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Systematic approaches for accurate repurposing of targeted therapies are needed. We developed and aimed to biologically validate our therapy predicting tool (TPT) for the repurposing of targeted therapies for specific tumor types by testing the role of Bromodomain and Extra-Terminal motif inhibitors (BETi) in inhibiting BRD4 function and downregulating Notch3 signaling in ovarian cancer.Utilizing established ovarian cancer preclinical models, we carried out in vitro and in vivo studies with clinically relevant BETis to determine their therapeutic effect and impact on Notch3 signaling.Treatment with BETis or siRNA-mediated BRD4 knockdown resulted in decreased cell viability, reduced cell proliferation, and increased cell apoptosis in vitro. In vivo studies with orthotopic mouse models demonstrated that treatment with BETi decreased tumor growth. In addition, knockdown of BRD4 with doxycycline-inducible shRNA increased survival up to 50% (P < 0.001). Treatment with either BETis or BRD4 siRNA decreased Notch3 expression both in vitro and in vivo. BRD4 inhibition also decreased the expression of NOTCH3 targets, including HES1. Chromatin immunoprecipitation revealed that BRD4 was present at the NOTCH3 promoter.Our findings provide biological validation for the TPT by demonstrating that BETis can be an effective therapeutic agent for ovarian cancer by downregulating Notch3 expression.The TPT could rapidly identify candidate drugs for ovarian or other cancers along with novel companion biomarkers.

Raw RPPA data for OVCAR 5.

File contains synthesis of CN210, sequences for siRNAs, supplementary tables 1 to 3, supplementary figures 1 through 8 and figure legends.

Raw RPPA data for OVCAR 4.

Inhibitors of bromodomain and extra-terminal motif (BET) proteins are emerging epigenetic therapeutics that suppress gene expressions that drive cancer and inflammation. The present study examined anti-inflammatory effects of a quinazoline-based BET inhibitor, CN210, in a murine ileitis model. CN210 was given orally 30 min before and 24 h after a subcutaneous administration of indomethacin. Macroscopic and histological evidences of ileitis, mucosal myeloperoxidase (MPO) activity and cytokine expressions were evaluated 48 h after the indomethacin administration. To further characterize the anti-inflammatory pathways modulated by CN210, its effects on RAW264 cells treated with lipopolysaccharide (LPS) were investigated. Competitive ligand binding and docking studies of CN210 to CREB-binding protein (CBP) and p300 were also performed. Oral administration of CN210 significantly reduced the severity of ileitis, normalized both proinflammatory MPO activity and concomitant cytokine expressions induced by indomethacin administration. Furthermore, CN210 attenuated the expression of cytokines and reversed the activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPK) induced by LPS. Competitive ligand binding assays showed that CN210 bound to the bromodomains of two paralogous histone acetyltransferases, CBP and p300, in addition to the bromodomains of BET proteins. Docking studies of CN210 to the bromodomains of CBP and p300 showed a similarity to the binding mode of SGC-CBP30, a specific CBP/p300 inhibitor. CN210 ameliorates indomethacin-induced ileitis by inhibiting the expression of inflammatory cytokines through the attenuation of NF-κB and MAPK pathways. CN210 thus represents a new mode of therapy for non-steroidal anti-inflammatory drug-induced ileitis and inflammatory bowel disease.

Schwannoma tumours typically arise on the eighth cranial nerve and are mostly caused by loss of the tumour suppressor Merlin (NF2). There are no approved chemotherapies for these tumours and the surgical removal of the tumour carries a high risk of damage to the eighth or other close cranial nerve tissue. New treatments for schwannoma and other NF2-null tumours such as meningioma are urgently required. Using a combination of human primary tumour cells and mouse models of schwannoma, we have examined the role of the Hippo signalling pathway in driving tumour cell growth. Using both genetic ablation of the Hippo effectors YAP and TAZ as well as novel TEAD palmitoylation inhibitors, we show that Hippo signalling may be successfully targeted in vitro and in vivo to both block and, remarkably, regress schwannoma tumour growth. In particular, successful use of TEAD palmitoylation inhibitors in a preclinical mouse model of schwannoma points to their potential future clinical use. We also identify the cancer stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) as a Hippo signalling target, driven by the TAZ protein in human and mouse NF2-null schwannoma cells, as well as in NF2-null meningioma cells, and examine the potential future role of this new target in halting schwannoma and meningioma tumour growth.

