Identifying novel radioprotective drugs via salivary gland tissue chip screening

During head and neck cancer treatment, off-target ionizing radiation damage to the salivary glands commonly causes a permanent loss of secretory function. Due to the resulting decrease in saliva production, patients have trouble eating, speaking and are predisposed to oral infections and tooth decay. While the radioprotective antioxidant drug Amifostine is FDA approved to prevent radiation-induced hyposalivation, it has intolerable side effects that limit its use, motivating the discovery of alternative therapeutics. To address this issue, we previously developed a salivary gland mimetic (SGm) tissue chip platform. Here, we leverage this SGm tissue chip for high-content drug discovery. First, we developed in-chip assays to quantify glutathione and cellular senescence (β-galactosidase), which are biomarkers of radiation damage, and we validated radioprotection using WR-1065, the active form of Amifostine. Other reported radioprotective drugs including Edaravone, Tempol, N-acetylcysteine (NAC), Rapamycin, Ex-Rad, and Palifermin were also tested to validate the ability of the assays to detect cell damage and radioprotection. All of the drugs except NAC and Ex-Rad exhibited robust radioprotection. Next, a Selleck Chemicals library of 438 FDA-approved drugs was screened for radioprotection. We discovered 25 hits, with most of the drugs identified exhibiting mechanisms of action other than antioxidant activity. Hits were down-selected using EC50 values and pharmacokinetic and pharmacodynamic data from the PubChem database. This led us to test Phenylbutazone (anti-inflammatory), Enoxacin (antibiotic), and Doripenem (antibiotic) for in vivo radioprotection in mice using retroductal injections. Results confirm that Phenylbutazone and Enoxacin exhibited radioprotection equivalent to Amifostine. This body of work demonstrates the development and validation of assays using a SGm tissue chip platform for high-content drug screening and the successful in vitro discovery and in vivo validation of novel radioprotective drugs with non-antioxidant primary indications pointing to possible, yet unknown novel mechanisms of radioprotection.


