Laurence Hurley

PMID: 2337606;Abstract:

Tomaymycin is a member of the pyrrolo[l,4]benzodiazepine [P(1,4)B] antitumor antibiotic group. This antibiotic is proposed to react with the exocyclic 2-amino group (N2) of guanine to form a covalent adduct that lies snugly within the minor groove of DNA. While DNA-footprinting experiments using methidiumpropyl-EDTA have revealed the favored bonding sequences for tomaymycin and related drugs on DNA, the stereochemistry at the covalent bonding site (C-11) and orientation in the minor groove were not established by these experiments. In previous studies using a combined fluorescence, high-field NMR, and molecular modeling approach, we have shown that for tomaymycin there are two diastereomeric species (11R and 11S) on both calf thymus DNA and d(ATGCAT)2. Although we were able to infer the identity (stereochemistry at C-11 and orientation in the minor groove) of the two species on d(ATGCAT)2 by high-field NMR and fluorescence studies, in combination with molecular mechanics calculations, definitive experimental evidence was lacking. We have designed and synthesized a self-complementary 12-mer [d(CICGAATTCICG)2] based on the Dickerson dodecamer [d(CGCGAATTCGCG)2] that bonds identically two tomaymycin molecules, each having a defined orientation and stereochemistry. Thus the bis(tomaymycin)-12-mer adduct maintains the self-complementarity of the original duplex molecule. Two-dimensional proton J-correlated spectroscopy (COSY) of the bis(tomaymycin)-d(CICGAATTCICG)2 adduct (I = inosine) unequivocally shows that C-11 of tomaymycin covalently bonds through N2 of guanine with an 11S stereochemistry in the sequence 5′-CGA-3′. Fluorescence studies confirm the "S" stereochemistry at C-11, and two-dimensional proton nuclear Overhauser (NOESY) experiments assign the orientation of the drug molecule in the minor groove of DNA, i.e., with the aromatic ring of tomaymycin to the 3′ side of covalently modified guanine. Molecular modeling experiments with AMBER are consistent with the identification of the species of tomaymycin (11S with 3′ orientation) bound to the 12-mer. This species and the other 11S species are favored over the two 11R species due to a combination of steric and electrostatic interactions. Analysis of two-dimensional COSY and NOESY experiments on the bis(tomaymycin)-d-(CICGAATTCICG)2 adducts reveals minimal effect of covalent bonding on local helix structure. From these experiments the modest but most pronounced distortion is at the deoxyribose attached to the modified guanine and both the phosphate and adjacent deoxyribose to the 5′ side. The distortion of this phosphate between the covalently modified guanine and the 5′ nucleoside is supported by its downfield-shifted phosphorus NMR resonance signal. The discrepancy between the pairs of most energetically favored species of tomaymycin-DNA adducts on d(ATGCAT)2 and the 12-mer is explained by examining individual drugnucleotide interactions. The results presented in this study together with previous investigations show that the orientation of the drug molecule in the minor groove, and stereochemistry at the covalent linkage site, is dependent upon both the flanking sequence and drug structure. This conclusion mandates caution be used in rationalizing the biochemical and biological effects of P(1,4)B bonding to DNA until precise structural information is established. © 1990 American Chemical Society.

PMID: 3346874;Abstract:

