ALDH1A1 Inhibitors Research Articles

Scaffold hopping based designing of selective ALDH1A1 inhibitors to overcome cyclophosphamide resistance: synthesis and biological evaluation.

Aldehyde dehydrogenase 1A1 (ALDH1A1) is an isoenzyme that catalyzes the conversion of aldehydes to acids. However, the overexpression of ALDH1A1 in a variety of malignancies is the major cause of resistance to an anti-cancer drug, cyclophosphamide (CP). CP is a prodrug that is initially converted into 4-hydroxycyclophosphamide and its tautomer aldophosphamide, in the liver. These compounds permeate into the cell and are converted as active metabolites, i.e., phosphoramide mustard (PM), through spontaneous beta-elimination. On the other hand, the conversion of CP to PM is diverted at the level of aldophosphamide by converting it into inactive carboxyphosphamide using ALDH1A1, which ultimately leads to high drug inactivation and CP resistance. Hence, in combination with our earlier work on the target of resistance, i.e., ALDH1A1, we hereby report selective ALDH1A1 inhibitors. Herein, we selected a lead molecule from our previous virtual screening and implemented scaffold hopping analysis to identify a novel scaffold that can act as an ALDH1A1 inhibitor. This results in the identification of various novel scaffolds. Among these, on the basis of synthetic feasibility, the benzimidazole scaffold was selected for the design of novel ALDH1A1 inhibitors, followed by machine learning-assisted structure-based virtual screening. Finally, the five best compounds were selected and synthesized. All synthesized compounds were evaluated using in vitro enzymatic assay against ALDH1A1, ALDH2, and ALDH3A1. The results disclosed that three molecules A1, A2, and A3 showed significant selective ALDH1A1 inhibitory potential with an IC50 value of 0.32 μM, 0.55 μM, and 1.63 μM, respectively, and none of the compounds exhibits potency towards the other two ALDH isoforms i.e. ALDH2 and ALDH3A1. Besides, the potent compounds (A1, A2, and A3) have been tested for in vitro cell line assay in combination with mafosfamide (analogue of CP) on two cell lines i.e. A549 and MIA-PaCa-2. All three compounds show significant potency to reverse mafosfamide resistance by inhibiting ALDH1A1 against these cell lines.

Flavonostilbenes natural hybrids from Rhamnoneuron balansae as potential antitumors targeting ALDH1A1: molecular docking, ADMET, MM-GBSA calculations and molecular dynamics studies

Several studies have linked Cancer stem cells (CSCs) to cancer resistance development to chemotherapy and radiotherapy. ALDH1A1 is a key enzyme that regulates the gene expression of CSCs and creates an immunosuppressive tumor microenvironment. It was reported that quercetin and resveratrol were among the inhibitors of ALDH1A1. In early 2022, it was reported that new 11 flavonostilbenes (rhamnoneuronal D–N) were isolated from Rhamnoneuron balansae as potential antiaging natural products. Rhamnoneuronal H (5) could be envisioned as a natural hybrid of quercetin and resveratrol. It was therefore hypothesized that 5 and its analogous isolates rhamnoneuronal D–G (1–4) and rhamnoneuronal I–N (6–11) would have potential ALDH1A1 inhibitory activity. To this end, all isolates were subjected to molecular docking, MM-GBSA, ADMET, and molecular dynamics simulations studies to assess their potential as new leads for cancer treatment targeting ALDH1A1. In silico findings revealed that natural hybrid 5 has a similar binding affinity, judged by MM-GBSA, to the ALDH1A1 active site when compared to the co-crystalized ligand (−64.71 kcal/mole and −64.12 kcal/mole, respectively). Despite having lesser affinity than that of the co-crystalized ligand, the rest of the flavonostilbenes, except 2–4, displayed better binding affinities (−37.55 kcal/mole to −58.6 kcal/mole) in comparison to either resveratrol (−34.44 kcal/mole) or quercetin (−36.48 kcal/mole). Molecular dynamic simulations showed that the natural hybrids 1, 5–11 are of satisfactory stability up to 100 ns. ADMET outcomes indicate that these hybrids displayed acceptable properties and hence could represent an ideal starting point for the development of potent ALDH1A1 inhibitors for cancer treatment. Communicated by Ramaswamy H. Sarma

