Computational Prediction and Experimental Validation of the Unique Molecular Mode of Action of Scoulerine.

Scoulerine is a natural compound that is known to bind to tubulin and has anti-mitotic properties demonstrated in various cancer cells. Its molecular mode of action has not been precisely known. In this work, we perform computational prediction and experimental validation of the mode of action of scoulerine. Based on the existing data in the Protein Data Bank (PDB) and using homology modeling, we create human tubulin structures corresponding to both free tubulin dimers and tubulin in a microtubule. We then perform docking of the optimized structure of scoulerine and find the highest affinity binding sites located in both the free tubulin and in a microtubule. We conclude that binding in the vicinity of the colchicine binding site and near the laulimalide binding site are the most likely locations for scoulerine interacting with tubulin. Thermophoresis assays using scoulerine and tubulin in both free and polymerized form confirm these computational predictions. We conclude that scoulerine exhibits a unique property of a dual mode of action with both microtubule stabilization and tubulin polymerization inhibition, both of which have similar affinity values.

The Effects of Ring Strain on Cyclic Tetraaryl[5]cumulenes.

Cyclic tetraaryl[5]cumulenes (1 a-f) have been synthesized and studied as a function of increasing ring strain. The magnitude of ring strain is approximated by the extent of bending of the cumulenic core as assessed by a combination of X-ray crystallographic analysis and DFT calculations. Trends are observed in 13 C NMR, UV-vis, and Raman spectra associated with ring strain, but the effects are small. In particular, the experimental HOMO-LUMO gap is not appreciably affected by bending of the [5]cumulene framework from ca. 174° (λmax =504 nm) in 1 a to ca. 178° (λmax =494 nm) in 1 f.

Mechanochemical synthesis of 0D and 3D cesium lead mixed halide perovskites.

A simplified mechanochemical synthesis approach for Cs-containing mixed halide perovskite materials of lower and higher dimensionality (0D and 3D, respectively) is presented with stoichiometric control from their halide salts, CsX and PbX2 (X = Cl, Br, I). Excellent optical bandgap tunability through halide substitution is supported by property measurements and changes to the materials' structure. Complementary NMR and XRD methods, along with support from DFT calculations, reveal highly crystalline 0D and 3D solid solutions with a complex arrangement of [PbX6-xXx']4- pseudooctahedra caused by halide distribution about the Pb centre.

Multireference Methods for Calculating the Dissociation Enthalpy of Tetrahedral P4 to Two P2.

The potential energy surface for the thermal decomposition of P4 → 2P2 was computed along the C2 v reaction trajectory. Single-reference methods were not suitable for describing this complex bond-breaking process, so two multiconfigurational methods, namely, multistate complete active space second-order perturbation theory (MS-CASPT2) and multiconfiguration pair-density functional theory (MC-PDFT), were used with the aim of determining the accuracy and efficiency of these methods for this process. Several active spaces and basis sets were explored. It was found that the multiconfiguration pair-density functional theory method was up to 900 times faster than multistate complete active space second-order perturbation theory while providing similar accuracy.

Explaining the Microtubule Energy Balance: Contributions Due to Dipole Moments, Charges, van der Waals and Solvation Energy.

Microtubules are the main components of mitotic spindles, and are the pillars of the cellular cytoskeleton. They perform most of their cellular functions by virtue of their unique dynamic instability processes which alternate between polymerization and depolymerization phases. This in turn is driven by a precise balance between attraction and repulsion forces between the constituents of microtubules (MTs)-tubulin dimers. Therefore, it is critically important to know what contributions result in a balance of the interaction energy among tubulin dimers that make up microtubules and what interactions may tip this balance toward or away from a stable polymerized state of tubulin. In this paper, we calculate the dipole-dipole interaction energy between tubulin dimers in a microtubule as part of the various contributions to the energy balance. We also compare the remaining contributions to the interaction energies between tubulin dimers and establish a balance between stabilizing and destabilizing components, including the van der Waals, electrostatic, and solvent-accessible surface area energies. The energy balance shows that the GTP-capped tip of the seam at the plus end of microtubules is stabilized only by - 9 kcal/mol, which can be completely reversed by the hydrolysis of a single GTP molecule, which releases + 14 kcal/mol and destabilizes the seam by an excess of + 5 kcal/mol. This triggers the breakdown of microtubules and initiates a disassembly phase which is aptly called a catastrophe.

