[This corrects the article DOI: 10.1371/journal.pbio.2001882.].

Dysregulation of hepatic lipid and cholesterol metabolism is a significant contributor to cardiometabolic health, resulting in excessive liver lipid accumulation and ultimately non-alcoholic steatohepatitis (NASH). Therapeutic activators of the AMP-Activated Protein Kinase (AMPK) have been proposed as a treatment for metabolic diseases; we show that the AMPK β1-biased activator PF-06409577 is capable of lowering hepatic and systemic lipid and cholesterol levels in both rodent and monkey preclinical models. PF-06409577 is able to inhibit de novo lipid and cholesterol synthesis pathways, and causes a reduction in hepatic lipids and mRNA expression of markers of hepatic fibrosis. These effects require AMPK activity in the hepatocytes. Treatment of hyperlipidemic rats or cynomolgus monkeys with PF-06409577 for 6 weeks resulted in a reduction in circulating cholesterol. Together these data suggest that activation of AMPK β1 complexes with PF-06409577 is capable of impacting multiple facets of liver disease and represents a promising strategy for the treatment of NAFLD and NASH in humans.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF06446846 reveals a previously unexpected tunability of the human ribosome that allows small molecules to specifically block translation of individual transcripts.

A powerful interplay exists between the recognition of gene families, sensitive techniques in proteomics, and the interrogation of protein function using chemical probes. The most prominent methods, such as affinity capture, activity-based protein profiling and photoaffinity labeling, are extensively reviewed in the literature. Here we briefly review additional methods developed in the past 15 years. These include "stability proteomics" methods such as proteomically analyzed cellular thermal shift assays and the use of chemical oxidation as a probe of structure, the use of multiple bead-linked kinase inhibitors to analyze inhibitor specificities, and advances in the use of proteolysis-targeting chimeras for selective protein elimination.

γ-Secretase, a four-subunit transmembrane aspartic proteinase, is a highly valued drug target in Alzheimer's disease and cancer. Despite significant progress in structural studies, the respective molecular mechanisms and binding modes of γ-secretase inhibitors (GSIs) and modulators (GSMs) remain uncertain. Here, we developed biotinylated cleavable-linker photoprobes based on the BMS-708163 GSI to study its interaction with γ-secretase. Comparison of four cleavable linkers indicated that the hydrazine-labile N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) linker was cleaved most efficiently to release photolabeled and affinity-captured presenilin-1 (PS1), the catalytic subunit of γ-secretase. Peptide mapping showed that the BMS-708163-based probe photoinserted at L282 of PS1. This insertion site was consistent with the results of molecular dynamics simulations of the γ-secretase complex with inhibitor. Taken together, this work reveals the binding site of a GSI and offers insights into the mechanism of action of this class of inhibitors.

Interleukin-17A (IL-17A) is a principal driver of multiple inflammatory and immune disorders. Antibodies that neutralize IL-17A or its receptor (IL-17RA) deliver efficacy in autoimmune diseases, but no small-molecule IL-17A antagonists have yet progressed into clinical trials. Investigation of a series of linear peptide ligands to IL-17A and characterization of their binding site has enabled the design of novel macrocyclic ligands that are themselves potent IL-17A antagonists.

Chemical modification of amino groups in proteins serves a diversity of preparative and analytical purposes. The most prominent is to attach nonpeptide groups with useful properties to proteins. Examples of these groups include biotin for affinity capture and fluorescent dyes for detectability. A widely applied chemistry, and one for which many reagents are available, is reaction of the activated ester of a carboxylic acid (often a succinimidyl ester) with amino groups at mildly basic pH. Reductive alkylation using a carbonyl compound and a hydride-donating reducing agent is another valued reaction with multiple applications. Most proteins contain more than one amino group, so the extent of reaction desired must be considered in advance and the result assessed experimentally after the fact. The distinctive environment of the α-amino group of a polypeptide sets it apart from the ɛ-amino groups of lysine side chains, and can afford useful specificity. © 2016 by John Wiley & Sons, Inc.

