Development of a Novel Multi-Isoform ALDH Inhibitor Effective As an Anti-Melanoma Agent

Author Manuscript Published OnlineFirst on November 21, 2019; DOI: 10.1158/1535-7163.MCT-19-0360 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Development of a novel multi-isoform ALDH inhibitor effective as an anti-melanoma agent

Saketh S. Dinavahi1,8,9, Raghavendra Gowda1,7,8,9, Krishne Gowda1, Christopher G. Bazewicz3,8,9, Venkat R. Chirasani1, Madhu Babu Battu10, Arthur Berg5, Nikolay V. Dokholyan1,6, Shantu Amin1, and Gavin P. Robertson1,2,3,4,7,8,9.

Departments of 1Pharmacology, 2Pathology, 3Dermatology, 4Surgery, 5Public Health Sciences, 6Biochemistry and Molecular Biology, 7Foreman Foundation for Melanoma Research, 8The Melanoma Center, 9The Melanoma Therapeutics Program, The Pennsylvania State University College of Medicine, Hershey, PA 17033; 10Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnostics, Uppal, Hyderabad, India 500039.

Running title: ALDH inhibition for melanoma.

Corresponding author: Gavin P. Robertson, Department of Pharmacology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033. Phone: (717) 531-8098; Fax: (717) 531-0480; E-mail: [email protected]

Conflicts of interest: The authors declare no potential conflicts of interest.

Downloaded from mct.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 21, 2019; DOI: 10.1158/1535-7163.MCT-19-0360 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

ABSTRACT

The aldehyde dehydrogenases (ALDHs) are a major family of detoxifying enzymes that contribute to cancer progression and therapy resistance. ALDH overexpression is associated with a poor prognosis in many cancer types. The use of multi-ALDH isoform or isoform-specific ALDH inhibitors as anti- cancer agents is currently hindered by the lack of viable candidates. Most multi-ALDH isoform inhibitors lack bioavailability, are non-specific or toxic while most isoform-specific inhibitors are not effective as monotherapy due to the overlapping functions of ALDH family members. The present study details the development of a novel, potent, multi-isoform ALDH inhibitor, called KS100. The rationale for drug development was that inhibition of multiple ALDH isoforms might be more efficacious for cancer compared to isoform-specific inhibition. Enzymatic IC50s of KS100 were 207, 1,410 and 240 nM towards ALDH1A1, 2 and 3A1, respectively. Toxicity of KS100 was mitigated by development of a nanoliposomal formulation, called NanoKS100. NanoKS100 had a loading efficiency of ~69% and was stable long-term. NanoKS100 was 5-fold more selective for killing melanoma cells compared to normal human fibroblasts. NanoKS100 administered intravenously at a submaximal dose (3-fold lower) was effective at inhibiting xenografted melanoma tumor growth by ~65% without organ related toxicity. Mechanistically, inhibition by KS100 significantly reduced total cellular ALDH activity to increase reactive oxygen species generation, lipid peroxidation and accumulation of toxic aldehydes leading to apoptosis and autophagy. Collectively, these data suggest the successful preclinical development of a non-toxic, bioavailable, nanoliposomal formulation containing a novel multi-ALDH isoform inhibitor effective in the treatment of cancer.

INTRODUCTION.

Malignant melanoma is an aggressive neoplasm accounting for the majority of skin cancer-related deaths (1). The outlook for metastatic disease remains poor, with current five-year survival rates of 20% (1). However, treatment strategies for malignant melanoma have vastly improved with the discovery of targeted therapies to BRAF and MEK along with the development of immune checkpoint inhibitors (2). Although current treatment strategies may kill the bulk of tumor cells, they often leave behind therapy-resistant cancer cells with a stem- cell-like phenotype, which serve as a reservoir for disease recurrence and metastasis (3).

Cancer cells with stem cell characteristics comprise a small subset of undifferentiated cells that initiate tumor formation and generate multipotent progenitors (3). They promote tumor aggressiveness, repopulation after injury and metastasis, having intrinsic resistance to radiation, chemotherapy and targeted therapies (4). A major mechanism by which these cells develop resistance is through upregulation of the aldehyde dehydrogenases (ALDHs), which has impaired the response to preoperative chemotherapy and radiation in esophageal carcinoma (5), conventional chemotherapy, erlotinib and gefitinib in lung carcinoma (6), olaparib in breast carcinoma (7) and cyclophosphamide in a myriad of carcinomas (8,9).

The 19 human ALDH isozymes are broadly defined as a superfamily of NAD(P)+-dependent enzymes that participate in aldehyde metabolism, catalyzing the oxidation of toxic aldehydes into carboxylic acids (10-13). ALDH activity within cells is generally a composite of the activities of multiple ALDH isoforms, which have overlapping substrate specificity (14,15). The ALDHs confer a survival advantage to metabolically active cancer cells, by oxidizing aldehydes that accumulate and cause oxidative damage, into less toxic, more soluble carboxylic acids (16,17). Accordingly, ALDH overexpression is linked to poorer survival in gastric, breast, lung, pancreatic and prostate carcinomas, as well as in head and neck squamous cell carcinomas (HNSCCs) (8,11,14,18,19). The ALDH1A1, 1A2, 1A3, 3A1 and 3A2 isozymes are particularly important in cancer progression and resistance to anti-cancer therapies (8,15,20,21).

Current ALDH inhibitors can be categorized into multi-ALDH isoform inhibitors and isoform-specific inhibitors, which primarily inhibit one isoform (11). Limitations of multi-ALDH isoform inhibitors, such as N,N- diethylaminobenzaldehyde (DEAB), which targets ALDH1A1, 1A2, 1A3, 1B1, 2 and 5A1, 4-dimethylamino-4- methyl–pent-2-ynthioic acid-S-methylester (DIMATE), which targets ALDH1A1 and 3A1, and citral, which targets ALDH1A1, 1A3 and 2, are lack of bioavailability or toxicity (11). DIMATE has tumor inhibitory efficacy when injected intraperitoneally (i.p.) but will require further preclinical evaluation (22). More recently, the ALDH inhibitors (aldis) -1, -2, -3, -4, and -6 have been developed, which target ALDH1A1, 2 and 3A1, and show efficacy in killing cultured cancer cells, particularly as combinatorial therapy (23-25). However, these compounds have mainly been tested in vitro and thus require further validation in preclinical models (23-25).

Isoform-specific inhibitors, such as Cpd 3 and CM037 (targeting ALDH1A1), CVT10216 (targeting ALDH2) and CB7 and CB29 (targeting ALDH3A1), have limited efficacy in killing cultured cancer cells, particularly when used as monotherapy, and have not been tested in animal cancer models (18,26,27). NCT-501, which targets ALDH1A1, has been shown to be effective in inhibiting HNSCC growth in animals via intra-tumoral injection, suggesting poor systemic bioavailability (28). Other more bioavailable ALDH1A1 specific inhibitors have been developed, such as the orally bioavailable compounds NCT-505 and NCT-506, and the i.p. available compounds 13g and 13h, but have not yet been evaluated in animals (29,30). Therefore, ALDH inhibitors are needed that inhibit the multiple functionally overlapping ALDH isoforms, with an acceptable pharmacological profile.

