Search

The central role of β-catenin in the Wnt pathway makes it an attractive therapeutic target for cancers driven by aberrant Wnt signaling. We recently developed a small-molecule inhibitor, BC-2059, that promotes apoptosis by disrupting the β-catenin/transducin β-like 1 (TBL1) complex through an unknown mechanism of action. In this study, we show that BC-2059 directly interacts with high affinity for TBL1 when in complex with β-catenin. We identified two amino acids in a hydrophobic pocket of TBL1 that are required for binding with β-catenin, and computational modeling predicted that BC-2059 interacts at the same hydrophobic pocket. Although this pocket in TBL1 is involved in binding with NCoR/SMRT complex members G Protein Pathway Suppressor 2 (GSP2) and SMRT and p65 NFκB subunit, BC-2059 failed to disrupt the interaction of TBL1 with either NCoR/SMRT or NFκB. Together, our results show that BC-2059 selectively targets TBL1/β-catenin protein complex, suggesting BC-2059 as a therapeutic for tumors with deregulated Wnt signaling pathway. SIGNIFICANCE STATEMENT This study reports the mechanism of action of a novel Wnt pathway inhibitor, characterizing the selective disruption of the transducin β-like 1/β-catenin protein complex. As Wnt signaling is dysregulated across cancer types, this study suggests BC-2059 has the potential to benefit patients with tumors reliant on this pathway.

Background: Breast cancer (BCa) is the most commonly diagnosed cancer and the second leading cause of cancer death in women. BCa is composed of distinct molecular subtypes, such as ER positive BCa (ER+ BCa) and triple negative BCa (TNBC). Development of novel effective therapies for patients with therapy resistant breast cancer (TR-BC) and TNBC remains the highest unmet need in patient treatment and survivorship. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) plays a critical role in multiple nuclear receptor functions leading to TR-BC and TNBC progression. PELP1 expression is dysregulated in BCa, is a prognostic indicator of poorer BCa survival, and its deregulation contributes to BCa therapy resistance. The objective of this study is development and characterization of a small molecule inhibitor of PELP1 (SMIP) as novel therapeutic for treating BCa. Methods: Using yeast two-hybrid screening, we identified PELP1 Inhibitory Peptide (PIP1) from a library of peptides. PIP1 binds PELP1 with high affinity and functions to inhibit PELP1 oncogenic activity. Direct binding of PIP1 to PELP1 was confirmed using biotin pull-down assays and inhibition of BCa proliferation confirmed using MTT assays. We used the Hit-Ligand interaction site with PIP1 hot spot residues based on 3D alignment and morphology to generate a library of peptidomimetics (small chemical molecules). In vitro activity was assessed using Celltiter Glo, MTT, and matrigel invasion chamber assays in multiple BCa models. Mechanistic studies were conducted using Western blot, reporter gene assays, and peptide competition assays. Xenograft and patient derived explant (PDEX) assays were used for preclinical evaluation and preliminary toxicity analysis. Results: Bioactivity screens revealed PELP1 Inhibitory Peptide (PIP1) significantly attenuates PELP1-mediated proliferation with an IC50 of 10µM across multiple BCa cell lines. We confirmed PIP1 binding to PELP1 using peptide pull-down assays with nuclear lysates from BCa cells. Using Hit-Ligand-Based interaction site with the PIP1 hot spot residues, we identified 61 potential hits using a 10,000 Diverse Set. Screening of these 61 potential hits using the MTT assays lead to the selection of SMIP34 (tetrahydropyrazolo [1,5a) pyrazole) as lead inhibitor of PELP1. SMIP34 treatment reduced proliferation at an IC50 of 3-10µM in ER+ BCa (ZR-75, MCF-7, and T- 47D); TR-BC (MCF-7-TamR, MCF-7-LTLT, ZR-75-MT-ER537S, and ZR-75-MT-ER538G); and TNBC (MDA- MB-231, and BT549) models. Additionally, SMIP34 showed no activity in human mammary epithelial cells. Specificity of SMIP34 was confirmed using PELP1 knockdown BCa cell lines. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation. In matrigel invasion chamber assays, SMIP34 significantly reduced the invasiveness of TR-BC and TNBC models. In combination studies, SMIP34 displayed synergy and enhanced the efficacy of current chemotherapeutics Cisplatin and Paclitaxel. In PDEX assays, Ki67 staining revealed SMIP34 significantly decreased tumor proliferation. In xenograft models, SMIP34 (10mg/kg/s.c.) treatment resulted in significant reduction in tumors compared to vehicle treatment. Furthermore, overall mouse body weight in both control and SMIP34 treated groups were similar, suggesting no overt signs of toxicity. Conclusion: We have developed a first-in-class small molecule inhibitor of PELP1 (SMIP) displaying effectivity against TR-BC and TNBC in vitro and in vivo. Supported by CPRIT Predoctoral Fellowship CPRIT RTA; RP170345 (K.A. Altwegg) Citation Format: Kristin A Altwegg, Suryavathi Viswanadhapalli, Monica Mann, Uday P Pratap, Mengxing Li, Junhao Liu, Yiliao Luo, Gangadhara R Sareddy, Hariprasad Vankayalapati, Ratna K. Vadlamudi. Development and characterization of a first-in-class small molecule inhibitor of PELP1 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-01.

Background RON (Recepteur d'Origine Nantais) receptor tyrosine kinase is a promising target for anti-cancer therapeutics. The aim of this study was to identify new RON inhibitors using virtual screening methods. Methods To this end, a ligand-based virtual screening approach was employed for screening of ZINC database on the homology model of RON receptor. All the selected hits were inspected in terms of drug-likeness, ADME properties, and toxicity profiles. Ligand-based similarity searches along with further filtering criteria led to the identification of two compounds, TKI1 and TKI2 that were evaluated using in vitro cell-based RON inhibition assays. Results The results showed that TKI1 and TKI2 could reduce phosphorylation of RON. Both compounds showed inhibitory activity of the downstream mTOR pathway with no apparent effects on other signaling mediators in a dose-dependent manner. Conclusion These compounds can provide a basis for developing novel anti-RON inhibitors applicable to cancer therapy using medicinal chemistry-oriented optimization strategies.

ETS transcription factors from the ERG and ETV1/4/5 subfamilies are overexpressed in the majority of prostate cancer patients and contribute to disease progression. Here, we have developed two in vitro assays for the interaction of ETS transcription factors with DNA that are amenable to high-throughput screening. Using ETS1 as a model, we applied these assays to screen 110 compounds derived from a high-throughput virtual screen. We found that the use of lower-affinity DNA binding sequences, similar to those that ERG and ETV1 bind to in prostate cells, allowed for higher inhibition from many of these test compounds. Further pilot experiments demonstrated that the in vitro assays are robust for ERG, ETV1, and ETV5, three of the ETS transcription factors that are overexpressed in prostate cancer.

Chromatin remodeling SWItch/Sucrose-NonFermentable (SWI/SNF) complexes, initially identified in yeast 20 years ago, are evolutionarily conserved multi-subunit protein complexes that use the energy from hydrolysis of adenosine triphosphate (ATP) to remodel nucleosome structure and modulate transcription. Mutations in proteins of SWI/SNF complexes occur in 20% of human cancers including ovarian cancer (OC). Approximately 50% of ovarian clear cell carcinoma (OCCC) carries mutations in the SWI/SNF subunit ARID1A while small cell carcinoma of the ovary hypercalcemic type (SCCOHT) is driven primarily by genetic inactivation of the SWI/SNF ATPase SMARCA4 (BRG1) alongside epigenetic silencing of the homolog ATPase SMARCA2 (BRM). Dual loss of these ATPases disrupts SWI/SNF chromatin remodeling activity and may also interfere with the function of other histone-modifying enzymes that associate with or are dependent on SWI/SNF activity. One such enzyme is lysine-specific histone demethylase 1 (LSD1/KDM1A) which regulates the chromatin landscape and gene expression by demethylating proteins, including histone H3. LSD1 associates with epigenetic complexes such as the nucleosome remodeling deacetylase complex (NuRD) and SWI/SNF to inhibit the transcription of genes involved in tumor suppression and cell differentiation. TGCA analysis of human cancers shows that LSD1 is highly expressed in SWI/SNF-mutated tumors. Further, SCCOHT and OCCC cell lines show low nM IC50s for the reversible LSD1 inhibitor SP-2577 (Seclidemstat, currently in clinical phase I trials), supporting that these SWI/SNF-deficient ovarian cancers are dependent on LSD1 activity. Recently, it has been also shown that inhibition of LSD1 stimulates interferon (IFN)-dependent anti-tumor immunity through induction of Endogenous Retroviruses Elements (ERVs) and may thereby overcome resistance to checkpoint blockade. Additionally, SCCOHTs have been shown to exhibit an immune-active tumor microenvironment with PD-L1 expression in both tumor and stromal cells that strongly correlated with T cell infiltration. Thus, in this study we investigated the ability of SP-2577 to promote anti-tumor immunity and T cell infiltration in SWI/SNF-mutant SCCOHT and OCCC models. Our data shows that the reversible LSD1 inhibitor SP-2577 stimulates IFN-dependent anti-tumor immunity in SCCOHT cells in vitro in a 3D immune-organoid platform. Additionally, SP-2577 promoted the expression of PD-L1 in both SCCOHT and OCCC models. Together our findings suggest that SP-2577 and checkpoint inhibitors as a therapeutic combination may induce or augment immunogenic responses in these tumors.

