William D. Kingsbury

Bio: William D. Kingsbury is an academic researcher from Smith, Kline & French. The author has contributed to research in topics: Peptide & Peptide transport. The author has an hindex of 9, co-authored 17 publications receiving 782 citations.

Modification of the hydroxy lactone ring of camptothecin: inhibition of mammalian topoisomerase I and biological activity.

Abstract: Several camptothecin derivatives containing a modified hydroxy lactone ring have been synthesized and evaluated for inhibition of topoisomerase I and cytotoxicity to mammalian cells. Each of the groups of the hydroxy lactone moiety, the carbonyl oxygen, the ring lactone oxygen, and the 20-hydroxy group, were shown to be critical for enzyme inhibition. For example the lactol, lactam, thiolactone, and 20-deoxy derivatives did not stabilize the covalent DNA-topoisomerase I complex. With a few exceptions, those compounds that did not inhibit topoisomerase I were not cytotoxic to mammalian cells. Two cytotoxic derivatives that did not inhibit topoisomerase I were shown to produce non-protein-associated DNA single-strand breaks and are likely to have a different mechanism of action. One of these compounds was tested for antitumor activity and was found to be inactive. The present findings, as well as other reports that the hydroxy lactone ring of camptothecin is critical for antitumor activity in vivo, correlate with the structure-activity relationships at the level of topoisomerase I and support the hypothesis that antitumor activity is related to inhibition of this target enzyme.

A novel peptide delivery system involving peptidase activated prodrugs as antimicrobial agents. Synthesis and biological activity of peptidyl derivatives of 5-fluorouracil

Abstract: As an approach to the development of antimicrobial agents, a novel peptide carrier system was designed, based on the chemical instability of alpha-substituted glycine analogues, with the explicit intent of actively transporting therapeutically useful compounds into microbial cells. Peptides containing 5-fluorouracil (5-FU) linked to the peptide backbone were selected to test the feasibility of this new delivery system. These peptide conjugates were designed such that they would be substrates for both the microbial peptide permeases and peptidases. After entry into cells, enzymatic hydrolysis of the peptide generates an unstable alpha-(5-FU)-glycine that spontaneously decomposes to release 5-FU. The 5-FU-peptide conjugates were tested for antifungal (Candida albicans) and antibacterial (Escherichia coli) activity and were found to have antimicrobial activities comparable to free 5-FU. Noninhibitory peptides antagonized the antimicrobial activities of the 5-FU-peptide conjugates but not of free 5-FU, a result consistent with peptide transport mediated entry of the peptide conjugates into cells. Further support for this conclusion was provided by the finding that biological activities were dependent upon peptide stereochemistry.

Synthesis of Water-Soluble (Aminoalkyl)camptothecin Analogues: Inhibition of Topoisomerase I and Antitumor Activity.

Abstract: Water-soluble analogues of the antitumor alkaloid camptothecin (1) were prepared in which aminoalkyl groups were introduced into ring A or B. Most of the analogues were prepared by oxidation of camptothecin to 10-hydroxycamptothecin (2) followed by a Mannich reaction to give N-substituted 9-(aminomethyl)-10-hydroxycamptothecins (4-12) or by subsequent modification of Mannich product 4 (13, 15, 17, 19, 21). Others were obtained by modification of the hydroxyl group of 2 (25,26) or by total synthesis (35,42,43). These analogues, as well as some of their synthetic precursors, were evaluated for inhibition of topoisomerase I, cytotoxicity, and antitumor activity. Although there was not a quantitative correlation between these assays, compounds that inhibited topoisomerase I were also cytotoxic and demonstrated antitumor activity in vivo. Further evaluation of the most active water-soluble analogue led to the selection of 9-[(dimethylamino)methyl]-10-hydroxycamptothecin (4, SK&F 104864) for development as an antitumor agent. In addition to its water solubility, ease of synthesis from natural camptothecin, and high potency, 4 demonstrated broad-spectrum activity in preclinical tumor models and is currently undergoing Phase I clinical trials in cancer patients.

Transport of antimicrobial agents using peptide carrier systems: anticandidal activity of m-fluorophenylalanine--peptide conjugates.

