Rational prioritization strategy allows the design of macrolide derivatives that overcome antibiotic resistance.

Antibiotic resistance is a major threat to global health; this problem can be addressed by the development of new antibacterial agents to keep pace with the evolutionary adaptation of pathogens. Computational approaches are essential tools to this end since their application enables fast and early strategical decisions in the drug development process. We present a rational design approach, in which acylide antibiotics were screened based on computational predictions of solubility, membrane permeability, and binding affinity toward the ribosome. To assess our design strategy, we tested all candidates for in vitro inhibitory activity and then evaluated them in vivo with several antibiotic-resistant strains to determine minimal inhibitory concentrations. The predicted best candidate is synthetically more accessible, exhibits higher solubility and binding affinity to the ribosome, and is up to 56 times more active against resistant pathogens than telithromycin. Notably, the best compounds designed by us show activity, especially when combined with the membrane-weakening drug colistin, against Acinetobacter baumanii, Pseudomonas aeruginosa, and Escherichia coli, which are the three most critical targets from the priority list of pathogens of the World Health Organization.

Molecular Dynamics Studies on the Bacterial Membrane Pore Formation by Small Molecule Antimicrobial Agents.

Antimicrobial peptides (AMPs) act on the membrane bilayer of pathogens, causing leakage in the membrane and cell death. Amphiphilic kaempferol derivatives possessing basic functional groups show excellent antibacterial activities, which has been proven through experimental techniques. These compounds are known to target negatively charged bacterial membranes. However, the detailed mechanism of action and their structure-activity relationship are not clear. In this work, we reported theoretical investigation on the mechanism of action of two previously reported kaempferol derivatives on a DMPC/DMPG mixed bilayer. Despite the rigid structure of the compounds when compared to AMPs, spontaneous pore formation in the membrane was not observed in 400 ns molecular dynamics (MD) simulations. MD simulations with biasing forces resulted in the formation of pores in the bilayer for the derivatives and not for kaempferol. The stability of the pores was assessed by pore closure timescales in unbiased MD simulations, which was found to be 5.3 and 17.0 ns for 2 and 3, respectively. Free energy change for the permeation into the bilayer for kaempferol (1), tertiary amine derivative (2), and arginine derivative (3) was calculated to be -1.5, -48.2, and -100.3 kJ/mol, respectively, which correlate with their antibacterial activity. Furthermore, our results indicate that compound 3 forms a stable toroidal pore in the membrane when multiple molecules are oriented in a transmembrane configuration. Our work sheds light on the mechanism of action of small molecule antimicrobial agents, which can be exploited for the rational design of drug candidates.

Solvent-Enhanced Conformational Flexibility of Cyclic Tetrapeptides.

Solvent and temperature can affect the structural properties of cyclic peptides by controlling their flexibility. Here, we investigate two cyclic peptides, featuring beta turns. Using temperature-dependent NMR and FT-IR, we observed a pronounced temperature effect on the conformation of the cyclic peptide D-1 in CHCl3 but a much smaller effect in CH3 CN. Almost no effect was observed for its diastereomer L-1 within a similar temperature range and using the same solvents. With the aid of Replica Exchange Molecular Dynamics simulations and Quantum Mechanics/Molecular Mechanics calculations, we were able to explain this behavior based on the increased flexibility of D-1 (in CHCl3 ) in terms of intramolecular hydrogen bonding. The largest temperature dependence is observed for D-1 in CHCl3 , while the temperature effect is less pronounced for L-1 in CHCl3 and for both peptides in CH3 CN. This work provides new insights into the role of the environment and temperature on the conformations of cyclic peptides.

How Protic Solvents Determine the Reaction Mechanisms of Diphenylcarbene in Solution.

We investigate the effects of small admixtures of protic solvent molecules, such as water and alcohols, on the ultrafast dynamics of diphenylcarbene in acetonitrile at room temperature. Broadband transient absorption measurements and quantum mechanics/molecular mechanics molecular dynamics simulations allow elucidating the dominant reaction mechanism of an intermediate hydrogen-bonded complex between singlet diphenylcarbene and a protic solvent molecule, thus competing with intersystem crossing. Analysis of the data indicates that complex formation is a diffusion-controlled process with orientational requirements. The reaction path involving a benzhydryl cation is less likely in neat bulkier alcohols, as it requires the interaction of the carbene with a protic solvent molecule being part of a hydrogen-bonded network. The simulations indicate a further reaction path toward O-H insertion and two side reactions depending on the involved protic solvent species. Thus, we established that not only the number but also the chemical nature of the protic solvent molecule determine which reaction path is pursued.

