Compositions and methods for treating airway injury

Abstract

Disclosed herein are compositions and methods of treating or preventing epithelial barrier dysfunction, epithelial inflammation, pulmonary inflammation, diminished mucociliary clearance, and / or smoke- or aerosol-induced inflammation in a subject in need thereof. Also disclosed are compositions and methods of improving or restoring mucociliary clearance in a subject in need thereof.

Description

[0001] Attorney Docket No.: UCH-43225

[0002] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0003] COMPOSITIONS AND METHODS FOR TREATING AIRWAY INJURY RELATED APPLICATIONS

[0004] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 755,026, filed February 6, 2025, the contents of which are incorporated herein by reference in their entirety.

[0005] STATEMENT OF GOVERNMENT SUPPORT

[0006] This invention was made with government support under ESO 15457 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] BACKGROUND

[0008] From the warmth of woodburning stoves or campfires to the devastation of wildfires, woodsmoke permeates both ambient and household environments, and potential health risks from these exposures are just beginning to be recognized. Despite the progress made in reducing air pollution in the United States over the last few decades, wildfire emissions have significantly worsened the quality of air humans breathe. Furthermore, climate change is projected to increase the frequency of wildfires in the future. Wildfire smoke produced from combustion of natural biomass contains thousands of compounds, including particulate matter, carbon dioxide, nitrogen oxides, and trace minerals. The emissions from wildfires are highly variable as they depend on the different kinds of burned fuel, the burning conditions, and environmental factors. In addition, as this complex mixture of pollutants moves away from the source of the fire, it can undergo physical and chemical transformations and become more toxic and more readily absorbed by the body. These factors cause the negative impact of wildfire smoke on public health. In recent epidemiological studies, components of wildfire smoke, such as the chemical components of particulate matter, have been shown to exacerbate respiratory diseases, such as asthma, and other conditions of the airway, such as epithelial barrier dysfunction, epithelial inflammation, pulmonary inflammation, diminished mucociliary clearance, or smoke- or aerosol-induced inflammation. In view of the foregoing, there is an unmet need for new therapeutics to treat conditions of the airway.

[0009] SUMMARY OF THE INVENTION

[0010] In some aspects, the present disclosure provides methods of treating or preventing:

[0011] (a) epithelial barrier dysfunction;Attorney Docket No.: UCH-43225

[0012] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0013] (b) ciliary dysfunction

[0014] (c) smoke- or aerosol-induced epithelial barrier dysfunction;

[0015] (d) environmental exposure-induced epithelial barrier dysfunction;

[0016] (e) epithelial inflammation;

[0017] (f) pulmonary inflammation; or

[0018] (g) smoke- or aerosol-induced inflammation; or

[0019] (h) environmental exposure-induced inflammation

[0020] in a subject in need thereof, comprising administering to the subject a modulator of protein phosphorylation.

[0021] In certain aspects, the present disclosure provides methods of improving or restoring mucociliary clearance in a subject in need thereof, comprising administering to the subject a modulator of protein phosphorylation. In certain embodiments, the subject suffers from diminished mucociliary clearance, for example, as a result of exposure to smoke or pollution or aerosols.

[0022] In some aspects, the present disclosure provides methods of treating or preventing lung cancer in a subject in need thereof, comprising administering to the subject a compound selected from:

[0023]

[0024] salt thereof.Attorney Docket No.: UCH-43225

[0025] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0026] In various embodiments, the modulator of protein phosphorylation can be a cyclin- dependent kinase (CDK) inhibitor, an alkaline phosphatase inhibitor, a tyrosine phosphatase inhibitor, a serine / threonine phosphatase inhibitor / activator or other phosphatase family member inhibitor / activator, a tyrosine kinase inhibitor, a serine kinase inhibitor, a threonine kinase inhibitor, a growth factor induced pathway inhibitor (e.g. Epidermal Growth Factor inhibitor), a JAK / STAT pathway inhibitor, or a mitogen-activated protein kinase inhibitor, such as a MAP kinase inhibitor e.g., a MEK1 or MEK2 inhibitor).

[0027] In certain embodiments, the modulator of protein phosphorylation is a MEK1 or MEK2

[0028]

[0029] pharmaceutically acceptable salt thereof.

[0030] In certain aspects, the present disclosure provides methods of:

[0031] (a) treating or preventing airway injury;

[0032] (b) treating or preventing lung cancer; or

[0033] (c) improving mucociliary clearance

[0034] in a subject in need thereof, comprising administering to the subject a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof:

[0035]

[0036] Attorney Docket No.: UCH-43225

[0037] UCLA Ref No.: [UCLA 2025-088-2] WO

[0038] (i); (ii);

[0039] wherein:

[0040] RA is selected from Ci-io hydrocarbyl, hydroxy Ci-io hydrocarbyl, amino Ci-io hydrocarbyl, nitro Ci-io hydrocarbyl, and halo Ci-io hydrocarbyl;

[0041] Ri, R2, R9, Rio, R11R12, R 13 and Rware independently selected from: hydrogen, halogen, cyano, nitro, azido, — OR3, — NR4C(O)OR6, — OC(O)R3, — NR4S(O)jR6, — S(O)jNR3R4, — S(O)jNR4C(O)R3, — C(O)NR4S(O)jR6, — S(O)jR6, — NR4C(O)R3, — C(O)NR3R4, — NR5C(O)NR3R4, — NR5C(NCN)NR3R4, NR3R4, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C 10 cycloalkyl, C3-C10 cycloalkylalkyl, — S(O)j(Ci-Ce alkyl), — S(O)j(CR4Rs)m-aryl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, — O(CR4Rs)m-aryl, — NR4(CR4Rs)m-aryl, — O(CR4R5)m-heteroaryl, — NR4(CR4R5)m, heteroaryl, — O(CR4R5)m-heterocyclyl, — NR4(CR4R5)m-heterocyclyl and — S(Ci-C2alkyl) substituted with 1 to 5 fluorines, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is independently substituted or unsubstituted;

[0042] Rsis selected from: hydrogen, trifluoromethyl, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkylalkyl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, and aryl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heteroaryl and heterocyclyl is independently substituted or unsubstituted; and wherein aryl is optionally substituted with 1 to 5 groups independently selected from: oxo, halogen, nitro, CF3, CHF2, CH2F, OCF3, OCHF2, OCH2F, azido, NR'SO2R'"', SO2NR", C(O)R', C(O)OR', OC(O)R', NR'C(O)OR'"', NR'C(O)R", C(O)NR'R", SR'"', S(O)R"", SO2R', NR'R", NR'C(O)NR''R"', NR'C(NCN)NR''R"', OR', aryl, heteroaryl, arylalkyl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl;

[0043] R4is selected from hydrogen or Ci-Ce alkyl, wherein alkyl may be substituted or unsubstituted; or

[0044] R3 and R4 can be taken together with the atom to which they are attached to form a 4 to 10 membered heteroaryl or heterocyclic ring, each of which is substituted or unsubstituted;

[0045] Rs is hydrogen or Ci-Ce alkyl, wherein alkyl may be substituted or unsubstituted; or R4 and Rs can be taken together with the atom to which they are attached to form a 4 to 10 membered carbocyclic, heteroaryl or heterocyclic ring, each of which is substituted or unsubstituted;Attorney Docket No.: UCH-43225

[0046] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0047] Reis selected from: trifluoromethyl, C1-C10 alkyl, C3-C10 cycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl, wherein each alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is independently substituted or unsubstituted;

[0048] R', R" and R'" are independently selected from: hydrogen, C1-C4 alkyl, C2-C4 alkenyl, aryl and arylalkyl;

[0049] R"" is selected from: C1-C4 alkyl, C1-C4 alkenyl, aryl and arylalkyl;

[0050] W is — C(0)0Ri5, — C(O)NR4Ri5, — C(O)NR4ORI5, — C(O)NR4S(O)jR6, — C(O)NR4NR4Ri5, — NR'C(O)R', — NR'S(O)jR', — NRC(O)NR'R", NR'S(O)jNR'R", or — C(O)NR4NR4C(O)RI5;

[0051] provided that W is not — C(O)OH;

[0052] Risis selected from: hydrogen, trifluoromethyl, C1-C10 alkyl, C2-C 10 alkenyl, C2-Cioalkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is independently substituted or unsubstituted;

[0053] Rieis selected from hydrogen or C1-C10 alkyl; or Risand Retaken together with the atom to which they are attached form a 4 to 10 membered cyclic ring with 1 or 2 nitrogen atoms and optionally an oxygen atom, said ring being substituted or unsubstituted;

[0054] X is N or N O ;

[0055] m is 0, 1, 2, 3, 4 or 5; and

[0056] j is 1 or 2;

[0057] with the proviso that 3-phenylamino-isonicotinic acid methyl ester, and 3-oxo-3-(3-phenylamino-pyridin-4-yl)-propionic acid ethyl ester are not included.

[0058] BRIEF DESCRIPTION OF THE DRAWINGS FIGs. 1A-1M show that ambient wildfire smoke exposure is associated with epithelial barrier loss and particulate matter infiltration in human sinonasal tissue. FIG. 1A shows a schematic of sinonasal tissue collection from Los Angeles residents undergoing functional endoscopic sinus surgery (FESS), grouped by air quality index (AQI) exposure relative to the January 2025 wildfires: pre-fire (AQI 0-100, n=4), active wildfire (AQI>300, n=6), and smoldering wildfire conditions (AQI 50-100, n=4). FIG. IB shows representative H&E-stained sections of sinonasal epithelium collected during pre-fire exposure windows. Arrows indicate airway epithelial surface. FIG. 1C shows representative H&E-stained sections of sinonasal epithelium collected during active wildfire exposure windows. Arrows indicate airway epithelial surface. FIG. ID shows representative H&E-stained sections of sinonasalAttorney Docket No.: UCH-43225

[0059] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0060] epithelium collected during smoldering wildfire exposure windows. Arrows indicate airway epithelial surface. FIG. IE shows the quantification of airway epithelial surface coverage and epithelial height across exposure groups (n=4 biological replicates, 3 technical replicates (prefire); n=6 biological replicates, 4 technical replicates (active-fire); n=4 biological replicates, 3 technical replicates (smoldering-fire)). Statistical significance was determined by one-way Anova; *p<0.05, **p<0.01, ***p<0.001. FIG. IF shows representative immunofluorescent images of Claudin-1 in pre-fire samples. FIG. 1G shows representative immunofluorescent images of Claudin-1 in active-fire samples. FIG. 1H shows representative immunofluorescent images of Claudin-1 in smoldering fire samples. FIG. II shows the quantification of Claudin-1 -positive airway epithelial coverage and epithelial height (n=4 biological replicates, 3 technical replicates (pre-fire); n=6 biological replicates, 4 technical replicates (active-fire); n=4 biological replicates, 3 technical replicates (smoldering-fire)). FIG. 1J shows transmission electron microscopy (TEM) images of sinonasal tissue collected under clean-air conditions (AQI 0-20) (n=l biological replicate, 17 technical replicates). FIG. IK shows TEM images of sinonasal tissue collected during active wildfire smoke exposure showing intracellular and intercellular particulate matter; insets show ultrafine particles (n=2 biological replicates, 17 technical replicates). FIG. IL shows a magnified view of the particulate matter imaged in FIG. IK. FIG. IM shows the quantification of intracellular particulate matter per image field of view in clean-air versus wildfire-exposed tissue. Statistical significance was determined by Mann-Whitney test; ****p<0.0001.

[0061] FIGs. 2A-2N show the development and characterization of a reproducible human primary airway epithelium woodfire smoke (WFS) exposure system. FIG. 2A shows a schematic of primary human airway basal stem cell isolation and differentiation at air-liquid interface (ALI). FIG. 2B shows a diagram of custom exposure chamber used to deliver woodfire smoke (WFS) to differentiated human ALI cultures. The smoke is generated from combustion of Pine Wood (Pinus canariensis (Canary Island Pine)) and pumped into the chamber via a microcontroller-regulated pump. FIG. 2C depicts the experimental timeline showing daily 3-minute WFS exposures for five consecutive days. FIG.2D shows the particle size distribution of WFS measured using scanning mobility particle sizer (SMPS) and aerosol particle sizer (APS). FIG. 2E shows the results of real-time monitoring of particulate matter concentrations (PM2.5 and PM10) and carbon dioxide within the exposure chamber during WFS delivery. FIG. 2F shows an excitation-emission matrix (EEM) fluorescence “thumbprint” of WFS particulate matter collected on filters. FIGs. 2G-2N show representativeAttorney Docket No.: UCH-43225

[0062] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0063] immunofluorescence images and fold change (FC) quantification of differentiated ALI cultures maintained in room air versus following five days of WFS exposure; Acetylated Tubulin (FIGs.2G-2H), SCGB1A1 (FIGs.2I-2J), Keratin 5 (FIGs.2K-2L), MUC5B (FIGs.2M-2N) (n=2 biological replicates, 3 technical replicates), p-values provided from unpaired t-tests. Scale bars = 50 pm. WFS: Woodfire Smoke.

[0064] FIGs. 3A-3F show that multi-omics integration reveals WFS-induced suppression of protein translation and altered phosphorylation of proteins involved in ciliary and cytoskeletal pathways. FIG.3A shows the experimental timeline showing RNA sequencing (n=2 biological replicates, 3 technical replicates), abundance proteomics, and phosphoproteomics (n=3 biological replicates, 3 technical replicates) on differentiated human airway ALI cultures following five consecutive days of WFS exposure. FIG. 3B shows the proportion of significantly regulated molecules identified by mRNA sequencing, abundance proteomics, and phosphoproteomics following WFS exposure at day 5 relative to room air controls. Significance thresholds were defined as |log2FC| >1 and adjusted p value < 0.05. FIG. 3C shows a comparison of RNA (x-axis) and protein abundance (y-axis) responses to WFS exposure, plotted as log2 fold change. Each point represents a donor-averaged gene-protein pair. Proteins exhibiting significant abundance changes that correlate with increased RNA expression are highlighted. FIG. 3D shows a gene ontology (GO) biological process-based gene set overrepresentation analysis (GSOA) of the 24 proteins identified in FIG. 3C. FIG.

[0065] 3E shows the GSOA of GO biological process terms for each dataset: RNA-seq, abundance proteomics, and phosphoproteomics. Heatmap shows top 15 most significantly enriched biological process terms, based on adjusted p-values, per datatype and change direction (up or down). Numbers within each cell depict genes that are both significantly changing and a member of the denoted term, with red numbers indicating significant enrichment. Bars at top indicate total number of genes that were found to be significantly regulated, up or down, per data type. For phosphorylation, sites were collapsed to the gene level. FIG. 3F shows that kinase activity was inferred from the phosphoproteomics data by quantifying shifts in phosphorylation of known kinase substrates using the OmniPath kinase-substrate interaction network. Phosphorylation site log2 fold changes were averaged across donors prior to kinase activity analysis (n=3 biological replicates and n=3 technical replicates). Only kinases with |z-score| >1 are shown. WFS: woodfire smoke.

[0066] FIGs. 4A-4E show that WFS exposure disrupts coordinated ciliary movement and compromises epithelial barrier integrity. FIG. 4A shows representative ciliary activity mapsAttorney Docket No.: UCH-43225

[0067] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0068] from human airway epithelial ALI cultures exposed to room air or WFS for 1, 3, or 5 days. Regions of active ciliary movement shown in light grey, regions of ciliary inactivity shown in dark grey (n=3 biological replicates, n=3 technical replicates, with 20 videos per well for n=l 80 videos per condition). FIG. 4B shows the quantification of ciliary movement from FIG. 4A. FIG. 4C shows representative IF images of tight junction protein TJP1 in room air and WFS exposed cultures. Scale bar = 50 pm. Insets show higher magnification views (300x) (n=3 biological replicates, n=2 technical replicates). FIG.4D shows the quantification data obtained using image! plugin, TIJOR, demonstrating tight junction organization following days of WFS exposure (*p<0.05) (n=3 biological replicates, n=2 technical replicates). FIG. 4E shows transepithelial electrical resistance (TEER) measurements collected over time in room air and WFS-exposed cultures. Data are presented as mean ± SEM. TEER measurements are reported as (Measurement-Blank)x(Surface area of transwell). Statistical significance indicated where applicable (n=2 biological replicates, n=3 technical replicates). WFS: woodfire smoke.

[0069] FIGs. 5A-5I show the pharmacologic modulation of MAPKZERK signaling during wildfire smoke exposure. FIG. 5A shows the timeline of experimental design including WFS exposure and addition of MEK inhibitor. FIG. 5B shows representative ciliary activity maps from human airway epithelial cultures exposed to room air, wildfire smoke (WFS) for 5 days without drug, or WFS for 5 days with pimasertib (100 nM). Regions of ciliary movement are shown in yellow, and regions of ciliary inactivity are shown in blue (n=2 biological replicates, with 20 videos per well for n=40 videos per condition). FIG. 5C shows the quantification of percent ciliary movement across conditions shown in FIG. 5B, including a pimasertib dose series administered during WFS exposure. Letters above represent significant changes compared to associated letters on the x-axis labels. Statistical significance was defined as p<0.05. Individual points represent individual videos (n=2 biological replicates, with 20 videos per well for n=40 videos per condition). FIG. 5D shows representative ciliary activity maps from cultures exposed to room air, WFS for 5 days without drug, or WFS for 5 days with the MEK inhibitor U0126 (n=3 biological replicates, n=20 technical replicates). FIG. 5E shows the quantification of percent ciliary movement for conditions shown in FIG. 5C, including a U0126 dose series administered during WFS exposure (n=3 biological replicates with 20 videos per well for n=60 videos per condition). FIG. 5F shows representative ciliary activity maps from cultures exposed to room air, WFS for 5 days without drug, or treated with the ERK activator BCI (n=3 biological replicates with 20 videos per well for n=60 videos per condition).

