PDB ID : 2ADU Title : Human Methionine Aminopeptidase Complex with 4-Aryl-1,2,3-triazole Inhibitor Authors : Kallander, L.S.; Lu, Q.; Chen, W.; Tomaszek, T.; Yang, G.; Tew, D.; Meek, T.D.; Hofmann, G.A.; Schulz-Pritchard, C.K.; Smith, W.W.; Janson, C.A.; Ryan, M.D.; Zhang, G.F.; Johanson, K.O.; Kirkpatrick, R.B.; Ho, T.F.; Fisher, P.W.; Mattern, M.R.; Johnson, R.K.; Hansbury, M.J.; Winkler, J.D.; Ward, K.W.; Veber, D.F.; Thompson, S.K. Deposited on : 2005-07-20 Resolution : 1.90 Å(reported)

As part of a search for novel inhibitors of cathepsin K, the MeOH extract of a Micronesian sponge of the order Haplosclerida was shown to be active. Bioassay-guided fractionation of the extract yielded halitoxins, tryptamine, and a novel tryptamine-derived alkaloid, haploscleridamine (1). The tetrahydro-beta-carboline structure of haploscleridamine (1) was elucidated through spectral techniques. Haploscleridamine (1) was found to be an inhibitor of cathepsin K with an IC(50) of 26 microM.

A nonpeptidyl small molecule SB 247464, capable of activating granulocyte-colony-stimulating factor (G-CSF) signal transduction pathways, was identified in a high-throughput assay in cultured cells. Like G-CSF, SB 247464 induced tyrosine phosphorylation of multiple signaling proteins and stimulated primary murine bone marrow cells to form granulocytic colonies in vitro. It also elevated peripheral blood neutrophil counts in mice. The extracellular domain of the murine G-CSF receptor was required for the activity of SB 247464, suggesting that the compound acts by oligomerizing receptor chains. The results indicate that a small molecule can activate a receptor that normally binds a relatively large protein ligand.

Bioassay-guided fractionation of the EtOAc extract of sterns and leaves of Piper ponapense yielded a novel tetrahydropyridine alkaloid, piperpense (1) whose structure was determined by interpretation of the spectral data and X-ray diffraction analysis. Alkaloid 1 was found to be active in a cell-based assay for DNA damaging activity.

As part of a search for novel inhibitors of Herpes Zoster protease, the MeOH extract of the green alga Tuemoya sp. was shown to be active, Bioassay-guided fractionation of the extract yielded a previously reported cycloartane-3,28-disulfate-23-ol plus a novel compound that was identified as cycloart-24-en-23-one-28-sulfate-3-ol (1). These compounds demonstrated activity against both VZV and CMV protease in the 4-7 mu M range.

The sponge Batzella sp, from Jamaica contains four new alkaloids, batzelladines F-I(1-4), that induce the p56(lck)-CD4 dissociation. Batzelladines F-I were isolated using a bioassay-directed fractionation scheme and were identified by interpretation of spectroscopic data, Batzelladines F, G, and H + I were active in the p56(lck)-CD4 dissociation assay at micromolar concentrations.

Bioassay-guided fractionation of the MeOH extract of the South African ascidian Lissoclinum sp. yielded three known compounds plus a novel alkaloid, lissoclin disulfoxide (1), whose structure was determined by interpretation of spectral data. Alkaloid 1 had IC50 values of 0.6 and 0.82 mu M against the inhibition of IL-8 R alpha and IL-8 R beta receptors, respectively.

Bioassay-guided fractionation of the EtOAc extract of sponge Plakortis halichondrioides yielded four novel bicyclic lactones, plakortones A, B, C, D and a novel acid, plakortide E (3, 4, 5, 6 and 7). The structures, including stereochemistry, of these compounds were established by interpretation of spectral data. Plakortones A-D (3–6) comprise a novel class of activators of cardiac SR-Ca2+-pumping ATPase which were found to be active at micromolar concentrations As part of an SAR study, the α and β diols 9 and 10 of plakortone D were prepared using Sharpless AD procedure.

The Caribbean sponge Batzella sp. contains a number of guanidine alkaloids, two of which, batzelladines A (1) and B (2), inhibit the binding of HIVgp-120 to CD4 and are therefore potential inhibitors of HIV. In addition to the known metabolites ptilomycalin A (6), ptilocaulin (7), crambescin A (8), crambescidin 800 (9), and crambescidin 816 (10), Batzella sp. contains five new alkaloids, batzelladines A-E (1-5), the structures of which were elucidated by interpretation of spectral data and chemical degradation.

