Drug Information Association Pharmacovigilance and Risk Management Strategies 2017: Overview of the Generic Drug Program and Surveillance.

The US Food and Drug Administration's (FDA's) generic drug program has dramatically increased the availability of affordable, high quality generic drugs. The foundation of generic drug approvals is a two-tiered regulatory framework of pharmaceutical equivalence and bioequivalence. Intrinsic to both of these is consideration of the clinical relevance of formulation and bioequivalence data to support an inference of therapeutic equivalence, based on clear evidence that there are no significant differences between the generic drug and the brand name drug. These analyses allow FDA to determine that the generic drug will perform in the patient in the same way, with the same safety and efficacy profiles, as the brand name drug. Allowable differences and the precise definition of what is meant by equivalence are critical to maintaining the quality, efficacy, and safety of generic drugs. The FDA Office of Generic Drugs' (OGD's) Clinical Safety Surveillance Staff (CSSS) has developed investigative processes that complement the broader FDA safety efforts that focus on the potential impact of allowable differences and equivalence determinations for generic drugs. Two recent examples of the CSSS's processes include a clonidine transdermal system and lansoprazole oral disintegrating tablet. Ongoing efforts of the CSSS result in improvements to the FDA's review processes and the quality of generic drugs in the US market.

Palmitoylation regulates regulator of G-protein signaling (RGS) 16 function. II. Palmitoylation of a cysteine residue in the RGS box is critical for RGS16 GTPase accelerating activity and regulation of Gi-coupled signalling.

Palmitoylation is a reversible post-translational modification used by cells to regulate protein activity. The regulator of G-protein signaling (RGS) proteins RGS4 and RGS16 share conserved cysteine (Cys) residues that undergo palmitoylation. In the accompanying article (Hiol, A., Davey, P. C., Osterhout, J. L., Waheed, A. A., Fischer, E. R., Chen, C. K., Milligan, G., Druey, K. M., and Jones, T. L. Z. (2003) J. Biol. Chem. 278, 19301-19308), we determined that mutation of NH2-terminal cysteine residues in RGS16 (Cys-2 and Cys-12) reduced GTPase accelerating (GAP) activity toward a 5-hydroxytryptamine (5-HT1A)/G alpha o1 receptor fusion protein in cell membranes. NH2-terminal acylation also permitted palmitoylation of a cysteine residue in the RGS box of RGS16 (Cys-98). Here we investigated the role of internal palmitoylation in RGS16 localization and GAP activity. Mutation of RGS16 Cys-98 or RGS4 Cys-95 to alanine reduced GAP activity on the 5-HT1A/G alpha o1 fusion protein and regulation of adenylyl cyclase inhibition. The C98A mutation had no effect on RGS16 localization or GAP activity toward purified G-protein alpha subunits. Enzymatic palmitoylation of RGS16 resulted in internal palmitoylation on residue Cys-98. Palmitoylated RGS16 or RGS4 WT but not C98A or C95A preincubated with membranes expressing 5-HT1a/G alpha o1 displayed increased GAP activity over time. These results suggest that palmitoylation of a Cys residue in the RGS box is critical for RGS16 and RGS4 GAP activity and their ability to regulate Gi-coupled signaling in mammalian cells.

Palmitoylation regulates regulators of G-protein signaling (RGS) 16 function. I. Mutation of amino-terminal cysteine residues on RGS16 prevents its targeting to lipid rafts and palmitoylation of an internal cysteine residue.

Regulators of G-protein signaling (RGS) proteins down-regulate signaling by heterotrimeric G-proteins by accelerating GTP hydrolysis on the G alpha subunits. Palmitoylation, the reversible addition of palmitate to cysteine residues, occurs on several RGS proteins and is critical for their activity. For RGS16, mutation of Cys-2 and Cys-12 blocks its incorporation of [3H]palmitate and ability to turn-off Gi and Gq signaling and significantly inhibited its GTPase activating protein activity toward aG alpha subunit fused to the 5-hydroxytryptamine receptor 1A, but did not reduce its plasma membrane localization based on cell fractionation studies and immunoelectron microscopy. Palmitoylation can target proteins, including many signaling proteins, to membrane microdomains, called lipid rafts. A subpopulation of endogenous RGS16 in rat liver membranes and overexpressed RGS16 in COS cells, but not the nonpalmitoylated cysteine mutant of RGS16, localized to lipid rafts. However, disruption of lipid rafts by treatment with methyl-beta-cyclodextrin did not decrease the GTPase activating protein activity of RGS16. The lipid raft fractions were enriched in protein acyltransferase activity, and RGS16 incorporated [3H]palmitate into a peptide fragment containing Cys-98, a highly conserved cysteine within the RGS box. These results suggest that the amino-terminal palmitoylation of an RGS protein promotes its lipid raft targeting that allows palmitoylation of a poorly accessible cysteine residue that we show in the accompanying article (Osterhout, J. L., Waheed, A. A., Hiol, A., Ward, R. J., Davey, P. C., Nini, L., Wang, J., Milligan, G., Jones, T. L. Z., and Druey, K. M. (2003) J. Biol. Chem. 278, 19309-19316) was critical for RGS16 and RGS4 GAP activity.

Src-mediated RGS16 tyrosine phosphorylation promotes RGS16 stability.

The amplitude of signaling evoked by stimulation of G protein-coupled receptors may be controlled in part by the GTPase accelerating activity of the regulator of G protein signaling (RGS) proteins. In turn, subcellular targeting, protein-protein interactions, or post-translational modifications such as phosphorylation may shape RGS activity and specificity. We found previously that RGS16 undergoes tyrosine phosphorylation on conserved tyrosine residues in the RGS box. Phosphorylation on Tyr(168) was mediated by the epidermal growth factor receptor (EGFR). We show here that endogenous RGS16 is phosphorylated after epidermal growth factor stimulation of MCF-7 cells. In addition, p60-Src or Lyn kinase phosphorylated recombinant RGS16 in vitro, and RGS16 underwent phosphorylation in the presence of constitutively active Src (Y529F) in EGFR(-) CHO-K1 cells. Blockade of endogenous Src activity by selective inhibitors attenuated RGS16 phosphorylation induced by pervanadate or receptor stimulation. Furthermore, the rate of RGS16 degradation was reduced in cells expressing active Src or treated with pervanadate or a G protein-coupled receptor ligand (CXCL12). Induction of RGS16 tyrosine phosphorylation was associated with increased RGS16 protein levels and enhanced GAP activity in cell membranes. These results suggest that Src mediates RGS16 tyrosine phosphorylation, which may promote RGS16 stability.