Interactions of linear dicationic molecules with lipid A: structural requisites for optimal binding affinity

The structural determinants of the binding affinity of linear dicationic molecules toward lipid A have been examined with respect to the distance between the terminal cationic functions, the basicity, and the type of cationic moieties using a series of spermidine derivatives and pentamidine analogs by fluorescence spectroscopic methods, The presence of two terminal cationic groups corresponds to enhanced affinity, A distinct sigmoidal relationship between the intercationic distance and affinity was observed with a sharp increase at 11 Angstrom, levelling off at about 13 Angstrom. The basicity (pK) and nature of the cationic functions are poor correlates of binding potency, since molecules bearing primary amino, imidazolino, or guanido termini are equipotent, The interaction of pentamidine, a bisamidine drug, with lipid A, characterized in considerable detail employing the putative intermolecular excimerization of the drug, suggests a stoichiometry of 1:1 in the resultant complex, The binding is driven almost exclusively by electrostatic forces, and is dependent on the ionization states of both lipid A and the drug, Under conditions when lipid A is highly disaggregated, pentamidine binds specifically to bis-phosphoryl- but not to monophosphoryl-lipid A indicating that both phosphate groups of lipid A are necessary for electrostatic interactions by the terminal amidininium groups of the drug, Based on these data, a structural model is proposed for the pentamidine-lipid A complex, which may be of value in designing endotoxin antagonists from first principles.

Phenyl carbohydrazone conjugated 2-oxoindoline as a new scaffold that augments the DNA and BSA binding affinity and anti-proliferative activity of a 1,10-phenanthroline based copper(II) complex

A new type of copper(II) complex, CuL(phen)(2)](NO3) (CuIP), where L ((E)-N'-(2-oxoindolin-3-ylidene) benzohydrazide) is a N donor ligand and phen is the N, N-donor heterocyclic 1,10-phenanthroline, has been synthesized. The phenyl carbohydrazone conjugated isatin-based ligand L and CuIP were characterized by elemental analysis, infrared, UV-Vis, H-1 and C-13 NMR and ESI-mass spectral data, as well as single-crystal X-ray diffraction. The interaction of calf thymus DNA (CT DNA) with L and CuIP has been investigated by absorption, fluorescence and viscosity titration methods. The complex CuIP displays better binding affinity than the ligand L. The observed DNA binding constant (K-b = 4.15(+/- 0.18) x 10(5) M-1) and binding site size (s = 0.19), viscosity data together with molecular docking studies of CuIP suggest groove binding and/or a partial intercalative mode of binding to CT DNA. In addition, CuIP shows good binding propensity to the bovine serum albumin (BSA) protein, giving a K-BSA value of 1.25(+/- 0.24) x 10(6) M-1. In addition, the docking studies on DNA and human serum albumin (HSA) CuIP interactions are consistent with the consequence of binding experiments. The in vitro anti-proliferative study establishes the anticancer potency of the CuIP against the human cervical (HeLa) and breast (MCF7) cancer cells; noncancer breast epithelial (MCF10a) cells have also been investigated. CuIP shows better cytotoxicity and sensitivity towards cancer cells over noncancer ones than L under identical conditions, with the appearance of apoptotic bodies. (C) 2014 Elsevier B.V. All rights reserved.

Increase and decrease in binding affinity of antibodies during the immune response

SCOPUS: ar.j

Statistical deconvolution of enthalpic energetic contributions to MHC-peptide binding affinity

Background: MHC Class I molecules present antigenic peptides to cytotoxic T cells, which forms an integral part of the adaptive immune response. Peptides are bound within a groove formed by the MHC heavy chain. Previous approaches to MHC Class I-peptide binding prediction have largely concentrated on the peptide anchor residues located at the P2 and C-terminus positions. Results: A large dataset comprising MHC-peptide structural complexes was created by remodelling pre-determined x-ray crystallographic structures. Static energetic analysis, following energy minimisation, was performed on the dataset in order to characterise interactions between bound peptides and the MHC Class I molecule, partitioning the interactions within the groove into van der Waals, electrostatic and total non-bonded energy contributions. Conclusion: The QSAR techniques of Genetic Function Approximation (GFA) and Genetic Partial Least Squares (G/PLS) algorithms were used to identify key interactions between the two molecules by comparing the calculated energy values with experimentally-determined BL50 data. Although the peptide termini binding interactions help ensure the stability of the MHC Class I-peptide complex, the central region of the peptide is also important in defining the specificity of the interaction. As thermodynamic studies indicate that peptide association and dissociation may be driven entropically, it may be necessary to incorporate entropic contributions into future calculations.

