Laurence Hurley

The GC-rich nuclease hypersensitivity element III1 (NHE III1) of the c-MYC promoter largely controls the transcriptional activity of the c-MYC oncogene. The C-rich strand in this region can form I-motif DNA secondary structures. We determined the folding pattern of the major I-motif formed in the NHE III1, which can be formed at near-neutral pH. While we find that the I-motif formed in the four 3' consecutive runs of cytosines appears to be the most favored, our results demonstrate that the C-rich strand of the c-MYC NHE III1 exhibits a high degree of dynamic equilibration. Using a trisubstituted oligomer of this region, we determined the formation of two equilibrating loop isomers, one of which contains a flipped-out cytosine. Our results indicate that the intercalative cytosine+-cytosine base pairs are not always necessary for an intramolecular I-motif. The dynamic character of the c-MYC I-motif is intrinsic to the NHE III1 sequence and appears to provide stability to the c-MYC I-motif.

PMID: 5095271;Abstract:

Indolmycin, an antibiotic produced by a strain of Streptomyces griseus, is formed from (S)-tryptophan, which loses from its side chain the amino nitrogen atom, the hydrogen atom from C-2, and one of the hydrogen atoms from C-3, two intact methyl groups of (S)-methionine, and the guanido carbon atom of (S)-arginine. (R)-β-Methylindolepyruvate and (2S,3R)-indolmycenic acid are intermediates in the biosynthesis. The absolute configuration of indolmycin has been determined by chemical correlation with (-)-(R)-indoleisopropionic acid. Studies with cell-free extracts of S. griseus revealed the presence of a transaminase which converts (S)-tryptophan into indolepyruvate and a methyltransferase which C-methylates indolepyruvate.

PMID: 19657049;PMCID: PMC2775253;Abstract:

A series of diaryl- and fluorenone-based analogs of the lead compound UA-62784 [4-(5-(4-methoxyphenyl)oxazol-2-yl)-9H-fluoren-9-one] was synthesized with the intention of improving upon the selective cytotoxicity of UA-62784 against human pancreatic cancer cell lines with a deletion of the tumor suppressor gene deleted in pancreas cancer locus 4 (DPC-4, SMAD-4). Over 80 analogs were synthesized and tested for antitumor activity against pancreatic cancer (PC) cell lines (the PC series). Despite a structural relationship to UA-62784, which inhibits the mitotic kinesin centromere protein E (CENP-E), none of the analogs was selective for DPC-4-deleted pancreatic cancer cell lines. Furthermore, none of the analogs was a potent or selective inhibitor of four different mitotic kinesins (mitotic kinesin-5, CENP-E, mitotic kinesin-like protein-1, and mitotic centromere-associated kinesin). Therefore, other potential mechanisms of action were evaluated. A diaryl oxazole lead analog from this series, PC-046 [5-(4-methoxyphenyl)-2-(3-(3-methoxyphenyl)pyridin-4-yl) oxazole], was shown to potently inhibit several protein kinases that are overexpressed in human pancreatic cancers, including tyrosine receptor kinase B, interleukin-1 receptor-associated kinase-4, and proto-oncogene Pim-1. Cells exposed to PC-046 exhibit a cell cycle block in the S-phase followed by apoptotic death and necrosis. PC-046 effectively reduced MiaPaca-2 tumor growth in severe combined immunodeficiency mice by 80% compared with untreated controls. The plasma half-life was 7.5 h, and cytotoxic drug concentrations of >3 μM were achieved in vivo in mice. The diaryl oxazole series of compounds represent a new chemical class of anticancer agents that inhibit several types of cancer-relevant protein kinases. Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics.

