Unraveling the allosteric mechanisms of prolyl endopeptidases for celiac disease therapy: Insights from molecular dynamics simulations.

Prolyl endopeptidases (PEP) from Sphingomonas capsulata (sc) and Myxococcus xanthus (mx) selectively degrade gluten peptides in vitro, offering a potential therapeutic strategy for celiac disease. However, the mechanisms governing the interaction of these enzymes with their substrates remain unclear. In this study, conventional molecular dynamics simulations with a microsecond timescale and targeted molecular dynamics simulations were performed to investigate the native states of mxPEP and scPEP enzymes, as well as their allosteric binding with a representative substrate, namely, Z-Ala-Pro-p-nitroanilide (pNA). The simulations reveal that the native scPEP is in an open state, while the native mxPEP is in a closed state. When pNA approaches a closed mxPEP, it binds to an allosteric pocket located at the first and second ß-sheet of the ß-propeller domain, inducing the opening of this enzyme. Neither enzyme is active in the open or partly-open states. Enzymatic activity is enabled only when the catalytic pocket in the closed state fully accommodates the substrates. The internal capacity of the catalytic pocket of PEP in the closed state determines the maximum size of the gluten peptides that the enzymes can catalyze. The present work provides essential molecular dynamics information for the redesign or engineering of PEP enzymes.

Crizotinib induces Par-4 secretion from normal cells and GRP78 expression on the cancer cell surface for selective tumor growth inhibition.

Lung cancer is the leading cause of cancer-related deaths. Lung cancer cells develop resistance to apoptosis by suppressing the secretion of the tumor suppressor Par-4 protein (also known as PAWR) and/or down-modulating the Par-4 receptor GRP78 on the cell surface (csGRP78). We sought to identify FDA-approved drugs that elevate csGRP78 on the surface of lung cancer cells and induce Par-4 secretion from the cancer cells and/or normal cells in order to inhibit cancer growth in an autocrine or paracrine manner. In an unbiased screen, we identified crizotinib (CZT), an inhibitor of activated ALK/MET/ROS1 receptor tyrosine kinase, as an inducer of csGRP78 expression in ALK-negative, KRAS or EGFR mutant lung cancer cells. Elevation of csGRP78 in the lung cancer cells was dependent on activation of the non-receptor tyrosine kinase SRC by CZT. Inhibition of SRC activation in the cancer cells prevented csGRP78 translocation but promoted Par-4 secretion by CZT, implying that activated SRC prevented Par-4 secretion. In normal cells, CZT did not activate SRC and csGRP78 elevation but induced Par-4 secretion. Consequently, CZT induced Par-4 secretion from normal cells and elevated csGRP78 in the ALK-negative tumor cells to cause paracrine apoptosis in cancer cell cultures and growth inhibition of tumor xenografts in mice. Thus, CZT induces differential activation of SRC in normal and cancer cells to trigger the pro-apoptotic Par-4-GRP78 axis. As csGRP78 is a targetable receptor, CZT can be repurposed to elevate csGRP78 for inhibition of ALK-negative lung tumors.

Novel 1,2,4-triazoles derived from Ibuprofen: synthesis and in vitro evaluation of their mPGES-1 inhibitory and antiproliferative activity.

Some novel triazole-bearing ketone and oxime derivatives were synthesized from Ibuprofen. In vitro cytotoxic activities of all synthesized molecules against five cancer lines (human breast cancer MCF-7, human lung cancer A549, human prostate cancer PC-3, human cervix cancer HeLa, and human chronic myelogenous leukemia K562 cell lines) were evaluated by MTT assay. In addition, mouse embryonic fibroblast cells (NIH/3T3) were also evaluated to determine the selectivity. Compounds 18, 36, and 45 were found to be the most cytotoxic, and their IC50 values were in the range of 17.46-68.76 µM, against the tested cancer cells. According to the results, compounds 7 and 13 demonstrated good anti-inflammatory activity against the microsomal enzyme prostaglandin E2 synthase-1 (mPGES-1) enzyme at IC50 values of 13.6 and 4.95 µM. The low cytotoxicity and non-mutagenity of these compounds were found interesting. Also, these compounds significantly prevented tube formation in angiogenesis studies. In conclusion, the anti-inflammatory and angiogenesis inhibitory activities of these compounds without toxicity suggested that they may be promising agents in anti-inflammatory treatment and they may be supportive agents for the cancer treatment.

