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PROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional 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." [ABSTRACT FROM AUTHOR]

The high variability in subcutaneous bioavailability of protein therapeutics is poorly understood, contributing to critical delays in patient access to new therapies. Preclinical animal and in vitro models fail to provide a physiologically relevant testbed to parse potential contributors to human bioavailability, therefore new strategies are necessary. Here, we present a microphysiological model of the human hypodermal vasculature at the injection site to study the interactions of administered protein therapeutics within the microenvironment that influence subcutaneous bioavailability. Our model combines human dermal endothelial cells, fibroblasts, and adipocytes, self-assembled into three-dimensional, perfusable microvessels that express relevant extracellular matrix. We demonstrate the utility of the model for measurement of biophysical parameters within the hypodermal microenvironment that putatively impact protein kinetics and distribution at the injection site. We propose that microphysiological models of the subcutaneous space have applications in preclinical development of protein therapeutics intended for subcutaneous administration with optimal bioavailability.

Therapeutic siRNA is a prodrug that requires Ago2-mediated site-specific hydrolysis of the sense strand before RNA interference can occur. Although this metabolic activation step was first described 15 years ago, the kinetics of this reaction, and its relationship to in vivo siRNA efficacy, remains unexplored in the literature. To provide a roadmap to address these gaps, we describe a liquid chromatography-mass spectrometry method to monitor formation of the cleaved sense-strand metabolites in a reconstituted system. In the absence of metabolite standards for quantitation, we apply an ionization efficiency correction across a panel of siRNA molecules and find that it improves in vitro-in vivo correlation in a transgenic mouse model. Finally, we lay out a case for why Michaelis-Menten kinetics will likely be inadequate for describing Ago2-mediated metabolic activation kinetics, and propose several alternative models that can be solved numerically and applied to quantitated kinetic data when it becomes available., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

There has been a renewed interest in therapeutic small interfering RNAs (siRNAs) over the past few years. This is particularly the result of successful and efficient delivery of N-acetylgalactosamine (GalNAc)-conjugated siRNAs to the liver. In general, the lead selection process for siRNA drugs is faster and more straightforward than traditional small molecules. Nevertheless, many siRNAs of different sequences and chemical modification patterns must still be evaluated before arriving at a final candidate. One of the major difficulties in streamlining this workflow is the well-known phenomenon that the in vitro data obtained from oligonucleotides transfected into cells are not directly predictive of their in vivo activity. Consequently, all oligonucleotides with some degree of in vitro activity are typically screened in vivo before final lead selection. Here, we demonstrate that the stability of liver-targeting GalNAc-conjugated siRNAs in a mouse liver homogenate shows an acceptable correlation to their in vivo target knockdown efficacy. Therefore, we suggest the incorporation of an in vitro liver homogenate stability assay during the lead optimization process for siRNAs. The addition of this assay to a flow scheme may decrease the need for animal studies, and it could bring cost savings and increase efficiency in siRNA drug development., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)