Tania Cordova-Sintjago

Drug discovery targeting human 5-HT2C receptors: Residues S3.36 and Y7.43 impact ligand—binding pocket structure via hydrogen bond formation

Specific activation of serotonin (5-HT) 5-HT(2C) G protein-coupled receptors may be therapeutic for obesity and neuropsychiatric disorders. Mutagenesis coupled with computational and molecular modeling experiments based on the human β₂ adrenergic receptor structure was employed to delineate the interactions of different ligands at human 5-HT(2C) residues D3.32, S3.36 and Y7.43. No binding of the tertiary amine radioligand ([³H]-mesulergine) could be detected when the 5-HT(2C) D3.32 residue was mutated to alanine (D3.32A). The S3.36A point-mutation greatly reduced affinity of primary amine ligands, modestly reduced affinity of a secondary amine, and except for the 5-HT(2C)-specific agonist N(CH₃)₂-PAT, affinity of tertiary amines was unaffected. Molecular modeling results indicated that the primary amines form hydrogen bonds with the S3.36 residue, whereas, with the exception of N(CH₃)₂-PAT, tertiary amines do not interact considerably with this residue. The Y7.43A point-mutation greatly reduced affinity of 5-HT, yet reduced to a lesser extent the affinity of tryptamine that lacks the 5-hydroxy moiety present in 5-HT; modeling results indicated that the 5-HT 5-hydroxy moiety hydrogen bonds with Y7.43 at the 5-HT(2C) receptor. Additional modeling results showed that 5-HT induced a hydrogen bond between Y7.43 and D3.32. Finally, modeling results revealed two low-energy binding modes for 5-HT in the 5-HT(2C) binding pocket, supporting the concept that multiple agonist binding modes may stabilize different receptor active conformations to influence signaling. Ligand potencies for modulating WT and point-mutated 5-HT(2C) receptor-mediated phospholipase C activity were in accordance with the affinity data. Ligand efficacies, however, were altered considerably by the S3.36A mutation only.

Aromatic interactions impact ligand binding and function at serotonin 5-HT2C G protein-coupled receptors: Receptor homology modeling, ligand docking, and molecular dynamics results validated by experimental studies.

The serotonin (5-hydroxytryptamine, 5-HT) 5-HT2 G protein-coupled receptor (GPCR) family consists of types 2A, 2B, and 2C that share ∼75% transmembrane (TM) sequence identity. Agonists for 5-HT2C receptors are under development for psychoses; whereas, at 5-HT2A receptors, antipsychotic effects are associated with antagonists – in fact, 5-HT2A agonists can cause hallucinations and 5-HT2B agonists cause cardiotoxicity. It is known that 5-HT2A TM6 residues W6.48, F6.51, and F6.52 impact ligand binding and function; however, ligand interactions with these residues at the 5-HT2C receptor have not been reported. To predict and validate molecular determinants for 5-HT2C-specific activation, results from receptor homology modelling, ligand docking, and molecular dynamics simulation studies were compared with experimental results for ligand binding and function at wild type and W6.48A, F6.51A, and F6.52A point-mutated 5-HT2C receptors.

Molecular pharmacology and ligand docking studies reveal a single amino acid difference between mouse and human serotonin 5-HT2A receptors that impacts behavioral translation of novel 4-phenyl-2-dimethylaminotetralin ligands

During translational studies to develop 4-phenyl-2-dimethylaminotetralin (PAT) compounds for neuropsychiatric disorders, the (2R,4S)-trans-(+)- and (2S,4R)-trans-(−)-enantiomers of the analog 6-hydroxy-7-chloro-PAT (6-OH-7-Cl-PAT) demonstrated unusual pharmacology at serotonin (5-HT) 5-HT2 G protein–coupled receptors (GPCRs). The enantiomers had similar affinities (Ki) at human (h) 5-HT2A receptors (∼70 nM). In an in vivo mouse model of 5-HT2A receptor activation [(±)-(2,5)-dimethoxy-4-iodoamphetamine (DOI)–elicited head twitch], however, (−)-6-OH-7-Cl-PAT was about 5-fold more potent than the (+)-enantiomer at attenuating the DOI-elicited response. It was discovered that (+)-6-OH-7-Cl-PAT (only) had ∼40-fold-lower affinity at mouse (m) compared with h5-HT2A receptors. Molecular modeling and computational ligand docking studies indicated that the 6-OH moiety of (+)- but not (−)-6-OH-7-Cl-PAT could form a hydrogen bond with serine residue 5.46 of the h5-HT2A receptor. The m5-HT2A as well as m5-HT2B, h5-HT2B, m5-HT2C, and h5-HT2C receptors have alanine at position 5.46, obviating this interaction; (+)-6-OH-7-Cl-PAT also showed ∼50-fold lower affinity than (−)-6-OH-7-Cl-PAT at m5-HT2C and h5-HT2C receptors. Mutagenesis studies confirmed that 5-HT2A S5.46 is critical for (+)- but not (−)-6-OH-7-Cl-PAT binding, as well as function. The (+)-6-OH-7-Cl-PAT enantiomer showed partial agonist effects at h5-HT2A wild-type (WT) and m5-HT2A A5.46S point-mutated receptors but did not activate m5-HT2A WT and h5-HT2A S5.46A point-mutated receptors, or h5-HT2B, h5-HT2C, and m5-HT2C receptors; (−)-6-OH-7-Cl-PAT did not activate any of the 5-HT2 receptors. Experiments also included the (2R,4S)-trans-(+)- and (2S,4R)-trans-(−)-enantiomers of 6-methoxy-7-chloro-PAT to validate hydrogen bonding interactions proposed for the corresponding 6-OH analogs. Results indicate that PAT ligand three-dimensional structure impacts target receptor binding and translational outcomes, supporting the hypothesis that GPCR ligand structure governs orthosteric binding pocket molecular determinants and resulting pharmacology.

