Carlos Kubli-Garfias

Amyloid-β Peptide Binds to Cytochrome C Oxidase Subunit 1

Extracellular and intraneuronal accumulation of amyloid-beta aggregates has been demonstrated to be involved in the pathogenesis of Alzheimers disease (AD). However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of macromolecules has deleterious effects on cellular functions. Mitochondria were found to be the target for amyloid-beta, and mitochondrial dysfunction is well documented in AD. In the present study we have shown for the first time that Aβ 1–42 bound to a peptide comprising the amino-terminal region of cytochrome c oxidase subunit 1. Phage clone, selected after screening of a human brain cDNA library expressed on M13 phage and bearing a 61 amino acid fragment of cytochrome c oxidase subunit 1, bound to Aβ 1–42 in ELISA as well as to Aβ aggregates present in AD brain. Aβ 1–42 and cytochrome c oxidase subunit 1 co-immunoprecipitated from mitochondrial fraction of differentiated human neuroblastoma cells. Likewise, molecular dynamics simulation of the cytochrome c oxidase subunit 1 and the Aβ 1–42 peptide complex resulted in a reliable helix-helix interaction, supporting the experimental results. The interaction between Aβ 1–42 and cytochrome c oxidase subunit 1 may explain, in part, the diminished enzymatic activity of respiratory chain complex IV and subsequent neuronal metabolic dysfunction observed in AD.

AB INITIO STUDY OF THE ELECTRONIC STRUCTURE OF PROGESTERONE AND RELATED PROGESTINS

Abstract The geometries and electronic structure of progesterone (P), one of the most important steroid hormones, and related compounds i.e. 20α-hydroxy-progesterone (20α-OHP), 20β-hydroxy-progesterone (20β-OHP) and 17α-hydroxy-progesterone (17α-OHP), were established by high-level ab initio methods using the 6-31G∗ basis set. In this way bond distances, valence angles and dihedral angles were measured. Likewise, total energy, frontier orbitals (i.e. highest occupied molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO)), dipole moments, electrostatic charges and electrostatic potentials were calculated. Ab initio results for P and 17α-OHP were compared with their crystal data, and showed good agreement between the ab initio level calculations and the crystal structures. However, differences were observed at the D-ring and the acetyl side chain at C17. The energy of progesterone was higher than its derivatives. Electrostatic charges and electronic density surfaces of the progestins showed different patterns according to the stereochemical arrangement of the carbonyl and hydroxyl functional groups at the acetyl C17 chain. In all the molecules the HOMO and LUMO were located between C4 and C5, along with a high electronic density. The direction of the dipole moment vector was similar in all the molecules, directed beneath the A-ring and with the positive pole pointing toward the methyl group at C18. The electrostatic potentials were observed emerging from the oxygens. This distribution of the intermolecular forces may explain to some extent the non-genomic effects of 4-en progestins. Likewise, the location of the frontier orbitals observed in this work agrees with the metabolic mechanism toward 5α and 5β-reduction of the studied hormones.

Ab initio comparative study of the electronic structure of testosterone, epitestosterone and androstenedione

Abstract The geometries and electronic structure of testosterone (T), epitestosterone (E) and androstenedione (A) were assessed at high ab initio level, using the 6-31 G∗ basis set. These androgens are related chemically, changing only one functional group, i.e. 17β-OH (T), 17α-OH (E), or 17-keto (A), but with different biological activity. Bond distances, valence angles and torsional angles were measured. In addition, total energy, valence orbitals, i.e. highest occupied molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO), electrostatic charges, dipole moment and electrostatic potentials were measured. Rotation barriers of the 17-OH functional group were also explored for T and E. Geometries showed good agreement with X-ray crystallography structures, except for some dihedral angles of the D-ring. Results showed high electronic density surfaces at C17, according with the stereochemical arrangement of the functional groups and the electrostatic potentials emerging from the lone pair electrons of the oxygens. HOMO and LUMO were located at the A-ring of the steroids, between C4 and C5 included in a high electronic density produced by the π electron delocalization along the O3C3C4C5 conjugation. These findings increase the feasibility of a chemical reaction at this point, explaining the metabolism of these androgens toward 5α and 5β derivatives. The dipole moment vector was observed traversing the molecules from the C3 carbonyl toward the C17 in T and E, changing slightly in androstenedione. The rotation barriers of the C17-OH group showed the interaction among geometry, enthalpy, electronic charge density, dipole moment and orbital energies. All of these features might explain to some extent the metabolism of androgens and the qualitatively different intermolecular forces at C17 which change the affinity for receptors and the biological actions.

Progesterone-like relaxant effect of RU 486 in the rat myometrium

The antihormone RU 486 is characterized by its antiprogesterone and antiglucocorticoid activities. In this work the likelihood of a non-genomic effect for this compound was assessed. Thus, RU 486 was compared with progesterone and the 5 beta-progestin pregnanolone, for its ability to modify the uterine contractility of the rat. An outstanding relaxant effect elicited by RU 486 was observed, slightly higher than that produced by progesterone but lower than pregnanolone. Moreover, calcium promoted contractions were antagonized by RU 486, in the same way as the endogenous steroids. The data suggest the capability of RU 486 to block the calcium channels. It is concluded that a non-genomic effect of RU 486 is produced before its journey into the cell for its genomic action.

