Effect of PAWI-2 on pancreatic cancer stem cell tumors.

Worldwide, pancreatic cancer (PC) is a major health problem and almost 0.5 million people were diagnosed with PC in 2020. In the United States, more than 64,000 adults will be diagnosed with PC in 2023. PC is highly resistant to currently available treatments and standard of care chemotherapies cause serious side effects. Most PC patients are resistant to clinical therapies. Combination therapy has showed superior efficacy over single-agent treatment. However, most therapy has failed to show a significant improvement in overall survival due to treatment-related toxicity. Developing efficacious clinically useful PC therapies remains a challenge. Herein, we show the efficacy of an innovative pathway modulator, p53-Activator Wnt Inhibitor-2 (PAWI-2) against tumors arising from human pancreatic cancer stem cells (i.e., hPCSCs, FGß3 cells). PAWI-2 is a potent inhibitor of tumor growth. In the present study, we showed PAWI-2 potently inhibited growth of tumors from hPCSCs in orthopic xenograft models of both male and female mice. PAWI-2 worked in a non-toxic manner to inhibit tumors. Compared to vehicle-treated animals, PAWI-2 modulated molecular regulators of tumors. Anti-cancer results showed PAWI-2 in vivo efficacy could be correlated to in vitro potency to inhibit FGß3 cells. PAWI-2 represents a safe, new approach to combat PC.

Human-induced pluripotent stem cell-derived cardiomyocytes: Cardiovascular properties and metabolism and pharmacokinetics of deuterated mexiletine analogs.

Prolongation of the cardiac action potential (AP) and early after depolarizations (EADs) are electrical anomalies of cardiomyocytes that can lead to lethal arrhythmias and are potential liabilities for existing drugs and drug candidates in development. For example, long QT syndrome-3 (LQTS3) is caused by mutations in the Nav 1.5 sodium channel that debilitate channel inactivation and cause arrhythmias. We tested the hypothesis that a useful drug (i.e., mexiletine) with potential liabilities (i.e., potassium channel inhibition and adverse reactions) could be re-engineered by dynamic medicinal chemistry to afford a new drug candidate with greater efficacy and less toxicity. Human cardiomyocytes were generated from LQTS3 patient-derived induced pluripotent stem cells (hIPSCs) and normal hIPSCs to determine beneficial (on-target) and detrimental effects (off-target) of mexiletine and synthetic analogs, respectively. The approach combined "drug discovery" and "hit to lead" refinement and showed that iterations of medicinal chemistry and physiological testing afforded optimized compound 22. Compared to mexiletine, compound 22 showed a 1.85-fold greater AUC and no detectable CNS toxicity at 100 mg/kg. In vitro hepatic metabolism studies showed that 22 was metabolized via cytochrome P-450, as previously shown, and by the flavin-containing monooxygenase (FMO). Deuterated-22 showed decreased metabolism and showed acceptable cardiovascular and physicochemical properties.

Human iPSC-derived cardiomyocytes and pyridyl-phenyl mexiletine analogs.

In the United States, approximately one million individuals are hospitalized every year for arrhythmias, making arrhythmias one of the top causes of healthcare expenditures. Mexiletine is currently used as an antiarrhythmic drug but has limitations. The purpose of this work was to use normal and Long QT syndrome Type 3 (LQTS3) patient-derived human induced pluripotent stem cell (iPSC)-derived cardiomyocytes to identify an analog of mexiletine with superior drug-like properties. Compared to racemic mexiletine, medicinal chemistry optimization of substituted racemic pyridyl phenyl mexiletine analogs resulted in a more potent sodium channel inhibitor with greater selectivity for the sodium over the potassium channel and for late over peak sodium current.

Antiarrhythmic Hit to Lead Refinement in a Dish Using Patient-Derived iPSC Cardiomyocytes.

