Integrated transcriptomic and proteomic analysis reveals Guizhi-Fuling Wan inhibiting STAT3-EMT in ovarian cancer progression.

BACKGROUND: Ovarian cancer (OC) is the most lethal gynecological malignancy. Frequent peritoneal dissemination is the main cause of low survival rate. Guizhi-Fuling Wan (GZFL) is a classical traditional Chinese herbal formula that has been clinically used for treating ovarian cancer with good outcome. However, its therapeutic mechanism for treating OC has not been clearly elucidated. PURPOSE: We aim to elucidate the potential mechanisms of GZFL in treating OC with a focus on STAT3 signaling pathway. METHODS: In vivo efficacy of GZFL was assessed using an OC xenograft mouse model. Proteomics analysis in OC cells and RNA-seq analysis in mice tumors were performed to fully capture the translational and transcriptional signature of GZFL. Effects of GZFL on proliferation, spheroid formation and reactive oxygen species (ROS) were assessed using wildtype and STAT3 knockout OC cells in vitro. STAT3 activation and transcription activity, hypoxia and EMT-related protein expression were assessed to validate the biological activity of GZFL. RESULTS: GZFL suppresses tumor growth with a safety profile in mice, while prevents cell growth, spheroid formation and accumulates ROS in a STAT3-dependent manner in vitro. GZFL transcriptionally and translationally affects genes involved in inflammatory signaling, EMT, cell migration, and cellular hypoxic stress response. In depth molecular study confirmed that GZFL-induced cytotoxicity and EMT suppression in OC cells are directly corelated to inhibition of STAT3 activation and transcription activity. CONCLUSION: Our study provides the first evidence that GZFL inhibits OC progression through suppressing STAT3-EMT signaling. These results will further support its potential clinical use in OC.

Inhibition of protein disulfide isomerase in glioblastoma causes marked downregulation of DNA repair and DNA damage response genes.

Aberrant overexpression of endoplasmic reticulum (ER)-resident oxidoreductase protein disulfide isomerase (PDI) plays an important role in cancer progression. In this study, we demonstrate that PDI promotes glioblastoma (GBM) cell growth and describe a class of allosteric PDI inhibitors that are selective for PDI over other PDI family members. Methods: We performed a phenotypic screening triage campaign of over 20,000 diverse compounds to identify PDI inhibitors cytotoxic to cancer cells. From this screen, BAP2 emerged as a lead compound, and we assessed BAP2-PDI interactions with gel filtration, thiol-competition assays, and site-directed mutagenesis studies. To assess selectivity, we compared BAP2 activity across several PDI family members in the PDI reductase assay. Finally, we performed in vivo studies with a mouse xenograft model of GBM combining BAP2 and the standard of care (temozolomide and radiation), and identified affected gene pathways with nascent RNA sequencing (Bru-seq). Results: BAP2 and related analogs are novel PDI inhibitors that selectively inhibit PDIA1 and PDIp. Though BAP2 contains a weak Michael acceptor, interaction with PDI relies on Histidine 256 in the b' domain of PDI, suggesting allosteric binding. Furthermore, both in vitro and in vivo, BAP2 reduces cell and tumor growth. BAP2 alters the transcription of genes involved in the unfolded protein response, ER stress, apoptosis and DNA repair response. Conclusion: These results indicate that BAP2 has anti-tumor activity and the suppressive effect on DNA repair gene expression warrants combination with DNA damaging agents to treat GBM.

TAIJI: approaching experimental replicates-level accuracy for drug synergy prediction.