Introduction
During head and neck cancer treatment, ionizing radiation damage to the salivary glands often causes a permanent loss of secretory function and reduced salivary ow.Due to decreased saliva production, patients have trouble eating, speaking, and swallowing 1,2 .Additionally, patients are at an increased risk of oral infections and tooth decay and suffer a reduced quality of life 2,3 .Current treatment options, including sialogogues, mouthwashes, and chewing gum, only provide temporary relief, and there is no cure 2 .Several strategies have been proposed to alleviate this damage, including cell transplantation [4][5][6][7] and gene therapy [8][9][10][11] .Despite promising results, these methods have remained experimental and are targeted toward patients already experiencing xerostomia.Hence, there is an unmet need to provide current and future head and neck cancer patients with preventative therapies to protect salivary gland function.
Intensity-modulated radiation therapy (IMRT) and the radioprotective drug Amifostine are used clinically to prevent salivary gland damage.IMRT involves using 3D imaging to target the radiation beams at the tumor and away from sensitive organs such as the salivary gland 12 .While this method can be bene cial in some cases, there are mixed results in patient-reported claims of dry mouth.Furthermore, IMRT is sometimes impossible to use due to tumor location 12,13 .The antioxidant Amifostine is the only FDAapproved drug to prevent radiation-induced xerostomia.However, its use is often discontinued during fractionated radiation regimens due to severe side effects, including nausea, vomiting, and hypotension 14, 15 .Additionally, its short half-life in vivo limits its e cacy, as the drug is cleared within minutes of administration 16 .These drawbacks highlight the critical need to discover new radioprotective drugs to prevent xerostomia.
To address this need, we developed a microbubble (MB) array-based tissue chip consisting of 3D salivary gland tissue mimetics (SGm) entrapped within a matrix metalloproteinase degradable poly(ethylene glycol) (PEG)-based hydrogel engineered extracellular matrix (eECM) that maintains secretory behavior 17 .
The spherical architecture of the MB combined with the cellularly degradable eECM creates a distinct niche that promotes cell viability and maintenance of the secretory acinar cells based on gene and protein expression as well as secretory function based on tissue architecture, gene expression, and secretory agonist-responsive calcium signaling 17 .In addition, this platform was validated for use in radioprotection studies using immunohistochemical staining of individual SGm to quantitate foci of DNA damage markers γH2AX and 53BP1 after irradiation.Analysis of control chips versus chips treated with WR-1065, the active form of Amifostine, showed a reduction in DNA damage with drug treatment 17 .
Array-based assays that can interrogate each SGm on the chip (~280 MBs/cm 2 ) are necessary to enable high-content drug screening.Based on literature, several assays commonly used to report radiationinduced cellular damage were tested in the tissue chip format.Assays tested included reactive oxygen species (ROS), apoptosis, secretion, and cytotoxicity at various time points post-radiation (Table S.1).
Based on signal-to-noise ratio and reproducibility, the glutathione 18 and cellular senescence 19 assays were selected for further development for high-content screening of radioprotective drugs.
Assays were tested with 0 Gy, 15 Gy, and 15 Gy + 4 mM WR-1065 to con rm usefulness for measuring radiation damage and radioprotection of individual SGm within the MB-hydrogel tissue chip (40-50 MBs per chip).The assays were further tested with other reported radioprotective drugs, including Tempol 20,21 , N-acetylcysteine 22 , Edaravone 23 , Rapamycin 24 , Ex-Rad 25,26 , and Palifermin 27,28 .Next, a library of 438 FDA-approved drugs was screened for radioprotection using both assays, identifying 25 double hits that were further investigated for suitability as potential therapeutics informed by data within the PubChem database and experimentally determined dose-response relationships.Three of the hits, Phenylbutazone (anti-in ammatory), Enoxacin (antibiotic), and Doripenem (antibiotic), were chosen for in vivo radioprotection testing of the mouse submandibular gland using retroductal injections.Results con rm that Phenylbutazone and Enoxacin exhibited equivalent radioprotection to Amifostine.

Materials
Detailed information on the drugs used for assay development and validation, including, Tempol, Nacetylcysteine, Edaravone, WR-1065, Rapamycin, Ex-Rad, and Palifermin is listed in Table S.2.Drug screening was completed using a 438 Selleck Chemicals library of FDA-approved drugs (Table S.3).The screen's 25 top drug hits (Table S.4) were purchased from Selleck Chemicals for dose-response studies.The drugs were prepared and stored per manufacturer's instructions.

Animals
Female SKH1 hairless mice, backcrossed 6 generations with C57BL/6J mice, aged 6-12 weeks were used in this study for in vitro assay development and drug discovery.Female C57BL/6J mice age 6-8 weeks were used for in vivo validation studies.Only female mice were used due to known sex differences in rodent salivary glands, with female glands more accurately emulating human salivary gland structure and function 29,30 .Animals were maintained on a 12 hr light/dark cycle and group-housed with food and water available ad libitum.All procedures were approved and conducted per the University Committee on Animal Resources at the University of Rochester Medical Center (UCAR #2010-024E, UCAR-2008-016E).

Microbubble (MB) array fabrication
Microbubble (MB) arrays were fabricated in poly(dimethyl) siloxane (PDMS) using gas expansion molding as previously described 17,31,32 .PDMS (Dow Corning Sylgard 184) was mixed in a 10:1 base-tocuring agent ratio and poured over a silicon wafer template consisting of deep etched cylindrical pits with a 200 µm diameter, spaced 600 µm apart on a square lattice.The PDMS was cured at 100 °C for 2 hrs before peeling off the template, resulting in an array of spherical cavities with 200 µm opening and ~350 µm diameter.Circular chips with 0.7 cm diameter (48 well plates) or 0.5 cm diameter (96 well plates) were punched from the PDMS cast and glued into well plates using a 5:1 ratio of PDMS cured at 60 °C for 8 hours.The MB arrays were primed in a desktop vacuum chamber with 70% ethanol to facilitate air removal from the MBs and replacement with uid.Ethanol was exchanged for PBS and arrays were incubated overnight before cell seeding.

