PubMed Health⌕ Search

Biomedical subjects

Craig M Crews

Publications and source records attributed to Craig M Crews.

At least 19 recordsLinked to original sources
PubMed2006

Targeted gene disruption of methionine aminopeptidase 2 results in an embryonic gastrulation defect and endothelial cell growth arrest.

The antiangiogenic agent fumagillin (Fg) and its analog TNP-470 bind to intracellular metalloprotease methionine aminopeptidase-2 (MetAP-2) and inhibit endothelial cell growth in a p53-dependent manner. To confirm the role of MetAP-2 in endothelial cell proliferation and to validate it as a physiological target for the Fg class of antiangiogenic agents, we have generated a conditional MetAP-2 knockout mouse. Ubiquitous deletion of the MetAP-2 gene (MAP2) resulted in an early gastrulation defect, which is bypassed in double MetAP-2/p53 knockout embryos. Targeted deletion of MAP2 specifically in the hemangioblast lineage resulted in abnormal vascular development, and these embryos die at the midsomite stage. In addition, knockdown of MetAP-2 using small interfering RNA or homologous recombination specifically suppresses the proliferation of cultured endothelial cells. Together, these results demonstrate an essential role for MetAP-2 in angiogenesis and indicate that MetAP-2 is responsible for the endothelial cell growth arrest induced by Fg and its derivatives.

Aminopeptidases↗
PubMed2006

Myriaporone 3/4 structure--activity relationship studies define a pharmacophore targeting eukaryotic protein synthesis.

Myriaporones are naturally occurring compounds which structurally resemble the southern hemisphere of the tedanolide family of macrolide antitumor agents. Despite the fact that myriaporone 3/4 represents only a portion of tedanolide, it nonetheless retains much of its biological activity. We show here that like tedanolide, myriaporone 3/4 inhibits protein synthesis and proliferation of mammalian cells with low nanomolar potencies but displays no prokaryotic growth inhibitory effect. Moreover, myriaporone 3/4 displays a very rapid, reversible and p21-independent activity to block S phase progression in mammalian cells. Structure-activity relationship studies revealed that the C18-C19 epoxide and the C14 hydroxymethyl group (tedanolide numbering) of myriaporone 3/4 are required for cell cycle inhibition. These constitute previously unidentified and/or novel pharmacophores for myriaporone 3/4. Our results show that the important biological activities associated with the structurally complex tedanolides are present and can be harnessed in the chemically much simpler myriaporones. This greatly increases the value of the latter as investigative tools for biochemical research as well as for development of potential therapeutics.

Animals↗
PubMed2006

Neurotrophic peptide aldehydes: solid phase synthesis of fellutamide B and a simplified analog.

A combination of solid phase and solution phase synthetic methods have been used to complete the total synthesis of the neurotrophic lipopeptide aldehyde fellutamide B (2). The beta-hydroxy aliphatic tail was prepared by regioselective reductive opening of a cyclic sulfate, and later coupled to a solid phase resin. The synthetic compound was then examined in cytotoxicity and nerve growth factor (NGF) induction assays. A simplified analog of fellutamide B also showed activity.

Aldehydes↗
PubMed2006

Synthetic studies on amphidinolide B1.

[structure: see text]. The syntheses of three fragments, 2, 3, and 4, of amphidinolide B1 have been accomplished. The 1,3-isomerization of allylic alcohol 10 was accomplished via rhenium oxo catalysis and has been applied successfully in the synthesis. (-)-MIB-catalyzed asymmetric vinylzinc addition to aldehyde 31 and the regio- and stereoselective epoxidation of unsymmetrical divinyl methanol 32 were key steps.

Animals↗
PubMed2006

A chemical and genetic approach to the mode of action of fumagillin.

Previous mode of action studies identified methionine aminopeptidase 2 (MetAP-2) as the target of the antiangiogenic natural product fumagillin and its drug candidate analog, TNP-470. We report here that TNP-470-mediated MetAP-2 inhibition blocks noncanonical Wnt signaling, which plays a critical role in development, cell differentiation, and tumorigenesis. Consistent with this finding, antisense MetAP-2 morpholino oligonucleotide injection in zebrafish embryos phenocopies gastrulation defects seen in noncanonical Wnt5 loss-of-function zebrafish mutants. MetAP-2 inhibition or depletion blocks signaling downstream of the Wnt receptor Frizzled, but upstream of Calmodulin-dependent Kinase II, RhoA, and c-Jun N-terminal Kinase. Moreover, we demonstrate that TNP-470 does not block the canonical Wnt/beta-catenin pathway. Thus, TNP-470 selectively regulates noncanonical over canonical Wnt signaling and provides a unique means to explore and dissect the biological systems mediated by these pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Developing microcolin A analogs as biological probes.