Raloxifene and bazedoxifene as selective ALDH1A1 inhibitors to ameliorate cyclophosphamide resistance: A drug repurposing approach

Cyclophosphamide (CP) is one of the most widely used anticancer drugs for various malignancies. However, its long-term use leads to ALDH1A1-mediated inactivation and subsequent resistance which necessitates the development of potential ALDH1A1 inhibitors. Currently, ALDH1A1 inhibitors from different chemical classes have been reported, but these failed to reach the market due to safety and efficacy problems. Developing a new treatment from the ground requires a huge amount of time, effort, and money, therefore it is worthwhile to improve CP efficacy by proposing better adjuvants as ALDH1A1 inhibitors. Herein, the database constituting the FDA-approved drugs with well-established safety and toxicity profiles was screened through already reported machine learning models by our research group. This model is validated for discriminating the ALDH1A1 inhibitors and non-inhibitors. Virtual screening protocol (VS) from this model identified four FDA-approved drugs, raloxifene, bazedoxifene, avanafil, and betrixaban as selective ALDH1A1 inhibitors. The molecular docking, dynamics, and water swap analysis also suggested these drugs to be promising ALDH1A1 inhibitors which were further validated for their CP resistance reversal potential by in-vitro analysis. The in-vitro enzymatic assay results indicated that raloxifene and bazedoxifene selectively inhibited the ALDH1A1 enzyme with IC50 values of 2.35 and 4.41 μM respectively, whereas IC50 values of both the drugs against ALDH2 and ALDH3A1 was >100 μM. Additional in-vitro studies with well-reported ALDH1A1 overexpressing A549 and MIA paCa-2 cell lines suggested that mafosfamide sensitivity was further ameliorated by the combination of both raloxifene and bazedoxifene. Collectively, in-silico and in-vitro studies indicate raloxifene and bazedoxifene act as promising adjuvants with CP that may improve the quality of treatment for cancer patients with minimal toxicities.

3D-QSAR and scaffold hopping based designing of benzo[d]ox-azol-2(3H)-one and 2-oxazolo[4,5-b]pyridin-2(3H)-one derivatives as selective aldehyde dehydrogenase 1A1 inhibitors: Synthesis and biological evaluation.

Aldehyde dehydrogenase 1 (ALDH1A1), an oxidoreductase class of enzymes, is overexpressed in various types of cancer cell lines and is the major cause of resistance to the Food and Drug Administration (FDA)-approved drug, cyclophosphamide (CP). In cancer conditions, CP undergoes a sequence of biotransformations to form an active metabolite, aldophosphamide, which further biotransforms to its putative cytotoxic metabolite, phosphoramide mustard. However, in resistant cancer conditions, aldophosphamide is converted into its inactive metabolite, carboxyphosphamide, via oxidation with ALDH1A1. Herein, to address the issue of ALDH1A1 mediated CP resistance, we report a series of benzo[d]oxazol-2(3H)-one and 2-oxazolo[4,5-b]pyridin-2(3H)-one derivatives as selective ALDH1A1 inhibitors. These inhibitors were designed using a validated 3D-quantitative structure activity relationship (3D-QSAR) model coupled with scaffold hopping. The 3D-QSAR model was developed using reported indole-2,3-diones based ALDH1A1 inhibitors, which provided field points in terms of electrostatic, van der Waals and hydrophobic potentials required for selectively inhibiting ALDH1A1. The most selective indole-2,3-diones-based compound, that is, cmp 3, was further considered for scaffold hopping. Two top-ranked bioisosteres, that is, benzo[d]oxazol-2(3H)-one and 2-oxazolo[4,5-b]pyridin-2(3H)-one, were selected for designing new inhibitors by considering the field pattern of 3D-QSAR. All designed molecules were mapped perfectly on the 3D-QSAR model and found to be predictive with good inhibitory potency (pIC50 range: 7.5-6.8). Molecular docking was carried out for each designed molecule to identify key interactions that are required for ALDH1A1 inhibition and to authenticate the 3D-QSAR result. The top five inhibitor-ALDH1A1 complexes were also submitted for molecular dynamics simulations to access their stability. In vitro enzyme assays of 21 compounds suggested that these compounds are selective towardALDH1A1 over the other two isoforms, that is,ALDH2 and ALDH3A1. All the compounds were found to be at least three and two times more selective towardALDH1A1 over ALDH2 and ALDH3A1, respectively. All the compounds showed an IC50 value in the range of 0.02-0.80 μM, which indicates the potential for these to be developed as adjuvant therapy for CP resistance.

