Relevant Protein Targets Research Articles

Diazo Reagents with Small Steric Footprints for Simultaneous Arming/SAR Studies of Alcohol-Containing Natural Products via O–H Insertion

Natural products are essential tools for basic cellular studies leading to the identification of medically relevant protein targets and the discovery of potential therapeutic leads. The development of methods that enable mild and selective derivatization of natural products continues to be of significant interest for mining their information-rich content. Herein, we describe novel diazo reagents for simultaneous arming and structure-activity relationship (SAR) studies of alcohol-containing natural products with a small steric footprint, namely, an α-trifluoroethyl (HTFB) substituted reagent. The Rh(II)-catalyzed O-H insertion reaction of several natural products, including the potent translation inhibitor lactimidomycin, was investigated, and useful reactivity and both chemo- and site (chemosite) selectivities were observed. Differential binding to the known protein targets of both FK506 and fumagillol was demonstrated, validating the advantage of the smaller steric footprint of α-trifluoroethyl derivatives. A p-azidophenyl diazo reagent is also described that will prove useful for photoaffinity labeling of low affinity small molecule protein receptors.

Mind Molecules

Scientific styles vary tremendously. For me, research is largely about the unfettered pursuit of novel ideas and experiments that can test multiple ideas in a day, not a year, an approach that I learned from my mentor Julius “Julie” Axelrod. This focus on creative conceptualizations has been my métier since working in the summers during medical school at the National Institutes of Health, during my two years in the Axelrod laboratory, and throughout my forty-five years at Johns Hopkins University School of Medicine. Equally important has been the “high” that emerges from brainstorming with my students. Nothing can compare with the eureka moments when, together, we sense new insights and, better yet, when high-risk, high-payoff experiments succeed. Although I have studied many different questions over the years, a common theme emerges: simple biochemical approaches to understanding molecular messengers, usually small molecules. Equally important has been identifying, purifying, and cloning the messengers' relevant biosynthetic, degradative, or target proteins, at all times seeking potential therapeutic relevance in the form of drugs. In the interests of brevity, this Reflections article is highly selective, and, with a few exceptions, literature citations are only of findings of our laboratory that illustrate notable themes.

Rational Methods for the Selection of Diverse Screening Compounds

Traditionally a pursuit of large pharmaceutical companies, high-throughput screening assays are becoming increasingly common within academic and government laboratories. This shift has been instrumental in enabling projects that have not been commercially viable, such as chemical probe discovery and screening against high-risk targets. Once an assay has been prepared and validated, it must be fed with screening compounds. Crafting a successful collection of small molecules for screening poses a significant challenge. An optimized collection will minimize false positives while maximizing hit rates of compounds that are amenable to lead generation and optimization. Without due consideration of the relevant protein targets and the downstream screening assays, compound filtering and selection can fail to explore the great extent of chemical diversity and eschew valuable novelty. Herein, we discuss the different factors to be considered and methods that may be employed when assembling a structurally diverse compound collection for screening. Rational methods for selecting diverse chemical libraries are essential for their effective use in high-throughput screens.

Avoiding Barriers to PET Radioligand Development: Cellular Assays of Brain Efflux Transporters

PET provides a uniquely sensitive and powerful means for studying a clinically relevant protein target within the brain, provided that an adequately specific and sensitive radiotracer is available. Interesting targets, which include neurotransmitter receptors, neurotransmitter transporters, enzymes

Identification of Clinically Relevant Protein Targets in Prostate Cancer with 2D-DIGE Coupled Mass Spectrometry and Systems Biology Network Platform

Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies.

Target highlights in CASP9: Experimental target structures for the critical assessment of techniques for protein structure prediction.

One goal of the CASP community wide experiment on the critical assessment of techniques for protein structure prediction is to identify the current state of the art in protein structure prediction and modeling. A fundamental principle of CASP is blind prediction on a set of relevant protein targets, that is, the participating computational methods are tested on a common set of experimental target proteins, for which the experimental structures are not known at the time of modeling. Therefore, the CASP experiment would not have been possible without broad support of the experimental protein structural biology community. In this article, several experimental groups discuss the structures of the proteins which they provided as prediction targets for CASP9, highlighting structural and functional peculiarities of these structures: the long tail fiber protein gp37 from bacteriophage T4, the cyclic GMP-dependent protein kinase Iβ dimerization/docking domain, the ectodomain of the JTB (jumping translocation breakpoint) transmembrane receptor, Autotaxin in complex with an inhibitor, the DNA-binding J-binding protein 1 domain essential for biosynthesis and maintenance of DNA base-J (β-D-glucosyl-hydroxymethyluracil) in Trypanosoma and Leishmania, an so far uncharacterized 73 residue domain from Ruminococcus gnavus with a fold typical for PDZ-like domains, a domain from the phycobilisome core-membrane linker phycobiliprotein ApcE from Synechocystis, the heat shock protein 90 activators PFC0360w and PFC0270w from Plasmodium falciparum, and 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae.