Ventricular cardiac arrhythmia (VA) arises in acquired or congenital heart disease. Long QT syndrome type-3 (LQT3) is a congenital form of VA caused by cardiac sodium channel (INaL) SCN5A mutations that prolongs cardiac action potential (AP) and enhances INaL current. Mexiletine inhibits INaL and shortens the QT interval in LQT3 patients. Above therapeutic doses, mexiletine prolongs the cardiac AP. We explored structure-activity relationships (SAR) for AP shortening and prolongation using dynamic medicinal chemistry and AP kinetics in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using patient-derived LQT3 and healthy hiPSC-CMs, we resolved distinct SAR for AP shortening and prolongation effects in mexiletine analogues and synthesized new analogues with enhanced potency and selectivity for INaL. This resulted in compounds with decreased AP prolongation effects, increased metabolic stability, increased INaL selectivity, and decreased avidity for the potassium channel. This study highlights using hiPSC-CMs to guide medicinal chemistry and "drug development in a dish".

Small-molecule probe reveals a kinase cascade that links stress signaling to TCF/LEF and Wnt responsiveness.

Wnt signaling plays a central role in tissue maintenance and cancer. Wnt activates downstream genes through ß-catenin, which interacts with TCF/LEF transcription factors. A major question is how this signaling is coordinated relative to tissue organization and renewal. We used a recently described class of small molecules that binds tubulin to reveal a molecular cascade linking stress signaling through ATM, HIPK2, and p53 to the regulation of TCF/LEF transcriptional activity. These data suggest a mechanism by which mitotic and genotoxic stress can indirectly modulate Wnt responsiveness to exert coherent control over cell shape and renewal. These findings have implications for understanding tissue morphogenesis and small-molecule anticancer therapeutics.

Pancreatic cancer drug-sensitivity predicted by synergy of p53-Activator Wnt Inhibitor-2 (PAWI-2) and protein biomarker expression.

Today, pancreatic cancer (PC) is a major health problem in the United States. It remains a challenge to develop efficacious clinically useful PC therapies. New avenues, based on translational approaches and innovative validated biomarkers could be a preclinical option to evaluate PC drug candidates or drug combinations before clinical trials. Herein, we describe evaluation of combination therapies by incorporating a novel pathway modulator, p53-Activator Wnt Inhibitor-2 (PAWI-2) with other FDA-approved cancer drugs that have been used in PC clinical trials. PAWI-2 is a potent inhibitor of drug-resistant PC cells that has been shown to selectively ameliorate human pancreatic cancer stem cells (i.e., hPCSCs, FGß3 cells). In the present study, we showed PAWI-2 produced therapeutic synergism with certain types of anti-cancer drugs. These drugs themselves oftentimes do not ameliorate PC cells (especially PCSCs) due to high levels of drug-resistance. PAWI-2 has the ability to rescue the potency of drugs (i.e., erlotinib, trametinib) and inhibit PC cell growth. Key molecular regulators of PAWI-2 could be used to predict synergistic/antagonistic effects between PAWI-2 and other anti-cancer drugs. Anti-cancer results showed potency could be quite accurately correlated to phosphorylation of optineurin (OPTN) in PC cells. Synergism/antagonism was also associated with inhibition of PCSC marker SOX2 that was observed in FGß3 cells. Synergism broadens the potential use of PAWI-2 as an adjunct chemotherapy in patients with PC that have developed resistance to first-line targeted therapies or chemotherapies.

A Method for Diagnosing Organophosphate Pesticide Exposure in Humans using Liquid Chromatography Coupled Tandem Mass Spectrometry.

Organophosphate (OP) pesticides are commonly utilized worldwide for agricultural purposes and pose a health threat through air, ground, and water contamination. Here, we present a convenient method for diagnosing exposure to OP pesticides in humans. This immunoprecipitation method relies on extraction of butyrylcholinesterase (BChE), a biomarker of OP poisoning that adducts OP compounds, from human serum using agarose beads conjugated to anti-BChE antibodies. Extracted BChE was then digested with pepsin and analyzed for unadducted and OP-adducted peptides by high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). To characterize and validate this method, pooled human plasma was exposed to parathion and dichlorvos to form diethoxyphospho, aged ethoxyphospho and dimethoxyphospho adducts with BChE. Untreated plasma was also analyzed for unadducted peptides. Additionally, samples were analyzed using Ellman's assay to measure BChE functional activity. The percent inhibition of BChE was 53.5±5.76 and 95.2±0.37%, respectively, for plasma treated with parathion for 1 hour and 24 hours. The percent inhibition was 97.2±0.98 for plasma treated with dichlorvos for 1 hour. The percent inhibition was 97.9±0.41% when the plasma treated with parathion for 1 hour, parathion for 24 hour and dichlorvos for 1 hour were mixed. Individual adducts were quantified in a single chromatographic run. Untreated plasma contained 26.4±1.87 ng/mL of unadducted BChE and no adducted peptides. In contrast, the plasma sample treated with both pesticides contained no unadducted BChE, but did contain 9.46±1.10, 10.9±0.98 and 14.1±1.10 ng/mL of diethoxyphospho, aged-ethoxy, and dimethoxyphospho peptides, respectively. The ability to identify and measure BChE and BChE adducts to parathion and dichlorvos is expected to be useful for diagnosing human exposure to multiple OP pesticides.

