GPR84 regulates pulmonary inflammation by modulating neutrophil functions.

Acute lung injury (ALI) is an acute, progressive hypoxic respiratory failure that could develop into acute respiratory distress syndrome (ARDS) with very high mortality rate. ALI is believed to be caused by uncontrolled inflammation, and multiple types of immune cells, especially neutrophils, are critically involved in the development of ALI. The treatment for ALI/ARDS is very limited, a better understanding of the pathogenesis and new therapies are urgently needed. Here we discover that GPR84, a medium chain fatty acid receptor, plays critical roles in ALI development by regulating neutrophil functions. GPR84 is highly upregulated in the cells isolated from the bronchoalveolar lavage fluid of LPS-induced ALI mice. GPR84 deficiency or blockage significantly ameliorated ALI mice lung inflammation by reducing neutrophils infiltration and oxidative stress. Further studies reveal that activation of GPR84 strongly induced reactive oxygen species production from neutrophils by stimulating Lyn, AKT and ERK1/2 activation and the assembly of the NADPH oxidase. These results reveal an important role of GPR84 in neutrophil functions and lung inflammation and strongly suggest that GPR84 is a potential drug target for ALI.

Strigolactone mimic 2-nitrodebranone is highly active in Arabidopsis growth and development.

Strigolactones play crucial roles in regulating plant architecture and development, as endogenous hormones, and orchestrating symbiotic interactions with fungi and parasitic plants, as components of root exudates. rac-GR24 is currently the most widely used strigolactone analog and serves as a reference compound in investigating the action of strigolactones. In this study, we evaluated a suite of debranones and found that 2-nitrodebranone (2NOD) exhibited higher biological activity than rac-GR24 in various aspects of plant growth and development in Arabidopsis, including hypocotyl elongation inhibition, root hair promotion and senescence acceleration. The enhanced activity of 2NOD in promoting AtD14-SMXL7 and AtD14-MAX2 interactions indicates that the molecular structure of 2NOD is a better match for the ligand perception site pocket of D14. Moreover, 2NOD showed lower activity than rac-GR24 in promoting Orobanche cumana seed germination, suggesting its higher ability to control plant architecture than parasitic interactions. In combination with the improved stability of 2NOD, these results demonstrate that 2NOD is a strigolactone analog that can specifically mimic the activity of strigolactones and that 2NOD exhibits strong potential as a tool for studying the strigolactone signaling pathway in plants.

DWARF14 is a non-canonical hormone receptor for strigolactone.

Classical hormone receptors reversibly and non-covalently bind active hormone molecules, which are generated by biosynthetic enzymes, to trigger signal transduction. The α/ß hydrolase DWARF14 (D14), which hydrolyses the plant branching hormone strigolactone and interacts with the F-box protein D3/MAX2, is probably involved in strigolactone detection. However, the active form of strigolactone has yet to be identified and it is unclear which protein directly binds the active form of strigolactone, and in which manner, to act as the genuine strigolactone receptor. Here we report the crystal structure of the strigolactone-induced AtD14-D3-ASK1 complex, reveal that Arabidopsis thaliana (At)D14 undergoes an open-to-closed state transition to trigger strigolactone signalling, and demonstrate that strigolactone is hydrolysed into a covalently linked intermediate molecule (CLIM) to initiate a conformational change of AtD14 to facilitate interaction with D3. Notably, analyses of a highly branched Arabidopsis mutant d14-5 show that the AtD14(G158E) mutant maintains enzyme activity to hydrolyse strigolactone, but fails to efficiently interact with D3/MAX2 and loses the ability to act as a receptor that triggers strigolactone signalling in planta. These findings uncover a mechanism underlying the allosteric activation of AtD14 by strigolactone hydrolysis into CLIM, and define AtD14 as a non-canonical hormone receptor with dual functions to generate and sense the active form of strigolactone.

GPR84 signaling promotes intestinal mucosal inflammation via enhancing NLRP3 inflammasome activation in macrophages.

