Large Hydrophobic Pocket Research Articles

Structure of WNT inhibitor adenomatosis polyposis coli down-regulated 1 (APCDD1), a cell-surface lipid-binding protein

Diverse extracellular proteins negatively regulate WNT signaling. One such regulator is adenomatosis polyposis coli down-regulated 1 (APCDD1), a conserved single-span transmembrane protein. In response to WNT signaling in a variety of tissues, APCDD1 transcripts are highly up-regulated. We have determined the three-dimensional structure of the extracellular domain of APCDD1, and this structure reveals an unusual architecture consisting of two closely apposed β-barrel domains (ABD1 and ABD2). ABD2, but not ABD1, has a large hydrophobic pocket that accommodates a bound lipid. The APCDD1 ECD can also bind to WNT7A, presumably via its covalently bound palmitoleate, a modification that is common to all WNTs and is essential for signaling. This work suggests that APCDD1 functions as a negative feedback regulator by titrating WNT ligands at the surface of responding cells.

A large conserved family of small-molecule carboxyl methyltransferases identified from microorganisms

Small-molecule carboxyl methyltransferases (CbMTs) constitute a small proportion of the reported methyltransferases, but they have received extensive attention due to their important physiological functions. Most of the small-molecule CbMTs isolated to date originate from plants and are members of the SABATH family. In this study, we identified a type of CbMT (OPCMT) from a group of Mycobacteria, which has a distinct catalytic mechanism from the SABATH methyltransferases. The enzyme contains a large hydrophobic substrate-binding pocket (~400Å3) and utilizes two conserved residues, Thr20 and Try194, to retain the substrate in a favorable orientation for catalytic transmethylation. The OPCMT_like MTs have a broad substrate scope and can accept diverse carboxylic acids enabling efficient production of methyl esters. They are widely (more than 10,000) distributed in microorganisms, including several well-known pathogens, whereas no related genes are found in humans. Invivo experiments implied that the OPCMT_like MTs was indispensable for M. neoaurum, suggesting that these proteins have important physiological functions.

Abstract 2717: A potent and highly tumor-selective KRAS inactivator for tumor targeting

Abstract Cancer driver mutations in the RAS-RAF-MEK-ERK pathway occur in 46% of all human cancers. This has inspired the successful development of many small molecule inhibitors of BRAF and MEK. However, development of RAS small molecule inhibitors has not been successful in large part due to the lack of a sufficiently large and deep hydrophobic pocket in RAS proteins to allow for small molecule binding. Notably, in this regard, many bacterial pathogens have evolved potent protein toxins to disrupt the RAS-RAF-MEK-ERK pathway. Fortunately, these naturally designed potent toxins, such as anthrax toxin, Fortunately, these potent, naturally designed toxins, such as anthrax toxin and DUF5, can be structurally modified to achieve high tumor specificity. DUF5 (amino acids Glu3581-Gln4089) is a toxin effector domain of MARTX (a multifunctional-autoprocessing repeats-in-toxin) from Vibrio vulnificus. DUF5 was recently identified to be a specific and potent endopeptidase that cleaves and inactivates RAS. To address the above critical unmet medical needs, in this work, we have generated an anthrax toxin/DUF5-based, highly tumor-selective RAS inactivator and evaluated its anti-tumor activity in a variety of tumor models. This tumor-selective RAS inactivator can specifically bind to the major anthrax toxin receptor CMG2 on tumor cells and tumor stromal cells, and strictly relies on the simultaneous presence of two distinct tumor-associated proteases, MMPs and urokinase, to gain entry into tumor cells and tumor stromal cells to proteolytically inactivate RAS signaling, achieving potent selective targeting. Our data show that our tumor-selective RAS inactivator displays potent cytotoxicity to cancer cells whose survival depends on oncogenic RAS signaling. Our engineered toxin displayed potent in vivo antitumor activity to a range of tumors with oncogenic RAS mutations in syngeneic and human xenograft mouse tumor models. Therefore, we have developed a tumor-selective, safe, and highly potent RAS inactivator that has high potential for targeting tumors with oncogenic RAS mutations. Citation Format: Shihui Liu, Zehua Zuo, Jie Liu. A potent and highly tumor-selective KRAS inactivator for tumor targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2717.