Abstract: A series of di- and tripeptides containing D- and L-m-fluorophenylalanine was prepared and tested in vitro for the ability to inhibit the growth of the yeast Candida albicans. The results demonstrate that peptides containing L-m-fluorophenylalanine inhibited the growth of C. albicans with minimum inhibitory concentrations (MIC's) ranging from 0.5 to 63 micrograms/mL. The parent L-m-fluorophenylalanine and peptides containing D-m-fluorophenylalanine were inactive (MIC greater than 250 micrograms/mL) in these tests. The results of competitive antagonism studies support peptide transport mediated entry of the inhibitory peptides, followed by release of L-m-fluorophenylalanine inside the cell.

Portage of various compounds into bacteria by attachment to glycine residues in peptides

Abstract: Synthetic di- and oligopeptides are described that contain nucleophilic moieties attached to the α carbon of a glycine residue. These peptides are accepted by the peptide transport systems of Escherichia coli (and other microorganisms) and are capable of being hydrolyzed by intracellular peptidases. After liberation of its amino group the α-substituted glycine is chemically unstable (although it is stable in peptide form) and decomposes, releasing the nucleophilic moiety. Thus, the combined result of peptide transport and peptidase action is the intracellular release of the nucleophile. Peptides containing glycine residues α-substituted with thiophenol, aniline, or phenol are used as models for this type of peptide-assisted entry and their metabolism by E. coli is described. Peptides of this type have broad applicability to the study of microbial physiology and the development of an additional class of antimicrobial agents.

Mesoporous Silica Nanoparticles as a Delivery System for Hydrophobic Anticancer Drugs

Abstract: A critical obstacle and challenge for cancer therapy concerns the limited availability of effective biocompatible delivery systems for most hydrophobic therapeutic anticancer drugs. It is particularly important to improve the aqueous solubility of drugs, as low drug solubility in aqueous media hampers the ability of drugs to be administered through the intravenous route. Since many important anticancer agents have poor water solubility, the development of novel delivery systems for these molecules without the use of organic solvents has received significant attention. Nanoparticles offer great potential and a promising approach to deliver therapeutic agents into targeted organs or cells and they have been actively developed for application in cancer therapy. We have incorporated a representative hydrophobic anticancer drug, camptothecin (CPT), into the pores of fluorescent mesoporous silica nanoparticles (FMSNs) and delivered the drug into a variety of human cancer cells to induce cell death, a procedure suggesting that the mesoporous silica nanoparticles might be used as a vehicle to overcome the insolubility problem of many anticancer drugs. CPT and its derivatives are considered to be among the most promising anticancer drugs of the 21st century. Although studies have demonstrated their effectiveness against carcinomas of the stomach, colon, neck, and bladder, as well as breast and small-cell lung cancers, and leukemia, in vitro, clinical application of CPT in humans has not been achieved to date because the poor water solubility of the drug requires changes to the physicochemical characteristics. The need to formulate water-soluble salts of CPT (that is, alkaline solutions for intravenous injections) led to chemical modifications of the molecule with loss of antiACHTUNGTRENNUNGtumor activity and significant alterations in the toxicological profile of the drug. Although derivatives such as irinotecan have produced good clinical results, irinotecan was shown to have far lower cytotoxicity to cancer cells than CPT (10%), and CPT remains the most potent compound. Among a variety of drug-delivery systems, mesoporous silica materials have several attractive features for use in the delivery of water-insoluble drugs. These particles have large surface areas and porous interiors that can be used as reservoirs for storing hydrophobic drugs. The pore size and environment can be tailored to selectively store different molecules of interest, while the size and shape of the particles can be tuned to maximize cellular uptake. Unlike polymer-based nanoparticles, these robust inorganic materials can tolerate many organic solvents. Silica-based materials have been successfully used as drug-delivery vectors, gene transfection reagents, cell markers, and carriers of molecules. Here, we describe the preparation of fluorescent mesoporous silica nanoparticles that are highly stable in aqueous solution and their use for the delivery of the hydrophobic anticancer drug CPT. The FMSNs were prepared by using a base-catalyzed sol–gel process at high temperature with a modification of published procedures. 25,26] In a typical synthesis, fluorescein isothiocyanate (FITC) was first treated with 3-aminopropyltriethoxysilane (APTS) in ethanol. The mixture was then added, along with tetraethylorthosilicate, to cetyltriACHTUNGTRENNUNGmethylammonium bromide solution at 80 8C. The surfactants were removed from the pores by refluxing the nanoparticles in acidic methanol, the success of which was confirmed by Fourier transform infrared spectroscopy (FTIR; see Supporting Information). Electron microscopy and Xray diffraction (XRD) analysis showed that the particle shape and hexagonal arrays of the pores in the FMSNs remained intact after the surfactant-removal process (Figure 1). The nanoparticles were roughly spherical in shape and smaller than 130 nm in diameter. An average pore diameter of around 2 nm was observed by using transmission electron microscopy (TEM) and an interplanar spacing of dACHTUNGTRENNUNG(100) 4 nm was calculated from the XRD pattern. It is necessary for efficient cellular uptake of the particles that the FMSNs remain dispersed and do not aggregate in the buffer solution. The observed aggregation is caused by interparticle hydrogen-bonding interactions between the amine groups (from the unreacted APTS) and the silanols (Scheme 1A). By modifying only the surfaces of the FMSNs with trihydroxysilylpropyl methylphosphonate (THMP) after particle formation, we reduced the aggregation and increased the stability of the particles in aqueous solution (Scheme 1B, see Supporting Information). [*] Dr. J. Lu, Prof. F. Tamanoi Department of Microbiology, Immunology, and Molecular Genetics California NanoSystems Institute, JCCC University of California, Los Angeles 609 Charles E. Young Drive East, Los Angeles, CA 90095 (USA) Fax: (+1)310-206-5231 E-mail: fuyut@microbio.ucla.edu

