Identifying Potential Human Monoacylglycerol Lipase Inhibitors from the Phytoconstituents of Morinda Citrifolia L. Fruits by in silico Pharmacology and in vitro Study.

Background: Human monoacylglycerol lipase (MGL) is accountable for the hydrolysis of 2-arachidonoylglycerol (2-AG), thus contributing pivotally to neuroprotection because 2-AG is the main source of arachidonic acid, the precursor of prostaglandins production. Inhibiting MGL reduces inflammatory damage in the ischemic brain and enhances cerebral blood flow. Plants have been reported for their neuroprotective effect, such as Morinda citrifolia on pentylenetetrazol (PTZ)-induced kindling seizures in mice, by reducing the seizures and restoring behavioral and biochemical changes, although the mechanism is not described. Purpose: To evaluate the binding affinity and stability of phytoconstituents in M. citrifolia fruits toward human MGL (PDB ID 3PE6), compared to the known MGL inhibitors (JZL195 and ZYH). The in silico pharmacology study was validated by an in vitro study of the phytosterols and the ethanol extract of M. citrifolia fruits (EEMC) towards MGL. Methods: Initially, nine phytoconstituents of M. citrifolia fruits were docked to the catalytic pocket of human MGL (PDB ID: 3PE6), and compounds with the best affinity were subjected to a molecular dynamic (MD) simulation. The in vitro study was performed using the MGL inhibitor screening assay kit. Results: The best binding affinity and stability toward human MGL were shown by stigmasterol and beta-sitosterol, and the MM-PBSA total binding energy of stigmasterol and beta-sitosterol to MGL is stronger than that of JZL195 and ZYH. Moreover, beta-sitosterol and EEMC inhibit MGL with an IC50 value of, respectively, 8.10 µg/mL and 196.20 µg/mL, while JZL195 shows an IC50 of 0.028 µg/mL. Conclusion: Beta-sitosterol of Morinda citrifolia fruits may have the potential to protect human neurons by occupying the catalytic site of human MGL, thus competitively inhibiting the substrate of the enzyme. However, the inhibitory activity towards human MGL is lower than JZL195.

Shifting Mycobacterial Serine Hydrolase Activity Visualized Using Multi-Layer In-Gel Activity Assays.

The ability of Mycobacterium tuberculosis to derive lipids from the host, store them intracellularly, and then break them down into energy requires a battery of serine hydrolases. Serine hydrolases are a large, diverse enzyme family with functional roles in dormant, active, and reactivating mycobacterial cultures. To rapidly measure substrate-dependent shifts in mycobacterial serine hydrolase activity, we combined a robust mycobacterial growth system of nitrogen limitation and variable carbon availability with nimble in-gel fluorogenic enzyme measurements. Using this methodology, we rapidly analyzed a range of ester substrates, identified multiple hydrolases concurrently, observed functional enzyme shifts, and measured global substrate preferences. Within every growth condition, mycobacterial hydrolases displayed the full, dynamic range of upregulated, downregulated, and constitutively active hydrolases independent of the ester substrate. Increasing the alkyl chain length of the ester substrate also allowed visualization of distinct hydrolase activity likely corresponding with lipases most responsible for lipid breakdown. The most robust expression of hydrolase activity was observed under the highest stress growth conditions, reflecting the induction of multiple metabolic pathways scavenging for energy to survive under this high stress. The unique hydrolases present under these high-stress conditions could represent novel drug targets for combination treatment with current front-line therapeutics. Combining diverse fluorogenic esters with in-gel activity measurements provides a rapid, customizable, and sensitive detection method for mycobacterial serine hydrolase activity.

Revision of the sophorolipid biosynthetic pathway in Starmerella bombicola based on new insights in the substrate profile of its lactone esterase.

