyl (-C6H5) group can be replaced with a methyl group to form a - C6H4CH3 group. Thus, the -C6H4CH3 group can be considered a substituted aryl group. As another example, two hydrogen atoms from the second carbon of a propyl (-CH2CH2CH3) group can be replaced with an oxygen atom to form a -CH2C(O)CH3group, which can be considered a substituted alkyl group. However, replacement of a hydrogen atom on a propyl (-CH2CH2CH3) group with a methyl group (e.g., giving -CH2CH(CH3)CH3) is not considered a “substitution” as used herein since the starting group and the ending group are both alkyl groups. However, if the propyl group was substituted with a methoxy group, thereby giving a -CH2CH(OCH3)CH3 group, the overall group can no longer be considered “alkyl”, and thus is “substituted alkyl”. Thus, in order to be considered a substituent, the replacement group is aAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 different type than the original group. In addition, groups are presumed to be unsubstituted unless described as substituted. For instance, the term “alkyl” and “unsubstituted alkyl” are used interchangeably herein.

[0045] The term “substituted versions thereof” refers to both substituted and unsubstituted categories being named. For instance, the recitation of “alkyl, aryl, heteroaryl, halo, nitro or a substituted version thereof” refers to the groups alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, and nitro.

[0046] Exemplary substituents include deuterium (D), alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, acyl, alkoxy, amino, azido, carbonyl, carboxy, cyano, ether, halo, hydroxy, nitro, thiol, and thioether, or a substituted version thereof.

[0047] In some cases, the substitutions can themselves be further substituted with one or more groups. For example, the group -C6H4CH2CH3 can be considered as substituted aryl, i.e., an aryl group substituted with the ethyl, which is an alkyl group. Furthermore, the ethyl group can itself be substituted with a pyridyl group to form -C6H4CH2CH2C5H5N, wherein - C6H4CH2CH2C5H5N can also be considered as a substituted aryl group as the term is used herein. In some cases, the substituents are not substituted with any other groups.

[0048] Divalent groups are also described herein. The term “alkylene” refers to the divalent version of an alkyl group, i.e., an alkylene group is a divalent, branched or linear, cyclic, or non-cyclic, saturated hydrocarbon group. Exemplary alkylene groups include diylmethane (- CH2-, which is also known as a methylene group), 1,2-diylethane (-CH2CH2-), and 1,1- diylethane (i.e., a CHCH3fragment where the first atom has two single bonds to other two different groups). The term “arylene” refers to the divalent version of an aryl group, e.g., 1,4- diylbenzene refers to a C6H4 fragment wherein two hydrogens that are located para to one another are removed and replaced with single bonds to other groups. The terms “alkenylene”, “alkynylene”, “heteroarylene”, and “heterocyclene” are also used herein.

[0049] “Acyl” refers to a group of formula -C(O)R wherein R is alkyl, alkenyl, alkynyl, or substituted versions thereof. For example, the acetyl group has formula -C(O)CH3. “Carbonyl” refers to a diradical group of formula -C(O)-.

[0050] “Alkoxy” refers to a group of formula -O(alkyl). Similar groups can be derived from alkenyl, alkynyl, aryl, heteroaryl, and other groups.Atty Dkt No.: STAN-2169WO Client Ref.: S23-492

[0051] “Amino” refers to the group -NRXRYwherein RXand RYare each independently H or a non-hydrogen substituent. Exemplary non-hydrogen substituents include alkyl groups (e.g., methyl, ethyl, and isopropyl).

[0052] “Carbonyl” refers to a diradical group of formula -C(O)-.

[0053] “Carboxy” is used interchangeably with carboxyl and carboxylate to refer to the - CO2H group and salts thereof.

[0054] “Ether” refers to a diradical group of formula -O-. For instance, if the ether group is connected to an alkyl group, then the overall group is an alkoxy group (e.g., -OCH3or methoxy). If the ether is connected to a carbonyl group, then the overall group is an ester group of formula -OC(O)-.

[0055] “Halo” and “halogen” refer to the fluoro, chloro, bromo, and iodo groups.

[0056] “Nitro” refers to the group of formula -NO2.

[0057] Unless otherwise specified, reference to an atom is meant to include all isotopes of that atom. For example, reference to H includes1H,2H (i.e., D or deuterium) and3H (i.e., tritium), and reference to C is includes both12C and all other isotopes of carbon (e.g.,13C). Unless specified otherwise, groups include all possible stereoisomers.

[0058] The term “covalent binding” means a chemical bond that involves the sharing of electrons between atoms.

[0059] The terms “non-covalent binding” or “non-covalent interaction” describe interactions that do not involve the sharing of electrons. Examples of non-covalent interactions include hydrophobic interactions, Van der Waals interactions, electrostatic interactions and ionic interactions.

[0060] The terms “subject” and “patient” are used interchangeably herein.

[0061] By “treatment” is meant that at least an amelioration of the symptoms associated with the condition afflicting the subject is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the condition being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g. terminated, such that the subject no longer suffers from the condition, or at least the symptoms that characterize the condition. Thus treatment includes: (i) prevention, that is, reducing the riskAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression to a harmful state; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease; and / or (iii) relief, that is, causing the regression of clinical symptoms. COMPOUNDS

[0062] As discussed above, the present disclosure provides compounds that covalently bind to mitochondrial fission protein 1 (Fis1). In certain embodiments, the compounds selectively bind to Fis1. In some embodiments, the compound binds to the cysteine (e.g., Cys41) of Fis1. In some embodiments, the compound selectively binds to the cysteine (e.g., Cys41) of Fis1.

[0063] In some embodiments, the compound is a compound of formula (I).

[0064] Provided are compounds of formula (I): (I) wherein: A is a tricyclic ring or a substituted version thereof; Z is a linker; and B is a reactive group that covalently binds to S.

[0065] As described above, A is a tricyclic ring or a substituted version thereof. In some cases, A is a tricyclic ring. In some cases, A is a substituted tricyclic ring. In some cases, the tricyclic ring is an aromatic tricyclic ring. In some cases, the tricyclic ring is a non-aromatic tricyclic ring. In some cases, the tricyclic ring is a heterocyclic ring. In some cases, the tricyclic is a carbocyclic ring. The tricyclic ring may comprise any combination of three fused 4-7- membered rings. In some cases, the tricyclic ring comprises three fused 6-membered rings. In some cases, the tricyclic ring comprises three fused 6-membered rings comprising at least 1 S and 1 N. In some cases, the tricyclic ring is a phenothiazine.

[0066] As described above, Z is a linker. Z may be any linker that is capable of linking A and B. Examples of linkers include, but are not limited to, -(CH2)nC(O)-, -C(O)(CH2)n-, - (CH2)nNHC(O)-, -C(O)NH(CH2)n-, -(CH2)nC(O)NH(CH2)n-, -(CH2)nNHC(O)(CH2)n-, -NH(CH2)nC(O)-, -C(O)(CH2)nNH-, and -(CH2)n- or a substituted version thereof, wherein each n is independentlyAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 selected from 0 or an integer from 1 to 10. In some cases, Z is -(CH2)nNHC(O)- or a substituted version thereof. In some cases, Z is -(CH2)n- or a substituted version thereof.

[0067] As described above, B is a reactive group that covalently binds to S. B may be any reactive group that is capable of covalently binding to S. By reactive group, it is meant a group that reacts with S. In some cases, B is selected from the group consisting of CH2Cl, CH2I, CH2Br, CH2OTs, CH2OTf, CH2OMs, and alkenyl. In some cases, B is CH2Cl. In some cases, B is CH2I. In some cases, B is CH2Br. In some cases, B is CH2OTs. In some cases, B is CH2OMs. In some cases, B is alkenyl, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, and tetracosenyl. In some cases, B is ethenyl.

[0068] In some embodiments, the compound is a compound of formula (II).

[0069] Provided are compounds of formula (II): wherein:Y1is a heteroatom or CH2; Y2is a heteroatom or CH; Z is a linker; and B is a reactive group that covalently binds to S.

[0070] As described above, Y1is a heteroatom or CH2. In some cases, Y1is a heteroatom, such as O, S or N. In some cases, Y1is CH2. In some cases, Y1is S.

[0071] As described above, Y2is a heteroatom or CH. In some cases, Y2is a heteroatom, such as O, S or N. In some cases, Y2is CH. In some cases, Y2is N.

[0072] As described above, Z is a linker. Z may be any linker that is capable of linking A and B. Examples of linkers include, but are not limited to, -(CH2)nC(O)-, -C(O)(CH2)n-, - (CH2)nNHC(O)-, -C(O)NH(CH2)n-, -(CH2)nC(O)NH(CH2)n-, -(CH2)nNHC(O)(CH2)n-, -NH(CH2)nC(O)-, -C(O)(CH2)nNH-, and -(CH2)n- or a substituted version thereof, wherein each n is independently selected from 0 or an integer from 1 to 10. In some cases, Z is -(CH2)nNHC(O)- or a substituted version thereof. In some cases, Z is -(CH2)n- or a substituted version thereof.Atty Dkt No.: STAN-2169WO Client Ref.: S23-492

[0073] As described above, B is a reactive group that covalently binds to S. B may be any reactive group that is capable of covalently binding to S. By reactive group, it is meant a group that reacts with S. In some cases, B is selected from the group consisting of CH2Cl, CH2I, CH2Br, CH2OTs, CH2OTf, CH2OMs, and alkenyl. In some cases, B is CH2Cl. In some cases, B is CH2I. In some cases, B is CH2Br. In some cases, B is CH2OTs. In some cases, B is CH2OMs. In some cases, B is alkenyl, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, and tetracosenyl. In some cases, B is ethenyl.

[0074] In some embodiments, the compound is a compound of formula (III).

[0075] Provided are compounds of formula (III): wherein:Y1is a heteroatom or CH2; Y2is a heteroatom or CH; each R1and R2is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, acyl, alkoxy, amino, azido, carbonyl, carboxy, cyano, ether, halo, hydroxy, and nitro, or a substituted version thereof; R3is selected from the group consisting of H, alkyl, alkenyl, alkynyl, acyl, carbonyl, and carboxy, or a substituted version thereof; B is a reactive group that covalently binds to S; and m is 0 or an integer from 1 to 10.

[0076] As described above, Y1is a heteroatom or CH2. In some cases, Y1is a heteroatom, such as O, S or N. In some cases, Y1is CH2. In some cases, Y1is S.

[0077] As described above, Y2is a heteroatom or CH. In some cases, Y2is a heteroatom, such as O, S or N. In some cases, Y2is CH. In some cases, Y2is N.

[0078] As described above, each R1and R2is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, acyl, alkoxy, amino, azido, carbonyl, carboxy, cyano, ether, halo, hydroxy, and nitro, or a substituted version thereof.Atty Dkt No.: STAN-2169WO Client Ref.: S23-492

[0079] In some cases, R1is selected from the group consisting of H, alkyl, alkenyl, alkynyl, acyl, alkoxy, amino, azido, carbonyl, carboxy, cyano, ether, halo, hydroxy, and nitro, or a substituted version thereof. In some cases, R1is H. In some cases, R1is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or C1-4 alkyl or C1-4 substituted alkyl, or C1-3 alkyl or C1-3substituted alkyl. In some cases, R1is alkenyl or substituted alkenyl, such as C2-6alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In some cases, R1is alkynyl or substituted alkynyl, such as C2-6 alkynyl or C2-6substituted alkynyl, or C2-4alkynyl or C2-4substituted alkynyl, or C2-3alkynyl or C2-3substituted alkynyl. In some cases, R1is acyl. In some cases, R1is alkoxy. In some cases, R1is amino. In some cases, R1is azido. In some cases, R1is carbonyl. In some cases, R1is carboxy. In some cases, R1is cyano. In some cases, R1is ether. In some cases, R1is halo. In some cases, R1is hydroxy. In some cases, R1is nitro.