Design, synthesis and biological evaluation of sulfonylamidines as potent c-Met inhibitors by enhancing hydrophobic interaction.

The dysregulation of c-Met kinase has emerged as a significant contributing factor for the occurrence, progression, poor clinical outcomes and drug resistance of various human cancers. In our ongoing pursuit to identify promising c-Met inhibitors as potential antitumor agents, a docking study of the previously reported c-Met inhibitor 7 revealed a large unoccupied hydrophobic pocket, which could present an opportunity for further exploration of structure-activity relationships to improve the binding affinity with the allosteric hydrophobic back pocket of c-Met. Herein we performed structure-activity relationship and molecular modeling studies based on lead compound 7. The collective endeavors culminated in the discovery of compound 21j with superior efficacy to 7 and positive control foretinib by increasing the hydrophobic interaction with the hydrophobic back pocket of c-Met active site.

Ostrinia furnacalis PBP2 solution NMR structure: Insight into ligand binding and release mechanisms.

Ostrinia furnacalis is an invasive lepidopteran agricultural pest that relies on olfaction for mating and reproduction. Male moths have an extremely sensitive olfactory system that can detect the sex pheromones emitted by females over a great distance. Pheromone-binding proteins present in the male moth antenna play a key role in the pheromone uptake, transport, and release at the dendritic membrane of the olfactory neuron. Here, we report the first high-resolution NMR structure of a pheromone-binding protein from an Ostrinia species at pH 6.5. The core of the Ostrinia furnacalis PBP2 (OfurPBP2) consists of six helices, α1a (2-14), α1b (16-22), α2 (27-37), α3 (46-60), α4 (70-80), α5 (84-100), and α6 (107-124) surrounding a large hydrophobic pocket. The structure is stabilized by three disulfide bridges, 19-54, 50-108, and 97-117. In contrast to the unstructured C-terminus of other lepidopteran PBPs, the C-terminus of OfurPBP2 folds into an α-helix (α7) at pH 6.5. The protein has nanomolar affinity towards both pheromone isomers. Molecular docking of both pheromones, E-12 and Z-12-tetradecenyl acetate, to OfurPBP2 revealed that the residues Met5, Lys6, Met8, Thr9, Phe12, Phe36, Trp37, Phe76, Ser115, Phe118, Lys119, Ile122, His123, and Ala128 interact with both isomers, while Thr9 formed a hydrogen bond with the acetate head group. NMR structure and thermal unfolding studies with CD suggest that ligand release at pH 4.5 is likely due to the partial unfolding of the protein.

Computational characterization of homologous ligands binding to a deep hydrophobic pocket in Shigella flexneri pilot protein MxiM.

The type III secretion system (T3SS) is an important molecular machinery in gram-negative bacteria Shigella flexneri as it provides ways for translocating virulence factors from the bacteria into host cells, eventually leading to severe disease symptoms such as bacillary dysentery. Due to the rising concerns of antibiotics resistance in bactericidal strategy, the anti-virulence strategy that primarily targets the T3SS components becomes an attractive alternative. MxiM, the secretin pilot protein of Shigella flexneri, binds the secretin MxiD and facilitates the formation of the secretin ring in outer membrane in T3SS assembly. MxiM harbors a large hydrophobic pocket that has been shown to be important in MxiM-MxiD interaction. In this work, I examined the ligand binding property of MxiM by performing molecular dynamics (MD) simulations of the association between MxiM and a series of hydrophobic ligands, with simulation time amounted to 30 μs. MD simulations successfully captured spontaneous ligand binding events in 153 of the 300 trajectories. The ligand binding can be categorized into two types: a fast type, in which the ligand binds quickly into the hydrophobic pocket and a slow type, in which the ligand forms an encounter complex with the protein before binding into the hydrophobic pocket. Using the MxiM-ligand binding poses captured in MD simulations, I additionally performed umbrella-sampling MD simulations with total simulation time amounted to 63 μs to obtain protein-ligand binding free energies. The relationship between the ligand binding free energy and ligand size appears to be nonlinear and exhibits an exponential decay pattern. In summary, I performed computational characterization of MxiM-hydrophobic ligand binding capabilities and properties, which may provide valuable insights into designing anti-bacterial medicine against antibiotics resistance in Shigella flexneri.

