Bogyo M

References (25)

Title : Mixed Alkyl\/Aryl Phosphonates Identify Metabolic Serine Hydrolases as Antimalarial Targets - Bennett_2024_bioRxiv__
Author(s) : Bennett JM , Narwal SK , Kabeche S , Abegg D , Hackett F , Yeo T , Li VL , Muir RK , Faucher FF , Lovell S , Blackman MJ , Adibekian A , Yeh E , Fidock DA , Bogyo M
Ref : Biorxiv , : , 2024
Abstract : Malaria, caused by Plasmodium falciparum, remains a significant health burden. A barrier for developing anti-malarial drugs is the ability of the parasite to rapidly generate resistance. We demonstrated that Salinipostin A (SalA), a natural product, kills parasites by inhibiting multiple lipid metabolizing serine hydrolases, a mechanism with a low propensity for resistance. Given the difficulty of employing natural products as therapeutic agents, we synthesized a library of lipidic mixed alkyl/aryl phosphonates as bioisosteres of SalA. Two constitutional isomers exhibited divergent anti-parasitic potencies which enabled identification of therapeutically relevant targets. We also confirm that this compound kills parasites through a mechanism that is distinct from both SalA and the pan-lipase inhibitor, Orlistat. Like SalA, our compound induces only weak resistance, attributable to mutations in a single protein involved in multidrug resistance. These data suggest that mixed alkyl/aryl phosphonates are a promising, synthetically tractable anti-malarials with a low-propensity to induce resistance.
ESTHER : Bennett_2024_bioRxiv__
PubMedSearch : Bennett_2024_bioRxiv__
PubMedID: 38260474

Title : Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of Staphylococcus aureus Infections - Jo_2024_J.Am.Chem.Soc__
Author(s) : Jo J , Upadhyay T , Woods EC , Park KW , Pedowitz NJ , Jaworek-Korjakowska J , Wang S , Valdez TA , Fellner M , Bogyo M
Ref : Journal of the American Chemical Society , : , 2024
Abstract : Staphylococcus aureus (S. aureus) is a major human pathogen that is responsible for a wide range of systemic infections. Since its propensity to form biofilms in vivo poses formidable challenges for both detection and treatment, tools that can be used to specifically image S. aureus biofilms are highly valuable for clinical management. Here, we describe the development of oxadiazolone-based activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologues in other bacteria, it is an ideal target for selective imaging of S. aureus infections. Using X-ray crystallography, direct cell labeling, and mouse models of infection, we demonstrate that oxadiazolone-based probes enable specific labeling of S. aureus bacteria through the direct covalent modification of the FphE active site serine. These results demonstrate the utility of the oxadizolone electrophile for activity-based probes and validate FphE as a target for the development of imaging contrast agents for the rapid detection of S. aureus infections.
ESTHER : Jo_2024_J.Am.Chem.Soc__
PubMedSearch : Jo_2024_J.Am.Chem.Soc__
PubMedID: 38411555
Gene_locus related to this paper: staau-SA2367

Title : Chemoproteomic identification of a DPP4 homolog in Bacteroides thetaiotaomicron - Keller_2023_Nat.Chem.Biol__
Author(s) : Keller LJ , Nguyen TH , Liu LJ , Hurysz BM , Lakemeyer M , Guerra M , Gelsinger DJ , Chanin R , Ngo N , Lum KM , Faucher F , Ipock P , Niphakis MJ , Bhatt AS , O'Donoghue AJ , Huang KC , Bogyo M
Ref : Nat Chemical Biology , : , 2023
Abstract : Serine hydrolases have important roles in signaling and human metabolism, yet little is known about their functions in gut commensal bacteria. Using bioinformatics and chemoproteomics, we identify serine hydrolases in the gut commensal Bacteroides thetaiotaomicron that are specific to the Bacteroidetes phylum. Two are predicted homologs of the human dipeptidyl peptidase 4 (hDPP4), a key enzyme that regulates insulin signaling. Our functional studies reveal that BT4193 is a true homolog of hDPP4 that can be inhibited by FDA-approved type 2 diabetes medications targeting hDPP4, while the other is a misannotated proline-specific triaminopeptidase. We demonstrate that BT4193 is important for envelope integrity and that loss of BT4193 reduces B. thetaiotaomicron fitness during in vitro growth within a diverse community. However, neither function is dependent on BT4193 proteolytic activity, suggesting a scaffolding or signaling function for this bacterial protease.
ESTHER : Keller_2023_Nat.Chem.Biol__
PubMedSearch : Keller_2023_Nat.Chem.Biol__
PubMedID: 37349583

