Unlocking the specificity of antimicrobial peptide interactions for membrane-targeted therapies.

Antimicrobial peptides (AMPs) are increasingly recognized as potent therapeutic agents, with their selective affinity for pathological membranes, low toxicity profile, and minimal resistance development making them particularly attractive in the pharmaceutical landscape. This study offers a comprehensive analysis of the interaction between specific AMPs, including magainin-2, pleurocidin, CM15, LL37, and clavanin, with lipid bilayer models of very different compositions that have been ordinarily used as biological membrane models of healthy mammal, cancerous, and bacterial cells. Employing unbiased molecular dynamics simulations and metadynamics techniques, we have deciphered the intricate mechanisms by which these peptides recognize pathogenic and pathologic lipid patterns and integrate into lipid assemblies. Our findings reveal that the transverse component of the peptide's hydrophobic dipole moment is critical for membrane interaction, decisively influencing the molecule's orientation and expected therapeutic efficacy. Our approach also provides insight on the kinetic and dynamic dependence on the peptide orientation in the axial and azimuthal angles when coming close to the membrane. The aim is to establish a robust framework for the rational design of peptide-based, membrane-targeted therapies, as well as effective quantitative descriptors that can facilitate the automated design of novel AMPs for these therapies using machine learning methods.

Spectroscopic approaches for studies of site-specific DNA base and backbone 'breathing' using exciton-coupled dimer-labeled DNA.

DNA regulation and repair processes require direct interactions between proteins and DNA at specific sites. Local fluctuations of the sugar-phosphate backbones and bases of DNA (a form of DNA 'breathing') play a central role in such processes. Here we review the development and application of novel spectroscopic methods and analyses - both at the ensemble and single-molecule levels - to study structural and dynamic properties of exciton-coupled cyanine and fluorescent nucleobase analogue dimer-labeled DNA constructs at key positions involved in protein-DNA complex assembly and function. The exciton-coupled dimer probes act as 'sensors' of the local conformations adopted by the sugar-phosphate backbones and bases immediately surrounding the dimer probes. These methods can be used to study the mechanisms of protein binding and function at these sites.

Deciphering the molecular choreography of Janus kinase 2 inhibition via Gaussian accelerated molecular dynamics simulations: a dynamic odyssey.

The Janus kinases (JAK) are crucial targets in drug development for several diseases. However, accounting for the impact of possible structural rearrangements on the binding of different kinase inhibitors is complicated by the extensive conformational variability of their catalytic kinase domain (KD). The dynamic KD contains mainly four prominent mobile structural motifs: the phosphate-binding loop (P-loop), the αC-helix within the N-lobe, the Asp-Phe-Gly (DFG) motif, and the activation loop (A-loop) within the C-lobe. These distinct structural orientations imply a complex signal transmission path for regulating the A-loop's flexibility and conformational preference for optimal JAK function. Nevertheless, the precise dynamical features of the JAK induced by different types of inhibitors still remain elusive. We performed comparative, microsecond-long, Gaussian accelerated molecular dynamics simulations in triplicate of three phosphorylated JAK2 systems: the KD alone, type-I ATP-competitive inhibitor (CI) bound KD in the catalytically active DFG-in conformation, and the type-II inhibitor (AI) bound KD in the catalytically inactive DFG-out conformation. Our results indicate significant conformational variations observed in the A-loop and αC helix motions upon inhibitor binding. Our studies also reveal that the DFG-out inactive conformation is characterized by the closed A-loop rearrangement, open catalytic cleft of N and C-lobe, the outward movement of the αC helix, and open P-loop states. Moreover, the outward positioning of the αC helix impacts the hallmark salt bridge formation between Lys882 and Glu898 in an inactive conformation. Finally, we compared their ligand binding poses and free energy by the MM/PBSA approach. The free energy calculations suggested that the AI's binding affinity is higher than CI against JAK2 due to an increased favorable contribution from the total non-polar interactions and the involvement of the αC helix. Overall, our study provides the structural and energetic insights crucial for developing more promising type I/II JAK2 inhibitors for treating JAK-related diseases.

Temperature-Dependent Paracrystalline Nucleation in Atomically Disordered Diamonds.