[0080] In some cases, R2is selected from the group consisting of H, alkyl, alkenyl, alkynyl, acyl, alkoxy, amino, azido, carbonyl, carboxy, cyano, ether, halo, hydroxy, and nitro, or a substituted version thereof. In some cases, R2is H. In some cases, R2is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or C1-4 alkyl or C1-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In some cases, R2is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6substituted alkenyl, or C2-4alkenyl or C2-4substituted alkenyl, or C2-3alkenyl or C2-3 substituted alkenyl. In some cases, R2is alkynyl or substituted alkynyl, such as C2-6 alkynyl or C2-6 substituted alkynyl, or C2-4 alkynyl or C2-4 substituted alkynyl, or C2-3 alkynyl or C2-3 substituted alkynyl. In some cases, R2is acyl. In some cases, R2is alkoxy. In some cases, R2is amino. In some cases, R2is azido. In some cases, R2is carbonyl. In some cases, R2is carboxy. In some cases, R2is cyano. In some cases, R2is ether. In some cases, R2is halo. In some cases, R2is hydroxy. In some cases, R2is nitro.

[0081] As described above, R3is selected from the group consisting of H, alkyl, alkenyl, alkynyl, acyl, carbonyl, and carboxy, or a substituted version thereof. In some cases, R3is H. In some cases, R3is alkyl or substituted alkyl, such as C1-6alkyl or C1-6substituted alkyl, or C1-4alkyl or C1-4substituted alkyl, or C1-3alkyl or C1-3substituted alkyl. In some cases, R3is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In some cases, R3is alkynyl or substituted alkynyl, such as C2-6alkynyl or C2-6substituted alkynyl, or C2-4alkynyl or C2-4Atty Dkt No.: STAN-2169WO Client Ref.: S23-492 substituted alkynyl, or C2-3alkynyl or C2-3substituted alkynyl. In some cases, R3is acyl. In some cases, R3is carbonyl. In some cases, R3is carboxy. In some cases, R3is methyl.

[0082] As described above, B is a reactive group that covalently binds to S. B may be any reactive group that is capable of covalently binding to S. By reactive group, it is meant a group that reacts with S. In some cases, B is selected from the group consisting of CH2Cl, CH2I, CH2Br, CH2OTs, CH2OTf, CH2OMs, and alkenyl. In some cases, B is CH2Cl. In some embodiments, B is CH2I. In some cases, B is CH2Br. In some cases, B is CH2OTs. In some cases, B is CH2OMs. In some cases, B is alkenyl, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, and tetracosenyl. In some cases, B is ethenyl.

[0083] As described above, m is 0 or an integer from 1 to 10. In some cases, m is 0. In some case, m is an integer from 1 to 10 including, e.g., an integer from 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some cases, m is 0. In some cases, m is 1. In some cases, m is 2. In some cases, m is 3. In some cases, m is 4. In some cases, m is 5. In some cases, m is 6. In some cases, m is 7. In some cases, m is 8. In some cases, m is 9. In some cases, m is 10.

[0084] In some embodiments, the compound has the structure .

[0085] In some embodiments, the compound has the .METHODS

[0086] As discussed above, the present disclosure provides methods of selectively inhibiting mitochondrial dysfunction. In embodiments, the method of selectively inhibiting mitochondrial dysfunction comprises covalently binding a compound to Fis1. Fis1 may be of any species. In some embodiments, Fis1 is mammalian Fis1 (e.g., human Fis1, mouse Fis1, rat Fis1, primate Fis1 and the like). In some embodiments, Fis1 is human Fis1. In someAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 embodiments, Fis1 is activated Fis1. Activated Fis1 comprises an exposed cysteine (e.g., Cys41). In some embodiments, the method comprises covalently binding a compound to a cysteine (e.g., Cys41) in Fis1. In some embodiments, the compound selectively covalently binds to the cysteine (e.g., Cys41) of Fis1. Compounds of interest that covalently bind to Fis1 are discussed in detail above. For instance, the compound can have formula (I), (II), or (III). In some embodiments, the compound is SP11 or SP22. In some embodiments, the compound is SP11. In some embodiments, the compound is SP22.

[0087] In some embodiments, covalently binding a compound to Fis1 occurs in vivo. In some embodiments, covalently binding a compound to Fis1 occurs in vitro. In some embodiments, covalently binding a compound to Fis1 occurs ex vivo.

[0088] In some cases, the mitochondrial dysfunction is caused by inflammatory cell stress. In some cases, the mitochondrial dysfunction is due to excessive fragmentation. In some cases, the mitochondrial dysfunction and fragmentation is due to increased oxidative stress. In some cases, oxidative stress is due to toxic agents. In some cases, the toxic agents can be aldehydes. In some cases, the toxic agents can be unfolded proteins.

[0089] In some embodiments, the method reduces mitochondrial fragmentation, reduces the production of reactive oxygen species (ROS), or a combination thereof.

[0090] In some embodiments, the method reduces mitochondrial fragmentation. By mitochondrial fragmentation, it is meant the process that occurs when mitochondria break down into smaller fragments. In some embodiments, mitochondrial fragmentation that occurs when a compound is covalently bound to Fis1 is reduced compared to the mitochondrial fragmentation that occurs when no compound is bound to Fis1. In some embodiments, mitochondrial fragmentation that occurs when a compound is covalently bound to Fis1 is reduced compared to the mitochondrial fragmentation that occurs when a compound is non-covalently bound to Fis1. Mitochondrial fragmentation may be measured with any convenient method including, but not limited to, microscopy methods of quantifying mitochondrial fragmentation (e.g., fluorescence staining, immunostaining, immunohistochemistry, etc.).

[0091] In some embodiments, the method reduces mitochondrial reactive oxygen species (ROS) production. In some embodiments, production of mitochondrial ROS that occurs when a compound is covalently bound to Fis1 is reduced compared to the production ofAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 mitochondrial ROS that occurs when no compound is bound to Fis1. In some embodiments, production of mitochondrial ROS that occurs when a compound is covalently bound to Fis1 is reduced compared to the production of mitochondrial ROS that occurs when a compound is non-covalently bound to Fis1. Production of mitochondrial ROS may be measured with any convenient method including, but not limited to, use of mitochondrial superoxide indicators (e.g., MitoSOX™) that can be detected using various detection methods (e.g., fluorescence microscopy, flow cytometry and the like).

[0092] Also provided are methods for treating mitochondrial dysfunction in a subject by administering an effective amount of a compound that covalently binds to Fis1. In some embodiments, the subject has a neurodegenerative disease, an inflammatory disease or a cardiovascular disease. In some cases, the subject has a neurodegenerative disease including, but not limited to, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS) and neuronopathic Gaucher’s disease. In some cases, the subject has an inflammatory disease including, but not limited to, sepsis, inflammatory bowel disease (IBD), graft-versus-host disease (GvHD) and acute kidney injury. In some cases, the subject has a cardiovascular disease including, but not limited to, cardiac ischemia reperfusion, stroke, Type 2 diabetes, myocardial infarction or stroke. In some cases, the subject has acute radiation syndrome (ARS), which is also referred to herein as radiation sickness. Such ARS can cause inflammation in some cases. In some cases, the patient has mitochondrial dysfunction from irradiation, e.g. from myeloablative conditioning, such as total body irradiation (TBI), e.g. as part of a preventative regimen for transplantation of hematopoietic stem cells or bone marrow.

[0093] An effective amount of a subject compounds may depend, at least, on the particular method of use, the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition. A “therapeutically effective amount” of a composition is a quantity of a compound sufficient to achieve a desired effect in a subject (e.g., patient) being treated. For example, this may be the amount of a subject compound necessary to prevent, inhibit, reduce or relieve a disease or disorder in a subject. Ideally, a therapeutically effective amount of a compound is an amount sufficient to prevent, inhibit, reduce or relieve a disease or disorder in a subject without causing a substantial cytotoxic effect on normal host cells in the subject.Atty Dkt No.: STAN-2169WO Client Ref.: S23-492

[0094] The route of administration may be selected according to a variety of factors including, but not limited to, the condition to be treated, the formulation and / or device used, the subject to be treated, and the like. Routes of administration useful in the disclosed methods include, but are not limited to, oral and parenteral routes, such as intravenous (iv), intraperitoneal (ip), rectal, topical, ophthalmic, nasal, otic, intrathecal, and transdermal. Formulations for these dosage forms are described herein.

[0095] The specific dose level and frequency of dosage for any particular subject may be varied and may depend upon a variety of factors, including the activity of the subject compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex and diet of the subject, mode and time of administration, rate of excretion, drug combination, and severity of the condition of the host undergoing therapy.

[0096] In some embodiments, multiple doses of a compound are administered. The frequency of administration of a compound can vary depending on any of a variety of factors, e.g., severity of the symptoms, condition of the subject, etc. For example, in some embodiments, a compound is administered once per month, twice per month, three times per month, every other week, once per week (qwk), twice per week, three times per week, four times per week, five times per week, six times per week, every other day, daily (qd / od), twice a day (bds / bid), or three times a day (tds / tid), etc. COMPOSITIONS

[0097] Provided are compositions comprising a compound as disclosed herein. For example, the compound can have formula (I), (II), or (III). In some embodiments, the compound is SP11 or SP22. In some embodiments, the compound is SP11. In some embodiments, the compound is SP22. In embodiments, the disclosed compounds are useful for the treatment of mitochondrial dysfunction. Accordingly, pharmaceutical compositions comprising at least one disclosed compound are described herein. For example, the present disclosure provides pharmaceutical compositions that include a therapeutically effective amount of a compound of the present disclosure (or a pharmaceutically acceptable salt or solvate or hydrate thereof) and a pharmaceutically acceptable excipient.

[0098] A pharmaceutical composition that includes a subject compound may be administered to a patient alone, or in combination with other supplementary active agents. For example, one or more compounds according to the present disclosure can beAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 administered to a patient with or without supplementary active agents. The pharmaceutical compositions may be manufactured using any of a variety of processes, including, but not limited to, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, lyophilizing, and the like. The pharmaceutical composition can take any of a variety of forms including, but not limited to, a sterile solution, suspension, emulsion, spray dried dispersion, lyophilisate, tablet, microtablets, pill, pellet, capsule, powder, syrup, elixir or any other dosage form suitable for administration.

[0099] A compound of the present disclosure may be administered to a subject using any convenient means capable of resulting in the desired reduction in disease condition or symptom. Thus, a compound can be incorporated into a variety of formulations for therapeutic administration. More particularly, a compound can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable excipients, carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, creams, gels, foams, solutions, suppositories, injections, inhalants, aerosols, and the like. In some embodiments, a compound of the present disclosure may be formulated for oral administration (e.g., as an oral dosage). In some embodiments, a compound of the present disclosure may be formulated for intravenous administration.