Inhibition of β-secretase BACE1 is considered one of the most promising approaches for treating Alzheimer's disease. Several structurally distinct BACE1 inhibitors have been withdrawn from development after inducing ocular toxicity in animal models, but the target mediating this toxicity has not been identified. Here we use a clickable photoaffinity probe to identify cathepsin D (CatD) as a principal off-target of BACE1 inhibitors in human cells. We find that several BACE1 inhibitors blocked CatD activity in cells with much greater potency than that displayed in cell-free assays with purified protein. Through a series of exploratory toxicology studies, we show that quantifying CatD target engagement in cells with the probe is predictive of ocular toxicity in vivo. Taken together, our findings designate off-target inhibition of CatD as a principal driver of ocular toxicity for BACE1 inhibitors and more generally underscore the power of chemical proteomics for discerning mechanisms of drug action.

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) plays a key role in regulating the levels of plasma low density lipoprotein cholesterol (LDL-C). Here we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome, which allows small molecules to specifically block translation of individual transcripts. One Sentence Summary A small-molecule PCSK9 inhibitor targets the human ribosome and selectively prevents PCSK9 synthesis.

PF-06651600, a newly discovered potent JAK3-selective inhibitor, is highly efficacious at inhibiting γc cytokine signaling, which is dependent on both JAK1 and JAK3. PF-06651600 allowed the comparison of JAK3-selective inhibition to pan-JAK or JAK1-selective inhibition, in relevant immune cells to a level that could not be achieved previously without such potency and selectivity. In vitro, PF-06651600 inhibits Th1 and Th17 cell differentiation and function, and in vivo it reduces disease pathology in rat adjuvant-induced arthritis as well as in mouse experimental autoimmune encephalomyelitis models. Importantly, by sparing JAK1 function, PF-06651600 selectively targets γc cytokine pathways while preserving JAK1-dependent anti-inflammatory signaling such as the IL-10 suppressive functions following LPS treatment in macrophages and the suppression of TNFα and IL-1β production in IL-27-primed macrophages. Thus, JAK3-selective inhibition differentiates from pan-JAK or JAK1 inhibition in various immune cellular responses, which could potentially translate to advantageous clinical outcomes in inflammatory and autoimmune diseases.

Lysophospholipase-like 1 (LYPLAL1) is an uncharacterized metabolic serine hydrolase. Human genome-wide association studies link variants of the gene encoding this enzyme to fat distribution, waist-to-hip ratio, and nonalcoholic fatty liver disease. We describe the discovery of potent and selective covalent small-molecule inhibitors of LYPLAL1 and their use to investigate its role in hepatic metabolism. In hepatocytes, selective inhibition of LYPLAL1 increased glucose production supporting the inference that LYPLAL1 is a significant actor in hepatic metabolism. The results provide an example of how a selective chemical tool can contribute to evaluating a hypothetical target for therapeutic intervention, even in the absence of complete biochemical characterization.

Crystals of phosphorylated JAK1 kinase domain were initially generated in complex with nucleotide (ADP) and magnesium. The tightly bound Mg2+-ADP at the ATP-binding site proved recalcitrant to ligand displacement. Addition of a molar excess of EDTA helped to dislodge the divalent metal ion, promoting the release of ADP and allowing facile exchange with ATP-competitive small-molecule ligands. Many kinases require the presence of a stabilizing ligand in the ATP site for crystallization. This procedure could be useful for developing co-crystallization systems with an exchangeable ligand to enable structure-based drug design of other protein kinases.

This unit describes group-specific modifications of amino groups. These reactions remain valid tools for early-stage evaluation of structure-function relationships, but are now valued even more for their applications in the preparation of bioconjugates, affinity columns, biosensors, and tagged macromolecules. Protocols are provided here for reaction of amino groups with succinimidyl esters and isothiocyanates. These methods are broadly useful for the stable coupling to proteins of groups with useful, non-native functional properties. These include biotin for detection or recovery, fluorescent groups for biophysics or cytochemistry, cross-linking reagents for making bioconjugates, or metal-chelators that permit proteins to be loaded with radioisotopes for medical imaging or antitumor therapy. These applications require accurate product characterization, which preferably is performed by mass spectrometry, as described in this unit as a support procedure. A protocol employing succinic or acetic anhydrides to change the charge state of protein amino groups is provided here, as is a procedure for reductive alkylation that leaves their charge unaltered but converts primary amines to secondary or tertiary amines.

