Mono-2-ethylhexylphthalate (MEHP) is a potent agonist of human TRPA1 channel.

Phthalates are extensively used as plasticizers in diverse consumer care products but have been reported to cause adverse health effects in humans. A commonly used phthalate, di-2-ethylhexylphthalate (DEHP) causes developmental and reproductive toxicities in humans, but the associated molecular mechanisms are not fully understood. Mono-2-ethylhexylphthalate (MEHP), a hydrolytic product of DEHP generated by cellular esterases, is proposed to be the active toxicant. We conducted a screen for sensory irritants among compounds used in consumer care using an assay for human Transient Receptor Potential A1 (hTRPA1). We have identified MEHP as a potent agonist of hTRPA1. MEHP-induced hTRPA1 activation was blocked by the TRPA1 inhibitor A-967079. Patch clamp assays revealed that MEHP induced inward currents in cells expressing hTRPA1. In addition, the N855S mutation in hTRPA1 associated with familial episodic pain syndrome decreased MEHP-induced hTRPA1 activation. In summary, we report that MEHP is a potent agonist of hTRPA1 which generates new possible mechanisms for toxic effects of phthalates in humans.

Inhibitors of human bitter taste receptors from the five-flavour berry, Schisandra chinensis.

The human bitter taste 2 receptor member 16 (TAS2R16) is one of 25 class A G-protein-coupled receptors (GPCRs) and responds to a variety of molecules responsible for the bitter taste sensation perceived in humans. TAS2R16 can be activated by ß-glucopyranosides, and its activation can be inhibited by probenecid, a synthetic drug compound used to treat gout. In this study we describe naturally derived compounds which can inhibit the activation of TAS2R16 by salicin in vitro. These compounds belong to the lignan class derived from the fruit of Schisandra chinensis, which is commonly known as the five-flavour berry. We further tested other analogs with this lignan scaffold, found their differential inhibitory activities towards TAS2R16 in vitro, and sought to rationalize the activity using molecular docking of these lignans on a computationally modelled structure of TAS2R16. Selected lignans with inhibitory activity against other TAS2Rs reveal sub-millimolar inhibitory activity towards TAS2R10, TAS2R14, and TAS2R43 in cell-based assays. These compounds with demonstrated in vitro inhibition of bitter taste receptors may serve as tool compounds to investigate the molecular mechanisms of hTAS2Rs biology in gustatory and non-gustatory tissues.

A sweeter future: Using protein language models for exploring sweeter brazzein homologs.

With growing concerns over the health impact of sugar, brazzein offers a viable alternative due to its sweetness, thermostability, and low risk profile. Here, we demonstrated the ability of protein language models to design new brazzein homologs with improved thermostability and potentially higher sweetness, resulting in new diverse optimized amino acid sequences that improve structural and functional features beyond what conventional methods could achieve. This innovative approach resulted in the identification of unexpected mutations, thereby generating new possibilities for protein engineering. To facilitate the characterization of the brazzein mutants, a simplified procedure was developed for expressing and analyzing related proteins. This process involved an efficient purification method using Lactococcus lactis (L. lactis), a generally recognized as safe (GRAS) bacterium, as well as taste receptor assays to evaluate sweetness. The study successfully demonstrated the potential of computational design in producing a more heat-resistant and potentially more palatable brazzein variant, V23.

Backbone resonance assignment for the full length tRNA-(N1G37) methyltransferase of Pseudomonas aeruginosa.

Bacterial tRNA (guanine37-N1)-methyltransferase (TrmD) plays important roles in translation, making it an important target for the development of new antibacterial compounds. TrmD comprises two domains with the N-terminal domain binding to the S-adenosyl-L-methionine (SAM) cofactor and the C-terminal domain critical for tRNA binding. Bacterial TrmD is functional as a dimer. Here we report the backbone NMR resonance assignments for the full length TrmD protein of Pseudomonas aeruginosa. Most resonances were assigned and the secondary structure for each amino acid was determined according to the assigned backbone resonances. The availability of the assignment will be valuable for exploring molecular interactions of TrmD with ligands, inhibitors and tRNA.