[0100] Formulations for pharmaceutical compositions are described in, for example, Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995, which describes examples of formulations (and components thereof) suitable for pharmaceutical delivery of the disclosed compounds. Pharmaceutical compositions that include at least one of the compounds can be formulated for use in human or veterinary medicine. Particular formulations of a disclosed pharmaceutical composition may depend, for example, on the mode of administration and / or on the location of the subject to be treated. In some embodiments, formulations include a pharmaceutically acceptable excipient in addition to at least one active ingredient, such as a compound of the present disclosure. In other embodiments, other medicinal or pharmaceutical agents, for example, with similar, related or complementary effects on the disease or condition being treated can also be included as active ingredients in a pharmaceutical composition.Atty Dkt No.: STAN-2169WO Client Ref.: S23-492

[0101] Pharmaceutically acceptable carriers useful for the disclosed methods and compositions may depend on the particular mode of administration being employed. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can optionally contain non-toxic auxiliary substances (e.g., excipients), such as wetting or emulsifying agents, preservatives, and pH buffering agents, and the like.

[0102] The dosage form of a disclosed pharmaceutical composition may be determined by the mode of administration chosen. For example, injectable fluids, topical or oral dosage forms may be employed. Topical preparations may include eye drops, ointments, sprays and the like. Oral formulations may be liquid (e.g., syrups, solutions or suspensions), or solid (e.g., powders, pills, tablets, or capsules). Formulations for intravenous administration may be used, wherein the formulation comprises a suitable carrier. Such suitable carriers include sterile saline although other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those of ordinary skill in the art. Methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.

[0103] Certain embodiments of the pharmaceutical compositions that include a subject compound may be formulated in unit dosage form suitable for individual administration of precise dosages. The amount of active ingredient administered may depend on the subject being treated, the severity of the affliction, and the manner of administration, and is known to those skilled in the art. In certain instances, the formulation to be administered contains a quantity of the compound disclosed herein in an amount effective to achieve the desired effect in the subject being treated.

[0104] A disclosed compound can be administered alone, as the sole active pharmaceutical agent, or in combination with one or more additional compound of the present disclosure or in conjunction with other agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are administered simultaneously or at different times, or the therapeutic agents can be administered together as a single composition combining two or more therapeutic agents. Thus, the pharmaceutical compositions disclosed herein containing a compound of the present disclosure optionally include other therapeutic agents. Accordingly, certain embodiments are directed to suchAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 pharmaceutical compositions, where the composition further includes a therapeutically effective amount of an agent selected as is known to those of skill in the art. UTILITY

[0105] Compounds that bind to Fis1 can be used to treat mitochondrial dysfunction associated pathologies. Mitochondrial dysfunction associated pathologies include, but are not limited to, neurodegeneration diseases such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and neuronopathic Gaucher’s disease, inflammatory diseases such as sepsis, inflammatory bowel disease, and acute kidney injury, and cardiovascular diseases such as ischemia reperfusion, stroke, and Type 2 diabetes.

[0106] Compounds provided herein (e.g., SP11 or SP22) can covalently bind to the cysteine of Fis1, thereby reducing its engagement with other targets. By binding to Fis1, the compound is able to disrupt pathological Fis1 interactions and promote mitochondrial integrity and health in tissues. Many modifications of phenothiazines are orally bioavailable, have longer half-lives and are blood brain barrier permeable. The compounds provided herein are irreversible inhibitors and have excellent potency, and therefore have beneficial effects even at lower dose, minimizing potential toxicological liabilities. Treatment with the compounds provided herein protect mitochondrial integrity and function under cellular stress. EMBODIMENTS 1. A method of selectively inhibiting mitochondrial dysfunction, the method comprising: covalently binding a compound to Fis1. 2. The method of clause 1, wherein the compound binds to Cys41 of Fis1. 3. The method of clause 1 or clause 2, wherein Fis1 is human Fis1. 4. The method of any one of clauses 1-3, wherein the method reduces mitochondrial fragmentation. 5. The method of any one of clauses 1-4, wherein the method reduces mitochondrial reactive oxygen species (ROS) production. 6. The method of any one of clauses 1-5, wherein the compound is of formula (I): (I) wherein: A is a tricyclic ring or a substituted version thereof;Atty Dkt No.: STAN-2169WO Client Ref.: S23-492 Z is a linker; and B is a reactive group that covalently binds to S. 7. The method of any one of clauses 1-6, wherein the compound is of formula (II): wherein:Y1is a heteroatom or CH2; Y2is a heteroatom or CH; Z is a linker; and B is a reactive group that covalently binds to S. 8. The method of any one of clauses 1-7, wherein the compound is of formula (III): wherein:Y1is a heteroatom or CH2; Y2is a heteroatom or CH; each R1and R2is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, acyl, alkoxy, amino, azido, carbonyl, carboxy, cyano, ether, halo, hydroxy, and nitro, or a substituted version thereof; R3is selected from the group consisting of H, alkyl, alkenyl, alkynyl, acyl, carbonyl, and carboxy, or a substituted version thereof; B is a reactive group that covalently binds to S; and m is 0 or an integer from 1 to 10. 9. The method of any one of clauses 6-8, wherein B is selected from the group consisting of CH2Cl, CH2I, CH2Br, CH2OTs, CH2OTf, CH2OMs, and alkenyl. 10. The method of any one of clauses 1-9, wherein the compound has structure:Atty Dkt No.: STAN-2169WO Client Ref.: S23-492 . 11. The method of any one compound has structure:. 12. A method for treatingsubject, the method comprising: administering an effective amount of a compound that covalently binds to Fis1 to the subject in need thereof. 13. The method of clause 12, wherein the mitochondrial dysfunction comprises mitochondrial fragmentation. 14. The method of clause 12 or clause 13, wherein the compound is of formula (I): (I) wherein: A is a tricyclic ring or a substituted version thereof; Z is a linker; and B is a reactive group that covalently binds to S. 15. The method of any one of clauses 12-14, wherein the compound is of formula (II): wherein:Y1is a heteroatom or CH2; Y2is a heteroatom or CH; Z is a linker; and B is a reactive group that covalently binds to S.Atty Dkt No.: STAN-2169WO Client Ref.: S23-492 16. The method of any one of clauses 12-16, wherein the compound is of formula (III): wherein:Y1is a heteroatom or Y2is a heteroatom or CH; each R1and R2is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, acyl, alkoxy, amino, azido, carbonyl, carboxy, cyano, ether, halo, hydroxy, and nitro, or a substituted version thereof; R3is selected from the group consisting of H, alkyl, alkenyl, alkynyl, acyl, carbonyl, and carboxy, or a substituted version thereof; B is a reactive group that covalently binds to S; and m is 0 or an integer from 1 to 10. 17. The method of any one of clauses 14-16, wherein B is selected from the group consisting of CH2Cl, CH2I, CH2Br, CH2OTs, CH2OTf, CH2OMs, and alkenyl. 18. The method of any one of clauses 12-17, wherein the compound has structure: . 19. The method of any onethe compound has structure: . 20. The method of any one ofsubject has a neurodegenerative disease.Atty Dkt No.: STAN-2169WO Client Ref.: S23-492 21. The method of clause 20, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amytrophic lateral sclerosis, and neuronopathic Gaucher’s disease. 22. The method of any one of clauses 12-19, wherein the subject has an inflammatory disease. 23. The method of clause 22, wherein the inflammatory disease is selected from the group consisting of sepsis, inflammatory bowel disease, and acute kidney injury. 24. The method of any one of clauses 12-19, wherein the subject has a cardiovascular disease. 25. The method of clause 24, wherein the cardiovascular disease is selected from the group consisting of cardiac ischemia reperfusion, stroke, and Type 2 diabetes. EXPERIMENTAL

[0107] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Numerous modifications and variations of the invention are possible in view of the teachings herein and, therefore, are within the scope of the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Example 1

[0108] The N-Terminal Region of Fis1 is dynamic.

[0109] To investigate the dynamic nature of the N-terminal region of Fis1, molecular dynamics (MD) simulations of the wildtype (WT) Fis1 were performed. Data obtained from 30 MD simulations of 1.2 µs of WT Fis1 showed that the first 10 amino acids of the N-terminus are extremely flexible with average root mean square fluctuations (RMSF) of backbone atoms (Cα, N and C) for these residues ranging from 2.3 to 13.6 Å (FIG.1A). Despite being structured, residues of α1 helix (aa 11-26) also showed high RMSF values (1.2-2.3 Å), compared toAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 residues that are part of the other helices in the protein (FIG.1A). Interestingly, the backbone atoms of the residues making the turn between α1-α2 helices (aa 27-31) also showed larger fluctuations (1.2-2.2 Å) compared to the rest of the protein suggesting the potential mobility of the entire α1 helix under perturbations (FIG. 1A). This high fluctuation of backbone residues of α1 helix suggests that perturbations such as phosphorylation events in this or neighboring helix could have large implications on the integrity of α1 helix.

[0110] The effect of Fis1 phosphorylation at Thr34 or Tyr38 on the dynamics of the N- terminus was determined. These phosphorylations were shown to increase Fis1-mediated Drp1 recruitment to mitochondria and increased mitochondrial fission. Thr34 and Tyr38 are on α2 helix in close proximity to Glu24 and Glu18 on the α1 helix, respectively, and the carboxylic acid side chains of these residues are even closer and face each other at a distance of 3.4Å and 2.7Å, respectively (FIG.1B). Based on the proximity of the phosphorylation sites on the α2 helix and the negatively charged residues on the α1 helix, the phosphorylation of Thr34 or Tyr38 could introduce significant charge repulsions between the phosphate group and the carboxylic acid side chain of Glu24 or Glu18, thus making the N-terminus even more flexible. This prediction was supported by 30 independent 1.2 μs-MD simulations of phosphorylated forms of Fis1, Thr34 or Tyr38 (FIG.1A). The effect of Tyr38 phosphorylation was much greater than that induced by Thr34 phosphorylation; the RMSF values for α1 helix (aa 11-26) and turn between α1-α2 helices (aa 27-31) were higher for the phosphorylated forms (FIG.1A). The N-terminus of the phosphorylated forms of Fis1 showed instances of α1 helix unfolding (FIG.1C).

[0111] The α1 helix of Fis1 unfolds and becomes disordered upon activation.

[0112] Using MD simulations, instances of disruption of integrity of the α1 helix were observed, particularly in the phosphorylated forms of Fis1. The average propensity of residues in α1 helix (aa 15-25) to form α-helical structure using the DSSP algorithm were calculated in 30 independent 1.2 μs-MD simulations for each of WT, pThr34, and pTyr38. Compared to unphosphorylated Fis1 (WT), the propensity of amino acid residues 15-25 to form α-helical structure in pThr34 and pTyr38 were lower by 0.19-2.22% and 1.91-6.44%, respectively (FIG. 1C, FIG. 8). φ and ψ angle analysis were also performed in these simulations. It was found that there were differences in standard deviations, but the differences in the average φ and ψ values were smaller (Table 1). MD simulation data indicates the potential fluctuation andAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 unfolding of the α1 helix, which could enable the exposure of concave hydrophobic surface in Fis1 for protein-protein interactions.

