Biochemical inhibition of the acetyltransferases ATase1 and ATase2 reduces ß-secretase (BACE1) levels and Aß generation.

The cellular levels of ß-site APP cleaving enzyme 1 (BACE1), the rate-limiting enzyme for the generation of the Alzheimer disease (AD) amyloid ß-peptide (Aß), are tightly regulated by two ER-based acetyl-CoA:lysine acetyltransferases, ATase1 and ATase2. Here we report that both acetyltransferases are expressed in neurons and glial cells, and are up-regulated in the brain of AD patients. We also report the identification of first and second generation compounds that inhibit ATase1/ATase2 and down-regulate the expression levels as well as activity of BACE1. The mechanism of action involves competitive and non-competitive inhibition as well as generation of unstable intermediates of the ATases that undergo degradation.

Drug screening to identify suppressors of GFAP expression.

Glial fibrillary acidic protein (GFAP) is the major intermediate filament protein of astrocytes in the vertebrate central nervous system. Increased levels of GFAP are the hallmark feature of gliosis, a non-specific response of astrocytes to a wide variety of injuries and disorders of the CNS, and also occur in Alexander disease where the initial insult is a mutation within the coding region of GFAP itself. In both settings, excess GFAP may cause or exacerbate astrocyte dysfunction. With the goal of finding drugs that reduce the expression of GFAP, we have devised screens to detect changes in GFAP promoter activity or protein levels in primary cultures of mouse astrocytes in a 96-well format. We have applied these screens to libraries enriched in compounds that are already approved for human use by the FDA. We report that several compounds are active at micromolar levels in suppressing the expression of GFAP. Treatment of mice for 3 weeks with one of these drugs, clomipramine, causes nearly 50% reduction in the levels of GFAP protein in brain.

Identification of novel small molecules that inhibit protein-protein interactions between MAGE and KAP-1.

The Class I MAGE proteins are normally expressed only in developing germ cells but are often aberrantly expressed in malignancies, particularly melanoma, making them good therapeutic targets. MAGE proteins promote tumor survival by binding to the RBCC region of KAP-1 and suppressing p53. Although, suppression of MAGE expression, by RNA interference, relieves p53 suppression and inhibits tumor growth, its therapeutic uses are limited by lack of methods for systemic delivery of small interfering RNA. To overcome this barrier, we sought to discover chemical compounds that inhibit binding between MAGE and KAP-1 proteins. Based on previously published effects of MAGE suppression, we developed a strategy for screening a small molecule library based on selective death of MAGE positive cells, activation of p53 and lack of caspase activity. We screened the Maybridge HitFinder library of compounds and eight compounds fulfilled these criteria. Seven of these compounds interfered with co-precipitation of MAGE and KAP-1, and three interfered with binding of MAGE and KAP-1 in a mammalian two hybrid assay. We now report identification of three potential compounds that interfere with MAGE/KAP-1 binding and can be developed as novel chemo-therapeutic agents for treatment of advanced melanoma and other cancers.

Functional characterization of TtnD and TtnF, unveiling new insights into tautomycetin biosynthesis.

The biosynthetic gene cluster for tautomycetin (TTN), a highly potent and selective protein phosphatase (PP) inhibitor isolated from Streptomyces griseochromogenes, has recently been cloned and sequenced. To better understand the transformations responsible for converting the post-polyketide synthase product into the exciting anticancer and immunosuppressive chemotherapeutic candidate TTN, we produced and characterized new analogues resulting from inactivation of two genes, ttnD and ttnF, in S. griseochromogenes. Inactivation of ttnD and ttnF, which encode for putative decarboxylase and dehydratase enzymes, respectively, afforded mutant strains SB13013 and SB13014. The DeltattnD mutant SB13013 accumulated four new TTN analogues, TTN D-1, TTN D-2, TTN D-3, and TTN D-4, whereas the DeltattnF mutant accumulated only one new TTN analogue, TTN F-1. The accumulation of these new TTN analogues defines the function of TtnD and TtnF and the timing of their chemistries in relation to installation of the C5 ketone moiety within TTN. Notably, all new analogues possess a structurally distinguishing carboxylic acid moiety, revealing that TtnD apparently cannot catalyze decarboxylation in the absence of TtnF. Additionally, cytotoxicity and PP inhibition assays reveal the importance of the functional groups installed by TtnDF and, consistent with earlier proposals, the C2''-C5 fragment of TTN to be a critical structural determinant behind the important and unique PP-1 selectivity displayed by TTN.