Atomically disordered diamonds with medium-range order realized in recent experiments extend our knowledge of atomic disorder in materials. However, the current understanding of amorphous carbons cannot answer why paracrystalline diamond (p-D) can be formed inherently different from other tetrahedral amorphous carbons (ta-Cs), and the emergence of p-D seems to be easily hindered by inappropriate temperatures. Herein, we performed atomistic-based simulations to shed light on temperature-dependent paracrystalline nucleation in atomically disordered diamonds. Using metadynamics and two carefully designed collective variables, reversible phase transitions among different ta-Cs can be presented under different temperatures, evidenced by corresponding local minima on the free energy surface and reaction path along the free energy gradient. We found that p-D is preferred in a narrow range of temperatures, which is comparable to real experimental temperatures under the Arrhenius framework. The insights and related methods should open up a perspective for investigating other amorphous carbons.

Insilico molecular modelling to identify PDK-1 targeting agents based on its protein-protein docking interaction.

PDK1, an attractive cancer target that downstreams 23 other kinases towards cell growth, survival and metabolism has gaining attention due to allosteric effect of ligands bound to it. Generally, the drug design strategy using pharmacophores is either a single protein structure or ensemble or ligand-based. Apart from these methods, yet another new approach of protein-protein docking with state of art computational tool like Schrodinger Suite to generate pharmacophores based on the interacting partners of the protein is proposed in this work. The structure-based pharmacophoric features were picked up from docking the ten interacting partners of PDK1 and screened against the Enamine libraries containing protein-protein interacting compound collection, advanced, protein mimetic and allosteric compounds. High throughput virtual screening against the PIF pocket of PDK1 yields an indole scaffold. The identified indole derivative is proposed to be a strong activator that binds in the protein-protein interaction site of PDK1 which was further confirmed by molecular metadynamics simulations, free energy surface analysis and MM-GBSA calculations. Thus, the pharmacophores generated by the interacting proteins for PPI can facilitate the virtual screening in structure-based drug discovery of similar therapeutic targets.Communicated by Ramaswamy H. Sarma.

Insight into the Binding of First- and Second-Generation PET Tracers to 4R and 3R/4R Tau Protofibrils.

Primary supranuclear palsy (PSP) is a rare neurodegenerative disease that perturbs body movement, eye movement, and walking balance. Similar to Alzheimer's disease (AD), the abnormal aggregation of tau fibrils in the central neuronal and glial cells is a major hallmark of PSP disease. In this study, we use multiple approaches, including docking, molecular dynamics, and metadynamics simulations, to investigate the binding mechanism of 10 first- and second-generations of PET tracers for PSP tau and compare their binding in cortical basal degeneration (CBD) and AD tauopathies. Structure-activity relationships, binding preferences, the nature of ligand binding in terms of basic intermolecular interactions, the role of polar/charged residues, induced-fit mechanisms, grove closures, and folding patterns for the binding of these tracers in PSP, CBD, and AD tau fibrils are evaluated and discussed in detail in order to build a holistic picture of what is essential for the binding and also to rank the potency of the different tracers. For example, we found that the same tracer shows different binding preferences for the surface sites of tau fibrils that are intrinsically distinct in the folding patterns. Results from the metadynamics simulations predict that PMPBB3 and PBB3 exhibit the strongest binding free energies onto the Q276[I277]I278, Q351[S352]K353, and N368[K369]K370 sites of PSP than the other explored tracers, indicating a solid preference for vdW and cation-π interactions. Our results also reproduced known preferences of tracers, namely, that MK6240 binds better to AD tau than CBD tau and PSP tau and that CBD2115, PI2620, and PMPBB3 are 4R tau binders. These findings fill in the well-sought-after knowledge gap in terms of these tracers' potential binding mechanisms and will be important for the design of highly selective novel PET tracers for tauopathies.

Concerted versus stepwise proton transfer reactions in the [2, 2'-bipyridyl]-3-3'-diol molecule: A static and dynamic ab-initio investigation.

The double proton transfer (PT) reaction has been investigated in the [2,2'-bipyridyl]-3-3'-diol, a complex molecule where the proton movements is coupled to significant rearrangement of the electronic structure. Moreover, the reaction could be concerted, that is the two protons are exchanged simultaneously, or stepwise, where the two protons are transferred sequentially. To this end, a static exploration of the potential energy surface (PES) was carried together with the analysis of the free-energy surface (FES), both surfaces being evaluated at density functional theory level and different exchange-correlation functionals. While the concerted mechanism has been clearly discharged, the characteristics of the stepwise PT significantly depends on the chosen functionals, some suggesting a clear stepwise mechanism characterized by a stable reaction intermediates and two transitions states, whereas other approaches propend for a asynchronous PT, with a single TS. These features appear on both PES and FES, albeit some differences appears due to their different nature.