Structure-Based Approach for the Study of Estrogen Receptor Binding Affinity and Subtype Selectivity

Estrogens exert important physiological effects through the modulation of two human estrogen receptor (hER) subtypes, alpa (hER alpha) and beta (hER beta). Because the levels and relative proportion of hER alpha and hER beta differ significantly in different target cells, selective hER ligands could target specific tissues or pathways regulated by one receptor subtype without affecting the other. To understand the structural and chemical basis by which small molecule modulators are able to discriminate between the two subtypes, we have applied three-dimensional target-based approaches employing a series of potent hER-ligands. Comparative molecular field analysis (CoMFA) studies were applied to a data set of 81 hER modulators, for which binding affinity values were collected for both hER alpha and hER beta. Significant statistical coefficients were obtained (hER alpha, q(2) = 0.76; hER beta, q(2) = 0.70), indicating the internal consistency of the models. The generated models were validated using external test sets, and the predicted values were in good agreement with the experimental results. Five hER crystal structures were used in GRID/PCA investigations to generate molecular interaction fields (MIF) maps. hER alpha and hER beta were separated using one factor. The resulting 3D information was integrated with the aim of revealing the most relevant structural features involved in hER subtype selectivity. The final QSAR and GRID/PCA models and the information gathered from 3D contour maps should be useful for the design or novel hER modulators with improved selectivity.

Impedance-derived electrochemical capacitance spectroscopy for the evaluation of lectin-glycoprotein binding affinity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A fragment-based approach for ligand binding affinity and selectivity for the liver X receptor beta

Selective modulation of liver X receptor beta (LXR beta) has been recognized as an important approach to prevent or reverse the atherosclerotic process. In the present work, we have developed robust conformation-independent fragment-based quantitative structure-activity and structure-selectivity relationship models for a series of quinolines and cinnolines as potent modulators of the two LXR sub-types. The generated models were then used to predict the potency of an external test set and the predicted values were in good agreement with the experimental results, indicating the potential of the models for untested compounds. The final 2D molecular recognition patterns obtained were integrated to 3D structure-based molecular modeling studies to provide useful insights into the chemical and structural determinants for increased LXR beta binding affinity and selectivity. (C) 2011 Elsevier Inc. All rights reserved.

Ring size of somatostatin analogues (ODT-8) modulates receptor selectivity and binding affinity

The synthesis, biological testing, and NMR studies of several analogues of H-c[Cys (3)-Phe (6)-Phe (7)-DTrp (8)-Lys (9)-Thr (10)-Phe (11)-Cys (14)]-OH (ODT-8, a pan-somatostatin analogue, 1) have been performed to assess the effect of changing the stereochemistry and the number of atoms in the disulfide bridge on binding affinity. Cysteine at positions 3 and/or 14 (somatostatin numbering) were/was substituted with d-cysteine, norcysteine, D-norcysteine, homocysteine, and/or D-homocysteine. The 3D structure analysis of selected partially selective, bioactive analogues (3, 18, 19, and 21) was carried out in dimethylsulfoxide. Interestingly and not unexpectedly, the 3D structures of these analogues comprised the pharmacophore for which the analogues had the highest binding affinities (i.e., sst 4 in all cases).