Cell isolation
Mice were euthanized and the submandibular glands were removed and chopped with a razor blade for 5 min.The tissue was then incubated in Hank's buffered salt solution (HBSS) containing 15 mM HEPES, 50 U/mL collagenase type II (Thermo Fisher 17101015), and 100 U/mL hyaluronidase (Sigma Aldrich H3506) at 37 °C for 30 min.Cells were centrifuged, resuspended in HBSS with 15 mM HEPES, and passed through 100 µm and 20 μm mesh lters to isolate cell clusters between 20-100 µm.The digestion protocol produces cell cluster sizes evenly distributed between 20 to 100 µm 33 .As described below, the isolated clusters were combined with hydrogel precursor solution and seeded into the primed MB array chips.

Glutathione assay
A glutathione assay was developed for in-chip measurements by adapting the Cellular Glutathione Detection Assay Kit (Cell Signaling Technology #13859).The monochlorobimane reagent was prepared by reconstitution in DMSO per manufacturer's directions.For 96 well plates, 10 µL of prepared reagent (1:50 ratio of monochlorobimane (MCB) to Tris assay buffer, per manufacturer's instructions) was added to wells containing 100 µL of culture media and incubated for 30 min at 37 °C, washed with PBS, and imaged using an Olympus IX70 microscope with a DAPI lter (Excitation/Emission: 358 nm/461 nm).

Senescence assay
A cellular senescence assay was developed for in-chip measurements by adapting the Cellular Senescence Detection Kit -SPiDER-βGal (Dojindo Molecular Technologies, Inc SG04).Balifomycin A1 and SPiDER-βGal stock solutions were prepared in DMSO per manufacturer's directions.The assay was performed by rst incubating the chips with Balifomycin A1 (1:1000 dilution in media) for 1 hr at 37 °C.
The solution was removed, replaced with 30 µL of media containing Balifomycin A1 (1:1000 dilution) and SPiDER-βGal (1:500 dilution), and incubated for 45 min at 37 °C.Chips were washed twice with media and imaged using a uorescence microscope with a Texas Red lter (Excitation/Emission: 580 nm/604 nm).

Drug mechanism meta-analysis
Drug interaction data was examined using PubChem BioAssays results for the hits discovered in the Selleck Chemicals drug library screen described below [36][37][38] .Pathway analysis was performed using the Drug Set Enrichment Analysis (DSEA) tool to identify pathways with gene expression patterns signi cantly impacted by treatment with the drug hits (p<0.05) 39.

Image quanti cation and statistical analysis
For the glutathione and senescence assays, images were quanti ed in ImageJ.Regions of interest (ROIs) were created by thresholding on the uorescence signal (localized to the SGm), and each ROI's mean intensity was measured.Data were graphed and statistical analyses (ANOVA with Tukey's post-hoc test) were performed using GraphPad Prism 9.

