Rapid screening of environmental chemicals for estrogen receptor binding capacity.

Over the last few years, an increased awareness of endocrine disrupting chemicals (EDCs) and their potential to affect wildlife and humans has produced a demand for practical screening methods to identify endocrine activity in a wide range of environmental and industrial chemicals. While it is clear that in vivo methods will be required to identify adverse effects produced by these chemicals, in vitro assays can define particular mechanisms of action and have the potential to be employed as rapid and low-cost screens for use in large scale EDC screening programs. Traditional estrogen receptor (ER) binding assays are useful for characterizing a chemical's potential to be an estrogen-acting EDC, but they involve displacement of a radioactive ligand from crude receptor preparations at low temperatures. The usefulness of these assays for realistically determining the ER binding interactions of weakly estrogenic environmental and industrial compounds that have low aqueous solubility is unclear. In this report, we present a novel fluorescence polarization (FP) method that measures the capacity of a competitor chemical to displace a high affinity fluorescent ligand from purified, recombinant human ER-[alpha] at room temperature. The ER-[alpha] binding interactions generated for 15 natural and synthetic compounds were found to be similar to those determined with traditional receptor binding assays. We also discuss the potential to employ this FP technology to binding studies involving ER-ss and other receptors. Thus, the assay introduced in this study is a nonradioactive receptor binding method that shows promise as a high throughput screening method for large-scale testing of environmental and industrial chemicals for ER binding interactions

Luminescence-based assay methods in drug discovery

The drug discovery process is facing new challenges in the evaluation process of the lead compounds as the number of new compounds synthesized is increasing. The potentiality of test compounds is most frequently assayed through the binding of the test compound to the target molecule or receptor, or measuring functional secondary effects caused by the test compound in the target model cells, tissues or organism. Modern homogeneous high-throughput-screening (HTS) assays for purified estrogen receptors (ER) utilize various luminescence based detection methods. Fluorescence polarization (FP) is a standard method for ER ligand binding assay. It was used to demonstrate the performance of two-photon excitation of fluorescence (TPFE) vs. the conventional one-photon excitation method. As result, the TPFE method showed improved dynamics and was found to be comparable with the conventional method. It also held potential for efficient miniaturization. Other luminescence based ER assays utilize energy transfer from a long-lifetime luminescent label e.g. lanthanide chelates (Eu, Tb) to a prompt luminescent label, the signal being read in a time-resolved mode. As an alternative to this method, a new single-label (Eu) time-resolved detection method was developed, based on the quenching of the label by a soluble quencher molecule when displaced from the receptor to the solution phase by an unlabeled competing ligand. The new method was paralleled with the standard FP method. It was shown to yield comparable results with the FP method and found to hold a significantly higher signal-tobackground ratio than FP. Cell-based functional assays for determining the extent of cell surface adhesion molecule (CAM) expression combined with microscopy analysis of the target molecules would provide improved information content, compared to an expression level assay alone. In this work, immune response was simulated by exposing endothelial cells to cytokine stimulation and the resulting increase in the level of adhesion molecule expression was analyzed on fixed cells by means of immunocytochemistry utilizing specific long-lifetime luminophore labeled antibodies against chosen adhesion molecules. Results showed that the method was capable of use in amulti-parametric assay for protein expression levels of several CAMs simultaneously, combined with analysis of the cellular localization of the chosen adhesion molecules through time-resolved luminescence microscopy inspection.Uusien johtoyhdisteiden etsintä lääkekehitysprosessissa on jatkuvasti uusien haasteiden edessä, koska tähän tarkoitukseen syntetisoitujen yhdisteiden määrä kasvaa jatkuvasti. Uusien yhdisteiden soveltuvuutta testataan yleensä määrittämällä yhdisteen sitoutumisvoimakkuus kohdemolekyyliin tai -reseptoriin tai toiminnallisilla testeillä mittaamalla yhdisteen aiheuttamia vaikutuksia kohdesoluissa tai -kudoksessa tai kohteeksi valitussa eliössä. Estrogeenireseptoriin (ER) sitoutuvia yhdisteitä eristetyillä reseptoreilla testattaessa käytetään usein homogeenisia luminesenssiin perustuvia tehoseulontamenetelmiä. Fluoresenssipolarisaatio (FP) on standardimetodeja mitattaessa ER:iin sitoutumista. Kaksifotoniviritteisen fluoresenssi -tekniikan soveltuvuutta perinteiseen yksifotoniviritteiseen verrattuna testattiin ER:n ligandin sitoutumisen FP-sovelluksella. Tuloksena todettiin kaksifotoniviritystekniikan olevan yhtäläinen yksifotoniviritykseen verrattuna sekä lisäksi havaittiin mahdollisuudet määritysdynamiikan tehostamiseen ja tehokkaaseen määritystilavuuden pienentämiseen. Jotkut luminesenssiin perustuvat ER:n ligandinsitoutumismenetelmät hyödyntävät energiansiirtoa pitkäikäisen luminoivan leiman kuten lantanidikelaatin (Eu, Tb) ja lyhytikäisen luminoivan leiman välillä. Vaihtoehtona näille menetelmille kehitettiin uusi yksileimamenetelmä (Eu), jossa leiman luminesenssi sammuu liukoisen sammutinmolekyylin johdosta silloin, kun sitoutumisesta kilpaileva leimaamaton ligandi syrjäyttää leimamolekyylin reseptorista liuokseen. Uutta menetelmää testattiin FP:tä verrokkimenetelmänä käyttäen ja tulokset osoittivat uudella menetelmällä saavutettavan huomattava signaali/tausta -suhteen parannus sekä menetelmän olevan vertailukelpoinen FP:n kanssa. Solupohjainen pintaproteiiniekspression määritys yhdistettynä mikroskooppianalyysiin antaisi arvokasta lisätietoa kohdeproteiinien olemuksesta verrattuna pelkkään ekspressiotason määritykseen. Tässä työssä immuunivastetta jäljiteltiin endoteelisolujen sytokiinistimulaatiolla, ja siitä seurannutta adheesiomolekyylien ekspressiotason nousua mitattiin fiksoiduista soluista immunosytokemian menetelmin käyttämällä pitkäikäisillä luminoivilla leimoilla varustettuja vasta-aineita, jotka spesifisesti tunnistivat valitut kohdeproteiinit. Tuloksena saavutettiin solun pintaproteiinien kvantitatiivinen määritysmenetelmä, jolla useita proteiineja voidaan määrittää samassa kokeessa ja lisäksi tarkastella näiden lokalisaatiota soluissa pitkäikäiseen luminesenssiin soveltuvalla mikroskoopilla.Siirretty Doriast