The putative medium-chain free fatty acid receptor GPR84 is a G protein-coupled receptor primarily expressed in myeloid cells that constitute the innate immune system, including neutrophils, monocytes, and macrophages in the periphery and microglia in the brain. The fact that GPR84 expression in leukocytes is remarkably increased under acute inflammatory stimuli such as lipopolysaccharide (LPS) and TNFα suggests that it may play a role in the development of inflammatory and fibrotic diseases. Here we demonstrate that GPR84 is highly upregulated in inflamed colon tissues of active ulcerative colitis (UC) patients and dextran sulfate sodium (DSS)-induced colitis mice. Infiltrating GPR84+ macrophages are significantly increased in the colonic mucosa of both the UC patients and the mice with colitis. Consistently, GPR84-/- mice are resistant to the development of colitis induced by DSS. GPR84 activation imposes pro-inflammatory properties in colonic macrophages through enhancing NLRP3 inflammasome activation, while the loss of GPR84 prevents the M1 polarization and properties of proinflammatory macrophages. CLH536, a novel GPR84 antagonist discovered by us, suppresses colitis by reducing the polarization and function of pro-inflammatory macrophages. These results define a unique role of GPR84 in innate immune cells and intestinal inflammation, and suggest that GPR84 may serve as a potential drug target for the treatment of UC.

High-Throughput Screening Campaign Identified a Potential Small Molecule RXFP3/4 Agonist.

Relaxin/insulin-like family peptide receptor 3 (RXFP3) belongs to class A G protein-coupled receptor family. RXFP3 and its endogenous ligand relaxin-3 are mainly expressed in the brain with important roles in the regulation of appetite, energy metabolism, endocrine homeostasis and emotional processing. It is therefore implicated as a potential target for treatment of various central nervous system diseases. Since selective agonists of RXFP3 are restricted to relaxin-3 and its analogs, we conducted a high-throughput screening campaign against 32,021 synthetic and natural product-derived compounds using a cyclic adenosine monophosphate (cAMP) measurement-based method. Only one compound, WNN0109-C011, was identified following primary screening, secondary screening and dose-response studies. Although displayed agonistic effect in cells overexpressing the human RXFP3, it also showed cross-reactivity with the human RXFP4. This hit compound may provide not only a chemical probe to investigate the function of RXFP3/4, but also a novel scaffold for the development of RXFP3/4 agonists.

Fluorescent Triazole Urea Activity-Based Probes for the Single-Cell Phenotypic Characterization of Staphylococcus aureus.

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.

Graft bending angle affects allograft tendon maturity early after anterior cruciate ligament reconstruction.

PURPOSE: The aim of this study was to clarify the association of the anterior cruciate ligament (ACL) graft bending angle and graft maturity of autograft and allograft tendons using high-resolution MRI. METHODS: Patients with unilateral ACL reconstruction were invited to participate in this study, and they were examined using a 3.0-T MRI scan at 3, 6 and 12 months after the operation. Anatomic single-bundle ACL reconstruction was performed on 48 patients using the trans-portal technique, including 28 with autograft hamstring tendons and 20 with allograft tendons. To evaluate graft healing, the signal/noise quotient (SNQ) was measured in four regions of interest (ROIs) of the femoral tunnel, proximal, midsubstance and distal ACL grafts. The graft bending angle was defined as the angle between the femoral bone tunnel and the line connecting the femoral and tibial tunnel apertures. Graft SNQ and graft bending angle were assessed at 3, 6 and 12 months postoperatively, and the association between SNQ and the average graft bending angle was analyzed. RESULTS: Generally, the mean graft bending angle of this cohort increased gradually with time. The SNQ value of each graft region increased from 3 to 6 months and then decreased from 6 to 12 months. In the whole cohort, the graft bending angle had a significant positive association with graft SNQ in the femoral tunnel or proximal site. In the allograft subgroup, the graft bending angle had a significant positive association with the graft SNQ in the femoral tunnel or proximal site at 6 months after surgery, while there was no association between the graft bending angle and SNQ at 12 months. In the autograft subgroup, the graft bending angle had a significant positive association with graft SNQ in the femoral tunnel or proximal site at 12 months after surgery. CONCLUSION: Generally, the graft bending angle was correlated with a high signal intensity of the proximal graft in the early postoperative period for allograft tendons and in the late postoperative period for allograft tendons. This suggests that the biomechanical effect from the graft bending angle on graft healing may be different for allografts and autografts after ACL reconstruction. LEVEL OF EVIDENCE: III.

Discovery of a novel calcium-sensitive fluorescent probe for α-ketoglutarate.

α-Ketoglutarate (α-KG), a pivotal metabolite in energy metabolism, has been implicated in nonalcoholic fatty liver disease (NAFLD) and several cancers. It is recently proposed that plasma α-KG is a surrogate biomarker of NAFLD. Here, we report the development of a novel "turn-on" chemosensor for α-KG that contains a coumarin moiety as a fluorophore. Using benzothiazole-coumarin (BTC) as inspiration, we designed a probe for calcium ion recognition that possesses a unique fluorophore compared with previously reported probes for α-KG measurement. This chemosensor is based on the specific Schiff base reaction and the calcium ion recognition property of the widely used calcium indicator BTC. The probe was synthesized, and a series of parallel experiments were conducted to optimize the chemical recognition process. Compared to the initial weak fluorescence, a remarkable 7.6-fold enhancement in fluorescence intensity (I/I0 at 495 nm) was observed for the conditions in which the probe (1 µmol/L), α-KG (50 µmol/L), and Ca2+ (100 µmol/L) were incubated at 30 °C in EtOH. The probe displayed good selectivity for α-KG even in an environment with an abundance of amino acids and other interfering species such as glutaric acid. We determined that the quantitative detection range of α-KG in EtOH was between 5 and 50 µmol/L. Finally, probe in serum loaded with α-KG (10 mmol/L) showed a 7.4-fold fluorescence enhancement. In summary, a novel probe for detecting the biomarker α-KG through a typical Schiff base reaction has been discovered. With further optimization, this probe may be a good alternative for detecting the physiological metabolite α-KG.

A negative allosteric modulator modulates GABAB-receptor signalling through GB2 subunits.

An γ-aminobutyric acid type B (GABAB)-receptor mediates slow and prolonged synaptic inhibition in the central nervous system, which represents an interesting target for the treatment of various diseases and disorders of the central nervous system. To date, only one activator of the GABAB-receptor, baclofen, is on the market for the treatment of spasticity. Inhibitors of the GABAB-receptor, such as antagonists, show anti-absence seizure activity and pro-cognitive properties. In a search for allosteric compounds of the GABAB-receptor, although several positive allosteric modulators have been developed, it is only recently that the first negative allosteric modulator (NAM), CLH304a (also named Compound 14), has been reported. In the present study, we provide further information on the mechanism of action of CLH304a, and also show the possibility of designing more NAMs, such as CLH391 and CLH393, based on the structure of CLH304a. First we show that CLH304a inhibits native GABAB-receptor activity in cultured cerebellar granular neurons. We then show that CLH304a has inverse agonist properties and non-competitively inhibits the effect of agonists, indicating that it binds at a different site to GABA. The GABAB-receptor is a mandatory heterodimer made of GB1 subunits, in which agonists bind, and GB2 subunits, which activate G-proteins. By using various combinations made up of wild-type and/or mutated GB1 and GB2 subunits, we show that CLH304a acts on the heptahelical domain of GB2 subunits. These data revealed the possibility of designing innovative NAMs acting in the heptahelical domain of the GB2 subunits, offering novel possibilities for therapeutic intervention based on GABAB-receptor inhibition.

Single-cell chemical proteomics with an activity-based probe: identification of low-copy membrane proteins on primary neurons.

We propose a novel single-cell chemical proteomics (SCCP) strategy to profile low-abundance membrane proteins in single cells. In this approach, the membrane protein GB1 and its splicing variants were targeted on cultured cell lines and primary neurons using a specifically designed activity-based probe. The functionally labeled single cells were encapsulated in individual buffer droplets on a PDMS microwell array, and were further picked up one at a time and loaded into a capillary electrophoresis system for cell lysis, separation, and laser-induced fluorescence detection of the targeted proteins. The results revealed the expression of GB1 splicing variants in HEK and MEF cells, which was previously only suggested at the transcriptional level. We further applied this method to investigate single primary cells and observed significant heterogeneity among individual mouse cerebellar granule neurons. Interference experiments with GB1 antagonist and agonist validated this observation.