The current study describes the design and development of a novel, potent, multi-isoform ALDH inhibitor, called KS100. KS100 was developed since ALDH1A1, 2 and 3A1 overexpression was observed in a cell line melanoma progression model and targeting these individual ALDH isoforms did not affect cultured cell growth. KS100 potently inhibited multiple ALDH isoforms with negligible toxicity when administered in a nanoliposomal form, called NanoKS100. NanoKS100 was bioavailable and inhibited melanoma tumor growth by ~65% at submaximal (3-fold lower) doses. Most importantly, KS100 significantly reduced total cellular ALDH activity compared to several ALDH inhibitors leading to enhanced reactive oxygen species (ROS) generation, lipid peroxidation and accumulation of toxic aldehydes causing increased apoptosis and autophagy.

MATERIALS AND METHODS.

Cell lines, culture conditions, and chemicals: Normal human fibroblasts (FF2441) were provided by Dr. Craig Myers, Penn State College of Medicine, Hershey, PA. The human melanoma cell lines WM35, WM115, WM278, WM3211, 1205 Lu, and A375M and normal melanocytes (NHEM) were provided by Dr. Herlyn, Wistar Institute, Philadelphia, PA. The human melanoma cell line UACC 903 was provided by Dr. Mark Nelson, University of Arizona, Tucson, AZ. The wildtype BRAF melanoma cell line C8161.Cl9 was provided by Dr. Danny Welch, University of Kansas, Kansas City, KS and MelJuSo was provided by Dr. Judith

Johnson, Institute for Immunology, Germany. Cell lines were maintained in a 37°C humidified 5% CO2 atmosphere incubator and periodically monitored for phenotypic and genotypic characteristics and tumorigenic potential to validate and confirm cell line identity. Cell lines were authenticated and tested for mycoplasma contaminations periodically and are used within 15 passages after authentication. The ALDH1A1 and 3A1 specific inhibitors, Cpd 3 (31,32) and CB7 (18,33) respectively, were synthesized in-house according to previously published procedures. The ALDH1A1 specific inhibitor, CM037 (34), and ALDH2 specific inhibitor, CVT10216 (34), were purchased from Tocris Biosciences. Isatin and the multi-ALDH isoform inhibitor DEAB were purchased from Sigma Aldrich. ALDH structure preparation: The structures of ALDH1A1, 2 and 3A1 bound to the inhibitors CM037, psoralen and CB7, respectively (4X4L, 5L13 and 4L2O), were retrieved from the protein data bank (PDB). The 3D structures of the protein complexes were prepared using the protein preparation wizard tool (Schrodinger, LLC, 2017); water molecules were deleted except those in the inhibitor binding pocket, bond orders were assigned, hydrogen atoms were added, and metal ions were treated as described previously (35-37). Next, the orientation of the side chain structures of Gln and Asn was flipped, if necessary, to provide the maximum degree of H-bond interactions. The charge state of His residues was optimized. Finally, a restrained minimization of the protein structure was performed using the OPLS force field with backbone atoms being fixed. The minimized protein was used for the docking analysis. The structure was validated using Gaia webserver (http://chiron.dokhlab.org). ALDH grid generation and ligand preparation: Prepared protein structures were used to generate scoring grids for subsequent docking calculations as described previously (35-37). To each protein crystal structure, a grid box of default size (20×20×20A⁰) was centered on the corresponding active site position. Default parameters were used, and no constraints were included during grid generation. The ligand preparation was then performed using the ligprep module in Schrodinger (35-37). Molecular docking modeling of ALDH with inhibitors:

The starting conformations of ligands were minimized using the OPLS 2005 force field until the energy difference between subsequent structures was 0.001 kJ/mol-A⁰. The docking study was performed using GLIDE 6.6 in Maestro 10.1 (35-37). The GLIDE (Grid Ligand Docking with Energetics) algorithm estimates a systematic search of positions, orientations and conformations of the ligand in the enzyme-binding pocket via a series of hierarchical filters. The shape and properties of the receptor are symbolized on a grid by various dissimilar sets of fields that furnish precise scoring of the ligand pose. The potential hit compounds with good fitness score were considered for docking analysis. All the hits were subjected to the extra precision (XP) mode of GLIDE. Default values were accepted for Van der Waals scaling and input partial charges were used. During the docking process, the GLIDE score was used to select the best conformation for each ligand (35-37). Modeling to assess specificity of ALDH inhibitor binding: All bound crystal water molecules and ligands were stripped out of the crystal structures of ALDH1A1, 2 and 3A1 prior to docking. Simultaneously, the structure of KS100 was built and optimized in Marvin sketch workspace. Since ALDH1A1, 2, and 3A1 are deposited in oligomeric states in the PDB database, monomeric conformations of respective structures were extracted, and missing atoms or residues were relocated through homology modeling. The structures were optimized using DMD software suite and subsequently, molecular docking using Medusadock suite (http://medusadock.dokhlab.org/) was employed, which is known for its rapid sampling efficiency and high prediction accuracy (38). Initially, molecular docking of KS100 to the active site of ALDH1A1 alone was attempted as the conformations of ALDH1A1, 2, and 3A1 are structurally identical (Fig. S1). From the ALDH1A1–KS100 docked complex, it was evident that the KS100 binding pocket in ALDH1A1 was lined by the residues: Ser-121, Phe-171, Val-174, Met-175, Trp-178, Glu-269, Phe- 290, His-293, Gly-294, Tyr-297, Cys-302, Cys-303, Ile-304, Tyr-457, Gly-458, Val-460, and Phe-466. To identify the off-target effects of KS100, the binding scaffold of KS100 as a substructure was extracted and employed in Erebus (http://erebus.dokhlab.org), a web-server that searches the entire PDB database for a given substructural scaffold (39). Erebus identifies off-target structures from the PDB database by matching substructures with the same amino acids and atoms segregated by identical distances (within a given tolerance) as the atoms of the query structure (39). Finally, the prediction accuracy of a match was evaluated by the root-mean-square deviation (RMSD) or by the normal weight with a given variance. Synthesis of KS100: KS100 was synthesized as shown in scheme 1 of Figure 2B. Briefly, 5,7- dibromoisatin (1) (10 mmol) was dissolved in anhydrous DMF (30 mL) and cooled on ice with stirring.

Solid K2CO3 (11 mmol) was added, and the dark-colored suspension was brought to room temperature and stirred for 1 hour. 1,4-bis(bromomethyl)benzene (40 mmol) was added slowly with constant stirring