Title : Trypanosoma brucei Acyl-Protein Thioesterase-like (TbAPT-L) Is a Lipase with Esterase Activity for Short and Medium-Chain Fatty Acids but Has No Depalmitoylation Activity - Brown_2022_Pathogens_11_1245
Author(s) : Brown RWB , Sharma AI , Villanueva MR , Li X , Onguka O , Zilbermintz L , Nguyen H , Falk BA , Olson CL , Taylor JM , Epting CL , Kathayat RS , Amara N , Dickinson BC , Bogyo M , Engman DM
Ref : Pathogens , 11 : , 2022
Abstract : Dynamic post-translational modifications allow the rapid, specific, and tunable regulation of protein functions in eukaryotic cells. S-acylation is the only reversible lipid modification of proteins, in which a fatty acid, usually palmitate, is covalently attached to a cysteine residue of a protein by a zDHHC palmitoyl acyltransferase enzyme. Depalmitoylation is required for acylation homeostasis and is catalyzed by an enzyme from the alpha/beta hydrolase family of proteins usually acyl-protein thioesterase (APT1). The enzyme responsible for depalmitoylation in Trypanosoma brucei parasites is currently unknown. We demonstrate depalmitoylation activity in live bloodstream and procyclic form trypanosomes sensitive to dose-dependent inhibition with the depalmitoylation inhibitor, palmostatin B. We identified a homologue of human APT1 in Trypanosoma brucei which we named TbAPT-like (TbAPT-L). Epitope-tagging of TbAPT-L at N- and C- termini indicated a cytoplasmic localization. Knockdown or over-expression of TbAPT-L in bloodstream forms led to robust changes in TbAPT-L mRNA and protein expression but had no effect on parasite growth in vitro, or cellular depalmitoylation activity. Esterase activity in cell lysates was also unchanged when TbAPT-L was modulated. Unexpectedly, recombinant TbAPT-L possesses esterase activity with specificity for short- and medium-chain fatty acid substrates, leading to the conclusion, TbAPT-L is a lipase, not a depalmitoylase.
ESTHER : Brown_2022_Pathogens_11_1245
PubMedSearch : Brown_2022_Pathogens_11_1245
PubMedID: 36364996
Gene_locus related to this paper: tryb2-q7yxi0

Title : Identification of covalent inhibitors that disrupt M. tuberculosis growth by targeting multiple serine hydrolases involved in lipid metabolism - Babin_2022_Cell.Chem.Biol_29_897
Author(s) : Babin BM , Keller LJ , Pinto Y , Li VL , Eneim AS , Vance SE , Terrell SM , Bhatt AS , Long JZ , Bogyo M
Ref : Cell Chemical Biology , 29 :897 , 2022
Abstract : The increasing incidence of antibiotic-resistant Mycobacterium tuberculosis infections is a global health threat necessitating the development of new antibiotics. Serine hydrolases (SHs) are a promising class of targets because of their importance for the synthesis of the mycobacterial cell envelope. We screen a library of small molecules containing serine-reactive electrophiles and identify narrow-spectrum inhibitors of M. tuberculosis growth. Using these lead molecules, we perform competitive activity-based protein profiling and identify multiple SH targets, including enzymes with uncharacterized functions. Lipidomic analyses of compound-treated cultures reveal an accumulation of free lipids and a substantial decrease in lipooligosaccharides, linking SH inhibition to defects in cell envelope biogenesis. Mutant analysis reveals a path to resistance via the synthesis of mycocerates, but not through mutations to SH targets. Our results suggest that simultaneous inhibition of multiple SH enzymes is likely to be an effective therapeutic strategy for the treatment of M. tuberculosis infections.
ESTHER : Babin_2022_Cell.Chem.Biol_29_897
PubMedSearch : Babin_2022_Cell.Chem.Biol_29_897
PubMedID: 34599874

Title : Chemiluminescent Protease Probe for Rapid, Sensitive, and Inexpensive Detection of Live Mycobacterium tuberculosis - Babin_2021_ACS.Cent.Sci_7_803
Author(s) : Babin BM , Fernandez-Cuervo G , Sheng J , Green O , Ordonez AA , Turner ML , Keller LJ , Jain SK , Shabat D , Bogyo M
Ref : ACS Cent Sci , 7 :803 , 2021
Abstract : Tuberculosis (TB) is a top-ten cause of death worldwide. Successful treatment is often limited by insufficient diagnostic capabilities, especially at the point of care in low-resource settings. The ideal diagnostic must be fast, be cheap, and require minimal clinical resources while providing high sensitivity, selectivity, and the ability to differentiate live from dead bacteria. We describe here the development of a fast, luminescent, and affordable sensor of Hip1 (FLASH) for detecting and monitoring drug susceptibility of Mycobacterium tuberculosis (Mtb). FLASH is a selective chemiluminescent substrate for the Mtb protease Hip1 that, when processed, produces visible light that can be measured with a high signal-to-noise ratio using inexpensive sensors. FLASH is sensitive to fmol of recombinant Hip1 enzyme in vitro and can detect as few as thousands of Mtb cells in culture or in human sputum samples within minutes. The probe is highly selective for Mtb compared to other nontuberculous mycobacteria and can distinguish live from dead cells. Importantly, FLASH can be used to measure antibiotic killing of Mtb in culture with greatly accelerated timelines compared to traditional protocols. Overall, FLASH has the potential to enhance both TB diagnostics and drug resistance monitoring in resource-limited settings.
ESTHER : Babin_2021_ACS.Cent.Sci_7_803
PubMedSearch : Babin_2021_ACS.Cent.Sci_7_803
PubMedID: 34079897
Gene_locus related to this paper: myctu-ym24