Role of Curcuminoids and Tricalcium Phosphate Ceramic in Rat Spinal Fusion.

Despite considerable research effort, there is a significant need for safe agents that stimulate bone formation. Treatment of large or complex bone defects remains a challenge. Implantation of small molecule-induced human bone marrow-derived mesenchymal stromal cells (hBMSCs) on an appropriate tricalcium phosphate (TCP) scaffold offers a robust system for noninvasive therapy for spinal fusion. To show the efficacy of this approach, we identified a small molecule curcuminoid that when combined with TCP ceramic in the presence of hBMSCs selectively induced growth of bone cells: after 8- or 25-day incubations, alkaline phosphatase was elevated. Treatment of hBMSCs with curcuminoid 1 and TCP ceramic increased osteogenic target gene expression (i.e., Runx2, BMP2, Osteopontin, and Osteocalcin) over time. In the presence of curcuminoid 1 and TCP ceramic, osteogenesis of hBMSCs, including proliferation, differentiation, and mineralization, was observed. No evidence of chondrogenic or adipogenic potential using this protocol was observed. Transplantation of curcuminoid 1-treated hBMSC/TCP mixtures into the spine of immunodeficient rats showed that it achieved spinal fusion and provided greater stability of the spinal column than untreated hBMSC-TCP implants or TCP alone implants. On the basis of histological analysis, greater bone formation was associated with curcuminoid 1-treated hBMSC implants manifested as contiguous growth plates with extensive hematopoietic territories. Stimulation of hBMSCs by administration of small molecule curcuminoid 1 in the presence of TCP ceramic afforded an effective noninvasive strategy that increased spinal fusion repair and provided greater stability of the spinal column after 8 weeks in immunodeficient rats. Impact statement Bone defects only slowly regenerate themselves in humans. Current procedures to restore spinal defects are not always effective. Some have side effects. In this article, a new method to produce bone growth within 8 weeks in rats is presented. In the presence of tricalcium phosphate ceramic, curcuminoid-1 small molecule-stimulated human bone marrow-derived mesenchymal stromal cells showed robust bone cell growth in vitro. Transplantation of this mixture into the spine showed efficient spinal fusion in rats. The approach presented herein provides an efficient biocompatible scaffold for delivery of a potentially clinically useful system that could be applicable in patients.

Reengineering an Antiarrhythmic Drug Using Patient hiPSC Cardiomyocytes to Improve Therapeutic Potential and Reduce Toxicity.

Modeling cardiac disorders with human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes is a new paradigm for preclinical testing of candidate therapeutics. However, disease-relevant physiological assays can be complex, and the use of hiPSC-cardiomyocyte models of congenital disease phenotypes for guiding large-scale screening and medicinal chemistry have not been shown. We report chemical refinement of the antiarrhythmic drug mexiletine via high-throughput screening of hiPSC-CMs derived from patients with the cardiac rhythm disorder long QT syndrome 3 (LQT3) carrying SCN5A sodium channel variants. Using iterative cycles of medicinal chemistry synthesis and testing, we identified drug analogs with increased potency and selectivity for inhibiting late sodium current across a panel of 7 LQT3 sodium channel variants and suppressing arrhythmic activity across multiple genetic and pharmacological hiPSC-CM models of LQT3 with diverse backgrounds. These mexiletine analogs can be exploited as mechanistic probes and for clinical development.