MOTIVATION: Combination therapy is widely used in cancer treatment to overcome drug resistance. High-throughput drug screening is the standard approach to study the drug combination effects, yet it becomes impractical when the number of drugs under consideration is large. Therefore, accurate and fast computational tools for predicting drug synergistic effects are needed to guide experimental design for developing candidate drug pairs. RESULTS: Here, we present TAIJI, a high-performance software for fast and accurate prediction of drug synergism. It is based on the winning algorithm in the AstraZeneca-Sanger Drug Combination Prediction DREAM Challenge, which is a unique platform to unbiasedly evaluate the performance of current state-of-the-art methods, and includes 160 team-based submission methods. When tested across a broad spectrum of 85 different cancer cell lines and 1089 drug combinations, TAIJI achieved a high prediction correlation (0.53), approaching the accuracy level of experimental replicates (0.56). The runtime is at the scale of minutes to achieve this state-of-the-field performance. AVAILABILITY AND IMPLEMENTATION: TAIJI is freely available on GitHub (https://github.com/GuanLab/TAIJI). It is functional with built-in Perl and Python. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Design, Synthesis, and Biological Evaluation of 4-Quinoline Carboxylic Acids as Inhibitors of Dihydroorotate Dehydrogenase.

We pursued a structure-guided approach toward the development of improved dihydroorotate dehydrogenase (DHODH) inhibitors with the goal of forming new interactions between DHODH and the brequinar class of inhibitors. Two potential residues, T63 and Y356, suitable for novel H-bonding interactions, were identified in the brequinar-binding pocket. Analogues were designed to maintain the essential pharmacophore and form new electrostatic interactions through strategically positioned H-bond accepting groups. This effort led to the discovery of potent quinoline-based analogues 41 (DHODH IC50 = 9.71 ± 1.4 nM) and 43 (DHODH IC50 = 26.2 ± 1.8 nM). A cocrystal structure between 43 and DHODH depicts a novel water mediated H-bond interaction with T63. Additional optimization led to the 1,7-naphthyridine 46 (DHODH IC50 = 28.3 ± 3.3 nM) that forms a novel H-bond with Y356. Importantly, compound 41 possesses significant oral bioavailability ( F = 56%) and an elimination t1/2 = 2.78 h (PO dosing). In conclusion, the data supports further preclinical studies of our lead compounds toward selection of a candidate for early-stage clinical development.

pH-responsive selenium nanoparticles stabilized by folate-chitosan delivering doxorubicin for overcoming drug-resistant cancer cells.

Herein, we first report pH-responsive SeNPs stabilized with modified folic acid-N-trimethyl chitosan (TMC-FA) as nanocarriers for delivery of doxorubicin (DOX) to overcome drug-resistant cancer cells, which could enhance the activity of DOX by approximately 10-fold for a reduced IC50 value compared to free DOX. When nanoparticles were taken up by cells, the DOX-loaded SeNPs@TMC-FA demonstrated a faster release rate under acidic conditions. The cumulative release amount of DOX at pH 5.3 was 54.1% within 2h and 95.5% at 6h, whereas the release rate at pH 7.4 was 12.3% in 2h and 42.2% for 6h; release was not completed at the end of the study, 72h. Mechanistic studies suggested that DOX-SeNPs@TMC-FA induced cell death through the apoptosis pathway by involvement of caspase-3 and PARP proteins. The results demonstrated that pH-responsive SeNPs@TMC-FA, as targeted nanocarriers, promoted the efficacy of DOX and overcame drug resistance in NCI/ADR-RES cells.

Design and discovery of novel quinazolinedione-based redox modulators as therapies for pancreatic cancer.