Prediction of dual NF-κB/IκB inhibitors using an integrative in-silico approaches.

Multiple lines of evidence indicate that the NF-κB signaling pathway plays a pivotal role in carcinogenesis; activation of NF-κB in cancer increases cell proliferation and suppresses apoptosis, both of which define tumor mass development. Inhibiting NF-κB leads to tumor suppression by blocking the IKK-α/ß enzymes, thus inhibiting its translocation. Furthermore, protecting p65 from acetylation and phosphorylation inhibits NF-κB through its active site. Some small molecules are assumed to inhibit NF-κB and IκB function separately. This study took one of the previously reported NF-κB inhibitors (compound D4) as a promising lead and predicted some dual NF-κB and IκB inhibitors. We performed a virtual screening (VS) workflow on a library with 186,146 compounds with 75% similarity to compound D4 on both NF-κB and IκB proteins. A total of 186 compounds were extracted from three steps of VS 36 were common in both proteins. These compounds were subjected to the quantum polarized ligand docking to elect potent compounds with the highest binding affinity for NF-κB and IκB proteins. The MM-GBSA method calculates the lowest binding free energy for eight selected compounds. These analyses found three top-ranked compounds for each protein with suitable pharmacokinetics properties and higher in-silico inhibitory ability. In the last screening, compound CID_4969 was introduced to a molecular dynamics (MDs) simulation study as a common inhibitor for both proteins. The MDs confirmed the main interactions between the final elected compound and NF-κB/IκB proteins. Consequently, the presented computational approaches could be used for designing promising anti-cancer agents.Communicated by Ramaswamy H. Sarma.

Identifying the Template for Oligomer to Fibril Conversion for Amyloid-ß (1-42) Oligomers using Hamiltonian Replica Exchange Molecular Dynamics.

The toxicity of amyloid-ß (Aß) oligomers has been known to be higher compared to mature fibrils. Yet the presence of plaques in Alzheimer's disease patients indicates the significance of oligomer to fibril conversion for Aß aggregates. In this study, we investigate Aß13-42 oligomers having two to five peptide chains using extensive all-atom molecular dynamics simulations to identify the on- or off-pathway intermediates in fibril formation pathway. Hamiltonian replica exchange method through solute tempering (REST2) has been employed to explore the different structures attained by these aggregates. Using intra-chain and inter-chain contacts as reaction coordinates, we obtain the free energy surface for the Aß13-42 oligomers. Consequently, their stable conformations and structural features have been identified. The found conformations belonging to most probable structures possess both parallel and anti-parallel ß-sheets, characteristic of on- and off-pathway intermediates, respectively. Further, we have measured the tendency to form fibril like interactions among the ß-sheet forming residues. Our analysis finds that residues 30-36 possess higher tendency to form fibril like contacts among all the residues. While we find stronger interaction among residues 30-36, these amino acids are also found to be more shielded from water compared to others. With previous experimental studies finding these residues to be more crucial for the stability of Aß42 oligomers, we propose that interactions within this patch could trigger seed formation that leads to conversion of on-pathway oligomers into disease relevant fibrils.

Investigation of the pH-dependent aggregation mechanisms of GCSF using low resolution protein characterization techniques and advanced molecular dynamics simulations.

Granulocyte-colony stimulating factor (GCSF) is a widely used therapeutic protein to treat neutropenia. GCSF has an increased propensity to aggregate if the pH is increased above 5.0. Although GCSF is very well experimentally characterized, the exact pH-dependent aggregation mechanism of GCSF is still under debate. This study aimed to model the complex pH-dependent aggregation behavior of GCSF using state-of-the-art simulation techniques. The conformational stability of GCSF was investigated by performing metadynamics simulations, while the protein-protein interactions were investigated using coarse-grained (CG) simulations of multiple GCSF monomers. The CG simulations were directly compared with small-angle X-ray (SAXS) data. The metadynamics simulations demonstrated that the orientations of Trp residues in GCSF are dependent on pH. The conformational change of Trp residues is due to the loss of Trp-His interactions at the physiological pH, which in turn may increase protein flexibility. The helical structure of GCSF was not affected by the pH conditions of the simulations. Our CG simulations indicate that at pH 4.0, the colloidal stability may be more important than the conformational stability of GCSF. The electrostatic potential surface and CG simulations suggested that the basic residues are mainly responsible for colloidal stability as deprotonation of these residues causes a reduction of the highly positively charged electrostatic barrier close to the aggregation-prone long loop regions.