Drug treatment and irradiation
For radioprotection experiments, SGm were cultured in MB-hydrogel chips for 4 days, then drugs were added to the chips 30 min before radiation and washed out with media 30 min post-radiation to parallel how Amifostine is used clinically 40 .A dose of 15 Gy ionizing radiation was delivered using a JL Shepherd 137 Cs irradiator.Drug treatment schemes and radiation doses were established in our previous work 17 .
The glutathione and senescence assays were performed at 4-and 5-days post-radiation.For assay validation experiments, at least 3 chips (N = 3) were used for each drug, corresponding to > 100 MBs (n > 100); these values are listed in the gure captions for each experiment.Mean, standard deviation, and statistics were calculated based on the number of MBs (n).
The same treatment scheme was used for screening the Selleck Chemicals library, with drugs administered at 100 µM.One MB chip (N = 1) was used per drug, with ~40-50 MBs per chip (n = 40-50); statistics were calculated using the number of MBs (n) and compared to 0 Gy controls.Drugs were rst screened using the glutathione assay.Drugs that exhibited statistically insigni cant differences compared to the 0 Gy control (hits) with the glutathione assay were then tested with the senescence assay to discover double hits.The glutathione assay was selected as the rst screen because it had higher signal-to-noise ratio, more rapid throughput (30 mins versus 2 hrs for completion of the senescent assay), lower assay kit cost, and a higher shelf-life of reagents in the kit after reconstitution.
To discriminate the effectiveness of drug candidates, for both the glutathione and senescence assays, we de ned a signal range bounded by the values of the assay result for 0 Gy unirradiated and 15 Gy irradiated controls.Full radioprotection would yield a signal for 15 Gy+drug that was statistically equivalent the 0 Gy control, or a recovery of 100%.

Retroductal injection and irradiation
Retroductal injection delivery of drugs to the murine submandibular gland has been described in detail 41,42 .Brie y, 6-10 week old female C57Bl/6J mice were anesthetized by intraperitoneal injection of sterile saline solution of 100 mg/kg ketamine and 10 mg/kg xylazine.Maxillary incisors were secured over a metal beam, while an elastic band provided tension from behind the mandibular incisors.The mouth was widened using a custom steel retractor to apply pressure to the buccal mucosa and the tongue was retracted and cotton placed in the oral cavity.The wire inset of a 32G intracranial catheter was cut at 45°t o create a bevel.The beveled wire created a shallow puncture in the left salivary papilla.A beveled catheter section containing the wire insert for support was gently inserted into the puncture site.The catheter was removed, and 1 mg/kg atropine was administered by intraperitoneal injection.After 10 min, the needle of a Hamilton syringe, loaded with vehicle or drug solution, was inserted into the catheter, and the catheter was inserted into the ori ce produced in the papilla.Drug solutions were injected by hand at 10 µl/min at a volume of 1 µl/g of body weight.Following injection, the pressure was maintained on the syringe for 1 min to ensure material retention before removal of the catheter.The cotton 43 and retractor were removed from the oral cavity, and the elastic band and metal beam were released from the incisors.The known radioprotectant, WR-1065 (50 mg/kg, saline) was used as a control for radioprotection.Phenylbutazone, Enoxacin, and Doripenem hydrate were dosed at 50, 0.3, and 26 mg/kg (N = 4-6 per group).Drug doses were chosen based on solubility and published in vivo studies 44,45 .Saline was used as the vehicle for all compounds, except Phenylbutazone, which required corn oil due to poor aqueous solubility.
Mice injected with vehicle controls (saline or corn oil) or drug were treated with 0 Gy or 15 Gy within 15-30 min of injection, the submandibular glands were irradiated as described previously [46][47][48] .The head and neck region was positioned over the slit of a custom collimator, which allowed body shielding.Mouse submandibular glands were exposed to a single dose of 15 Gy gamma radiation delivered by a 137 Cs radiation source.This single dose in mice recapitulates human sequalae of salivary gland radiation damage 17,49 .Animals were allowed to recover for 48 hours to measure persistent long-lived DNA damage, after which, the submandibular glands were harvested, xed, sectioned, and analyzed using immunohistochemistry.

