Computational and biochemical docking of the irreversible cocaine analog RTI 82 directly demonstrates ligand positioning in the dopamine transporter central substrate-binding site.

The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3ß-(p-chlorophenyl)tropane-2ß-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([(125)I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [(125)I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors.

Crystal structure of (E)-N-[(E)-3-(4-meth-oxy-phen-yl)allyl-idene]naphthalen-1-amine.

In the title compound, C20H17NO, the dihedral angle between the mean planes of the 4-meth-oxy-phenyl ring and the naphthalene ring is 69.50 (7)°. The meth-oxy group is almost coplanar with the benzene ring to which it is connected [Cb-Cb-Om-Cm torsion angle of -7.9 (2)°; b = benzene and m = meth-oxy] and the imine group displays a C-C-N=C torsion angle is -57.2 (2)°. The imine (C=N) group has an E conformation. In the crystal, weak π-π inter-actions between the benzene rings [centroid-centroid distance = 3.7781 (10) Å] are observed.

(E)-2,2'-[3-(4-Fluoro-phen-yl)prop-2-ene-1,1-di-yl]bis-(3-hy-droxy-5,5-dimethyl-cyclo-hex-2-en-1-one).

In the title compound, C25H29FO4, each cyclo-hexenone ring has an envelope conformation with the dimethyl-substituted atom as the flap. The hy-droxy and carbonyl groups form two intra-molecular O-H⋯O hydrogen bonds, as is typical for xanthene derivatives. In the crystal, very weak C-H⋯O hydrogen bonds link mol-ecules into dimers.

(E)-N-[(E)-3-(4-Nitro-phen-yl)allyl-idene]naphthalen-1-amine.

In the title compound, C19H14N2O2, the dihedral angle between the mean planes of the 4-nitro-phenyl ring and the naphthalene ring system is 12.79 (2)°. The imine group displays a C-C-N=C torsion angle of 41.0 (2)° and the C=N group has an E conformation. In the crystal, weak C-H⋯O hydrogen bonds link molecules into layers parallel to the b axis.

(E)-2,2'-[3-(4-Chloro-phen-yl)prop-2-ene-1,1-di-yl]bis-(3-hy-droxy-5,5-di-methyl-cyclo-hex-2-en-1-one).

The title compound, C25H29ClO4, adopts a trans conformation about the C=C double bond and the di-methyl-cyclo-hexenone rings both show an envelope conformation with the dimethyl-substituted C atom as the flap. In the mol-ecule, the hy-droxy and carbonyl groups form two intra-molecular O-H⋯O hydrogen bonds typical for xanthene derivatives. In the crystal, weak C-H⋯O hydrogen bonds link the mol-ecules into chains running parallel to the a-axis direction.

(E)-9-(4-Fluoro-styr-yl)-3,3,6,6-tetra-methyl-3,4,5,6,7,9-hexa-hydro-2H-xanthene-1,8-dione.

In the title compound, C25H27FO3, each of the cyclo-hexenone rings adopts a half-chair conformation, whereas the six-membered pyran ring adopts a flattened boat conformation, with the O and methine C atoms deviating by 0.0769 (15) and 0.196 (2) Å, respectively, from the plane of the other four atoms (r.m.s. deviation = 0.004 Å). The C=C double bond adopts an E conformation. The dihedral angle between the benzene and pyran (all atoms) rings is 89.94 (10)°. In the crystal, weak C-H⋯O hydrogen bonds link the mol-ecules into chains running parallel to the b axis.

(E)-N-(3,3-Di-phenyl-allyl-idene)-2-(tri-fluoro-meth-yl)aniline.

In the title compound, C22H16F3N, the C=N bond of the central imine group adopts an E conformation. The dihedral angles between the 2-(tri-fluoro-meth-yl)phenyl ring and the benzene rings are 9.34 (1) and 68.8 (1)°. The imine group displays a C-C-N=C torsion angle of 41.6 (3)°. In the crystal, weak C-H⋯F hydrogen bonds link the mol-ecules into chains parallel to the b-axis direction.

(E)-N-(3,3-Di-phenyl-allyl-idene)naph-thal-en-1-amine.

