Unleashing the Potential of 1,3-Diketone Analogues as Selective LH2 Inhibitors.

Lysyl hydroxylase 2 (LH2) catalyzes the formation of highly stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs), thus promoting lung cancer metastasis through its capacity to modulate specific types of collagen cross-links within the tumor stroma. Using 1 and 2 from our previous high-throughput screening (HTS) as lead probes, we prepared a series of 1,3-diketone analogues, 1-18, and identified 12 and 13 that inhibit LH2 with IC50's of approximately 300 and 500 nM, respectively. Compounds 12 and 13 demonstrate selectivity for LH2 over LH1 and LH3. Quantum mechanics/molecular mechanics (QM/MM) modeling indicates that the selectivity of 12 and 13 may stem from noncovalent interactions like hydrogen bonding between the morpholine/piperazine rings with the LH2-specific Arg661. Treatment of 344SQ WT cells with 13 resulted in a dose-dependent reduction in their migration potential, whereas the compound did not impede the migration of the same cell line with an LH2 knockout (LH2KO).

Computational Investigation of a Series of Small Molecules as Potential Compounds for Lysyl Hydroxylase-2 (LH2) Inhibition.

The catalytic function of lysyl hydroxylase-2 (LH2), a member of the Fe(II)/αKG-dependent oxygenase superfamily, is to catalyze the hydroxylation of lysine to hydroxylysine in collagen, resulting in stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs). Reports show that high amounts of LH2 lead to the accumulation of HLCCs, causing fibrosis and specific types of cancer metastasis. Some members of the Fe(II)/αKG-dependent family have also been reported to have intramolecular O2 tunnels, which aid in transporting one of the required cosubstrates into the active site. While LH2 can be a promising target to combat these diseases, efficacious inhibitors are still lacking. We have used computational simulations to investigate a series of 44 small molecules as lead compounds for LH2 inhibition. Tunneling analyses indicate the existence of several intramolecular tunnels. The lengths of the calculated O2-transporting tunnels in holoenzymes are relatively longer than those in the apoenzyme, suggesting that the ligands may affect the enzyme's structure and possibly block (at least partially) the tunnels. The sequence alignment analysis between LH enzymes from different organisms shows that all of the amino acid residues with the highest occurrence rate in the oxygen tunnels are conserved. Our results suggest that the enolate form of diketone compounds establishes stronger interactions with the Fe(II) in the active site. Branching the enolate compounds with functional groups such as phenyl and pyridinyl enhances the interaction with various residues around the active site. Our results provide information about possible leads for further LH2 inhibition design and development.

Molecular Dynamics Simulations of Protein RNA Complexes by Using an Advanced Electrostatic Model.

Protein-RNA interactions are integral to the biological functions of RNA. It is well recognized that molecular dynamics (MD) simulations of protein-RNA complexes are more challenging than those of each component. The difficulty arises from the strong electrostatic interactions and the delicate balance between various types of physical forces at the interface. Previously, MD simulations of protein-RNA complexes have predominantly employed fixed-charge force fields. Although force field modifications have been developed to address problems identified in the simulations, some protein-RNA structures are still hard to reproduce by simulations. Here, we present MD simulations of two representative protein-RNA complexes using the AMOEBA polarizable force field. The van der Waals parameters were refined to reproduce accurate quantum-mechanical data of base-base and base-amino acid interactions. It was found that the refined parameters produced a more stable hydrogen-bond network in the interface. One of the complexes remained stable during the short simulations, whereas it could quickly break down in previous simulations using fixed-charge force fields. There was reversible breaking and formation of hydrogen bonds that are observed in the crystal structure, which may indicate the difference in solution and crystal structures. While further improvement and validation of the force fields are still needed, this work demonstrates that polarizable force fields are promising for the study of protein-RNA complexes.

Computationally driven discovery of SARS-CoV-2 Mpro inhibitors: from design to experimental validation.