Modulation of MicroRNA Expression In Sickle Reticulocytes Is Associated with Hydroxyurea Treatment and Fetal Hemoglobin Induction

AbstractAbstract 2650 Background:Hydroxyurea has both laboratory and clinical efficacy for children, adolescents, and adults with Sickle Cell Anemia (SCA), and its benefits are primarily due to its ability to increase fetal hemoglobin (HbF). However, HbF induction by hydroxyurea is highly variable among patients, and its mechanism of HbF reactivation remains unclear. MicroRNAs (miRNA) are small non-coding RNAs that can regulate gene expression by inhibiting transcription or translation of targeted proteins. Recently, miRNAs have been implicated in cellular regulation and differentiation including hematopoiesis and hemoglobin switching. In an effort to elucidate the mechanisms behind hydroxyurea-mediated HbF induction, we tested the hypothesis that hydroxyurea modulates miRNA expression in sickle reticulocytes in vivo and this modulation is associated with changes in HbF levels. Methods:As part of the prospective Hydroxyurea Study of Long-term Effects (HUSTLE, NCT00305175), total RNA was purified from CD71+ reticulocytes isolated from the peripheral blood of SCA patients, either prior to hydroxyurea treatment or after reaching stable maximum tolerated dose (MTD); non-SCA adults were included as controls. Initially, differential miRNA expression associated with disease and/or hydroxyurea exposure was determined by microarray and then confirmed by real-time PCR in a cross-sectional analysis of hydroxyurea-treated (n=13) and untreated (n=22) SCA patients, plus controls (n=8). Subsequently to identify hydroxyurea-mediated changes in miRNA expression and its association with HbF induction, miRNA levels were measured by real-time PCR in paired samples collected from patient reticulocytes at baseline and at hydroxyurea MTD (n=41). Statistical correlation to HbF levels used Spearman correlation coefficient and mixed model analysis. Results:Microarray analysis identified 108 miRNAs expressed in CD71+ reticulocytes, 10 of which were significantly different in SCA patients with or without hydroxyurea exposure and normal controls. Real-time PCR confirmed that miRNA expression of human miRNA (hsa-mir) 29a, 130b, 215, and 494 were upregulated, while hsa-mir-223 was downregulated, in untreated SCA patients compared to non SCA controls. Additional analysis of 41 paired samples showed that 3 miRNAs, hsa-mir 148a, 151-3p, and 494, were significantly upregulated with hydroxyurea treatment (Table 1). At MTD, HbF levels increased an average of 17% from a mean of 8.9 ± 6.1% at baseline (range 0.0–22.9%) to a mean of 25.9 ± 9.0% at MTD (range 9.4–55.9%).Significant associations between hsa-mir 26b miRNA expression and HbF levels were identified, both at baseline (rs= -.34; p=0.03) and at MTD (rs= -.32; p=0.04). Using mixed model analysis, change in hsa-mir 151-3p expression was significantly associated with the change in HbF (p=0.047) from baseline to MTD. Conclusion:We identified specific miRNAs that are significantly associated with sickle cell anemia, hydroxyurea treatment, and hydroxyurea-mediated HbF induction. These studies suggest that miRNA regulation, specifically hsa-mir 26b and 151-3p, may be involved in hydroxyurea-mediated HbF induction in patients with sickle cell anemia. Future studies to identify the relevant protein targets may lead to a better understanding of hydroxyurea's mechanisms of action and patient response to the drug including the observed inter-patient variability in HbF response.Table 1Modulation of miRNA expression in SCA patients and by hydroxyurea treatment.Control v Untreated SCAFold Changep-valuemir 29a1.840.035mir 130b1.740.018mir 2152.30.009mir 223−1.790.015mir 4941.870.026Baseline v MTDmir 148a1.690.0002mir 151-3p1.640.004mir 4943.060.029Results from real-time PCR shows differential miRNA expression in non-SCA controls compared to untreated SCA patients from cross-sectional analysis, and upregulation of 3 miRNAs in SCA patients at baseline compared to MTD from paired-sample analysis. Negative fold change indicates a downregulation. Disclosure:Off Label Use: Hydroxyurea used to treat sickle cell anemia in children. Disclosures:Off Label Use: Hydroxyurea used to treat sickle cell anemia in children.