To minimize treatment toxicities, recent anti-cancer research efforts have switched from broad-based chemotherapy to targeted therapy, and emerging data show that altered cellular metabolism in cancerous cells can be exploited as new venues for targeted intervention. In this study, we focused on, among the altered metabolic processes in cancerous cells, altered glycosylation due to its documented roles in cancer tumorigenesis, metastasis and drug resistance. We hypothesize that the enzymes required for the biosynthesis of UDP-hexoses, glycosyl donors for glycan synthesis, could serve as therapeutic targets for cancers. Through structure-based virtual screening and kinetic assay, we identified a drug-like chemical fragment, GAL-012, that inhibit a small family of UDP-hexose pyrophosphorylases-galactose pyro-phosphorylase (GALT), UDP-glucose pyrophosphorylase (UGP2) and UDP-N-acetylglucosamine pyrophosphorylase (AGX1/UAP1) with an IC50 of 30 &micro, M. The computational docking studies supported the interaction of GAL-012 to the binding sites of GALT at Trp190 and Ser192, UGP2 at Gly116 and Lys127, and AGX1/UAP1 at Asn327 and Lys407, respectively. One of GAL-012 derivatives GAL-012-2 also demonstrated the inhibitory activity against GALT and UGP2. Moreover, we showed that GAL-012 suppressed the growth of PC3 cells in a dose-dependent manner with an EC50 of 75 &micro, M with no effects on normal skin fibroblasts at 200 &micro, M. Western blot analysis revealed reduced expression of pAKT (Ser473), pAKT (Thr308) by 77% and 72%, respectively in the treated cells. siRNA experiments against the respective genes encoding the pyrophosphorylases were also performed and the results further validated the proposed roles in cancer growth inhibition. Finally, synergistic relationships between GAL-012 and tunicamycin, as well as bortezomib (BTZ) in killing cultured cancer cells were observed, respectively. With its unique scaffold and relatively small size, GAL-012 serves as a promising early chemotype for optimization to become a safe, effective, multi-target anti-cancer drug candidate which could be used alone or in combination with known therapeutics.

N-Glycanase deficiency, or NGLY1 deficiency, is an extremely rare human genetic disease. N-Glycanase, encoded by the gene NGLY1, is an important enzyme involved in protein deglycosylation of misfolded proteins. Deglycosylation of misfolded proteins precedes the endoplasmic reticulum (ER)-associated degradation (ERAD) process. NGLY1 patients produce little or no N-glycanase (Ngly1), and the symptoms include global developmental delay, frequent seizures, complex hyperkinetic movement disorder, difficulty in swallowing/aspiration, liver dysfunction, and a lack of tears. Unfortunately, there has not been any therapeutic option available for this rare disease so far. Recently, a proposed molecular mechanism for NGLY1 deficiency suggested that endo-β-N-acetylglucosaminidase (ENGase) inhibitors may be promising therapeutics for NGLY1 patients. Herein, we performed structure-based virtual screening utilizing FDA-approved drug database on this ENGase target to enable repurposing of existing drugs. Several Proton Pump Inhibitors (PPIs), a series of substituted 1H-benzo [d] imidazole, and 1H-imidazo [4,5-b] pyridines, among other scaffolds, have been identified as potent ENGase inhibitors. An electrophoretic mobility shift assay was employed to assess the inhibition of ENGase activity by these PPIs. Our efforts led to the discovery of Rabeprazole Sodium as the most promising hit with an IC50 of 4.47±0.44μM. This is the first report that describes the discovery of small molecule ENGase inhibitors, which can potentially be used for the treatment of human NGLY1 deficiency.

In patients with metastatic castration-resistant prostate cancers (mCRPC) who have previously been treated with abiraterone or enzalutamide, the median overall survival is only 14 months. Unlike many other cancers, immunotherapies have had limited successes, due to the fact that there are very few T cells in the tumor microenvironment of prostate cancer (PC) patients. Identifying ways to boost immunotherapy responses could change the paradigm of mCRPC, a disease still difficult to treat. The highly proliferative nature of tumor cells, along with infiltration of myeloid cells into the tumors, leads to depletion of nutrients such as functional/natural amino acids. This metabolically stressful milieu causes activation of nutrient stress pathways, autophagy, and repressed immune responses. A key meditator of this nutrient stress pathway is a cytoplasmic Ser/Thr protein kinase called General Control Nonderepressible 2 (GCN2), also called EIF2AK4. GCN2 switches on by a reduction of amino acids, and its activity results in T cell inactivation, T cell death, regulatory T cell expansion, and the potentiation of myeloid-derived suppressor cells (MDSCs). We have developed and synthesized a series of novel small molecule immunotherapeutic agents that reversibly bind to GCN2 kinase, competitively block the ATP site, and elicit pharmacological responses in immune cells. GCN2 cell-free kinase binding, kinome selectivity, pGCN2, pEIF2α, ATF-4 phosphorylation inhibition assays were performed and confirmed it's on-target efficacy and potency of lead GCN2 inhibitor HCI-1046. In these studies, our lead GCN2 kinase inhibitor HCI-1046 demonstrated potent activity with an IC50 of 36 nM in inhibiting GCN2. GCN2 expression has been detected in PC-3, DU-145, and LNCap cell lines, and HCI-1046 exhibited cellular efficacy with an IC50 of 0.9 to 8 μM range, reduced phosphorylation of GCN2, ATF-4 significantly and its downstream target eIF2α. HCI-1046 inhibited pGCN2 and pEIF2α in human prostate cancer clinical patient samples derived MDSC cultured in amino-acid starved conditions. HCI-1046, in a dose-dependent fashion, restored CD8+ T cell proliferation and function in clinical samples of PC patients. Our preliminary results support the hypothesis that inhibition of GCN2 reinstates anti-tumor immunity and blocks tumor progression in PC cellular models. In vivo PK studies of HCI-1046 in rodent species showed excellent PK properties; 55% oral bioavailability, low clearance, and >5 hours half-life. HCI-1046 is nominated as a pre-clinical agent. Additional 3D cellular efficacy studies, FACS for cellular apoptosis, cell migration, live PC cells including immuno-ELISA, ELISpot experimental results for MDSCs suppressive function of T cells, and restoration by HCI-1046 will be presented at the conference. PC mouse model efficacy studies will also be discussed. Citation Format: Kyle Medley, Zhaoliang Li, Dongqing Yan, Umang Swami, Neeraj Agarwal, Hariprasad Vankayalapati. Targeting GCN2 kinase-driven stress response inactivation by orally available small molecules to restore immune tumor microenvironment in prostate cancers [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 LB136.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked inherited enzymopathic disorder that may lead to transfusion-requiring acute hemolytic anemia (AHA) triggered by fava beans ingestion, infection or some drugs. The gene encoding for G6PD carries a large number of genetic variants that have varying pathogenicity. We reported on three G6PD variants in the Gaza Strip Palestinian population with differing clinical impacts and frequencies: G6PD Mediterraneanc.563T, African G6PD A−c.202A/c.376G, and G6PD Cairoc.404C. We also identified a novel G6PD missense (Ser179Asn) mutation c.536G > A “G6PD Gaza”. In this work we explore the effect of these four genetic variants on the structural and substrate (NADP+ and G6P) binding characteristics of the G6PD enzyme using the Monte Carlo (MC) flexible docking and molecular dynamics (MD) simulation approaches. We report that G6PD A−c.202A/c.376G, G6PD Mediterraneanc.563T, G6PD Cairoc.404C and G6PD Gazac.536A mutations cause significant structural changes in G6PD enzyme to induce conformational instability leading to the loss of binding of one or both substrates and are causative of G6PD deficiency.