BACKGROUND: Sophorolipids (SLs) are a class of natural, biodegradable surfactants that found their way as ingredients for environment friendly cleaning products, cosmetics and nanotechnological applications. Large-scale production relies on fermentations using the yeast Starmerella bombicola that naturally produces high titers of SLs from renewable resources. The resulting product is typically an extracellular mixture of acidic and lactonic congeners. Previously, we identified an esterase, termed Starmerella bombicola lactone esterase (SBLE), believed to act as an extracellular reverse lactonase to directly use acidic SLs as substrate. RESULTS: We here show based on newly available pure substrates, HPLC and mass spectrometric analysis, that the actual substrates of SBLE are in fact bola SLs, revealing that SBLE actually catalyzes an intramolecular transesterification reaction. Bola SLs contain a second sophorose attached to the fatty acyl group that acts as a leaving group during lactonization. CONCLUSIONS: The biosynthetic function by which the Starmerella bombicola 'lactone esterase' converts acidic SLs into lactonic SLs should be revised to a 'transesterase' where bola SL are the true intermediate. This insights paves the way for alternative engineering strategies to develop designer surfactants.

[Structure motif guided mining of MHET hydrolase and development of a two-enzyme cascade for plastics depolymerization at mild temperature].

The utilization of polyethylene terephthalate (PET) has caused significant and prolonged ecological repercussions. Enzymatic degradation is an environmentally friendly approach to addressing PET contamination. Hydrolysis of mono(2-hydroxyethyl) terephthalate (MHET), a competitively inhibited intermediate in PET degradation, is catalyzed by MHET degrading enzymes. Herein, we employed bioinformatic methods that combined with sequence and structural information to discover an MHET hydrolase, BurkMHETase. Enzymatic characterization showed that the enzyme was relatively stable at pH 7.5-10.0 and 30-45 ℃. The kinetic parameters kcat and Km on MHET were (24.2±0.5)/s and (1.8±0.2) µmol/L, respectively, which were similar to that of the well-known IsMHETase with higher substrate affinity. BurkMHETase coupled with PET degradation enzymes improved the degradation of PET films. Structural analysis and mutation experiments indicated that BurkMHETase may have evolved specific structural features to hydrolyze MHET. For MHET degrading enzymes, aromatic amino acids at position 495 and the synergistic interactions between active sites or distal amino acids appear to be required for MHET hydrolytic activity. Therefore, BurkMHETase may have substantial potential in a dual-enzyme PET degradation system while the bioinformatic methods can be used to broaden the scope of applicable MHETase enzymes.

Metabolism of host lysophosphatidylcholine in Plasmodium falciparum-infected erythrocytes.

The human malaria parasite Plasmodium falciparum requires exogenous fatty acids to support its growth during the pathogenic, asexual erythrocytic stage. Host serum lysophosphatidylcholine (LPC) is a significant fatty acid source, yet the metabolic processes responsible for the liberation of free fatty acids from exogenous LPC are unknown. Using an assay for LPC hydrolysis in P. falciparum-infected erythrocytes, we have identified small-molecule inhibitors of key in situ lysophospholipase activities. Competitive activity-based profiling and generation of a panel of single-to-quadruple knockout parasite lines revealed that two enzymes of the serine hydrolase superfamily, termed exported lipase (XL) 2 and exported lipase homolog (XLH) 4, constitute the dominant lysophospholipase activities in parasite-infected erythrocytes. The parasite ensures efficient exogenous LPC hydrolysis by directing these two enzymes to distinct locations: XL2 is exported to the erythrocyte, while XLH4 is retained within the parasite. While XL2 and XLH4 were individually dispensable with little effect on LPC hydrolysis in situ, loss of both enzymes resulted in a strong reduction in fatty acid scavenging from LPC, hyperproduction of phosphatidylcholine, and an enhanced sensitivity to LPC toxicity. Notably, growth of XL/XLH-deficient parasites was severely impaired when cultured in media containing LPC as the sole exogenous fatty acid source. Furthermore, when XL2 and XLH4 activities were ablated by genetic or pharmacologic means, parasites were unable to proliferate in human serum, a physiologically relevant fatty acid source, revealing the essentiality of LPC hydrolysis in the host environment and its potential as a target for anti-malarial therapy.

A conserved but structurally divergent loop in acyl protein thioesterase 1 regulates its catalytic activity, ligand binding, and folded stability.