Title : Blocking Palmitoylation of Toxoplasma gondii Myosin Light Chain 1 Disrupts Glideosome Composition but Has Little Impact on Parasite Motility - Rompikuntal_2021_mSphere_6_
Author(s) : Rompikuntal PK , Kent RS , Foe IT , Deng B , Bogyo M , Ward GE
Ref : mSphere , 6 : , 2021
Abstract : Toxoplasma gondii is a widespread apicomplexan parasite that causes severe disease in immunocompromised individuals and the developing fetus. Like other apicomplexans, T. gondii uses an unusual form of substrate-dependent gliding motility to invade cells of its hosts and to disseminate throughout the body during infection. It is well established that a myosin motor consisting of a class XIVa heavy chain (TgMyoA) and two light chains (TgMLC1 and TgELC1/2) plays an important role in parasite motility. The ability of the motor to generate force at the parasite periphery is thought to be reliant upon its anchoring and immobilization within a peripheral membrane-bound compartment, the inner membrane complex (IMC). The motor does not insert into the IMC directly; rather, this interaction is believed to be mediated by the binding of TgMLC1 to the IMC-anchored protein, TgGAP45. Therefore, the binding of TgMLC1 to TgGAP45 is considered a key element in the force transduction machinery of the parasite. TgMLC1 is palmitoylated, and we show here that palmitoylation occurs on two N-terminal cysteine residues, C8 and C11. Mutations that block TgMLC1 palmitoylation completely abrogate the binding of TgMLC1 to TgGAP45. Surprisingly, the loss of TgMLC1 binding to TgGAP45 in these mutant parasites has little effect on their ability to initiate or sustain movement. These results question a key tenet of the current model of apicomplexan motility and suggest that our understanding of gliding motility in this important group of human and animal pathogens is not yet complete.IMPORTANCE Gliding motility plays a central role in the life cycle of T. gondii and other apicomplexan parasites. The myosin motor thought to power motility is essential for virulence but distinctly different from the myosins found in humans. Consequently, an understanding of the mechanism(s) underlying parasite motility and the role played by this unusual myosin may reveal points of vulnerability that can be targeted for disease prevention or treatment. We show here that mutations that uncouple the motor from what is thought to be a key structural component of the motility machinery have little impact on parasite motility. This finding runs counter to predictions of the current, widely held "linear motor" model of motility, highlighting the need for further studies to fully understand how apicomplexan parasites generate the forces necessary to move into, out of, and between cells of the hosts they infect.
ESTHER : Rompikuntal_2021_mSphere_6_
PubMedSearch : Rompikuntal_2021_mSphere_6_
PubMedID: 34011689

Title : Toxoplasma gondii serine hydrolases regulate parasite lipid mobilization during growth and replication within the host - Onguka_2021_Cell.Chem.Biol__
Author(s) : Onguka O , Babin BM , Lakemeyer M , Foe IT , Amara N , Terrell SM , Lum KM , Cieplak P , Niphakis MJ , Long JZ , Bogyo M
Ref : Cell Chemical Biology , : , 2021
Abstract : The intracellular protozoan parasite Toxoplasma gondii must scavenge cholesterol and other lipids from the host to facilitate intracellular growth and replication. Enzymes responsible for neutral lipid synthesis have been identified but there is no evidence for enzymes that catalyze lipolysis of cholesterol esters and esterified lipids. Here, we characterize several T. gondii serine hydrolases with esterase and thioesterase activities that were previously thought to be depalmitoylating enzymes. We find they do not cleave palmitoyl thiol esters but rather hydrolyze short-chain lipid esters. Deletion of one of the hydrolases results in alterations in levels of multiple lipids species. We also identify small-molecule inhibitors of these hydrolases and show that treatment of parasites results in phenotypic defects reminiscent of parasites exposed to excess cholesterol or oleic acid. Together, these data characterize enzymes necessary for processing lipids critical for infection and highlight the potential for targeting parasite hydrolases for therapeutic applications.
ESTHER : Onguka_2021_Cell.Chem.Biol__
PubMedSearch : Onguka_2021_Cell.Chem.Biol__
PubMedID: 34043961
Gene_locus related to this paper: toxgo-TgASH3 , toxgo-TgASH2 , toxgo-s4ug63 , toxgo-a0a125yz56