Acute lymphoblastic leukemia with chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene (MLL-r ALL) remains an incurable disease. Thus, development of a safe and effective therapeutic agent to treat this disease is crucial to address this unmet medical need. BRD4, a member of the bromodomain and extra-terminal domain (BET) protein family, and cyclic AMP response element binding protein binding protein (CBP) and p300, two paralogous histone acetyltransferases, are all considered cancer drug targets and simultaneous targeting of these proteins may have therapeutic advantages. Here, we demonstrate that a BET/CBP/p300 multi-bromodomain inhibitor, CN470, has anti-tumor activity against MLL-r ALL in vitro and in vivo. CN470, potently inhibited ligand binding to the bromodomains of BRD4, CBP, and p300 and suppressed the growth of MLL-r ALL cell lines and patient-derived cells with MLL rearrangements. CN470 suppressed mRNA and protein expression of MYC and induced apoptosis in MLL-r ALL cells, following a cell cycle arrest in the G1 phase. Moreover, CN470 reduced BRD4 binding to acetylated histone H3. The in vivo effects of CN470 were investigated using SEM(Luc/GFP) cells expressing luminescent markers in an orthotopic mouse model. Mice administered CN470 daily had prolonged survival compared to the vehicle group. Further, CN470 also showed anti-tumor effects against an MLL-r ALL patient-derived xenograft model. These findings suggest that inhibition of BET/CBP/p300 by the multi-bromodomain inhibitor, CN470, represents a promising therapeutic approach against MLL-r ALL.

The estrogen receptor α (ERα) is a target of intense pharmacological intervention and toxicological biomonitoring. Current methods to directly quantify cellular levels of ERα involve antibody-based assays, which are labor-intensive and of limited throughput. In this study, we generated a post-translational reporter cell line, referred to as MCF7-ERα-HiBiT, by fusing a small pro-luminescent nanoluciferase (NLuc) tag (HiBiT) to the C-terminus of endogenous ERα in MCF7 cells. The tag allows the luminescent detection and quantification of endogenous ERα protein by addition of the complementary NLuc enzyme fragment. This MCF7-ERα-HiBiT cell line was optimized for quantitative high-throughput screening (qHTS) to identify compounds that reduce ERα levels. In addition, the same cell line was optimized for a qHTS cellular thermal shift assay to identify compounds that bind and thermally stabilize ERα. Here, we interrogated the MCF7-ERα-HiBiT assay against the NCATS Pharmacological Collection (NPC) of 2,678 approved drugs and identified compounds that potently reduce and thermally stabilize ERα. Our novel post-translational reporter cell line provides a unique opportunity for profiling large pharmacological and toxicological compound libraries for their effect on ERα levels as well as for assessing direct compound binding to the receptor, thus facilitating mechanistic studies by which compounds exert their biological effects on ERα.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell lymphoproliferative malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). ATL is an orphan disease with no curative drug treatment regimens urgently needing new combination therapy. HTLV-1-infected cells rely on viral proteins, Tax and HBZ (HTLV-1-b-ZIP factor), to activate the transcription of various host genes that are critical for promoting leukemic transformation. Inhibition of bromodomain and extraterminal motif (BET) protein was previously shown to collapse the transcriptional network directed by BATF3 super-enhancer and thereby induced ATL cell apoptosis. In the current work, by using xenograft, ex vivo, and in vitro models, we demonstrated that I-BET762 (BETi) synergized with copanlisib (PI3Ki) and bardoxolone methyl (NF-κBi) to dramatically decrease the growth of ATL cells. Mechanistically, the triple combination exhibited synergistic activity by down-regulating the expression of c-MYC while upregulating the level of the glucocorticoid-induced leucine zipper (GILZ). The triple combination also enhanced apoptosis induction by elevating the expression of active caspase-3 and cleaved PARP. Importantly, the triple combination prolonged the survival of ATL-bearing xenograft mice and inhibited the proliferation of ATL cells from peripheral blood mononuclear cells (PBMCs) of both acute and smoldering/chronic ATL patients. Therefore, our data provide the rationale for a clinical trial exploring the multiagent combination of BET, PI3K/AKT, and NF-κB inhibitors for ATL patients and expands the potential treatments for this recalcitrant malignancy.

