Ilda D'Annessa

Structural and dynamical effects induced by the anticancer drug topotecan on the human topoisomerase I - DNA complex

Background Human topoisomerase I catalyzes the relaxation of DNA supercoils in fundamental cell processes like transcription, replication and chromosomal segregation. It is the only target of the camptothecin family of anticancer drugs. Among these, topotecan has been used to treat lung and ovarian carcinoma for several years. Camptothecins reversibly binds to the covalent intermediate DNA-enzyme, stabilizing the cleavable complex and reducing the religation rate. The stalled complex then collides with the progression of the replication fork, producing lethal double strand DNA breaks and eventually cell death. Methodology/Principal Findings Long lasting molecular dynamics simulations of the DNA-topoisomerase I binary complex and of the DNA-topoisomerase-topotecan ternary complex have been performed and compared. The conformational space sampled by the binary complex is reduced by the presence of the drug, as observed by principal component and cluster analyses. This conformational restraint is mainly due to the reduced flexibility of residues 633–643 (the region connecting the linker to the core domain) that causes an overall mobility loss in the ternary complex linker domain. During the simulation, DNA/drug stacking interactions are fully maintained, and hydrogen bonds are maintained with the enzyme. Topotecan keeps the catalytic residue Lys532 far from the DNA, making it unable to participate to the religation reaction. Arg364 is observed to interact with both the B and E rings of topotecan with two stable direct hydrogen bonds. An interesting constrain exerted by the protein on the geometrical arrangement of topotecan is also observed. Conclusions/Significance Atomistic-scale understanding of topotecan interactions with the DNA-enzyme complex is fundamental to the explaining of its poisonous effect and of the drug resistance observed in several single residue topoisomerase mutants. We observed significant alterations due to topotecan in both short-range interactions and long-range protein domain communications.

Erybraedin C, a natural compound from the plant Bituminaria bituminosa, inhibits both the cleavage and religation activities of human topoisomerase I

The interaction of human topoisomerase I and erybraedin C, a pterocarpan purified from the plant Bituminaria bituminosa, that was shown to have an antitumour activity, was investigated through enzymatic activity assays and molecular docking procedures. Erybraedin C is able to inhibit both the cleavage and the religation steps of the enzyme reaction. In both cases, pre-incubation of the drug with the enzyme is required to produce a complete inhibition. Molecular docking simulations indicate that, when interacting with the enzyme alone, the preferential drug-binding site is localized in proximity to the active Tyr723 residue, with one of the two prenilic groups close to the active-site residues Arg488 and His632, essential for the catalytic reaction. When interacting with the cleavable complex, erybraedin C interacts with both the enzyme and DNA in a way similar to that found for topotecan. This is the first example of a natural compound able to act on both the cleavage and religation reaction of human topoisomerase I.

Molecular mechanism of the camptothecin resistance of Glu710Gly topoisomerase IB mutant analyzed in vitro and in silico

BackgroundDNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase IB can be inhibited by several compounds that act through different mechanisms, including clinically used drugs, such as the derivatives of the natural compound camptothecin that reversibly bind the covalent topoisomerase-DNA complex, slowing down the religation of the cleaved DNA strand, thus inducing cell death. Three enzyme mutations, which confer resistance to irinotecan in an adenocarcinoma cell line, were recently identified but the molecular mechanism of resistance was unclear.MethodsThe three resistant mutants have been investigated in S. cerevisiae model system following their viability in presence of increasing amounts of camptothecin. A systematical analysis of the different catalytic steps has been made for one of these mutants (Glu710Gly) and has been correlated with its structural-dynamical properties studied by classical molecular dynamics simulation.ResultsThe three mutants display a different degree of camptothecin resistance in a yeast cell viability assay. Characterization of the different steps of the catalytic cycle of the Glu710Gly mutant indicated that its resistance is related to a high religation rate that is hardly affected by the presence of the drug. Analysis of the dynamic properties through simulation indicate that the mutant displays a much lower degree of correlation in the motion between the different protein domains and that the linker almost completely loses its correlation with the C-terminal domain, containing the active site tyrosine.ConclusionsThese results indicate that a fully functional linker is required to confer camptothecin sensitivity to topoisomerase I since the destabilization of its structural-dynamical properties is correlated to an increase of religation rate and drug resistance.