Molecular basis and dual ligand regulation of tetrameric Estrogen Receptor α/14-3-3ζ protein complex

Therapeutic strategies targeting Nuclear Receptors (NRs) beyond their endogenous ligand binding pocket have gained significant scientific interest, driven by a need to circumvent problems associated with drug resistance and pharmacological profile. The hub protein 14-3-3 is an endogenous regulator of various NRs, providing a novel entry point for small molecule modulation of NR activity. Exemplified, 14-3-3 binding to the C-terminal F-domain of the Estrogen Receptor alpha (ERα), and small molecule stabilization of the ERα/14-3-3ζ protein complex by the natural product Fusicoccin A (FC-A), was demonstrated to downregulate ERα-mediated breast cancer proliferation. This presents a novel drug discovery approach to target ERα, however, structural and mechanistic insights into ERα/14-3-3 complex formation are lacking. Here, we provide an in-depth molecular understanding of the ERα/14-3-3ζ complex by isolating 14-3-3ζ in complex with an ERα protein construct comprising its Ligand Binding Domain (LBD) and phosphorylated F-domain. Bacterial co-expression and co-purification of the ERα/14-3-3ζ complex, followed by extensive biophysical and structural characterization, revealed a tetrameric complex between the ERα homodimer and the 14-3-3ζ homodimer. 14-3-3ζ binding to ERα, and ERα/14-3-3ζ complex stabilization by FC-A, appeared to be orthogonal to ERα endogenous agonist (E2) binding, E2-induced conformational changes, and cofactor recruitment. Similarly, the ERα antagonist 4-hydroxytamoxifen inhibited cofactor recruitment to the ERα LBD while ERα was bound to 14-3-3ζ. Furthermore, stabilization of the ERα/14-3-3ζ protein complex by FC-A was not influenced by the disease-associated and 4-hydroxytamoxifen resistant ERα-Y537S mutant. Together, these molecular and mechanistic insights provide direction for targeting ERα via the ERα/14-3-3 complex as an alternative drug discovery approach.</p

Identification and characterization of a small molecule inhibitor of IMP-1 that decreases expression of IMP-1 target mRNAs and inhibits proliferation of IMP-1 positive cancer cells