Dissecting the Binding Profile of PET Tracers to Corticobasal Degeneration Tau Fibrils.

Alzheimer's disease and primary tauopathies are characterized by the presence of tau pathology in brain. Several tau positron emission tomography (PET) tracers have been developed and studied in Alzheimer's disease (AD), but there is still a lack of 4R-tau specific tracers for non-AD tauopathies. We here present the first computational study on the binding profiles of four tau different PET tracers, PI2620, CBD2115, PM-PBB3, and MK6240, to corticobasal degeneration (CBD) tau. The in silico results showed different preferences for the various binding sites on the 4R fibril, and especially an entry site, a concave site, and a core site showed high binding affinity to these tracers. The core site and entry site both showed higher binding affinity than the surface sites, but the tracers were less likely to enter these sites. PI2620, CBD2115, and PM-PBB3 all showed higher binding affinities to CBD tau than the 3R/4R tracer MK6240. The same strategy has also been applied to AD tau fibrils, and significant differences in selectivity of binding sites were also observed. A higher binding affinity was observed for CBD2115 and PM-PBB3 to AD tau compared to PI2620. None of the studied tracers showed a selectivity for 4R compared to 3R/4R tau. This study clearly shows that identified binding sites from cryo-EM with low resolution can be further refined by metadynamics simulations in order to provide atomic resolution of the binding modes as well as of the thermodynamic properties.

Permeation of ß-Lactamase Inhibitors through the General Porins of Gram-Negative Bacteria.

Modern medicine relies upon antibiotics, but we have arrived to the point where our inability to come up with new effective molecules against resistant pathogens, together with the declining private investment, is resulting in the number of untreatable infections increasing worldwide at worrying pace. Among other pathogens, widely recognized institutions have indicated Gram-negative bacteria as particularly challenging, due to the presence of the outer membrane. The very first step in the action of every antibiotic or adjuvant is the permeation through this membrane, with small hydrophilic drugs usually crossing through protein channels. Thus, a detailed understanding of their properties at a molecular level is crucial. By making use of Molecular Dynamics simulations, we compared the two main porins of four members of the Enterobacteriaceae family, and, in this paper, we show their shared geometrical and electrostatic characteristics. Then, we used metadynamics simulations to reconstruct the free energy for permeation of selected diazobicyclooctans through OmpF. We demonstrate how porins features are coupled to those of the translocating species, modulating their passive permeation. In particular, we show that the minimal projection area of a molecule is a better descriptor than its molecular mass or the volume. Together with the magnitude and orientation of the electric dipole moment, these are the crucial parameters to gain an efficient compensation between the entropic and enthalpic contributions to the free energy barrier required for permeation. Our results confirm the possibility to predict the permeability of molecules through porins by using a few molecular parameters and bolster the general model according to which the free energy increase is mostly due to the decrease of conformational entropy, and this can be compensated by a favorable alignment of the electric dipole with respect to the channel intrinsic electric field.

On the Catalytic Activity of the Engineered Coiled-Coil Heptamer Mimicking the Hydrolase Enzymes: Insights from a Computational Study.