Disulfiram Transcends ALDH Inhibitory Activity When Targeting Ovarian Cancer Tumor-Initiating Cells.

Epithelial ovarian cancer (EOC) is a global health burden and remains the fifth leading cause of cancer related death in women worldwide with the poorest five-year survival rate of the gynecological malignancies. EOC recurrence is considered to be driven by the survival of chemoresistant, stem-like tumor-initiating cells (TICs). We previously showed that disulfiram, an ALDH inhibitor, effectively targeted TICs compared to adherent EOC cells in terms of viability, spheroid formation, oxidative stress and also prevented relapse in an in vivo model of EOC. In this study we sought to determine whether specific targeting of ALDH isoenzyme ALDH1A1 would provide similar benefit to broader pathway inhibition by disulfiram. NCT-505 and NCT-506 are isoenzyme-specific ALDH1A1 inhibitors whose activity was compared to the effects of disulfiram. Following treatment with both the NCTs and disulfiram, the viability of TICs versus adherent cells, sphere formation, and cell death in our in vitro relapse model were measured and compared in EOC cell lines. We found that disulfiram decreased the viability of TICs significantly more effectively versus adherent cells, while no consistent trend was observed when the cells were treated with the NCTs. Disulfiram also affected the expression of proteins associated with NFκB signaling. Comparison of disulfiram to the direct targeting of ALDH1A1 with the NCTs suggests that the broader cellular effects of disulfiram are more suitable as a therapeutic to eradicate TICs from tumors and prevent EOC relapse. In addition to providing insight into a fitting treatment for TICs, the comparison of disulfiram to NCT-505 and -506 has increased our understanding of the mechanism of action of disulfiram. Further elucidation of the mechanism of disulfiram has the potential to reveal additional targets to treat EOC TICs and prevent disease recurrence.

Abstract 1011: Disulfiram superior to ALDH1A1 inhibitor analogs in targeting ovarian cancer stem cells

Abstract Epithelial ovarian cancer (EOC) is a global health burden and remains the fifth leading cause of cancer related death in women worldwide with the poorest five-year survival rate of the gynecological malignancies. Disease recurrence is the major cause of morbidity and mortality of EOC, considered to be driven by the survival of chemoresistant, stem-like tumor-initiating cells (TICs). TIC populations in EOC can be identified by the expression of molecular markers such as aldehyde dehydrogenase (ALDH), CD133, Nanog and Sox2. Although the TICs have been characterized, little progress has been made in finding drugs to specifically target this population, which has driven the research and development of novel therapies to prevent relapse. We previously showed that disulfiram, an ALDH inhibitor, effectively targeted TICs in terms of viability, spheroid formation, oxidative stress and also prevented relapse in an in vivo model of EOC. In this study we sought to determine whether specific targeting of ALDH isoenzyme ALDH1A1 would provide similar benefit as the broader pathway inhibition by disulfiram. NCTs 505 and 506 are isoenzyme specific ALDH1A1 inhibitors whose activity was compared to the effects of disulfiram. Following treatment with either the NCTs or disulfiram, the viability of TICs versus adherent cells, sphere formation, oxidative stress and cell death in our in vitro relapse model were measured and compared in OC cell lines. We found that disulfiram decreased the viability of TICs significantly more effectively versus non-TIC cells, while no trend was observed when the cells were treated with the NCTs. Disulfiram also induced greater amounts of ROS than the NCTs and affected the expression of proteins associated with the NFkB and JNK signaling pathways. Comparison of disulfiram to the direct targeting of ALDH1A1 with the NCTs suggests that disulfiram's broader cellular effects are more suitable as a therapeutic to eradicate TICs from tumors and prevent OC relapse. In addition to providing insight into a fitting treatment for TICs, the comparison of disulfiram to NCTs 505 and 506 has increased our understanding of disulfiram's mechanism of action. Further elucidation of disulfiram's mechanism has the potential to reveal additional targets to treat OC TICs and prevent disease recurrence. Citation Format: Michael Caminear, Brittney Harrington, Christina Annunziata. Disulfiram superior to ALDH1A1 inhibitor analogs in targeting ovarian cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1011.

