TREM1 activation of myeloid cells promotes antitumor immunity.

Myeloid cells in the tumor microenvironment (TME) can exist in immunosuppressive and immunostimulatory states that impede or promote antitumor immunity, respectively. Blocking suppressive myeloid cells or increasing stimulatory cells to enhance antitumor immune responses is an area of interest for therapeutic intervention. Triggering receptor expressed on myeloid cells-1 (TREM1) is a proinflammatory receptor that amplifies immune responses. TREM1 is expressed on neutrophils, subsets of monocytes and tissue macrophages, and suppressive myeloid populations in the TME, including tumor-associated neutrophils, monocytes, and tumor-associated macrophages. Depletion or inhibition of immunosuppressive myeloid cells, or stimulation by TREM1-mediated inflammatory signaling, could be used to promote an immunostimulatory TME. We developed PY159, an afucosylated humanized anti-TREM1 monoclonal antibody with enhanced FcγR binding. PY159 is a TREM1 agonist that induces signaling, leading to up-regulation of costimulatory molecules on monocytes and macrophages, production of proinflammatory cytokines and chemokines, and enhancement of T cell activation in vitro. An antibody against mouse TREM1, PY159m, promoted antitumor efficacy in syngeneic mouse tumor models. These results suggest that PY159-mediated agonism of TREM1 on tumoral myeloid cells can promote a proinflammatory TME and offer a promising strategy for immunotherapy.

Biophysical Approaches to the Study of LeuT, a Prokaryotic Homolog of Neurotransmitter Sodium Symporters.

Ion-coupled secondary transport is utilized by multiple integral membrane proteins as a means of achieving the thermodynamically unfavorable translocation of solute molecules across the lipid bilayer. The chemical nature of these molecules is diverse and includes sugars, amino acids, neurotransmitters, and other ions. LeuT is a sodium-coupled, nonpolar amino acid symporter and eubacterial member of the solute carrier 6 (SLC6) family of Na(+)/Cl(-)-dependent neurotransmitter transporters. Eukaryotic counterparts encompass the clinically and pharmacologically significant transporters for γ-aminobutyric acid (GABA), glycine, serotonin (5-hydroxytryptamine, 5-HT), dopamine (DA), and norepinephrine (NE). Since the crystal structure of LeuT was first solved in 2005, subsequent crystallographic, binding, flux, and spectroscopic studies, complemented with homology modeling and molecular dynamic simulations, have allowed this protein to emerge as a remarkable mechanistic paradigm for both the SLC6 class as well as several other sequence-unrelated SLCs whose members possess astonishingly similar architectures. Despite yielding groundbreaking conceptual advances, this vast treasure trove of data has also been the source of contentious hypotheses. This chapter will present a historical scientific overview of SLC6s; recount how the initial and subsequent LeuT structures were solved, describing the insights they each provided; detail the accompanying functional techniques, emphasizing how they either supported or refuted the static crystallographic data; and assemble these individual findings into a mechanism of transport and inhibition.

Structure and regulatory interactions of the cytoplasmic terminal domains of serotonin transporter.

Uptake of neurotransmitters by sodium-coupled monoamine transporters of the NSS family is required for termination of synaptic transmission. Transport is tightly regulated by protein-protein interactions involving the small cytoplasmic segments at the amino- and carboxy-terminal ends of the transporter. Although structures of homologues provide information about the transmembrane regions of these transporters, the structural arrangement of the terminal domains remains largely unknown. Here, we combined molecular modeling, biochemical, and biophysical approaches in an iterative manner to investigate the structure of the 82-residue N-terminal and 30-residue C-terminal domains of human serotonin transporter (SERT). Several secondary structures were predicted in these domains, and structural models were built using the Rosetta fragment-based methodology. One-dimensional (1)H nuclear magnetic resonance and circular dichroism spectroscopy supported the presence of helical elements in the isolated SERT N-terminal domain. Moreover, introducing helix-breaking residues within those elements altered the fluorescence resonance energy transfer signal between terminal cyan fluorescent protein and yellow fluorescent protein tags attached to full-length SERT, consistent with the notion that the fold of the terminal domains is relatively well-defined. Full-length models of SERT that are consistent with these and published experimental data were generated. The resultant models predict confined loci for the terminal domains and predict that they move apart during the transport-related conformational cycle, as predicted by structures of homologues and by the "rocking bundle" hypothesis, which is consistent with spectroscopic measurements. The models also suggest the nature of binding to regulatory interaction partners. This study provides a structural context for functional and regulatory mechanisms involving SERT terminal domains.

