Mechanism of inhibition of human glucose transporter GLUT1 is conserved between cytochalasin B and phenylalanine amides.

Cancerous cells have an acutely increased demand for energy, leading to increased levels of human glucose transporter 1 (hGLUT1). This up-regulation suggests hGLUT1 as a target for therapeutic inhibitors addressing a multitude of cancer types. Here, we present three inhibitor-bound, inward-open structures of WT-hGLUT1 crystallized with three different inhibitors: cytochalasin B, a nine-membered bicyclic ring fused to a 14-membered macrocycle, which has been described extensively in the literature of hGLUTs, and two previously undescribed Phe amide-derived inhibitors. Despite very different chemical backbones, all three compounds bind in the central cavity of the inward-open state of hGLUT1, and all binding sites overlap the glucose-binding site. The inhibitory action of the compounds was determined for hGLUT family members, hGLUT1-4, using cell-based assays, and compared with homology models for these hGLUT members. This comparison uncovered a probable basis for the observed differences in inhibition between family members. We pinpoint regions of the hGLUT proteins that can be targeted to achieve isoform selectivity, and show that these same regions are used for inhibitors with very distinct structural backbones. The inhibitor cocomplex structures of hGLUT1 provide an important structural insight for the design of more selective inhibitors for hGLUTs and hGLUT1 in particular.

A gene-expression screen identifies a non-toxic sumoylation inhibitor that mimics SUMO-less human LRH-1 in liver.

SUMO-modification of nuclear proteins has profound effects on gene expression. However, non-toxic chemical tools that modulate sumoylation in cells are lacking. Here, to identify small molecule sumoylation inhibitors we developed a cell-based screen that focused on the well-sumoylated substrate, human Liver Receptor Homolog-1 (hLRH-1, NR5A2). Our primary gene-expression screen assayed two SUMO-sensitive transcripts, APOC3 and MUC1, that are upregulated by SUMO-less hLRH-1 or by siUBC9 knockdown, respectively. A polyphenol, tannic acid (TA) emerged as a potent sumoylation inhibitor in vitro (IC50 = 12.8 µM) and in cells. TA also increased hLRH-1 occupancy on SUMO-sensitive transcripts. Most significantly, when tested in humanized mouse primary hepatocytes, TA inhibits hLRH-1 sumoylation and induces SUMO-sensitive genes, thereby recapitulating the effects of expressing SUMO-less hLRH-1 in mouse liver. Our findings underscore the benefits of phenotypic screening for targeting post-translational modifications, and illustrate the potential utility of TA for probing the cellular consequences of sumoylation.

Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells.

NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutants based on the protein-DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings demonstrate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering.

ß-Lactam estrogen receptor antagonists and a dual-targeting estrogen receptor/tubulin ligand.

Twelve novel ß-lactams were synthesized and their antiproliferative effects and binding affinity for the predominant isoforms of the estrogen receptor (ER), ERα and ERß, were determined. ß-Lactams 23 and 26 had the strongest binding affinities for ERα (IC50 values: 40 and 8 nM, respectively) and ERß (IC50 values: 19 and 15 nM). ß-Lactam 26 was the most potent in antiproliferative assays using MCF-7 breast cancer cells, and further biochemical analysis showed that it caused accumulation of cells in G2/M phase (mitotic blockade) and depolymerization of tubulin in MCF-7 cells. Compound 26 also induced apoptosis and downregulation of the expression of pro-survival proteins Bcl-2 and Mcl-1. Computational modeling predicted binding preferences for the dual ER/tubulin ligand 26. This series is an important addition to the known pool of ER antagonists and ß-lactam 26 is the first reported compound that has dual-targeting properties for both the ER and tubulin.

Molecular topology applied to the discovery of 1-benzyl-2-(3-fluorophenyl)-4-hydroxy-3-(3-phenylpropanoyl)-2H-pyrrole-5-one as a non-ligand-binding-pocket antiandrogen.

We report the discovery of 1-benzyl-2-(3-fluorophenyl)-4-hydroxy-3-(3-phenylpropanoyl)-2H-pyrrole-5-one as a novel non-ligand binding pocket (non-LBP) antagonist of the androgen receptor (AR) through the application of molecular topology techniques. This compound, validated through time-resolved fluorescence resonance energy transfer and fluorescence polarization biological assays, provides the basis for lead optimization and structure-activity relationship analysis of a new series of non-LBP AR antagonists. Induced-fit docking and molecular dynamics studies have been performed to establish a consistent hypothesis for the interaction of the new active molecule on the AR surface.

Retinazone inhibits certain blood-borne human viruses including Ebola virus Zaire.