A Multiperspective Approach to Solvent Regulation of Enzymatic Activity: HMG-CoA Reductase.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was investigated in different organic cosolvents by means of kinetic and calorimetric measurements, molecular dynamics simulations, and small-angle X-ray scattering. The combined experimental and theoretical techniques were essential to complement each other's limitations in the investigation of the complex interaction pattern between the enzyme, different solvent types, and concentrations. In this way, the underlying mechanisms for the loss of enzyme activity in different water-miscible solvents could be elucidated. These include direct inhibitory effects onto the active center and structural distortions.

The Interaction Modes of Haloimidazolium Salts in Solution.

We performed a comparative study on the interaction modes of 2-haloimidazolium salts with anions in solution, particularly with regard to halogen bonding, hydrogen bonding and anion-π interactions. The syntheses and solid-state analyses of a series of sterically and electronically modified 2-haloimidazolium structures are presented. Detailed isothermal titration calorimetry (ITC) measurements, quantum mechanics/molecular mechanics (QM/MM), classical molecular dynamics simulations (MD) and free-energy calculations together with NMR spectroscopy were used to elucidate the binding modes in solution. Our work reveals the absence of a potential anion-π interaction between the cationic imidazolium ring and the Lewis basic counteranion, and corroborates a formation of halogen bonding via the Lewis acidic iodine moiety and hydrogen bonding via the backbone hydrogen atoms, with repercussions in the field of organocatalysis.

Switching the Spin State of Diphenylcarbene via Halogen Bonding.

The interactions between diphenylcarbene DPC and the halogen bond donors CF3I and CF3Br were investigated using matrix isolation spectroscopy (IR, UV-vis, and EPR) in combination with QM and QM/MM calculations. Both halogen bond donors CF3X form very strong complexes with the singlet state of DPC, but only weakly interact with triplet DPC. This results in a switching of the spin state of DPC, the singlet complexes becoming more stable than the triplet complexes. CF3I forms a second complex (type II) with DPC that is thermodynamically slightly more stable. Calculations predict that in this second complex the DPC···I distance is shorter than the F3C···I distance, whereas in the first (type I) complex the DPC···I distance is, as expected, longer. CF3Br only forms the type I complex. Upon irradiation I or Br, respectively, are transferred to the DPC carbene center and radical pairs are formed. Finally, on annealing, the formal C-X insertion product of DPC is observed. Thus, halogen bonding is a powerful new principle to control the spin state of reactive carbenes.

The effect of double (S238F/W159H) mutations on the structure and dynamics of PET degrading enzyme.

Polyethylene terephthalate (PET) is one of the highly produced synthetic polymers worldwide and had acquired attention due to its impact resistance, high clarity, and light weight. PET has become the first choice in making disposable bottles, leading to massive scales of production resulting in very high utilization across various facets of our daily life. Unfortunately, PET accumulates as waste and is highly resistant to biodegradation, thus presenting a serious threat to the ecosystem. Degradation of PET by enzymatic hydrolysis is a promising strategy to depolymerize the PET into its monomers. In recent studies, a plastic-degrading enzyme known as PETase (IsPETase) from the Ideonella sakaiensis has been identified to hydrolyze PET. The wild-type enzyme from Ideonella sp., has been engineered to improve the catalytic activity. While the IsPETase and its variants have been the subject of extensive structural and biochemical studies, the corresponding computational studies to support the improved activity of the mutant enzyme is not fully understood. In this work, we employed all-atom classical molecular dynamics simulations of the wild-type and double mutant IsPETase enzymes to investigate the underlying reason for the improved catalytic activity in the double mutant by means of structure-dynamics-function relationship. Our results show that the engineered mutations reshape the active site structure, volume, and dynamics of the protein loops which is crucial for substrate binding. We also demonstrate that addition of aromatic and hydrogen bond-forming residues near catalytic site improves binding affinity. This work will enable the rational design of mutants for enhanced PET degrading activity.Communicated by Ramaswamy H. Sarma.

Effect of Substituents on the Photodynamic Action of Anthraquinones: EPR, Computational and In Vitro Studies.

