Insights into Allosteric Inhibition of the AcrB Efflux Pump: Role of Distinct Binding Pockets, Protomer Preferences, and Crosstalk Disruption.

AcrB, a key component in bacterial efflux processes, exhibits distinct binding pockets that influence inhibitor interactions. In addition to the well-known distal binding pocket within the periplasmic domain, a noteworthy pocket amidst the transmembrane (TM) helices serves as an alternate binding site for inhibitors. The bacterial efflux mechanism involves a pivotal functional rotation of the TM protein, inducing conformational changes in each protomer and propelling drugs toward the outer membrane domain. Surprisingly, inhibitors binding to the TM domain display a preference for L protomers over T protomers. Metadynamics simulations elucidate that Lys940 in the TM domain of AcrB can adopt two conformations in L protomers, whereas the energy barrier for such transitions is higher in T protomers. This phenomenon results in stable inhibitor binding in l protomers. Upon a detailed analysis of unbinding pathways using random accelerated molecular dynamics and umbrella sampling, we have identified three distinct routes for ligand exit from the allosteric site, specifically involving regions within the TM domains─TM4, TM5, and TM10. To explore allosteric crosstalk, we focused on the following key residues: Val452 from the TM domain and Ala831 from the porter domain. Surprisingly, our findings reveal that inhibitor binding disrupts this communication. The shortest path connecting Val452 and Ala831 increases upon inhibitor binding, suggesting sabotage of the natural interdomain communication dynamics. This result highlights the intricate interplay between inhibitor binding and allosteric signaling within our studied system.

Exploring transmembrane allostery in the MexB: DB08385 variant as a promising inhibitor-like candidate against Pseudomonas aeruginosa antibiotic resistance: a computational study.

Pseudomonas aeruginosa, a formidable pathogen renowned for its antimicrobial resistance, poses a significant threat to immunocompromised individuals. In this regard, the MexAB-OprM efflux pump acts as a pivotal line of defense by extruding antimicrobials from bacterial cells. The inner membrane homotrimeric protein MexB captures antibiotics and translocates them into the outer membrane OprM channel protein connected through the MexA adaptor protein. Despite extensive efforts, competitive inhibitors targeting the tight (T) protomer of the MexB protein have not received FDA approval for medical use. Over the past few years, allosteric inhibitors have become popular as alternatives to the classical competitive inhibitor-based approach because of their higher specificity, lower dosage, and reduced toxicological effects. Hence, in this study, we unveiled the existence of a transmembrane allosteric binding pocket of MexB inspired by the recent discovery of an important allosteric inhibitor, BDM88855, for the homolog AcrB protein. While repurposing BDM88855 proved ineffective in controlling the MexB loose (L) protomer, our investigation identified a promising alternative: a chlorine-containing variant of DB08385 (2-Cl DB08385 or Variant 1). Molecular dynamics simulations, including binding free energy estimation coupled with heterogeneous dielectric implicit membrane model (implicit-membrane MM/PBSA), interaction entropy (IE) analysis and potential of mean force (PMF) calculation, demonstrated Variant 1's superior binding affinity to the transmembrane pocket, displaying the highest energy barrier in the ligand unbinding process. To elucidate the allosteric crosstalk between the transmembrane and porter domain of MexB, we employed the 'eigenvector centrality' measure in the linear mutual information obtained from the protein correlation network. Notably, this study confirmed the presence of an allosteric transmembrane site in the MexB L protomer. In addition to this, Variant 1 emerged as a potent regulator of allosteric crosstalk, inducing an 'O-L intermediate state' in the MexB L protomer. This induced state might hold the potential to diminish substrate intake into the access pocket, leading to the ineffective efflux of antibiotics.

Decoding Inhibitor Egression from Wild-Type and G2019S Mutant LRRK2 Kinase: Insights into Unbinding Mechanisms for Precision Drug Design in Parkinson's Disease.

