Biological RNAs that bind small-molecules have been implicated in a variety of regulatory and catalytic processes. Inspired by these examples, we used in vitro selection to search a pool of genomeencoded RNA fragments for naturally occurring GTP aptamers. Several classes of aptamers were identified, including one ("the G motif") with a G-quadruplex structure. Further analysis revealed that most RNA and DNA G-quadruplexes bind GTP. The G motif is abundant in eukaryotes, and the human genome contains ∼75,000 examples with dissociation constants comparable to the GTP concentration of a eukaryotic cell (∼300 µM). G-quadruplexes play roles in diverse cellular processes, and our findings raise the possibility that GTP may play a role in the function of these elements. Consistent with this possibility, the sequence requirements of several classes of regulatory G-quadruplexes parallel those of GTP binding.
Cancer cells hijack BCL-2 family survival proteins to suppress the death effectors and thereby enforce an immortal state. This is accomplished biochemically by an anti-apoptotic surface groove that neutralizes the pro-apoptotic BH3 α-helix of death proteins. Anti-apoptotic MCL-1 in particular has emerged as a ubiquitous resistance factor in cancer. Whereas targeting the BCL-2 anti-apoptotic subclass effectively restores the death pathway in BCL-2-dependent cancer, the development of molecules tailored to the binding specificity of MCL-1 has lagged. We previously discovered that a hydrocarbon-stapled MCL-1 BH3 helix is an exquisitely selective MCL-1 antagonist. By deploying this unique reagent in a competitive screen, we identified an MCL-1 inhibitor molecule that selectively targets the BH3-binding groove of MCL-1, neutralizes its biochemical lockhold on apoptosis, and induces caspase activation and leukemia cell death in the specific context of MCL-1 dependence.
Proliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM2 isoform, effectively constraining lower glycolysis. Here, we report the discovery of PKM2 activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM2 activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the nonessential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM2 activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM2 expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress.
Semiconductor quantum dots are quickly becoming a critical diagnostic tool for discerning cellular function at the molecular level. Their high brightness, long-lasting, sizetunable, and narrow luminescence set them apart from conventional fluorescence dyes. Quantum dots are being developed for a variety of biologically oriented applications, including fluorescent assays for drug discovery, disease detection, single protein tracking, and intracellular reporting. This review introduces the science behind quantum dots and describes how they are made biologically compatible. Several applications are also included, illustrating strategies toward target specificity, and are followed by a discussion on the limitations of quantum dot approaches. The article is concluded with a look at the future direction of quantum dots.
Azaphilones are a class of fungal metabolites characterized by a highly oxygenated pyrano-quinone bicyclic core and exhibits a broad range of bioactivities. While widespread among various fungi, their biosynthesis has not been thoroughly elucidated. By activation of a silent (aza) gene cluster in Aspergillus niger ATCC 1015, we have discovered six new azaphilone compounds, azanigerones A-F (1, 3-7). Transcriptional analysis and deletion of a key polyketide synthase (PKS) gene further confirmed the involvement of the aza gene cluster. The biosynthetic pathway was shown to involve the convergent actions of a highly-reducing and a non-reducing PKSs. Most significantly, in vitro reaction of a key flavin-dependent monooxygenase encoded in the cluster with an early benzaldehyde intermediate revealed its roles in hydroxylation and pyran-ring formation to afford the characteristic bicylic core shared by azaphilones.
Firefly luciferase (FLuc) is frequently used as a reporter in high-throughput screening assays owing to the exceptional sensitivity, dynamic range, and rapid measurement that bioluminescence affords. However, interaction of small molecules with FLuc has, to some extent, confounded its use in chemical biology and drug discovery. To identify and characterize chemotypes interacting with FLuc, we determined potency values for 360,864 compounds, found in the NIH Molecular Libraries Small Molecule Repository, available in PubChem. FLuc inhibitory activity was observed for 12% of this library with discernible SAR. Characterization of 151 inhibitors demonstrated a variety of inhibition modes including FLuc-catalyzed formation of multisubstrate-adduct enzyme inhibitor complexes. As in some cell-based FLuc reporter assays compounds acting as FLuc inhibitors yield paradoxical luminescence increases, data on compounds acquired from FLuc-dependent assays requires careful analysis as described in this report.
