Detecting DNA Mismatches with Metallo-Insertors: A Molecular Simulation Study

Molecules that selectively recognize DNA mismatches (MMs) play a key role as nucleic acids probes and as chemotherapeutic agents. Metallo-insertors bind to the minor groove (mG) of double strand (ds) DNA, expelling the mismatched base pairs and acting as their pi-stacking replacement. In contrast, metallo-intercalators bind to the major groove (MG) of ds DNA and pi-stack to adjacent base pairs. In this study we focused on structural and energetic properties of Delta-[Rh(bpy)(2)(chrysi)](3+) (1), Delta-[Ru(bpy)(2)(ddpz)](2+) (2), and Delta-[Ru(bpy)(2)(eilatin)](2+) (3) as prototypical examples of metallo-insertors and intercalators. For all molecules we characterized both insertion and intercalation into a DNA dodecamer via force field based molecular dynamics (MD) and hybrid quantum-classical (QM/MM) MD simulations. A structural analysis of the 1-3/DNA noncovalent adducts reveals that the insertion provokes an untwist of the DNA, an opening of the mG and of the phosphate backbone in proximity of the mismatch, while the intercalation induces smaller changes of these structural parameters. This behavior appears to be correlated with the size of the inserting/intercalating ligand in proximity of the metal coordination site. Moreover, our simulations show that the different selectivity of 1 toward distinct MM types may be correlated with the thermodynamic stability of the MMs in the free DNA and with that of the corresponding insertion adduct. Understanding the factors which tune a specific insertion is of crucial importance for designing specific luminescent probes that selectively recognize MMs, as well as for developing more effective anticancer drugs active in MM repair of deficient cells lines

The molecular mechanism of secondary sodium symporters elucidated through the lens of the computational microscope

Transport of molecules across cellular membranes is a key biological process for normal cell function. As such, secondary active transporters exploit electrochemical ion gradients to carry out fundamental processes, i.e. nutrients uptake, ion regulation, neurotransmission, and substrate extrusion. Despite their modest sequence similarity, several Na+ symporters share the same fold of LeuT (leucine transporter), a prokaryotic member of the neurotransmitter-sodium symporter family, pinpointing to a common structural/functional mechanism of transport. This is associated with specific conformational transitions occurring along a so-called alternating access mechanism. Thanks to recent advances in computer simulation techniques and the ever-increasing computational power that has become available in the last decade, molecular dynamics (MD) simulations have been largely employed to provide atomistic insights into mechanistic, kinetic, and thermodynamic aspects of this family of transporters. Here we report a detailed overview of selected Na+-symporters belonging to the LeuT-fold superfamily for which different aspects of the transport mechanism have been addressed using both experimental and computational studies. The aim of this review is to describe current state-of-the-art knowledge on the mechanism of these transporters showing how molecular simulations have contributed to elucidate mechanistic aspects and can provide nowadays a spatial and temporal resolution, allowing the interpretation of experimental findings, complementing biophysical methods, and filling the gaps in fragmentary experimental information

Theoretical Studies of Homogeneous Catalysts Mimicking Nitrogenase

The conversion of molecular nitrogen to ammonia is a key biological and chemical process and represents one of the most challenging topics in chemistry and biology. In Nature the Mo-containing nitrogenase enzymes perform nitrogen 'fixation' via an iron molybdenum cofactor (FeMo-co) under ambient conditions. In contrast, industrially, the Haber-Bosch process reduces molecular nitrogen and hydrogen to ammonia with a heterogeneous iron catalyst under drastic conditions of temperature and pressure. This process accounts for the production of millions of tons of nitrogen compounds used for agricultural and industrial purposes, but the high temperature and pressure required result in a large energy loss, leading to several economic and environmental issues. During the last 40 years many attempts have been made to synthesize simple homogeneous catalysts that can activate dinitrogen under the same mild conditions of the nitrogenase enzymes. Several compounds, almost all containing transition metals, have been shown to bind and activate N(2) to various degrees. However, to date Mo(N(2))(HIPTN)(3)N with (HIPTN)(3)N= hexaisopropyl-terphenyl-triamidoamine is the only compound performing this process catalytically. In this review we describe how Density Functional Theory calculations have been of help in elucidating the reaction mechanisms of the inorganic compounds that activate or fix N(2). These studies provided important insights that rationalize and complement the experimental findings about the reaction mechanisms of known catalysts, predicting the reactivity of new potential catalysts and helping in tailoring new efficient catalytic compounds

