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Dive into the research topics where Richard W. Fitch is active.

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Featured researches published by Richard W. Fitch.


Proceedings of the National Academy of Sciences of the United States of America | 2003

Membrane potential fluorescence: A rapid and highly sensitive assay for nicotinic receptor channel function

Richard W. Fitch; Yingxian Xiao; Kenneth J. Kellar; John W. Daly

Seven cell lines expressing native and transfected nicotinic receptor subtypes were evaluated functionally by using fluorescent assays based on membrane potential and calcium dynamics with “no-wash” dye systems. Both assays provided the same rank orders of potency for (±)-epibatidine, 2S-(−)-nicotine, 7R,9S-(−)-cytisine, and 1,1-dimethyl-4-phenylpiperazinium in a cell line expressing rat α3β4 receptors. Nicotinic antagonists mecamylamine and dihydro-β-erythroidine inhibited responses in both assays. Both agonist and antagonist activity were assessed within the same experiment. Agonists seemed more potent in the membrane potential assay than in the calcium assay, whereas the converse was true for antagonists. The membrane potential assay afforded robust responses in K-177 cells expressing human α4β2 receptors, in IMR-32 and SH-SY5Y cells expressing human ganglionic receptors, and in TE-671 cells expressing human neuromuscular receptors. These lines gave weak to modest calcium responses. Moreover, membrane potential responses were obtained in cell lines expressing rat α4β2 and α4β4 receptors, which were devoid of calcium responses. Thus, membrane potential serves as a sensitive measure of nicotinic activity, and the resulting depolarization may be as important as calcium in cell signaling.


Organic Letters | 2008

N,N-Acetals as N-acyliminium ion precursors: Synthesis and absolute stereochemistry of epiquinamide

Wijdeven; Roel Wijtmans; R.J.F. van den Berg; W.L. Noorduin; Hans E. Schoemaker; Theo Sonke; F.L. van Delft; R. H. Blaauw; Richard W. Fitch; Thomas F. Spande; John W. Daly; Floris P. J. T. Rutjes

A stereoselective synthesis of (+)-epiquinamide is presented in combination with determination of the absolute configuration of the natural product. Key steps in the sequence involved chemoenzymatic formation of an enantiomerically pure cyanohydrin, reductive cyclization to the corresponding cyclic N,N-acetal, and subsequent conversion into a suitable N-acyliminium ion precursor to enable construction of the second ring.


Journal of Natural Products | 2009

Epiquinamide: a poison that wasn't from a frog that was.

Richard W. Fitch; Gordon D. Sturgeon; Shaun R. Patel; Thomas F. Spande; H. Martin Garraffo; John W. Daly; Richard H. Blaauw

In 2003, we reported the isolation, structure elucidation, and pharmacology of epiquinamide (1), a novel alkaloid isolated from an Ecuadoran poison frog, Epipedobates tricolor. Since then, several groups, including ours, have undertaken synthetic efforts to produce this compound, which appeared initially to be a novel, beta2-selective nicotinic acetylcholine receptor agonist. Based on prior chiral GC analysis of synthetic and natural samples, the absolute structure of this alkaloid was established as (1S,9aS)-1-acetamidoquinolizidine. We have synthesized the (1R*,9aS*)-isomer (epi-epiquinamide) using an iminium ion nitroaldol reaction as the key step. We have also synthesized ent-1 semisynthetically from (-)-lupinine. Synthetic epiquinamide is inactive at nicotinic receptors, in accord with recently published reports. We have determined that the activity initially reported is due to cross-contamination from co-occurring epibatidine in the isolated material.


Molecular Cancer Therapeutics | 2011

Novel Irreversible Small Molecule Inhibitors of Replication Protein A Display Single Agent Activity and Synergize with Cisplatin.