RNA-binding proteins control a variety of biological processes ranging from messenger RNA splicing to transport and translation. These post-transcriptional events are critical for proper cell function. One emerging class of proteins functions in several of these capacities. The VICKZ family of RNA-binding proteins is involved in translation control, mRNA localization and mRNA stability. I have studied the Insulin-like Growth Factor-2 mRNA-Binding Protein 1 (IGF2BP1/IMP-1/CRD-BP). IMP-1 exhibits an oncofetal pattern of expression, where it is expressed in embryonic development and its expression is repressed shortly after birth. However, the IMP-1 gene is reactivated in many different human cancers. Overexpression of IMP-1 leads to increased levels of proteins that promote tumor growth, metastasis, and resistance to anticancer drugs and is associated with a poor prognosis. IMP-1 enhances proliferation and migration of cancer cells by binding to and stabilizing mRNAs important in cancer, such as c-Myc. Although the role of c-Myc in cancer has been well established, it has remained an elusive therapeutic target because of its role as a transcription factor in non-neoplastic proliferating cells. Given its oncofetal pattern of expression, targeting IMP-1 presents a novel approach to targeting c-Myc. To identify new chemical entities with therapeutic potential in IMP-1 positive cancer, we carried out a pilot screen using an in vitro fluorescence anisotropy microplate assay (FAMA) and found that this approach was robust and appropriate for high throughput screening. We then carried out a high throughput screen of approximately 150,000 small molecules. Reported here is BTYNB, the first small molecule inhibitor of IMP-1, BTYNB decreases levels of IMP-1 target mRNAs, inhibits proliferation of IMP-1 positive cancer cells, and functions through the unique mechanism of decreasing oncogene mRNA stability. We believe that BTYNB not only can be developed as a potential therapeutic agent, but also serves as a useful molecular tool, with which we can probe the actions of IMP-1 in cancer cells. In addition to identifying and characterizing the first small molecule inhibitor of IMP-1, we were also interested in identifying novel molecular targets of IMP-1. Using in silico analysis of publicly available microarrays where IMP-1 was knocked down, we identified a panel of candidate target genes. Using qRTPCR and Western blot analysis, we then confirmed whether or not the mRNAs of candidate genes were decreased with IMP-1 knockdown and identified Protein Kinase C a (PKCa) as a new molecular target of IMP-1. Overall, this work has led to the identification and characterization of the first small molecule inhibitor of IMP-1 and has demonstrated that despite the fact that studies of the role of IMP-1 in cancer are rapidly expanding, there still remain novel molecular targets, such as PKCa, which may play critical roles in IMP-1 action in cancer cells

Protein Disulfide Isomerase Modulates the Activation of Thyroid Hormone Receptors

Thyroid hormone receptors (TRs) are responsible for mediating thyroid hormone (T3 and T4) actions at a cellular level. They belong to the nuclear receptor (NR) superfamily and execute their main functions inside the cell nuclei as hormone-regulated transcription factors. These receptors also exhibit so-called “non-classic” actions, for which other cellular proteins, apart from coregulators inside nuclei, regulate their activity. Aiming to find alternative pathways of TR modulation, we searched for interacting proteins and found that PDIA1 interacts with TRβ in a yeast two-hybrid screening assay. The functional implications of PDIA1—TR interactions are still unclear; however, our co-immunoprecipitation (co-IP) and fluorescence assay results showed that PDI was able to bind both TR isoforms in vitro. Moreover, T3 appears to have no important role in these interactions in cellular assays, where PDIA1 was able to regulate transcription of TRα and TRβ-mediated genes in different ways depending on the promoter region and on the TR isoform involved. Although PDIA1 appears to act as a coregulator, it binds to a TR surface that does not interfere with coactivator binding. However, the TR:PDIA1 complex affinity and activation are different depending on the TR isoform. Such differences may reflect the structural organization of the PDIA1:TR complex, as shown by models depicting an interaction interface with exposed cysteines from both proteins, suggesting that PDIA1 might modulate TR by its thiol reductase/isomerase activity

DISCOVERY, STRUCTURE-FUNCTION CHARACTERIZATION AND ASSESSMENT OF POLYOXOMETALATES AS MODULATORS OF THE DNA BINDING ACTIVITY OF THE SOX-HMG FAMILY OF TRANSCRIPTION FACTORS