Investigating the correlations among the chemical structures, bioactivity profiles and molecular targets of small molecules

Motivation: Most of the previous data mining studies based on the NCI-60 dataset, due to its intrinsic cell-based nature, can hardly provide insights into the molecular targets for screened compounds. On the other hand, the abundant information of the compound–target associations in PubChem can offer extensive experimental evidence of molecular targets for tested compounds. Therefore, by taking advantages of the data from both public repositories, one may investigate the correlations between the bioactivity profiles of small molecules from the NCI-60 dataset (cellular level) and their patterns of interactions with relevant protein targets from PubChem (molecular level) simultaneously.Results: We investigated a set of 37 small molecules by providing links among their bioactivity profiles, protein targets and chemical structures. Hierarchical clustering of compounds was carried out based on their bioactivity profiles. We found that compounds were clustered into groups with similar mode of actions, which strongly correlated with chemical structures. Furthermore, we observed that compounds similar in bioactivity profiles also shared similar patterns of interactions with relevant protein targets, especially when chemical structures were related. The current work presents a new strategy for combining and data mining the NCI-60 dataset and PubChem. This analysis shows that bioactivity profile comparison can provide insights into the mode of actions at the molecular level, thus will facilitate the knowledge-based discovery of novel compounds with desired pharmacological properties.Availability: The bioactivity profiling data and the target annotation information are publicly available in the PubChem BioAssay database (ftp://ftp.ncbi.nlm.nih.gov/pubchem/Bioassay/).Contact: ywang@ncbi.nlm.nih.gov; bryant@ncbi.nlm.nih.govSupplementary information: Supplementary data are available at Bioinformatics online.

Anti-glioma effect of combination of bFGF-siRNA and Vpr in nude mice

To study the anti-glioma effect of recombinant adenovirus mediated combined gene therapy of bFGF-siRNA and HIV1-Vpr in vivo. Mouse glioma model was established by injecting 5 × 10(6) LN229 cells into BALB/c-nu nude mice. 30 nude mice were randomly divided into 5 groups: the negative control group, mock group, bFGF-siRNA group, Vpr group and combined therapy group, which at regular intervals were injected with PBS, rAd5-null, rAd5-bFGF-siRNA, rAd5-Vpr, rAd5-bFGF-siRNA plus rAd5-Vpr, respectively. The tumor volume was recorded every third day to draw a growth curve. After four weeks treatment, the mice were killed and specimens were taken. HE, immunohistochemical and TUNEL staining were performed to observe the cell morphology, detect the changes of relevant target proteins and cell apoptosis, respectively. Also the ultrastructural changes were observed by electron microscopy. The tumor growth inhibition rates were 36.9%, 37.2% and 58.6% in the bFGF-siRNA group, Vpr group and combined therapy group, respectively, and the combined therapy group showed the most significant effect (P < 0.05). Also the results of HE, immunohistochemical and TUNEL staining revealed that the combined therapy group had the best effects on proliferation inhibition and apoptosis induced in glioma cells (P < 0.05). The most significant ultrastructural changes were observed in the combined therapy group. The combined gene therapy of bFGF-siRNA with Vpr shows a prominent and synergistic anti-glioma effect compared with that of mono-gene therapy in nude mice.

Conformationally constrained NR2B selective NMDA receptor antagonists derived from ifenprodil: Synthesis and biological evaluation of tetrahydro-3-benzazepine-1,7-diols

NR2B selective NMDA receptor antagonists with tetrahydro-3-benzazepine-1,7-diol scaffold have been designed by formal cleavage and reconstitution of the piperidine ring of the lead compound ifenprodil ( 1). The secondary amine 10 represents the central building block for the synthesis of more than 25 tetrahydro-3-benzazepin-1-ols. Generally 7-hydroxy derivatives display higher NR2B receptor affinities than the corresponding 7-benzyloxy compounds. A distance of four atoms (five bond lengths) between the basic amino group and the terminal aryl moiety led to highest NR2B affinity. 3-(4-Phenylbutyl)-2,3,4,5-tetrahydro-1 H-3-benzazepine-1,7-diol (WMS-1410, 25) represents the most promising NR2B antagonist of this series showing a K i-value of 14 nM. Compound 25 reveals excellent selectivity over more than 100 further relevant target proteins, antagonizes glutamate induced excitotoxicity (IC 50 = 18.4 nM) and is metabolically more stable than ifenprodil. Up to a dose of 100 mg/kg 25 is well tolerated by mice and it shows dose dependent analgesic activity in the late neuropathic pain phase of the formalin assay.

