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A novel series of histone deacetylase (HDAC) inhibitors lacking a zinc-binding moiety has been developed and described herein. HDAC isozyme profiling and kinetic studies indicate that these inhibitors display a selectivity preference for HDACs 1, 2, 3, 10, and 11 via a rapid equilibrium mechanism, and crystal structures with HDAC2 confirm that these inhibitors do not interact with the catalytic zinc. The compounds are nonmutagenic and devoid of electrophilic and mutagenic structural elements and exhibit off-target profiles that are promising for further optimization. The efficacy of this new class in biochemical and cell-based assays is comparable to the marketed HDAC inhibitors belinostat and vorinostat. These results demonstrate that the long-standing pharmacophore model of HDAC inhibitors requiring a metal binding motif should be revised and offers a distinct class of HDAC inhibitors.

[Image: see text] PKG1α is a central node in cGMP signaling. Current therapeutics that look to activate this pathway rely on elevation of cGMP levels and subsequent activation of PKG1α. Direct activation of PKG1α could potentially drive additional efficacy without associated side effects of blanket cGMP elevation. We undertook a high-throughput screen to identify novel activators. After triaging through numerous false positive hits, attributed to compound mediated oxidation and activation of PKG1α, a piperidine series of compounds was validated. The hit 1 was a weak activator with EC(50) = 47 μM. The activity could be improved to single digit micromolar, as seen in compounds 21 and 25 (7.0 and 3.7 μM, respectively). Several compounds were tested in a pVASP cell-based assay, and for compounds with moderate permeability, good agreement was observed between the biochemical and functional assays. These compounds will function as efficient tools to further interrogate PKG1α biology.

[Image: see text] By employing a phenotypic screen, a set of compounds, exemplified by 1, were identified which potentiate the ability of histone deacetylase inhibitor vorinostat to reverse HIV latency. Proteome enrichment followed by quantitative mass spectrometric analysis employing a modified analogue of 1 as affinity bait identified farnesyl transferase (FTase) as the primary interacting protein in cell lysates. This ligand-FTase binding interaction was confirmed via X-ray crystallography and temperature dependent fluorescence studies, despite 1 lacking structural and binding similarity to known FTase inhibitors. Although multiple lines of evidence established the binding interaction, these ligands exhibited minimal inhibitory activity in a cell-free biochemical FTase inhibition assay. Subsequent modification of the biochemical assay by increasing anion concentration demonstrated FTase inhibitory activity in this novel class. We propose 1 binds together with the anion in the active site to inhibit farnesyl transferase. Implications for phenotypic screening deconvolution and HIV reactivation are discussed.

Polysorbate is widely used to maintain stability of biotherapeutic proteins in formulation development. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual enzymes such as lipases and esterases can cause polysorbate degradation. Their high activity, even at low concentration, constitutes a major analytical challenge. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using chemical probes to enrich active serine hydrolases, more than 80 proteins were identified in harvested cell culture fluid (HCCF) from monoclonal antibodies (mAb) production. A total of 8 known lipases were identified by ABPP, while only 5 lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study, phospholipase A2 group VII (PLA2G7) and sialic acid acetylesterase (SIAE) were identified by ABPP as possible root causes of polysorbate-80 degradation. The established ABBP approach can fill the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.

Mutations in the lysosomal enzyme glucocerebrosidase (GCase, GBA1 gene) are the most common genetic risk factor for developing Parkinson's disease (PD). GCase metabolizes the glycosphingolipids glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Mutations in GBA1 reduce enzyme activity and the resulting accumulation of glycosphingolipids may contribute to the underlying pathology of PD, possibly via altering lysosomal function. While reduction of GCase activity exacerbates α-synuclein (α-syn) aggregation, it has not been determined that this effect is the result of altered glycosphingolipid levels and lysosome function or some other effect of altering GCase. The glycosphingolipid GlcCer is synthesized by a single enzyme, glucosylceramide synthase (GCS), and small molecule inhibitors (GCSi) reduce cellular glycosphingolipid levels. In the present studies, we utilize a preformed fibril (PFF) rodent primary neuron in vitro model of α-syn pathology to investigate the relationship between glycosphingolipid levels, α-syn pathology, and lysosomal function. In primary cultures, pharmacological inhibition of GCase and D409V GBA1 mutation enhanced accumulation of glycosphingolipids and insoluble phosphorylated α-syn. Administration of a novel small molecule GCSi, benzoxazole 1 (BZ1), significantly decreased glycosphingolipid concentrations in rodent primary neurons and reduced α-syn pathology. BZ1 rescued lysosomal deficits associated with the D409V GBA1 mutation and α-syn PFF administration, and attenuated α-syn induced neurodegeneration of dopamine neurons. In vivo studies revealed BZ1 had pharmacological activity and reduced glycosphingolipids in the mouse brain to a similar extent observed in neuronal cultures. These data support the hypothesis that reduction of glycosphingolipids through GCS inhibition may impact progression of synucleinopathy and BZ1 is useful tool to further examine this important biology.