Background: Breast cancer (BC) is the most commonly diagnosed cancer and the second leading cause of cancer death in American women. BC is composed of distinct molecular subtypes, such as ER positive BC (ER+) and triple negative BC (TNBC). Development of novel effective therapies for patients with therapy resistant BC (TR-BC) and TNBC remains the highest unmet need in patient treatment and survivorship. Proline, glutamic acid-, and leucine-rich protein 1 (PELP1) plays a critical role in multiple nuclear receptor functions leading to TR-BC and TNBC progression. PELP1 expression is dysregulated in BC, is a prognostic indicator of poorer BC survival, and its deregulation contributes to TR-BC. The objective of this study is characterization of a small molecule inhibitor of PELP1 (SMIP34) as novel therapeutic for treating advanced BC. Methods: Yeast two-hybrid screening was used to identify PELP1 Inhibitory Peptide 1 (PIP1). Direct binding of PIP1 to PELP1 was confirmed using biotin pull-down assays and inhibition of BC proliferation confirmed using MTT assays. Hit-Ligand interaction site with PIP1 hot spot residues based on 3D alignment and morphology was used to generate a library of small chemical molecules that function as peptidomimetics of PIP1. In vitro activity was assessed using CellTiter Glo, MTT, colony formation and matrigel invasion assays in multiple BC models. Mechanistic studies were conducted using Western blot, and reporter gene assays. Xenograft and patient derived explant (PDEX) assays were used for preclinical evaluation. Results: Screening utilizing MTT assays lead to the selection of SMIP34 as an inhibitor of PELP1. SMIP34 treatment reduced cell viability at an IC50 of 3-10µM in a panel of BC cells. Additionally, SMIP34 showed no activity in human mammary epithelial cells. Knockdown of PELP1 in BC cells significantly reduced the SMIP34 activity, confirming target specificity. Furthermore, SMIP34 treatment significantly reduced the invasiveness and colony formation of TR-BC and TNBC cells. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation. In combination studies, SMIP34 displayed synergy and enhanced the efficacy of current chemotherapeutics Cisplatin and Paclitaxel. In PDEX assays, SMIP34 significantly decreased tumor proliferation as measured by Ki67 staining. In xenograft models, SMIP34 (10mg/kg/s.c.) treatment resulted in significant reduction in tumor volume compared to vehicle control. Furthermore, overall mouse body weight in both control and SMIP34 treated groups were similar, suggesting no overt signs of toxicity. Conclusion: We have developed a first-in-class small molecule inhibitor of PELP1 (SMIP34) displaying therapeutic efficacy against TR-BC and TNBC in vitro, ex vivo, and in vivo. Supported by CPRIT Predoctoral Fellowship CPRIT RTA; RP170345 (K.A. Altwegg) and VA grant I01BX004545 (R.K.V) Citation Format: Kristin Ann Altwegg, Suryavathi Viswanadhapalli, Monica Mann, Uday P. Pratap, Mengxing Li, Junhao Liu, Yiliao Luo, Gangadhara R. Sareddy, Hariprasad Vankayalapati, Ratna K. Vadlamudi. Characterization of small molecule inhibitors of PELP1 for treating advanced breast cancer [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 6403.

Genomic instability is a recognized hallmark of cancer. Germline mutations in critical DNA-repair and DNA-damage response genes predispose to cancer development, but also create vulnerabilities that can be exploited for cancer therapy. Poly (ADP-ribose) polymerase (PARP) inhibitors are selectively active in cells with homologous recombination deficiency (HRD) caused by mutations in BRCA1, BRCA2, and other DNA repair genes. PARP inhibitors elicit significant responses in ovarian and breast cancers from BRCA1 or BRCA2 mutation carriers. However, many cancers that initially respond to PARP inhibitors eventually develop drug resistance. Thus, it is important to develop new strategies to enhance PARP inhibitor sensitivity and to increase the duration of response. We have identified Salt Induced Kinase 2 (SIK2) inhibitors (ARN3236 and ARN3261) that induce double strand breaks (DSBs) in DNA of HR-competent cells and produce synthetic lethality with multiple PARP inhibitors. SIK2 is an AMP-activated protein kinase (AMPK)-related protein kinase that is required for ovarian cancer cell proliferation and metastasis. SIK2 is overexpressed and correlates with poor prognosis in patients with high-grade serous ovarian carcinoma and triple negative breast cancer. SIK2 inhibition enhances paclitaxel sensitivity in both cancer types. We have demonstrated that olaparib-induced-growth inhibition was significantly enhanced by concurrent treatment with either ARN3236 or ARN3261 in each of 12 ovarian and breast cancer cell lines tested, but not in 3 non-tumorigenic cell lines. Co-administration of olaparib with SIK2 inhibitors suppressed tumor growth and increased the survival of mice with human ovarian (OC316) and breast (MDA-MB-231) cancer xenografts without affecting animal weight. ARN3261 produced little toxicity in preclinical toxicology studies. SIK2 inhibitors decrease the phosphorylation of class-IIa HDAC4/5/7 and abolish class-IIa HDAC 4/5/7-associated transcriptional activity of Myocyte Enhancer Factor 2D (MEF2D). Genome-wide chromatin immunoprecipitation (CHIP) sequencing revealed that SIK2 inhibitors reduce MEF2D binding to regulatory regions with high-chromatin accessibility in DNA repair genes, including FANCD2, EXO1 and XRCC4, resulting in repression of critical genes in DNA DSB repair pathway and induction of apoptosis. In addition, SIK2 inhibitors significantly decreased olaparib-mediated PARP activity and enhanced olaparib-induced cytotoxicity. Together, our data argue that the combination of a SIK2 inhibitor and a PARP inhibitor has the potential to increase the magnitude and duration of PARP inhibitor activity with tolerable toxicity. Use of a SIK2 inhibitor in combination with a PARP inhibitor provides a novel therapeutic strategy for ovarian and triple negative breast cancers with or without BRCA gene mutation. Citation Format: Zhen Lu, Weiqun Mso, Janice M. Santiago-O'Farrill, Hailing Yang, Lan Pang, Ahmed A. Ahmed, Hariprasad Vankayalapati, Robert C. Bast Jr. Novel SIK2 inhibitors sensitize ovarian and breast cancer to PARP inhibitors [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 5210.

Xylosides are small molecules that serve as primers of glycosaminoglycan biosynthesis. Xyloside mediated modulation of biological functions depends on the extent of priming activity and fine structures of primed GAG chains. In earlier studies, copper (Cu) catalyzed synthesis of click-xylosides and their priming activity were extensively documented. In the current study, ruthenium (Ru) mediated catalysis was employed to synthesize xylosides with a 1,5-linkage between the xylose and the triazole ring instead of a 1,4-linkage as found in Cu-catalyzed click-xyloside synthesis. Mono- and bis-click-xylosides were synthesized using each catalytic method and their glycosaminoglycan priming activity was assessed in vitro using a cellular system. Ru-catalyzed click-xylosides showed a higher priming activity as measured by incorporation of radioactive sulfate into primed glycosaminoglycan chains. This study demonstrates that altering the linkage of the aglycone to the triazole ring changes the priming activity. Computational modeling provides a molecular rationale for higher priming ability of Ru-mediated click-xylosides. Higher GAG priming activity is attributed to the formation of more stable interactions between the 1,5-linked xylosides and β-1,4-galactosyltransferase 7 (β4GalT7).

In this work, we describe the use of the rule of 3 fragment-based strategies from biochemical screening data of 1100 in-house, small, low molecular weight fragments. The sequential combination of in silico fragment hopping and fragment linking based on S160/Y161/A162 hinge residues hydrogen bonding interactions leads to the identification of novel 1 H -benzo[ d ]imidazol-2-yl)-1 H -indazol class of Phosphoinositide-Dependent Kinase-1 (PDK1) inhibitors. Consequent SAR and follow-up screening data led to the discovery of two potent PDK1 inhibitors: compound 32 and 35 , with an IC 50 of 80 nM and 94 nM, respectively. Further biological evaluation showed that, at the low nanomolar concentration, the drug had potent ability to inhibit phosphorylation of AKT and p70S6, and selectively kill the cancer cells with mutations in both PTEN and PI3K. The microarray data showed that DUSP6, DUSP4, and FOSL1 were down-regulated in the sensitive cell lines with the compound treatment. The in vivo test showed that 35 can significantly inhibit tumor growth without influencing body weight growth. Our results suggest that these compounds, especially 35 , merit further pre-clinical evaluation.