Interaction between the DNA model base 9-ethylguanine and a group of ruthenium polypyridyl complexes: Kinetics and conformational temperature dependence

The binding capability of three ruthenium polypyridyl compounds of structural formula [Ru(apy)(tpy)Ln-](ClO4)((2-n)) [1a-c; apy = 2,2'-azobis(pyridine), tpy = 2,2':6',2 ''-terpyridine, L = Cl, H2O, CH3CN] to a fragment of DNA was studied. The interaction between each of these complexes and the DNA model base 9-ethylguanine (9-EtGua) was followed by means of H-1 NMR studies. Density functional theory calculations were carried out to explore the preferential ways of coordination between the ruthenium complexes and guanine. The ruthenium-9-EtGua adduct formed was isolated and fully characterized using different techniques. A variable-temperature H-1 NMR experiment was carried out that showed that while the 9-EtGua fragment was rotating fast at high temperature, a loss of symmetry was suffered by the model base adduct as the temperature was lowered, indicating restricted rotation of the guanine residue

Cancer-Related Mutations Alter RNA-Driven Functional Cross-Talk Underlying Premature-Messenger RNA Recognition by Splicing Factor SF3b

The pillar of faithful premature-messenger (pre-mRNA)splicingis the precise recognition of key intronic sequences by specific splicingfactors. The heptameric splicing factor 3b (SF3b) recognizes the branchpoint sequence (BPS), a key part of the 3 & PRIME; splice site. SF3bcontains SF3B1, a protein holding recurrent cancer-associated mutations.Among these, K700E, the most-frequent SF3B1 mutation, triggers aberrantsplicing, being primarily implicated in hematologic malignancies.Yet, K700E and the BPS recognition site are 60 & ANGS; apart, suggestingthe existence of an allosteric cross-talk between the two distal spots.Here, we couple molecular dynamics simulations and dynamical networktheory analysis to unlock the molecular terms underpinning the impactof SF3b splicing factor mutations on pre-mRNA selection. We establishthat by weakening and remodeling interactions of pre-mRNA with SF3b,K700E scrambles RNA-mediated allosteric cross-talk between the BPSand the mutation site. We propose that the altered allostery contributesto cancer-associated missplicing by mutated SF3B1. This finding broadensour comprehension of the elaborate mechanisms underlying pre-mRNAmetabolism in eukaryotes

Enantioselective palladium-catalyzed hydrosilylation of styrene: Detailed reaction mechanism from first-principles and hybrid QM/MM molecular dynamics simulations

The mechanism of the enantioselective hydrosilylation of styrene catalyzed by Pd-0 species generated in situ from dichloro {1-{(R)-1-[(S)-2(diphenylphosphino-kappaP)ferrocenyl]ethyl}-3-trimethylphenyl-5-1H-pyrazole-kappaN}palladium, 1, has been investigated in detail through ab initio molecular dynamics and hybrid ab initio molecular dynamics/molecular mechanics (QM/MM) calculations. Different QM/MM models have been adopted in order to probe the specific steric and electronic contributions of different substituents. The catalytic cycle is initiated by the formation of a weakly bound pi-complex (DeltaE approximate to -5.4 kcal/mol) under simultaneous detachment of the pyrazole ligand. In agreement with a Chalk-Harrod mechanism, this is followed by the migratory insertion of the hydride, which leads to a eta(3)-coordination mode of the benzylic fragment. The significant stabilization of the allylic intermediate (DeltaE approximate to -11 kcal/mol) is responsible for the high regioselectivity of the reaction (as well as for its enantioselectivity). The rate-determining step with an activation barrier of 16 kcal/mol is the migration of the silyl ligand to the a-carbon of the substrate with concomitant closure of the ligand chelate ring. This step leads to the formation of an intermediate in which the phenyl moiety of the product remains coordinated in an eta(2)-mode to the palladium. The addition of trichlorosilane leads to product formation and hence to the regeneration of the catalyst. A unimolecular reaction pathway on the other hand, in which the transfer of the silyl ligand to the benzylic fragment is concerted with the addition of a molecule of HSiCl3 to the catalyst, is disfavored by an activation barrier of similar to30 kcal/mol