[0070] FIG. 5G shows the quantification of percent ciliary movement for conditions shown in FIG.Attorney Docket No.: UCH-43225

[0071] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0072] 5E (with 20 videos per well for n=60 videos per condition). FIG. 5H shows representative immunofluorescence images of the tight junction protein TJP1 in cultures exposed to room air, WFS for 5 days without drug, or WFS for 5 days with pimasertib. Scale bars = 50 pm. Inset at 300x. (n=3 biological replicates, n=3 technical replicates). FIG. 51 shows the quantification of TJP1 organization using imageJ, TIJOR. Statistical annotations are shown as indicated. WFS: woodfire smoke.

[0073] FIGs. 6A-6I show that MEK inhibition modulates kinase signaling and phosphorylation of cytoskeletal and ciliary proteins associated with axonemal integrity. FIG.

[0074] 6A shows the results of kinase activity profiling depicting relative kinase activity (z-score) in cultures exposed to wildfire smoke (WFS) compared with room air, and WFS with pimasertib compared with room air. Kinases are grouped by signaling pathway as indicated. Only kinases with an absolute z-score change > 1.5 are shown. FIG. 6B shows a scatter plot comparing log2 fold changes in phosphorylation (PH) following WFS relative to room air (x-axis) versus phosphorylation changes following WFS with pimasertib relative to WFS alone (y-axis) for cytoskeletal-associated proteins. (n=3 biological replicate, n=3 technical replicates). Linear regression lines and correlation statistics are shown. FIG. 6C shows a scatter plot comparing log2 fold changes in phosphorylation (PH) following WFS relative to room air (x-axis) versus phosphorylation changes following WFS with pimasertib relative to WFS alone (y-axis) for cilia-associated proteins. (n=3 biological replicate, n=3 technical replicates). Linear regression lines and correlation statistics are shown. FIG. 6D shows a heat map of selected phosphorylated proteins involved in cytoskeletal organization, cilia structure, and axonemal assembly identified from phosphoproteomic analysis. Phosphorylation changes are shown for WFS versus room air and WFS with pimasertib versus WFS, grouped by functional category.

[0075] FIG. 6E shows a schematic representation of motile cilia ultrastructure depicting a healthy axonemal arrangement with a central pair (9+2) and an altered configuration lacking the central pair. FIG. 6F shows representative transmission electron microscopy (TEM) images of motile cilia from room air-exposed cultures, with magnified views highlighting 9+2 axonemal structure (n=3 biological replicates, n=4-9 technical replicates). FIG. 6G shows representative TEM images of motile cilia from cultures exposed to WFS for 5 days without drug (n=3 biological replicates, n=4-9 technical replicates). FIG. 6H shows representative TEM images of motile cilia from cultures exposed to WFS for 5 days with pimasertib or U0126 treatment (n=2 biological replicates, n=4-9 technical replicates). FIG. 61 shows the quantification of theAttorney Docket No.: UCH-43225

[0076] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0077] percentage of cilia exhibiting abnormal axonemal structure across conditions shown in FIGs.

[0078] 6F-6H. Individual points represent each image scored per condition. WFS: woodfire smoke.

[0079] FIGs. 7A-7H show additional images from WFS-exposed tissues. FIG. 7A shows uncropped H&E-stained images from pre-fire sinonasal tissue samples corresponding to FIG. IB. FIG. 7B shows uncropped H&E-stained images from active wildfire sinonasal tissue samples corresponding to FIG. 1C. FIG. 7C shows uncropped H&E-stained images from smoldering wildfire sinonasal tissue samples corresponding to FIG. ID. FIG. 7D shows uncropped transmission electron microscopy (TEM) image of sinonasal tissue collected under clean-air conditions. FIG. 7E shows uncropped TEM images from active wildfire smoke-exposed sinonasal tissue showing widespread intracellular and intercellular particulate matter.

[0080] FIG. 7F shows a TEM image showing particulate matter localized within disrupted tight junction regions in wildfire smoke-exposed tissue. FIG. 7G shows a TEM image showing dense, metal-containing particles within the epithelium of wildfire smoke-exposed tissue. FIG.

[0081] 7H shows the inset of metal particulate from FIG. 7G.

[0082] FIGs. 8A-8I show additional data related to development of the human primary airway epithelium woodfire smoke exposure system. FIG. 8A shows the particulate mass collected on 19 Teflon filters distributed across inlet (1), middle (2), and outlet (3) positions across four independent WFS exposures, demonstrating uniform particulate distribution across the chamber. FIG. 8B shows the unedited excitation-emission matrix (EEM) fluorescence “thumbprint” of WFS particulate matter corresponding to FIG. 2F and shown with label overlay. FIG. 8C shows representative DAPI-stained images of differentiated ALI cultures maintained under room air conditions or following five days of WFS exposure. Scale bar = 50 pm. FIG. 8D shows the quantification of DAPI-positive cell number in room air and WFS-exposed ALI cultures. FIG. 8E shows the quantification of nuclear size in room air and WFS-exposed ALI cultures. FIGs. 8F-8G show immunofluorescence staining and quantification of Caspase-3 in room air and WFS-exposed cultures. Scale bar = 50 pm. FIGs. 8H-8I show immunofluorescence staining and quantification of PCNA-positive nuclei in room air and WFS-exposed cultures. Statistical significance was determined by unpaired t-tests; *p<0.05,**p<0.01, ***p<0.001, ***p<0.0001. Scale bar = 50 pm. WFS: woodfire smoke.

[0083] FIGs. 9A-9K show additional data related to the multi-omic characterization of wildfire smoke exposure. FIG. 9A shows a principal component analysis (PCA) of RNA sequencing data showing separation by exposure condition (n=2 biological replicates, n=3 technical replicates). FIGs. 9B-9E show quality control metrics for abundance proteomicsAttorney Docket No.: UCH-43225

[0084] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0085] (AB) (n=3 biological replicates, n=3 technical replicates). FIG.9B shows the number of unique peptides identified per donor under room air and WFS conditions. FIG. 9C shows the number of unique proteins identified per donor under each condition. FIG. 9D shows pairwise Pearson correlation coefficients for abundance proteomic datasets across technical replicates. FIG. 9E shows a PCA of abundance proteomic data showing separation by exposure condition and donor. FIGs.9F-9H show quality control metrics for phosphoproteomics (PH). FIG.9F shows the number of unique phosphopeptides and non-phosphorylated peptides identified per donor under room air and WFS conditions. FIG. 9G shows pairwise Pearson correlation coefficients for phosphoproteomic datasets across technical replicates. FIG. 9H shows a PCA of phosphoproteomic data showing separation by exposure condition and donor. FIG. 91 shows the overlap of significantly regulated phosphoproteins and phosphorylation sites across donors following WFS exposure, indicating donor concordance. FIG. 9 J shows the gene ontology (GO) enrichment analysis of proteins exhibiting a significant decrease in abundance but significant increase in RNA following WFS exposure. FIG.9K shows the gene set enrichment analysis of PID pathways using kinases up- or down-regulated in response to WFS exposure.

[0086] FIGs. 10A-10C show that MEK1 / 2 Inhibition (Pimasertib) Improves Recovery of Ciliary Function Following WFS Exposure in ALI Cell Cultures. FIG. 10A shows the experimental timeline. FIG. 10B shows a comparison of ciliary activity (light grey) and ciliary inactivity (dark grey) in room air, following a 5-day exposure to wildfire smoke plus a 3 -day recovery, or following a 5-day exposure to wildfire smoke plus a 3 -day recovery in the presence of 100 nM pimasertib. FIG. 10C shows the quantification of ciliary activity from FIG. 10B.

[0087] FIGs. 11A-11B show that pimasertib treatment protects ciliary movement following exposure to plastic combustion. FIG. HA shows a comparison of ciliary activity (light grey) and ciliary inactivity (dark grey) in room air, following an exposure to plastic combustion smoke, or following an exposure to plastic combustion smoke in the presence of 100 nM pimasertib. FIG. 11B shows the quantification of ciliary activity from FIG. 11 A.

[0088] FIGs. 12A-12B show that pimasertib treatment protects ciliary movement following exposure to hay combustion. FIG. 12A shows a comparison of ciliary activity (light grey) and ciliary inactivity (dark grey) in room air, following an exposure to hay combustion smoke, or following an exposure to hay combustion smoke in the presence of 100 nM pimasertib. FIG.

[0089] 12B shows the quantification of ciliary activity from FIG. 12A.Attorney Docket No.: UCH-43225

[0090] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0091] DETAILED DESCRIPTION OF THE INVENTION

[0092] Despite substantial progress in reducing air pollutants in the United States over recent decades, wildfire smoke has emerged as an increasingly important contributor to poor air quality. Smoke generated from the combustion of natural biomass contains thousands of compounds, including particulate matter, carbon monoxide, nitrogen oxides, and trace metals. The composition and toxicity of wildfire smoke vary widely depending on fuel type, combustion conditions, and environmental factors. Epidemiologic studies have consistently linked particulate matter exposure to exacerbations of respiratory disease, including asthma and chronic rhinosinusitis, as well as adverse clinical and surgical outcomes. However, the cellular and molecular mechanisms by which wildfire smoke directly injures airway epithelial cells remain poorly understood. Other combustion-derived smoke exposures such as military bum pits and occupational smoke exposure among firefighters have been associated with chronic respiratory symptoms, airway inflammation, and long-term pulmonary dysfunction, highlighting the broader relevance of complex smoke inhalation to human health.

[0093] The lung is the gateway to the body, and it is protected by the action of mucociliary clearance (MCC), the primary innate defense mechanism of the upper and lower airways. MCC involves the production of mucus, which traps inhaled pathogens, pollutants, and particulate matter, and the coordinated, unidirectional beating of motile cilia on multi-ciliated epithelial cells, which move the mucus up and out of the airways. This process is essential for preventing retention of toxins within the respiratory tract and for limiting their penetration across the airway epithelial barrier into the systemic circulation. Motile cilia are highly specialized organelles whose function depends on precise structural organization. The axoneme consists of nine outer microtubule doublets surrounding a central pair complex that coordinates ciliary beating. Cilia are anchored at the apical cell surface by the transition zone and basal body, structures that are mechanically supported by both the actin and microtubule cytoskeleton. Accordingly, effective ciliary motility depends on cytoskeletal integrity, which also maintains epithelial organization and barrier function through tight junctions. Loss of epithelial barrier integrity, sometimes referred to as epithelial unjamming, allows inhaled particles and toxins to traverse the airway epithelium, providing a direct route from the external environment into the body. Ciliary dysfunction may arise from inherited defects, as in primary ciliary dyskinesia, or be acquired following environmental exposures such as diesel exhaust particles and wood smoke. Impaired ciliary function is linked to asthma, chronic obstructive pulmonary disease, respiratory infections, and bronchiectasis.Attorney Docket No.: UCH-43225

[0094] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0095] Changes in protein phosphorylation — chemical modifications consisting of phosphate groups added to serine, threonine, or tyrosine residues on proteins — are dynamic, regulated by kinases and phosphatases, and play a central role in how cells sense and respond to their environment. Protein phosphorylation is the most ubiquitous regulatory post-translational modification, known to regulate a multitude of cellular processes, including cytoskeletal dynamics, junctional stability, and ciliary motility. The Ras-Raf-MEK-ERK signaling cascade, one of the mitogen activated protein kinase (MAPK) pathways, transduces signals through Ras, a small GTPase, which results in the sequential activation of Raf, MEK, and ERK kinases through phosphorylation. This pathway is known to regulate cell cycle progression, cytoskeletal remodeling, and ciliary structure and function. Furthermore, woodsmoke exposure has been shown to activate MAPKs in airway epithelial cells, contributing to inflammatory responses, and has been linked to alterations in distal airway epithelial integrity. However, the global impact of wildfire smoke on kinase activities and protein phosphorylation, and how these changes affect airway barrier integrity and ciliary function, has not been studied. Importantly, kinases represent attractive therapeutic targets for mitigating the adverse effects of wildfire smoke exposure.

[0096] The present disclosure examines upper airway samples from patients exposed to the 2025 Los Angeles wildfires, which were found to have undergone a loss of airway epithelial barrier integrity. These observations motivated the development of a controlled, reproducible wood combustion smoke exposure system to define the mechanisms by which wildfire smoke injures the human airway epithelium and to test strategies aimed at preserving airway epithelial barrier function and mucociliary clearance. Transcriptomic changes were identified in the airway epithelium in response to the woodfire smoke exposure, accompanied by the activation of PERK as part of the integrated stress response and a concomitant block in protein translation. MAPK activity was further identified as a driver of altered phosphorylation of proteins regulating motile cilia central pair function and tight junction-associated cytoskeletal architecture and demonstrated that pharmacological perturbation of the ERK pathway could either restore or exacerbate disease-associated defects in barrier integrity and ciliary function. The present disclosure provides a mechanistic framework for understanding wildfire smoke-induced airway dysfunction and provides a rationale for therapeutic strategies aimed at preserving or restoring mucociliary clearance and epithelial barrier function following toxic smoke inhalation. To understand how the airway epithelial integrity is lost and the mucociliary clearance of PM is impaired, the direct effects of woodfire smoke on the airway epitheliumAttorney Docket No.: UCH-43225

[0097] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0098] were studied. We found that woodfire smoke induces a transcriptional remodeling in the airway epithelium, but it is uncoupled to changes in protein levels due to a block in translation mediated by the activation of PERK. Importantly, this response is likely dynamic, and our analysis represents a snapshot of a temporally evolving signaling landscape. It is possible, and likely, that oxidative stress induced by smoke exposure triggers transient PERK activation, and that different phases of the stress response would be observed at earlier or later time points.

[0099] On the other hand, wildfire smoke activates MAPK, which results in phosphorylation changes on ciliary and cytoskeletal proteins that leads to ciliary paralysis and breakdown of airway barrier integrity. These phosphorylation changes were particularly enriched on proteins associated with the central pair apparatus, radial spokes, and axonemal organization, all of which are essential for motile cilia function. These molecular alterations were accompanied by ultrastructural loss of the ciliary central pair, supporting a model in which altered phosphorylation disrupts the spatial organization and function of the ciliary motility machinery rather than depleting ciliary axonemal components. The phosphorylation changes were also identified on microtubule proteins, which are a central component of the cytoskeleton as well as cilia. Together, these data identify phosphorylation-dependent regulation of ciliary and cytoskeletal architecture as a mechanistic link between environmental exposure and loss of mucociliary clearance, providing a direct molecular explanation for how wildfire smoke enables particulate matter to breach epithelial junctions, accumulate within airway cells and enter the systemic circulation.

[0100] Our findings establish phosphorylation remodeling, rather than overt cytotoxicity or cell loss, as a central feature of woodfire smoke-induced airway injury. This distinction is critical, as it suggests that airway epithelial injury from woodfire smoke exposure can be mitigated by targeting specific kinases that are responsible for the phosphorylation changes on ciliary and cytoskeletal proteins. MAPK signaling emerged as a dominant kinase regulating this phosphorylation and the pharmacologic inhibition of MEK during woodfire smoke exposure preserved ciliary movement in a dose-dependent manner, maintained junctional organization, and protected epithelial barrier integrity. Importantly, this mechanistic framework identified kinase signaling as a therapeutic entry point for preserving epithelial barrier function and preventing particulate persistence in response to woodfire smoke exposure, with implications for reducing long-term respiratory morbidity.

[0101] The implications of these findings extend beyond wildfire events alone. Firefighters, first responders, and military personnel experience repeated high-intensity inhalationAttorney Docket No.: UCH-43225

[0102] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0103] exposures from wildfires, structural fires, and bum pits, placing them at elevated risk for acute and chronic respiratory diseases. In addition, populations worldwide are increasingly exposed to extreme air pollution events, including prolonged periods of hazardous particulate exposure in densely populated regions. These data suggest that acute kinase-driven disruption of ciliary function and epithelial integrity may represent a shared mechanism underlying airway vulnerability across these exposure scenarios, highlighting the potential value of targeted airway-protective strategies during periods of extreme air pollution.

[0104] Together, this work demonstrates the power of integrating multi-omics measurements across transcription, translation, and phosphorylation to capture regulation across multiple molecular layers. By linking kinase signaling activities to ciliary and barrier function and architecture, a phosphorylation-centric framework is defined for woodfire smoke-induced lung injury. Furthermore, this systems-level, signaling-centric approach reveals actionable therapeutic targets for preserving epithelial defense in the face of an escalating global health threat.

[0105] In some aspects, the present disclosure provides methods of treating or preventing: (a) epithelial barrier dysfunction;

[0106] (b) ciliary dysfunction

[0107] (c) smoke- or aerosol-induced epithelial barrier dysfunction;

[0108] (d) environmental exposure-induced epithelial barrier dysfunction;

[0109] (e) epithelial inflammation;

[0110] (f) pulmonary inflammation; or

[0111] (g) smoke- or aerosol-induced inflammation; or

[0112] (h) environmental exposure-induced inflammation

[0113] in a subject in need thereof, comprising administering to the subject a modulator of protein phosphorylation.

[0114] In some embodiments, the modulator of protein phosphorylation is a kinase inhibitor (e.g., a serine kinase inhibitor, a tyrosine kinase inhibitor, or a threonine kinase inhibitor) or a phosphatase inhibitor (e.g., a serine / threonine phosphatase inhibitor, a tyrosine phosphatase inhibitor, or an alkaline phosphatase inhibitor).Attorney Docket No.: UCH-43225

[0115] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0116] In some embodiments, the modulator of protein phosphorylation is selected from:

[0117]

[0118] salt thereof.