Anesthetic Alterations of Collective Terahertz Oscillations in Tubulin Correlate with Clinical Potency: Implications for Anesthetic Action and Post-Operative Cognitive Dysfunction.

Anesthesia blocks consciousness and memory while sparing non-conscious brain activities. While the exact mechanisms of anesthetic action are unknown, the Meyer-Overton correlation provides a link between anesthetic potency and solubility in a lipid-like, non-polar medium. Anesthetic action is also related to an anesthetic's hydrophobicity, permanent dipole, and polarizability, and is accepted to occur in lipid-like, non-polar regions within brain proteins. Generally the protein target for anesthetics is assumed to be neuronal membrane receptors and ion channels, however new evidence points to critical effects on intra-neuronal microtubules, a target of interest due to their potential role in post-operative cognitive dysfunction (POCD). Here we use binding site predictions on tubulin, the protein subunit of microtubules, with molecular docking simulations, quantum chemistry calculations, and theoretical modeling of collective dipole interactions in tubulin to investigate the effect of a group of gases including anesthetics, non-anesthetics, and anesthetic/convulsants on tubulin dynamics. We found that these gases alter collective terahertz dipole oscillations in a manner that is correlated with their anesthetic potency. Understanding anesthetic action may help reveal brain mechanisms underlying consciousness, and minimize POCD in the choice and development of anesthetics used during surgeries for patients suffering from neurodegenerative conditions with compromised cytoskeletal microtubules.

Engaging dual donor sites within an N-heterocyclic olefin phosphine ligand.

By investigating the coordination chemistry of a neutral N-heterocyclic olefin phosphine ligand, a new digold(i) chloride complex was discovered, demonstrating that mixed element (P/C) donor sites can be accessed at the same time. However attempts to extend this strategy for the preparation of heterobimetallic complexes featuring copper(i) and gold(i) centers with this mixed donor ligand were unsuccessful. The related monometallic copper(i) and gold(i) iodide complexes were discovered to be emissive in the solid state.

Antitumor Activity of Lankacidin Group Antibiotics Is Due to Microtubule Stabilization via a Paclitaxel-like Mechanism.

Lankacidin group antibiotics show strong antimicrobial activity against various Gram-positive bacteria. In addition, they were shown to have considerable antitumor activity against certain cell line models. For decades, the antitumor activity of lankacidin was associated with the mechanism of its antimicrobial action, which is interference with peptide bond formation during protein synthesis. This, however, was never confirmed experimentally. Due to significant similarity to paclitaxel-like hits in a previous computational virtual screening study, we suggested that the cytotoxic effect of lankacidin is due to a paclitaxel-like action. In this study, we tested this hypothesis computationally and experimentally and confirmed that lankacidin is a microtubule stabilizer that enhances tubulin assembly and displaces taxoids from their binding site. This study serves as a starting point for optimization of lankacidin derivatives for better antitumor activities. It also highlights the power of computational predictions and their aid in guiding experiments and formulating rigorous hypotheses.

Analysis of the binding mode of laulimalide to microtubules: Establishing a laulimalide-tubulin pharmacophore.

Laulimalide (LA) is a microtubule-stabilizing agent, currently in preclinical studies. However, studying the binding of this species and successfully synthesizing potent analogues have been challenging. The LA binding site is located between tubulin protofilaments, and therefore LA is in contact with two adjacent [Formula: see text]-tubulin units. Here, an improved model of the binding mode of LA in microtubules is presented, using the newly available crystal structure pose and an extended tubulin heterodimer complex, as well as molecular dynamics simulations. With this model, a series of LA analogues developed by Mooberry and coworkers are also analyzed in order to establish important pharmacophores in LA binding and cytotoxicity. In the side chain, [Formula: see text]-[Formula: see text] interactions are important contributors to LA binding, as are water-mediated hydrogen bonds. An intramolecular hydrogen bond is correlated with high cytotoxicity, and is dependent on macrocycle conformation. Therefore, while the epoxide and olefin groups in the macrocycle do not engage in specific interactions with the protein, they are essential contributions to an active macrocycle conformation, and therefore potency. Calculations reveal that a balance in binding affinity is important for LA activity, where the more potent compounds have larger interactions with the adjacent tubulin unit than the less-active analogs. Several modifications are suggested for the rational design of LA analogues that should not disrupt the active macrocycle conformation.

