Roughly half of the drug overdose-related deaths in the United States are related to synthetic opioids represented by fentanyl which is a potent agonist of mu-opioid receptor (mOR). In recent years, X-ray crystal structures of mOR in complex with morphine derivatives have been determined; however, structural basis of mOR activation by fentanyl-like opioids remains lacking. Exploiting the X-ray structure of BU72-bound mOR and several molecular simulation techniques, we elucidated the detailed binding mechanism of fentanyl. Surprisingly, in addition to the salt-bridge binding mode common to morphinan opiates, fentanyl can move deeper and form a stable hydrogen bond with the conserved His2976.52, which has been suggested to modulate mOR’s ligand affinity and pH dependence by previous mutagenesis experiments. Intriguingly, this secondary binding mode is only accessible when His2976.52 adopts a neutral HID tautomer. Alternative binding modes may represent a general mechanism in G protein-coupled receptor-ligand recognition.

Several epidemiological studies on ataxia-telangiectasia families indicate that obligate ATM heterozygotes display an elevated risk for developing breast cancer. However, a molecular basis for a potential link between diminished ATM function and sporadic breast malignancy remains elusive. Here, we show that 78% (18 out of a panel of 23) of surgically removed breast tumors (stage II or greater) displayed aberrant methylation of the ATM proximal promoter region as judged by methylation-specific PCR. Aberrant methylation of the ATM promoter was independently confirmed in several tumors by bisulfite sequencing. Moreover, bisulfite sequencing indicated that this region of the genome is subject to dense methylation. Further, we found a highly significant correlation (P ¼ 0.0006) between reduced ATM mRNA abundance, as measured by realtime RT-PCR, and aberrant methylation of the ATM gene promoter. These findings indicate that epigenetic silencing of ATM expression occurs in locally advanced breast tumors, and establish a link at the molecular level between reduced ATM function and sporadic breast malignancy. Oncogene (2004Oncogene ( ) 23, 9432-9437. doi:10.1038 Published online 1 November 2004Keywords: ATM; epigenetics; promoter hypermethylation; breast cancer Germline mutation of the ATM gene is the underlying cause of the cancer-prone disorder ataxia-telangiectasia (A-T) (Rotman and Shiloh, 1998). As outlined in the seminal work of Swift et al. (1987), and validated in subsequent studies (Easton, 1994;Athma et al., 1996), obligate ATM heterozygotes display an approximate fourfold elevated risk for developing breast cancer. Following the cloning of the ATM gene, several groups (FitzGerald et al., 1997;Bebb et al., 1999;Shafman et al., 2000) studied large cohorts of sporadic breast cancer patients and age-matched controls for nonsense or frame-shift mutations within the ATM gene. Neither group found evidence for a higher incidence of defective ATM alleles in the cancer patient cohort. Furthermore, the correlation between breast cancer and dominantnegative forms of ATM arising from defined missense or intronic mutations within the ATM gene remains controversial (Chenevix-Trench et al., 2002;Bernstein et al., 2003). Thus, a potential role for diminished ATM function in sporadic breast cancer remains unresolved.We have reported that, in cultured tumor cells, the ATM gene is subject to epigenetic silencing attributable to aberrant methylation of its proximal promoter region (Kim et al., 2002). More recently, we determined that aberrant methylation of the ATM promoter occurs in a subset of head and neck tumors (Ai et al., 2004). Further, several reports showed that reduced ATM expression, as judged by immunohistochemical staining, occurs in a significant portion of breast tumors (Kairouz et al., 1999;Angele et al., 2000). Collectively, these findings led us to hypothesize that epigenetic events, rather than somatic mutation of the ATM gene itself, link reduced ATM function to sporadic breast cancer.The putative proximal pro...

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