In vitro selection of a DNA-templated small-molecule library reveals a class of macrocyclic kinase inhibitors.

DNA-templated organic synthesis enables the translation of DNA sequences into synthetic small-molecule libraries suitable for in vitro selection. Previously, we described the DNA-templated multistep synthesis of a 13 824-membered small-molecule macrocycle library. Here, we report the discovery of small molecules that modulate the activity of kinase enzymes through the in vitro selection of this DNA-templated small-molecule macrocycle library against 36 biomedically relevant protein targets. DNA encoding selection survivors was amplified by PCR and identified by ultra-high-throughput DNA sequencing. Macrocycles corresponding to DNA sequences enriched upon selection against several protein kinases were synthesized on a multimilligram scale. In vitro assays revealed that these macrocycles inhibit (or activate) the kinases against which they were selected with IC50 values as low as 680 nM. We characterized in depth a family of macrocycles enriched upon selection against Src kinase, and showed that inhibition was highly dependent on the identity of macrocycle building blocks as well as on backbone conformation. Two macrocycles in this family exhibited unusually strong Src inhibition selectivity even among kinases closely related to Src. One macrocycle was found to activate, rather than inhibit, its target kinase, VEGFR2. Taken together, these results establish the use of DNA-templated synthesis and in vitro selection to discover small molecules that modulate enzyme activities, and also reveal a new scaffold for selective ATP-competitive kinase inhibition.

Specific Adducts Formed through a Radical Reaction between Peptides and Contact Allergenic Hydroperoxides

The first step in the development of contact allergy (allergic contact dermatitis) includes the penetration of an allergy-causing chemical (hapten) into the skin, where it binds to macromolecules such as proteins. The protein-hapten adduct is then recognized by the immune system as foreign to the body. For hydroperoxides, no relevant hapten target proteins or protein-hapten adducts have so far been identified. In this work, bovine insulin and human angiotensin I were used as model peptides to investigate the haptenation mechanism of three hydroperoxide haptens: (5R)-5-isopropenyl-2-methyl-2-cyclohexene-1-hydroperoxide (Lim-2-OOH), cumene hydroperoxide (CumOOH), and 1-(1-hydroperoxy-1-methylethyl) cyclohexene (CycHexOOH). These hydroperoxides are expected to react via a radical mechanism, for which 5,10,15,20-tetraphenyl-21H,23H-porphine iron(III) chloride (Fe(III)TPPCl) was used as a radical initiator. The reactions were carried out in 1:1 ethanol/10 mM ammonium acetate buffer pH 7.4, for 3 h at 37 degrees C, and the reaction products were either enzymatically digested or analyzed directly by MALDI/TOF-MS, HPLC/MS/MS, and 2D gel electrophoresis. Both hydroperoxide-specific and unspecific reaction products were detected, but only in the presence of the iron catalyst. In the absence of catalyst, the hydroperoxides remained unreacted. This suggests that the hydroperoxides can enter into the skin and remain inert until activated. Through the detection of a Lim-2-OOH adduct bound at the first histidine (of two) of angiotensin I, it was confirmed that hydroperoxides have the potential to form specific antigens in contact allergy.

A Unitary Anesthetic Binding Site at High Resolution

Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABA(A) receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABA(A) receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABA(A) receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.

Chemical Approaches to a Novel Antitumor Natural Product Pladienolide

Pladienolide, a naturally occurring antitumor molecule discovered by a cell–based reporter gene expression assay, shows prominent antitumor activities in diverse in vitro and in vivo systems based on a unique mechanism of action. The first total synthesis of pladienolides B and D was accomplished to confirm absolute configuration of ten asymmetric centers of pladienolides. Despite the unique mechanism of action, the target protein of pladienolide remained unclear. Binding protein identification by using several types of pladienolide’s derivatives as ‘chemical probes’ demonstrated that splicing factor SF3b is a pharmacologically relevant protein target of pladienolide.