Detailed Per-residue Energetic Analysis Explains the Driving Force for Microtubule Disassembly.

Microtubules are long filamentous hollow cylinders whose surfaces form lattice structures of αß-tubulin heterodimers. They perform multiple physiological roles in eukaryotic cells and are targets for therapeutic interventions. In our study, we carried out all-atom molecular dynamics simulations for arbitrarily long microtubules that have either GDP or GTP molecules in the E-site of ß-tubulin. A detailed energy balance of the MM/GBSA inter-dimer interaction energy per residue contributing to the overall lateral and longitudinal structural stability was performed. The obtained results identified the key residues and tubulin domains according to their energetic contributions. They also identified the molecular forces that drive microtubule disassembly. At the tip of the plus end of the microtubule, the uneven distribution of longitudinal interaction energies within a protofilament generates a torque that bends tubulin outwardly with respect to the cylinder's axis causing disassembly. In the presence of GTP, this torque is opposed by lateral interactions that prevent outward curling, thus stabilizing the whole microtubule. Once GTP hydrolysis reaches the tip of the microtubule (lateral cap), lateral interactions become much weaker, allowing tubulin dimers to bend outwards, causing disassembly. The role of magnesium in the process of outward curling has also been demonstrated. This study also showed that the microtubule seam is the most energetically labile inter-dimer interface and could serve as a trigger point for disassembly. Based on a detailed balance of the energetic contributions per amino acid residue in the microtubule, numerous other analyses could be performed to give additional insights into the properties of microtubule dynamic instability.

Inactivation of Protein Tyrosine Phosphatases by Peracids Correlates with the Hydrocarbon Chain Length.

BACKGROUND/AIMS: Protein tyrosine phosphatases are crucial enzymes controlling numerous physiological and pathophysiological events and can be regulated by oxidation of the catalytic domain cysteine residue. Peracids are highly oxidizing compounds, and thus may induce inactivation of PTPs. The aim of the present study was to evaluate the inhibitory effect of peracids with different length of hydrocarbon chain on the activity of selected PTPs. METHODS: The enzymatic activity of human CD45, PTP1B, LAR, bacterial YopH was assayed under the cell-free conditions, and activity of cellular CD45 in human Jurkat cell lysates. The molecular docking and molecular dynamics were performed to evaluate the peracids binding to the CD45 active site. RESULTS: Here we demonstrate that peracids reduce enzymatic activity of recombinant CD45, PTP1B, LAR, YopH and cellular CD45. Our studies indicate that peracids are more potent inhibitors of CD45 than hydrogen peroxide (with an IC50 value equal to 25 nM for peroctanoic acid and 8 µM for hydrogen peroxide). The experimental data show that the inactivation caused by peracids is dependent on hydrocarbon chain length of peracids with maximum inhibitory effect of medium-chain peracids (C8-C12 acyl chain), which correlates with calculated binding affinities to the CD45 active site. CONCLUSION: Peracids are potent inhibitors of PTPs with the strongest inhibitory effect observed for medium-chain peracids.

Designing and Testing of Novel Taxanes to Probe the Highly Complex Mechanisms by Which Taxanes Bind to Microtubules and Cause Cytotoxicity to Cancer Cells.

Our previous work identified an intermediate binding site for taxanes in the microtubule nanopore. The goal of this study was to test derivatives of paclitaxel designed to bind to this intermediate site differentially depending on the isotype of ß-tubulin. Since ß-tubulin isotypes have tissue-dependent expression--specifically, the ßIII isotype is very abundant in aggressive tumors and much less common in normal tissues--this is expected to lead to tubulin targeted drugs that are more efficacious and have less side effects. Seven derivatives of paclitaxel were designed and four of these were amenable for synthesis in sufficient purity and yield for further testing in breast cancer model cell lines. None of the derivatives studied were superior to currently used taxanes, however computer simulations provided insights into the activity of the derivatives. Our results suggest that neither binding to the intermediate binding site nor the final binding site is sufficient to explain the activities of the derivative taxanes studied. These findings highlight the need to iteratively improve on the design of taxanes based on their activity in model systems. Knowledge gained on the ability of the engineered drugs to bind to targets and bring about activity in a predictable manner is a step towards personalizing therapies.