BACKGROUND: Altered cellular bioenergetics and oxidative stress are emerging hallmarks of most cancers including pancreatic cancer. Elevated levels of intrinsic reactive oxygen species (ROS) in tumors make them more susceptible to exogenously induced oxidative stress. Excessive oxidative insults overwhelm their adaptive antioxidant capacity and trigger ROS-mediated cell death. Recently, we have discovered a novel class of quinazolinediones that exert their cytotoxic effects by modulating ROS-mediated signaling. METHODS: Cytotoxic potential was determined by colorimetric and colony formation assays. An XF24 Extracellular Flux Analyzer, and colorimetric and fluorescent techniques were used to assess the bioenergetics and oxidative stress effects, respectively. Mechanism was determined by Western blots. RESULTS: Compound 3a (6-[(2-acetylphenyl)amino]quinazoline-5,8-dione) was identified through a medium throughput screen of ~1000 highly diverse in-house compounds and chemotherapeutic agents for their ability to alter cellular bioenergetics. Further structural optimizations led to the discovery of a more potent analog, 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) that displayed anti-proliferative activities in low micromolar range in both drug-sensitive and drug-resistant cancer cells. Treatment with 3b causes Akt activation resulting in increased cellular oxygen consumption and oxidative stress in pancreatic cancer cells. Moreover, oxidative stress induced by 3b promoted activation of stress kinases (p38/JNK) resulting in cancer cell death. Treatment with antioxidants was able to reduce cell death confirming ROS-mediated cytotoxicity. CONCLUSION: In conclusion, our novel quinazolinediones are promising lead compounds that selectively induce ROS-mediated cell death in cancer cells and warrant further preclinical studies. GENERAL SIGNIFICANCE: Since 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) exerts Akt-dependent ROS-mediated cell death, it might provide potential therapeutic options for chemoresistant and Akt-overexpressing cancers.

Discovery of novel inhibitors of LEDGF/p75-IN protein-protein interactions.

Human lens epithelium-derived growth factor (LEDGF)/p75 plays an important role in the HIV life cycle by stimulating integrase (IN)-led viral DNA integration into cellular chromosomes. Mechanistic studies show the majority of IN inhibitors chelate magnesium ions in the catalytic active site, a region topologically distant from the LEDGF/p75 binding site. Compounds disrupting the formation of LEDGF/p75 and IN complexes serve as a novel mechanistic approach different from current antiretroviral therapies. We previously built pharmacophore models mimicking LEDGF/p75 residues and identified four classes of LEDGF/p75-IN inhibitors. Substructure and similarity searches yielded additional LEDGF/p75-IN inhibitors containing an acylhydrazone moiety. The most potent of the acylhydrazones inhibited LEDGF/p75-IN interaction with an IC(50) value of 400nM. We explored structure-activity relationships (SAR) and identified new acylhydrazones, hydrazines, and diazenes as lead molecules for further optimization. Two lead LEDGF/p75-IN inhibitors showed antiviral activity.

Discovery and preclinical evaluation of a novel class of cytotoxic propynoic acid carbamoyl methyl amides (PACMAs).

Herein, we discovered a series of propynoic acid carbamoyl methyl-amides (PACMAs) with potent cytotoxicity against a panel of cancer cell lines. These compounds interrupted cell cycle progression at low micromolar concentrations and induced early and late stage apoptosis. A representative compound suppressed tumor growth without apparent toxicity in an MDA-MB-435 mouse xenograft model. We used a Kinexus 628-antibody microarray and the Ingenuity Pathway Analysis (IPA) bioinformatics tools to better understand their mechanisms. The IPA analysis revealed the initiation of Nrf2-mediated oxidative stress through modulating the expression of SOD1 and STIP1 by compound 1. The involvement of the oxidative stress pathway was further validated by measuring the levels of the PACMA-induced mitochondrial superoxide species. To our knowledge, this is the first report on the discovery and biological evaluations of PACMAs as anticancer agents. Their broad-spectrum in vitro cytotoxicity, possibly through an oxidative stress-mediated pathway, and in vivo efficacy warrant further preclinical investigations.

Synthesis and anti-HIV-1 integrase activity of cyano pyrimidinones.

A series of 2-phenethyl/benzylthio-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitrile were synthesized and tested against recombinant HIV-1 integrase in an enzyme assay. 2-(Phenethylthio)-4-(4-chlorophenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile 4m and 2-(phenethylthio)-4-(3-chlorophenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile 4o showed significant inhibition against integrase in the assay (strand transfer: IC(50) values of 16 and 17 microM, respectively).

Synthesis and biological evaluation of novel hydrazide based cytotoxic agents.