Targeting the Bacterial Epitranscriptome for Antibiotic Development: Discovery of Novel tRNA-(N1G37) Methyltransferase (TrmD) Inhibitors.

Bacterial tRNA modification synthesis pathways are critical to cell survival under stress and thus represent ideal mechanism-based targets for antibiotic development. One such target is the tRNA-(N1G37) methyltransferase (TrmD), which is conserved and essential in many bacterial pathogens. Here we developed and applied a widely applicable, radioactivity-free, bioluminescence-based high-throughput screen (HTS) against 116350 compounds from structurally diverse small-molecule libraries to identify inhibitors of Pseudomonas aeruginosa TrmD ( PaTrmD). Of 285 compounds passing primary and secondary screens, a total of 61 TrmD inhibitors comprised of more than 12 different chemical scaffolds were identified, all showing submicromolar to low micromolar enzyme inhibitor constants, with binding affinity confirmed by thermal stability and surface plasmon resonance. S-Adenosyl-l-methionine (SAM) competition assays suggested that compounds in the pyridine-pyrazole-piperidine scaffold were substrate SAM-competitive inhibitors. This was confirmed in structural studies, with nuclear magnetic resonance analysis and crystal structures of PaTrmD showing pyridine-pyrazole-piperidine compounds bound in the SAM-binding pocket. Five hits showed cellular activities against Gram-positive bacteria, including mycobacteria, while one compound, a SAM-noncompetitive inhibitor, exhibited broad-spectrum antibacterial activity. The results of this HTS expand the repertoire of TrmD-inhibiting molecular scaffolds that show promise for antibiotic development.

Backbone resonance assignment for the N-terminal region of bacterial tRNA-(N1G37) methyltransferase.

Bacterial tRNA (guanine37-N1)-methyltransferase (TrmD) is an important antibacterial target due to its essential role in translation. TrmD has two domains connected with a flexible linker. The N-terminal domain (NTD) of TrmD contains the S-adenosyl-L-methionine (SAM) cofactor binding site and the C-terminal domain is critical for tRNA binding. Here we report the backbone NMR resonance assignments for NTD of Pseudomonas aeruginosa TrmD. Its secondary structure was determined based on the assigned resonances. Relaxation analysis revealed that NTD existed as dimers in solution. NTD also exhibited thermal stability in solution. Its interactions with SAM and other compounds suggest it can be used for evaluating SAM competitive inhibitors by NMR.

Phenotypic Screening for Inhibitors of a Mutant Thrombopoietin Receptor.

An inhibitor for the thrombopoietin receptor (TpoR) would be more specific for the treatment of myeloproliferative neoplasms (MPNs) due to constitutively active mutant TpoR compared to the current treatment approach of inhibiting Janus kinase 2 (JAK2). We describe a cell-based high-throughput phenotypic screening approach to identify inhibitors for constitutively active mutant TpoR. A stepwise elimination process is used to differentiate generally cytotoxic compounds from compounds that specifically inhibit growth of cells expressing wild-type TpoR and/or mutant TpoR. We have systematically optimized the phenotypic screening assay and documented in this chapter critical parameters for a successful phenotypic screen, such as cell growth and seeding optimization, plate reproducibility and uniformity studies, and an assay robustness analysis with a pilot screen.

Applications of NMR and ITC for the Study of the Kinetics of Carbohydrate Binding by AMPK ß-Subunit Carbohydrate-Binding Modules.