Multiple machine learning models combined with virtual screening and molecular docking to identify selective human ALDH1A1 inhibitors

Aldehyde dehydrogenases (ALDHs) are the enzymes of oxidoreductase family that are responsible for the aldehyde metabolism. The unbalanced expression of these enzymes may be associated with a variety of disease conditions including cancers. ALDH1A1 is one of the isoform of ALDHs majorly overexpressed in a variety of tumors and responsible for the anti-cancer drug resistance. This makes ALDH1A1 as a specific target to develop small molecule ALDH1A1 inhibitors for resistant cancer condition. Number of ALDH1A1 inhibitors have been developed and reported in the literature, but because of non-selectivity and inappropriate pharmacokinetic properties till now none of these have reached in the market for clinical use. Therefore, multiple machine learning models of different isoforms of ALDHs are integrated with in-silico techniques including virtual screening, docking, ADMET profiling, and MD simulation to identify selective ALDH1A1 inhibitors. Total ten selective ALDH1A1 inhibitors with diverse scaffolds and appropriate ADMET were identified that can be further developed as adjuvant therapy in cyclophosphamide and cisplatin resistance cancer.

Development of new disulfiram analogues as ALDH1a1-selective inhibitors

Disulfiram is an FDA-approved drug used to treat chronic alcoholism. This drug works by blocking the second step of ethanol metabolism by inhibiting aldehyde dehydrogenase-2 (ALDH2), the enzyme responsible for acetaldehyde oxidation into acetic acid. This leads to the accumulation of acetaldehyde in the blood following alcohol ingestion and to highly unpleasant symptoms known as acetaldehyde syndrome. Disulfiram also inhibits ALDH1a1, another member of the aldehyde dehydrogenases that catalyzes the oxidation of retinal into retinoic acid. ALDH1a1 represents a key therapeutic target for the treatment of important diseases such as cancer and obesity. The substrate tunnel is larger in ALDH1a1 than in ALDH2; therefore. Thus, replacing disulfiram ethyl groups with larger groups will yield selective ALDH1a1 inhibitors. In this work, we successfully synthesized derivative 2b, in which two ethyl groups were replaced by two para fluorobenzyl groups. The 2b derivative showed a comparable activity to disulfiram against ALDH1a1; however, it was completely devoid of inhibitory activity against ALDH2.

Design, synthesis characterization and biological evaluation of novel multi-isoform ALDH inhibitors as potential anticancer agents

The aldehyde dehydrogenases (ALDHs) are a family of detoxifying enzymes that are overexpressed in various cancers. Increased expression of ALDH is associated with poor prognosis, stemness, and drug resistance. Because of the critical role of ALDH in cancer stem cells, several ALDH inhibitors have been developed. Nonetheless, all these inhibitors either lack efficacy or are too toxic or have not been tested extensively. Thus, the continued development of ALDH inhibitors is warranted. In this study, we designed and synthesized potent multi-ALDH isoform inhibitors based on the isatin backbone. The early molecular docking studies and enzymatic tests revealed that 3(a–l) and 4(a–l) are the potent ALDH1A1, ALDHA2, and ALDH3A1 inhibitors. ALDH inhibitory IC50s of 3(a–l) and 4(a–l) were 230 nM to >10,000 nM for ALDH1A1, 939 nM to >10,000 nM for ALDH2 and 193 nM to >10,000 nM for ALDH3A1. The most potent compounds 3(h–l) had IC50s for killing melanoma cells ranged from 2.1 to 5.7 μM, while for colon cancer cells, it ranged from 2.5 to 5.8 μM and for multiple myeloma cells ranging from 0.3 to 4.7 μM. Toxicity studies of 3(h–l) revealed that 3h to be the least toxic multi-ALDH isoform inhibitor. Mechanistically, 3(h–l) caused increased ROS activity, lipid peroxidation, and toxic aldehyde accumulation, secondary to potent multi-ALDH isoform inhibition leading to increased apoptosis and G2/M cell cycle arrest. Together, the study details the design, synthesis, and evaluation of potent, multi-isoform ALDH inhibitors to treat cancers.