Ph.DDOCTOR OF PHILOSOPH

Multifunctional Scaffolds for Selective Protein-Protein Inhibition

Protein-protein interactions (PPIs) play an important role in numerous biological processes. Consequently, modulating PPIs is fundamental for understanding and manipulating mechanisms that govern many diseases. Among the wide range of topographies that PPIs display, the α-helix is the most common secondary structure in nature and thus represents a good generic template for inhibitor design.1 Some of the most relevant approaches in this field are the proteomimetic approach, which recapitulate the key binding residues of an α-helix on a non-peptidic scaffold; and the constrained peptides, which aim to reproduce the helical structure by stabilising a helical peptide. Both approaches have generated potent inhibitors of a great diversity of α-helix mediated PPIs. However, developing a better understanding of the key features that govern the modulation of protein recognition is necessary to further advance the field and fully exploit each class of foldamer. In that context, we developed functionalised aromatic oligoamide backbones to mimic residues located on multiple faces of an -helix to target the ER/co-activator PPI. The novel scaffolds are based on bis-benzamide and N-(4-aminophenyl)terephthalamidic acid backbones functionalised with isobutyl groups to reproduce the key side chains of the co-activator α-helix. Conformational studies in combination with molecular modeling and docking analysis provide evidence that the new oligomers can adopt conformations that mimic the residues at i, i+3 and i+4 positions of the native co-activator α-helix. In addition, the rules that govern molecular recognition of protein surfaces were further investigated through the optimisation of the oligobenzamide hybrid scaffold using a structure-activity relationship (SAR) study. A library of compound analogues has been synthesised incorporating five variable sites. The modifications focused on size, polarity and stereochemistry to obtain more potent and selective proteomimetic inhibitors of the p53/hDM2 and Mcl-1/NOXA B PPIs. Finally, using existing methodologies a 3-O-alkylated proteomimetic scaffold and hydrocarbon stapling peptide strategy, have been used to design inhibitors of the Asf1/H3 interaction. The application of both approaches allowed the different inhibitor designs to be directly compared when targeting the same PPI

Cytohesins are cytoplasmic ErbB receptor activators

Signaling by ErbB receptors requires the activation of their cytoplasmic tyrosine kinase domains which is initiated by ligand binding to the receptor ectodomains. Up to now, cytoplasmic factors contributing to the activation of ErbB receptors have been unknown. This thesis introduces members of the cytohesin family of guanine nucleotide exchange factors as the first cytoplasmic ErbB receptor activators. Chemical inhibition or knock-down of cytohesins decreased ErbB receptor autophosphorylation and signaling whereas cytohesin overexpression stimulated receptor activation. Crosslinking experiments and studies with a constitutively dimerized EGFR construct showed that cytohesins act on already dimerized receptors. Cell-free reconstitution of cytohesin-dependent receptor autophosphorylation as well as monitoring EGFR conformation by fluorescence anisotropy microscopy indicate that cytohesins facilitate conformational rearrangements in the intracellular domains of dimerized receptors. Thus cytohesins may represent an additional layer of regulation of ErbB receptor activation which would allow the cell to modulate the number of activated ligand-bound receptors according to cellular needs. Consistent with cytohesins playing a prominent role in ErbB receptor signaling, cytohesins were found to be overexpressed in human lung adenocarcinomas. Furthermore, the overexpression correlated with increased activation of the EGF signaling pathway. Inhibition of cytohesins by the cytohesin-specific small-molecule inhibitor SecinH3 resulted in reduced proliferation and induction of apoptosis in EGFR-dependent lung cancer cells in vitro, as well as in tumor xenografts in mice. These findings underline the functional importance of cytohesin-dependent EGFR activation for tumor growth. Furthermore, chemical inhibition of cytohesins led to a strong reduction of glioblastoma cell proliferation. In order to optimize the inhibitory potential of SecinH3 , new Secin chemotypes were analyzed. In addition, a new assay for the screening of small molecules that interfere with cytohesin-catalyzed guanine nucleotide exchange on ARFs was established. The work in hand establishes cytohesins as an unprecedented, pathophysiologically relevant class of cytoplasmic conformational ErbB receptor activators and opens up new, entirely unexplored avenues for fighting ErbB receptor-dependent cancers by targeting not the receptors themselves but their activators

Characterization of TSG101 Induced Allostery within Glucocorticoid Receptor and Computational Drug-Lead Targeting of the Glucocorticoid Receptor DNA-Binding Domain

Glucocorticoid Receptor (GR) is a typical steroid hormone receptor. GR has a disordered N-terminal domain that binds transcriptional cofactors, a DNA-binding domain, and a steroid binding domain. Most work for the past forty years has been focused on the latter two domains that are structured and amenable to characterization. Yet, the disordered N-terminus makes up more than half of the protein and is absolutely required for transcriptional activation (repression can occur regardless). There are also translational isoforms with truncated N-termini that alter the resulting activity of GR. How the disordered isoforms of the N-terminus couple to cofactors and transcriptional activity is currently unclear. In this thesis, I sought organizing principles to explain the varying activities of GR translational isoforms. I found that a transcriptional cofactor, tumor susceptibility gene 101 (TSG101), can bind and fold the disordered N-terminus of GR. TSG101's binding is allosterically coupled to binding of DNA, but the exact mechanism differs between different isoforms of GR. In this thesis I also characterized the thermodynamic stability of the TSG101 coiled-coil, which binds GR, and I used computational docking to develop novel drug-leads targeted against the GR DNA-binding domain

Probing the Functional Impact of Sequence Variation on p53-DNA Interactions Using a Novel Microsphere Assay for Protein-DNA Binding with Human Cell Extracts

The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs). Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation—including polymorphisms—and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD) for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt) variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs) was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks