Explore the vast range of titles available.

The exploration of biologically relevant chemical space for the discovery of small bioactive molecules present in marine organisms has led not only to important advances in certain therapeutic areas, but also to a better understanding of many life processes. The still largely untapped reservoir of countless metabolites that play biological roles in marine invertebrates and microorganisms opens new avenues and poses new challenges for research. Computational technologies provide the means to (i) organize chemical and biological information in easily searchable and hyperlinked databases and knowledgebases; (ii) carry out cheminformatic analyses on natural products; (iii) mine microbial genomes for known and cryptic biosynthetic pathways; (iv) explore global networks that connect active compounds to their targets (often including enzymes); (v) solve structures of ligands, targets, and their respective complexes using X-ray crystallography and NMR techniques, thus enabling virtual screening and structure-based drug design; and (vi) build molecular models to simulate ligand binding and understand mechanisms of action in atomic detail. Marine natural products are viewed today not only as potential drugs, but also as an invaluable source of chemical inspiration for the development of novel chemotypes to be used in chemical biology and medicinal chemistry research.

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.

Bending of double-stranded (ds) DNA plays a crucial role in many important biological processes and is relevant for nanotechnological applications. Among all the elements that have been studied in relation to dsDNA bending, A-tracts stand out as one of the most controversial. The “ApA wedge” theory was disproved when a series of linear polynucleotides containing phased 5′-A4T4-3′ or 5′-T4A4-3′ runs were shown to be bent or straight, respectively, and crystallographic evidence revealed that A-tracts are unbent. Furthermore, some of the smallest dsDNA minicircles described to date (~ 100 bp in size) lack A-tracts and are subjected to varying levels of torsional stress. Representative DNA sequences from this experimental background were modeled in atomic detail and their dynamic behavior was simulated over hundreds of nanoseconds using the AMBER force field ParmBSC1. Subsequent analysis of the resulting trajectories allowed us to (i) unambiguously establish the location of the bends in all cases; (ii) identify the structural elements that are directly responsible for the macroscopically detected curvature; and (iii) reveal the importance not only of coherently summing the effects of the bending elements when they are in synchrony with the natural repeat of the helix (i.e. separated by an integral number of helical turns) but also when alternated with a half-integral separation of opposite effects. We conclude that the major determinant of the macroscopically observed bending is the proper grouping and phasing of the positive roll imposed by pyrimidine-purine (YR) steps and the negative or null roll characteristic of RY steps and A-tracts, respectively. This conclusion is in very good agreement with the structural parameters experimentally derived for much smaller DNA molecules either alone or as found in DNA–protein complexes. We expect that this work will pave the way for future studies on drug-induced DNA bending, DNA shape readout by transcription factors, structure of circular extrachromosomal DNA, and custom design of curved DNA origami scaffolds.

The experimental construction of a double-stranded DNA microcircle of only 42 base pairs entailed a great deal of ingenuity and hard work. However, figuring out the three-dimensional structures of intermediates and the final product can be particularly baffling. Using a combination of model building and unrestrained molecular dynamics simulations in explicit solvent we have characterized the different DNA structures involved along the process. Our 3D models of the single-stranded DNA molecules provide atomic insight into the recognition event that must take place for the DNA bases in the cohesive tail of the hairpin to pair with their complementary bases in the single-stranded loops of the dumbbell. We propose that a kissing loop involving six base pairs makes up the core of the nascent dsDNA microcircle. We also suggest a feasible pathway for the hybridization of the remaining complementary bases and characterize the final covalently closed dsDNA microcircle as possessing two well-defined U-turns. Additional models of the pre-ligation complex of T4 DNA ligase with the DNA dumbbell and the post-ligation pre-release complex involving the same enzyme and the covalently closed DNA microcircle are shown to be compatible with enzyme recognition and gap ligation.

