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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.

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.

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.

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.

SummaryStaphylococcus aureus is a major cause of infection in the community and in hospitals. Serine hydrolases play key roles in bacterial homeostasis, in particular biofilms. Activity-based profiling has previously identified a family of serine hydrolases, designated fluorophosphonate-binding hydrolases (Fphs), which contribute to virulence of S. aureus in the biofilm niche. Here we report structures of the putative tributyrin esterase FphF, alone and covalently bound by a substrate analog, and small molecule inhibitors that occupy the hydrophobic substrate-binding pocket. We show that FphF has promiscuous esterase activity. Building from this, we extended our analysis to the wider Fph protein family using homology modeling and docking tools. We predict that other Fph enzymes, including FphB which was linked directly to virulence, may be more specific than FphF. This study provides insight into Fph function and a template for designing new imaging agents, diagnostic probes, and inhibitors to treat S. aureus infections.

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.

Phenotypically distinct cellular (sub)populations are clinically relevant for virulence and antibiotic resistance of a bacterial pathogen, but functionally different cells are usually indistinguishable from each other. Here, we introduce fluorescent activity-based probes as chemical tools for 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 chemical probe labeling of enzymatic activities as a generalizable method for phenotyping of bacterial cells at the population and single-cell level.

With the growth of population and the reduction of arable land, breeding has been considered as an effective way to solve the food crisis. As an important part in breeding, high-throughput phenotyping can accelerate the breeding process effectively. Light detection and ranging (LiDAR) is an active remote sensing technology that is capable of acquiring three-dimensional (3D) data accurately, and has a great potential application in crop phenotyping. Given that crop phenotyping based on LiDAR technology is not common in China, we developed a high-throughput crop phenotyping platform, named Crop 3D, which integrated LiDAR, high-resolution camera, thermal camera and hyperspectral imager. Compared with traditional crop phenotyping techniques, Crop 3D can acquire the multi-source phenotypic data in the whole crop growing period and extract plant height, plant width, leaf length, leaf width, leaf area, leaf inclination angle and other parameters for plant biology and genomics analysis. In this paper, we described the designs, functions and testing results of the Crop 3D platform. Then, the potential applications and future development of the platform in phenotyping were briefly discussed. We concluded that platforms integrating LiDAR and traditional remote sensing techniques might be the future trend of crop high-throughput phenotyping.

Classical hormone receptors reversibly and non-covalently bind active hormone molecules, which are generated by biosynthetic enzymes, to trigger signal transduction. The α/β 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.

An γ-aminobutyric acid type B (GABA B )-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 GABA B -receptor, baclofen, is on the market for the treatment of spasticity. Inhibitors of the GABA B -receptor, such as antagonists, show anti-absence seizure activity and pro-cognitive properties. In a search for allosteric compounds of the GABA B -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 GABA B -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 GABA B -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 GABA B -receptor inhibition.

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.

A series of alkylpyrimidine-4,6-diol derivatives were designed and synthesized as novel GRP84 agonists based on a high-throughput screening (HTS) hit 1. 6-Nonylpyridine-2,4-diol was identified as the most potent agonist of GPR84 reported so far, with an EC50 of 0.189 nM. These novel GPR84 agonists will provide valuable tools for the study of the physiological functions of GPR84.

Initialized from the scaffold of CGP7930, an allosteric agonist of GABAB receptors, a series of noncompetitive antagonists were discovered. Among these compounds, compounds 3, 6, and 14 decreased agonist GABA-induced maximal effect of IP3 production in HEK293 cells overexpressing GABAB receptors and Gqi9 proteins without changing the EC50. Compounds 3, 6, and 14 not only inhibited agonist baclofen-induced ERK1/2 phosphorylation but also blocked CGP7930-induced ERK1/2 phosphorylation in HEK293 cells overexpressing GABAB receptors. The results suggested that compounds 3, 6, and 14 are negative allosteric modulators of GABAB receptors. The representative compound 14 decreased GABA-induced IP3 production with IC50 of 37.9 μM and had no effect on other GPCR Class C members such as mGluR1, mGluR2, and mGluR5. Finally, we showed that compound 14 did not bind to the orthosteric binding sites of GABAB receptors, demonstrating that compound 14 negatively modulated GABAB receptors activity as a negative allosteric modulator.

Three novel tetrahydroisoquinoline tetracyclic core frameworks were stereoselectively synthesized. The key steps included a ring-closing metathesis (RCM)-mediated cyclization and a subsequent intramolecular SN2 N(O)-substituted reaction. This simple method for tetracyclic core synthesis facilitates the further exploration of the chemical space of tetrahydroisoquinoline alkaloids.

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 GABAB (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 GABAB receptor. This probe enables us first to lock the GABAB 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 GABAB 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 GABAB 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.