A Study of the Binding of Estradiol and 8-Isoestradiol to the Estrogen α-Receptor by Molecular Modeling

The complexes of the estrogen -receptor with estradiol and 8-isoestradiol were comparatively analyzed. The computations of ligand–receptor complexes, carried out using the FLEXX program, allowed us to propose a model for the binding of the analogues of 8-isoestradiol. It was found that rings Cand D of estradiol and 8-isoestradiol are similarly arranged in the ligand-binding pocket and coincide upon the superposition of the corresponding ligand–receptor complexes, whereas rings A and B do not coincide. The oxygen functions in position 17 of the estradiol analogues of both series coincide upon superposition, whereas the phenol 3-hydroxyl groups are 0.05 Å apart. A comparison of the predicted biological properties of modified estradiol analogues of the natural and 8-iso-series with the available experimental data revealed their similarity. Synthetic 2-acetyl analogues of 8-isoestrogens were found to have no uterotropic activity, which is also consistent with the proposed model.     

Synthesis and study of B-nor-8-isoanalogs of steroid estrogens

The study of the binding of estradiol B-nor-8-isonalogues to estrogen receptors from the rat uterus helped create the proposed model of the corresponding ligand-receptor complexes. The use of this model ensured the choice of such micromodifications in this steroid group that sharply decreased their hormonal activity. By the example of 16,16-dimethyl-D-homo-B-nor-8-isoestrone, we demonstrated the possibility of the synthesis of the estrogen analogues devoid of uterotropic activity but retaining immunosuppressive activity.     

Enhancing lipophilicity as a strategy to overcome resistance against platinum complexes?

Decreased influx represents one of the major resistance mechanisms of platinum complexes. In order to address the question if this mechanism of resistance can be overcome by enhancing the lipophilicity of platinum complexes, we investigated the influence of lipophilicity on cellular accumulation and cytotoxicity in a panel of oxaliplatin analogues with different carrier ligands. Cellular accumulation, DNA platination and cytotoxicity were measured in a cisplatin-sensitive and -resistant ovarian carcinoma (A2780/A2780cis) and in an oxaliplatin-sensitive and -resistant ileocecal colorectal adenocarcinoma (HCT-8/HCT-8ox) cell line pair. Platinum concentrations were determined by flameless atomic absorption spectrometry or adsorptive stripping voltammetry. Passive diffusion represented the main influx mechanism of oxaliplatin analogues during the first minutes of incubation as indicated by a correlation between lipophilicity and early influx rate. Afterwards, the predominant influx mechanism was lipophilicity-independent. More lipophilic complexes showed a reduced cytotoxic activity, although the early influx rate was increased. The resistance profiles of the two cell line pairs were found to be different: HCT-8ox cells were less resistant against more lipophilic complexes, whereas A2780cis cells exhibited a comparable degree of resistance against all investigated compounds. However, the reduction in resistance factor of HCT-8ox cells cannot be explained by increased influx suggesting that other resistance mechanisms are circumvented upon exposure to more lipophilic compounds. Though resistance against more lipophilic platinum complexes analogues is lower we conclude that enhancing lipophilicity is not a successful strategy to overcome platinum resistance as higher lipophilicity is also associated with lower cytotoxic activity.     

Exploring the relationship between binding modes of 9-(aminomethyl)-9,10-dihydroanthracene and cyproheptadine analogues at the 5-HT2A serotonin receptor

Comparison of the serotonin 5-HT2A receptor affinities of a parallel series of structural analogues of the novel ligand 9-aminomethyl-9,10-dihydroanthracene (AMDA) and a structurally similar prototypical tricyclic amine cyproheptadine suggests that the two agents bind to the receptor in different fashions. Examination of ligand-receptor model complexes supports the experimental data and suggests a potential origin for the differences in binding modes.     

QSAR study on adenosine kinase inhibition of pyrrolo[2,3-d]pyrimidine nucleoside analogues using the hansch approach

QSAR studies on series of pyrrolo[2,3-d]pyrimidine nucleoside analogues were performed for their adenosine kinase (AK) inhibitory activity using the Hansch approach. Significant correlations were obtained with hydrophobic parameter at position 'X'. Electronic and steric parameters on pyrimidine and pyrrole rings found to play an important role in the ligand-receptor interactions with the active sites of the enzyme. Presence of bulkier groups at 'X' and 'Y' positions seems to protect the title compounds from biodegradation, as is evident from their positive sterimol steric parameter B1 at these positions.     

