A high-resolution structure of a DNA-chromomycin-Co(II) complex determined from pseudocontact shifts in nuclear magnetic resonance

BACKGROUND: The drug chromomycin-A(3) binds to the minor groove of DNA and requires a divalent metal ion for complex formation. (1)H, (31)P and (13)C pseudocontact shifts occurring in the presence of a tightly bound divalent cobalt ion in the complex between d(TTGGCCAA)(2) and chromomycin-A(3) have been used to determine the structure of the complex. The accuracy of the structure was verified by validation with nuclear Overhauser enhancements (NOEs) and J-coupling constants not used in the structure calculation. RESULTS: The final structure was determined to 0.7 A resolution. The structure was compared with a structure obtained in an earlier study using NOEs, in order to assess the accuracy of NOEs in giving global structural information for a DNA complex. Although some basic features of the structures agreed, they differed substantially in the fine structural details and in the DNA axis curvature generated by the drug. The distortion of base-pair planarity that was observed in the NOE structure was not seen in our structure. Differences in drug orientation and hydrogen bonding also occurred. The curvature and elongation of the DNA that was obtained previously was not found to occur in our study. CONCLUSIONS: The use of pseudocontact shifts has enabled us to obtain a high-precision global structure of the chromomycin-DNA complex, which provides an accurate template on which to consider targeting minor groove binding drugs. The effect of such binding is not propagated far along the helix but is restricted to a local kink in the axis that reverts to its original direction within four base pairs.     

Structure of a bis-amidinium derivative of hoechst 33258 complexed to dodecanucleotide d(CGCGAATTCGCG)2: the role of hydrogen bonding in minor groove drug-DNA recognition.

The crystal structure is reported of a complex between the dodecanucleotide sequence d(CGCGAATTCGCG)2and an analogue of the DNA binding drug Hoechst 33258, in which the piperazine ring has been replaced by an amidinium group and the phenol ring by a phenylamidinium group. The structure has been refined to an R factor of 19.5% at 2.2 A resolution. The drug is held in the minor groove by five strong hydrogen bonds, together with bridging water molecules at both ends. There are few other contacts with the floor of the groove, indicating a lack of isohelicity with the groove and suggesting (i) that the observed high DNA affinity of this drug is primarily due to the array of hydrogen bonds and (ii) that these more than compensate for its poor isohelicity.     

Crystal structure of a berenil-dodecanucleotide complex: the role of water in sequence-specific ligand binding.

The three-dimensional structure of a complex between the dodecanucleotide d(CGCGAATTCGCG) and the anti-trypanocidal drug berenil, has been determined to a resolution of 2.5 A. The structure has been solved by molecular replacement and refined to an R factor of 0.177. A total of 49 water molecules have been located. The drug is bound at the 5'-AAT-3' region of the oligonucleotide. At one end of the drug the amidinium group is in hydrogen-bonded contact with N3 of the adenine base complementary to the thymine of the AAT. The other amidinium group does not make direct interactions with the DNA. Instead, a water molecule mediates between them. This is in hydrogen-bonded contact with an amidinium nitrogen atom, N3 of the 5' end adenine base and the ring oxygen atom of an adjacent deoxyribose. Molecular mechanics calculations have been performed on this complex, with the drug at various positions along the sequence. These show that the observed position is only 0.8 kcal/mol higher in energy than the best position. It is suggested that there is a broad energy well in the AATT region for this drug, and that water molecules as well as the neighbouring sequence, will determine precise positioning. More general aspects of minor groove binding are discussed.     

Three-dimensional structure of a complex of galanthamine (Nivalin) with acetylcholinesterase from Torpedo californica: implications for the design of new anti-Alzheimer drugs

The 3D structure of a complex of the anti-Alzheimer drug galanthamine with Torpedo californica acetylcholinesterase is reported. Galanthamine, a tertiary alkaloid extracted from several species of Amarylidacae, is so far the only drug that shows a dual activity, being both an acetylcholinesterase inhibitor and an allosteric potentiator of the nicotinic response induced by acetylcholine and competitive agonists. The X-ray structure, at 2.5A resolution, shows an unexpected orientation of the ligand within the active site, as well as unusual protein-ligand interactions. The inhibitor binds at the base of the active site gorge, interacting with both the acyl-binding pocket and the principal quaternary ammonium-binding site. However, the tertiary amine group of galanthamine does not directly interact with Trp84. A docking study using the program AUTODOCK correctly predicts the orientation of galanthamine in the active site. The docked lowest-energy structure has a root mean square deviation of 0.5A with respect to the corresponding crystal structure of the complex. The observed binding mode explains the affinities of a series of structural analogs of galanthamine and provides a rational basis for structure-based drug design of synthetic derivatives with improved pharmacological properties. Proteins 2001;42:182-191.     