Three microcolin A and B analogs have been synthesized. Their biological activity profiles were evaluated against several cell lines, revealing the existence of a structural determinant whose role in mediating the antiproliferative effect of the microcolins has heretofore gone unrecognized. While previously described as potent immunosuppressive natural products, we found that these microcolin analogs possessed no selective cytotoxicity when comparing immune cell versus nonimmune cell proliferation. In addition, the amino-terminus of microcolin A has been modified to incorporate a biotinylated polyethylene glycol linker. The biological activity of this biotinylated microcolin A analog was determined. This affinity reagent was shown to retain limited biological activity and thus can serve as a useful probe for elucidating the mechanism of action of microcolin A.

Animals↗
PubMed2005

Stereoselective assembly of a 1,3-diene via coupling between an allenic acetate and a (B)-alkylborane: synthetic studies on amphidinolide B1.

The preparation of three fragments for the total synthesis of amphidinolide B1 has been described. The C16 stereochemistry was set by asymmetric allylic alkylation. C21 and C25 stereogenic centers were set by an enantioselective/diastereoselective double allylation reaction. The C9 configuration was set by an asymmetric heteroene reaction. A differentially substituted stereodefined 1,3-diene iodide was synthesized by iodide-mediated S(N)2' reaction. A novel stereoselective method to assemble a 1,3-diene by coupling an allenic acetate and (B)-alkylborane is also reported. [structure: see text]

Acetates↗
PubMed2005

Studies on calcium dependence reveal multiple modes of action for triptolide.

Triptolide, a diterpene triepoxide isolated from the traditional Chinese medicinal vine Trypterygium wilfordii hook f., has been shown to induce rapid apoptosis in a myriad of cancer cell lines and inhibit NFkappaB transactivation. To understand further the general cellular mechanisms for this therapeutically relevant natural product, binding and biological activities were assessed. Studies showed that triptolide binding was saturable, reversible, and primarily localized to cell membranes. Depletion of calcium enhanced overall binding while differentially modulating biological function. Furthermore, triptolide's structural moieties demonstrated variability in the regulation of cell death versus inhibition of NFkappaB transactivation. These results implicate triptolide in the manipulation of at least two distinct cellular pathways with differing requirements for calcium and effective triptolide concentration in order to elicit each particular biological function.

Antineoplastic Agents, Alkylating↗
PubMed2005

Development and characterization of proteasome inhibitors.

Although many proteasome inhibitors have been either synthesized or identified from natural sources, the development of more sophisticated, selective proteasome inhibitors is important for a detailed understanding of proteasome function. We have found that antitumor natural product epoxomicin and eponemycin, both of which are linear peptides containing a alpha,beta-epoxyketone pharmacophore, target proteasome for their antitumor activity. Structural studies of the proteasome-epoxomicin complex revealed that the unique specificity of the natural product toward proteasome is due to the alpha,beta-epoxyketone pharmacophore, which forms an unusual six-membered morpholino ring with the amino terminal catalytic Thr-1 of the 20S proteasome. Thus, we believe that a facile synthetic approach for alpha,beta-epoxyketone linear peptides provides a unique opportunity to develop proteasome inhibitors with novel activities. In this chapter, we discuss the detailed synthetic procedure of the alpha',beta'-epoxyketone natural product epoxomicin and its derivatives.

Chromatography, High Pressure Liquid↗
PubMed2004

Total synthesis of TMC-95A and -B via a new reaction leading to Z-enamides. Some preliminary findings as to SAR.

A full account of the total syntheses of proteasome inhibitors TMC-95A and -B is provided. A key feature of the syntheses involved installation of a cis-propenylamide moiety by a thermal rearrangement of an alpha-silylallyl amide. The scope and mechanism of the enamide-forming reaction are discussed. Also provided are some preliminary results from SAR studies. It was found that simplified analogues can retain the full potency of proteasome inhibition.

Amides↗
PubMed2004

Chemical genetic control of protein levels: selective in vivo targeted degradation.

Genetic loss of function analysis is a powerful method for the study of protein function. However, some cell biological questions are difficult to address using traditional genetic strategies often due to the lack of appropriate genetic model systems. Here, we present a general strategy for the design and syntheses of molecules capable of inducing the degradation of selected proteins in vivo via the ubiquitin-proteasome pathway. Western blot and fluorometric analyses indicated the loss of two different targets: green fluorescent protein (GFP) fused with FK506 binding protein (FKBP12) and GFP fused with the androgen receptor (AR), after treatment with PROteolysis TArgeting Chimeric moleculeS (PROTACS) incorporating a FKBP12 ligand and dihydrotestosterone, respectively. These are the first in vivo examples of direct small molecule-induced recruitment of target proteins to the proteasome for degradation upon addition to cultured cells. Moreover, PROTAC-mediated protein degradation offers a general strategy to create "chemical knockouts," thus opening new possibilities for the control of protein function.

Cell Line↗
PubMed2003

A single amino acid residue defines the difference in ovalicin sensitivity between type I and II methionine aminopeptidases.