Lactimidomycin, iso-migrastatin and related glutarimide-containing 12-membered macrolides are extremely potent inhibitors of cell migration.

Migrastatin (1), iso-migrastatin (5) and lactimidomycin (7) are all glutarimide-containing polyketides known for their unique structures and cytotoxic activities against human cancer cell lines. Migrastatin, a strong inhibitor of tumor cell migration, has been an important lead in the development of antimetastatic agents. Yet studies of the related 12-membered macrolides iso-migrastatin, lactimidomycin, and related analogues have been hampered by their limited availability. We report here the production, isolation, structural characterization, and biological activities of iso-migrastatin, lactimidomycin, and 23 related congeners. Our studies showed that, as a family, the glutarimide-containing 12-membered macrolides are extremely potent cell migration inhibitors with some members displaying activity on par or superior to that of migrastatin as exemplified by compounds 5, 7, and 9-12. On the basis of these findings, the structures and activity of this family of compounds as cell migration inhibitors are discussed.

MAGE-A, mMage-b, and MAGE-C proteins form complexes with KAP1 and suppress p53-dependent apoptosis in MAGE-positive cell lines.

The MAGE-A, MAGE-B, and MAGE-C protein families comprise the class-I MAGE/cancer testes antigens, a group of highly homologous proteins whose expression is suppressed in all normal tissues except developing sperm. Aberrant expression of class I MAGE proteins occurs in melanomas and many other malignancies, and MAGE proteins have long been recognized as tumor-specific targets; however, their functions have largely been unknown. Here, we show that suppression of class I MAGE proteins induces apoptosis in the Hs-294T, A375, and S91 MAGE-positive melanoma cell lines and that members of all three families of MAGE class I proteins form complexes with KAP1, a scaffolding protein that is known as a corepressor of p53 expression and function. In addition to inducing apoptosis, MAGE suppression decreases KAP1 complexing with p53, increases immunoreactive and acetylated p53, and activates a p53 responsive reporter gene. Suppression of class I MAGE proteins also induces apoptosis in MAGE-A-positive, p53wt/wt parental HCT 116 colon cancer cells but not in a MAGE-A-positive HCT 116 p53-/- variant, indicating that MAGE suppression of apoptosis requires p53. Finally, treatment with MAGE-specific small interfering RNA suppresses S91 melanoma growth in vivo, in syngenic DBA2 mice. Thus, class I MAGE protein expression may suppress apoptosis by suppressing p53 and may actively contribute to the development of malignancies and by promoting tumor survival. Because the expression of class I MAGE proteins is limited in normal tissues, inhibition of MAGE antigen expression or function represents a novel and specific treatment for melanoma and diverse malignancies.

Evaluation of new migrastatin and dorrigocin congeners unveils cell migration inhibitors with dramatically improved potency.

Lactimidomycin (LTM, 1), iso-migrastatin (iso-MGS, 2) and migrastatin (MGS, 3) are macrolide antitumor antibiotics differing in macrolide ring size but all bearing a glutarimide side chain. To further develop these natural products and related analogs as drug candidates we have produced and evaluated the biological activities of a small library of iso-MGS and LTM-derived agents; congeners evaluated bear either the MGS scaffold or related acyclic (dorrigocin) scaffolds. Scratch wound-healing (SWH) assays with 4T1 mouse and MDA-MB-231 human mammary tumor cell lines, respectively, reveal structural elements crucial to inhibition of cell migration by these compounds. Moreover, two substances, 14 and 17, with activity far superior to that of MGS are unveiled by SWH assays.

Chimeric autologous/allogeneic constructs for skin regeneration.