We report a fast-track computationally driven discovery of new SARS-CoV-2 main protease (Mpro) inhibitors whose potency ranges from mM for the initial non-covalent ligands to sub-µM for the final covalent compound (IC50 = 830 ± 50 nM). The project extensively relied on high-resolution all-atom molecular dynamics simulations and absolute binding free energy calculations performed using the polarizable AMOEBA force field. The study is complemented by extensive adaptive sampling simulations that are used to rationalize the different ligand binding poses through the explicit reconstruction of the ligand-protein conformation space. Machine learning predictions are also performed to predict selected compound properties. While simulations extensively use high performance computing to strongly reduce the time-to-solution, they were systematically coupled to nuclear magnetic resonance experiments to drive synthesis and for in vitro characterization of compounds. Such a study highlights the power of in silico strategies that rely on structure-based approaches for drug design and allows the protein conformational multiplicity problem to be addressed. The proposed fluorinated tetrahydroquinolines open routes for further optimization of Mpro inhibitors towards low nM affinities.

Molecular dynamics free energy simulations of ATP:Mg2+ and ADP:Mg2+ using the polarizable force field AMOEBA.

ATPases and GTPases are two important classes of protein that play critical roles in energy transduction, cellular signaling, gene regulation and catalysis. These proteins use cofactors such as nucleoside di and tri-phosphates (NTP, NDP) and can detect the difference between NDP and NTP which then induce different protein conformations. Mechanisms that drive proteins into the NTP or NDP conformation may depend on factors such as ligand structure and how Mg2+ coordinates with the ligand, amino acids in the pocket and water molecules. Here, we have used the advanced electrostatic and polarizable force field AMOEBA and molecular dynamics free energy simulations (MDFE) to examine the various binding mechanisms of ATP:Mg2+ and ADP:Mg2+.We compared the ATP:Mg2+ binding with previous studies using non-polarizable force fields and experimental data on the binding affinity. It was found that the total free energy of binding for ATP:Mg2+ (-7.00 ± 2.13 kcal/mol) is in good agreement with experimental values (-8.6 ± .2 kcal/mol)1. In addition, parameters for relevant protonation states of ATP, ADP, GTP and GDP have been derived. These parameters will allow for researchers to investigate biochemical phenomena involving NTP's and NDP's with greater accuracy than previous studies involving non-polarizable force fields.

Thermodynamics of ion binding and occupancy in potassium channels.

Potassium channels modulate various cellular functions through efficient and selective conduction of K+ ions. The mechanism of ion conduction in potassium channels has recently emerged as a topic of debate. Crystal structures of potassium channels show four K+ ions bound to adjacent binding sites in the selectivity filter, while chemical intuition and molecular modeling suggest that the direct ion contacts are unstable. Molecular dynamics (MD) simulations have been instrumental in the study of conduction and gating mechanisms of ion channels. Based on MD simulations, two hypotheses have been proposed, in which the four-ion configuration is an artifact due to either averaged structures or low temperature in crystallographic experiments. The two hypotheses have been supported or challenged by different experiments. Here, MD simulations with polarizable force fields validated by ab initio calculations were used to investigate the ion binding thermodynamics. Contrary to previous beliefs, the four-ion configuration was predicted to be thermodynamically stable after accounting for the complex electrostatic interactions and dielectric screening. Polarization plays a critical role in the thermodynamic stabilities. As a result, the ion conduction likely operates through a simple single-vacancy and water-free mechanism. The simulations explained crystal structures, ion binding experiments and recent controversial mutagenesis experiments. This work provides a clear view of the mechanism underlying the efficient ion conduction and demonstrates the importance of polarization in ion channel simulations.

Advanced Electrostatic Model for Monovalent Ions Based on Ab Initio Energy Decomposition.

Ions play important roles in the structures and functions of biomolecules. In biomolecular simulations, ions either directly interact with biomolecules or provide an ionic environment that influences electrostatic interactions of solutes. The AMOEBA+ water model has demonstrated significant advancement of the classical force field for describing molecular interactions due to its improvements on the functional forms to account for essential physics. This work expands the applicability of the AMOEBA+ model toward alkali metal (Li, Na, K, Rb, and Cs) and halogen (F, Cl, Br, and I) ions. Various quantum chemical data on ion-ion and ion-water interactions, experimental ion hydration free energies, and lattice energies of salt crystals are used in the parametrization. The final parameters are verified with other properties outside of the parametrization data, including lattice energies of additional salt crystals and ionic activity coefficients in solution. The new model captures a wide range of ion properties from the gas phase to solution phase and crystals. More importantly, AMOEBA+ provides energy components that are consistent with ab initio energy decomposition. Thus, we expect AMOEBA+ to be more general, transferable, and valuable for the interpretation of intermolecular forces in efficient classical simulations.