profiling; PubChem; luciferase; quantitative high-throughput screening; qHTS; firefly luciferase; reporter-gene assays; adenylate forming enzymes
Pinpointing a specific cell from within a relatively uniform cell population to determine its chemical content presents a challenging bioanalytical task. Immunocytochemistry is the classical method used to localize specific molecules and hence, selected cells. Mass spectrometry also probes endogenous molecules such as neuropeptides within a cell. Here, these two approaches are hyphenated to allow microchemical analysis of immunocytochemical-selected peptidergic neurons. This two-step strategy utilizes antibody-based localization of cells containing selected biomarkers to isolate the cell(s) of interest, followed by peptidomic analysis via mass spectrometry. Applicable to a broad range of analyte and cell types, the strategy was used to successfully profile neuropeptides from individual immunostained insect neurons stored for up to two weeks as well as from tissues preserved for 42 weeks.
peptidomics; paraformaldehyde-fixed immunolabeled (PFIL) samples; single-cell analysis; MALDI-TOF mass spectrometry; cell culture; neuropeptides; insect
The Hedgehog signaling pathway is linked to a variety of diseases, notably a range of cancers. The first generation of drug screens identified Smoothened (Smo), a membrane protein essential for signaling, as an attractive drug target. Smo localizes to the primary cilium upon pathway activation, and this transition is critical for the response to Hedgehog ligands. In a high content screen directly monitoring Smo distribution in Hedgehog responsive cells, we identified different glucocorticoids as specific modulators of Smo ciliary accumulation. One class promoted Smo accumulation, conferring cellular hypersensitivity to Hedgehog stimulation. In contrast, a second class inhibited Smo ciliary localization and signaling activity by both wildtype Smo, and mutant forms of Smo, SmoM2 and SmoD473H, that are refractory to previously identified Smo antagonists. These findings point to the potential for developing glucocorticoid-based pharmacological modulation of Smo signaling to treat mutated drug-resistant forms of Smo, an emerging problem in long-term cancer therapy. They also raise a concern about potential crosstalk of glucocorticoid drugs in the Hedgehog pathway, if therapeutic administration exceeds levels associated with on-target transcriptional mechanisms of glucocorticoid action.
Whereas intracellular carbon metabolism has emerged as an attractive drug target, the carbon sources of intracellularly replicating pathogens, such as the tuberculosis bacillus Mycobacterium tuberculosis, which causes long-term infections in one-third of the world’s population, remain mostly unknown. We used a systems-based approach—13C-flux spectral analysis (FSA) complemented with manual analysis—to measure the metabolic interaction between M. tuberculosis and its macrophage host cell. 13C-FSA analysis of experimental data showed that M. tuberculosis obtains a mixture of amino acids, C1 and C2 substrates from its host cell. We experimentally confirmed that the C1 substrate was derived from CO2. 13C labeling experiments performed on a phosphoenolpyruvate carboxykinase mutant revealed that intracellular M. tuberculosis has access to glycolytic C3 substrates. These findings provide constraints for developing novel chemotherapeutics.
•The intracellular metabolism of Mycobacterium tuberculosis was directly measured•A tool for analyzing metabolic interactions between host and pathogen was developed•Amino acids C1, C2, and C3 are intracellular substrates for M. tuberculosis•CO2 was identified as an intracellular carbon source for M. tuberculosis
Despite being a potential drug target, intracellular metabolism of Mycobacterium tuberculosis (Mtb) is one of the least understood aspects of host pathogen biology. Beste et al. probe the metabolism of the pathogen directly growing inside the macrophage and see that Mtb has access to a diverse diet and nutrients.
Iron delivery by transferrin (Tf) is accomplished through clathrin-mediated endocytosis of Tf receptors. The small molecule NSC306711 inhibits iron uptake from the Tf-TfR pathway. Here we show that the drug’s mechanism of action is to induce internalization and degradation of unoccupied Tf receptors through an unexpected endocytic pathway. Unlike classical clathrin-mediated Tf receptor endocytosis, internalization promoted by NSC306711 is independent of clathrin and dynamin, and is sensitive to the cholesterol-depleting agents filipin and nystatin. The finding of this cholesterol-dependent Tf receptor internalization pathway through use of the small-molecule inhibitor sheds light on the pleiotropic nature of membrane trafficking dynamics and adds a complex dimension to our understanding of receptor regulation. Because of its unusual properties to inhibit iron uptake, we refer to NSC306711 as “ferristatin.”