Facile access of stable divalent tin compounds with terminal methyl, amide, fluoride, and iodide substituents.

The stable beta-diketiminate tin(II) complexes LSnX [L = HC(CMeNAr)2, Ar = 2,6-iPr2C6H3] with terminal methyl, amide, fluoride, and iodide (X = Me, N(SiMe3)2, F, I) are described. LSnMe (2) is synthesized by salt metathesis reaction of LSnCl (1) with MeLi and can be isolated in the form of yellow crystals in 88% yield. Compound LSnN(SiMe3)2 (3) was obtained by treatment of LH with 2 equiv of KN(SiMe3)2 in THF followed by adding 1 equiv of SnCl2. Reaction of 2 and 3 respectively with Me3SnF in toluene provided the tin(II)fluoride LSnF (4) with a terminal fluorine as colorless crystals in 85% yield. 4 is highly soluble in common organic solvents. The reaction of LLi(OEt2) with 1 equiv of SnI2 in diethyl ether afforded the LSnI (5). Compounds 2, 3, 4, and 5 were characterized by microanalysis, multinuclear NMR spectroscopy, and X-ray structural analysis. Single crystal X-ray structural analyses indicate that all the compounds (2, 3, 4, 5) are monomeric and the tin center resides in a trigonal-pyramidal environment.

Structure of tripeptidyl-peptidase I provides insight into the molecular basis of late infantile neuronal ceroid lipofuscinosis.

Late infantile neuronal ceroid lipofuscinosis, a fatal neurodegenerative disease of childhood, is caused by mutations in the TPP1 gene that encodes tripeptidyl-peptidase I. We show that purified TPP1 requires at least partial glycosylation for in vitro autoprocessing and proteolytic activity. We crystallized the fully glycosylated TPP1 precursor under conditions that implied partial autocatalytic cleavage between the prosegment and the catalytic domain. X-ray crystallographic analysis at 2.35 angstroms resolution reveals a globular structure with a subtilisin-like fold, a Ser475-Glu272-Asp360 catalytic triad, and an octahedrally coordinated Ca2+-binding site that are characteristic features of the S53 sedolisin family of peptidases. In contrast to other S53 peptidases, the TPP1 structure revealed steric constraints on the P4 substrate pocket explaining its preferential cleavage of tripeptides from the unsubstituted N terminus of proteins. Two alternative conformations of the catalytic Asp276 are associated with the activation status of TPP1. 28 disease-causing missense mutations are analyzed in the light of the TPP1 structure providing insight into the molecular basis of late infantile neuronal ceroid lipofuscinosis.

Structures of viscotoxins A1 and B2 from European mistletoe solved using native data alone.

Crystals of the cytotoxic thionin proteins viscotoxins A1 and B2 extracted from mistletoe diffracted to high resolution (1.25 and 1.05 A, respectively) and are excellent candidates for testing crystallographic methods. Ab initio direct methods were only successful in solving the viscotoxin B2 structure, which with 861 unique non-H atoms is one of the largest unknown structures without an atom heavier than sulfur to be solved in this way, but sulfur-SAD phasing provided a convincing solution for viscotoxin A1. Both proteins form dimers in the crystal and viscotoxin B2 (net charge +4 per monomer), but not viscotoxin A1 (net charge +6), is coordinated by sulfate or phosphate anions. The viscotoxin A1 crystal has a higher solvent content than the viscotoxin B2 crystal (49% as opposed to 28%) with solvent channels along the crystallographic 4(3) axes.

From unstable to stable: half-metallocene catalysts for olefin polymerization.

The reaction of LAlMeOH [L = CH(N(Ar)(CMe))2, Ar = 2,6-i-Pr2C6H3] with CpTiMe3, Cp*TiMe3, and Cp*ZrMe3 was investigated to yield LAlMe(mu-O)TiMe2Cp (2), LAlMe(mu-O)TiMe2Cp* (3), and LAlMe(mu-O)ZrMe2Cp* (4), respectively. The resulting compounds 2-4 are stable at elevated temperatures, in contrast to their precursors such as CpTiMe3 and Cp*ZrMe3, which already decompose below room temperature. Compounds 2-4 were characterized by single-crystal X-ray structural analysis. Compounds 2 and 3 were tested for ethylene polymerization in the presence of methylaluminoxane. The half-metallocene complex 3 has higher activity compared to 2. The polydispersities are in the range from 2.8 to 4.2. A copolymerization with styrene was not observed.

A reactivity change of a strontium monohydroxide by umpolung to an acid.