Homogeneous catalysis on the gas-phase dehydration reaction of tertiary alcohols by hydrogen bromide. Density functional theory calculation

The gas-phase thermal dehydration mechanism of tert-butanol, 2-methyl-2-butanol, 2-methyl-2-pentanol and 2,3-dimethyl-2-butanol by homogeneous catalysis of hydrogen bromide was examined by density functional theory calculations with the hybrid functionals: M062X, CAMB3LYP and WB97XD. Reasonable agreements were found between theoretical and experimental enthalpy values at the WB97XD/6-311++G(d,p) level. The dehydration mechanism of tert-butanol with and without catalysis was evaluated in order to examine the catalyst effect on the mechanism. The elimination reaction without catalysis involves a four-membered transition state (TS), while the reaction with catalysis involves a six-membered TS. The mechanism without catalysis has enthalpy activation over 150 kJ mol–1 greater than the catalysed reaction. In all these reactions, the elongation of the C–O bond is significant in the TS. The un-catalysed reaction is controlled by breaking of C–O bond, and it was found to be more synchronous (Sy ≈ 0.91) than the hydrogen bromide catalysed reactions (Sy ≈ 0.75–0.78); the latter reactions are dominated by the three reaction coordinates associated with water formation. No significant effect on the enthalpies of activation was observed when the size of the alkyl chain was increased.

Electrocyclic [1,5] hydrogen shift in the thermal elimination kinetics of phenyl acetate and p-tolyl acetate in the gas phase: a density functional theory study

The kinetics and mechanisms of thermal decomposition of phenyl acetate and p-tolyl acetate in the gas phase were studied by means of electronic structure calculations using density functional theory methods: B3LYP/6-31G(d,p), B3LYP/6-31++G(d,p), B3PW91/6-31G(d,p), B3PW91/6-31++G(d,p), MPW1PW91/6-31G(d,p), MPW1PW91/6-31++G(d,p), PBE/6-31G(d,p) and PBE/6-31++G(d,p). Two possible mechanisms have been considered: mechanism A is a stepwise process involving electrocyclic [1,5] hydrogen shift to eliminate ketene through concerted six-membered cyclic transition-state structure, followed by tautomerisation of cyclohexadienone or by 4-methyl cyclohexadienone intermediate to give the corresponding phenol. Mechanism B is a one-step concerted [1,3] hydrogen shift through a four-membered cyclic transition-state geometry, to produce ketene and phenol or p-cresol. Theoretical calculations showed reasonable agreement with experimental activation parameters when using the Perdew, Burke and Ernserhof (PBE)functional, through the stepwise [1,5] hydrogen-shift mechanism. For mechanism B, large deviation for the entropy of activation was observed. No experimental data were available for p-tolyl acetate; however, theoretical calculations showed similar results to phenyl acetate, thus supporting the stepwise mechanism for both phenyl acetate and p-tolyl acetate.

Assessment in the competition between steric and electronic effects in the elimination kinetic of hydrogen in 1,4-cyclohexadienes in the gas phase. Quantum chemical theory calculations

The mechanisms of gas-phase thermal decomposition of alkyl-substituted cyclohexadienes were studied by the means of quantum chemical calculations with theory levels Møller-Plesset pertubation theory (MP2) and density functional theory (DFT) (B3LYP, MPW1PW91, PBEPBE, ωB97XD, CAM-B3LYP, M06, and M062X) with 6-31G(d,p), 6-31++G(d,p) basis sets. The examination of the reaction pathways of each substrate demonstrated a molecular mechanism through six-membered cyclic boat-like transition state (TS) structure. An alkyl group substituent causes a detrimental effect on the reaction rate, compared to the parent compound 1,4-cyclohexadiene; however, the reaction was favoured in the case of 3,6-dimethyl substitution. The 3,6-dimethyl-1,4-cyclohexadiene compound has activation energy 11.2 kJ/mol lower than the reference compound, which overcomes the effect of the most negative entropy of activation in the series. The effects of alkyl substituents in these reactions suggest a complex combination of electronic and steric influence. These reactions are characterised as highly synchronous concerted, with small predominance of C–H bond breaking in the TS.