Structural basis for mitoguardin-2 mediated lipid transport at ER-mitochondrial membrane contact sites

The endoplasmic reticulum (ER)-mitochondria contact site (ERMCS) is crucial for exchanging biological molecules such as phospholipids and Ca2+ ions between these organelles. Mitoguardin-2 (MIGA2), a mitochondrial outer membrane protein, forms the ERMCS in higher eukaryotic cells. Here, we report the crystal structures of the MIGA2 Lipid Droplet (LD) targeting domain and the ER membrane protein VAPB bound to the phosphorylated FFAT motif of MIGA2. These structures reveal that the MIGA2 LD targeting domain has a large internal hydrophobic pocket that accommodates phospholipids and that two phosphorylations of the FFAT motif are required for tight interaction of MIGA2 with VAPB, which enhances the rate of lipid transport. Further biochemical studies show that MIGA2 transports phospholipids between membranes with a strong preference for binding and trafficking phosphatidylserine (PS). These results provide a structural and molecular basis for understanding how MIGA2 mediates the formation of ERMCS and facilitates lipid trafficking at the ERMCS.

Interaction of Carrier Protein with Potential Metallic Drug Candidate N-Glycoside 'GATPT': Validation by Multi-Spectroscopic and Molecular Docking Approaches.

Lysozyme is often used as a model protein to study interaction with drug molecules and to understand biological processes which help in illuminating the therapeutic effectiveness of the drug. In the present work, in vitro interaction studies of 1-{(2-hydroxyethyl)amino}-2-amino-1,2-dideoxy-d-glucose triphenyl tin (IV) (GATPT) complex with lysozyme were carried out by employing various biophysical methods such as absorption, fluorescence, and circular dichroism (CD) spectroscopies. The experimental results revealed efficient binding affinity of GATPT with lysozyme with intrinsic binding (Kb) and binding constant (K) values in the order of 105 M−1. The number of binding sites and thermodynamic parameters ΔG, ΔH, and ΔS at four different temperatures were also calculated and the interaction of GATPT with lysozyme was found to be enthalpy and entropy driven. The CD spectra revealed alterations in the population of α–helical content within the secondary structure of lysozyme in presence of GATPT complex. The morphological analysis of the complex with lysozyme and lysozyme-DNA condensates was carried out by employing confocal and SEM studies. Furthermore, the molecular docking studies confirmed the interaction of GATPT within the larger hydrophobic pocket of the lysozyme via several non-covalent interactions.

Cryo-EM Structure of Mechanosensitive Channel YnaI Using SMA2000: Challenges and Opportunities.

Mechanosensitive channels respond to mechanical forces exerted on the cell membrane and play vital roles in regulating the chemical equilibrium within cells and their environment. High-resolution structural information is required to understand the gating mechanisms of mechanosensitive channels. Protein-lipid interactions are essential for the structural and functional integrity of mechanosensitive channels, but detergents cannot maintain the crucial native lipid environment for purified mechanosensitive channels. Recently, detergent-free systems have emerged as alternatives for membrane protein structural biology. This report shows that while membrane-active polymer, SMA2000, could retain some native cell membrane lipids on the transmembrane domain of the mechanosensitive-like YnaI channel, the complete structure of the transmembrane domain of YnaI was not resolved. This reveals a significant limitation of SMA2000 or similar membrane-active copolymers. This limitation may come from the heterogeneity of the polymers and nonspecific interactions between the polymers and the relatively large hydrophobic pockets within the transmembrane domain of YnaI. However, this limitation offers development opportunities for detergent-free technology for challenging membrane proteins.

Arabidopsis Carboxylesterase 20 Binds Strigolactone and Increases Branches and Tillers When Ectopically Expressed in Arabidopsis and Maize.