Tungsten Oxide Nanodots Exhibit Mild Interactions with WW and SH3 Modular Protein Domains.

Tungsten oxide nanodot (WO3-x ) is an active photothermal nanomaterial that has recently been discovered as a promising candidate for tumor theranostics and treatments. However, its potential cytotoxicity remains elusive and needs to be evaluated to assess its biosafety risks. Herein, we investigate the interactions between WO3-x and two ubiquitous protein domains involved in protein-protein interactions, namely, WW and SH3 domains, using atomistic molecular dynamics simulations. Our results show that WO3-x interacts only weakly with the key residues at the putative proline-rich motif (PRM) ligand-binding site of both domains. More importantly, our free energy landscape calculations reveal that the binding strength between WO3-x and WW/SH3 is weaker than that of the native PRM ligand with WW/SH3, implying that WO3-x has a limited inhibitory effect over PRM on both the WW and SH3 domains. These findings suggest that the cytotoxic effects of WO3-x on the key modular protein domains could be very mild, which provides new insights for the future potential biomedical applications of this nanomaterial.

Molecular Dynamics Study of the Hybridization between RNA and Modified Oligonucleotides.

MicroRNAs (miRNAs) are attractive drug candidates for many diseases as they can modulate the expression of gene networks. Recently, we discovered that DNAs targeting microRNA-22-3p (miR-22-3p) hold the potential for treating obesity and related metabolic disorders (type 2 diabetes mellitus, hyperlipidemia, and nonalcoholic fatty liver disease (NAFLD)) by turning fat-storing white adipocytes into fat-burning adipocytes. In this work, we explored the effects of chemical modifications, including phosphorothioate (PS), locked nucleic acid (LNA), and peptide nucleic acid (PNA), on the structure and energy of DNA analogs by using molecular dynamics (MD) simulations. To achieve a reliable prediction of the hybridization free energy, the AMOEBA polarizable force field and the free energy perturbation technique were employed. The calculated hybridization free energies are generally compatible with previous experiments. For LNA and PNA, the enhanced duplex stability can be explained by the preorganization mechanism, i.e., the single strands adopt stable helical structures similar to those in the duplex. For PS, the S and R isomers (Sp and Rp) have preferences for C2'-endo and C3'-endo sugar puckering conformations, respectively, and therefore Sp is less stable than Rp in DNA/RNA hybrids. In addition, the solvation penalty of Rp accounts for its destabilization effect. PS-LNA is similar to LNA as the sugar puckering is dominated by the locked sugar ring. This work demonstrated that MD simulations with polarizable force fields are useful for the understanding and design of modified nucleic acids.

Computational and Experimental Studies of Inhibitor Design for Aldolase A.

Glycolytic enzyme fructose-bisphosphate aldolase A is an emerging therapeutic target in cancer. Recently, we have solved the crystal structure of murine aldolase in complex with naphthalene-2,6-diyl bisphosphate (ND1) that served as a template of the design of bisphosphate-based inhibitors. In this work, a series of ND1 analogues containing difluoromethylene (-CF2), methylene (-CH2), or aldehyde substitutions were designed. All designed compounds were studied using molecular dynamics (MD) simulations with the AMOEBA force field. Both energetics and structural analyses have been done to understand the calculated binding free energies. The average distances between ligand and protein atoms for ND1 were very similar to those for the ND1 crystal structure, which indicates that our MD simulation is sampling the correct conformation well. CF2 insertion lowers the binding free energy by 10-15 kcal/mol, while CF2 substitution slightly increases the binding free energy, which matches the experimental measurement. In addition, we found that NDB with two CF2 insertions, the strongest binder, is entropically driven, while others including NDA with one CF2 insertion are all enthalpically driven. This work provides insights into the mechanisms underlying protein-phosphate binding and enhances the capability of applying computational and theoretical frameworks to model, predict, and design diagnostic strategies targeting cancer.

