Experimental versus predicted affinities for ligand binding to estrogen receptor: iterative selection and rescoring of docked poses systematically improves the correlation.

The computational determination of binding modes for a ligand into a protein receptor is much more successful than the prediction of relative binding affinities (RBAs) for a set of ligands. Here we consider the binding of a set of 26 synthetic A-CD ligands into the estrogen receptor ERα. We show that the MOE default scoring function (London dG) used to rank the docked poses leads to a negligible correlation with experimental RBAs. However, switching to an energy-based scoring function, using a multiple linear regression to fit experimental RBAs, selecting top-ranked poses and then iteratively repeating this process leads to exponential convergence in 4-7 iterations and a very strong correlation. The method is robust, as shown by various validation tests. This approach may be of general use in improving the quality of predicted binding affinities.

Evolutionarily conserved paired immunoglobulin-like receptor α (PILRα) domain mediates its interaction with diverse sialylated ligands.

Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ∼22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.

A-CD estrogens. I. Substituent effects, hormone potency, and receptor subtype selectivity in a new family of flexible estrogenic compounds.

Long-term use of estrogen supplements by women leads to an increased risk of breast and uterine cancers. Possible mechanisms include metabolism of estradiol and compounds related to tumor-initiating quinones, and ligand-induced activation of the estrogen receptors ERα and ERß which can cause cancer cell proliferation, depending on the ratio of receptors present. One therapeutic goal would be to create a spectrum of compounds of variable potency for ERα and ERß, which are resistant to quinone formation, and to determine an optimum point in this spectrum. We describe the synthesis, modeling, binding affinities, hormone potency, and a measure of quinone formation for a new family of A-CD estrogens, where the A-C bond is formed by ring coupling. Some substituents on the A-ring increase hormone potency, and one compound is much less quinone-forming than estradiol. These compounds span a wide range of receptor subtype selectivities and may be useful in hormone replacement therapy.

Design and synthesis of 2-phenoxynicotinic acid hydrazides as anti-inflammatory and analgesic agents.

A series of 2-phenoxynicotinic acid hydrazides were synthesized and evaluated for their analgesic and anti-inflammatory activities. Several compounds having an unsubstituted phenyl/4-pyridyl or C-4 methoxy substituent on the terminal phenyl ring showed moderate to high analgesic or anti-inflammatory activity in comparison to mefenamic acid as the reference drug. The compounds with highest anti-inflammatory activity were subjected to in vitro COX-1/COX-2 inhibition assays and showed moderate to good COX-1 and weak COX-2 inhibition activities.

Rational design, synthesis, and optical properties of film-forming, near-infrared absorbing, and fluorescent chromophores with multidonors and large heterocyclic acceptors.

A new series of film-forming, low-bandgap chromophores (1 a,b and 2 a,b) were rationally designed with aid of a computational study, and then synthesized and characterized. To realize absorption and emission above the 1000 nm wavelength, the molecular design focuses on lowering the LUMO level by fusing common heterocyclic units into a large conjugated core that acts an electron acceptor and increasing the charge transfer by attaching the multiple electron-donating groups at the appropriate positions of the acceptor core. The chromophores have bandgap levels of 1.27-0.71 eV, and accordingly absorb at 746-1003 nm and emit at 1035-1290 nm in solution. By design, the relatively high molecular weight (up to 2400 g mol(-1)) and non-coplanar structure allow these near-infrared (NIR) chromophores to be readily spin-coated as uniform thin films and doped with other organic semiconductors for potential device applications. Doping with [6,6]-phenyl-C(61) butyric acid methyl ester leads to a red shift in the absorption only for 1 a and 2 a. An interesting NIR electrochromism was found for 2 a, with absorption being turned on at 1034 nm when electrochemically switched (at 1000 mV) from its neutral state to a radical cation state. Furthermore, a large Stokes shift (256-318 nm) is also unique for this multidonor-acceptor type of chromophore, indicating a significant structural difference between the ground state and the excited state. Photoluminescence of the film of 2 a was further probed at variable temperatures and the results strongly suggest that the restriction of bond rotations certainly helps to diminish non-radiative decay and thus enhance the luminescence of these large chromophores.

Investigation of residues Lys112, Glu136, His138, Gly247, Tyr248, and Asp249 in the active site of yeast cystathionine beta-synthase.