Crystal Structures of Human Topoisomerase I in Covalent and Noncovalent Complexes with DNA

Abstract: Topoisomerases I promote the relaxation of DNA superhelical tension by introducing a transient single-stranded break in duplex DNA and are vital for the processes of replication, transcription, and recombination. The crystal structures at 2.1 and 2.5 angstrom resolution of reconstituted human topoisomerase I comprising the core and carboxyl-terminal domains in covalent and noncovalent complexes with 22-base pair DNA duplexes reveal an enzyme that "clamps" around essentially B-form DNA. The core domain and the first eight residues of the carboxyl-terminal domain of the enzyme, including the active-site nucleophile tyrosine-723, share significant structural similarity with the bacteriophage family of DNA integrases. A binding mode for the anticancer drug camptothecin is proposed on the basis of chemical and biochemical information combined with these three-dimensional structures of topoisomerase I-DNA complexes.

DNA Topoisomerases: Essential Enzymes and Lethal Targets

Abstract: INTRODUCTION In 1971, Wang (1) discovered the first DNA topoisomerase in Escherichia coli. The enzyme (E. coli DNA topoisomerase I, or w protein) catalyzed relaxation of negatively supercoiled DNA in the absence of any energy cofactor (1). Wang proposed that this enzyme also catalyzed transient nicking of the DNA double helix and possessed both DNase and ligase activity in one polypeptide (1). The lack of any energy cofactor for the reaction also led Wang to the proposal that the enzyme may form a high-energy covalent bond between itself and the transiently broken DNA phosphodiester bond (1). Both of these predictions have turned out to be correct (2). Since the discovery of E. coli topoisomerase I, investigators have isolated many other DNA topoisomerases from both prokaryotes and eukaryotes. In 1972, Champoux & Dulbecco isolated an enzyme with activity similar to that of E. coli topoisomerase I from mouse embryo cells (3). In 1976, Gellert and his colleagues identified an enzyme activity opposing E. coli DNA topoisomerase I (4). They demonstrated that this enzyme (E. coli DNA topoisomerase II, or gyrase) catalyzed the conversion of relaxed DNA into negatively supercoiled DNA in a reaction requiring ATP hydrolysis (4). These two opposing activities are important for maintaining the super­ helical state of the chromosomal DNA during various DNA transactions (5-8). In 1979, Liu et al (9) isolated an enzyme from bacteriophage

The mechanism of topoisomerase I poisoning by a camptothecin analog

Abstract: We report the x-ray crystal structure of human topoisomerase I covalently joined to double-stranded DNA and bound to the clinically approved anticancer agent Topotecan. Topotecan mimics a DNA base pair and binds at the site of DNA cleavage by intercalating between the upstream (−1) and downstream (+1) base pairs. Intercalation displaces the downstream DNA, thus preventing religation of the cleaved strand. By specifically binding to the enzyme–substrate complex, Topotecan acts as an uncompetitive inhibitor. The structure can explain several of the known structure–activity relationships of the camptothecin family of anticancer drugs and suggests that there are at least two classes of mutations that can produce a drug-resistant enzyme. The first class includes changes to residues that contribute to direct interactions with the drug, whereas a second class would alter interactions with the DNA and thereby destabilize the drug-binding site.