Immunohistochemical analysis
Submandibular glands and SGm were isolated and xed in 4% paraformaldehyde overnight at 4 °C.Tissues were para n-embedded and then cut into 5 μm sections.Slides were treated with HIER buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) for antigen retrieval in a pressure cooker for 10 minutes then sections were blocked in CAS-block histochemical reagent (Thermo Fisher Scienti c, 008120).Permeabilization was performed with 0.5% Triton X-100 in PBS for 5 minutes.Immunostaining was performed overnight (at 4 °C) with primary antibody for γH2AX (EMD Millipore, 05-636).Alexa-Fluor 594conjugated donkey anti-mouse IgG was diluted 1:500 (Invitrogen, A21203) as secondary antibody and applied on sections for 1 hour at room temperature.Following a PBS rinse, 10 μg/ml DAPI (Invitrogen, Carlsbad, CA) in PBS was applied to sections for 5 minutes.Sections were washed thrice in PBS for 5 minutes and the slides were mounted using Immu-Mount mounting medium (Thermo).Microscopic images were acquired using a Leica TCS SP5 confocal microscope with a 100X oil immersion objective and Argon laser.Analysis of images was performed in ImageJ.

Results and Discussion
Salivary Gland Tissue Chip Our previously reported salivary gland tissue chip 17 was leveraged for high-content drug screening to identify novel radioprotective compounds.The chip platform consists seeding primary salivary gland cell clusters 20-100 µm (Fig. 1A), suspended in a poly(ethylene glycol) (PEG) hydrogel precursor and MMPdegradable peptide crosslinker solution together with the photoinitiator LAP (Fig. 1B), into an array of near-spherical microbubble (MB) cavities (Fig. 1C) formed in poly(dimethylsiloxane) (PDMS).Each chip, containing 40-50 MBs, is a xed within wells of a 96-well plate.In situ polymerization of the hydrogel was achieved using long-wave, low intensity UV light.Over time, the cell clusters aggregate and proliferate to form SGm (Fig. 1D).