until the starting material had been consumed (monitored by TLC). The reaction mixture was poured into cold water and extracted with ethyl acetate. The ethyl acetate layer was washed with water, brine and dried over MgSO4. The solvent was removed, and the crude product was purified by silica gel column chromatography using 80:20 hexanes/ethyl acetate as the eluent to yield the intermediate 5,7- dibromo-1-(4-bromomethylbenzyl)-1H-indole-2,3-dione (2) as orange-red crystals. To the intermediate compound (1.02 mmol), thiourea (1.02 mmol) and ethanol (25 ml) were added and heated to reflux until the starting material had been consumed (monitored by TLC). The solvent was removed under vacuum. The final product (2-[4-(5,7-dibromo-2,3-dioxo-2,3-dihydro-indol-1- ylmethyl)benzyl]isothiourea) (3) was recrystallized in ethanol-ethyl acetate to afford KS100 (yield 70%). The identity of KS100 was confirmed by nuclear magnetic resonance as well as mass spectra analysis, and purity (>99%) was quantified by high-performance liquid chromatography analysis. Preparation of NanoKS100: KS100 was encapsulated into a nanoliposome by first combining L-α-Phosphatidylcholine and 1,2- Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000] ammonium salt in chloroform at 80:20 mol % for a final lipid concentration of 25 mg/mL (Avanti Polar Lipids) (40,41). 5 mg of KS100 (in methanol) was then added to the lipid mixture, dried under nitrogen gas and re-suspended in saline at 60°C. The mixture was then sonicated at 60°C for 30 minutes followed by extrusion through a 100-nm polycarbonate membrane using Avanti Mini-Extruder (Avanti Polar Lipids Inc-Alabaster, AL). The particle size, charge and liposomal characteristics were determined as described in the supplementary methods (40,41). Characterization of nanoparticle based KS100 called, NanoKS100: (a) Drug encapsulation. Efficiency of encapsulation of KS100 in the nanoliposomal formulation was estimated by UV-visible spectrophotometry (SPECTRAmax M2 plate reader; Molecular devices) (40,41). Specifically, 1 mL of NanoKS100 solution was added to a 10 kDa Centricon filter tube (Millipore) and centrifuged at 3,750 rpm for 30 minutes to remove free KS100. Next, 0.5 mL of purified NanoKS100 was combined with 0.5 mL of a 1:1 solution of chloroform to methanol to destroy the nanoliposomal structure and release the drug into the solution. The precipitated lipids were separated via centrifugation at 10,000 rpm for 15 minutes. The supernatant was then used to measure KS100 concentration, calculated from a standard curve of KS100 from 0.01 to 1 mg/mL. A 1:1 solution of chloroform to methanol was used as the reference blank. The percentage of KS100 incorporated into nanoliposomes was calculated as (incorporated KS100/total KS100) X 100 (40,41). (b) Stability. Stability of NanoKS100 stored at 4oC was assessed weekly by comparing size and zeta potential using the Malvern Zetasizer and measuring IC50 efficacy for killing UACC 903 melanoma cells by MTS assay and comparing these values to that of freshly manufactured NanoKS100 (40,41).

(c) In vitro drug-release kinetics. Drug release kinetics of KS100 from the liposomes were measured using 1 mL of purified NanoKS100 by dialysis in 1 L of 10 mM reduced glutathione at room temperature through a molecular weight cut off 25 kDa membrane (Spectra Por). 0.05 mL NanoKS100 in the dialysis bag was removed at 0.5, 1, 2, 4, 8, 12, 24, 36, 48 and 72 hours and the amount of KS100 released at each time point was estimated using UV-visible spectrophotometry as detailed previously (40,41). (d) Hemolytic activity. The hemolytic activity assay was performed as described previously (41,42). Briefly, fresh mouse and rat blood were drawn and placed into an EDTA test tube. Erythrocytes were separated from plasma by centrifugation at 1,500 rpm for 10 minutes at 4˚C using PBS. Erythrocyte pellets were diluted with 50 mL PBS in centrifuge tubes to give a 5% v/v solution and then treated with 5 µM KS100 in DMSO, NanoKS100 (10–40 µM) in PBS, empty liposome or 1% Triton X-100 (positive control). Samples were incubated at 37oC for 60 minutes and then centrifuged at 12,000 rpm for 10 minutes. Next, supernatants were transferred to a 96-well plate and absorption measured at 540 nm. The amount of hemoglobin released in the presence of 1% Triton X-100 was set as 100% lysis, and % hemolysis was calculated as: (absorbance of the samples at 540 nm/absorbance of the positive control) X 100. siRNA transfections: Duplex stealth siRNA sequences for scrambled and ALDH1A1, 1A2, 1A3, 1L1, 2, 3A1, 5A1, 18A1 and BRAF were obtained from Invitrogen. Individual siRNAs were introduced into UACC 903 cells via nucleofection using an Amaxa nucleofector with solution R / program K-17. Nucleofection efficiency was >90% with 80– 90% cell viability. siRNA knockdown was confirmed either by qRT-PCR or by western blots. qRT-PCR analysis: Total RNA was extracted by Trizol (Sigma) and cDNA was generated by reverse transcription kit (Applied Biosystems). qRT-PCR was performed using a SYBR green kit (Qiagen). Expression of each isoform in melanoma cell lines were normalized to corresponding expression in fibroblasts. Primers for the ALDH isoforms used as described previously (15). ALDH isoform-specific enzyme assays: ALDH1A1, 2 and 3A1 enzyme assays were performed as described by the manufacturer (R & D systems). Isoform-specific aldehydes were converted to their respective carboxylic acids along with the conversion of NAD+ to NADH (absorbance at 340 nm). Specifically, 1 µg/mL of ALDH1A1 was treated with various concentrations of ALDH inhibitors for 15 minutes followed by addition of substrate mixture (10 mM propionaldehyde; 100 mM KCl; 1 mM NAD; 2 mM DTT; 50 mM Tris pH 8.5) and the absorbance of NADH was measured in kinetic mode for 5 minutes. Similarly, 0.5 µg/mL of ALDH2 was used with 2 mM of acetaldehyde as the substrate, and 0.2 µg/mL of ALDH3A1 was used with 1 mM of 4-nitrobenzaldehyde as the substrate following the addition of ALDH inhibitors.

Total cellular ALDH activity assay: Briefly, total cellular ALDH activity assays were performed on cell lysates. 20 µg of cell lysate was treated with 1 µM of ALDH inhibitors or DMSO for 15 minutes followed by addition of substrate mixture (2 mM acetaldehyde; 100 mM KCl; 1 mM NAD; 2 mM DTT; 50 mM Tris pH 8.5) and the absorbance of NADH was measured in kinetic mode for 5 minutes. Cell viability assays: Cell viability assays of UACC 903 cells transfected with siRNA, and melanoma cells (UACC 903, 1205 Lu, C8161.CI9, MelJuSo), normal human fibroblasts (FF2441) and melanocytes (NHEM) treated with ALDH inhibitors were performed as described previously (43-45). Briefly, 5,000 cells per well were plated in a 96- well plate and incubated overnight at 37˚C in a 5% CO2 atmosphere. For the siRNA knockdown experiment, cells were incubated for another 72 hours. For the ALDH inhibitor experiments, cells were treated with agents at various concentrations and incubated for 72 hours. 20 µL of MTS reagent was then added into each well and formation of tetrazolium was measured by absorbance after 1 hour at 492 nm. IC50 values or % cells for each experimental group were measured in three independent experiments. Toxicity and maximum tolerated dose animal studies: All the animal experiments were conducted according to the guidelines of Penn State, Hershey IACUC. To determine the effective dose for in vivo efficacy studies, KS100 and NanoKS100 were injected i.p. and i.v., respectively, into Swiss Webster mice (Jackson labs) once daily for 7 days (40,41). Animals were monitored for changes in body weight, behavior and physical distress compared to control (DMSO for KS100, empty liposomes for NanoKS100). Dose escalation was performed to identify the maximum tolerated dose. AldeRed ALDH detection assay: The AldeRed ALDH detection assay (Millipore) was used to distinguish ALDH+ cells from the ALDH- cells. Briefly, cells were incubated with 5 µM of ALDH inhibitor or DMSO for 24 hours. Cells were washed with PBS and stained with AldeRed reagent (AldeRed 588-A) for 1 hour as per the manufacturer’s instructions. Cells were acquired by BD Fortessa flow cytometer and gated for ALDH+ cells using DEAB as a negative control. ROS assay: To quantify ROS levels, the non-fluorescent dye DCFDA (Sigma) was used (43). DCFDA turns to highly fluorescent 2′,7′-dichlorofluorescein upon oxidation by ROS generated in cells (43). Briefly, cells were treated with 5 µM of ALDH inhibitor or DMSO for 24 hours. 10 µM of DCFDA was then added and incubated for 30 minutes before measuring fluorescence at 485 nm excitation and 520 nm emission. Lipid peroxidation: Lipid peroxidation was measured using the thiobarbituric acid reactive substances kit according to the manufacturer’s instructions (Cayman Chemicals). Briefly, cells were treated with 5 µM of ALDH inhibitor or DMSO for 24 hours. Cell pellets were lysed in PBS by sonication on ice. Lipids in the lysates were

