Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here, we describe a proteomics method to quantitatively profile the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyperreactivity was a rare feature among cysteines and found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyperreactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and involved in iron-sulfur protein biogenesis. Finally, we demonstrate that quantitative reactivity profiling can also form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs.

We have developed a general synthetic route to encapsulate small molecules in monodisperse zeolitic imid-azolate framework-8 (ZIF-8) nanospheres for drug delivery. Electron microscopy, powder X-ray diffraction, and elemental analysis show that the small-molecule-encapsulated ZIF-8 nanospheres are uniform 70 nm particles with single-crystalline structure. Several small molecules, including fluorescein and the anticancer drug camptothecin, were encapsulated inside of the ZIF-8 framework. Evaluation of fluorescein-encapsulated ZIF-8 nanospheres in the MCF-7 breast cancer cell line demonstrated cell internalization and minimal cytotoxicity. The 70 nm particle size facilitates cellular uptake, and the pH-responsive dissociation of the ZIF-8 framework likely results in endosomal release of the small-molecule cargo, thereby rendering the ZIF-8 scaffold an ideal drug delivery vehicle. To confirm this, we demonstrate that camptothecin encapsulated ZIF-8 particles show enhanced cell death, indicative of internalization and intracellular release of the drug. To demonstrate the versatility of this ZIF-8 system, iron oxide nanoparticles were also encapsulated into the ZIF-8 nanospheres, thereby endowing magnetic features to these nanospheres.

Endocannabinoids are lipid signaling molecules that regulate a wide range of mammalian behaviors, including pain, inflammation, and cognitive/emotional state. The endocannabinoid anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH), and there is currently much interest in developing FAAH inhibitors to augment endocannabinoid signaling in vivo. Here we report the discovery and detailed characterization of a highly efficacious and selective FAAH inhibitor PF-3845. Mechanistic and structural studies confirm that PF-3845 is a covalent inhibitor that carbamylates FAAH's serine nucleophile. PF-3845 selectively inhibits FAAH in vivo as determined by activity-based protein profiling and raises brain anandamide levels for up to 24 hrs, resulting in profound cannabinoid receptor-dependent reductions in inflammatory pain. These data thus designate PF-3845 as a valuable pharmacological tool for in vivo characterization of the endocannabinoid system.

This article presents several covalent inhibitors, including examples of successful drugs, as well as highly selective, irreversible inhibitors of emerging therapeutic targets, such as fatty acid amide hydolase. Covalent inhibitors have many desirable features, including increased biochemical efficiency of target disruption, less sensitivity toward pharmacokinetic parameters and increased duration of action that outlasts the pharmacokinetics of the compound. Safety concerns that must be mitigated include lack of specificity and the potential immunogenicity of protein-inhibitor adduct(s). Particular attention will be given to recent technologies, such as activity-based protein profiling, which allow one to define the proteome-wide selectivity patterns for covalent inhibitors in vitro and in vivo. For instance, any covalent inhibitor can, in principle, be modified with a 'clickable' tag to generate an activity probe that is almost indistinguishable from the original agent. These probes can be applied to any living system across a broad dose range to fully inventory their on and off targets. The substantial number of drugs on the market today that act by a covalent mechanism belies historical prejudices against the development of irreversibly acting therapeutic small molecules. Emerging proteomic technologies offer a means to systematically discriminate safe (selective) versus deleterious (nonselective) covalent inhibitors and thus should inspire their future design and development. Brief history & examples of covalent inhibitorsThe design of selective covalent inhibitors is conceptually very attractive but in practice hard to achieve. That is because it is difficult to strike the right balance between reactivity and selectivity. In many cases, a highly electrophilic species (e.g., α-halo ketone, α,β-unsaturated ketone, fluorophosphonate (FP) or cyanamide) needs to be incorporated into the inhibitor to achieve covalent modification of a protein target [1]. Alkylation of other macromolecules can take place in vivo, leading to deleterious effects, or the reactive species may be scavenged by ubiquitous low-molecular-weight nucleophiles such as glutathione. †

Cells produce electrophilic products with the potential to modify and affect the function of proteins. Chemoproteomic methods have provided a means to qualitatively inventory proteins targeted by endogenous electrophiles; however, ascertaining the potency and specificity of these reactions to identify the most sensitive sites in the proteome to electrophilic modification requires more quantitative methods. Here, we describe a competitive activity-based profiling method for quantifying the reactivity of electrophilic compounds against 1000+ cysteines in parallel in the human proteome. Using this approach, we identify a select set of proteins that constitute “hot spots” for modification by various lipid-derived electrophiles, including the oxidative stress product 4-hydroxynonenal (HNE). We show that one of these proteins, ZAK kinase, is labeled by HNE on a conserved, active site-proximal cysteine, resulting in enzyme inhibition to create a negative feedback mechanism that can suppress the activation of JNK pathways by oxidative stress.