Anthraquinone class of compounds possesses a broad spectrum of therapeutic applications. Cancer cell targeting ability, together with photogeneration of reactive oxygen species, renders anthraquinones an interesting class of photosensitizers for photodynamic therapy (PDT). Screening of newer compounds for better singlet oxygen generation is of current interest to improve the practical usability in PDT. In this study, we investigate the photodynamic activity of nine commercially available anthraquinones, using EPR spectroscopy and computational techniques, to identify the role of substituents on singlet oxygen yield. Three anthraquinone derivatives, 1,5-diaminoanthraquinone, 15-dihydroxyanthraquinone and 1,2,7-trihydroxyanthraquinone, showed highest singlet oxygen quantum yield (0.21, 0.18 and 0.15, respectively) relative to Rose Bengal. Time-dependent density functional theory calculations indicate the singlet oxygen quantum yield of anthraquinones inversely correlate well with the excited singlet-triplet (S1-T1) energy gap. Electron-donating substituents present at positions 1, 2 and 5 of anthraquinone seem to reduce the S1-T1 energy gap, facilitating inter-system crossing and the production of singlet oxygen. This would greatly aid in the design of newer anthraquinone-based photosensitizers. This study also highlights the suitability of 1,5-diaminoanthraquinone for PDT applications as demonstrated by in vitro experiments of photoinduced DNA cleavage and photocytotoxicity in Dalton's lymphoma ascites.

Computational modeling and experimental validation of the EPI-X4/CXCR4 complex allows rational design of small peptide antagonists.

EPI-X4, a 16-mer fragment of albumin, is a specific endogenous antagonist and inverse agonist of the CXC-motif-chemokine receptor 4 (CXCR4) and thus a key regulator of CXCR4 function. Accordingly, activity-optimized synthetic derivatives of EPI-X4 are promising leads for the therapy of CXCR4-linked disorders such as cancer or inflammatory diseases. We investigated the binding of EPI-X4 to CXCR4, which so far remained unclear, by means of biomolecular simulations combined with experimental mutagenesis and activity studies. We found that EPI-X4 interacts through its N-terminal residues with CXCR4 and identified its key interaction motifs, explaining receptor antagonization. Using this model, we developed shortened EPI-X4 derivatives (7-mers) with optimized receptor antagonizing properties as new leads for the development of CXCR4 inhibitors. Our work reveals the molecular details and mechanism by which the first endogenous peptide antagonist of CXCR4 interacts with its receptor and provides a foundation for the rational design of improved EPI-X4 derivatives.

An optimized derivative of an endogenous CXCR4 antagonist prevents atopic dermatitis and airway inflammation.

Aberrant CXCR4/CXCL12 signaling is involved in many pathophysiological processes such as cancer and inflammatory diseases. A natural fragment of serum albumin, named EPI-X4, has previously been identified as endogenous peptide antagonist and inverse agonist of CXCR4 and is a promising compound for the development of improved analogues for the therapy of CXCR4-associated diseases. To generate optimized EPI-X4 derivatives we here performed molecular docking analysis to identify key interaction motifs of EPI-X4/CXCR4. Subsequent rational drug design allowed to increase the anti-CXCR4 activity of EPI-X4. The EPI-X4 derivative JM#21 bound CXCR4 and suppressed CXCR4-tropic HIV-1 infection more efficiently than the clinically approved small molecule CXCR4 antagonist AMD3100. EPI-X4 JM#21 did not exert toxic effects in zebrafish embryos and suppressed allergen-induced infiltration of eosinophils and other immune cells into the airways of animals in an asthma mouse model. Moreover, topical administration of the optimized EPI-X4 derivative efficiently prevented inflammation of the skin in a mouse model of atopic dermatitis. Thus, rationally designed EPI-X4 JM#21 is a novel potent antagonist of CXCR4 and the first CXCR4 inhibitor with therapeutic efficacy in atopic dermatitis. Further clinical development of this new class of CXCR4 antagonists for the therapy of atopic dermatitis, asthma and other CXCR4-associated diseases is highly warranted.

Competitive solvent-molecule interactions govern primary processes of diphenylcarbene in solvent mixtures.