hydrolyzed in the presence of acetic acid and sodium hydroxide. Free malondialdehyde (MDA) was measured by the reaction to thiobartituric acid colorimetrically at 530 nm. Apoptosis assay: The Annexin-V-PE/7-AAD kit (Millipore) was used to distinguish live cells from apoptotic cells as described previously (43). Briefly, cells were incubated with 5 µM of ALDH inhibitor or DMSO for 24 hours. Cells were washed with PBS and stained with Annexin-V-PE and 7-AAD solution per the manufacturer’s instructions. Cells were acquired by BD Fortessa flow cytometer and gated for four distinct regions, namely, live cells (Annexin V-7-AAD-), early apoptotic (Annexin V-7+AAD-), late apoptotic (Annexin V-7+AAD+) and necrotic (Annexin V-7-AAD+) cells. Western blot analysis: Cells were harvested by the addition of RIPA lysis buffer and samples were processed as previously 6 described (46,47). Briefly, 1-2 X 10 cells were incubated overnight at 37˚C in a 5% CO2 atmosphere. For experiments with KS100, the agent was added and protein lysates collected following 24 hours of treatment. Blots were probed with antibodies according to each supplier’s recommendations: antibodies to cleaved PARP and LC3B from Cell Signaling Technology; alpha-enolase, ALDH1A1, 2, 3A1, 18A1, BRAF, and secondary antibodies conjugated with horseradish peroxidase from Santa Cruz Biotechnology. Immunoblots were developed using the enhanced chemiluminescence detection system (Thermo Fisher Scientific). Alpha- enolase served as the loading control. Tumor efficacy and toxicity assessment: Efficacy and toxicity studies were performed in nude mice as described previously (40,46,48,49). Briefly, 1 million cells were injected in both flanks of 4-6 week old female nude Balb/c mice (Envigo). After a week, when the tumors were vascularized, animals were either treated with NanoKS100 (at various doses) or empty liposomes. Tumor volumes, animal weight and behavior were monitored continuously every other day. Animals were sacrificed after tumor volumes in the empty liposome groups exceeded 2,500 mm3 and tumors were subsequently collected. Assessment of serum biomarkers of major organ toxicity: At the end of the UACC 903 xenograft study, serum samples were analyzed for levels of ALT (alanine aminotransferase), ALKP (alkaline phosphatase), ALB (albumin), GLOB (globulin), TP (total protein), TBIL (total bilirubin), BUN (blood urea nitrogen), GLU (glucose), CREA (creatinine), AMYL (amylase) and CAL (calcium) (40,46,48,49). Serum analysis was performed at the Centralized Biological Laboratory, Penn State, University Park. Empty liposomes served as the control. Statistics:

Statistical analysis was undertaken using the one-way/two-way ANOVA GraphPad PRISM Version 7.04 software. Dunnett’s as post hoc analysis was performed when there was a significant difference. Results were considered significant at a p-value of <0.05.

RESULTS ALDH overexpression occurs in melanoma and is associated with disease progression. Cancer cell expression of ALDHs often increases with disease progression, as oxidative stress secondary to high metabolic demands leads to ROS generation, lipid peroxidation and the accumulation of toxic aldehydes, which can inhibit cancer cells (17,50). Western blot analysis of ALDH1A1, 2 and 3A1 in melanoma cells revealed that ALDH overexpression occurs in melanoma compared to control fibroblast (FF2441) and melanocyte (NHEM) cells (Fig. 1A). Further, the degree of ALDH expression correlated with melanoma stage such that metastatic melanomas exhibited the highest ALDH expression levels, followed by vertical growth phase and finally radial growth phase melanomas. ALDH expression was not dependent on BRAF mutational status, as ALDH levels were similar between mutant V600EBRAF and wildtype BRAF cells. qRT-PCR analysis of the ALDH isoforms demonstrated that the isoforms overexpressed in UACC 903 and 1205 Lu melanoma cells compared to control fibroblasts are ALDH1A1, 1A2, 1A3, 1L1, 2, 3A1, 5A1 and 18A1 (Supplementary Table 1). Expectedly, when cells were stained with Aldered dye to isolate the ALDH+ (cells with high levels of ALDH) cells and ALDH- cells using flow sorting, the ALDH+ cells had a high expression of these isoforms compared to ALDH- cells (Supplementary Table 1). Subsequent analysis of the TCGA database to determine the relationship of ALDH overexpression on melanoma patient survival yielded variable results. Specifically, overexpression of ALDH1A1 and 2 was associated with slightly improved survival (Fig. 1B) while high ALDH3A1 expression was associated with lower survival (Fig. 1C). However, these data are measurements of RNA expression and thus do not take into account overall protein or ALDH activity in cancer cells. To functionally determine whether targeting ALDH1A1, 2 or 3A1 in melanoma effects cell proliferation, a rapid siRNA screen was undertaken (Fig. 1D). siRNA for ALDH18A1, a unique ALDH isoform that promotes melanoma cell survival through proline synthesis (51), and V600EBRAF were used as positive controls. Knockdown of each respective protein by its siRNA is shown in Figure 1E. Similar to the scrambled siRNA, individual siRNA knockdown of ALDH1A1, 2 and 3A1 did not affect UACC 903 cell survival up to 72 hours compared to the positive control siRNAs, which caused a ~50% reduction in cell survival (Fig. 1D). Pharmacological inhibition of ALDH1A1, 2 and 3A1 by isoform-specific inhibitors also had no effect on cell survival, even when 100 µM concentrations were used for 72 hours (Fig. 1F). In contrast, DEAB, a multi- ALDH isoform inhibitor, reduced UACC 903 cell survival by 30% at a 100 µM concentration after 72 hours. These results suggested that targeting multiple ALDH isoforms with overlapping function may be more effective for melanoma therapy specifically and anti-cancer therapy in general. Additionally, that inhibiting individual ALDH isoforms may lead to resistance mediated through upregulation of functionally similar ALDHs.