The proximal promoter region of the human vascular endothelial growth factor (VEGF) gene contains a polypurine/polypyrimidine tract that serves as a multiple binding site for Sp1 and Egr-1 transcription factors. This tract contains a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif for the formation of an intramolecular G-quadruplex. In this study, we observed the progressive unwinding of the oligomer duplex DNA containing this region into single-stranded forms in the presence of KCl and the G-quadruplex-interactive agents TMPyP4 and telomestatin, suggesting the dynamic nature of this tract under conditions which favor the formation of the G-quadruplex structures. Subsequent footprinting studies with DNase I and S1 nucleases using a supercoiled plasmid DNA containing the human VEGF promoter region also revealed a long protected region, including the guanine-rich sequences, in the presence of KCl and telomestatin. Significantly, a striking hypersensitivity to both nucleases was observed at the 3'-side residue of the predicted G-quadruplex-forming region in the presence of KCl and telomestatin, indicating altered conformation of the human VEGF proximal promoter region surrounding the guanine-rich sequence. In contrast, when specific point mutations were introduced into specific guanine residues within the G-quadruplex-forming region (Sp1 binding sites) to abolish G-quadruplex-forming ability, the reactivity of both nucleases toward the mutated human VEGF proximal promoter region was almost identical, even in the presence of telomestatin with KCl. This comparison of wild-type and mutant sequences strongly suggests that the formation of highly organized secondary structures such as G-quadruplexes within the G-rich region of the human VEGF promoter region is responsible for observed changes in the reactivity of both nucleases within the polypurine/polypyrimidine tract of the human VEGF gene. The formation of the G-quadruplex structures from this G-rich sequence in the human VEGF promoter is further confirmed by the CD experiments. Collectively, our results provide strong evidence that specific G-quadruplex structures can naturally be formed by the G-rich sequence within the polypurine/polypyrimidine tract of the human VEGF promoter region, raising the possibility that the transcriptional control of the VEGF gene can be modulated by G-quadruplex-interactive agents.

Abstract:

The pluramycin antitumor antibiotics, which include the altromycins, pluramycin, hedamycin, and rubiflavin, are a group of highly evolved DNA-reactive compounds that have structural features reminiscent of both nogalamycin and the aflatoxins. As such, they are characterized as "threading intercalators" with the added ability to alkylate N7 of guanine (see preceding article in this issue). In this article we have demonstrated that different members of this group of antibiotics have sequence specificities that differ for the base pair to the 5′ side of the alkylated guanine and also have a range of reactivities with susceptible sequences. Subsequent experiments were designed to determine the molecular origin for both these observed contrasting sequence specificities and covalent reactivities. First, neopluramycin, an analog of pluramycin that lacks the epoxide, and thus is unable to covalently modify DNA, but is in other respects structurally similar, exhibits no discernible sequence selectivity. This suggests that the sequence selectivity of the pluramycins is determined at the covalent bonding step rather than the precovalent binding interactions. Second, using An tracts of varying length (n = 1-5) to modulate the minor groove geometry to the 5′ side of the covalent alkylation site, this structural parameter has been shown to have a major effect on both sequence specificity and alkylation reactivity. Last, the electronegativity of the N7 position of the alkylated base can also affect reactivity and, to a lesser extent, sequence specificity. In order to determine the molecular details of the interactions in the minor and major grooves, which could give rise to the different sequence specificities of the nonclassical (typified by altromycin B) and classical (typified by hedamycin) pluramycins, we have used molecular models of the altromycin B and hedamycin-DNA adducts that are derived from high-field NMR data of their 10-mer duplex diadducts. These studies demonstrate that it is likely that the sequence-dependent reactivities of the epoxide of the pluramycin to N7 of guanine are dependent upon the relative extent of a "proximity effect". The magnitude of the proximity effect is determined by a "steering reaction", which takes place in the minor and major grooves due to the different placement of the carbohydrate substituents on the pluramycins and their hydrogen bonding and van der Waals interactions with the base pairs to the 5′ side of the alkylation site. This is proposed to be a novel mechanism for sequence recognition, where cooperative interactions in the minor and major grooves transmitted via the intercalation moiety dictate the positioning of the epoxide in the major groove and, thus, sequence reactivity. Finally, we propose that the increased reactivity of the classical pluramycins in contrast to the altromycins is at least partially determined by the "reach" of the reactive epoxide in the major groove, which varies from one group to another. The molecular mechanisms for sequence recognition described here provide a new paradigm for sequence recognition by minor and major groove interactions mediated by intercalactive binding but achieved at the covalent bonding step. * Address correspondence to this author.