Nicotine has no significant cytoprotective activity against SARS-CoV-2 infection.

When coronavirus disease 2019 (COVID-19) became a pandemic, one of most important questions was whether people who smoke are at more risk of COVID-19 infection. A number of clinical data have been reported in the literature so far, but controversy exists in the collection and interpretation of the data. Particularly, there is a controversial hypothesis that nicotine might be able to prevent SARS-CoV-2 infection. In the present study, motivated by the reported controversial clinical data and the controversial hypothesis, we carried out cytotoxicity assays in Vero E6 cells to examine the potential cytoprotective activity of nicotine against SARS-CoV-2 infection and demonstrated for the first time that nicotine had no significant cytoprotective activity against SARS-CoV-2 infection in these cells.

Replenishing HDL with synthetic HDL has multiple protective effects against sepsis in mice.

Sepsis is a major health issue with mortality exceeding 30% and few treatment options. We found that high-density lipoprotein cholesterol (HDL-C) abundance was reduced by 45% in septic patients compared to that in nonseptic patients. Furthermore, HDL-C abundance in nonsurviving septic patients was substantially lower than in those patients who survived. We therefore hypothesized that replenishing HDL might be a therapeutic approach for treating sepsis and found that supplementing HDL with synthetic HDL (sHDL) provided protection against sepsis in mice. In mice subjected to cecal ligation and puncture (CLP), infusing the sHDL ETC-642 increased plasma HDL-C amounts and improved the 7-day survival rate. Septic mice treated with sHDL showed improved kidney function and reduced inflammation, as indicated by marked decreases in the plasma concentrations of blood urea nitrogen (BUN) and the cytokines interleukin-6 (IL-6) and IL-10, respectively. We found that sHDL inhibited the ability of the endotoxins LPS and LPA to activate inflammatory pathways in RAW264.7 cells and HEK-Blue cells expressing the receptors TLR4 or TLR2 and NF-κB reporters. In addition, sHDL inhibited the activation of HUVECs by LPS, LTA, and TNF-α. Together, these data indicate that sHDL treatment protects mice from sepsis in multiple ways and that it might be an effective therapy for patients with sepsis.

Generalized Methodology for the Quick Prediction of Variant SARS-CoV-2 Spike Protein Binding Affinities with Human Angiotensin-Converting Enzyme II.

Variants of the SARS-CoV-2 virus continue to remain a threat 2 years from the beginning of the pandemic. As more variants arise, and the B.1.1.529 (Omicron) variant threatens to create another wave of infections, a method is needed to predict the binding affinity of the spike protein quickly and accurately with human angiotensin-converting enzyme II (ACE2). We present an accurate and convenient energy minimization/molecular mechanics Poisson-Boltzmann surface area methodology previously used with engineered ACE2 therapeutics to predict the binding affinity of the Omicron variant. Without any additional data from the variants discovered after the publication of our first model, the methodology can accurately predict the binding of the spike/ACE2 variant complexes. From this methodology, we predicted that the Omicron variant spike has a Kd of ∼22.69 nM (which is very close to the experimental Kd of 20.63 nM published during the review process of the current report) and that spike protein of the new "Stealth" Omicron variant (BA.2) will display a Kd of ∼12.9 nM with the wild-type ACE2 protein. This methodology can be used with as-yet discovered variants, allowing for quick determinations regarding the variant's infectivity versus either the wild-type virus or its variants.

Mutations of Human DopamineTransporter at Tyrosine88, Aspartic Acid206, and Histidine547 Influence Basal and HIV-1 Tat-inhibited Dopamine Transport.