Identification and development of the novel, potent, multi-isoform ALDH inhibitor, called KS100. To create a multi-ALDH isoform inhibitor, an in silico screen was initially undertaken based on the x-ray crystal structure of ALDH1A1 using various natural products. Isatin was identified during this screen as weakly binding to ALDH1A1 compared to the ALDH1A1 specific inhibitors Cpd 3 and CM037 (Fig. 2A). A medicinal chemistry approach was subsequently undertaken to design compounds that would bind and interact more effectively in the ligand-binding pocket of the ALDHs, using the backbones of Isatin and Cpd 3. A series of compounds were tested through in silico modeling to determine whether they had optimal docking in the ligand-binding pocket of ALDH1A1, and KS100 was selected as the best candidate (Fig. 2A). It was also found to fit well into the ligand-binding pockets of ALDH2 and 3A1. KS100 had docking scores of -10.247, - 8.716 and -13.851 for ALDH1A1, 2 and 3A1, respectively (Table 1), compared to -11.276, -11.809 and - 14.576 for CM037 bound to ALDH1A1, CVT10216 bound to ALDH2 and CB7 bound to ALDH3A1, respectively. Docking scores indicated strong binding of KS100 to ALDH1A1, 2 and 3A1. KS100 had a π-π interaction with the W178 residue and an H-bond with the free amine group within the ALDH1A1 ligand-binding pocket (Fig. 2A). Similarly, KS100 had π-π interactions with the F459 and F465 residues along with an H-bond interaction between the free amine group and L269 residue within the ALDH2 ligand-binding pocket. Further, KS100 had a π-π interaction with the R292 residue and an H-bond interaction with the G187 residue in ALDH3A1 ligand- binding pocket (Fig. 2A). Due to strong ALDH1A1, 2 and 3A1 binding, KS100 was then synthesized through scheme 1 shown in Figure 2B. Inhibition of ALDH1A1, 2 and 3A1 by KS100 was compared to Isatin, the ALDH1A1 specific inhibitors Cpd 3 and CM037, the ALDH2 specific inhibitor CVT10216, the ALDH3A1 specific inhibitor CB7, and the multi-ALDH isoform inhibitor, DEAB (Table 1). Isatin was a relatively ineffective inhibitor of all ALDH isoforms, having

IC50s of 15.6 µM for ALDH1A1, >160 µM for ALDH2 and 5 µM for ALDH3A1. KS100 was an effective inhibitor of ALDH1A1 activity, having an IC50 of 207 nM compared to 44 nM and 98 nM for Cpd 3 and CM037, respectively. KS100 was an effective inhibitor of ALDH2 activity, having an IC50 of 1,410 nM compared to 53 nM for CVT10216. KS100 effectively inhibited ALDH3A1 activity, having an IC50 of 240 nM compared to 298 nM for CB7. DEAB was slightly superior to KS100 in inhibiting ALDH1A1 and ALDH2 activity, having IC50s of 89 nM and 833 nM, respectively, for these isoforms. However, DEAB was inferior to KS100 in inhibiting

ALDH3A1, having an IC50 of 15.1 µM. To evaluate the specificity of KS100 to inhibit various ALDH isoforms, the isoforms overexpressed in melanoma cells (Supplementary Table 1) were knockdown in UACC 903 cells using siRNA and the effect of KS100 on cell survival was evaluated. The potency of KS100 was reduced when there was lower levels of ALDH1A1, 1A2, 1A3, 2 and 3A1 while the potency did not significantly vary when ALDH1L1, 5A1 and 18A1 were knocked down (Fig. S1). However, due to the overlapping roles of ALDH1A1, 1A2 and 1A3, the precise

effect of KS100 to inhibit 1A2 and 1A3 needs to be characterized by enzymatic studies. Knockdown of RNA levels was verified by qRT-PCR (Fig. S1B). Collectively, these results suggest the successful development of a novel, potent, multi-ALDH inhibitor. Specificity and toxicity of KS100 To identify potential off-target effects and ALDH binding specificity of KS100, the binding scaffold of KS100 as a substructure was extracted and employed in Erebus (Fig. S2). To precisely determine the most similar binding scaffolds to our query structure, we imposed a cut-off RMSD of ≤ 7Å to the query during the substructural search against the PDB database. The subsequent hits are listed in Supplementary Table 2. The identification of ALDH1A1 as the primary hit highlights the accuracy of the Erebus algorithm. The RMSD of ~2.24Å between the query and the primary hit is likely due to the flexible docking approach used during initial docking of KS100 to ALDH1A1. NiFe-hydrogenase from Desulfovibrio fructosivorans was also identified as having a similar substructural scaffold. Similar studies were conducted with the active site pockets of ALDH2 and 3A1. These isoforms were identified to be the primary hits, with no observed off-target effects of KS100. Thus, KS100 appears to have no off-target effects in humans based on the Erebus algorithm, indicating the specificity of KS100 binding to human ALDHs. To evaluate the toxicity of KS100, Swiss Webster mice (n=3) were treated with daily i.p. administration of KS100 at 5, 10 and 15 mg/kg and compared to DMSO (Fig. 2C). A 16.6% decrease in animal body weight, on average, along with hunched backs and lethargy were observed at day 7 in the 5 mg/kg group. All animals treated with 10 and 15 mg/kg KS100 died before day 7, indicating significant toxicity. Thus, the toxicity associated with multi-ALDH isoform inhibition by KS100 necessitated the development of a formulation with controlled release of the drug to eliminate these effects. Developing a nontoxic, effective, stable nanoliposomal formulation of KS100, called NanoKS100. Nanoliposomal formulations can overcome drug toxicity (40-42,52). Therefore, KS100 was loaded into a nanoliposomal formulation, called NanoKS100, and the physiochemical properties of NanoKS100 were analyzed. A schematic representation of NanoKS100 is shown in Figure 3A where KS100 is trapped in the phospholipid bilayer of the nanoliposome. The maximum loading efficiency of KS100 into nanoliposomes was 68.6% (Fig. 3B). The size of NanoKS100 was 78.5 nm, with an average charge of +0.54 eV in saline at the day of manufacture (Figs. 3F-G). Release kinetics of NanoKS100 revealed continuous release of the agent over 48 hours with maximal release of 70% occurring by 48 hours (Fig. 3C). The efficacy and specificity of NanoKS100 for killing cultured melanoma cells were examined by MTS assay and compared to KS100. The IC50 killing efficacy of NanoKS100 on FF2441 and NHEM cells was 11.5 μM compared to 2.3 μM across all melanoma cells, irrespective of BRAF mutational status, amounting to a killing selectivity index of ~5-fold higher for melanoma cells, similar to that of KS100 (Fig. 3D). Thus, KS100 maintained its melanoma cell killing efficacy and selectivity in the NanoKS100 formulation. The cell killing