Purpose: Small cell carcinoma of the ovary-hypercalcemic type (SCCOHT) is a rare ovarian cancer of young women characterized by SWI/SNF ATPase SMARCA4 (BRG1) genetic inactivation alongside epigenetic silencing of the homolog ATPase SMARCA2 (BRM). Because the SWI/SNF complex associates with histone modifying complexes (HMCs) containing targetable epigenetic enzymes, the lack of SWI/SNF ATPase activity is hypothesized to interrupt normal function of HMCs that result in altered epigenetic landscape. Lysine-specific histone demethylase 1 (LSD1 or KDM1) regulates the chromatin landscape and gene expression by removing specific methyl groups from methylated proteins including histone H3. It associates with a number of transcriptional regulatory complexes including SWI/SNF and acts in a context-dependent manner. We and others have found that LSD1 is among the most highly expressed histone modifiers in SCCOHT. SCCOHT cell lines are very sensitive to the reversible LSD1 inhibitor SP-2509 compared to other epigenetic agents like EZH2 and bromodomain inhibitors, suggesting that LSD1 dependence is a defining feature of these SWI/SNF-deficient ovarian cancers. Recently it has been shown that LSD1 inhibition stimulates an INF-dependent anti-tumor immunity and overcomes resistance to checkpoint blockade through induction of Endogenous Retroviruses (ERVs) in breast cancer. Interestingly, SCCOHT have been shown to exhibit an immune-active tumor microenvironment with PD-L1 expression in both tumor and stromal cells that strongly correlated with T-cell infiltration (TIL). In this study we investigate the ability of SP-2509 to promote anti-tumor immunity and T-cell infiltration in SCCOHT. Experimental Design: SCCOHT cell line BIN67, COV434 and SCCOHT1 were subjected to RT-PCR to evaluate ERVs induction and INF expression in response to SP-2509 treatment. SCCOHT cells were also co-cultured with peripheral blood mononuclear cells (PBMCs) in a 3D platform to evaluate T-cell infiltration following SP-2509 treatment. Results: SP-2509 promotes ERV-mediated immune response in SCCOHT cell lines. Additionally, SP-2509 stimulates T-cell infiltration in SCCOHT in a spheroid platform. Conclusion: Our data suggest that the reversible LSD1 inhibitor SP-2509 stimulates an INF-dependent anti-tumor immunity in SCCOHT cells in vitro and in 3D platform. Given that LSD-1 is a potential therapeutic target in SWI/SNF mutated SCCOHT, the combination of SP-2509 and PD-1 inhibitor represents a plausible combination to induce or augment immunogenic responses in these tumors. Citation Format: Raffaella Soldi, Alexis Weston, Trason Thode, Rhonda Lewis, Mohan Kaadige, Hariprasad Vankayalapati, William Hendricks, Sunil Sharma. The reversible LSD1 inhibitor SP-2509 promotes anti-tumor immunity in small cell carcinoma of the ovary-hypercalcemic type (SCCOHT) [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 3869.

Lysine specific demethylase 1 (LSD1) plays an important role in regulating histone lysine methylation at residues K4 and K9 on histone H3 and is an attractive therapeutic target in multiple malignancies. Here we report a structure-based virtual screen of a compound library containing ∼2 million small molecular entities. Computational docking and scoring followed by biochemical screening led to the identification of a novel N'-(1-phenylethylidene)-benzohydrazide series of LSD1 inhibitors with hits showing biochemical IC50s in the 200-400 nM range. Hit-to-lead optimization and structure-activity relationship studies aided in the discovery of compound 12, with a Ki of 31 nM. Compound 12 is reversible and specific for LSD1 as compared to the monoamine oxidases shows minimal inhibition of CYPs and hERG and inhibits proliferation and survival in several cancer cell lines, including breast and colorectal cancer. Compound 12 may be used to probe LSD1's biological role in these cancers.

Purpose: Salt-inducible kinase 2 (SIK2) is a centrosome kinase required for mitotic spindle formation and a potential target for ovarian cancer therapy. Here, we examine the effects of a novel small-molecule SIK2 inhibitor, ARN-3236, on sensitivity to paclitaxel in ovarian cancer. Experimental Design: SIK2 expression was determined in ovarian cancer tissue samples and cell lines. ARN-3236 was tested for its efficiency to inhibit growth and enhance paclitaxel sensitivity in cultures and xenografts of ovarian cancer cell lines. SIK2 siRNA and ARN-3236 were compared for their ability to produce nuclear–centrosome dissociation, inhibit centrosome splitting, block mitotic progression, induce tetraploidy, trigger apoptotic cell death, and reduce AKT/survivin signaling. Results: SIK2 is overexpressed in approximately 30% of high-grade serous ovarian cancers. ARN-3236 inhibited the growth of 10 ovarian cancer cell lines at an IC50 of 0.8 to 2.6 μmol/L, where the IC50 of ARN-3236 was inversely correlated with endogenous SIK2 expression (Pearson r = −0.642, P = 0.03). ARN-3236 enhanced sensitivity to paclitaxel in 8 of 10 cell lines, as well as in SKOv3ip (P = 0.028) and OVCAR8 xenografts. In at least three cell lines, a synergistic interaction was observed. ARN-3236 uncoupled the centrosome from the nucleus in interphase, blocked centrosome separation in mitosis, caused prometaphase arrest, and induced apoptotic cell death and tetraploidy. ARN-3236 also inhibited AKT phosphorylation and attenuated survivin expression. Conclusions: ARN-3236 is the first orally available inhibitor of SIK2 to be evaluated against ovarian cancer in preclinical models and shows promise in inhibiting ovarian cancer growth and enhancing paclitaxel chemosensitivity. Clin Cancer Res; 23(8); 1945–54. ©2016 AACR.

Classic Galactosemia is an autosomal recessive disorder caused by the deficiency of galactose-1-phosphate uridylyltransferase (GALT), one of the key enzymes in the Leloir pathway of galactose metabolism. While the neonatal morbidity and mortality of the disease are now mostly prevented by newborn screening and galactose restriction, long-term outcome for older children and adults with this disorder remains unsatisfactory. The pathophysiology of Classic Galactosemia is complex, but there is convincing evidence that galactose-1-phosphate (gal-1P) accumulation is a major, if not the sole pathogenic factor. Galactokinase (GALK) inhibition will eliminate the accumulation of gal-1P from both dietary sources and endogenous production, and efforts toward identification of therapeutic small molecule GALK inhibitors are reviewed in detail. Experimental and computational high-throughput screenings of compound libraries to identify GALK inhibitors have been conducted, and subsequent studies aimed to characterize, prioritize, as well as to optimize the identified positives have been implemented to improve the potency of promising compounds. Although none of the identified GALK inhibitors inhibits glucokinase and hexokinase, some of them cross-inhibit other related enzymes in the GHMP small molecule kinase superfamily. While this finding may render the on-going hit-to-lead process more challenging, there is growing evidence that such cross-inhibition could also lead to advances in antimicrobial and anti-cancer therapies.

