Structural and mechanistic insight into DNA bending by antitumour calicheamicins.

Among the class of enediyne antibiotics endowed with potent antitumour activities, the calicheamicin derivative known as ozogamicin is selectively targeted to several leukaemia cell types by means of tailor-made immunoconjugates. Binding of these drugs to the DNA minor groove in a sequence-specific fashion eventually causes double-stranded cleavage that results in cell death. Use of calicheamicin ε, an unreactive analogue of calicheamicin γ1I, has demonstrated that these structurally sophisticated molecules inflict bending on certain DNA oligonucleotides of defined sequence to the extent that they increase their circularization ratio upon ligation into multimers. By modelling and simulating several linear and circular DNA constructs containing high-affinity 5'-TCCT-3' and low-affinity 5'-TTGT-3' target sites in the presence and absence of calicheamicin ε, we have shed light into the structural distortions introduced by the drug upon binding to DNA. This new insight not only informs about the direction and magnitude of the DNA curvature but also provides a rationale for an improved understanding of the preferred structural and dynamic features associated with DNA target selection by calicheamicins.

Insight into the sequence-specific elements leading to increased DNA bending and ligase-mediated circularization propensity by antitumor trabectedin.

DNA curvature is the result of a combination of both intrinsic features of the double helix and external distortions introduced by the environment and the binding of proteins or drugs. The propensity of certain double-stranded DNA (dsDNA) sequences to bend is essential in crucial biological processes, such as replication and transcription, in which proteins are known to either recognize noncanonical DNA conformations or promote their formation upon DNA binding. Trabectedin (Yondelis®) is a clinically used antitumor drug which, following covalent bond formation with the 2-amino group of guanine, induces DNA curvature and enhances the circularization ratio, upon DNA ligation, of several dsDNA constructs but not others. By means of unrestrained molecular dynamics simulations using explicitly solvated all-atom models, we rationalize these experimental findings in structural terms and shed light on the crucial, albeit possibly underappreciated, role played by T4 DNA ligase in stabilizing a bent DNA conformation prior to cyclization. Taken together, our results expand our current understanding on how DNA shape modification by trabectedin may affect both the sequence-specific recognition by transcription factors to promoter sites and RNA polymerase II binding.

Structural Cues for Understanding eEF1A2 Moonlighting.

Spontaneous mutations in the EEF1A2 gene cause epilepsy and severe neurological disabilities in children. The crystal structure of eEF1A2 protein purified from rabbit skeletal muscle reveals a post-translationally modified dimer that provides information about the sites of interaction with numerous binding partners, including itself, and maps these mutations onto the dimer and tetramer interfaces. The spatial locations of the side chain carboxylates of Glu301 and Glu374, to which phosphatidylethanolamine is uniquely attached via an amide bond, define the anchoring points of eEF1A2 to cellular membranes and interorganellar membrane contact sites. Additional bioinformatic and molecular modeling results provide novel structural insight into the demonstrated binding of eEF1A2 to SH3 domains, the common MAPK docking groove, filamentous actin, and phosphatidylinositol-4 kinase IIIß. In this new light, the role of eEF1A2 as an ancient, multifaceted, and articulated G protein at the crossroads of autophagy, oncogenesis and viral replication appears very distant from the "canonical" one of delivering aminoacyl-tRNAs to the ribosome that has dominated the scene and much of the thinking for many decades.

Tuning melatonin receptor subtype selectivity in oxadiazolone-based analogues: Discovery of QR2 ligands and NRF2 activators with neurogenic properties.

New multi-target indole and naphthalene derivatives containing the oxadiazolone scaffold as a bioisostere of the melatonin acetamido group have been developed. The novel compounds were characterized at melatonin receptors MT1R and MT2R, quinone reductase 2 (QR2), lipoxygenase-5 (LOX-5), and monoamine oxidases (MAO-A and MAO-B), and also as radical scavengers. We found that selectivity within the oxadiazolone series can be modulated by modifying the side chain functionality and co-planarity with the indole or naphthalene ring. In phenotypic assays, several oxadiazolone-based derivatives induced signalling mediated by the transcription factor NRF2 and promoted the maturation of neural stem-cells into a neuronal phenotype. Activation of NRF2 could be due to the binding of indole derivatives to KEAP1, as deduced from surface plasmon resonance (SPR) experiments. Molecular modelling studies using the crystal structures of QR2 and the KEAP1 Kelch-domain, as well as the recently described X-ray free-electron laser (XFEL) structures of chimeric MT1R and MT2R, provided a rationale for the experimental data and afforded valuable insights for future drug design endeavours.

Structure-Guided Tuning of a Selectivity Switch towards Ribonucleosides in Trypanosoma brucei Purine Nucleoside 2'-Deoxyribosyltransferase.