IC50s (Fig. 3E), size (Fig. 3F) and charge (Fig. 3G) of NanoKS100 did not vary significantly over a 12-month period when stored at 4˚C, indicating stability of the formulation. Since intravenous (i.v.) dosing of nanoliposomes can trigger injection site hemolysis (40-42), the effect of NanoKS100 on red blood cell (RBC) lysis was examined. RBCs from mice and rats were incubated with KS100 or NanoKS100 for 1 hour, and the amount of hemolysis was quantified. KS100 caused 27% and 19% hemolysis of mouse and rat RBCs, respectively, compared to 100% hemolysis with the Triton X-100 positive control (Fig. 3H). However, NanoKS100 lysed <5% of RBCs in both groups indicating a protective effect of the nanoliposomal formulation. Toxicity of NanoKS100 was further examined in Swiss Webster mice (n=3) treated with i.v. NanoKS100 at 5- 60 mg/kg for 7 days and compared to empty liposomes. Results revealed negligible weight loss on average (0.6 to 2.5%), with no mortality or abnormal behavioral changes seen in any treatment group (Fig. 4A). The maximum dose that could be administered to animals was 60 mg/kg as the nanoliposomes were not stable above this loaded concentration. Thus, a maximum tolerated dose of NanoKS100 could not be attained, as doses above 60 mg/kg could not be tested. NanoKS100 inhibits melanoma tumor development with no apparent toxicity. Having identified the safe dose range of NanoKS100, 3 submaximal doses (10, 20 and 30 mg/kg) were selected for tumor inhibitory studies. UACC 903 melanoma cells were injected into the flanks of nude mice (n=6) and once vascularized tumors had formed, mice were treated with daily i.v. NanoKS100 for 20 days. All 3 treatment groups showed significant inhibition of melanoma xenograft growth compared to empty liposomes (Fig. 4B). No significant differences in toxicity and tumor volumes between groups were observed. Based on these findings, treatment with daily i.v. 20 mg/kg NanoKS100 was selected for further xenograft experiments (n=8). A >65% reduction in tumor volumes was observed for NanoKS100 in both UACC 903 (Fig. 4C) and 1205 Lu (Fig. 4D) xenografts at days 20-22 with no significant reduction in animal weights compared to the empty liposomes (insets of Figs. 4C-D). The blood of the mice with UACC 903 xenografted tumors was collected at day 20 and no significant differences in serum biomarkers between NanoKS100 and empty liposomes were observed (Fig. 4E). Collectively, these data suggest that daily i.v. administration of a submaximal dose of NanoKS100 (3-fold lower) is safe and effective in this mouse melanoma model. KS100 inhibits total cellular ALDH activity to increase ROS, lipid peroxidation, toxic aldehyde accumulation, apoptosis and autophagy. The ALDHs reduce ROS, lipid peroxidation and toxic aldehyde accumulation, which can lead to cell damage and apoptosis as shown in Figure 5A (16,17). To evaluate the effects of KS100 on total cellular ALDH activity, UACC 903 (Fig. 5B) and 1205 Lu (Fig. 5C) cells were treated with 5 µM of ALDH inhibitor or DMSO for 24 hours, stained with Aldered reagent, and ALDH+ cells (cells with high levels of ALDH) were identified using flow cytometry. KS100 was the only ALDH inhibitor that significantly reduced ALDH+ cells in UACC 903

(52% reduction) and 1205 Lu (57% reduction) cells (representative dot plots in Figs. S3 and S4). Additionally, UACC 903 (Fig. S5A) and 1205 Lu (Fig. S5B) cell lysates were treated with 1 µM of ALDH inhibitor or DMSO for 15 minutes followed by the addition of aldehyde substrate mixture. KS100 was the most effective at reducing total cellular ALDH activity in both UACC 903 (75% reduction) and 1205 Lu (73% reduction) cells. The remaining ALDH inhibitors significantly reduced total cellular ALDH activity compared to controls, particularly CM037 and DEAB, while isatin was ineffective. Levels of ROS were measured in UACC 903 (Fig. 5D) and 1205 Lu (Fig. 5E) cells and compared to FF2441 cells (Fig. 5F) following treatment with 5 μM of ALDH inhibitor or DMSO for 24 hours. No ALDH inhibitor had an effect on ROS levels in FF2441 cells (Fig. 5F). KS100 was the most effective at increasing ROS levels in both cell lines (Figs. 5D-E). DEAB and CM037 were the only other agents that significantly increased ROS levels in either cell line. Subsequently, levels of lipid peroxidation and toxic aldehyde accumulation were measured in UACC 903 (Fig. 5G) and 1205 Lu (Fig. 5H) cells following treatment with 5 μM of ALDH inhibitor or DMSO for 24 hours. Consistent with the ROS assay, KS100 was the most effective at increasing lipid peroxidation and toxic aldehyde accumulation in both cell lines (Figs. 5G-H). DEAB and CM037 were the only other inhibitors that significantly increased lipid peroxidation and toxic aldehyde accumulation in either cell line. Flow cytometric analysis showed that 5 μM KS100 significantly increased Annexin-V positive UACC 903 and 1205 Lu cells compared to 5 μM of the other ALDH inhibitors after 24 hours (representative dot plots are shown in Figs. S6 and S7). Specifically, KS100 increased the early apoptotic cell fraction (Annexin-V+7-AAD- ) from 9.5% to 22.4% in UACC 903 cells (Fig. 5I) and from 12.5% to 60.4% in 1205 Lu cells (Fig. 5J). Western blot analysis of cultured UACC 903 cells following treatment with increasing concentrations (2-6 μM) of KS100 for 24 hours (Fig. 5K) showed increased apoptosis and autophagy, exemplified by elevated levels of cleaved PARP and LC3B, respectively. Collectively, these data demonstrate that KS100 significantly reduces total cellular ALDH activity to increase ROS generation, lipid peroxidation and accumulation of toxic aldehydes leading to increased apoptosis and autophagy.

DISCUSSION.

Increased metabolism of toxic aldehydes through ALDH upregulation can facilitate cancer progression and therapy resistance (9,17,53). Thus, numerous ALDH inhibitors have been developed as anti-cancer agents, and show variable efficacy in the treatment of breast (54-56), lung (18,23,25,33), hepatocellular (57), ovarian (26,29,30,58,59), gastric (25), colon (25), prostate (60), and head and neck squamous cell carcinoma (24,25,28,61) as well as glioblastoma (18,33), leukemia (22) and melanoma (62). Elevated ALDH activity is typically a composite of multiple ALDH isoforms (14,15). The major isoforms whose overexpression is implicated in cancer progression and drug resistance include the ALDH1A and 3A family (9,11,17,20,21,53) (15). ALDH2 has also been extensively characterized and implicated in various disease states, including alcohol-based cancers (11,15,17). Thus, ALDH1A1, 2 and 3A1 were selected to be the focus of this study. Here, we show that ALDH1A1, 2 and 3A1 overexpression is associated with melanoma progression and that targeting multiple ALDH isoforms is more effective for melanoma treatment, likely due to the overlapping ability of ALDH isoforms to metabolize toxic aldehydes (11). These data are consistent with previous reports in which knockdown of ALDH1A1, 2 and 3A1 had minimal effect on cancer cell proliferation (63,64). Subsequently, we undertook a structure-based drug designing campaign using the backbones of isatin and Cpd 3 to identify a novel, potent, multi-ALDH isoform inhibitor, called KS100, in an attempt to increase anti- cancer efficacy and reduce resistance development mediated by the ALDHs. KS100 was evaluated for ALDH inhibitory activity and found to be a potent ALDH1A1, 2 and 3A1 inhibitor. Currently available multi-ALDH isoform inhibitors include DEAB, DIMATE, citral and aldis-1, -2, -3, -4, and -6 (11,23-25). DEAB inhibits cultured ovarian cancer and melanoma cells, but minimal in vivo studies have been undertaken (11,58). DIMATE has an IC50 of 5 μM towards ALDH1A1 and 3A1, inhibits cultured prostate cancer cells, and administered at 14 mg/kg i.p. daily, reduces growth of melanoma xenografts (22,60,62). However, oral bioavailability of DIMATE and its evaluation in clinical trials has not yet been reported. Citral inhibits cultured breast cancer cells and xenografts, particularly when encapsulated into nanoparticles; however, it has a wide range of off-target activities leading to toxicity (55,56).