Locating an antagonist in the 5-HT3 receptor binding site using modeling and radioligand binding

We have used a homology model of the extracellular domain of the 5-HT(3) receptor to dock granisetron, a 5-HT(3) receptor antagonist, into the binding site using AUTODOCK. This yielded 13 alternative energetically favorable models. The models fell into 3 groups. In model type A the aromatic rings of granisetron were between Trp-90 and Phe-226 and its azabicyclic ring was between Trp-183 and Tyr-234, in model type B this orientation was reversed, and in model type C the aromatic rings were between Asp-229 and Ser-200 and the azabicyclic ring was between Phe-226 and Asn-128. Residues located no more than 5 A from the docked granisetron were identified for each model; of 26 residues identified, 8 were found to be common to all models, with 18 others being represented in only a subset of the models. To identify which of the docking models best represents the ligand-receptor complex, we substituted each of these 26 residues with alanine and a residue with similar chemical properties. The mutant receptors were expressed in human embryonic kidney (HEK)293 cells and the affinity of granisetron determined using radioligand binding. Mutation of 2 residues (Trp-183 and Glu-129) ablated binding, whereas mutation of 14 other residues caused changes in the [(3)H]granisetron binding affinity in one or both mutant receptors. The data showed that residues both in and close to the binding pocket can affect antagonist binding and overall were found to best support model B.     

The Synthesis and Properties of B-Nor-8-Isoanalogues of Steroid Estrogens

The study of the binding of estradiol B-nor-8-isonalogues to estrogen receptors from the rat uterus helped create the proposed model of the corresponding ligand–receptor complexes. The use of this model ensured the choice of such micromodifications in this steroid group that sharply decreased their hormonal activity. By the example of 16,16-dimethyl-D-homo-B-nor-8-isoestrone, we demonstrated the possibility of the synthesis of the estrogen analogues devoid of uterotropic activity but retaining immunosuppressive activity.     

Dependence of the redistribution of ligand-receptor complexes in cultured cells on the type of growth and on the cell cycle phase

The process of active redistribution of ligand-receptor complexes on the surface of both suspension (LS) and adapted for growing in monolayer (LMS) sublines of mouse fibroblasts L was studied. The binding of ligands to its specific receptors on the surface of the LS cells induced an accumulation of ligand-receptor complexes on one pole of a cell with the formation of a typical cap. Under the same conditions in the LSM cells the cleaning of processes and lamello-plasma surfaces from the ligand-receptor complexes was registered. The binding of ligands to the surface of the LSM cells, detached with EDTA, induced the same capping process, as in the case of the LS cells. No differences were found in the redistribution capacity of the ligand-receptor complexes in synchronized cultures of the LS and LSM cells in the G1 and S phase of the cycle. But such a redistribution was not registered on the surface of cells in metaphase and anaphase. The accumulation of ligand-receptor complexes was found in the region of cleavage between daughter cells in telophase. These results are in a good agreement with the well-known data on the changes in the cytoskeleton organization during transition from a monolayer to a suspension state and during mitosis.     

Interaction of synthetic analogues of distamycin with poly(dA-dT): role of the conjugated N-methylpyrrole system

Two synthetic analogues of distamycin (Dst), PPA and PAP, containing a saturated beta-alanine moiety substituting for an N-methylpyrrole chromophore were studied for their interactions with the double-stranded alternating copolymer poly(dA-dT).poly(dA-dt) [abbreviated as poly(dA-dT)], with UV absorption and circular dichroism (CD) spectroscopy. The distinctive feature of these analogues is the difference in the extents of extended conjugation due to contiguous pyrrole rings: it decreases in the order Dst greater than PPA greater than PAP. Both these analogues bind to poly(dA-dT) in a way similar to Dst, as suggested from the observed red shift in the UV spectra of the ligands upon complexation and the appearance of induced Cotton effects (in the 290-350-nm region) in the CD spectra of the complexes. A comparative study of (i) the spectral features of the complexes between these ligands, Dst and netrospin (Nt) and poly(dA-dT), and (ii) the binding parameters for the association with the polynucleotide suggests that the number and relative positions of the pyrrole moieties influence the spectral features and thermodynamic stabilities of the complexes, and the latter show a progressive decrease in the order Dst greater than Nt greater than PPA greater than PAP. Implications of these results vis-à-vis the molecular basis of Dst-DNA interaction are discussed.     