To generate drug molecules of desired properties with computational methods is the holy grail in pharmaceutical research. Here we describe an AI strategy, retro drug design, or RDD, to generate novel small molecule drugs from scratch to meet predefined requirements, including but not limited to biological activity against a drug target, and optimal range of physicochemical and ADMET properties. Traditional predictive models were first trained over experimental data for the target properties, using an atom typing based molecular descriptor system, ATP. Monte Carlo sampling algorithm was then utilized to find the solutions in the ATP space defined by the target properties, and the deep learning model of Seq2Seq was employed to decode molecular structures from the solutions. To test feasibility of the algorithm, we challenged RDD to generate novel drugs that can activate {\mu} opioid receptor (MOR) and penetrate blood brain barrier (BBB). Starting from vectors of random numbers, RDD generated 180,000 chemical structures, of which 78% were chemically valid. About 42,000 (31%) of the valid structures fell into the property space defined by MOR activity and BBB permeability. Out of the 42,000 structures, only 267 chemicals were commercially available, indicating a high extent of novelty of the AI-generated compounds. We purchased and assayed 96 compounds, and 25 of which were found to be MOR agonists. These compounds also have excellent BBB scores. The results presented in this paper illustrate that RDD has potential to revolutionize the current drug discovery process and create novel structures with multiple desired properties, including biological functions and ADMET properties. Availability of an AI-enabled fast track in drug discovery is essential to cope with emergent public health threat, such as pandemic of COVID-19., Comment: 27 pages, 6 figures

Endoplasmic reticulum (ER) dysregulation is associated with pathologies including neurodegenerative, muscular, and diabetic conditions. Depletion of ER calcium can lead to the loss of resident proteins in a process termed exodosis. To identify compounds that attenuate the redistribution of ER proteins under pathological conditions, we performed a quantitative high-throughput screen using the Gaussia luciferase (GLuc)-secreted ER calcium modulated protein (SERCaMP) assay, which monitors secretion of ER-resident proteins triggered by calcium depletion. We identify several clinically used drugs, including bromocriptine, and further characterize them using assays to measure effects on ER calcium, ER stress, and ER exodosis. Bromocriptine elicits protective effects in cell-based models of exodosis as well as in vivo models of stroke and diabetes. Bromocriptine analogs with reduced dopamine receptor activity retain similar efficacy in stabilizing the ER proteome, indicating a non-canonical mechanism of action. This study describes a strategic approach to identify small-molecule drugs capable of improving ER proteostasis in human disease conditions.

Emerging evidence has established primary nephrotic syndrome (NS), including focal segmental glomerulosclerosis (FSGS), as a primary podocytopathy. Despite the underlying importance of podocyte endoplasmic reticulum (ER) stress in the pathogenesis of NS, no treatment currently targets the podocyte ER. In our monogenic podocyte ER stress-induced NS/FSGS mouse model, the podocyte type 2 ryanodine receptor (RyR2)/calcium release channel on the ER was phosphorylated, resulting in ER calcium leak and cytosolic calcium elevation. The altered intracellular calcium homeostasis led to activation of calcium-dependent cytosolic protease calpain 2 and cleavage of its important downstream substrates, including the apoptotic molecule procaspase 12 and podocyte cytoskeletal protein talin 1. Importantly, a chemical compound, K201, can block RyR2-Ser2808 phosphorylation-mediated ER calcium depletion and podocyte injury in ER-stressed podocytes, as well as inhibit albuminuria in our NS model. In addition, we discovered that mesencephalic astrocyte-derived neurotrophic factor (MANF) can revert defective RyR2-induced ER calcium leak, a bioactivity for this ER stress-responsive protein. Thus, podocyte RyR2 remodeling contributes to ER stress-induced podocyte injury. K201 and MANF could be promising therapies for the treatment of podocyte ER stress-induced NS/FSGS.