[0113] Table 1. Average φ and ψ values from the dihedral data sets in 30 independent 1.2μs MD simulations for WT, pT34, and pY38. GluFis1 and Tyr38Glu Fis1, were crystallized and X-ray structures were determined (FIG.2A). Each of these mutants was previously shown to be sufficient to increase Drp1 recruitment to mitochondria and subsequent mitochondrial fission in cell-based studies. These mutants provided information about the structural changes that activate Fis1. Consistently, it was observed that the first 30 residues including the α1 helix were missing from the structure providing strong evidence that the α1 helix is unfolded and disordered in the phosphorylated forms, whereas it was intact in the WT (FIG.2A). In one of the instances, Tyr38Glu Fis1 mutant was crystallized in an intermediate state, where the α1-helix was still intact, but was shifted away from the rest of the protein, as compared to the WT (FIG.9). This observation further corroborates the results from the MD simulations and the dynamic nature of the N-terminus of Fis1.

[0115] To confirm that these experimental observations are not crystallographic artifacts, and that similar differences in the structures can be observed in solution, size exclusion chromatography coupled with small angle X-ray scattering was used (FIG. 2B, FIG. 16 and Tables 2A, 2B). In solution, Thr34Asp Fis1 and Tyr38Glu Fis1 had different structures compared to the WT Fis1 (FIG.2B). If the α1 helix is unfolded and away from the rest of the protein, then the structures of the Fis1 phosphorylation mimic mutants should appear larger compared to WT Fis1. Indeed, based on the SAXS data, the Porod volumes for the WT, Thr34Asp and Tyr38Glu Fis1 were 24,400Å3, 25,900Å3and 29,900Å3, respectively. These dataAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 are consistent with the MD simulations, where Tyr38Glu Fis1 had the biggest difference followed by Thr34Asp Fis1 and WT Fis1 (FIGS.1A, 1C).

[0116] α1-helix unfolding and Fis1 activation expose the one and only cysteine, Cys41, in Fis1.

[0117] Human Fis1 protein contains a single cysteine, Cys41, in the entire protein. Cys41 and a stretch of hydrophobic amino acids around this residue is conserved in all vertebrates suggesting a critical functional role (FIG. 3A). A previous study showed that Fis1 forms a Cys41-mediated covalent dimer in vitro, but the formation of the dimer was minimal, and the propensity of dimerization increased after unfolding and refolding. These data suggest that Cys41 is not exposed in the basal state but can be exposed and can mediate dimerization upon unfolding. Additionally, Fis1 has been shown to be activated through phosphorylation under oxidative stress induced by cisplatin, lipopolysaccharide (LPS), rotenone or hydrogen peroxide treatments in cells in culture and oxidizable cysteine residues are well known to act as redox sensors. Hence, the Cys41 residue could work in a similar way, with the unfolding of N-terminus exposing the thiol group of Cys41, thus making it available for oxidation.

[0118] All-atom MD simulations of unphosphorylated Fis1, pThr34 and pTyr38 were performed and the solvent-accessible surface area (SASA) was calculated for Cys41 based on the LCPO method. Cys41 in both pThr34 and pTyr38 were more exposed to the solvent than that in the WT-Fis1, as expected (FIG. 3B). The average SASAs of the Cys41 in the unphosphorylated Fis1, pThr34 and pTyr38 during the 1.2 μs × 30 MD simulations were 12.5±8.5 Å2, 13.9±10.0 Å2and 29.2±14.7 Å2, respectively (FIG. 3B). These data indicate that Cys41 in pThr34 and pTyr38 are more exposed to the solvent than in the unphosphorylated Fis1. This trend of Cys41 exposure is consistent with the trend seen in N-terminus fluctuation, propensity of α1-helix unfolding, and the SAXS data, where pTyr38 has the greatest effect followed by pThr34 and unphosphorylated Fis1.

[0119] To corroborate these findings from the MD simulations, it was investigated biochemically whether Cys41 residue in the phosphorylation mimic-mutants, Thr34Asp Fis1 and Tyr38Glu Fis1, is more exposed compared to the WT Fis1. To this end, a thiol reactive maleimide-based fluorescent probe, CPM (7-diethylamino-3-(4-maleimidylphenyl)-4- methylcoumarin), was used to measure the cysteine reactivity in solution (FIG. 3C). To establish that all the fluorescence signal in the system is coming from CPM reaction with theAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 thiol group of Cys41, a Cys41Ser Fis1 mutant, which has no thiol groups, was used. Co- incubation of Cys41Ser Fis1 and CPM probe produced very low fluorescence signal (FIG.3D). WT Fis1 showed minimal fluorescence signal, but the phosphorylation mimic-mutants had approximately over 12-fold higher fluorescence signal, suggesting that the cysteine is more exposed in the phosphorylation-mimic mutants compared to the WT (FIG. 3D). These data also support the observations from molecular dynamics simulations and SAXS, where the N- terminal region was found to be dynamic, and it unfolded and moved away from the concave hydrophobic surface, thus exposing the normally hidden Cys41 under conditions that mimic its activation. There was a difference in Cys41 exposure in MD simulations data in the phosphorylated forms of Thr34 and Tyr38 but there was no major difference in the amount of chemical modification after 30 min incubation of these protein constructs with CPM.

[0120] Cys41 can be oxidized, when exposed, to mediate covalent Fis1-Fis1 homodimerization.

[0121] As mentioned previously, human Fis1 protein was reported to have propensity to form a small number of covalent dimers, mediated by Cys41. It was difficult to rationalize this observation because of the unexposed position of Cys41 residue, buried in between α1-α2 helices, thus making it unavailable for disulfide bridge formation with another Fis1 protomer (FIG.10). However, in light of the evidence that the α1 helix can unfold and move away from the concave hydrophobic surface, it is conceivable that Cys41 can form a disulfide bridge and hence mediate Fis1 covalent homodimer. The phosphorylation-mimic mutants, Thr34Asp, Thr34Glu and Tyr38Glu Fis1, were crystallized and it was observed that in all the cases, these mutants crystallized as covalent dimers mediated by Cys41 disulfide bridge between two Fis1 protomers (FIG.4A, FIGS.11A, 11B). As Thr34Asp, Thr34Glu and Tyr38Glu Fis1 have a greater propensity for a disordered N-terminus (FIG.1, FIG.2) and hence have more exposed Cys41, they crystallized as covalent dimers.

[0122] In cells, Fis1 is localized to mitochondrial outer membrane via a transmembrane anchoring sequence, but all the in vitro data presented in this study used only the cytosolic domain of Fis1 protein. To overcome this limitation, the cytosolic domain of Fis1 was tethered to His-tag affinity magnetic beads could better resemble the biological context of Fis1 by restricting the degrees of freedom when Fis1 is tethered to a membrane. When the cytosolic domain of Fis1 was tethered to His-tag affinity magnetic beads, the propensity ofAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 phosphorylation mimic constructs to form dimer increased under non-reducing condition (FIG. 4B). This dimer could be reversed to monomer in reducing condition using 2- mercaptoethanol (2-ME), suggesting that Fis1 homodimer formation was mediated by Cys41 (FIG. 4B). The propensity of dimerization of immobilized phosphorylation mimic Fis1 constructs increased with time, with more dimer than monomer at longer time points, while the WT Fis1 construct yielded no dimer. (FIG.12).

[0123] Upon closer examination of the crystal structures, it was observed that the concave hydrophobic surface in the covalent dimers of all the phosphorylation-mimic mutants was exposed (FIG. 4C). Such exposed concave hydrophobic surfaces were reported to be critical for scaffolding proteins to mediate homo-oligomerization or interaction with other protein partners.

[0124] Unlike phosphorylation mimic mutants, the WT Fis1 crystallized as a non-covalent dimer. A similar non-covalent Fis1 dimer was also reported previously. It was rationalized that the α1-helix and N-terminus structure is intact in WT Fis1, making Cys41 unavailable for disulfide bridge formation and hence WT Fis1 could not form a covalent dimer (FIG. 10). However, due to the inherent dynamic nature of N-terminus of Fis1 as seen in MD simulations and CPM fluorescence assay (FIG. 1A, FIG. 3D), WT Fis1 showed some propensity to form covalent dimers in solution as reported previously. It was observed that the concave hydrophobic surface that is known to mediate protein-protein interactions in TPR motif- containing proteins is between the interface of the dimer in both the current and the previously reported structure (FIG. 4D). However, in the active form, the concave hydrophobic surface should be exposed to mediate homo-oligomerization or interaction with other proteins. Yeast Fis1 interaction with Caf4 and bacterial secretion system protein PscG interaction with PscE both require concave hydrophobic surfaces (not sufficient in the case of Fis1 / Caf4). This WT dimer observed by X-ray crystallography is likely an artifact due to crystal packing and the dimer of Fis1 should be different in solution. Indeed, the dimer structure determined from crystallography did not fit the SAXS curve (FIG.4E), suggesting that the Fis1 dimer in solution is structurally different than that observed in the crystal. When the structure of Fis1 WT dimer using the Thr34Asp Fis1 covalent dimer with modeled the missing unstructured first 30 residues, the Fis1 covalent WT dimer perfectly fit the SAXS curve for WT Fis1 dimer (FIG. 4F). Ab initio density and envelopes using experimental SAXS curve wereAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 generated, implementing both P1 and P2 symmetry. No significant differences in the shape of the envelope were observed when either P1 or P2 symmetry was enforced. The modeled WT Fis1 covalent dimer fit both the P1 and P2 symmetry enforced ab initio envelops independently calculated from experimental SAXS curve (FIG.4G). This indicates that the Fis1 dimer that is present in solution is not related to the WT crystallographic dimer. These data combined with the data from the partial native SDS-PAGE gel (FIG.4B) suggest that the small amount of Fis1 dimer in solution is indeed a covalent dimer. However, it is also possible that the differences of WT Fis1 dimers in solution and crystals may represent different types of dimers that enable interactions with different protein partners.

[0125] Structural insights guide assay design for screening specific inhibitor of activated Fis1.

[0126] As discussed above, Cys41 is exposed when Thr34 or Tyr38 is phosphorylated, and these phosphorylations mediate Fis1 activation and subsequent pathological mitochondrial fission and dysfunction. Therefore, compounds that bind to Cys41 should inhibit Fis1 function to induce a protective effect during oxidative stress. Structural insights from the X-ray crystallography, SAXS and MD simulations were used to design small molecules that can specifically inhibit activated Fis1 without interfering with the Fis1 in the basal state with intact α1-helix structure.

[0127] Cys41 is exposed only in the activated Fis1 but is occluded in basal state (FIG.3D). Therefore, small molecules that bind to Fis1 by tightly engaging Cys41 should be specific for the activated Fis1 as this residue is unavailable in the Fis1 in basal state. Here, a small molecule with cysteine reactive warheads would be an ideal candidate for this purpose. A CPM probe, a fluorescently labelled thiol reactive maleimide, was used to confirm binding to Cys41 in activated Fis1 and if this binding can be protected by incubating the protein with unlabeled maleimide (0-50 µM) (FIG. 5A). Phosphorylation mimic-mutants of Fis1, Thr34Asp and Tyr38Glu, with exposed Cys41 were used as models for activated Fis1. These mutants produced robust fluorescence signal with CPM and this signal was inhibited by preincubating the protein with unlabeled maleimide (FIG. 5B), suggesting the suitability of the assay to screen for covalent inhibitors that can bind to Cys41 only in activated Fis1. Thr34Asp Fis1 was used as a model for activated Fis1 in the high throughput screen and was preincubated with cysteine reactive small molecules followed by incubation with CPM. When the smallAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 molecules were covalently bound to Cys41, fluorescence signal of thiol bound CPM decreased as thiol group of Cys41 was already occupied. This reduction in fluorescence signal was used as a readout of Cys41 binding (FIG.5A).