Genomic instability is a recognized hallmark of cancer. Germline mutations in critical DNA-repair and DNA-damage response genes, including BRCA1and BRCA2, predispose to cancer development, but also create vulnerabilities that can be exploited for cancer therapy. The successful development of PARP inhibitors for cancers with BRCA1/2 mutations and a deficiency in homologous recombination DNA repair has provided proof of concept. Inhibition of kinases that promote transcription of DNA repair genes might provide targets that enhance the activity of PARP-inhibitors. One novel candidate, salt-inducible kinase 2 (SIK2), is overexpressed in approximately 30% of high grade serous ovarian cancers. Inhibition of SIK2 induces tetraploidy, triggers apoptotic cell death, prevents metastasis, reduces AKT/survivin signaling and decreases phosphorylation of class-IIa histone deacetylases (HDACs) enhancing inhibition of gene expression. In contrast to other HDACs, class-IIa HDACs function as signal-responsive transcriptional corepressors for the myocyte enhancer factor-2 (MEF2) family of transcription factors. MEF2s play important roles in regulating growth factor responses, neuronal survival, T-cell apoptosis and, importantly, B-cell DNA double strand break (DSB) repair. Here we ask whether inhibition of SIK2 can enhance HDAC class-IIa inhibition of MEF activity, decrease DNA repair and enhance sensitivity to the PARP inhibitor Olaparib in ovarian cancer cells. Treatment with SIK2 inhibitors (ARN3236 or ARN3261) enhanced sensitivity to Olaparib in each of 8 ovarian cancer cell lines without BRCA1/2 mutations (OC316, OVCAR8, IGROV1, A2780, HCC5030, HCC5032, SKOv3 and OVCAR5) and significantly reduced the IC50 of Olaparib in two PARP inhibitor-resistant ovarian cancer cell lines (A2780CP-R and UWB1-289-10R). Synergistic cytotoxicity, judged with a Chou-Talaylay combination index (CI), was observed in all 8 cell lines. Treatment with a SIK2 inhibitor decreased the phosphorylation of class-IIa HDAC4/5/7, abolished class-IIa HDAC4/5/7-associated transcriptional activity of MEF2 and decreased MEF2 binding to regulatory regions with high-chromatin accessibility in DNA repair genes, resulting in repression of critical gene expression in DNA repair pathways. DNA damage, judged by rH2AX expression was enhanced by SIK2 inhibition. These observations not only provide new insights into the transcriptional regulation of DNA repair gene expression, but also have important implications for enhancing sensitivity of high grade serous ovarian cancers to PARP inhibition, with or without BRCA1 or BRCA2 germline mutations. Citation Format: Zhen Lu, Wequn Mao, Lan Pang, Janice M. Santiago-O'Farrill, Haling Yang, Ahmed Ahmed, Hariprasad Vankayalapati, Robert C. Bast. SIK2 inhibitors regulate DNA repair pathway and sensitize ovarian cancer to PARP1 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 324.

The Wnt/β-catenin signaling pathway plays a vital role in cell growth, the regulation, cell development, and the differentiation of normal stem cells. Constitutive activation of the Wnt/β-catenin signaling pathway is found in many human cancers, and thus, it is an attractive target for anticancer therapy. Specific inhibitors of this pathway have been keenly researched and developed. Cell based screening of compounds library, hit-to-lead optimization, computational and structure-based design strategies resulted in the design and synthesis of a series of anthracene-9,10-dione dioxime series of compounds demonstrated potent inhibition of β-catenin in vitro (IC50 < 10 nM, 14) and the growth of several cancer cell lines. This article discusses the potential of inhibiting the Wnt/β-catenin signaling pathway as a therapeutic approach for cancer along with an overview of the development of specific inhibitors.

Aurora A and Aurora B are potential targets for anticancer drug development due to their roles in tumorigenesis and disease progression. To identify small-molecule inhibitors of the Aurora kinases, we undertook a structure-based design approach that used three-dimensional structural models of the Aurora A kinase and molecular docking simulations of chemical entities. Based on these computational methods, a new generation of inhibitors derived from quinazoline and pyrimidine-based tricyclic scaffolds were synthesized and evaluated for Aurora A kinase inhibitory activity, which led to the identification of 4-(6,7-dimethoxy-9H-1,3,9-triaza-fluoren-4-yl)-piperazine-1-carbothioic acid [4-(pyrimidin-2-ylsulfamoyl)-phenyl]-amide. The lead compound showed selectivity for the Aurora kinases when it was evaluated against a panel of diverse kinases. Additionally, the compound was evaluated in cell-based assays, showing a dose-dependent decrease in phospho-histone H3 levels and an arrest of the cell cycle in the G2-M fraction. Although biological effects were observed only at relatively high concentrations, this chemical series provides an excellent starting point for drug optimization and further development. [Mol Cancer Ther 2006;5(7):1764–72]

Psorospermin is a natural product that has been shown to have activity against drug-resistant leukemia lines and AIDS-related lymphoma. It has also been shown to alkylate DNA through an epoxide-mediated electrophilic attack, and this alkylation is greatly enhanced at specific sites by topoisomerase II. In this article, we describe the synthesis of the two diastereomers of O5-methyl psorospermin and their in vitro activity against a range of solid and hematopoietic tumors. The diastereomeric pair (±)-(2′R,3′R) having the naturally occurring enantiomer (2′R,3′R) is the most active across all the cell lines and shows approximately equal activity in both drug-sensitive and drug-resistant cell lines. In subsequent studies using all four enantiomers of O5-methyl psorospermin, the order of biological potency is (2′R,3′R) > (2′R,3′S) = (2′S,3′R) > (2′S,3′S). This order of potency is also found in the topoisomerase II–induced alkylation of O5-methyl psorospermin and can be rationalized by molecular modeling of the psorospermin-duplex binding complex. Therefore, this study defines the optimum stereochemical requirements for both the topoisomerase II–induced alkylation of DNA and the biological activity by psorospermin and its O5-methyl derivatives. Finally, (2′R,3′R) psorospermin was found to be as effective as gemcitabine in slowing tumor growth in vivo in a MiaPaCa pancreatic cancer model. In addition, (2′R,3′R) psorospermin in combination with gemcitabine was found to show an at least additive effect in slowing tumor growth of MiaPaCa.

The antileukemic xanthone psorospermin is a topoisomerase II-dependent DNA alkylator in advanced preclinical development. Efforts have been made to further understand the structural requirements of its mechanism of action through the synthesis of ring-constrained analogues, based on the skeleton of the bisfuranoxanthone natural products. Molecules were designed that contain the bisfuran and xanthone portions of naturally occurring psorofebrins, and molecular modeling was used to assess their DNA alkylating potential and to refine the structures. A short, diastereoselective synthetic process to access bisfuranoxanthones was developed, culminating in the first total synthesis of (+/-)-isohydroxypsorofebrin. Two compounds designed and synthesized were of particular interest, chlorohydrin 7 and epoxide 6, which are reactive analogues of the natural product isohydroxypsorofebrin. The chlorohydrin retains the psorospermin-like DNA alkylation characteristics despite its rigid structure and high innate affinity for DNA. Molecular modeling has been used to rationalize the increased activity of the chlorohydrin. The chlorohydrin and epoxide show increased cytotoxicity compared to isohydroxypsorofebrin against a range of human tumor cell lines.

Glycosyl phosphates have emerged as useful glycosyl donors for stereoselective glycosidic bond formation in the synthe- sis of oligosaccharides. In this account, we describe some of our work in developing and further expanding this methodology, partic- ularly with the use of propane-1,3-diyl phosphate as the anomeric leaving group. We have studied the reactions of several glycosyl propane-1,3-diyl phosphates with or without C-2 participating groups with a range of glycosyl acceptors in the presence of trime- thylsilyl triflate in either stoichiometric or catalytic amount, and demonstrated their application in the synthesis of several disaccha- rides and also the synthesis of some trisaccharides.

ARN-3261 is an orally bioavailable small molecule inhibitor of the Salt Inducible Kinase 2 (SIK2, 11 nM) and SIK3 (19 nM). Three isoforms of SIK (SIKs) proteins have been reported: SIK1 (SNF1LK), SIK2 (QIK), and SIK3 (QSK). They are the Ser/Thr centrosome kinase family members required for bipolar mitotic spindle formation. The overexpression of SIK2 kinase in 30% of ovarian cancer specimens allows a novel, clinically important new method of treating ovarian cancer by blocking SIK2 kinase activity. In addition to a role in ovarian cancer, SIK2 and SIK3 are prevalent in several other tumor types, including breast, prostate diffuse large B-cell lymphoma, and melanoma cancers. Inhibition of SIK2 has been reported to cause centrosome splitting in interphase, while SIK2 depletion blocked centrosome separation in mitosis and sensitized ovarian cancers to paclitaxel in culture and in vivo Xenograft models. Depletion of SIK2 also delayed G1/S transition and reduced AKT phosphorylation. Higher levels of expression of SIK2 have been shown to be highly correlated with poor survival in patients with high-grade serous ovarian cancers. Using the homology structure of SIK2, fragment-based lead optimization strategies, and screening and structure-activity relationship efforts, we have discovered ARN-3261, a first-in-class novel, selective inhibitor of SIK2 that could prove useful in treating ovarian, endometrial, primary peritoneal, fallopian tube, and triple negative breast cancers. ARN-3261 specifically inhibited SIK2-expressed SKOv3 cells with an IC50 of 92 nM. ARN3261 was effective against ovarian, breast cancer cell lines alone and in combination with Paclitaxel and Cisplatin. ARN-3261 also inhibited ovarian tumor growth significantly at 70% in SKOv3 human ovarian cancer Xenografts in Ncr nu/nu mice in a dose dependent manner at 20, 40, 60, and 100 mg/kg orally. Moreover, ARN-3261 has exhibited excellent in vivo pharmacokinetic, pharmacodynamics, and correlative PK/PD and ADME characteristics. Preliminary in vitro and in vivo tumor up-take studies suggest that ARN-3261 blocks centrosome separation by inhibiting SIK2, thereby enhancing the sensitivity of Paclitaxel. Encouraged by these results, we initiated the IND enabling GLP-Toxicology and safety studies to bring ARN-3261 to the clinic for First-In-Human (FIH) Phase 1 trials. The non-clinical pharmacology data along with Phase 1 POC clinical trial plans will be presented. Citation Format: Hariprasad Vankayalapati, Venkatakrishnareddy Yerramreddy, Jinhua Zhou, Jeffrey A. Handler, Rajendra P. Appalaneni, Ramamohan R. Kancherla, Roy J. Wu, Hiroshi Takemori, Angelique Whitehurst, Amir Anthony Jazaeri, Robert L. Coleman, Zhen Lu, Robert C. Bast. Discovery of ARN-3261 as a potent, selective, orally available SIK2 inhibitor for treating ovarian, endometrial, primary peritoneal, fallopian tube, and triple negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-296. doi:10.1158/1538-7445.AM2017-LB-296

