NMR techniques for characterization of ligand binding: utility for lead generation and optimization in drug discovery.

Over the last ten years, nmr spectroscopy has evolved into an important discipline in drug discovery. Initially, nmr was most useful as a technique to provide structural information regarding protein drug targets and target-ligand interactions. More recently, it has been shown that nmr may be used as an alternative method for identification of small molecule ligands that bind to protein drug targets. High throughput implementation of these experiments to screen small molecule libraries may lead to identification of potent and novel lead compounds. In this review, we will use examples from our own research to illustrate how nmr experiments to characterize ligand binding may be used to both screen for novel compounds during the process of lead generation, as well as provide structural information useful for lead optimization during the latter stages of a discovery program.     

Disaccharide conformational flexibility. II. Molecular dynamics simulations of sucrose

Molecular dynamics simulations have been used to study the motions in vacuum of the disaccharide sucrose. Ensembles of trajectories were calculated for each of the five local minimum energy conformations identified in the adiabatic conformational energy mapping of this molecule. The model sucrose molecules were found to exhibit a variety of motions, although the global minimum energy conformation was found to be dynamically stable, and no transitions away from this structure were observed to occur spontaneously. In all but one of these vacuum trajectories, the intramolecular hydrogen bond between residues was maintained, in accord with recent nmr studies of this molecule in aqueous solution. Considerable flexibility of the furanoid ring was found in the trajectories. No "flips" to the opposite puckering for this ring were found in the simulations starting from the global minimum, although such a transition was observed for a trajectory initiated with one of the higher local minimum energy conformations. Overall, the observed structural fluctuations were consistent with the experimental picture of sucrose as a relatively rigid molecule.     

Substituent activity relationship studies on new azolo benzoxazepinyl oxazolidinones

In an effort to discover potent antibacterials based on the entropically favored 'bioactive conformation' approach, a series of novel tricyclic molecules mimicking the conformationally constrained structure of Linezolid is reported. Based on the initial tricyclic molecule 1, the benzazepine derivative 2 was designed where the tricyclic structure had more flexibility around C-N bond compared to 1. While, the molecule 2 was less active, the molecule 3 showed promising antibacterial activity presumably after having obtained rigidity due to pyrrole ring. The syntheses, SAR studies, and evaluation of 3 as a lead compound are reported.     

Melting behavior of a covalently closed, single-stranded, circular DNA

We synthesized the 26-residue deoxynucleotide sequence d(TTCCT5GGAATTCCT5GGAA) which folds intramolecularly to form a dumbbell-shaped, double-hairpin structure with a gap between the 3' and the 5' ends. We used T4 polynucleotide kinase to phosphorylate the 5' end followed by T4 DNA ligase to close the 3' and 5' ends. Melting of the dumbbell structure formed by this ligated sequence produces a covalently closed, single-stranded, circular final state. We employed calorimetric and spectroscopic techniques to characterize thermodynamically the melting behavior of the ligated molecule and compared it with the corresponding melting behavior of its unligated precursor. This comparison allowed us to characterize uniquely the influence of single-stranded ring closure on intramolecular duplex melting. The data reveal that ring closure produces a thermally more stable structure which exhibits significantly altered melting thermodynamics. We rationalize these thermodynamic differences in terms of differential solvation and differential counterion association between the ligated and unligated molecules. We also note the importance of such constrained dumbbell structures as models for hairpins, cruciforms, and locally melted domains within naturally occurring DNA polymers.     

Theoretical conformational analysis on Cyclo(prolyl-phenylalanyl) peptides

Empirical energy calculations on cyclo(L -Pro-L -Phe) and cyclo(L -Pro-D-Phe) indicate that different conformations are possible for each molecule. The theoretical results are compared to ir, nmr, and crystallographic data. The interdependence between diketopiperazine ring and side-chain conformations is also discussed.     