Asparagine-linked protein glycosylation is a prevalent protein modification reaction in eukaryotic systems. This process involves the co-translational transfer of a pre-assembled tetradecasaccharide from a dolichyl-pyrophosphate donor to the asparagine side chain of nascent proteins at the endoplasmic reticulum (ER) membrane. Recently, the first such system of N-linked glycosylation was discovered in the Gram-negative bacterium, Campylobacter jejuni. Glycosylation in this organism involves the transfer of a heptasaccharide from an undecaprenyl-pyrophosphate donor to the asparagine side chain of proteins at the bacterial periplasmic membrane. Here we provide a detailed comparison of the machinery involved in the N-linked glycosylation systems of eukaryotic organisms, exemplified by the yeast Saccharomyces cerevisiae, with that of the bacterial system in C. jejuni. The two systems display significant similarities and the relative simplicity of the bacterial glycosylation process could provide a model system that can be used to decipher the complex eukaryotic glycosylation machinery.

Activity-based protein profiling (ABPP) utilizes active site-directed chemical probes to monitor the functional state of enzymes directly in native biological systems. Identification of the specific sites of probe labeling on enzymes remains a major challenge in ABPP experiments. In this protocol, we describe an advanced ABPP platform that utilizes a tandem orthogonal proteolysis (TOP) strategy coupled with mass spectrometric analysis to simultaneously identify probe-labeled proteins together with their exact sites of probe modification. Elucidation of probe modification sites reveals fundamental insights into the molecular basis of specific probe-protein interactions. The TOP-ABPP method can be applied to any type of proteomic sample, including those derived from in vitro or in vivo labeling experiments, and is compatible with a variety of chemical probe structures. Completion of the entire protocol, including chemical synthesis of key reagents, requires approximately 8-10 days.

Insights into the proteome reactivity of electrophiles are crucial for designing activity-based probes for enzymes lacking cognate affinity labels. Here, we show that different classes of carbon electrophiles exhibit markedly distinct amino acid labeling profiles in proteomes, ranging from selective reactivity with cysteine to adducts with several amino acids. These data thus specify electrophilic chemotypes with restricted and permissive reactivity profiles to guide the tailored design of next-generation functional proteomics probes.The field of activity-based protein profiling (ABPP) applies reactive chemical probes to profile the functional state of enzymes in native proteomes1. Original ABPP probes incorporated well-defined affinity labels as reactive groups to target enzyme classes such as the serine2 and cysteine3 hydrolases. Many enzymes, however, do not possess cognate affinity labels, and the design of ABPP probes for these proteins remains challenging. Structural insights into the substrate-binding pocket of enzyme classes can reveal nucleophilic residues for targeting with appropriate electrophiles. Recent work in the design of protein kinase probes positioned α-fluoromethyl ketone and acyl-phosphate electrophiles within an adenosine triphosphate (ATP) scaffold to exploit the nucleophilicity of proximal cysteine4 and lysine5 residues respectively. Differentiating among electrophilic chemotypes that show restricted and permissive amino acid reactivity profiles should streamline such endeavors to design ABPP probes for a wide range of enzyme classes.A variety of electrophiles are available for incorporation into ABPP probes. The proteome reactivity profiles of iodoacetamide and maleimide reactive groups have been extensively investigated 6 . Here, we expand on these studies by investigating the reactivity of a panel of carbon electrophiles (Fig. 1a), comprising a phenylsulfonate ester (SE, 1), linear-(EP, 2) and spiro-epoxides (SP, 5), an α-chloroacetamide (CA, 3) and an α,β-unsaturated ketone (UK, 4) in complex proteomes. An alkyne was incorporated into these electrophilic frameworks to provide a click chemistry handle for gel and mass spectrometric analysis 7 . Application of these electrophiles to a soluble mouse proteome, followed by click chemistry with a rhodamine azide (Rh-N 3 ) reporter tag and visualization of labeled proteins by SDS-PAGE and in-gel fluorescence scanning, demonstrated that the panel of electrophiles exhibit a range of protein reactivities (see Supplementary Information Fig. 1). Highest reactivity was observed for the UK probe, which demonstrated substantial protein labeling at concentrations as low as 1 μM. The CA and SE electrophiles demonstrated moderate levels of reactivity, whereas, the EP and SP probes displayed little to no protein labeling even at concentrations up to 20 μM. We then examined in greater depth the protein and amino-acid labeling profiles for the three probes that displayed the highest levels of proteome reactivity (SE, CA and UK). To address this ...