Discovery of GPR84 Fluorogenic Probes Based on a Novel Antagonist for GPR84 Bioimaging.

GPR84 is a promising therapeutic target and biomarker for a range of diseases. In this study, we reported the discovery of BINOL phosphate (BINOP) derivatives as GPR84 antagonists. By investigating the structure-activity relationship, we identified 15S as a novel GPR84 antagonist. 15S exhibits low nanomolar potency and high selectivity for GPR84, while its enantiomer 15R is less active. Next, we rationally designed and synthesized a series of GPR84 fluorogenic probes by conjugating Nile red and compound 15S. The leading hybrid, probe F8, not only retained GPR84 activity but also exhibited low nonspecific binding and a turn-on fluorescent signal in an apolar environment. F8 enabled visualization and detection of GPR84 in GPR84-overexpressing HEK293 cells and lipopolysaccharide-stimulated neutrophils. Furthermore, we demonstrated that F8 can detect upregulated GPR84 protein levels in mice models of inflammatory bowel disease and acute lung injury. Thus, compound F8 represents a promising tool for studying GPR84 functions.

An activity-based probe reveals dynamic protein-protein interactions mediating IGF-1R transactivation by the GABA(B) receptor.

Many GPCRs (G-protein-coupled receptors) can activate RTKs (receptor tyrosine kinases) in the absence of RTK ligands, a phenomenon called transactivation. However, the underlying molecular mechanisms remain undefined. In the present study we investigate the molecular basis of GABA(B) (γ-aminobutyric acid B) receptor-mediated transactivation of IGF-1R (insulin-like growth factor type I receptor) in primary neurons. We take a chemical biology approach by developing an activity-based probe targeting the GABA(B) receptor. This probe enables us first to lock the GABA(B) receptor in an inactive state and then activate it with a positive allosteric modulator, thereby permitting monitoring of the dynamic of the protein complex associated with IGF-1R transactivation. We find that activation of the GABA(B) receptor induces a dynamic assembly and disassembly of a protein complex, including both receptors and their downstream effectors. FAK (focal adhesion kinase), a non-RTK, plays a key role in co-ordinating this dynamic process. Importantly, this dynamic of the GABA(B) receptor-associated complex is critical for transactivation and transactivation-dependent neuronal survival. The present study has identified an important mechanism underlying GPCR transactivation of RTKs, which was enabled by a new chemical biology tool generally applicable for dissecting GPCR signalling.

A Study on Origin Traceability of White Tea (White Peony) Based on Near-Infrared Spectroscopy and Machine Learning Algorithms.

Identifying the geographical origins of white tea is of significance because the quality and price of white tea from different production areas vary largely from different growing environment and climatic conditions. In this study, we used near-infrared spectroscopy (NIRS) with white tea (n = 579) to produce models to discriminate these origins under different conditions. Continuous wavelet transform (CWT), min-max normalization (Minmax), multiplicative scattering correction (MSC) and standard normal variables (SNV) were used to preprocess the original spectra (OS). The approaches of principal component analysis (PCA), linear discriminant analysis (LDA) and successive projection algorithm (SPA) were used for features extraction. Subsequently, identification models of white tea from different provinces of China (DPC), different districts of Fujian Province (DDFP) and authenticity of Fuding white tea (AFWT) were established by K-nearest neighbors (KNN), random forest (RF) and support vector machine (SVM) algorithms. Among the established models, DPC-CWT-LDA-KNN, DDFP-OS-LDA-KNN and AFWT-OS-LDA-KNN have the best performances, with recognition accuracies of 88.97%, 93.88% and 97.96%, respectively; the area under curve (AUC) values were 0.85, 0.93 and 0.98, respectively. The research revealed that NIRS with machine learning algorithms can be an effective tool for the geographical origin traceability of white tea.

Unsymmetrical Phosphodiesters as GPR84 Antagonists with High Blood Exposure for the Treatment of Lung Inflammation.