Protein-glycan interactions are important regulators for a variety of biological processes, ranging from immune recognition to anticoagulation. An important area of active research is directed towards understanding the role of host cell surface glycans as recognition sites for pathogen protein receptors. Recognition of cell surface glycans is a widely employed strategy for a variety of pathogens, including bacteria, parasites, and viruses. We present here a representative example of such an interaction: the binding of influenza A hemagglutinin (HA) to specific sialylated glycans on the cell surface of human upper airway epithelial cells, which initiates the infection cycle. We detail a generalizable strategy to understand the nature of protein-glycan interactions both structurally and biochemically, using HA as a model system. This strategy combines a top-down approach using available structural information to define important contacts between glycans and HA, with a bottom-up approach using data mining and informatics approaches to identify the common motifs which distinguish glycan binders from non-binders. By probing protein-glycan interactions simultaneously through top-down and bottom-up approaches we can scientifically validate a series of observations. This in turn provides additional confidence and surmounts known challenges in the study of protein-glycan interactions, such as accounting for multivalency, and thus truly defines concepts such as specificity, affinity, and avidity. With the advent of new technologies for glycomics—including glycan arrays, data mining solutions, and robust algorithms to model protein-glycan interactions—we anticipate that such combination approaches will become tractable for a wide variety of protein-glycan interactions.
A desire to better understand the role of voltagegated sodium channels (NaVs) in signal conduction and their dysregulation in specific disease states motivates the development of high precision tools for their study. Nature has evolved a collection of small molecule agents, including the shellfish poison (+)-saxitoxin, that bind to the extracellular pore of select NaV isoforms. As described in this report, de novo chemical synthesis has enabled the preparation of fluorescently labeled derivatives of (+)-saxitoxin, STX-Cy5, and STX-DCDHF, which display reversible binding to NaVs in live cells. Electrophysiology and confocal fluorescence microscopy studies confirm that these STX-based dyes function as potent and selective NaV labels. The utility of these probes is underscored in single-molecule and super-resolution imaging experiments, which reveal NaV distributions well beyond the optical diffraction limit in subcellular features such as neuritic spines and filopodia.
Clofarabine (ClF) is a drug used in the treatment of leukemia. One of its primary targets is human ribonucleotide reductase (hRNR), a dual-subunit, (α2)m(β2)n, regulatory enzyme indispensable in de novo dNTP synthesis. We report that in live mammalian cells, ClF targets hRNR by converting its α-subunit into kinetically-stable hexamers. We established mammalian expression platforms that enabled isolation of functional α and characterization of its altered oligomeric associations in response to ClF treatment. Size exclusion chromatography and electron microscopy documented persistence of in-cell-assembled-α6. Our data validate hRNR as an important target of ClF, provide previously-unreported evidence that in vivo α’s quaternary structure can be perturbed by a non-natural ligand, and suggest small-molecule-promoted, persistent hexamerization as a new strategy to modulate hRNR activity. These studies lay foundations for documentation of RNR oligomeric state within a cell.
Protease inhibitor discovery has focused almost exclusively on compounds that bind to the active site. Inhibitors targeting protease exosites, regions outside of the active site that influence catalysis, offer potential advantages of increased specificity but are difficult to systematically discover. Here we describe an assay suitable for detecting exosite-targeting inhibitors of the metalloproteinase anthrax lethal factor (LF) based on cleavage of a full length mitogen-activated protein kinase kinase (MKK) substrate. We used this assay to screen a small molecule library, and then subjected hits to a secondary screen to exclude compounds that efficiently blocked cleavage of a peptide substrate. We identified a compound that preferentially inhibited cleavage of MKKs compared with peptide substrates and could suppress LF-induced macrophage cytolysis. This approach should be generally applicable to the discovery of exosite-targeting inhibitors of many additional proteases.