[0119] In certain preferred embodiments, the modulator of protein phosphorylation is

[0120]

[0121] pharmaceutically acceptable salt thereof.

[0122] In certain preferred embodiments, the modulator of protein phosphorylation is

[0123]

[0124] pharmaceutically acceptable salt thereof.Attorney Docket No.: UCH-43225

[0125] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0126] In certain embodiments, the modulator of protein phosphorylation i

[0127]

[0128] ; or a pharmaceutically acceptable salt thereof.

[0129] In some embodiments, the modulator of protein phosphorylation is

[0130]

[0131] In certain embodiments, the smoke- or aerosol-induced inflammation is wildfire smoke- or aerosol-induced inflammation, tobacco smoke- or aerosol-induced inflammation, cooking smoke- or aerosol-induced inflammation, industrial smoke- or aerosol-induced inflammation, or waste disposal smoke- or aerosol-induced inflammation.

[0132] In some preferred embodiments, the smoke- or aerosol-induced inflammation is wildfire smoke- or aerosol -induced inflammation. In certain such embodiments, the smoke- or aerosol-induced inflammation occurs in an airway of the subject.Attorney Docket No.: UCH-43225

[0133] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0134] In some embodiments, the smoke- or aerosol-induced inflammation is induced following a chronic exposure to a smoke or an aerosol for about 1-120 minutes per day over a period of about 1-10 days.

[0135] In some embodiments, the smoke- or aerosol-induced inflammation is induced following an acute exposure to a smoke or an aerosol for about 1-120 minutes.

[0136] In certain embodiments, the modulator of protein phosphorylation is administered prior to the exposure to the smoke or the aerosol. In some such embodiments, the modulator of protein phosphorylation is administered about 1-12 hours prior to the exposure to the smoke or the aerosol. In some embodiments, the modulator of protein phosphorylation is administered about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours prior to the exposure to the smoke or the aerosol.

[0137] In some embodiments, the modulator of protein phosphorylation is administered daily for a period of about 1-5 days prior to the exposure to the smoke or the aerosol. In certain such embodiments, the modulator of protein phosphorylation is administered daily for a period of about 1 day, about 2 days, or about 3 days prior to the exposure to the smoke or the aerosol.

[0138] In certain embodiments, the modulator of protein phosphorylation is administered following the exposure to the smoke or the aerosol. In certain such embodiments, the modulator of protein phosphorylation is administered about 1-12 hours following the exposure to the smoke or the aerosol. In some embodiments, the modulator of protein phosphorylation is administered about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours following the exposure to the smoke or the aerosol.

[0139] In certain embodiments, the modulator of protein phosphorylation is administered daily for a period of about 1-5 days following the exposure to the smoke or the aerosol. In certain such embodiments, the modulator of protein phosphorylation is administered daily for a period of about 1 day, about 2 days, or about 3 days following the exposure to the smoke or the aerosol.

[0140] In some embodiments, the modulator of protein phosphorylation is administered to an airway of the subject (e.g., via inhaler).

[0141] In some embodiments, the modulator of protein phosphorylation is formulated for administration as an aerosol.

[0142] In certain aspects, the present disclosure provides methods of improving or restoring mucociliary clearance in a subject in need thereof, comprising administering to the subject aAttorney Docket No.: UCH-43225

[0143] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0144] modulator of protein phosphorylation. In certain embodiments, the subject suffers from diminished mucociliary clearance, for example, as a result of exposure to smoke or aerosols.

[0145] In some embodiments, the modulator of protein phosphorylation is a kinase inhibitor (e.g., a serine kinase inhibitor, a tyrosine kinase inhibitor, or a threonine kinase inhibitor) or a phosphatase inhibitor (e.g., a serine / threonine phosphatase inhibitor, a tyrosine phosphatase inhibitor, or an alkaline phosphatase inhibitor).

[0146] In some embodiments, the modulator of protein phosphorylation is selected from:

[0147]

[0148] salt thereof.

[0149] In certain preferred embodiments, the modulator of protein phosphorylation is

[0150]

[0151] pharmaceutically acceptable salt thereof.Attorney Docket No.: UCH-43225

[0152] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0153] In certain preferred embodiments, the modulator of protein phosphorylation is

[0154]

[0155] pharmaceutically acceptable salt thereof.

[0156] In some embodiments, the modulator of protein phosphorylation

[0157]

[0158] or a pharmaceutically acceptable salt thereof.

[0159] In some embodiments, the modulator of protein phosphorylation is

[0160]

[0161] In some embodiments, prior to the administration of the modulator of protein phosphorylation, the subject suffers from epithelial barrier dysfunction.Attorney Docket No.: UCH-43225

[0162] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0163] In certain embodiments, prior to the administration of the modulator of protein phosphorylation, the subject has suffered an exposure to a smoke or an aerosol. In certain such embodiments, the subject has suffered smoke- or aerosol -induced inflammation following the exposure to the smoke or the aerosol. In some embodiments, the smoke- or aerosol -induced inflammation is wildfire smoke- or aerosol-induced inflammation, tobacco smoke- or aerosol-induced inflammation, cooking smoke- or aerosol-induced inflammation, industrial smoke- or aerosol-induced inflammation, or waste disposal smoke- or aerosol-induced inflammation.

[0164] In some embodiments, the smoke- or aerosol-induced inflammation is wildfire smoke-or aerosol-induced inflammation. In some embodiments, the smoke- or aerosol-induced inflammation occurs in an airway of the subject.

[0165] In certain embodiments, the modulator of protein phosphorylation is administered to an airway of the subject (e.g., via inhaler).

[0166] In some embodiments, the modulator of protein phosphorylation is formulated for administration as an aerosol.

[0167] In some aspects, the present disclosure provides methods of treating or preventing lung cancer in a subject in need thereof, comprising administering to the subject a compound selected from:

[0168]

[0169] salt thereof.Attorney Docket No.: UCH-43225

[0170] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0171] In certain embodiments, the lung cancer is characterized by an upregulation of a gene, wherein the gene is selected from MAPK14, MAPKAPK2, MAPKAPK3, MAPK10, MAPK12, MAPK13, MAP2K3, and MAP2K6; or any combinations thereof.

[0172] In some embodiments, the lung cancer is characterized by a downregulation of a gene, wherein the gene is MAPK37.

[0173] In certain aspects, the present disclosure provides methods of:

[0174] (a) treating or preventing airway injury;

[0175] (b) treating or preventing lung cancer; or

[0176] (c) improving mucociliary clearance

[0177] in a subject in need thereof, comprising administering to the subject a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof:

[0178]

[0179] (i); Qi);

[0180] wherein:

[0181] RA is selected from Ci-io hydrocarbyl, hydroxy Ci-io hydrocarbyl, amino Ci-io hydrocarbyl, nitro Ci-io hydrocarbyl, and halo Ci-io hydrocarbyl;

[0182] Ri, R2, R9, Rio, R11R12, R 13 and Rware independently selected from: hydrogen, halogen, cyano, nitro, azido, — OR3, — NR4C(O)OR6, — OC(O)R3, — NR4S(O)jR6, — S(O)jNR3R4, — S(O)jNR4C(O)R3, — C(O)NR4S(O)jR6, — S(O)jR6, — NR4C(O)R3, — C(O)NR3R4, — NR5C(O)NR3R4, — NR5C(NCN)NR3R4, NR3R4, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C 10 cycloalkyl, C3-C10 cycloalkylalkyl, — S(O)j(Ci-Ce alkyl), — S(O)j(CR4Rs)m-aryl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, — O(CR4Rs)m-aryl, — NR4(CR4Rs)m-aryl, — O(CR4R5)m-heteroaryl, — NR4(CR4R5)m, heteroaryl, — O(CR4R5)m-heterocyclyl, — NR4(CR4R5)m-heterocyclyl and — S(Ci-C2alkyl) substituted with 1 to 5 fluorines, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is independently substituted or unsubstituted;

[0183] Rsis selected from: hydrogen, trifluoromethyl, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkylalkyl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, and aryl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl,Attorney Docket No.: UCH-43225

[0184] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0185] heteroaryl and heterocyclyl is independently substituted or unsubstituted; and wherein aryl is optionally substituted with 1 to 5 groups independently selected from: oxo, halogen, nitro, CF3, CHF2, CH2F, OCF3, OCHF2, OCH2F, azido, NR'SO2R'"', SO2NR", C(O)R', C(O)OR', OC(O)R', NR'C(O)OR'"', NR'C(O)R", C(O)NR'R", SR'"', S(O)R"", SO2R', NR'R", NR'C(O)NR''R"', NR'C(NCN)NR''R"', OR', aryl, heteroaryl, arylalkyl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl;

[0186] RUs selected from hydrogen or Ci-Ce alkyl, wherein alkyl may be substituted or unsubstituted; or

[0187] R3 and R4 can be taken together with the atom to which they are attached to form a 4 to 10 membered heteroaryl or heterocyclic ring, each of which is substituted or unsubstituted;

[0188] Rs is hydrogen or Ci-Ce alkyl, wherein alkyl may be substituted or unsubstituted; or R4 and Rs can be taken together with the atom to which they are attached to form a 4 to 10 membered carbocyclic, heteroaryl or heterocyclic ring, each of which is substituted or unsubstituted;

[0189] Reis selected from: trifluoromethyl, C1-C10 alkyl, C3-C 10 cycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl, wherein each alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is independently substituted or unsubstituted;

[0190] R', R" and R'" are independently selected from: hydrogen, C1-C4 alkyl, C2-C4 alkenyl, aryl and arylalkyl;

[0191] R"" is selected from: C1-C4 alkyl, C1-C4 alkenyl, aryl and arylalkyl;

[0192] W is — C(O)ORi5, — C(O)NR4Ri5, — C(O)NR4ORI5, — C(O)NR4S(O)jR6, — C(O)NR4NR4Ri5, — NR'C(O)R', — NR'S(O)jR', — NRC(O)NR'R'', NR'S(O)jNR'R", or — C(O)NR4NR4C(O)Ri5;

[0193] provided that W is not — C(O)OH;

[0194] Risis selected from: hydrogen, trifluoromethyl, C1-C10 alkyl, C2-C 10 alkenyl, C2-Cioalkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is independently substituted or unsubstituted;

[0195] Rieis selected from hydrogen or C1-C10 alkyl; or Risand Retaken together with the atom to which they are attached form a 4 to 10 membered cyclic ring with 1 or 2 nitrogen atoms and optionally an oxygen atom, said ring being substituted or unsubstituted;

[0196] X isN orN+CT;

[0197] m is 0, 1, 2, 3, 4 or 5; andAttorney Docket No.: UCH-43225

[0198] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0199] j is 1 or 2;

[0200] with the proviso that 3-phenylamino-isonicotinic acid methyl ester, and 3-oxo-3-(3-phenylamino-pyridin-4-yl)-propionic acid ethyl ester are not included.

[0201] In some embodiments, the compound is a compound of Formula (I),

[0202] wherein:

[0203] Ri, R2, R9, Rio, R11, R12, R 13 and Rware independently selected from: hydrogen, halogen, cyano, nitro, azido, — OR3, — NR4C(O)ORe, — OC(O)R3, — NR4S(O)jRe, — S(O)jNR3R4, — S(O)jNR4C(O)R3, — C(O)NR4S(O)jR6, — S(0)jR6, — NR4C(O)R3, — C(O)NR3R4, — NR5C(O)NR3R4, — NR5C(NCN)NR3R4, — NR3R4, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkylalkyl, — S(O)j(Ci-Ce alkyl), — S(O)j(CR4Rs)m-aryl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, — O(CR4Rs)m-aryl, — NFUfCFURUni-aryl, — O(CR4R5)m-heteroaryl, — NR4(CR4R5)m, heteroaryl, — O(CR4R5)m-heterocyclyl, — NR4(CR4Rs)m-heterocyclyl and — S(C1-C2alkyl) substituted with 1 to 5 fluorines; wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is independently substituted or unsubstituted;

[0204] Rsis selected from: hydrogen, trifluoromethyl, C1-C10 alkyl, C2-10 alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkylalkyl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, aryl and heterocyclylalkyl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heteroaryl and heterocyclyl is independently substituted or unsubstituted; and wherein aryl is optionally substituted with 1 to 5 groups independently selected from: oxo, halogen, nitro, CF3, CHF2, CH2F, OCF3, OCHF2, OCH2F, azido, NR'SO2R'"', SO2NR", C(O)R', C(O)OR', OC(O)R', NR'C(O)OR'"', NR'C(O)R", C(O)NR'R", SR'"', S(O)R"", SO2R', NR'R", NR'C(O)NR''R"', NR'C(NCN)NR''R"', OR', aryl, heteroaryl, arylalkyl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl;

[0205] R4 is selected from hydrogen or Ci-Ce alkyl, wherein alkyl may be substituted or unsubstituted; or

[0206] R3 and R4 can be taken together with the atom to which they are attached to form a 4 to 10 membered heteroaryl or heterocyclic ring, each of which is substituted or unsubstituted;

[0207] Rs is selected from hydrogen or Ci-Ce alkyl, wherein alkyl may be substituted or unsubstituted; or

[0208] R4 and Rs can be taken together with the atom to which they are attached to form a 4 to 10 membered carbocyclic, heteroaryl or heterocyclic ring, each of which is substituted or unsubstituted;Attorney Docket No.: UCH-43225

[0209] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0210] Reis selected from trifluoromethyl, C1-C10 alkyl, C3-C10 cycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is independently substituted or unsubstituted;

[0211] R', R" and R'" are independently selected from: hydrogen, C1-C4 alkyl, C2-C4 alkenyl, aryl and arylalkyl;

[0212] R"" is selected from: C1-C4 alkyl, C1-C4 alkenyl, aryl and arylalkyl;

[0213] W is — C(O)ORi5, — C(O)NR4Ri5, — C(O)NR4ORI5, or — C(O)NR4S(O)jR6; provided that W is not C(O)OH;

[0214] Risis selected from: hydrogen, trifluoromethyl, C1-C10 alkyl, C2-C 10 alkenyl, C2-Cioalkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is substituted or unsubstituted;

[0215] Rieis selected from hydrogen or C1-C10 alkyl; or Risand Retaken together with the nitrogen atom to which they are both attached form a 4 to 10 membered cyclic ring with 1 or 2 nitrogen atoms and optionally an oxygen atom, said ring being substituted or unsubstituted;

[0216] X isN orN+CT;

[0217] m is 0, 1, 2, 3, 4 or 5; and

[0218] j is 1 or 2.

[0219] In certain embodiments, the compound is a compound of Formula (I),

[0220] wherein:

[0221] Ri, R2, R9, Rn are independently selected from: hydrogen, halo, C1-C4 alkyl, C3-C4 cycloalkyl, C2-C4 alkenyl, C2-C4 alkynyl, cyano, nitro, — OR3 and — NR3R4; wherein each alkyl, alkenyl, alkynyl, cycloalkyl is independently and optionally substituted with one to five halogens;

[0222] Rio and Rnare independently selected from: hydrogen, halo, C1-C10 alkyl, C3-C10 cycloalkyl, C2-C10 alkenyl, C2-C10 alkynyl, cyano, nitro, azido, — NR4SO2R6, — SO2NR3R4, — SO2R6, — C(O)NR3R4, — S(O)jNR4C(O)R3, — C(O)NR4S(O)jR6, — OR3, — NR3R4and — S(Ci-C2 alkyl) substituted with 1 to 5 fluorines; wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is independently substituted or unsubstituted;

[0223] Rnand Rnare independently selected from: H, F, Cl, C1-C4 alkyl, C3-C4 cycloalkyl, C2-C4 alkenyl, and C2-C4 alkynyl; wherein each alkyl, alkenyl, cycloalkyl, and alkynyl is independently and optionally substituted with one to five halogens;Attorney Docket No.: UCH-43225

[0224] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0225] W is — C(O)ORi5, — C(O)NR4Ri5, — C(O)NR4ORI5,— C(0)(C2-CIO alkyl), or — C(O)NR4S(O)jR6;

[0226] Risis selected from: hydrogen, Ci-C4alkyl, Ci-C4alkenyl, and C4-Ce cycloalkylalkyl; wherein each alkyl or alkenyl is independently and optionally substituted by 1 or 2 OH, Ci-C4alkoxy or NR'R";

[0227] Ri6 is hydrogen or Ci-C4alkyl; and

[0228] R' and R" are each independently selected from: hydrogen, Ci-C4alkyl, C2-C4alkenyl, aryl and arylalkyl.

