NMR approaches in structure-based lead discovery: recent developments and new frontiers for targeting multi-protein complexes.

This is the final version. It was first published by Elsevier at http://www.sciencedirect.com/science/article/pii/S007961071400087X.Nuclear magnetic resonance (NMR) spectroscopy is a pivotal method for structure-based and fragment-based lead discovery because it is one of the most robust techniques to provide information on protein structure, dynamics and interaction at an atomic level in solution. Nowadays, in most ligand screening cascades, NMR-based methods are applied to identify and structurally validate small molecule binding. These can be high-throughput and are often used synergistically with other biophysical assays. Here, we describe current state-of-the-art in the portfolio of available NMR-based experiments that are used to aid early-stage lead discovery. We then focus on multi-protein complexes as targets and how NMR spectroscopy allows studying of interactions within the high molecular weight assemblies that make up a vast fraction of the yet untargeted proteome. Finally, we give our perspective on how currently available methods could build an improved strategy for drug discovery against such challenging targets.The authors are very grateful to the organizations that funded their research: the UK Biotechnology and Biological Sciences Research Council (BBSRC, grants BB/J001201/1 and David Phillips Fellowship BB/G023123/1 to A.C.), the European Research Council (ERC-2012-StG-311460 DrugE3CRLs, Starting Grant to A.C.) the European Commission (Bio-NMR, Project 261863), and the Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/81735/2011 Studentship to D.M.D.)

Discovery of small molecule ligands for the von Hippel-Lindau (VHL) E3 ligase and their use as inhibitors and PROTAC degraders

The von Hippel-Lindau (VHL) Cullin RING E3 ligase is an essential enzyme in the ubiquitin-proteasome system that recruits substrates such as the hypoxia inducible factor for ubiquitination and subsequent proteasomal degradation. The ubiquitin-proteasome pathway can be hijacked toward non-native neo-substrate proteins using proteolysis targeting chimeras (PROTACs), bifunctional molecules designed to simultaneously bind to an E3 ligase and a target protein to induce target ubiquitination and degradation. The availability of high-quality small-molecule ligands with good binding affinity for E3 ligases is fundamental for PROTAC development. Lack of good E3 ligase ligands as starting points to develop PROTAC degraders was initially a stumbling block to the development of the field. Herein, the journey towards the design of small-molecule ligands binding to VHL is presented. We cover the structure-based design of VHL ligands, their application as inhibitors in their own right, and their implementation into rationally designed, potent PROTAC degraders of various target proteins. We highlight the key findings and learnings that have provided strong foundations for the remarkable development of targeted protein degradation, and that offer a blueprint for designing new ligands for E3 ligases beyond VHL

RNA-seq analysis of PHD and VHL inhibitors reveals differences and similarities to the hypoxia response

Background: Hypoxia-inducible factor (HIF) transcription factors are well known to control the transcriptional response to hypoxia. Given the importance of cellular response to hypoxia, a number of pharmacological agents to interfere with this pathway have been developed and entered pre-clinical or clinical trial phases. However, how similar or divergent the transcriptional response elicited by different points of interference in cells is currently unknown. Methods: We performed RNA-sequencing to analyse the similarities and differences of transcriptional response in HeLa cells treated with hypoxia or chemical agents that stabilise HIF by inhibiting components of the hypoxia signalling pathway - prolyl hydroxylase (PHD) inhibitor or von Hippel-Lindau (VHL) inhibitor. Results: This analysis revealed that hypoxia produces the highest changes in gene transcription, with activation and repression of genes being in large numbers. Treatment with the PHD inhibitor IOX2 or the VHL inhibitor VH032 led mostly to gene activation, majorly via a HIF-dependent manner. These results were also confirmed by qRT-PCR using more specific and/or efficient inhibitors, FG-4592 (PHDs) and VH298 (VHL). Conclusion: PHD inhibition and VHL inhibition mimic gene activation promoted by hypoxia via a HIF-dependent manner. However, gene repression is mostly associated with the hypoxia response and not common to the response elicited by inhibitors of the pathway