The small intestine is the primary site of dietary lipid absorption in mammals. The balance of nutrients, microorganisms, bile, and mucus that determine intestinal luminal environment cannot be recapitulated ex vivo, thus complicating studies of lipid absorption. We show that fluorescently labeled lipids can be used to visualize and study lipid absorption in live zebrafish larvae. We demonstrate that the addition of BODIPY-fatty acid to a diet high in atherogenic lipids enables imaging of enterocyte lipid droplet dynamics in real time. We find that a lipid-rich meal promotes BODIPY-cholesterol absorption into an endosomal compartment distinguishable from lipid droplets. We also show that dietary fatty acids promote intestinal cholesterol absorption by rapid relocalization of NPC1L1 to intestinal brush border. These data illustrate the power of the zebrafish system to address longstanding questions in vertebrate digestive physiology.
Whole-cell screening of Mycobacterium tuberculosis (Mtb) remains a mainstay of drug discovery but subsequent target elucidation often proves difficult. Conditional mutants that under-express essential genes have been used to identify compounds with known mechanism of action by target-based whole-cell screening (TB-WCS). Here, the feasibility of TB-WCS in Mtb was assessed by generating mutants that conditionally express pantothenate synthetase (panC), diaminopimelate decarboxylase (lysA) and isocitrate lyase (icl1). The essentiality of panC and lysA, and conditional essentiality of icl1 for growth on fatty acids, was confirmed. Depletion of PanC and Icl1 rendered the mutants hypersensitive to target-specific inhibitors. Stable reporter strains were generated for use in high-throughput screening, and their utility demonstrated by identifying compounds that display greater potency against a PanC-depleted strain. These findings illustrate the power of TB-WCS as a tool for tuberculosis drug discovery.
Chemical inhibitors can help analyze dynamic cellular processes, particularly when probes are active in genetically tractable model systems. Although fission yeast has served as an important model system, which shares more cellular processes (e.g., RNAi) with humans than budding yeast, its use for chemical biology has been limited by its multidrug resistance (MDR) response. Using genomics and genetics approaches, we identified the key transcription factors and drug-efflux transporters responsible for fission yeast MDR and designed strains sensitive to a wide-range of chemical inhibitors, including commonly used probes. We used this strain, along with acute chemical inhibition and high-resolution imaging, to examine metaphase spindle organization in a “closed” mitosis. Together, our findings suggest that our fission yeast strains will allow the use of several inhibitors as probes, discovery of new inhibitors, and analysis of drug action.
New tools are needed for managing celiac sprue, a lifelong immune disease of the small intestine. Ongoing drug trials are also prompting a search for noninvasive biomarkers of gluten-induced intestinal change. We have synthesized and characterized noninflammatory gluten peptide analogs in which key Gln residues are replaced by Asn or His. Like their proinflammatory counterparts, these biomarkers are resistant to gastrointestinal proteases, susceptible to glutenases, and permeable across enterocyte barriers. Unlike gluten peptides, however, they are not appreciably recognized by transglutaminase, HLA-DQ2, or disease-specific T cells. In vitro and animal studies show that the biomarkers can detect intestinal permeability changes as well as glutenase-catalyzed gastric detoxification of gluten. Accordingly, controlled clinical studies are warranted to evaluate the use of these peptides as probes for abnormal intestinal permeability in celiac patients and for glutenase efficacy in clinical trials and practice.
The extensive physiological influence of transmission through the CB2 cannabinoid receptor makes this G protein-coupled receptor (GPCR) a promising therapeutic target for treating neuropathic pain, inflammation, and immune disorders. However, there is little direct structural information pertaining to either GPCR or CB2-receptor ligand recognition and activation. The present work helps characterize experimentally the ligand-binding interactions of the human CB2 (hCB2) receptor. This study illustrates how our overall experimental approach, “ligand-assisted protein structure” (LAPS), affords direct determination of the requirements for ligand binding to the hCB2 receptor and discrimination among the binding motifs for ligands that activate therapeutically relevant GPCRs.