Robust Antibacterial Activity of Tungsten Oxide (WO3-x) Nanodots.

Antibacterial agents are an important tool in the prevention of bacterial infections. Inorganic materials are attractive due to their high stability under a variety of conditions compared to organic antibacterial agents. Herein tungsten oxide nanodots (WO3-x), synthesized by a simple one-pot synthetic approach, were found to exhibit strong antibacterial capabilities. The analyses with colony-forming units (CFU) showed an excellent antibacterial activity of WO3-x against both Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) strains. The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images revealed clear damages to the bacterial cell membranes, which was further confirmed by molecular dynamics simulations. Additionally, exposure to simulated sunlight was found to further increase the germicidal activity of WO3-x nanodots, a 30 min exposure to sunlight combined with 50 µg/mL WO3-x nanodots showed a 70% decrease in E. coli viability compared to without exposure. Electron spin resonance spectroscopy (ESR) was used to elucidate the underlying mechanism of this photocatalytic activity through the generation of hydroxyl radical species. The cell counting kit-8 (CCK-8) and the live/dead assay were further employed to evaluate the cytotoxicity of WO3-x nanodots on eukaryotic cells, which demonstrated their general biocompatibility. In summary, our results suggest WO3-x nanodots have considerable potential in antibacterial applications, while also being biocompatible at large.

Polarizable Force Fields for Biomolecular Simulations: Recent Advances and Applications.

Realistic modeling of biomolecular systems requires an accurate treatment of electrostatics, including electronic polarization. Due to recent advances in physical models, simulation algorithms, and computing hardware, biomolecular simulations with advanced force fields at biologically relevant timescales are becoming increasingly promising. These advancements have not only led to new biophysical insights but also afforded opportunities to advance our understanding of fundamental intermolecular forces. This article describes the recent advances and applications, as well as future directions, of polarizable force fields in biomolecular simulations.

Investigating the Association Mechanism between Rafoxanide and Povidone.

The low aqueous solubility of most hydrophobic medications limits their oral absorption. An approach to solve this problem is to make a drug-polymer association. Herein, we investigated the association between rafoxanide (RAF), a surface-active, poorly water-soluble drug, with a commercial hydrophilic polymer povidone. We found that the association is a function of medium composition and could only take place in polar media, such as water. The association is favored by the hydrogen-bond formation between the amide group in RAF and the carbonyl group in povidone. In addition, the association is also favored by the self-association of RAF through π-π interaction between the benzene rings in adjacent RAF molecules. Two-dimensional nuclear magnetic resonance has been applied to investigate the interactions and has confirmed our hypotheses. Geometry optimization confirmed that RAF exists primarily in the antiparallel configuration in the RAF aggregates. This study provides critical information for designing suitable drug-vehicle complexes and engineering the interactions between them to maximize the oral absorption. Our results shed light on drug design and delivery, drug molecule structure-functionality relationship, as well as efficacy enhancement toward interaction engineering.

Many-body effect determines the selectivity for Ca2+ and Mg2+ in proteins.

Calcium ion is a versatile messenger in many cell-signaling processes. To achieve their functions, calcium-binding proteins selectively bind Ca2+ against a background of competing ions such as Mg2+ The high specificity of calcium-binding proteins has been intriguing since Mg2+ has a higher charge density than Ca2+ and is expected to bind more tightly to the carboxylate groups in calcium-binding pockets. Here, we showed that the specificity for Ca2+ is dictated by the many-body polarization effect, which is an energetic cost arising from the dense packing of multiple residues around the metal ion. Since polarization has stronger distance dependence compared with permanent electrostatics, the cost associated with the smaller Mg2+ is much higher than that with Ca2+ and outweighs the electrostatic attraction favorable for Mg2+ With the AMOEBA (atomic multipole optimized energetics for biomolecular simulation) polarizable force field, our simulations captured the relative binding free energy between Ca2+ and Mg2+ for proteins with various types of binding pockets and explained the nonmonotonic size dependence of the binding free energy in EF-hand proteins. Without electronic polarization, the smaller ions are always favored over larger ions and the relative binding free energy is roughly proportional to the net charge of the pocket. The many-body effect depends on both the number and the arrangement of charged residues. Fine-tuning of the ion selectivity could be achieved by combining the many-body effect and geometric constraint.