Abstract 3855: Preclinical investigation of novel ALDH1A1 inhibitors in pancreatic cancer

Abstract Introduction: Pancreatic adenocarcinoma remains a devastating disease, with a predicted 5-year survival at the time of diagnosis of less than 10%, and likely the number 2 cause of U.S. cancer deaths in the next decade. The lack of effective therapies and resistance to cytotoxic agents contribute to the poor outcomes in this patient population. Aldehyde dehydrogenases (ALDHs) are a family of enzymes that play important biological and metabolic roles in the human body. ALDH activity has been identified in many human malignancies as a marker of tumor initiating cells. In pancreatic cancer, ALDH1A1 has been shown to be upregulated in cancer stem cells (CSCs) and has been attributed to tumorigenesis and chemotherapeutic resistance. Methods: Six novel ALDH1A1 inhibitor drugs from the National Center for Advancing Translational Sciences (NCATS) were screened on human pancreatic cancer cell lines using the Cell Titer-Glo Assay. Proliferation assays were also performed with the three best performing inhibitors on a total of eight human pancreatic cancer cell lines. The best performing ALDH1A1 inhibitor drug were chosen and tested in combination with gemcitabine and paclitaxel on patient-derived tumor organoids (PDTOs). The combination effects were assessed over a 7-day period using the IncuCyte ZOOM live cell imager and CellTiter-Glo 3D Assay. Results: We assessed the anti-proliferative effects of 6 novel ALDH1A1 inhibitors on 4 pancreatic cancer cell lines. Two out of 4 pancreatic cell lines showed moderate treatment effects to 3 ALDH1A1 inhibitors. Further evaluation of one of the ALDH1A1 inhibitor on 8 additional pancreatic cancer cell lines revealed similar results. While several pancreatic cell lines showed some moderate activity at higher levels of drug exposure, most of the pancreatic cancer cell lines treated were more resistant. We next investigated the treatment effects of ALDH1A1 inhibitor in combination with gemcitabine or paclitaxel on PDTO 269 and 272. A combination effect with the ALDH1A1 inhibitor and paclitaxel was observed after 72 hours of drug exposure in both PDTO 269 and 272. In contrast, treatment with the ALDH1A1 inhibitor with gemcitabine did not further decrease growth when compared to single agent ALDH1A1 and gemcitabine. Conclusions: Although single agent activity of novel ALDH1A1 inhibitors is limited at significantly reducing cellular proliferation, inhibition of ALDH1A1 in combination with paclitaxel appears to have potent anti-tumor activity in PDTOs. These findings support further investigation of this combinational therapy for the treatment of pancreatic cancer. Additional studies are currently underway to evaluate this combinational activity in more preclinical pancreatic cancer models and to elucidate the underlying mechanisms whereby the inhibition of ALDH1A1 enhances the anti-tumor potential of paclitaxel. Citation Format: Betelehem W. Yacob, John J. Arcaroli, Maraake Taddese, Todd M. Pitts, Stacey M. Bagby, Sarah J. Hartman, S.Lindsey Davis, Christopher H. Lieu, Alexis D. Leal, Wells A. Messersmith. Preclinical investigation of novel ALDH1A1 inhibitors in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3855.

Discovery of Orally Bioavailable, Quinoline-Based Aldehyde Dehydrogenase 1A1 (ALDH1A1) Inhibitors with Potent Cellular Activity.

Aldehyde dehydrogenases (ALDHs) are responsible for the metabolism of aldehydes (exogenous and endogenous) and possess vital physiological and toxicological functions in areas such as CNS, inflammation, metabolic disorders, and cancers. Overexpression of certain ALDHs (e.g., ALDH1A1) is an important biomarker in cancers and cancer stem cells (CSCs) indicating the potential need for the identification and development of small molecule ALDH inhibitors. Herein, a newly designed series of quinoline-based analogs of ALDH1A1 inhibitors is described. Extensive medicinal chemistry optimization and biological characterization led to the identification of analogs with significantly improved enzymatic and cellular ALDH inhibition. Selected analogs, e.g., 86 (NCT-505) and 91 (NCT-506), demonstrated target engagement in a cellular thermal shift assay (CETSA), inhibited the formation of 3D spheroid cultures of OV-90 cancer cells, and potentiated the cytotoxicity of paclitaxel in SKOV-3-TR, a paclitaxel resistant ovarian cancer cell line. Lead compounds also exhibit high specificity over other ALDH isozymes and unrelated dehydrogenases. The in vitro ADME profiles and pharmacokinetic evaluation of selected analogs are also highlighted.