We report a new strategy for the generation of heterodimeric protein conjugates using an unnatural amino acid with orthogonal reactivity. This paper addresses the challenges of site-specificity and homogeneity with respect to the synthesis of bivalent proteins and antibody-drug conjugates. There are numerous antibody-drug conjugates in preclinical and clinical development, yet these are based either on nonspecific lysine coupling chemistry or on disulfide modification made difficult by the large number of cysteines in antibodies. Here we describe a recombinant approach that can be used to rapidly generate a variety of constructs with defined conjugation sites. Moreover, this methodology results in homogeneous antibody conjugates whose biological, physical and pharmacological properties can be quantitatively assessed and subsequently optimized. As proof of concept, we have generated anti-Her2 Fab-Saporin conjugates that demonstrate excellent potency in vitro.
Cytokine-induced beta-cell apoptosis is important to the etiology of type-1 diabetes. Although previous reports have shown that general inhibitors of histone deacetylase (HDAC) activity, such as suberoylanilide hydroxamic acid and trichostatin A, can partially prevent beta-cell death, they do not fully restore beta-cell function. To understand HDAC isoform selectivity in beta cells, we measured the cellular effects of eleven structurally diverse HDAC inhibitors on cytokine-induced apoptosis in the rat INS-1E cell line. All eleven compounds restored ATP levels and reduced nitrite secretion. However, caspase-3 activity was reduced only by MS-275 and CI-994, both of which target HDAC1, 2, and 3. Importantly, both MS-275 and genetic knock-down of Hdac3 alone were sufficient to restore glucose-stimulated insulin secretion in the presence of cytokines. These results suggest that HDAC3-selective inhibitors may be effective in preventing cytokine-induced beta-cell apoptosis.
Zampanolide and its less active analog dactylolide compete with paclitaxel for binding to microtubules and represent a new class of microtubule-stabilizing agent (MSA). Mass spectrometry demonstrated that the mechanism of action of both compounds involved covalent binding to β-tubulin at residues N228 and H229 in the taxane site of the microtubule. Alkylation of N228 and H229 was also detected in α,β-tubulin dimers. However, unlike cyclostreptin, the other known MSA that alkylates β-tubulin, zampanolide was a strong MSA. Modeling the structure of the adducts, using the NMR-derived dactylolide conformation, indicated that the stabilizing activity of zampanolide is likely due to interactions with the M-loop. Our results strongly support the existence of the luminal taxane site of microtubules in tubulin dimers and that microtubule nucleation induction by MSAs may proceed through an allosteric mechanism.
NADPH-oxidases are a primary source of reactive oxygen species (ROS), which function in normal physiology and, when overproduced, in pathophysiology. Recent studies using mice deficient in Nox2 identify this isoform as a novel target against Nox2-implicated inflammatory diseases. Nox2 activation depends on the binding of the proline rich domain of its heterodimeric partner p22phox to p47phox. A high-throughput screen that monitored this interaction via fluorescence polarization identified ebselen and several of its analogs as inhibitors. Medicinal chemistry was performed to explore structure-activity relationships and to optimize potency. Ebselen and analogs potently inhibited Nox1 and Nox2 activity but were less effective against other isoforms. Ebselen also blocked translocation of p47phox to neutrophil membranes. Thus, ebselen and its analogs represent a class of compounds that inhibit ROS generation by interrupting the assembly of Nox2-activating regulatory subunits.
Rho GTPases have been implicated in diverse cellular functions and are potential therapeutic targets. By virtual screening, we have identified a Rho specific inhibitor, Rhosin. Rhosin contains two-aromatic rings tethered by a linker, and it binds to the surface area sandwiching Trp58 of RhoA with a submicromolar Kd and effectively inhibits GEF-catalyzed RhoA activation. In cells Rhosin specifically inhibited RhoA activity and RhoA-mediated cellular function without affecting Cdc42 or Rac1 signaling activities. By suppressing RhoA or RhoC activity Rhosin could inhibit mammary sphere formation by breast cancer cells, suppress invasion of mammary epithelial cells, and induce neurite outgrowth of PC12 cells in synergy with NGF. Thus, the rational designed RhoA subfamily specific small molecule inhibitor is useful for studying the physiological and pathologic roles of Rho GTPase.
Rho GTPases; RhoA; signaling; small molecule; inhibitor; rational drug design; targeting