Recently major advances were gained on the designed proteins aimed to generate biomolecular mimics of proteases. Although such enzyme-like catalysts must still suffer refinements for improving the catalytic activity, at the moment, they represent a good example of artificial enzymes to be tested in different fields. Herein, a de novo designed homo-heptameric peptide assembly (CC-Hept) where the esterase activity towards p-nitro-phenylacetate was obtained for introduction of the catalytic triad (Cys-His-Glu) into the hydrophobic matrix, is the object of the present combined molecular dynamics and quantum mechanics/molecular mechanics investigation. Constant pH Molecular Dynamics simulations on the apoform of CC-Hept suggested that the Cys residues are present in the protonated form. Molecular dynamics (MD) simulations of the enzyme-substrate complex evidenced the attitude of the enzyme-like system to retain water molecules, necessary in the hydrolytic reaction, in correspondence of the active site, represented by the Cys-His-Glu triad on each of the seven chains, without significant structural perturbations. A detailed reaction mechanism of esterase activity of CC-Hept-Cys-His-Glu was investigated on the basis of the quantum mechanics/molecular mechanics calculations employing a large quantum mechanical (QM) region of the active site. The proposed mechanism is consistent with available esterases kinetics and structural data. The roles of the active site residues were also evaluated. The deacylation phase emerged as the rate-determining step, in agreement with esterase activity of other natural proteases.

Atropisomerism in Diarylamines: Structural Requirements and Mechanisms of Conformational Interconversion.

In common with other hindered structures containing two aromatic rings linked by a short tether, diarylamines may exhibit atropisomerism (chirality due to restricted rotation). Previous examples have principally been tertiary amines, especially those with cyclic scaffolds. Little is known of the structural requirement for atropisomerism in structurally simpler secondary and acyclic diarylamines. In this paper we describe a systematic study of a series of acyclic secondary diarylamines, and we quantify the degree of steric hindrance in the ortho positions that is required for atropisomerism to result. Through a detailed experimental and computational analysis, the role of each ortho-substituent on the mechanism and rate of conformational interconversion is rationalised. We also present a simple predictive model for the design of configurationally stable secondary diarylamines.

Probing the effect of polyethene glycol on the adsorption mechanisms of Gem on the hexagonal boron nitride as a highly efficient polymer-based drug delivery system: DFT, classical MD and Well-tempered Metadynamics simulations.

The stability of Gemcitabine (Gem) anticancer drug on the hexagonal boron nitride (h-BN) and functionalized h-BN with polyethylene glycol (PEG-h-BN) as drug delivery carriers (DDSs) is investigated. The density functional theory (DFT) calculations, molecular dynamics (MD) simulation and Metadynamics simulations are used to study the nature of h-BN-Gem interactions as well as the role of PEG group to increase the efficiency of the DDS. The results of DFT calculations reveal that the drug physisorbed on the h-BN surface through the formation of π-π stacking with an adsorption energy range -15.08 kJ/mol to -90.74 kJ/mol. Moreover, the obtained results show that the grafting the PEG group to h-BN cause to π-π stacking is reinforced by the formation of strong HBs and leads to increase adsorption energy about 20%. There is a good agreement between DFT calculation and MD simulation results. Also, The MD simulations demonstrate in adsorption of the drug on the carriers, the contribution of van der Waals energy is more than the electrostatic energy. The well-tempered metadynamics simulations are performed to find the free energy surface (FES) of the studied systems. The FES for the Gem/h-BN and Gem/PEG-h-BN interfaces show the global minimum at around 3.0-6.0 Å and 1.2 Å, respectively. The orientational analysis proves that the global minimum can be related to the formation of π-π stacking and HB interaction.

On the Chemistry and Diffusion of Hydrogen in the Interstitial Space of Layered Crystals h-BN, MoS2 , and Graphite.

Recent experiments have demonstrated transport and separation of hydrogen isotopes through the van der Waals gap in hexagonal boron nitride and molybdenum disulfide bulk layered materials. However, the experiments cannot distinguish if the transported particles are protons (H+ ) or protium (H) atoms. Here, reported are the theoretical studies, which indicate that protium atoms, rather than protons, are transported through the gap. First-principles calculations combined with well-tempered metadynamics simulations at finite temperature reveal that for h-BN and MoS2 , the diffusion mechanism of both protons and protium (H) atoms involves a hopping process between adjacent layers. This process is assisted by low-energy phonon shear modes. The extracted diffusion coefficient of protium matches the experiment, while for protons it is several orders of magnitude smaller. This indicates that protium atoms are responsible for the experimental observations. These results allow for a comprehensive interpretation of experimental results on the transport of hydrogen isotopes through van der Waals gaps and can help identify other materials for hydrogen isotope separation applications.

Finite-Temperature Dimer Method for Finding Saddle Points on Free Energy Surfaces.