Aldis-1, -2, -3 and -4 have IC50 values of 2.2-7.9 μM for ALDH1A1, 5.4-8.6 μM for ALDH2 and 1.7-12 μM for ALDH3A1 (23). They inhibit cultured lung cancer and HNSCC cells, particularly as combinatorial therapy

(23,25). Aldi-6 has superior multi-ALDH isoform potency, with an IC50 of 600 nM for ALDH1A1, 800 nM for ALDH2 and 1 μM for ALDH3A1 (24). Aldi-6 inhibits cultured HNSCC cells as monotherapy and in combination with cisplatin (24). Further, administration at 24 mg/kg/day using implantable osmotic mini pumps led to 60% tumor reduction in HNSCC xenograft models, an effect enhanced with cisplatin, with no toxicity observed (24). However, due to its recent development, it has not been evaluated further. Thus, there is a continued need to develop multi-ALDH isoform inhibitors as anti-cancer agents, with KS100 being a potentially useful drug in this therapeutic field.

Enzymatic IC50s for KS100 were 207, 1,410 and 240 nM towards ALDH1A1, 2 and 3A1, respectively. KS100 had similar potency towards ALDH1A1 and 2 compared to DEAB. Importantly, KS100 exhibited a >60-fold increased potency towards ALDH3A1 compared to DEAB. Further, KS100 has a ~24-fold and ~21-fold increased potency towards ALDH1A1 and 3A1, respectively, compared to DIMATE (22,60,62). Finally, KS100 has a ~3-fold and 5-fold increased potency towards ALDH1A1 and 3A1, respectively, compared to aldi-6, while having slightly less potency towards ALDH2 (24). ALDH1A family and 3A1 overexpression are important in cancer progression and therapy resistance, while ALDH2 inhibition could potentially lead to toxicity through disruption of ethanol metabolism (11,17,20,21). Thus, KS100 is likely a more efficacious anti- cancer ALDH inhibitor than any currently reported multi-ALDH isoform inhibitors. KS100 showed efficacy and selectivity for killing cultured melanoma cells. Off-target interactions of KS100 were minimal based on the Erebus algorithm. However, studies in mice revealed KS100 to be toxic starting at 5 mg/kg/day. Therefore, a nanoliposomal formulation, called NanoKS100, was developed to overcome toxicity, as nanoliposomal formulations have been shown to decrease toxicity and increase the bioavailability of compounds (40-42,52). A PEGylated liposomal formulation was chosen as it increases the bioavailability of nanoliposomes by reducing drug accumulation in the liver and spleen, thus bypassing reticulo-endothelial elimination (40,42). NanoKS100 had a 68.6% loading efficiency and remained stable for at least 12 months when stored at 4˚C. NanoKS100 showed similar efficacy and selectivity for killing cultured melanoma cells compared to KS100. Importantly, NanoKS100 did not exhibit toxicity even at 60 mg/kg/day, the maximum dosage that could be manufactured based on the loading efficiency and stability of the formulation. NanoKS100 was significantly more effective at inhibiting melanoma tumor growth compared to empty liposomes at 10-30 mg/kg/day i.v. No significant difference in tumor killing efficacy was observed among treatment groups, so 20 mg/kg/day i.v. was selected for further evaluation in multiple xenograft models. NanoKS100 at 20 mg/kg/day i.v. led to a 65% reduction in UACC 903 and 1205 Lu xenografts compared to empty liposomes. It also caused no significant reduction in animal weight or increase in serum biomarkers of major organ function. Collectively, these results indicate efficacy with no apparent toxicity of NanoKS100 in inhibiting melanoma, which are similar to results of aldi-6 in HNSCC xenograft models at 24 mg/kg/day using osmostic mini-pumps (24). However, NanoKS100 was evaluated at a submaximal dose (3-fold lower), and the nanoliposomal formulation abrogated the need for osmotic mini-pumps, which are costly and invasive. Mechanistically, KS100 was the most effective ALDH inhibitor at reducing total cellular ALDH activity in UACC 903 and 1205 cells. Specifically, KS100 was the only ALDH inhibitor that significantly reduced ALDH+ cells in UACC 903 (52% reduction) and 1205 Lu (57% reduction) cell lines, and was at least 2-fold more effective at reducing total cellular ALDH activity in cell lysates compared to the other ALDH inhibitors. Further, KS100 significantly increased ROS activity only in melanoma cells. Specifically, KS100 was 2.8-fold and 4.8-fold

more effective in increasing ROS levels compared to DEAB and CM037, respectively, in both cell lines. The remaining ALDH inhibitors had no significant effect. Similarly, KS100 was 5.6-fold and 11.5-fold more effective in increasing lipid peroxidation and the accumulation of toxic aldehydes compared to DEAB and CM037, respectively, in both cell lines, while the other ALDH inhibitors had no significant effect. KS100 also led to increased apoptosis and autophagy, as exemplified by elevated levels of cleaved PARP and LC3B, respectively. KS100-induced apoptosis was further verified by flow cytometry using Annexin V staining. Specifically, KS100 induced a ~2-5-fold increase in the early apoptotic cell fraction (Annexin-V+7- AAD-) in both cell lines. All other ALDH inhibitors had no effect on apoptosis. Thus, reduced cellular ALDH activity as well as increased ROS generation, lipid peroxidation, toxic aldehyde accumulation and apoptosis were substantially higher with KS100 in UACC 903 and 1205 cells, confirming the superiority of KS100 at inhibiting multiple ALDH isoforms and its utility as an anti-cancer agent. In conclusion, this study demonstrates the association of ALDH overexpression in melanoma progression and that targeting multiple ALDH isoforms with overlapping functions may be necessary for effective anti-cancer therapy as well as for preventing resistance. A novel, potent, multi-isoform ALDH inhibitor, called KS100, was successfully synthesized and characterized. A nanoliposomal formulation of KS100, called NanoKS100, was developed to minimize toxicity and exhibited significant tumor inhibitory activity in melanoma xenograft models. KS100 efficiently inhibited ALDH1A1, 2 and 3A1 enzymatic activity, leading to increased ROS generation, lipid peroxidation and toxic aldehyde levels, as well as apoptosis and autophagy. A limitation of the study was that KS100 could not be evaluated for its inhibitory activity against other ALDH isoforms due to lack of commercially available enzymes and x-ray crystal structures. Thus, a siRNA screen to evaluate the specificity of KS100 on other ALDH isoforms was performed showing inhibition of ALDH1A1, 1A2, 1A3, 2 and 3A1. Future studies could look at the efficacy of NanoKS100 in other ALDH overexpressing cancers and its potential as combinatorial therapy with agents having resistance mechanisms through the ALDHs.

ACKNOWLEDGEMENTS.

G.P.Robertson received research grants from The Foreman Foundation for Melanoma Research, The Geltrude Foundation, The Penn State Chocolate Tour Cancer Research Fund and The Melanoma Research Alliance to support this project. N.V.Dokholyan received grants from Passan Foundation, Bridge V grant from Penn State University, and NIH grant GM114015 to support this project.

