Myeloperoxidase fluorescence imaging

Abstract

The present application relates to compounds which are imaging probe for MPO activity and methods of using the compounds.

Description

[0001] MYELOPEROXIDASE FLUORESCENCE IMAGING

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 638,888, filed April 25, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant numbers R01NS103998, RF1 AG075055, and K25HL150305 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD

[0007] The present application relates to compounds which are imaging probes for myeloperoxidase (MPO) activity and methods of using the compounds.

[0008] BACKGROUND

[0009] One of the key products of an abnormal immune response is elevated oxidative stress, which can result in the accumulation of oxidative damage to lipids, DNA, and proteins by reactive oxygen / nitrogen species that over time may contribute to aging. On the other hand, oxidative stress also plays roles in homeostasis and cell signaling and can sometimes be protective. It is when oxidative species are uncontrollably elevated and overwhelm the antioxidant balance that excess oxidants can contribute to aging and disease.

[0010] One of the key producers of oxidative stress is myeloperoxidase (MPO). MPO is a highly oxidative enzyme, capable of generating both oxidative and nitrosative stress. After activation with H2O2, MPO oxidizes substrates (e.g., chloride (Cl ), bromide (Br ), nitrite (NO2 ), tyrosine (Tyr)) to potent oxidants (hypochlorous acid (HOC1) or hypobromous acid (HOBr)) and free radicals (nitrogen dioxide (*NO2) or tyrosyl radical (Tyr*)). These free radicals and oxidants are more potent than O2 * and H2O2 for oxidizing biomolecules and inducing cellular injury. Thus, elevated MPO activity is indicative of increased oxidant production and the presence of excessive oxidative stress. To clearly identify the changes in tissue MPO activity, a new fluorescent imaging probe for MPO activity called a myeloperoxidase activatable fluorescent agent (MAFA) to image tissue MPO activity in the ischemic brain was developed. SUMMARY

[0011] Provided herein are compounds of Formula (IA): or a pharmaceutically acceptable salt thereof, wherein:

[0012] R1A, R2A, R3A, R4A, R1B, R2B, R3B, and R4Bare each independently H, -OH, -OCH3, - CN, halogen, -NH2, or -CH3;

[0013] R5A, R5B, and Rcare each independently H or -CH3;

[0014] RDis -Ci-10 alkyl substituted with REwherein the -C1-10 alkyl of RDis optionally substituted with 1, 2, 3, or 4 substituents independently selected from -OH, -OCH3, -CN, halogen, and NH2 and 1 or 2 non-terminal carbons of the -C1-10 alkyl of RDare independently optionally replaced with O, -NH, or -NCH3; and

[0015] REcomprises a fluorescent group.

[0016] Also provided herein are pharmaceutical compositions comprising a compound provided herein and a pharmaceutically acceptable carrier.

[0017] Also provided herein are methods of imaging a cell or tissue sample.

[0018] Also provided herein are methods of diagnosing a disease or disorder associated with abnormal myeloperoxidase (MPO) activity in a subject.

[0019] Also provided herein are methods of imaging myeloperoxidase (MPO) activity in a cell or tissue sample.

[0020] Also provided herein are methods of detecting myeloperoxidase (MPO) activity in a cell or tissue sample.

[0021] Also provided herein are methods of detecting myeloperoxidase activity in a subject.

[0022] Also provided herein are methods of monitoring treatment of a disease or disorder associated with abnormal myeloperoxidase (MPO) activity in a subject.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 A and FIG. IB shows immunofluorescent images and quantification of fresh frozen brains. Imaging of MPO activity with myeloperoxidase activatable fluorescent agent (MAFA) revealed a significantly higher fluorescent signal in wild-type as compared to MPO- KO mice on day 3 after stroke (n = 5 wild-type mice, n = 3 MPO-KO mice, the scale bar = 100 pm). Correspondingly, chlorotyrosine expression was remarkedly higher in the wild type as compared to MPO-KO mice (n = 5 wild-type mice, n = 3 MPO-KO mice, Mann-Whitney test) the scale bar = 100 pm). *, / ? <0.05.

[0025] FIG. 2 shows MAFA signals increased with aging. (FIG. 2A) Representative immunofluorescent images illustrating MAFA and Ibal signal in the thalamus region of aged and young adult brains prior to stroke (n = 4 mice per group, the scale bar = 100 pm). (FIG. 2B) Quantification showed that MAFA and Ibal signals were significantly higher in aged brains as compared to those of young adults. MPO ELISA also showed higher amount of MPO protein in the aged brains compared to that in the young adult brains (*, p < 0.05, Mann-Whitney test).

[0026] FIG. 3 shows immunofluorescent imaging of MPO activity compared to Ibal, MPO protein, and neuronal integrity at the early subacute stage (day 3) after stroke. (FIG. 3 A, FIG. 3C, FIG. 3E) Representative immunofluorescent images depicting MAFA, Ibal, MPO protein and NeuN staining of the infarct area of aged and young adult brains on day 3 after stroke (respectively n = 4 mice per group, the scale bars = 100 pm). (FIG. 3 A, FIG. 3B) The integrated density of MAFA signal was significantly higher in aged brains as compared to young adults on day 3 after stroke. (FIG. 3 A, FIG. 3B) The integrated density of Ibal signal also was higher in aged brains as compared to young adults. (FIG. 3E, FIG. 3F) NeuN expression in aged brains is significantly lower on day 3 after stroke as compared to young adult brains. (FIG. 3D, FIG. 3F) These results were corroborated with MPO and beta-3 tubulin assays (n = 6 mice per group). *, p < 0.05, **, p < 0.01). Mann-Whitney test was performed to determine the differences between aged and young adults.

[0027] FIG. 4 shows immunofluorescent imaging of MPO activity compared to Ibal, MPO protein, and neuronal integrity at the late subacute stage (day 10) after stroke. (FIG. 4 A, FIG. 4C, FIG. 4E) Representative immunofluorescent images showing MAFA, Ibal, MPO protein and NeuN staining of the infarct area of aged and young adult brains on day 10 after stroke (respectively n = 4-5 mice per group, the scale bar = 100 pm). (FIG. 4A, FIG. 4B) The integrated density of MAFA was significantly higher in aged brains as compared to young adults on day 10 after stroke. (FIG. 4A, FIG. 4B) The integrated density of Ibal also was higher in aged brains as compared to young adults. (FIG. 4E, FIG. 4F) NeuN expression in aged brains trended lower on day 10 after stroke as compared to young adult brains. (FIG. 4D, FIG. 4F) These results were corroborated with MPO and beta-3 tubulin assays (n = 5 mice per group). *, p < 0.05; ns, not significant. Mann-Whitney test was performed to determine the differences between aged and young adults.

[0028] FIG. 5 shows neurobehavioral evaluation and survival rate show a beneficial effect of MPO inhibitor on stroke outcome in the aged group. (FIG. 5C) Behavior and functional tests of aged ABAH-treated and aged untreated and young adult stroke mice during the early subacute stage after stroke (n = 6 ABAH-treated, n = 11 untreated, and n = 6 young adult) and survival (log-rank) curve of aged ABAH-treated (n = 12) and aged untreated mice (n = 55) after stroke. (FIG. 5 A and FIG. 5B) Representative immunofluorescence images showing MAFA and Ibal signals of infarct areas in ABAH-treated and untreated aged brains on day 3 following stroke (n = 4-5 per group, the scale bar = 100 pm). *, p < 0.05. Mann-Whitney test was performed to evaluate the differences of Ibal and MAFA signals between ABAH-treated and untreated aged stroke mice. Neurological deficit score and hanging wire test between the aged untreated and ABAH-treated groups were analyzed using the unpaired two-tailed t-test.

[0029] FIG. 6 shows aging worsens neurological deficits and behavior outcomes in ischemic stroke. (FIG. 6A) Neurological deficits scores were significantly higher in aged mice compared to young adults after stroke (n = 11 for the aged and n = 6 for young adults for all behavior tests and neurological deficits scores). (FIG. 6B) Forelimb grip strength was measured by the hanging wire test and the latency to fall was quantified. (FIG. 6C) The ratio of using the contralateral limb was measured with cylinder tests. Significant differences were found in both hanging wire and cylinder tests between aged and young adults after stroke. (FIG. 6D and FIG. 6E) The mortality rate was higher in (log-rank) curve of aged and young adult mice after stroke. Statistical values were determined by unpaired t-tests.

[0030] FIG. 7 shows aging changes myeloid cell dynamics following ischemic stroke. (FIG. 7 A, FIG. 7B) Analysis of flow cytometry data showed that aged mice have significantly fewer MP0+ cells in ischemic brains and more MP0+ cells in blood as compared to young adults as compared to young adult brains on day 3 after stroke (n = 4 mice per group). (FIG. 7C, FIG. 7D) on day 10th after stroke, aged mice have significantly more MP0+ cells in the brain and fewer MP0+ cells in the blood as compared to young adults (n = 5 mice per group). (FIG. 7E, FIG. 7F, FIG. 7G, FIG. 7H) Notably, the analysis also found a significant increase in MP0+ infiltrating M<p / activated Mg in the aged brains compared to young adult brains on both day 3 and day 10 post-stroke. Flow cytometry results were quantified as a percentage of total cells and MP0+ cells. *, p < 0.05, **, p < 0.01. Mann-Whitney test was performed to determine the differences between aged and young adults. FIG. 8 shows MPO activity was increased in human postmortem XDP PFC. (FIG. 8 A) Representative images of MPO activity in control and XDP postmortem PFC. (FIG. 8B) MPO activity was significantly increased in XDP PFC (n = 25) compared with controls (n = 7) (Mann-Whitney U test = 28, p = 0.0051). **p < 0. 01. Scale bar: 100 pm; scale bar insert: 25 pm.

[0031] FIG. 9 shows There were no correlations between MPO activity and XDP clinical parameters. There was no impact of (FIG. 9A) age at disease onset (Spearman correlation, r = 0.05046, p = 0.8107), (FIG. 9B) age at death (Spearman correlation, r = 0.09327, p = 0.6574), (FIG. 9C) disease duration (in months) (Spearman correlation, r = 0.1537, p = 0.4633), and (FIG. 9D) repeat size within the SVA (Spearman correlation, r = 0.09051, p = 0.6670) on MPO activity in XDP PFC (n = 25).

[0032] FIG. 10 shows ROS levels were increased in XDP- derived fibroblasts and XDP- treated SH- SY5Y cells. (FIG. 10A) Two- way ANOVA demonstrated a significant effect of genotype ([F (2, 24) = 244.5], p < 0.0001), and time ([F (12, 24) = 47.84], p < 0.0001) on ROS levels in XDP fibroblasts. As revealed by Tukey's test, ROS levels were increased in antimycin A-treated fibroblasts compared with vehicle-treated control fibroblasts (p < 0.0001). Additionally, there was a significant increase in ROS in vehicle- treated XDP fibroblasts compared with control fibroblasts (p < 0.0001). XY graph represents the average ROS measured in fibroblasts derived from two individuals living with XDP (33,109 and 35,883) and two controls (36,175 and 33,362). (FIG. 10B) Two- way ANOVA demonstrated a significant effect of treatment ([F (2, 24) = 25.66], p < 0.0001), and time ([F (12, 24) = 8.994], p < 0.0001) on ROS levels in SH- SY5Y cells. As expected, Tukey's test revealed a significant increase in ROS in antimycin A- treated cells compared with vehicle- treated cells (p < 0.0001). Importantly, there was a significant increase in ROS in XDP- treated cells compared with vehicle- treated cells (p < 0.0001). ****p < 0.0001.