Computational study of glyceraldehyde-3-phosphate dehydrogenase of Entamoeba histolytica: implications for structure-based drug design

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of the pathogenic protozoa Entamoeba histolytica (Eh) is a major glycolytic enzyme and an attractive drug target since this parasite lacks a functional citric acid cycle and is dependent solely on glycolysis for its energy requirements. The three-dimensional structure of dimeric EhGAPDH in complex with cofactor NAD(+) has been generated by homology modeling based on the crystal structure of human liver GAPDH. Our refined model indicates the presence of a parasite specific disulfide bond between two cysteine residues of adjacent monomers in the EhGAPDH dimer, which may be an important target for future drug design. Flexible docking with the substrate glyceraldehyde-3-phosphate (G3P) shows that Cys151, His178, Thr210, and Arg233 are important residues in G3P binding. The inorganic phosphate-binding site of EhGAPDH has been determined by docking study. The binding mode of a natural GAPDH inhibitor, chalepin to EhGAPDH has also been predicted. In search for a better inhibitor for EhGADPH, in silico modification of chalepin has been carried out to form an additional specific polar interaction with Asp194 of EhGAPDH whose equivalent is Leu195 in human GAPDH. In the absence of a crystal structure, our study provides an early insight into the structure of major drug target EhGAPDH, thus, facilitating the inhibitor design.     

Medical implications from the crystal structure of a copper-containing amine oxidase complexed with the antidepressant drug tranylcypromine

The X-ray crystal structure of the copper-containing quinoprotein amine oxidase from E. coli has been determined in complex with the antidepressant drug tranylcypromine to 2.4 A resolution. The drug is a racemic mix of two enantiomers, but only one is seen bound to the enzyme. The other enantiomer is not acting as a substrate for the enzyme as no catalytic activity was detected when the enzyme was initially exposed to the drug. The inhibition of human copper amine oxidases could be a source of side-effects in its use as an antidepressant to inhibit the flavin-containing monoamine oxidases in the brain.     

The molecular structure of the complex of Hoechst 33258 and the DNA dodecamer d(CGCGAATTCGCG).

The crystal structure of the complex between the dodecamer d(CGCGAATTCGCG) and a synthetic dye molecule Hoechst 33258 was solved by X-ray diffraction analysis and refined to an R-factor of 15.7% at 2.25 A resolution. The crescent-shaped Hoechst compound is found to bind to the central four AATT base pairs in the narrow minor groove of the B-DNA double helix. The piperazine ring of the drug has its flat face almost parallel to the aromatic bisbenzimidazole ring and lies sideways in the minor groove. No evidence of disordered structure of the drug is seen in the complex. The binding of Hoechst to DNA is stabilized by a combination of hydrogen bonding, van der Waals interaction and electrostatic interactions. The binding preference for AT base pairs by the drug is the result of the close contact between the Hoechst molecule and the C2 hydrogen atoms of adenine. The nature of these contacts precludes the binding of the drug to G-C base pairs due to the presence of N2 amino groups of guanines. The present crystal structural information agrees well with the data obtained from chemical footprinting experiments.     

Structural insights into how irreversible inhibitors can overcome drug resistance in EGFR

Resistance to kinase-targeted cancer drugs has recently been linked to a single point mutation in the ATP binding site of the kinase. In EGFR, the crucial Thr790 gatekeeper residue is mutated to a Met and prevents reversible ATP competitive inhibitors from binding. Irreversible 4-(phenylamino)quinazolines have been shown to overcome this drug resistance and are currently in clinical trials. In order to obtain a detailed structural understanding of how irreversible inhibitors overcome drug resistance, we used Src kinase as a model system for drug resistant EGFR-T790M. We report the first crystal structure of a drug resistant kinase in complex with an irreversible inhibitor. This 4-(phenylamino)quinazoline inhibits wild type and drug resistant EGFR in vitro at low nM concentrations. The co-crystal structure of drug resistant cSrc-T338M kinase domain provides the structural basis of this activity.     