[0229] In certain embodiments, the compound is a compound of Formula (I),

[0230] wherein:

[0231] Ri is selected from H and F;

[0232] R2is selected from: hydrogen, F, Cl, and Me; wherein the methyl group is optionally substituted with one to three fluorines;

[0233] R9is selected from: H, F, and Cl;

[0234] Rio is selected from: H, F, Cl, Br, nitro, — SO2NR3R4or — C(O)NR3R4, -Me, and — OMe, wherein the methyl groups are optionally substituted with one to three fluorines;

[0235] Rnis selected from: H, F, Cl, Br, Me, and — OMe; wherein the methyl groups are optionally substituted with one to three fluorines;

[0236] Ri2is selected from: H, F, Cl, Br, nitro, Me, — SCF3, — SCHF2, — SCH2F, — SO2NR3R4, — C(O)NR3R4and — OMe; wherein the methyl groups are optionally substituted with one to three fluorines;

[0237] R3 and R4are Ci-Ce alkyl, independently and optionally substituted by 1 or 2 alkyl amino or alkoxy groups; or R3 and R4taken together with the nitrogen atom to which they are attached form a cyclic ring with 1 or 2 nitrogen atoms and optionally an oxygen atom, said ring being optionally substituted by 1 or 2 alkyl amino or alkoxy groups;

[0238] Rnis H or F;

[0239] Ri4is H or F;

[0240] W is — C(O)NR4ORI5;

[0241] Risis Ci-C4alkyl or C1-C4 alkenyl; wherein each is independently and optionally substituted with 1 to 3 — OH, — OMe, — NH2, — N(methyl)2or — N(ethyl)2;

[0242] Ri6 is hydrogen or C1-C4 alkyl; or Ri6 and R15 taken together with the nitrogen atom to which they are attached form a 4 to 10 membered cyclic ring with 1 or 2 nitrogen atoms andAttorney Docket No.: UCH-43225

[0243] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0244] optionally an oxygen atom, said ring being optionally substituted by 1 or 2 alkyl amino, amino, hydroxy or alkoxy groups; and

[0245] Yis O, S orNR'.

[0246] In certain embodiments, the compound is a compound of Formula (I),

[0247] wherein:

[0248] W is — C(O)NR4ORI5;

[0249] R4is hydrogen;

[0250] Ris is selected from: Ci-C4alkyl and Ci-C4alkenyl; wherein each is independently and optionally substituted by 1 or 2 — OH, Ci-C4alkoxy or — NR'R";

[0251] R' and R" are independently hydrogen, methyl or ethyl; and

[0252] Y is O.

[0253] In certain embodiments, the compound of Formula (I) is selected from the group consisting of:

[0254] N-(2,3-Dihydroxy-propoxy)-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; N-{[(4R)-2,2-dimethyl-l,3-dioxolan-4-yl]methoxy}-3-[(2-fluoro-4-iodophenyl)amino]isonicotinamide; 3-[(2-chloro-4-iodophenyl)amino]-N-{[(4R)-2,2-dimethyl-l,3-dioxolan-4-yl]methoxy}isonicotinamide; N-(2,3-Dihydroxy-propoxy)-3-(4-iodo-2-methyl-phenylamino)-isonicotinamide; 3-(2-Chloro-4-iodo-phenylamino)-isonicotinic acid methyl ester; 3-(2-Chloro-4-iodo-phenylamino)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(2-morpholin-4-yl-ethyl)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(2-hydroxy-propyl)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(2-hydroxy-ethyl)-isonicotinamide; [3-(2-Fluoro-4-iodo-phenylamino)-pyridin-4-yl]-morpholin-4-yl-methanone; N-Ethyl-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-piperidin-l-yl-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(3-imidazol-l-yl-propyl)-isonicotinamide; N-Benzyl-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; 3-(2-Chloro-4-iodo-phenylamino)-N-methyl-isonicotinamide; 3-(2-Chloro-4-iodo-phenylamino)-N,N-dimethyl-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(2-methoxy-ethyl)-N-methyl-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-morpholin-4-yl -isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(2-phenoxy-ethyl)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-[2-(2-methoxy-phenyl)-ethyl]-isonicotinamide; N-[2-(3-Chloro-phenyl)-ethyl]-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-[3-(2-oxo-pyrrolidin-l-yl)-propyl]-isonicotinamide; 2-Chl oro-3 -(2-fluoro-4-iodo-Attorney Docket No.: UCH-43225

[0255] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0256] phenylamino)-isoni cotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-isonicotinic acid N'-phenyl-hydrazide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(2-piperidin-l-yl-ethyl)-isonicotinamide; { 1-[3-(2-Fluoro-4-iodo-phenylamino)-pyridine-4-carbonyl]-piperidin-4-yl}-carbamic acid tertbutyl ester; 3-(2-Fluoro-4-iodo-phenylamino)-N-(3-morpholin-4-yl-propyl)-isonicotinamide; 3-(2-Chloro-4-iodo-phenylamino)-N-(5-hydroxy-pentyl)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(2-hydroxy-ethyl)-N-methyl-isonicotinamide; 2-Chloro-N-(2,2-dimethyl-[l,3]dioxolan-4-ylmethoxy)-3-(2-fluoro-4-iodo-phenylamino) -isonicotinamide; 3-(2 -Fluoro-4-iodo-phenylamino)-N-(4-hydroxy-butyl)-isoni cotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-pyridin-2-ylmethyl-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(2-hydroxy-propyl)-isonicotinamide; N-Azepan-l-yl-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; 2-Chloro-N-(2,3-dihydroxy-propoxy)-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; (4-Amino-piperidin-l-yl)-[3-(2-fluoro-4-iodo-phenylamino)-pyridin-4-yl]-methanone; N'-[3-(2-Fluoro-4-iodo-phenylamino)-pyridine-4-carbonyl]-hydrazinecarboxylic acid tert-butyl ester; 4-({[3-(2-Fluoro-4-iodo-phenylamino)-pyridine-4-carbonyl]-amino}-methyl)-benzoic acid; N-Cyclopropyl-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(2-hydroxy-propyl)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-isonicotinic acid N'-pyridin-2-yl-hydrazide; 3-(2-Fluoro-4-iodo-phenylamino)-isonicotinic acid N'-(4-trifluoromethyl-pyrimidin-2-yl)-hydrazide; 3-(2-Fluoro-4-iodo-phenylamino)-isonicotinic acid hydrazide; N-Cyclopropylmethyl-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; 3-(2-Chloro-4-ethynyl-phenylamino)-N-(2,3-dihydroxy-propoxy)-isonicotinamide; 3 -Methoxy -benzoic acid N'-[3-(2-fluoro-4-iodo-phenylamino)-pyridine-4-carbonyl]-hydrazide; 3-(2-Fluoro-4-iodo-phenylamino)-isonicotinic acid N'-(7-chloro-quinolin-4-yl) -hydrazide; 2-(4-Dimethylamino-phenyl)-5-(2-fluoro-4-iodo-phenylamino)-isonicotinic acid; N-Cyclobutyl-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-indan-l-yl-isonicotinamide; N-Cyclopentyl-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; N-Cyclohexyl-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; N-(l,2-Dimethyl-propyl)-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; N-(2,2-Dimethyl-[l,3]dioxolan-4-ylmethoxy)-3-(2-fluoro-4-iodo-phenylamino) -isonicotinamide; N-(2-Acetylamino-ethyl)-3-(2-chloro-4-iodo-phenylamino)-isonicotinamide; N-tert-Butoxy-3-(2-chloro-4-iodo-phenylamino)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-hydroxy-isonicotinamide; 3-(4-Iodo-phenylamino)-isonicotinamide; 2-Bromo-5-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; 2-Bromo-N-([l,3]dioxolan-4-ylmethoxy)-5-(2-fluoro-4-iodo-phenylamino) -isonicotinamide;Attorney Docket No.: UCH-43225

[0257] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0258] 2-Bromo-5-(2-fluoro-4-iodo-phenylamino)-N-(3-hydroxy-propyl)-isonicotinamide; 2-Bromo-N-(2,3-dihydroxy-propoxy)-5-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; 2-Bromo-5-(2-fluoro-4-iodo-phenylamino)-N-(3-imidazol-l-yl-propyl)-isonicotinamide; 2-Bromo-5-(2-fluoro-4-iodo-phenylamino)-N-(2-hydroxy-ethyl)-isonicotinamide; N-(2, 3 -Dihydroxy -propoxy)-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; 3-(2-Chloro-4-iodo-phenylamino)-N-ethoxy-isonicotinamide; N-Allyloxy-3-(2-chloro-4-iodo-phenylamino)-isonicotinamide; 3-(2-Chloro-4-iodo-phenylamino)-N-isobutoxy-isoni cotinamide; N-(3-Chloro-propyl)-3-(2-fluoro-4-iodo-phenylamino)-isoni cotinamide; 3-(2-Chloro-4-iodo-phenylamino)-N-methoxy-isonicotinamide; N-Benzyloxy-3-(2-chloro-4-iodo-phenylamino)-isonicotinamide; N-Bicyclo[2.2.1]hept-2-yl-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; 3-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyphenoxypropyl)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(tetrahydro-pyran-2-yloxy)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(2-p-tolyl-ethyl)-isonicotinamide; N-{l-[3-(2-Fluoro-4-iodo-phenylamino)-pyridine-4-carbonyl]-piperidin-4-yl}-2-p-tolyl acetamide; 2-Bromo-5-(2-fluoro-4-iodo-phenylamino)-N-(2-methoxy-ethyl)-isonicotinamide; 2-Bromo-5-(2-fluoro-4-iodo-phenylamino)-N-(2-morpholin-4-yl-ethyl)-isonicotinamide; N-(2,2-Dimethyl-[l,3]dioxolan-4-ylmethoxy)-3-(2-fluoro-4-iodo-phenylamino)-l-oxy isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-isonicotinic acid N'-m-tolyl-hydrazide; N-Benzyloxy-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; {[3-(2-Fluoro-4-iodo-phenylamino)-pyridine-4-carbonyl]-aminooxy}-acetic acid; N-(2,4-Difluoro-benzyl)-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(3-iodo-benzyl)-isonicotinamide; N-(2, 3 -Dihydroxy -propoxy)-3-(4-iodo-phenylamino)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-l-oxy-isonicotinamide; N-(2,2-Diethoxy-ethyl)-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-isonicotinic acid N'-p-tolyl-hydrazide; 3-(2-Fluoro-4-iodo-phenylamino)-2-methyl-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-isonicotinic acid N'-(3,5-bis-trifluoromethyl-phenyl) -hydrazide; 4-(2-{ [3-(2-Fluoro-4-iodo-phenylamino)-pyridine-4-carbonyl]-amino}-ethyl)-benzoic acid; 3-(2-Fluoro-4-iodo-phenylamino)-N-pentafluorophenylmethoxy -isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(3-methoxy-phenyl)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(3-fluoro-5-trifluoromethyl-benzyl) -isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(3-hydroxy-benzyl)-isonicotinamide; N-(4,4-Diethoxy-butyl)-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; N-(4-Fluoro-benzyl)-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; 3-Attorney Docket No.: UCH-43225

[0259] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0260] (2-Fluoro-4-iodo-phenylamino)-N-(2,2,2-trifluoro-ethyl)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(l-hydroxymethyl-cyclopentyl)-isonicotinamide; N-(l-Carbamoyl-2-hydroxy-ethyl)-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(2-hydroxy-cyclohexyl)-isonicotinamide; N-(l,l-Bis-hydroxymethyl-propyl)-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; N-(2,3-Dihydroxy-propyl)-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(3-piperazin-l-yl-propyl)-isonicotinamide; 3-(2-Fluoro-4-iodo-phenylamino)-N-(3-fluoro-5-trifluoromethyl-benzyl)-iso -nicotinamide; 3-[(4-Bromo-2-fluorophenyl)amino]-N-ethoxyisonicotinamide; 3-[(4-Iodo-2-fluorophenyl)amino]-N-ethoxyisonicotinamide; N-[3-(4-Iodo-2-methyl-phenylamino)-pyridine-4-carbonyl]-methanesulfonamide; N — ((S)-2,3- Dihydroxy-propoxy)-3-(4-iodo-2-methyl-phenylamino)-isoni cotinamide; or a pharmaceutically acceptable salt thereof.

[0261] In some embodiments, the compound of Formula (II) is a substituted butadiene in which each of the central carbon atoms of the butadiene nucleus bears a cyano group and each, of the terminal carbon atoms of the butadiene nucleus bears an amino group and a hydrocarbyl-substituted mercapto group of not more than ten carbon atoms.

[0262] In some embodiments, the compound of Formula (II) is a substituted butadiene in which each of the central carbon atoms of the butadiene nucleus bears a cyano group and each of the terminal carbon atoms of the butadiene nucleus bears an amino group and a hydroxy Ci-io hydrocarbyl-substituted mercapto group of not more than ten carbon atoms.

[0263] In some embodiments, the compound of Formula (II) is a substituted butadiene in which each of the central carbon atoms of the butadiene nucleus bears a cyano group and each of the terminal carbon atoms of the butadiene nucleus bears an amino group and an amino Ci-io hydrocarbyl-substituted mercapto group of not more than ten carbon atoms.

[0264] In some embodiments, the compound of Formula (II) is a substituted butadiene in which each of the central carbon atoms of the butadiene nucelus bears a cyano group and each of the terminal carbon atoms of the butadiene nucleus bears an amino group and an alkylmercapto group of not more than ten carbon atoms.

[0265] In some embodiments, the compound of Formula (II) is a substituted butadiene in which each of the central carbon atoms of the butadiene nucelus bears a cyano group and each of the terminal carbon atoms of the butadiene nucleus bears an amino group and an arylmercapto group of not more than ten carbon atoms.Attorney Docket No.: UCH-43225

[0266] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0267] In some embodiments, the compound of Formula (II) is a substituted butadiene in which each of the central carbon atoms, of the butadiene nucleus bears a cyano group and each of the terminal carbon atoms of the butadiene nucelus bears an amino group and a monohydroxyalkylmercapto group of not more than ten carbon atoms.

[0268] In some embodiments, the compound of Formula (II) is a substituted butadiene in which each of the central carbon atoms of the butadiene nucleus bears a cyano group and each of the terminal carbon atoms of the butadiene nucleus bears an amino group and a monoaminoarylmercapto group of not more than ten carbon atoms.

[0269] In some embodiments, the compound of Formula (II) is 1,4 diamino-2,3-dicyano-l,4-bis(m ethylmercapto)- butadiene.

[0270] In some embodiments, the compound of Formula (II) is 1,4 diamino 2,3-dicyano-l,4-bis(ethylmercapto)- butadiene.

[0271] In some embodiments, the compound of Formula (II) is 1,4 diamino2,3-dicyano-l,4-bis (phenylmercapto) butadiene.

[0272] In some embodiments, the compound of Formula (II) is 1,4 diamino-Z,3-dicyano-l,4-bis(beta-hydroxyethylmercapto)butadiene.

[0273] In certain embodiments, the compound is

[0274]

[0275] pharmaceutically acceptable salt thereof.

[0276] In certain embodiments, the compound

[0277]

[0278] or a pharmaceutically acceptable salt thereof.

[0279] In some embodiments, the methods comprise treating or preventing airway injury in a subject in need thereof.

[0280] In some embodiments, the airway injury is selected from epithelial barrier dysfunction, ciliary dysfunction, smoke- or aerosol-induced epithelial barrier dysfunction, environmental exposure-induced epithelial barrier dysfunction, epithelial inflammation, pulmonaryAttorney Docket No.: UCH-43225

[0281] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0282] inflammation, smoke- or aerosol-induced inflammation, and environmental exposure-induced inflammation.

[0283] In certain embodiments, the smoke- or aerosol-induced inflammation is wildfire smoke- or aerosol-induced inflammation, tobacco smoke- or aerosol-induced inflammation, cooking smoke- or aerosol-induced inflammation, industrial smoke- or aerosol-induced inflammation, or waste disposal smoke- or aerosol-induced inflammation.

[0284] In some embodiments, the compound is administered prior to the airway injury. In certain such embodiments, the compound is administered about 1-12 hours prior to the airway injury. In some embodiments, the compound is administered about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours prior to the airway injury.

[0285] In some embodiments, the compound is administered daily for a period of about 1-5 days prior to the airway injury. In certain such embodiments, the compound is administered daily for a period of about 1 day, about 2 days, or about 3 days prior to the airway injury.

[0286] In certain embodiments, the compound is administered following the airway injury. In certain such embodiments, the compound is administered about 1-12 hours following the airway injury. In some embodiments, the compound is administered about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours following the airway injury.

[0287] In some embodiments, the compound is administered daily for a period of about 1-5 days following the airway injury. In certain such embodiments, the compound is administered daily for a period of about 1 day, about 2 days, or about 3 days following the airway injury.

[0288] In some embodiments, the methods comprise treating or preventing lung cancer in a subject in need thereof.

[0289] In some embodiments, the methods comprise improving mucociliary clearance in a subject in need thereof.

[0290] In some embodiments, the compound is administered to an airway of the subject (e.g., via inhaler).

[0291] In some embodiments, the compound is formulated for administration as an aerosol. In various embodiments, the modulator of protein phosphorylation can be a cyclin-dependent kinase (CDK) inhibitor, an alkaline phosphatase inhibitor, a tyrosine phosphatase inhibitor, a serine / threonine phosphatase inhibitor / activator or other phosphatase family member inhibitor / activator, a tyrosine kinase inhibitor, a serine kinase inhibitor, a threonineAttorney Docket No.: UCH-43225

[0292] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0293] kinase inhibitor, a growth factor induced pathway inhibitor (e.g., Epidermal Growth Factor inhibitor), a JAK / STAT pathway inhibitor, or a mitogen-activated protein kinase inhibitor, such as a MAP kinase inhibitor e.g., a MEK1 or MEK2 inhibitor).

[0294] In certain embodiments, the modulator of protein phosphorylation is a MEK1 or MEK2

[0295]

[0296] pharmaceutically acceptable salt thereof.

[0297] In some preferred embodiments, the modulator of protein phosphorylation is

[0298]

[0299] pharmaceutically acceptable salt thereof. In some preferred

[0300] embodiments, the modulator of protein phosphorylation

[0301]

[0302] pharmaceutically acceptable salt thereof.

[0303] Pimasertib and additional illustrative MEK inhibitors are described, for example, in U.S. Pat. No. 7,956,191, the contents of which are incorporated herein by reference in theirAttorney Docket No.: UCH-43225

[0304] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0305] entirety. U0126 is described, for example, in U.S. Pat. No. 2,779,780, the contents of which are incorporated herein by reference in their entirety.

