Scaffold For Molecules Research Articles

Myeloproliferative Disease or Myeloid Leukemia Caused by Cbl Mutants in a Mouse Transplantation Model.

Abstract 3973Poster Board III-909 PurposeSomatic mutations of Kit have been found in leukemias and gastrointestinal stromal tumors. The proto-oncogene c-Cbl negatively regulates Kit and Flt3 by its E3 ligase activity and acts as a scaffold for several signaling adaptor molecules. We recently identified the first c-Cbl mutation in human disease in an AML patient, called Cbl-R420Q. ResultsWe transduced primary murine bone marrow retrovirally with c-Cbl mutants and transplanted it into lethally irradiated mice. Almost all recipients of bone marrow cells transduced with Cbl mutants developed a lethal hematologic disorder with a mean latency of 341 days in the Cbl-R420Q group and 395 days in the Cbl-70Z group. Eleven out of 13 mice and 8 out of 11 mice died in the Cbl-R420Q group and Cbl-70Z group, respectively. Two animals succumbed to a myeloid leukemia, the other mice developed a myeloproliferative disease. The leukemic mice showed a leukocytosis of up to 140.000/μL. They developed a splenomegaly with massive expansion of myeloid cells in liver and spleen. Histology sections of spleen, liver and bone marrow and FACS analyses of spleen, bone marrow and peripheral blood showed extensive infiltration of myeloid cells. ConclusionThus, transplantation of bone marrow cells expressing Cbl mutants leads to a myeloid leukemia or to a myeloproliferative disease with long latency and high penetrance. Disclosures:No relevant conflicts of interest to declare.

A Single Molecule Scaffold for the Maize Genome

About 85% of the maize genome consists of highly repetitive sequences that are interspersed by low-copy, gene-coding sequences. The maize community has dealt with this genomic complexity by the construction of an integrated genetic and physical map (iMap), but this resource alone was not sufficient for ensuring the quality of the current sequence build. For this purpose, we constructed a genome-wide, high-resolution optical map of the maize inbred line B73 genome containing >91,000 restriction sites (averaging 1 site/∼23 kb) accrued from mapping genomic DNA molecules. Our optical map comprises 66 contigs, averaging 31.88 Mb in size and spanning 91.5% (2,103.93 Mb/∼2,300 Mb) of the maize genome. A new algorithm was created that considered both optical map and unfinished BAC sequence data for placing 60/66 (2,032.42 Mb) optical map contigs onto the maize iMap. The alignment of optical maps against numerous data sources yielded comprehensive results that proved revealing and productive. For example, gaps were uncovered and characterized within the iMap, the FPC (fingerprinted contigs) map, and the chromosome-wide pseudomolecules. Such alignments also suggested amended placements of FPC contigs on the maize genetic map and proactively guided the assembly of chromosome-wide pseudomolecules, especially within complex genomic regions. Lastly, we think that the full integration of B73 optical maps with the maize iMap would greatly facilitate maize sequence finishing efforts that would make it a valuable reference for comparative studies among cereals, or other maize inbred lines and cultivars.

Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects.

Sarcoglycanopathies are a group of autosomal recessive muscle-wasting disorders caused by genetic defects in one of four cell membrane glycoproteins, α-, β-, γ- or δ-sarcoglycan. These four sarcoglycans form a subcomplex that is closely linked to the major dystrophin-associated protein complex, which is essential for membrane integrity during muscle contraction and provides a scaffold for important signalling molecules. Proper assembly, trafficking and targeting of the sarcoglycan complex is of vital importance, and mutations that severely perturb tetramer formation and localisation result in sarcoglycanopathy. Gene defects in one sarcoglycan cause the absence or reduced concentration of the other subunits. Most genetic defects generate mutated proteins that are degraded through the cell's quality control system; however, in many cases, conformational modifications do not affect the function of the protein, yet it is recognised as misfolded and prematurely degraded. Recent evidence shows that misfolded sarcoglycans could be rescued to the cell membrane by assisting their maturation along the ER secretory pathway. This review summarises the etiopathogenesis of sarcoglycanopathies and highlights the quality control machinery as a potential pharmacological target for therapy of these genetic disorders.