Title : Structural basis for the inhibitor and substrate specificity of the unique Fph serine hydrolases of Staphylococcus aureus - Fellner_2020_ACS.Infect.Dis_6_2771
Author(s) : Fellner M , Lentz CS , Jamieson SA , Brewster JL , Chen L , Bogyo M , Mace PD
Ref : ACS Infect Dis , 6 :2771 , 2020
Abstract : Staphylococcus aureus is a prevalent bacterial pathogen in both community and hospital settings, and its treatment is made particularly difficult by resilience within biofilms. Within this niche, serine hydrolase enzymes play a key role in generating and maintaining the biofilm matrix. Activity-based profiling has previously identified a family of serine hydrolases, designated fluorophosphonate-binding hydrolases (Fphs), some of which contribute to the virulence of S. aureus in vivo. These ten Fph proteins have limited annotation, and have few, if any, characterized bacterial or mammalian homologs. This suggests unique hydrolase functions even within bacterial species. Here we report structures of one of the most abundant Fph family members, FphF. Our structures capture FphF alone, covalently bound to a substrate analog, and bound to small molecule inhibitors that occupy the hydrophobic substrate-binding pocket. In line with these findings, we show that FphF has promiscuous esterase activity towards hydrophobic lipid substrates. We present docking studies that characterize interactions of inhibitors and substrates within the active site environment, which can be extended to other Fph family members. Comparison of FphF to other esterases and the wider Fph protein family suggest that FphF forms a new esterase subfamily. Our data suggest that other Fph enzymes, including the virulence factor FphB, are likely to have more restricted substrate profiles than FphF. This work demonstrates a clear molecular rationale for the specificity of fluorophosphonate probes that target FphF and provides a structural template for the design of enhanced probes and inhibitors of the Fph family of serine hydrolases.
ESTHER : Fellner_2020_ACS.Infect.Dis_6_2771
PubMedSearch : Fellner_2020_ACS.Infect.Dis_6_2771
PubMedID: 32865965
Gene_locus related to this paper: staau-MW0741 , staau-MW2456 , staau-SA1143 , staau-SA2240 , staau-SA2306 , staau-SA2367 , staau-SA2422 , staau-SAV0457 , staau-SAV1793 , staau-SAV2188

Title : A Protease-Activated Fluorescent Probe Allows Rapid Visualization of Keratinocyte Carcinoma during Excision - Walker_2020_Cancer.Res_80_2045
Author(s) : Walker E , Liu Y , Kim I , Biro M , Iyer SR , Ezaldein H , Scott J , Merati M , Mistur R , Zhou B , Straight B , Yim JJ , Bogyo M , Mann M , Wilson DL , Basilion JP , Popkin DL
Ref : Cancer Research , 80 :2045 , 2020
Abstract : Keratinocyte carcinomas, including basal and squamous cell carcinomas, are the most common human cancers worldwide. While 75% of all keratinocyte carcinoma (4 million annual cases in the United States) are treated with conventional excision, this surgical modality has much lower cure rates than Mohs micrographic surgery, likely due to the bread-loaf histopathologic assessment that visualizes <1% of the tissue margins. A quenched protease-activated fluorescent probe 6qcNIR, which produces a signal only in the protease-rich tumor microenvironment, was topically applied to 90 specimens ex vivo immediately following excision. "Puzzle-fit" analysis was used to correlate the fluorescent images with histology. Probe-dependent fluorescent images correlated with cancer determined by conventional histology. Point-of-care fluorescent detection of skin cancer had a clinically relevant sensitivity of 0.73 and corresponding specificity of 0.88. Importantly, clinicians were effectively trained to read fluorescent images within 15 minutes with reliability and confidence, resulting in sensitivities of 62%-78% and specificities of 92%-97%. Fluorescent imaging using 6qcNIR allows 100% tumor margin assessment by generating en face images that correlate with histology and may be used to overcome the limitations of conventional bread-loaf histology. The utility of 6qcNIR was validated in a busy real-world clinical setting, and clinicians were trained to effectively read fluorescent margins with a short guided instruction, highlighting clinical adaptability. When used in conventional excision, this approach may result in higher cure rates at a lower cost by allowing same-day reexcision when needed, reducing patient anxiety and improving compliance by expediting postsurgical specimen assessment. SIGNIFICANCE: A fluorescent-probe-tumor-visualization platform was developed and validated in human keratinocyte carcinoma excision specimens that may provide simple, rapid, and global assessment of margins during skin cancer excision, allowing same-day reexcision when needed.
ESTHER : Walker_2020_Cancer.Res_80_2045
PubMedSearch : Walker_2020_Cancer.Res_80_2045
PubMedID: 32132111

