High-throughput identification of inhibitors of human mitochondrial peptide deformylase

The human mitochondrial peptide deformylase (HsPDF) provides a potential new target for broadly acting antiproliferative agents. To identify novel nonpeptidomimetic and nonhydroxamic acid-based inhibitors of HsPDF, the authors have developed a high-throughput screening (HTS) strategy using a fluorescence polarization (FP)-based binding assay as the primary assay for screening chemical libraries, followed by an enzymatic-based assay to confirm hits, prior to characterization of their antiproliferative activity against established tumor cell lines. The authors present the results and performance of the established strategy tested in a pilot screen of 2880 compounds and the identification of the 1st inhibitors. Two common scaffolds were identified within the hits. Furthermore, cytotoxicity studies revealed that most of the confirmed hits have antiproliferative activity. These findings demonstrate that the designed strategy can identify novel functional inhibitors and provide a powerful alternative to the use of functional assays in HTS and support the hypothesis that HsPDF inhibitors may constitute a new class of antiproliferative agent.     

Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain

The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans - stereoconfiguration in the beta-position are described. The water- soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis -isoprene unit in the beta-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc- P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis -isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water- soluble analog may provide an experimental approach to assay the hypothetical "flippase" proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.      

A protecting group-free synthesis of deazathiamine: a step toward inhibitor design

The discovery of 3-deazathiamine diphosphate (deazaThDP) as a potent inhibitor analog of the cofactor thiamine diphosphate (ThDP) has highlighted the need for an efficient and scalable synthesis of deazaThDP. Such a method would facilitate development of analogs with the ability to inhibit individual ThDP-dependent enzymes selectively. Toward the goal of developing selective inhibitors of the mycobacterial enzyme 2-hydroxy-3-oxoadipate synthase (HOAS), we report an improved synthesis of deazaThDP without use of protecting groups. Tribromo-3-methylthiophene served as a versatile starting material whose selective functionalization permitted access to deazaThDP in five steps, with potential to make other analogs accessible in substantial amounts.     

Synthesis and evaluation of geldanamycin-testosterone hybrids

Geldanamycin (GDM) binds to the Hsp90 chaperone protein resulting in the degradation of several important signaling proteins. A series of GDM-testosterone linked hybrids has been synthesized and evaluated for activity against prostate cancer cell lines. The hybrid with the greatest activity exhibits potent and selective cytotoxicity against prostate cancer cells containing the androgen receptor.     

Structure and Activity of Human Mitochondrial Peptide Deformylase, a Novel Cancer Target

Peptide deformylase proteins (PDFs) participate in the N-terminal methionine excision pathway of newly synthesized peptides. We show that the human PDF (HsPDF) can deformylate its putative substrates derived from mitochondrial DNA-encoded proteins. The first structural model of a mammalian PDF (1.7 Å), HsPDF, shows a dimer with conserved topology of the catalytic residues and fold as non-mammalian PDFs. The HsPDF C-terminus topology and the presence of a helical loop (H2 and H3), however, shape a characteristic active site entrance. The structure of HsPDF bound to the peptidomimetic inhibitor actinonin (1.7 Å) identified the substrate-binding site. A defined S1′ pocket, but no S2′ or S3′ substrate-binding pockets, exists. A conservation of PDF-actinonin interaction across PDFs was observed. Despite the lack of true S2′ and S3′ binding pockets, confirmed through peptide binding modeling, enzyme kinetics suggest a combined contribution from P2′and P3′ positions of a formylated peptide substrate to turnover.     

Stereo-controlled synthesis of novel photoreactive γ-secretase inhibitors

The stereoselective synthesis of novel photoreactive γ-secretase inhibitors 2 and 3 has been achieved. Key steps of the strategy involve preparation of α-N-Boc-epoxide 8 and formation of lactone 14 in a practical and stereo-controlled fashion. Compounds 2 and 3 are potent γ-secretase inhibitors and directly interact with presenilin-1, a catalytic subunit of γ-secretase.     

