Laurence Hurley

PMID: 1085163;Abstract:

11-Demethyltomaymycin, an antitumor antibiotic produced by Streptomyces achromogenes, and its biologically inactive metabolite oxotomaymycin are biosynthesized from L-tyrosine, DL-tryptophan, and L-methionine. The anthranilate part of 11-demethyltomaymycin is derived from tryptophan probably via the kynurenine pathway. The predominant loss of tritium from DL-[5-³H]tryptophan, during its conversion to 11-demethyltomaymycin and oxotomaymycin is interpreted to mean by NIH shift rules, that the main pathway to the 5-methoxy-4-hydroxy anthranilate moiety is through hydroxylation at C-8 prior to hydroxylation at C-7. The methoxy carbon is derived from the S-methyl group of methionine by transfer of an intact methyl group. The ethylideneproline moiety of 11-demethyltomaymycin is biosynthesized from tyrosine, without a 1-carbon unit from methionine. The results of biosynthetic feeding experiments with L-[1-¹⁴C, 3- or 5-3H] tyrosine are consistent with a "meta" or extradiol cleavage of 6, 7-dihydroxycyclodopa as has also been demonstrated previously for anthramycin and lincomycin A. An experiment in which L-[1-¹⁴C, Ala-2,3-³H]tyrosine was fed showed that both of the β hydrogens of this amino acid are retained in 11-demethyltomaymycin. It has been demonstrated in cultures and washed cell preparations that 11-demethyltomaymycin is enzymatically converted to oxotomaymycin by an intracellular constitutive enzyme. Conversion of oxotomaymycin to 11-demethyltomaymycin by these same preparations could not be demonstrated. The enzymatic activity associated with the conversion of 11 -demethyltomaymycin to oxotomaymycin is not limited to the 11-demethyltomaymycin production phase, since trophophase cells and even cells from 11-demethyltomaymycin nonproducing cultures of S. achromogenes were equally active in converting 11-demethyltomaymycin to oxotomaymycin.

PMID: 11990855;Abstract:

DNA is the molecular target for many of the drugs that are used in cancer therapeutics, and is viewed as a non-specific target of cytotoxic agents. Although this is true for traditional chemotherapeutics, other agents that were discovered more recently have shown enhanced efficacy. Furthermore, a new generation of agents that target DNA-associated processes are anticipated to be far more specific and effective. How have these agents evolved, and what are their molecular targets?

PMID: 15984871;Abstract:

The human telomeric sequence d[T2AG3]4 has been demonstrated to form different types of G-quadruplex structures, depending upon the incubation conditions. For example, in sodium (Na⁺), a basket-type G-quadruplex structure is formed. In this investigation, using circular dichroism (CD), biosensor-surface plasmon resonance (SPR), and a polymerase stop assay, we have examined how the addition of different G-quadruplex-binding ligands affects the conformation of the telomeric G-quadruplex found in solution. The results show that while telomestatin binds preferentially to the basket-type G-quadruplex structure with a 2:1 stoichiometry, 5,10,15,20-[tetra-(N-methyl-3-pyridyl)]-26-28-diselena sapphyrin chloride (Se2SAP) binds to a different form with a 1:1 stoichiometry in potassium (K⁺). CD studies suggest that Se2SAP binds to a hybrid G-quadruplex that has strong parallel and antiparallel characteristics, suggestive of a structure containing both propeller and lateral, or edgewise, loops. Telomestatin is unique in that it can induce the formation of the basket-type G-quadruplex from a random coil human telomeric oligonucleotide, even in the absence of added monovalent cations such as K⁺ or Na ⁺. In contrast, in the presence of K⁺, Se2SAP was found to convert the preformed basket G-quadruplex to the hybrid structure. The significance of these results is that the presence of different ligands can determine the type of telomeric G-quadruplex structures formed in solution. Thus, the biochemical and biological consequences of binding of ligands to G-quadruplex structures found in telomeres and promoter regions of certain important oncogenes go beyond mere stabilization of these structures. © 2005 American Chemical Society.

Abstract:

The cationic porphyrins TMPyP4 and TMPyP2 possess similar structures but have strikingly different potencies for telomerase inhibition. To rationalize this difference, the interactions of TMPyP4 and TMPyP2 with an antiparallel quadruplex DNA were investigated. A single-stranded DNA oligonucleotide (G4A) containing four human telomere repeats of GGGTTA has been designed to form an intramolecular quadruplex DNA and was confirmed to form such a structure under 100 mM KCl by a DNA ligase assay, DMS footprinting, and CD spectrum analysis. By carrying out UV spectroscopic studies of the thermal melting profiles of G4A-porphyrin complexes, we provide evidence that TMPyP4 and TMPyP2 both stabilized quadruplex DNA to about the same extent. A photocleavage assay was used to determine the precise location for TMPyP4 and TMPyP2 in their interactions with quadruplex DNA. The results show that TMPyP4 binds to the intramolecular quadruplex DNA by stacking externally to the guanine tetrad at the GT step, while TMPyP2 binds predominantly to the same G4 DNA structure via external binding to the TTA loop. We propose that the inability of TMPyP2 to bind to the G4A by stacking externally to the guanine tetrad accounts for the differential effects on telomerase inhibition by TMPyP4 and TMPyP2.