https://www.cell.com/cell/pdf/0092-8674(91)90035-W.pdf

Membrane organization of the dystrophin-glycoprotein complex

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

Mesoporous Silica Nanoparticles as a Delivery System for Hydrophobic Anticancer Drugs

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.

https://science.sciencemag.org/content/sci/279/5356/1504.full.pdf

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

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

DNA Topoisomerases: Essential Enzymes and Lethal Targets

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.

https://www.pnas.org/content/pnas/99/24/15387.full.pdf

The mechanism of topoisomerase I poisoning by a camptothecin analog

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.

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

The Synthesis of Prodigiosin

The DNA alkylation and sequence specificity of a group of natural and synthetic pyrrolo-[1,4]benzodiazepines [P(1,4)Bs] were evaluated by using an exonuclease III stop assay, and the results were compared with in vitro and in vivo biological potency and antitumor activity. The P(1,4)B antibiotics are potent antitumor agents produced by various Actinomycetes, which are believed to mediate their cytotoxic effects by covalent bonding through N-2 of guanine in the minor groove of DNA. In this article we describe the results of a sensitive DNA alkylation assay using exonuclease III which permits both estimation of the extent of DNA modification as well as location of the precise guanines to which the drugs are covalently bound. Using this assay, we have evaluated a series of natural and synthetic compounds of the P(1,4)B class for their ability to bind to DNA and also determined their DNA sequence preference. The compounds included in this study are P(1,4)Bs carrying different substituents in the aromatic ring, having varying degrees of saturation in the five-membered ring, or differing in the stereochemistry at C-11a. These same compounds were evaluated for in vitro cytotoxic activity against B16 melanoma cells, for potency in vivo in B6D2F1 mice (LD50), and for antitumor activity (ILSmax) against P388 leukemia cells. A good correlation was found between extent of DNA alkylation and in vitro and in vivo potency. Furthermore, on the basis of electronic and steric considerations, it was possible to rationalize why those compounds that showed negligible biological activity were unable to bond covalently to DNA. Last, we have determined that the degree of saturation in the five-membered ring of the P(1,4)Bs has a significant effect on the DNA bonding reactivity and biological activity of this class of compounds.

Pyrrolo[1,4]benzodiazepine antitumor antibiotics: relationship of DNA alkylation and sequence specificity to the biological activity of natural and synthetic compounds.

Kenneth G. Holden

Papers

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

The Synthesis of Prodigiosin

Pyrrolo[1,4]benzodiazepine antitumor antibiotics: relationship of DNA alkylation and sequence specificity to the biological activity of natural and synthetic compounds.

Identification and quantitation of alditol acetates of neutral and amino sugars from mucins by automated gas-liquid chromatography.

Nuclear analogues of beta-lactam antibiotics. 2 The total synthesis of 8-oxo-4-thia-z-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acids.