High-throughput screening (HTS) is a widespread method in early drug discovery for identifying promising chemical matter that modulates a target or phenotype of interest. Because HTS campaigns involve screening millions of compounds, it is often desirable to initiate screening with a subset of the full collection. Subsequently, virtual screening methods prioritize likely active compounds in the remaining collection in an iterative process. With this approach, orthogonal virtual screening methods are often applied, necessitating the prioritization of hits from different approaches. Here, we introduce a novel method of fusing these prioritizations and benchmark it prospectively on 17 screening campaigns using virtual screening methods in three descriptor spaces. We found that the fusion approach retrieves 15% to 65% more active chemical series than any single machine-learning method and that appropriately weighting contributions of similarity and machine-learning scoring techniques can increase enrichment by 1% to 19%. We also use fusion scoring to evaluate the tradeoff between screening more chemical matter initially in lieu of replicate samples to prevent false-positives and find that the former option leads to the retrieval of more active chemical series. These results represent guidelines that can increase the rate of identification of promising active compounds in future iterative screens.

Though phenotypic and target-based high-throughput screening approaches have been employed to discover new antibiotics, the identification of promising therapeutic candidates remains challenging. Each approach provides different information, and understanding their results can provide hypotheses for a mechanism of action (MoA) and reveal actionable chemical matter. Here, we describe a framework for identifying efficacy targets of bioactive compounds. High throughput biophysical profiling against a broad range of targets coupled with machine learning was employed to identify chemical features with predicted efficacy targets for a given phenotypic screen. We validate the approach on data from a set of 55 000 compounds in 24 historical internal antibacterial phenotypic screens and 636 bacterial targets screened in high-throughput biophysical binding assays. Models were built to reveal the relationships between phenotype, target, and chemotype, which recapitulated mechanisms for known antibacterials. We also prospectively identified novel inhibitors of dihydrofolate reductase with nanomolar antibacterial efficacy against Mycobacterium tuberculosis. Molecular modeling provided structural insight into target-ligand interactions underlying selective killing activity toward mycobacteria over human cells.

Summary Proprotein convertase substilisin-like/kexin type 9 (PCSK9) is a serine protease involved in a protein-protein interaction with the low-density lipoprotein (LDL) receptor that has both human genetic and clinical validation. Blocking this protein-protein interaction prevents LDL receptor degradation and thereby decreases LDL cholesterol levels. Our pursuit of small-molecule direct binders for this difficult to drug PPI target utilized affinity selection/mass spectrometry, which identified one confirmed hit compound. An X-ray crystal structure revealed that this compound was binding in an unprecedented allosteric pocket located between the catalytic and C-terminal domain. Optimization of this initial hit, using two distinct strategies, led to compounds with high binding affinity to PCSK9. Direct target engagement was demonstrated in the cell lysate with a cellular thermal shift assay. Finally, ligand-induced protein degradation was shown with a proteasome recruiting tag attached to the high-affinity allosteric ligand for PCSK9.

Since the initial report of the novel Coronavirus Disease 2019 (COVID-19) emanating from Wuhan, China, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally. While the effects of SARS-CoV-2 infection are not completely understood, there appears to be a wide spectrum of disease ranging from mild symptoms to severe respiratory distress, hospitalization, and mortality. There are no Food and Drug Administration (FDA)-approved treatments for COVID-19 aside from remdesivir; early efforts to identify efficacious therapeutics for COVID-19 have mainly focused on drug repurposing screens to identify compounds with antiviral activity against SARS-CoV-2 in cellular infection systems. These screens have yielded intriguing hits, but the use of nonhuman immortalized cell lines derived from non-pulmonary or gastrointestinal origins poses any number of questions in predicting the physiological and pathological relevance of these potential interventions. While our knowledge of this novel virus continues to evolve, our current understanding of the key molecular and cellular interactions involved in SARS-CoV-2 infection is discussed in order to provide a framework for developing the most appropriate in vitro toolbox to support current and future drug discovery efforts.