The structure of the antitumor complex cis-(diammino) (1,1-cyclobutanedicarboxylato)-Pt(II): X ray and nmr studies

X-ray crystallography shows that Pt(NH₃)₂(CBDCA) is a square-planar complex with the dicarboxylate chelate ring in the boat conformation and a planar cyclobutane ring. ¹H and ¹³C nmr studies suggest that rapid chelate ring flipping occurs in solution. The value of ¹⁹⁵Pt nmr combined with ¹⁵N labeling as an informative new method of studying carboxylate coordination is illustrated. nmr results are also reported for the analogous ethylmalonate complex.     

Investigation of the base-pairing structure of the anticodon hairpin from E. coli initiator tRNA by high-resolution nmr

The high-resolution nmr spectrum of the anticodon hairpin from E. coli tRNA^fMet has been obtained at a number of different temperatures. The positions of the resonances from interior Watson-Crick base pairs are well accounted for (within 0.1 ppm) by a semi-empirical ring current shift theory, but the terminal base pairs are susceptible to the exact orientation of adjacent bases in single-stranded regions. From a careful examination of the exact way in which resonances disappear at elevated temperatures, we conclude that melting in the nmr experiments occurs when the lifetime of a base pair is reduced to several milliseconds. On the basis of these experiments we are able to assign an nmr T_m to each individual base pair and these should be useful in interpreting the melting behavior of the intact molecule. An “extra” resonance is observed at ～11.3 ppm and, on the basis of its position and temperature sensitivity, it is tentatively assigned to the ring nitrogen proton of a “protected” U residue in the anticodon loop. A strong preference for stacking of a nonbase-paired A residue on an adjacent GC base pair is observed even at temperatures in excess of 52°C.     

Combined use of molecular dynamics simulations and NMR to explore peptide bond isomerization and multiple intramolecular hydrogen-bonding possibilities in a cyclic pentapeptide, cyclo(Gly-Pro-D-Phe-Gly-Val).

The conformational behavior of a model cyclic pentapeptide--cyclo(Gly-L-Pro-D-Phe-Gly-L-Val)--has been explored through the combined use of in vacuo molecular dynamics simulations and a range of nmr experiments (preceding paper). The molecular dynamics analysis suggests that, despite the conformational constraints imposed by formation of the pentapeptide cycle, this pentapeptide undergoes conformational transitions between various hydrogen-bonded conformations, characterized by low energy barriers. An inverse gamma turn with Pro in position i + 1 and a gamma turn with D-Phe in position i + 1 are two alternatives occurring frequently. Like other DLDDL cyclic pentapeptides, cyclo(Gly-Pro-D-Phe-Gly-Val) is also stabilized by an inverse gamma-turn structure with the beta-branched Val residue in position i + 1, and this hydrogen bond is retained in the different conformational families. The gamma-turn around D-Phe3 and the inverse gamma turn around Val5 are consistent with the nmr observations. 3JNH-CH alpha coupling constants of the all-trans forms were calculated from one of the molecular dynamics trajectories and are comparable to nmr experimental data, suggesting that the conformational states visited during the simulation are representative of the conformational distribution in solution. In addition to the equilibrium among various hydrogen-bonded all-trans conformers, the observation in nmr spectra of two sets of resonances for all peptide protons indicated a slow conformational interconversion of the Gly-Pro peptide bond between trans and cis isomers. The activation energy between these two conformers was determined experimentally by magnetization transfer and was calculated by high temperature constrained molecular dynamics simulation. Both methods yield a free energy of activation of ca. 20 kcal/mol. Furthermore, the free energy of activation is dependent on the direction of rotation of the Gly-Pro peptide bond.     

Conformer equilibria of triostin A and its conformer-specific interaction with nucleic acid bases

A quinoxaline antibiotic triostin A has a bicyclic octadepsipeptide structure. Proton and carbon-13 nmr spectra showed the presence of two symmetrical conformations favoring polar and nonpolar solvents, respectively. They interconvert slowly on the nmr time scale, and this slow interconversion is due to the cooperative effects of the presence of the quinoxaline ring and the N-methyl peptide bonds. Reversal of the chirality of the disulfide bond as the origin of the slow exchange was excluded by the presence of two conformers for S-benzyltriostin A. Conformer 2, which favors the polar solvent, can form hydrogen-bonded complexes with purine nucleoside derivatives in organic solvents, but conformer 1 does not. The binding sites were elucidated and a mode of interaction with DNA proposed.     