Elucidating the mechanism of action of the clinically approved taxanes: a comprehensive comparison of local and allosteric effects.

The clinically approved taxanes (paclitaxel, docetaxel and cabazitaxel) target the tubulin protein in microtubules. Despite the clinical success of these agents, the mechanism of action of this class of drugs remains elusive, making rational design of taxanes difficult. Molecular dynamics simulations of these three taxanes with the αß-tubulin heterodimer examine the similarities and differences in the effects of the drugs on tubulin, probing both local and allosteric effects. Despite their structural similarity, the drugs adopt different conformations in the binding site on ß-tubulin. The taxanes similarly increase the helical character of α- and ß-tubulins. No correlations are found between microtubule assembly and (i) binding affinity or (ii) the role of the M-loop in enhancing lateral contacts. Instead, changes in intra- and interdimer longitudinal contacts are indicative of the mechanism of action of the taxanes. We find ß:H1-S1', and more importantly ß:H9 and ß:H10, play a role translating the effect of local drug binding in ß-tubulin to an allosteric effect in α-tubulin and propose that the displacement of these secondary structures towards α-tubulin may be used as a predictor of the effect of taxanes on the tubulin heterodimers in rational drug design approaches.

Anesthetics act in quantum channels in brain microtubules to prevent consciousness.

The mechanism by which anesthetic gases selectively prevent consciousness and memory (sparing non-conscious brain functions) remains unknown. At the turn of the 20(th) century Meyer and Overton showed that potency of structurally dissimilar anesthetic gas molecules correlated precisely over many orders of magnitude with one factor, solubility in a non-polar, 'hydrophobic' medium akin to olive oil. In the 1980s Franks and Lieb showed anesthetics acted in such a medium within proteins, suggesting post-synaptic membrane receptors. But anesthetic studies on such proteins yielded only confusing results. In recent years Eckenhoff and colleagues have found anesthetic action in microtubules, cytoskeletal polymers of the protein tubulin inside brain neurons. 'Quantum mobility' in microtubules has been proposed to mediate consciousness. Through molecular modeling we have previously shown: (1) olive oil-like non-polar, hydrophobic quantum mobility pathways ('quantum channels') of tryptophan rings in tubulin, (2) binding of anesthetic gas molecules in these channels, and (3) capabilities for π-electron resonant energy transfer, or exciton hopping, among tryptophan aromatic rings in quantum channels, similar to photosynthesis protein quantum coherence. Here, we show anesthetic molecules can impair π-resonance energy transfer and exciton hopping in tubulin quantum channels, and thus account for selective action of anesthetics on consciousness and memory.

The Unique Binding Mode of Laulimalide to Two Tubulin Protofilaments.

Laulimalide, a cancer chemotherapeutic in preclinical development, has a unique binding site located on two adjacent ß-tubulin units between tubulin protofilaments of a microtubule. Our extended protein model more accurately mimics the microtubule environment, and together with a 135 ns molecular dynamics simulation, identifies a new binding mode for laulimalide, which differs from the modes presented in work using smaller protein models. The new laulimalide-residue interactions that are computationally revealed explain the contacts observed via independent mass shift perturbation experiments. The inclusion of explicit solvent shows that many laulimalide-tubulin interactions are water mediated. The new contacts between the drug and the microtubule structure not only improve our understanding of laulimalide binding but also will be essential for efficient derivatization and optimization of this prospective cancer chemotherapy agent. Observed changes in secondary protein structure implicate the S7-H9 loop (M-loop) and H1'-S2 loop in the mechanism by which laulimalide stabilizes microtubules to exert its cytotoxic effects.

Mathematical and computational modeling in biology at multiple scales.

A variety of topics are reviewed in the area of mathematical and computational modeling in biology, covering the range of scales from populations of organisms to electrons in atoms. The use of maximum entropy as an inference tool in the fields of biology and drug discovery is discussed. Mathematical and computational methods and models in the areas of epidemiology, cell physiology and cancer are surveyed. The technique of molecular dynamics is covered, with special attention to force fields for protein simulations and methods for the calculation of solvation free energies. The utility of quantum mechanical methods in biophysical and biochemical modeling is explored. The field of computational enzymology is examined.