Severe drought stress can delay maize silk emergence relative to the pollen shedding period, resulting in poor fertilization and reduced grain yield. Methods to minimize the delay in silking could thus improve yield stability. An Arabidopsis enhancer-tagged carboxylesterase 20 (AtCXE20) line was identified in a drought tolerance screen. Ectopic expression of AtCXE20 in Arabidopsis and maize resulted in phenotypes characteristic of strigolactone (SL)-deficient mutants, including increased branching and tillering, decreased plant height, delayed senescence, hyposensitivity to ethylene, and reduced flavonols. Maize silk growth was increased by AtCXE20 overexpression, and this phenotype was partially complemented by exogenous SL treatments. In drought conditions, the transgenic maize plants silked earlier than controls and had decreased anthesis-silking intervals. The purified recombinant AtCXE20 protein bound SL in vitro, as indicated by SL inhibiting AtCXE20 esterase activity and altering AtCXE20 intrinsic fluorescence. Homology modeling of the AtCXE20 three-dimensional (3D) protein structure revealed a large hydrophobic binding pocket capable of accommodating, but not hydrolyzing SLs. The AtCXE20 protein concentration in transgenic maize tissues was determined by mass spectrometry to be in the micromolar range, well-above known endogenous SL concentrations. These results best support a mechanism where ectopic expression of AtCXE20 with a strong promoter effectively lowers the concentration of free SL by sequestration. This study revealed an agriculturally important role for SL in maize silk growth and provided a new approach for altering SL levels in plants.

Dynamic interaction between lysozyme and ceftazidime: Experimental and molecular simulation approaches

The interaction of an important antibiotic drug, ceftazidime, with an imperative globular protein, lysozyme (which also possesses antibacterial properties), was studied. The formation of a complex between ceftazidime and lysozyme was verified by ultraviolet–visible (UV–Vis) spectroscopy. Intrinsic fluorescence spectroscopy (FS) was primarily employed to obtain information about the binding mechanism. The fluorescence data were corrected for the inner filter effect (IFE) before the analysis because of the strong absorption of ceftazidime at the excitation wavelength and the emission range of lysozyme. The apparent fluorescence quenching parameters, which were obtained after the IFE correction, exhibited the weak affinity between lysozyme and ceftazidime. The interaction was due to the dynamic quenching with a binding ratio of 1:1. Ceftazidime exerted small effect on the secondary structure of lysozyme. The binding site of ceftazidime in lysozyme was determined by molecular docking. The large hydrophobic pocket of lysozyme was the most preferred binding site for ceftazidime. The interaction was, primarily, reinforced by hydrophobic forces and hydrogen bonding with a small participation of electrostatic forces. Molecular dynamics (MD) simulations were also performed to theoretically understand the behavior of the drug–protein complex. The stability of lysozyme changed slightly in the presence of ceftazidime. The pattern of the radius of gyration (Rg) in the absence and presence of ceftazidime also exhibited a slight change. There was a slight decrease in the residual fluctuation of residues 50–90, whereas a significant increase was observed in the residual fluctuations of residues 100–120. In the 100 ns simulation of the formation of lysozyme and ceftazidime, the disruption of hydrogen bonding occurred with the probability of forming three to six hydrogen bonds during most of the time evolutions of the simulation. The density-functional theory (DFT) calculations indicated that the stability of ceftazidime decreased after binding with lysozyme.

Design, synthesis, and biological studies of novel 3-benzamidobenzoic acid derivatives as farnesoid X receptor partial agonist.

Farnesoid X receptor (FXR), a bile acid-activated nuclear receptor, regulates the metabolism of bile acid and lipids as well as maintains the stability of internal environment. FXR was considered as a therapeutic target of liver disorders, such as drug-induced liver injury, fatty liver and cholestasis. The previous reported FXR partial agonist 6 was a suitable lead compound in terms of its high potent and low molecular size, while the docking study of compound 6 suggested a large unoccupied hydrophobic pocket, which might be provided more possibility of structure-activity relationship (SAR) study. In this study, we have performed comprehensive SAR and molecular modeling studies based on lead compound 6. All of these efforts resulted in the identification of a novel series of FXR partial agonists. In this series, compound 41 revealed the best activity and strong interaction with binding pocket of FXR. Moreover, compound 41 protected mice against acetaminophen-induced hepatotoxicity by the regulation of FXR-related gene expression and improving antioxidant capacity. In summary, these results suggest that compound 41 is a promising FXR partial agonist suitable for further investigation.

Pyrethroid Carboxylesterase PytH from Sphingobium faniae JZ-2: Structure and Catalytic Mechanism.