Atomic force and electron microscopy of high molecular weight circular DNA complexes with synthetic oligopeptide trivaline

Intramolecular compact structures formed by high molecular weight circular superhelical DNA molecules due to interaction with synthetic oligopeptide trivaline (1) were studied by atomic force and electron microscopy. Three DNA preparations were used: plasmids pTbol, pRX10 and cosmid 27,877, with sizes 6,120 bp, 10,500 bp and 44,890 bp respectively. Plasmid pTbo1 and pRX10 preparations along with monomers contained significant amount of dimers and trimers. Main structures in all preparations observed were compact particles, which coincide in their appearance and compaction coefficient (3,5-3,7) with triple rings described earlier. The size and structure characteristics of triple rings and other compact particles on atomic force images in general coincide with those obtained by EM (2). AFM (3) images allow to get additional information about the ultrastructural organization and arrangement of DNA fibers within the compact structures. Along with triple rings in pTbol and pRX10-TVP complexes significant amount of compact structures were observed having the shape of two or three compact rings attached to each other by a region of compact fibre. Basing on the data of contour length measurements and the shape of the particles it was concluded that these structures were formed due to compaction of dimeric and trimeric circular DNA molecules. Structures consisting of several attached to each other triple rings were not found for pTbol, pRX10 monomers or cosmid preparations--TVP complexes where only single triple rings were observed. The conclusion is made that initiation of compact fibre formation within the circular molecules depends on the primary structure and for dimeric or trimeric circular molecules two or three compaction initiation points are present, located in each monomer unit within one circular DNA molecule. The nucleotide sequence dependent compaction mechanism providing independent compaction of portions of one circular molecule can be of interest for understanding of DNA compaction processes in vivo.     

Synthesis and SAR exploration of dinapsoline analogues

Dinapsoline is a full D(1) dopamine receptor agonist that produces robust rotational activity in the unilateral 6-OHDA rat model. This compound is orally active, and shows a low tendency to cause tolerance in rat models. The active enantiomer was determined to have the S-(+) configuration, and the opposite enantiomer is essentially devoid of biological activity. Taken together, dinapsoline has significant metabolic and pharmacological advantages over previous D(1) agonists. In an attempt to define the structure-activity relationships (SARs) and to map out the key elements surrounding the unique structure of dinapsoline, core analogues and substitution analogues of the parent tetracyclic condensed ring structure were prepared. Based on a recently developed synthesis of dinapsoline and its enantiomers, both core and substitution analogues on all four rings (A, B', C and D ring) of dinapsoline were synthesized. It was found that affinity for both D(1)and D(2) receptors was decreased by most substituents on the A, B', and C rings, whereas D ring substitutions preserved much of the dopamine receptor binding activity.     

Enzyme inhibition potency enhancement by active site metal chelating and hydrogen bonding induced conformation-restricted cyclopropanecarbonyl derivatives

Two cyclopropanecarbonyl derivatives were independently found to be 15 and 14 times more potent than the corresponding isopropylcarbonyl analogues as inhibitors of 4-hydroxyphenylpyruvate dioxygenase and dihydroorotate dehydrogenase, respectively. A thorough examination of the co-crystal structures of available enzyme inhibitor complexes and the conformation of X-ray crystal structures of several synthesized cyclopropanecarbonyl derivatives revealed that this enhancement by one order of magnitude of inhibition potency exhibited by cyclopropanecarbonyl derivatives in both enzymes is probably caused by respective metal chelating and hydrogen bonding interactions at the ligand-receptor binding site. These specific interactions subsequently cause the cyclopropyl group of the molecules to adopt a fixed bisected conformation, which is unavailable for isopropylcarbonyl derivatives.     