Leucine-rich repeat kinase 2 (LRRK2) remains a viable target for drug development since the discovery of the association of its mutations with Parkinson's disease (PD). G2019S (in the kinase domain) is the most common mutation for LRRK2-based PD. Though various types of inhibitors have been developed for the kinase domain to reduce the effect of the mutation, understanding the working of these inhibitors at the molecular level is still ongoing. This study focused on the exploration of the dissociation mechanism (pathways) of inhibitors from (WT and G2019S) LRRK2 kinase (using homology model CHK1 kinase), which is one of the crucial aspects in drug discovery. Here, two ATP-competitive type I inhibitors, PF-06447475 and MLi-2 (Comp1 and Comp2 ), and one non-ATP-competitive type II inhibitor, rebastinib (Comp3), were considered for this investigation. To study the unbinding process, random accelerated molecular dynamics simulations were performed. The binding free energies of the three inhibitors for different egression paths were determined using umbrella sampling. This work found four major egression pathways that were adopted by the inhibitors Comp1 (path1, path2, and path3), Comp2 (path1, path2 and path3), and Comp3 (path3 and path4). Also, the mechanism of unbinding for each path and key residues involved in unbinding were explored. Mutation was not observed to impact the preference of the particular egression pathways for both LRRK2-Comp1 and -Comp2 systems. However, the findings suggested that the size of the inhibitor molecules might have an effect on the preference of the egression pathways. The binding energy and residence time of the inhibitors followed a similar trend to experimental observations. The findings of this work might provide insight into designing more potent inhibitors for the G2019S LRRK2 kinase.

Elucidating the Molecular Basis of 14-3-3 Interaction with α-Synuclein: Insights from Molecular Dynamics Simulations and the Design of a Novel Protein-Protein Interaction Inhibitor.

Parkinson's disease is a widespread age-related neurodegenerative disorder characterized by the loss of dopaminergic neurons in the midbrain along with the appearance of protein aggregates, termed as "Lewy bodies" in the surviving neuronal cells. The components of Lewy bodies include proteins such as α-synuclein, 14-3-3, Parkin, and LRRK2, along with other cellular organelles, which, in their native state, perform a plethora of vital biological functions within the human biome. Formation of these aggregates renders these components inactive, thereby interfering with homeostasis. In this regard, the current study attempts to investigate the complexation behavior of all human-based 14-3-3 isoforms with α-synuclein via a combination of classical and enhanced sampling techniques and thereby determine the causality of these protein-protein interactions. The study indicated that upon complexation, the aggregation propensity of both 14-3-3 and α-synuclein increases, and this increment is propelled by the interfacial residues on either protein. Furthermore, mutagenesis studies revealed that Lys214 of 14-3-3 (henceforth termed K214A) is crucial for the formation of this binary complex. Principal component analysis combined with clustering studies unveiled the stability of these complexes in terms of their conformational distribution across the entire MD trajectory. For K214A, these clustered states were sparsely located, thereby making the transitions between them slightly difficult. Dynamic cross-correlation maps (DCCM) revealed the role of residues in the range 80-130 of 14-3-3 having a potential allosteric role in driving this complexation process. Finally, a novel peptide-based supramolecular inhibitor was designed, which exhibited higher proficiency in limiting the 14-3-3/α-synuclein interaction compared to the previous inhibitor model. It was also revealed that the presence of this inhibitor induces structural rigidity in α-synuclein, making changes in its conformations extremely difficult, as observed through Umbrella Sampling studies. Based on available information, the current study provides an insight into the molecular-level understanding of protein-protein interactions underlying Parkinson's disease and adds on to the methods of devising novel therapeutic approaches to treat the same.

Elucidating Daptomycin's Antibacterial Efficacy: Insights into the Tripartite Complex with Lipid II and Phospholipids in Bacterial Septum Membrane.