Carboxylesterase PytH, isolated from the pyrethroid-degrading bacterium Sphingobium faniae JZ-2, could rapidly hydrolyze the ester bond of a wide range of pyrethroid pesticides, including permethrin, fenpropathrin, cypermethrin, fenvalerate, deltamethrin, cyhalothrin, and bifenthrin. To elucidate the catalytic mechanism of PytH, we report here the crystal structures of PytH with bifenthrin (BIF) and phenylmethylsulfonyl fluoride (PMSF) and two PytH mutants. Though PytH shares low sequence identity with reported α/β-hydrolase fold proteins, the typical triad catalytic center with Ser-His-Asp triad (Ser78, His230, and Asp202) is present and vital for the hydrolase activity. However, no contact was found between Ser78 and His230 in the structures we solved, which may be due to the fact that the PytH structures we determined are in their inactive or low-activity forms. The structure of PytH is composed of a core domain and a lid domain; some hydrophobic amino acid residues surrounding the substrate from both domains form a deeper and wider hydrophobic pocket than its homologous structures. This indicates that the larger hydrophobic pocket makes PytH fit for its larger substrate binding; both lid and core domains are involved in substrate binding, and the lid domain-induced core domain movement may make the active center correctly positioned with substrates.IMPORTANCE Pyrethroid pesticides are widely applied in agriculture and household; however, extensive use of these pesticides also causes serious environmental and health problems. The hydrolysis of pyrethroids by carboxylesterases is the major pathway of microbial degradation of pyrethroids, but the structure of carboxylesterases and its catalytic mechanism are still unknown. Carboxylesterase PytH from Sphingobium faniae JZ-2 could effectively hydrolyze a wide range of pyrethroid pesticides. The crystal structures of PytH are solved in this study. This showed that PytH belongs to the α/β-hydrolase fold proteins with typical catalytic Ser-His-Asp triad, though PytH has a low sequence identity (about 20%) with them. The special large hydrophobic binding pocket enabled PytH to bind bigger pyrethroid family substrates. Our structures shed light on the substrate selectivity and the future application of PytH and deepen our understanding of α/β-hydrolase members.

Design and Discovery of Covalent α-GalCer Derivatives as Potent CD1d Ligands.

CD1d is a nonpolymorphic antigen-presenting protein responsible for the regulation of natural killer T (NKT) cell activation. α-Galactosyl ceramide (α-GalCer, KRN7000) is the representative CD1d ligand that can bind to the CD1d protein. The resulting complex is recognized by the T cell receptors of the NKT cell, inducing various immune responses. Previous structure-activity relationship studies of α-GalCer have revealed that the ability of NKT cells to induce cytokines depends on the ligand structure, and in particular, ligands that form more stable complexes with CD1d display potent activity. We focused on the Cys residue of the large hydrophobic pockets of CD1d (A' pocket) and developed α-GalCer derivatives containing groups that can form covalent bonds. The assay results revealed that these ligands displayed higher levels of cytokine production and Th2 cell-type cytokine polarization response. Furthermore, the LC-MS/MS analysis indicated that the chloroacetylamide-containing ligand was covalently bound to Cys12 of CD1d, which suggests that the enhanced activities result from the formation of a stable CD1d-ligand complex. To our knowledge, this is the first ligand that allows covalent bond formation to CD1d under physiological conditions.