We show that cell lines derived from childhood alveolar rhabdomyosarcoma (RMS) are very sensitive to the growth-inhibitory effects of the immunosuppressive agent rapamycin (RAP), compared to other human cell lines (50% inhibitory concentration range of 0.1-8 ng/ml, compared to 1280 to > 10,000 ng/ml). Our data suggest that the sensitivity of RMS lines is due to RAP inhibition of insulin-like growth factor 1 receptor-mediated signaling, which is essential for continued proliferation of RMS cells. The embryonal RMS line Rh1, which was resistant to RAP in serum-containing medium (50% inhibitory concentration, 4180 ng/ml), was highly sensitive under autocrine conditions of growth, indicating that resistance was due to paracrine signaling pathways insensitive to RAP action. FK506 reversed RAP action in all cell lines, indicating a dependence on complexing with the cytosolic FK506-binding protein for activity.

The binding of FK506 and rapamycin to their cytosolic receptor FKBP12 is an intermediate step in the paths leading to their potent immunosuppressive properties. One of the amino acids defining the hydrophobic binding cleft for the macrocycles is Tyr82, which is thought to form a hydrogen bond with the amide oxygens of the common pipecolyl structural element within the two macrolides. To understand better the influence of this amino acid residue in catalytic activity (cis-trans peptidyl prolyl isomerization) and ligand binding properties, a Tyr82 to Leu site-specific modification of FKBP12 was prepared, purified and characterized. Kinetic experiments have demonstrated that the Tyr82 to Leu modification has a greater effect on catalytic properties than on ligand binding affinities, a result which indicates that these inhibitors may not be binding as true transition-state analogues. In an additional test for cellular function, expression of both wild-type and mutant human FKBP12 in a strain of Saccharomyces cerevisiae rendered resistant to rapamycin by deletion of the gene encoding a cytosolic rapamycin binding protein (RPB1), the yeast homologue of FKBP12, restored wild-type drug sensitivity.

Inophyllums are novel non-nucleoside inhibitors of human immunodeficiency virus (HIV) type 1 reverse transcriptase identified through an enzyme screening program and isolated from the plant Calophyllum inophyllum. The kinetics of reverse transcriptase inhibition by inophyllum B were characterized using recombinant purified enzyme, a heteropolymeric RNA template, and a scintillation proximity assay. Preincubation of inhibitor with the enzyme-template-primer complex for 11 min was required for maximal inhibition of reverse transcriptase to occur, suggesting that inophyllum B had a slow on-rate and that template-primer must bind to reverse transcriptase prior to inhibitor binding. Inhibition of reverse transcriptase by inophyllums was shown to be reversible. When thymidine triphosphate was the variable substrate, inophyllum B inhibited reverse transcriptase noncompetitively with a K-i of 42 nM. Enzyme inhibition with respect to template-primer was uncompetitive with a K-i of 26 nM. Reverse transcriptase enzymes containing point mutations in which tyrosine 181 was changed to either cysteine or isoleucine exhibited marginal resistance to inophyllums but were resistant to (+)-(5S) 4,5,6,7-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)- imidazo[4,5,1-j,k][1,4]benzodiazepin-2-(1H)-thione (TIBO RS2913). A mutant enzyme in which tyrosine 188 was changed to leucine was cross-resistant to both inophyllum B and TIBO RS2913, as was HIV type 2 reverse transcriptase. These studies suggest that inophyllum B and TIBO R82913 bind to distinct but overlapping sites. Inhibition of avian myeloblastosis virus reverse transcriptase and Moloney murine leukemia virus reverse transcriptase by inophyllum B was detectible, suggesting that these inhibitors may be more promiscuous than other previously described non-nucleoside inhibitors. Inophyllums were active against HIV type 1 in cell culture with IC50 values of approximately 1.5 mu M. These studies imply that the inophyllums have a novel mechanism of interaction with reverse transcriptase and as such could conceivably play a role in combination therapy.

FK506 and cyclosporin A (CsA) are potent immunosuppressive agents that display antifungal activity. They act by blocking a Ca2+-dependent signal transduction pathway leading to interleukin-2 transcription. Each drug forms a complex with its cognate cytosolic immunophilin receptor (i.e., FKBP12-FK506 and cyclophilin-CsA) which acts to inhibit the Ca2+/calmodulin-dependent protein phosphatase 2B, or calcineurin (CN). We and others have defined the Saccharomyces cerevisiae FKS1 gene by recessive mutations resulting in 100–1000-fold hypersensitivity to FK506 and CsA (as compared to wild type), but which do not affect sensitivity to a variety of other antifungal drugs. The fks1 mutant also exhibits a slow-growth phenotype that can be partially alleviated by exogenously added Ca2+ [Parent et al., J. Gen. Microbiol. 139 (1993) 2973–2984]. We have cloned FKS1 by complementation of the drug-hypersensitive phenotype. It contains a long open reading frame encoding a novel 1876-amino-acid (215 kDa) protein which shows no similarity to CN or to other protein phosphatases. The FKS1 protein is predicted to contain 10 to 12 transmembrane domains with a structure resembling integral membrane transporter proteins. Genomic disruption experiments indicate that FKS1 encodes a nonessential function; fks1::LEU2 cells exhibit the same growth and recessive drug-hypersensitive phenotypes observed in the original fks1 mutants. Furthermore, the fks1::LEU2 allele is synthetically lethal in combina- tion with disruptions of both of the nonessential genes encoding the alternative forms of the catalytic A subunit of CN (CNA1 and CNA2). These data suggest that FKS1 provides a unique cellular function which, when absent, increases FK506 and CsA sensitivity by making the CNs (or a CN-dependent function) essential.