Title : Characterization of Serine Hydrolases Across Clinical Isolates of Commensal Skin Bacteria Staphylococcus epidermidis Using Activity-Based Protein Profiling - Keller_2020_ACS.Infect.Dis_6_930
Author(s) : Keller LJ , Lentz CS , Chen YE , Metivier RJ , Weerapana E , Fischbach MA , Bogyo M
Ref : ACS Infect Dis , 6 :930 , 2020
Abstract : The bacterial genus Staphylococcus comprises diverse species that colonize the skin as commensals but can also cause infection. Previous work identified a family of serine hydrolases termed fluorophoshonate-binding hydrolases (Fphs) in the pathogenic bacteria Staphylococcus aureus, one of which, FphB, functions as a virulence factor. Using a combination of bioinformatics and activity-based protein profiling (ABPP), we identify homologues of these enzymes in the related commensal bacteria Staphylococcus epidermidis. Two of the S. aureus Fph enzymes were not identified in S. epidermidis. Using ABPP, we identified several candidate hydrolases that were not previously identified in S. aureus that may be functionally related to the Fphs. Interestingly, the activity of the Fphs vary across clinical isolates of S. epidermidis. Biochemical characterization of the FphB homologue in S. epidermidis (SeFphB) suggests it is a functional homologue of FphB in S. aureus, but our preliminary studies suggest it may not have a role in colonization in vivo. This potential difference in biological function between the Fphs of closely related staphylococcal species may provide mechanisms for specific inhibition of S. aureus infection without perturbing commensal communities of related bacteria.
ESTHER : Keller_2020_ACS.Infect.Dis_6_930
PubMedSearch : Keller_2020_ACS.Infect.Dis_6_930
PubMedID: 32298574
Gene_locus related to this paper: staep-GEHD , staep-lipas , staep-SE0226 , staep-SE0245 , staep-SE0424 , staep-SE0564 , staep-SE0714 , staep-SE0980 , staep-SE1460 , staep-SE1780 , staep-SERP2035 , staep-SE2095 , staep-SE2213 , staep-SE2328 , staep-SE2403

Title : Strategies for Tuning the Selectivity of Chemical Probes that Target Serine Hydrolases - Faucher_2020_Cell.Chem.Biol_27_937
Author(s) : Faucher F , Bennett JM , Bogyo M , Lovell S
Ref : Cell Chemical Biology , 27 :937 , 2020
Abstract : Serine hydrolases comprise a large family of enzymes that have diverse roles in key cellular processes, such as lipid metabolism, cell signaling, and regulation of post-translation modifications of proteins. They are also therapeutic targets for multiple human pathologies, including viral infection, diabetes, hypertension, and Alzheimer disease; however, few have well-defined substrates and biological functions. Activity-based probes (ABPs) have been used as effective tools to both profile activity and screen for selective inhibitors of serine hydrolases. One broad-spectrum ABP containing a fluorophosphonate electrophile has been used extensively to advance our understanding of diverse serine hydrolases. Due to the success of this single reagent, several robust chemistries have been developed to further diversify and tune the selectivity of ABPs used to target serine hydrolases. In this review, we highlight approaches to identify selective serine hydrolase ABPs and suggest new synthetic methodologies that could be applied to further advance probe development.
ESTHER : Faucher_2020_Cell.Chem.Biol_27_937
PubMedSearch : Faucher_2020_Cell.Chem.Biol_27_937
PubMedID: 32726586

Title : The Antimalarial Natural Product Salinipostin A Identifies Essential alpha\/beta Serine Hydrolases Involved in Lipid Metabolism in P. falciparum Parasites - Yoo_2020_Cell.Chem.Biol_27_143
Author(s) : Yoo E , Schulze CJ , Stokes BH , Onguka O , Yeo T , Mok S , Gnadig NF , Zhou Y , Kurita K , Foe IT , Terrell SM , Boucher MJ , Cieplak P , Kumpornsin K , Lee MCS , Linington RG , Long JZ , Uhlemann AC , Weerapana E , Fidock DA , Bogyo M
Ref : Cell Chemical Biology , 27 :143 , 2020
Abstract : Salinipostin A (Sal A) is a potent antiplasmodial marine natural product with an undefined mechanism of action. Using a Sal A-derived activity-based probe, we identify its targets in the Plasmodium falciparum parasite. All of the identified proteins contain alpha/beta serine hydrolase domains and several are essential for parasite growth. One of the essential targets displays a high degree of homology to human monoacylglycerol lipase (MAGL) and is able to process lipid esters including a MAGL acylglyceride substrate. This Sal A target is inhibited by the anti-obesity drug Orlistat, which disrupts lipid metabolism. Resistance selections yielded parasites that showed only minor reductions in sensitivity and that acquired mutations in a PRELI domain-containing protein linked to drug resistance in Toxoplasma gondii. This inability to evolve efficient resistance mechanisms combined with the non-essentiality of human homologs makes the serine hydrolases identified here promising antimalarial targets.
ESTHER : Yoo_2020_Cell.Chem.Biol_27_143
PubMedSearch : Yoo_2020_Cell.Chem.Biol_27_143
PubMedID: 31978322
Gene_locus related to this paper: plaf7-q8ii19 , plafa-a0a143zya4 , plaf7-q8iik5 , plafa-MAL8P1.38 , plafa-PF07.0040 , plafa-PF10.0020 , plafa-PF10.0379 , plafa-PF13.0153