[0306] In some embodiments, non-limiting examples of kinase inhibitors may include such compounds as sunitinib, sorafenib, dasatinib, cobimetinib, afatinib, bosutinib, cetuximab, crizotinib, cabozantinib, dacomitinib, entrectinib, erdafitinib, fostamatinib, gefitinib, ibrutinib, lapatinib, and pazopanib. In certain embodiments, non-limiting examples of phosphatase inhibitors may include such compounds as bromotetramisole, levamisole, imidazole, okadaic acid, microcystin-LR, cantharidin, ethylenediaminetetraacetic acid, cyclosporine, FK-506, vanadate salts, such as sodium orthovanadate, and molybdate salts, such as sodium molybdate.

[0307] In certain embodiments, epithelial inflammation, pulmonary inflammation, and smoke-or aerosol-induced inflammation result from dysfunction of the epithelial barrier in the airway. In certain such embodiments, the airway is considered to comprise the nose and sinuses in addition to the trachea and lungs. In some embodiments, dysfunction of the epithelial barrier in the airway results from exposure to irritants, such as smoke or aerosols, including wildfire smoke or aerosols.

[0308] In some embodiments, epithelial barrier dysfunction may be treated or prevented through the administration of a modulator of protein phosphorylation, such as a kinase inhibitor or phosphatase inhibitor.

[0309] In certain embodiments, epithelial barrier dysfunction may lead to decreased mucociliary clearance in the airway. In some embodiments, mucociliary clearance may be improved or restored through the administration of a modulator of protein phosphorylation, such as a kinase inhibitor or a phosphatase inhibitor.

[0310] In some embodiments, kinases involved in regulating the epithelial barrier in the airway may include, but are not limited to, MEK1, MEK2, GSK3B, MAPK14, RPS6KA3, MAPKAPK2, CDK1, RPS6KB1, MY03A, PRKCD, MAPK12, MAPK13, CDK2, PRKG2, MAPKAPK3, MAP2K3, MAP2K6, AKT1, NLK, BCR, AKT3, SGK1, MAPK10, PRKG1, CDK6, EIF2AK2, RIPK3, PRKD1 ROCK2, CSF1R, IGF1R, NUAK2, DAPK3, PRKX, PRKY, MET, PTK2, CDKN1A, SRC, EGFR, LATS1, MAP3K7, WNK1, WNK4, and FYN. In certain such embodiments, kinases may be up- or down-regulated following exposure to irritants, such as smoke or aerosols, including wildfire smoke or aerosols.

[0311] Pharmaceutical Compositions

[0312] The compositions and methods of the present disclosure may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as aAttorney Docket No.: UCH-43225

[0313] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0314] human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound described herein and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0315] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound described herein. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a selfemulsifying drug delivery system or a selfmicroemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound described herein. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0316] 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 animalsAttorney Docket No.: UCH-43225

[0317] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0318] without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0319] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0320] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

[0321] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage formAttorney Docket No.: UCH-43225

[0322] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0323] will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

[0324] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound described herein, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0325] Formulations suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a compound described herein as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

[0326] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-Attorney Docket No.: UCH-43225

[0327] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0328] filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0329] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surfaceactive or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0330] The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

[0331] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.Attorney Docket No.: UCH-43225

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[0333] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0334] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0335] Dosage forms for the airway, topical or transdermal administration include powders, sprays, nebulizers, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

[0336] The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0337] Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0338] Transdermal patches have the added advantage of providing controlled delivery of a compound described herein to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

[0339] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or moreAttorney Docket No.: UCH-43225

[0340] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0341] pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0342] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0343] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0344] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0345] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.Attorney Docket No.: UCH-43225

[0346] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0347] For use in the methods described herein, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0348] Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.

[0349] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0350] The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0351] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound described herein. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996)Attorney Docket No.: UCH-43225

[0352] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0353] Harrison’s Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).

[0354] In general, a suitable daily dose of an active compound used in the compositions and methods described herein will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0355] If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments, the active compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily.

[0356] The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.

[0357] In certain embodiments, compounds described herein may be used alone or conjointly administered with another type of therapeutic agent.

[0358] The present disclosure includes the use of pharmaceutically acceptable salts of compounds described herein in the compositions and methods described herein. In certain embodiments, contemplated salts described herein include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts include, but are not limited to, L-arginine, benethamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, IH-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, l-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, 1-ascorbic acid, 1-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor- 10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-di sulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid,Attorney Docket No.: UCH-43225

[0359] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0360] gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, 1-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthal ene-l,5-disulfonic acid, naphthal ene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, 1 -pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, 1-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid salts.

[0361] The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

[0362] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0363] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0364] Definitions

[0365] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well known and commonly used in the art.

[0366] The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout thisAttorney Docket No.: UCH-43225

[0367] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0368] specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed ”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed ”, Sinauer Associates, Inc., Sunderland, MA (2000).

[0369] Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

[0370] All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

[0371] The term “agent” is used herein to denote a chemical compound (such as an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents whose structure is known, and those whose structure is not known.

[0372] A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).

[0373] “Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0374] As used herein, the terms “restore” and “restoring” refer to improving the condition of a bodily or physiological function to that of the wild-type level, or partially to that of the wildtype level. As an illustrative example, restoring mucociliary clearance refers to improving diminished mucociliary clearance e.g., following exposure to wildfire smoke) to a levelAttorney Docket No.: UCH-43225

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[0376] comparable to wild-type mucociliary clearance (e.g., having no exposure to wildfire smoke), or to a level that is partial to that of wild-type mucociliary clearance (e.g., 50%).

[0377] The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0378] “Administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intratracheally (by inhalation), via nebulizer, intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0379] Appropriate methods of administering a substance, a compound or an agent to a subject will also depend, for example, on the age and / or the physical condition of the subject and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, a compound or an agent is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.

[0380] As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the bodyAttorney Docket No.: UCH-43225

[0381] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0382] (e.g., the two agents are simultaneously effective in the patient, which may include synergistic effects of the two agents). For example, the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.

[0383] A “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject’s size, health and age, and the nature and extent of the condition being treated, such as cancer or MDS. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.

[0384] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. For example, “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted.

[0385] The phrase “pharmaceutically acceptable” is art-recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers and other materials 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.

[0386] “Pharmaceutically acceptable salt” or “salt” is used herein to refer to an acid addition salt or a basic addition salt which is suitable for or compatible with the treatment of patients.

[0387] The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any base compounds described herein. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylicAttorney Docket No.: UCH-43225

[0388] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0389] acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of compounds described herein are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, e.g., oxalates, may be used, for example, in the isolation of compounds described herein for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0390] The term “pharmaceutically acceptable basic addition salt” as used herein means any non-toxic organic or inorganic base addition salt of any acid compounds described herein or any of their intermediates. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art.

[0391] Many of the compounds useful in the methods and compositions of this disclosure have at least one stereogenic center in their structure. This stereogenic center may be present in a R or a S configuration, said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates all stereoisomeric forms such as enantiomeric and diastereoisomeric forms of the compounds, salts, prodrugs or mixtures thereof (including all possible mixtures of stereoisomers). See, e.g., WO 01 / 062726.

[0392] Furthermore, certain compounds which contain alkenyl groups may exist as Z (zusammen) or E (entgegen) isomers. In each instance, the disclosure includes both mixture and separate individual isomers.

[0393] “Prodrug” or “pharmaceutically acceptable prodrug” refers to a compound that is metabolized, for example hydrolyzed or oxidized, in the host after administration to form the compound of the present disclosure. Typical examples of prodrugs include compounds that have biologically labile or cleavable (protecting) groups on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound. Examples of prodrugs using ester or phosphoramidate as biologically labile or cleavable (protecting)Attorney Docket No.: UCH-43225

[0394] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0395] groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of this disclosure are metabolized to produce a compound described herein. The present disclosure includes within its scope, prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in “Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985.

[0396] The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use.

[0397] The term “Log of solubility”, “LogS” or “logS” as used herein is used in the art to quantify the aqueous solubility of a compound. The aqueous solubility of a compound significantly affects its absorption and distribution characteristics. A low solubility often goes along with a poor absorption. LogS value is a unit stripped logarithm (base 10) of the solubility measured in mol / liter.

[0398] EXAMPLES

[0399] The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.

[0400] Example 1: Exemplary Materials and Methods of the Disclosure

[0401] Human tissue procurement

[0402] Large airways and bronchial tissues were acquired from de-identified human donors after lung transplantations at Ronald Reagan UCLA Medical Center and from the International Institute for the Advancement of Medicine (IIAM). De-identified tissues were procured under Institutional Review Board-approved protocols at the David Geffen School of Medicine at UCLA, IRB exemption #21-000390. ABSCs from two to three donors were used for all experiments. Information about each donor tissue and experiment included in FIG. 2 and Tables 1-2Attorney Docket No.: UCH-43225

[0403] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0404] Table 2, Donor Demographics

[0405]

[0406] Sinonasal tissue was obtained from de-identified patients undergoing functional endoscopic sinus surgery for Chronic Rhinosinusitis under the Institutional Review Board approved protocol: IRB exemption #24-5970.

[0407] Hematoxylin and Eosin (H&E) Staining / Imaging

[0408] H&E staining was performed per standard protocols. Whole-slide imaging was conducted using a Leica Aperio Versa high-throughput scanning system. Quantitative image analysis was performed using the Definiens’ Tissue Studio Software.

[0409] Epithelium Measurement

[0410] Measurement of the epithelium coverage and height was blindly scored using Fiji / ImageJ. Each image was imported and analyzed using the straight-line tool to create line segments. The entire tissue edge at the basement membrane was outlined and summed to measure the total length. The same line tool was then used to measure line segments of where actual epithelial tissue was. The criteria for identifying intact epithelial tissue included the presence of a continuous cell layer and its attachment to the underlying basement membrane. Epithelial tissue percentage coverage was calculated as the ratio of the actual epithelial tissueAttorney Docket No.: UCH-43225

[0411] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0412] length / total tissue length. Epithelial height measurement was measured from three randomly selected locations of the tissue where the epithelial tissue was intact to measure height, using the same line segment tool.

[0413] Airway Basal Stem Cell (AB SC) isolation

[0414] Human Airway basal stem cells (ABSCs) were isolated following previously published methods. All steps were performed with the tracheal tissue maintained in cold phosphate buffered saline supplemented with antimicrobials. Briefly, airway issue was dissected, cleaned, and incubated in 50U / mL dispase for 30 minutes at room temperature. Tissues were then incubated in lOmg / mL DNAse for 30 minutes at room temperature. Epithelium was stripped and incubated in 0.25% Trypsin-EDTA for 30 minutes at 37*C with agitation to generate a single cell suspension. Isolated cells were passed through a 40 pm strainer and plated for expansion. All cells used in these experiments were P2-P3 at Air-Liquid Interface culture.

[0415] (FIG. 2A)

[0416] Air-liquid Interface Cultures

[0417] 24-well 6.5mm transwells or 96 well 0.32mm transwell with 0.4 pm pore polyester membrane inserts were coated with 0.5mg / ML collagen type IV from human placenta dissolved in acetic acid and diluted in cell grade water at a ratio of 1:10. 100 pl was added to each transwell, allowed to air dry and cross-linked with UV light for 30 minutes prior to cell seeding. ABSC’s Passages 2-3 were seeded at 80,000 cells per 24-well transwell and 8,000 cells per 96-well transwell and grown in the submerged phase (Pneumacult Ex Plus) for 5 days with 500 pl in basal chamber and 250 pl in apical chamber (24-well), 180 ul in basal chamber and 75 pl in apical chamber (96-well). Confluent ABSCs were lifted to ALI day 0 and cultured with 500 pl Pneumacult ALI in the basal chamber (24-well) and 180 pl in the basal chamber (96-well). ALIs are considered mature at ALI day 21. Cultures were exposed to woodfire smoke on ALI day 21 and harvested for further experiments on each day until ALI day 25. Woodfire Smoke in vitro Exposures

[0418] Primary human ALI cell cultures were placed uncovered in a 3 -liter airtight chamber equipped with an inlet and an outlet (Kent Scientific). An air pump (Adafruit Industries) connected to the inlet was controlled by an Arduino UNO microcontroller fitted with a relay shield mod (Seed Technology Co). One outlet remained open to prevent vacuum formation.

[0419] Pinus canariensis (Canary Island Pine) bark was finely ground and filtered through a 100 pm filter to remove fine dust. After pre-treatment (12 hour drying at 37*C), 0.3g of bark was rolled in burnable paper and connected to the inlet pump. The Arduino was programmedAttorney Docket No.: UCH-43225

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[0421] to activate the air pump for 3 seconds which pulls the smoke generated from the pine bark and paper combustion into the chamber. The outlet was then closed, and ALI cultures were exposed to smoke for 3 minutes. After exposure, the chamber lid was removed to release smoke. This exposure was repeated once daily for five consecutive days.

[0422] Immunofluorescence, Confocal Imaging, and Cell Countins

[0423] ALI transwell inserts were fixed with cold 100% MEOH for 15 minutes. Briefly, transwells were rehydrated with MEOH / PBT, then permeabilized with cold acetone. Transwells were blocked for 1 hour in appropriate blocking solution and primary antibodies were added overnight at 4*C. The following day, respective secondary antibodies were added and co-stained with DAPI (ThermoFisher D3571). Transwells were clarified in a series of PBT / Glycerol incubations and mounted with VectaShield Vibrance Antifade Mounting Medium (H-1700-10, Vector Laboratories) mounting media.

[0424] Imaging was performed using a Zeiss LSM700 confocal microscope at 20X and 63X Oil objectives. Three random z-stack images were acquired per sample. Cell counts, fluorescence intensity, and protein marker quantification was done using Halo Image Analysis Platform Version 3.5.3577 (Indica Labs Inc).

[0425] RNA Extraction

[0426] RNA was extracted using Qiagen RNeasy Mini Kit (Cat. 74104) following manufacturer’s protocol. RNA concentration and purity were assessed using a NanoDrop Spectrophotometer (ThermoFisher).

[0427] Global RNA Sequencing Experimental Methods

[0428] Libraries for RNA-Seq were prepared with KAPA Stranded RNA-Seq Kit with RiboErase Kit. The workflow consists of depletion of rRNA by hybridization of complementary DNA oligonucleotides, followed by treatment with RNase H and DNase and RNA fragmentaion. First strand cDNA synthesis using random priming followed by second strand synthesis converting cDNA:RNA hybrid to double-stranded cDNA (ds cDNA), and incorporates dUTP into the second cDNA strand. cDNA generation is followed by A-tailing, adaptor ligation and PCR amplification. Different adapters were used for multiplexing samples in one lane. Sequencing was performed on Illumina NovaSeq X Plus for PE 2x50 run. Data quality check was done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq v2.19.1.403 software.

[0429] Global RNA Sequencing Bioinformatics MethodsAttorney Docket No.: UCH-43225

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[0431] Paired-fastq files were initially assessed for quality with fastqc v 0.12.1. Trimgalore v 0.6.10 with GNU Parallel v3 was then used to trim 12 base pairs from the 5’ end and 3 base pairs from the 3’ end from each fastq and remove adapter sequences. This was done as there was evidence of random hexamer bias and some slight overhang. Then each fastq was aligned to GRCh38.pl4 Ensembl genome with GRCh38.112 GTF file using STAR v 2.7.10b. Salmon v 1.10.3 was then used to quantify the bam files. Further analysis was carried out using R v 4.4.1 and RStudio 2024.04.2-764. Salomon quantification files were uploaded into R using ensembld v 2.28.1 and txtimport v 1.32.0. This was then used to create a DESEQ2 v 1.44.0 object for differential expression analysis. The model used was -Sample + Condition to account for differences between sample and analyze the effect of woodfire smoke. DEGs were filtered by adjusted p-value < 0.05. Gene symbols were added to DEG files using biomaRt v 2.60.1. Ontology pathway analysis was then performed using ToppFunn with DEGs with padj < 0.05 with a log 2-fold change > 1 for up-regulated and < -1 for down-regulated pathways. RNA sequencing volcano and column plots were made with tidyverse 2.0.0 and heatmaps with pheatmap 1.0.12.

[0432] Proteomics sample processing

[0433] Samples were lysed in 6M guanidine hydrochloride, boiled at 95 °C for 5 min, and kept on ice prior to sonication. Lysates were sonicated using a probe sonicator (2 x 10 s pulses at 10% amplitude), after which protein concentration was determined by Bradford assay. Approximately 150 pg of total protein was used for downstream processing. Samples were reduced with tris(2-carboxyethyl)phosphine (TCEP; 10 mM final concentration) at 58°C for 30 min with shaking at 1500 rpm, followed by alkylation with 2-chloroacetamide (40 mM final concentration) at 25°C for 30 min with shaking at 1,500 rpm. Prior to proteolytic digestion, guanidine hydrochloride was diluted 6-fold with 100 mM Tris-HCl (pH 8.0). Proteins were digested with trypsin (1:100, enzyme: protein) and Lys-C (1:50, enzyme :protein) at 37°C for 16 hr with shaking at 800 rpm. Following digestion, 10% trifluoroacetic acid (TFA) was added to each sample to a final pH of -2.