The ideal treatment for severe cutaneous injuries would eliminate the need for autografts and promote fully functional, aesthetically pleasing autologous skin regeneration. NIKS progenitor cell-based skin tissues have been developed to promote healing by providing barrier function and delivering wound healing factors. Independently, a device has recently been created to "copy" skin by harvesting full-thickness microscopic tissue columns (MTCs) in lieu of autografts traditionally harvested as sheets. We evaluated the feasibility of combining these two technologies by embedding MTCs in NIKS-based skin tissues to generate chimeric autologous/allogeneic constructs. Chimeric constructs have the potential to provide immediate wound coverage, eliminate painful donor site wounds, and promote restoration of a pigmented skin tissue possessing hair follicles, sweat glands, and sebaceous glands. After MTC insertion, chimeric constructs and controls were reintroduced into air-interface culture and maintained in vitro for several weeks. Tissue viability, proliferative capacity, and morphology were evaluated after long-term culture. Our results confirmed successful MTC insertion and integration, and demonstrated the feasibility of generating chimeric autologous/allogeneic constructs that preserved the viability, proliferative capacity, and structure of autologous pigmented skin. These feasibility studies established the proof-of-principle necessary to further develop chimeric autologous/allogeneic constructs for the treatment of complex skin defects.

A cell-based screening system for influenza A viral RNA transcription/replication inhibitors.

Although two classes of antivirals, NA inhibitors and M2 ion channel blockers, are licensed for influenza treatment, dual resistant mutants, including highly pathogenic H5N1 viruses, have appeared. Alternative treatment options are, therefore, needed. Influenza A viral RNA (vRNA) transcription/replication is a promising target for antiviral development, since it is essential for virus replication. Accordingly, an efficient and reliable method to identify vRNA transcription/replication inhibitors is desirable. Here, we developed a cell-based screening system by establishing a cell line that stably expresses influenza viral ribonucleoprotein complex (vRNP). Compound library screening using this cell line allowed us to identify a compound that inhibits vRNA transcription/replication by using reporter protein expression from virus-like RNA as a readout and virus replication in vitro. vRNP-expressing cells have potential as a simple and convenient high-throughput screening (HTS) system, and, thus, are promising to identify vRNA transcription/replication inhibitors for various RNA viruses, especially for primary screens.

Endoperoxide synthesis by photocatalytic aerobic [2 + 2 + 2] cycloadditions.

Structurally novel endoperoxides can be sythesized by the photocatalytic cyclotrimerization of bis(styrene) substrates with molecular oxygen. The optimal catalyst for this process is Ru(bpz)(3)(2+), which is a markedly more efficient catalyst for these photooxygention reactions than conventional organic photosensitizers. The 1,2-dioxolane products are amenable to synthetic manipulation and can be easily processed to 1,4-diols and γ-hydroxyketones. An initial screen of the biological activity of these compounds reveals promising inhibition of cancer cell growth.

Pretreatment with anti-oxidants sensitizes oxidatively stressed human cancer cells to growth inhibitory effect of suberoylanilide hydroxamic acid (SAHA).

PURPOSE: Most prostate, colon and breast cancer cells are resistant to growth inhibitory effects of suberoylanilide hydroxamic acid (SAHA). We have examined whether the high oxidative stress in these cells causes a loss of SAHA activity and if so, whether pretreatment with an anti-oxidant can sensitize these cells to SAHA. METHODS: A DNA-Hoechst dye fluorescence measured cell growth and dichlorfluorescein-diacetate (DCF-DA) dye fluorescence measured reactive oxygen species (ROS). Growth inhibitory and ROS-generating activities of SAHA in androgen-treated or untreated LNCaP cells and PC-3 prostate cancer cells, HT-29 and HCT-115 colon cancer cells, MDA-MB231 breast cancer cells and A549 and NCI-H460 lung cancer cells with or without pretreatment with an anti-oxidant Vitamin E was determined. SAHA activity against LNCaP cells treated with another anti-oxidant N-acetyl cysteine (NAC) was also determined. Liquid chromatography-mass spectrometry (LC-MS) was used to determine intracellular SAHA level. RESULTS: SAHA treatment markedly inhibits LNCaP cell growth, when the cells are at a low ROS level. SAHA is, however, inactive against the same cell line, when the cells are at a high ROS level. A significant decrease in SAHA level was observed in LNCaP cells with high ROS after 24- and 72-h treatment when compared to cells with low ROS. Vitamin E pretreatment that reduces cellular ROS, synergistically sensitizes oxidatively stressed LNCaP, PC-3, HT-29, HCT-115 and MDA-MB231 cells, but not the A-549 and NCI-H460 cells with low ROS to SAHA. NAC treatment also sensitized androgen-treated LNCaP cells to the growth inhibitory effects of SAHA. CONCLUSION: Response to SAHA could be improved by combining anti-oxidants such as Vitamin E with SAHA for the treatment of oxidatively stressed human malignancies that are otherwise resistant to SAHA.