[0128] To avoid molecules with excessively strong electrophilic warheads, like the maleimide, which can bind to any protein with cysteines due to intrinsic reactivity to thiol, a secondary screen was used. In the secondary screen, a new cysteine residue, Val56Cys, was introduced on the surface of Cys41Ser Fis1. This construct has a similar fluorescence profile when treated with CPM and its fluorescence can be inhibited with unlabeled maleimide like Thr34Asp or Tyr38Glu Fis1 (FIG.5B).6000 molecules from Enamine (Kyiv, Ukraine) with thiol reactive covalent warheads were screened in a 384 well format to identify molecules that bind to Thr34Asp Fis1 and not the counter screen, Cys41Ser Val56Cys Fis1 double mutant. One molecule identified from the screen was SP11. SP11 is a molecule with phenothiazine core and methyl substituted chloroacetamide warhead, which binds to Cys41 of Thr34Asp Fis1 and not to Cys56 of Cys41Ser, Val56Cys Fis1 double mutant counter screen (FIGS.5C, 5D).

[0129] SP11 covalently binds specifically to Cys41 of activated Fis1.

[0130] SP11 inhibited CPM fluorescence of Thr34Asp Fis1 with an IC50 of 9.4 µM (FIG.5C). As the binding of CPM dye to Thr34Asp Fis1 is 1:1, a high concentration of Fis1 (4 µM) was used to achieve robust fluorescence signal and good separation of signal to noise. SP11 binding to Thr34Asp Fis1 is also 1:1 and therefore an IC50 of 9.4 µM translates to about 2.5 molecules of SP11 for every half molecule of Thr34Asp Fis1, suggesting that SP11 is a potent inhibitor. SP11 did not bind to the counter screen Cys41Ser, Val56Cys Fis1 double mutant even at a high concentration (FIG.5C). Inhibition by SP11 plateaued at about 55% inhibition, which may reflect solubility issue at the higher concentrations. It may also reflect the dynamic nature of the α1-helix and N-terminus; not all the Thr34Asp Fis1 proteins have homogenous SP11 binding pocket or Cys41 exposure. Indeed, incubation of Thr34Asp Fis1 with SP11 for longer time period increased percent inhibition of CPM fluorescence and approached almost 100% at 52 hours (FIG. 17). As expected, preincubation of Fis1 with SP11 inhibited the dimerization of immobilized Thr34Asp Fis1 (FIG.13).

[0131] Next, MS / MS-based peptide mapping was used and it was found that the Fis1- derived peptides are labeled by SP11 at Cys41, leading to a mass shift by 310 Da (FIG.6A, FIG. 18). To confirm that the inhibition of Fis1 dimerization with SP11 is not an artifact of theAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 covalent warhead, an analogue, SP22, with the same phenothiazine core but with acrylamide warhead was tested (FIG. 14A). Similar to SP11, SP22 also bound to Cys41 of Thr34Asp Fis1 and not to Cys56 of Cys41Ser, Val56Cys Fis1 double mutant counter screen, but with a slightly higher IC50 of 15.4 µM (FIGS.14B, 14C). This data with SP22 establishes that the binding of these molecules to Thr34Asp Fis1 is driven by the binding of phenothiazine core to the binding pocket of Thr34Asp Fis1 and not just due to the intrinsic reactivity of the chloroacetamide warhead.

[0132] Co-crystal structure of Thr34Asp Fis1 with SP11.

[0133] Co-crystal structure of Thr34Asp with SP11 was solved at 1.9Å resolution (FIG.6B). Consistent with the previous Thr34Asp Fis1 structure, the first 30 residues in the N-terminus were also missing in this structure (FIG.2, FIG.6B). A clear and continuous density for SP11 arising from Cys41 residue was also observed, indicative of a covalent bond between Cys41 and SP11 (FIG. 6B). Besides the covalent bond, the two benzene rings of the phenothiazine core of SP11 made two CH-Pi interactions with Tyr38 (FIGS. 6C, 6D). Other interactions of SP11 with Fis1 are exclusively hydrophobic and the phenothiazine core of SP11 assumes a slightly bent conformation to complement with the topology of hydrophobic surface formed by Tyr38 (FIG.6D).

[0134] To validate the significance of the CH-Pi interaction with Tyr38 in the crystal structure, the Tyr38 residue in Fis1 was mutated to a Glu residue which also has an exposed Cys41 and was compatible with CPM probe-based fluorescence assay (FIG.3D, FIG.5B). SP11 did not bind to the Tyr38Glu Fis1 even at 100 µM, suggesting that interaction with Tyr38 is required for SP11 binding, validating the crystal structure (FIGS. 6D, 6E). SP22 also did not bind to Fis1 with Tyr38Glu mutation (FIG.14D).

[0135] SP11 inhibits Fis1-mediated pathological mitochondrial fission and dysfunction during oxidative stress.

[0136] The effect of SP11 in Fis1-mediated mitochondrial fission and mitochondrial reactive oxygen species (ROS) production during oxidative stress was tested using HK-2 cells in culture. Previously, Thr34 residue was shown to be phosphorylated in HK-2 under cellular stress. Using MitoSOX, it was found that 250 nM of SP11 inhibited mitochondrial ROS generation when cells were treated with 50 µM hydrogen peroxide compared to the DMSO only treatment (FIG.7A). Cells treated with 100 µM hydrogen peroxide showed mitochondrialAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 fragmentation, and this fragmentation was also prevented by SP11 treatment (FIGS.7B-7D). There was a drastic reduction in the average mitochondrion size per cell in hydrogen peroxide treated cells and this was prevented by SP11 treatment (FIG.7C). More than half of the cells showed fragmented mitochondria when treated with hydrogen peroxide but only 8% of the cells had fragmented mitochondria in control.250 nM of SP11 treatment reduced the fraction of cells with fragmented mitochondria from 52% in hydrogen peroxide treated cells back to 8%, similar to control cells (FIG.7D). SP11 treatment also increased the fraction of cells with fused mitochondria by about 50% compared to hydrogen peroxide only treated cells. The amounts of mitochondria associated Drp1 in HK-2 cells was determined in the presence of 100 µM hydrogen peroxide. Peroxide-induced Drp1 mitochondrial localization was inhibited by treatment with 250 nM of SP11 (FIG. 7E). These data taken together suggest that SP11 treatment inhibits the ability of Fis1 to recruit Drp1 to mitochondria thus protecting mitochondrial integrity and function under cellular stress. Role of the Cys41 residue of Fis1 was further confirmed using Cys41Ser knock-in homozygous Be(2)-M17 cells. The mutant cells with Cys41Ser Fis1 showed less sensitivity to 50 µM hydrogen peroxide treatment in MitoSOX assay (FIG.7F). SP11 did not show any effect in non-stimulated cells (FIG.15).

[0137] Discussion

[0138] This study shows that the N-terminal region of Fis1 is dynamic and that perturbations affecting the N-terminal region due to cellular stress such as phosphorylation of tyrosine or threonine on the α2-helix increases the fluctuation and causes unfolding of the α1-helix. The crystal structure not only shows the absence of α1 helix but also shows that there is no space for structured α1 helix in the covalent dimer as this space is occupied by α2 helix of another monomer (FIG. 10). This observation was consistent across all modeled phosphorylated Fis1 structures and phosphorylation mimic-mutants, using different biochemical, biophysical and structural biology experiments, including MD simulations, small angle X-ray scattering in solution and X-ray crystallography of WT and phosphorylation mimic mutants.

[0139] The unwinding and displacement of the α1-helix activates Fis1 protein and exposes the one and only cysteine residue, Cys41, in Fis1. Oxidizable cysteine residues that perceive oxidative stress and function as redox sensors are found ubiquitously in many proteins. Here, it was shown that Cys41 is oxidizable and mediates Fis1 covalent dimerization throughAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 disulfide linkage. This oxidation-induced Fis1 dimerization was obvious in the crystal structures of Thr34Asp, Thr34Glu and Tyr38Glu Fis1, where a clear electron density for the disulfide linkage was shown. The covalent dimers in Fis1 have concave hydrophobic surfaces exposed, which is conducive for self-interaction or interaction with other proteins. Higher R- free values were observed for the phosphorylation mimic mutant crystal structures. R-free values vary depending on several key factors such as resolution and the number of residues are disordered in the crystal. The latter is particularly important in this case since all the mutants and Fis1-SP11 structures lack α1 helix, i.e., 1 / 6th of the total amino acids. The atoms of the α1 helix are included in the protein structure but are disordered and not visible. This contributed significantly to high R-free values in the structures reported in this study.

[0140] The non-covalent nature of the Fis1 dimer was reported in yeast where it is the only mitochondrial adapter that recruits Drp1. In mammals, Fis1 recruits Drp1 only under pathological conditions. Therefore, the effect of dimerization in mammals (exclusively under pathological conditions) and in yeast (under physiological condition) leads to different functional effects on the ability of the protein to recruit Fis1. Cys41 in Fis1 is conserved in all vertebrates, suggesting a critical function. In birds, however, even though the stretch of hydrophobic amino acids (36-FEYAWXLVRS-45 (Seq ID NO: 19)) is conserved, like in other vertebrates, birds have glycine residue instead of Cys41. It is interesting to note that birds have high metabolic rates and have high energy demands and it is likely that cells have higher ROS burden and oxidative stress. Along with other adaptations, such as higher levels of antioxidants e.g., glutathione and fewer mitochondria per cell in birds with longer lifespan, this divergence in Fis1 sequence could also be protective against oxidative stress. Additionally, birds are known to be relatively insensitive to LPS even at high concentrations while in other vertebrates LPS has been shown to be one of the stress agents that causes Fis1 phosphorylation at Thr34 position and causes excessive mitochondrial fragmentation and dysfunction.

[0141] Small molecule inhibitors for small, membrane-bound, scaffolding proteins have rarely been described previously; such proteins that mediate protein-protein interaction are considered ‘undruggable’, partly because of the solvent exposed inconspicuous “pockets”. Also, the lack of enzymatic assays for structural proteins makes it difficult to screen for small molecule binders or inhibitors. In this study, using structural insights, an assay method wasAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 developed to find specific inhibitors of activated Fis1, using the maleimide-based fluorescent probe, CPM. The fluorescence signal as a result of direct covalent binding of CPM to Cys41 and the decrease of this signal due to small molecule binding served as a readout for small molecule binding. Using this strategy, a first in class covalent inhibitor that binds to activated Fis1 was identified. As discussed above, SP11 inhibits Thr34Asp Fis1 CPM fluorescence in vitro with an IC50 of 9.4 µM. As SP11 binding to Thr34Asp Fis1 is 1:1 and irreversible, this IC50 is underestimated, as 4 µM Thr34Asp Fis1 was used. This half inhibitory concentration of 9.4 µM at 4 µM Thr34Asp Fis1 effectively translates to 5 molecules of SP11 binding 1 molecule of Fis1, indicating excellent potency. SP11 was co-crystallized with activated Fis1 and was found to bind covalently to the Cys41 residue. The phenothiazine moiety of SP11 adopted a very slightly bent conformation to make two CH-Pi interactions with the Tyr38 residue. No SP11 binding was observed when the Tyr38 residue is mutated, thus validating the crystal structure. Importantly, it was shown that 250 nM SP11 inhibits Fis1-mediated mitochondrial localization of Drp1, mitochondrial fission and dysfunction induced by oxidative stress, in culture, supporting the efficacy of SP11.