[0434] Peptides were desalted using 30 mg Oasis HLB cartridges (Waters) on a vacuum manifold. Cartridges were activated with 1 mL of 80% acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA) and equilibrated with 3 >< 1 mL of 0.1% TFA. Samples were loaded onto each cartridge twice, followed by washing with 3 x 1 mL of 0.1% TFA. Peptides were eluted with 0.8 mL of 50% ACN containing 0.25% formic acid (FA). Approximately 10% of the desalted peptides were separated for global proteomics analysis, while the remaining 90%Attorney Docket No.: UCH-43225

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[0436] were used for phosphopeptide enrichment. Both fractions were dried via vacuum centrifugation. The Ti-IMAC HP beads (Resyn Biosciences) were used according to the manufacturer's protocol for phosphopeptide enrichment. After the elution of phosphopeptides from the beads, the pH was brought down immediately to pH ~3 with 10% FA. Samples were dried by vacuum centrifugation and stored at -80°C until further analysis.

[0437] Mass spectrometry proteomics acquisition

[0438] Dried peptides were resuspended in 0.1% FA in MS-grade water and analyzed using a timsTOF HT mass spectrometer coupled to a Vanquish Neo UHPLC system. Mobile phase A consisted of 0.1% FA in MS-grade water, and mobile phase B consisted of 0.1% FA in 100% MS-grade ACN. Liquid chromatography was performed in trap-and-elute mode, in which peptides were first captured on a PepMap Neo Trap column (5 mm length, 100 A pore size, 5 pm particle size) and subsequently separated by reversed-phase chromatography on an Aurora Elite Cl 8 column (15 cm length, 100 A pore size, 1.5 pm particle size; lonOpticks) using the gradients described below. The analytical column was maintained at 50°C using a column oven compatible with the Bruker CaptiveSpray source (Sonation Lab Solutions), and peptides were ionized via a CaptiveSpray source (Bruker Daltonics) operated at 1700 V.

[0439] For the global proteome analysis, peptides were separated using a 45 min LC gradient as follows: 5-35% mobile phase B over 37 min, 35-45% B over 4 min, 45-60% B over 1 min, and 60-95% B over 1 min, all at a flow rate of 300 nL / min. The flow rate was increased to 400 nL / min for the final 2 min of the run. On the timsTOF HT, raw data was acquired in dia-PASEF mode. Equal-size windows of 24 Da were designed with an overlap of 1 Da to maximize precursors ion coverage for further MS / MS. The ion accumulation time and ramp times in the dual TIMS analyzer were set to 100 ms each. In the ion mobility (1 / K0) range 0.6 to 1.6 Vs cm-2, the collision energy was linearly decreased from 59 eV at 1 / K0 = 1.41 Vs cm-2 to 20 eV at 1 / K0 = 0.7 Vs cm-2 to collect the MS / MS spectra in the mass range 250.3 to 1240.3 Da. The estimated mean cycle time was 1.38 s.

[0440] For the phosphoproteome analysis, peptides were separated using a 45 min LC gradient as follows: 3-16% mobile phase B over 26 min, 16-30% B over 11.5 min, 30-45% B over 4 min, 45-60% B over 1 min, and 60-95% B over 0.5 min, all at a flow rate of 300 nL / min. The flow rate was increased to 400 nL / min for the final 2 min of the run. On the timsTOF HT, raw data was acquired in dia-PASEF mode. Equal-size windows of 25 Da were designed with an overlap of 1 Da to maximize precursors ion coverage for further MS / MS. The ion accumulation time and ramp times in the dual TIMS analyzer were set to 100 ms each. In the ion mobilityAttorney Docket No.: UCH-43225

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[0442] (1 / K0) range 0.6 to 1.6 Vs cm-2, the collision energy was linearly decreased from 59 eV at 1 / K0 = 1.41 Vs cm-2 to 20 eV at 1 / K0 = 0.71 Vs cm-2 to collect the MS / MS spectra in the mass range 274.4 to 1283.4 Da. The estimated mean cycle time was 1.38 s.

[0443] Mass spectrometry proteomics data analysis

[0444] Raw DIA files were processed using Spectronaut (Biognosys, version 20.2) with the directDIA+ (Deep) search algorithm, which performs library-free, in silico-derived DIA analysis. Carbamidom ethylation (cysteine) was specified as a fixed modification, while acetylation (protein N-terminus), oxidation (methionine), and phosphorylation (serine, threonine, and tyrosine) were included as variable modifications. Reviewed human protein sequences downloaded from UniProt (October 6, 2023) were used for peptide and protein identification. False discovery rates (FDRs) at the PSM, peptide, and protein group levels were controlled at 1%. The minimum PTM localization threshold was set to zero, and no probability cutoff was applied for PTM site localization during quantification.

[0445] For MS2-level, area-based quantification, Spectronaut’s cross-run normalization was enabled. This normalization approach corrects for systematic technical variation between LC-MS runs by aligning intensity distributions across samples using a global scaling strategy based on the assumption that the majority of quantified features do not change between conditions. Statistical testing was performed within Spectronaut using its built-in, group-wise comparison framework, which applies a moderated t-test and controls for multiple hypothesis testing using FDR correction.

[0446] Missing values were handled using the Spectronaut imputation strategy “Use Background Signal,” which replaces missing peptide or fragment ion intensities with values sampled from the measured background noise distribution. This approach assumes that missing values primarily reflect signals below the detection limit rather than true absence and preserves variance structure while avoiding artificial inflation of fold changes.

[0447] Downstream quantitative analyses were performed in the R statistical programming language (version 4.3.2). Initial quality control assessments including inter-run clustering, correlation analyses, principal component analysis (PCA), peptide and protein identification counts, and intensity distributions were conducted using in-house R scripts.

[0448] Gene Set Overr epresentation Analysis

[0449] Gene sets exhibiting significant up- or down-regulation were tested for enrichment of Gene Ontology (GO) Biological Process terms. Genes were included if they showed a significant change in RNA expression, protein abundance, or protein phosphorylation in at leastAttorney Docket No.: UCH-43225

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[0451] one donor. Gene set overrepresentation analysis (GSOA) was performed using the GO Biological Process gene sets obtained from MSigDB. GO terms with an adjusted p value < 0.05 were considered significantly enriched.

[0452] Gene Set Overr epresentation analysis

[0453] Gene sets exhibiting significant up- or down-regulation were tested for enrichment of Gene Ontology (GO) Biological Process terms. Genes were included if they showed a significant change in RNA expression, protein abundance, or protein phosphorylation in at least one donor. Gene set overrepresentation analysis (GSOA) was performed using the GO Biological Process gene sets obtained from MSigDB. GO terms with an adjusted p value < 0.05 were considered significantly enriched.

[0454] Kinase activity analysis of phosphoproteomics data

[0455] Kinase activities were estimated using curated kinase-substrate relationships from OmniPath. For each kinase, activity was inferred by calculating a z-score based on the mean log2fold change (log2FC) of its phosphorylated substrates relative to the global distribution of phosphosite log2FC values across the sample (z = (M - p) / s.e.). Statistical significance was assessed using a two-tailed z-test. This approach has previously been previously shown to robustly estimate kinase activity from phosphoproteomic data.

[0456] Transmission Electron Microscopy

[0457] Cultures were fixed in 2% glutaraldehyde and 4% paraformaldehyde in 0.1M Sodium Cacodylate buffer, pH 7.4 for 15 minutes at room temperature, then changed to fresh fixative and left at 4°C overnight. Samples were osmicated, stained with uranyl acetate, then dehydrated with a graded ethanol series. Filters were cut out of the plastic supports, then infiltrated with EMBed 812 (Electron Microscopy Sciences). Ultrathin sections (80 nm) were mounted onto copper grids and analyzed with a JEOL JEM-1400plus Transmission Electron Microscope (JEOL).

[0458] Real-Time High-Speed Cilia Videography

[0459] According to previously published protocols, high-speed microscopy videos of live beating cilia were acquired. On the day of ALI collection, transwells were cut and mounted on a glass microscopy slide with a 1.5mm coverslip (12-5410-55, Fisher Scientific). A Zeiss Axiom Observer 7 fully motorized inverted microscope with a motorized scanning stage and a Plan- Apochromat 63x oil immersion objective (1.4 NA) and a Hamamatsu Flash 4.0 version 3 camera was used for imaging. Utilizing this setup, the entire height of the epithelium and ciliaAttorney Docket No.: UCH-43225

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[0461] was visible and captured. Zen 3.3 software was used to acquire DIC videos for 3 seconds at 400 frames / sec.

[0462] To measure motile ciliary coverage, videos were acquired from 20 fields of view (FOVs) per ALI membrane in a predetermined, unbiased layout in the shape of a plus sign. This approach captures data from both central and peripheral regions, minimizing selection bias. A MATLAB-based analysis pipeline was used to characterize ciliary activity across all videos in all experimental and Control groups.

[0463] Real-Time High-Speed Microscopy Algorithm

[0464] Cilia movement was analyzed using a previously published MATLAB analysis pipeline to characterize ciliary activity across all high-speed microscopy videos. Briefly, videos from 20 FOVs per ALI membrane were acquired in a predetermined unbiased layout in the shape of a plus sign. The MATLAB algorithm automated the detection of motile cilia and calculated ciliary movement coverage (light grey) and ciliary inactivity coverage (dark grey). The algorithm normalizes video intensity, applies Fourier transform analysis to identify areas with ciliary movement, and generates a frequency map for the entire field of view (FOV) to calculate the percentage of motile cilia. This approach minimizes user intervention, reduces bias, and allows for comprehensive analysis of multiple FOVs.

[0465] Transepithelial Electrical Resistance

[0466] Transepithelial electrical resistance (TEER) was measured to assess barrier integrity of the ALI cell monolayer using EV0M3 voltohmmeter with STX100C96 electrodes (World Precision Instruments). Measurements were obtained from 2 biological replicates and 3 technical replicates for control and each day of woodfire exposure cell cultures. The EVOM instrument was calibrated prior to each experiment. Measurements were performed in a 96-well format, with probes equilibrized for 15 minutes in Pneumacult ALI media. 75ul of media was added to the apical chamber and 200ul of media to the basal chamber for measurements. Teer measurements are reported as (Measurement-Blank)x(Surface area of transwell).

[0467] Scanning Mobility Particle Sizer and Aerosol Particle Sizer

[0468] During combustion, particle sizes between 0.5 and 25 pm were measured in the chamber with a TSI Aerosol Particle Sizer (APS) (Shoreview, MN, USA). Particle sizes 10-450 nm were measured in the chamber using a Scanning Mobility Particle Sizer (SMPS), which consisted of a TSI (Shoreview MN, USA) Differential Mobility Analyzer (DMA) model 3080 partnered with a Condensation Particle Counter (CPS) model 3787. The size ranges for SMPS (10-445 nm) and APS (542 nm to 20 pm) cover the range of wildfire particle emissions thatAttorney Docket No.: UCH-43225

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[0470] have been found to be the most deleterious to human health. Both the APS and SMPS sampling was conducted in parallel to ensure consistency in emission plume characteristics. Particle size distribution data from both APS and SMPS were then integrated. Each experiment consisted of between three and five measurements (60-second sampling time), which were averaged to represent each combustion experiment. Each sample was combusted three separate times for statistical analysis.

[0471] Mass and Black Carbon (BC) Measurements

[0472] Particle mass was determined by weighing the filters using a microbalance (Sartorius ME-5) in a temperature and humidity controlled weighting room before and after sample collection. BC concentration on the filters was measured by an OT21 dual wavelength optical transmissometer (Magee Scientific) using optical absorption at 370 nm and 880 nm. Light absorption at 370 and 880 nm is specific to BC and brown carbon, or Humic Like Substances (HULIS) respectively. Detailed information about the loading and scattering corrections for the BC measurements is explained in previously published manuscripts. The attenuation-corrected BC concentration is calculated using the following expression:

[0473]

[0474] Where Io and I are the incident and transmitted light, respectively. S is the filter collection area, ox is the mass absorption cross-section. The manufacturer default value 7.77 m2g-1was used for o at 880 nm. Cref is a constant and 1.2626 was used for Teflon filters. The coefficient fx is 1.064 at 880 nm.

[0475] Excitation-Emission Matrix (EEM) Measurements

[0476] Fluorescence features of the chamber biomass burning samples were analyzed using Excitation-Emission Matrix (EEM) spectroscopy with a Lumina fluorometer (Thermo Scientific). The samples were extracted in phosphate buffer (pH 7.4) with a mass concentration of 5 pg / mL. EEM scans were performed across excitation wavelengths (1 ex) from 200-450 nm and emission wavelengths (1 em) 250-600 nm with intervals of 20 nm. Excitation and emission slit widths were set to 10 nm and scanning at 60 nm / s with an integration time of 10 ms. The diagonal line across the entire EEM spectrum is due to Rayleigh scattering and is considered an instrument artifact. Spectra were processed using the 3D and contour graph function in OriginLab software.

[0477] MEK drug studyAttorney Docket No.: UCH-43225

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[0479] Prevention: Cell cultures were treated with U0126 (Cell Signaling Technologies 99035, Danvers, MA) or pimasertib (Selleck Chemicals S1475, Houston, TX) 30 min prior to each daily woodfire exposure for 5 days. Cultures received 500 pL of media in the basal chamber and 30 pL in the apical chamber for drug treatment at indicated doses. U0126 doses included 10 nM, 50 nM, 100 nM, and 10 pM. Pimasertib doses included 10 nM, 50 nM, 100 nM, and 1 pM. Control samples received DMSO at matched concentrations.

[0480] ERK Activation: ERK signaling was activated using BCI (B4313 BCI Hydrochloride Millipore Sigma) at 30 pM each day for 5 days.

[0481] Statistical analyses

[0482] Immunofluorescent data are presented as the mean ± standard error of the mean (SEM) values. Due to a limited amount of human donor samples, Kruskal -Wallis or non-paired t-tests were used when appropriate. All results were considered significant if p<0.05 and absLOG2Fold Change >1. Graphs made in prism software, letters are used to denote statistical groupings in comparison tests; groups sharing the same letter are not significantly different from each other, while groups with different letters indicate statistically significant differences. Cilia movement video statistical analysis were done through R software. Values presented are means with 95% confidence intervals. Between condition differences were assessed using individualized fixed effects modeling to account for time-invariant confounding between donors. Letter values above each figure correspond to detecting a statistically significant difference in comparison to another group (see x-axis for lettering / labels).

[0483] Example 2; Exposure to ambient wildfire smoke is associated with epithelial barrier loss and infiltration of ultrafine particulate matter in human upper airway tissue

[0484] Wildfires are increasing globally, yet the direct cellular and molecular effects of wildfire smoke on the human airway remain poorly defined. In January 2025, the Palisades and Eaton wildfires led to hazardous air quality across Los Angeles County. To study the effects of wildfire smoke on the human upper airway, sinonasal tissue was collected from patients undergoing routine functional endoscopic sinus surgery (FESS) for chronic rhinosinusitis (CRS). Samples were obtained across three air quality index (AQI)-defined exposure time windows: a pre-fire baseline (AQI 0-100, n=4), active wildfire conditions during the LA wildfires (AQI >300, n=6) and smoldering wildfire conditions characterized by persistent smoke exposure for the 2 weeks after the LA wildfires with partially improved AQI (AQI 50-100, n=4) (FIG. 1A). Patient demographic characteristics are provided in Table 1.Attorney Docket No.: UCH-43225

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[0486] Blinded histologic evaluation (BG, AG) of Hematoxylin and Eosin (H&E)-stained sections revealed pre-fire samples all displayed an intact pseudostratified epithelium with baseline inflammatory features that are typical of CRS (FIG. IB, FIG.7A). In contrast, tissues collected during the active wildfire smoke exposure timeframe all exhibited focal epithelial sloughing, reduced epithelial coverage, and dense inflammatory infiltration, including regions of complete epithelial loss (FIG. 1C, FIG. 7B). During the smoldering phase, the airway epithelium recovered partially, with improved coverage, but failure to regain full epithelial height (FIGs. 1D-1E and FIG. 7C).

[0487] To assess airway epithelial barrier integrity, Claudin-1 immunofluorescence (IF) staining was performed and quantified in these same samples. Pre-fire samples displayed continuous, apically localized Claudin-1 positive tight junctions (FIG. IF). During active wildfire smoke exposure, Claudin-1 staining was markedly fragmented and discontinuous within the airway epithelium (FIG. 1G). This junctional defect persisted during the smoldering phase, with reductions in Claudin-1 expression in terms of the airway epithelial height and percentage epithelial cell coverage relative to the pre-fire tissue (p<0.0001; FIGs. 1H-1I).

[0488] Given the loss of airway epithelial barrier integrity, transmission electron microscopy (TEM) was used to evaluate particulate matter (PM) infiltration. Control tissue obtained under clean-air conditions from Ann Arbor, Michigan (AQI 0-20) contained no detectable intracellular PM (FIG. 1 J, FIG. 7D). In contrast, tissue collected during active wildfire smoke exposure contained abundant intracellular and intercellular PM, including ultrafine PM (200-1,100 nm) (FIGs. 1K-1L, FIG. 7E) which was quantified in FIG. IM. PM was observed within disrupted tight junctions (FIG. 7F), and dense, metal-containing particles were also detected within the airway epithelial cells (FIGs. 7G-7H).

[0489] The epithelial barrier disruption and PM infiltration observed under active and smoldering wildfire smoke exposure conditions led us to study the cellular and molecular mechanisms by which wildfire smoke impacts the airway. Therefore, to directly investigate these mechanisms, a controlled system to expose differentiated human airway cultures to wildfire smoke was developed.