Identification and validation of Notch pathway activating compounds through a novel high-throughput screening method.

BACKGROUND: Carcinoids are neuroendocrine (NE) tumors with limited treatment options. Notch activation has been shown to suppress growth and hormone production in carcinoid cells. METHODS: The purpose of this study was to provide a process for identifying Notch activating compounds via high-throughput screening (HTS) and to validate the effects of the strongest hit from the 7264 compounds analyzed: resveratrol (RESV). RESULTS: Treatment of carcinoid cells with RESV resulted in up-regulation of the Notch signaling pathway as measured by suppression of its downstream target achaete-scute complex-like 1. Luciferase reporter assays incorporating the centromere-binding factor 1 binding site also confirmed the functional activity of RESV-induced Notch. Because activation of the Notch pathway has been shown to suppress carcinoid proliferation, RESV treatment of carcinoid cells led to a dose-dependent inhibition of cellular growth. Immunoblotting revealed phosphorylation of cdc2 (Tyr15) and up-regulation of p21Cip1/Waf, markers of cell cycle arrest, with RESV treatment. Flow cytometry confirmed the mechanism of RESV-induced growth inhibition is S phase cell cycle arrest. Furthermore, because Notch has been shown to inhibit bioactive hormone production from NE tumors, RESV also suppressed expression of the NE peptides/hormones chromogranin A and serotonin. RNA interference assays demonstrated that the hormone suppressing capacity of RESV was due to up-regulation of the Notch2 isoform. CONCLUSIONS: HTS can be used to identify novel Notch activating compounds, which may have the potential to suppress carcinoid tumor growth and the associated endocrinopathies. Cancer 2011. © 2010 American Cancer Society.

Identification and characterization of a novel estrogenic ligand actinopolymorphol A.

Xenoestrogenic compounds are abundant in the modern environment including phytoestrogens from plants, chemical by-products from industry, and secondary metabolites from microbes; all can profoundly affect human health. Consequently mechanism-based screens are urgently needed to improve the rate at which the xenoestrogens are discovered. Estrogen Receptor (ER) dimerization is required for target gene transcription. The three ER dimer pairs (ERalpha/alpha homodimers, ERbeta/beta homodimers, and ERalpha/beta heterodimers) exhibit diverse physiological responses in response to ligand-dependent activation with ERalpha/alpha homodimers being pro-proliferative and ERbeta/beta homodimers being anti-proliferative. The biological role of the ERalpha/beta heterodimer remains unclear. We previously developed a cell-based, bioluminescence resonance energy transfer (BRET) assay that can distinguish natural estrogenic compounds based on their abilities to activate the three diverse ER dimer pairs. Using BRET assays, we sought to identify novel xenoestrogens from soil bacteria that preferentially activate ERalpha/beta heterodimer with hopes of shedding light on the biological function of this elusive dimer pair. Here we describe the application of BRET assays in high throughput screens of crude bacterial extracts not previously screened for ER modulatory function and originating from unique ecological niches. Here we report the discovery and biological evaluation of a new natural product, actinopolymorphol A (1), that preferentially induces ERalpha/beta dimerization. Actinopolymorphol A represents the first representative of a new ER modulatory scaffold.

Functional characterization of ttmM unveils new tautomycin analogs and insight into tautomycin biosynthesis and activity.

The biosynthetic gene cluster for tautomycin (TTM), a potent protein phosphatase (PP) inhibitor has recently been characterized. Inactivation of ttmM, which encodes a putative C3' hydroxylase, afforded mutant SB6005 which accumulated three new 3'-deshydroxy TTM analogs, supporting the function of TtmM and the previously proposed linear pathway for TTM biosynthesis. Bioassays reveal the importance of the C3' OH moiety in PP inhibition and that PP inhibition is not the exclusive mechanism driving TTM-induced cell death.