Theoretical study on the mechanism of the gas-phase elimination kinetics of alkyl chloroformates

ABSTRACT The theoretical calculations on the mechanism of the homogeneous and unimolecular gas-phase elimination kinetics of alkyl chloroformates– ethyl chloroformate (ECF), isopropyl chloroformate (ICF), and sec-butyl chloroformate (SCF) – have been carried out by using CBS-QB3 level of theory and density functional theory (DFT) functionals CAM-B3LYP, M06, MPW1PW91, and PBE1PBE with the basis sets 6-311++G(d,p) and 6-311++G(2d,2p). The chlorofomate compounds with alkyl ester Cβ–H bond undergo thermal decomposition producing the corresponding olefin, HCl and CO2. These homogeneous eliminations are proposed to undergo two different types of mechanisms: a concerted process, or via the formation of an unstable intermediate chloroformic acid (ClCOOH), which rapidly decomposes to HCl and CO2 gas. Since both elimination mechanisms may occur through a six-membered cyclic transition state structure, it is difficult to elucidate experimentally which is the most reasonable reaction mechanism. Theoretical calculations show that the stepwise mechanism with the formation of the unstable intermediate chloroformic acid from ECF, ICF, and SCF is favoured over one-step elimination. Reasonable agreements were found between theoretical and experimental values at the CAM-B3LYP/6-311++G(d,p) level. GRAPHICAL ABSTRACT

Molecular interactions of agonist and inverse agonist ligands at serotonin 5-HT2C G protein-coupled receptors: computational ligand docking and molecular dynamics studies validated by experimental mutagenesis results

To understand molecular determinants for ligand activation of the serotonin 5-HT2C G protein-coupled receptor (GPCR), a drug target for obesity and neuropsychiatric disorders, a 5-HT2C homology model was built according to an adrenergic β2 GPCR (β2AR) structure and validated using a 5-HT2B GPCR crystal structure. The models were equilibrated in a simulated phosphatidyl choline membrane for ligand docking and molecular dynamics studies. Ligands included (2S, 4R)-(–)-trans-4-(3’-bromo- and trifluoro-phenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalene-2-amine (3’-Br-PAT and 3’-CF3-PAT), a 5-HT2C agonist and inverse agonist, respectively. Distinct interactions of 3’-Br-PAT and 3’-CF3-PAT at the wild-type (WT) 5-HT2C receptor model were observed and experimental 5-HT2C receptor mutagenesis studies were undertaken to validate the modelling results. For example, the inverse agonist 3’-CF3-PAT docked deeper in the WT 5-HT2C binding pocket and altered the orientation of transmembrane helices (TM) 6 in comparison to the agonist 3’-Br-PAT, suggesting that changes in TM orientation that result from ligand binding impact function. For both PATs, mutation of 5-HT2C residues S3.36, T3.37, and F5.47 to alanine resulted in significantly decreased affinity, as predicted from modelling results. It was concluded that upon PAT binding, 5-HT2C residues T3.37 and F5.47 in TMs 3 and 5, respectively, engage in inter-helical interactions with TMs 4 and 6, respectively. The movement of TMs 5 and 6 upon agonist and inverse agonist ligand binding observed in the 5-HT2C receptor modelling studies was similar to movements reported for the activation and deactivation of the β2AR, suggesting common mechanisms among aminergic neurotransmitter GPCRs.

A new insight on the gas phase retro-Diels–Alder reaction of bicyclic compounds: density functional theory calculations

The mechanisms of the gas-phase thermal decomposition of bicyclo[2.2.1]heptadiene and 3,7,7-trimethylbicyclo[2.2.1]hept-2-ene were examined by density functional theory calculations with the hybrid functionals: B3LYP, CAM-B3LYP, MPW1PW91, and PBEPBE. Reasonable agreements were found between theoretical and experimental values with the B3LYP hybrid functional. Three molecular concerted pathways for bicyclo[2.2.1]heptadiene decomposition are proposed. The retro-Diels–Alder (retro-DA) pathway yields cyclopentadiene and acetylene through a nearly synchronous transition state structure (Sy = 0.97). The other two reaction channels are stepwise with a common step with the formation of the intermediate bicyclo[4.1.0] heptadiene. This reaction is dominated by C–C bond breaking leading to the methylene migration by an early transition state in the reaction coordinate (Sy = 0.91). The rearrangements of the latter intermediate producing toluene were also studied. The retro-DA elimination of 3,7,7-trimethylbicyclo[2.2.1]hept-2-ene gives 1,5,5-trimethyl-cyclopenta-1,3-diene in a less synchronous process (Sy = 0.77). This fact may be due to the electronic effects of the methyl substituent. The latter product is unstable and undergoes methyl migrations to give a more stable isomer 1,2,3-trimethylcyclopenta-1,3-diene. The stepwise mechanism for the retro-DA reaction through a biradical intermediate appears to be unfavourable because the barrier is bigger than that for the concerted reaction.