Controlled hydrolysis of strontium amide LSrN(SiMe 3) 2(thf) (L = CH(CMe2,6- i-Pr 2C 6H 3N) 2) ( 1) gave an unprecedented example of a hydrocarbon-soluble strontium hydroxide, [LSr(thf)(mu-OH) 2Sr(thf) 2L] ( 2). In compound 2, the tetrahydrofuran (THF) molecules can easily replaced by benzophenone and triphenylphosphine oxide to yield [LSr(mu-OH)(OCPh 2)] 2 ( 3) and [LSr(mu-OH)(OPPh 3)] 2 ( 4) compounds. Among the two strontium atoms of 2, one is coordinated to a single THF molecule, while the other is coordinated to two THF molecules. Interestingly, strontium hydroxide complex 2 behaves as an acid in its reaction with Zr(NMe 2) 4 and results in a heterobimetallic oxide, [LSr(mu-O)Zr(NMe 2) 3] 2 ( 5). Compound 5 is dimeric in the solid state and contains a Sr 2Zr 2O 2 core.

Molybdenum oxo and imido complexes of beta-diketiminate ligands: synthesis and structural aspects.

Treatment of [MoO2(eta2-Pz)2] (Pz = 3,5-di-tert-butylpyrazolate) with the diketiminate ligand NacNacH (NacNac = CH[C(Me)NAr]2-, Ar = 2,6-Me2C6H3) at 55 degrees C leads under reduction of the metal to the formation of the dimeric molybdenum(V) compound [{MoO2(NacNac)}2] (1). The compound was characterized by spectroscopic means and by X-ray crystal structure analysis. The dimer consists of a [Mo2O4]2+ core with a short Mo-Mo bond (2.5591(5) A) and one coordinated diketiminate ligand on each metal atom. The reaction of [MoO2(eta2-Pz)2] with NacNacH in benzene at room temperature leads to a mixture of 1 and the monomeric molybdenum(VI) compound [MoO2(NacNac)(eta2-Pz)] (2). From such solutions, yellow crystals of 2 suitable for X-ray structural analysis were obtained revealing the coordination of one bidentate NacNac and one eta2-coordinate Pz ligand. This renders the two oxo groups inequivalent. Further high oxidation state molybdenum compounds containing the NacNac ligand were obtained by the reaction of [Mo(NAr)2Cl2(dme)] (Ar = 2,6-Me2C6H3) and [Mo(N-t-Bu)2Cl2(dme)] (dme = dimethoxyethane) with 1 equiv of the potassium salt NacNacK forming [Mo(NAr)2Cl(NacNac)] (3) and [Mo(N-t-Bu)2Cl(NacNac)] (4), respectively, in good yields. The X-ray structure analysis of 3 revealed a penta-coordinate compound where the geometry is best described as trigonal-bipyramidal.

Pseudohelical and helical primary structures of 1,2-spiroannelated four- and five-membered rings: syntheses and chiroptical properties.

The pseudohelical hydrocarbons (R)-6, (S)-7, and (R)-8 and the helical hydrocarbon (P)-9, formally derived from the helical hydrocarbon (P)-4 by stepwise replacement of each of the four-membered rings by a five-membered ring, have been prepared. Their optical rotations vary systematically, both in magnitude and sign. Of the extremes, (P)-4 represents the usual case of a right-handed dextrorotatory helix, while (P)-9 represents the unusual case of a right-handed levorotatory helix. To rationalize these facts, DFT calculations of the rotatory power of (P)-helices of three-, four-, and five-membered rings have been performed. The results show a very good agreement with the experimental data for the rigid helices of three-membered rings and always show the correct sign and order of magnitude for the flexible helices of four- and five-membered rings for which Boltzmann-averaged optical rotations of up to six conformers had to be used. Within the conformers of the latter, a set of large dihedral angles for the bonds of the inner sphere correspond to a high specific rotation, and a set of small dihedral angles correspond to a low specific rotation. As a consequence, the Boltzmann-averaged values markedly depend on the geometry and weight of the conformers involved.

Rhenium(V) oxo complexes with acetylacetone derived Schiff bases: structure and catalytic epoxidation.