A new drug binding subsite on human serum albumin and drug-drug interaction studied by X-ray crystallography

3'-Azido-3'-deoxythymidine (AZT) is the first clinically effective drug for the treatment of human immunodeficiency virus infection. The drug interaction with human serum albumin (HSA) has been an important component in understanding its mechanism of action, especially in drug distribution and in drug-drug interaction on HSA in the case of multi-drug therapy. We present here crystal structures of a ternary HSA-Myr-AZT complex and a quaternary HSA-Myr-AZT-SAL complex (Myr, myristate; SAL, salicylic acid). From this study, a new drug binding subsite on HSA Sudlow site 1 was identified. The presence of fatty acid is needed for the creation of this subsite due to fatty acid induced conformational changes of HSA. Thus, the Sudlow site 1 of HSA can be divided into three non-overlapped subsites: a SAL subsite, an indomethacin subsite and an AZT subsite. Binding of a drug to HSA often influences simultaneous binding of other drugs. From the HSA-Myr-AZT-SAL complex structure, we observed the coexistence of two drugs (AZT and SAL) in Sudlow site 1 and the competition between these two drugs in subdomain IB. These results provide new structural information on HSA-drug interaction and drug-drug interaction on HSA.     

Molecular modeling,in silico screening and molecular dynamics of PfPRL-PTP of P. falciparum for identification of potential anti-malarials

Millions of deaths occur every year due to malaria. Growing resistance against existing drugs for treatment of malaria has exaggerated the problem further. There is an intense demand of identifying drug targets in malaria parasite. PfPRL-PTP protein is PRL group of phosphatase, and one of the interesting drug targets being involved in three important pathways of malaria parasite (secretion, phosphorylation, and prenylation). Therefore, in this study, we have modeled three-dimensional structure of PfPRL-PTP followed by validation of 3D structure using RAMPAGE, verify3D, and other structure validation tools. We could identify 12 potential inhibitory compounds using in silico screening of NCI library against PfPRL-PTP with Glide. The molecular dynamics simulation was also performed using GROMACS on PfPRL-PTP model alone and PfPRL-PTP-inhibitor complex. This study of identifying potential drug-like molecules would add up to the process of drug discovery against malaria parasite.     

Crystal structure of the 2:1 complex between d(GAAGCTTC) and the anticancer drug actinomycin D.

The crystal structures of the 2:1 complex of the self-complementary DNA octamer d(GAAGCTTC) with actinomycin D has been determined at 3.0 A resolution. This is the first example of a crystal structure of a DNA-drug complex in which the drug intercalates into the middle of a relatively long DNA segment. The results finally confirmed the DNA-actinomycin intercalation model proposed by Sobell & co-workers in 1971. The DNA molecule adopts a severely distorted and slightly kinked B-DNA-like structure with an actinomycin D molecule intercalated in the middle sequence, GC. The two cyclic depsipeptides, which differ from each other in overall conformation, lie in the minor groove. The complex is further stabilized by forming base-peptide and chromophore-backbone hydrogen bonds. The DNA helix appears to be unwound by rotating one of the base-pairs at the intercalation site. This single base-pair unwinding motion generates a unique asymmetrically wound helix at the binding site of the drug, i.e. the helix is loosened at one end of the intercalation site and tightened at the other end. The large unwinding of the DNA by the drug intercalation is absorbed mostly in a few residues adjacent to the intercalation site. The asymmetrical twist of the DNA helix, the overall conformation of the two cyclic depsipeptides and their interaction mode with DNA are correlated to each other and rationally explained.     

The antimalarial and cytotoxic drug cryptolepine intercalates into DNA at cytosine-cytosine sites.

Cryptolepine, a naturally occurring indoloquinoline alkaloid used as an antimalarial drug in Central and Western Africa, has been found to bind to DNA in a formerly unknown intercalation mode. Evidence from competition dialysis assays demonstrates that cryptolepine is able to bind CG-rich sequences containing nonalternating CC sites. Here we show that cryptolepine interacts with the CC sites of the DNA fragment d(CCTAGG)(2) in a base-stacking intercalation mode. This is the first DNA intercalator complex, from approximately 90 solved by X-ray crystallography, to bind a nonalternating (pyrimidine-pyrimidine) DNA sequence. The asymmetry of the drug induces a perfect stacking with the asymmetric site, allowing for the stability of the complex in the absence of hydrogen bonding interactions. The crystal structure of this antimalarial drug-DNA complex provides evidence for the first nonalternating intercalation and, as such, provides a basis for the design of new anticancer or antimalarial drugs.     