The use of nucleoside 2'-deoxyribosyltransferases (NDTs) as biocatalysts for the industrial synthesis of nucleoside analogues is often hindered by their strict preference for 2'-deoxyribonucleosides. It is shown herein that a highly versatile purine NDT from Trypanosoma brucei (TbPDT) can also accept ribonucleosides as substrates; this is most likely because of the distinct role played by Asn53 at a position that is usually occupied by Asp in other NDTs. Moreover, this unusual activity was improved about threefold by introducing a single amino acid replacement at position 5, following a structure-guided approach. Biophysical and biochemical characterization revealed that the TbPDTY5F variant is a homodimer that displays maximum activity at 50 °C and pH 6.5 and shows a remarkably high melting temperature of 69 °C. Substrate specificity studies demonstrate that 6-oxopurine ribonucleosides are the best donors (inosine>guanosine≫adenosine), whereas no significant preferences exist between 6-aminopurines and 6-oxopurines as base acceptors. In contrast, no transferase activity could be detected on xanthine and 7-deazapurines. TbPDTY5F was successfully employed in the synthesis of a wide range of modified ribonucleosides containing different purine analogues.

Unravelling the covalent binding of zampanolide and taccalonolide AJ to a minimalist representation of a human microtubule.

Many natural products target mammalian tubulin but only a few can form a covalent bond and hence irreversibly affect microtubule function. Among them, zampanolide (ZMP) and taccalonolide AJ (TAJ) stand out, not only because they are very potent antitumor agents but also because the adducts they form with ß-tubulin have been structurally characterized in atomic detail. By applying model building techniques, molecular orbital calculations, molecular dynamics simulations and hybrid QM/MM methods, we have gained insight into the 1,2- and 1,4-addition reactions of His229 and Asp226 to ZMP and TAJ, respectively, in the taxane-binding site of ß-tubulin. The experimentally inaccessible precovalent complexes strongly suggest a water-mediated proton shuttle mechanism for ZMP adduct formation and a direct nucleophilic attack by the carboxylate of Asp226 on C22 of the C22R,C23R epoxide in TAJ. The M-loop, which is crucially important for interprotofilament interactions, is structured into a short helix in both types of complexes, mostly as a consequence of the fixation of the phenol ring of Tyr283 and the guanidinium of Arg284. As a side benefit, we obtained evidence supporting the existence of a commonly neglected intramolecular disulfide bond between Cys241 and Cys356 in ß-tubulin that contributes to protein compactness and is absent in the ßIII isotype associated with resistance to taxanes and other drugs.

(F)uridylylated Peptides Linked to VPg1 of Foot-and- Mouth Disease Virus (FMDV): Design, Synthesis and X-Ray Crystallography of the Complexes with FMDV RNA-Dependent RNA Polymerase.

Foot-and-mouth disease virus (FMDV) is an RNA virus belonging to the Picornaviridae family that contains three small viral proteins (VPgs), named VPg1, VPg2 and VPg3, linked to the 5'-end of the viral genome. These VPg proteins act as primers for RNA replication, which is initiated by the consecutive binding of two UMP molecules to the hydroxyl group of Tyr3 in VPg. This process, termed uridylylation, is catalyzed by the viral RNA-dependent RNA polymerase named 3Dpol. 5-Fluorouridine triphosphate (FUTP) is a potent competitive inhibitor of VPg uridylylation. Peptide analysis showed FUMP covalently linked to the Tyr3 of VPg. This fluorouridylylation prevents further incorporation of the second UMP residue. The molecular basis of how the incorporated FUMP blocks the incorporation of the second UMP is still unknown. To investigate the mechanism of inhibition of VPg uridylylation by FUMP, we have prepared a simplified 15-mer model of VPg1 containing FUMP and studied its x-ray crystal structure in complex with 3Dpol. Unfortunately, the fluorouridylylated VPg1 was disordered and not visible in the electron density maps; however, the structure of 3Dpol in the presence of VPg1-FUMP showed an 8 Å movement of the ß9-α11 loop of the polymerase towards the active site cavity relative to the complex of 3Dpol with VPg1-UMP. The conformational rearrangement of this loop preceding the 3Dpol B motif seems to block the access of the template nucleotide to the catalytic cavity. This result may be useful in the design of new antivirals against not only FMDV but also other picornaviruses, since all members of this family require the uridylylation of their VPg proteins to initiate the viral RNA synthesis.

Synthesis and Cytotoxicity of 7,9-O-Linked Macrocyclic C-Seco Taxoids.

A series of novel 7,9-O-linked macrocyclic taxoids together with modification at the C2 position were synthesized, and their cytotoxicities against drug-sensitive and P-glycoprotein and ßIII-tubulin overexpressed drug-resistant cancer cell lines were evaluated. It is demonstrated that C-seco taxoids conformationally constrained via carbonate containing-linked macrocyclization display increased cytotoxicity on drug-resistant tumors overexpressing both ßIII and P-gp, among which compound 22b, bearing a 2-m-methoxybenzoyl group together with a five-atom linker, was identified as the most potent. Molecular modeling suggested the improved cytotoxicity of 22b results from enhanced favorable interactions with the T7 loop region of ßIII.