The current predominant theapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target’s ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.

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In format provided by Birmingham et al. (JUNE 2014) NATURE REVIEWS | DRUG DISCOVERY www.nature.com/reviews/drugdisc Box S1 | Bioelectronic medicines: the detailed research roadmap Creation of a visceral nerve atlas Structural mappingOverall objective: Create organ-centric wiring diagrams in models representative of human anatomy. Research imperatives:• Generate tools for high-resolution tracing of the fibre anatomy and taxonomy to and from individual organs o Build a library of tracers to visualize the full length of pre-ganglionic axons, ganglionic cell bodies, post-ganglionic axons, and intrinsic neurons 1 , with particular focus on tracing that starts at the target organ 2-5 o Advance approaches for high-resolution labelling of peripheral neurotransmitters, their receptors and co-receptors, and for imaging of myelination, peri-and epineurium o Develop and adapt micrometer-resolution imaging and 3D reconstruction techniques for visceral organs and peripheral nerves (for example, automated tissue slicing, clearing, in situ hybridization, multi-photon imaging) 6-12 • Explore inter-and intra-species variation in neuroanatomy and establish the optimal animal models for detailed mapping of each organ o Update and extend the macro-level innervation map of the major visceral organs in key animal model species and establish the extent to which this map is conserved in humans o Characterise the variability in different parts of these maps between individuals • Build organ-centric high-resolution maps for each visceral organ in their most representative animal model o Conduct high-resolution nerve tracing, labelling, and imaging in the animal model of choice, taking the organ as the starting point o Standardise and coordinate mapping, data management and 3D visualisation across organs • Establish methods to image and find nerves in the clinical setting o Develop tracers and associated imaging techniques that can be used in human preoperative and intra-operative settings to identify and localize peripheral nerves o Identify anatomical landmarks associated with putative intervention points for surgery Functional mappingOverall objective: Map the neural signalling patterns that control individual organ functions. Research imperatives:• Generate simultaneous recordings of neural signal pattern and organ function o Record both afferent (sensory) and efferent (motor) neural signals and associated end-organ biomarkers at a range of physiological stimuli

We describe a method for selecting aggregation-resistant proteins by heat denaturation. This is illustrated with antibody heavy chain variable domains (dAbs), which are prone to aggregate. The dAbs were displayed multivalently at the infective tip of filamentous bacteriophage, and heated transiently to induce unfolding and to promote aggregation of the dAbs. After cooling, the dAbs were selected for binding to protein A (a ligand common to these folded dAbs). Phage displaying dAbs that unfold reversibly were thereby enriched with respect to those that do not. From a repertoire of phage dAbs, six dAbs were characterized after selection; they all resisted aggregation, and were soluble, well expressed in bacteria and could be purified in good yields. The method should be useful for making aggregation-resistant proteins and for helping to identify features that promote or prevent protein aggregation, including those responsible for misfolding diseases.

Aims/hypothesis A new class of treatments termed bioelectronic medicines are now emerging that aim to target individual nerve fibres or specific brain circuits in pathological conditions to repair lost function and reinstate a healthy balance. Carotid sinus nerve (CSN) denervation has been shown to improve glucose homeostasis in insulin-resistant and glucose-intolerant rats; however, these positive effects from surgery appear to diminish over time and are heavily caveated by the severe adverse effects associated with permanent loss of chemosensory function. Herein we characterise the ability of a novel bioelectronic application, classified as kilohertz frequency alternating current (KHFAC) modulation, to suppress neural signals within the CSN of rodents. Methods Rats were fed either a chow or high-fat/high-sucrose (HFHSu) diet (60% lipid-rich diet plus 35% sucrose drinking water) over 14 weeks. Neural interfaces were bilaterally implanted in the CSNs and attached to an external pulse generator. The rats were then randomised to KHFAC or sham modulation groups. KHFAC modulation variables were defined acutely by respiratory and cardiac responses to hypoxia (10% O 2 + 90% N 2 ). Insulin sensitivity was evaluated periodically through an ITT and glucose tolerance by an OGTT. Results KHFAC modulation of the CSN, applied over 9 weeks, restored insulin sensitivity (constant of the insulin tolerance test [K ITT ] HFHSu sham, 2.56 ± 0.41% glucose/min; K ITT HFHSu KHFAC, 5.01 ± 0.52% glucose/min) and glucose tolerance (AUC HFHSu sham, 1278 ± 20.36 mmol/l × min; AUC HFHSu KHFAC, 1054.15 ± 62.64 mmol/l × min) in rat models of type 2 diabetes. Upon cessation of KHFAC, insulin resistance and glucose intolerance returned to normal values within 5 weeks. Conclusions/interpretation KHFAC modulation of the CSN improves metabolic control in rat models of type 2 diabetes. These positive outcomes have significant translational potential as a novel therapeutic modality for the purpose of treating metabolic diseases in humans.