A molecular guidance system based upon target genes, nuclear receptors and ligands applied to drug discovery and prediction of toxicity.

A molecular guidance system useful in drug design is described in which nuclear receptors position ligands in intercalation sites in responsive genes. Evidence is based upon positions of agonists in receptors and the transcriptional activity of a designed estrogen that is 3 times more potent than the steroid hormone estradiol.

Small molecule intercalation with double stranded DNA: implications for normal gene regulation and for predicting the biological efficacy and genotoxicity of drugs and other chemicals.

The binding of small molecules to double stranded DNA including intercalation between base pairs has been a topic of research for over 40 years. For the most part, however, intercalation has been of marginal interest given the prevailing notion that binding of small molecules to protein receptors is largely responsible for governing biological function. This picture is now changing with the discovery of nuclear enzymes, e.g. topoisomerases that modulate intercalation of various compounds including certain antitumor drugs and genotoxins. While intercalators are classically flat, aromatic structures that can easily insert between base pairs, our laboratories reported in 1977 that a number of biologically active compounds with greater molecular thickness, e.g. steroid hormones, could fit stereospecifically between base pairs. The hypothesis was advanced that intercalation was a salient feature of the action of gene regulatory molecules. Two parallel lines of research were pursued: (1) development of technology to employ intercalation in the design of safe and effective chemicals, e.g. pharmaceuticals, nutraceuticals, agricultural chemicals; (2) exploration of intercalation in the mode of action of nuclear receptor proteins. Computer modeling demonstrated that degree of fit of certain small molecules into DNA intercalation sites correlated with degree of biological activity but not with strength of receptor binding. These findings led to computational tools including pharmacophores and search engines to design new drug candidates by predicting desirable and undesirable activities. The specific sequences in DNA into which ligands best intercalated were later found in the consensus sequences of genes activated by nuclear receptors implying intercalation was central to their mode of action. Recently, the orientation of ligands bound to nuclear receptors was found to match closely the spatial locations of ligands derived from intercalation into unwound gene sequences suggesting that nuclear receptors may be guiding ligands to DNA with remarkable precision. Based upon multiple lines of experimental evidence, we suggest that intercalation in double stranded DNA is a ubiquitous, natural process and a salient feature of the regulation of genes. If double stranded DNA is proven to be the ultimate target of genomic drug action, intercalation will emerge as a cornerstone of the future discovery of safe and effective pharmaceuticals.

Transcriptional inhibition of the estrogen response element by antiestrogenic piperidinediones correlates with intercalation into DNA measured by energy calculations.

The energy of interaction of antiestrogenic ligands bound to DNA derived from molecular modeling was compared to the capacity of the ligands to directly inhibit the transcriptional activity of an estrogen responsive gene. 3-Phenylacetylamino-2,6-piperidinedione (A10) and related compounds were intercalated into a partially unwound DNA site in a canonical estrogen response element (ERE). The piperidinedione/ERE complexes were subjected to energy minimization and the strength of interaction of the ligands with the DNA was measured. The ability of the ligands to inhibit transactivation was assessed using a reporter gene constructed with the ERE of the vitellogenin gene promoter (ERE(v)-tk-Luc) transiently transfected into the human estrogen receptor-positive MCF-7 breast cancer cell line. The results demonstrate a direct correlation between the calculated energetic fit of the compounds in the ERE and inhibition of ERE(v) transactivation. The order of potency of the compounds to suppress estrogen-dependent reporter gene activity was identical to that previously shown for inhibiting the growth of MCF-7 cells. To our knowledge, these results provide the first direct experimental evidence that the predicted fit of a class of compounds into a defined DNA binding site correlates with the ability of the compounds to modulate specific gene functions regulated at that site.

Evaluation of DNA intercalation potential of pharmaceuticals and other chemicals by cell-based and three-dimensional computational approaches.

To what extent noncovalent chemical-DNA interactions, in particular weak nonbonded DNA intercalation, contribute to genotoxic responses in mammalian cells has not been fully elucidated. Moreover, with the exception of predominantly flat, multiple-fused-ring structures, our ability to predict intercalation ability of novel compounds is nearly completely lacking. Computational programs such as DEREK and MCASE recognize primarily those molecules that can form irreversible covalent adducts with DNA since their learning sets, for the most part, have not been populated by compounds for which a relationship between noncovalent interaction and genotoxicity exists. We describe here a novel three-dimensional (3D) computational DNA-docking model for prediction of DNA intercalative activity of molecules with both classical and nonclassical intercalating structures. The 3D docking results show a remarkable concordance with results obtained from testing these molecules directly in the Chinese hamster V79 cell-based bleomycin amplification system suggesting that either or both of these approaches may have utility in defining noncovalent chemical-DNA interactions. The ability to predict and/or demonstrate cellular DNA intercalation of novel molecules may well provide fresh insights into the nature and mechanistic basis of structurally unexpected genotoxicity observed during safety testing.