Kinases are principal components of signal transduction pathways and the focus of intense basic and drug discovery research. Irreversible inhibitors that covalently modify non-catalytic cysteines in kinase active-sites have emerged as valuable probes and approved drugs. Many protein classes, however, possess functional cysteines and therefore understanding the proteome-wide selectivity of covalent kinase inhibitors is imperative. Here, we accomplish this objective using activity-based protein profiling coupled with quantitative mass spectrometry to globally map the targets, both specific and non-specific, of covalent kinase inhibitors in human cells. Many of the specific off-targets represent non-kinase proteins that, interestingly, possess conserved, active-site cysteines. We define windows of selectivity for covalent kinase inhibitors and show that, when these windows are exceeded, rampant proteome-wide reactivity and kinase target-independent cell death conjointly occur. Our findings, taken together, provide an experimental roadmap to illuminate opportunities and surmount challenges for the development of covalent kinase inhibitors.

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Structurally disparate molecules reportedly engage and activate Toll-like receptor (TLR) 4 and other TLRs, yet the interactions that mediate binding and activation by dissimilar ligands remain unknown. We describe Neoseptins, chemically synthesized peptidomimetics that bear no structural similarity to the established TLR4 ligand, lipopolysaccharide (LPS), but productively engage the mouse TLR4 (mTLR4)/ myeloid differentiation factor 2 (MD-2) complex. Neoseptin-3 activates mTLR4/MD-2 independently of CD14 and triggers canonical myeloid differentiation primary response gene 88 (MyD88)-and Toll-interleukin 1 receptor (TIR) domain-containing adaptor inducing IFN-beta (TRIF)-dependent signaling. The crystal structure mTLR4/MD-2/Neoseptin-3 at 2.57-Å resolution reveals that Neoseptin-3 binds as an asymmetrical dimer within the hydrophobic pocket of MD-2, inducing an active receptor complex similar to that induced by lipid A. However, Neoseptin-3 and lipid A form dissimilar molecular contacts to achieve receptor activation; hence strong TLR4/MD-2 agonists need not mimic LPS.neoseptins | peptidomimetic compounds | innate immunity | proinflammatory response | crystal structure

Alpha/beta-hydrolase domain containing 6 (ABHD6) is a transmembrane serine hydrolase that hydrolyzes the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) to regulate certain forms of cannabinoid receptor-dependent signaling in the nervous system. The full spectrum of ABHD6 metabolic activities and functions is currently unknown and would benefit from selective, in vivo-active inhibitors. Here, we report the development and characterization of an advanced series of irreversible (2-substituted)-piperidyl-1,2,3-triazole urea inhibitors of ABHD6, including compounds KT182 and KT203, which show exceptional potency and selectivity in cells (< 5 nM) and, at equivalent doses in mice (1 mg kg-1), served as systemic and peripherally-restricted ABHD6 inhibitors, respectively. We also describe an orally-bioavailable ABHD6 inhibitor KT185 that displays excellent selectivity against other brain and liver serine hydrolases in vivo. We thus describe several chemical probes for biological studies of ABHD6, including brain-penetrant and peripherally-restricted inhibitors that should prove of value for interrogating ABHD6 function in animal models.

-The design and solution-phase synthesis of an α-helix mimetic library as an integral component of a small molecule library targeting protein-protein interactions are described. The iterative design, synthesis, and evaluation of the candidate α-helix mimetic was initiated from a precedented triaryl template and refined by screening the designs for inhibition of MDM2/p53 binding. Upon identifying a chemically and biologically satisfactory design and consistent with the screening capabilities of academic collaborators, the corresponding complete library was assembled as 400 mixtures of 20 compounds (20 × 20 × 20-mix), where the added subunits are designed to mimic all possible permutations of the naturally occurring i, i+4, i+7 amino acid side chains of an α-helix. The library (8000 compounds) was prepared using a solution-phase synthetic protocol enlisting acid/base liquid-liquid extractions for purification on a scale that insures its long term availability for screening campaigns. Screening of the library for inhibition of MDM2/p53 binding not only identified the lead α-helix mimetic upon which the library was based, but also suggests that a digestion of the initial screening results that accompany the use of such a comprehensive library can provide insights into the nature of the interaction (e.g., an α-helix mediated protein-protein interaction) and define the key residues and their characteristics responsible for recognition.