This study elucidated the mechanism of formation of a tripartite complex containing daptomycin (Dap), lipid II, and phospholipid phosphatidylglycerol in the bacterial septum membrane, which was previously reported as the cause of the antibacterial action of Dap against gram-positive bacteria via molecular dynamics and enhanced sampling methods. Others have suggested that this transient complex ushers in the inhibition of cell wall synthesis by obstructing the downstream polymerization and cross-linking processes involving lipid II, which is absent in the presence of cardiolipin lipid in the membrane. In this work, we observed that the complex was stabilized by Ca2+-mediated electrostatic interactions between Dap and lipid head groups, hydrophobic interaction, hydrogen bonds, and salt bridges between the lipopeptide and lipids and was associated with Dap concentration-dependent membrane depolarization, thinning of the bilayer, and increased lipid tail disorder. Residues Orn6 and Kyn13, along with the DXDG motif, made simultaneous contact with constituent lipids, hence playing a crucial role in the formation of the complex. Incorporating cardiolipin into the membrane model led to its competitively displacing lipid II away from the Dap, reducing the lifetime of the complex and the nonexistence of lipid tail disorder and membrane depolarization. No evidence of water permeation inside the membrane hydrophobic interior was noted in all of the systems studied. Additionally, it was shown that using hydrophobic contacts between Dap and lipids as collective variables for enhanced sampling gave rise to a free energy barrier for the translocation of the lipopeptide. A better understanding of Dap's antibacterial mechanism, as studied through this work, will help develop lipopeptide-based antibiotics for rising Dap-resistant bacteria.

Mononuclear organogermanium(IV) catalysts for a [3 + 2] cycloaddition reaction.

Three mononuclear Ge(IV) compounds, [(C6H5)2Ge(C13H8N2O4)] (1), [(C6H5)2Ge(C14H10N2O5)] (2), and [(C6H5)2Ge(C14H11NO3)] (3), have been synthesized by the reaction of pro-ligands H2L1 (C13H10N2O4), H2L2 (C14H12N2O5), and H2L3 (C14H13NO3) with (C6H5)2GeCl2 in the presence of triethylamine. All compounds were characterized by FT-IR spectroscopy and NMR spectroscopy. Single crystal X-ray diffraction analysis shows that the germanium(IV) atom exhibits a five-coordinated geometry in compounds 1 and 2. All compounds were screened as Lewis acid catalysts in the [3 + 2] cycloaddition reaction between sodium azide and various nitriles. The reactions resulted in the formation of 5-substituted 1H-tetrazoles with yields of up to 96%. Based on the experimental findings and DFT calculations, a plausible mechanism is proposed for the [3 + 2] cycloaddition reaction.

Design of saccharide based organic binder for low-grade iron ore pelletization using atomistic simulations and machine learning methods.

Inorganic binders like bentonite, used for pelletization of low-grade iron ore, generate iron ore slimes with comparatively high silica and alumina content necessitating extra steps for their removal during iron making process. This demands the usage of organic binders as full or partial replacement of bentonite for iron ore pelletization. In this work, adsorption of organic binders with saccharides skeleton and -H, -OH, -CH2OH and -CH2CH2OH as polar substituents, on goethite surface was studied using density functional theory, molecular dynamics and machine learning. It was observed that adsorption energy of binders on goethite surface had weak dependence on number of hydrogen bonds between them. With this favorable interaction in mind, a library containing 64 organic binders was constructed and adsorption energy of 30 of these binders was computed using molecular dynamics, followed by training of a linear regression model, which was then used to predict the adsorption energy of rest of the binders in the library. It was found that the introduction of -CH2CH2OH at R2 position resulted in statistically significant higher adsorption energy. Binder34 and Binder44 were identified as viable candidates for both goethite and hematite ore pelletization and adsorption of their n-mers on goethite and hematite surfaces was also quantified.

Machine Learning-Guided Discovery of AcrB and MexB Efflux Pump Inhibitors.