Metadynamics Simulations Reveal a Na+ Independent Exiting Path of Galactose for the Inward-Facing Conformation of vSGLT

Sodium-Galactose Transporter (SGLT) is a secondary active symporter which accumulates sugars into cells by using the electrochemical gradient of Na+ across the membrane. Previous computational studies provided insights into the release process of the two ligands (galactose and sodium ion) into the cytoplasm from the inward-facing conformation of Vibrio parahaemolyticus sodium/galactose transporter (vSGLT). Several aspects of the transport mechanism of this symporter remain to be clarified: (i) a detailed kinetic and thermodynamic characterization of the exit path of the two ligands is still lacking; (ii) contradictory conclusions have been drawn concerning the gating role of Y263; (iii) the role of Na+ in modulating the release path of galactose is not clear. In this work, we use bias-exchange metadynamics simulations to characterize the free energy profile of the galactose and Na+ release processes toward the intracellular side. Surprisingly, we find that the exit of Na+ and galactose is non-concerted as the cooperativity between the two ligands is associated to a transition that is not rate limiting. The dissociation barriers are of the order of 11-12 kcal/mol for both the ion and the substrate, in line with kinetic information concerning this type of transporters. On the basis of these results we propose a branched six-state alternating access mechanism, which may be shared also by other members of the LeuT-fold transporters

Unusual Ar-H/Rh-H J(HH) NMR coupling in complexes of rhodium(III): experimental evidence and theoretical support for an eta1-arene structure

The synthesis and structural properties of three new hydridorhodium(III) complexes are reported. Hydrogenolysis of the cyclometalated rhodium dichloride complexes [RhCl2{(S,S)-benbox(Me-2)}] (2a-c) leads to formation of the new complexes [RhCl2(H){(S,S)-ip-benbox(Me-2)H}] (3a-c) in 45% to 85% yield. Compounds 3a-c were found to have unusual features by NMR spectroscopy: in particular, downfield shifted aryl proton resonances (8.88-9.03 ppm) that were coupled to the rhodium hydride resonances. Using X-ray crystallographic studies, a variety of solid- and solution-state characterization techniques, and DFT calculations, these features were attributed to the presence of weak pi-type eta(1)-arene interactions in 3a-c

Exploring the Anticancer Activity of Tamoxifen-Based Metal Complexes Targeting Mitochondria

Two new 'hybrid' metallodrugs of Au(III)(AuTAML)and Cu(II) (CuTAML) were designed featuring a tamoxifen-derived pharmacophoreto ideally synergize the anticancer activity of both the metal centerand the organic ligand. The compounds have antiproliferative effectsagainst human MCF-7 and MDA-MB 231 breast cancer cells. Moleculardynamics studies suggest that the compounds retain the binding activityto estrogen receptor (ER & alpha;). In vitro and in silico studies showed that the Au(III) derivative isan inhibitor of the seleno-enzyme thioredoxin reductase, while theCu(II) complex may act as an oxidant of different intracellular thiols.In breast cancer cells treated with the compounds, a redox imbalancecharacterized by a decrease in total thiols and increased reactiveoxygen species production was detected. Despite their different reactivitiesand cytotoxic potencies, a great capacity of the metal complexes toinduce mitochondrial damage was observed as shown by their effectson mitochondrial respiration, membrane potential, and morphology