Laurence Hurley

PMID: 11731295;Abstract:

Background: Ecteinascidin 743 (Et 743), a natural product derived from a marine tunicate, is a potent antitumor agent presently in phase II clinical trials. Et 743 binds in the minor groove of DNA and alkylates N2 of guanine via a unique mechanism involving catalytic activation. The sequence selectivity of Et 743 is governed by different patterns of hydrogen-bonding to DNA, which results in differential reversibility of the covalent adducts. As determined by nuclear magnetic resonance spectroscopy, the preferred sequences 5′-PuGC and 5′-PyGG are stabilized by a hydrogen-bonding network, while the non-preferred sequences 5′-NG(A/T) are much less stabilized due to the lack of a key hydrogen bond to the GC base pair on the 3′-side of the alkylated guanine. Results: Mammalian cell lines (XPB, XPD, XPF, XPG, and ERCC1) deficient in the nucleotide excision repair (NER) gene products show resistance to Et 743. The recognition and subsequent incision of Et 743-DNA adducts by the bacterial multisubunit endonuclease UvrABC were used to evaluate DNA repair-mediated toxicity as a rationale for the resistance of NER-defective cell lines and the antitumor activity of Et 743. The Et 743-DNA adducts are indeed recognized and incised by the UvrABC repair proteins; however, the pattern of incision indicated that the non-preferred, and less stable, sequences (i.e. 5′-NG(A/T)) modified with Et 743 are generally incised at a much higher efficiency than the preferred, more stable sequences (i.e. 5′-PuGC or 5′-PyGG). In addition, within the same Et 743 recognition sequence, the level of incision varies, indicating that flanking regions also contribute to the differential incision frequency. Conclusions: The inefficient repair incision by the UvrABC nuclease of Et 743-DNA adducts provides a basis for rationalizing the observed repair-dependent cytotoxicities of these DNA adducts, if other associated structural properties of Et 743-DNA adducts are taken into account. In particular, the wedge-shaped Et 743, which forces open the minor groove of DNA, introducing a major groove bend, and the extrahelical protrusion of the C-subunit of Et 743 provide unique characteristics alongside the hydrogen-bonding stabilization of a covalent DNA adduct, which we propose traps an intermediate in NER processing of Et 743-DNA adducts. This trapped intermediate protein-Et 743-DNA adduct complex can be considered analogous to a poisoned topoisomerase I- or topoisomerase II-DNA complex. In the absence of an intact NER nuclease complex, this toxic lesion is unable to form, and the Et 743-DNA adducts, although not repaired by the NER pathway, are less toxic to cells. Conversely, elevated levels of either of these nucleases should lead to enhanced Et 743 toxicity. © 2001 Elsevier Science Ltd. All right reserved.

PMID: 6473105;PMCID: PMC320064;Abstract:

CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. The drug binds covalently through N-3 of adenine and lies within the minor groove of DNA. Previous studies indicated that CC-1065 reacted with adenine in DNA to yield a thermally labile product that could be used to reveal its sequence specificity. These studies also provided insight into a DNA sequence (5′-CGGAGTTAGGGGCG-3′) which should bind one molecule of CC-1065 in an unambiguous manner. This sequence, which contains the CC-1065 adenine binding site within the sequence 5′-TTA-3′ was chemically synthesized together with the complementary strand. CC-1065 reacted with the oligoduplex to give an adduct that maintained the B-DNA form and had a final CD spectrum similar to those of the CC-1065 comp1exes formed with calf thymus DNA. The above l4mer was 5′ end-labelled with ³²P, annealed with its complementary strand, reacted with CC-1065 and heated. Drug-mediated strand breakage was evaluated on a sequencing gel. A single break occurred in the labelled strands to give a fragment that migrated as an 8.5mer; subsequent piperidine treatment produced a fragment that migrated as a 7mer, which is the size expected from the known binding of CC-1065 at adenine in 5′-TTA-3′ sequences. © 1984 IRL Press Limited.

Abstract:

The monoalkylation and cross-linking reactivities of a group of four structurally related DNA-DNA interstrand cross-linkers have been determined on restriction enzyme fragments and select oligomers. These highly potent cytotoxic DNA-DNA cross-linkers consist of two cyclopropa[c]pyrrolo[3,4-3]indol-4(5H)-ones indoles [(+)-CPI-I] joined by a urea (Bizelesin) or a bisamido furan, bisamido pyrrole, or bisamido N-methylpyrrole linker. Using a thermal cleavage assay in combination with radio-labeled restriction enzyme fragments, we have shown that these compounds cross-link duplex DNA six or seven base pairs apart on opposite strands, but they differ among themselves for both alkylation reactivity and DNA sequence selectivity. Bizelesin and the [(+)-CPI-I]2 bisamido furan and [(+)-CPI-I]2 bisamido N-methyl pyrrole compounds prefer purely AT-rich sequences (e.g., 5'-T(A/T)(4 or 5)A*-3', where T represents the cross-strand adenine alkylation and A* represents an adenine alkylation), while the [(+)-CPI-I]2 bisamido pyrrole requires a centrally positioned GC base pair for high cross-linking reactivity (i.e., 5'-T(A/T)2G(A/T)2A*-3'). By comparison of the cross-linking reactivity of the four compounds in 21-mer duplex oligomers containing strategically placed GC or IC base pairs, the sequence and linker requirements for high reactivity of the six- and seven-base-pair cross-linkers in 5'-T(N)(4 or 5)5A*-3' sequences were determined. In the duplex, to attain highest reactivity, a centrally placed GC base pair and the exocyclic 2-amino group were required, while for the linker in the bisamido pyrrole compound, an unsubstituted amine in the pyrrole ring was necessary. On the basis of the known requirements for monoalkylation of duplex DNA by (+)-CPI-derived compounds and the structural consequences of monoalkylation, together with the information gleaned from this study, we are able to provide a rationale for the structural requirements for the specific sequence cross-linked with high reactivity by the pyrrole compound. We propose that, because monoalkylation of the duplex produced a bent DNA duplex that is unsuitable for cross-linking, the duplex has to first undergo a ligand-induced rearrangement involving two hydrogen-bonding donor-acceptor pairs, which reinstates the requirements necessary for the second alkylation reaction.

The majority of kinase inhibitors developed to date are competitive inhibitors that target the ATP binding site; however, recent crystal structures of Gleevec (imatinib mesylate, STI571, PDB: 1IEP), Nexavar (Sorafenib tosylate, BAY 43-9006, PDB: 1UWJ), and BIRB-796 (PDB: 1KV2) have revealed a secondary binding site adjacent to the ATP binding site known as the DFG-out allosteric binding site. The recent successes of Gleevec and Nexavar for the treatment of chronic myeloid leukemia and renal cell carcinoma has generated great interest in the development of other kinase inhibitors that target this secondary binding site. Here, we present a structural comparison of the important and similar interactions necessary for Gleevec(R), Nexavar, and BIRB-796 to bind to their respective DFG-out allosteric binding pockets and the selectivity of each with respect to c-Abl, B-Raf, and p38alpha. A structural analysis of their selectivity profiles has been generated from the synthesis and evaluation of 8 additional DFG-out allosteric inhibitors that were developed directly from fragments of these successful scaffolds.

PMID: 956946;Abstract:

The long-range ¹³C-¹H couplings of anthramycin and pyrrolnitrin are utilized to locate the ¹³C-enriched carbon atoms of the biosynthetically labeled antibiotics which were isolated from feeding experiments with L-[Me-¹³C]methionine and DL-[alanine-3-¹³C]tryptophan.