Multidrug efflux pump is one of the reasons behind the antimicrobial inactivity related to infection caused by Gram-negative pathogens. The inner membrane resistance-nodulation-cell division transporter proteins, AcrB and MexB, in association with outer membrane proteins, TolC and OprM, are responsible for the extrusion of a broad range of substrates, followed by recognizing them. Although various inhibitors were proposed to stop the efflux activity of the transporter protein, none of them had been approved clinically. Our study aims to identify potent inhibitor-like molecules employing supervised classification models trained upon the molecular descriptors of previously known inhibitors. Based on the intrinsic minimum inhibitory concentration (MIC) values of the reported inhibitors, they were classified into highly potent and less potent categories. A total of 10 different classification models were built using various molecular descriptors; among them, support vector machine, Random Forest, AdaBoost, and LightGBM models appeared to deliver promising results with >80% accuracy. These top four models were implemented on a library of 5043 to obtain 8 hit molecules after the multistep filtering process. To assess their activity toward AcrB and MexB, several molecular dynamics simulations of their ligand-bound structures were performed. We also calculated the binding free-energy values and analyzed other structural properties. Mol.3488 of the unknown molecules showed higher binding affinities for both AcrB and MexB. Also, the presence of "pyridopyrimidone" and "benzothiazole" moieties in the molecules and "V"-shaped orientation of ligands inside the deep binding pocket increase the binding affinity, thereby higher inhibitory properties.

Probing the pH Sensitivity of OprM: Insights into Metastable States and Semi-Open Conformation.

Efflux pumps are specialized transport proteins that play a key role in the bacterial defense against a wide spectrum of antibiotics. Hence, understanding the biophysical mechanism associated with this complex system of drug expulsion becomes crucial. This work deals with some vital aspects of the outer membrane factor (OMF) of MexAB-OprM. After being passed through MexB and MexA, efflux substrates have to go through OprM for their final judgment. Thus, it is very important to understand the periplasmic pore opening mechanism and the associated biophysical changes during this process. Our study captures a detailed analysis of the pore opening mechanism involving OprM. With powerful molecular dynamics (MD) techniques such as well-tempered metadynamics, the presence of metastable states in between open and closed states was confirmed. Also, upon mutating R376, the energy barrier for the conversion of the close to open conformation decreases, indicating an important role played by the residue. Further, constant pH MD was performed to capture the effect of pH in both conformations. OprM exhibits distinct conformational states at pH values greater than 5.5 and lower than 5.5, suggesting its pH-responsive characteristics. Overall, our study elucidates a crucial undertaking toward discovering potential inhibitors for MexAB-OprM efflux pumps.

Formate dehydrogenase activity by a Cu(II)-based molecular catalyst and deciphering the mechanism using DFT studies.

Due to the requirement to establish renewable energy sources, formic acid (FA), one of the most probable liquid organic hydrogen carriers (LOHCs), has received great attention. Catalytic formic acid dehydrogenation in an effective and environmentally friendly manner is still a challenge. The N3Q3 ligand (N3Q3 = N,N-bis(quinolin-8-ylmethyl)quinolin-8-amine) and the square pyramidal [Cu(N3Q3)Cl]Cl complex have been synthesised in this work and characterised using several techniques, such as NMR spectroscopy, mass spectrometry, EPR spectroscopy, cyclic voltammetry, X-ray diffraction and DFT calculations. This work investigates the dehydrogenation of formic acid using a molecular and homogeneous catalyst [Cu(N3Q3)Cl]Cl in the presence of HCOONa. The mononuclear copper complex exhibits catalytic activity towards the dehydrogenation of formic acid in H2O with the evolution of a 1 : 1 CO2 and H2 mixture. The activation energy of formic acid dehydrogenation was calculated to be Ea = 86 kJ mol-1, based on experiments carried out at various temperatures. The Gibbs free energy was found to be 82 kJ at 298 K for the decomposition of HCOOH. The DFT studies reveal that [Cu(N3Q3)(HCOO-)]+ undergoes an uphill process of rearrangement followed by decarboxylation to generate [Cu(N3Q3)(H-)]+. The initial uphill step for forming a transition state is the rate-determining step. The [Cu(N3Q3)(H-)]+ follows an activated state in the presence of HCOOH to liberate H2 and generate the [Cu(N3Q3)(OH2)]2+.

Uncovering the Structural and Binding Insights of Dual Inhibitors Simultaneously Targeting Two Distinct Sites on EGFR Kinase.