[0033] FIG. 11 shows MPO immunodepletion decreased ROS levels in SH- SY5Y cells. (FIG. 11 A) One- way ANOVA revealed a significant effect of immunodepletion ([F (2, 9) = 24.7], p = 0.0002) on MPO activity in SH- SY5Y cells. Specifically, Tukey's test revealed a significant decrease in MPO activity in XDP(-) treated cells compared with both XDP (p = 0.011) and XDP(+) treated cells (p = 0.0003). (FIG. 1 IB) One- way ANOVA demonstrated a significant effect of treatment ([F (3, 12) = 9.763], p = 0.0015) on MPO activity in SH- SY5Y cells. Tukey's test demonstrated a significant increase in MPO activity in XDP- treated cells compared with vehicle- treated cells (p = 0.0460) as well as a significant decrease in MPO activity in XDP(-) cells compared with both XDP (p = 0.0020) and XDP(+) treated cells (p = 0.0066). (FIG. 11C) Two- way ANOVA revealed a significant effect of immunodepletion ([F (4, 48) = 32.85], p < 0.0001) and time ([F (12, 48) = 16.96], p < 0.0001) on ROS levels in SH- SY5Y cells. As expected, there was an increase in ROS in antimycin A treated cells compared with vehicle treated cells (Tukey's test, p < 0.0001). Additionally, Tukey's test demonstrated a significant increase in ROS in XDP (p < 0.0001) and XDP(+) treated cells (p < 0.0001) compared with vehicle treated cells. Importantly, Tukey's test also revealed a significant decrease in ROS in XDP(-) treated cells compared with both XDP (p < 0.0001) and XDP(+) treated cells (p < 0.0001). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0034] FIG. 12 shows verdiperstat decreased MPO activity and ROS levels in XDP fibroblasts. (A) Two- way ANOVA revealed a significant effect of treatment ([F (5, 36) = 20.08], p < 0.0001), genotype ([F (1, 36) = 2890], p < 0.0001), and treatment x XDP interaction ([F (5, 36) = 22.71], p < 0.0001) on MPO activity in fibroblasts. Tukey's' test demonstrated a significant increase in MPO activity in XDP- compared with control- derived fibroblasts (p < 0.0001) as well as a significant increase in MPO activity in XDP- derived fibroblasts treated with either 0.1 (p < 0.0001), 0.5 (p < 0.0001), 1 (p < 0.0001), 5 (p < 0.0001), or 10 pg / mL of verdiperstat (p < 0.0001) compared with control- derived fibroblasts treated with the same doses of verdiperstat. Lastly, Tukey's test revealed a significant decrease in MPO activity in XDP- derived fibroblasts treated with either 5 (p < 0.0001) or 10 pg / mL of verdiperstat (p < 0.0001) compared with vehicle- treated XDP- derived fibroblasts. Bar graph represents the average MPO activity measured in two different XDP fibroblast lines (33109 and 35883) and two different control fibroblasts (36175 and 33362). (FIG. 12B) Two- way ANOVA revealed a significant effect of genotype ([F (4, 48) = 120.3], p < 0.001), and time ([F (12, 48) = 61.32], p < 0.0001) on ROS levels in fibroblasts. Tukey's test revealed an expected significant increase in ROS in antimycin A- treated fibroblasts compared to vehicle- treated control fibroblasts (p < 0.0001). There was also a significant with in ROS in XDP fibroblasts compared to control fibroblasts (p < 0.0001). Importantly, Tukey's test demonstrated a significant decrease in ROS in both control- and XDP- derived fibroblasts treated with verdiperstat compared with vehicle- treated control (p = 0.052) and XDP fibroblasts (p < 0.0001). (FIG. 12C) Two- way ANOVA demonstrated a significant effect of genotype ([F (4, 48) = 91.45], p < 0.0001), and time ([F (12, 48) = 3.579], p = 0.0008) on ROS levels in a second set of fibroblasts. As expected, there was a significant increase in antimycin A- treated fibroblasts compared with vehicle- treated control fibroblasts (p < 0.0001). Furthermore, Tukey's test demonstrated a significant increase in ROS in XDP fibroblasts compared with control fibroblasts (p = 0.0025). Importantly, Tukey's test revealed a significant decrease in ROS levels in both control- and XDP- derived fibroblasts treated with verdiperstat compared with vehicle- treated control (p < 0.0001) and XDP fibroblasts (p = 0.0002). **p < 0.001; ****p < 0.0001.

[0035] FIG. 13 shows verdiperstat decreased MPO activity and ROS levels in XDP- treated SH- SY5Y cells. (FIG. 13 A) One- way ANOVA revealed a significant effect of treatment ([F (2, 9) = 29.19], p = 0.0001) on MPO activity in SH- SY5Y cells. Tukey's test revealed a significant increase in MPO activity in XDP treated cells compared with both vehicle (p = 0.0012) and XDP+verdiperstat treated cells (p = 0.0001). (FIG. 13B) Two- way ANOVA revealed a significant effect of treatment ([F (4, 48) = 16.35], p < 0.0001), and time ([F (12, 48) = 31.68], p < 0.001) on ROS levels in SH- SY5Y cells. As expected, ROS levels were increased in antimycin A treated cells compared with vehicle treatment (Tukey's test, p = 0.0003). Additionally, Tukey's test demonstrated a significant increase in ROS in XDP treated cells compared with both vehicle (p = 0.0014) and XDP+verdiperstat treated cells (p < 0.0001, respectively). **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0036] DETAILED DESCRIPTION OF THE INVENTION

[0037] Compounds

[0038] Provided herein is a compound of Formula (IA): or a pharmaceutically acceptable salt thereof, wherein: CN, halogen, -NH2, or -CH3;

[0039] RSA R5B,anj RCare each in(iepen(ientiy H or -CH3;

[0040] RDis -Ci-10 alkyl substituted with REwherein the -C1-10 alkyl of RDis optionally substituted with 1, 2, 3, or 4 substituents independently selected from -OH, -OCH3, -CN, halogen, and NH2 and 1 or 2 non-terminal carbons of the -C1-10 alkyl of RDare independently optionally replaced with O, -NH, or -NCH3; and REcomprises a fluorescent group.

[0041] In some embodiments, R1Ais H. In some embodiments, R1Ais -OH. In some embodiments, R1Ais -OCH3. In some embodiments, R1Ais -CN. In some embodiments, R1Ais halogen. In some embodiments, R1Ais -NH2. In some embodiments, R1Ais -CH3.

[0042] In some embodiments, R2Ais H. In some embodiments, R2Ais -OH. In some embodiments, R2Ais -OCH3. In some embodiments, R2Ais -CN. In some embodiments, R2Ais halogen. In some embodiments, R2Ais -NH2. In some embodiments, R2Ais -CH3.

[0043] In some embodiments, R3Ais H. In some embodiments, R3Ais -OH. In some embodiments, R3Ais -OCH3. In some embodiments, R3Ais -CN. In some embodiments, R3Ais halogen. In some embodiments, R3Ais -NH2. In some embodiments, R3Ais -CH3.

[0044] In some embodiments, R4Ais H. In some embodiments, R4Ais -OH. In some embodiments, R4Ais -OCH3. In some embodiments, R4Ais -CN. In some embodiments, R4Ais halogen. In some embodiments, R4Ais -NH2. In some embodiments, R4Ais -CH3.

[0045] In some embodiments, R1Bis H. In some embodiments, R1Bis -OH. In some embodiments, R1Bis -OCH3. In some embodiments, R1Bis -CN. In some embodiments, R1Bis halogen. In some embodiments, R1Bis -NH2. In some embodiments, R1Bis -CH3.

[0046] In some embodiments, R2Bis H. In some embodiments, R2Bis -OH. In some embodiments, R2Bis -OCH3. In some embodiments, R2Bis -CN. In some embodiments, R2Bis halogen. In some embodiments, R2Bis -NH2. In some embodiments, R2Bis -CH3.

[0047] In some embodiments, R3Bis H. In some embodiments, R3Bis -OH. In some embodiments, R3Bis -OCH3. In some embodiments, R3Bis -CN. In some embodiments, R3Bis halogen. In some embodiments, R3Bis -NH2. In some embodiments, R3Bis -CH3.

[0048] In some embodiments, R4Bis H. In some embodiments, R4Bis -OH. In some embodiments, R4Bis -OCH3. In some embodiments, R4Bis -CN. In some embodiments, R4Bis halogen. In some embodiments, R4Bis -NH2. In some embodiments, R4Bis -CH3.

[0049] In some embodiments, R3Ais -OH and R1A, R2A, and R4Aare each H. In some embodiments, R3Bis -OH and R1B, R2B, and R4Bare each H. In some embodiments, R3Ais - OH and R3Bis -OH. In some embodiments, R1A, R2A, R4A, R1B, R2B, and R4Bare each H. In some embodiments, R1A, R2A, R4A, R1B, R2B, and R4Bare each H, R3Ais -OH, and R3Bis -OH.

[0050] In some embodiments, R5Ais H. In some embodiments, R5Ais -CH3.

[0051] In some embodiments, R5Bis H. In some embodiments, R5Bis -CH3.

[0052] In some embodiments, R3Ais -OH and R5Ais H. In some embodiments, R3Bis -OH and R5Bis H. In some embodiments, R3Ais -OH, R5Ais H, R3Bis -OH, and R5Bis H. In some embodiments, R3Ais -OH and R1A, R2A, R4A, and R5Aare each H. In some embodiments, R3B is -OH and R1B, R2B, R4B, and R5Bare each H. In some embodiments, R3Ais -OH, R3Bis -OH, and R1A, R2A, R4A, R5A, R1B, R2B, R4B, and R5Bare each H.

[0053] In some embodiments, Rcis H. In some embodiments, Rcis -CH3.

[0054] In some embodiments, RDis -C1-10 alkyl substituted with RE. In some embodiments, RDis -C2-8 alkyl substituted with RE. In some embodiments, RDis -C3-7 alkyl substituted with RE. In some embodiments, RDis -C4-6 alkyl substituted with RE. In some embodiments, RDis -C5 alkyl substituted with RE. In some embodiments, RDis .

[0055] In some embodiments, REcomprises Cy3, Cy5, FITC, Rhodamine red, Rhodamine, Rhodamine B, or BODIPY. In some embodiments, REcomprises Cy3, Cy5, Cy7, FITC, Rhodamine red, Rhodamine, Rhodamine B, or BODIPY. In some embodiments, REcomprises Cy3, Cy5, Cy7, Alexa647, FITC, Rhodamine red, Rhodamine, Rhodamine B, or BODIPY.

[0056] In some embodiments, wherein

[0057] REis C1-4 alkyl optionally substituted with SO3H, OH, or NH2. In some embodiments, REis C1-4 alkyl. In some embodiments, REis -CH3. In some embodiments, REis -CH2CH3. In some embodiments, REis C1-4 alkyl substituted with SO3H, OH, or NH2. In some embodiments, REis C1-4 alkyl substituted with SO3H. In some embodiments, REis C1-4 alkyl substituted with OH. In some embodiments, REis C1-4 alkyl substituted with NH2. In some embodiments, REis C3 alkyl substituted with SO3H, OH, or NH2. In some embodiments, REis C3 alkyl substituted with SO3H. In some embodiments, REis C3 alkyl substituted with OH. In some embodiments, REis C3 alkyl substituted with NH2.

[0058]

[0059]

[0060] In some embodiments, the compound of Formula (IA) is a compound of formula

[0061] pharmaceutically acceptable salt thereof.

[0062] In some embodiments, the compound of Formula (IA) is a compound of formula pharmaceutically acceptable salt thereof.

[0063] Also provided herein is a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein: Ring A is selected from the group consisting of Ce-io aryl, 4-16 membered heterocycloalkyl, and 5-16 membered heteroaryl;

[0064] Ring B is selected from the group consisting of Ce-io aryl, 4-16 membered heterocycloalkyl, and 5-16 membered heteroaryl; L1is selected from the group consisting of NHC(O)L2, NHC(O)-L2-C(O)NH, L2- NHC(O)-L2, L2-NHC(O)-L2-NHC(O), and L2-NHC(O)-L2-NHC(O)-L2-; each L2is independently selected from the group consisting of C1-4 alkylene, C1-4 alkyleneoxy, and C1-4 alkenylene;

[0065] R1comprises a fluorescent moiety;

[0066] R2are R3are each independently selected from the group consisting of ORa, C(O)Ra, and OC(O)Ra; each Rais independently selected from the group consisting of H and C1-4 alkyl; m is 0, 1, 2, 3, or 4; and n is 0, 1, 2, 3, or 4.

[0067] In some embodiments, Ring A is selected from the group consisting of phenyl, bicyclic 8-16 membered heterocycloalkyl, tricyclic 8-16 membered heterocycloalkyl, bicyclic 8-16 membered heteroaryl, and tricyclic 8-16 membered heteroaryl.