α- and β-Glycosyl cyanides, per-O-acetyl-1,2-O-1-cyanoethylidenes and C-allyl glycopyranosides were efficiently prepared by treatment of 2,3,4-tri-O-acetyl-α,β- l -rhamno-, l -fuco- and 2,3,4,6-tetra-O-acetyl-α,β- d -galactopyranosyl propane-1,3-diyl phosphates with trimethylsilyl cyanide (TMSCN) and allyltrimethylsilane in the presence of trimethylsilyl triflate (TMSOTf). Similarly 2,3,4,6-tetra-O-benzyl-α,β- d -manno- and d -glucopyranosyl propane-1,3-diyl phosphates were employed in the synthesis of C-glycosides.

Glycosyl donors having a diphenylphosphinate and a propane-1,3-diyl phosphate leaving group were easily prepared by the addition of the anomeric hydroxyl group of 2,3,4,6-tetra- O -benzyl-α,β- d -glucopyranose to diphenylphosphinic and propane-1,3-diyldioxyphosphoryl chlorides. These glycosyl donors were selectively glycosylated with a number of primary and secondary oxygen nucleophiles in the presence of trimethylsilyl triflate (TMSOTf). The use of 1,3-diyl phosphate resulted in the stereoselective formation of β- O -linked glycosides.

The synthesis of the disaccharides α-L-Fucp-(1,2)- and α-L-Fucp-(1,3)-β-D-Galp(1)-4-methylumbelliferone as fucosidase substrates was accomplished by activation of the anomeric centre of 2,3,4-tri-O-acetyl- and 2,3,4,6-tetra-O-acetyl-α,β-L-fuco- and -D-galactopyranosyl propane-1,3-diyl phosphates with TMSOTf and with minimal protection of 4-methylumbelliferyl β-D-galactopyranoside.

Activation of the anomeric centre of 1- O -2,3,4,6-tetra- O -benzyl- d -mannopyranosyl propane-1,3-diyl phosphate in the presence of trimethylsilyl triflate allowed the preparation of α- or β-manno-linked glycosides.

Activation of the anomeric centre of suitably protected l -fucopyranose and d -galactopyranose with propane-1,3-diyl phosphate allowed the synthesis of the disaccharide α- l -Fucp-(1,2)-β- d -Galp(1)-4-methylumbelliferone as a fucosidase substrate.

We present the discovery and optimization of a novel series of inhibitors of bacterial UDP-N-acetylglucosamine 2-epimerase (called 2-epimerase in this paper). Starting from virtual screening hits, the activity of various inhibitory molecules was optimized using a combination of structure-based and rational design approaches. We successfully designed and identified a 2-epimerase inhibitor (compound 12-ES-Na, that we named Epimerox) which blocked the growth of methicillin-resistant Staphylococcus aureus (MRSA) at 3.9 μM MIC (minimum inhibitory concentration) and showed potent broad-range activity against all Gram-positive bacteria that were tested. Additionally a microplate coupled assay was performed to further confirm that the 2-epimerase inhibition of Epimerox was through a target-specific mechanism. Furthermore, Epimerox demonstrated in vivo efficacy and had a pharmacokinetic profile that is consonant with it being developed into a promising new antibiotic agent for treatment of infections caused by Gram-positive bacteria.

We identified an essential cell wall biosynthetic enzyme in Bacillus anthracis and an inhibitor thereof to which the organism did not spontaneously evolve measurable resistance. This work is based on the exquisite binding specificity of bacteriophage-encoded cell wall-hydrolytic lysins, which have evolved to recognize critical receptors within the bacterial cell wall. Focusing on the B. anthracis-specific PlyG lysin, we first identified its unique cell wall receptor and cognate biosynthetic pathway. Within this pathway, one biosynthetic enzyme, 2-epimerase, was required for both PlyG receptor expression and bacterial growth. The 2-epimerase was used to design a small-molecule inhibitor, epimerox. Epimerox prevented growth of several Gram-positive pathogens and rescued mice challenged with lethal doses of B. anthracis. Importantly, resistance to epimerox was not detected (

Ovarian carcinomas account for 4% of total cancers in women in the United States. Improved outcomes might be attained if sensitivity to primary chemotherapy were enhanced. A recent study discovered that the salt inducible kinase 2 (SIK2) plays a key role in the initiation of mitosis and regulates paclitaxel sensitivity. Here we show that SIK2 is overexpressed in 30% ovarian cancer specimens, associated with a poor prognosis. ARN-3236, a selective, highly potent small molecule SIK2 inhibitor with function similar to SIK2 siRNA, blocks cell proliferation in a panel of ovarian cancer cell lines, where the IC50 of ARN-3236 was inversely correlated with endogenous SIK2 expression. ARN-3236 also enhanced response to paclitaxel in ovarian cancer cells (OC316, SKOv3, A2780, HEY, ES2 and UPN251). Similar to the function of SIK2 siRNA, ARN-3236 effectively inhibits AKT phosphorylation at Ser473 and Tyr308, as well as the expression of the downstream effector survivin. ARN-3236 uncouples the centrosome from the nucleus in interphase, and blocks centrosome separation in mitosis, resulting in the accumulation of cells in prometaphase. In addition, ARN-3236 enhances tumor growth inhibition of paclitaxel in ovarian cancer xenograft models. Thus, functional inhibition of SIK2 with ARN-3236 had the same effects on ovarian cancer cell function as knockdown of SIK2 with siRNA. ARN-3236 deserves further study as a SIK2 inhibitor that significantly improves the sensitivity to paclitaxel for treatment of human ovarian cancer cells and xenografts. Citation Format: Jinhua Zhou, Albandri Alfredi, Shu Zhang, Janice M. Santiago-O’Farrill, Ahmed A. Ahmed, Hailing Yang, Weiqun Mao, Yan Wang, Hariprasad Vankayalapati, Zhen Lu, Robert C. Bast Jr. A novel compound ARN-3236 inhibits SIK2 and sensitizes ovarian cancer to paclitaxel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3032.

The biosyntheses of isoprenoids is essential for the survival in all living organisms, and requires one of the two biochemical pathways: (a) Mevalonate (MVA) Pathway or (b) Methylerythritol Phosphate (MEP) Pathway. The latter pathway, which is used by all Gram-negative bacteria, some Gram-positive bacteria and a few apicomplexan protozoa, provides an attractive target for the development of new antimicrobials because of its absence in humans. In this report, we describe two different approaches that we used to identify novel small molecule inhibitors of Escherichia coli and Yersinia pestis 4-diphosphocytidyl-2-C-methyl D-erythritol (CDP-ME) kinases, key enzymes of the MEP pathway encoded by the E. coli ispE and Y. pestis ipk genes, respectively. In the first approach, we explored existing inhibitors of the GHMP kinases while in the second approach; we performed computational high-throughput screening of compound libraries by targeting the CDP-ME binding site of the two bacterial enzymes. From the first approach, we identified two compounds with 6-(benzylthio)-2-(2-hydroxyphenyl)-4-oxo-3,4-dihydro-2H-1,3-thiazine-5-carbonitrile and (Z)-3-methyl-4-((5-phenylfuran-2-yl)methylene)isoxazol-5(4H)-one scaffolds which inhibited Escherichia coli CDP-ME kinase in vitro. We then performed substructure search and docking experiments based on these two scaffolds and identified twenty three analogs for structure-activity relationship (SAR) studies. Three new compounds from the isoxazol-5(4H)-one series have shown inhibitory activities against E. coli and Y. pestis CDP-ME kinases with the IC50 values ranging from 7μM to 13μM. The second approach by computational high-throughput screening (HTS) of two million drug-like compounds yielded two compounds with benzenesulfonamide and acetamide moieties which, at a concentration of 20μM, inhibited 80% and 65%, respectively, of control CDP-ME kinase activity.