A high-throughput method to identify novel senescence-inducing compounds.

Cellular senescence is a persistently growth-arrested phenotype in normal and transformed cells induced by noncytotoxic stress. Cytostasis as a method of cancer treatment has recently generated significant interest. Research into the induction of cellular senescence as cancer therapy has been hindered by a lack of compounds that efficiently induce this response. The authors describe a semiautomated high-throughput method to identify library compounds that induce senescence using prostate cancer cells cultured in 96-well plates. Primary hits are identified by low cell numbers after 3 days in culture, measured by Hoechst 33342 fluorescence. A secondary visual assessment of senescence-associated beta-galactosidase staining and cellular morphology in the same wells distinguishes senescence from quiescence, apoptosis, and other false positives. This method was used to screen a 4160-compound library of known bioactive compounds and natural products at a 10-microM dose. Candidate compounds were further selected based on persistent growth arrest after drug removal and increased expression of previously described senescence marker genes. Four lead compounds not previously associated with senescence were identified for further investigation. This is the first successful assay to identify novel agents from compound libraries based on senescence induction in cancer cells.

Colchicine glycorandomization influences cytotoxicity and mechanism of action.

The reaction of 70 unprotected, diversely functionalized free reducing sugars with methoxyamine-appended colchicine led to the production of a 58-member glycorandomized library. High-throughput cytotoxicity assays revealed glycosylation to modulate specificity and potency. Library members were also identified which, unlike the parent natural product (a destabilizer), stabilized in vitro tubulin polymerization in a manner similar to taxol. This study highlights a simple extension of neoglycorandomization toward amine-bearing scaffolds and the potential benefit of glycosylating nonglycosylated natural products.

RebG- and RebM-catalyzed indolocarbazole diversification.

Rebeccamycin and staurosporine represent two broad classes of indolocarbazole glycoside natural products with antitumor properties. Based upon previous sequence annotation and in vivo studies, rebG encodes for the rebeccamycin N-glucosyltransferase, and rebM for the requisite 4'-O-methyltransferase. In the current study, an efficient in vivo biotransformation system for RebG was established in both Streptomyces lividans and Escherichia coli. Bioconversion experiments revealed RebG to glucosylate a set of indolocarbazole surrogates, the products of which could be further modified by in vitro RebM-catalyzed 4'-O-methylation. Both RebG and RebM displayed substrate promiscuity, and evidence for a remarkable lack of RebG regioselectivity in the presence of asymmetric substrates is also provided. In the context of the created indolocarbazole analogues, cytotoxicity assays also highlight the importance of 4'-O-methylation for their biological activity.

Enhancing the anticancer properties of cardiac glycosides by neoglycorandomization.

Glycosylated natural products are reliable platforms for the development of many front-line drugs, yet our understanding of the relationship between attached sugars and biological activity is limited by the availability of convenient glycosylation methods. When a universal chemical glycosylation method that employs reducing sugars and requires no protection or activation is used, the glycorandomization of digitoxin leads to analogs that display significantly enhanced potency and tumor specificity and suggests a divergent mechanistic relationship between cardiac glycoside-induced cytotoxicity and Na+/K+-ATPase inhibition. This report highlights the remarkable advantages of glycorandomization as a powerful tool in glycobiology and drug discovery.

Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain.

To identify previously unknown small molecules that inhibit cell cycle machinery, we performed a chemical genetic screen in Xenopus extracts. One class of inhibitors, termed ubistatins, blocked cell cycle progression by inhibiting cyclin B proteolysis and inhibited degradation of ubiquitinated Sic1 by purified proteasomes. Ubistatins blocked the binding of ubiquitinated substrates to the proteasome by targeting the ubiquitin-ubiquitin interface of Lys(48)-linked chains. The same interface is recognized by ubiquitin-chain receptors of the proteasome, indicating that ubistatins act by disrupting a critical protein-protein interaction in the ubiquitin-proteasome system.