Substitution reactions of rhenium(V) oxo precursors [ReOCl3(PPh3)2] or [NBu4][ReOCl4] with the bidentate acetylacetone-derived ketoamine ligands APOH = 4-anilino-3-penten-2-one, DPOH = 4-[2,6-dimethylanilino]-3-penten-2-one, and MTPOH = 4-[2-(methylthio)anilino]-3-penten-2-one gave the complexes [ReO(APO)Cl2(PPh3)] (1), [ReO(DPO)Cl2(PPh3)] (2), and [NBu4][ReOLCl3] (3, L = APO; 4, L = DPO; 5, L = MTPO), respectively. All complexes exhibit only one ketoamino chelate, independent of the amount of ligand added to the rhenium precursors. The complexes were characterized by 1H and 13C NMR spectroscopy. X-ray crystal structures of the complexes 1, 2, 4, and 5, including that of MTPOH, were determined, revealing the trans position of the two oxygen atoms and the trans-Cl,Cl conformation in 1 and 2, in contrast to most other rhenium complexes of this type where the cis-Cl,Cl conformation is observed. Coordination of the potentially tridentate ligand MTPOH in 5 is bidentate with a dangling thioether substituent. Compound 2 shows catalytic activity in the oxidation of cis-cyclooctene with tert-butylhydroperoxide.

Molecular oxygen activation by a molybdenum(IV) monooxo bis(beta-ketiminato) complex.

Molybdenum(IV) monooxo compound that contains bis(beta-ketiminato) ligands activates molecular oxygen forming a molybdenum(VI) monooxo peroxo compound, representing a new entry into molybdenum peroxo derivatives.

Synthesis, structural characterization, catalytic properties, and theoretical study of compounds containing an Al-O-M (M = Ti, Hf) core.

Two single oxygen-bridged heterobimetallic oxides of Al(III) with group 4 metals (Ti, Hf) have been prepared. The reaction of LAlMeOH (1) [L = CH(N(Ar)(CMe))2, Ar = 2,6-iPr2C6H3] with dimethylmetallocenes of Ti and Hf in toluene (80 degrees C) and ether (room temperature), respectively, resulted in the formation of LAl(Me)(mu-O)M(Me)Cp2 [M = Ti (2), Hf (3)] in moderate to good yield. Compounds 2 and 3 were characterized by elemental analysis, IR, NMR (1H and 13C), EI-MS, and single-crystal X-ray structural analysis. Furthermore, compound 2 showed good catalytic activity in ethylene and styrene homopolymerization, while compound 3 is less active in ethylene polymerization. The styrene polymerization yields atactic polystyrene.

Base free lithium-organoaluminate and the gallium congener: potential precursors to heterometallic assemblies.

The first examples of base free lithium-organoaluminate and the corresponding gallium compound [LM(Me)OLi]3 (M = Al (3), Ga (4); L = HC{C(Me)N-2,6-iPr2C6H3}2) have been prepared by the reaction of Li[N(SiMe3)2] with the corresponding metal hydroxides LM(Me)OH (M = Al (1), Ga(2)); the oxygen atom in the M-O-Li fragment exists as oxide ion and is involved in the central Li3O3 six-membered ring formation.

Synthesis and characterization of [[PhC(NBu(t))2]Si(S)SBu(t)]: a silicon thioester analogue with the Si(=S)-S-skeleton.

A silicon thioester analogue with the Si(=S)-S-skeleton [[PhC(NBu(t))2]Si(S)SBu(t)] (3) has been prepared from the reaction of [[PhC(NBu(t))2]SiCl2SBu(t)] (2) with potassium.

Soluble molecular compounds with the Mg-O-Al structural motif: a model approach for the fixation of organometallics on a MgO surface.

We report a facile route to the molecular compounds with the Mg-O-Al structural motif. The reaction of Mg[N(SiMe3)2]2 (1) with a stoichiometric amount of LAlOH(Me) (2) [L = CH{(CMe)(2,6-iPr2C6H3N)}2] in THF/n-hexane at 0 degrees C results in the formation of the heterobimetallic compound (Me3Si)2NMg(THF)2-O-Al(Me)L (3) in high yield. The similar reaction of 1 equiv of Mg[N(SiMe3)2]2 and 2 equiv of LAlOH(Me) results in the formation of trimetallic compound L(Me)Al-O-Mg(THF)2-O-Al(Me)L (4). Structural analyses of 3 and 4 have been carried out, revealing the presence of the Mg-O-Al motif. A tentative assignment of the Mg-O-Al vibrations has been made and was supported by calculations.

Crystal structures of the complexes of a group IIA phospholipase A2 with two natural anti-inflammatory agents, anisic acid, and atropine reveal a similar mode of binding.