A peptidomimetic siRNA transfection reagent for highly effective gene silencing

RNA interference (RNAi) techniques hold forth great promise for therapeutic silencing of deleterious genes. However, clinical applications of RNAi require the development of safe and efficient methods for intracellular delivery of small interfering RNA (siRNA) oligonucleotides specific to targeted genes. We describe the use of a lipitoid, a cationic oligopeptoid-phospholipid conjugate, for non-viral transfection of synthetic siRNA oligos in cell culture. This peptidomimetic delivery vehicle allows for efficient siRNA transfection in a variety of human cell lines with negligible toxicity and promotes extensive downregulation of the targeted genes at both the protein and the mRNA level. We compare the lipitoid reagent to a standard commercial transfection reagent. The lipitoid is highly efficient even in primary IMR-90 human lung fibroblasts in which other commercial reagents are typically ineffective.     

Chemical inhibition of N-WASP by stabilization of a native autoinhibited conformation

Current drug discovery efforts focus primarily on proteins with defined enzymatic or small molecule binding sites. Autoregulatory domains represent attractive alternative targets for small molecule inhibitors because they also occur in noncatalytic proteins and because allosteric inhibitors may avoid specificity problems inherent in active site-directed inhibitors. We report here the identification of wiskostatin, a chemical inhibitor of the neural Wiskott-Aldrich syndrome protein (N-WASP). Wiskostatin interacts with a cleft in the regulatory GTPase-binding domain (GBD) of WASP in the solution structure of the complex. Wiskostatin induces folding of the isolated, unstructured GBD into its autoinhibited conformation, suggesting that wiskostatin functions by stabilizing N-WASP in its autoinhibited state. The use of small molecules to bias conformational equilibria represents a potentially general strategy for chemical inhibition of autoinhibited proteins, even in cases where such sites have not been naturally evolved in a target.     

Radiosynthesis of the tumor hypoxia marker [18F]TFMISO via O-[18F]trifluoroethylation reveals a striking difference between trifluoroethyl tosylate and iodide in regiochemical reactivity toward oxygen nucleophiles

The MRI hypoxia marker trifluoromisonidazole (TFMISO) [1-(2-nitro-1H-imidazol-1-yl)-3-(2,2,2-trifluoroethoxy)propan-2-ol] was successfully labeled with (18)F to expand its role into a bimodal PET/MRI probe. (18)F-Labeling was achieved via a three-step procedure in which 2,2,2-[(18)F]trifluoroethyl p-toluenesulfonate prepared by (18)F-(19)F exchange served as the [(18)F]trifluoroethylating agent. The O-[(18)F]trifluoroethylation reaction proceeded efficiently to give the intermediate 1,2-epoxy-3-(2,2,2-[(18)F]trifluoroethoxy)propane, with approximately 60% of (18)F incorporated from the tosylate precursor, which was condensed with 2-nitroimidazole to yield [(18)F]TFMISO. Approximately 40% of the [(18)F]trifluoroethyl tosylate precursor was converted into the final product. In stark contrast, 2,2,2-[(18)F]trifluoroethyl iodide failed to produce [(18)F]TFMISO, giving instead 1,1-[(18)F]difluoro-2-iodoethoxy and 1-[(18)F]fluoro-2-iodovinyloxy analogs of [(18)F]TFMISO. Thus, this investigation has identified 2,2,2-[(18)F]trifluoroethyl tosylate as an excellent [(18)F]trifluoroethylating agent, which can convert efficiently an alcohol into the corresponding [(18)F]trifluoroethyl ether.     

Substitutions of potentially phosphorylatable serine residues of Bax reveal how they may regulate its interaction with mitochondria

During apoptosis, the pro-apoptotic protein Bax relocalizes from the cytosol to the mitochondrial outer membrane. This relocalization is associated to major conformational changes, namely at the N- and C-terminal ends of the protein. Substitution of residues located at critical positions within the protein potentially stimulates or inhibits this process. In the present study, we investigated the hypothesis that phosphorylation of serine residues might trigger these conformational changes, with a focus on Ser(163) and Ser(184), which have been shown to be phosphorylatable by protein kinases GSK3beta and Akt/PKB, respectively, and on Ser(60), which is located in a consensus target sequence for PKA. Substitutions of these serine residues by alanine or aspartate were done in wild type or previously characterized Bax mutants, and the capacity of the resulting proteins to interact with mitochondria and to release cytochrome c was assayed in yeast, which provides a tool to study the function of Bax, independently of the rest of the apoptotic network. We conclude that sequential phosphorylation of these serine residues might participate in the triggering of the different conformational changes associated with Bax activation during apoptosis.