Structural dynamics of the mitochondrial ADP/ATP carrier revealed by molecular dynamics simulation studies

The mitochondrial adenosine diphosphate/adenosine triphosphate (ADP/ATP) carrier has been recently crystallized in complex with its specific inhibitor carboxyatractyloside (CATR). In the crystal structure, the six‐transmembrane helix bundle that defines the nucleotide translocation pathway is closed on the matrix side due to sharp kinks in the odd‐numbered helices. The closed conformation is further sealed by the loops protruding into the matrix that interact through an intricate network of charge‐pairs. To gain insight into its structural dynamics we performed molecular dynamics (MD) simulation studies of the ADP/ATP carrier with and without its cocrystallized inhibitor. The two trajectories sampled a conformational space around two different configurations characterized by distinct salt‐bridge networks with a significant shift from inter‐ to intrarepeat bonding on the matrix side in the absence of CATR. Analysis of the geometrical parameters defining the transmembrane helices showed that even‐numbered helices can undergo a face rotation, whereas odd‐numbered helices can undergo a change in the wobble angle with a conserved proline acting as molecular hinge. Our results provide new information on the dynamical properties of the ADP/ATP carrier and for the first time yield a detailed picture of a stable carrier conformation in absence of the inhibitor. Proteins 2006.

Simulations of DNA topoisomerase 1B bound to supercoiled DNA reveal changes in the flexibility pattern of the enzyme and a secondary protein–DNA binding site

Human topoisomerase 1B has been simulated covalently bound to a negatively supercoiled DNA minicircle, and its behavior compared to the enzyme bound to a simple linear DNA duplex. The presence of the more realistic supercoiled substrate facilitates the formation of larger number of protein–DNA interactions when compared to a simple linear duplex fragment. The number of protein–DNA hydrogen bonds doubles in proximity to the active site, affecting all of the residues in the catalytic pentad. The clamp over the DNA, characterized by the salt bridge between Lys369 and Glu497, undergoes reduced fluctuations when bound to the supercoiled minicircle. The linker domain of the enzyme, which is implicated in the controlled relaxation of superhelical stress, also displays an increased number of contacts with the minicircle compared to linear DNA. Finally, the more complex topology of the supercoiled DNA minicircle gives rise to a secondary DNA binding site involving four residues located on subdomain III. The simulation trajectories reveal significant changes in the interactions between the enzyme and the DNA for the more complex DNA topology, which are consistent with the experimental observation that the protein has a preference for binding to supercoiled DNA.

The human topoisomerase 1B Arg634Ala mutation results in camptothecin resistance and loss of inter-domain motion correlation

Human topoisomerase 1B, the unique target of the natural anticancer compound camptothecin, catalyzes the unwinding of supercoiled DNA by introducing transient single strand nicks and providing covalent protein-DNA adducts. The functional properties and the drug reactivity of the single Arg634Ala mutant have been investigated in comparison to the wild type enzyme. The mutant is characterized by an identical relaxation and cleavage rate but it displays resistance to camptothecin as indicated by a viability assay of the yeast cells transformed with the mutated protein. The mutant also displays a very fast religation rate that is only partially reduced by the presence of the drug, suggesting that this is the main reason for its resistance. A comparative analysis of the structural-dynamical properties of the native and mutant proteins by molecular dynamics simulation indicates that mutation of Arg634 brings to a loss of motion correlation between the different domains and in particular between the linker and the C-terminal domain, containing the catalytic tyrosine residue. These results indicate that the loss of motion correlation and the drug resistance are two strongly correlated events.

Role of Flexibility in Protein-DNA-Drug Recognition: The Case of Asp677Gly-Val703Ile Topoisomerase Mutant Hypersensitive to Camptothecin

Topoisomerases I are ubiquitous enzymes that control DNA topology within the cell. They are the unique target of the antitumor drug camptothecin that selectively recognizes the DNA-topoisomerase covalent complex and reversibly stabilizes it. The biochemical and structural-dynamical properties of the Asp677Gly-Val703Ile double mutant with enhanced CPT sensitivity have been investigated. The mutant displays a lower religation rate of the DNA substrate when compared to the wild-type protein. Analyses of the structural dynamical properties by molecular dynamics simulation show that the mutant has reduced flexibility and an active site partially destructured at the level of the Lys532 residue. These results demonstrate long-range communication mechanism where reduction of the linker flexibility alters the active site geometry with the consequent lowering of the religation rate and increase in drug sensitivity.