BACKGROUND: Human HBV and HIV integrate their retro-transcribed DNA proviruses into the human host genome. Existing antiretroviral drug regimens fail to directly target these intrachromosomal xenogenomes, leading to persistence of viral genetic information. Retinazone (RTZ) constitutes a novel vitamin A-derived (retinoid) thiosemicarbazone derivative with broad-spectrum antiviral activity versus HIV, HCV, varicella-zoster virus and cytomegalovirus. METHODS: The in vitro inhibitory action of RTZ on HIV-1 strain LAI, human HBV strain ayw, HCV-1b strain Con1, enhanced green fluorescent protein-expressing Ebola virus Zaire 1976 strain Mayinga, wild-type Ebola virus Zaire 1976 strain Mayinga, human herpesvirus 6B and Kaposi's sarcoma-associated herpesvirus replication was investigated. The binding of RTZ to human glucocorticoid receptor was determined. RESULTS: RTZ inhibits blood-borne human HBV multiplication in vitro by covalent inactivation of intragenic and intraexonic viral glucocorticoid response elements, and, in close analogy, RTZ suppresses HIV-1 multiplication in vitro. RTZ disrupts the multiplication of blood-borne human HCV and Ebola Zaire virus at nanomolar concentrations in vitro. RTZ has the capacity to bind to human glucocorticoid receptor, to selectively and covalently bind to intraexonic viral glucocorticoid response elements, and thereby to inactivate human genome-integrated proviral DNA of human HBV and HIV. CONCLUSIONS: RTZ represents the first reported antiviral agent capable of eradicating HIV and HBV proviruses from their human host. Furthermore, RTZ represents a potent and efficacious small-molecule in vitro inhibitor of Ebola virus Zaire 1976 strain Mayinga replication.

Structure-activity relationships in non-ligand binding pocket (non-LBP) diarylhydrazide antiandrogens.

We report the synthesis and a study of the structure-activity relationships of a new series of diarylhydrazides as potential selective non-ligand binding pocket androgen receptor antagonists. Their biological activity as antiandrogens in the context of the development of treatments for castration resistant prostate cancer was evaluated using in vitro time resolved fluorescence resonance energy transfer and fluorescence polarization on target assays. Additionally, a theoretical study combining docking and molecular dynamics methods was performed to provide insight into their mechanism of action as a basis for further lead optimization studies.

Beyond the ligand-binding pocket: targeting alternate sites in nuclear receptors.

Nuclear receptors (NRs) are a family of ligand-modulated transcription factors with significant therapeutic relevance from metabolic disorders and inflammation to cancer, neurodegenerative, and psychiatric disorders. Drug discovery efforts are typically concentrated on modulating the natural ligand action within the ligand-binding pocket (LBP) in the C-terminal ligand-binding domain (LBD). Drawbacks of LBP-based strategies include physiological alterations due to disruption of ligand binding and difficulties in achieving tissue specificity. Furthermore, the lack of a "pure" and predictable mechanism of action predisposes such intervention toward drug resistance. Recent outstanding progress in our understanding of NR biology has shifted the focus of drug discovery efforts from inside to outside the LBP, affording consideration to the interaction between NRs and coactivator proteins, the interaction between NRs and DNA and the NRs' ligand-independent functions. This review encompasses such currently available NR non-LBP-based interventions and their potential application in therapy or as specific tools to probe NR biology.

Study of E/Z isomerization in a series of novel non-ligand binding pocket androgen receptor antagonists.

We report the conformational analysis of a series of 3-hydroxy-N'-((naphthalen-2-yl)methylene)naphthalene-2-carbohydrazides. This class of compounds has recently been reported as androgen receptor (AR)-coactivator disruptors for potential application in prostate cancer therapy. Definition of the E/Z isomerism around the imine linker group (hydrazide) is significant from a mechanistic point of view. A detailed study using theoretical calculations coupled with experimental techniques has allowed us determine an initial preference for the E isomer. The biological activity of newly synthesized compounds at the androgen receptor, along with a series of structural analogs, was determined and provides the basis for preliminary qualitative structure-activity relationship analysis.

"True" antiandrogens-selective non-ligand-binding pocket disruptors of androgen receptor-coactivator interactions: novel tools for prostate cancer.

Prostate cancer (PCa) therapy typically involves administration of "classical" antiandrogens, competitive inhibitors of androgen receptor (AR) ligands, dihydrotestosterone (DHT) and testosterone (tes), for the ligand-binding pocket (LBP) in the ligand-binding domain (LBD) of AR. Prolonged LBP-targeting leads to resistance, and alternative therapies are urgently required. We report the identification and characterization of a novel series of diarylhydrazides as selective disruptors of AR interaction with coactivators through application of structure and ligand-based virtual screening. Compounds demonstrate full ("true") antagonism in AR with low micromolar potency, selectivity over estrogen receptors α and ß and glucocorticoid receptor, and partial antagonism of the progesterone receptor. MDG506 (5) demonstrates low cellular toxicity in PCa models and dose responsive reduction of classical antiandrogen-induced prostate specific antigen expression. These data provide compelling evidence for such non-LBP intervention as an alternative approach or in combination with classical PCa therapy.

Consensus Computational Ligand-Based Design for the Identification of Novel Modulators of Human Estrogen Receptor Alpha.

We describe the first targeted validation of fFLASH, a molecular similarity program from IBM that has been previously proposed as suitable for the virtual screening (VS) of compound libraries based on explicit 3D flexible superimpositions, as part of its deployment within a novel consensus ligand-based virtual screening cascade. A virtual screening protocol using fFLASH for the human estrogen receptor alpha (ERα) was advanced and benchmarked against screens completed using established commercial screening softwares - Catalyst and ROCS. The optimised protocol was applied to a ∼6000 member physical screening collection and virtual 'hits' sourced and biologically assayed. The approach identified a novel, potent and highly selective partial antagonist of the ERα. This study firstly validates the clique detection algorithm utilised by fFLASH and secondly, emphasises the benefits of the consensus approach of employing more than one program in a VS protocol.