A High-Content Assay Enables the Automated Screening and Identification of Small Molecules with Specific ALDH1A1-Inhibitory Activity.

Aldehyde dehydrogenase enzymes (ALDHs) have a broad spectrum of biological activities through the oxidation of both endogenous and exogenous aldehydes. Increased expression of ALDH1A1 has been identified in a wide-range of human cancer stem cells and is associated with cancer relapse and poor prognosis, raising the potential of ALDH1A1 as a therapeutic target. To facilitate quantitative high-throughput screening (qHTS) campaigns for the discovery, characterization and structure-activity-relationship (SAR) studies of small molecule ALDH1A1 inhibitors with cellular activity, we show herein the miniaturization to 1536-well format and automation of a high-content cell-based ALDEFLUOR assay. We demonstrate the utility of this assay by generating dose-response curves on a comprehensive set of prior art inhibitors as well as hundreds of ALDH1A1 inhibitors synthesized in house. Finally, we established a screening paradigm using a pair of cell lines with low and high ALDH1A1 expression, respectively, to uncover novel cell-active ALDH1A1-specific inhibitors from a collection of over 1,000 small molecules.

Obesity: Current Treatment and Future Horizons.

Obesity is growing at an alarming rate with huge consequences on health and economy. The worldwide prevalence of obesity has nearly doubled in less than 35 years. Obesity causes many physiological dysfunctions that affect nearly every organ producing multiple morbidities. Despite these facts, there is still no clear, well-defined solution. Notwithstanding the devastating prevalence and consequences of obesity, today only five medicines, orlistat, lorcaserin, phentermine-topiramate, bupropion-naltrexone and liraglutide, are approved by the FDA for long-term treatment of obesity. In this review, the current approaches to treat obesity such as the development of diacylglycerol Oacyltransferase- 1 inhibitors, growth hormone secretagogue receptor-1a antagonists/inverse agonists, melanocortin-3 receptors agonists and melanin concentrating hormone receptor-1 antagonists, will be discussed. The main focus will be on the molecules that were able to reach clinical trials. The last section is dedicated to the "browning" phenomenon of white adipose tissues and the potential of Aldh1a1 inhibitors to treat obesity.

Inhibition of ALDH1A1 activity decreases expression of drug transporters and reduces chemotherapy resistance in ovarian cancer cell lines

The high mortality of ovarian cancer patients results from the failure of treatment caused by the inherent or acquired chemotherapy drug resistance. It was reported that overexpression of aldehyde dehydrogenase A1 (ALDH1A1) in cancer cells can be responsible for the development of drug resistance. To add the high expression of the drug transporter proteins the ALDHA1 is considered as a molecular target in cancer therapy. Therefore, we analysed drug-resistant ovarian cancer cell lines according to ALDHA1 expression and the association with drug resistance.The expression of ALDH1A1, P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP) was determined using a microarray and confirmed by Q-PCR, western blot and fluorescence analysis. ALDH1A1 activity was determined using an Aldefluor assay. The impact of all-trans retinoic acid (ATRA) and diethylaminobenzaldehyde (DEAB) on chemotherapy resistance was assessed by the MTT chemosensitivity assay.The most abundant expression of ALDH1A1 was noted in paclitaxel- and topotecan-resistant cell lines where two populations of ALDH-positive and ALDH-negative cells could be observed. Those cell lines also revealed the overexpression of P-gp and BCRP respectively, and were able to form spheres in non-adherent conditions. Pre-treatment with ATRA and DEAB reduced chemotherapy resistance in both cell lines. ATRA treatment led to downregulation of the ALDH1A1, P-gp and BCRP proteins. DEAB treatment led to downregulation of the P-gp protein and BCRP transcript and protein.Our results indicate that ALDH1A1-positive cancer cells can be responsible for drug resistance development in ovarian cancer. Developing more specific ALDH1A1 inhibitors can increase chemotherapy effectiveness in ovarian cancer.

Insight into the Structural Requirements of Theophylline-Based Aldehyde Dehydrogenase lAl (ALDHlAl) Inhibitors Through Multi-QSAR Modeling and Molecular Docking Approaches.