[0068] In some embodiments, Ring A is selected from the group consisting of: wherein indicates the bond between Ring A and L1.

[0069] In some embodiments, m is 1 or 2. In some embodiments, m is 1.

[0070] In some embodiments, each R2is independently selected from the group consisting of OH, OCH3, C(O)CH3, and OC(O)CH3. In some embodiments, each R2is OH.

[0071] In some embodiments, Ring A is selected from the group consisting of: wherein indicates the bond between Ring A and L1

[0072] In some embodiments, Ring A is selected from the group consisting of: wherein indicates the bond between Ring A and L1.

[0073] In some embodiments, Ring B is selected from the group consisting of phenyl, bicyclic 8-16 membered heterocycloalkyl, tricyclic 8-16 membered heterocycloalkyl, bicyclic 8-16 membered heteroaryl, and tricyclic 8-16 membered heteroaryl.

[0074] In some embodiments, Ring B is selected from the group consisting of: wherein indicates the bond between Ring B and L1. n some embodiments, n is 1 or 2. In some embodiments, n is 1. In some embodiments, each R3is independently selected from the group consisting of OH, OCH3, C(O)CH3, and OC(O)CH3. In some embodiments, each R3is OH.

[0075] In some embodiments, Ring B is selected from the group consisting of: wherein In some embodiments, Ring B is selected from the group consisting of: wherein indicates the between Ring B and L1.

[0076] In some embodiments, Ring A and Ring B are the same. In some embodiments, Ring A and Ring B are different. In some embodiments, Ring A and Ring B are each In some embodiments, L1is L2-NHC(O)-L2-NHC(O)-L2-, and each L2is an independently selected C1-4 alkylene.

[0077] In some embodiments, L1is: wherein: indicates the bond between L1and Ring A;

[0078] - indicates the bond between L1and Ring B; and

[0079] — indicates the bond between L1and R1.

[0080] In some embodiments, R1is a fluorescent moiety. In some embodiments, the fluorescent moiety is Cy3, Cy5, FITC, Rhodamine red, or BODIPY.

[0081] In some embodiments, the compound of Formula (I) is a compound of Formula (II): or a pharmaceutically acceptable salt thereof.

[0082] In some embodiments, the compound of Formula (I) is a compound of Formula (III): or a pharmaceutically acceptable salt thereof.

[0083] In some embodiments, the compound of Formula (I) is a compound of Formula (IV):

[0084] or a pharmaceutically acceptable salt thereof.

[0085] Treatment Methods

[0086] The present application further provides a method of imaging a cell or tissue sample. In some embodiments, the method comprises: i) administering to the subject a compound provided herein (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof); ii) waiting a time sufficient to allow the compound to accumulate at the cell or tissue sample; and iii) imaging the cell or tissue sample with an imaging technique. In some embodiments, the method further comprises imaging the cell or tissue sample prior to step i).

[0087] The present application further provides a method of diagnosing a disease or disorder associated with abnormal myeloperoxidase (MPO) activity in a subject. In some embodiments, the method comprises: i) administering to the subject a compound provided herein (e.g., a compound of Formula (I), or a pharmaceutically acceptable salt thereof); ii) waiting a time sufficient to allow the compound to accumulate at a cell or tissue site associated with the disease; and iii) imaging the cell or tissue with an imaging technique. In some embodiments, the method further comprises imaging the subject prior to step i).

[0088] In some embodiments, the method is an in vitro method. In some embodiments, the method is an in vivo method.

[0089] In some embodiments, the time sufficient is from about 5 minutes to about 6 hours, for example, from about 5 minutes to about 6 hours, about 5 minutes to about 4 hours, about 5 minutes to about 2 hours, about 5 minutes to about 1 hour, about 5 minutes to about 30 minutes, about 30 minutes to about 6 hours, about 30 minutes to about 4 hours, about 30 minutes to about 2 hours, about 30 minutes to about 1 hour, about 1 hour to about 6 hours, about 1 hour to about 4 hours, about 1 hour to about 2 hours, about 2 hours to about 6 hours, about 2 hours to about 4 hours, or from about 4 hours to about 6 hours.

[0090] The present application further provides a method of imaging myeloperoxidase (MPO) activity in a cell. In some embodiments, the method comprises: i) contacting the cell with a compound provided herein; and ii) imaging the cell with an imaging technique.

[0091] The present application further provides a method of imaging myeloperoxidase (MPO) activity in a tissue sample. In some embodiments, the method comprises: i) contacting the tissue sample with a compound provided herein; and ii) imaging the tissue sample with an imaging technique.

[0092] The present application further provides a method of detecting myeloperoxidase (MPO) activity in a cell or tissue sample. In some embodiments, the method comprises: i) contacting the cell or tissue sample with a compound provided herein; and ii) imaging the cell or tissue sample with an imaging technique.

[0093] The present application further provides a method of detecting myeloperoxidase activity in a subject. In some embodiments, the method comprises: i) administering to the subject a compound provided herein; and ii) imaging the subject with an imaging technique.

[0094] The present application further provides a method of monitoring treatment of a disease or disorder associated with abnormal myeloperoxidase (MPO) activity in a subject, the method comprising: i) administering to the subject a compound provided herein; ii) imaging the subject with an imaging technique; iii) administering to the subject a therapeutically effective amount of a therapeutic compound to treat the disease or disorder; iv) imaging the cell or tissue in the subject with an imaging technique; and v) comparing the image of step i) and the image of step iv).

[0095] In some embodiments, the method further comprises administering to the subject a compound provided herein after the administering of step iii) and prior to the imaging of step iv).

[0096] In some embodiments, the therapeutic compound is a compound useful in the treatment of a disease or disorder associated with abnormal myeloperoxidase (MPO) activity. In some embodiments, the therapeutic compound is an MPO inhibitor. In some embodiments, the therapeutic compound is verdiperstat (l-(2 -isopropoxy ethyl)-2-thioxo-l, 2, 3, 5-tetrahydro- 4H-pyrrolo[3,2-d]pyrimidin-4-one).

[0097] In some embodiments, the imaging technique is fluorescence imaging.

[0098] In some embodiments, the disease or disorder associated with abnormal myeloperoxidase activity is selected from the group consisting of nonalcoholic steatohepatitis (NASH), cancer, a rheumatic disease, an infectious disease, a disease of the central nervous system, a cardiovascular disorder, an autoimmune disorder, and inflammation associated with one or more of a cancer, a rheumatic disease, an infectious disease, a disease of the central nervous system, a cardiovascular disorder, and an autoimmune disorder. In some embodiments, the disease or disorder associated with abnormal myeloperoxidase activity is selected from the group consisting of cancer, a rheumatic disease, an infectious disease, a disease of the central nervous system, a cardiovascular disorder, and an autoimmune disorder. In some embodiments, the disease or disorder associated with abnormal myeloperoxidase activity is selected from the group consisting of inflammation associated with one or more of a cancer, a rheumatic disease, an infectious disease, a disease of the central nervous system, a cardiovascular disorder, and an autoimmune disorder.

[0099] In some embodiments, the disease or disorder associated with abnormal myeloperoxidase activity is nonalcoholic steatohepatitis (NASH).

[0100] In some embodiments, the disease or disorder associated with abnormal myeloperoxidase activity is a cancer. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from the group consisting of bladder cancer, breast cancer, carcinoma, cervical cancer, colorectal cancer, endometrial cancer, glioma, cancer of the head and neck, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, stomach cancer, testicular cancer, leukemia, and thyroid cancer. In some embodiments, the cancer is a solid tumor associated with one or more of bladder cancer, breast cancer, carcinoma, cervical cancer, colorectal cancer, endometrial cancer, glioma, cancer of the head and neck, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, or any combination thereof. In some embodiments, the disease or disorder associated with abnormal myeloperoxidase activity is inflammation associated with one or more cancers selected from the group consisting of bladder cancer, breast cancer, carcinoma, cervical cancer, colorectal cancer, endometrial cancer, glioma, cancer of the head and neck, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, leukemia, or any combination thereof.

[0101] In some embodiments, the disease or disorder associated with abnormal myeloperoxidase activity is a disease of the central nervous system. In some embodiments, the disease of the central nervous system is selected from the group consisting of Alzheimer’s disease, stroke, epilepsy, Parkinson’s disease, and inflammation associated with Alzheimer’s disease, stroke, epilepsy, and Parkinson’s disease. In some embodiments, the disease of the central nervous system is selected from the group consisting of Alzheimer’s disease, stroke, epilepsy, and Parkinson’s disease. In some embodiments, the disease of the central nervous system is inflammation associated with one or more of Alzheimer’s disease, and stroke, epilepsy, and Parkinson’s disease.

[0102] In some embodiments, the disease or disorder associated with abnormal myeloperoxidase activity is a cardiovascular disorder. In some embodiments, the cardiovascular disorder is selected from the group consisting of atherosclerosis, myocardial infarction, atrial fibrillation, vasculitis, and inflammation associated with one or more of atherosclerosis, myocardial infarction, atrial fibrillation, and vasculitis. In some embodiments, the cardiovascular disorder is selected from the group consisting of atherosclerosis, myocardial infarction, atrial fibrillation, and vasculitis. In some embodiments, the cardiovascular disorder is inflammation associated with one or more of atherosclerosis, myocardial infarction, atrial fibrillation, and vasculitis.

[0103] In some embodiments, the disease or disorder associated with abnormal myeloperoxidase activity is an autoimmune disorder. In some embodiments, the autoimmune disorder is selected from the group consisting of multiple sclerosis, meningitis, encephalitis, and inflammation associated with one or more of multiple sclerosis, meningitis, and encephalitis. In some embodiments, the autoimmune disorder is inflammation associated with one or more of multiple sclerosis, meningitis, and encephalitis.

[0104] In some embodiments, the disease or disorder associated with abnormal myeloperoxidase activity is a rheumatic disease. In some embodiments, the rheumatic disease is selected from the group consisting of rheumatoid arthritis, osteoarthritis, and inflammatory arthritis. In some embodiments, the rheumatic disease is inflammatory arthritis. In some embodiments, the inflammatory arthritis is selected from the group consisting of gout and calcium pyrophosphate deposition disease (CPPD). In some embodiments, the disease or disorder associated with abnormal myeloperoxidase activity is inflammation associated with one or more of rheumatoid arthritis, osteoarthritis, and inflammatory arthritis. In some embodiments, the disease or disorder associated with abnormal myeloperoxidase activity is an infectious disease. In some embodiments, the infectious disease is a fungal disease or a bacterial disease. In some embodiments, the fungal disease is a disease associated with C. albicans. In some embodiments, the infectious disease comprises a yeast infection. In some embodiments, the yeast infection is an infection associated with C. tropicalis. In some embodiments, the disease or disorder associated with abnormal myeloperoxidase activity is inflammation associated with an infectious disease or a bacterial disease.

[0105] In some embodiments, the disease or disorder associated with abnormal myeloperoxidase activity is parkinsonism. In some embodiments, the disease or disorder associated with abnormal myeloperoxidase activity Parkinson’s disease. In some embodiments, the disease or disorder associated with abnormal myeloperoxidase activity is X-linked dystonia parkinsonism (XDP). In some embodiments, the disease or disorder associate with abnormal myeloperoxidase activity is multiple system atrophy. In some embodiments, the disease or disorder associated with abnormal myeloperoxidase activity is Alzheimer's disease, Huntington's disease, Parkinson's disease, or amyotrophic lateral sclerosis.

[0106] Pharmaceutical Formulations

[0107] In some embodiments, the compounds described herein are administered alone or as part of a pharmaceutically acceptable composition comprising the compound and a pharmaceutically acceptable carrier. The relative amounts of the compound and the pharmaceutically acceptable carrier will vary depending upon the identity, size, and condition of the subject and further depending upon the route by which the composition is to be administered. In some embodiments, the composition is administered to a subject parenterally, for example, intravenously, intramuscularly, subcutaneously, intracerebrally, or intrathecally. In some embodiments, the composition is administered to a subject intravenously.

[0108] In some embodiments, the pharmaceutically acceptable carrier comprises a liquid medium suitable for parenteral administration. In some embodiments, the pharmaceutically acceptable carrier comprises water. In some embodiments, the pharmaceutically acceptable carrier further comprises additional components such as buffers and / or tonicity modifiers.