HIV-1 transactivator of transcription (Tat) has a great impact on the development of HIV-1 associated neurocognitive disorders through disrupting dopamine transmission. This study determined the mutational effects of human dopamine transporter (hDAT) on basal and Tat-induced inhibition of dopamine transport. Compared to wild-type hDAT, the maximal velocity (Vmax) of [3H]dopamine uptake was decreased in D381L and Y88F/D206L/H547A, increased in D206L/H547A, and unaltered in D206L. Recombinant TatR1 - 86 inhibited dopamine uptake in wild-type hDAT, which was attenuated in either DAT mutants (D206L, D206L/H547A, and Y88F/D206L/H547A) or mutated TatR1 - 86 (K19A and C22G), demonstrating perturbed Tat-DAT interaction. Mutational effects of hDAT on the transporter conformation were evidenced by attenuation of zinc-induced increased [3H]WIN35,428 binding in D206L/H547A and Y88F/D206A/H547A and enhanced basal MPP+ efflux in D206L/H547A. H547A-induced outward-open transport conformational state was further validated by enhanced accessibility to MTSET ([2-(trimethylammonium)ethyl]-methanethiosulfonate) of an inserted cysteine (I159C) on a hDAT background.. Furthermore, H547A displayed an increase in palmitoylation inhibitor-induced inhibition of dopamine uptake relative to wide-type hDAT, indicating a change in basal palmitoylation in H547A. These results demonstrate that Y88F, D206L, and H547A attenuate Tat inhibition while preserving DA uptake, providing insights into identifying targets for improving DAT-mediated dopaminergic dysregulation. HIV-1 Tat inhibits dopamine uptake through human dopamine transporter (hDAT) on the presynaptic terminal through a direct allosteric interaction. Key hDAT residues D-H547, D-Y88, and D-D206 are predicted to be involved in the HIV-1 Tat-DAT binding. Mutating these residues attenuates this inhibitory effect by disrupting the Tat-hDAT interaction.

Clinical data mining reveals analgesic effects of lapatinib in cancer patients.

Microsomal prostaglandin E2 synthase 1 (mPGES-1) is recognized as a promising target for a next generation of anti-inflammatory drugs that are not expected to have the side effects of currently available anti-inflammatory drugs. Lapatinib, an FDA-approved drug for cancer treatment, has recently been identified as an mPGES-1 inhibitor. But the efficacy of lapatinib as an analgesic remains to be evaluated. In the present clinical data mining (CDM) study, we have collected and analyzed all lapatinib-related clinical data retrieved from clinicaltrials.gov. Our CDM utilized a meta-analysis protocol, but the clinical data analyzed were not limited to the primary and secondary outcomes of clinical trials, unlike conventional meta-analyses. All the pain-related data were used to determine the numbers and odd ratios (ORs) of various forms of pain in cancer patients with lapatinib treatment. The ORs, 95% confidence intervals, and P values for the differences in pain were calculated and the heterogeneous data across the trials were evaluated. For all forms of pain analyzed, the patients received lapatinib treatment have a reduced occurrence (OR 0.79; CI 0.70-0.89; P = 0.0002 for the overall effect). According to our CDM results, available clinical data for 12,765 patients enrolled in 20 randomized clinical trials indicate that lapatinib therapy is associated with a significant reduction in various forms of pain, including musculoskeletal pain, bone pain, headache, arthralgia, and pain in extremity, in cancer patients. Our CDM results have demonstrated the significant analgesic effects of lapatinib, suggesting that lapatinib may be repurposed as a novel type of analgesic.

Catalytic Roles of Coenzyme Pyridoxal-5'-phosphate (PLP) in PLP-dependent Enzymes: Reaction Pathway for Methionine-γ-lyase-catalyzed L-methionine Depletion.