Small Molecule Regulation of Stem Cells that Generate Bone, Chondrocyte, and Cardiac Cells.

Embryonic stem cells (ESCs) are stem cells (SCs) that can self-renew and differentiate into a myriad of cell types. The process of developing stemness is determined by signaling molecules that drive stem cells to a specific lineage. For example, ESCs can differentiate into mature cells (e.g., cardiomyocytes) and mature cardiomyocytes can be characterized for cell beating, action potential, and ion channel function. A goal of this Perspective is to show how small molecules can be used to differentiate ESCs into cardiomyocytes and how this can reveal novel aspects of SC biology. This approach can also lead to the discovery of new molecules of use in cardiovascular disease. Human induced pluripotent stem cells (hiPSCs) afford the ability to produce unlimited numbers of normal human cells. The creation of patient-specific hiPSCs provides an opportunity to study cell models of human disease. The second goal is to show that small molecules can stimulate hiPSC commitment to cardiomyocytes. How iPSCs can be used in an approach to discover new molecules of use in cardiovascular disease will also be shown in this study. Adult SCs, including mesenchymal stem cells (MSCs), can likewise participate in self-renewal and multilineage differentiation. MSCs are capable of differentiating into osteoblasts, adipocytes or chondrocytes. A third goal of this Perspective is to describe differentiation of MSCs into chondrogenic and osteogenic lineages. Small molecules can stimulate MSCs to specific cell fate both in vitro and in vivo. In this Perspective, some recent examples of applying small molecules for osteogenic and chondrogenic cell fate determination are summarized. Underlying molecular mechanisms and signaling pathways involved are described. Small molecule-based modulation of stem cells shows insight into cell regulation and potential approaches to therapeutic strategies for MSC-related diseases.

PAWI-2 overcomes tumor stemness and drug resistance via cell cycle arrest in integrin ß3-KRAS-dependent pancreatic cancer stem cells.

Today, pancreatic cancer (PC) remains a major health problem in the US. The fact that cancer stem cells (CSCs) become enriched in humans following anti-cancer therapy implicates CSCs as key contributors to tumor dormancy, metastasis, and relapse in PC. A highly validated CSC model (FGß3 cells) was used to test a novel compound (PAWI-2) to eradicate CSCs. Compared to parental bulk FG cells, PAWI-2 showed greater potency to inhibit cell viability and self-renewal capacity of FGß3 cells. For FGß3 cells, dysregulated integrin ß3-KRAS signaling drives tumor progression. PAWI-2 inhibited ß3-KRAS signaling independent of KRAS. This is clinically relevant. PAWI-2 targeted the downstream TBK1 phosphorylation cascade that was negatively regulated by optineurin phosphorylation via a feedback mechanism. This was confirmed by TBK1 genetic knockdown or co-treatment with TBK1-specific inhibitor (MRT67307). PAWI-2 also overcame erlotinib (an EGFR inhibitor) resistance in FGß3 cells more potently than bortezomib. In the proposed working model, optineurin acts as a key regulator to link inhibition of KRAS signaling and cell cycle arrest (G2/M). The findings show PAWI-2 is a new approach to reverse tumor stemness that resensitizes CSC tumors to drug inhibition.

N-Oxygenation of Oxycodone and Retro-reduction of Oxycodone N-Oxide.