Understanding the kinetics of proteins interacting with their ligands is important for characterizing molecular mechanism. However, it can be difficult to determine the extent and nature of these interactions for weakly formed protein-ligand complexes that have lifetimes of micro- to milliseconds. Nuclear magnetic resonance (NMR) spectroscopy is a powerful solution-based method for the atomic-level analysis of molecular interactions on a wide range of timescales, including micro- to milliseconds. Recently the combination of thermodynamic experiments using isothermal titration calorimetry (ITC) with kinetic measurements using ZZ-exchange and CPMG relaxation dispersion NMR spectroscopy have been used to determine the kinetics of weakly interacting protein systems. This chapter describes the application of ITC and NMR to understand the differences in the kinetics of carbohydrate binding by the ß1- and ß2-carbohydrate-binding modules of AMP-activated protein kinase.

A Phenotypic Screen for Small-Molecule Inhibitors of Constitutively Active Mutant Thrombopoietin Receptor Implicated in Myeloproliferative Neoplasms.

BACKGROUND: Rather than a Janus Kinase 2 inhibitor (ruxolitinib), a specific thrombopoietin receptor (TpoR) inhibitor would be more specific for the treatment of myeloproliferative neoplasms due to TpoR mutations. OBJECTIVE: A cell-based phenotypic approach to identify specific TpoR inhibitors was implemented and a library of 505,483 small molecules was screened for inhibitory effects on cells transformed by TpoR mutants. RESULTS: Among the identified hits are two analogs of 3-(4-piperidinyl) indole. The analogs showed about five-fold preferential inhibition of cell viability towards Ba/F3 cells expressing the TpoR W515L mutation compared to the parental cells. There was no significant difference in inhibition of cell viability between the TpoR wild type and the TpoR W515L mutant cells. Preferential inhibition of viability was observed in Ba/F3 cells expressing erythropoietin receptor (EpoR) when stimulated with Epo compared to stimulation with interleukin-3 (IL3). The indole analog inhibited ex vivo colony formations of primary bone marrow cells from heterozygous JAK2 V617F knock-in mice. Drug combination treatment study was performed using ruxolitinib and the indole analog. Drug synergistic effects were observed when cells were stimulated to proliferate through both the IL3 and TpoR pathways. Our compound specifically targets monoamine receptors in the rhodopsin-like receptor family of G protein-coupled receptor. CONCLUSION: This screen has identified a monoamine receptor inhibitor that can inhibit viability of cells with active TpoR or EpoR signalings. Drug synergism with ruxolitib is demonstrated.

Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants.

Mutations in the calreticulin gene (CALR) represented by deletions and insertions in exon 9 inducing a -1/+2 frameshift are associated with a significant fraction of myeloproliferative neoplasms (MPNs). The mechanisms by which CALR mutants induce MPN are unknown. Here, we show by transcriptional, proliferation, biochemical, and primary cell assays that the pathogenic CALR mutants specifically activate the thrombopoietin receptor (TpoR/MPL). No activation is detected with a battery of type I and II cytokine receptors, except granulocyte colony-stimulating factor receptor, which supported only transient and weak activation. CALR mutants induce ligand-independent activation of JAK2/STAT/phosphatydylinositol-3'-kinase (PI3-K) and mitogen-activated protein (MAP) kinase pathways via TpoR, and autonomous growth in Ba/F3 cells. In these transformed cells, no synergy is observed between JAK2 and PI3-K inhibitors in inhibiting cytokine-independent proliferation, thus showing a major difference from JAK2V617F cells where such synergy is strong. TpoR activation was dependent on its extracellular domain and its N-glycosylation, especially at N117. The glycan binding site and the novel C-terminal tail of the mutant CALR proteins were required for TpoR activation. A soluble form of TpoR was able to prevent activation of full-length TpoR provided that it was N-glycosylated. By confocal microscopy and subcellular fractionation, CALR mutants exhibit different intracellular localization from that of wild-type CALR. Finally, knocking down either MPL/TpoR or JAK2 in megakaryocytic progenitors from patients carrying CALR mutations inhibited cytokine-independent megakaryocytic colony formation. Taken together, our study provides a novel signaling paradigm, whereby a mutated chaperone constitutively activates cytokine receptor signaling.