Over expression of aldehyde dehydrogenase (ALDH1A1) is one of the vital hallmarks of the self-renewal and differentiational cancer stem cells (CSCs). Till now, no selective ALDH1A1 inhibitor is commercially available in the market. So there is an urgent need to explore some novel molecules which can selectively inhibit ALDH1A1 to combat cancer. Presently, our work deals with the development of QSAR models of some theophylline-based molecules by conventional 2D-QSAR, hologram QSAR (HQSAR), and Bayesian classification modeling. The descriptors identified from these QSAR models give avenues to modulate the structure of theophylline-based compounds to a desirable biological end point. Molecular docking study reveals the selectivity of these molecules towards ALDH1A1 (PDB: 4WP7) and important binding residues (GLY 125, 458; THR 129; TRP 178; TYR 297; PHE 171, 466; VAL 174, 460; MET 175; HIS 293 etc.) for the interaction with the receptors. The current study may help to design novel compounds as selective ALDH1A1 inhibitors.

Abstract POSTER-BIOL-1309: Beta-catenin regulated ALDH1A1 is a target in ovarian cancer stem cells

Abstract Spheroids are 3D multi-cell aggregates formed in non-adherent culture conditions. In ovarian cancer (OC), they serve as a vehicle for cancer cell dissemination in the peritoneal cavity and protect cells from extracellular stress-induced anoikis. We hypothesized that spheroids are enriched in cancer stem cells and investigated pathways activated under these conditions. Affymetrix-based gene expression profiling of OC cells grown as spheroids vs. monolayers identified β-catenin pathway being upregulated in 3D cultures. Increased expression of β-catenin and target genes (cyclin D1, c-myc) was demonstrated in spheroids vs. monolayers and in successive spheroid generations using OC cell lines and primary cultures. B-catenin function measured as TCF/LEF1 reporter activity was augmented in spheroids vs. monolayers and siRNA mediated β-catenin knock down decreased the number of spheroids (p<0.001). Along with β-catenin, the expression level of ALDH1A1, an OC stem cell marker, was increased in successive spheroid generations. The percentage of ALDH+ cells was significantly higher in spheroids vs. monolayers in IGROV1, A2780, SKOV3, and primary OC cells. B-catenin knock-down decreased ALDH1A1 expression, suggesting that it is a β-catenin target. The percentage of ALDH+ cells and the number of spheroids formed were increased in the cell population dissociated from OC xenografts treated with carboplatin compared to control (44% vs. 2.3%), supporting that ALDH1A1 is a stem cell marker. A37 is a novel ALDH1A1 enzymatic inhibitor (Ki of 5nM). A37 decreased cell viability and spheroid formation by IGROV1 cells (p<0.001). A37 also decreased the percentage of ALDH+ cells under 3D culture conditions. These data support the role of β-catenin regulated ALDH1A1 in the maintenance of OC spheroids and of a stem cell phenotype and propose new ALDH1A1 inhibitors targeting this cell population. (Funding: US Department of Veterans Affairs and Ovarian Cancer Research Fund) Citation Format: Salvatore Condello, Sarbajeet Nagdas, Cynthia Morgan, John Turek, Thomas Hurley, Daniela Matei. Beta-catenin regulated ALDH1A1 is a target in ovarian cancer stem cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1309.

Discovery of NCT-501, a Potent and Selective Theophylline-Based Inhibitor of Aldehyde Dehydrogenase 1A1 (ALDH1A1).

Aldehyde dehydrogenases (ALDHs) metabolize reactive aldehydes and possess important physiological and toxicological functions in areas such as CNS, metabolic disorders, and cancers. Increased ALDH (e.g., ALDH1A1) gene expression and catalytic activity are vital biomarkers in a number of malignancies and cancer stem cells, highlighting the need for the identification and development of small molecule ALDH inhibitors. A new series of theophylline-based analogs as potent ALDH1A1 inhibitors is described. The optimization of hits identified from a quantitative high throughput screening (qHTS) campaign led to analogs with improved potency and early ADME properties. This chemotype exhibits highly selective inhibition against ALDH1A1 over ALDH3A1, ALDH1B1, and ALDH2 isozymes as well as other dehydrogenases such as HPGD and HSD17β4. Moreover, the pharmacokinetic evaluation of selected analog 64 (NCT-501) is also highlighted.