Oxycodone is used as a potent analgesic medication. Oxycodone is extensively metabolized. To fully describe its metabolism, the oxygenation of oxycodone to oxycodone N-oxide was investigated in hepatic preparations. The hypothesis tested was that oxycodone N-oxygenation was enzymatic and the amount of N-oxide detected was a consequence of both oxygenation and retro-reduction. Methods for testing the hypothesis included both in vitro and in vivo studies. Results indicated that oxycodone was N-oxygenated by the flavin-containing monooxygenase. Oxycodone N-oxide is chemically quite stable but in the presence of hepatic preparations and NADPH was retro-reduced to its parent compound oxycodone. Subsequently, oxycodone was metabolized to other metabolites including noroxycodone, noroxymorphone, and oxymorphone via cytochrome P-450. Retro-reduction of oxycodone N-oxide to oxycodone was facilitated by quinone reductase, aldehyde oxidase, and hemoglobin but not to a great extent by cytochrome P-450 or the flavin-containing monooxygenase. To confirm the in vitro observations, oxycodone was administered to rats and humans. In good agreement with in vitro results, substantial oxycodone N-oxide was observed in urine after oxycodone administration to rats and humans. Administration of oxycodone N-oxide to rats showed substantial amount of recovered oxycodone N-oxide. In vivo, noroxycodone was formed as a major rat urinary metabolite from oxycodone N-oxide presumably after retro-reduction to oxycodone and oxidative N-demethylation. To a lesser extent, oxycodone, noroxymorphone, and oxymorphone were observed as urinary metabolites. SIGNIFICANCE STATEMENT: This manuscript describes the N-oxygenation of oxycodone in vitro as well as in small animals and humans. A new metabolite was quantified as oxycodone N-oxide. Oxycodone N-oxide undergoes extensive retro-reduction to oxycodone. This re-establishes the metabolic profile of oxycodone and introduces new concepts about a metabolic futile cycle related to oxycodone metabolism.

A Novel Small Molecule Inhibits Tumor Growth and Synergizes Effects of Enzalutamide on Prostate Cancer.

Prostate cancer (PCa) is the second leading cause of cancer-related death for men in the United States. Approximately 35% of PCa recurs and is often transformed to castration-resistant prostate cancer (CRPCa), the most deadly and aggressive form of PCa. However, the CRPCa standard-of-care treatment (enzalutamide with abiraterone) usually has limited efficacy. Herein, we report a novel molecule (PAWI-2) that inhibits cellular proliferation of androgen-sensitive and androgen-insensitive cells (LNCaP and PC-3, respectively). In vivo studies in a PC-3 xenograft model showed that PAWI-2 (20 mg/kg per day i.p., 21 days) inhibited tumor growth by 49% compared with vehicle-treated mice. PAWI-2 synergized currently clinically used enzalutamide in in vitro inhibition of PCa cell viability and resensitized inhibition of in vivo PC-3 tumor growth. Compared with vehicle-treated mice, PC-3 xenograft studies also showed that PAWI-2 (20 mg/kg per day i.p., 21 days) and enzalutamide (5 mg/kg per day i.p., 21 days) inhibited tumor growth by 63%. Synergism was mainly controlled by the imbalance of prosurvival factors (e.g., Bcl-2, Bcl-xL, Mcl-1) and antisurvival factors (e.g., Bax, Bak) induced by affecting mitochondrial membrane potential/mitochondria dynamics. Thus, PAWI-2 utilizes a distinct mechanism of action to inhibit PCa growth independently of androgen receptor signaling and overcomes enzalutamide-resistant CRPCa. SIGNIFICANCE STATEMENT: Castration-resistant prostate cancer (CRPCa) is the most aggressive human prostate cancer (PCa) but standard chemotherapies for CRPCa are largely ineffective. PAWI-2 potently inhibits PCa proliferation in vitro and in vivo regardless of androgen receptor status and uses a distinct mechanism of action. PAWI-2 has greater utility in treating CRPCa than standard-of-care therapy. PAWI-2 possesses promising therapeutic potency in low-dose combination therapy with a clinically used drug (e.g., enzalutamide). This study describes a new approach to address the overarching challenge in clinical treatment of CRPCa.

Disruption of NOTCH signaling by a small molecule inhibitor of the transcription factor RBPJ.

NOTCH plays a pivotal role during normal development and in congenital disorders and cancer. γ-secretase inhibitors are commonly used to probe NOTCH function, but also block processing of numerous other proteins. We discovered a new class of small molecule inhibitor that disrupts the interaction between NOTCH and RBPJ, which is the main transcriptional effector of NOTCH signaling. RBPJ Inhibitor-1 (RIN1) also blocked the functional interaction of RBPJ with SHARP, a scaffold protein that forms a transcriptional repressor complex with RBPJ in the absence of NOTCH signaling. RIN1 induced changes in gene expression that resembled siRNA silencing of RBPJ rather than inhibition at the level of NOTCH itself. Consistent with disruption of NOTCH signaling, RIN1 inhibited the proliferation of hematologic cancer cell lines and promoted skeletal muscle differentiation from C2C12 myoblasts. Thus, RIN1 inhibits RBPJ in its repressing and activating contexts, and can be exploited for chemical biology and therapeutic applications.