Pyridoxal-5'-phosphate (PLP), the active form of vitamin B6, is an important and versatile coenzyme involved in a variety of enzymatic reactions, accounting for about 4% of all classified activities. However, the detailed catalytic reaction pathways for PLP-dependent enzymes remain to be explored. Methionine-γ-lyase (MGL), a promising alternative anti-tumor agent to conventional chemotherapies whose catalytic mechanism is highly desired for guiding further development of re-engineered enzymes, was used as a representative PLP-dependent enzyme, and the catalytic mechanism for L-Met elimination by MGL was explored at the first-principles quantum mechanical/molecular mechanical (QM/MM) level with umbrella sampling. The QM/MM calculations revealed that the enzymatic reaction pathway consists of 4 stages for a total of 19 reaction steps with five intermediates captured in available crystal structures. Furthermore, the more comprehensive role of PLP was revealed. Besides the commonly known role of "electron sink", coenzyme PLP can also assist proton transfer and temporarily store the excess proton generated in some intermediate states by using its hydroxyl group and phosphate group. Thus, PLP is participated in most of the 19 steps. This study not only provided a theoretical basis for further development and re-engineering MGL as a potential anti-tumor agent, but also revealed the comprehensive role of PLP which could be used to explore the mechanisms of other PLP-dependent enzymes.

Epigenetic Regulation of Wnt Signaling by Carboxamide-Substituted Benzhydryl Amines that Function as Histone Demethylase Inhibitors.

Aberrant activation of Wnt signaling triggered by mutations in either Adenomatous Polyposis Coli (APC) or CTNNB1 (ß-catenin) is a hallmark of colorectal cancers (CRC). As part of a program to develop epigenetic regulators for cancer therapy, we developed carboxamide-substituted benzhydryl amines (CBAs) bearing either aryl or heteroaryl groups that selectively targeted histone lysine demethylases (KDMs) and functioned as inhibitors of the Wnt pathway. A biotinylated variant of N-((5-chloro-8-hydroxyquinolin-7-yl) (4-(diethylamino)phenyl)-methyl)butyramide (CBA-1) identified KDM3A as a binding partner. KDM3A is a Jumonji (JmjC) domain-containing demethylase that is significantly upregulated in CRC. KDM3A regulates the demethylation of histone H3's lysine 9 (H3K9Me2), a repressive marker for transcription. Inhibiting KDM3 increased H3K9Me2 levels, repressed Wnt target genes, and curtailed in vitro CRC cell proliferation. CBA-1 also exhibited in vivo inhibition of Wnt signaling in a zebrafish model without displaying in vivo toxicity.

Identify potent SARS-CoV-2 main protease inhibitors via accelerated free energy perturbation-based virtual screening of existing drugs.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global crisis. There is no therapeutic treatment specific for COVID-19. It is highly desirable to identify potential antiviral agents against SARS-CoV-2 from existing drugs available for other diseases and thus repurpose them for treatment of COVID-19. In general, a drug repurposing effort for treatment of a new disease, such as COVID-19, usually starts from a virtual screening of existing drugs, followed by experimental validation, but the actual hit rate is generally rather low with traditional computational methods. Here we report a virtual screening approach with accelerated free energy perturbation-based absolute binding free energy (FEP-ABFE) predictions and its use in identifying drugs targeting SARS-CoV-2 main protease (Mpro). The accurate FEP-ABFE predictions were based on the use of a restraint energy distribution (RED) function, making the practical FEP-ABFE-based virtual screening of the existing drug library possible. As a result, out of 25 drugs predicted, 15 were confirmed as potent inhibitors of SARS-CoV-2 Mpro The most potent one is dipyridamole (inhibitory constant Ki = 0.04 µM) which has shown promising therapeutic effects in subsequently conducted clinical studies for treatment of patients with COVID-19. Additionally, hydroxychloroquine (Ki = 0.36 µM) and chloroquine (Ki = 0.56 µM) were also found to potently inhibit SARS-CoV-2 Mpro We anticipate that the FEP-ABFE prediction-based virtual screening approach will be useful in many other drug repurposing or discovery efforts.

Develop a High-Throughput Screening Method to Identify C-P4H1 (Collagen Prolyl 4-Hydroxylase 1) Inhibitors from FDA-Approved Chemicals.