[0142] SP11 contains a phenothiazine moiety with a chloroacetamide warhead connected by a three-carbon chain. Phenothiazine is a pharmacologically active molecule and several molecules with phenothiazine substitutions have been already approved by the FDA for different indications including nausea, vomiting, allergy and neurological diseases. This scaffold is known to have poly pharmacology by engaging many targets. The addition of a covalent warhead to phenothiazine in SP11 may reduce its engagement with other targets. With fragments of smaller size, specificity is always an issue and one rarely gets away without elaborate medicinal chemistry to achieve selectivity. However, poly pharmacology may also be beneficial for SP11 in certain indications; phenothiazines by themselves have been shown to have beneficial effect in certain neurodegenerative diseases and SP11 with its covalent warhead can disrupt pathological Fis1-Drp1 interaction and promote mitochondrial integrity and health in nervous tissues, as shown before for other inhibitors of Drp1 / Fis1 binding. Many phenothiazine-containing drugs are orally bioavailable, have longer half-lives and are blood brain barrier permeable. SP11 is an irreversible inhibitor and also has excellent potency and therefore should have beneficial effect even at lower dose, minimizing potential toxicological liabilities.Atty Dkt No.: STAN-2169WO Client Ref.: S23-492

[0143] The Fis1-Drp1 interaction has been recognized for its role in driving mitochondrial fragmentation and dysfunction in diseases, such as Parkinson’s disease, Huntington’s disease, cardiac ischemia and reperfusion injury, endotoxemia, cancer metastasis etc. P110, a peptide inhibitor of Fis1-Drp, was efficacious in preclinical mouse models of diseases by improving mitochondrial integrity, function and reducing disease-associated pathologies. These data indicated that disruption of Fis1-Drp1 interaction is a viable and effective strategy to correct dysregulated mitochondrial dynamics to alleviate diseases. Chemical agents, peptides and small molecules known thus far that disrupt Fis1-Drp1 interaction, exert their effect by engaging and reducing GTPase activity of Drp1, which is also involved in Mff-mediated physiological fission and in several other cellular processes. Therefore, inhibitors of Fis1-Drp1 interaction that bind Fis1 are more likely to be safer as therapeutic agents and perhaps more beneficial for chronic diseases, especially those in fast proliferating tissues (e.g., wound healing) and in pediatric diseases with high metabolic demands.

[0144] Sequences

[0145] Table 2. Sequences used for Fis1 alignment. Name Species Sequence Seq ID NOAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 DGLVGMAIVGGVVVGLAGLAALAIHKGTR XP_030398511.1 Gopherus MESVLSEVVAVEDLLRFEKKYNAELSGGTVSKGTQFEYAW 6 mit h ndril v di CLVRSKYNDDIRKGIVLLED rayCrystallography. Name Sequence Seq ID NO WT Fis1 MEAVLNELVSVEDLLKFEKKFQSEKAAGSVSKSTQFEYAWCLVRSKYN 7ion assay. Name Sequence Seq ID NO. g . Seq ID NOAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 Donor Sequence GAAGGCAGCAGGCTCGGTGTCCAAGAGCACGCAGTTT 16 GAGTACGCCTGGTCCCTGGTGCGCAGCAAGTACAATGA TGACATCCGTAAAGGCATCGTGCTGC

[0150] CPM probe and small molecules. 7-Diethylamino-3-(4-maleimidylphenyl)-4- methylcoumarin, CPM probe, was obtained from Abcam (ab145275; Cambridge, UK). Small molecules SP11 and SP22 used in this study were purchased off the shelf initially from Enamine (Kyiv, Ukraine). Independent batches of SP11 and SP22 were also synthesized, characterized and verified. Chemical characterization of the compounds were performed at Enamine (Kyiv, Ukraine) and are provided below in FIG.19 and FIG.20.

[0151] N-(3-(10H-phenothiazin-10-yl)propyl)-2-chloro-N-methylacetamide (SP11).1H NMR (DMSO-d6, 500 MHz) δ 7.23 – 7.18 (2H, m), 7.18 – 7.11 (2H, m), 7.07 (1H, d), 7.00 (1H, d), 6.94 (2H, dtd, J=10.76, 7.49, 1.11 Hz), 4.23 (2H, d, J=68.78 Hz), 3.87 (2H, dt, J=26.19, 6.87 Hz), 3.44 – 3.33 (2H, m), 2.82 (3H, d, J=87.89 Hz), 1.91 (2H, dp, J=35.03, 6.94 Hz).13C NMR (DMSO-d6, 151 MHz) δ 166.37, 166.03, 145.11, 128.08, 128.05, 127.69, 127.61, 123.18, 123.01, 116.61, 116.28, 46.94, 44.48, 41.99, 33.60, 25.57. HRMS (m / z): calc. for C18H19ClN2OS (M+) 346.09057, obs.346.09806.

[0152] N-(3-(10H-phenothiazin-10-yl)propyl)-N-methylacrylamide (SP22).1H NMR (DMSO- d6, 500 MHz) δ 7.25 – 7.10 (4H, m), 7.01 (2H, dd, J=17.73, 8.07 Hz), 6.94 (2H, dt, J=11.62, 7.42 Hz), 6.59 (1H, ddd, J=82.58, 16.59, 10.35 Hz), 5.99 (1H, ddd, J=53.22, 16.65, 2.53 Hz), 5.48 (1H, ddd, J=131.90, 10.37, 2.54 Hz), 4.02 – 3.77 (2H, m), 3.55 – 3.36 (2H, m), 2.87 (3H, d, J=81.60 Hz), 1.90 (2H, p, J=6.74 Hz).13C NMR (DMSO-d6, 126 MHz) δ 164.99, 144.67, 144.63, 127.72, 127.56, 127.17, 127.11, 122.66, 122.50, 116.18, 115.76, 46.09, 45.20, 25.59, 24.45. HRMS (m / z): calc. for C19H20N2OS (M+) 324.12834, obs.325.13679.

[0153] Molecular dynamics (MD) simulations. The systems for MD simulations were constructed using the wild-type structure of Fis1 (residue 1-123) revealed by X-ray crystallography published in this study. The structures of pThr34 and pTyr38 were modeled using molecular operating environment (MOE; Chemical Computing Group, Montreal, Canada). Protonation states were determined by Adaptive Poisson-Boltzmann Solver (APBS)-Atty Dkt No.: STAN-2169WO Client Ref.: S23-492 PDB2PQR which is a module solvation force library package with pH 7.5. The structures of WT- Fis1, pThr34 and pTyr38 were solvated in TIP3P water using Amber 18. In each system, NaCl was added to neutralize the charge for the Fis1 and to reach a concentration of 150 mM. All MD simulations were performed using Amber 18, with Amber ff14SB and phosaa14SB as the force field for the protein and phosphorylated amino acids, respectively. MD simulations were performed under periodic boundary conditions in a rectangular box. For simulations, the time step was 2 fs, and the trajectory interval was 10 ps. The temperature was maintained at room temperature (298.15 K) using a Langevin thermostat, and the pressure was maintained at 1 bar using a Berendsen barostat. The shake method was also employed. Short-range electrostatic and van der Waals forces were cut off at 1 nm. Finally, 30 MD simulations of 1.2 μs were performed for the WT-Fis1, the pThr34 and the pTyr38 (1.2 μs × 90 MD simulations in total). Since the atomic positional fluctuation of the α1 helix were focused on, the root mean square fluctuation (RMSF) of backbone atoms (Cα, N and C) was calculated by fitting to the average structure except for the α1 helix (aa 31-123). The average α-helix propensity for each amino acid residue was calculated based on the DSSP algorithm. The solvent-accessible surface area (SASA) for Cys41 was calculated based on the LCPO method.

[0154] Recombinant protein production. Fis1 codon optimized sequence in frame with HRV 3C protease cleavage site and hexa-histidine tag at the C-terminus was cloned into a pET-28a vector (Genscript, NJ, USA). BL21 (DE3) E. coli cells transformed with Fis1 expression plasmid was grown in LB media at 37°C in a shaking incubator (200 rpm) to an OD600 of 0.6. Cells were then induced with 0.5 mM IPTG (GoldBio, MO, USA) and grown for 16 h at 18°C. The cells were then collected as pellets by centrifuging the culture for 15 min at 3000×g. All steps after this were performed at 4°C. E. coli pellet was resuspended in lysis buffer (50 mM Tris pH 8, 150 mM NaCl). Resuspended cells were sonicated for 2.5 m (1 s on and 4 s off) at 70% amplitude using a QSONICA SONICATORS instrument (500 Watts). The whole cell lysate was then centrifuged at 20442×g for 80 min and the soluble fraction was loaded onto a Ni-NTA Agarose (Qiagen, MD, USA) gravity column pre-equilibrated with lysis buffer. The Ni-NTA column was then washed with 50 column volumes of wash buffer (50 mM Tris pH 8, 150 mM NaCl and 40 mM Imidazole). Fis1 protein bound to Ni-NTA was then eluted with elution buffer (50 mM Tris pH 8, 150 mM NaCl and 400 mM Imidazole). Eluted Fis1 protein was then buffer exchanged using Zeba™ Spin Desalting Column, 7K MWCO, 10 mL (89894; Thermo FisherAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 Scientific, MA, USA) into lysis buffer (50 mM Tris pH 8, 150 mM NaCl). Buffer exchanged Fis1 was mixed with 6xHis-tagged HRV 3C protease in 40:1 (W / W), incubated overnight at 4°C and was passed through Ni-NTA Agarose (Qiagen) gravity column to remove un-cleaved hexa- histidine tagged impurities including the HRV 3C protease. Hexa-histidine tag free Fis1 was obtained as the flowthrough was flash frozen and stored at −80°C. This protocol was also followed to express and purify the Fis1 mutants used in this study. Fis1 sequences are provided in Tables 3 and 4.

[0155] CPM fluorescence assay. Recombinant Fis1 protein from above was diluted to 4 µM concentration in PBS and 48 µL of this solution was added to each well in a 96 well black flat bottom chimney plate. 1 µL of test small molecule dissolved in DMSO was added to the protein solution to obtain desired final concentration. The test small molecule and protein were incubated at room temperature for 30 min.1 µL of CPM in DMSO to achieve 10 µM final concentration was added to each well with the protein and a test molecule. This reaction mixture was incubated at room temperature for 30 min and fluorescence readout (Ex: 391 nm / Em: 472 nm) was taken using Tecan M1000 microplate reader (Tecan, Männedorf, Switzerland). No test small molecule control was taken as 100% signal and the precent inhibition of this signal was calculated at different concentrations of the test small molecule.