Cystathionine beta-synthase (CBS), the first enzyme of the reverse transsulfuration pathway, catalyzes the pyridoxal 5'-phosphate-dependent condensation of l-serine and l-homocysteine to form l-cystathionine (l-Cth). A model of the l-Cth complex of the truncated form of yeast CBS (ytCBS), comprising the catalytic core, was constructed to identify residues involved in the binding of l-homocysteine and the distal portion of l-Cth. Residue K112 was selected for site-directed mutagenesis based on the results of the in silico docking of l-Cth to the modeled structure of ytCBS. Residues E136, H138, Y248, and D249 of ytCBS were also targeted as they correspond to identical polar residues lining the mouth of the active site in the structure of human CBS. A series of 8 site-directed mutants was constructed, and their order of impact on the ability of ytCBS to catalyze the beta-replacement reaction is G247S asymptotically equal to K112Q > K112L asymptotically equal to K112R >> Y248F > D249A asymptotically equal to H138F > E136A. The beta-replacement activity of G247S, which corresponds to the homocystinuria-associated G307S mutant of human CBS, is undetectable. The Kml-Ser of the K112L and K112R mutants is increased by 50- and 90-fold, respectively, while Kml-Hcys increases by only 2- and 4-fold, respectively. The Kml-Hcys of H138F and Y248F is increased by 8- and 18-fold, respectively. These results indicate that, while the targeted residues are not direct determinants of l-Hcys binding, G307, Y248, and K112 play essential roles in the maintenance of appropriate active-site conformation.

Deconstructing estradiol: removal of B-ring generates compounds which are potent and subtype-selective estrogen receptor agonists.

Estradiol and related estrogens have been widely used as supplements to relieve menopausal symptoms, but they lead to an increased risk of breast and endometrial cancer. Here we report the synthesis of a new family of compounds where we have removed the B-ring from the steroid ABCD structure, and functionalized the A-ring. These A-CD compounds show a preferential affinity for the estrogen receptor subtype ERbeta. Some show binding affinities which are greater than estradiol. The presence of electron-withdrawing substituents on the A-ring should reduce the tendency of these compounds to form carcinogenic metabolites, so they might lead to a safer approach to hormone replacement therapy.

Interaction force diagrams: new insight into ligand-receptor binding.

A method is described to calculate and visualize the interaction forces of ligand-receptor complexes. Starting from an X-ray crystallographic structure, a "thawing" procedure results in a force-field energy-minimized geometry which is close to the crystallographic starting point. By subtracting non-bonded interactions of the ligand with each amino acid residue and using the resulting force vectors to describe the slope of the remaining potential, two types of interaction force diagrams are created; the first shows the direction of the force vectors in 3D and the second shows the magnitude of the force vectors. The latter representation leads to definition of an 'Interaction Force Fingerprint' (IFFP) which is characteristic of the ligand-receptor binding. IFFPs are used to discuss ligand binding in the human estrogen receptors ERalpha and ERbeta, and provide new insight into ligand selectivity between receptor isoforms.

Stability of carbon-centered radicals: effect of functional groups on the energetics of addition of molecular oxygen.

In this paper we examine a series of hydrocarbons with structural features which cause a weakening of the C-H bond. We use theoretical calculations to explore whether the carbon-centered radicals R(*) which are created after breaking the bond can be stabilized enough so that they resist the addition of molecular oxygen, i.e. where the reaction R(*) + O(2) --> ROO(*) becomes energetically unfavorable. Calculations using a B3LYP-based method provide accurate bond dissociation enthalpies (BDEs) for R-H and R-OO(*) bonds, as well as Gibbs free energy changes for the addition reaction. The data show strong correlations between R-OO(*) and R-H BDEs for a wide variety of structures. They also show an equally strong correlation between the R-OO(*) BDE and the unpaired spin density at the site of addition. Using these data we examine the major functional group categories proposed in several experimental studies, and assess their relative importance. Finally, we combine effects to try to optimize resistance to the addition of molecular oxygen, an important factor in designing carbon-based antioxidants.

Using Molecular Strain and Aromaticity To Create Ultraweak C-H Bonds and Stabilized Carbon-Centered Radicals.

An approach based on relief of molecular strain in the parent hydrocarbon, extended conjugation in the radical, and the driving force toward aromaticity is used to design molecules with ultraweak C-H bonds. The molecular strain is generated by two fused rings containing (5,5)-, (5,6)-, or (6,6)-membered ring structures. Homodesmotic reactions are used to calculate the molecular strain enthalpy (MSE) of the parent hydrocarbons and the corresponding radicals, and to analyze how it changes through these reactions. B3LYP calculations are used to obtain the bond dissociation enthalpies (BDEs) for breaking one or more C-H bonds as well as the C-O bond formed after oxygen addition to the radical. Loss of a second H-atom can lead to very low R-H BDE values, especially when the ultimate product is aromatic. Molecular structures based on these ideas may be of interest as novel antioxidants based on carbon-centered radicals.

Computational modeling of substituent effects on phenol toxicity.