Structural features which distinguish estrogen agonists and antagonists

The molecular structures of compounds that compete for the estrogen binding site suggest that when estradiol is bound to the receptor, there is a close fit only at the A-ring end of the steroid. The most potent antagonists have phenolic rings capable of mimicking the estradiol A-ring in promoting high affinity binding to the receptor. They fail to stabilize the conformational change or molecular interaction needed to achieve hormonal response because they either lack an essential functional group (hydrogen bond donor) or they present a steric block or topological feature incompatible with transformation or interaction subsequent to the initiation of binding by the A-ring or A-ring analogues. X-Ray analyses of 12 triphenylethylene derivatives demonstrate the stability of a specific conformation that is unaffected by crystal packing forces. This conformation has a pinwheel-like orientation of phenyl rings, the direction of which is correlated with a 10 degrees twist about the central double bond and appears to depend upon the orientation of the non-phenyl substituent relative to the double bond. Empirical energy calculations are insensitive to this intramolecular structural dependence and incorrectly predict that the pinwheel of the opposite direction is of lower energy.     

Protein structural changes accompanying formation of enzymatic transition states: tryptophan environment in ground-state and transition-state analogue complexes of adenosine deaminase

The accessibility of protein tryptophan fluorescence to the quenching agent acrylamide has been studied in adenosine deaminase and in binary complexes of the enzyme with ground-state or transition-state analogues. Although the enzyme contains three tryptophan residues, Stern-Volmer plots are linear with all the fluorescence quenchable at high acrylamide concentrations. Tryptophan fluorescence is less easily quenched in the binary complexes than in the free enzyme, indicating a decrease in the accessibility of these residues. The greatest decrease in accessibility is found for the transition-state analogue complexes. Although the affinities of the transition-state analogues studied span a range of 10(6), the Stern-Volmer constants of the complexes are the same within experimental error. Thus, as measured by this technique, changes in enzyme conformation accompanying formation of these complexes are similar for all transition-state analogues. Resonance energy transfer from tryptophan as donor to ligand as acceptor successfully explains the differing abilities of ligands to quench the enzyme's intrinsic fluorescence upon formation of complexes in the absence of acrylamide. On the basis of Forster distance calculations, it is likely that the residues partially quenched upon formation of transition-state analogue complexes are distant from the active site.     

Probabilistic description of the system "ligand-receptor"

The theory of probabilities was used to describe the ligand-receptor interaction. Mean and variance of number ligand-receptor complexes are calculated. It is shown that the mean number of ligand-receptor complexes coincides with that obtained from the law of conservation masses. Proceeding from a ratio of mean and expectation it is shown that the variance of the number of ligand-receptor complexes should be taken into account with concentration of ligand-receptor complexes component less than 1 fmol.     

A kinetic model of the transient phase in the response of olfactory receptor neurons

A model is presented that predicts the instantaneous spike rate of an olfactory receptor neuron (ORN) in response to the quality and concentration of an odor stimulus. The model accounts for the chemical kinetics of ligand-receptor binding and activation processes, and implicitly the initiation of second messenger cascades that lead to depolarization and/or hyperpolarization of the ORN membrane. Both of these polarizing processes are included in the most general form of the model, as well as a process that restores the voltage to its negative resting state. The spike rate is assumed to be linearly proportional to the level of voltage depolarization above a critical negative voltage level. The model includes the simplifying assumption that activation of bound ligand-receptor complexes by G-proteins and other enabling molecules follows a Monod function that has the ratio of enabling molecules to bound unactivated ligand-receptor complexes as its argument. Parameters are selected that provide an excellent fit of the model to previously published empirical data on the response of cockroach ORNs to pulsed 1-hexanol stimuli. The sensitivity of model output to various model parameters is investigated and changes to parameters are discussed that would improve the ability of ORNs to follow rapidly pulsed stimuli.     

Three-dimensional structure of the complexes of ribonuclease A with 2',5'-CpA and 3',5'-d(CpA) in aqueous solution, as obtained by NMR and restrained molecular dynamics.