A structural model for the ternary cleavable complex formed between human topoisomerase I, DNA, and camptothecin

Using the X-ray crystal structure of the human topoisomerase I (TOP1)-DNA cleavable complex, we have developed a general model for the ternary drug-DNA-TOP1 cleavable complex formed with camptothecin (CPT) and its analogues. This model has the drug intercalated between the -1 and +1 base pairs, with the E-ring pointing into the minor groove and the A-ring directed toward the major groove. The ternary complex is stabilized by an array of hydrogen bonding and hydrophobic interactions between the drug and both the enzyme and the DNA. Significantly, the proposed model is consistent with the current body of experimental mutation, cross-linking, and structure-activity data. In addition, the model reveals potential sites of interaction that can provide a rational basis for the design of next generation compounds as well as for de novo drug design.     

Evaluation of the utility of NMR structures determined from minimal NOE-based restraints for structure-based drug design, using MMP-1 as an example

The application of deuterium labeling and residual dipolar coupling constants in combination with other structural information has demonstrated the potential for significantly expanding the range of viable protein targets for structural analysis by NMR. A previous study by Clore et al. [(1999) J. Am. Chem. Soc. 121, 6513-6514] demonstrated that a significant improvement in the overall protein structure occurs with the combination of residual dipolar coupling constants and minimal tertiary long-range distance restraints. The analysis of NMR protein structures determined with minimal structural information is extended with a particular interest in the utility of these structures for a structure-based drug design program. As an example, the catalytic fragment of human fibroblast collagenase (MMP-1) was used to follow the effect of minimal restraint sets on the protein structure and its utility in drug design with a particular interest in the effect on the active site conformation. An MMP-1 structure that was calculated with the maximal number of restraints attainable with the constraint of a deuterated protein was shown to be very similar to a high-quality MMP-1 structure that was calculated from a complete set of restraints. The superposition of the active site backbone atoms for the high-quality and minimal restraint MMP-1 structures yielded an rmsd of 0.68 A where the size and shape of the S1' pocket are nearly identical. Additionally, an MMP-1-CGS-27023A complex based on a minimal set of NOE-based restraints reliably reproduced the structure of the complex, establishing the usefulness of the structures for drug design.     

A proposed molecular model for the interaction of calcineurin with the cyclosporin A-cyclophilin A complex.

Cyclosporin A (CsA) and FK506 are potent natural product immunosuppressants that induce their biological effects by forming an initial complex with cytosolic proteins termed immunophilins. These drug immunophilin complexes then bind to and inhibit the serine/threonine protein phosphatase calcineurin (CN). Two classes of immunophilin have been identified with cyclophilins (CyP's) being proteins specifically binding CsA and FKBPs specifically binding FK506. Solution and crystal structures of various CsA-CyP and FK506-FKBP complexes have been determined and show no apparent structural similarity between the two classes of drug protein complexes. These findings raise the question as to how, given their structural differences, these two complexes can both inhibit CN. While the crystal structure of the FK506-FKBP12-CN complex has been reported, no structure for a CsA-CyP CN complex has been determined. Here are reported studies that use various modelling strategies to construct a model for the interaction of the cyclosporin A- cyclophilin A complex with calcineurin. The first stage of constructing this model consisted of using conformational comparison of CsA and FK506, GRID and GROUP analysis and restrained molecular dynamics to dock CsA into the FK506 binding site of the FK506-FKBP12-CN structure. An initial model for the CsA-CyPA-CN complex was then constructed by superimposing the structure of the CsA-CyPA complex onto the docked CsA molecule. This model was then optimised with molecular dynamics simulations run on sterically clashing regions. The validity of the model for the CsA-CyPA-CN complex was then examined with respect to the effect of chemical modifications to CsA and amino acid substitutions within CyPA on the ability of the drug-immunophilin complex to inhibit calcineurin.     

The crystal structure of the complex between a disaccharide anthracycline and the DNA hexamer d(CGATCG) reveals two different binding sites involving two DNA duplexes

The crystal structure of the complex formed between the anthracycline antibiotic 3'-deamino-3'- hydroxy-4'-(O-L-daunosaminyl)-4-demethoxydoxo rubicin (MEN 10755), an active disaccharide analogue of doxorubicin, and the DNA hexamer d(CGATCG) has been solved to a resolution of 2.1 A. MEN 10755 exhibits a broad spectrum of antitumor activities, comparable with that of the parent compound, but there are differences in the mechanism of action as it is active in doxorubicin-resistant tumors and is more effective in stimulating topoisomerase DNA cleavage. The structure is similar to previously crystallised anthracycline- DNA complexes. However, two different binding sites arise from drug intercalation so that the two halves of the self-complementary duplex are no longer equivalent. In one site both sugar rings lie in the minor groove. In the other site the second sugar protrudes out from the DNA helix and is linked, through hydrogen bonds, to guanine of a symmetry-related DNA molecule. This is the first structure of an anthracycline-DNA complex where an interaction of the drug with a second DNA helix is observed. We discuss the present findings with respect to the relevance of the amino group for DNA binding and to the potential role played by the second sugar in the interactions with topoisomerases or other cellular targets.     