Epidermal growth factor receptor (EGFR) is the first growth factor receptor identified in normal cells that is related to the receptor tyrosine kinase, which causes regular cell division. A point mutation in EGFR intracellular kinase domain forces the abnormal cell divisions throughout time, leading to non-small cell lung cancer (NSCLC) transformation. Thus, competitive inhibitors that bind to the ATP binding pocket have been developed as a targeted therapy for NSCLC. The third-generation kinase inhibitor Osimertinib is currently playing a very vital role in the treatment of NSCLC. However, it is not effective toward the C797S kinase domain mutation. For this reason, fourth-generation kinase noncompetitive inhibitors are introduced which work through binding to an allosteric pocket near the ATP binding region and act as a better binding agent for this mutated kinase domain. However, the problem is that these single fourth-generation kinase inhibitors may not be as effective as a single agent. The aim of this work was to apply combinations of these two inhibitors together in different binding regions of EGFR without overlapping the resistance mechanism to obtain the key direct and indirect interactions occurring between them. Moreover, the free energy of dissociation of an inhibitor from its binding sites in the presence of a second inhibitor immobilized in another binding site was also the focus of the study. To realize this aim, we performed conventional molecular dynamics simulations and principal component analysis and dynamic cross-correlation matrices along with umbrella sampling. Our results demonstrated that binding of dual inhibitors triggered conformational changes of the protein more toward the inactive state. Furthermore, allosteric inhibitors bound more strongly to protein kinase EGFR than the orthosteric inhibitors in the presence of dual inhibitors. Finally, the binding mechanism and important hydrogen-bonding residues during unbinding of the inhibitors were fully elucidated. This study provides insight into the binding of the receptor-orthosteric inhibitor-allosteric inhibitor, which can be helpful for further design of novel inhibitors that have a better inhibitory action.

Structural insight into G2019S mutated LRRK2 kinase and brain-penetrant type I inhibitor complex: a molecular dynamics approach.

More than 40 mutations in the multidomain leucine-rich repeat kinase 2 (LRRK2) are found and mutation G2019S in the kinase domain is the most concerned with Parkinson's disease (PD). The discovery of the various types of inhibitors has largely emerged recently. However, the comparative study on molecular insight in WT and G2019S LRRK2 kinase domain upon binding of the inhibitors has not yet been explored in detail. This work considered five ATP-competitive Type I inhibitors complexed with WT and mutated LRRK2 kinase. Three reported potent and brain-penetrant inhibitors, GNE-7915, PF-06447475 and MLi-2 (comp1, comp2 and comp3 respectively) and also, another two inhibitors, Pyrrolo[2,3-b] pyridine derivative (comp4) and Pyrrolo[2,3-d] pyrimidine derivative (comp5), were used. In this work, classical and accelerated molecular dynamics (cMD and aMD) simulations were performed for a total of 12 systems (apo and holo). This study found structural and thermodynamic stability for all the inhibitors. Comparatively larger molecules (size 15.3 - 15.4 Å), comp1, comp3 and comp5, showed more selectivity towards mutated LRRK2 kinase in terms of flexibility of residues, compactness and dynamics of kinase, the stability inside the binding-pocket. Also, inhibitors comp3 and comp5 showed higher binding affinity towards G2019S LRRK2 among the five. Residues, E1948 and A1950 (in hinge region) were observed mainly to form hydrogen bonds with inhibitors. Finally, MLi-2 showed a conformational rearrangement by dihedral flipping in both WT and mutated systems but got stability in G2019S LRRK2. This work could potentially help design more improved and effective Type I inhibitors for G2019S LRRK2 kinase.

Enhanced Uptake of Anticancer C6-Pep Dimer in a Model Membrane Caused by Differential pKa in Acidic Microenvironment.