The title compound, C25H19N, adopts an E conformation about the C=N bond. The naphthalene ring system and the phenyl rings form dihedral angles 38.1 (1), 46.9 (8) and 48.5 (1)°, respectively, with the mean plane of the central enimino fragment. The crystal packing exhibits no directional close contacts.

Nanostructure and nanoindentation study of pulse electric-current sintered TiB2-SiC-Cf composite.

A carbon-fiber (Cf) doped TiB2-SiC composite was prepared and investigated to determine its densification behavior, micro/nanostructural properties, and mechanical characteristics. TiB2-25 vol% SiC-2 wt% Cf was prepared at 40 MPa and 1800 °C for 7 min using the pulsed electric-current sintering technique, and a relative density of 98.5% was realized. The as-sintered composite was characterized using advanced techniques, e.g., X-ray diffractometry, energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, field-emission scanning electron microscopy, high-resolution transmission electron microscopy, field-emission electron probe micro-analysis, and nanoindentation. The Cf additive could remove the surface oxide layers from the TiB2 and SiC domains, thus transforming them into TiB2 and SiC. According to micro/nanostructural studies, Cf could not retain its initial structure and was eventually converted into graphite nanosheets. In addition, the prepared composite was examined using the nanoindentation technique, and the following results were obtained for the calculated hardness, elastic modulus, and stiffness values: TiB2 > SiC > TiB2/SiC interface.

(E)-N-(3,3-Diphenyl-allyl-idene)-3-nitro-aniline.

In the title compound, C(21)H(16)N(2)O(2), the 3-nitro-phenyl and two phenyl rings are twisted from the mean plane of the enimino fragment by 44.4 (1), 37.2 (1) and 74.1 (1)°, respectively. The crystal packing exhibits no classical inter-molecular contacts.

(E)-N-(3,3-Diphenyl-allyl-idene)-4-nitro-aniline.

In the title compound, C(21)H(16)N(2)O(2), the dihedral angles between the mean planes of the 4-nitro-phenyl ring and the two phenyl rings are 57.3 (5) and 16.8 (6)°. The imine group displays a C-C-N-C torsion angle of -24.9 (3)°.

(E)-2,2'-[3-(2-Nitro-phen-yl)prop-2-ene-1,1-di-yl]bis-(3-hy-droxy-cyclo-hex-2-en-1-one).

In the title compound, C(21)H(21)NO(6), each of the cyclo-hexenone rings adopts a half-chair conformation. Each of the pairs of hy-droxy and carbonyl O atoms are oriented to allow for the formation of intra-molecular O-H⋯O hydrogen bonds, which are typical of xanthene derivatives.

[2-(4-Chloro-phen-yl)-5-phenyl-oxolan-3-yl](cyclo-penten-yl)methanone.

In the title compound, C(22)H(21)ClO(2), the oxolane ring adopts a twisted conformation. The dihedral angles between the mean plane of the oxolane ring and the mean planes of the 4-chloro-phenyl, phenyl and cyclo-pentenyl rings are 71.81 (18), 76.9 (18) and 82.08 (18)°, respectively.

2,2'-[(E)-3-(4-Nitro-phen-yl)prop-2-ene-1,1-di-yl]bis-(3-hy-droxy-5,5-dimethyl-cyclo-hex-2-en-1-one).

In the title compound, C(25)H(29)NO(6), each of the cyclo-hexenone rings adopts a half-chair conformation. The hy-droxy and carbonyl O atoms face each other and are oriented to allow for the formation of two intra-molecular O-H⋯O hydrogen bonds. In the crystal, weak C-H⋯O hydrogen bonds are formed between molecules, generating a two-dimensional supramolecular structure.

9-[(E)-2-(2-Meth-oxy-phen-yl)ethen-yl]-3,4,5,6,7,9-hexa-hydro-2H-xanthene-1,8-dione.

In the title compound, C(22)H(22)O(4), the two cyclo-hexenone rings adopt half-chair conformations, whereas the six-membered pyran ring adopts a flattened boat conformation, with the O and methine C atoms deviating from the plane of the other four atoms by 0.142 (2) and 0.287 (2)Å, respectively. In the crystal, weak C-H⋯O hydrogen bonds link mol-ecules into chains running parallel to the a axis.