Title : Activity-based protein profiling in bacteria: Applications for identification of therapeutic targets and characterization of microbial communities - Keller_2020_Curr.Opin.Chem.Biol_54_45
Author(s) : Keller LJ , Babin BM , Lakemeyer M , Bogyo M
Ref : Curr Opin Chemical Biology , 54 :45 , 2020
Abstract : Activity-based protein profiling (ABPP) is a robust chemoproteomic technique that uses activity-based probes to globally measure endogenous enzymatic activity in complex proteomes. It has been utilized extensively to characterize human disease states and identify druggable targets in diverse disease conditions. ABPP has also recently found applications in microbiology. This includes using activity-based probes (ABPs) for functional studies of pathogenic bacteria as well as complex communities within a microbiome. This review will focus on recent advances in the use of ABPs to profile enzyme activity in disease models, screen for selective inhibitors of key enzymes, and develop imaging tools to better understand the host-bacterial interface.
ESTHER : Keller_2020_Curr.Opin.Chem.Biol_54_45
PubMedSearch : Keller_2020_Curr.Opin.Chem.Biol_54_45
PubMedID: 31835131

Title : Synthetic Fluorogenic Peptides Reveal Dynamic Substrate Specificity of Depalmitoylases - Amara_2019_Cell.Chem.Biol_26_35
Author(s) : Amara N , Foe IT , Onguka O , Garland M , Bogyo M
Ref : Cell Chemical Biology , 26 :35 , 2019
Abstract : Palmitoylation is a post-translational modification involving the thioesterification of cysteine residues with a 16-carbon-saturated fatty acid. Little is known about rates of depalmitoylation or the parameters that dictate these rates. Here we report a modular strategy to synthesize quenched fluorogenic substrates for the specific detection of depalmitoylase activity and for mapping the substrate specificity of individual depalmitoylases. We demonstrate that human depalmitoylases APT1 and APT2, and TgPPT1 from the parasite Toxoplasma gondii, have distinct specificities that depend on amino acid residues distal to the palmitoyl cysteine. This information informs the design of optimal and non-optimal substrates as well as isoform-selective substrates to detect the activity of a specific depalmitoylase in complex proteomes. In addition to providing tools for studying depalmitoylases, our findings identify a previously unrecognized mechanism for regulating steady-state levels of distinct palmitoylation sites by sequence-dependent control of depalmitoylation rates.
ESTHER : Amara_2019_Cell.Chem.Biol_26_35
PubMedSearch : Amara_2019_Cell.Chem.Biol_26_35
PubMedID: 30393067

Title : Synthetic and biological approaches to map substrate specificities of proteases - Chen_2019_Biol.Chem_401_165
Author(s) : Chen S , Yim JJ , Bogyo M
Ref : Biol Chem , 401 :165 , 2019
Abstract : Proteases are regulators of diverse biological pathways including protein catabolism, antigen processing and inflammation, as well as various disease conditions, such as malignant metastasis, viral infection and parasite invasion. The identification of substrates of a given protease is essential to understand its function and this information can also aid in the design of specific inhibitors and active site probes. However, the diversity of putative protein and peptide substrates makes connecting a protease to its downstream substrates technically difficult and time-consuming. To address this challenge in protease research, a range of methods have been developed to identify natural protein substrates as well as map the overall substrate specificity patterns of proteases. In this review, we highlight recent examples of both synthetic and biological methods that are being used to define the substrate specificity of protease so that new protease-specific tools and therapeutic agents can be developed.
ESTHER : Chen_2019_Biol.Chem_401_165
PubMedSearch : Chen_2019_Biol.Chem_401_165
PubMedID: 31639098

Title : Fluorescent Triazole Urea Activity-Based Probes for the Single-Cell Phenotypic Characterization of Staphylococcus aureus - Chen_2019_Angew.Chem.Int.Ed.Engl_58_5643
Author(s) : Chen L , Keller LJ , Cordasco E , Bogyo M , Lentz CS
Ref : Angew Chem Int Ed Engl , 58 :5643 , 2019
Abstract : Phenotypically distinct cellular (sub)populations are clinically relevant for the virulence and antibiotic resistance of a bacterial pathogen, but functionally different cells are usually indistinguishable from each other. Herein, we introduce fluorescent activity-based probes as chemical tools for the single-cell phenotypic characterization of enzyme activity levels in Staphylococcus aureus. We screened a 1,2,3-triazole urea library to identify selective inhibitors of fluorophosphonate-binding serine hydrolases and lipases in S. aureus and synthesized target-selective activity-based probes. Molecular imaging and activity-based protein profiling studies with these probes revealed a dynamic network within this enzyme family involving compensatory regulation of specific family members and exposed single-cell phenotypic heterogeneity. We propose the labeling of enzymatic activities by chemical probes as a generalizable method for the phenotyping of bacterial cells at the population and single-cell level.
ESTHER : Chen_2019_Angew.Chem.Int.Ed.Engl_58_5643
PubMedSearch : Chen_2019_Angew.Chem.Int.Ed.Engl_58_5643
PubMedID: 30768830