The bacterial cell-wall peptidoglycan is made of glycan strands crosslinked by short peptide stems. Crosslinks are catalyzed by DD-transpeptidases (4,3-crosslinks) and LD-transpeptidases (3,3-crosslinks). However, recent research on non-model species has revealed novel crosslink types, suggesting the existence of uncharacterized enzymes. Here, we identify an LD-transpeptidase, LDT Go , that generates 1,3-crosslinks in the acetic-acid bacterium Gluconobacter oxydans . LDT Go -like proteins are found in Alpha- and Betaproteobacteria lacking LD3,3-transpeptidases. In contrast with the strict specificity of typical LD- and DD-transpeptidases, LDT Go can use non-terminal amino acid moieties for crosslinking. A high-resolution crystal structure of LDT Go reveals unique features when compared to LD3,3-transpeptidases, including a proline-rich region that appears to limit substrate access, and a cavity accommodating both glycan chain and peptide stem from donor muropeptides. Finally, we show that DD-crosslink turnover is involved in supplying the necessary substrate for LD1,3-transpeptidation. This phenomenon underscores the interplay between distinct crosslinking mechanisms in maintaining cell wall integrity in G. oxydans . The cell-wall peptidoglycan in model bacteria typically includes 4,3- and 3,3-crosslinks, catalysed by DD- and LD-transpeptidases, respectively. Here, the authors identify and characterise the activity and structure of an LD-transpeptidase that generates a new type of crosslink (1,3).

Paclitaxel (Taxol) is a taxane and a chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect its mechanism of action. Here, we solved the crystal structure of baccatin III the core moiety of paclitaxel-tubulin complex at 1.9 Å resolution. Based on this information, we engineered taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects on microtubules (X-ray fiber diffraction), along with those of paclitaxel, docetaxel, and baccatin III. Further comparison of high-resolution structures and microtubules’ diffractions with the apo forms and molecular dynamics approaches allowed us to understand the consequences of taxane binding to tubulin in solution and under assembled conditions. The results sheds light on three main mechanistic questions: (1) taxanes bind better to microtubules than to tubulin because tubulin assembly is linked to a βM-loopconformational reorganization (otherwise occludes the access to the taxane site) and, bulky C13 side chains preferentially recognize the assembled conformational state; (2) the occupancy of the taxane site has no influence on the straightness of tubulin protofilaments and; (3) longitudinal expansion of the microtubule lattices arises from the accommodation of the taxane core within the site, a process that is no related to the microtubule stabilization (baccatin III is biochemically inactive). In conclusion, our combined experimental and computational approach allowed us to describe the tubulin-taxane interaction in atomic detail and assess the structural determinants for binding.

Hyperekplexia (OMIM 149400), a sensorimotor syndrome of perinatal clinical relevance, causes newborns to display an energic startle reflex in response to certain trivial stimuli. This condition can be lethal due to apnea episodes. The disease is caused by a blockade of glycinergic neurotransmission. Glycinergic interneurons preserve their identity by the activity of the surface glycine transporter GlyT2, which supplies glycine to presynaptic terminals to maintain glycine content in synaptic vesicles. Loss-of-function mutations in the GlyT2 gene (SLC6A5) cause a presynaptic form of human hyperekplexia. Here, we describe a new GlyT2 variant found in an infantile patient diagnosed with hyperekplexia. A missense mutation in the open reading frame of the GlyT2 gene inherited in homozygosity caused the substitution G449E in a residue highly conserved across the phylogenetic scale. The sequences of the glycine receptor genes GLRA1 and GLRB did not show abnormalities. We expressed the recombinant GlyT2 variant in heterologous cells and analyzed its pathogenic mechanism. The transporter was totally inactive, behaving as a bona fide loss-of-function mutant. Furthermore, the mutation promoted the abnormal insertion of the protein into the membrane, leading to its large incorporation into lipid rafts. However, there was no apparent alteration of wild-type trafficking upon mutant coexpression, as the mutant was prematurely degraded from the endoplasmic reticulum. Rescue with chemical chaperones was not possible for this mutant. Proteomics demonstrated that the expression of the mutant induced the unfolded protein response and interfered with raft-dependent processes. Therefore, the new variant causes a loss of function regarding GlyT2 activity but a gain of function as a cell proteostasis disturber.