Semiempirical assessment of changes on the electronic structure of androstene with the addition of carbonyl and hydroxyl groups

Abstract A systematic study on the impact of carbonyl and hydroxyl functional groups on the electronic structure of the steroid, androst-4-ene (androstene) was carried out by the semiempirical methods, AM1 and PM3. A total of 16 molecules from androstene were studied with mono and disubstituted hydroxyl and carbonyl groups at C3 and C17. Results showed the lowering of total energies and enthalpies of formation with the addition of the functional groups. HOMO was located always along of the C4C5 π-bond. The LUMO occupied the same place except for two 17-keto androstene structures which showed the LUMO at C17 and degeneracy at LUMO and LUMO + 1. Structures with 3-keto conformation were the most conspicuous showing higher dipole moment, lower energy in both HOMO and LUMO, some degeneracy with HOMO and HOMO-1 and more impact than the hydroxyl groups in the charges of neighbor carbon atoms. Interestingly, this conformation which conjugates O  C3C4  C5, belongs to the ring A conformation of testosterone, epitestosterone and androstenedione, three well known androgens. It is concluded that the double bond of androstenes at C4C5 when conjugated with a carbonyl group at C3 enhances the likelihood of biological activity.

Ion trap MS/MS of intact testosterone and epitestosterone conjugates--adducts, fragile ions and the advantages of derivatisation.

In ion trap mass spectrometry, fragile ions may fragment under the application of resonance ejection during precursor mass isolation, reducing MS/MS spectral intensity. In this study the steroidal epimers testosterone glucuronide (TG) and epitestosterone glucuronide (EG) have been chosen as a model for exploring whether compound structure is linked to ion trap fragility. Both compounds form multiple adducts by ESI-MS, namely protonation, ammonium and sodium, however, the mass spectrum of EG displays a more intense ammonium adduct peak than TG. [TG+NH(4)](+), [EG+NH(4)](+) and [EG+H](+) were found to be fragile ions. To explain the differences in adduct formation and fragility, molecular modelling was employed. Ammonium adduction was localised to the glucuronide ring oxygens and while EG has eight possible adduction sites, only seven were located for TG explaining the increased ammonium adduct abundance with EG. In EG the bond between the steroid and the glucuronide was slightly longer and the oxygen in this bond was more basic than TG. This shows that the EG bond is weaker which may contribute to the fact that [EG+H](+) but not [TG+H](+) is fragile. To investigate whether stability could be restored by chemical means, EG was derivatised with tris(trimethoxyphenyl)phosphonium chloride or methylated on the carboxylic acid and Girard P or methoxylamine on the 3-keto group. Derivatisation of the steroid rather than the glucuronide eliminated fragility and using a charged derivative eliminated adduct formation. This work demonstrates the importance of carefully considering the nature of the derivative and the site of derivatisation.

AB INITIO CALCULATIONS OF THE ELECTRONIC STRUCTURE OF GLUCOCORTICOIDS

Abstract Glucocorticoids promote the conversion of protein into glucose and glycogen, act as anti-inflammatory and block the immune response. Progesterone may follow two metabolic routes: one to corticosterone and the other to cortisol. Because their biological importance and their interesting metabolism the geometry and electronic structure, at ab initio level with the 6-31G* basis set, of the following glucocorticoids: deoxycorticosterone (DOC), corticosterone (C), deoxycortisol, cortisol and cortisone, were studied. The precursors progesterone (P) and 17α-hydroxy-progesterone (17α-OHP) were included for comparison. Likewise, the X-ray crystal conformers of all the steroids were submitted to single point calculations with the 6-31G* basis set. Interatomic distances and valence angles were almost similar for all the studied molecules. However, dihedral angles were different in the C/D-ring system. X-ray crystal conformers also showed some differences of the dihedral angles when compared with their respective theoretical molecules. Progesterone showed the highest total energy, followed by DOC and 17α-OHP. The addition of two hydroxyl groups (C and deoxycortisol) lowered still more the total energy but it was almost similar in both steroids. Cortisone was followed by cortisol which showed the lower energy among all the studied molecules. X-ray crystal conformers showed always higher energy than their corresponding theoretical molecules. Cortisol showed also the higher HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) energies followed by 17α-OHP, P, deoxycortisol, C, DOC and cortisone. This sequence was similar for both HOMO and LUMO energy values. In all the cases both HOMO and LUMO were located along the π-double bond between C4 and C5, including also C6 and the carbonyl at C3. Dipole moments were larger for the compounds bearing the 17α-hydroxy functional group, i.e. 17α-OHP, deoxycortisol, cortisol and cortisone. The electrostatic potentials were associated directly with the lone pairs of oxygen atoms of the hydroxyl and carbonyl functional groups. By the results it is concluded that the 17α-hydroxy functional group enhances the dipole moment and elicit an extra electrostatic potential increasing the glucocorticoid action. Besides, this functional group might be important for receptor-glucocorticoid recognition and genomic interaction.