[0109] In some embodiments, the pharmaceutical composition is formulated as a solid to be dissolved in a carrier such as water prior to administration to a subject. In such embodiments, the solid formulation further comprises additional components such as buffers and / or tonicity modifiers.

[0110] In some embodiments, the pharmaceutical composition comprises a mixture of the compounds described herein.

[0111] Definitions

[0112] The term “n-membered” where n is an integer typically describes the number of ringforming atoms in a moiety where the number of ring-forming atoms is n. For example, phenyl is an example of a 6-membered aryl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, and pyridyl is an example of a 6-membered heteroaryl ring.

[0113] Throughout the definitions, the term “Cn-m” indicates a range which includes the endpoints, wherein n and m are integers and indicate the number of carbons. Examples include Ci-4, Ci-6, and the like.

[0114] As used herein, the term “Cn-m alkyl” refers to a saturated hydrocarbon group that may be straight-chain or branched, having n to m carbons. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, w-propyl, isopropyl, w-butyl, tertbutyl, isobutyl, ec-butyl, and the like. In some embodiments, the alkyl group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

[0115] As used herein, the term “Cn-m alkylene” refers to a divalent alkyl linking group having n to m carbons (e.g, -CH2-, ethan-l,2-diyl, propan- 1,3 -diyl, and the like). In some embodiments, the alkylene moiety contains 2 to 6, 2 to 4, 2 to 3, 1 to 6, 1 to 4, or 1 to 2 carbon atoms.

[0116] As used herein, the term “Cn-m alkenylene” refers to a divalent alkene linking group having n to m carbons. In some embodiments, the alkenylene moiety contains 2 to 6, 2 to 4, or 2 to 3, carbon atoms.

[0117] As used herein, the term “Cn-m alkyleneoxy” refers to a divalent alkoxy linking group having n to m carbons (z.e., “-O-Cn-m alkylene-”). In some embodiments, the alkyleneoxy moiety contains 1 to 2 to 6, 2 to 4, 2 to 3, 1 to 6, 1 to 4, or 1 to 2 carbon atoms.

[0118] As used herein, the term “aryl” refers to an aromatic hydrocarbon group, which may be monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings). The term “Cn-m aryl” refers to an aryl group having from n to m ring carbon atoms. Aryl groups include, e.g, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms, from 6 to about 15 carbon atoms, or from 6 to about 10 carbon atoms. In some embodiments, the aryl group is phenyl.

[0119] As used herein, “heteroaryl” refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, any ring-forming N in a heteroaryl moiety can be an N-oxide. In some embodiments, the heteroaryl has 5-20 ring atoms and 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl has 5-16 ring atoms and 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl is a bicyclic heteroaryl (e.g., a fused bicyclic heteroaryl) having 5-16 ring atoms and 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl is a tricyclic heteroaryl (e.g., a fused tricyclic heteroaryl) having 5-16 ring atoms and 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen.

[0120] As used herein, “heterocycloalkyl” refers to non-aromatic monocyclic or polycyclic heterocycles having one or more ring-forming heteroatoms selected from O, N, or S. Ringforming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally substituted by oxo or sulfido (e.g., C(O), S(O), C(S), or S(O)2, etc.). The heterocycloalkyl group can be attached through a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds or 0 to 2 double bonds. A heterocycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. In some embodiments, the heterocycloalkyl has 4-20, 4-16, 8-20, or 8-16 ring atoms with 1 or 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and optionally having one or more oxidized ring members. In some embodiments, the heterocycloalkyl is a bicyclic heterocycloalkyl (e.g., a fused bicyclic heterocycloalkyl) having 4-16 ring atoms and 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen, and optionally having one or more oxidized ring members. In some embodiments, the heterocycloalkyl is a tricyclic heterocycloalkyl (e.g., a fused tricyclic heterocycloalkyl) having 4-16 ring atoms and 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen, and optionally having one or more oxidized ring members. At certain places, the definitions or embodiments refer to specific rings (e.g., an azetidine ring, a pyridine ring, etc.). Unless otherwise indicated, these rings can be attached to any ring member provided that the valency of the atom is not exceeded. For example, a pyridinyl ring may be attached at any position of the ring, whereas a pyridin-3-yl ring is attached at the 3 -position.

[0121] At various places in the present specification, divalent linking substituents are described. It is specifically intended that each divalent linking substituent include both the forward and backward forms of the linking substituent. For example, -NR(CR’R”)n- includes both -NR(CR’R”)n- and -(CR’R”)nNR-; and NHC(O)L2includes both -NHC(O)L2- and - L2C(O)NH-. Where the structure clearly requires a linking group, the Markush variables listed for that group are understood to be linking groups.

[0122] The term “compound” as used herein is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. Compounds herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.

[0123] Compounds provided herein also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

[0124] Unless specifically defined, compounds provided herein can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. Unless otherwise stated, when an atom is designated as an isotope or radioisotope, the atom is understood to comprise the isotope or radioisotope in an amount at least greater than the natural abundance of the isotope or radioisotope. For example, when an atom is designated as “D” or “deuterium”, the position is understood to have deuterium at an abundance that is at least 3000 times greater than the natural abundance of deuterium, which is 0.015% (i.e., at least 45% incorporation of deuterium). The reactions for preparing compounds and salts described herein can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, (e.g., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature). A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected by the skilled artisan.

[0125] Preparation of compounds and salts described herein can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rdEd., Wiley & Sons, Inc., New York (1999).

[0126] Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g.,JH or13C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or by chromatographic methods such as high- performance liquid chromatography (HPLC), liquid chromatography-mass spectroscopy (LCMS), or thin layer chromatography (TLC). Compounds can be purified by those skilled in the art by a variety of methods, including high performance liquid chromatography (HPLC) and normal phase silica chromatography.

[0127] All compounds, and pharmaceutically acceptable salts thereof, can be found together with other substances such as water and solvents (e.g., hydrates and solvates) or can be isolated.

[0128] In some embodiments, preparation of compounds can involve the addition of acids or bases to affect, for example, catalysis of a desired reaction or formation of salt forms such as acid addition salts.

[0129] Example acids can be inorganic or organic acids and include, but are not limited to, strong and weak acids. Some example acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, / ?-toluenesulfonic acid, 4 -nitrobenzoic acid, methanesulfonic acid, benzenesulfonic acid, trifluoroacetic acid, and nitric acid. Some weak acids include, but are not limited to acetic acid, propionic acid, butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid. Example bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and sodium bicarbonate. Some example strong bases include, but are not limited to, hydroxide, alkoxides, metal amides, metal hydrides, metal dialkylamides and arylamines, wherein; alkoxides include lithium, sodium and potassium salts of methyl, ethyl and t-butyl oxides; metal amides include sodium amide, potassium amide and lithium amide; metal hydrides include sodium hydride, potassium hydride and lithium hydride; and metal dialkylamides include lithium, sodium, and potassium salts of methyl, ethyl, n-propyl, / .w-propyl, n-butyl, tert-butyl, trimethyl silyl and cyclohexyl substituted amides.

[0130] In some embodiments, the compounds provided herein, or salts thereof, are substantially isolated. By “substantially isolated” is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compounds provided herein. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compounds provided herein, or salt thereof. Methods for isolating compounds and their salts are routine in the art.

[0131] As used herein, the term “room temperature” or “rt” is understood in the art, and refers generally to a temperature, e.g., a reaction temperature, that is about the temperature of the room in which the reaction is carried out, for example, a temperature from about 20 °C to about 30 °C.

[0132] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0133] The present application also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present application include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol, or butanol) or acetonitrile (MeCN) are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977). Conventional methods for preparing salt forms are described, for example, in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, 2002.

[0134] As used herein, the phrase “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician.

[0135] As used herein, the term “treating” or “treatment” refers to one or more of (1) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and / or symptomatology); and (2) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and / or symptomatology) such as decreasing the severity of disease or reducing or alleviating one or more symptoms of the disease.

[0136] As used herein, the term “subject,” refers to any animal, including mammals. For example, mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, primates, and humans. In some embodiments, the subject is a human.

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[0238] EXAMPLES

[0239] The following examples are illustrative and not intended to be limiting. Example 1: Synthesis of myeloperoxidase activatable fluorescent agent (MAE A)

[0240] Step 1.

[0241] Intermediate 2

[0242] Starting from 5-hydroxytryptaphan, the Intermediate 2 was obtained in three steps in a total yield of 51% as described previously (Wang et al, J Med Chem 2021, 64 (9), 5874- 5885). To a solution of Intermediate 2 (8.4 mg, 1.2 equiv.) in DMSO (1 mL) was added triethylamine (6 qL). The resulting mixture was stirred for 20 min at room temperature. A solution of cy5-NHS ester (Intermediate 3) in DMSO (0.5 mL) was added to the above solution. The resulting solution stirred for another 1 h at the same temperature. The reaction solution underwent high performance liquid chromatography (HPLC) to give the desired Compound 4 (MAFA) in the yield of 48%. High resolution mass spectrometry (HRMS) of MPO-disero-cy5: found 1017.3888 (Calc. 1017.3885).

[0243] Example A: Aging Intensifies Myeloperoxidase Activity after Ischemic Stroke

[0244] Animal model of focal cerebral ischemia

[0245] Animal experiments were approved by the local institutional animal care. All mice were maintained in a federally approved animal facility at our institution and allowed to acclimate for one week prior to the start of the experiments. Transient focal cerebral ischemia was induced in 60 male and female aged C57BL / 6J mice (10-14 months old) and 46 male and female young adult C57BL / 6J mice (2-3 months old). To investigate the effects MPO inhibition has on aged mice after stroke, 14 mice from the aged group were treated intraperitoneally twice daily with 80 mg / kg of 4-aminobenzoic acid hydrazide (AB AH), a specific and irreversible MPO inhibitor.

[0246] Mice were anesthetized with 2% isoflurane. A rectal temperature probe was inserted to monitor and maintain a constant animal core temperature of 37 ± 0.5 °C using a temperature controller (TC-1000, CWE INC, Ardmore, PA), was induced by MCAO as described by Li et al. Briefly, an 8-0 nylon monofilament suture (ETHICON LLC, Puerto Rico, USA) coated with silicone rubber (Heraeus Kulzer LLC, South Bend, IN) and hardener was inserted into the left internal carotid artery and advanced approximately 10 mm distal to the carotid bifurcation to occlude the origin of the middle cerebral artery. The thread was carefully withdrawn 30 min after MCAO to induce I / R injury. On day 3 and day 10 after surgery, blood was collected for flow cytometry and brains were collected for flow cytometry, ELISA, and immunofluorescent staining. To investigate MAFA specificity, focal cerebral ischemia was induced in three C57BL / 6J male and female aged mice (10-14 months old, Jackson Laboratories) and three aged-matched (10-14 months old), MPO knockout mice (B6.129Xl-MpotmlLus / J, Jackson Laboratories). Fresh frozen brains were collected on day 3 after induction for immunofluorescent staining. Animals with intracranial hemorrhages were excluded from the study (n = 5 from aged n = 2 from young adults and n = 2 from aged ABAH-treated) because hemorrhages can exaggerate the inflammatory and immune response in ischemic areas.

[0247] Behavior tests

[0248] The following three types of behavior tests were performed with eleven male and female aged C57BL / 6J mice (10-14 months old) and six male and female young adult C57BL / 6J mice (2-3 months old).

[0249] Neurological deficit score: Neurologic deficit score (NDS) was scored into four categories (modified from Clark and colleagues) daily for up to 10 days after MCAO stroke induction.

[0250] Hanging wire test: Mice were placed on a metal wire (60 cm long) suspended 48 cm above a foam pad.; the latency to when the animal falls within a 60-sec period is recorded every day after MCAO.

[0251] Cylinder test: Mice were placed in a glass cylinder (10 cm diameter and 15 cm height). The number of times the animal used each forelimb or both forelimbs to contact the wall of the cylinder within 10 minutes were counted for each animal daily for 10 days after MCAO and a percentage relative to the total number of contacts was computed.