Crystal structure of the topoisomerase II poison 9-amino-[N-(2-dimethylamino)ethyl]acridine-4-carboxamide bound to the DNA hexanucleotide d(CGTACG)2.

The structure of the complex formed between d(CGTACG)(2) and the antitumor agent 9-amino-[N-(2-dimethylamino)ethyl]acridine-4-carboxamide has been solved to a resolution of 1.6 A using X-ray crystallography. The complex crystallized in space group P6(4) with unit cell dimensions a = b = 30.2 A and c = 39.7 A, alpha = beta = 90 degrees, gamma = 120 degrees. The asymmetric unit contains a single strand of DNA, 1. 5 drug molecules, and 29 water molecules. The final structure has an overall R factor of 19.3%. A drug molecule intercalates between each of the CpG dinucleotide steps with its side chain lying in the major groove, and the protonated dimethylamino group partially occupies positions close to ( approximately 3.0 A) the N7 and O6 atoms of guanine G2. A water molecule forms bridging hydrogen bonds between the 4-carboxamide NH and the phosphate group of the same guanine. Sugar rings adopt the C2'-endo conformation except for cytosine C1 which moves to C3'-endo, thereby preventing steric collision between its C2' methylene group and the intercalated acridine ring. The intercalation cavity is opened by rotations of the main chain torsion angles alpha and gamma at guanines G2 and G6. Intercalation perturbs helix winding throughout the hexanucleotide compared to B-DNA, steps 1 and 2 being unwound by 8 degrees and 12 degrees, respectively, whereas the central TpA step is overwound by 17 degrees. An additional drug molecule, lying with the 2-fold axis in the plane of the acridine ring, is located at the end of each DNA helix, linking it to the next duplex to form a continuously stacked structure. The protonated N,N-dimethylamino group of this "end-stacked" drug hydrogen bonds to the N7 atom of guanine G6. In both drug molecules, the 4-carboxamide group is internally hydrogen bonded to the protonated N-10 atom of the acridine ring. The structure of the intercalated complex enables a rationalization of the known structure-activity relationships for inhibition of topoisomerase II activity, cytotoxicity, and DNA-binding kinetics for 9-aminoacridine-4-carboxamides.     

Design and synthesis of novel benzofurans as a new class of antifungal agents targeting fungal N-myristoyltransferase. Part 2

Modification of the C-2 position of a benzofuran derivative 6 (RO-09-4609), an N-myristoyltransferase (Nmt) inhibitor, has led us to discover antifungal agents that are active in a murine systemic candidiasis model. The drug design is based on the analysis of a crystal structure of a Candida Nmt complex with 2. The optimization has been guided by various biological evaluations including a quasi in vivo assay and pharmacokinetic analysis.     

Structure and biological properties of the copper(II) complex with the quinolone antibacterial drug N-propyl-norfloxacin and 2,2'-bipyridine

The neutral mononuclear copper complex with the quinolone antibacterial drug N-propyl-protected norfloxacin, Hpr-norfloxacin, in the presence of the nitrogen donor heterocyclic ligand 2,2'-bipyridine has been prepared and characterized. The crystal structure of (chloro)(2,2'-bipyridine)(pr-norfloxacinato)copper(II), 1, has been determined and refined with X-ray crystallography. X-band electron paramagnetic resonance (=EPR) spectroscopy at liquid helium temperatures from powdered samples indicates the presence of dimeric units in consistency with the crystal structure. In aqueous solutions of 1 the EPR behavior indicates mixture of dimeric and monomeric species. The antimicrobial activity of the complex has been tested on three different microorganisms and the best inhibition (MIC=0.25mugmL(-1)) has been exhibited against Escherichia coli. The study of the interaction of the complex with calf-thymus DNA has been performed with diverse spectroscopic techniques and has shown that complex 1 is bound to calf-thymus DNA by the intercalative mode. Potential anticancer cytostatic and cytotoxic effects of complex 1 on human promyelocytic leukemia HL-60 and human chronic myelogenous leukemia K562 cell lines have been investigated. Complex 1 shows an increased antiproliferative and necrotic effect on both HL-60 and K562 human leukemia cells in comparison to the free pr-norfloxacin.