HIV-1 protease (PR) represents one of the primary targets for developing antiviral agents for the treatment of HIV-infected patients. To identify novel PR inhibitors, a label-free, high-throughput mass spectrometry (HTMS) assay was developed using the RapidFire platform and applied as an orthogonal assay to confirm hits identified in a fluorescence resonance energy transfer (FRET)-based primary screen of1 million compounds. For substrate selection, a panel of peptide substrates derived from natural processing sites for PR was evaluated on the RapidFire platform. As a result, KVSLNFPIL, a new substrate measured to have a ~ 20- and 60-fold improvement in k cat/K m over the frequently used sequences SQNYPIVQ and SQNYPIV, respectively, was identified for the HTMS screen. About 17% of hits from the FRET-based primary screen were confirmed in the HTMS confirmatory assay including all 304 known PR inhibitors in the set, demonstrating that the HTMS assay is effective at triaging false-positives while capturing true hits. Hence, with a sampling rate of ~7 s per well, the RapidFire HTMS assay enables the high-throughput evaluation of peptide substrates and functions as an efficient tool for hits triage in the discovery of novel PR inhibitors.

Allosteric integrase inhibitors (ALLINIs) bind to the lens epithelial-derived growth factor (LEDGF) pocket on HIV-1 integrase (IN) and possess potent antiviral effects. Rather than blocking proviral integration, ALLINIs trigger IN conformational changes that have catastrophic effects on viral maturation, rendering the virions assembled in the presence of ALLINIs noninfectious. A high-throughput screen for compounds that disrupt the IN·LEDGF interaction was executed, and extensive triage led to the identification of a t-butylsulfonamide series, as exemplified by 1. The chemical, biochemical, and virological characterization of this series revealed that 1 and its analogs produce an ALLINI-like phenotype through engagement of IN sites distinct from the LEDGF pocket. Key to demonstrating target engagement and differentiating this new series from the existing ALLINIs was the development of a fluorescence polarization probe of IN (FLIPPIN) based on the t-butylsulfonamide series. These findings further solidify the late antiviral mechanism of ALLINIs and point toward opportunities to develop structurally and mechanistically novel antiretroviral agents with unique resistance patterns.

Affinity-based selection strategies have recently emerged as a complement to traditional high throughput screening for the rapid discovery of lead compounds for the large number of protein targets emerging from--omics technologies. Herein, we describe a method for the ranking of mixtures of ligands by affinity selection and apply it to rank order a set of inhibitors for the enzyme dipeptidyl peptidase IV.

Genome sequencing projects have uncovered many novel enzymes and enzyme classes for which knowledge of active site structure and mechanism is limited. To facilitate mechanistic investigations of the numerous enzymes encoded by prokaryotic and eukaryotic genomes, new methods are needed to analyze enzyme function in samples of high biocomplexity. Here, we describe a general strategy for profiling enzyme active sites in whole proteomes that utilizes activity-based chemical probes coupled with a gel-free analysis platform. We apply this gel-free strategy to identify the sites of labeling on enzymes targeted by sulfonate ester probes. For each enzyme examined, probe labeling was found to occur on a conserved active site residue, including catalytic nucleophiles (e.g., C32 in glutathione S-transferase omega) and bases/acids (e.g., E269 in aldehyde dehydrogenase-1; D204 in enoyl CoA hydratase-1), as well as residues of unknown function (e.g., D127 in 3 beta-hydroxysteroid dehydrogenase/isomerase-1). These results reveal that sulfonate ester probes are remarkably versatile activity-based profiling reagents capable of labeling a diversity of catalytic residues in a range of mechanistically distinct enzymes. More generally, the gel-free strategy described herein, by consolidating into a single step the identification of both protein targets of activity-based probes and the specific residues labeled by these reagents, provides a novel platform in which the proteomic comparison of enzymes can be accomplished in unison with a mechanistic analysis of their active sites.