A unique binding mode of the eukaryotic translation initiation factor 4E for guiding the design of novel peptide inhibitors

The interaction between the eukaryotic translation initiation factor 4E (eIF4E) and eIF4E binding proteins (4E‐BP) is a promising template for the inhibition of eIF4E and the treatment of diseases such as cancer and a spectrum of autism disorders, including the Fragile X syndrome (FXS). Here, we report an atomically detailed model of the complex between eIF4E and a peptide fragment of a 4E‐BP, the cytoplasmic Fragile X interacting protein (CYFIP1). This model was generated using computer simulations with enhanced sampling from an alchemical replica exchange approach and validated using long molecular dynamics simulations. 4E‐BP proteins act as post‐transcriptional regulators by binding to eIF4E and preventing mRNA translation. Dysregulation of eIF4E activity has been linked to cancer, FXS, and autism spectrum disorders. Therefore, the study of the mechanism of inhibition of eIF4E by 4E‐BPs is key to the development of drug therapies targeting this regulatory pathways. The results obtained in this work indicate that CYFIP1 interacts with eIF4E by an unique mode not shared by other 4E‐BP proteins and elucidate the mechanism by which CYFIP1 interacts with eIF4E despite having a sequence binding motif significantly different from most 4E‐BPs. Our study suggests an alternative strategy for the design of eIF4E inhibitor peptides with superior potency and specificity than currently available.

Mutation of Gly717Phe in human topoisomerase 1B has an effect on enzymatic function, reactivity to the camptothecin anticancer drug and on the linker domain orientation

Human topoisomerase 1B controls the topological state of supercoiled DNA allowing the progression of fundamental cellular processes. The enzyme, which is the unique molecular target of the natural anticancer compound camptothecin, acts by cleaving one DNA strand and forming a transient protein-DNA covalent adduct. In this work the role of the Gly717 residue, located in a α-helix structure bridging the active site and the linker domain, has been investigated mutating it in Phe. The mutation gives rise to drug resistance in vivo as observed through a viability assay of yeast cells. In vitro activity assays show that the mutant is characterized by a fast religation rate, only partially reduced by the presence of the drug. Comparative molecular dynamics simulations of the native and mutant proteins indicate that the mutation of Gly717 affects the motion orientation of the linker domain, changing its interaction with the DNA substrate, likely affecting the strand rotation and religation rate. The mutation also causes a slight rearrangement of the active site and of the drug binding site, providing an additional explanation for the lowered effect of camptothecin toward the mutant.

Development of Derivatives of 3, 3′-Diindolylmethane as Potent Leishmania donovani Bi-Subunit Topoisomerase IB Poisons

Background The development of 3, 3′-diindolyl methane (DIM) resistant parasite Leishmania donovani (LdDR50) by adaptation with increasing concentrations of the drug generates random mutations in the large and small subunits of heterodimeric DNA topoisomerase I of Leishmania (LdTOP1LS). Mutation of large subunit of LdTOP1LS at F270L is responsible for resistance to DIM up to 50 µM concentration. Methodology/Principal Findings In search of compounds that inhibit the growth of the DIM resistant parasite and inhibit the catalytic activity of mutated topoisomerase I (F270L), we have prepared three derivatives of DIM namely DPDIM (2,2′-diphenyl 3,3′-diindolyl methane), DMDIM (2,2′-dimethyl 3,3′-diindolyl methane) and DMODIM (5,5′-dimethoxy 3,3′-diindolyl methane) from parent compound DIM. All the compounds inhibit the growth of DIM resistant parasites, induce DNA fragmentation and stabilize topo1-DNA cleavable complex with the wild type and mutant enzyme. Conclusion The results suggest that the three derivatives of DIM can act as promising lead molecules for the generation of new anti-leishmanial agents.