[0156] Fis1 immobilization experiment. 20 µg of recombinant Fis1 protein in 100µL lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl and 10mM 2-ME) was added to 20 µL of HisPurTMNi-NTA magnetic beads (Thermo Fisher Scientific) washed three times with wash A buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10 mM 2-ME and 0.1% Tween-20). The mixture was left shaking at room temperature for 1 h. The beads with bound Fis1 protein were separated from the elution using magnetic rack and washed 3 times with wash B buffer (50 mM Tris pH 8.0, 150 mM NaCl and 0.1% Tween-20). Beads with bound protein was resuspended in 100µL wash B buffer and incubated at room temperature while shaking for time period as indicated. Beads were then separated from the elution using magnetic rack and resuspended in buffer containing 50 mM Tris pH 8.0 and 150 mM NaCl and analyzed using SDS-PAGE (in the presence or absence of 2-ME as indicated). Wherever partial native SDS-PAGE is indicated, protein samples were loaded into 4-20% SDS-PAGE gel in the absence of reducing agent and without boiling. For SP11 treatment, Thr34Asp Fis1 protein was diluted to 4µM in lysis buffer without 10 mM 2-ME and preincubated with 50 µM SP11 for 1 h at room temperature beforeAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 adding to the beads. The experiment was performed similarly as above except 2-ME was not added in any buffer. No pre-existing Thr34Asp Fis1 dimer was broken and inhibition of new Fis1 dimer formation by SP11 was monitored (FIG.13).

[0157] Crystallization, data collection and structure determination. Tag-free recombinant Fis1 protein / mutant was concentrated to 8 mg / ml using 3-kDa molecular weight cutoff Amicon Ultra15 ultrafiltration membrane. For Thr34Asp Fis1-SP11 complex crystallization, Thr34Asp-Fis1 was diluted to 4 µM in 50 mM Tris pH 8, 150 mM NaCl buffer and 10-fold molar excess of SP11 was added and incubated for 1 h at room temperature. Thr34Asp Fis1-SP11 complex was then concentrated with a 3-kDa molecular weight cutoff Amicon Ultra15 ultrafiltration membrane to 8 mg / ml.

[0158] Each of the protein sample went through screening with around 960 conditions at two different protein concentrations using Oryx 8 crystallization robot in a sitting drop setup. The WT protein was crystallized from screen condition C9 (25% PEG 5KMMe, 0.15 M NaCl, 0.033 M HEPES (pH 7.5), 0.033 M ADA (pH 6.5) and 0.033 M Tris.HCl (pH 8.0)) of a screen developed at SSRL and the crystals cryo-cooled with well solution supplemented with 20% glycerol. The Thr34Asp mutant was crystallized from Pact screen condition E2 (0.2 M sodium bromide, 20% w / v PEG 3350). The crystal was cryo-cooled with well-solution supplemented with 10% glycerol. The Thr34Glu mutant was crystallized from Top96 crystallization solution F2 (0.2 M ammonium acetate, 0.1 M HEPES (pH 7.5) and 25% PEG 3350) and the crystals cryo- cooled with well solution supplemented with 5% glycerol. The Tyr38Glu mutant was crystallized from Grass2 crystallization solution F11 (0.1 M sodium phosphate dibasic dihydrate, 20% PEG 3350) and the crystals cryo-cooled with well solution supplemented with 10% glycerol. The Tyr38Glu mutant that showed movement of the N-terminal helix was crystallized from BCS screen condition A1 (0.1 M sodium acetate (pH 4.5) and 30% PEG smear low) and cryo-cooled with well solution supplemented with 3% glycerol. The SP11 covalent complex was crystallized from MemGold crystallization screen G12 (0.001 M zinc sulfate, 0.05 M HEPES, 28% PEG 600) and the crystal was cryo-cooled directly from the drop.

[0159] The diffraction data for wild type, Thr34Asp, Thr34Glu, and the two Tyr38Glu mutants were collected at 100 K at the BL12-2 beamline of the Stanford Synchrotron Radiation Lightsource using PILATUS 6 M detectors. The diffraction data for the SP11 covalent complex was collected at the same beamline using the EIGERX 16M detector. All diffractionAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 data were collected with 360° of data per crystal and 0.2° oscillation per image. For each crystal, diffraction data were merged and processed with XDS. The structures were solved by molecular replacement with MOLREP or PHASER using the coordinates of cytosolic domain of human Fis1 (PDB code: 1NZN) as the search model. Iterative rounds of model-building and refinement were performed with the programs COOT and REFMAC. The final rounds of refinements were performed by REFMAC or PHENIX. The details of data collection and refinement for the higher resolution data are presented in Tables 6A and 6B.

[0160] Table 6A. Crystallographic parameters, data collection and refinement statistics.Atty Dkt No.: STAN-2169WO Client Ref.: S23-492 Fis1-WT Fis1-T34D Fis1-T34E Crystallographic parameters 1Å 7)bR factor = Ʃ|Fp -Fpcalc| / ƩFp, where Fp and Fpcalc are the observed and calculated structure factors; Rfree is calculated with 5% of the data.

[0161] Table 6B. Crystallographic parameters, data collection and refinement statistics.Atty Dkt No.: STAN-2169WO Client Ref.: S23-492 Fis1-Y38E Fis1-Y38E (Move) Fis1-SP11 Crystallographic parameters Å 0)bR factor = Ʃ|Fp -Fpcalc| / ƩFp, where Fp and Fpcalc are the observed and calculated structure factors; Rfree is calculated with 5% of the data.

[0162] Peptide mapping and MS analysis. Thr34Asp Fis1 was diluted to 1 mg / ml in 50 mM Tris pH 8, 150 mM NaCl buffer and SP11 was added to the protein solution at 100 µM final concentration. The reaction mixture was incubated at room temperature for 1 h and was flash frozen. Peptide mapping and MS analysis was performed at BGI Global Genomic Services (CA, USA).

[0163] Samples (25 μg) were denatured with the treatment of 9M urea followed by the reduction with dithiothreitol (DTT) and alkylation with 2-iodoacetamide (IAM). 10 μLAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 Trypsin / LysC mix stock (0.2 μg / μL) was added to the sample for digestion overnight at 37°C. The reaction was then quenched with 10% TFA. Digested samples were desalted and eluted by the standard C18 Stage-tip Clean Up protocol. Peptides were then dried by SpeedVac. Samples were reconstituted with H2O containing 0.1% Formic acid. An aliquot of 10 μL was subjected to the LC-MS / MS system equipped with Thermo Fisher Vanquish LC and Q Exactive HF-X mass spectrometer. MS Data was analyzed with PMI Byos (Protein Metrics, CA, USA) software and sequence provided to reveal peptides coverage on the Fis1 protein.

[0164] SEC-SAXS experiments and analyses. The SEC-SAXS experiments were performed at the Stanford Synchrotron Radiation Lightsource (SSRL) Bio-SAXS beamline 4-2. SEC-SAXS data were collected using a Superdex 75 Increase 3.2 / 300 column (Cytiva, MA, USA) using SEC running buffer (50 mM Tris pH 8.0, 150 mM NaCl), with samples concentrated with the buffer at 6 or 5 mg / ml for Fis 1 wild-type dimer and others, respectively.500 images were acquired with 1 s exposure every 5 s at a flow rate of 0.05 ml / min. After the 100th image (blank data collection), the x-ray shutter was closed until just before sample elution to keep the sample cell clean. Data reduction and initial analyses were performed using the BL4-2 automated SEC- SAXS data processing and analysis pipeline, SECPipe (https: / / www- ssrl.slac.stanford.edu / smb-saxs / node / 1860). It implements the programs SASTOOL (https: / / www-ssrl.slac.stanford.edu / smb-saxs / node / 1914) and ATSAS AUTORG. The data were plotted as I(q) versus q, where q = 4πsin(θ) / λ, 2θ is the scattering angle, and λ is the wavelength of the X-ray. After careful manual inspection, a total of 5 images were selected to generate the average profile for further analysis. Experimental and analytical details are summarized in Tables 7A and 7B.Atty Dkt No.: STAN-2169WO Client Ref.: S23-492

[0165] Table 7A. SAXS data collection and analysis. WT Dimer WT Thr34Asp Tyr38Glu Data collection parameters Instrument SSRL BL4-2 Type of Experiment SEC-SAXS Beam Current (mA) 500 Defining slits size (H mm × V mm) 0.3 × 0.30 Detector distance (m) 1.1 Detector Pilatus3 X 1M Beam energy (keV) 11.0 q range (Å− 1) 0.0111-0.732 0.010–0.594 0.010–0.594 0.010–0.594 Sample cell Quartz capillary (ID=~1.3mm) Temperature (K) 295 Exposure time / frame (s) 1 Frames per SEC-SAXS data set 500 Number of blank images used for averaging 100 Number of sample images used for averaging 15 5 5 5 Image numbers used for averaging 285-299 305-309 306-310 307-311 SEC column Superdex 75 Increase 3.2 / 300 HPLC flow rate (mL / min) 0.05 Sample concentration (mg / ml) 5 6 6 6 SEC injection volume (µL) 100 30 30 30 Buffer 50 mM Tris / HCl pH 8.0 and 150 mM NaCl Software employed Primary data reduction SasTool / SECPip e Data processing PRIMUS P(r) analysis GNOM Atomistic modeling CORAL ab initio modeling Dammif, DENSS Structural parameters Guinier analysis I(0) 0.013 0.017 0.023 0.015 ± 0.000058 ± 0.000059 ± 0.000063 ± 0.000054 Rg (Å) 25.36 ± 0.17 17.91 ± 0.10 17.55 ± 0.08 17.12 ± 0.10Atty Dkt No.: STAN-2169WO Client Ref.: S23-492

[0166] Table 7B. SAXS data collection and analysis. WT Dimer WT Thr34ASP Tyr38Glu Data collection parameters. type dimer was calculated and fitted to the experimental data using the program CRYSOL. SAXS modeling of the wild-type dimer was performed using the program CORAL. A covalent dimer in the Thr34Asp crystal structure (aa: 31-129) was employed as the initial model and the first 30 residues were reconstructed by CORAL. The initial model was fixed during modeling. 20 independent runs with P2 symmetry were performed. The model with the lowest χ2 value was selected as the best model.

[0168] An ab initio electron density map of the wild-type dimer was calculated using the program DENSS, with a final map calculated by averaging 20 independent runs (the final resolution = 20.75 Å). Ab initio shape determination was also performed by the program DAMMIF. 20 independent models with P2 symmetry were generated. The resulting models were averaged (ICP = 2.46 + / - 1.89) and filtered using the program DAMAVER. Docking of the CORAL model to the SAXS envelopes was performed manually using ChimeraX.

[0169] Cell cultures. HK-2 cells were purchased from American Type Culture Collection (VA, USA). The cells were kept in their growth medium DMEM / F12 (SH30023.01; Cytiva) supplemented with 10% fetal bovine serum (FBS; 100-500-500; GeminiBio, CA, USA), 1% penicillin / streptomycin (P / S; 15140-122; Thermo Fisher Scientific), and 5 ng / ml human epidermal growth factor (hEGF; PHG0311; Thermo Fisher Scientific). Wild-type and genetically engineered Be(2)-M17 cells were purchased from Synthego (CA, USA). The cells were kept in growth medium which was 1:1 mixture of MEM (10370-201; Thermo Fisher Scientific) and Ham’s F-12 Nutrient Mix (31756035; Thermo Fisher Scientific) supplemented with 10% FBS and 1% P / S. For the validation of gene editing, DNA was extracted from the cells using Quick-DNATMMicroprep Plus Kit (D4074; Zymo Research, CA, USA) according to theAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 manufacturer’s instructions. Then the gene sequencing was performed by Elim Biopharmaceuticals (CA, USA). All cells were kept in a 95% air and 5% CO2atmosphere at 37ºC. Gene editing information for Be(2)-M17 cells is provided in FIG.24 and Table 5.

