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.

Chemical strategies to using small molecules to stimulate hypoxia inducible factors (HIFs) activity and trigger a hypoxic response under normoxic conditions, such as iron chelators and inhibitors of prolyl hydroxylase domain (PHD) enzymes, have broad-spectrum activities and off-target effects. Here we disclose VH298, a potent VHL inhibitor that stabilizes HIF-α and elicits a hypoxic response via a different mechanism, that is the blockade of the VHL:HIF-α protein–protein interaction downstream of HIF-α hydroxylation by PHD enzymes. We show that VH298 engages with high affinity and specificity with VHL as its only major cellular target, leading to selective on-target accumulation of hydroxylated HIF-α in a concentration- and time-dependent fashion in different cell lines, with subsequent upregulation of HIF-target genes at both mRNA and protein levels. VH298 represents a high-quality chemical probe of the HIF signalling cascade and an attractive starting point to the development of potential new therapeutics targeting hypoxia signalling.

Following correct folding/assembly, many eukaryotic proteins undergo single or multiple endoproteolytic cleavages during transport through the secretory pathway, resulting in the release of smaller, bioactive products. The proprotein convertases (PCs) 1 are a family of calcium-dependent serine endoproteases that catalyze these cleavages at sites containing doublets or clusters of basic amino acids. The PC family is evolutionarily related to bacterial subtilisin and includes seven members expressed in secretory compartments of mammalian cells (see Refs. 1-4 for reviews). Furin, the most intensively studied member of the PC family, is a type I membrane protein localized primarily to the TGN (5-8). Furin is not statically retained in this compartment, but rather it traffics between two local cycling loops, one at the TGN and the other at the cell surface (9, 10). The dynamic trafficking of furin enables it to cleave and activate numerous cellular and pathogen proproteins in both the biosynthetic and endocytic pathways (reviewed in Refs. 1 and 4). Endoproteolysis of these substrates occurs primarily at the consensus furin cleavage site, -Arg-X-Lys/Arg-Arg2-, containing a P1 and P4 Arg. However, in some cases at acidic pH, furin cleaves substrates at the motif -Arg-X-X-X-Lys/Arg-Arg2-, in which a P6 Arg is present in place of the P4 Arg (11, 12).Before it can act on proprotein substrates, furin itself must go through a complex process of activation. Furin is translated as an inactive zymogen with an 83-amino acid N-terminal propeptide. Attempts to eliminate or substitute the native furin proregion produced inactive enzyme, suggesting that the furin propeptide may play a critical role in folding and activation (13)(14)(15). This is consistent with research that shows that the folding of many evolutionarily unrelated classes of protease (e.g. serine-, aspartyl-, cysteinyl-and metalloproteases) is mediated by (typically N-terminal) propeptides that act as "intramolecular chaperones" (IMCs). IMC-mediated folding has been most thoroughly investigated in the secreted bacterial serine endoproteases ␣-lytic protease and subtilisin. These IMCs apparently increase the folding rate of their cognate protease domains by lowering a specific kinetic barrier very late in the folding pathway (reviewed in . IMC propeptides are autoproteolytically excised when their cognate enzymes have only partially folded. In subtilisin, propeptide excision initiates significant conformational changes that result in the loss of hydrophobic surface exposure to solvent (21-23). Although no longer covalently attached, these postexcision conformational changes are mediated by the IMC (22). In the absence of the IMC propeptide, the enzyme folds into an inactive and kinetically stable "molten globule"-like intermediate (16,25). The addition of the propeptide in trans causes rapid conversion of this folding intermediate to the native state (16,25,27,28).Following excision, IMCs remain noncovalently bound to

We devised a high-throughput chemoproteomics method that enabled multiplexed screening of 16,000 compounds against native protein and lipid kinases in cell extracts. Optimization of one chemical series resulted in CZC24832, which is to our knowledge the first selective inhibitor of phosphoinositide 3-kinase γ (PI3Kγ) with efficacy in in vitro and in vivo models of inflammation. Extensive target- and cell-based profiling of CZC24832 revealed regulation of interleukin-17-producing T helper cell (T(H)17) differentiation by PI3Kγ, thus reinforcing selective inhibition of PI3Kγ as a potential treatment for inflammatory and autoimmune diseases.

ATP-sensitive K؉ channels (K ATP channels) of pancreatic ␤-cells play key roles in glucose-stimulated insulin secretion by linking metabolic signals to cell excitability. Membrane phosphoinositides, in particular phosphatidylinositol 4,5-bisphosphates (PIP 2 ), stimulate K ATP channels and decrease channel sensitivity to ATP inhibition; as such, they have been postulated as critical regulators of K ATP channels and hence of insulin secretion in ␤-cells. Here, we tested this hypothesis by manipulating the interactions between K ATP channels and membrane phospholipids in a ␤-cell line, INS-1, and assessing how the manipulations affect membrane excitability and insulin secretion. We demonstrate that disruption of channel interactions with PIP 2 by overexpressing PIP 2 -insensitive channel subunits leads to membrane depolarization and elevated basal level insulin secretion at low glucose concentrations. By contrast, facilitation of channel interactions with PIP 2 by upregulating PIP 2 levels via overexpression of a lipid kinase, phosphatidylinositol 4-phosphate 5 kinase, decreases the ATP sensitivity of endogenous K ATP channels by ϳ26-fold and renders INS-1 cells hyperpolarized, unable to secrete insulin properly in the face of high glucose. Our results establish an important role of the interaction between membrane phosphoinositides and K ATP channels in regulating insulin secretion. Diabetes 54:2852-2858, 2005 P ancreatic ␤-cells secrete insulin in response to glucose stimulus. The ATP-sensitive K ϩ (K ATP ) channel, a complex of four inwardly rectifying K ϩ channel Kir6.2 subunits and four sulfonylurea receptor 1 (SUR1) subunits, is a key component in this stimulus-secretion coupling process (1-3). The hallmark features of K ATP channels are their sensitivities to intracellular nucleotides ATP and ADP, the derivatives of glucose metabolism (1,2). ATP inhibits channel activity, whereas ADP, in complex with Mg 2ϩ , stimulates channel activity. It is now generally accepted that the physiological activity of K ATP channels is regulated primarily by the relative concentrations of ATP and ADP (1,4,5). As plasma glucose increases, ATP concentration increases and ADP concentration decreases, resulting in K ATP channel closure, membrane depolarization, Ca 2ϩ influx, and insulin release. Conversely, when glucose decreases, the concentration ratio of ATP to ADP decreases, leading to K ATP channel opening, membrane hyperpolarization, and termination of insulin secretion. The importance of ATP and ADP in regulating K ATP channels in vivo has been confirmed by the finding that mutations that reduce channel sensitivity to ATP or MgADP are causative in permanent neonatal diabetes or congenital hyperinsulinism, respectively (5-9).The discovery that membrane phosphoinositides, in particular the most abundant phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP 2 ) (10), stimulate K ATP channel activity and antagonize the inhibitory effect of ATP in isolated membrane patches (11,12) has led to the proposal that in addition ...