Acidic tumor microenvironment (TME) presents a challenge for the action of antitumor drugs by acting as an additional barrier for the passive crossing of the cell membrane by chemotherapic agents playing a critical role in the proliferation of tumor cells. Anticancer lipopeptide C6-Pep dimer containing the leucine zipper motif shows an increased uptake into the model tumor membrane in TME, and application of external heat might lead to the uncoiling of the zipper, which could result in cell lysis. This work investigated the cause of this increased uptake of C6-Pep dimer into the bilayer model in TME. Accurate protonation states of all the titratable residues of the C6-Pep dimer in TME were determined using constant pH molecular dynamics. In TME, except for two terminal Glu5 residues, all other Glu residues in the C6-Pep dimer were permanently protonated. The remaining Glu5 residues had differential pKa values, leading to the construction of four possible dimers with different fixed protonation states, and molecular dynamics was used to study their interaction with the anionic bilayer. Except for the dimer at a physiological pH, the other dimers were positively charged and could readily adsorb on the membrane surface. The free energy of insertion of these dimers in the bilayer was lower for single and double protonated Glu5-containing dimers than for the others. After the insertion of the lipopeptides into the membrane, thinning of the bilayer in the vicinity of dimers and an increase in area per lipid of the bilayer were observed for all systems, indicating destabilization of the bilayer due to this intercalation. This study shows that the anticancer lipopeptide C6-Pep utilizes the TME around a tumor cell for insertion into the membrane.

In silico structural and mechanical insights into bedaquiline resistance associated with high-grade non-synonymous mutations in atpE, mmpR5, and pepQ.

Clinical resistance against bedaquiline (BDQ) remains intractable to anti-tuberculosis therapies since its introduction to the market over a decade ago. Herein, we investigated the structural and mechanical aspects of BDQ resistance in AtpE, MmpR5, and PepQ. The known target-specific resistant single non-synonymous mutations were refined to high-grade candidates. Thus, 7 (AtpE), 5 (MmpR5), and 1 (PepQ) single nucleotide polymorphisms (SNPs) and one insertion frameshift mutation in MmpR5 were recreated at the molecular level, and these phenotypic models were then directed to stringent dynamics to define time-scaled changes. The AtpE variants destabilized the structure; mainly, L59V, E61D, and I66M were detrimental to the complex fitness, while L74V and L114P boosted the BDQ binding to MmpR5. The first three and last two alterations gave rise to loss- and gain-of-function to AtpE and MmpR5, respectively. Hence, these five mutants are functionally relevant and therapeutically targetable hotspots of BDQ resistance. There were no noticeable changes in PepQ data analysis. The present study revealed that MmpR5 mutations confer BDQ resistance, whereas AtpE and PepQ SNPs display low susceptibility. These results were tallied with the published findings, which testified to the pursued method's reliability and accuracy. We hope these data and inferences could be helpful for the futuristic design of novel TB drugs.Communicated by Ramaswamy H. Sarma.

Telescoping-Solvation-Box Protocol-Based Umbrella Sampling Method for Potential Mean Force Estimation.

Previously, it was shown that the telescoping box scheme, in combination with adaptive steered molecule dynamics (ASMD), can be used to estimate the potential of mean force (PMF) with a decrease in computational cost associated with large solvation boxes. Since ASMD reduces to umbrella sampling (US) in the limit of infinitely slow pulling velocity, a hypothesis was made that the telescoping box scheme can be extended to include the US method. The hypothesis was tested using the unfolding pathway of a polyalanine peptide in a water box and translocation of α-tocopherol through the human membrane. Two different approaches were tried: telescoping US (TELUS), in which the number of solvent molecules was linearly coupled to the reaction coordinate, and Block-TELUS, which was a compromise between the fixed solvation box of the US and the window-wise variable solvation box of TELUS. In the case of polyalanine peptide in a water box, both approaches gave overlapping potential of mean force (PMF) concerning the benchmark US-PMF. Window-wise comparison of properties like root-mean-square inner product, Ramachandran plot, α-helix content, and hydrogen bond formation was used to verify that both approaches captured the same dynamics as the US method. Applying the Block-TELUS protocol in the system with diffusing α-tocopherol through the bilayer resulted in overlapping PMF to its US benchmark. A comparison between the window-wise orientation of the chromanol headgroup also produced almost identical results. This study concluded that with the careful application of telescoping solvation boxes, a less computationally expensive US could be performed for systems where the effect of distant solvent molecules on the configurational space sampled in the window depends on the value of the reaction coordinate.