Inhibition of invasive pancreatic cancer: restoring cell apoptosis by activating mitochondrial p53.

Pancreatic ductal adenocarcinoma (PDAC), constitutes >90% of pancreatic cancers (PC) and is one of the most aggressive human tumors. Standard chemotherapies for PDAC (e.g., gemcitabine, FOLFIRINOX, etc.) has proven to be largely ineffective. Herein, we report a novel molecule (i.e., compound 1) that potently inhibits proliferation and induces apoptosis of PDAC cells. As we observed in other cancer types (i.e., colorectal, breast cancer), the effect of 1 against PDAC cells is also related to microtubule destabilization and DNA damage checkpoint activation. However, in PDAC cells, the inhibitory effect of 1 was mainly controlled by mitochondrial p53-dependent apoptosis. Compound 1 worked with cells of different p53 mutant status and affected p53 activation/phosphorylation not simply by stabilizing p53 protein but through antagonizing anti-apoptotic effects of Bcl-xL and restoring p53 to activate mitochondrial-apoptotic pathways (i.e., cytochrome c release, caspase activation and PARP cleavage). Compound 1 was more efficient than a typical PDAC combination therapy (i.e., gemcitabine with paclitaxel) and showed synergism in inhibiting PDAC cell proliferation with gemcitabine (or gemcitabine with paclitaxel). This synergism varied between different types of PDAC cells and was partially controlled by the phosphorylation of p53 on Serine15 (phospho-Ser15-p53). In vivo studies in an orthotopic syngeneic murine model showed that 1 (20 mg/kg/day, 28 days, i.p.) inhibited tumor growth by 65% compared to vehicle-treated mice. No apparent acute or chronic toxicity was observed. Thus, compound 1 utilizes a distinct mechanism of action to inhibit PC growth in vitro and in vivo and is a novel anti-PDAC compound.

Curcumin and Novel Synthetic Analogs in Cell-Based Studies of Alzheimer's Disease.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder that is associated with the most common type of dementia and is characterized by the presence of deposits of the protein fragment amyloid beta (Aß) in the brain. The natural product mixture of curcuminoids that improves certain defects in innate immune cells of AD patients may selectively enhance Aß phagocytosis by alteration of gene transcription. In this work, we evaluated the protective effects of curcuminoids in cells from AD patients by investigating the effect on NF-κB and BACE1 signaling pathways. These results were compared to the gene expression profile of the clearance of Aß. The minor curcumin constituent, bisdemethoxycurcumin (BDC) showed the most potent protective action to decrease levels of NF-κB and BACE1, decrease the inflammatory cascade and diminish Aß aggregates in cells from AD patients. Moreover, mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase (MGAT3) and vitamin D receptor (VDR) gene mRNAs were up-regulated in peripheral blood mononuclear cells from AD patients treated with BDC. BDC treatment impacts both gene expression including Mannosyl (Beta-1,4-)-Glycoprotein Beta-1,4-N-Acetylglucosaminyltransferase, Vitamin D and Toll like receptor mRNA and Aß phagocytosis. The observation of down-regulation of BACE1 and NF-κB following administration of BDC to cells from AD patients as a model system may have utility in the treatment of asymptomatic AD patients.

b-Annulated 1,4-dihydropyridines as Notch inhibitors.

The Notch signaling pathway is involved in cell proliferation and differentiation, and has been recognized as an active pathway in regenerating tissue and cancerous cells. Notch signaling inhibition is considered a viable approach to the treatment of a variety of conditions including colorectal cancer, pancreatic cancer, breast cancer and metastatic melanoma. The discovery that the b-annulated dihydropyridine FLI-06 (1) is an inhibitor of the Notch pathway with an EC50 ≈ 2.5 µM prompted us to screen a library of related analogs. After structure activity studies were conducted, racemic compound 7 was identified with an EC50 = 0.36 µM. Synthesis of individual enantiomers provided (+)-7 enantiomer with an EC50 = 0.13 µM, or about 20-fold the potency of 1.

Novel tertiary sulfonamides as potent anti-cancer agents.