Tracy M. Neher; Diane Bodenmiller; Richard W. Fitch; Shadia I. Jalal; John J. Turchi

Replication protein A (RPA) is a single-strand DNA-binding protein with essential roles in DNA replication, recombination, and repair. It is necessary for the formation of the preincision complex that is required for proper incision of damaged DNA nucleotides during DNA repair. We have previously identified small molecule inhibitors (SMI) with the ability to disrupt RPA-binding activity to ssDNA. Further characterization of these RPA inhibitors was done using both lung and ovarian cancer cell lines. Lung cancer cell lines showed increased apoptotic cell death following treatment with the SMI MCI13E, with IC50 values of approximately 5 μmol/L. The ovarian cancer cell line A2780 and the p53-null lung cancer cell line H1299 were particularly sensitive to MCI13E treatment, with IC50 values less than 3 μmol/L. Furthermore, a cell-cycle effect was observed in lung cancer cell lines that resulted in a lengthening of either G1 or S-phases of the cell cycle following single-agent treatment. Sequential treatment with MCI13E and cisplatin resulted in synergism. Overall, these data suggest that decreasing DNA-binding activity of RPA via a SMI may disrupt the role of RPA in cell-cycle regulation. Thus, SMIs of RPA hold the potential to be used as single-agent chemotherapeutics or in combination with current chemotherapeutic regimens to increase efficacy. Mol Cancer Ther; 10(10); 1796–806. ©2011 AACR.


Journal of Natural Products | 2010

Phantasmidine: an epibatidine congener from the ecuadorian poison frog Epipedobates anthonyi.

Richard W. Fitch; Thomas F. Spande; H. Martin Garraffo; Herman J. C. Yeh; John W. Daly

The skin of the Ecuadorian poison frog Epipedobates anthonyi contains the potent nicotinic agonists epibatidine (1) and N-methylepibatidine (3). In addition, a condensed tetracyclic epibatidine congener has been identified with activity at nicotinic acetylcholine receptors, but different selectivity than epibatidine. This rigid tetracycle has been named phantasmidine (4). Phantasmidine has a molecular formula of C(11)H(11)N(2)OCl, shares a chloropyridine moiety with 1, and also contains furan, pyrrolidine, and cyclobutane rings. A combination of GC-MS and GC-FTIR analysis with on-column derivatization, 1D NMR spectroscopy with selective irradiation, and spectral simulation, along with 2D NMR, were used to elucidate the structure from a total sample of approximately 20 microg of HPLC-purified 4 and its corresponding acetamide (5). After synthesis, this novel rigid agonist may serve as a selective probe for beta4-containing nicotinic receptors and potentially lead to useful pharmaceuticals.


Journal of Nucleic Acids | 2010

Targeting the OB-Folds of Replication Protein A with Small Molecules

Victor J. Anciano Granadillo; Jennifer N. Earley; Sarah C. Shuck; Millie M. Georgiadis; Richard W. Fitch; John J. Turchi

Replication protein A (RPA) is the main eukaryotic single-strand (ss) DNA-binding protein involved in DNA replication and repair. We have identified and developed two classes of small molecule inhibitors (SMIs) that show in vitro inhibition of the RPA-DNA interaction. We present further characterization of these SMIs with respect to their target binding, mechanism of action, and specificity. Both reversible and irreversible modes of inhibition are observed for the different classes of SMIs with one class found to specifically interact with DNA-binding domains A and B (DBD-A/B) of RPA. In comparison with other oligonucleotide/oligosaccharide binding-fold (OB-fold) containing ssDNA-binding proteins, one class of SMIs displayed specificity for the RPA protein. Together these data demonstrate that the specific targeting of a protein-DNA interaction can be exploited towards interrogating the cellular activity of RPA as well as increasing the efficacy of DNA-damaging chemotherapeutics used in cancer treatment.


Tetrahedron Letters | 1994

The aluminum amalgam reduction of 2-nitroalkanols promoted by ultrasound

Richard W. Fitch; Frederick A. Luzzio

Abstract The sonochemical promoted aluminum amalgam reduction of 2-nitroalkanols provides an improved yield and accelerated conversion to the corresponding amino alcohols when compared to the nonultrasound (benchtop) reductions. The appearance of by-product hydroxylamines is minimized during the ultrasound promoted reaction. The product amino alcohols were conveniently acylated in situ with promotion by ultrasound thus affording the N-acyl derivatives in the same operation.