Recently, we designed and synthesized a series of pyrroloquinoxaline compounds with hydrazine moiety linking a nitrogen-containing polycyclic group to a heteroaroyl system. Several derivatives, with attractive drug-like properties, were identified as promising cytotoxic agents, showing excellent potency in a panel of cancer cell lines. In the current study, we synthesized a further 19 new analogues to optimize their physicochemical properties and assess a coherent mechanism of action. Several chemical modifications were made to the reference compounds by varying the fused-ring system and/or the heteroacyl moiety. To evaluate their in vitro activity, we tested these compounds in six human cancer cell lines derived from different origins. Among them, two compounds ( 9 and 12 ) showed similar potency as the reference compounds with IC(50) values in the sub-micromolar range in all cell lines tested. Furthermore, compound 12 showed excellent in vivo efficacy in our preliminary human ovarian cancer mouse xenograft studies. Flow cytometric studies indicated that both derivatives interrupted cell cycle progression in colorectal cancer HCT116 cell lines and ovarian cancer SKOV-3 cells. Further mechanistic studies revealed that 9 and 12 were able to induce reactive oxygen species in SKOV-3 cells with apparently different kinetic patterns. Considering their cytotoxicity profiles in a variety of in vitro and in vivo cancer models, these hydrazide based compounds seem to have considerable potentials as novel chemotherapeutics.

Increased levels of copper efflux transporter ATP7B are associated with poor outcome in colorectal cancer patients receiving oxaliplatin-based chemotherapy.

Recently, the copper efflux transporters ATP7B and ATP7A have been implicated in the transport of and resistance to platinum drugs in breast and ovarian cancers. Because of the extensive use of oxaliplatin in colorectal cancer (CRC), we examined the expression of both transporters in tumors from CRC patients treated with oxaliplatin/5FU and sought to determine whether their expression can predict clinical outcome in these patients. ATP7B and ATP7A levels were determined by quantitative real-time PCR in 50 primary tumors of previously untreated patients with advanced colorectal adenocarcinoma who were subsequently treated with oxaliplatin/5FU. Additionally, ATP7B protein expression was assessed by immunohistochemical staining using a tissue microarray. Patients with the lowest mRNA expression levels of ATP7B had a significantly longer time to progression (TTP) (p = 0.0009) than patients with the highest levels (12.14 months vs. 6.43 months) who also had an increased risk of progression (HR = 3.56; 95% CI, 1.6-7.9; p = 0.002). Furthermore, patients with low levels of both protein and mRNA of ATP7B derived the maximum benefit from oxaliplatin/5FU with the longest TTP as compared with patients with high levels of ATP7B protein and mRNA (14.64 months vs. 4.63 months, respectively, p = 0.01) and showed a nonsignificant trend toward a lower response rate (37.5% and 75%, respectively). In conclusion, ATP7B mRNA and protein expression in colorectal tumors is associated with clinical outcome to oxaliplatin/5FU. Prospective studies are required to evaluate the role of this marker in tailoring chemotherapy.

Pharmacophore guided discovery of small-molecule human apurinic/apyrimidinic endonuclease 1 inhibitors.

Human apurinic/apyrimidinic endonuclease 1 (APE1) is an important enzyme in the base excision repair (BER) pathway that is essential for the repair of abasic sites in the genome. Evidence for APE1 as an attractive therapeutic target in anticancer drug development has been demonstrated by studies that link overexpression of APE1 in many cancers to resistance of tumor cells to radio- and chemotherapy. APE1 also shows a protective effect in several cancer cell models to a variety of DNA damaging agents. This study represents the first rational design of selective small-molecule APE1 inhibitors utilizing a three-dimensional interaction-based pharmacophore perception. All of our most potent molecules show inhibitory activity below 10 muM and are selective for APE1 inhibition.

HIV-1 integrase inhibitors: an emerging clinical reality.