SnRK1 from Arabidopsis thaliana is an atypical AMPK.

SNF1-related protein kinase 1 (SnRK1) is the plant orthologue of the evolutionarily-conserved SNF1/AMPK/SnRK1 protein kinase family that contributes to cellular energy homeostasis. Functional as heterotrimers, family members comprise a catalytic α subunit and non-catalytic ß and γ subunits; multiple isoforms of each subunit type exist, giving rise to various isoenzymes. The Arabidopsis thaliana genome contains homologues of each subunit type, and, in addition, two atypical subunits, ß(3) and ßγ, with unique domain architecture, that are found only amongst plants, suggesting atypical heterotrimers. The AtSnRK1 subunit structure was determined using recombinant protein expression and endogenous co-immunoprecipitation, and six unique isoenzyme combinations were identified. Each heterotrimeric isoenzyme comprises a catalytic α subunit together with the unique ßγ subunit and one of three non-catalytic ß subunits: ß(1), ß(2) or the plant-specific ß(3) isoform. Thus, the AtSnRK1 heterotrimers contain the atypical ßγ subunit rather than a conventional γ subunit. Mammalian AMPK heterotrimers are phosphorylated on the T-loop (pThr175/176) within both catalytic a subunits. However, AtSnRK1 is insensitive to AMP and ADP, and is resistant to T-loop dephosphorylation by protein phosphatases, a process that inactivates other SNF1/AMPK family members. In addition, we show that SnRK1 is inhibited by a heat-labile, >30 kDa, soluble proteinaceous factor that is present in the lysate of young rosette leaves. Finally, none of the three SnRK1 carbohydrate-binding modules, located in the ß(1), ß(2) and ßγ subunits, associate with various carbohydrates, including starch, the plant analogue of glycogen to which AMPK binds in vitro. These data clearly demonstrate that AtSnRK1 is an atypical member of the SNF1/AMPK/SnRK1 family.

Determinants of oligosaccharide specificity of the carbohydrate-binding modules of AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) is an αßγ heterotrimer that is important in regulating energy metabolism in all eukaryotes. The ß-subunit exists in two isoforms (ß1 and ß2) and contains a carbohydrate-binding module (CBM) that interacts with glycogen. The two CBM isoforms (ß1- and ß2-CBM) are near identical in sequence and structure, yet show differences in carbohydrate-binding affinity. ß2-CBM binds linear carbohydrates with 4-fold greater affinity than ß1-CBM and binds single α1,6-branched carbohydrates up to 30-fold tighter. To understand these affinity differences, especially for branched carbohydrates, we determined the NMR solution structure of ß2-CBM in complex with the single α1,6-branched carbohydrate glucosyl-ß-cyclodextrin (gBCD) which supported the dynamic nature of the binding site, but resonance broadening prevented defining where the α1,6 branch bound. We therefore solved the X-ray crystal structures of ß1- and ß2-CBM, in complex with gBCD, to 1.7 and 2.0 Å (1 Å=0.1 nm) respectively. The additional threonine (Thr101) of ß2-CBM expands the size of the surrounding loop, creating a pocket that accommodates the α1,6 branch. Hydrogen bonds are formed between the α1,6 branch and the backbone of Trp99 and Lys102 side chain of ß2-CBM. In contrast, the α1,6 branch could not be observed in the ß1-CBM structure, suggesting that it does not form a specific interaction. The orientation of gBCD bound to ß1- and ß2-CBM is supported by thermodynamic and kinetic data obtained through isothermal titration calorimetry (ITC) and NMR. These results suggest that AMPK containing the muscle-specific ß2-isoform may have greater affinity for partially degraded glycogen.

AMP-activated protein kinase ß-subunit requires internal motion for optimal carbohydrate binding.

AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the ß-subunit, for which there are two isoforms (ß(1) and ß(2)). Muscle-specific ß(2)-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous ß(1)-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of ß(2)-CBM is concurrent with an increase in flexibility in ß(2)-CBM, which may account for the affinity differences between the two isoforms. In contrast to ß(1)-CBM, unbound ß(2)-CBM showed microsecond-to-millisecond motion at the base of a ß-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this ß-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from ß(2)-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the ß(2)-CBM mutant. Insertion of a threonine into the background of ß(1)-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms.

AMPK beta subunits display isoform specific affinities for carbohydrates.

AMP-activated protein kinase (AMPK) is a heterotrimer of catalytic (alpha) and regulatory (beta and gamma) subunits with at least two isoforms for each subunit. AMPK beta1 is widely expressed whilst AMPK beta2 is highly expressed in muscle and both beta isoforms contain a mid-molecule carbohydrate-binding module (beta-CBM). Here we show that beta2-CBM has evolved to contain a Thr insertion and increased affinity for glycogen mimetics with a preference for oligosaccharides containing a single alpha-1,6 branched residue. Deletion of Thr-101 reduces affinity for single alpha-1,6 branched oligosaccharides by 3-fold, while insertion of this residue into the equivalent position in the beta1-CBM sequence increases affinity by 3-fold, confirming the functional importance of this residue.

Thienopyridone drugs are selective activators of AMP-activated protein kinase beta1-containing complexes.

The AMP-activated protein kinase (AMPK) is an alphabetagamma heterotrimer that plays a pivotal role in regulating cellular and whole-body metabolism. Activation of AMPK reverses many of the metabolic defects associated with obesity and type 2 diabetes, and therefore AMPK is considered a promising target for drugs to treat these diseases. Recently, the thienopyridone A769662 has been reported to directly activate AMPK by an unexpected mechanism. Here we show that A769662 activates AMPK by a mechanism involving the beta subunit carbohydrate-binding module and residues from the gamma subunit but not the AMP-binding sites. Furthermore, A769662 exclusively activates AMPK heterotrimers containing the beta1 subunit. Our findings highlight the regulatory role played by the beta subunit in modulating AMPK activity and the possibility of developing isoform specific therapeutic activators of this important metabolic regulator.

Oligosaccharide recognition and binding to the carbohydrate binding module of AMP-activated protein kinase.

The AMP-activated protein kinase (AMPK) contains a carbohydrate-binding module (beta1-CBM) that is conserved from yeast to mammals. Beta1-CBM has been shown to localize AMPK to glycogen in intact cells and in vitro. Here we use Nuclear Magnetic Resonance spectroscopy to investigate oligosaccharide binding to 15N labelled beta1-CBM. We find that beta1-CBM shows greatest affinity to carbohydrates of greater than five glucose units joined via alpha,1-->4 glycosidic linkages with a single, but not multiple, glucose units in an alpha,1-->6 branch. The near identical chemical shift profile for all oligosaccharides whether cyclic or linear suggest a similar binding conformation and confirms the presence of a single carbohydrate-binding site.

AMP-activated protein kinase does not associate with glycogen alpha-particles from rat liver.

The AMP-activated protein kinase (AMPK) is heterotrimer consisting of alpha catalytic subunit and beta/gamma regulatory subunits. It acts as a critical focal point for whole body and cellular mechanisms maintaining energy homeostasis by regulating carbohydrate and lipid metabolism, food intake, gene transcription, and protein synthesis. The AMPK beta subunit contains a glycogen-binding domain that has been shown to associate with glycogen particles in vitro and glycogen phosphorylase and glycogen synthase in cultured cells. To determine whether AMPK associates with glycogen particles in vivo, we developed a procedure to purify glycogen alpha-particles to apparent homogeneity from rat liver. Using immunoreactivity and mass spectrometry we determined that AMPK does not associate with the glycogen particle in livers from random-fed rats. This surprising finding indicates that the glycogen-binding properties of the AMPK beta subunit are likely regulated and responsive to the metabolic status of the hepatocyte.