Standard computational models of cytotoxicity of substituted phenols relate the toxicity to a set of quatitative structure-activity relationship (QSAR) descriptors such as log P, p K a, OH bond dissociation enthalpy (BDE), etc. Implicit in this approach is the idea that the phenoxyl radical is disruptive to the cell and factors increasing its production rate will enhance the toxicity. To improve the QSAR correlations, substituents are usually divided into electron-donating groups (EDG) and electron-withdrawing groups (EWG), which are treated separately and thought to follow different mechanisms of toxicity. In this paper, we focus on one important aspect of toxicity, the rate constant for production of phenoxyl radical. Activation energies are obtained for the reaction of X-phenol with peroxyl radical by using the Evans-Polanyi principle, giving rate constants as a function of DeltaBDE values for both EDG and EWG sets. We show that (i) a plot of log k for phenoxyl formation vs DeltaBDE shows a double set of straight lines with different slopes, justifying the usual EDG and EWG separation but without requiring any change in mechanism; (ii) the same method can be effectively used for different target radicals (e.g., tert-butoxyl) or different sets of parent compounds (e.g., substituted catechols), thus giving a useful general approach to analysis of toxicity data; (iii) regions of constant toxicity in all cases are predicted; and (iv) we argue that competing parallel mechanisms of toxicity are likely to be dominant for EWG-substituted phenols.

Understanding the toxicity of phenols: using quantitative structure-activity relationship and enthalpy changes to discriminate between possible mechanisms.

Experimental studies of the "extended toxicity" of substituted phenols are mainly of two types: the toxicity due to phenoxyl radical formation and the toxicity caused by metabolites, for example, the formation of quinones. Quantitative structure-activity relationship (QSAR) studies of phenol toxicity have dealt with the formation of phenoxyl radicals using bond dissociation enthalpy (BDE) of parent phenols, have obtained good correlations with experimental data, and have concluded that phenoxyl radicals are the toxic agent. However, the actual toxic mechanism has remained poorly defined. In this study, we follow the metabolic pathways of monosubstituted phenols to their quinone end products and calculate enthalpy changes for all relevant reactions. These enthalpy changes are first used as descriptors for a QSAR analysis. Many of these new descriptors, including some relevant to quinone formation, are highly correlated with the BDE values of the parent phenols. Therefore, a QSAR analysis by itself is inconclusive as to the mechanism of toxicity. To better define the problem, we have returned to a detailed analysis of net enthalpy changes. We show that the formation of phenoxyl radical is the rate-determining step: This step is slow for electron-withdrawing group substituted phenols (EWG-phenols), whereas it is fast for electron-donating group substituted phenols (EDG-phenols). The study of net enthalpy changes of reactions reveals that once the phenoxyl radical is present, the corresponding quinone is rapidly formed, so that quinone formation may be ultimately responsible for toxicity of EDG-phenols. We then demonstrate how the suggested mechanism (quinone formation) is successful in predicting the toxicity of some complex phenols, which are predicted poorly using the phenoxyl radical argument. We also discuss the toxicities of some estrogens in light of the quinone mechanism.

Synthesis, antibacterial activity, and quantitative structure-activity relationships of new (Z)-2-(nitroimidazolylmethylene)-3(2H)-benzofuranone derivatives.

A new series of (Z)-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranones (11a-p) and (Z)-2-(1-methyl-4-nitroimidazole-5-ylmethylene)-3(2H)-benzofuranones (12a-m) were synthesized and assayed for their antibacterial activity against Gram-positive and Gram-negative bacteria. Most of the 5-nitroimidazole analogues (11a-p) showed a remarkable inhibition of a wide spectrum of Gram-positive bacteria (Staphylococcus aureus, Streptococcus epidermidis, MRSA, and Bacillus subtilis) and Gram-negative Klebsiella pneumoniae, whereas 4-nitroimidazole analogues (12a-m) were not effective against selected bacteria. The quantitative structure-activity relationship investigations were applied to find out the correlation between the experimentally evaluated activities with various parameters of the compounds studied. The QSAR models built in this work had reasonable predictive power and could be explained by the observed trends in activities.

Design, synthesis, and biological evaluation of substituted 2-alkylthio-1,5-diarylimidazoles as selective COX-2 inhibitors.

A new type of 1-aryl-5-(4-methylsulfonylphenyl)imidazoles, possessing C-2 alkylthio (SMe or SEt) substituents, were designed and synthesized for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors with in vivo anti-inflammatory activity. The compound, 1-(4-bromophenyl)-5-(4-methylsulfonylphenyl)-2-methylthioimidazole (11g), was the most potent and selective COX-2 inhibitor (COX-2 IC50=0.43 microM with no inhibition of COX-1 up to 25 microM) relative to the reference drug celecoxib (COX-2 IC50=0.21 microM with no inhibition of COX-1 up to 25 microM) and also showed very good anti-inflammatory activity compared to celecoxib in carrageenan-induced rat paw edema assay.