From the discovery of HIV-1 integrase (IN) inhibitors using enzyme-based assays in 1992, it has taken 15 years to achieve success in human clinical trials. Currently available antiretroviral drugs set high clinical standards in efficacy and long-term safety for upcoming novel HIV/AIDS therapeutic agents. The results from advanced stages of human clinical trials with IN inhibitors indicate a promising future for these compounds as a novel class of antiretroviral drugs. Success and failure of previously discovered antiretroviral drugs have taught us that there are no magic bullets in eradicating HIV. However, approval of drugs selectively targeting IN has long been awaited. There is once again a surge of interest in the field focusing on clinical development of IN inhibitors. Here, we summarise the current status of IN inhibitors under clinical development. These agents include S-1360, GSK-364735, L-870,810, L-870,812, MK-0518, GS-9137, L-900564, GS-9224, and BMS-707035. Promising antiviral activity has already been achieved with MK-0518 and GS-9137 in late-stage clinical studies.

Ring substituent effects on biological activity of vinyl sulfones as inhibitors of HIV-1.

In a previous study, we prepared a small library of chicoric acid analogs that possessed both potent anti-integrase and antiviral activity. It was also shown that active compounds fell into one of two groups: those that inhibited an early stage in viral replication and those that inhibited at a later stage. In this study, a series of vinyl geminal disulfone-containing compounds possessing a range of ring substituents has been synthesized to probe the impact of structure on inhibitory mechanisms. Four active compounds were identified using HIV drug susceptibility assays. Three of the inhibitors possessing either no substituents or electron-withdrawing substituents on the aromatic rings led to high levels of cytotoxicity and antiviral activity. Intrigued by the potential implications of electronic effects on activity, we probed whether the active compounds could be nonspecifically reacting via 1,4-addition. To investigate this hypothesis, the compounds were incubated with glutathione and upon LC/MS analysis, molecular ion peaks corresponding to both mono and double addition adducts were identified. Second, we synthesized analogs lacking the ability to participate in 1,4-addition and tested them for antiviral activity and cytotoxicity, and found the compounds inactive for both activities. Taken together, the studies reported herein suggest that compounds lacking electron-donating substituents on the aromatic ring are promiscuous acceptors of biological nucleophiles, whereas compounds possessing electron-donating substituents seem to resist addition or at least be more selective and significantly less toxic.

Dynamic pharmacophore model optimization: identification of novel HIV-1 integrase inhibitors.

We extended the previously described dynamic pharmacophore model studies of HIV-1 integrase (IN) by considering more key residues in the active site, including Mg2+. First, we applied a Monte Carlo sampling method to map the complementary features of the IN binding surface. Two types of dynamic pharmacophore models were generated. One considers Mg2+ as part of the IN and therefore as an excluded volume, and the other treats Mg2+ as a positively charged feature, representing a new type of pharmacophore model aimed to identify compounds potentially preventing Mg2+ binding. Second, we validated the models with 385 known active (IC50 < 20 microM) and 235 (IC50 > 100 microM) inactive IN inhibitors. Third, we used the derived models to screen our small molecule database. Twenty-two structurally novel compounds were tested in an in vitro assay specific for IN, and two of them showed IC50 < or = 10 microM for strand transfer reaction.

Synthesis, antitumor and anti-HIV activities of benzodithiazine-dioxides.

Several 8-chloro-7-R1-6-R2-3-R3-imidazo[1,2-b][1,4,2]benzodithiazine 5,5-dioxide derivatives (9-11, 16-19, and 21-24) have been synthesized as potential antitumor or anti-HIV agents. The in vitro antitumor and anti-HIV-1 activities of the compounds were determined in a panel of cell lines. The benzodithiazine-dioxide 10 showed 50% growth inhibitory activity in low micromolar against most cells. It was particularly effective in leukemia, lung, melanoma, ovarian, and renal cancer cells with GI50 values of 1-2 microM. Interestingly, benzodithiazine-dioxide 16 showed remarkable anti-HIV-1 activity with 50% effective concentration EC50 value of 0.94 microM and no significant cytotoxicity at 200.0 microM.