Systematic approaches for accurate repurposing of targeted therapies are needed. We developed and aimed to biologically validate our therapy predicting tool (TPT) for the repurposing of targeted therapies for specific tumor types by testing the role of Bromodomain and Extra-Terminal motif inhibitors (BETi) in inhibiting BRD4 function and downregulating Notch3 signaling in ovarian cancer. Utilizing established ovarian cancer preclinical models, we carried out in vitro and in vivo studies with clinically relevant BETis to determine their therapeutic effect and impact on Notch3 signaling. Treatment with BETis or siRNA-mediated BRD4 knockdown resulted in decreased cell viability, reduced cell proliferation, and increased cell apoptosis in vitro. In vivo studies with orthotopic mouse models demonstrated that treatment with BETi decreased tumor growth. In addition, knockdown of BRD4 with doxycycline-inducible shRNA increased survival up to 50% (P < 0.001). Treatment with either BETis or BRD4 siRNA decreased Notch3 expression both in vitro and in vivo. BRD4 inhibition also decreased the expression of NOTCH3 targets, including HES1. Chromatin immunoprecipitation revealed that BRD4 was present at the NOTCH3 promoter. Our findings provide biological validation for the TPT by demonstrating that BETis can be an effective therapeutic agent for ovarian cancer by downregulating Notch3 expression. The TPT could rapidly identify candidate drugs for ovarian or other cancers along with novel companion biomarkers.

A new series of quinazoline-based analogs as potent bromodomain-containing protein 4 (BRD4) inhibitors is described. The structure-activity relationships on 2- and 4-position of quinazoline ring, and the substitution at 6-position that mimic the acetylated lysine are discussed. A co-crystallized structure of 48 (CN750) with BRD4 (BD1) including key inhibitor-protein interactions is also highlighted. Together with preliminary rodent pharmacokinetic results, a new lead (65, CN427) is identified which is suitable for further lead optimization.

Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate, with concomitant oxidation of reduced nicotinamide adenine dinucleotide as the final step in the glycolytic pathway. Glycolysis plays an important role in the metabolic plasticity of cancer cells and has long been recognized as a potential therapeutic target. Thus, potent, selective inhibitors of LDH represent an attractive therapeutic approach. However, to date, pharmacological agents have failed to achieve significant target engagement in vivo, possibly because the protein is present in cells at very high concentrations. We report herein a lead optimization campaign focused on a pyrazole-based series of compounds, using structure-based design concepts, coupled with optimization of cellular potency, in vitro drug–target residence times, and in vivo PK properties, to identify first-in-class inhibitors that demonstrate LDH inhibition in vivo. The lead compounds, named NCATS-SM1440 (43) and NCATS-SM1441 (52), possess desirable attributes for further studying the effect of in vivo LDH inhibition.

Cancer stem cells (CSCs), representing the root of many solid tumors including ovarian cancer, have been implicated in disease recurrence, metastasis, and therapeutic resistance. Our previous study has demonstrated that the CSC subpopulation in ovarian cancer can be limited by DNA damage-binding protein 2 (DDB2). Here, we demonstrated that the ovarian CSC subpopulation can be maintained via cancer cell dedifferentiation, and DDB2 is able to suppress this non-CSC-to-CSC conversion by repression of ALDH1A1 transcription. Mechanistically, DDB2 binds to the ALDH1A1 gene promoter, facilitating the enrichment of histone H3K27me3, and competing with the transcription factor C/EBPβ for binding to this region, eventually inhibiting the promoter activity of the ALDH1A1 gene. The de-repression of ALDH1A1 expression contributes to DDB2 silencing-augmented non-CSC-to-CSC conversion and expansion of the CSC subpopulation. We further showed that treatment with a selective ALDH1A1 inhibitor blocked DDB2 silencing-induced expansion of CSCs, and halted orthotopic xenograft tumor growth. Together, our data demonstrate that DDB2, functioning as a transcription repressor, can abrogate ovarian CSC properties by downregulating ALDH1A1 expression.

