Modeling the CRL4A ligase complex to predict target protein ubiquitination induced by cereblon-recruiting PROTACs.

PROteolysis TArgeting Chimeras (PROTACs) are hetero-bifunctional small molecules that can simultaneously recruit target proteins and E3 ligases to form a ternary complex, promoting target protein ubiquitination and degradation via the Ubiquitin-Proteasome System (UPS). PROTACs have gained increasing attention in recent years due to certain advantages over traditional therapeutic modalities and enabling targeting of previously "undruggable" proteins. To better understand the mechanism of PROTAC-induced Target Protein Degradation (TPD), several computational approaches have recently been developed to study and predict ternary complex formation. However, mounting evidence suggests that ubiquitination can also be a rate-limiting step in PROTAC-induced TPD. Here, we propose a structure-based computational approach to predict target protein ubiquitination induced by cereblon (CRBN)-based PROTACs by leveraging available structural information of the CRL4A ligase complex (CRBN/DDB1/CUL4A/Rbx1/NEDD8/E2/Ub). We generated ternary complex ensembles with Rosetta, modeled multiple CRL4A ligase complex conformations, and predicted ubiquitination efficiency by separating the ternary ensemble into productive and unproductive complexes based on the proximity of the ubiquitin to accessible lysines on the target protein. We validated our CRL4A ligase complex models with published ternary complex structures and additionally employed our modeling workflow to predict ubiquitination efficiencies and sites of a series of cyclin-dependent kinases (CDKs) after treatment with TL12-186, a pan-kinase PROTAC. Our predictions are consistent with CDK ubiquitination and site-directed mutagenesis of specific CDK lysine residues as measured using a NanoBRET ubiquitination assay in HEK293 cells. This work structurally links PROTAC-induced ternary formation and ubiquitination, representing an important step toward prediction of target "degradability."

Solution-State (17)O†Quadrupole Central-Transition NMR Spectroscopy in the Active Site of Tryptophan Synthase.

Oxygen is an essential participant in the acid-base chemistry that takes place within many enzyme active sites, yet has remained virtually silent as a probe in NMR spectroscopy. Here, we demonstrate the first use of solution-state (17)O quadrupole central-transition NMR spectroscopy to characterize enzymatic intermediates under conditions of active catalysis. In the 143 kDa pyridoxal-5'-phosphate-dependent enzyme tryptophan synthase, reactions of the α-aminoacrylate intermediate with the nucleophiles indoline and 2-aminophenol correlate with an upfield shift of the substrate carboxylate oxygen resonances. First principles calculations suggest that the increased shieldings for these quinonoid intermediates result from the net increase in the charge density of the substrate-cofactor π-bonding network, particularly at the adjacent α-carbon site.

Occurrence of halogenated transformation products of selected pharmaceuticals and personal care products in secondary and tertiary treated wastewaters from southern California.

Final effluent samples from 10 southern California (United States) wastewater treatment facilities, employing four distinct treatment schemes, were surveyed for selected pharmaceuticals, personal care products (PPCPs), alkylphenols, and 21 of their halogenated disinfection byproducts. Chlorinated and brominated standards and isotopically labeled internal standards were synthesized and purified to confirm and more accurately quantify selected disinfection byproducts of salicylic acid, bisphenol A, gemfibrozil, naproxen, diclofenac, technical 4-nonylphenol, and 4-tert-octylphenol using high-performance liquid chromatography and tandem mass spectrometry. Concentrations of parent compounds ranged from <10 to 3830 ng/L (gemfibrozil), and those of chloro/bromo byproducts ranged from <4 to 370 ng/L (dibromo nonylphenol). The highest concentrations of parent compounds were measured in effluent that was not subjected to tertiary treatment. The chlorinated and brominated byproduct concentration may be affected by the influent concentration of parent compounds, hydraulic retention times, and chlorine contact times. Salicylic acid was readily halogenated, which is evident from the ratio of halogenated to nonhalogenated species. There were no measured chlorinated byproducts of bisphenol A despite occasionally high concentrations of the parent compound. Not surprisingly, higher concentrations of most brominated species were measured in the treatment plant with the highest bromide concentrations. These results demonstrate the occurrence of novel halogenated byproducts of PPCPs that have limited toxicological data and significant uncertainty with regard to their risk to ecological systems.

Analytical and biological characterization of halogenated gemfibrozil produced through chlorination of wastewater.

The cholesterol-lowering pharmaceutical gemfibrozil is a relevant environmental contaminant because of its frequency of detection in U.S. wastewaters at concentrations which have been shown to disrupt endocrine function in aquatic species. The treatment of gemfibrozil solutions with sodium hypochlorite yielded a 4'-chlorinated gemfibrozil analog (chlorogemfibrozil). In the presence of bromide ion, as is often encountered in municipal wastewater, hypobromous acid generated through a halogen exchange reaction produced an additional 4'-brominated gemfibrozil product (bromogemfibrozil). Standards of chloro- and bromogemfibrozil were synthesized, isolated and characterized using mass spectrometry and NMR spectroscopy. Mass spectrometry was used to follow the in situ halogenation reaction of gemfibrozil in deionized water and wastewater matrices, and to measure levels of gemfibrozil (254 ± 20 ng/L), chlorogemfibrozil (166 ± 121 ng/L), and bromogemfibrozil (50 ± 11 ng/L) in advanced primary wastewater treatment effluent treated by chlorination. Chlorogemfibrozil demonstrated a significant (p < 0.05) reduction in the levels of 11-ketotestosterone at 55.1 µg/L and bromogemfibrozil demonstrated a significant (p < 0.05) reduction in the levels of testosterone at 58.8 µg/L in vivo in Japanese medaka in a 21 day exposure. These results indicated that aqueous exposure to halogenated degradates of gemfibrozil enhanced the antiandrogenicity of the parent compound in a model fish species, demonstrating that chlorination may increase the toxicity of pharmaceutically active compounds in surface water.