Beta-diketo acid pharmacophore hypothesis. 1. Discovery of a novel class of HIV-1 integrase inhibitors.

HIV-1 Integrase (IN) is an essential enzyme for viral replication. The discovery of beta-diketo acids was crucial in the validation of IN as a legitimate target in drug discovery against HIV infection. In this study, we discovered a novel class of IN inhibitors using a 3D pharmacophore guided database search. We used S-1360 (1), the first IN inhibitor to undergo clinical trials, and three other analogues to develop a common feature pharmacophore hypothesis. Testing this four-featured pharmacophore against a multiconformational database of 150,000 structurally diverse small molecules yielded 1,700 compounds that satisfied the 3D query. Subsequently, all 1,700 compounds were docked into the active site of IN. On the basis of docking scores, Lipinski's rule-of-five, and structural novelty, 110 compounds were selected for biological screening. We found that compounds that contain both salicylic acid and a 2-thioxo-4-thiazolidinone (rhodanine) group (e.g. 5-13) showed significant inhibitory potency against IN, while the presence of either salicylic acid or a rhodanine group alone did not. Although some of the compounds containing only a salicylic acid showed inhibitory potency against IN, none of the compounds containing only rhodanine exhibited considerable potency. Of the 52 compounds reported in this study, 11 compounds (5, 6, 8, 10-13, 32-33, 51, and 53) inhibited 3'-processing or strand transfer activities of IN with IC(50) < or = 25 microM. This is the first reported use of S-1360 and its analogues as leads in developing a pharmacophore hypothesis for IN inhibition and for identification of new compounds with potent inhibition of this enzyme.

Design and synthesis of novel dihydroxyindole-2-carboxylic acids as HIV-1 integrase inhibitors.

In a search for new HIV-1 integrase (IN) inhibitors, we synthesized and evaluated the biological activity of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and a series of its derivatives. These compounds were designed as conformationally constrained analogues of the acrylate moiety of caffeic acid phenethyl ester (CAPE). DHICA, an intermediate in the biosynthesis of melanins, was prepared as a monomeric unit by a novel synthetic route. In order to perform coherent SAR studies, two series of DHICA amides were synthesized. First, to validate the utility of a previously identified three-point pharmacophore based on CAPE in inhibitor design, we prepared a series of benzyl- or phenylethylamine substituted derivatives lacking and containing hydroxyl groups. Second, dimers of DHICA containing various aminoalkylamine linkers were also prepared with a goal to increase potency. All compounds were tested against purified IN and the C65S mutant in enzyme-based assays. They were also tested for cytotoxicity in an ovarian carcinoma cell line and antiviral activity in HIV-1-infected CEM cells. Seven compounds inhibited catalytic activities of purified IN with IC50 values below 10 microM. Further computational docking studies were performed to determine the title compounds' mode of interaction with the IN active site. The residues K156, K159 and D64 were the most important for potency against purified IN.

New paradigms in drug design and discovery.

The new millennium has ushered in an era of science that will revolutionize a great majority of our daily activities. That revolution is being experienced by a growing number of the population who are pushing the average life expectancy closer to the 80-year mark. The primary reason for this increase is the changes we have made in the last 2-3 decades both in how we live our lives as well as how we treat our maladies when they arise. The advent of new techniques in diagnostics and surgery have allowed many to survive debilitating illnesses when their chances would have been slim only a few years ago. In addition, several new therapeutic agents have been developed in the latter part of the 20th century that have improved our quality of life and increased our overall survival time. New medicines to treat cardiovascular, degenerative, infectious, and neoplastic diseases are rapidly being discovered in an effort to further lengthen our lifetimes. The processes used by academic and industrial scientist to discover new drugs has recently experienced a true renaissance with many new and exciting techniques being developed in only the past 5-10 years. In this review, we will attempt to outline these latest protocols that chemists and biomedical scientist are currently employing to rapidly bring new drugs to the clinic.