[0252] Flow cytometry

[0253] Mice were transcardially perfused with 20-30 mL ice-cold PBS. The ischemic hemisphere of brains was collected and stored on ice in PBS. Then the brains were mechanically disrupted with a glass homogenizer and passed through a 40 pm nylon cell strainer (BD Biosciences, San Jose, CA), and single cells were isolated from 30 / 70 Percoll (GE Healthcare, Boston, MA) gradient, centrifuged for 5 minutes at 350 G and resuspended in FACS buffer (PBS with 0.5% BSA) for surface antibody staining. Antibody panels used were as described below. Briefly, cells were first stained with anti-CD16 / CD32 (Bio Legend, San Diego, CA, 1 :200) to block Fc binding sites for 20 minutes, washed with FACS buffer 3 times and then stained with anti-mouse antibodies against anti-NKl. l-PE / Cy7 (Bio Legend); anti-B220 / CD45-PE / Cy7 (Bio Legend); anti-CD3-PE / Cy7 (Invitrogen); anti-CDl Ib-PE (Bio Legend); anti-Ly6G-FITC (Bio Legend); anti-CD45.2-PB (Bio Legend); anti-MPO-Biotin (Hycult). For intracellular staining, cells were fixed and permeabilized using Cytofix / Cy toperm (BD Bioscience) after staining for cell surface markers. Streptavidin- conjugated Brilliant Violet 605 secondary antibody (Bio Legend) was used to label biotinylated anti-MPO. For MPO-positive cells analysis in blood, blood was lysed in red blood cell lysis buffer, and staining was performed in a similar setup as that for brain staining once cells were in a single-cell suspension. On flow cytometry, CD1 lb+CD45highcells represented peripherally derived CNS -infiltrating M<p / activated Mg while CD1 lb+CD45 mtermediate / iowrepresented resident Mg. These cells were then further identified as MPO+ and MPO- cells. Data were acquired with a flow cytometer (Fortessa X-20; BD Biosciences and Aurora) and analyzed with BD Flow Jo software (version 10.4).

[0254] Enzyme-linked immunosorbent assay

[0255] Ischemic brain hemispheres were homogenized separately in 500 pL of cetyltrimethylammonium buffer (50 mM potassium phosphate at pH 6.0 with 50 mM CTAB) by a tissue homogenizer (Fisher Scientific). The samples were then sonicated for 30 seconds and centrifuged at 5668 G for 15 minutes. The supernatant was used for protein analysis with a BCA protein assay kit (Thermo Scientific). The supernatant was then used for MPO (R&D system, Cat# dy3667) and beta-3 tubulin (MyBioSource, Cat# mbs9398627), a neuronal integrity marker ELISA following manufacture instructions.

[0256] Immunofluor e scent staining

[0257] Fresh-frozen brains were cut in serial cross sections (8-10 pm thick). The sections were fixed with 4% PFA for 2-3 minutes at room temperature (RT). The slides were incubated in blocking buffer (5% Donkey serum (EMD Millipore Cat# 3915503) and 0.3% triton x-100 (Millipore Cat# t8787-50 ml in PBS) at RT for 1 h, followed by incubating in blocking buffer containing the primary antibody overnight at 4 °C. Primary antibodies were rabbit anti-MPO (1 :300, Thermo Fisher, Cat#PA5- 16672), rat anti-NeuN (1 :500, Abeam Cat# ab279297), rat anti-Ibal(l :300, Abeam Cat# ab 283346) and for MAFA validation, Rabbit anti-chlorotyrosine (1 :500, Hycult, Cat# HP5002). On the second day, slides were rinsed with PBS and then incubated with secondary antibodies at RT for 1 h. Secondary antibodies were anti-rabbit Alexa flour 555 (1 :300, Invitrogen Cat# xf347096) and anti-rat Dy Light 488 (1 :300, Invitrogen, Cat# xg3652581). The sections were washed 3 times with PBS following incubating with MAFA (1 :300 of stock solution 10 mM in DMSO and 1 mM of 3% H2O2) for 30 minutes at RT. The sections were mounted in an antifade mounting medium (Vectashield, Cat# zjO8O8) and DAPI. Images were captured with the Nikon DS-Ri2 model microscope connected to Prime BSI Express. Statistical analysis

[0258] Results were reported as mean ± standard error of mean (SEM). Neurological deficit scores and behavior tests between the aged and young adult groups were analyzed using unpaired two-tailed t-test. Log-rank (Mantel-Cox test) was applied to compare the mortality rate between the aged untreated and aged ABAH-treated group.

[0259] Differences between the two groups for flow cytometry, ELISA, and immunostaining were assessed by the Mann-Whitney test. Three to four regions of interest within the infarct area were chosen for each mouse to calculate the integrated density of immunofluorescent images using ImageJ (v 2.1.0). GraphPad Prism (v.9.5.1, GraphPad Software) were used for statistical analysis. P < 0.05 was considered statistically significant.

[0260] Aged mice exhibit significantly worse neurological deficits scores and behavior outcomes compared to young adult mice

[0261] To assess neurological outcomes after stroke in both aged and young adult mice, the neurological deficit scores were recorded daily after stroke induction. Both young adults and aged mice showed clear neurological deficits on the 1st day after surgery. However, young adult mice showed rapid improvement in their deficits post-stroke whereas aged mice exhibited a slower recovery rate and continued to show functional deficits (FIG. 6A). Additional tests evaluating motor coordination similarly revealed a larger motor deficit in the aged mice compared to the young adult mice: in the hanging wire test young adult mice could hold on to a wire longer compared to aged mice after stroke, and in the cylinder test aged mice showed more asymmetric limb preference compared to young mice (FIG. 6B and FIG. 6C). These findings revealed that functional damage was exacerbated in aged mice compared to young mice after stroke. Aged mice not only had significantly worse functional outcomes compared to young mice but also had a higher stroke-induced mortality rate and lower survival rate than young adult mice after stroke (FIG. 6D and FIG. 6E). Notably, mortality was highest in the aged group within the first three days after stroke induction.

[0262] Validation ofMAFA tissue imaging in ischemic brain tissues

[0263] MAFA was designed as an activatable fluorescent probe for the detection of MPO activity. As a substrate for MPO, MPO oxidizes MAFA leading to the formation of radicals that can chemically link to nearby proteins containing similar moieties, such as tyrosine or tryptophan. Activated agents thus remain bound to tissue sections, while unactivated agents are easily removed through gentle washing steps. This enables activated MAFA to identify areas of elevated MPO activity. To validate the specificity of MAFA for MPO, experiments were conducted using brains harvested from aged wild-type and MPO-knockout (MPO-KO) mice three days post-ischemic stroke. MAFA was employed to detect MPO activity in the ischemic areas of brain sections, and the obtained signal was compared with chlorotyrosine immunostaining — a specific product of MPO activity. In wild-type mouse brains, a significant elevation in MPO activity was observed within the ischemic regions that correlated well with the chlorotyrosine signal. In contrast, virtually no signal was detected by either MAFA or chlorotyrosine immunostaining in the ischemic areas from MPO-KO mouse brains (p = 0.0357, FIG. 1 A and FIG. IB). This highlights the specificity of MAFA to MPO activity, as its signal matched well with that of chlorotyrosine in both wild-type and MPO- KO mice tissue sections. Given that young naive mouse brain sections exhibit a mean MAFA signal of 2.3 x 109(FIG. 2A and FIG. 2B), slightly above that of MPO-KO mice sections (7.5 x 108, considered to be background), the sensitivity or detection threshold of MAFA was estimated to be approximately ~2 x 109in this in vitro / ex vivo setting.

[0264] MPO activity in the brain increases with aging

[0265] MPO activity was next assessed in both aged and young adult naive brains without stroke using MAFA. Amarkedly increased MAFA signal was found in the thalamus region of aged brains compared to that of young adult brains (p = 0.0286, FIG. 2A and FIG. 2B).

[0266] Similarly, higher Ibal+ signals were found in the thalamus region of aged brains as compared to young adult brains (p = 0.0286, FIG. 2A and FIG. 2B). MPO ELISA also showed a higher amount of MPO protein in the aged brain compared to that in the young adult brains (p = 0.0286, FIG. 2B).

[0267] Recruitment ofMPO+ cells was altered by aging after an ischemic stroke

[0268] The effect of aging on the immune response was next assessed on day 3 (early subacute) and day 10 (late subacute) after ischemic stroke by flow cytometry. A significantly decreased percentage of MPO+ cells was observed in the brains of aged mice as compared to that of young adult mice on day 3 after surgery (p = 0.0286, FIG. 7A). In contrast, in the blood, a higher percentage of MPO+ cells were found in aged mice as compared to those of young adult mice on day 3 after stroke (p = 0.0286, FIG. 7B). Interestingly, a significantly increased percentage of MP0+ infiltrating macrophages M<p) / activated microglia (Mg) was observed in the brains of aged mice as compared to that of young adult mice on day 3 after surgery (p = 0.028, FIG. 7E). On day 10 post-stroke, a further elevation in the percentage of MP0+ cells was evident in the brains of aged mice compared to young mice (p = 0.0317, FIG. 7C). Conversely, a decreased percentage of MP0+ cells was observed in the blood of aged mice compared to young adult mice on day 10 after stroke (p = 0.0317, FIG. 7D). Although both groups experienced a decline in the percentage of MP0+ infiltrating M<p / activated Mg on day 10 after stroke, the levels remained elevated in the brains of aged mice compared to young adult mice (p = 0.0079, FIG. 7E and FIG. 7G). Interestingly, the percentage of MP0+ resident Mg remained consistently elevated in the aged compared to that of young ischemic brains over time (FIG. 7F and FIG. 7H).

[0269] Aging enhanced the MPO activity after ischemic stroke

[0270] Since it was demonstrated herein that aging affected immune cell recruitment and altered neurological outcomes after stroke was demonstrated, it was next investigated how MPO activity changes in aged and young adult brains on days 3 and 10 after stroke using MAFA. Aged brains were found to display significantly increased MAFA signal on day 3 after stroke compared to that of young adult mice (p = 0.0286, FIG. 3 A and FIG. 3B). Correspondingly, higher Ibal+ signals in aged brains were found as compared to young adult brains on day 3 after stroke, and colocalized with MAFA signals (p = 0.0286, FIG. 3 A and FIG. 3B). Interestingly, double immunofluorescent staining of MPO protein and MAFA showed that aged brains have less MPO protein compared to those of young adult brains on day 3 after stroke (p = 0.0286, FIG. 3B and FIG. 3D). In addition, the MPO protein foci in the aged brains are much smaller in size compared to those in the young adult brains, revealing that these smaller foci in the aged brain represent granules or lysosomes that have degranulated and secreted MPO. On the other hand, while there were more and larger foci of MPO protein in the young adult brain, there was comparably less MAFA signal, indicating that these MP0+ cells have less degree of degranulation. MPO immunostaining results were confirmed by MPO enzyme-linked immunosorbent assay (ELISA) (p = 0.0043, FIG. 3D). As expected, the neuronal nuclear protein (NeuN) staining revealed a significant decrease in neuronal integrity in aged brains on day 3 compared to that of younger brains on day 3 (p = 0.0286, FIG. 3E and FIG. 3F). Similarly, P-3 tubulin ELISA confirmed a similar decrease in the aged brains compared to those from young adult brains on day 3 after stroke (p = 0.026, FIG. 3F).

[0271] On day 10 after the stroke, while MAFA signal markedly decreased from that of day 3, higher MAFA signal and MPO protein were still detected in the aged brains compared to those of the young adult (FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D). Concordant with MPO immunostaining results, the amount of MPO protein was higher in the aged brain as compared to that of the young adult on day 10 after stroke (p = 0.0159 and p = 0.0317 respectively, FIG. 4B, FIG. 4D). Similar to MAFA results it was found that the Ibal signal also decreased on day 10 as compared to day 3 in both groups and the Ibal signal remained significantly higher in aged brains as compared with young adult brains (p = 0.0159, FIG. 4A, FIG. 3B). Notably, only a subset of Ibal+ cells showed MAFA signal, indicating that on day 10 not all Ibal+ cells are secreting MPO. Similar to day 3 results, a discordance was also found between MPO immunostaining and MPO activity (MAFA) imaging, again indicating that MPO immunostaining predominately identifies cells that have not secreted MPO. There were no significant differences between aged and young adult brains in NeuN immunostaining and P-3 tubulin assays on day 10 after stroke (FIG. 4E and FIG. 4F).