Despite its diverse applications, such as identification of the protein binding partners of small molecules and investigation of intracellular drug-target engagement, photoaffinity labelling (PAL) is intrinsically challenging, primarily due to the difficulty in discovering functionally active photoaffinity probes. Here we describe the creation of a chemoproteomic library to discover a novel photoaffinity probe for DcpS, an mRNA decapping enzyme that is a putative target for Spinal Muscular Atrophy. This library approach expedites the discovery of photoaffinity probes and expands the chemical biology toolbox to include RNA cap-binding proteins.

This work describes the first rational targeting of tyrosine residues in a protein binding site by small-molecule covalent probes. Specific tyrosine residues in the active site of the mRNA-decapping scavenger enzyme DcpS were modified using reactive sulfonyl fluoride covalent inhibitors. Structure-based molecular design was used to create an alkyne-tagged probe bearing the sulfonyl fluoride warhead, thus enabling the efficient capture of the protein from a complex proteome. Use of the probe in competition experiments with a diaminoquinazoline DcpS inhibitor permitted the quantification of intracellular target occupancy. As a result, diaminoquinazoline upregulators of survival motor neuron protein that are used for the treatment of spinal muscular atrophy were confirmed as inhibitors of DcpS in human primary cells. This work illustrates the utility of sulfonyl fluoride probes designed to react with specific tyrosine residues of a protein and augments the chemical biology toolkit by these probes uses in target validation and molecular pharmacology.

RATIONALE: The covalent modification of proteins by toxicants, new chemical entities or drug molecules, either by metabolic activation or the presence of inherently reactive functional groups, is commonly implicated in organ toxicity and idiosyncratic reactions. In efforts to better prosecute protein modifications, we investigated a tag-free technique capable of detecting protein-small molecule adducts based solely on the collision-induced dissociation (CID) of the protein-small molecule complex. Detection of proteins using unique CID small molecule (SM) product ions would mitigate common issues associated with tagging technologies (e.g., altered reactivity/affinity of the protein-SM complex). METHODS: A Waters SYNAPT G2 mass spectrometer (MS) was operated in MSe mode with appropriate collision energy conditions during the MS2 acquisition for fragmentation of protein-small molecule adducts to generate characteristic small molecule product ions. RESULTS: Ibrutinib, an acrylamide-containing small molecule drug, was shown to form adducts with rat serum albumin in ex vivo experiments and these adducts were detected by relying solely on the CID product ions generated from ibrutinib. Additionally, ibrutinib produced three CID product ions, one of which was a selective protein-ibrutinib fragment ion not produced by the compound alone. CONCLUSIONS: Herein we describe a tag-free mass spectral detection technique for protein-small molecule conjugates that relies on the unique product ion fragmentation profile of the small molecule. This technique allows the detection of macromolecular ions containing the adducted small molecule from complex protein matrices through mass range selection for the unique product ions in the CID spectra. Copyright (c) 2015 John Wiley & Sons, Ltd.

Mitogen-activated :protein kinase 4 (MAP4K4) regulates the MEK kinase cascade and is implicated in cytoskeletal rearrangement and migration; however, identifying MAP4K4 substrates has remained a challenge. To ascertain MAP4K4-dependent phosphorylation events, we combined phosphoproteomic studies of MAP4K4 inhibition with in vitro assessment of its kinase specificity. We identified 235 phosphosites affected by MAP4K4 inhibition in cells and found that pTP and pSP Motifs were predominant among them. In contrast, in vitro assessment of kinase specificity showed that MAP4K4 favors a pTL motif. We showed that MAP4K4 directly phosphorylates and coimmunoprecipitates with FERM, Rho GEF, and pleckstrin domain-containing protein 1 (FARP1). MAP4K4 inhibition in SH-SY5Y cells increases neurite outgrowth, a process known to involve FARP1. As FARP1 and MAP4K4 both contribute to cytoskeletal rearrangement, the results suggest that MAP4K4 exerts some of its effects on the cytoskeleton via phosphorylation of FARP1.