GPR84 is a proinflammatory G protein-coupled receptor that mediates myeloid immune cell functions. Blocking GPR84 with antagonists is a promising approach for treating inflammatory and fibrotic diseases. Previously, a GPR84 antagonist 604c, with a symmetrical phosphodiester structure, has displayed promising efficacy in a mouse model of ulcerative colitis. However, the low blood exposure resulting from physicochemical properties prevented its uses in other inflammatory diseases. In this study, a series of unsymmetrical phosphodiesters with lower lipophilicity were designed and tested. The representative compound 37 exhibited a 100-fold increase in mouse blood exposure compared to 604c while maintaining in vitro activity. In a mouse model of acute lung injury, 37 (30 mg/kg, po) significantly reduced the infiltration of proinflammatory cells and the release of inflammatory cytokines and ameliorated pathological changes equally or more effectively than N-acetylcysteine (100 mg/kg, po). These findings suggest that 37 is a promising candidate for treating lung inflammation.

Phosphodiesters as GPR84 Antagonists for the Treatment of Ulcerative Colitis.

GPR84 is a proinflammatory G protein-coupled receptor associated with several inflammatory and fibrotic diseases. GPR84 antagonists have been evaluated in clinical trials to treat ulcerative colitis, idiopathic pulmonary fibrosis, and nonalcoholic steatohepatitis. However, the variety of potent and selective GPR84 antagonists is still limited. Through high-throughput screening, a novel phosphodiester compound hit 1 was identified as a GPR84 antagonist. The subsequent structural optimization led to the identification of compound 33 with improved potency in the calcium mobilization assay and the ability to inhibit the chemotaxis of neutrophils and macrophages upon GPR84 activation. In a DSS-induced mouse model of ulcerative colitis, compound 33 significantly alleviated colitis symptoms and reduced the disease activity index score at oral doses of 25 mg/kg qd, with an efficacy similar to that of positive control 5-aminosalicylic acid (200 mg/kg, qd, po), suggesting that compound 33 is a promising candidate for further drug development.

Anatomy and relationships of forelimb lymph nodes in Sprague-Dawley rats: A detailed dissecting approach.

Background: Constructing a reliable animal model for preclinical treatment of secondary lymphedema is challenging because the anatomical characteristics near the lymph nodes are understudied. Therefore, this study examined the detailed anatomical relationship between the axillary lymph node flaps (ALNFs) and brachial lymph node flaps (BLNFs) in the forelimb of Sprague-Dawley (SD) rats. Materials and methods: Ten male rats, weighing 250-300 g, were used. The ALNFs and BLNFs on either side of the rat forelimbs were dissected. The two lymph node flaps (LNFs) were immediately harvested to analyze their physical characteristics (via imaging process software) and microscopic structure (via histology examinations). Results: A total of 20 ALNFs and BLNFs from 10 rats were harvested and analyzed. ALNF dissection was simpler and lasted a shorter time than BLNF dissection (p < 0.0001). The left LNFs were more difficult to dissect than the right LNFs (p < 0.0001). In physical characteristics of LNFs, the area (p < 0.001) of LNFs and the number of lymph nodes (p < 0.0001) associated with ALNFs were greater than those associated with BLNFs, but the pedicle lengths of ALNFs were shorter than that of BLNFs (p < 0.0001). No significant difference in the diameter of the venous and arterial pedicles was noted between the two LNFs (p > 0.05). Conclusion: This study reported detailed physical characteristics of ALNFs and BLNFs in SD rat forelimbs, assessing the respective area of LNFs, number of lymph nodes, and lengths and diameters of vascular pedicles. Moreover, this study suggested an efficient method to perform a study of LNFs by describing the operation process and repeatedly measuring the operation time.

Identification of key candidate genes in local dorsal root ganglion inflammation by integrated bioinformatics analysis.

The purpose of the present study was to identify potential markers of local dorsal root ganglion (DRG) inflammation to aid diagnosis, treatment and prognosis evaluation of DRG pain. A localized inflammation of the DRG (LID) rat model was used to study the contribution of inflammation to pain. The dataset GSE38859 was obtained from the Gene Expression Omnibus database. Pre-treatment standardization of gene expression data for each experiment was performed using the R/Bioconductor Limma package. Differentially expressed genes (DEGs) were identified between a LID model and a sham surgery control group. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of DEGs and gene set enrichment analysis (GSEA) were carried out using the 'clusterProfiler' package in R. Using the Search Tool for Retrieval of Interacting Genes, a protein-protein interaction network was constructed and visualized. Candidate genes with the highest potential validity were validated using reverse transcription-quantitative PCR and western blotting. In total, 66 DEGs were enriched in GO terms related to inflammation and the immune response processes. KEGG analysis revealed 14 associated signaling pathway terms. Protein-protein interaction network analysis revealed 9 node genes, 3 of which were among the top 10 DEGs. Matrix metallopeptidase 9, chemokine CXCL9, and complement component 3 were identified as key regulators of DRG inflammatory pain progression.