Conformational analysis of dolastatin 10: An nmr and theoretical approach

A solution conformational analysis of dolastatin 10, a powerful antineoplaslic agent, has been carried out by means of nmr techniques and theoretical calculations. ¹H mono- and bidimensional nmr experiments, as well as ¹H-¹³C heterocorrelated spectra, have been performed on CD₂Cl₁₂ solutions. The most interesting nmr data is a huge shielding of the aCH (25) proton of the Dov residue, suggesting the presence of an interaction between the N-terminal and the aromatic C-terminal ends of the molecule. The possibility of a head-to-tail intermolecular association having been discarded, the presence of a series of preferred folded conformation has been hypothesized. Conformational theoretical analysis supports the nmr hypothesis of a folded peptide-like molecule, and a series of possible conformers in good agreement with the experimental data have been analyzed. © 1995 John Wiley & Sons, Inc.     

Crystal structure and nmr conformation of a cyclic pseudotetrapeptide containing urethane backbone linkages

Urethane bonds, derived from the hydroxyl group of the tyrosine side chain, have been investigated as a new type of amide bond mimetic in the design of pseudopeptides. The structure of a representative cyclic pseudotetrapeptide that consists of an — Ala — Tyr(urethane) Ala — Tyr (urethane) sequence fused into a rigid ring has been studied in the solid state by x-ray crystallography and in solution by two-dimensional nmr techniques. The cyclic pseudotetrapeptide has an oblong shape. The backbone urethane bonds assume a trans–trans conformation. The carbonyl groups in the ring have an alternating pattern of down, up, down, up with respect to the average ring plane. Solution nmr studies give observed nuclear Overhauser effects and coupling constants largely in agreement with the crystal structure. However, in solution the observed structure is likely to be conformationally averaged, and in the averaged structure, the urethane bond is perpendicular to the plane of the aromatic ring of the tyrosine, while in the crystal it is close to this plane. These differences may be explained by intermolecular hydrogen-bonding interactions. Four aspects of the conformation of the cyclic pseudotetrapeptide were investigated in detail: the tyrosine residue with the attached side-chain urethane bond (the tyrosine-urethane unit), the conformation of the two urethane backbone linkages, the conformation of the two conventional peptide bonds within this unusual ring structure, and the tight turns within the cyclic pseudotetrapeptide. The conformation of the tight turns present in the cyclic pseudotetrapeptide is very similar to that of a β-bend of type II. Intermolecular hydrogen bonding, joining adjacent layers of the cyclic pseudotetrapeptide in the solid state, resemble a parallel β-pleated sheet. The presence of these structural motifs in the cyclic pseudotetrapeptide indicates that the tyrosine urethane unit may find applications in peptide and protein engineering. © 1994 John Wiley & Sons, Inc.     

Effects of distance constraints on macromolecular conformation. II. Simulation of experimental results and theoretical predictions

By generating classes of random structures for trypsin inhibitor and carp myogen, each consistent with a given set of experimental or theoretical information, we have assessed the relative utility of various experiments and theories in deducing the conformation of macromolecules. We compare the calculated structures with known x-ray coordinates and compute for each class an average error. Small errors mean that the experimental or theoretical constraints limit the structures to the vicinity of the crystal structure, whereas large errors show that the constraints permit a wide variety of tertiary conformations. We find the following points to hold true: (1) Qualitative information on all the distances, as might be obtained from the correct prediction of interresidue contacts, effectively determines the structure (error approximately 1 Å). (2) Quantitative information on a limited number of distances, as might be obtained from nmr or crosslinking experiments, significantly restricts the range of possible structures only when the number of distances given is comparable to the number of residues (error approximately 3 Å). (3) Quantitative information on the distances of each residue to the center of mass of the molecule, as might in part be obtained from solvent accessibility and solution x-ray studies, is not particularly restrictive by itself (error approximately 5 Å). (4) Complete qualitative local distance information, as might be obtained from secondary prediction and CD/ORD studies, is clearly consistent with a wide variety of tertiary structures (error approximately 7 Å).     