As a label-free technology, mass spectrometry (MS) enables assays to be generated that monitor the conversion of substrates with native sequences to products without the requirement for substrate modifications or indirect detection methods. Although traditional liquid chromatography (LC)-MS methods are relatively slow for a high-throughput screening (HTS) paradigm, with cycle times typically ≥ 60 s per sample, the Agilent RapidFire High-Throughput Mass Spectrometry (HTMS) System, with a cycle time of 5-7 s per sample, enables rapid analysis of compound numbers compatible with HTS. By monitoring changes in mass directly, HTMS assays can be used as a triaging tool by eliminating large numbers of false positives resulting from fluorescent compound interference or from compounds interacting with hydrophobic fluorescent dyes appended to substrates. Herein, HTMS assays were developed for multiple protease programs, including cysteine, serine, and aspartyl proteases, and applied as a confirmatory assay. The confirmation rate for each protease assay averaged30%, independent of the primary assay technology used (i.e., luminescent, fluorescent, and time-resolved fluorescent technologies). Importantly,99% of compounds designed to inhibit the enzymes were confirmed by the corresponding HTMS assay. Hence, HTMS is an effective tool for removing detection-based false positives from ultrahigh-throughput screening, resulting in hit lists enriched in true actives for downstream dose response titrations and hit-to-lead efforts.

Background: The field of proteomics aims to characterize dynamics in protein function on a global level. However, several classes of proteins, in particular low abundance proteins, remain difficult to characterize using standard proteomics technologies. Recently, chemical strategies have emerged that profile classes of proteins based on activity rather than quantity, thereby greatly facilitating the analysis of low abundance constituents of the proteome. Results: In order to expand the classes of proteins susceptible to analysis by activity-based methods, we have synthesized a library of biotinylated sulfonate esters and applied its members to complex proteomes under conditions that distinguish patterns of specific protein reactivity. Individual sulfonates exhibited unique profiles of proteome reactivity that in extreme cases appeared nearly orthogonal to one another. A robustly labeled protein was identified as a class I aldehyde dehydrogenase and shown to be irreversibly inhibited by members of the sulfonate library. Conclusions: Through screening the proteome with a non-directed library of chemical probes, diverse patterns of protein reactivity were uncovered. These probes labeled protein targets based on properties other than abundance, circumventing one of the major challenges facing contemporary proteomics research. Considering further that the probes were found to inhibit a target enzyme's catalytic activity, the methods described herein should facilitate the identification of compounds possessing both selective proteome reactivities and novel bioactivities.

Penicillin sulfones, which structurally incorporate both a 6-position alkylidene substituent and a 2'beta substituent, have been synthesized and evaluated as inhibitors of class C and class A serine beta-lactamases. Incorporation of the 2'beta-substituent generally improves inhibitory activity. Substituents that improve transport across the bacterial cell membrane have also been incorporated.

A series of 7-alkylidenecephalosporins and 7-vinylidenecephalosporins, as their benzhydryl esters, have been tested as inhibitors of both porcine pancreatic elastase and human leukocyte elastase. Selected 7-alkylidenecephalosporin esters are found to be potent inhibitors of HLE. One category of new inhibitors is the 7-(haloalkylidene)cephalosporins. In contrast to previously reported cephalosporin-based elastase inhibitors, these haloalkylidene cephems show optimum inhibitory activity as sulfides, rather than as sulfones. They are efficient and irreversible inhibitors. A second class of active compounds is represented by the benzhydryl ester 7-(cyanomethylidene)cephalosporin sulfone. In contrast to the activity of these new inhibitors, the benzhydryl ester of the mechanism-based beta-lactamase inhibitor, 7-[(2'-pyridyl)methylidene]-cephalosporin sulfone showed little inhibitory activity as an elastase inhibitor. 7-Vinylidenecephalosporins were also relatively poor inhibitors, although the terminally unsubstituted allene sulfide showed activity as an inhibitor of PPE. A modeling analysis suggests the 7-alkylidene substituents can be readily accommodated in the S1 pocket. A potential mechanism of inhibition is proposed.