Cellular Immunology | 2011

Synthetic adjuvants for vaccine formulations: Evaluation of new phytol derivatives in induction and persistence of specific immune response

Youssef Aachoui; Michael L. Schulte; Richard W. Fitch; Swapan K. Ghosh

Terpenoids are ubiquitous natural compounds that have been shown to improve vaccine efficacy as adjuvants. To gain an understanding of the structural features important for adjuvanticity, we studied compounds derived from a diterpene phytol and assessed their efficacy. In a previous report, we showed that phytol and one of its derivatives, PHIS-01 (a phytol-derived immunostimulant, phytanol), are excellent adjuvants. To determine the effects of varying the polar terminus of PHIS-01, we designed amine and mannose-terminated phytol derivatives (PHIS-02 and PHIS-03, respectively). We studied their relative efficacy as emulsions with soluble proteins, ovalbumin, and a hapten-protein conjugate phthalate-KLH. Immunological parameters evaluated consisted of specific antibody responses in terms of titers, specificities and isotype profiles, T cell involvement and cytokine production. Our results indicate that these new isoprenoids were safe adjuvants with the ability to significantly augment immunogen-specific IgG1 and IgG2a antibody responses. Moreover, there was no adverse phthalate cross-reactive anti-DNA response. Interestingly, PHIS-01 and PHIS-03 influenced differentially T-helper polarization. We also observed that these compounds modulated the immune response through apoptotic/necrotic effects on target tumor cells using murine lymphomas. Finally, unlike squalene and several other terpenoids reported to date, these phytol derivatives did not appear arthritogenic in murine models.


Cellular Immunology | 2011

Molecular signatures of phytol-derived immunostimulants in the context of chemokine-cytokine microenvironment and enhanced immune response.

Youssef Aachoui; Roshni Roy Chowdhury; Richard W. Fitch; Swapan K. Ghosh

In a previous report, we observed that the phytol-derived immunostimulant, PHIS-01 (phytanol), is a nontoxic oil-in-water adjuvant which is superior to most commercial adjuvants. In contrast, the parent diterpene alcohol phytol, though highly effective as an adjuvant, is relatively toxic. To assess the importance of the polar functional group in PHIS-01, we prepared two new compounds PHIS-02 (phytanyl amine) and PHIS-03 (phytanyl mannose). All three phytol derivatives proved to be excellent adjuvants, but differed in solubility and mode of action. To delineate their molecular signatures in the local microenvironment, we performed inflammasome and cytokine microarray analyses with the peritoneal fluid of mice treated with alum or the phytol compounds above, in the presence or absence of soluble protein antigens. We report here that the phytol derivatives had a significant time-dependent impact on the host chemokine-cytokine microenvironment and subsequently on specific humoral responses. Moreover, the inclusion of protein immunogens induced further changes in host microenvironments, including rapid (<2h) expression of cytokines and chemotactic factors (IL-6, MCP-1, KC, MIP-1, and LIX), implying mobilization and activation of neutrophils, and monocytes. PHIS-01 proved to be the most effective in this regard. Inflammatory cytokine cascades were dominant even after 24h possibly to facilitate involvement of the acquired immune system with the release of B-lymphocyte chemo-attractant BLC, T-cell activation-3 chemokines TCA, IL-4, IL-12, and TIMP-1. We also noted enhanced expression of NLRP genes including NLRP3 with both alum and phytol derivatives (particularly PHIS-01).


Bioorganic & Medicinal Chemistry | 2008

Polyethylene glycol-based homologated ligands for nicotinic acetylcholine receptors

Bradley A. Scates; Bethany L. Lashbrook; Benjamin C. Chastain; Kaoru Tominaga; Brandon T. Elliott; Nicholas J. Theising; Thomas A. Baker; Richard W. Fitch

A homologous series of polyethylene glycol (PEG) monomethyl ethers were conjugated with three ligand series for nicotinic acetylcholine receptors. Conjugates of acetylaminocholine, the cyclic analog 1-acetyl-4,4-dimethylpiperazinium, and pyridyl ether A-84543 were prepared. Each series was found to retain significant affinity at nicotinic receptors in rat cerebral cortex with tethers of up to six PEG units. Such compounds are hydrophilic ligands which may serve as models for fluorescent/affinity probes and multivalent ligands for nAChR.

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John W. Daly

National Institutes of Health

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Thomas F. Spande

National Institutes of Health

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H. Martin Garraffo

National Institutes of Health

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Herman J. C. Yeh

National Institutes of Health

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Mary D. Menachery

University of Pennsylvania

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