For adult women in the United States, breast cancer is the most prevalent form of cancer. Compounds that target dysregulated signal transduction can be efficacious anti-cancer therapies. A prominent signaling pathway frequently dysregulated in breast cancer cells is the Wingless-related integration site (Wnt) pathway. The purpose of the work was to optimize a "hit" from a screening campaign. 76,000 compounds were tested in a Wnt transcription assay and revealed potent and reproducible "hit," compound 1. Medicinal chemistry optimization of 1 led to more potent and drug-like molecules, 19, 24 and 25 (i.e., Wnt pathway IC50 values = 11, 18 and 7 nM, respectively). The principal results showed compounds 19, 24 and 25 were potent anti-proliferative agents in breast cancer cell lines, MCF-7 (i.e., IC50 values = 10, 7 and 4 nM, respectively) and MDA-MB 231 (i.e., IC50 values = 13, 13 and 16 nM, respectively). Compound 19 synergized anti-proliferation with chemotherapeutic Doxorubicin in vitro. A major conclusion was that compound 19 enhanced anti-proliferation of Doxorubicin in vitro and in a xenograft animal model of breast cancer.

A Novel Inhibitor Targets Both Wnt Signaling and ATM/p53 in Colorectal Cancer.

For 2017, the estimated lifetime risk of developing colorectal cancer was 1 in 22. Even though preventative colonoscopy screening and standard-of-care surgery, radiation, and chemotherapy have decreased the death rate from colorectal cancer, new therapies are needed for metastatic colorectal cancer. Here, we developed a novel small molecule, compound 2, that inhibited proliferation and viability of human colorectal cancer cells (HCT-116, DLD-1, SW480, and 10.1). Compound 2 inhibited cell migration, invasion, and epithelial-mesenchymal transition processes and potently increased cell apoptosis in human colorectal cancer cells. Compound 2 also modulated mitotic stress signaling, leading to both inhibition of Wnt responsiveness and stabilization and activation of p53 to cause cell-cycle arrest. In mouse xenografts, treatment with compound 2 (20 mg/kg/day, i.p.) induced cell death and inhibited tumor growth more than four-fold compared with vehicle at day 34. Neither acute cytotoxicity nor toxicity in animals (up to 1,000 mg/kg, i.p.) were observed for compound 2 To our knowledge, compound 2 is the first reported potent small molecule that inhibits Wnt/ß-catenin signaling, activates p53 signaling regardless of p53 mutation status, and binds microtubules without detectable toxicity. Thus, compound 2 offers a novel mechanism of action and a new strategy to treat colorectal cancer.Significance: These findings identify a potent small molecule that may be therapeutically useful for colon cancer that works by inhibiting Wnt/ß-catenin signaling, activating p53, and binding microtubules without detectable toxicity. Cancer Res; 78(17); 5072-83. ©2018 AACR.

Use of Hupresin To Capture Red Blood Cell Acetylcholinesterase for Detection of Soman Exposure.

Toxicity from acute exposure to nerve agents and organophosphorus toxicants is due to irreversible inhibition of acetylcholinesterase (AChE) in the nervous system. AChE in red blood cells is a surrogate for AChE in the nervous system. Previously we developed an immunopurification method to enrich red blood cell AChE (RBC AChE) as a biomarker of exposure. The goal of the present work was to provide an alternative RBC AChE enrichment strategy, by binding RBC AChE to Hupresin affinity gel. AChE was solubilized from frozen RBC by addition of 1% Triton X-100. Insoluble debris was removed by centrifugation. The red, but not viscous, RBC AChE solution was loaded on a Hupresin affinity column. Hemoglobin and other proteins were washed off with 3 M NaCl, while retaining AChE bound to Hupresin. Denatured AChE was eluted with 1% trifluoroacetic acid. The same protocol was used for 20 mL of RBC AChE inhibited with a soman model compound. The acid denatured protein was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry on a 6600 Triple-TOF mass spectrometer. A targeted method identified the aged soman adduct on serine 203 in peptide FGESAGAAS. It was concluded that Hupresin can be used to enrich soman-inhibited AChE solubilized from 8 mL of frozen human erythrocytes, yielding a quantity sufficient for detecting soman exposure.