Trends In Binding Affinity Research Articles

DNA Structure Dependence of DNA Binding by Escherichia coli and Thermus aquaticus DNA Polymerase I

Understanding substrate selection by DNA Polymerase I is important for the balance between repair and replication for this enzyme in vivo. Due to the sequence and structural homologies, all DNA Pol I are considered functional homologues. We have examined the DNA structural preferences of Klenow and Klentaq, the “large fragment” of DNA polymerase I of Escherichia coli and Thermus aquaticus. The binding affinity trend for Klentaq is pt-DNA ≈ ds-DNA >> ss-DNA, while Klenow has long been known to bind ds-DNA very weakly. Temperature dependent examination of Klentaq DNA binding shows that it binds ds-DNA and pt-DNA with parallel affinities across a wide range of temperatures. These non-sequence specific binding proteins exhibit relatively large heat capacity changes (ΔCp) upon DNA binding that reflects significant hydrophobic interactions in the interface of the complex. The magnitude of the ΔCp is also DNA structure dependent as well as being non-homologous for these polymerases. Since these polymerases do not exhibit significant DNA sequence dependence, we conclude that the nature of the “structural site” determines the specific binding thermodynamics. The thermodynamic differences among the different DNA structural substrates for the two polymerases suggest that the in vivo functions of these two supposedly homologous polymerases are different. Funded by the NSF and the Louisiana Biomedical Research Network.

Large-Scale Validation of a Quantum Mechanics Based Scoring Function: Predicting the Binding Affinity and the Binding Mode of a Diverse Set of Protein−Ligand Complexes

Computational methods to calculate binding affinity in protein-ligand interaction are of immense interest because of obvious practical applications in structure-based drug design. Scoring functions attempt to calculate the variation in binding affinity of ligands-inhibitors bound to protein targets at various levels of theory. In this study we use semiempirical quantum mechanics to design a scoring function that can calculate the electrostatic interactions and solvation free energy expected during complexation. This physically based approach has the ability to capture binding affinity trends in a diverse range of protein-ligand complexes. We also show the predictive power of this scoring function within protein targets and its ability to score ligand poses docked to a protein target. We also demonstrate the ability of this scoring function to discriminate between native and decoy poses and highlight the crucial role played by electrostatic interactions in molecular recognition. Finally we compare the performance of our scoring function with other available scoring functions in the literature.

Recognition of Specific DNA Sequences by Stacked Pyrrole- and Imidazole- Containing Polyamides: An Efficient Screening Method Based on Competitive Dialysis

A competitive dialysis method has been developed to screen compounds for their DNA binding properties, and it is based on directly comparing the binding of polyamide molecules to a series of distinctively varied, short, synthetic deoxyribonucleotides. Relative binding ratios for each polyamideoligonucleotide pairing were calculated from the concentrations of free polyamide and total polyamide in order to quantitatively compare binding to different DNA sequences. This approach works well as a preliminary screen to determine the viability of novel small molecules, prior to investing significant resources in further characterization and development of possible sequence specific DNA targeted therapeutic agents. The trends in binding affinities of the four triamide molecules (f-ImPyIm, distamycin A, f-PyPyPy and f-ImImPy; where Im is imidazole and Py is pyrrole) correlated well with data obtained from surface plasmon resonance (SPR) studies. Results from circular dichroism studies confirmed the minor groove side-by-side stacked binding motif of the triamides, and thermal stability experiments corroborated the improved DNA stability of promising polyamide-DNA complexes. The affinity of distamycin A for its cognate DNA sequence (A3T3) was unambiguously selected over the other DNA sequences tested. Alternatively, as expected from SPR, circular dichroism and thermal melting experiments, f-ImImPy showed very poor affinity for DNA sequences tested, including its cognate DNA, TCGA. Thus, the very good (distamycin A) and very poor (f-ImImPy) DNA binders were effectively screened. Keywords: competitive dialysis, dna minor groove binder, polyamide, screen

Design of novel N-(2,4-dioxo-1,2,3,4-tetrahydro-thieno[3,2- d]pyrimidin-7-yl)-guanidines as thymidine phosphorylase inhibitors, and flexible docking to a homology model

A novel class of thymidine phosphorylase (TP) inhibitors has been designed based on analogy to the enzyme substrate as well as known inhibitors. Flexible docking studies, using a homology model of human TP, of the designed N-(2,4-dioxo-1,2,3,4-tetrahydro-thieno[3,2- d]pyrimidin-7-yl)-guanidines as well as their synthetic precursors provide insight into the observed experimental trends in binding affinity.