The oxoaporphine alkaloids oxophoebine [1] and liriodenine [2] have been isolated from Xylopia aethiopica (Annonaceae). Both showed selective toxicity against DNA repair and recombination deficient mutants of the yeast Saccharomyces cerevisae. Three related but inactive compounds, oxoglaucine [3], O-methylmoschatoline [4], and lysicamine [5], were also isolated from this plant. Selective toxicity was also observed for 10-methoxyliriodenine (lauterine) [6] and 10-hydroxyliriodenine [7], two oxoaporphine alkaloids isolated from Miliusa cf. banacea (Annonaceae). The structure of 10-hydroxyliriodenine [7], a novel oxoaporphine, was determined by spectroscopic methods and chemical conversion to compound 6. The role of the bioactive oxoaporphine alkaloids as DNA topoisomerase inhibitors is discussed.

The yeast TOR1 and TOR2 proteins were previously discovered as putative targets of the immunosuppressive drug rapamycin, Although their cellular function is unknown, they are predicted to be at least 215 kDa in size and possess a C-terminal phosphatidylinositol (PI) kinase-related domain. We previously identified a conserved Ser residue, within the PI kinase-related domain of both yeast TOR proteins (Ser(1972) in TOR1; Ser(1975) in TOR2), as being the site of missense mutations conferring dominant rapamycin resistance. The Ser(1972/1975) res. residue of yeast TOR is conserved in mammalian TOR homologs. One possibility is that this residue is critical for a direct interaction between TOR and the FKBP12-rapamycin complex. There is very recent biochemical evidence for an interaction between mammalian TOR and FKBP12-rapamycin (Brown, E. J., Albers, M. W., Shin, T. B., Ichikawa, K., Keith, C. T., Lane, W. S., and Schreiber, S. L. (1994) Nature 369, 756-758; Sabatini, D. M., Erdjument-Bromage, H., Lui, M., Tempst, P., and Snyder, S. H. (1994) Cell 78, 35-43). Using the yeast two-hybrid system, we now have obtained genetic proof of a physical interaction between FKBP12-rapamycin and TOR and have demonstrated that this interaction requires the conserved Ser residue. We have found that a small fragment of wild-type yeast TOR2 spanning Ser(1975) is capable of interacting with human FKBP12 in the presence of rapamycin, whereas an Arg(1975) mutant fails to interact, This effect is dependent upon rapamycin and is antagonized by FK506.

Twelve coumarins isolated from plants of the Rutaceae collected in Sri Lanka have been subjected to a mechanism-based anticancer bioassay employing DNA repair-deficient and repair-proficient yeasts. Of these, seselin [10] and xanthyletin [11] were found to be active. Seselin also exhibited moderate cytotoxicity.

The C-7 methoxy group in rapamycin has been found to be labile toward acidic reagents. Conditions have been developed to replace this group with a number of different nucleophiles, such as alcohols, thiols, and electron-rich aromatic systems. This novel, efficient transformation allows the selective manipulation of the rapamycin effector domain.

The yeast TOR1 (DRR1) and TOR2 (DRR2) proteins are putative targets of the immunosuppressive drug rapamycin (Rm), defined by dominant drug-resistance mutations. They share a large C-terminal domain that exhibits sequence similarity to the 110-kDa subunit of phosphatidylinositol (PI) 3-kinases. In this report, we present an amino acid (aa) sequence alignment of TOR1 (DRR1) and TOR2 (DRR2) and identify conserved and nonconserved motifs within the N-terminal domain that are indicative of possible nuclear localization. We also show that the mutations responsible for Rm resistance in four independent drr 2(dom) alleles alter the identical aa (Ser(1975)-->Arg) previously identified in drr1(dom) mutants (Ser(1972)-->Arg or Asn). Models for TOR (DRR) protein function are discussed.

Bioactivity-directed fractionation of the CHCl3 extract of the bark of Erythrina burana afforded phaseollidin [1] and cristacarpin [2]. Both 1 and 2 exhibited moderate but selective activity towards DNA repair-deficient yeast mutants, whereas only 1 was found to be cytotoxic. 13C-nmr spectra of both compounds were assigned.