Analysis of the strength of interfacial hydrogen bonds between tubulin dimers using quantum theory of atoms in molecules.

Microtubules are key structural elements that, among numerous biological functions, maintain the cytoskeleton of the cell and have a major role in cell division, which makes them important cancer chemotherapy targets. Understanding the energy balance that brings tubulin dimers, the building blocks of microtubules, together to form a microtubule is especially important for revealing the mechanism of their dynamic instability. Several studies have been conducted to estimate various contributions to the free energy of microtubule formation. However, the hydrogen-bond contribution was not studied before as a separate component. In this work, we use concepts such as the quantum theory of atoms in molecules to estimate the per-residue strength of hydrogen bonds contributing to the overall stability that brings subunits together in pair of tubulin heterodimers, across both the longitudinal and lateral interfaces. Our study shows that hydrogen bonding plays a major role in the stability of tubulin systems. Several residues that are crucial to the binding of vinca alkaloids are shown to be strongly involved in longitudinal microtubule stabilization. This indicates a direct relation between the binding of these agents and the effect on the interfacial hydrogen-bonding network, and explains the mechanism of their action. Lateral contacts showed much higher stability than longitudinal ones (-462 ± 70 vs. -392 ± 59 kJ/mol), which suggests a dramatic lateral stabilization effect of the GTP cap in the ß-subunit. The role of the M-loop in lateral stability in absence of taxol was shown to be minor. The B-lattice lateral hydrogen bonds are shown to be comparable in strength to the A-lattice ones (-462 ± 70 vs. -472 ± 46 kJ/mol). These findings establish the importance of hydrogen bonds to the stability of tubulin systems.

Similarity-based virtual screening for microtubule stabilizers reveals novel antimitotic scaffold.

Microtubules are among the most studied and best characterized cancer targets identified to date. Many microtubule stabilizers have been introduced so far that work by disrupting the dynamic instability of microtubules causing mitotic block and apoptosis. However, most of these molecules, especially taxol and epothilone, suffer absorption, toxicity and/or resistance problems. Here we employ a novel similarity-based virtual screening approach in the hope of finding other microtubule stabilizers that perform better and have lower toxicity and resistance. Epothilones, discodermolide, eleutherobin and sarcodictyin A have been found to compete with taxanes for the ß-tubulin binding site, which suggests common chemical features qualifying for that. Our approach was based on similarity screening against all these compounds and other microtubule stabilizers, followed by virtual screening against the taxol binding site. Some novel hits were found, together with a novel highly rigid molecular scaffold. After visual manipulations, redocking and rescoring of this novel scaffold, its affinity dramatically increased in a promising trend, which qualifies for biological testing.

Natural Spinors Reveal How the Spin-Orbit Coupling Affects the Jahn-Teller Distortions in the Hexafluorotungstate(V) Anion.

We investigate the Jahn-Teller distortions in the hexafluorotungstate(V) anion (WF6(-)) by applying the recently developed concept of natural spinors (spin-orbitals) and show that they are a very powerful tool providing simple and clear pictorial explanation for the spin-orbit effect in determining the structure of the anion. The calculations are performed at the levels of spin-orbit configuration interaction and multiconfigurational quasi-degenerate perturbation theory. The hexafluorotungstate(V) anion represents a very rare example of spin-orbit coupling enhancing the Jahn-Teller distortion, and the natural spinor analysis gives a clear interpretation of this enhancement. Advantages of using the natural spinors are explored and explained in detail in this case study.

Two-component natural spinors from two-step spin-orbit coupled wave functions.

We developed an algorithm to obtain the natural orbitals (natural spinors) from the two-step spin-orbit coupled wave functions. These natural spinors are generally complex-valued, mixing two spin components, and they can have similar symmetry properties as the j-j spinors from the one-step spin-orbit coupling calculations, if the reduced density equally averages all the components of a multi-dimensional irreducible representation. Therefore, the natural spinors can serve as an approximation to the j-j spinors and any wave function analysis based on the j-j spinors can also be performed based on them. The comparison between the natural spinors and the j-j spinors of three representative atoms, Tl, At, and Lu, shows their close similarity and demonstrates the ability of the natural spinors to approximate the j-j spinors.