The discovery of 1,3,8-triazaspiro[4.5]decane-2,4-diones (spirohydantoins) as a structural class of pan-inhibitors of the prolyl hydroxylase (PHD) family of enzymes for the treatment of anemia is described. The initial hit class, spirooxindoles, was identified through affinity selection mass spectrometry (AS-MS) and optimized for PHD2 inhibition and optimal PK/PD profile (short-acting PHDi inhibitors). 1,3,8-Triazaspiro[4.5]decane-2,4-diones (spirohydantoins) were optimized as an advanced lead class derived from the original spiroindole hit. A new set of general conditions for C-N coupling, developed using a high-throughput experimentation (HTE) technique, enabled a full SAR analysis of the spirohydantoins. This rapid and directed SAR exploration has resulted in the first reported examples of hydantoin derivatives with good PK in preclinical species. Potassium channel off-target activity (hERG) was successfully eliminated through the systematic introduction of acidic functionality to the molecular structure. Undesired upregulation of alanine aminotransferese (ALT) liver enzymes was mitigated and a robust on-/off-target margin was achieved. Spirohydantoins represent a class of highly efficacious, short-acting PHD1-3 inhibitors causing a robust erythropoietin (EPO) upregulation in vivo in multiple preclinical species. This profile deems spirohydantoins as attractive short-acting PHDi inhibitors with the potential for treatment of anemia.

A series of 7-alkylidenecephalosporins and 7-vinylidenecephalosporins, as their benzhydryl esters, have been tested as inhibitors of both porcine pancreatic elastase and human leukocyte elastase. Selected 7-alkylidenecephalosporin esters are found to be potent inhibitors of HLE. One category of new inhibitors is the 7-(haloalkylidene)cephalosporins. In contrast to previously reported cephalosporin-based elastase inhibitors, these haloalkylidene cephems show optimum inhibitory activity as sulfides, rather than as sulfones. They are efficient and irreversible inhibitors. A second class of active compounds is represented by the benzhydryl ester 7-(cyanomethylidene)cephalosporin sulfone. In contrast to the activity of these new inhibitors, the benzhydryl ester of the mechanism-based beta-lactamase inhibitor, 7-[(2'-pyridyl)methylidene]-cephalosporin sulfone showed little inhibitory activity as an elastase inhibitor. 7-Vinylidenecephalosporins were also relatively poor inhibitors, although the terminally unsubstituted allene sulfide showed activity as an inhibitor of PPE. A modeling analysis suggests the 7-alkylidene substituents can be readily accommodated in the S1 pocket. A potential mechanism of inhibition is proposed.

Penicillin sulfones, which structurally incorporate both a 6-position alkylidene substituent and a 2'β substituent, have been synthesized and evaluated as inhibitors of class C and class A serine β-lactamases. Incorporation of the 2'β-substituent generally improves inhibitory activity. Substituents that improve transport across the bacterial cell membrane have also been incorporated.

A series of 3-substituted-7-(alkylidene)cephaloporin sulfones were prepared and evaluated as inhibitors of representative class A and class C serine beta-lactamase. Appropriate substituents resulted in a 1000-fold improvement in the inhibition of the class A enzymes and a simultaneous 20-fold improvement in the inhibition of class C. These new compounds have achieved the goal of creating broad scale inhibitors in the cephalosporin series.

To discover antifungal treatments that possess the desired characteristics of broad spectrum activity, a strong safety profile, and oral bioavailability, new discovery strategies must be implemented to identify structural classes of molecules capable of combating these microorganisms. One such technique that has been implemented is the Candida albicans Fitness Test, a whole cell screening platform capable of delineating the mechanism of action of compounds that demonstrate activity against the clinically relevant pathogenic fungus, C. albicans. Screening crude natural product extracts with this technology has resulted in the identification of a novel family of antifungal natural products, named the parnafungins, which inhibit the enzyme polyadenosine polymerase (PAP), a key component of the mRNA cleavage and polyadenylation complex. Owing to the rapid interconversion of the structural and stereoisomers of the parnafungins at neutral pH, the determination of the structural isomer with the highest affinity for PAP with standard biochemical assays has not been possible. Herein, we present an application of affinity-selection/mass spectrometry (AS-MS) to determine that the "straight" parnafungin structural isomer (parnafungin A) binds preferentially to PAP compared to the "bent" structural isomer (parnafungin B).