Activation of the anomeric centre of 1- O -2,3,4,6-tetra- O -benzyl- d -mannopyranosyl propane-1,3-diyl phosphate in the presence of trimethylsilyl triflate allowed the preparation of α- or β-manno-linked glycosides.

α- and β-Glycosyl cyanides, per-O-acetyl-1,2-O-1-cyanoethylidenes and C-allyl glycopyranosides were efficiently prepared by treatment of 2,3,4-tri-O-acetyl-α,β- l -rhamno-, l -fuco- and 2,3,4,6-tetra-O-acetyl-α,β- d -galactopyranosyl propane-1,3-diyl phosphates with trimethylsilyl cyanide (TMSCN) and allyltrimethylsilane in the presence of trimethylsilyl triflate (TMSOTf). Similarly 2,3,4,6-tetra-O-benzyl-α,β- d -manno- and d -glucopyranosyl propane-1,3-diyl phosphates were employed in the synthesis of C-glycosides.

Glycosyl phosphates have emerged as useful glycosyl donors for stereoselective glycosidic bond formation in the synthe- sis of oligosaccharides. In this account, we describe some of our work in developing and further expanding this methodology, partic- ularly with the use of propane-1,3-diyl phosphate as the anomeric leaving group. We have studied the reactions of several glycosyl propane-1,3-diyl phosphates with or without C-2 participating groups with a range of glycosyl acceptors in the presence of trime- thylsilyl triflate in either stoichiometric or catalytic amount, and demonstrated their application in the synthesis of several disaccha- rides and also the synthesis of some trisaccharides.

Topoisomerase II, an enzyme that catalyzes changes in the topology of DNA, plays several key roles in DNA metabolism and chromosome structure, and it is the primary cytotoxic target for a number of clinically important DNA intercalating agents such as doxorubicin. It seems likely that if these intercalating topoisomerase II poisons are structurally modified to also be DNA alkylating agents, they will have increased dwell time on the topoisomerase II-DNA complex and increased potency and selectivity for cancer cells. On the basis of insights into the mechanisms of action of psorospermin and the quinobenzoxazine A-62176 and molecular modeling studies of these compounds with duplex DNA, we have designed and synthesized a series of novel hybrid DNA-interactive compounds that alkylate DNA most efficiently at sequences directed by topoisomerase II. The epoxydihydrofuran ring of psorospermin was used as a DNA alkylating moiety, and this was fused to the pyridobenzophenoxazine ring of A-62176. The chlorohydrin ring opened form of the epoxide was also prepared and tested. These hybrid compounds showed enhanced DNA alkylating activity in the presence of topoisomerase II, exhibited significant activity against all the cancer cells tested at submicromolar concentrations, and were more potent than both parent compounds. However, the biochemical assays indicated that they lost some of the topoisomerase II and Mg(2+) dependency for reaction with DNA that is associated with psorospermin and A-62176, respectively.

Salt Inducible Kinase 2 (SIK2) is a Ser/Thr kinase required for centrosome splitting and bipolar mitotic spindle formation. SIK2 is overexpressed in 30% of ovarian cancers associated with a poor prognosis. Knockdown of SIK2 with siRNA has inhibited ovarian cancer cell growth and enhanced paclitaxel sensitivity in cell culture and in xenografts. Given the challenges of delivering siRNA to cancer cells in vivo, we have sought small molecule inhibitors of SIK2 to permit clinical trials, alone and in combination with paclitaxel. Fragment-based design strategies utilizing SIK2 ATP-binding site residues, scaffold-hopping, SIK2 binding assay and subsequent SAR have led to the discovery of potent, selective, orally bioavailable first-in-class SIK2 inhibitors. Here we describe the identification and the preclinical characterization of ARN-3236 and its novel analogue ARN-3252 belonging to the same chemical class, suitable for oral administration. ARN-3236 and ARN-3252 compounds are highly potent (IC50: 1 and 5 nM), ATP competitive SIK2 inhibitors, characterized by high selectivity when tested against a panel of more than 456 kinases. ARN-3236 and ARN-3252 potently blocked proliferation of human ovarian cancer cell lines with endogenous SIK2 activation including OVCAR-3, SKOv3, HEY and ES-2 with an IC50 between 0.5 to 3.0 nM. Inhibition of proliferation has been observed in 9 additional ovarian cancer cell lines. The ARN-3236 and ARN-3252 exhibit a promising in vitro ADME profile and favorable in vivo PK parameters in mice and rats, including oral bioavailability. When tested in vivo in three murine subcutaneous xenograft models employing OVCAR-3, SKOv3 and ES-2 cells, ARN-3236 displayed dose-dependent tumor growth inhibition following daily oral administration at 30, 60 and 100 mg/Kg, with tumor regression observed at these dose levels. The lead identification, optimization, SAR, PK, single agent efficacy, efficacy in combination with paclitaxel and dose-dependent target modulation and the maintenance of SIK2 phosphorylation inhibition data will be presented. Given these specific small molecule inhibitors, SIK2 may provide a valuable target for treatment of a subset of human ovarian cancers. Citation Format: Albandri Alfredi, Shu Zhang, Weiqu Mao, Yan Wang, Hiroshi Takemori, Zhen Lu, Robert C. Bast, Hariprasad Vankayalapati. Highly potent and orally available SIK2 inhibitors block growth of human ovarian cancer cells in culture and xenografts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 749. doi:10.1158/1538-7445.AM2014-749

The synthesis of the disaccharides α-L-Fucp-(1,2)- and α-L-Fucp-(1,3)-β-D-Galp(1)-4-methylumbelliferone as fucosidase substrates was accomplished by activation of the anomeric centre of 2,3,4-tri-O-acetyl- and 2,3,4,6-tetra-O-acetyl-α,β-L-fuco- and -D-galactopyranosyl propane-1,3-diyl phosphates with TMSOTf and with minimal protection of 4-methylumbelliferyl β-D-galactopyranoside.

Synthesis of octyl-O-beta-D-mannopyranoside, a caloporoside analogue was achieved by the activation of 2,3,4,6-rerra-O-benzyl-1-O-1',3'2'-dioxaphosphacyclohexane-a lpha,beta-D-mannopyranosyl-2-oxide with TMSOTf (Trimethyl silyl triflate) and subsequent debenzylation. At 100 microM the molecule significantly and reversibly increased the magnitude of GABA(A) currents evoked in cultured rat pyramidal neurones whilst concomitantly reducing the incidence of spontaneous synaptic activity. These results contradict earlier proposals that such molecules bind to the TBPS (tert-Butylbicyclophosphorothionate) site to block the chloride channel.

Telomestatin is a natural product isolated from Streptomyces anulatus 3533-SV4 and has been shown to be a very potent telomerase inhibitor. The structural similarity between telomestatin and a G-tetrad suggested to us that the telomerase inhibition might be due to its ability either to facilitate the formation of or trap out preformed G-quadruplex structures, and thereby sequester single-stranded d[T(2)AG(3)](n) primer molecules required for telomerase activity. Significantly, telomestatin appears to be a more potent inhibitor of telomerase (5 nM) than any of the previously described G-quadruplex-interactive molecules. In this communication we provide the first experimental evidence that telomestatin selectively facilitates the formation of or stabilizes intramolecular G-quadruplexes, in particular, that produced from the human telomeric sequence d[T(2)AG(3)](4). A simulated annealing (SA) docking approach was used to study the binding interactions of telomestatin with the intramolecular antiparallel G-quadruplex structure. Each intramolecular G-quadruplex molecule was found to bind two telomestatin molecules (unpublished results). A 2:1 model for the telomestatin bound in the external stacking mode in an energy minimized complex with the human telomeric basket-type G-quadruplex was constructed. Our observation that a G-quadruplex-interactive molecule without significant groove interactions is able to reorient in a G-quadruplex structure proints to the importance of core interaction with an asymmetric G-quadruplex structure in producing selective binding. Furthermore, the G-quadruplex interactions of telomestatin are more selective for the intramolecular structure in contrast to other G-quadruplex-interactive agents, such as TMPyP4.