Photochemical reactions in solution often proceed via competing reaction pathways comprising intermediates that capture a solvent molecule. A disclosure of the underlying reaction mechanisms is challenging due to the rapid nature of these processes and the intricate identification of how many solvent molecules are involved. Here combining broadband femtosecond transient absorption and quantum mechanics/molecular mechanics simulations, we show for one of the most reactive species, diphenylcarbene, that the decision-maker is not the nearest solvent molecule but its neighbour. The hydrogen bonding dynamics determine which reaction channels are accessible in binary solvent mixtures at room temperature. In-depth analysis of the amount of nascent intermediates corroborates the importance of a hydrogen-bonded complex with a protic solvent molecule, in striking analogy to complexes found at cryogenic temperatures. Our results show that adjacent solvent molecules take the role of key abettors rather than bystanders for the fate of the reactive intermediate.

Hybrid Quantum Mechanics/Molecular Mechanics/Coarse Grained Modeling: A Triple-Resolution Approach for Biomolecular Systems.

We present a hybrid quantum mechanics/molecular mechanics/coarse-grained (QM/MM/CG) multiresolution approach for solvated biomolecular systems. The chemically important active-site region is treated at the QM level. The biomolecular environment is described by an atomistic MM force field, and the solvent is modeled with the CG Martini force field using standard or polarizable (pol-CG) water. Interactions within the QM, MM, and CG regions, and between the QM and MM regions, are treated in the usual manner, whereas the CG-MM and CG-QM interactions are evaluated using the virtual sites approach. The accuracy and efficiency of our implementation is tested for two enzymes, chorismate mutase (CM) and p-hydroxybenzoate hydroxylase (PHBH). In CM, the QM/MM/CG potential energy scans along the reaction coordinate yield reaction energies that are too large, both for the standard and polarizable Martini CG water models, which can be attributed to adverse effects of using large CG water beads. The inclusion of an atomistic MM water layer (10 Å for uncharged CG water and 5 Å for polarizable CG water) around the QM region improves the energy profiles compared to the reference QM/MM calculations. In analogous QM/MM/CG calculations on PHBH, the use of the pol-CG description for the outer water does not affect the stabilization of the highly charged FADHOOH-pOHB transition state compared to the fully atomistic QM/MM calculations. Detailed performance analysis in a glycine-water model system indicates that computation times for QM energy and gradient evaluations at the density functional level are typically reduced by 40-70% for QM/MM/CG relative to fully atomistic QM/MM calculations.

Poly-cyclodextrin and poly-paclitaxel nano-assembly for anticancer therapy.

Effective anticancer therapy can be achieved by designing a targeted drug-delivery system with high stability during circulation and efficient uptake by the target tumour cancer cells. We report here a novel nano-assembled drug-delivery system, formed by multivalent host-guest interactions between a polymer-cyclodextrin conjugate and a polymer-paclitaxel conjugate. The multivalent inclusion complexes confer high stability to the nano-assembly, which efficiently delivers paclitaxel into the targeted cancer cells via both passive and active targeting mechanisms. The ester linkages between paclitaxel and the polymer backbone permit efficient release of paclitaxel within the cell by degradation. This novel targeted nano-assembly exhibits significant antitumour activity in a mouse tumour model. The strategy established in this study also provides knowledge for the development of advanced anticancer drug delivery.

Simple Method for Simulating the Mixture of Atomistic and Coarse-Grained Molecular Systems.

Combining fine-grained (FG) all-atom and coarse-grained (CG) systems in a single simulation in a hybrid manner is of immense interest in recent times, owing to the possibility of overcoming the limitations of both FG simulations as well as CG simulations. The existing methods for combining these two resolutions tend to require heavy parametrizations or sometimes lack in transferability to other systems of interest, and further developments toward such directions are highly required. We report here a simple protocol to combine CG and FG systems in a single simulation, using the standard FG and CG force field models by adopting a series of small proteins as test cases. Our method makes use of virtual sites as reported earlier for relatively simple butane and dialaine systems (Rzepiela et al. Phys. Chem. Chem. Phys. 2011, 13, 10437-10448), to bridge the interaction between FG protein atoms and CG water. We find that the conventional CG model (MARTINI potentials) couples too strongly with the FG model and that it leads to complete unfolding of a test protein within very short time. We find that reducing the Lennard-Jones potential between CG atoms and virtual site atoms stabilizes the secondary and tertiary structures, sometimes almost to a comparable level with the fully atomistic simulations. However, detailed inspection reveals that this reduction is not enough for satisfactory consistency of the hybrid scheme against the FG simulation. As a remedy, we observe that the addition of as small as 4 Å thick position-restrained FG water layer in the hybrid simulation can further improve the structural behaviors in many respects, with its results closely mimicking those of the FG-only simulations. However, free energy landscapes reveal that this agreement with a restrained solvent layer is still accompanied by the overstabilization of the protein native structure, which will likely pose limitations for studying protein dynamics with the scheme. We show various test results that we have tried in optimizing the FG-CG mixing scheme over the course and discuss future prospects as concluding remarks of the present work.