High-throughput methods to assess drug radioprotection
In prior studies 17 , immunohistochemical (IHC) staining was used to quantify the number of γH2AX and 53BP1 puncta within nuclei, which are sensitive markers for double-stranded DNA breaks 50 and direct measures of radiation damage.IHC staining is, however, a laborious time and resource-consuming process requiring retrieval of the SGm from the MB array chip, tissue sectioning, staining, and imaging.
While IHC enables sensitive analyses of radiation damage, it completely abrogates the goal of in situ high-content screening for which the tissue chip was developed.As mentioned above, several assays commonly used to assess radiation-induced cellular damage were tested in the tissue chip format at various time points post-radiation (Table S.1).Based on signal-to-noise ratio and reproducibility, the glutathione 18 and senescence 19 assays were selected for further development.The glutathione assay was used to measure glutathione levels at various time points post-radiation to determine the optimal time for detecting differences between 0 Gy and 15 Gy.Based on the data, the greatest signal separation was measured at 4 days post-irradiation (Fig. S1).This time point is similar to previous reports on decreases in glutathione post-radiation 18 and was used for all experiments moving forward.
Next, WR-1065 was tested to identify effective concentration(s) that prevented changes in glutathione levels post-radiation.A SGm tissue chip was cultured for 4 days to allow spheres to form 17 .The chip was then treated with WR-1065 30 min before and during radiation, followed by drug wash out with media 30 min post-radiation (Fig. 2A).This dosing scheme is consistent with the use of Amifostine clinically 14 and with our previous work 17 .The glutathione assay was performed 4 days post-radiation (Fig. 2A).Example images show high levels of glutathione at 0 Gy (Fig. 2B) that is decreased by 15 Gy radiation (Fig. 2C) and maintained with 4 mM WR-1065 (Fig. 2D).Quanti cation shows that 0.1 mM and 0.4 mM WR-1065 were ineffective at preventing radiation damage.In contrast, 1 mM and 4 mM WR-1065 provided signi cant protection (Fig. 2E).The 4 mM dose corresponds with our previous work on DNA damage markers γH2AX and 53BP1 17 and values from literature 51 and establishes the range of effective concentrations for WR-1065 treatment in vitro.Moreover, it is clinically relevant as 15-30 minutes before radiation, patients are administered Amifostine intravenously at 200 mg/m 2 15, 40 .Assuming an average adult body surface area of 17,000 cm 2 and a blood volume of 1.35 gallons (5.1 liters) 52,53 , Amifostine is administered at 300 µM.
Since WR-1065 is an antioxidant and mediates radioprotective effects through free radical scavenging and induction of superoxide dismutase expression 54 , we tested other antioxidants implicated as radioprotective (Tempol, Edaravone, N-acetylcysteine).Tempol exhibited excellent radioprotection at both 1 and 4 mM (Fig. 2F) which is consistent with prior studies 20,49,55 .Edaravone showed complete radioprotection at 1 mM but not at 4 mM (Fig. 2G).Edaravone maintained ~43% of glutathione levels at 0.1 mM (Fig. S2A), suggesting that the optimal concentration range for Edaravone might be lower than WR-1065, which is consistent with literature indicating radio-protective dose ranges are 0.1-1 mM 23,56,57 .
For N-acetylcysteine (NAC), no improvement in glutathione levels was observed at 1 or 4 mM compared to untreated SGm (Fig. 2H); however,glutathione levels were rescued by 56% when treated with 10 mM NAC (Fig. S2B), also consistent with literature 22 .
Drugs reported with non-antioxidant radioprotective mechanisms were also tested using the glutathione assay.Rapamycin is an mTOR inhibitor reported to restore salivary ow rate post-irradiation in swine 24 .
For senescence, a protocol similar to the glutathione assay was followed to determine the optimal time point post-radiation for detecting a change in senescence between 0 Gy and 15 Gy, as measured by senescence-associated β-galactosidase activity.A time point of 5 days post-radiation was optimal (Fig. S3), similar to a previous study 19 .WR-1065 radioprotection was tested by adding drug to the chips 30 min before radiation followed by wash out 30 min post-radiation (Fig. 3A).An expected increase in senescence was detected for SGm exposed to 15 Gy compared to 0 Gy (Figs. 3B,C) , which was restored to levels equivalent to 0 Gy with the addition of WR-1065 (Fig. 3D).Quanti cation shows that both 1 mM and 4 mM WR-1065 treatment resulted in complete radioprotection (Fig. 3E), consistent with the glutathione assay results.
A summary of results from the glutathione and senescence assays shows similar trends for radioprotection (Table S.5).The few differences may result from different mechanisms of action and/or assay targets.Logically, the glutathione assay may be more sensitive to antioxidant function (NAC, Tempol, Edaravone), while the senescence assay may be more appropriate for drugs such as rapamycin, which has anti-senescence properties 58 .These results highlight the trade-offs in developing screening assays and point to the bene t of screening with two assays.Although the assays developed are indirect measures of radiation-induced DNA damage, they nonetheless were validated to detect radiation induced cell-damage and drug radioprotection.More importantly, these assays can be used for in situ highcontent drug screening with multiple replicates (40-50) per test and enhanced throughput compared to IHC staining for γH2AX.

Drug library screening identi ed several promising radioprotective compounds
The glutathione and senescence assays were used to screen a library of FDA-approved drugs (Selleck Chemicals) at 100 µM.Drugs were rst screened using the glutathione assay according to the timeline in Fig. 2A.Any compound resulting in statistically equivalent glutathione levels compared to the 0 Gy control was considered a hit (Fig. 4, orange circles).Hits with the glutathione assay were then tested uisng the senescence assay and considered a "double hit" if senescence levels were statistically equivalent to levels at 0 Gy (Fig. 4, blue circles).A list of the 438 drugs screened and relevant statistics are shown in Table S.3.Overall, 438 drugs from the library were tested with a hit rate of 5.7%, for a total of 25 double hits (Fig. 4B) listed in Table 1.While this hit rate is higher than many other drug screening reports (0.1 -0.3%) 59,60 , this may be due to the high statistical rigor afforded by the tissue chip format.Additionally, phenotypic screens generally have higher hit rates than target-based screens and they maybe a more successful strategy for discovery of rst-in-class medicines (> 1%) [61][62][63] .