The PDK1 is a monomeric serine/threonine kinase and a promising oncology target of significant current interest for drug development due to its central role in the PI3K/AKT/mTOR pathway. It phosphorylates highly conserved Ser or Thr residues in the activation loop of several AGC super family kinases including PKC, SGK, PKB/Akt, p70S6K, and PDK-1 itself. Approximately, 40-50% of all tumors involve mutations in the PTEN protein, which results in elevated levels of PIP3 and enhanced activation of PKB/AKT, p70S6K, and SGK. It has been proposed that inhibitors of PDK-1 could provide a valuable therapeutic approach to targeting cancer, particularly those with PTEN deficiencies. Using a fragment-based design strategy, we screened a collection of 1100 low molecular weight (< 250 MW) fragments against the PDK1 kinase and identified 9 fragments with moderate inhibitory activity against PDK1 (IC50 values from 45-82 μM). Of these fragments, those based off of a 5-Br,4-I-1H-indazol-3-amine scaffold seemed the most promising based on initial activity and in keeping with the “rule of 3” for fragment-based drug design. Subsequent molecular docking studies using a crystal structure of PDK1 allowed for the structural rationalization of how these fragments bound in the ATP-binding pocket (hydrogen bonding to S160/A162 hinge residues) and provided insight for further optimization. Concurrently, we carried out scaffold-hopping searches at 2-site points for hydrophobic and solvent pocket fragments. With the addition of one fused heterocyclic ring, the potency increased to 8.8 and 10.9 μM. Our systematic fragment based workflow led to the preparation of target molecules in 4 steps beginning with the condensation, cyclization, and reduction and finally installing the hydrophobic binding site fragments under normal amide coupling. Subsequent SAR and follow-up screening led to the discovery of HCI-1680, a potent PDK1 inhibitor with an IC50 of 97 nM. Additional productive interactions sites with PDK1 were introduced to further improve both biochemical and cellular activities in panel of cancer cells. In cell-based assays HCI-1680 demonstrated enhanced activity in PTEN-deficient cell lines compared to PTEN-wild type lines. HCI-1680 and additional compounds from this series were also shown to inhibit the activation of AKT and other downstream signaling molecules. Kinase selectivity profiling, additional cell-based assays, and animal studies are ongoing. The details of these studies will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1368. doi:10.1158/1538-7445.AM2011-1368

Axl and Mer kinases belong to the TAM family that was first identified as a transforming gene in chronic myeloid leukemia. Its intracellular region has the typical receptor tyrosine kinase (RTK) structure and its extracellular domain is similar to cadherin-type adhesion molecules in that it is composed of fibronectin type II and immunoglobulin (Ig) motifs. Axl binds various growth factors, with vitamin K-dependent protein growth-arrest-specific gene 6 (GAS6) being the best studied. Axl is involved in mesenchymal and neuronal development as well as in cell survival, adhesion and blood vessel function. High levels of Axl are found in various tumors, including malignant glioma and metastatic colon, ovarian and breast cancers, and it plays a role in cancer invasion. Recent development of Axl inhibitors suggested that inhibition of Axl activity may be impact both tumor angiogenesis and tumor growth. We describe here the design, synthesis, molecular modeling, and biological evaluation of a series of small molecule, inhibitors of Axl and Mer kinases. Our initial lead compound was identified via cross-docking experiments utilizing the homology model of Axl kinase and screening of a diverse in-house chemical library. We subsequently carried out structure-activity relationship studies and optimized the lead structure which has 2 to 6.1 μM inhibition activity to 2-fold improvement in the Axl and Mer kinase activities to 730 nM. As a starting point for further optimization, it was considered that the modest of these series could be improved by introducing a spacer at the aryl piperazine moiety and focused on maximizing the in vitro potency, addressing the SAR and molecular properties. We further explored Leu620, hydrophobic, Gly543, Phe547 and DFG motif sites through scaffold hoping lead to the identification of an electron-withdrawing functional groups lead to the compound HCI-2084 and HCI-2091 which exhibited potent Axl kinase inhibition activity of 7 and 12 nM in Axl kinase assay and 30-50 nM is panel of cancer cell lines. These novel series of compounds were synthesized in two-step procedure via a standard cross-coupling reaction or with various substituted aryl amine derivatives under amination conditions. In a subsequent step we employed Buchwald-Hartwig amination reaction to prepare target molecules. Due to the ready synthesis accessibility, we extensively investigated the SAR improving the RO5, solubility and permeability parameters. The details of these results will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3609. doi:10.1158/1538-7445.AM2011-3609

Bruton's tyrosine kinase (BTK) is a cytoplasmic nonreceptor tyrosine kinase belonging to the Tec family of kinases. Critical for its function, BTK contains a pleckstrin homology (PH) domain and Src homology SH3 and SH2 domains. It signals downstream of the B-cell receptor (BCR) and is centrally involved in B-cell development. Activation of B-cells by various ligands is accompanied by the translocation of BTK to the cell membrane where it binds phosphatidylinositol-3,4,5-trisphosphate through its PH domain. Activation of BTK results in downstream signaling through the PI3K/AKT, PLCγ, NFκB, and other signaling pathways important for B-cell development and function. Recent reports have shown the aberrant expression and function of BTK in some cancers, including B-cell malignancies. We describe here the design, synthesis, molecular modeling, and biological evaluation of a series of small molecule, inhibitors of BTK kinase. Our initial lead compounds were identified via cross-docking experiments utilizing the crystal structure of BTK kinase and screening a previously in-house explored kinase inhibitor scaffold. We subsequently carried out structure-activity relationship studies and optimized the lead structures, which have IC50 activities in the range of 1 to 10μM against BTK. A critical step in the optimization of this chemical series against BTK was to explore the possibility of adding a Michael's acceptor group to react with Cys481 in the ATP-binding pocket of BTK. Optimization efforts yielded the currently best leads, HCI-1684 and HCI-1685, which inhibit BTK with IC50 values of 12 and 45 nM, respectively. Modeling data suggest these compounds irrepressibly bind in the ATP-binding pocket of BTK. HCI-1684, HCI-1685 and other lead compounds were further evaluated in cell-based assays and were demonstrated to inhibit BTK function downstream of BCR activation. This series of BTK inhibitors were shown to decrease phospho-PLCγ1/2 levels and other downstream phosphorylation events in malignant B-cell cell lines, such as Ramos B. Compound optimization and biological evaluation of this chemical series and evaluation in animal pharmacodynamic endpoint studies will be presented. Taken together, these results suggest BTK is a potential therapeutic target in cancer and that HCI-1684 is an exciting agent to potentially treat B-cell malignancies Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2788. doi:10.1158/1538-7445.AM2011-2788

JAK2 is an intracellular protein tyrosine kinase whose dysregulation has been implicated in leukemia, lymphoma, and myeloproliferative disorders (MPD). Increased kinase activity of JAK2, caused by point mutation of the JH2 autoinhibitory region or formation of JAK2 fusion proteins, causes increased activation of downstream signaling pathways affecting cell differentiation, proliferation, migration, and apoptosis. Through the use of CLIMB™, our proprietary drug discovery process, the published JAK2 crystal structure was used to build several models that were then used as a substrate for in silico docking of 2.3 million virtual small molecule compounds to generate a subset of leads based on calculated binding energies. These leads were further screened using a number of in silico physicochemical and ADMET prediction algorithms to determine “druggable” leads which were most likely to be successful in a biological context. Lead JAK2 inhibitor candidates exhibit low nanomolar IC50 activity against the JAK2 and JAK2 V617F mutant enzymes. Cancer cell lines expressing either the wild-type or mutant JAK2 enzyme demonstrate sensitivity to these inhibitors resulting in IC50 values in low micromolar to nanomolar range. Consistent with the inhibition of the JAK2 enzyme, activity of downstream signaling partners are severely decreased. The phosphorylation level of STAT5, a downstream modulator of JAK2 signaling, in treated HEL cell lysates was analyzed by western blot analysis. These results showed that lead JAK2 candidates caused an inhibition of STAT5 phosphorylation at a low nanomolar EC50. This series of compounds are currently being tested in in vivo xenograft models. Evaluation of lead candidates in biochemical assays against the hERG and CYP450 enzymes showed that these compounds have little inhibitory activity against these enzymes. SuperGen’s lead selective JAK2 inhibitors exhibit potent inhibition of wild-type and mutant JAK2 kinase activity translating into potent inhibition of cellular signaling pathways and cancer cell proliferation.