Cancer Chemoprevention: Selective Inhibition of Cytochrome P450 2A13

4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) is one of the most prevalent and carcinogenic compounds in tobacco. NNK is selectively activated by human cytochrome P450 2A13 (CYP2A13) to form two carcinogens that generate DNA adducts. CYP2A13 is found in the respiratory tract where NNK‐derived adducts can result in lung cancer. CYP2A13 is 93.5% identical to the hepatic cytochrome P450 2A6 (CYP2A6), which does not efficiently metabolize NNK but does metabolize other xenobiotic compounds. Thus a selective inhibitor of CYP2A13 could be used as a chemopreventative for tobacco users. High throughput screening was used to identify small molecule scaffolds that selectively bound CYP2A13 over CYP2A6. One such scaffold was benzylmorpholine. A small library of benzylmorpholine analogs was generated and has been evaluated for differential ligand binding (Kd) and inhibition of human cytochrome P450 2A enzymes. Examination of these results is revealing selective compounds that could be used for the reduction of lung cancer related to DNA damage in tobacco users.

The crystal structure of mouse VDAC1 at 2.3 Å resolution reveals mechanistic insights into metabolite gating

The voltage-dependent anion channel (VDAC) constitutes the major pathway for the entry and exit of metabolites across the outer membrane of the mitochondria and can serve as a scaffold for molecules that modulate the organelle. We report the crystal structure of a beta-barrel eukaryotic membrane protein, the murine VDAC1 (mVDAC1) at 2.3 A resolution, revealing a high-resolution image of its architecture formed by 19 beta-strands. Unlike the recent NMR structure of human VDAC1, the position of the voltage-sensing N-terminal segment is clearly resolved. The alpha-helix of the N-terminal segment is oriented against the interior wall, causing a partial narrowing at the center of the pore. This segment is ideally positioned to regulate the conductance of ions and metabolites passing through the VDAC pore.

Diarylethynyl amides that recognize the parallel conformation of genomic promoter DNA G-quadruplexes.

We report bis-phenylethynyl amide derivatives as a potent G-quadruplex binding small molecule scaffold. The amide derivatives were efficiently prepared in 3 steps by employing Sonogashira coupling, ester hydrolysis and a chemoselective amide coupling. Ligand-quadruplex recognition has been evaluated using a fluorescence resonance energy transfer (FRET) melting assay, surface plasmon resonance (SPR), circular dichroism (CD) and (1)H nuclear magnetic resonance (NMR) spectroscopy. While most of the G-quadruplex ligands reported so far comprise a planar, aromatic core designed to stack on the terminal tetrads of a G-quadruplex, these compounds are neither polycyclic, nor macrocyclic and have free rotation around the triple bond enabling conformational flexibility. Such molecules show very good binding affinity, excellent quadruplex:duplex selectivity and also promising discrimination between intramolecular promoter quadruplexes. Our results indicate that the recognition of the c-kit2 quadruplex by these ligands is achieved through groove binding, which favors the formation of a parallel conformation.

Expression of Lewy body protein septin 4 in postmortem brain of Parkinson's disease and control subjects

In Parkinson's disease (PD) neuronal degeneration is associated with abnormal protein aggregation in various forms including Lewy bodies (LBs). A major component of LBs is alpha-synuclein; septin 4 (SEPT4), a polymerizing GTP-binding protein that serves as scaffold for diverse molecules has been found to colocalize with alpha-synuclein in LBs. The central role of SEPT4 in the etiopathogenesis of PD has been suggested since SEPT4 also shows a physiological association with alpha-synuclein and serves as a substrate for parkin. To this end, we studied the expression of septin 4 and alpha-synuclein in postmortem human substantia nigra (SN) and amygdala from patients with PD and healthy controls. Twenty patients (14 men : 6 women, onset 63.0 +/- 11.4 years, age 77.3 +/- 7.6 years, Hoehn and Yahr 4.05/5) and 9 neurologically healthy controls (4 men/5 women, age at death 80.1 +/- 8.6 years) were studied. Sporadic PD cases showed a statistically significant decrease of the fold change (FC) of SNCA (FC = 0.31, P = 0.00001) and SEPT4 (FC = 0.67, P = 0.054) gene expressions in the SN and the amygdala (SNCA: FC = 0.49, P = 0.02; SEPT4: FC = 0.32, P = 0.007) versus healthy controls. However, an increase of both proteins in PD versus control subjects was observed with immunoblotting. The semi-quantitative protein ratio calculations revealed more than 10-fold increases for both SEPT4 and alpha-synuclein in PD versus control subjects. We present for the first time similar signal expression patterns and parallel accumulation of SEPT4 and alpha-synuclein in well-characterized postmortem PD brain. Considering the heterogeneous etiology of sporadic PD and the variability of individual human samples, SEPT4 accumulation may be regarded as one of the common pathological changes in PD and should therefore be further explored.