We report the discovery and medicinal chemistry optimization of a novel series of pyrazole-based inhibitors of human lactate dehydrogenase (LDH). Utilization of a quantitative high-throughput screening paradigm facilitated hit identification, while structure-based design and multiparameter optimization enabled the development of compounds with potent enzymatic and cell-based inhibition of LDH enzymatic activity. Lead compounds such as 63 exhibit low nM inhibition of both LDHA and LDHB, submicromolar inhibition of lactate production, and inhibition of glycolysis in MiaPaCa2 pancreatic cancer and A673 sarcoma cells. Moreover, robust target engagement of LDHA by lead compounds was demonstrated using the cellular thermal shift assay (CETSA), and drug-target residence time was determined via SPR. Analysis of these data suggests that drug-target residence time (off-rate) may be an important attribute to consider for obtaining potent cell-based inhibition of this cancer metabolism target.

Lecithin:cholesterol acyltransferase (LCAT) and LCAT-activating compounds are being investigated as treatments for coronary heart disease (CHD) and familial LCAT deficiency (FLD). Herein we report the crystal structure of human LCAT in complex with a potent piperidinylpyrazolopyridine activator and an acyl intermediate-like inhibitor, revealing LCAT in an active conformation. Unlike other LCAT activators, the piperidinylpyrazolopyridine activator binds exclusively to the membrane-binding domain (MBD). Functional studies indicate that the compound does not modulate the affinity of LCAT for HDL, but instead stabilizes residues in the MBD and facilitates channeling of substrates into the active site. By demonstrating that these activators increase the activity of an FLD variant, we show that compounds targeting the MBD have therapeutic potential. Our data better define the substrate binding site of LCAT and pave the way for rational design of LCAT agonists and improved biotherapeutics for augmenting or restoring reverse cholesterol transport in CHD and FLD patients., eLife digest Cholesterol is a fatty substance found throughout the body that is essential to our health. However, if too much cholesterol builds up in our blood vessels, it can cause blockages that lead to heart and kidney problems. The body removes excess cholesterol by sending out high-density lipoproteins (HDL) that capture the fatty molecules and carry them to the liver where they are eliminated. The first step in this process requires an enzyme called LCAT, which converts cholesterol into a form that HDL particles can efficiently pack and transport. The enzyme acts by interacting with HDL particles, and chemically joining cholesterol with another compound. Finding ways to make LCAT perform better and produce more HDL could improve treatments for heart disease. This could be particularly helpful to people with genetic changes that make LCAT defective. Several small molecules that ‘dial up’ the activity of LCAT have been identified, but how they act on the enzyme is not always well understood. Manthei et al. therefore set out to determine precisely how one such small activator promotes LCAT function. The experiments involved using a method known as crystallography to look at the structure of LCAT when it is attached to the small molecule. They also evaluated the activity of the enzyme and other aspects of the protein in the presence of the small molecule and HDL particles. Taken together, the results led Manthei et al. to suggest that the small molecule works by more efficiently bringing into LCAT the materials that this enzyme needs to create the transport-ready form of cholesterol. The small molecule also partially restored the activity of mutant LCAT found in human disease. This knowledge may help to design more drug-like chemicals to ‘boost’ the activity of LCAT and prevent heart and kidney disease, especially in people who carry a defective version of the enzyme.