[0272] MPO inhibition decreased MAFA signal and improved behavior outcomes in aged in the early subacute stage following stroke

[0273] Next was investigated the impact of 4-aminobenzoic acid hydrazide (AB AH), an irreversible and specific MPO inhibitor, on neurobehavior outcomes during the early subacute stage following stroke in aged mice. As expected, MPO tissue imaging on day 3 after stroke revealed decreased MAFA signals in ABAH-treated brains compared to those in untreated brains (p = 0.0159, FIG. 5A and FIG. 5B). Similarly, a decrease in Ibal+ immunostaining in the ABAH-treated group was observed compared to that of the untreated group (p = 0.0159, FIG. 5A and FIG. 5B). Consistent with the decreased inflammatory response, it was found that ABAH-treated group exhibited notable improvement in functional outcomes compared to untreated group, approaching the levels of untreated young adult mice after stroke (FIG. 5C). Consequently, a significant improvement in mortality in the ABAH- treated group was observed compared to the control-aged group during the early subacute phase after stroke (p = 0.04, FIG. 5C).

[0274] Discussion

[0275] While aging is an unmodifiable risk factor for stroke, the adverse changes resulting from aging may be potentially altered. In this study, the new MAFA agent was developed and utilized to track MPO activity on ischemic tissues longitudinally in aged and young adult mice after stroke. Examining how MPO activity in thalamus and cortex regions changed from naive (pre-stroke), day 3 (post-stroke), and day 10 (post-stroke) brains in aged and young adults, it was found that while aging primarily impacts the thalamus and spares the cortex in naive mice with elevated MPO activity, MPO activity increased in both thalamus and cortex regions after stroke regardless of age. However, there was a much sharper increase in MPO activity in the aged animals in both regions within the first 3 days after ischemia, while the increase in the young adult animals was more modest. Although MPO activity decreased markedly in both aged and young adult mice by day 10 after stroke, it remained elevated in the aged brains compared to the young adult brains. Importantly, this rise in MPO activity after ischemic stroke in the aged group corresponded to the time of highest mortality in this group (within 48 hours). Notably, the administration of AB AH, a specific and irreversible inhibitor of MPO, exhibited remarkable outcomes in mitigating the inflammatory response, reducing MPO activity, enhancing neurological outcomes, and improving the mortality rate in the aged group after stroke. These positive effects played a pivotal role in ameliorating neurobehavior, with noticeable improvement apparent as early as 48 hours post-stroke. These observations suggest that modulating MPO activity during the initial phase has the potential to significantly improve post-stroke outcomes in the elderly population.

[0276] MAFA was designed to be an efficient and specific fluorescent probe to detect MPO activity. MAFA is activated by MPO and hydrogen peroxide to form radicals and chemically links to nearby proteins containing tyrosine or tryptophan. In comparison to other methods, such as MABS, which serves as a versatile imaging platform but is not specifically designed for fluorescent imaging and requires a secondary reporter imaging probe linked to streptavidin for reporting on MPO activity, MAFA stands out as a self-contained probe that does not require a secondary reporter. This characteristic makes MAFA more convenient for in vitro / ex vivo applications, requiring fewer washing steps. Additionally, in vivo administration requires only a single injection, streamlining the process compared to the two injections needed with MABS. Several other optical imaging agents that may report on MPO have been described but are not specific to MPO activity, including bioluminescent agents luminol and L-012 (designed to detect NADPH oxidase), SNAPF (designed to detect hypochlorous acid and other oxidants but not MPO activity directly). In this study, MAFA demonstrated the superiority of detecting MPO activity over antibody -based MPO protein immunostaining in identifying the underlying biological process. This is especially crucial in cases where enzymatically active extracellular MPO may not co-localize with MPO protein stored in intracellular granules or lysosomes.

[0277] As shown in FIG. 3 and FIG. 4, these apparently incongruent results between MPO activity and MPO protein levels are likely because during acute inflammation there is massive degranulation to secrete MPO into the extracellular space. As a result, less MPO is stored intracellularly in the active immune cells to be detected by immunohistochemical techniques as the harsher reagent treatments usually make detecting extracellular targets challenging if not impossible. On the other hand, when MAFA was used to detect MPO activity, it only involved gentle washing steps and the activated MAFA binds to surrounding proteins, which enables detection of extracellular MPO activity. Indeed, similar results were found in a mouse model of cerebritis using an MPO activatable biotinylated sensor. Thus, the aged mice on day 3, with a high MAFA signal but low MPO protein expression in the ischemic brain revealed a high degree of oxidative stress and damaging inflammation. On the other hand, in the young adult mice on day 3, there was a much lower MAFA signal but a high amount of MPO protein, signifying that most of the immune cells were primed but did not release MPO extracellularly to cause damage. These findings were corroborated by NeuN and P-3-tubulin results that showed more damage in the aged mice brains compared with young adult mice brains.

[0278] During the late subacute phase (day 10), inflammation has greatly subsided as revealed by the >4-5 folds decrease in MAFA signal on day 10 compared to day 3 for both age groups. However, MPO protein levels were only slightly decreased in the aged brains but markedly decreased in the young adult brains. Together these results revealed that young adults are more resilient toward inflammatory damage after ischemic stroke, with less secreted MPO during the early subacute phase of stroke and a faster resolution as stroke evolved. On the other hand, in the aged mice, ischemic stroke elicited a much larger MPO- mediated response that even during the resolution stage there continued to be MPO+ cells in the ischemic area even if they are not actively degranulating.

[0279] An increase in MPO activity was observed within the thalamus region in aged brains compared to young adult brains. Previous studies have shown that aging contributes to the heightened vulnerability of both aged mice and elderly humans to microbleeds and thalamic degeneration. These results provide potential mechanistic insight into the cause of thalamic degeneration. It was also found that the increased MPO activity in the thalamus in naive aged brains colocalized with Ibal+ cells, which are likely activated microglia given the lack of injury to recruit peripheral macrophages. After ischemic stroke, more Ibal+ cells are recruited to the ischemic sites in both aged and young adult brains, which are likely a mixture of microglia and macrophages. On day 3, Ibal+ cells colocalized with MAFA signal, indicating that these cells are predominately MPO-secreting cells and Ml -like. By day 10, only a small subset of Ibal+ cells colocalized with MAFA signal, most Ibal+ cells at this time point are non-MPO-secreting, M2-like, cells performing repair.

[0280] Aged mice demonstrated worse initial neurological deficits and exhibited a slower recovery compared to young adult mice after stroke. Nonetheless, aged mice eventually recovered to almost equivalent levels seen in young adult mice by day 10, similar to a previous study. Despite this apparent eventual recovery, aged mice had an increased mortality rate and a decreased survival after stroke, which is also consistent with previous studies. Notably, most deaths in the aged occurred within the first few days after stroke, which is likely related to the markedly elevated MPO activity observed between days 0 (naive) to day 3 in the aged group. MPO, as an enzyme known for its high oxidative activity, plays a pivotal role in generating both oxidative and nitrosative stress. When activated by H2O2, MPO catalyzes the oxidation of substrates (e.g., chloride (Cl ), bromide (Br ), nitrite (NO2 ), tyrosine (Tyr)), resulting in the production of potent oxidants (hypochlorous acid (HOC1) or hypobromous acid (HOBr)) and free radicals (nitrogen dioxide (*NO2) or tyrosyl radical (Tyr*)). These highly reactive species surpass the potency of O2_* and H2O2, leading to cellular injury and increased oxidative stress. Thus, elevated MPO activity serves as an indicator of heightened oxidant production, indicating an excess of oxidative stress. Previous studies suggested that aging involves alterations in membrane fatty acid composition, including a decrease in polyunsaturated fatty acids (PUFAs) and an increase in monounsaturated fatty acids. PUFAs, like arachidonic acid (AA), abundant in the aging brain, are susceptible to free radical attack, and the oxidative depletion of AA levels contributes to cognitive deficits in aged rats. Other studies indicated that protein oxidation, reflected in elevated protein 3 -nitro-tyrosine (3 -NT) (a downstream effect of MPO activation through the oxidation of nitrite) levels in different regions of the brain of aged animals and white matter of aging monkeys, is implicated in the decline of physiological functioning during aging. Additionally, an increased percentage of MP0+ cells was found in the blood of aged mice following stroke during the early subacute phase, highlighting an age-related amplification of the bone marrow response. This observation aligns with previous studies that established a significant correlation between elevated blood neutrophil counts with more severe neurological deficit scores and higher mortality rates. In addition to increased innate immune response, increased MPO activity has been found to decrease neurogenesis and neuroprotection, which likely also contributes to increased mortality in the aged. As MPO activity increases oxidative stress and elevated oxidative stress is associated with aging, aging-related changes and elevated abnormal MPO activity, as found in the aged mice after stroke, may form a vicious cycle that worsens each other and leads to higher mortality. Thus, MPO activity may have both direct and indirect effects on mortality.

[0281] Notably, on the 3rdday post-stroke, a marked decrease was found in both MAFA and Ibal+ signals in the aged group after treatment with AB AH, revealing that inhibition of MPO activity resulted in a reduced recruitment of Ibal+ cells to the infarcted area. Furthermore, this reduction in MPO activity translated into significant improvement in neurob ehavi oral performance and mortality rate within the aged group approaching the level seen in young adult stroke mice. This work has revealed an association between increased MPO activity, particularly in the early subacute phase post-stroke, and neuronal damage in aged mice. Previous findings demonstrated that MPO inhibition, coupled with its impact on oxidative stress and inflammation, fosters an environment that activates crucial endogenous resources, including promoting neurogenesis and neuroprotection. These findings provide evidence linking MPO inhibition with protection against damage and improving repair after stroke. Thus, the aged mice with elevated MPO activity compared to those of young adult mice likely have decreased neuroprotection and neurogenesis capabilities. Future studies investigating the association between MPO activity and neurogenesis and neuroprotection in different age groups will contribute to a more nuanced understanding of the impact of MPO activity across the aging spectrum.

[0282] Elevated systemic levels of MPO have been found to increase mortality in elderly individuals. This study provides a potential mechanism for this observation in aging humans, bridging a gap in knowledge. Unraveling the relationship between aging, MPO activity, and stroke outcomes could allow more rational design of more effective therapeutic interventions that benefit different age groups after stroke. This understanding becomes crucial in the context of anti -oxi dative therapy trials, potentially offering insights to identify drug targets for the elderly and aiding in the identification of suitable patients. Multiple MPO inhibitors are in different stages of clinical development to mitigate oxidative stress and inflammation associated with various diseases. However, the translation of MPO inhibition into a viable therapeutic strategy for clinical applications presents its own set of challenges. Critical considerations include determining optimal dosage and managing potential off-target effects. Additionally, the inherent variability among individuals, given that there are different MPO genotypes in humans and likely differential responses to MPO inhibition, complicates accurate predictions of responses to MPO inhibition in the absence of a prognostic biomarker to select patients who will most benefit from the therapy. Imaging MPO activity could offer such a biomarker to improve patient selection and track outcomes from MPO inhibition. Through longitudinal studies between two different age groups, it was demonstrated that elevated MPO activity in aging markedly worsened neurological outcomes after ischemic stroke. It was observed that the administration of AB AH, a specific irreversible MPO inhibitor, mitigated the inflammatory response, reduced MPO activity, and improved neurob ehavi oral scores, contributing to a significant improvement in survival rates by day 3 after stroke in the aged group. These findings underscore the critical role of modulating MPO activity during the initial phase post stroke, emphasizing its potential to significantly enhance post-stroke outcomes in elderly patients. These findings showed that sustained MPO activity in the aged if left unmitigated, also worsened longer-term outcomes by day 10 and possibly beyond.

[0283] Example B: Targeting Myeloperoxidase to Reduce Neuroinflammation in X- Linked Dystonia Parkinsonism

[0284] Human Brain Tissue Samples

[0285] Postmortem XDP PFC were provided by the Massachusetts General Hospital Collaborative Center for XDP (CCXDP) with approval of the Institutional Review Boards (IRB) at Massachusetts General Hospital (Boston, USA) and Makati Medical Center (Makati City, Philippines). Detailed methods on donor consent, tissue collection, and processing, as well as quality control metrics, have been previously reported. Postmortem control PFC were provided by the Massachusetts Alzheimer's Disease Research Center (ADRC) with approval from the Massachusetts General Hospital IRB. This study included only control and XDP PFC derived from male subjects. Demographic information about the samples is provided in Table 1.