Discovery of benzamide tetrahydro-4 H-carbazol-4-ones as novel small molecule inhibitors of Hsp90

Hsp90 maintains the conformational stability of multiple proteins implicated in oncogenesis and has emerged as a target for chemotherapy. We report here the discovery of a novel small molecule scaffold that inhibits Hsp90. X-ray data show that the scaffold binds competitively at the ATP site on Hsp90. Cellular proliferation and client assays demonstrate that members of the series are able to inhibit Hsp90 at nanomolar concentrations.

Triarylpyridines: a versatile small molecule scaffold for G-quadruplex recognition

The triarylpyridines are potent G-quadruplex ligands that are highly discriminating against duplex DNA and show promising selectivity between intramolecular quadruplexes.

G-quadruplex recognition by bis-indole carboxamides.

Herein we report the de novo design and synthesis of a geometrically flexible bis-indole carboxamide and a constrained derivative, as a novel class of small molecule scaffold that exhibits high stabilization potential for DNA G-quadruplex sequences associated with the promoters of c-kit2 and c-myc.

A Novel Role for Caveolin-1 in B Lymphocyte Function and the Development of Thymus-Independent Immune Responses

Caveolin-1 (Cav-1) functions as a scaffold or platform for many molecules involved in signal transduction. However, the expression and function of Cav-1 in the immune system has been controversial. Here, we show that Cav-1 mRNA and protein is indeed expressed in murine B-lymphocytes in a regulated manner. Cav-1 deficient mice displayed reduced levels of antibody in their serum. In order to examine the role of Cav-1 in the development of immunoglobulin-mediated immune responses, we immunized wild-type and Cav-1 deficient mice with thymus-dependent and thymus independent antigens. Our results show that Cav-1 deficient mice have a normal response to thymus-dependent antigens, but have a reduced response to both type I and type II thymus independent antigens. However, lymphocyte populations in the spleen and peritoneum were not altered and no changes were observed in splenic architecture. Caveolin-1 deficient B-lymphocytes did not display altered proliferation in response to different stimuli. However, we found that Cav-1 deficient B cells have reduced IgG3 secretion in vitro in response to LPS. Finally, we also demonstrate that human plasma cells (mature B lymphocytes) express Cav-1 in vivo. Taken, together these results provide convincing evidence for expression of Cav-1 in activated B-lymphocytes and demonstrate a role for Cav-1 in the development of thymus-independent immune responses.

Stereochemical Promiscuity in Artificial Transcriptional Activators

Small molecule replacements of transcriptional activation domains are highly desirable targets due to their utility as mechanistic tools and their long-term therapeutic potential for a variety of human diseases. Here, we examine the ability of amphipathic isoxazolidines differing only in the placement of constituent side chains to function as transcriptional activation domains. The results reveal that precise positioning of functional groups within a conformationally constrained small molecule scaffold is not required for transcription function; rather, the balance of polarity and hydrophobicity within the scaffold is the more important determinant of transcription function. This suggests that a number of different organic molecule scaffolds should function as transcriptional activator domains when appropriately functionalized, a hypothesis currently under investigation.

Quality, not Quantity: The Role of Natural Products and Chemical Proteomics in Modern Drug Discovery

Chemical genetics and reverse chemical genetics parallel classical genetics but target genes at the protein level and have proven useful in recent years for screening combinatorial libraries for compounds of biological interest. However, the performance of combinatorial chemistry in filling pharmaceutical pipelines has been lower than anticipated and the tide may be turning back to Nature in the search for new drug candidates. Even though diversity oriented synthesis is now producing molecules that are natural product-like in terms of size and complexity, these molecules have not evolved to interact with biomolecules. Natural products, on the other hand, have evolved to interact with biomolecules, which is why so many can be found in pharmacopoeias. However, the cellular targets and modes of action of these fascinating compounds are seldom known, hindering the drug development process. This review focuses on the emergence of chemical proteomics and reverse chemical proteomics as tools for the discovery of cellular receptors for natural products, thereby generating protein/ligand pairs that will prove useful in identifying new drug targets and new biologically active small molecule scaffolds. Such a system-wide approach to identifying new drugable targets and their small molecule ligands will help unblock the pharmaceutical product pipelines by speeding the process of target and lead identification.