The three-dimensional structure of the complexes of ribonuclease A with cytidyl-2',5'-adenosine (2',5'-CpA) and deoxycytidyl-3',5'-deoxyadenosine [3',5'-d(CpA)] in aqueous solution has been determined by 1H NMR methods in combination with restrained molecular dynamics calculations. Twenty-three intermolecular NOE cross-corrections for the 3',5'-d(CpA) complex and 19 for the 2',5'-CpA, together with about 1,000 intramolecular NOEs assigned for each complex, were translated into distance constraints and used in the calculation. No significant changes in the global structure of the enzyme occur upon complex formation. The side chains of His 12, Thr 45, His 119, and the amide backbone group of Phe 120 are involved directly in the binding of the ligands at the active site. The conformation of the two bases is anti in the two complexes, but differs from the crystal structure in the conformation of the two sugar rings in 3',5'-d(CpA), shown to be in the S-type region, as deduced from an analysis of couplings between the ribose protons. His 119 is found in the two complexes in only one conformation, corresponding to position A in the free protein. Side chains of Asn 67, Gln 69, Asn 71, and Glu 111 from transient hydrogen bonds with the adenine base, showing the existence of a pronounced flexibility of these enzyme side chains at the binding site of the downstream adenine. All other general features on the structures coincide clearly with those observed in the crystal state.     

The effects of sex hormones on the synthesis of prostacyclin (PGI2) by vascular tissues

The effects of estradiol and testosterone on prostacyclin (PGI2) release (measured as 6-keto-PGF1 alpha) by vascular tissues using rat aortic rings and cultured rabbit aortic smooth muscle cells (SMC) were investigated. Aortic SMC were prepared from either explants of atherosclerotic intima or those of normal media. Aortic rings obtained from male and female rats which had been treated with estradiol resulted in increased PGI2 synthesis. Furthermore, PGI2 synthesis by cultured medial SMC was significantly increased in the presence of estradiol (10(-7), 10(-9) M). An increased tendency in PGI2 synthesis was also observed in intimal SMC. On the other hand, aortic rings obtained from female rats treated with testosterone resulted in a significant decrease in PGI2 synthesis. However, aortic rings from testosterone-treated male rats and cultured medial and intimal SMC treated with testosterone (10(-6), 10(-8) M) for 48 hr did not show any significant changes in PGI2 synthesis. We also found greater PGI2 synthesis by intimal SMC compared with that by medial SMC. These results suggest that estradiol and testosterone may have opposite functions in the development of atherosclerosis, that is, estradiol for anti-atherosclerotic and testosterone for atherogenic, by modulating PGI2 synthesis by vascular tissues.     

Spectra and structure of complexes formed by sodium fusidate and potassium helvolate with beta- and gamma-cyclodextrin

The complexation of two steroid antibiotics of the fusidane family, sodium fusidate and potassium helvolate, by beta-CD and gamma-CD has been studied by using 1D and 2D-NMR techniques. Both guests form 1:1 complexes with gamma-CD and 1:2 (guest:cyclodextrin) complexes with beta-CD. Thus, both antibiotics behave as monotopic and ditopic guests when they are complexed by gamma-CD and beta-CD, respectively. Both steroids enter into the cavity of the gamma-CD by the side chain, reaching the central region of the steroid (rings C and D), whereas the A and B (partially) rings remain outside. For beta-CD complexes, ROESY spectra show a remarkable absence of interactions of the protons of the C and D rings, whereas clear interactions corresponding to the side chain, and A and B rings are observed. The obtained equilibrium constants (see previous paper) are discussed in terms of the structures proposed for the complexes. NMR spectra of sodium fusidate are revised, and a full assignment of the 1H and 13C NMR spectra is presented for potassium helvolate.     

NMR studies of CCK-8/CCK1 complex support membrane-associated pathway for ligand-receptor interaction

The interaction of peptide ligands with their associated G-protein-coupled receptors has been examined by a number of different experimental approaches over the years. We have been developing an approach utilizing high-resolution NMR to determine the structural features of the peptide ligand, well-designed fragments of the receptor, and the ligand-receptor complexes formed upon titration of the peptide hormone. The results from these investigations provide evidence for a membrane-associated pathway for the initial interaction of peptide ligands with the receptor. Here, our results from the investigation of the interaction of CCK-8 with the CCK1 receptor are described. Our spectroscopic results clearly show that both CCK-8 and the regions of CCK1 with which it interacts are closely associated with the zwitterionic interface of the lipids utilized in our solution spectroscopic studies.