Somatic mutations in the KEAP1 ubiquitin ligase or its substrate NRF2 (NFE2L2) commonly occur in human cancer, resulting in constitutive NRF2-mediated transcription of cytoprotective genes. However, many tumors display high NRF2 activity in the absence of mutation, supporting the hypothesis that alternative mechanisms of pathway activation exist. Previously, we and others discovered that via a competitive binding mechanism, the proteins WTX (AMER1), PALB2 and SQSTM1 bind KEAP1 to activate NRF2. Proteomic analysis of the KEAP1 protein interaction network revealed a significant enrichment of associated proteins containing an ETGE amino acid motif, which matches the KEAP1 interaction motif found in NRF2. Like WTX, PALB2, and SQSTM1, we found that the dipeptidyl peptidase 3 (DPP3) protein binds KEAP1 via an ‘ETGE’ motif to displace NRF2, thus inhibiting NRF2 ubiquitination and driving NRF2-dependent transcription. Comparing the spectrum of KEAP1 interacting proteins with the genomic profile of 178 squamous cell lung carcinomas characterized by The Cancer Genome Atlas revealed amplification and mRNA over-expression of the DPP3 gene in tumors with high NRF2 activity but lacking NRF2 stabilizing mutations. We further show that tumor-derived mutations in KEAP1 are hypomorphic with respect to NRF2 inhibition and that DPP3 over-expression in the presence of these mutants further promotes NRF2 activation. Collectively, our findings further support the competition model of NRF2 activation and suggest that ‘ETGE’-containing proteins like DPP3 contribute to NRF2 activity in cancer.

Salvinorin A (1), from the sage Salvia divinorum, is a potent and selective kappa opioid receptor (KOR) agonist. We screened other salvinorins and derivatives for binding affinity and functional activity at opioid receptors. Our results suggest that the methyl ester and furan ring are required for activity, but that the lactone and ketone functionalities are not. Other salvinorins showed negligible binding affinity at the KOR. None of the compounds bound to mu or delta opioid receptors.Salvinorin A (1) 1 was isolated from Salvia divinorum, a traditional medicine of the Mazatec Indians of Oaxaca, Mexico. Infusions of the leaves induce visions, and 1 is a potent hallucinogen in humans. 2 1 is a selective agonist at the κ opioid receptor (KOR), 3 with comparable potency and efficacy to the synthetic KOR agonists U50488 and U69593. 4 KOR ligands appear to have therapeutic potential against a range of conditions, including pain, 5 nausea, 6 depression, 7 and HIV infection. 8 1 is the only non-nitrogenous KOR agonist known, with no structural similarity to other such compounds. 3 Given this, 1 represents a valuable lead for the development of more potent and selective KOR ligands. At present, little is known of the compound's structure-activity relationships. The deacetyl compound salvinorin B (2), also isolated from S. divinorum ,9 is inactive at the KOR, while substitution of more hindered esters at the 2-position reduces or abolishes activity. 4 To probe the importance of other functional groups in 1, we set out to test related compounds from this plant, as well as semisynthetic derivatives of 1, for binding affinity and functional activity at cloned opioid receptors.Salvinorins A (1), D (4), and E (5) were isolated as described previously. 10 Acetylation of 4 gave salvinorin C (6). 11 The known 1α-hydroxy derivative 8 9,12a and diacetate 9 11 were prepared by published procedures (Figure 1).A number of new derivatives of 1 were also synthesized, and the structures verified by NMR analysis, including DEPT, COSY, HMQC, HMBC and nOe experiments. As a preliminary step, 1 itself was reexamined using these techniques. This confirmed the original 1 H and 13 C assignments (based on decoupling experiments) 9 in all cases except the overlapping multiplets of H-6 and H-7. The HMQC spectrum showed crosspeaks from C-6 (δ 38.1 ppm) to multiplets *Corresponding author. Tel: +61-3-8344-6488. Fax: +61-3-9347-5180. E-mail: masr@unimelb.edu.au.. Supporting Information Available: Experimental procedures, characterization data, 1 H and 13 C NMR spectra, and IUPAC/NIST Chemical Identifiers (INChIs) of the target compounds (the spectra of 4 and 5 were published previously). 10 This material is available free of charge via the Internet at http://pubs.acs.org. Until recently, no satisfactory procedure for deacetylation of 1 had been published. Brown reported that KCN in refluxing MeOH/THF gave 2 in quantitative yield, 13a but in our hands 8-epi-2 was the major product. Additionally, the epimers were not resolved on silica using Br...