TNP-470, the first anti-angiogenic small molecule to enter clinical trials, targets methionine aminopeptidase-2 (MetAP-2), a metalloprotease that cleaves the N-terminal methionine of proteins. Previously, biochemical binding, in vivo yeast studies, and structural studies of human methionine aminopeptidase-2 bound to TNP-470 and its analogs fumagillin and ovalicin revealed that these compounds exhibit specificity for MetAP-2 over its family member MetAP-1. To further elucidate the nature of this specificity, we developed a yeast-based screen for human MetAP-2 mutations that confer ovalicin resistance. Of the three resistant alleles, A362T appeared in the majority of clones and was found to be the most resistant to the ovalicin class of inhibitors. Alignment of human MetAP-2 with human MetAP-1, which is naturally ovalicin-resistant, revealed that the analogous residue in MetAP-1 is also a threonine. Mutation of this residue to alanine resulted in an ovalicin-sensitive MetAP-1 allele, demonstrating that an alanine at this position is critical for inhibition by ovalicin. These results provide a molecular explanation for the specificity exhibited by this class of anti-angiogenic agents for MetAP-2 over MetAP-1 and may prove useful in the development of additional MetAP-2-specific therapeutic agents.

Alanine↗
PubMed2003

Development of Protacs to target cancer-promoting proteins for ubiquitination and degradation.

The proteome contains hundreds of proteins that in theory could be excellent therapeutic targets for the treatment of human diseases. However, many of these proteins are from functional classes that have never been validated as viable candidates for the development of small molecule inhibitors. Thus, to exploit fully the potential of the Human Genome Project to advance human medicine, there is a need to develop generic methods of inhibiting protein activity that do not rely on the target protein's function. We previously demonstrated that a normally stable protein, methionine aminopeptidase-2 or MetAP-2, could be artificially targeted to an Skp1-Cullin-F-box (SCF) ubiquitin ligase complex for ubiquitination and degradation through a chimeric bridging molecule or Protac (proteolysis targeting chimeric molecule). This Protac consisted of an SCF(beta-TRCP)-binding phosphopeptide derived from IkappaBalpha linked to ovalicin, which covalently binds MetAP-2. In this study, we employed this approach to target two different proteins, the estrogen (ER) and androgen (AR) receptors, which have been implicated in the progression of breast and prostate cancer, respectively. We show here that an estradiol-based Protac can enforce the ubiquitination and degradation of the alpha isoform of ER in vitro, and a dihydroxytestosterone-based Protac introduced into cells promotes the rapid disappearance of AR in a proteasome-dependent manner. Future improvements to this technology may yield a general approach to treat a number of human diseases, including cancer.

Breast Neoplasms↗
PubMed2003

Simplified synthetic TMC-95A/B analogues retain the potency of proteasome inhibitory activity.

The proteasome regulates diverse intracellular processes, including cell-cycle progression, antigen presentation, and inflammatory response. Selective inhibitors of the proteasome have great therapeutic potential for the treatment of cancer and inflammatory disorders. Natural cyclic peptides TMC-95A and B represent a new class of noncovalent, selective proteasome inhibitors. To explore the structure-activity relationship of this class of proteasome inhibitors, a series of TMC-95A/B analogues were prepared and analyzed. We found that the unique enamide functionality at the C8 position of TMC-95s can be replaced with a simple allylamide. The asymmetric center at C36 that distinguishes TMC-95A from TMC-95B but which necessitates a complicated separation of the two compounds can be eliminated. Therefore, these findings could lead to the development of more accessible simple analogues as potential therapeutic agents.

Animals↗
PubMed2003

Feeding the machine: mechanisms of proteasome-catalyzed degradation of ubiquitinated proteins.

The proteasome plays a role in a myriad of intracellular processes from cell-cycle control to antigen presentation. Central to these processes is the targeting of selected proteins for proteasomal degradation via their conjugation to ubiquitin. The mechanisms by which the ubiquitin-dependent proteasomal proteolysis occurs can be divided into four steps: first, substrate protein recognition by its cognate E3 ubiquitin ligase; second, polyubiquitinated protein substrate recruitment to the proteasome; third, protein substrate deubiquitination; and four, proteolytic chamber pore opening/substrate translocation followed by proteolysis. Recent advances include the identification of novel E3 ubiquitin ligase recognition determinants, a new isopeptidase activity, and a better understanding of how the proteasome's axial channels are gated.

Carbon-Nitrogen Lyases↗
PubMed2003

Chemical genetics: adding to the developmental biology toolbox.

Recent advances in cell and molecular biology have generated important tools to probe developmental questions. In addition, new genetic model systems such as Danio rerio now make large-scale vertebrate early developmental mutant screens feasible. Nonetheless, some developmental questions remain difficult to study because of the need for finer temporal, spatial, or tuneable control of gene function within a developmental system. New uses for old teratogens as well as novel chemical modulators of development have begun to fill this void.

Animals↗
PubMed2003

Small-molecule inhibitors of the cell cycle: an overview.

Potent and selective small-molecule mediated inhibition of the cell's replication machinery remains a principal aim in the development of novel therapeutics and biological probes. Recent efforts have identified small molecules capable of arresting the cell cycle via specific interaction with a variety of intracellular protein targets. Advances in combinatorial and diversity oriented synthetic methods, coupled with a continued effort to identify sources of bioactive natural products, promise to contribute to the growing library of small-molecule inhibitors of the cell cycle.

Adenosine Triphosphatases↗