[0286] Table 1. Postmortem prefrontal cortex (BA9) sample information.

[0287] Although XDP is mainly characterized by loss of medium spiny neurons in the striatum, previous studies have provided evidence demonstrating that other brain areas such as frontal and temporal cortices, pallidum, and cerebellum also degenerate in XDP. Previous findings from these labs support these studies and demonstrated a significant increase in neuroinflammation and MPO in XDP PFC. Therefore, the work assessing and understanding the precise molecular mechanisms that occur in the PFC in XDP has continued. Ex Vivo Detection of MP O Activity Using MAFA

[0288] As a substrate for MPO, MPO can oxidize MAFA to form radicals that bind to nearby proteins containing phenolic or indolic moieties. Activated MAFA would then remain bound to tissues after washing, while inactivated agents would be washed away, enabling the identification of areas with elevated MPO activity. Fresh-frozen human brain sections (8 pm thickness) were fixed in 4% paraformaldehyde (PF A) for 5 min at room temperature (RT) and then incubated in blocking solution containing 1% fetal bovine serum (FBS; #A5670701, Gibco, Thermo Fisher Scientific, MA), and 0.3% Triton X- 100 (#T8787- 50 mL, MilliporeSigma, MA) at RT for 1 h. Following three washes in phosphate buffer saline (PBS; #10010049, Gibco, Thermo Fisher Scientific, MA), the sections were incubated with MAFA (1 :300 of stock solution 10 mM dimethyl- sulfoxide DMSO; #D1391, Thermo Fisher Scientific, MA) and 1 mM of 3% hydrogen peroxide (H2O2; #BP2633500, Thermo Fisher Scientific, MA) for 30 min at RT. Lastly, the sections were mounted in an antifade mounting medium (#ZJ0808, Vectashield, Vector Laboratories, CA) containing the nuclear stain DAPI. Images were captured with a Nikon DS- Ri2 model microscope connect to Prime BSI Express. Intensity of fluorescence was measured by using Imaged 1.53 t (National Institute of Health, Bethesda, MD).

[0289] Meso Scale Discovery (MSD) Assay

[0290] Cytokines levels were assessed using the human proinflammatory panel- 1 10-p lex kits to detect 10 cytokines, including interferon y (INF- y), interleukin (IL)- ip, IL- 2, IL- 4, IL- 5, IL- 6, IL- 8, IL- 10, IL- 12p70, IL- 13, and tumor necrosis factor- a (TNF- a). Specifically, cytokines levels were measured using an electrochemiluminescence-based multiarray method through the Quickplex SQ 120 system (Meso Scale Diagnostics LLC, MD) following previously reported methods. Briefly, both experimental samples and protein standards provided by the manufacturer were incubated on a shaker at 4 °C overnight. Next, experimental samples and protein standards were washed off the plates and then incubated with the detection antibodies provided by the kit at room temperature while shaking for 2 h, followed by washing and the addition of the reading buffer. Lastly, the electrochemiluminescence signals were captured by the SQ 120 system, and cytokines concentrations (pg / mL) were calculated following manufacturer's instruction using the standard concentrations provided by the kit. Human Fibroblast Cultures

[0291] Human fibroblasts were provided by the CCXDP at Massachusetts General Hospital. In this study, two fibroblast lines derived from individuals who lived with XDP and their unaffected family members were used. All fibroblasts were grown in Dulbecco's Modified medium (DMEM; #11965118, Gibco, Thermo Fisher Scientific, MA) supplemented with 20% FBS (#A5670701, Gibco, Thermo Fisher Scientific, MA), and lx penicillin / streptomycin / L- glutamine (#10378016, Thermo Fisher Scientific, MA) and kept in an incubator at 37°C, 5% CO2. Specifically, this study was per-formed in fibroblasts derived from two individuals living with XDP (33109 and 35883) and two controls (36175 and 33362). Human neuroblastoma SH- SY5Y cells (ATCC CRL- 2266) were cultured in DMEM / Nutrient Mixture F- 12 (1 : 1) supplemented with 10% inactivated FBS (#A5670701, Gibco, Thermo Fisher Scientific, MA), 2 mM L-glutamine, 50 pg / mL streptomycin, and 50 lU / mL penicillin (#A5670701, Gibco, Thermo Fisher Scientific, MA). The cells were kept at 5% CO2at 37 °C.

[0292] Oxidative Stress Detection

[0293] ROS levels were measured in both fibroblasts and SH- SY5Y cells by using CellROX Orange reagent (#010443; Thermo Fisher Scientific, MA), a fluorogenic probe for measuring oxidative stress in live cells, as previously reported. Briefly, follow-ing specific treatments, the cells were incubated with 2.5 pM CellROX for 30 min at 37 °C in the dark. Then, the medium was replaced with fresh culture media before imaging on a BioTek Cytation 5 imaging reader (BioTek, VT). Images were captured from 6 random areas of each well every 10 min for 2 h. To analyze data generated by Cytation, images were processed using a custom Fiji macro to automatically calculate mean fluorescent intensity (MFI) for each image. The MFI values were then grouped by using a custom Python script, as previously reported. Statistics and XY curves were generated using GraphPad Prism 10.2.0.

[0294] MPO Activity Assay

[0295] MPO activity was measured by using the Oxi Select Myeloperoxidase Chlorination Activity Assay Kit (#STA- 803; Cell Biolabs, CA), according to manufacturer's protocol. Briefly, samples were incubated in 1 mM hydrogen peroxide (H2O2) solution for 1 h at RT. Next, samples were incubated in lx stop solution for 15 min at RT before incubating in 1 mM chromogen working solution for additional 15 min in the dark at RT. All solutions were provided by the kit. At the end of the last incubation, a standard curve was generated follow- ing manufacturer's instruction, and absorbance was read at 405 nm. MPO activity in milliunits / mL (mU / mL) was deter-mined for each sample by dividing the quantity of chromogen consumed by the reaction time as indicated by the kit's instruction.

[0296] MPO Immunodepletion

[0297] MPO content was immunodepleted from four XDP PFC homogenates that contained the highest MPO levels, as determined by previously published studies. Specifically, 150 pg of proteins from each XDP PFC was incubated with 15 pL anti- MPO anti-body (#A1374, ABclonal Technology, MA) in GAL4 immunoprecipitation buffer, composed of 250 mM sodium chloride (NaCl; #S9888, Sigma- Aldrich, MA), 5 mM ethylenediaminetetraacetic acid (EDTA; #AM9260G, Thermo Fisher Scientific, MA), 1% Nonidet P-4 0 (#J19628.K2, Thermo Fisher Scientific, MA), 50 mM Tris, pH7.5 (#AM9850G, Thermo Fisher Scientific, MA). After the incubation at 4 °C for 3.5 h, magnetic protein A beads (Invitrogen, Thermo Fisher, MA) were incubated (20 pL per sample) in agitation overnight at 4 °C. At the end of the incubation, all samples were placed on a magnetic rack to separate the supernatants, depleted of MPO. The supernatants were then collected and saved in new tubes labeled as XDP(-). The magnetic beads bound to MPO were, instead, resuspended in GAL4 buffer (50 pL per sample), boiled at 95 °C for 5 min and magnetized again to remove the magnetic beads and save the supernatants, enriched in MPO, in new tubes labeled as XDP(+). The efficiency of the immunodepletion was determined by measuring MPO activity in whole cell extract from XDP, XDP(-), and XDP(+) PFC.

[0298] Cell Cultures Treatment

[0299] SH-S Y5Y cells were incubated with 10 ng / mL of homogenates from either XDP PFC (n = 4), XDP(-) (n = 4) or XDP(+) (n = 4) for 24 h before measuring MPO activity and ROS as de-scribed above.

[0300] Control- and XDP- derived fibroblasts were treated with either 0.1, 0.5, 1, 5, or 10 pg / mL of verdiperstat for 24 h before measuring MPO activity. ROS levels were measured in control- and XDP- derived fibroblasts in the absence or in the presence of 5 pg / mL of verdiperstat. Similarly, MPO activity and ROS were assessed in XDP- treated SH- SY5Y cells in the absence or in the presence of 5 pg / mL of verdiperstat. Statistics

[0301] Normal distribution of data was not assumed regardless of sample size or variance. Individual value plots with the central line representing the median and the whiskers representing the interquartile range, box plot with the central line represent-ing the median, the edges representing the interquartile range, and the whiskers representing the minimum and maximum values, and XY curves were used for graphical representation. Comparisons between groups were performed using a non- parametric Mann-Whitney U test, a one- way ANOVA followed by Tukey's test, and a two- way ANOVA followed by Tukey's test. Correlation of MPO levels with XDP clinical features, including age at disease onset, age at death, disease duration, and repeat size within the SVA, were performed as non- parametric Spearman correlations. All tests were two- sided with a significance level of 0.05, and exact p values were reported. GraphPad Prism 10.2.0 was used to perform statistical analyses and generate graphs.

[0302] Study Approval

[0303] The study was approved by the Partners Healthcare Institutional Review Board at Massachusetts General Hospital (Boston, MA, USA) and by the Makati Medical Center (Makati City, Philippines). Postmortem consent was obtained from the appropriate representative (next of kin or health care proxy) prior to autopsy.

[0304] MPO Activity Was Increased in XDP Postmortem PFC

[0305] Given the previously reported increase in MPO levels in XDP PFC, MPO activity in brain tissue sections ex-vivo was measured using a specific MPO activity fluorescent imaging probe (MAFA) in a large cohort of postmortem XDP PFC. These results revealed a significant increase in MPO activity in XDP PFC compared with controls (FIG. 8A and FIG. 8B), consistent with these lab’s previously published findings.

[0306] To determine whether there was an association between known XDP clinical parameters and MPO activity, MPO activity in PFC was correlated with age at disease onset, age at death, disease duration, and the size of the hexameric repeat expansion within the SVA in TAF1 for all XDP samples. The results demonstrated no impact or correlation of any of the clinical parameters with MPO activity in XDP (FIG. 9A, FIG. 9B, FIG. 9C, and FIG. 9C). Cytokines Levels Were Not Changed in XDP Postmortem PFC

[0307] Given that increases in MPO result in increases in proinflammatory cytokines, alterations in a panel of 10 cytokines were measured in control and XDP postmortem PFC using an MSD assay. The results demonstrated that the levels of INF- y, TNF- a, IL- ip, IL- 2, IL- 4, IL- 6, IL- 8, IL- 10, IL- 12p70, and IL- 13 were not significantly different between XDP and control PFC.

[0308] ROS Levels were Increased in XDP

[0309] Increases in MPO activity are also directly linked to increases in ROS by generating hypochlorous acid. Therefore, ROS in XDP- derived fibroblasts were measured by using CellROX and live cell imaging. Antimycin A, an inhibitor of mitochondrial complex III capable of increasing ROS, was used as positive control. As expected, ROS levels were significantly increased in antimycin A-treated cells compared with control- derived fibroblasts. Importantly, these results demonstrated a significant increase in ROS in XDP- derived fibroblasts compared with control- derived fibroblasts (FIG. 10A).

[0310] Next, it was sought to determine whether the treatment of SH- SY5Y cells with whole cell extracts derived from XDP PFC could recapitulate pathological features of XDP, such as increases in oxidative stress, as results have recently demonstrated in ALS. SH- SY5Y cells were treated with XDP PFC (10 ng / mL) for 24 h before ROS levels were measured as described above. As expected, antimycin A induced a significant increase in ROS compared with vehicle treated cells. Furthermore, there was a significant increase in ROS in XDP treated SH- SY5Y cells com-pared with vehicle treated cells (FIG. 10B).