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IC s (nM) Docking scores 50 Compound ALDH1A1 ALDH2 ALDH3A1 ALDH1A1 ALDH2 ALDH3A1 Isatin -5.46 -6.398 -5.819 15,635±1,821 168,661±28,679 5,047±304 Cpd 3 -7.686 -9.839 -7.695 44±12 72,136±1,640 11,866±548 CM037 -11.276 -7.137 -8.137 98±34 2,278±250 1,774±303 CVT10216 -7.892 -11.809 -8.924 2,427±194 53±2 2,719±608 CB7 -8.159 -7.846 -14.576 139,016±16,934 144,409±11,470 298±29 DEAB -9.154 -10.026 -11.211 89±23 833±277 15,119±4,091 KS100 -10.247 -8.716 -13.851 207±10 1,410±248 240±50

Table 1. Docking scores and ALDH inhibitory activity for KS100 and other ALDH inhibitors. Isatin, Cpd 3, CM037, CVT10216, CB7, DEAB, and KS100 were docked into the active site pockets of ALDH1A1, 2 and 3A1. Docking scores were calculated using GLIDE 6.6 in Maestro 10.1. Enzyme inhibition studies for ALDH1A1, 2 and 3A1 were performed as described in the

materials and methods. KS100 displayed low IC50 values for all three ALDH isoforms tested. Preexisting isoform-specific ALDH inhibitors (Cpd 3 and CM037 for ALDH1A1, CVT10216 for ALDH2, CB7 for ALDH3A1) and the multi-ALDH isoform inhibitor, DEAB, were used as positive controls.

FIGURE LEGENDS.

1. The ALDH family is collectively important in melanoma. Western blot showing ALDH1A1, 2 and 3A1 expression levels in normal human fibroblasts (FF2441), melanocytes (NHEM), radial growth phase (RGP), vertical growth phase (VGP) and metastatic melanoma cell lines. ALDH expression in general increased during disease progression and was not dependent on BRAF mutational status. Alpha-enolase served as the loading control (1A). Data from the TCGA database showing slightly better survival with ALDH1A1 and 2 overexpression (1B) and worse survival with ALDH3A1 overexpression (1C) in melanoma patients. The data are available through the UCSC Xena Cancer Browser. Individual siRNA knockdown of ALDH1A1, 2 and 3A1 did not significantly reduce the growth survival of UACC 903 cells after 72 hours in an MTS assay. siRNA to BRAF and ALDH18A1 served as positive controls. Scrambled siRNA served as the negative control (1D). siRNA knockdown of ALDH1A1, 2, 3A1, 18A1 and BRAF in UACC 903 cells was confirmed via western blot. Alpha-enolase served as loading control (1E). Pharmacological inhibition of ALDH1A1, 2 and 3A1 using ALDH isoform-specific inhibitors (Cpd 3, CVT10216 and CB7, respectively) and the multi-ALDH isoform inhibitor, DEAB, revealed multi-ALDH isoform inhibition was most effective in inhibiting UACC 903 cell survival (1F). 2. Design, synthesis and toxicity analysis of the novel, ALDH1A1, 2 and 3A1 inhibitor, called KS100. Based on the structure and binding of Isatin, Cpd 3 and CM037, a medicinal chemistry approach was undertaken to design KS100, which exhibited more effective binding to ALDH1A1, 2 and 3A1 (2A). KS100 was synthesized from 5,7-dibromoisatin followed by benzylation as detailed in the supplementary materials and methods (2B). A 7-day repeated dose study was conducted for KS100. KS100 was administered i.p. daily, while animal body weight, physical and behavioral changes, and mortality were monitored. KS100 was toxic starting at 5 mg/kg (2C). 3. Development and characterization of the nanoliposomal formulation of KS100, called NanoKS100. NanoKS100 consists of an aqueous core surrounded by a phospholipid bilayer. KS100 is contained within the phospholipid bilayer (3A). NanoKS100 was manufactured with a 68.6% loading efficiency of KS100 into nanoliposomes (3B). KS100 is released from the nanoliposomal

formulation continuously for 48 hours with the maximal release of 70% (3C). Cell killing IC50s for KS100 and NanoKS100 against BRAF mutant (UACC 903, 1205 Lu) and wildtype (C8161.CI9, MelJuSo) melanoma cell lines were calculated and compared to that of normal human fibroblasts (FF2441) and melanocytes (NHEM) (3D). KS100 was ~4.5-fold, and NanoKS100 was ~5-fold more selective for killing melanoma cells compared to FF2441 and NHEM cells. NanoKS100 is stable for

at least 12 months when stored at 4˚C with no significant changes in IC50s (3E), size (3F) or charge

(3G). NanoKS100 causes significantly lower hemolysis compared to KS100 in both mouse and rat red blood cells. Triton X-100 served as the positive control (3H). 4. NanoKS100 inhibited melanoma tumor growth with negligible toxicity. A 7-day repeated dose study was conducted for NanoKS100. NanoKS100 was administered i.v. daily at various doses, while animal body weight, physical and behavioral changes, and mortality were monitored (4A). NanoKS100 significantly inhibited tumor growth of UACC 903 xenografts compared to empty liposome vehicle control following 20 days of treatment. No significant difference in tumor growth was seen between the NanoKS100 treatment groups (4B). NanoKS100 at 20 mg/kg body weight administered daily i.v. led to a ~65% reduction in tumor growth in UACC 903 (4C) and 1205 Lu (4D) xenografts following 20-22 days of treatment. NanoKS100 did not significantly affect animal body weight (4C, D-insets) or serum biomarkers of toxicity (4E) compared to empty liposome vehicle control. Normal reference ranges for serum biomarkers are included. 5. KS100 reduced total cellular ALDH activity to increase ROS generation, lipid peroxidation and toxic aldehyde accumulation leading to apoptosis and autophagy. The ALDHs reduce ROS generation, lipid peroxidation and toxic aldehyde accumulation, the latter of which can lead to cell damage and apoptosis (5A). KS100 was the only ALDH inhibitor that significantly reduced ALDH+ cells in both UACC 903 (5B) and 1205 Lu (5C) cells. ALDH+ cells were analyzed by flow cytometry following staining with Aldered. DMSO served as the control. UACC 903 (5D) and 1205 Lu (5E) cells treated with KS100 had increased ROS activity compared to the other ALDH inhibitors tested. DMSO served as control. No ALDH inhibitor significantly increased ROS activity in normal human fibroblasts (FF2441) compared to the DMSO control (5F). UACC 903 (5G) and 1205 Lu (5H) cells treated with KS100 had increased lipid peroxidation and toxic aldehyde accumulation compared to the other ALDH inhibitors tested. DMSO served as the control. Flow cytometric analysis of apoptosis in UACC 903 (5I) and 1205 Lu (5J) cells treated with 5 μM of ALDH inhibitor for 24 hours showed significantly increased apoptosis with KS100 compared the other ALDH inhibitors tested in both cell lines. DMSO served as the control. Western blot of increasing concentrations of KS100 (2, 4 and 6 μM) showed increased apoptosis (cleaved-PARP) and autophagy (LC3B) in UACC 903 cells after 24 hours of treatment (5K).

Development of a novel multi-isoform ALDH inhibitor effective as an anti-melanoma agent

Saketh S Dinavahi, Raghavendra Gowda, Krishne Gowda, et al.

Mol Cancer Ther Published OnlineFirst November 21, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-19-0360

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