Human acyl protein thioesterases (APTs) catalyze the depalmitoylation of S-acylated proteins attached to the plasma membrane, facilitating reversible cycles of membrane anchoring and detachment. We previously showed that a bacterial APT homologue, FTT258 from the gram-negative pathogen Francisella tularensis, exists in equilibrium between a closed and open state based on the structural dynamics of a flexible loop overlapping its active site. Although the structural dynamics of this loop are not conserved in human APTs, the amino acid sequence of this loop is highly conserved, indicating essential but divergent functions for this loop in human APTs. Herein, we investigated the role of this loop in regulating the catalytic activity, ligand binding, and protein folding of human APT1, a depalmitoylase connected with cancer, immune, and neurological signaling. Using a combination of substitutional analysis with kinetic, structural, and biophysical characterization, we show that even in its divergent structural location in human APT1 that this loop still regulates the catalytic activity of APT1 through contributions to ligand binding and substrate positioning. We confirmed previously known roles for multiple residues (Phe72 and Ile74) in substrate binding and catalysis while adding new roles in substrate selectivity (Pro69), in catalytic stabilization (Asp73 and Ile75), and in transitioning between the membrane binding ß-tongue and substrate-binding loops (Trp71). Even conservative substitution of this tryptophan (Trp71) fulcrum led to complete loss of catalytic activity, a 13°C decrease in total protein stability, and drastic drops in ligand affinity, indicating that the combination of the size, shape, and aromaticity of Trp71 are essential to the proper structure of APT1. Mixing buried hydrophobic surface area with contributions to an exposed secondary surface pocket, Trp71 represents a previously unidentified class of essential tryptophans within α/ß hydrolase structure and a potential allosteric binding site within human APTs.

Identification of sequence determinants for the ABHD14 enzymes.

Over the course of evolution, enzymes have developed remarkable functional diversity in catalyzing important chemical reactions across various organisms, and understanding how new enzyme functions might have evolved remains an important question in modern enzymology. To systematically annotate functions, based on their protein sequences and available biochemical studies, enzymes with similar catalytic mechanisms have been clustered together into an enzyme superfamily. Typically, enzymes within a superfamily have similar overall three-dimensional structures, conserved catalytic residues, but large variations in substrate recognition sites and residues to accommodate the diverse biochemical reactions that are catalyzed within the superfamily. The serine hydrolases are an excellent example of such an enzyme superfamily. Based on known enzymatic activities and protein sequences, they are split almost equally into the serine proteases and metabolic serine hydrolases. Within the metabolic serine hydrolases, there are two outlying members, ABHD14A and ABHD14B, that have high sequence similarity, but their biological functions remained cryptic till recently. While ABHD14A still lacks any functional annotation to date, we recently showed that ABHD14B functions as a lysine deacetylase in mammals. Given their high sequence similarity, automated databases often wrongly assign ABHD14A and ABHD14B as the same enzyme, and therefore, annotating functions to them in various organisms has been problematic. In this article, we present a bioinformatics study coupled with biochemical experiments, which identifies key sequence determinants for both ABHD14A and ABHD14B, and enable better classification for them. In addition, we map these enzymes on an evolutionary timescale and provide a much-wanted resource for studying these interesting enzymes in different organisms.

Short synthesis of a broadly Reactive, cell permeable serine hydrolase Fluorophosphonate-Alkyne probe.

An abbreviated synthesis of the cell permeable fluorophosphonate-alkyne probe (FP-alkyne) for the broad assessment of serine hydrolase activity has been developed. While FP-alkyne has proven pivotal in numerous chemical biology studies access has relied on a lengthy preparation over nine steps. We have developed a four-step synthesis, starting from commercially available compounds, with three purification steps to provide a new expedited route allowing easy access to a useful tool compound for exploring serine hydrolases chemistry and biology. This route was used in our own studies to generate FP-alkyne which in turn was used to identify the enzyme responsible for Fatty Acid Esters of Hydroxy Fatty Acids (FAHFA) biosynthesis. The use of this route can enable the syntheses of new tool compounds in addition to improving accessibility to FP-alkyne.

Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of S. aureus Infections.

Staphylococcus aureus is a major human pathogen 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 oxadiazolonebased activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologs 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 (ABPs) and validate FphE as a target for development of imaging contrast agents for the rapid detection of S. aureus infections.

2.1 Å crystal structure of the Mycobacterium tuberculosis serine hydrolase, Hip1, in its anhydro-form (Anhydrohip1).

The 2.6 Å crystal structure of the apo form of Hip1 (hydrolase important for pathogenesis) has been previously reported. However, very little is known about the active site architecture of this M. tuberculosis (Mtb), serine hydrolase drug target. To begin mapping the active site of Hip1, we cocrystallized Hip1 with the irreversible serine protease inhibitor, 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF). We chose AEBSF for cocrystallization with Hip1 since the similar inhibitor, phenylmethylsulfonyl fluoride (PMSF), interestingly exhibited no activity against Hip1. We obtained crystals that diffracted to 2.1 Å but to our bewilderment, we did not observe any electron density for the inhibitor in the omit map for the Hip1-AEBSF complex. Rather, in the active site, dehydroalanine (dAla) was found to occupy the expected position of the catalytic Ser228, thus yielding anhydrohip1. Here we present a comparative analysis of the crystal structures of anhydrohip1 and Hip1 and provide a mechanism for the conversion of the enzyme to the anhydro-form through reaction with AEBSF. With the aid of molecular docking, we propose an explanation for the differential inhibition of Hip1 by AEBSF and PMSF. We also present a preliminary definition of the S1 and S2 pockets of the protease's active site and propose a mechanism for a ligand-induced conformational change within the S2 pocket. Finally, we expand upon the previous demarcation of the putative lipid binding pocket in the α-domain of the enzyme. We believe that this detailed analysis of the structures of anhydrohip1 and Hip1 provides valuable information useful for the structure-based drug design of novel Hip1-directed Mtb therapeutics.

The unusual convergence of steroid catabolic pathways in Mycobacterium abscessus.

Mycobacterium abscessus, an opportunistic pathogen responsible for pulmonary infections, contains genes predicted to encode two steroid catabolic pathways: a cholesterol catabolic pathway similar to that of Mycobacterium tuberculosis and a 4-androstenedione (4-AD) catabolic pathway. Consistent with this prediction, M. abscessus grew on both steroids. In contrast to M. tuberculosis, Rhodococcus jostii RHA1, and other Actinobacteria, the cholesterol and 4-AD catabolic gene clusters of the M. abscessus complex lack genes encoding HsaD, the meta-cleavage product (MCP) hydrolase. However, M. abscessus ATCC 19977 harbors two hsaD homologs elsewhere in its genome. Only one of the encoded enzymes detectably transformed steroid metabolites. Among tested substrates, HsaDMab and HsaDMtb of M. tuberculosis had highest substrate specificities for MCPs with partially degraded side chains thioesterified with coenzyme A (kcat/KM = 1.9 × 104 and 5.7 × 103 mM-1s-1, respectively). Consistent with a dual role in cholesterol and 4-AD catabolism, HsaDMab also transformed nonthioesterified substrates efficiently, and a ΔhsaD mutant of M. abscessus grew on neither steroid. Interestingly, both steroids prevented growth of the mutant on acetate. The ΔhsaD mutant of M. abscessus excreted cholesterol metabolites with a fully degraded side chain, while the corresponding RHA1 mutant excreted metabolites with partially degraded side chains. Finally, the ΔhsaD mutant was not viable in macrophages. Overall, our data establish that the cholesterol and 4-AD catabolic pathways of M. abscessus are unique in that they converge upstream of where this occurs in characterized steroid-catabolizing bacteria. The data further indicate that cholesterol is a substrate for intracellular bacteria and that cholesterol-dependent toxicity is not strictly dependent on coenzyme A sequestration.

Unveiling a novel serpinB2-tripeptidyl peptidase II signaling axis during senescence.