Title : Development of an activity-based probe for acyl-protein thioesterases - Garland_2018_PLoS.One_13_e0190255
Author(s) : Garland M , Schulze CJ , Foe IT , van der Linden WA , Child MA , Bogyo M
Ref : PLoS ONE , 13 :e0190255 , 2018
Abstract : Protein palmitoylation is a dynamic post-translational modification (PTM) important for cellular functions such as protein stability, trafficking, localization, and protein-protein interactions. S-palmitoylation occurs via the addition of palmitate to cysteine residues via a thioester linkage, catalyzed by palmitoyl acyl transferases (PATs), with removal of the palmitate catalyzed by acyl protein thioesterases (APTs) and palmitoyl-protein thioesterases (PPTs). Tools that target the regulators of palmitoylation-PATs, APTs and PPTs-will improve understanding of this essential PTM. Here, we describe the synthesis and application of a cell-permeable activity-based probe (ABP) that targets APTs in intact mammalian cells and the parasite Toxoplasma gondii. Using a focused library of substituted chloroisocoumarins, we identified a probe scaffold with nanomolar affinity for human APTs (HsAPT1 and HsAPT2) and synthesized a fluorescent ABP, JCP174-BODIPY TMR (JCP174-BT). We use JCP174-BT to profile HsAPT activity in situ in mammalian cells, to detect an APT in T. gondii (TgPPT1). We show discordance between HsAPT activity levels and total protein concentration in some cell lines, indicating that total protein levels may not be representative of APT activity in complex systems, highlighting the utility of this probe.
ESTHER : Garland_2018_PLoS.One_13_e0190255
PubMedSearch : Garland_2018_PLoS.One_13_e0190255
PubMedID: 29364904

Title : Identification of a S. aureus virulence factor by activity-based protein profiling (ABPP) - Lentz_2018_Nat.Chem.Biol_14_609
Author(s) : Lentz CS , Sheldon JR , Crawford LA , Cooper R , Garland M , Amieva MR , Weerapana E , Skaar EP , Bogyo M
Ref : Nat Chemical Biology , 14 :609 , 2018
Abstract : Serine hydrolases play diverse roles in regulating host-pathogen interactions in a number of organisms, yet few have been characterized in the human pathogen Staphylococcus aureus. Here we describe a chemical proteomic screen that identified ten previously uncharacterized S. aureus serine hydrolases that mostly lack human homologs. We termed these enzymes fluorophosphonate-binding hydrolases (FphA-J). One hydrolase, FphB, can process short fatty acid esters, exhibits increased activity in response to host cell factors, is located predominantly on the bacterial cell surface in a subset of cells, and is concentrated in the division septum. Genetic disruption of fphB confirmed that the enzyme is dispensable for bacterial growth in culture but crucial for establishing infection in distinct sites in vivo. A selective small molecule inhibitor of FphB effectively reduced infectivity in vivo, suggesting that it may be a viable therapeutic target for the treatment or management of Staphylococcus infections.
ESTHER : Lentz_2018_Nat.Chem.Biol_14_609
PubMedSearch : Lentz_2018_Nat.Chem.Biol_14_609
PubMedID: 29769740
Gene_locus related to this paper: staau-MW2456 , staau-SA2422

Title : The Toxoplasma gondii Active Serine Hydrolase 4 Regulates Parasite Division and Intravacuolar Parasite Architecture - Foe_2018_mSphere_3_
Author(s) : Foe IT , Onguka O , Amberg-Johnson K , Garner RM , Amara N , Beatty W , Yeh E , Bogyo M
Ref : mSphere , 3 : , 2018
Abstract : Hydrolase are enzymes that regulate diverse biological processes, including posttranslational protein modifications. Recent work identified four active serine hydrolases (ASHs) in Toxoplasma gondii as candidate depalmitoylases. However, only TgPPT1 (ASH1) has been confirmed to remove palmitate from proteins. ASH4 (TgME49_264290) was reported to be refractory to genetic disruption. We demonstrate that recombinant ASH4 is an esterase that processes short acyl esters but not palmitoyl thioesters. Genetic disruption of ASH4 causes defects in cell division and premature scission of parasites from residual bodies. These defects lead to the presence of vacuoles with a disordered intravacuolar architecture, with parasites arranged in pairs around multiple residual bodies. Importantly, we found that the deletion of ASH4 correlates with a defect in radial dispersion from host cells after egress. This defect in dispersion of parasites is a general phenomenon that is observed for disordered vacuoles that occur at low frequency in wild-type parasites, suggesting a possible general link between intravacuolar organization and dispersion after egress.IMPORTANCE This work defines the function of an enzyme in the obligate intracellular parasite Toxoplasma gondii We show that this previously uncharacterized enzyme is critical for aspects of cellular division by the parasite and that loss of this enzyme leads to parasites with cell division defects and which also are disorganized inside their vacuoles. This leads to defects in the ability of the parasite to disseminate from the site of an infection and may have a significant impact on the parasite's overall infectivity of a host organism.
ESTHER : Foe_2018_mSphere_3_
PubMedSearch : Foe_2018_mSphere_3_
PubMedID: 30232166
Gene_locus related to this paper: toxgo-a0a125yz56