Design and synthesis of conformationally constrained N,N-disubstituted 1,4-diazepanes as potent orexin receptor antagonists

Orexins are neuropeptides that regulate wakefulness and arousal. Small molecule antagonists of orexin receptors may provide a novel therapy for the treatment of insomnia and other sleep disorders. In this Letter we describe the design and synthesis of conformationally constrained N,N-disubstituted 1,4-diazepanes as orexin receptor antagonists. The design of these constrained analogs was guided by an understanding of the preferred solution and solid state conformation of the diazepane central ring.     

Conformational analysis of a dinucleotide photodimer with the aid of the genetic algorithm.

The solution structure of the photodimer cis,syn-dUp[]dT is derived with the aid of the genetic algorithm. The conformational space available for the molecule is sampled efficiently using the computer program DENISE and tested against a set of constraints available from nmr experiments. The dominant conformation in solution found with this approach can be described by the following combinations of sugar-phosphate backbone torsion angles: epsilon(t), zeta(t), alpha(+), beta(-ac), and gamma(t). The conformation of the sugars and glycosidic torsion angles are S type and syn, respectively. The cyclobutane ring and pyrimidines are puckered. In addition, other conformations that exist in equilibrium with the first are found. It is concluded that the cyclobutane-pyrimidine system is rigid, whereas the sugar-phosphate backbone is flexible. The solution structures are compared with the crystal structure of the strongly related cyano-ethyl ester of cis,syn-dTp[]dT.     

Crystal structure,conformational analysis,and molecular dynamics of tetra-O-methyl-(+) -catechin

The structure of tetra-O-methyl- (+) -catechin has been determined in the crystalline state. Two independent molecules, denoted structure A and structure B, exist in the unit cell. Crystals are triclinic, space group P1, a = 4.8125(2) Å, b = 12.9148(8) Å, c = 13.8862(11) Å, α = 86.962(6) °, β = 89.120(5)°, γ = 88.044(5)°, Z = 2, D_c = 1.336 g cm^?3, R = 0.033 for 6830 observations. The heterocyclic rings of the crystal structures are compared to previous results for 8-bromotetra-O-methyl-(+)-catechin, penta-O-acetyl-(+)-catechin, and (?) -epicatechin. One of the two molecules has a heterocyclic ring conformation similar to that observed previously for (?)-epicatechin, and the other has a heterocyclic ring conformation similar to one predicted earlier in a theoretical analysis of dimers of (+)-catechin and (?) -epicatechin. Both structure A and structure B in the crystal have heterocyclic ring conformations that place the dimethoxyphenyl substituent at C(2) in the equatorial position. However, this heterocyclic ring conformation does not explain the proton nmr coupling constant measured in solution. Molecular dynamics simulations show an equatorial ? axial interconversion of the heterocyclic ring, which can explain the nmr results. © 1993 John Wiley & Sons, Inc.     

Demonstration of a 7-nm RNP fiber as the basic structural element in a premessenger RNP particle

Balbiani ring granules in Chironomus salivary glands represent premessenger ribonucleoprotein (RNP) particles, each containing a 35- to 40-kb message for a secretory polypeptide. Their gross structure can be described as an RNP ribbon bent into a toroid. We now demonstrate that an unfolded, thin RNP fiber is observed after low salt treatment of isolated Balbiani ring granules. Moreover, the thin RNP fiber, 7 nm in diameter, can be revealed as the main structural element in Balbiani ring granules studied in situ in 3-D with electron microscope tomography. It is proposed that the thin RNP fiber consists of a premessenger RNA molecule coiled around a filamentous core of polymeric proteins, which has functional implications for processes such as assembly of RNP, intranuclear degradation of RNA, and delivery of RNA through the nuclear pores.     