Computational modeling on the recognition of the HRE motif by HIF-1: molecular docking and molecular dynamics studies.

Hypoxia inducible factor-1 (HIF-1) is a bHLH-family transcription factor that controls genes involved in glycolysis, angiogenesis, migration, as well as invasion factors that are important for tumor progression and metastasis. HIF-1, a heterodimer of HIF-1α and HIF-1ß, binds to the hypoxia responsive element (HRE) present in the promoter regions of hypoxia responsive genes, such as vascular endothelial growth factor (VEGF). Neither the structure of free HIF-1 nor that of its complex with HRE is available. Computational modeling of the transcription factor-DNA complex has always been challenging due to their inherent flexibility and large conformational space. The present study aims to model the interaction between the DNA-binding domain of HIF-1 and HRE. Experiments showed that rigid macromolecular docking programs (HEX and GRAMM-X) failed to predict the optimal dimerization of individually modeled HIF-1 subunits. Hence, the HIF-1 heterodimer was modeled based on the phosphate system positive regulatory protein (PHO4) homodimer. The duplex VEGF-DNA segment containing HRE with flanking nucleotides was modeled in the B form and equilibrated via molecular dynamics (MD) simulation. A rigid docking approach was used to predict the crude binding mode of HIF-1 dimer with HRE, in which the putative contacts were found to be present. An MD simulation (5 ns) of the HIF-1-HRE complex in explicit water was performed to account for its flexibility and to optimize its interactions. All of the conserved amino acid residues were found to play roles in the recognition of HRE. The present work, which sheds light on the recognition of HRE by HIF-1, could be beneficial in the design of peptide or small molecule therapeutics that can mimic HIF-1 and bind with the HRE sequence.

Multiple templates-based homology modeling enhances structure quality of AT1 receptor: validation by molecular dynamics and antagonist docking.

We present a comparative account on 3D-structures of human type-1 receptor (AT1) for angiotensin II (AngII), modeled using three different methodologies. AngII activates a wide spectrum of signaling responses via the AT1 receptor that mediates physiological control of blood pressure and diverse pathological actions in cardiovascular, renal, and other cell types. Availability of 3D-model of AT1 receptor would significantly enhance the development of new drugs for cardiovascular diseases. However, templates of AT1 receptor with low sequence similarity increase the complexity in straightforward homology modeling, and hence there is a need to evaluate different modeling methodologies in order to use the models for sensitive applications such as rational drug design. Three models were generated for AT1 receptor by, (1) homology modeling with bovine rhodopsin as template, (2) homology modeling with multiple templates and (3) threading using I-TASSER web server. Molecular dynamics (MD) simulation (15 ns) of models in explicit membrane-water system, Ramachandran plot analysis and molecular docking with antagonists led to the conclusion that multiple template-based homology modeling outweighs other methodologies for AT1 modeling.

Glycine in the conserved motif III modulates the thermostability and oxidative stress resistance of peptide deformylase in Mycobacterium tuberculosis.

Peptide deformylase (PDF) catalyses the removal of the N-formyl group from the nascent polypeptide during protein maturation. The PDF of Mycobacterium tuberculosis H37Rv (MtbPDF), overexpressed and purified from Escherichia coli, was characterized as an iron-containing enzyme with stability towards H(2) O(2) and moderate thermostability. Substitution of two conserved residues (G49 and L107) from MtbPDF with the corresponding residues found in human PDF affected its deformylase activity. Among characterized PDFs, glycine (G151) in motif III instead of conserved aspartate is characteristic of M. tuberculosis. Although the G151D mutation in MtbPDF increased its deformylase activity and thermostability, it also affected enzyme stability towards H(2) O(2) . Molecular dynamics and docking results confirmed improved substrate binding and catalysis for the G151D mutant and the study provides another possible molecular basis for the stability of MtbPDF against oxidizing agents.