Extensive optimization of quinazoline-based lead 8 is described. The structure-activity relationship studies indicate the S-configuration is preferred for the phenylmorpholine substitution. Together with incorporation of a (2-hydroxyl-2-methylpropyl)pyrazole moiety at the 2-position leads to analogs with comparable potency and marked improvement in the pharmacokinetic profile over our previously reported lead compounds. Further in vivo efficacy studies in Kasumi-1 xenograft mouse model demonstrates that the selected inhibitors are well tolerated and highly efficacious in the inhibition of tumor growth. Additionally, the representative analog 19 also demonstrated significant improvement of arthritis severity in a collagen-induced arthritis (CIA) mouse model. These results indicate potential use of these quinazoline-based BET inhibitors for treatment of cancer and inflammatory diseases. A brief discussion of the co-crystallized structure of 19 with BRD4 (BD1) is also highlighted.

Acute lymphoblastic leukemia (ALL) is the most common form of childhood leukemia. While the prognosis of patients with ALL has improved significantly, ALL with chromosomal rearrangements involving mixed-lineage leukemia (MLL) gene remains an incurable disease. Thus, development of a safe and efficacious therapeutic agent based on the biology of MLL rearrangement is of high importance to address this unmet medical need. Bromodomains are protein motifs that bind to acetylated histones and are recognized as new epigenetic drug targets in recent years. Bromodomain and extra-terminal motif proteins (“BET proteins”) are epigenetic ‘reader' proteins that are characterized by containing two of such bromodomains. BRD4, a member of BET protein family, is a key component of multi-protein complex that induce oncogene transcription at Super-Enhancers. Currently, small-molecule BET inhibitors are being investigated in the clinic as potential therapeutics for hematological cancers. CBP and p300, two paralogous histone acetyltransferases, also contain bromodomains and are considered as drug targets for cancer. Because BET and CBP/p300 proteins are key components of the etiology of MLL-rearranged (MLL-r) leukemia, an agent that simultaneously target both classes of proteins may have therapeutic advantages. We previously demonstrated that a novel BET inhibitor, CG13250, suppressed multiple myeloma cell proliferation in vitro and in vivo (Biochem Biophys Res Commun, 484:262-268, 2017) and subsequently synthesized additional BET inhibitors (Bioorg Med Chem Lett, 29:1220-1226, 2019). In the present study, we demonstrate that some of our BET inhibitors can also inhibit bromodomains of CBP/p300 proteins. One such compound, CN470, potently suppresses BRD4, CBP, and p300 with BROMOscan KD values of 33, 32, and 20 nM, respectively, and suppressed mRNA and protein expression of c-MYC as expected. CN470 potently inhibited the growth of MLL-r ALL cell lines in a dose-dependent manner and flow cytometric analyses showed that CN470 caused apoptosis in MLL-r ALL cells following a cell cycle arrest at the G1 phase. Moreover, co-immunoprecipitation assay demonstrated that CN470 reduced the binding of BRD4 to acetylated H3 (H3K27), an effect absent with a reference BET inhibitor, OTX-015. Taken together, CN470 may represent an effective strategy against MLL-r ALL cells. As a confirmatory study, we investigated the in vivo effects of CN470 by using SEMLuc/GFP cells in an orthotopic mouse model of MLL-AF4 leukemia. Mice were orally administered with CN470 (10 mg/kg) once daily resulting in a prolonged survival compared to vehicle control group (mean 37 days vs. 28 days; p=0.0246). In conclusion, simultaneous inhibition of BET/CBP/p300 with a multi-bromodomain inhibitor represents a promising novel therapeutic approach against MLL-r ALL. Citation Format: Natsuki Imayoshi, Makoto Yoshioka, Shyh-Ming Yang, Koshi Akahane, Yuki Toda, Shigekuni Hosogi, Takeshi Inukai, Seiji Okada, David J. Maloney, Jefferey W. Strovel, Eishi Ashihara. Application of BET/CBP/p300 multi-bromodomain inhibitors as a novel therapeutic strategy for MLL-rearranged acute lymphoblastic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1745.