Ring size in octreotide amide modulates differently agonist versus antagonist binding affinity and selectivity

H-DPhe (2)-c[Cys (3)-Phe (7)-DTrp (8)-Lys (9)-Thr (10)-Cys (14)]-Thr (15)-NH2 (1) (a somatostatin agonist, SRIF numbering) and H-Cpa (2)-c[DCys (3)-Tyr (7)-DTrp (8)-Lys (9)-Thr (10)-Cys (14)]-Nal (15)-NH2 (4) (a somatostatin antagonist) are based on the structure of octreotide that binds to three somatostatin receptor subtypes (sst 2/3/5) with significant binding affinity. Analogues of 1 and 4 were synthesized with norcysteine (Ncy), homocysteine (Hcy), or D-homocysteine (DHcy) at positions 3 and/or 14. Introducing Ncy at positions 3 and 14 constrained the backbone flexibility, resulting in loss of binding affinity at all sst s. The introduction of Hcy at positions 3 and 14 improved selectivity for sst 2 as a result of significant loss of binding affinity at the other sst s. Substitution by DHcy at position 3 in the antagonist scaffold (5), on the other hand, resulted in a significant loss of binding affinity at sst 2 and sst 3 as compared to the different affinities of the parent compound (4). The 3D NMR structures of the analogues in dimethylsulfoxide are consistent with the observed binding affinities.

Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity

Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant Kd = 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin–biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 105–107 yeast surface-displayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wild-type complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.

Correlation between Binding Affinity and Necrosis-Inducing Activity of Mutant AVR9 Peptide Elicitors1

The race-specific peptide elicitor AVR9 of the fungus Cladosporium fulvum induces a hypersensitive response only in tomato (Lycopersicon esculentum) plants carrying the complementary resistance gene Cf-9 (MoneyMaker-Cf9). A binding site for AVR9 is present on the plasma membranes of both resistant and susceptible tomato genotypes. We used mutant AVR9 peptides to determine the relationship between elicitor activity of these peptides and their affinity to the binding site in the membranes of tomato. Mutant AVR9 peptides were purified from tobacco (Nicotiana clevelandii) inoculated with recombinant potato virus X expressing the corresponding avirulence gene Avr9. In addition, several AVR9 peptides were synthesized chemically. Physicochemical techniques revealed that the peptides were correctly folded. Most mutant AVR9 peptides purified from potato virus X::Avr9-infected tobacco contain a single N-acetylglucosamine. These glycosylated AVR9 peptides showed a lower affinity to the binding site than the nonglycosylated AVR9 peptides, whereas their necrosis-inducing activity was hardly changed. For both the nonglycosylated and the glycosylated mutant AVR9 peptides, a positive correlation between their affinity to the membrane-localized binding site and their necrosis-inducing activity in MoneyMaker-Cf9 tomato was found. The perception of AVR9 in resistant and susceptible plants is discussed.

Major histocompatibility complex binding affinity of an antigenic determinant is crucial for the differential secretion of interleukin 4/5 or interferon gamma by T cells.

Differential activation of CD4+ T-cell precursors in vivo leads to the development of effectors with unique patterns of lymphokine secretion. To investigate whether the differential pattern of lymphokine secretion is influenced by factors associated with either the display and/or recognition of the ligand, we have used a set of ligands with various class II binding affinities but unchanged T-cell specificity. The ligand that exhibited approximately 10,000-fold higher binding to I-Au considerably increased the frequency of interferon gamma-producing but not interleukin (IL) 4- or IL-5-secreting cells in vivo. Using an established ligand-specific, CD4+ T-cell clone secreting only IL-4, we also demonstrated that stimulation with the highest affinity ligand resulted in interferon gamma production in vitro. In contrast, ligands that demonstrated relatively lower class II binding induced only IL-4 secretion. These data suggest that the major histocompatibility complex binding affinity of antigenic determinants, leading to differential interactions at the T cell-antigen-presenting cell interface, can be crucial for the differential development of cytokine patterns in T cells.

Inference of binding affinity from neuronal receptors in humans

Tese de mestrado, Bioinformática e Biologia Computacional (Bioinformática), Universidade de Lisboa, Faculdade de Ciências, 2016

Static energy analysis of MHC class I and class II peptide-binding affinity

Antigenic peptide is presented to a T-cell receptor (TCR) through the formation of a stable complex with a major histocompatibility complex (MHC) molecule. Various predictive algorithms have been developed to estimate a peptide's capacity to form a stable complex with a given MHC class II allele, a technique integral to the strategy of vaccine design. These have previously incorporated such computational techniques as quantitative matrices and neural networks. A novel predictive technique is described, which uses molecular modeling of predetermined crystal structures to estimate the stability of an MHC class II-peptide complex. The structures are remodeled, energy minimized, and annealed before the energetic interaction is calculated.