[0490] Table 1. Patient Demographics from Functional Endoscopic Sinus Surgery

[0491]

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[0494]

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[0497]

[0498] Example 3: A human primary air-liquid interface model recapitulated woodfire smoke exposure in the airway epithelium

[0499] Primary human airway basal stem cells were expanded and differentiated at the airliquid interface (ALI) for 21 days to generate a mature mucociliary airway epithelium containing ciliated, goblet, club, and basal cells (FIG. 2A). All airway experiments were performed using primary human airway basal stem cells derived from donors with no prior history of lung disease. These fully differentiated human airway epithelial cultures were placed inside a sealed 6-L custom exposure chamber connected to a microcontroller-regulated pump that delivered smoke generated from combustion of 0.3 grams of pine bark (woodfire smoke) (FIG. 2B). Woodfire smoke was pumped into the exposure chamber for 3 minutes per day for five days, resulting in a cumulative exposure time of 15 minutes (FIG. 2C). Combustion was produced using a butane flame (1,316-2610 °C), generating high-temperature combustion conditions comparable to the upper range of wildfire combustion environments, including crown fires (982-1200 °C)49, 50. This exposure chamber-pump configuration enabled stable, programmable woodfire smoke delivery and highly reproducible exposures across experiments.

[0500] Physical characterization of the woodfire smoke demonstrated particle-size features consistent with PM found in aerosols from the burning of biomass. Measurements obtained using a scanning mobility particle sizer (SMPS) and aerosol particle sizer (APS) indicated that smoke was generated with a particle size distribution spanning fine and coarse modes, with a number-mode diameter of -210-220 nm and a long tail extending into the micrometer range (FIG. 2D). This distribution closely aligns with woodfire smoke spectra measured in ambient plume studies. To assess spatial uniformity of PM exposure within the chamber, nineteen Teflon filters were placed at defined positions throughout the chamber, and the pre- and post-Attorney Docket No.: UCH-43225

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[0502] bum masses of the filters were compared across four independent bums. All the filters accumulated similar amounts of particulate mass (mean 189 ± 13.82 pg), indicating uniform PM distribution across the volume of the chamber (FIG. 8A).

[0503] Real-time chamber monitoring recorded short-duration PM2.5 and PM10 spikes reaching approximately 1000pg / m3immediately after smoke introduction, followed by rapid declines within 60 seconds (FIG. 2E). These transient, high-intensity peaks fall within the upper concentration range documented during severe wildfire smoke events, including urban and near-plume conditions where PM2.5 has been shown to rapidly rise to >1000 pg / m3or higher and near-source occupational exposures where personal PM2.5 measurements frequently exceeded 1000 pg / m3and can reach >2000pg / m3. Carbon dioxide concentrations remained stable throughout each exposure (FIG. 2E).

[0504] Chemical analysis of the woodfire smoke further supported a biomass-burning profile. Black carbon attenuation at 370 nm and 880 nm (16.4 ± 8.2 and 7.3 ± 3.8, respectively) reflected contributions from both brown and black carbon, consistent with incomplete combustion, which is typical of wildfire emissions. To chemically fingerprint the smoke, excitation-emission matrix (EEM) fluorescence was performed on PM collected after five bums. EEM spectra contained humic-like substance peaks (320-350 nm / 405-420 nm and 345-355 nm / 460-465 nm), along with a 250-260 nm / 380-480 nm peak commonly associated with combustion of ambient organic matter. Spectral features were consistent across multiple sample dilutions, and a fluorescent “thumbprint” panel closely matched published signatures of ambient woodfire smoke, supporting the chemical fidelity of this in vitro exposure (FIG.

[0505] 2F). The corresponding raw EEM fluorescence map with overlay is shown in FIG. 7B.

[0506] After confirming that the exposure system produced compositionally representative and reproducible woodfire smoke, differentiated human ALI cultures were evaluated after 5 days of woodfire smoke exposure. DAPI staining revealed a significant reduction in cell number in woodfire smoke-exposed ALI cultures relative to room air controls, indicating that there was cell loss from the woodfire smoke exposure (FIGs. 8C-8D). The DAPI dye also showed an increase in nuclear size in surviving cells, consistent with early stress-associated epithelial changes (FIG. 8C and FIG. 8E). Although woodfire smoke exposure resulted in a significant reduction in total epithelial cell number, as reflected by decreased DAPI positive nuclei, celltype quantification was normalized to the remaining epithelial cells, and no preferential depletion or expansion of specific epithelial cell types was observed following woodfire smoke exposure. Specifically, the relative abundance of acetylated tubulin-expressing ciliated cellsAttorney Docket No.: UCH-43225

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[0508] (FIGs. 2G-2H), secretoglobulinlAl (SCGB1A1) positive club cells (FIGs. 2I-2J), and Keratin 5 (KRT5) positive airway basal stem cells (FIGs.2K-2L) remained unchanged relative to room air controls. A trend towards an increase in Mucin 5B (MUC5B) positive mucusproducing cells was observed, although this did not reach statistical significance (p=0.056) (FIGs. 2M-2N). Woodfire smoke exposure was also associated with a trend towards increased apoptosis by nuclear Caspase-3 immunostaining (p=0.056) (FIGs. 8F-8G), and a significant increase in Proliferating Cell Nuclear Antigen (PCNA)-positive cells, suggesting an increased proportion of cells in S phase undergoing DNA repair (p=0.0088; FIGs. 8H-8I). These observations motivated the use of an unbiased multi-omics approach to identify global molecular changes underlying woodfire smoke-induced airway epithelial injury.

[0509] Example 4: Multi-omics revealed dramatic remodeling of mRNA levels and protein phosphorylation, which was uncoupled from changes in protein abundance

[0510] To define molecular pathways disrupted by cumulative woodfire smoke exposure, mRNA and protein was isolated from human ALI cultures exposed to woodfire smoke in the system disclosed herein and global RNA-sequencing (RNA-seq) was performed (2 donors, 3 technical replicates each) combined with abundance proteomics and phosphoproteomics (3 donors, 3 technical replicates each) using the experimental timeline shown in FIG. 3A. Each dataset compared ALI cultures exposed to 3 -minute woodfire smoke exposures for five consecutive days with matched room air controls harvested on Day 25. RNA-seq data showed a clear separation of samples by treatment condition in principal component analysis (PCA), with strong reproducibility among replicates (FIG. 9A). Abundance proteomics detected 108,746-143,292 peptides (FIG. 9B) corresponding to 8,124-8,556 proteins (FIG. 9C) High reproducibility among samples was confirmed by pairwise Pearson correlation analysis (FIG.

[0511] 9D), despite minor changes between conditions as evidenced by the PCA (FIG. 9E). This phosphoproteomics analysis detected 18,404-22,177 phosphorylated peptides per sample (FIG. 9F) and replicate consistency was similarly supported by strong pairwise correlations in peptide intensities (FIG. 9G) and clustering in the PCA (FIG. 9H). While donor-specific phosphorylation sites were common, enhanced overlap was observed at the protein level: 42% of significant phosphoproteins were shared by at least two donors, compared with only 13.8% overlap at the phosphorylation site level, indicating conserved targeting of protein despite site¬ level heterogeneity (FIG. 91).

[0512] Across datasets, woodfire smoke induced widespread changes in mRNA and phosphorylation levels, but comparatively few alterations in total protein abundance (FIG.3B).Attorney Docket No.: UCH-43225

[0513] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0514] This divergence between transcript and protein changes suggested that woodfire smoke induced a global suppression of translation mediated by post-translational modifications (FIG.

[0515] 3C). However, a small subset of genes did increase at both the mRNA and protein levels in response to woodfire smoke, which were enriched for oxidative stress, detoxification, and metabolic-related pathways, consistent with a stress response signature (FIG. 3D). Together, these results reveal that wildfire smoke drives dramatic remodeling of phosphorylation signaling in airway epithelial cells, coupled to oxidative stress, inhibition of translation, and reprogramming of transcriptional programs.

[0516] A global gene set overrepresentation analysis (GSOA) was next performed on significantly differentially regulated mRNAs, proteins, or phosphorylated proteins using Gene Ontology (GO) biological process terms (FIG. 3E). GSOA across the multi-omics data revealed strong convergence on pathways related to ciliary structure, microtubule organization, and cytoskeletal regulation (FIG. 3E). These pathways were predominantly enriched in the phosphoproteomic and transcriptomic datasets, with minimal changes observed at the level of protein abundance, suggesting that woodfire smoke alters ciliary and cytoskeletal function through post-translational modifications rather than changes in protein expression. A notable feature of woodfire smoke-exposed ALI cultures was a discordance in innate immune pathways: although transcripts associated with innate immune GO terms were induced, the corresponding protein programs were unchanged or reduced in abundance (FIG. 9J). This uncoupling suggests that woodfire smoke exposure suppresses effective airway innate immune defenses. Together, these data identified a translational block and phosphorylation-dependent regulation of ciliary and cytoskeletal proteins as the central features of airway epithelial response to woodfire smoke exposure. It was therefore speculated that these molecular alterations could lead to functional changes in the airway epithelium.

[0517] Example 5: Woodfire smoke activates ERK signaling while suppressing kinases that regulate cilia and cytoskeletal function

[0518] Kinase activity analysis was then performed using the phosphoproteomics dataset to evaluate how woodfire smoke altered kinase signaling activities. Briefly, log2 fold changes in phosphosite intensities between woodfire smoke exposure and room air, averaged across donors, were used to infer kinase activity states based on prior knowledge networks of known kinase-substrate interactions within the OmniPath database (FIG.3F, see Methods in Example 1). Eukaryotic initiation factor 2 alpha kinase 3 (EIF2AK3 or PERK) emerged as the most activated kinase following woodfire smoke exposure (FIG. 3F). PERK is a central regulatorAttorney Docket No.: UCH-43225

[0519] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0520] of the integrated stress response and translation initiation; its activation is known to lead to phosphorylation of EIF2a, which results in the global inhibition of cap-dependent translation. This result provides mechanistic insight into our observation of uncoupled transcriptomic and proteomic changes (FIG. 3B).

[0521] In addition, the activation of multiple components of the ERK signaling pathway was observed, including BRAF, RAS1, KSR1, RET, and MAP3K8, consistent with pathway-level enrichment of Ras-RAF-MEK-ERK signaling among upregulated kinases identified by PID-based gene set over-representation analysis (FIG. 9K). Activation of proteins including the NF-KB protein, MAP3K14 (NIK), as well as protein kinase C, casein kinases, PIK3C3, PLK1, and LIMK2 was also observed. Inactivated kinases included those known to regulate cilia function (e.g., NEKs, LMTKs, protein kinase A, and CAMK2B), cytoskeleton (e.g., PTKs, EPHB1, and ABL2), PI3K-AKT signaling, and immune signaling, among others (FIG.3F). In summary, the kinase activity analysis indicated activation of MAPKs and NF-KB alongside inhibition of kinases regulating cilia, cytoskeletal organization, and immune signaling.

[0522] Example 6: Wildfire smoke disrupts airway ciliary motility and epithelial barrier integrity To determine whether the molecular changes identified by multi-omics translated into functional airway impairment, ciliary motility and epithelial barrier integrity was assessed following cumulative woodfire smoke exposure. Differentiated human airway epithelial ALI cultures were exposed for 3 minutes per day, as described above (FIG. 2C), and 20 videos were taken in an unbiased grid pattern across each transwell. High-speed videography combined with unbiased MATLAB-based segmentation analysis revealed a progressive loss of coordinated ciliary motion with successive woodfire smoke exposures (n=3 biological replicates, n=3 technical repeats, with 20 videos per well for n=180 videos; FIG. 4A).

[0523] Strikingly, a single 3 -minute woodfire smoke exposure was sufficient to reduce ciliary motility compared with room air controls, indicating rapid ciliary dysfunction. Daily exposures led to a cumulative decline in ciliary motility, with near-complete paralysis observed by day 5 (FIG.

[0524] 4A), which was quantified in FIG. 4B. Room air control cultures exhibited a mean of 71.8% (range of 67.4-76.1%) ciliary movement across each high-power field, which declined to 58.9% (range of 52.2-65.7%) after one woodfire smoke exposure, 50.7% (range of 45.5-56.0%) after three exposures, and 26.1% (range of 21.6-30.6%) after five exposures (FIG. 4B).

[0525] Notably, the ALI cultures were returned to room air between woodfire smoke exposures, yet ciliary motility continued to deteriorate across the exposure days, indicating that repeated,Attorney Docket No.: UCH-43225

[0526] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0527] short-duration woodfire smoke exposures are sufficient to drive progressive ciliary dysfunction.

[0528] Next, based on enrichment of pathways exhibiting altered phosphorylation in the phosphoproteomics dataset, we examined epithelial barrier integrity following woodfire smoke exposure. IF staining for Tight Junction Protein 1 (TJP1) revealed marked disruption of tight junction organization in cultures exposed to woodfire smoke for five days compared to room air controls (FIG. 4C). Quantitative tight junction organization analysis (TIJOR), an imagebased method that quantifies the continuity and organization of tight junction proteins to assess epithelial barrier integrity, demonstrated a significant reduction in junctional organization following woodfire smoke exposure (FIG. 4D), consistent with altered cytoskeletal regulation. To assess the functional consequences of this TJP1 loss, we measured trans-epithelial electrical resistance (TEER), which quantifies electrical resistance across the cell layer, longitudinally throughout the exposure period. While TEER values were maintained after each woodfire smoke exposure on days 1 through 4, a significant loss of epithelial resistance was observed after 5 days of woodfire smoke exposure (FIG. 4E), indicating that the airway barrier function was likely compromised by cumulative woodfire smoke induced injury, possibly driven by changes in phosphorylation signaling.

[0529] Example 7: MEK inhibition preserves ciliary motility and epithelial barrier integrity during woodfire smoke exposure

[0530] Phosphoproteomics analysis identified enrichment of MAPK signaling following woodfire smoke exposure (FIG. 3F), prompting an investigation of whether pharmacologic inhibition of this pathway could mitigate the woodfire smoke induced airway dysfunction. The effects of MEK inhibition were therefore evaluated on the loss of ciliary motility after woodfire smoke exposure using two MEK inhibitors: pimasertib, and U0126.

[0531] To assess ciliary function, ALI cultures were exposed to daily 3 -minute woodfire smoke pulses for five consecutive days in the presence or absence of MEK inhibitors, followed by high-speed videography and quantitative ciliary motion analysis (FIG. 5A). Woodfire smoke exposure alone resulted in marked loss of ciliary motility, whereas co-treatment with pimasertib substantially preserved coordinated ciliary movement (FIGs. 5B-5C). Ciliary movement in room air control cultures was 60.3% (range of 55.9-64.7%), whereas five days of woodfire smoke exposure reduced motility to 9.1% (range of 4.6-13.6%) (FIG. 5C).

[0532] Preventative treatment with the MEK inhibitor pimasertib preserved ciliary movement during woodfire smoke exposure in a dose-dependent manner. At 10 nM and 50 nM, ciliary movementAttorney Docket No.: UCH-43225

[0533] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0534] remained low at 19.9% (range of 14.3-25.6) and 19.8% (range of 14.1-25.4), respectively. In contrast, higher doses improved motility, with 33.5% (range of 27.7-39.3) movement at 100 nM and 41.2 (range of 35.4-46.9) at 1 pM (FIG. 5C). Next, U0126, a structurally distinct MEK1 / 2 inhibitor, was tested using the same preventative strategy. Consistent with the effects observed with pimasertib, U0126 significantly preserved ciliary movement during woodfire smoke exposure (FIGs. 5D-5E). Woodfire smoke alone reduced ciliary movement to 9.1% (range of 4.6-13.6), while daily treatment with 10 pM U0126 maintained motility at 47.9% (range of 43.7-52.2). Intermediate protection was observed at 100 nM (26.0% [range of 20.7-31.3]), whereas lower doses of 10 nM and 50 nM did not significantly improve ciliary movement (15.0% [range of 9.7-20.3] and 13.8% [range of 8.4-19.2], respectively. To test whether activation of ERK signaling is sufficient to impair ciliary function, ALI cultures were treated with the ERK agonist BCI-215 in the absence of woodfire smoke exposure. BCI enhances ERK signaling by inhibiting ERK phosphatases, thereby preventing ERK dephosphorylation and prolonging its activation. Ciliary movement in room air controls was 60.3% (range of 55.9-64.7) and was reduced to 47.4% (range of 41.9-52.9) following BCI treatment (FIGs. 5F-5G). This reduction was less severe than the impairment observed following woodfire smoke exposure, which reduced ciliary movement to 9.1% (range of 4.6-13.6).

[0535] Given the cytoskeletal and junctional disruptions observed following woodfire smoke exposure, it was next examined whether MEK inhibition could also preserve epithelial barrier organization. IF staining for Tight Junction Protein 1 (TJP1) revealed disrupted junctional architecture following woodfire smoke exposure, which was significantly improved by pimasertib co-treatment (FIG.5H). Quantitative analysis demonstrated a significant difference between room air and woodfire smoke-exposed cultures, as well as between woodfire smoke and woodfire smoke plus pimasertib conditions, while no significant difference was observed between room air controls and woodfire smoke cultures treated with pimasertib (FIG. 51).

[0536] Together, these results demonstrate that MEK inhibition preserved ciliary motility and epithelial junctional integrity during woodfire smoke exposure.