Our lab is interested in the development and growth of schwannoma and meningioma tumors of the nervous system. Approximately 70% of these tumors arise due to loss of the tumor suppressor Merlin. In a mouse model of schwannoma (P0-CRE NF2fl/fl), we observed increased mRNA levels of the cancer stem cell marker Aldehyde dehydrogenase 1A1 (Aldh1A1) in Merlin-null Schwann cells. Immunostaining showed elevated levels of Aldh1A1 protein in this mouse model and in another mouse model (Postn-CRE NF2fl/fl), in which schwannoma tumors arise spontaneously within the vestibulocochlear nerve and dorsal root ganglia of the nervous system. Similarly, staining of human schwannoma samples also showed a strong elevation of Aldh1A1 protein in all tumor cells. Parallel analysis of meningioma tumor samples, in which Merlin loss also occurs, also showed a strong increase of Aldh1A1 protein levels. Western blotting enabled identification of the mechanism by which Merlin loss regulates Aldh1A1. Merlin/TAZ double knockout, but not Merlin/YAP double knockout, sciatic nerves show reduced Aldh1A1 expression. This implicates Hippo signalling through the TAZ effector as the mechanism of Aldh1A1 upregulation both in vitro and in vivo in both tumor types. Chemical inhibition with Aldh1a1-specific inhibitor (NCT-505) or shRNA knockdown of Aldh1A1 in Merlin-null schwannoma and meningioma cells in culture attenuated tumor cell growth, proving Aldh1A1 has a role in tumor cell proliferation; studies using Aldh1A1 null mice to test this in vivo are ongoing. A major target of Aldh1A1 signalling is the regulation of retinoic acid (RA) signalling. We are examining expression and activation of components of RA signalling in both schwannoma and meningioma tumor cells to identify whether this may be of therapeutic use in the future for these two tumor types. In conclusion, the TAZ-dependent upregulation of Aldh1A1 in Merlin-null schwannoma and meningioma tumor cells has a functional role in driving cell proliferation in these two clinically important tumor types. Further work will seek to identify the mechanism by which Aldh1A1 activity contributes to tumor growth. Citation Format: Lily Hillson, Liyam Laraba, Aditya G. Shivane, Philip Edwards, Ganesha Bantukallu, Anton Simeonov, Natalia J. Martinez, Shyh-Ming Yang, David B. Parkinson. Roles for the cancer stem cell marker Aldh1A1 in Merlin null nervous system tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5957.

Assessment of the interactions between a drug and its protein target in a physiologically relevant cellular environment constitutes a major challenge in the pre-clinical drug discovery space. The Cellular Thermal Shift Assay (CETSA) enables such an assessment by quantifying the changes in the thermal stability of proteins upon ligand binding in intact cells. Here, we present the development and validation of a homogeneous, standardized, target-independent, and high-throughput (384- and 1536-well formats) CETSA platform that uses a split Nano Luciferase approach (SplitLuc CETSA). The broad applicability of the assay was demonstrated for diverse targets, and its performance was compared with independent biochemical and cell-based readouts using a set of well-characterized inhibitors. Moreover, we investigated the utility of the platform as a primary assay for high-throughput screening. The SplitLuc CETSA presented here enables target engagement studies for medium and high-throughput applications. Additionally, it provides a rapid assay development and screening platform for targets where phenotypic or other cell-based assays are not readily available.

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.

Design, synthesis, and biological evaluation of pyridazine-based, 4-bicyclic heteroaryl-piperidine derivatives as potent stearoyl-CoA desaturase-1 (SCD1) inhibitors are described. In a chronic study of selected analog (3e) in Zucker fa/fa (ZF) rat, dose-dependent decrease of body weight gain and plasma fatty acid desaturation index (DI) in both C16 and C18 are also demonstrated. The results indicate that the plasma fatty acid DI may serve as an indicator for direct target engagement and biomarker for SCD1 inhibition.

A new series of urea-based, 4-bicyclic heteroaryl-piperidine derivatives as potent SCD1 inhibitors is described. The structure-activity relationships focused on bicyclic heteroarenes and aminothiazole-urea portions are discussed. A trend of dose-dependent decrease in body weight gain in diet-induced obese (DIO) mice is also demonstrated.