Elucidating the Phosphate Binding Mode of Phosphate-Binding Protein: The Critical Effect of Buffer Solution.

Phosphate is an essential component of cell functions, and the specific transport of phosphorus into a cell is mediated by phosphate-binding protein (PBP). The mechanism of PBP-phosphate recognition remains controversial: on the basis of similar binding affinities at acidic and basic pHs, it is believed that the hydrogen network in the binding site is flexible to adapt to different protonation states of phosphates. However, only hydrogen (1H) phosphate was observed in the sub-angstrom X-ray structures. To address this inconsistency, we performed molecular dynamics simulations using the AMOEBA polarizable force field. Structural and free energy data from simulations suggested that 1H phosphate was the preferred bound form at both pHs. The binding of dihydrogen (2H) phosphate disrupted the hydrogen-bond network in the PBP pocket, and the computed affinity was much weaker than that of 1H phosphate. Furthermore, we showed that the discrepancy in the studies described above is resolved if the interaction between phosphate and the buffer agent is taken into account. The calculated apparent binding affinities are in excellent agreement with experimental measurements. Our results suggest the high specificity of PBP for 1H phosphate and highlight the importance of the buffer solution for the binding of highly charged ligands.

Tinker-HP: a massively parallel molecular dynamics package for multiscale simulations of large complex systems with advanced point dipole polarizable force fields.

We present Tinker-HP, a massively MPI parallel package dedicated to classical molecular dynamics (MD) and to multiscale simulations, using advanced polarizable force fields (PFF) encompassing distributed multipoles electrostatics. Tinker-HP is an evolution of the popular Tinker package code that conserves its simplicity of use and its reference double precision implementation for CPUs. Grounded on interdisciplinary efforts with applied mathematics, Tinker-HP allows for long polarizable MD simulations on large systems up to millions of atoms. We detail in the paper the newly developed extension of massively parallel 3D spatial decomposition to point dipole polarizable models as well as their coupling to efficient Krylov iterative and non-iterative polarization solvers. The design of the code allows the use of various computer systems ranging from laboratory workstations to modern petascale supercomputers with thousands of cores. Tinker-HP proposes therefore the first high-performance scalable CPU computing environment for the development of next generation point dipole PFFs and for production simulations. Strategies linking Tinker-HP to Quantum Mechanics (QM) in the framework of multiscale polarizable self-consistent QM/MD simulations are also provided. The possibilities, performances and scalability of the software are demonstrated via benchmarks calculations using the polarizable AMOEBA force field on systems ranging from large water boxes of increasing size and ionic liquids to (very) large biosystems encompassing several proteins as well as the complete satellite tobacco mosaic virus and ribosome structures. For small systems, Tinker-HP appears to be competitive with the Tinker-OpenMM GPU implementation of Tinker. As the system size grows, Tinker-HP remains operational thanks to its access to distributed memory and takes advantage of its new algorithmic enabling for stable long timescale polarizable simulations. Overall, a several thousand-fold acceleration over a single-core computation is observed for the largest systems. The extension of the present CPU implementation of Tinker-HP to other computational platforms is discussed.

AMOEBA Polarizable Atomic Multipole Force Field for Nucleic Acids.