Tomaymycin is a member of the pyrrolo[1,4] benzodiazepine antitumor-antibiotic group that binds covalently to the exocyclic 2-amino group of guanine in DNA. Previous correlation of fluorescence and NMR data suggested that the 11R,11aS and the 11S,11aS diastereomers of tomaymycin could bind to DNA in two orientations relative to the covalently modified guanine (Barkley, M. D.; Cheatham, S.; Thurston, D. E.; Hurley, L. H. Biochemistry 1986, 25, 3021-3031). We now report on fluorescence, one- and two-dimensional proton NMR, and molecular modeling studies of the tomaymycin-d(ATGCAT)2 adduct, which corroborate these earlier observations. Fluorescence measurements show that there are two species of tomaymycin bound to d(ATGCAT)2, which are tentatively identified as the 11R,11aS and 11S,11aS diastereomers. Two distinct sets of signals for the tomaymycin molecule are present in the proton NMR spectrum of the tomaymycin-d(ATGCAT)2 duplex adduct. Two-dimensional correlation spectroscopy (2D-COSY) studies also show connectivities for four cytosine H5-H6 and eight thymine methyl-H6 protons and thus clearly establish the presence of two distinct species of tomaymycin-d(ATGCAT)2 adducts in solution. A single scalar 11-11a ¹H NMR coupling in the 2D-COSY spectrum is indicative of an adduct species that has an S configuration at the C-11 position. Two-dimensional nuclear Overhauser effect (NOESY) spectra of the tomaymycin-d(ATGCAT)2 duplex adduct show that the adducts are relatively nondistortive. In a NOESY experiment, cross-peaks were identified between both the aromatic H9 proton and the ethylidine methyl protons of tomaymycin and two different adenine H2 protons of d(ATGCAT)2. Molecular mechanics calculations with AMBER show that the two species with the thermodynamically most favorable binding energies are the 11R,11aS and 11S,11aS isomers with their aromatic rings to the 5′ and 3′ sides of the covalently bound guanine, respectively. The NOEs observed between tomaymycin protons and adenine H2 protons are in accord with molecular modeling studies. Taken together, these results strongly suggest that the two forms of tomaymycin bound to d(ATGCAT)2 are the 11S,11aS and 11R,11aS species, oriented with their aromatic rings to the 3′ and 5′ sides, respectively, of the covalently modified guanines. © 1988 American Chemical Society.

PMID: 1547223;Abstract:

(+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have shown that the potent cytotoxic and antitumor activities of (+)-CC-1065 are due to the ability of this compound to covalently modify DNA. (+)-CC-1065 reacts with duplex DNA to form an N3- adenine DNA adduct which lies in the minor groove of the DNA helix overlapping with a 5-base-pair region. As a consequence of covalent modification with (+)-CC-1065, the DNA helix bends into the minor groove and also undergoes winding and stiffening [Lee, C.-S., Sun, D., Kizu, R., and Hurley, L. H. (1991) Chem. Res. Toxicol. 4, 203-213]. In the studies described here, in which we have constructed site-directed DNA adducts on single-stranded DNA templates, we have shown that (+)-CC-1065 and select synthetic analogues, which have different levels of cytotoxicity, all show strong blocks against progression of Klenow fragment, E. coli DNA polymerase, and T4 DNA polymerase. The inhibition of bypass of drug lesions by polymerase could be partially alleviated by increasing the concentration of dNTPs and, to a small extent, by increasing polymerase levels. Klenow fragment binds equally well to a DNA template adjacent to a drug modification site and to unmodified DNA. These results taken together lead us to suspect that it is primarily inhibition of base pairing around the drug modification site and not prevention of polymerase binding that leads to blockage of DNA synthesis. Unexpectedly, the exact termination site of the in vitro DNA synthesis by Klenow fragment is not dependent on the species of covalently bound drug molecule but on the sequence to the 5' side of the drug-modified adenine. Misincorporation of dA for dG by Klenow fragment occurred at the secondary pausing site specifically for (+)-CC-1065 contained within the covalently modified sequence 5'-GATTA-3'. Although (+)-CC-1065 and its analogues evaluated in this study did not produce dramatically different effects on DNA polymerases when the drugs were bound to a single-stranded template, polymerization from a primer site containing a drug lesion in the duplex region did show a selective inhibitory effect with (+)-CC-1065 and (+)-AB'C'. When this observation is considered alongside results of experiments showing selective inhibition by these same compounds of T4 ligase and helicase II, the winding phenomena uniquely found with these compounds may be associated with the potent biological effect known as delayed lethality.