Growth-promoting effect of muscarinic acetylcholine receptors in colon cancer cells.

G-protein-coupled receptors are known to mediate cell growth via divergent signaling pathways. It has been reported that colon cancer cells express muscarinic acetylcholine receptor (mAChR) although their functional role is largely unknown. The aim of this study is to elucidate possible mechanisms responsible for the growth-promoting effect of mAChRs in colon cancer cells by using colon cancer cell line T84. Carbachol, a stable mAChR agonist, dose-dependently induced cell growth with a maximal effect observed at 100 microM, equipotent with 1 nM EGF. 4-DAMP, a specific antagonist of subtype 3 mAChR, inhibited the stimulatory effect by carbachol, suggesting that the growth-promoting effect was receptor-mediated. Carbachol also dose-dependently stimulated extracellular signal-regulated protein kinase (ERK) activation. This effect was inhibited by PD98059, an inhibitor of extracellular signal-regulated protein kinase kinase, which also blocked carbachol activation of cell proliferation, indicating that the p21Ras-ERK pathway is an important signaling cascade in the mitogenic effect. To investigate how mAChR activated the p21Ras-ERK pathway, transactivation of epidermal growth factor receptor (EGFR) was examined. Carbachol induced tyrosine phosphorylation of EGFR, which was abolished by an EGFR tyrosine kinase inhibitor AG1478. Transactivation by carbachol was also abrogated by a metalloproteinases (MMPs) inhibitor GM6001 or an EGFR-blocking antibody (LA-1), suggesting that binding of EGFR ligand(s) produced by MMPs may initiate transactivation in a manner dependent on EGFR tyrosine kinase. The tyrosine-phosphorylated EGFR was immunoprecipitated together with GRB2 and tyrosine-phosphorylated Shc, indicating that transactivated EGFR is able to generate downstream signals. AG 1478 and LA-1 inhibited carbachol stimulation of cell growth. Taken together, our results indicate that the growth-promoting effect of subtype 3 mAChR in colon cancer cells may depend on transactivated EGFR-ERK pathways. EGFR not only receives external stimuli but also serves as a scaffold for downstream signaling molecules.

Discovery of nonpeptide, peptidomimetic peptidase inhibitors that target alternate enzyme active site conformations.

Structure-generating programs provide rational methods to rapidly design novel scaffolds targeting the biologic receptor of choice. Recent research has demonstrated proteins equilibrate between families of conformations (ensembles) for which drug design may target. New methods are currently being developed utilizing structure-generating programs to target alternate enzyme conformations in an attempt to overcome the challenge of developing therapeutically useful molecules. These new methods provide the potential to overcome bioavailability problems encountered with peptide and peptide-like molecules by identifying novel small molecule scaffolds.

Function of the cytoskeleton in human neutrophils and methods for evaluation

The cytoskeleton plays a critical role in the determination of cell shape and serves as a scaffold for critical cellular enzymes and adhesion molecules. It provides structural integrity for the cell and regulates the function of many biochemical events that are critical to cellular function. The microfilamentous cytoskeleton participates in force generation necessary for shape change and motion. In neutrophils and other motile cells, polymerization of actin likely drives extension of the lamellae and participates in force generation through interaction with myosin, by polymerization alone and by osmotic mechanisms. Here, we will focus on the microfilamentous cytoskeleton in the neutrophil and briefly review its function as well as some direct and indirect methods that have been used to asses its role in neutrophil function. The discussion will address general approaches and leaves the details of the methods to the references.

Role for alpha-dystrobrevin in the pathogenesis of dystrophin-dependent muscular dystrophies.

A dystrophin-containing glycoprotein complex (DGC) links the basal lamina surrounding each muscle fibre to the fibre's cytoskeleton, providing both structural support and a scaffold for signalling molecules. Mutations in genes encoding several DGC components disrupt the complex and lead to muscular dystrophy. Here we show that mice deficient in alpha-dystrobrevin, a cytoplasmic protein of the DGC, exhibit skeletal and cardiac myopathies. Analysis of double and triple mutants indicates that alpha-dystrobrevin acts largely through the DGC. Structural components of the DGC are retained in the absence of alpha-dystrobrevin, but a DGC-associated signalling protein, nitric oxide synthase, is displaced from the membrane and nitric-oxide-mediated signalling is impaired. These results indicate that both signalling and structural functions of the DGC are required for muscle stability, and implicate alpha-dystrobrevin in the former.