A theoretical model of the catalytic mechanism of the Δ5-3-ketosteroid isomerase reaction

The present paper describes a theoretical approach to the catalytic reaction mechanism involved in the conversion of 5-androstene-3,17-dione to 4-androstene-3,17-dione. The model incorporates the side chains of the residues tyrosine (Tyr(14)), aspartate (Asp(38)) and aspartic acid (Asp(99)) of the enzyme Delta(5)-3-ketosteroid isomerase (KSI; EC 5.3.3.1). The reaction involves two steps: first, Asp(38) acts as a base, abstracting the 4beta-H atom (proton) from C-4 of the steroid to form a dienolate as the intermediate; next, the intermediate is reketonized by proton transfer to the 6beta-position. Each step goes through its own transition state. Functional groups of the Tyr(14) and Asp(99) side chains act as hydrogen bond donors to the O1 atom of the steroid, providing stability along the reaction coordinate. Calculations were assessed at high level Hartree-Fock theory, using the 6-31G(*) basis set and the most important physicochemical properties involved in each step of the reaction, such as total energy, hardness, and dipole moment. Likewise, to explain the mechanism of reaction, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), atomic orbital contributions to frontier orbitals formation, encoded electrostatic potentials, and atomic charges were used. Energy minima and transition state geometries were confirmed by vibrational frequency analysis. The mechanism described herein accounts for all of the properties, as well as the flow of atomic charges, explaining both catalytic mechanism and proficiency of KSI.

Ab initio electronic structure of the progestogen norethisterone and its 5α-derivatives

Abstract The steroid 17α-ethynyl-19-nor-4-androsten-17β-ol, 3-one (Norethisterone; NET) and its 5α-dihydro (5α-NET), 3α- and 3β-tetrahydro derivatives (3α,5α- and 3β,5α-NET), were comparatively studied by the ab initio quantum mechanics theory. Additionally, 5α-androstan-3β,17β-diol (ADIOL) was also studied. The Hartree–Fock method and the 6-31G∗ basis set were used to obtain the lowest energy conformation, geometries, electronic structure and physicochemical properties of the steroids. The results showed bond distances and valence angles similar among all steroids, but some differences in dihedral angles in the A–B-ring system were observed. The electronic structure analysis showed that NET has both frontier orbitals that is, the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) located at the C4–C5 π-bond. In A-ring reduced derivatives, the HOMO was found at the 17β-OH and ethynyl groups. In the case of 5α-NET, the LUMO was confined to the A-ring and its C3 carbonyl group while the two NET tetrahydro-reduced derivatives showed the LUMO at the 17β-OH and ethynyl groups. The energy changes of the rotational barrier of the17β-OH group suggest that its movement is somewhat restricted by the 17α-ethynyl group. Interestingly both groups at C17 form a single electrostatic potential with high electronic density. On the other side, the 19-nor condition increases the A-ring mobility. However, the 3β-OH group of 3β,5α-NET may rotate without significant energy differences as compared to the same group in ADIOL. The electronic structure of NET and its A-ring reduced derivatives explains in some extent their interaction with androgen and progesterone receptors as well as their selectivity for the estrogen α-receptor.

Austin Model 1 study of the effect of carbonyl and hydroxyl functional groups on the electronic structure of androstane

Abstract Androgens are steroid hormones with a wide variety of biological actions. Biochemically, testosterone the main hormone is reduced at C5 yielding 5α and 5β-reduced androgens. Frequently, those 5-reduced androgens show biological actions different to testosterone. Besides, these compounds have either carbonyl and/or hydroxyl groups placed at carbons 3 and 17. The present work was aimed to explore the importance of such functional groups in 5α-androstane. Carbonyl or hydroxyl groups alone or combined at C3 and C17 were systematically included to 5α-androstane resulting a set of 16 different structures. The electronic structure of the 16 structures was studied using the Austin Model 1 (AM1) semi-empirical method. The results showed, that hydroxyl groups decreased the enthalpy of formation of 5α-androstane almost two-fold than carbonyl groups. Higher energy values for the HOMO and lower energy values for the LUMO were observed in structures bearing carbonyl groups. The 3-D picture of the HOMO and LUMO for the template (5α-androstane) as well as the mono- and di-hydroxy substituted structures were observed widespread along the molecules in a ‘sausage-like’ or ‘ribbon-like’ fashion. However mono- and di-keto-substituted structures showed that both HOMO and LUMO were located closer to the carbonyl groups. Carbonyl-substituted structures showed also the higher dipole moments and higher electrostatic charges. The lower values of enthalpy, dipole moments and electrostatic charges of hydroxylated molecules suggest a higher stability and lower reactivity of these compounds. Moreover, keto and di-keto structures which have the frontier orbitals at the neighborhood of these groups might facilitate a nucleophilic attack at the HOMO. It may be concluded that carbonyl groups increase the intermolecular forces of androstanes enhancing the probability to exert a biological action. Likewise, the metabolism of androstanes might be facilitated by the presence of carbonyl groups.