[0537] Example 8: MEK inhibition restores cytoskeletal and ciliary phosphorylation and preserves ciliary ultrastructure after woodfire smoke exposure

[0538] To define the molecular mechanism by which MEK inhibition protects airway structure and function during woodfire smoke exposure, it was examined how pimasertib alters the phosphorylation landscape induced by woodfire smoke. Kinase activity inference comparingAttorney Docket No.: UCH-43225

[0539] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0540] woodfire smoke-exposed cultures with and without pimasertib revealed widespread reprogramming of signaling networks, including reversal of woodfire smoke-associated MAPK activity, and coordinated shifts across multiple kinase families (FIG. 6A). These changes indicate that MEK inhibition alters the broader phosphorylation landscape perturbed by woodfire smoke exposure rather than solely acting through MAPK suppression. To determine whether MEK inhibition actively reverses woodfire smoke-induced phosphorylation changes on structural proteins, phosphosite differences were compared in woodfire smoke-exposed ALI cultures with and without pimasertib. For cytoskeletal phosphosites, comparison of log2 fold changes for woodfire smoke plus pimasertib versus woodfire smoke with those for woodfire smoke versus room air revealed a strong negative correlation (R = -0.364, p < 0.001), indicating that MEK inhibition reverses woodfire smoke-induced phosphorylation changes (FIG. 6B). An analogous analysis restricted to cilia-associated proteins demonstrated the same inverse relationship (R=-0.370, p<0.001) (FIG. 6C). This supports the direct role for MEK inhibition in preserving phosphosites that are key for cytoskeletal organization and ciliary motility.

[0541] GSEA analysis was next performed on phosphorylated proteins that were significantly altered by woodfire smoke and subsequently shifted in the opposite direction following pimasertib treatment (FIGs. 6B-6C). These GO terms were enriched for pathways related to cytoskeletal organization, intermediate filament regulation, actin dynamics, tight junction assembly, and microtubule stability, as well as multiple cilia-specific processes including centriole assembly, axonemal docking, and motile ciliogenesis (FIG. 6D). These findings indicate that MEK inhibition counteracts woodfire smoke-induced phosphorylation remodeling that disrupts airway epithelial architecture and ciliary function.

[0542] Given the phosphorylation patterns on cilia proteins seen with MEK inhibition during woodfire smoke exposure, it was next examined whether this translated to preservation of ciliary ultrastructure. Motile cilia rely on conserved 9+2 axonemal organization, in which the central pair is essential for coordinated beating (FIG. 6E). Transmission electron microscopy of ALI cultures revealed intact 9+2 architecture under room air conditions (FIG. 6F), whereas woodfire smoke exposure resulted in marked loss of the central pair, consistent with severe ultrastructural disruption (FIG. 6G). In contrast, cultures exposed to woodfire smoke in the presence of either pimasertib or U0126 retained their ciliary central pair structures (FIG. 6H).

[0543] Quantification of ciliary ultrastructural outer doublets and central pairs confirmed these observations (FIG.61). The proportion of abnormal cilia was significantly increased followingAttorney Docket No.: UCH-43225

[0544] UCLA Ref. No.: [UCLA 2025-088-2] WO

[0545] woodfire smoke exposure compared with room air controls (p=0.001), whereas no significant difference was observed between room air controls and woodfire smoke cultures treated with pimasertib (p=0.181) or U0126 (p=0.999) (FIG. 61). Together, the present disclose demonstrates that MEK inhibition reverses woodfire smoke-induced phosphorylation of cytoskeletal and ciliary proteins and preserves the ultrastructural integrity required for motile ciliary function.

[0546] INCORPORATION BY REFERENCE

[0547] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0548] EQUIVALENTS

[0549] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

Attorney Docket No.: UCH-43225UCLA Ref No.: [UCLA 2025-088-2] WOWe claim:

1. A method of treating or preventing:(a) epithelial barrier dysfunction;(b) ciliary dysfunction(c) smoke- or aerosol-induced epithelial barrier dysfunction;(d) environmental exposure-induced epithelial barrier dysfunction;(e) epithelial inflammation;(f) pulmonary inflammation; or(g) smoke- or aerosol-induced inflammation; or(h) environmental exposure-induced inflammationin a subject in need thereof, comprising administering to the subject a modulator of protein phosphorylation.

2. The method of claim 1, wherein the modulator of protein phosphorylation is a kinase inhibitor (e.g., a serine kinase inhibitor, a tyrosine kinase inhibitor, or a threonine kinase inhibitor) or a phosphatase inhibitor (e.g., a serine / threonine phosphatase inhibitor, a tyrosine phosphatase inhibitor, or an alkaline phosphatase inhibitor).

3. The method of claim 1 or 2, wherein the modulator of protein phosphorylation isselected from:Attorney Docket No.: UCH-43225UCLA Ref. No.: [UCLA 2025-088-2] WOsalt thereof.

4. The method of claim 3, wherein the modulator of protein phosphorylation ispharmaceutically acceptable salt thereof.

5. The method of claim 3, wherein the modulator of protein phosphorylation ispharmaceutically acceptable salt thereof.

6. The method of claim 3, wherein the modulator of protein phosphorylation ispharmaceutically acceptable salt thereof.Attorney Docket No.: UCH-43225UCLA Ref. No.: [UCLA 2025-088-2] WO7. The method of claim 3, wherein the modulator of protein phosphorylation is9. The method of any one of claims 1-8, wherein the smoke- or aerosol-induced inflammation is wildfire smoke- or aerosol-induced inflammation, tobacco smoke- or aerosol-induced inflammation, cooking smoke- or aerosol-induced inflammation, industrial smoke- or aerosol-induced inflammation, or waste disposal smoke- or aerosol-induced inflammation.

10. The method of claim 9, wherein the smoke- or aerosol-induced inflammation is wildfire smoke- or aerosol-induced inflammation.

11. The method of claim 9 or 10, wherein the smoke- or aerosol-induced inflammation occurs in an airway of the subject.

12. The method of claim 11, wherein the smoke- or aerosol-induced inflammation is induced following a chronic exposure to a smoke or an aerosol for about 1-120 minutes per day over a period of about 1-10 days.Attorney Docket No.: UCH-43225UCLA Ref. No.: [UCLA 2025-088-2] WO13. The method of claim 11, wherein the smoke- or aerosol-induced inflammation is induced following an acute exposure to a smoke or an aerosol for about 1-120 minutes.

14. The method of claim 11, wherein the modulator of protein phosphorylation is administered prior to the exposure to the smoke or the aerosol.

15. The method of claim 14, wherein the modulator of protein phosphorylation is administered about 1-12 hours prior to the exposure to the smoke or the aerosol.

16. The method of claim 15, wherein the modulator of protein phosphorylation is administered about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours prior to the exposure to the smoke or the aerosol.

17. The method of claim 14, wherein the modulator of protein phosphorylation is administered daily for a period of about 1-5 days prior to the exposure to the smoke or the aerosol.

18. The method of claim 17, wherein the modulator of protein phosphorylation is administered daily for a period of about 1 day, about 2 days, or about 3 days prior to the exposure to the smoke or the aerosol.

19. The method of any one of claims 1-13, wherein the modulator of protein phosphorylation is administered following the exposure to the smoke or the aerosol.

20. The method of claim 19, wherein the modulator of protein phosphorylation is administered about 1-12 hours following the exposure to the smoke or the aerosol.

21. The method of claim 20, wherein the modulator of protein phosphorylation is administered about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours following the exposure to the smoke or the aerosol.Attorney Docket No.: UCH-43225UCLA Ref. No.: [UCLA 2025-088-2] WO22. The method of claim 19, wherein the modulator of protein phosphorylation is administered daily for a period of about 1-5 days following the exposure to the smoke or the aerosol.

23. The method of claim 22, wherein the modulator of protein phosphorylation is administered daily for a period of about 1 day, about 2 days, or about 3 days following the exposure to the smoke or the aerosol.

24. The method of any one of claims 1-23, wherein the modulator of protein phosphorylation is administered to an airway of the subject (e.g., via inhaler).

25. The method of any one of claims 1-24, wherein the modulator of protein phosphorylation is formulated for administration as an aerosol.

26. A method of improving mucociliary clearance in a subject in need thereof, comprising administering to the subject a modulator of protein phosphorylation.

27. The method of claim 26, wherein the modulator of protein phosphorylation is a kinase inhibitor e.g., a serine kinase inhibitor, a tyrosine kinase inhibitor, or a threonine kinase inhibitor) or a phosphatase inhibitor (e.g., a serine / threonine phosphatase inhibitor, a tyrosine phosphatase inhibitor, or an alkaline phosphatase inhibitor).

28. The method of claim 26 or 27, wherein the modulator of protein phosphorylation is selected from:Attorney Docket No.: UCH-43225UCLA Ref. No.: [UCLA 2025-088-2] WOsalt thereof.

29. The method of claim 28, wherein the modulator of protein phosphorylation ispharmaceutically acceptable salt thereof.

30. The method of claim 28, wherein the modulator of protein phosphorylation ispharmaceutically acceptable salt thereof.Attorney Docket No.: UCH-43225UCLA Ref. No.: [UCLA 2025-088-2] WO31. The method of claim 28, wherein the modulator of protein phosphorylation ispharmaceutically acceptable salt thereof.

32. The method of claim 28, wherein the modulator of protein phosphorylation is34. The method of any one of claims 28-33, wherein prior to the administration of the modulator of protein phosphorylation, the subject suffers from epithelial barrier dysfunction.

35. The method of any one of claims 28-34, wherein prior to the administration of the modulator of protein phosphorylation, the subject has suffered an exposure to a smoke or an aerosol.Attorney Docket No.: UCH-43225UCLA Ref. No.: [UCLA 2025-088-2] WO36. The method of claim 35, wherein the subject has suffered smoke- or aerosol-induced inflammation following the exposure to the smoke or the aerosol.

37. The method of claim 36, wherein the smoke- or aerosol-induced inflammation is wildfire smoke- or aerosol-induced inflammation, tobacco smoke- or aerosol-induced inflammation, cooking smoke- or aerosol-induced inflammation, industrial smoke- or aerosol-induced inflammation, or waste disposal smoke- or aerosol-induced inflammation.

38. The method of claim 37, wherein the smoke- or aerosol-induced inflammation is wildfire smoke- or aerosol-induced inflammation.

39. The method of claim 37 or 38, wherein the smoke- or aerosol -induced inflammation occurs in an airway of the subject.

40. The method of any one of claims 28-39, wherein the modulator of protein phosphorylation is administered to an airway of the subject (e.g., via inhaler).

41. The method of any one of claims 28-40, wherein the modulator of protein phosphorylation is formulated for administration as an aerosol.

42. A method of treating or preventing lung cancer in a subject in need thereof, comprising administering to the subject a compound selected from:-n -Attorney Docket No.: UCH-43225UCLA Ref. No.: [UCLA 2025-088-2] WOsalt thereof.

43. The method of claim 42, wherein the lung cancer is characterized by an upregulation of a gene, wherein the gene is selected from MAPK14, MAPKAPK2, MAPKAPK3, MAPK10, MAPK12, MAPK13, MAP2K3, and MAP2K6; or any combinations thereof44. The method of claim 42 or 43, wherein the lung cancer is characterized by a downregulation of a gene, wherein the gene is MAPK37.

45. A method of:(a) treating or preventing airway injury;(b) treating or preventing lung cancer; or(c) improving mucociliary clearancein a subject in need thereof, comprising administering to the subject a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof:Attorney Docket No.: UCH-43225UCLA Ref No.: [UCLA 2025-088-2] WOwherein:R\ is selected from Ci-io hydrocarbyl, hydroxy Ci-iohydrocarbyl, amino Ci-iohydrocarbyl, nitro Ci-io hydrocarbyl, and halo Ci-io hydrocarbyl;Ri, R2, R9, Rio, R11 R12, R13 and R14 are independently selected from: hydrogen, halogen, cyano, nitro, azido, — OR3, — NR4C(O)OR6, — OC(O)R3, — NR4S(O)jRe, — S(O)jNR3R4, — S(O)jNR4C(O)R3, — C(O)NR4S(O)jR6, — S(O)jR6, — NR4C(O)R3, — C(O)NR3R4, — NR5C(O)NR3R4, — NR5C(NCN)NR3R4, NR3R4, C1-C10 alkyl, C2- C10 alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl, C3-C 10 cycloalkylalkyl, — S(O)j(Ci- Ce alkyl), — S(O)j(CR4Rs)m-aryl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, — O(CR4Rs)m-aryl, — NR4(CR4Rs)m-aryl, — O(CR4Rs)m-heteroaryl, — NR4(CR4R s)m, heteroaryl, — O(CR4R5)m-heterocyclyl, — NR4(CR4R5)m-heterocyclyl and — S(Ci-C2 alkyl) substituted with 1 to 5 fluorines, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is independently substituted or unsubstituted;Rais selected from: hydrogen, trifluoromethyl, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkylalkyl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, and aryl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heteroaryl and heterocyclyl is independently substituted or unsubstituted; and wherein aryl is optionally substituted with 1 to 5 groups independently selected from: oxo, halogen, nitro, CF3, CHF2, CH2F, OCF3, OCHF2, OCH2F, azido, NR'SO2R"", SO2NR", C(O)R', C(O)OR', OC(O)R', NR'C(O)OR"", NR'C(O)R", C(O)NR'R", SR"", S(O)R"", SO2R', NR'R", NR'C(O)NR"R'", NR'C(NCN)NR"R'", OR', aryl, heteroaryl, arylalkyl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl; R4is selected from hydrogen or Ci-Ce alkyl, wherein alkyl may be substituted or unsubstituted; orAttorney Docket No.: UCH-43225UCLA Ref. No.: [UCLA 2025-088-2] WOR3 and R4 can be taken together with the atom to which they are attached to form a 4 to 10 membered heteroaryl or heterocyclic ring, each of which is substituted or unsubstituted;Rs is hydrogen or Ci-Ce alkyl, wherein alkyl may be substituted or unsubstituted; orR4 and Rs can be taken together with the atom to which they are attached to form a 4 to 10 membered carbocyclic, heteroaryl or heterocyclic ring, each of which is substituted or unsubstituted;Reis selected from: trifluoromethyl, C1-C10 alkyl, C3-C 10 cycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl, wherein each alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is independently substituted or unsubstituted;R', R" and R'" are independently selected from: hydrogen, C1-C4 alkyl, C2-C4 alkenyl, aryl and arylalkyl;R"" is selected from: C1-C4 alkyl, C1-C4 alkenyl, aryl and arylalkyl;W is — C(O)ORi5, — C(O)NR4Ri5, — C(O)NR4ORI5, — C(O)NR4S(O)jR6, — C(O)NR4NR4Ri5, — NR'C(O)R', — NR'S(O)jR', — NRC(O)NR'R", NR'S(O)jNR'R", or — C(O)NR4NR4C(O)Ri5;provided that W is not — C(O)OH;Risis selected from: hydrogen, trifluoromethyl, C1-C10 alkyl, C2-C 10 alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl, C3-C10 cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is independently substituted or unsubstituted;Ri6 is selected from hydrogen or C1-C10 alkyl; or R15 and Ri6 taken together with the atom to which they are attached form a 4 to 10 membered cyclic ring with 1 or 2 nitrogen atoms and optionally an oxygen atom, said ring being substituted or unsubstituted; XisN orN+CT;m is 0, 1, 2, 3, 4 or 5; andj is 1 or 2;with the proviso that 3-phenylamino-isonicotinic acid methyl ester, and 3-oxo-3-(3- phenylamino-pyridin-4-yl)-propionic acid ethyl ester are not included.Attorney Docket No.: UCH-43225UCLA Ref. No.: [UCLA 2025-088-2] WO46. The method of claim 45, wherein the compound ipharmaceutically acceptable salt thereof.

47. The method of claim 45, wherein the compoundpharmaceutically acceptable salt thereof.

48. The method of any one of claims 45-47, wherein the method comprises treating or preventing airway injury in a subject in need thereof.

49. The method of any one of claims 45-48, wherein the airway injury is selected from epithelial barrier dysfunction, ciliary dysfunction, smoke- or aerosol-induced epithelial barrier dysfunction, environmental exposure-induced epithelial barrier dysfunction, epithelial inflammation, pulmonary inflammation, smoke- or aerosol-induced inflammation, and environmental exposure-induced inflammation.

50. The method of claim 49, wherein the smoke- or aerosol-induced inflammation is wildfire smoke- or aerosol-induced inflammation, tobacco smoke- or aerosol-induced inflammation, cooking smoke- or aerosol-induced inflammation, industrial smoke- or aerosol-induced inflammation, or waste disposal smoke- or aerosol-induced inflammation.

51. The method of any one of claims 45-50, wherein the compound is administered prior to the airway injury.

52. The method of claim 51, wherein the compound is administered about 1-12 hours prior to the airway injury.Attorney Docket No.: UCH-43225UCLA Ref. No.: [UCLA 2025-088-2] WO53. The method of claim 52, wherein the compound is administered about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours prior to the airway injury.

54. The method of claim 51, wherein the compound is administered daily for a period of about 1-5 days prior to the airway injury.

55. The method of claim 54, wherein the compound is administered daily for a period of about 1 day, about 2 days, or about 3 days prior to the airway injury.

56. The method of any one of claims 45-50, wherein the compound is administered following the airway injury.

57. The method of claim 56, wherein the compound is administered about 1-12 hours following the airway injury.

58. The method of claim 57, wherein the compound is administered about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours following the airway injury.

59. The method of claim 56, wherein the compound is administered daily for a period of about 1-5 days following the airway injury.

60. The method of claim 59, wherein the compound is administered daily for a period of about 1 day, about 2 days, or about 3 days following the airway injury.

61. The method of any one of claims 45-47, wherein the method comprises treating or preventing lung cancer in a subject in need thereof.

62. The method of any one of claims 45-47, wherein the method comprises improving mucociliary clearance in a subject in need thereof.

63. The method of any one of claims 45-62, wherein the compound is administered to an airway of the subject (e.g., via inhaler).Attorney Docket No.: UCH-43225UCLA Ref. No.: [UCLA 2025-088-2] WO64. The method of any one of claims 45-63, wherein the compound is formulated for administration as an aerosol.

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