The AMOEBA polarizable atomic multipole force field for nucleic acids is presented. Valence and electrostatic parameters were determined from high-level quantum mechanical data, including structures, conformational energy, and electrostatic potentials, of nucleotide model compounds. Previously derived parameters for the phosphate group and nucleobases were incorporated. A total of over 35 µs of condensed-phase molecular dynamics simulations of DNA and RNA molecules in aqueous solution and crystal lattice were performed to validate and refine the force field. The solution and/or crystal structures of DNA B-form duplexes, RNA duplexes, and hairpins were captured with an average root-mean-squared deviation from NMR structures below or around 2.0 Å. Structural details, such as base pairing and stacking, sugar puckering, backbone and χ-torsion angles, groove geometries, and crystal packing interfaces, agreed well with NMR and/or X-ray. The interconversion between A- and B-form DNAs was observed in ethanol-water mixtures at 328 K. Crystal lattices of B- and Z-form DNA and A-form RNA were examined with simulations. For the RNA tetraloop, single strand tetramers, and HIV TAR with 29 residues, the simulated conformational states, 3 J-coupling, nuclear Overhauser effect, and residual dipolar coupling data were compared with NMR results. Starting from a totally unstacked/unfolding state, the rCAAU tetranucleotide was folded into A-form-like structures during ∼1 µs molecular dynamics simulations.

Study of interactions between metal ions and protein model compounds by energy decomposition analyses and the AMOEBA force field.

The interactions between metal ions and proteins are ubiquitous in biology. The selective binding of metal ions has a variety of regulatory functions. Therefore, there is a need to understand the mechanism of protein-ion binding. The interactions involving metal ions are complicated in nature, where short-range charge-penetration, charge transfer, polarization, and many-body effects all contribute significantly, and a quantitative description of all these interactions is lacking. In addition, it is unclear how well current polarizable force fields can capture these energy terms and whether these polarization models are good enough to describe the many-body effects. In this work, two energy decomposition methods, absolutely localized molecular orbitals and symmetry-adapted perturbation theory, were utilized to study the interactions between Mg2+/Ca2+ and model compounds for amino acids. Comparison of individual interaction components revealed that while there are significant charge-penetration and charge-transfer effects in Ca complexes, these effects can be captured by the van der Waals (vdW) term in the AMOEBA force field. The electrostatic interaction in Mg complexes is well described by AMOEBA since the charge penetration is small, but the distance-dependent polarization energy is problematic. Many-body effects were shown to be important for protein-ion binding. In the absence of many-body effects, highly charged binding pockets will be over-stabilized, and the pockets will always favor Mg and thus lose selectivity. Therefore, many-body effects must be incorporated in the force field in order to predict the structure and energetics of metalloproteins. Also, the many-body effects of charge transfer in Ca complexes were found to be non-negligible. The absorption of charge-transfer energy into the additive vdW term was a main source of error for the AMOEBA many-body interaction energies.

Biocompatible and blood-brain barrier permeable carbon dots for inhibition of Aß fibrillation and toxicity, and BACE1 activity.

Amyloid-ß peptide (Aß) fibrillation is pathologically associated with Alzheimer's disease (AD), and this has resulted in the development of an Aß inhibitor which is essential for the treatment of AD. However, the design of potent agents which can target upstream secretases, inhibit Aß toxicity and aggregation, as well as cross the blood-brain barrier remains challenging. In, this research carbon dots for AD treatment were investigated in vitro using experimental and computational methods for the first time. The results presented here demonstrate a novel strategy for the discovery of novel antiamyloidogenic agents for AD treatments.

Calculating binding free energies of host-guest systems using the AMOEBA polarizable force field.

Molecular recognition is of paramount interest in many applications. Here we investigate a series of host-guest systems previously used in the SAMPL4 blind challenge by using molecular simulations and the AMOEBA polarizable force field. The free energy results computed by Bennett's acceptance ratio (BAR) method using the AMOEBA polarizable force field ranked favorably among the entries submitted to the SAMPL4 host-guest competition [Muddana, et al., J. Comput.-Aided Mol. Des., 2014, 28, 305-317]. In this work we conduct an in-depth analysis of the AMOEBA force field host-guest binding thermodynamics by using both BAR and the orthogonal space random walk (OSRW) methods. The binding entropy-enthalpy contributions are analyzed for each host-guest system. For systems of inordinate binding entropy-enthalpy values, we further examine the hydrogen bonding patterns and configurational entropy contribution. The binding mechanism of this series of host-guest systems varies from ligand to ligand, driven by enthalpy and/or entropy changes. Convergence of BAR and OSRW binding free energy methods is discussed. Ultimately, this work illustrates the value of molecular modelling and advanced force fields for the exploration and interpretation of binding thermodynamics.