Lead Finder: An Approach To Improve Accuracy of Protein−Ligand Docking, Binding Energy Estimation, and Virtual Screening

An innovative molecular docking algorithm and three specialized high accuracy scoring functions are introduced in the Lead Finder docking software. Lead Finder's algorithm for ligand docking combines the classical genetic algorithm with various local optimization procedures and resourceful exploitation of the knowledge generated during docking process. Lead Finder's scoring functions are based on a molecular mechanics functional which explicitly accounts for different types of energy contributions scaled with empiric coefficients to produce three scoring functions tailored for (a) accurate binding energy predictions; (b) correct energy-ranking of docked ligand poses; and (c) correct rank-ordering of active and inactive compounds in virtual screening experiments. The predicted values of the free energy of protein-ligand binding were benchmarked against a set of experimentally measured binding energies for 330 diverse protein-ligand complexes yielding rmsd of 1.50 kcal/mol. The accuracy of ligand docking was assessed on a set of 407 structures, which included almost all published test sets of the following programs: FlexX, Glide SP, Glide XP, Gold, LigandFit, MolDock, and Surflex. rmsd of 2 A or less was observed for 80-96% of the structures in the test sets (80.0% on the Glide XP and FlexX test sets, 96.0% on the Surflex and MolDock test sets). The ability of Lead Finder to distinguish between active and inactive compounds during virtual screening experiments was benchmarked against 34 therapeutically relevant protein targets. Impressive enrichment factors were obtained for almost all of the targets with the average area under receiver operator curve being equal to 0.92.

Abstract ED05-02: Discovering new drugs to inhibit prostate cancer metastasis

Abstract ED05-02 Cancer chemoprevention offers a unique drug discovery opportunity. This is because at its core, chemoprevention involves the interface between pharmacologic agents that induce perturbations in biological systems and early stage cancer biology. The molecular aberrations in early stage cancer are more limited than those in advanced stage cancer. This provides a more permissive environment for understanding the complex relationships between drug, pharmacologic target, and therapeutic result. Thus, agents with therapeutically relevant activity represent important investigative tools that can be used to reveal important underlying biology, to identify new pharmacologic targets, and to develop pathophysiologically relevant models. Initial agents can then serve as starting blocks for new chemical synthesis, and these new compounds in turn can be evaluated for bioactivity in relevant model systems. The same considerations in the field of chemoprevention that facilitate this conventional bench-to-man approach to drug discovery can also be applied to the more novel approach of beginning drug discovery endeavors in man. The impetus for undertaking such an approach stems from the facts that our preclinical models have severe restrictions in emulating the biology in man, that the transition from preclinical to clinical represents a frequent point of failure, that the time to drug approval is not improving, and that the cost of drug discovery continues to rise. With the advent of new methods that permit robust system-wide measurement of cell and molecular parameters on limiting amounts of tissue, a practical barrier to beginning drug discovery efforts in man is now being lifted. Here too, chemoprevention offers a unique opportunity. This is because chemopreventive agent candidates are relatively non-toxic, and because they are tested in well defined risk cohorts. Thus, taken together, it now becomes possible to prospectively administer relatively non-toxic agents to at risk cohorts, and to screen for cell and molecular effects in a target organ harboring a limited spectrum of molecular aberrations. By definition, the resultant findings will have direct relevance to man, as well as to relevant underlying biology. An example of how an integrated approach was pursued by a single research group will be provided, and will serve to highlight the efficiency of interfacing chemoprevention with drug discovery. In particular, a natural product that possesses anti-motility properties has fostered the elucidation of pathways which regulated prostate cancer cell motility, has led to the identification of a pharmacologically relevant target protein, and has provided the chemical scaffold which supported new agent discovery. By coupling administration of the natural product to man, with gene expression profiling of target cells in man, new regulators of prostate cell motility have been identified and then confirmed by in vitro studies. Citation Information: Cancer Prev Res 2008;1(7 Suppl):ED05-02.