Modulation of the G-Protein-Coupled Receptor 84 (GPR84) by Agonists and Antagonists.

Since the discovery of medium-chain fatty acids as GPR84 ligands, significant advancements have been made in the development of GPR84 agonists and antagonists. Most agonists have lipid-like structures except for 3,3'-diindolylmethane (DIM), which acts as an allosteric agonist. GPR84 activation in macrophages leads to increased cytokine secretion, chemotaxis, and phagocytosis, revealing the proinflammatory role of GPR84 associated with various inflammatory responses. Three GPR84 antagonists (S)-2-((1,4-dioxan-2-yl)methoxy)-9-(cyclopropylethynyl)-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (GLPG1205), sodium 2-(3-pentylphenyl)acetate (PBI-4050), and sodium 2-(3,5-dipentylphenyl)acetate (PBI-4547) have displayed therapeutic effects in animal models of several inflammatory and fibrotic diseases and are being evaluated in clinical studies. Although GLPG1205 has failed in a clinical trial for ulcerative colitis, it is undergoing another phase II clinical study for idiopathic pulmonary fibrosis. Further studies are needed to resolve the GPR84 structure, identify more endogenous ligands, elucidate their physiological and pathological roles, and fulfill the therapeutic potential of GPR84 antagonists and agonists.

[The clinical study of partial perineum area and wound repair in males].

OBJECTIVE: To explore the suitable division of male genitalia subunits and the effectiveness of large-area perineum defect repair under its guidance. METHODS: According to the anatomical and functional characteristics of male genitalia, the subunit division scheme was proposed: area Ⅰ, glans penis; area Ⅱ, body of penis; area Ⅲ, scrotum; area Ⅳ, scrotum. Between April 2017 and July 2019, 12 patients with large genitalia defects were treated, with an average age of 60.9 years (range, 57-66 years) and an average disease duration of 2.7 years (range, 2-5 years). The defect area involved area Ⅰ in 1 case, area Ⅱ in 7 cases, area Ⅲ in 5 cases, and area Ⅳ in 8 cases; the size of area ranged from 6 cm×4 cm to 23 cm×16 cm. The causes of defect included 3 cases of trauma, 6 cases of Paget disease, 2 cases of squamous cell carcinoma, 1 case of spindle cell tumor. According to the design of the corresponding repair scheme, the main repair methods were to rotate and advance the skin flap and pedicled skin flap in the same area. When the defect was large, the free skin flap transplantation, free skin grafting, and free mucosa transplantation were used to repair the defect. RESULTS: All the patients were followed up 6-13 months with an average of 8.6 months. Skin flap, skin graft, and mucosa survived in one stage in 10 patients; infection occurred in 1 case after the scrotal flap of area Ⅲ was transferred to repair the defect in area Ⅱ, 1 case had distal venous crisis at 2 days after repair area Ⅲ defect used free anterolateral thigh flap, and after active treatment, the condition improved. The appearance of the receiving area and the supplying area was good, and the local feeling was recovered satisfactorily. The range of motion of hip joint was good in 10 cases, and 2 cases were slightly stretched but did not affect normal life. All patients had normal urination and defecation function, and were satisfied with the treatment effectiveness. CONCLUSION: The subunits of male genitalia can be used to guide the repair of the defect, which can better restore the physiological appearance and function, and has positive clinical significance.

Structural Basis for the Inhibitor and Substrate Specificity of the Unique Fph Serine Hydrolases of Staphylococcus aureus.

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 (Fph's), some of which contribute to the virulence of S. aureus in vivo. These 10 Fph proteins have limited annotation and have few, if any, characterized bacterial or mammalian homologues. 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 analogue 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 toward 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.