[0311] MPO Immunodepletion Reduced MPO Activity and ROS In Vitro

[0312] To determine whether increases in MPO directly induce an increase in ROS in XDP, MPO was removed from XDP postmortem PFC homogenates by performing an immunodepletion experiment. To determine the efficiency of immunodepletion, MPO activity was measured in whole cell extracts from XDP PFC, XDP PFC depleted of MPO [XDP(-)], and XDP PFC enriched in MPO [XDP(+)] by using a commercially available kit. The results revealed that MPO activity was significantly decreased in XDP(-) compared with both XDP(+) and XDP PFC, confirming the successful removal of MPO from the samples (FIG. 11 A).

[0313] Next, SH-S Y5Y cells were treated with XDP, XDP(-), and XDP(+) PFC (10 ng / mL) for 24 h before measuring MPO activity using a commercially available kit. These findings indicated a significant increase in MPO activity in XDP and XDP(+) treated cells compared with vehicle treated cells. Importantly, MPO activity was significantly decreased in XDP(-) treated cells (FIG. 1 IB).

[0314] Lastly, to determine whether MPO causes increase in ROS in XDP, ROS levels were measured in SH- SY5Y cells following treatment with either XDP, XDP(-), or XDP(+) PFC (10 ng / mL / 24 h) by using CellROX and live cell imaging. As expected, the positive control antimycin A induced a significant increase in ROS. A similar increase was measured in XDP and XDP(+) treated cells compared with vehicle treated cells. Importantly, there was a significant decrease in ROS in XDP(-) treated cells compared with both XDP and XDP(+) treated cells (FIG. 11C).

[0315] Verdiperstat Reduced MPO Activity and ROS in XDP- Derived Fibroblasts

[0316] Next, the effects of a potent and selective MPO inhibitor, verdiperstat, on MPO activity were assessed in control- and XDP- derived fibroblasts. The cells were treated with verdiperstat (0.1, 0.5, 1, 5, and 10 pg / mL), for 24 h and MPO activity was measured with a commercially available kit. Verdiperstat treatment significantly decreased MPO activity in a dose- dependent manner. Specifically, there was a significant de-crease in MPO activity in XDP- derived fibroblasts treated with either 1, 5 or 10 pg / mL of verdiperstat compared to vehicle- treated XDP- derived fibroblasts (FIG. 12A). Based on these results, 5 pg / mL of verdiperstat was used for the next set of experiments outlined below given that this dose decreased MPO activity by 50%.

[0317] Next, the effect of verdiperstat on ROS levels was measured in control- and XDP- derived fibroblasts using CellROX and live cell imaging as described above. The results confirmed a significant increase in ROS in XDP- derived fibroblasts compared with control- derived fibroblasts (FIG. 12B and FIG. 12C). Importantly, verdiperstat treatment reduced ROS in both control- and XDP- derived fibroblasts (FIG. 12B and FIG. 12C).

[0318] Verdiperstat Reduced MPO Activity and ROS in XDP- Treated SH- SY5Y

[0319] To verify whether verdiperstat was also able to reduce MPO activity and ROS in SH- SY5Y cells treated with postmortem XDP PFC, the cells were treated with XDP PFC homogenates (10 ng / mL) for 24 h in the absence or in the presence of verdiperstat (5 pg / mL) and MPO activity was measured as previously described. The results confirmed a significant increase in MPO activity in XDP treated SH- SY5Y cells compared with vehicle treated cells Importantly, there was a significant decrease in MPO activity in XDP+verdiperstat treated SH- SY5Y cells compared with XDP treated SH- SY5Y cells (FIG. 13 A).

[0320] Lastly, ROS in SH- SY5Y cells was measured following treatment with XDP (10 ng / mL) for 24 h in the absence or in the presence of verdiperstat (5 pg / mL) using CellROX and live cell imaging. The results demonstrated a significant increase in ROS in SH- SY5Y cells treated with the positive control antimycin A as well as in XDP treated cells compared with vehicle treated cells. Importantly, there was a significant decrease in ROS in XDP+verdiperstat treated SH- SY5Y cells compared with XDP treated cells (FIG. 13B).

[0321] Conclusion

[0322] In this study, a significant increase in MPO activity ex-vivo was demonstrated in a large cohort of XDP human postmortem PFC using a novel MPO fluorescent imaging probe. While these results revealed that increases in MPO did not alter pro- and anti- inflammatory cytokine levels, it contributed to a significant increase in ROS in XDP. Both immunodepletion of MPO as well as inhibition of MPO using verdiperstat reduced MPO activity and resulted in significant decreases in ROS in XDP- derived fibroblasts and SH- SY5Y cells treated with postmortem XDP PFC. Collectively, these findings suggest that inhibiting MPO dampens oxidative stress in XDP.

[0323] Recently, results demonstrated a significant increase in neuroinflammation and MPO in XDP postmortem PFC, highlighting MPO as a contributor to this neuroinflammatory process similar to other neurodegenerative diseases. Indeed, increases in MPO levels, astrogliosis and microgliosis have been described in several neurodegenerative diseases, including AD, PD, and multiple sclerosis (MS).

[0324] MPO plays a critical role in the innate immune response as it contributes to both neutrophil antimicrobial activity and phago-cytosis; however, a prolonged increase in the systemic levels of MPO could cause extravasation into extracellular spaces, causing tissue damage during chronic inflammation. One consequence of the sustained increase in MPO is the rise in the expression and release of proinflammatory cytokines. However, these findings suggest that increases in MPO levels and activity in XDP, specifically, did not alter cytokine levels. It should be noted that one caveat of the current study is that only a panel of 10 cytokines was assessed and thatthe effects of MPO on a complete panel of inflammatory markers were not fully characterized. Therefore, it is possible that increases in MPO may alter the expression and release of other cytokines or chemokines such as CXCL13 and CX3CL1 as have previously demonstrated in XDP- derived fibroblasts. Another downstream consequence of increased MPO is oxidative stress and an increase in ROS production through the generation of hypochi orous acid. Herein is reported a significant increase in ROS in both XDP- derived fibroblasts and XDP PFC-treated SH- SY5Y cells. Furthermore, the increase in ROS is a direct consequence of increased MPO levels in XDP as it was mitigated by decreasing MPO by either immunodepletion or treatment with verdiperstat. Together these findings demonstrate that inhibiting MPO could represent a viable therapeutic strategy to dampen oxidative stress in XDP.

[0325] Verdiperstat is a first-in-class, potent, selective, brain-permeable MPO inhibitor produced by Biohaven Pharmaceuticals Inc. Treatment with verdiperstat has been shown to decrease microglia activation in multiple system atrophy (MSA) and PD. Additionally, increases in MPO levels and activity have been described in human postmortem brain from people who lived with MSA as well as in a transgenic MSA mouse model. Of note, the treatment of MSA mice with verdiperstat reduced MPO activity and ameliorated the disease phenotype, thus providing a strong rational for assessing the potential beneficial effect of verdiperstat in a clinical trial (NCT03952806 or M- STAR study). Similarly, increases in MPO were reported in the SOD1G93A animal model of ALS, another neurodegenerative disease in which microglia activation plays a critical pathogenic role, thus suggesting that verdiperstat could exert a therapeutic effect in ALS and providing the groundwork for assessing verdiperstat in a clinical trial for ALS (NCT04436510 or Regimen B in the Healey ALS Platform Trial in ALS). Although both clinical trials demonstrated safety and tolerability in people, they failed to meet the primary endpoints. Nevertheless, the potential beneficial effect of verdiperstat in treating neurodegenerative diseases is still under investigation and a third clinical trial (Veri- T) is currently ongoing to determine the therapeutic efficacy of verdiperstat for the treatment of semantic variant primary progressive aphasia (svPPA) (NCT05184569). Therefore, these findings in XDP may pave the way for further assessing the potential beneficial therapeutic effect of verdiperstat in dampening neurodegeneration in XDP.

[0326] In summary, these findings demonstrate that increases in MPO may contribute to XDP pathogenesis, as revealed by a significant increase in MPO activity ex -vivo in human postmortem XDP PFC. Additionally, our results indicate that MPO causes oxidative stress in XDP, as demonstrated by increases in ROS in cellular models of XDP. Importantly, depleting MPO or using a selective and potent MPO inhibitor mitigated ROS levels in XDP and lays the foundation for future studies to assess the neuroprotective effects of MPO inhibition in XDP. OTHER EMBODIMENTS

[0327] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMS1. A compound of Formula (IA):or a pharmaceutically acceptable salt thereof, wherein:CN, halogen, -NH2, or -CH3;RSA R5B,anj RCare each in(iepen(ientiy H or -CH3;RDis -Ci-10 alkyl substituted with REwherein the -C1-10 alkyl of RDis optionally substituted with 1, 2, 3, or 4 substituents independently selected from -OH, -OCH3, -CN, halogen, and NH2 and 1 or 2 non-terminal carbons of the -C1-10 alkyl of RDare independently optionally replaced with O, -NH, or -NCH3; andREcomprises a fluorescent group.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R3Ais OH.

3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R3Bis OH.

4. The compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein R1A, R2A, R4A, R1B, R2B, and R4Bare each H.

5. The compound of any one of claims 1-4, or a pharmaceutically acceptable salt thereof, wherein R5Ais H.

6. The compound of any one of claims 1-5, or a pharmaceutically acceptable salt thereof, wherein R5Bis H.

7. The compound of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein Rcis H.

8. The compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein RDis -C1-10 alkyl substituted with RE.

9. The compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein RDis -C5 alkyl substituted with RE.

10. The compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein11. The compound of any one of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein REcomprises Cy3, Cy5, Cy7, Alexa647, FITC, Rhodamine red, Rhodamine, Rhodamine B, or BODIPY.

12. The compound of any one of claims 1-10, or a pharmaceutically acceptable salt thereof, whereinoptionally substituted with SO3H, OH, or NH2.

13. The compound of any one of claims 1-10, or a pharmaceutically acceptable salt14. The compound of any one of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein15. The compound of any one of claims 1-10, or a pharmaceutically acceptable salt16. The compound of any one of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein REis17. The compound of any one of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein REis18. The compound of claim 1, wherein the compound of Formula (IA) is a compound of formulapharmaceutically acceptable salt thereof.

19. A pharmaceutical composition comprising a compound of any one of claims 1-18, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

20. A method of imaging a cell or tissue sample, the method comprising: i) administering to the subject a compound any one of claims 1-18, or a pharmaceutically acceptable salt thereof; ii) waiting a time sufficient to allow the compound to accumulate at the cell or tissue sample; and iii) imaging the cell or tissue sample with an imaging technique.

21. A method of diagnosing a disease or disorder associated with abnormal myeloperoxidase (MPO) activity in a subject, the method comprising: i) administering to the subject a compound of any one of claims 1-18, or a pharmaceutically acceptable salt thereof; ii) waiting a time sufficient to allow the compound to accumulate at a cell or tissue site associated with the disease; and iii) imaging the cell or tissue with an imaging technique. In some embodiments, the method further comprises imaging the subject prior to step i).

22. A method of imaging myeloperoxidase (MPO) activity in a cell or tissue sample, the method comprising: i) contacting the cell or tissue sample with a compound of any one of claims 1-18, or a pharmaceutically acceptable salt thereof; and ii) imaging the cell with an imaging technique.

23. A method of detecting myeloperoxidase (MPO) activity in a cell or tissue sample, the method comprising: i) contacting the cell or tissue sample with a compound of any one of claims 1-18, or a pharmaceutically acceptable salt thereof; and ii) imaging the cell or tissue sample with an imaging technique.

24. A method of detecting myeloperoxidase activity in a subject, the method comprising: i) administering to the subject a compound of any one of claims 1-18, or a pharmaceutically acceptable salt thereof; and ii) imaging the subject with an imaging technique.

25. A method of monitoring treatment of a disease or disorder associated with abnormal myeloperoxidase (MPO) activity in a subject, the method comprising: i) administering to the subject a compound of any one of claims 1-18, or a pharmaceutically acceptable salt thereof; ii) imaging the subject with an imaging technique; iii) administering to the subject a therapeutically effective amount of a therapeutic compound to treat the disease or disorder; iv) imaging the cell or tissue in the subject with an imaging technique; and v) comparing the image of step i) and the image of step iv).

26. The method of claim 25, wherein the therapeutic compound is an MPO inhibitor.

27. The method of claim 25, wherein the therapeutic compound is verdiperstat.

28. The method of any one of claims 20-27, wherein the imagining technique is fluorescence imaging.

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