Identi cation of potential radioprotective drug mechanisms
Of the 25 potential radioprotective compounds, 20 have known interactions with proteins involved in calcium signaling identi ed within the BioAssay database in PubChem (Fig. 5A).These compounds may impact secretory signaling in the salivary gland, which can be radioprotective 64,65 .While degranulation may not be key to radioprotection, secretory stimulation may play a role in proliferation and survival of the secretory cells 64 .Similarly, using the Drug Set Enrichment Analysis (DSEA) tool to identify pathways, the Reactome analysis related to secretion appear to be upregulated by many of the identi ed drugs, supporting this potential mechanism (Fig. 5B,C, and Fig. S5).Interestingly, only 9 of the 25 compounds have known antioxidant properties, and 12 are anti-in ammatory.This is critical data indicating that alternate mechanisms of radioprotection may be achievable and represented in the identi ed hits.A reduction in pathway activity related to cell adhesion, cell-cell and cell-matrix interactions also represents a potential area of exploration.Multiple studies have identi ed changes in integrin expression, cell adhesion and matrix interactions upon radiation exposure, which may be linked to cell response 1,[66][67][68][69] .Manipulation of these mechanisms in the salivary gland may convey radioprotection.
Hit down-selection using drug promiscuity data and EC 50 values A systematic approach was used to down selection the 25 double hits for in vivo testing.Since drugs within the library are FDA-approved, considerable information on their pharmacology in mice and humans is readily available through resources such as PubChem.Within PubChem, the BioAssay database was created by the National Institute of Health (NIH) as an open repository containing results of small molecule screening data 38 .We used the BioAssay data to analyze drug promiscuity, which refers to the ability of a drug to bind multiple molecular targets with distinct pharmacological outcomes, often causing unwanted side effects 108 .The drugs exhibiting bioactivity in a large number of assays were deprioritized.Data for each double hit was obtained from the database and promiscuity was calculated as the percent of assays reported as "active" (Table 1).Drugs with high activity (>10%) were excluded from further testing.Additionally, Etidronate, Melatonin, and Albendazole were excluded due to poor bioavailability [109][110][111] , and Eplerenone was excluded due to solubility concerns 112 .
The radioprotection trends based on the senescence assay (Fig. 7) differed somewhat, with Phenylbutazone (Fig. 7B) and Meropenem (Fig. 7C) showing only partial protection and Diethylstilbestrol (Fig. 7D) exhibiting radioprotection equivalent to 0 Gy between 50-100 µM concentrations whereas Prazosin (Fig. 7E) showed complete protection between 0.1-100 µM.Glipizide (Fig. 7G) and Doripenem (Fig. 7H) also showed variable protection.EC 50 values extrapolated from dose response curves are shown in Table 2. Phenylbutazone showed the most promising results, with low EC 50 values for both the glutathione (0.08 µM) and senescence (0.05 µM) assays.Phenylbutazone is a non-steroidal anti-in ammatory drug (NSAID) that inhibits cyclooxygenases (COX-1 and COX-2), enzymes that produce prostaglandins 113 .Prostaglandins, speci cally PGE 2 signaling, have been shown to increase in irradiated salivary glands, and mitigation of salivary gland damage was achieved through treatment with the anti-in ammatory drug Indomethacin 1,114 .Indomethacin also showed radioprotection in our drug screen but was ineffective at concentrations lower than 100 µM.Indomethacin only blocks COX-1, underscoring the greater e cacy of Phenylbutazone.Phenylbutazone was originally developed for chronic pain for conditions such as arthritis but has since been restricted to treating ankylosing spondylitis due to induction of rare but severe blood disorders, including anemia and leukopenia 113 .However, doses ranged from 300-1000 mg, generating a plasma concentration of 30-50 µg/mL 113 .In contrast, the EC 50 of 0.08 µM for protecting against radiation-induced glutathione changes established in this study equates to a 26 ng/mL concentration.Thus, the risk of severe adverse effects may be greatly diminished for doses necessary for radioprotection.Additionally, Phenylbutazone has excellent bioavailability (up to 90%) 113,115 and long half-life (50-105 hrs) 113 , which may enable dose de-escalation, further decreasing risks.