The dimer method and its variants have been shown to be efficient in finding saddle points on potential surfaces. In the dimer method, the most unstable direction is approximately obtained by minimizing the total potential energy of the dimer. Then, the force in this direction is reversed to move the dimer toward saddle points. When the finite-temperature effect is important for a high-dimensional system, one usually needs to describe the dynamics in a low-dimensional space of reaction coordinates. In this case, transition states are collected as saddle points on the free energy surface. The traditional dimer method cannot be directly employed to find saddle points on a free energy surface since the surface is not known a priori. Here, we develop a finite-temperature dimer method for searching saddle points on the free energy surface. In this method, a constrained rotation dynamics of the dimer system is used to sample dimer directions and an efficient average method is used to obtain a good approximation of the most unstable direction. This approximated direction is then used in reversing the force component and evolving the dimer toward saddle points. Our numerical results suggest that the new method is efficient in finding saddle points on free energy surfaces. © 2019 Wiley Periodicals, Inc.

Networks of electrostatic and hydrophobic interactions modulate the complex folding free energy surface of a designed ßα protein.

The successful de novo design of proteins can provide insights into the physical chemical basis of stability, the role of evolution in constraining amino acid sequences, and the production of customizable platforms for engineering applications. Previous guanidine hydrochloride (GdnHCl; an ionic denaturant) experiments of a designed, naturally occurring ßα fold, Di-III_14, revealed a cooperative, two-state unfolding transition and a modest stability. Continuous-flow mixing experiments in our laboratory revealed a simple two-state reaction in the microsecond to millisecond time range and consistent with the thermodynamic results. In striking contrast, the protein remains folded up to 9.25 M in urea, a neutral denaturant, and hydrogen exchange (HDX) NMR analysis in water revealed the presence of numerous high-energy states that interconvert on a time scale greater than seconds. The complex protection pattern for HDX corresponds closely with a pair of electrostatic networks on the surface and an extensive network of hydrophobic side chains in the interior of the protein. Mutational analysis showed that electrostatic and hydrophobic networks contribute to the resistance to urea denaturation for the WT protein; remarkably, single charge reversals on the protein surface restore the expected urea sensitivity. The roughness of the energy surface reflects the densely packed hydrophobic core; the removal of only two methyl groups eliminates the high-energy states and creates a smooth surface. The design of a very stable ßα fold containing electrostatic and hydrophobic networks has created a complex energy surface rarely observed in natural proteins.

Efficient Computation of Free Energy Surfaces of Diels⁻Alder Reactions in Explicit Solvent at Ab Initio QM/MM Level.

For Diels⁻Alder (DA) reactions in solution, an accurate and converged free energy (FE) surface at ab initio (ai) quantum mechanical/molecular mechanical (QM/MM) level is imperative for the understanding of reaction mechanism. However, this computation is still far too expensive. In a previous work, we proposed a new method termed MBAR+wTP, with which the computation of the ai FE profile can be accelerated by several orders of magnitude via a three-step procedure: (I) an umbrella sampling (US) using a semi-empirical (SE) QM/MM Hamiltonian is performed; (II) the FE profile is generated using the Multistate Bennett Acceptance Ratio (MBAR) analysis; and (III) a weighted Thermodynamic Perturbation (wTP) from the SE Hamiltonian to the ai Hamiltonian is performed to obtain the ai QM/MM FE profile using weight factors from the MBAR analysis. In this work, this method is extended to the calculations of two-dimensional FE surfaces of two Diels⁻Alder reactions of cyclopentadiene with either acrylonitrile or 1-4-naphthoquinone at ai QM/MM level. The accurate activation free energies at the ai QM/MM level, which are much closer to the experimental measurements than those calculated by other methods, indicate that this MBAR+wTP method can be applied in the studies of complex reactions in condensed phase with much-enhanced efficiency.

Intrinsic map dynamics exploration for uncharted effective free-energy landscapes.

We describe and implement a computer-assisted approach for accelerating the exploration of uncharted effective free-energy surfaces (FESs). More generally, the aim is the extraction of coarse-grained, macroscopic information from stochastic or atomistic simulations, such as molecular dynamics (MD). The approach functionally links the MD simulator with nonlinear manifold learning techniques. The added value comes from biasing the simulator toward unexplored phase-space regions by exploiting the smoothness of the gradually revealed intrinsic low-dimensional geometry of the FES.