Tripeptidyl peptidase II (TPPII or TPP2) degrades N-terminal tripeptides from proteins and peptides. Studies in both humans and mice have shown that TPPII deficiency is linked to cellular immune-senescence, lifespan regulation and the aging process. However, the mechanism of how TPPII participates in these processes is less clear. In this study, we established a chemical probe-based assay and found that although the mRNA and protein levels of TPPII were not altered during senescence, its enzymatic activity was reduced in senescent human fibroblasts. We also showed that elevation of the levels of the serine protease inhibitor serpinB2 reduced TPPII activity in senescent cells. Moreover, suppression of TPPII led to elevation in the amount of lysosomal contents as in well as TPPI (TPP1) and ß-galactosidase activities, suggesting that lysosome biogenesis is induced to compensate for the reduction of TPPII activity in senescent cells. Together, this study discloses a critical role of the serpinB2-TPPII signaling pathway in proteostasis during senescence. Since serpinB2 levels can be increased by a variety of cellular stresses, reduction of TPPII activity through activation of serpinB2 might represent a common pathway for cells to respond to different stress conditions. This article has an associated First Person interview with the first author of the paper.

Activity-based Protein Profiling of Serine Hydrolase Superfamily Enzymes.

Activity-based protein profiling (ABPP) is a chemoproteomics platform to assess the functional state of enzymes in complex biological systems. Over the two decades, ABPP has emerged from a gel-based to gel-free platform, for in-depth proteome analysis with enhanced resolution, sensitivity for target detection, and discovery of small molecule inhibitors. The gel-free format of ABPP coupled with advanced mass spectrometry is highly sensitive and provides more comprehensive knowledge for the targeted enzyme family than the gel-based method. ABPP strategy is applied across microbe, plant, and animal models. It can be performed both in vitro and in vivo studies, and there is no limitation on sample origin. Here, we report an ultrasensitive, gel-free format of ABPP called active site peptide profiling. This protocol describes the identification of authentic functional proteins, by tagging their active sites in a native biological system. It is high throughput in nature and helps enrich even low abundance functional proteins. Since protein identification is virtually based on a single peptide, the identified peptide should be a unique peptide to identify its parent protein. It can be performed in a facile manner and offers to consolidate identification of protein targets as well as the site of probe modification. We have validated this approach using a fluorophosphonate (FP) serine hydrolase probe in the native proteome of the cereal crop Oryza sativa. Graphic abstract: Serine hydrolase active site peptide profiling.

Identification of cell wall synthesis inhibitors active against Mycobacterium tuberculosis by competitive activity-based protein profiling.

The identification and validation of a small molecule's targets is a major bottleneck in the discovery process for tuberculosis antibiotics. Activity-based protein profiling (ABPP) is an efficient tool for determining a small molecule's targets within complex proteomes. However, how target inhibition relates to biological activity is often left unexplored. Here, we study the effects of 1,2,3-triazole ureas on Mycobacterium tuberculosis (Mtb). After screening ∼200 compounds, we focus on 4 compounds that form a structure-activity series. The compound with negligible activity reveals targets, the inhibition of which is functionally less relevant for Mtb growth and viability, an aspect not addressed in other ABPP studies. Biochemistry, computational docking, and morphological analysis confirms that active compounds preferentially inhibit serine hydrolases with cell wall and lipid metabolism functions and that disruption of the cell wall underlies biological activity. Our findings show that ABPP identifies the targets most likely relevant to a compound's antibacterial activity.

Transition metal cation inhibition of Mycobacterium tuberculosis esterase RV0045C.