[0170] Measurement of mitochondrial reactive oxygen species. HK-2 cells were plated in 96-well clear bottom black polystyrene microplates (3904; Corning, NY, USA) at 3.0 × 104cells / well. The cells were incubated overnight in growth medium supplemented with 10% FBS, 1% P / S and 5 ng / ml. Then their media were removed and replaced with growth medium with or without 50 µM H2O2(H1009; Sigma-Aldrich, MO, USA) with or without 250 nM SP11 or vehicle control (DMSO) and incubated for additional 24 h. Be(2)-M17 cells were plated in 96- well clear bottom black polystyrene microplates at a density of 1.0 × 104cells / well. The cells were incubated overnight in growth medium supplemented with 10% FBS and 1% P / S. Then their media were removed, and replaced with serum-free MEM / F-12 with or without 50 µM H2O2 with or without 250 nM SP11 or vehicle control (DMSO) and incubated for additional 24h. Cells were washed with PBS and incubated in FluoroBriteTMDMEM (A18967-01; Thermo Fisher Scientific) containing 1 µM MitoSOXTMRed Mitochondrial Superoxide Indicators (M36008; Thermo Fisher Scientific) and 1 µg / mL Hoechst 33342 (H3570; Thermo Fisher Scientific) for 30 min at 37°C and then washed with PBS and placed in FluoroBriteTMDMEM. Fluorescence was measured for MitoSOXTM(Ex 510 nm and Em 580 nm) and Hoechst 33342 (Ex 350 nm and Em 470 nm) using a fluorescent microplate reader SpectraMax M2 (Molecular Devices, CA, USA).

[0171] Live-cell imaging for mitochondrial morphology. HK-2 cells were plated in 35-mm glass bottom dish (P35GC-1.5-10-C; MatTek, MA, USA) at 2.0 × 105cells / dish and were incubated overnight in growth medium. Cell media were then removed and replaced with glucose-free, serum-free, and galactose-supplemented (10 mM) DMEM / F12 medium (A2494301; Thermo Fisher Scientific) in the presence or absence of 100 µM H2O2and in the presence or absence of SP11 or DMSO vehicle control, as above and incubated for 24 h. Cells were washed with PBS and incubated in FluoroBriteTMDMEM containing 100 nM MitoTracker Deep Red FM (M22426; Thermo Fisher Scientific) and 1 µg / mL Hoechst 33342 for 27 min at 37°C. After PBS wash, cells were placed in FluoroBriteTMDMEM and Z-stacked images were randomly taken using a fluorescence microscope BX-X700 (Keyence, Osaka, Japan) outfitted with a stage top incubator (Tokai Hit, Shizuoka, Japan) using a 60X oil objective lensAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 (MRD01605; Nikon, Tokyo, Japan). The Z-stacked images were further processed for the Z- projection and the haze reduction using Keyence BZ-Analyzer. The images from MitoTracker channel were binarized using ImageJ (version 1.54f). Particles of each cell greater than 4 pixels were analyzed using ImageJ to exclude noise. The analysis was performed by a person blinded to the conditions.

[0172] Isolating of mitochondria-enriched fractions. HK-2 cells were plated in 10 cm tissue culture plate (Genesee Scientific, CA, USA) at a density of 1.0 × 106cells / plate and were incubated overnight in their growth medium. Cells were further incubated with serum-free growth medium for 24 h. Cell media were then removed and replaced with glucose-free, serum-free, galactose-supplemented (10 mM) DMEM / F12 medium with or without SP11 or vehicle control (DMSO) for 30 min. Then the cells were treated with 100 µM H2O2in the presence of SP11 or vehicle control and incubated for additional 24 h. Cells were washed with cold phosphate-buffered saline (PBS, pH 7.4) and harvested with a cell scraper. The collected cells were centrifuged briefly at 10,000 × g for 1 min and the supernatant was discarded. The pellet was re-suspended with a 27-gauge 1 / 2-inch needle for lysis in mannitol-sucrose (MS) buffer, containing 210 mM mannitol, 70 mM sucrose, 5 mM MOPS (3-(N-morpholino) propane-sulfonic acid), 1 mM EDTA, and protease inhibitor cocktail (11697498001; Roche, Basel, Switzerland), followed by centrifugation at 800 × g to remove nuclei pellet. The supernatant was further centrifuged at 10,000 × g for 20 min. The supernatant was collected for cytosolic fraction. The pellet was washed twice by gently re-suspending in MS buffer, and spinning down at 10,000 × g for 5 min. This last supernatant was discarded, and the mitochondrial pellet was re-suspended in MS buffer containing 1% Triton X-100 (X100; Sigma- Aldrich), as before. Note that in some experiments, there was no significant increased Drp1 association with the mitochondria 24 h after H2O2treatment, relative to vehicle control. Thus, data presented in FIG. 14E, determining the effect of SP11 on Drp1 association with the mitochondria, include only experiments in which H2O2-induced Drp1 translocation to the mitochondria was noted at that time point.

[0173] Immunoblotting. Protein concentrations were determined using PierceTMBCA Protein Assay Kit (23225; Thermo Fisher Scientific) following the manufacturer’s instruction. Cell fractions from above were diluted in 4X Laemmli buffer containing DTT, heated at 70°C for 10 min, loaded in 4–20% Mini-PROTEAN TGXTMPrecast Protein Gels (Bio-Rad, CA, USA),Atty Dkt No.: STAN-2169WO Client Ref.: S23-492 and transferred to PVDF membrane (Bio-Rad). Membranes were incubated with indicated antibody and visualized by ECL (PI34095; Thermo Fisher Scientific). The images were acquired using Azure c600 imager (Azure Biosystems, CA, USA). The antibodies used in this study are: Anti-Drp1 (#8570; Cell Signaling Technology, MA, USA) at 1:500; Anti-VDAC (ab15895; Abcam, Cambridge, UK) at 1:1000; Anti-α / β Tubulin (#2148; Cell Signaling Technology) at 1:2000; Anti- Rabbit IgG (NA934V; Cytiva) at 1:5000; Anti-Mouse IgG (NA931V; Cytiva) at 1:5000.

[0174] Statistical Analysis. Prism was used to analyze all the data and generate graphs. T- test was used to determine the statistical difference between two groups and a one-way ANOVA with Dunnett's or Tukey’s multiple comparisons test was used for multiple groups.

[0175] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0176] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should beAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0177] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0178] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges whichAtty Dkt No.: STAN-2169WO Client Ref.: S23-492 can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0179] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0180] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0181] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

Atty Dkt No.: STAN-2169WO Client Ref.: S23-492 What Is Claimed Is:

1. A method of selectively inhibiting mitochondrial dysfunction, the method comprising: covalently binding a compound to Fis1.

2. The method of claim 1, wherein the compound binds to Cys41 of Fis1.

3. The method of claim 1 or claim 2, wherein Fis1 is human Fis1.

4. The method of any one of claims 1-3, wherein the method reduces mitochondrial fragmentation.

5. The method of any one of claims 1-4, wherein the method reduces mitochondrial reactive oxygen species (ROS) production.

6. The method of any one of claims 1-5, wherein the compound is of formula (I): (I) wherein: A is a tricyclic ring or a substituted version thereof; Z is a linker; and B is a reactive group that covalently binds to S.

7. The method of any one of claims 1-6, wherein the compound is of formula (II): wherein:Y1is a heteroatom or CH2; Y2is a heteroatom or CH;Atty Dkt No.: STAN-2169WO Client Ref.: S23-492 Z is a linker; and B is a reactive group that covalently binds to S.

8. The method of any one of claims 1-7, wherein the compound is of formula (III): wherein:Y1is a heteroatom or CH2; Y2is a heteroatom or CH; each R1and R2is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, acyl, alkoxy, amino, azido, carbonyl, carboxy, cyano, ether, halo, hydroxy, and nitro, or a substituted version thereof; R3is selected from the group consisting of H, alkyl, alkenyl, alkynyl, acyl, carbonyl, and carboxy, or a substituted version thereof; B is a reactive group that covalently binds to S; and m is 0 or an integer from 1 to 10.

9. The method of any one of claims 6-8, wherein B is selected from the group consisting of CH2Cl, CH2I, CH2Br, CH2OTs, CH2OTf, CH2OMs, and alkenyl.

10. The method of any one of claims 1-9, wherein the compound has structure: .

11. The method of any one of claims 1-9, wherein the compound has structure:Atty Dkt No.: STAN-2169WO Client Ref.: S23-492 .

12. A method for treating mitochondrial dysfunction in a subject, the method comprising: administering an effective amount of a compound that covalently binds to Fis1 to the subject in need thereof.

13. The method of claim 12, wherein the mitochondrial dysfunction comprises mitochondrial fragmentation.

14. The method of claim 12 or claim 13, wherein the compound is of formula (I): (I) wherein: A is a tricyclic ring or a substituted version thereof; Z is a linker; and B is a reactive group that covalently binds to S.

15. The method of any one of claims 12-14, wherein the compound is of formula (II): wherein:Y1is a heteroatom or CH2; Y2is a heteroatom or CH; Z is a linker; and B is a reactive group that covalently binds to S.Atty Dkt No.: STAN-2169WO Client Ref.: S23-492 16. The method of any one of claims 12-16, wherein the compound is of formula (III): wherein:Y1is a heteroatom or Y2is a heteroatom or CH; each R1and R2is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, acyl, alkoxy, amino, azido, carbonyl, carboxy, cyano, ether, halo, hydroxy, and nitro, or a substituted version thereof; R3is selected from the group consisting of H, alkyl, alkenyl, alkynyl, acyl, carbonyl, and carboxy, or a substituted version thereof; B is a reactive group that covalently binds to S; and m is 0 or an integer from 1 to 10.

17. The method of any one of claims 14-16, wherein B is selected from the group consisting of CH2Cl, CH2I, CH2Br, CH2OTs, CH2OTf, CH2OMs, and alkenyl.

18. The method of any one of claims 12-17, wherein the compound has structure: .

19. The method of any one of claims 12-17, wherein the compound has structure: .Atty Dkt No.: STAN-2169WO Client Ref.: S23-492 20. The method of any one of claims 12-19, wherein the subject has a neurodegenerative disease.

21. The method of claim 20, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amytrophic lateral sclerosis, and neuronopathic Gaucher’s disease.

22. The method of any one of claims 12-19, wherein the subject has an inflammatory disease.

23. The method of claim 22, wherein the inflammatory disease is selected from the group consisting of sepsis, inflammatory bowel disease, and acute kidney injury.

24. The method of any one of claims 12-19, wherein the subject has a cardiovascular disease.

25. The method of claim 24, wherein the cardiovascular disease is selected from the group consisting of cardiac ischemia reperfusion, stroke, and Type 2 diabetes.

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