eEF1A2 is one of the isoforms of the alpha subunit of the eukaryotic Elongation Factor 1. It is overexpressed in human tumors and is endowed with oncogenic properties, favoring tumor cell proliferation while inhibiting apoptosis. We demonstrate that plitidepsin, an antitumor agent of marine origin that has successfully completed a phase-III clinical trial for multiple myeloma, exerts its antitumor activity by targeting eEF1A2. The drug interacts with eEF1A2 with a K D of 80 nM and a target residence time of circa 9 min. This protein was also identified as capable of binding [ 14 C]-plitidepsin in a cell lysate from K-562 tumor cells. A molecular modelling approach was used to identify a favorable binding site for plitidepsin at the interface between domains 1 and 2 of eEF1A2 in the GTP conformation. Three tumor cell lines selected for at least 100-fold more resistance to plitidepsin than their respective parental cells showed reduced levels of eEF1A2 protein. Ectopic expression of eEF1A2 in resistant cells restored the sensitivity to plitidepsin. FLIM-phasor FRET experiments demonstrated that plitidepsin localizes in tumor cells sufficiently close to eEF1A2 as to suggest the formation of drug-protein complexes in living cells. Altogether, our results strongly suggest that eEF1A2 is the primary target of plitidepsin.

Cetirizine, a major metabolite of hydroxyzine, became a marketed second-generation H1 antihistamine that is orally active and has a rapid onset of action, long duration of effects and a very good safety record at recommended doses. The approved drug is a racemic mixture of (S)-cetirizine and (R)-cetirizine, the latter being the levorotary enantiomer that also exists in the market as a third-generation, non-sedating and highly selective antihistamine. Both enantiomers bind tightly to the human histamine H1 receptor (hH1R) and behave as inverse agonists but the affinity and residence time of (R)-cetirizine are greater than those of (S)-cetirizine. In blood plasma, cetirizine exists in the zwitterionic form and more than 90% of the circulating drug is bound to human serum albumin (HSA), which acts as an inactive reservoir. Independent X-ray crystallographic work has solved the structure of the hH1R:doxepin complex and has identified two drug-binding sites for cetirizine on equine serum albumin (ESA). Given this background, we decided to model a membrane-embedded hH1R in complex with either (R)- or (S)-cetirizine and also the complexes of both ESA and HSA with these two enantiomeric drugs to analyze possible differences in binding modes between enantiomers and also among targets. The ensuing molecular dynamics simulations in explicit solvent and additional computational chemistry calculations provided structural and energetic information about all of these complexes that is normally beyond current experimental possibilities. Overall, we found very good agreement between our binding energy estimates and extant biochemical and pharmacological evidence. A much higher degree of solvent exposure in the cetirizine-binding site(s) of HSA and ESA relative to the more occluded orthosteric binding site in hH1R is translated into larger positional fluctuations and considerably lower affinities for these two nonspecific targets. Whereas it is demonstrated that the two known pockets in ESA provide enough stability for cetirizine binding, only one such site does so in HSA due to a number of amino acid replacements. At the histamine-binding site in hH1R, the distinct interactions established between the phenyl and chlorophenyl moieties of the two enantiomers with the amino acids lining up the pocket and between their free carboxylates and Lys179 in the second extracellular loop account for the improved pharmacological profile of (R)-cetirizine.

Enterovirus A71 (EV-A71) is a non-polio neurotropic enterovirus with pandemic potential. There are no antiviral agents approved to prevent or treat EV-A71 infections. We here report on the molecular mechanism by which a novel class of tryptophan dendrimers inhibits (at low nanomolar to high picomolar concentration) EV-A71 replication in vitro. A lead compound in the series (MADAL385) prevents binding and internalization of the virus but does not, unlike classical capsid binders, stabilize the particle. By means of resistance selection, reverse genetics and cryo-EM, we map the binding region of MADAL385 to the 5-fold vertex of the viral capsid and demonstrate that a single molecule binds to each vertex. By interacting with this region, MADAL385 prevents the interaction of the virus with its cellular receptors PSGL1 and heparan sulfate, thereby blocking the attachment of EV-A71 to the host cells.