Enoxacin is an antibacterial agent used for treating urinary tract infections 116 that was previously identi ed as radioprotective 117 .Using a high-throughput screening method with the viability of lymphocytes as the primary readout, two classes of antibiotics (tetracyclines and uoroquinolones) were identi ed as robust radioprotectors, including Enoxacin 117 .This observation corroborates our drug screening results, in which several antibiotics were identi ed as double hits, including Enoxacin, Meropenem, Doripenem hydrate, Rifampin, and Rifabutin.The Enoxacin EC 50 of 2.4 µM for the glutathione assay is similar to the 13 µM EC 50 reported for viability of lymphocyte cells 117 .Notably, ve other uoroquinolones reported as radioprotectors (Levo oxacin, Gati oxacin, O oxacin, Moxi oxacin, and Nor oxacin) 117 were not hits in our drug screen (Table S3).These disparities may be related to differences in cell type (salivary gland versus lymphocyte) or readouts (glutathione/senescence versus viability).
Based upon drug down selection data and measured EC 50 values, Phenylbutazone, Enoxacin, and Doripenem hydrate were selected for in vivo validation in mice with γH2AX foci per nucleus IHC staining as an outcome measure consistent with prior work showing correlation with the development of xerostomia 17,41,42,46 .Vehicle-treated SGm exposed to 15 Gy radiation exhibited a 3.3-fold increase in the number of γH2AX foci per nucleus, indicating a signi cant increase in double-stranded DNA breaks due to radiation exposure (Figs.8A-D, I).Treatment with WR-1065 resulted in a 0.5-fold reduction in γH2AX foci per nucleus compared to 15 Gy controls (Figs.8D, E, I).No signi cant differences existed between 0 Gy controls and WR-1065 treated SGm exposed to 15 Gy (Figs. 8C, E, I).These results are similar to prior studies utilizing WR-1065 in vitro and in vivo via retrograde ductal injection 17,42 .Treatment with the test compounds, Phenylbutazone, and Enoxacin resulted in 0.4-and 0.5-fold reduction in γH2AX foci per nucleus compared to 15 Gy controls, respectively (Figs. 8E-G, I).Results observed after Phenylbutazone and Enoxacin treatment were not signi cantly different from 0 Gy controls (Figs.8C, F, G, I).Treatment with Doripenem hydrate did not reduce γH2AX foci per nucleus relative to 15 Gy controls and showed a 2.7-fold increase compared to 0 Gy controls (Figs.8C, D, H, I).

Conclusions
To overcome long-standing challenges associated with off-target radiation damage resulting in life-long dry mouth and poor quality of life, we sought to leverage our salivary gland tissue chip technology 17 to identify novel radioprotective drugs.First, we investigated several assays that detect cell damage following radiation exposure (Table S1).We identi ed reduced glutathione and β-galactosidase (cell     The drug screen identi ed 25 compounds that protect against post-radiation changes in glutathione and senescence.A) Each circle represents the normalized uorescence intensity for the glutathione assay for each compound.White circles are compounds that were not hits.Orange circles are compounds that were hits with the glutathione assay only.Blue circles are compounds that were hits with both assays.The green dotted line represents 0 Gy, used for normalization of the data, the red line represents 15 Gy untreated controls using the glutathione assay (A).Table representing the total and percent-positive compounds using the initial glutathione assay and combined glutathione and senescence assays (B).

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Figure 3 See
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Table 2 :
Calculated EC 50 values for the top radioprotective drugs.Values were estimated using a nonlinear t in Prism."Undetermined" indicates that the software could not accurately t a curve to the data.