Collagen prolyl 4-hydroxylase 1 (C-P4H1) is an α-ketoglutarate (α-KG)-dependent dioxygenase that catalyzes 4-hydroxylation of proline on collagen. C-P4H1-induced prolyl hydroxylation is required for proper collagen deposition and cancer metastasis. Therefore, targeting C-P4H1 is considered a potential therapeutic strategy for collagen-related cancer progression and metastasis. However, no C-P4H1 inhibitors are available for clinical testing, and the high content assay is currently not available for C-P4H1 inhibitor screening. In the present study, we developed a high-throughput screening assay by quantifying succinate, a byproduct of C-P4H-catalyzed hydroxylation. C-P4H1 is the major isoform of collagen prolyl 4-hydroxylases (CP4Hs) that contributes the majority prolyl 4-hydroxylase activity. Using C-P4H1 tetramer purified from the eukaryotic expression system, we showed that the Succinate-GloTM Hydroxylase assay was more sensitive for measuring C-P4H1 activity compared with the hydroxyproline colorimetric assay. Next, we performed high-throughput screening with the FDA-approved drug library and identified several new C-P4H1 inhibitors, including Silodosin and Ticlopidine. Silodosin and Ticlopidine inhibited C-P4H1 activity in a dose-dependent manner and suppressed collagen secretion and tumor invasion in 3D tissue culture. These C-P4H1 inhibitors provide new agents to test clinical potential of targeting C-P4H1 in suppressing cancer progression and metastasis.

A plant-derived cocaine hydrolase prevents cocaine overdose lethality and attenuates cocaine-induced drug seeking behavior.

Cocaine use disorders include short-term and acute pathologies (e.g. overdose) and long-term and chronic disorders (e.g. intractable addiction and post-abstinence relapse). There is currently no available treatment that can effectively reduce morbidity and mortality associated with cocaine overdose or that can effectively prevent relapse in recovering addicts. One recently developed approach to treat these problems is the use of enzymes that rapidly break down the active cocaine molecule into inactive metabolites. In particular, rational design and site-directed mutagenesis transformed human serum recombinant butyrylcholinesterase (BChE) into a highly efficient cocaine hydrolase with drastically improved catalytic efficiency toward (-)-cocaine. A current drawback preventing the clinical application of this promising enzyme-based therapy is the lack of a cost-effective production strategy that is also flexible enough to rapidly scale-up in response to continuous improvements in enzyme design. Plant-based expression systems provide a unique solution as this platform is designed for fast scalability, low cost and the advantage of performing eukaryotic protein modifications such as glycosylation. A Plant-derived form of the Cocaine Super Hydrolase (A199S/F227A/S287G/A328W/Y332G) we designate PCocSH protects mice from cocaine overdose, counters the lethal effects of acute cocaine overdose, and prevents reinstatement of extinguished drug-seeking behavior in mice that underwent place conditioning with cocaine. These results demonstrate that the novel PCocSH enzyme may well serve as an effective therapeutic for cocaine use disorders in a clinical setting.

OleD Loki as a Catalyst for Hydroxamate Glycosylation.

Herein we describe the ability of the permissive glycosyltransferase (GT) OleD Loki to convert a diverse set of >15 histone deacetylase (HDAC) inhibitors (HDACis) into their corresponding hydroxamate glycosyl esters. Representative glycosyl esters were subsequently evaluated in assays for cancer cell line cytotoxicity, chemical and enzymatic stability, and axolotl embryo tail regeneration. Computational substrate docking models were predictive of enzyme-catalyzed turnover and suggest certain HDACis may form unproductive, potentially inhibitory, complexes with GTs.

Pictet-Spengler condensations using 4-(2-aminoethyl)coumarins.