Interpreting trends in the binding of cyclic ureas to HIV-1 protease

The design of new HIV protease inhibitors requires an improved understanding of the physical basis of inhibitor/protein binding. Here, the binding affinities of seven aliphatic cyclic ureas to HIV-1 protease are calculated using a predominant states method and an implicit solvent model based upon finite difference solutions of the Poisson-Boltzmann equation. The calculations are able to reproduce the observed U-shaped trend of binding free energy as a function of aliphatic chain length. Interestingly, the decrease in affinity for the longest chains is attributable primarily to the energy cost of partly desolvating charged aspartic and arginine groups at the mouths of the active site. Even aliphatic chains too short to contact these charged groups directly are subject to considerable desolvation penalties. We are not aware of other systems where binding affinity trends have been attributed to long-ranged electrostatic desolvation of ionized groups. A generalized Born/surface area solvation model yields a much smaller change in desolvation energy with chain length and, therefore, does not reproduce the experimental binding affinity trends. This result suggests that the generalized Born model should be used with caution for complex, partly desolvated systems like protein binding sites. We also find that changing the assumed protonation state of the active site aspartyl dyad significantly affects the computed binding affinity trends. The protonation state of the aspartyl dyad in the presence of cyclic ureas is discussed in light of the observation that the monoprotonated state reproduces the experimental results best.

Improved convergence of binding affinities with free energy perturbation: application to nonpeptide ligands with pp60src SH2 domain.

Free Energy Perturbations (FEP) in the context of Monte Carlo (MC) simulations were conducted to predict the relative free energies of binding for a series of human Src SH2 domain ligands. Two procedures for disappearing atoms during a single-topology FEP are investigated and dramatic differences in free energy convergence behavior are seen. Comparison of these two protocols suggests that the coupling of the removal of angular constraints with the disappearance of an atom may significantly slow free energy convergence. The series of ligands under investigation here cover a range of modifications at the 3-position of 4-([[4-(cyclohexyl methoxy)benzyl]amino]carbonyl) phenyl phosphate. Unlike any other compound in this study, the 3-amide analog can form two hydrogen bonds within the region of the perturbation, one to a backbone amide hydrogen and one to a highly coordinated water molecule. Agreement with experimental trends in binding affinity is seen, although the computed relative free energy of binding of the amido compound is underestimated. These results are reconciled by examination of the hydration energies of model systems, which predict primary amides as too hydrophilic.

Phenylalanine in the second membrane-spanning domain of alpha 1A-adrenergic receptor determines subtype selectivity of dihydropyridine antagonists.

The alpha 1-adrenergic receptors (alpha 1-AR) belong to the G-protein coupled seven-transmembrane biogenic amine receptor family. Three subtypes have been successfully cloned in the alpha 1-adrenergic receptor family, and they share 50% identical amino acid sequences and 70% similarity. We have constructed seven chimeric receptors of the alpha 1A-AR. Each of the chimeras contains alpha 1D-subtype amino acid sequences within the membrane-spanning domains. Comparisons of ligand affinities with these chimeras has provided information on the importance of certain amino acid residues in determining receptor subtype specificity in the alpha 1A- and alpha 1D-ARs. With ligands in the dihydropyridine series, the niguldipine analog 1 was found to have respective pKi's of 9.32 +/- 0.17 for alpha 1A-AR; 6.84 +/- 0.24 for alpha 1D-AR; and 6.76 +/- 0.28 for alpha 1A/D(TM2), respectively. This trend was also exhibited by two other niguldipine analogs, 2 and 3, which had similar pKi's toward alpha 1D-AR and alpha 1A/D(TM2). This subtype selectivity was also maintained in the piperdine derivative, 4, and alpha 1A-AR selective ligand, which showed the same parallel trends in binding affinities with alpha 1A-AR and the six chimeras as the niguldipine analogs. Since in considering the second membrane-spanning domain, the alpha 1A- and alpha 1D-ARs only differ at positions 76, 77, 85, and 86, we were able to show through mutational studies that phenylalanine 86 is solely responsible for the selectivity found in the chimeric receptor alpha 1A/D(TM2) exhibited against the ligands 1-4 used in this study. A model based on the rhodopsin structure places the amino acid at position 86 in the final turn toward the extracellular region. This is four helical turns above aspartic acid-79, a conserved amino acid in the second membrane-spanning domain. This is the first report that suggests a significant involvement of the second membrane-spanning domain in antagonist binding in the biogenic amines class of the superfamily of seven-transmembrane receptors.

Conformation and electrostatic potential surfaces of opiates: Relationship to µ- and λ-site binding.

Molecular mechanics energy minimization and electrostatic potential surface calculations have been used to examine a series of opioid compounds that interact with the µ opoid receptor and a recently discovered high-affinity naloxone binding site (the λ site). The compounds studied include members of the morphinan, 4,5-epoxymorphinan, and benzomorphan families. All compounds bind with a high affinity at the µ opiate receptor site, but only the 4,5-epoxymorphinans bind tightly at the λ site. The results suggest that conformational differences in the various families do not satisfactorily explain the observed trends in binding affinity at the λ site. However, electrostatic potential surfaces for a representative subset of these opioids exhibit patterns that allow us to classify members as high-affinity or low-affinity λ-site ligands in good agreement with the experimental results. The procedures used in this work may be useful in defining characteristics that impart selectivity for opiate receptor subtypes such as the µ, δ, and κ receptors.