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Covalent inhibitors are viable therapeutics. However, off-target reactivity challenges the field. Chemists have attempted to solve this issue by varying the reactivity attributes of electrophilic warheads. Here, we report the development of an approach to increase the selectivity of covalent molecules that is independent of warhead reactivity features and can be used in concert with existing methods. Using the scaffold of the Bruton's tyrosine kinase (BTK) inhibitor Ibrutinib for our proof-of-concept, we reasoned that increasing the steric bulk of fumarate-based electrophiles on Ibrutinib should improve selectivity via the steric exclusion of off-targets but retain rates of cysteine reactivity comparable to that of an acrylamide. Using chemical proteomic techniques, we demonstrate that elaboration of the electrophile to a

Borrelia burgdorferi(Bb), the causative agent of Lyme disease, adapts to vastly different environments as it cycles between tick vector and vertebrate host. During a tick bloodmeal,Bbalters its gene expression to prepare for vertebrate infection; however, the full range of transcriptional changes that occur over several days inside of the tick are technically challenging to capture. We developed an experimental approach to enrichBbcells to longitudinally define their global transcriptomic landscape inside nymphalIxodes scapularisticks during a transmitting bloodmeal. We identified 192Bbgenes that substantially change expression over the course of the bloodmeal from one to four days after host attachment. The majority of upregulated genes encode proteins found at the cell envelope or proteins of unknown function, including 45 outer surface lipoproteins embedded in the unusual protein-rich coat ofBb. As these proteins may facilitateBbinteractions with the host, we utilized mass spectrometry to identify candidate tick proteins that physically associate withBb. TheBbenrichment methodology along with theex vivo Bbtranscriptomes and candidate tick interacting proteins presented here provide a resource to facilitate investigations into key determinants ofBbpriming and transmission during the tick stage of its unique transmission cycle.

The microbiome is an underappreciated contributor to intestinal drug metabolism with broad implications for drug efficacy and toxicity. While considerable progress has been made towards identifying the gut bacterial genes and enzymes involved, the role of environmental factors in shaping their activity remains poorly understood. Here, we focus on the gut bacterial reduction of azo bonds (R-N=N-R’), found in diverse chemicals in both food and drugs. Surprisingly, the canonicalazoRgene inEscherichia coliwas dispensable for azo bond reduction. Instead, azo reductase activity was controlled by the fumarate and nitrate reduction (fnr) regulator, consistent with a requirement for the anoxic conditions found within the gastrointestinal tract. Paired transcriptomic and proteomic analysis of thefnrregulon revealed that in addition to altering the expression of multiple reductases, FNR is necessary for the metabolism of L-Cysteine to hydrogen sulfide, enabling the degradation of azo bonds. Taken together, these results show how gut bacteria sense and respond to their intestinal environment to enable the metabolism of chemical motifs found in both dietary and pharmaceutical compounds.IMPORTANCEThis work has broad relevance due to the ubiquity of dyes containing azo bonds in food and drugs. We report that azo dyes can be degraded by human gut bacteria through both enzymatic and non-enzymatic mechanisms, even from a single gut bacterial species. Furthermore, we revealed that environmental factors, oxygen and cysteine, control the ability ofE. colito degrade azo dyes due to their impacts on bacterial transcription and metabolism. These results open up new opportunities to manipulate the azoreductase activity of the gut microbiome through the manipulation of host diet, suggest that azoreductase potential may be altered in patients suffering from gastrointestinal disease, and highlight the importance of studying bacterial enzymes for drug metabolism in their natural cellular and ecological context.

Macrophages play a vital role in the innate immune system, identifying and destroying unwanted cells. However, it has been difficult to attain a comprehensive understanding of macrophage protein abundance due to technical limitations. In addition, it remains unclear how changes in proteome composition are linked to phagocytic activity. In this study we developed methods to derive human macrophages and prepare them for mass spectrometry analysis in order to more-deeply understand the proteomic consequences of macrophage stimulation. Interferon gamma (IF-g), an immune stimulating cytokine, was used to induce macrophage activation, increasing phagocytosis of cancer cells by 2-fold. These conditions were used to perform comparative shotgun proteomics between resting macrophages and stimulated macrophages with increased phagocytic activity. Our analysis revealed that macrophages bias their protein production toward biological processes associated with phagocytosis and antigen processing in response to stimulation. We confirmed our findings by antibody-based western blotting experiments, validating both previously reported and novel proteins of interest. In addition to whole protein changes, we evaluated active protein synthesis by treating cells with the methionine surrogate probe homopropargylglycine (HPG). We saw increased rates of HPG incorporation during phagocytosis-inducing stimulation, suggesting protein synthesis rates are altered by stimulation. Together our findings provide the most comprehensive proteomic insight to date into primary human macrophages. We anticipate that this data can be used as a launchpoint to generate new hypotheses about innate immune function.

Invasive fungal pathogens are major causes of human mortality and morbidity

Viruses are responsible for some of the most deadly human diseases, yet available vaccines and antivirals address only a fraction of the potential viral human pathogens. Here, we provide a methodology for managing human herpesvirus (HHV) infection by covalently inactivating the HHV maturational protease via a conserved, non-catalytic cysteine (C161). Using human cytomegalovirus protease (HCMV Pr) as a model, we screened a library of disulfides to identify molecules that tether to C161 and inhibit proteolysis, then elaborated hits into irreversible HCMV Pr inhibitors that exhibit broad-spectrum inhibition of other HHV Pr homologs. We further developed an optimized tool compound targeted toward HCMV Pr and used an integrative structural biology and biochemical approach to demonstrate inhibitor stabilization of HCMV Pr homodimerization, exploiting a conformational equilibrium to block proteolysis. Irreversible HCMV Pr inhibition disrupts HCMV infectivity in cells, providing proof of principle for targeting proteolysis via a non-catalytic cysteine to manage viral infection.