Identification of a Heme Activation Site on the MD-2/TLR4 Complex

Lipopolysaccharide (LPS), the first-identified TLR4 agonist, binds myeloid differentiation factor-2 (MD-2) in association with TLR4 to initiate TLR4 signaling. LPS binds to a large hydrophobic pocket in MD-2 and directly bridges the MD-2/TLR4 heterodimer. The MD-2/TLR4 complex also recognizes a diverse number of endogenous molecules released from injured cells called damage-associated molecular patterns or DAMPs. One such DAMP is heme. Large amounts of heme can be released intravascularly by trauma, sepsis, malaria and red blood cell disorders such as sickle cell disease (SCD). Recent studies underscore the importance of heme-mediated MD-2/TLR4 activation in inflammation, vessel occlusion, lethality and pulmonary injury in SCD. Therefore, we examined human MD-2 for potential heme activation sites. Recombinant MD-2 (rMD-2) was produced by transfecting Chinese hamster ovary (CHO) cells with human MD-2 plasmids. After 72 hours, Western blots of the CHO-conditioned media demonstrated soluble rMD-2 was present. Heme was shown to bind rMD-2 using pull-down assays utilizing heme-agarose or biotin-heme with streptavidin-agarose coupled with MD-2 Western blots of the pellet. These pull-down assays of rMD2 were inhibited by excess heme, indicating specific binding of heme to rMD-2. UV/visible scanning spectroscopy (250 - 550 nm) of purified rMD-2 in the presence or absence of heme, confirmed specific rMD-2-heme binding. In silico analyses combining both structure and sequence-based methods, identified two potential heme docking sites on MD-2 near conserved amino acids W23/S33/Y34 and Y36/C37/I44 (Figure 1). To determine whether MD-2 mutations at these two sites affect heme-MD-2/TLR4 signaling, HEK293 cells were transfected with plasmids encoding human MD-2, TLR4, CD14 and an NF-κB luciferase reporter. After 24 hours, transfected cells were stimulated with heme (10 μM) or LPS (10 ng/ml) for 6 hours and NF-κB luciferase reporter activity was measured. Heme or LPS treatment elicited robust luciferase activity. The addition of both heme and LPS had an additive effect on NF-κB luciferase activity. Absence of an MD-2, TLR4 or CD14 plasmid abolished NF-κB luciferase reporter responses to heme and/or LPS. When plasmids encoding MD-2 point mutants W23A or Y34A were introduced into MD-2, heme-induced NF-κB luciferase activity was inhibited 91-92% compared to WT-MD-2. The S33A MD-2 mutant stimulated NF-κB luciferase activity by 40%. NF-κB activation by LPS was marginally affected by the same mutants. Biotin-heme/streptavidin-agarose pulled down 68% less W23A mutant MD-2 and 80% less W23A/S33A/Y34A mutant MD-2 than WT-MD-2. In contrast, at the other potential heme binding site, heme-induced NF-κB luciferase activity was increased in mutants Y36A (120%), C37A (121%) and I44A (230%) compared to WT-MD-2. These data suggest that amino acids W23 and Y34 on MD-2 are specific for heme binding and TLR4 signaling. This heme activation site was targeted for potential inhibitors using virtual screening. The virtual screen identified 60 potential inhibitors for screening in heme-stimulated primary human umbilical vein endothelial cells (HUVEC) and a human U-937 monocyte cell line. Four of these molecules inhibited Weibel-Palade body P-selectin and von Willebrand factor expression in HUVEC and IL-8 secretion by U-937 cells stimulated with heme. We conclude that heme activates MD-2/TLR4 signaling at residues W23 and Y34 on MD-2, which might be a drugable target in SCD and other hemolytic diseases. Disclosures Belcher: Mitobridge, an Astellas Company: Consultancy, Research Funding. Vercellotti:Mitobridge, an Astellas Company: Consultancy, Research Funding.

Structural analysis of a Vibrio phospholipase reveals an unusual Ser–His–chloride catalytic triad

Phospholipases can disrupt host membranes and are important virulence factors in many pathogens. VvPlpA is a phospholipase A2 secreted by Vibrio vulnificus and essential for virulence. Its homologs, termed thermolabile hemolysins (TLHs), are widely distributed in Vibrio bacteria, but no structural information for this virulence factor class is available. Herein, we report the crystal structure of VvPlpA to 1.4-Å resolution, revealing that VvPlpA contains an N-terminal domain of unknown function and a C-terminal phospholipase domain and that these two domains are packed closely together. The phospholipase domain adopts a typical SGNH hydrolase fold, containing the four conserved catalytic residues Ser, Gly, Asn, and His. Interestingly, the structure also disclosed that the phospholipase domain accommodates a chloride ion near the catalytic His residue. The chloride is five-coordinated in a distorted bipyramid geometry, accepting hydrogen bonds from a water molecule and the amino groups of surrounding residues. This chloride substitutes for the most common Asp/Glu residue and forms an unusual Ser-His-chloride catalytic triad in VvPlpA. The chloride may orient the catalytic His and stabilize the charge on its imidazole ring during catalysis. Indeed, VvPlpA activity depended on chloride concentration, confirming the important role of chloride in catalysis. The VvPlpA structure also revealed a large hydrophobic substrate-binding pocket that is capable of accommodating a long-chain acyl group. Our results provide the first structure of the TLH family and uncover an unusual Ser-His-chloride catalytic triad, expanding our knowledge on the biological role of chloride.