Androgen-deprivation therapy (ADT) is only a palliative measure, and prostate cancer invariably recurs in a lethal, castration-resistant form (CRPC). Prostate cancer resists ADT by metabolizing weak, adrenal androgens to growth-promoting 5α-dihydrotestosterone (DHT), the preferred ligand for the androgen receptor (AR). Developing small-molecule inhibitors for the final steps in androgen metabolic pathways that utilize 17-oxidoreductases required probes that possess fluorescent groups at C-3 and intact, naturally occurring functionality at C-17. Application of the Pictet-Spengler condensation to substituted 4-(2-aminoethyl)coumarins and 5α-androstane-3-ones furnished spirocyclic, fluorescent androgens at the desired C-3 position. Condensations required the presence of activating C-7 amino or N,N-dialkylamino groups in the 4-(2-aminoethyl)coumarin component of these condensation reactions. Successful Pictet-Spengler condensation, for example, of DHT with 9-(2-aminoethyl)-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one led to a spirocyclic androgen, (3R,5S,10S,13S,17S)-17-hydroxy-10,13-dimethyl-1,2,2',3',4,5,6,7,8,8',9,9',10,11,12,12',13,13',14,15,16,17-docosahydro-7'H,11'H-spiro-[cyclopenta[a]phenanthrene-3,4'-pyrido[3,2,1-ij]pyrido[4',3':4,5]pyrano[2,3-f]quinolin]-5'(1'H)-one. Computational modeling supported the surrogacy of the C-3 fluorescent DHT analog as a tool to study 17-oxidoreductases for intracrine, androgen metabolism.

Development of a novel prostate apoptosis response-4 (Par-4) protein entity with an extended duration of action for therapeutic treatment of cancer.

Prostate apoptosis response-4 (Par-4) is a tumor suppressor which protects against neoplastic transformation. Remarkably, Par-4 is capable of inducing apoptosis selectively in cancer cells without affecting the normal cells. In this study, we found that recombinant Par-4 protein had limited serum persistence in mice that may diminish its anti-tumor activity in vivo. To improve the in vivo performance of the short-lived Par-4 protein, we aimed to develop a novel, long-lasting form of Par-4 with extended sequence, denoted as Par-4Ex, without affecting the desirable molecular function of the natural Par-4. We demonstrate that the Par-4Ex protein entity, produced by using the Escherichia coli expression system suitable for large-scale production, fully retains the desirable pro-apoptotic activity of Par-4 protein, but with ~7-fold improved biological half-life. Further in vivo tests confirmed that, due to the prolonged biological half-life, the Par-4Ex protein is indeed more potent in suppressing metastatic tumor growth in mice.

An Underlying Mechanism of Dual Wnt Inhibition and AMPK Activation: Mitochondrial Uncouplers Masquerading as Wnt Inhibitors.

The importance of upregulated Wnt signaling in colorectal cancers led to efforts to develop inhibitors that target ß-catenin in this pathway. We now report that several "Wnt inhibitors" that allegedly target ß-catenin actually function as mitochondrial proton uncouplers that independently activate AMPK and concomitantly inhibit Wnt signaling. As expected for a process in which mitochondrial uncoupling diminishes ATP production, a mitochondrial proton uncoupler, FCCP, and a glucose metabolic inhibitor, 2-DG, activated AMPK and inhibited Wnt signaling. Also consistent with these findings, a well-known "Wnt inhibitor", FH535, functioned as a proton uncoupler, and in support of this finding, the N-methylated analog, 2,5-dichloro-N-methyl-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535-M), was inactive as an uncoupler and Wnt inhibitor. Apart from suggesting an opportunity to develop dual Wnt inhibitors and AMPK activators, these findings provide a cautionary tale that claims for Wnt inhibition alone require scrutiny as possible mitochondrial proton uncouplers or inhibitors of the electron transport chain.

In Silico Observation of the Conformational Opening of the Glutathione-Binding Site of Microsomal Prostaglandin E2 Synthase-1.