Microbial bile salt hydrolases (BSHs) found in the intestine catalyze the deconjugation of taurine- and glycine-linked bile salts produced in the liver. The resulting bile salts are biological detergents and are critical in aiding lipophilic nutrient digestion. Therefore, the activity of BSHs in the gut microbiome is directly linked to human metabolism and overall health. Bile salt metabolism has also been associated with disease phenotypes such as liver and colorectal cancer. In order to reshape the gut microbiome to optimize bile salt metabolism, tools to characterize and quantify these processes must exist to enable a much-improved understanding of how metabolism goes awry in the face of disease, and how it can be improved through an altered lifestyle and environment. Furthermore, it is necessary to attribute metabolic activity to specific members and BSHs within the microbiome. To this end, we have developed activity-based probes with two different reactive groups to target bile salt hydrolases. These probes bind similarly to the authentic bile salt substrates, and we demonstrate enzyme labeling of active bile salt hydrolases by using purified protein, cell lysates, and in human stool.

Glutathione S-transferases (GSTs) comprise a diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione (GSH) to endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured, the isoform-specific contribution to the metabolism of xenobiotics in complex biological samples has not been possible. We have developed two activity-based probes (ABPs) that characterize active GSTs in mammalian tissues. The GST active site is composed of a GSH binding “G site” and a substrate binding “H site”. Therefore, we developed (1) a GSH-based photoaffinity probe (GSTABP-G) to target the “G site”, and (2) an ABP designed to mimic a substrate molecule and have “H site” activity (GSTABP-H). The GSTABP-G features a photoreactive moiety for UV-induced covalent binding to GSTs and GSH-binding enzymes. The GSTABP-H is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competition experiments in the liver. Herein, we present robust tools for the characterization of enzyme- and active site-specific GST activity in mammalian model systems.

Acute and chronic exposure to organophosphates (OPs), including agricultural pesticides, industrial chemicals, and chemical warfare agents, remain a significant worldwide health risk. The mechanisms by which OPs alter development and cognition in exposed individuals remain poorly understood, in part due to the large number of structurally diverse OPs and the wide range of affected proteins and signaling pathways. To investigate the influence of structure on OP targets in mammalian systems, we have developed a series of probes for activity-based protein profiling (ABPP) featuring two distinct reactive groups that mimic OP chemical reactivity. FOP features a fluorophosphonate moiety, and PODA and CODA utilize a dialkynyl phosphate ester; both reactive group types target serine hydrolase activity. As the oxon represents the highly reactive and toxic functional group of many OPs, the new probes described herein enhance our understanding of tissue-specific reactivity of OPs. Chemoproteomic analysis of mouse tissues treated with the probes revealed divergent protein profiles, demonstrating the influence of probe structure on protein targeting. These targets also vary in sensitivity towards different OPs. The simultaneous use of multiple probes in ABPP experiments may therefore offer more comprehensive coverage of OP targets; FOP consistently labeled more targets in both brain and liver than PODA or CODA, suggesting the dialkyne warhead is more selective for enzymes in major signaling pathways than the more reactive fluorophosphonate warhead. Additionally, the probes can be used to assess reactivation of OP-inhibited enzymes by N-oximes and may serve as diagnostic tools for screening of therapeutic candidates in a panel of protein targets. These applications will help clarify the short- and long-term effects of OP toxicity beyond acetylcholinesterase inhibition, investigate potential points of convergence for broad spectrum therapeutic development, and support future efforts to screen candidate molecules for efficacy in various model systems.

Commensal microorganisms in the mammalian gut play important roles in host health and physiology, but a central challenge remains in achieving a detailed mechanistic understanding of specific microbial contributions to host biochemistry. New function-based approaches are needed that analyze gut microbial function at the molecular level by coupling detection and measurements of in situ biochemical activity with identification of the responsible microbes and enzymes. We developed a platform employing β-glucuronidase selective activity-based probes to detect, isolate, and identify microbial subpopulations in the gut responsible for this xenobiotic metabolism. We find that metabolic activity of gut microbiota can be plastic and that between individuals and during perturbation, phylogenetically disparate populations can provide β-glucuronidase activity. Our work links biochemical activity with molecular-scale resolution without relying on genomic inference.

Lung diseases and disorders are a leading cause of death among infants. Many of these diseases and disorders are caused by premature birth and underdeveloped lungs. In addition to developmentally related disorders, the lungs are exposed to a variety of environmental contaminants and xenobiotics upon birth that can cause breathing issues and are progenitors of cancer. In order to gain a deeper understanding of the developing lung, we applied an activity-based chemoproteomics approach for the functional characterization of the xenometabolizing cytochrome P450 enzymes, active ATP and nucleotide binding enzymes, and serine hydrolases using a suite of activity-based probes (ABPs). We detected P450 activity primarily in the postnatal lung; using our ATP-ABP, we characterized a wide range of ATPases and other active nucleotide- and nucleic acid-binding enzymes involved in multiple facets of cellular metabolism throughout development. ATP-ABP targets include kinases, phosphatases, NAD- and FAD-dependent enzymes, RNA/DNA helicases, and others. The serine hydrolase-targeting probe detected changes in the activities of several proteases during the course of lung development, yielding insights into protein turnover at different stages of development. Select activity-based probe targets were then correlated with RNA in situ hybridization analyses of lung tissue sections.