XII. Conformational studies of histidine-containing peptides in solution

The spectroscopic properties (uv, CD, nmr) of histidine, glycylhistidine, histidylglycine, glycylhistidylglycine have been investigated in water and methanol in the temperature range 200–320 K in order to obtain information about their conformational equilibria. This analysis has been carried out for the different ionic forms of the compounds, in order to evaluate the influence of the ionization state of the carboxyl, histidyl, and amino groups on the rotamer distribution of the histidyl side chain (as evaluated from proton nmr analysis) and on the overall molecule (as judged from CD spectra). On the basis of certain approximations and from the temperature dependence of the proton nmr resonance, the thermodynamic parameters (ΔH° and ΔS°) characterizing the conformational equilibrium of the hystidyl side chain have been evaluated for the different structures and ionization states. Relatively large entropy differences between the rotamers are obtained in some cases. The data of the sidechain rotamer population, as determined by nmr, have been analytically correlated with the CD data, and in the case of hystidine and histidylglycine in basic solution, first-approximation values for the ellipticity of the single conformers have been evaluated. Finally, in the example of glycylhistidine and histidylglycine in basic solution, it is shown how the data obtained from the different experimental approaches (nmr and CD), as well as from theoretical energy calculations, converge to characterize the most stable conformation in solution.     

Self-assembly of Congo Red—A theoretical and experimental approach to identify its supramolecular organization in water and salt solutions

The supramolecular organization of Congo Red molecules was studied to approach an understanding of the unusual complexation characteristics associated with the liquid crystalline nature of this dye. Differential scanning calorimetry (DSC) and nmr data indicate that Congo Red assembly arrangements differ in water and salt solutions. Compact, highly ordered material with a distinct melting transition is created, but not below 0.3% sodium chloride concentration. The twist in the assembly arrangement of Congo Red molecules, caused in water by repulsion, decreases when the charges are shielded, allowing for more overlapping of the naphthalene rings and their engagement in stacking interaction. The crystallization transition observed in DSC analysis of Congo Red fast-assembled by cooling in salt solutions indicates that the formation of compact crystalline mesophase material is a time-consuming process in which coplanarity and a highly ordered organization must be achieved. Two different superposition variants, called “direct” and “reversed” here, were considered fundamental to compact Congo Red organization. They correspond to optimal face-to-face ring stackings, and are formed by simple direct translation or alternative imposition of reversed (180° rotated) molecules, respectively. In NaCl solution (2.8%) there is a significant downfield chemical shift alteration of the nmr signal related to proton 8, which is in the naphthalene ring on the side opposite to the charged sulfonic group. It was associated selectively with the transition of Congo Red to compact form. This effect confirms the close approach of the sulfonic groups and proton 8, and indicates that formation of the reversed arrangement is favored in the Congo Red supramolecular organization. Molecular dynamics simulation based on AMBER 4.1 force field and analysis of electrostatic field densities around the molecule were used for comparative modeling. Molecular dynamics (150 ps) were simulated for two eight-molecule micelle models constructed to reflect direct and reversed arrangements of Congo Red molecules. Although both versions generally preserved their initial assembly structure in the simulations, the reversed version proved more stable. The proximity of the sulfonic group and proton 8, confirmed by computer analysis, explains the correlation between the formation of Congo Red micellar organization and the distinct shift alteration related to this proton, as found by nmr. © 1998 John Wiley & Sons, Inc. Biopoly 46: 267–281, 1998     

Solid-state 13C NMR and X-ray diffraction of dermatan sulfate

Dermatan sulfate in the solid state has been studied by 13C CP/MAS nmr and X-ray diffraction in order to establish the ring conformation of the L-iduronate moiety. The solid state nmr spectrum is similar to the solution spectrum obtained previously, indicating that a ring conformation at least approximating to 1C4 predominates in the solid state. X-ray powder diffraction data from the same sample indicate the presence of the 8-fold helix form previously observed by fiber diffraction, and interpreted in terms of a 4C1 ring form. A likely explanation of the results is that a distorted 1C4 L-iduronate ring conformation, not considered in the initial X-ray analysis, may emerge to provide a satisfactory interpretation of all available physical-chemical data.     

Structure and antibacterial activity of the silver(I) complex of 2-aminophenoxazine-3-one

The crystal structure and antibacterial activity of the first metal complex of 2-aminophenoxazine-3-one is reported. We describe the silver(i) complex of this important biological molecule and show that the binding mode is through the phenoxazine ring nitrogen. A new synthetic route to 2-aminophenoxazine-3-one is also reported.