Secretory low molecular weight phospholipase A(2)s (PLA(2)s) are believed to be involved in the release of arachidonic acid, a precursor for the biosynthesis of pro-inflammatory eicosanoids. Therefore, the specific inhibitors of these enzymes may act as potent anti-inflammatory agents. Similarly, the compounds with known anti-inflammatory properties should act as specific inhibitors. Two plant compounds, (a) anisic acid (4-methoxy benzoic acid) and (b) atropine (8-methyl-8-azabicyclo oct-3-hydroxy-2-phenylpropanoate), have been used in various inflammatory disorders. Both compounds (a) and (b) have been found to inhibit PLA(2) activity having binding constants of 4.5 x 10(-5) M and 2.1 x 10(-8) M, respectively. A group IIA PLA(2) was isolated and purified from the venom of Daboia russelli pulchella (DRP) and its complexes were made with anisic acid and atropine. The crystal structures of the two complexes (i) and (ii) of PLA(2) with compounds (a) and (b) have been determined at 1.3 and 1.2 A resolutions, respectively. The high-quality observed electron densities for the two compounds allowed the accurate determinations of their atomic positions. The structures revealed that these compounds bound to the enzyme at the substrate - binding cleft and their positions were stabilized by networks of hydrogen bonds and hydrophobic interactions. The most characteristic interactions involving Asp 49 and His 48 were clearly observed in both complexes, although the residues that formed hydrophobic interactions with these compounds were not identical because their positions did not exactly superimpose in the large substrate-binding hydrophobic channel. Owing to a relatively small size, the structure of anisic acid did not alter upon binding to PLA(2), while that of atropine changed significantly when compared with its native crystal structure. The conformation of the protein also did not show notable changes upon the bindings of these ligands. The mode of binding of anisic acid to the present group II PLA(2) is almost identical to its binding with bovine pancreatic PLA(2) of group I. On the other hand, the binding of atropine to PLA(2) is similar to that of another plant alkaloid aristolochic acid.

Dioxomolybdenum(VI) and dioxotungsten(VI) complexes supported by an amido ligand.

Stepwise addition of one equivalent of n-butyllithium and trimethylsilyl chloride to 2-tert-butylmercaptoaniline affords the new ligand 1-(Me3SiNH)-2-(t-BuS)C6H4 (LH), that reacts with one equivalent of butyllithium to its lithium salt LLi. Dioxodichloromolybdenum [MoO2Cl2] and dioxodichlorotungsten dimethoxyethane [WO2Cl2(dme)] react in tetrahydrofuran solution at low temperature with two equivalents LLi to monomeric dioxomolybdenum(VI) [MoO2L2] (1) and dioxotungsten(VI) complex [WO2L2] (2) employing two bidentate amido thioether ligands. The crystallographic determination of the molecular structures of 1 and 2 show evidence for M...S contacts. The reaction of [MoO2Cl2] with LLi in tetrahydrofuran solution at room temperature leads next to 1 to two compounds where silyl group migration from nitrogen to oxygen atoms occurs forming [Mo(=NL')2(OSiMe)2] (3) and [Mo(=NL')2(OSiMe3)L] (4, L' = N-2-t-BuSC6H4) as determined by NMR spectroscopy. Compound 4 was isolated in low yield and its molecular structure determined by X-ray crystallography. Higher yields of a bisimido complex can be obtained by the direct reaction of one equivalent of LLi with [Mo(NAr)2Cl2(dme)] (Ar = 2,6-Me2C6H4) forming [Mo(NAr)2LCl] (5).

Syntheses, characterization, and X-ray crystal structures of beta-diketiminate group 13 hydrides, chlorides, and fluorides.

A series of organometallic compounds of group 13 metals supported by the sterically encumbered beta-diketiminate ligand containing hydrides, fluorides, chlorides, and bromide have been synthesized and structurally characterized. The synthetic strategy applied utilizes halide metathesis and reduction of metal chlorides to the corresponding hydrides. Thus, the reaction of LLi.OEt2 with MeMCl2 affords LM(Me)Cl (M = Al (1), Ga (2), In (3)) and LGaBr2 (4) with GaBr3. Reduction of LGa(Me)Cl with LiH.BEt3 leads to the formation of LGa(Me)H (10). Synthesis of LGaH(2) (12) has been accomplished by reacting LGaI2 (8) with LiH.BEt3. LAl(Me)Cl (1) and LAlH2 (6) have been converted to LAl(Me)F (5) and LAlF2 (7), respectively. The former was obtained in a reaction of LAl(Me)Cl with Me3SnF while the latter was isolated in a reaction of LAlH2 with BF3.OEt2. Similarly reaction of LGaI2 (8) with Me3SnF affords LGaF2 (9). Compounds reported herein have been characterized by elemental analyses, IR, NMR, EI-MS, and single-crystal X-ray diffraction techniques.