Chemical probes that covalently modify the active sites of enzymes in complex proteomes are useful tools for identifying enzyme activities associated with discrete (patho) physiological states. Researchers in proteomics typically use two types of activity-based probes to fulfill complementary objectives: fluorescent probes for rapid and sensitive target detection and biotinylated probes for target purification and identification. Accordingly we hypothesized that a strategy in which the target detection and target isolation steps of activity-based proteomic experiments were merged might accelerate the characterization of differentially expressed protein activities. Here we report the synthesis and application of trifunctional chemical proteomic probes in which elements for both target detection (e.g. rhodamine) and isolation (e.g. biotin) are appended to a sulfonate ester reactive group, permitting the consolidated visualization and affinity purification of labeled proteins by a combination of in-gel fluorescence and avidin chromatography procedures. A trifunctional phenyl sulfonate probe was used to identify several technically challenging protein targets, including the integral membrane enzyme 3beta-hydroxysteroid dehydrogenase/Delta5-isomerase and the cofactor-dependent enzymes platelet-type phosphofructokinase and type II tissue transglutaminase. The latter two enzyme activities were significantly up-regulated in the invasive estrogen receptor-negative (ER(-)) human breast cancer cell line MDA-MB-231 relative to the non-invasive ER(+) breast cancer lines MCF7 and T-47D. Collectively these studies demonstrate that chemical proteomic probes incorporating elements for both target detection and target isolation fortify the important link between the visualization of differentially expressed enzyme activities and their subsequent molecular identification, thereby augmenting the information content achieved in activity-based profiling experiments.

With complete genome sequences now available for several prokaryotic and eukaryotic organisms, biological researchers are charged with the task of assigning molecular and cellular functions to thousands of predicted gene products. To address this problem, the field of proteomics seeks to develop and apply methods for the global analysis of protein expression and protein function. Here we review a promising new class of proteomic strategies that utilizes synthetic chemistry to create tools and assays for the characterization of protein samples of high complexity. These approaches include the development of chemical affinity tags to measure the relative expression level and post-translational modification state of proteins in cell and tissue proteomes. Additionally, we discuss the emerging field of activity-based protein profiling, which aims to synthesize and apply small molecule probes that monitor dynamics in protein function in complex proteomes.

Proteomics research requires methods to characterize the expression and function of proteins in complex mixtures. Toward this end, chemical probes that incorporate known affinity labeling agents have facilitated the activity-based profiling of certain enzyme families. To accelerate the discovery of proteomics probes for enzyme classes lacking cognate affinity labels, we describe here a combinatorial strategy. Members of a probe library bearing a sulfonate ester chemotype were screened against complex proteomes for activity-dependent protein reactivity, resulting in the labeling of at least six mechanistically distinct enzyme classes. Surprisingly, none of these enzymes represented targets of previously described proteomics probes. The sulfonate library was used to identify an omega-class glutathione S-transferase whose activity was upregulated in invasive human breast cancer lines. These results indicate that activity-based probes compatible with whole-proteome analysis can be developed for numerous enzyme classes and applied to identify enzymes associated with discrete pathological states.

A series of 3-substituted-7-(alkylidene)cephaloporin sulfones were prepared and evaluated as inhibitors of representative class A and class C serine beta-lactamase. Appropriate substituents resulted in a 1000-fold improvement in the inhibition of the class A enzymes and a simultaneous 20-fold improvement in the inhibition of class C. These new compounds have achieved the goal of creating broad scale inhibitors in the cephalosporin series.

Toward the goal of assigning function to the tens of thousands of protein products encoded by eukaryotic and prokaryotic genomes, the field of proteomics requires new technologies that can functionally characterize proteins within the dynamic environment of the cell, where these biomolecules are subject to myriad posttranslational modifications and the actions of endogenous activators and inhibitors. Here, we report an advanced strategy for activity-based protein profiling (ABPP) that addresses this important need. We show that several enzymes can be labeled in an activity-based manner both in vitro and in vivo by an azido-sulfonate ester probe and that these labeling events can be detected in whole proteomes by copper-catalyzed ligation with a rhodamine-alkyne reagent. This click chemistry-based strategy for ABPP represents a unique and versatile method for functional proteome analysis.

SummaryResistance to existing classes of antibiotics drives the need for discovery of novel compounds with unique mechanisms of action. Nargenicin A1, a natural product with limited antibacterial spectrum, was rediscovered in a whole-cell antisense assay. Macromolecular labeling in both Staphylococcus aureus and an Escherichia coli tolC efflux mutant revealed selective inhibition of DNA replication not due to gyrase or topoisomerase IV inhibition. S. aureus nargenicin-resistant mutants were selected at a frequency of ∼1 × 10−9, and whole-genome resequencing found a single base-pair change in the dnaE gene, a homolog of the E. coli holoenzyme α subunit. A DnaE single-enzyme assay was exquisitely sensitive to inhibition by nargenicin, and other in vitro characterization studies corroborated DnaE as the target. Medicinal chemistry efforts may expand the spectrum of this novel mechanism antibiotic.