No abstract

We have previously shown that redox agents including superoxide anion radical and nitrogen dioxide can react with GXXXXGK(S/T)C motif-containing GTPases (i.e., Rac1, Cdc42, and RhoA) to stimulate guanine nucleotide release. We now show that the reaction of RhoA with redox agents leads to different functional consequences from that of Rac1 and Cdc42 due to the presence of an additional cysteine (GXXXCGK(S/T)C) in the RhoA redox-active motif. While reaction of redox agents with RhoA stimulates guanine nucleotide dissociation, RhoA is subsequently inactivated through formation of an intramolecular disulfide that prevents guanine nucleotide binding thereby causing RhoA inactivation. Thus, redox agents may function to downregulate RhoA activity under conditions that stimulate Rac1 and Cdc42 activity. The opposing functions of these GTPases may be due in part to their differential redox regulation. In addition, the results presented herein suggest that the platinated-chemotherapeutic agent, cisplatin, which is known for targeting nucleic acids, reacts with RhoA to produce a RhoA thiol-cisplatin-thiol adduct, leading to inactivation of RhoA. Similarly, certain arsenic complexes (i.e., arsenate and arsenic trioxide) may inactivate RhoA by bridging the cysteine residues in the GXXXCGK(S/T)C motif. Thus, in addition to redox agents, platinated-chemotherapeutic agents and arsenic complexes may modulate the activity of GTPases containing the GXXXCGK(S/T)C motif (i.e., RhoA and RhoB).

Salvinorin A, the most potent naturally occurring hallucinogen, has gained increasing attention since the κ-opioid receptor (KOR) was identified as its principal molecular target by us (Roth et al, PNAS, 2002). Here we report the design, synthesis and biochemical characterization of novel, irreversible, salvinorin A-derived ligands suitable as active state probes of the KOR. Based on prior substituted cysteine accessibility and molecular modeling studies, C315 7.38 was chosen as a potential anchoring point for covalent labeling of salvinorin A-derived ligands. Automated docking of a series of potential covalently-bound ligands suggested that either a haloacetate moiety or other similar electrophilic groups could irreversibly bind with C315 7.38 . 22-thiocyanatosalvinorin A (RB-64) and 22-chlorosalvinorin A (RB-48) were both found to be extraordinarily potent and selective KOR agonists in vitro and in vivo. As predicted based on molecular modeling studies, RB-64 induced wash-resistant inhibition of binding with a strict requirement for a free cysteine in or near the binding pocket. Mass spectrometry (MS) studies utilizing synthetic KOR peptides and RB-64 supported the hypothesis that the anchoring residue was C315 7.38 and suggested one biochemical mechanism for covalent binding. These studies provide direct evidence for the presence of a free cysteine in the agonist-bound state of KOR and provide novel insights into the mechanism by which salvinorin A binds to and activates KOR.Salvinorin A, the active ingredient of the hallucinogenic plant Salvia divinorum, is the most potent known naturally-occurring hallucinogen (1,2). In 2002, we discovered that the κ-opioid receptor (KOR) was the molecular target for the actions of salvinorin A in vitro (3). Studies with KOR knock-out mice (4) unequivocally demonstrated that the KOR was also the site of † This research was supported in part by NIH R01DA017204 (to B . NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 July 28. Published in final edited form as:Biochemistry. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript action of salvinorin A in vivo-a finding which has been widely replicated (see (5) and (6) for reviews). Subsequently, salvinorin A emerged as an attractive lead compound for drug discovery and during the past few years, hundreds of salvinorin A derivates have been synthesized (6). Some of these analogues present interesting pharmacological profiles, from full KOR agonist to partial δ-opioid receptor (DOR) or µ-opioid receptor (MOR) agonists and antagonists (7) (8) (9) (10) (11). However, most of the hundreds of analogues displayed decreased affinity (or even no affinity) to KOR. The present challenge now is to use the knowledge about salvinorin A-KOR interactions (12) (13) to design unique salvinorin A derivatives with novel pharmacological profiles and therapeutic potential. In recent years, covalently-bound ligands emerged as a new class of receptor ligands with unique pharmacologi...

SummaryRegulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits and are thus crucial to the timing of G protein-coupled receptor (GPCR) signaling. Small molecule inhibition of RGS proteins is an attractive therapeutic approach to diseases involving dysregulated GPCR signaling. Methyl-N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986) was reported as a selective RGS4 inhibitor, but with an unknown mechanism of action (Roman et al., 2007 Mol Pharmacol. 71:169−75). Here, we describe its mechanism of action as covalent modification of RGS4. Mutant RGS4 proteins devoid of surface-exposed cysteine residues were characterized using surface plasmon resonance and FRET assays of Gα binding, as well as singleturnover GTP hydrolysis assays of RGS4 GAP activity, demonstrating that cysteine-132 within RGS4 is required for sensitivity to CCG-4986 inhibition. Sensitivity to CCG-4986 can be engendered within RGS8 by replacing the wildtype residue found in this position to cysteine. Mass spectrometry analysis identified a 153 dalton fragment of CCG-4986 as being covalently attached to the surfaceexposed cysteines of the RGS4 RGS domain. We conclude that the mechanism of action of the RGS protein inhibitor CCG-4986 is via covalent modification of Cys-132 of RGS4, likely causing steric hinderance with the all-helical domain of the Gα substrate.

The oxidation of guanine to 5-carboxamido-5-formamido-2-iminohydantoin (2-Ih) is shown to be a major transformation in the oxidation of the single-stranded DNA 5-mer d(TTGTT) by m-CPBA and DMDO as a model for peracid oxidants and in the oxidation of the 5-base pair duplex d[(TTGTT)·(AACAA)] with DMDO. 2-Ih has not been reported as an oxidative lesion at the level of single/double-stranded DNA or at the nucleoside/nucleotide level. The lesion is stable to DNA digestion and chromatographic purification suggesting that 2-Ih may be a stable biomarker in vivo. The oxidation products have been structurally characterized and the reaction mechanism probed by oxidation of the monomeric species dGuo, dGMP and dGTP. DMDO selectively oxidizes the guanine moiety of dGuo, dGMP and dGTP to 2-Ih, and both peracetic and m-chloroperbenzoic acids exhibit the same selectivity. The presence of the glycosidic bond results in the stereoselective induction of an asymmetric center at the spiro carbon to give a mixture of diastereomers, with each diastereomer in equilibrium with a minor conformer through rotation about the formamido C-N bond. Labeling studies with 18O2-m-CPBA and H218O to determine the source of the added oxygen atoms have established initial epoxidation of the guanine 4-5 bond with pyrimidine ring contraction by an acyl 1,2-migration of guanine carbonyl C6 to form a transient dehydrodeoxyspiroiminodihydantoin followed by hydrolytic ring opening of the imidazolone ring. Consistent with the proposed mechanism, no 8-oxoguanine was detected as a product of the oxidations of the oligonucleotides or monomeric species mediated by DMDO or the peracids. The 2-Ih base thus appears to be a pathway-specific lesion generated by peracids and possibly other epoxidizing agents and holds promise as a potential biomarker.