Microsomal prostaglandin E2 synthase-1 (mPGES-1) is known as an ideal target for next-generation anti-inflammatory drugs to effectively and safely treat a variety of inflammation-related diseases. High-resolution X-ray crystal structures are available for human mPGES-1, but all in a closed conformation for a glutathione (GSH)-binding site. Here, we report an in silico observation of the desirable open conformation of mPGES-1 using a simple computational strategy with fully relaxed molecular dynamics simulations starting a high-resolution X-ray crystal structure in the closed conformation. The open conformation mainly exists in the apo-form. Once GSH enters the binding site, the binding site is closed and, thus, mPGES-1 becomes the closed conformation. According to the determined free energy profile, both the open and closed conformations can co-exist in solution with a thermodynamic equilibrium, and the conformational distribution is dependent on the GSH concentration. In addition, the cap domain responsible for the conformational transition is located right on the crystal packing interface, showing that only closed conformation is suitable for the crystal packing. All of the computational insights are consistent with reported experimental observations. The computationally simulated open conformation of mPGES-1 may serve as a new target state for the rational design of novel inhibitors of mPGES-1. We anticipate that a computational strategy similar to the one used in this study may also be used to explore open conformation starting from a crystal structure of the corresponding closed conformation with a ligand bound for other proteins.

Semisynthetic aurones inhibit tubulin polymerization at the colchicine-binding site and repress PC-3 tumor xenografts in nude mice and myc-induced T-ALL in zebrafish.

Structure-activity relationships (SAR) in the aurone pharmacophore identified heterocyclic variants of the (Z)-2-benzylidene-6-hydroxybenzofuran-3(2H)-one scaffold that possessed low nanomolar in vitro potency in cell proliferation assays using various cancer cell lines, in vivo potency in prostate cancer PC-3 xenograft and zebrafish models, selectivity for the colchicine-binding site on tubulin, and absence of appreciable toxicity. Among the leading, biologically active analogs were (Z)-2-((2-((1-ethyl-5-methoxy-1H-indol-3-yl)methylene)-3-oxo-2,3-dihydrobenzofuran-6-yl)oxy)acetonitrile (5a) and (Z)-6-((2,6-dichlorobenzyl)oxy)-2-(pyridin-4-ylmethylene)benzofuran-3(2H)-one (5b) that inhibited in vitro PC-3 prostate cancer cell proliferation with IC50 values below 100 nM. A xenograft study in nude mice using 10 mg/kg of 5a had no effect on mice weight, and aurone 5a did not inhibit, as desired, the human ether-à-go-go-related (hERG) potassium channel. Cell cycle arrest data, comparisons of the inhibition of cancer cell proliferation by aurones and known antineoplastic agents, and in vitro inhibition of tubulin polymerization indicated that aurone 5a disrupted tubulin dynamics. Based on molecular docking and confirmed by liquid chromatography-electrospray ionization-tandem mass spectrometry studies, aurone 5a targets the colchicine-binding site on tubulin. In addition to solid tumors, aurones 5a and 5b strongly inhibited in vitro a panel of human leukemia cancer cell lines and the in vivo myc-induced T cell acute lymphoblastic leukemia (T-ALL) in a zebrafish model.

Phenylethynyl-substituted Heterocycles Inhibit Cyclin D1 and Induce the Expression of Cyclin-dependent Kinase Inhibitor p21Wif1/Cip1 in Colorectal Cancer Cells.

Fluorinated, phenylethynyl-substituted heterocycles that possessed either an N-methylamino or N,N-dimethylamino group attached to heterocycles including pyridines, indoles, 1H-indazoles, quinolines, and isoquinolines inhibited the proliferation of LS174T colon cancer cells in which the inhibition of cyclin D1 and induction of the cyclin-dependent kinase inhibitor-1 (i.e., p21Wif1/Cip1) served as a readout for antineoplastic activity at a cellular level. On a molecular level, these agents, particularly 4-((2,6-difluorophenyl)ethynyl)-N-methylisoquinolin-1-amine and 4-((2,6-difluorophenyl)ethynyl)-N,N-dimethylisoquinolin-1-amine, bound and inhibited the catalytic subunit of methionine S-adenosyltransferase-2 (MAT2A).