Phosphonium salt-activated, Pd-catalyzed Suzuki–Miyaura and Sonogashira cross-coupling reactions of cyclic 1,3-diones in the synthesis of β-substituted cyclic enones are described. These transformations exhibit good isolated yield and high generality with respect to both substrates and coupling partners. Extension of the substrate scope to cyclic 1,3-dione equivalents, such as 2-cyanocyclohexanone (4), is also briefly examined.

Phosphonium salt-activated, Pd-catalyzed Suzuki–Miyaura and Sonogashira cross-coupling reactions of cyclic 1,3-diones in the synthesis of β-substituted cyclic enones are described. These transformations exhibit good isolated yield and high generality with respect to both substrates and coupling partners. Extension of the substrate scope to cyclic 1,3-dione equivalents, such as 2-cyanocyclohexanone (4), is also briefly examined.

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.

In an effort to identify novel Janus kinase 3 inhibitors, a sequential focused screening approach was adopted to search our in-house chemical database. By biologically testing only 79 selected compounds, we successfully identified 19 compounds showing IC 50 < 20 microM, with four of them in the nanomolar range. Particularly, a 3,5-disubstituted pyrazolo[4,3- d]pyrimidine scaffold emerged as a promising candidate for further lead optimization. With the advantages of efficiency and flexibility, this approach may be utilized to identify leads for other therapeutic targets.

A new series of β-N-biaryl ether sulfonamide hydroxamates as novel gelatinase inhibitors is described. These compounds exhibit good potency for MMP-2 and MMP-9 without inhibiting MMP-1. The structure–activity relationships (SAR) reveal the biaryl ether type P1′ moiety together with methanesulfonamide is the optimal combination that provides inhibitory activity of MMP-9 in the single-digit nanomolar range.

Several classes of arylsulfone-based MMP-2/-9 inhibitors utilizing 6- to 8-membered heterocyclic rings as zinc-binding groups (ZBGs) have been synthesized and their enzyme inhibitory activities were evaluated. Although a number of 6- and 7-membered heterocycles were effective, the most potent arylsulfone inhibitors are based on the rigid 1- or 3-hydroxypyridone ZBG.

A microwave accelerated epoxide ring-opening process with N-biaryl sulfonamides is described. Under this mild, highly efficient condition, an α-hydroxy-β-N-biaryl sulfonamide skeleton is rapidly assembled leading ultimately to a novel series of matrix metalloproteinase-9 inhibitors with single digit nanomolar activities.

Simplification of bottom ring and regioselective functionalization of the indolocarbazole unit of staurosporine (2) are described. The modification led to a new series of simplified staurosporine analogs, which exhibited significant inhibitory activity against Janus kinase 3 (JAK3). The structure-activity relationships (SAR) are discussed and a proposed binding model is also highlighted.

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.

The SmI 2 -promoted three-component coupling reaction of thiophene-2-carboxylate, indole-2-carbaldehyde and acetophenone provides an expedient route to a series of tetracyclic carbazolothiophene compounds bearing the indole and thiophene rings. Among these samples, 9-benzyl-4-methyl-4-(4-hydroxyphenyl)-10-oxo-4,10-dihydrocarbazolo[2,3- b ]thiophene-2-carboxylic acid ( 18 ) shows the most potent inhibition against the endothelin-1 induced increase of intracellular calcium ion concentration.

The coupling reactions of 2-thiophenecarbaldehyde with aromatic or aliphatic aldehydes were promoted by samarium diiodide in the presence of hexamethylphosphoramide to give C-5 hydroxyalkylation products. The coupling reactions of 3-thiophenecarbaldehyde occurred at C-2, and the subsequent alkylations occurred at the sulfur atom, accompanied by a concurrent opening of the thiophene ring to afford γ-lactols. Double hydroxyalkylations of 2- and 3-thiophenecarbaldehydes were also carried out under appropriate reaction conditions. Synthetic applications of these thiophenecarbonyl coupling products were demonstrated, for example, by elaboration to furans, butenolides, and thiophene-fused polycyclic compounds.