Adult drug metabolism is dominated by cytochrome P450 3A4 (CYP3A4), which is often inhibited by antifungal azole drugs, resulting in potential alterations in drug metabolism and adverse drug/drug interactions. In the fetal and neonatal stages of life, the 87%-identical cytochrome P450 3A7 (CYP3A7) is expressed but not CYP3A4. Azole antifungals developed for adults are also used in neonates assuming they interact similarly with both enzymes, but systematic information is lacking. Herein a method was developed for generating recombinant purified CYP3A7. Thirteen different azoles were then evaluated for binding and inhibition of purified human CYP3A4 vs. CYP3A7. All imidazole-containing azoles bound both enzymes via coordination to the heme iron and inhibited both with IC50 values ranging from 180 nM for clotrimazole to the millimolar range for imidazole itself. Across this wide range of potencies, CYP3A4 was consistently inhibited more strongly than CYP3A7, with clotrimazole being the least selective (1.5-fold) inhibitor and econazole the most selective (12-fold). Observations for 1,2,4-triazole-containing azoles were more varied. Most bound to CYP3A4 via coordination to the heme iron, but several also demonstrated evidence of a distinct binding mode at low concentrations.However, only posaconazole inhibited CYP3A4. Of the triazoles only posaconazole inhibited CYP3A7, again less potently than CYP3A4. Spectral evidence for binding was weak or nonexistent for all triazoles. Overall, while the details of binding interactions do vary, the same azole compounds inhibit both enzymes, albeit with weaker interactions with CYP3A7 compared to CYP3A4.

The human cytochrome P450 enzymes are membrane-bound monooxygenases that perform diverse roles. Xenobiotic-metabolizing P450 enzymes are generalists responsible for the first step in the elimination of hydrophobic drugs and toxins, but can also activate protoxins, procarcinogens, and prodrugs. With the propensity of each to act on many different substrates, one small molecule drug or xenobiotic can often alter the metabolism of a second, causing adverse interactions. Other human P450 enzymes play more specific roles in the production and interconversions of key endobiotics including steroids, vitamins, fatty acids, and eicosanoids. These latter P450 enzymes are often drug targets. While poor solubility and stability prevented any membrane P450 X-ray structures until 2000, since then at least one structure has been determined for 22 enzymes of the 57 human genes. Within the conserved P450 fold, variations of secondary structure placement and side chain accommodations are apparent, both between and within a given P450 enzyme. In some cases, these structures provide a logical framework for observed function and mutational effects, but in other cases these structures raise additional questions. Regardless, enough structural information is available to begin guiding drug design, whether to target specific P450 enzymes or to avoid undesirable P450 inhibition or metabolism.

The Grb7 protein is an adaptor protein that is often co-amplified with the ErbB2 receptor in 20–30% of breast cancer patients. Grb7 overexpression has been linked to increased cell migration and cancer metastasis. The RA and PH domain region of Grb7 has been reported to interact with various other downstream signaling proteins such as four and half LIM domains isoform 2 (FHL2) and filamin α. These interactions are believed to play a role in regulating Grb7 mediated cell migration function. The full-length Grb7 protein has been shown to dimerize, and the oligomeric state of the Grb7SH2 domain has been extensively studied; however, the oligomerization state of the RA and PH domains, and the importance of this oligomerization in Grb7 function is yet to be fully known. In this study, we characterize the oligomeric state of the Grb7RA domain using size exclusion chromatography, NMR nuclear relaxation studies, glutaraldehyde cross linking and dynamic light scattering. We report the Grb7RA domain can exist in transient multimeric forms, and, based upon modeling results, postulate the potential role of Grb7RA domain oligomerization in Grb7 function.