Induction of Apoptosis in Murine Neuroblastoma by Systemic Delivery of Transferrin-Shielded siRNA Polyplexes for Downregulation of Ran

The polymer, OEI-HD, based on beta-propionamide-cross-linked oligoethylenimine and its chemical transferrin conjugate were evaluated for siRNA delivery into murine Neuro2A neuroblastoma cells in vitro and in vivo. An 80% silencing of luciferase expression in neuroblastoma cells, stably transfected with a luciferase gene, was obtained using standard OEI-HD polyplexes or transferrin-conjugated shielded OEI-HD polyplexes. The Ras-related nuclear protein Ran was selected as a therapeutically relevant target protein. Systemic delivery of transferrin-conjugated OEI-HD/RAN siRNA formulations (three intravenous applications at 3 days interval) resulted in >80% reduced Ran protein expression, apoptosis, and a reduced tumor growth in Neuro2A tumors of treated mice. The treatment was not associated with signs of acute toxicity or significant changes in weight, hematology parameters, or liver enzymes (AST, ALT, or AP) of mice. All our results demonstrate that OEI-HD/siRNA formulations can knockdown genes in tumor cells in vitro and in vivo in mice in the absence of unspecific toxicity.

Protein phosphatase 4 regulates apoptosis, proliferation and mutation rate of human cells

The proteins which regulate apoptosis are of great importance both in normal cell biological processes and in the development of pathology in the diverse diseases which involve apoptosis dysfunction. The activity of many of these proteins is controlled by reversible phosphorylation, so that the relevant kinases and phosphatases play crucial roles in apoptosis control. Here we report the analysis of the role of the serine/threonine protein phosphatase, protein phosphatase 4, in controlling the apoptosis of HEK 293 T cells, using the complementary techniques of gene over-expression and down regulation through RNA interference. This analysis has demonstrated that PP4 regulates both apoptosis and proliferation in human cells and has also shown that the level of PP4 has a strong influence on gene mutation rate, which is crucial to oncogenesis. A parallel proteomic analysis has shown that the phosphorylation status of many relevant protein targets is affected by changes in PP4 and has focused attention particularly on the critical apoptosis regulators Bad and PEA-15. The phosphorylation of both of these proteins is increased when PP4 levels are suppressed, and is reduced when PP4 levels are increased, with striking consequences for the fate of the cell.

Simultaneous Arming and Structure/Activity Studies of Natural Products Employing O−H Insertions: An Expedient and Versatile Strategy for Natural Products-Based Chemical Genetics

The identification of "druggable" targets is an immediate opportunity and challenge in the post-genomic era. Natural products are enduring tools for basic cellular studies and leads for identifying medically relevant protein targets. However, their use for these studies is often hampered by limited quantities and a lack of selective and mild monofunctionalization reactions. The development of selective methods that could simultaneously equip the natural product with a reactive group for subsequent conjugation to reporter tags and provide important structure-activity relationship (SAR) information, requiring only a knowledge of functional groups present in the natural product, could significantly decrease the time between bioactive natural product isolation and target identification. Herein, we report such a strategy that enables simultaneous arming and SAR studies of alcohol-containing natural products involving both chemo- and site-selective ("chemosite" selective) and site-nonselective O-H insertion reactions with rhodium carbenoids derived from alkynyl diazo acetates. This strategy was applied to a diverse set of natural products, and general guidelines for predicting chemosite selectivity were formulated. A subsequent Sharpless-Hüisgen [3 + 2] cycloaddition reaction with the appended alkyne allows for attachment of a variety of reporter tags. Using this strategy, we synthesized a novel FK506-biotin conjugate that enabled pull-down of the entire "immunosuppressive complex" including FKBP12, calcineurins A and B, and calmodulin. In addition, the potential for a chemoselective but site-nonselective process was shown with both gibberellic acid methyl ester and brefeldin A using only achiral rhodium catalysts.

Splicing factor SF3b as a target of the antitumor natural product pladienolide

Pladienolide is a naturally occurring antitumor macrolide that was discovered by using a cell-based reporter gene expression assay controlled by the human vascular endothelial growth factor promoter. Despite the unique mechanisms of action and prominent antitumor activities of pladienolides B and D in diverse in vitro and in vivo systems, their target protein has remained unclear. We used 3H-labeled, fluorescence-tagged and photoaffinity/biotin (PB)-tagged 'chemical probes' to identify a 140-kDa protein in splicing factor SF3b as the binding target of pladienolide. Immunoblotting of an enhanced green fluorescent protein fusion protein of SF3b subunit 3 (SAP130) revealed direct interaction between the PB probe and SAP130. The binding affinities of pladienolide derivatives to the SF3b complex were highly correlated with their inhibitory activities against reporter gene expression and cell proliferation. Furthermore, pladienolide B impaired in vivo splicing in a dose-dependent manner. Our results demonstrate that the SF3b complex is a pharmacologically relevant protein target of pladienolide and suggest that this splicing factor is a potential antitumor drug target.