Characterization of two distinct structural classes of selective aldehyde dehydrogenase 1A1 inhibitors.

Aldehyde dehydrogenases (ALDH) catalyze the irreversible oxidation of aldehydes to their corresponding carboxylic acid. Alterations in ALDH1A1 activity are associated with such diverse diseases as cancer, Parkinson's disease, obesity, and cataracts. Inhibitors of ALDH1A1 could aid in illuminating the role of this enzyme in disease processes. However, there are no commercially available selective inhibitors for ALDH1A1. Here we characterize two distinct chemical classes of inhibitors that are selective for human ALDH1A1 compared to eight other ALDH isoenzymes. The prototypical members of each structural class, CM026 and CM037, exhibit submicromolar inhibition constants but have different mechanisms of inhibition. The crystal structures of these compounds bound to ALDH1A1 demonstrate that they bind within the aldehyde binding pocket of ALDH1A1 and exploit the presence of a unique glycine residue to achieve their selectivity. These two novel and selective ALDH1A1 inhibitors may serve as chemical tools to better understand the contributions of ALDH1A1 to normal biology and to disease states.

Development of a high-throughput in vitro assay to identify selective inhibitors for human ALDH1A1

The human aldehyde dehydrogenase (ALDH) superfamily consists of at least 19 enzymes that metabolize endogenous and exogenous aldehydes. Currently, there are no commercially available inhibitors that target ALDH1A1 but have little to no effect on the structurally and functionally similar ALDH2. Here we present the first human ALDH1A1 structure, as the apo-enzyme and in complex with its cofactor NADH to a resolution of 1.75 and 2.1Å, respectfully. Structural comparisons of the cofactor binding sites in ALDH1A1 with other closely related ALDH enzymes illustrate a high degree of similarity. In order to minimize discovery of compounds that inhibit both isoenzymes by interfering with their conserved cofactor binding sites, this study reports the use of an in vitro, NAD+-independent, esterase-based high-throughput screen (HTS) of 64,000 compounds to discover novel, selective inhibitors of ALDH1A1. We describe 256 hits that alter the esterase activity of ALDH1A1. The effects on aldehyde oxidation of 67 compounds were further analyzed, with 30 selectively inhibiting ALDH1A1 compared to ALDH2 and ALDH3A1. One compound inhibited ALDH1A1 and ALDH2, while another inhibited ALDH1A1, ALDH2, and the more distantly related ALDH3A1. The results presented here indicate that this in vitro enzyme activity screening protocol successfully identified ALDH1A1 inhibitors with a high degree of isoenzyme selectivity. The compounds identified via this screen plus the screening methodology itself represent a starting point for the development of highly potent and selective inhibitors of ALDH1A1 that may be utilized to better understand the role of this enzyme in both normal and disease states.

β-Catenin-regulated ALDH1A1 is a target in ovarian cancer spheroids.

Cancer cells form three dimensional (3D) multicellular aggregates (or spheroids) under non-adherent culture conditions. In ovarian cancer (OC), spheroids serve as a vehicle for cancer cell dissemination in the peritoneal cavity, protecting cells from environmental stress-induced anoikis. To identify new targetable molecules in OC spheroids, we investigated gene expression profiles and networks upregulated in three dimensional (3D) versus traditional monolayer culture conditions. We identified ALDH1A1, a cancer stem cell marker as being overexpressed in OC spheroids and directly connected to key elements of the β-catenin pathway. B-catenin function and ALDH1A1 expression were increased in OC spheroids vs. monolayers and in successive spheroid generations, suggesting that 3D aggregates are enriched in cells with stem cell characteristics. B-catenin knockdown decreased ALDH1A1 expression levels and β-catenin coimmunoprecipitated with the ALDH1A1 promoter, suggesting that ALDH1A1 is a direct β-catenin target. Both siRNA mediated β-catenin knockdown and A37, a novel ALDH1A1 small molecule enzymatic inhibitor described here for the first time, disrupted OC spheroid formation and cell viability (p<0.001). B-catenin knockdown blocked tumor growth and peritoneal metastasis in an OC xenograft model. These data strongly support the role of β-catenin regulated ALDH1A1 in the maintenance of OC spheroids and propose new ALDH1A1 inhibitors targeting this cell population.