Structure-activity relationship studies of Bz amide-containing α-GalCer derivatives as natural killer T cell modulators

AbstractCD1d is a non-polymorphic antigen-presenting glycoprotein that recognizes glycolipids as ligands. Ligands bind to the hydrophobic grooves of CD1d, and the resulting ligand-CD1d complexes activate natural killer T (NKT) cells by means of T cell receptor recognition, leading to the secretion of various cytokines. However, details of the ligand recognition mechanism of a large hydrophobic ligand binding pocket and the relationship between cytokine induction and ligand structure are unclear. We report the synthesis of α-GalCer derivatives containing a Bz amide group having various substituting groups in the ceramide moiety, and the analysis of the structure-activity relationships. The assays reveal that the Bz amide-containing CD1d ligands function as NKT cell modulators displaying Th2 cytokine biasing responses. Furthermore, molecular dynamics simulation studies suggest that the phenyl groups can interact with the aromatic amino acid residues in the lipid binding pocket of CD1d.

Probing the Anticancer Action of Novel Ferrocene Analogues of MNK Inhibitors.

Two novel ferrocene-containing compounds based upon a known MNK1/2 kinase (MAPK-interacting kinase) inhibitor have been synthesized. The compounds were designed to use the unique shape of ferrocene to exploit a large hydrophobic pocket in MNK1/2 that is only partially occupied by the original compound. Screening of the ferrocene analogues showed that both exhibited potent anticancer effects in several breast cancer and AML (acute myeloid leukemia) cell lines, despite a loss of MNK potency. The most potent ferrocene-based compound 5 was further analysed in vitro in MDA-MB-231 (triple negative breast cancer cells). Dose–response curves of compound 5 for 2D assay and 3D assay generated IC50 values (half maximal inhibitory concentration) of 0.55 µM and 1.25 µM, respectively.

Site-specific effect of polar functional group-modification in lipids of TLR2 ligands for modulating the ligand immunostimulatory activity

Toll-like receptor 2 (TLR2), a member of the TLR innate immune receptor family, recognizes lipoproteins from bacteria and modulates the immune response by inducing the expression of various cytokines. TLR2 has a large hydrophobic pocket that recognizes long fatty acyl groups on TLR2 ligands. However, few studies have focused on the property of the hydrophobic TLR2 pocket. Based on the X-ray crystal structure of TLR2, small polar regions were found in the hydrophobic TLR2 pocket. Interactions between the polar residues and ligands were explored here by designing and synthesizing a Pam2CSK4 derivative of the TLR2 ligands, containing an amide group within the lipid moiety. We evaluated the binding affinities and immunomodulatory activities of these ligands. Results suggested that the amide groups in the lipid chain interacted with the polar residues in the hydrophobic lipid-binding pocket of TLR2.

Crystal structure of the mammalian lipopolysaccharide detoxifier

LPS is a potent bacterial endotoxin that triggers the innate immune system. Proper recognition of LPS by pattern-recognition receptors requires a full complement of typically six acyl chains in the lipid portion. Acyloxyacyl hydrolase (AOAH) is a host enzyme that removes secondary (acyloxyacyl-linked) fatty acids from LPS, rendering it immunologically inert. This activity is critical for recovery from immune tolerance that follows Gram-negative infection. To understand the molecular mechanism of AOAH function, we determined its crystal structure and its complex with LPS. The substrate's lipid moiety is accommodated in a large hydrophobic pocket formed by the saposin and catalytic domains with a secondary acyl chain inserted into a narrow lateral hydrophobic tunnel at the active site. The enzyme establishes dispensable contacts with the phosphate groups of LPS but does not interact with its oligosaccharide portion. Proteolytic processing allows movement of an amphipathic helix possibly involved in substrate access at membranes.