Title : An in vivo multiplexed small-molecule screening platform - Gruner_2016_Nat.Methods_13_883
Author(s) : Gruner BM , Schulze CJ , Yang D , Ogasawara D , Dix MM , Rogers ZN , Chuang CH , McFarland CD , Chiou SH , Brown JM , Cravatt BF , Bogyo M , Winslow MM
Ref : Nat Methods , 13 :883 , 2016
Abstract : Phenotype-based small-molecule screening is a powerful method to identify molecules that regulate cellular functions. However, such screens are generally performed in vitro under conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of small-molecule libraries. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed toward hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo.
ESTHER : Gruner_2016_Nat.Methods_13_883
PubMedSearch : Gruner_2016_Nat.Methods_13_883
PubMedID: 27617390

Title : Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis - Lentz_2016_ACS.Infect.Dis_2_807
Author(s) : Lentz CS , Ordonez AA , Kasperkiewicz P , La Greca F , O'Donoghue AJ , Schulze CJ , Powers JC , Craik CS , Drag M , Jain SK , Bogyo M
Ref : ACS Infect Dis , 2 :807 , 2016
Abstract : Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb "Hydrolase important for pathogenesis 1" (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections.
ESTHER : Lentz_2016_ACS.Infect.Dis_2_807
PubMedSearch : Lentz_2016_ACS.Infect.Dis_2_807
PubMedID: 27739665
Gene_locus related to this paper: myctu-ym24

Title : Global Analysis of Palmitoylated Proteins in Toxoplasma gondii - Foe_2015_Cell.Host.Microbe_18_501
Author(s) : Foe IT , Child MA , Majmudar JD , Krishnamurthy S , van der Linden WA , Ward GE , Martin BR , Bogyo M
Ref : Cell Host Microbe , 18 :501 , 2015
Abstract : Post-translational modifications (PTMs) such as palmitoylation are critical for the lytic cycle of the protozoan parasite Toxoplasma gondii. While palmitoylation is involved in invasion, motility, and cell morphology, the proteins that utilize this PTM remain largely unknown. Using a chemical proteomic approach, we report a comprehensive analysis of palmitoylated proteins in T. gondii, identifying a total of 282 proteins, including cytosolic, membrane-associated, and transmembrane proteins. From this large set of palmitoylated targets, we validate palmitoylation of proteins involved in motility (myosin light chain 1, myosin A), cell morphology (PhIL1), and host cell invasion (apical membrane antigen 1, AMA1). Further studies reveal that blocking AMA1 palmitoylation enhances the release of AMA1 and other invasion-related proteins from apical secretory organelles, suggesting a previously unrecognized role for AMA1. These findings suggest that palmitoylation is ubiquitous throughout the T. gondii proteome and reveal insights into the biology of this important human pathogen.
ESTHER : Foe_2015_Cell.Host.Microbe_18_501
PubMedSearch : Foe_2015_Cell.Host.Microbe_18_501
PubMedID: 26468752

Title : Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion - Child_2013_Nat.Chem.Biol_9_651
Author(s) : Child MA , Hall CI , Beck JR , Ofori LO , Albrow VE , Garland M , Bowyer PW , Bradley PJ , Powers JC , Boothroyd JC , Weerapana E , Bogyo M
Ref : Nat Chemical Biology , 9 :651 , 2013
Abstract : Although there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical-genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma ortholog of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small-molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii.
ESTHER : Child_2013_Nat.Chem.Biol_9_651
PubMedSearch : Child_2013_Nat.Chem.Biol_9_651
PubMedID: 23934245
Gene_locus related to this paper: toxgo-s4ug63

Title : Identification of proteases that regulate erythrocyte rupture by the malaria parasite Plasmodium falciparum - Arastu-Kapur_2008_Nat.Chem.Biol_4_203
Author(s) : Arastu-Kapur S , Ponder EL , Fonovic UP , Yeoh S , Yuan F , Fonovic M , Grainger M , Phillips CI , Powers JC , Bogyo M
Ref : Nat Chemical Biology , 4 :203 , 2008
Abstract : Newly replicated Plasmodium falciparum parasites escape from host erythrocytes through a tightly regulated process that is mediated by multiple classes of proteolytic enzymes. However, the identification of specific proteases has been challenging. We describe here a forward chemical genetic screen using a highly focused library of more than 1,200 covalent serine and cysteine protease inhibitors to identify compounds that block host cell rupture by P. falciparum. Using hits from the library screen, we identified the subtilisin-family serine protease PfSU B1 and the cysteine protease dipeptidyl peptidase 3 (DPAP3) as primary regulators of this process. Inhibition of both DPAP3 and PfSUB1 caused a block in proteolytic processing of the serine repeat antigen (SERA) protein SERA5 that correlated with the observed block in rupture. Furthermore, DPAP3 inhibition reduced the levels of mature PfSUB1. These results suggest that two mechanistically distinct proteases function to regulate processing of downstream substrates required for efficient release of parasites from host red blood cells.
ESTHER : Arastu-Kapur_2008_Nat.Chem.Biol_4_203
PubMedSearch : Arastu-Kapur_2008_Nat.Chem.Biol_4_203
PubMedID: 18246061