Mycobacterium tuberculosis virulence is highly metal-dependent with metal availability modulating the shift from the dormant to active states of M. tuberculosis infection. Rv0045c from M. tuberculosis is a proposed metabolic serine hydrolase whose folded stability is dependent on divalent metal concentration. Herein, we measured the divalent metal inhibition profile of the enzymatic activity of Rv0045c and found specific divalent transition metal cations (Cu2+ ≥ Zn2+ > Ni2+ > Co2+ ) strongly inhibited its enzymatic activity. The metal cations bind allosterically, largely affecting values for kcat rather than KM . Removal of the artificial N-terminal 6xHis-tag did not change the metal-dependent inhibition, indicating that the allosteric inhibition site is native to Rv0045c. To isolate the site of this allosteric regulation in Rv0045c, the structures of Rv0045c were determined at 1.8 Å and 2.0 Å resolution in the presence and absence of Zn2+ with each structure containing a previously unresolved dynamic loop spanning the binding pocket. Through the combination of structural analysis with and without zinc and targeted mutagenesis, this metal-dependent inhibition was traced to multiple chelating residues (H202A/E204A) on a flexible loop, suggesting dynamic allosteric regulation of Rv0045c by divalent metals. Although serine hydrolases like Rv0045c are a large and diverse enzyme superfamily, this is the first structural confirmation of allosteric regulation of their enzymatic activity by divalent metals.

The activity and stability of a cold-active acylaminoacyl peptidase rely on its dimerization by domain swapping.

The study of enzymes from extremophiles arouses interest in Protein Science because of the amazing solutions these proteins adopt to cope with extreme conditions. Recently solved, the structure of the psychrophilic acyl aminoacyl peptidase from Sporosarcina psychrophila (SpAAP) pinpoints a mechanism of dimerization unusual for this class of enzymes. The quaternary structure of SpAAP relies on a domain-swapping mechanism involving the N-terminal A1 helix. The A1 helix is conserved among homologous mesophilic and psychrophilic proteins and its deletion causes the formation of a monomeric enzyme, which is inactive and prone to aggregate. Here, we investigate the dimerization mechanism of SpAAP through the analysis of chimeric heterodimers where a protomer lacking the A1 helix combines with a protomer carrying the inactivated catalytic site. Our results indicate that the two active sites are independent, and that a single A1 helix is sufficient to partially recover the quaternary structure and the activity of chimeric heterodimers. Since catalytically competent protomers are unstable and inactive unless they dimerize, SpAAP reveals as an "obligomer" for both structural and functional reasons.

Proteoform of Arabidopsis seed storage protein identified by functional proteomics approach exhibits acyl hydrolase activity during germination.

Lipases play a crucial role in the life cycle of seed plants and the oil content of the seed is highly regulated by the lipase activity. Hence, understanding the role of lipases during germination and post-germination will provide insights into lipid mobilization. However, to date, no lipase gene has been identified in seeds except, Sugar-dependent-1 in Arabidopsis. Hence, in the present study, we employed a functional proteomic approach for the identification of seed-specific lipase. Activity-Based Proteome Profiling (ABPP) of Arabidopsis mature and germinating seeds revealed the expression of a functional serine hydrolase exclusively during germination. The mass-spectrometry analysis reveals the identity and amino acid sequence of the protein correspond to AT4G28520 gene, a canonical 12S Seed Storage Protein (SSP). Interestingly, the identified SSP was a proteoform of AT4G28520 (SL-AT4G28520) and exhibited >90% identity with the canonical AT4G28520 (FL-AT4G28520). Heterologous expression and enzyme assays indicated that SL-AT4G28520 protein indeed possesses monoacylglycerol lipase activity, while the FL-AT4G28520 protein didn't exhibit any detectable activity. Functional proteomics and lipidomics analysis demonstrated a catalytic function of this SSP. Collectively, this is the first report, which suggests that SL-AT4G28520 encodes a lipase, and the activity is depending on the physiological condition.

Characterization and engineering of a two-enzyme system for plastics depolymerization.

Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all nonzero MHETase concentrations tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics.

Heteroarylureas with fused bicyclic diamine cores as inhibitors of fatty acid amide hydrolase.

A series of mechanism-based heteroaryl urea fatty acid amide hydrolase (FAAH) inhibitors with fused bicyclic diamine cores is described. In contrast to compounds built around a piperazine core, most of the fused bicyclic diamine bearing analogs prepared exhibited greater potency against rFAAH than the human enzyme. Several compounds equipotent against both species were identified and profiled in vivo.

Strategies for Tuning the Selectivity of Chemical Probes that Target Serine Hydrolases.

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.