(E)-9-(4-Chloro-styr-yl)-3,4,5,6,7,9-hexa-hydro-2H-xanthene-1,8-dione.

In the title compound, C(21)H(19)ClO(3), the two cyclo-hexenone rings adopt half-chair conformations, whereas the pyran ring adopts a boat conformation. The 4-chloro-phenyl ring is almost perpendicular to the plane through the four C atoms of the pyran ring [dihedral angle = 87.97 (6)°]. In the crystal, weak C-H⋯O hydrogen bonds link the mol-ecules into a chain parallel to the a-axis.

(E)-3,3,6,6-Tetra-methyl-9-(2-nitro-styr-yl)-3,4,5,6,7,9-hexa-hydro-1H-xanthene-1,8(2H)-dione.

In the title compound, C(25)H(27)NO(5), each of the cyclo-hexenone rings adopts a half-chair conformation, whereas the six-membered pyran ring adopts a flattened boat conformation, with the O and methine C atoms deviating from the plane of the other four atoms. In the crystal, weak C-H⋯O hydrogen bonds link mol-ecules into chains parallel to the c axis.

Microstructural and nanoindentation study of TaN incorporated ZrB2 and ZrB2-SiC ceramics.

This study assessed the sinterability and microstructure of ZrB2-SiC-TaN and ZrB2-TaN ceramics. Spark plasma sintering at 2000 °C and 30 MPa for 5 min produced both ceramics. The relative density of ZrB2 ceramic containing TaN was 95.3%; the addition of SiC increased this value to 98.1%. SiC's contribution to the elimination of ZrB2 surface oxides was the primary factor in the advancement of densification. The in situ formation of hexagonal boron nitride at the interface of TaN and ZrB2 was confirmed by high-resolution transmission electron microscopy, field emission-electron probe microanalyzer, X-ray diffractometry, and field emission scanning electron microscopy. Moreover, the in situ graphite might be produced as a byproduct of the SiC-SiO2 process, hence boosting the reduction of oxide compounds in the ternary system. The SiC compound had the highest hardness (29 ± 3 GPa), while the ZrB2/TaN interface exhibited the greatest values of elastic modulus (473 ± 26 GPa) and stiffness (0.76 ± 0.13 mN/nm).

The N terminus of monoamine transporters is a lever required for the action of amphetamines.

The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). We explored the functional importance of the N terminus in mediating the action of amphetamines by focusing initially on the highly conserved threonine residue at position 81, a candidate site for phosphorylation by protein kinase C. Molecular dynamics simulations of the wild type SERT, compared with its mutations SERT(T81A) and SERT(T81D), suggested structural changes in the inner vestibule indicative of an opening of the inner vestibule. Predictions from this model (e.g. the preferential accumulation of SERT(T81A) in the inward conformation, its reduced turnover number, and a larger distance between its N and C termini) were verified. Most importantly, SERT(T81A) (and the homologous mutations in noradrenaline and dopamine) failed to support amphetamine-induced efflux, and this was not remedied by aspartate at this position. Amphetamine-induced currents through SERT(T81A) were comparable with those through the wild type transporter. Both abundant Na(+) entry and accumulation of SERT(T81A) in the inward facing conformation ought to favor amphetamine-induced efflux. Thus, we surmised that the N terminus must play a direct role in driving the transporter into a state that supports amphetamine-induced efflux. This hypothesis was verified by truncating the first 64 amino acids and by tethering the N terminus to an additional transmembrane helix. Either modification abolished amphetamine-induced efflux. We therefore conclude that the N terminus of monoamine transporters acts as a lever that sustains reverse transport.

3-Hy-droxy-2-[(2E)-1-(2-hy-droxy-6-oxocyclo-hex-1-en-1-yl)-3-(2-meth-oxy-phen-yl)prop-2-en-1-yl]cyclo-hex-2-en-1-one.

In the title compound, C(22)H(24)O(5), each of the cyclo-hexenone rings adopts a half-chair conformation. The hy-droxy and carbonyl O atoms face each other and are orientated to allow for the formation of the two intra-molecular O-H⋯O hydrogen bonds which are typical of xanthene derivatives. In the crystal, weak inter-molecular C-H⋯O hydrogen bonds link mol-ecules into layers parallel to the ab plane.