Role of the Residue Q1919 in Increasing Kinase Activity of G2019S LRRK2 Kinase: A Computational Study.

Mutations in multi-domain leucine-rich repeat kinase 2 (LRRK2) have been an interest to researchers as these mutations are associated with Parkinson's disease. G2019S mutation in LRRK2 kinase domain leads to the formation of additional hydrogen bonds by S2019 which results in stabilization of the active state of the kinase, thereby increasing kinase activity. Two additional hydrogen bonds of S2019 are reported separately. Here, a mechanistic picture of the formation of additional hydrogen bonds of S2019 with Q1919 (also with E1920) is presented using 'active' Roco4 kinase as a homology model and its relationship with the stabilization of the 'active' G2019S LRRK2 kinase. A conformational flipping of residue Q1919 was found which helped to form stable hydrogen bond with S2019 and made 'active' state more stable in G2019S LRRK2. Two different states were found within the 'active' kinase with respect to the conformational change (flipping) in Q1919. Two doubly-mutated systems, G2019S/Q1919A and G2019S/E1920 K, were studied separately to check the effect of Q1919 and E1920. For both cases, the stable S2 state was not formed, leading to a decrease in kinase activity. These results indicate that both the additional hydrogen bonds of S2019 (with Q1919 and E1920) are necessary to stabilize the active G2019S LRRK2.

Self-Uptake Mechanism of Polymyxin-Based Lipopeptide against Gram-Negative Bacterial Membrane: Role of the First Adsorbed Lipopeptide.

Research in the continuously increasing threat of polymyxin-resistant multidrug-resistant Pseudomonas aeruginosa, which causes severe infection in immunocompromised patients, has resulted in the development of several polymyxin-derived cyclic lipopeptides containing l-α-γ- diamino butyric acid-like FADDI-019 (F19). In this work, F19's insertion into a minimal model of the asymmetric outer membrane of the bacterium, which contained only penta-acylated lipid A (LipA) and lacked keto-d-octulosonic acid and O-antigens, in the top leaflet and phospholipids in the bottom leaflet, was studied. F19 exhibited all of the hallmarks of the self-uptake mechanism into the asymmetric bilayer. While a single monomer of the lipopeptide did not get partitioned into the inside of the bilayer, it competitively displaced Ca2+ from the membrane surface, observed as a decrease in Ca2+ coordination number with phosphate groups (1.89 vs 1.718), resulting in membrane destabilization. This resulted in an increment of the average defect size and the probability of interplay between lipid tails and hydrophobic residues of another F19. When more than one monomer was present in the system, the first monomer remained docked on the surface, while other monomers intercalated into the bilayer interior with their hydrophobic moieties "sleeved" by lipid acyl chains. The free energy barrier for partial insertion of the lipopeptide into a bilayer in the presence of surface-docked second F19 was recorded at ∼1.3 kcal/mol using two-dimensional (2D) well-tempered metadynamics, making it a low barrier process at 300 K. This study is an attempt to demonstrate the self-uptake mechanism of F19 during intercalation process into the bilayer interior, which may help in the design of better alternates for polymyxins to work against polymyxin resistance.

Prediction of COMT Inhibitors Using Machine Learning and Molecular Dynamics Methods.

Catechol O-methyltransferase (COMT) plays a vital role in deactivating neurotransmitters like dopamine, norepinephrine, etc., by methylating those compounds. However, the deactivation of an excess amount of neurotransmitters leads to serious mental ailments such as Parkinson's disease. Molecules that bind inside the enzyme's active site inhibit this methylation mechanism by methylating themselves, termed COMT inhibitors. Our study is focused on designing these inhibitors by various machine learning methods. First, we have developed a classification model with experimentally available COMT inhibitors, which helped us generate a new data set of small inhibitor-like molecules. Then, to predict the activity of the new molecules, we have applied regression techniques such as Random Forest, AdaBoost, gradient boosting, and support vector machines. Each of the regression models yielded an R2 value > 70% for both training and test data sets. Finally, to validate our models, 200 ns long molecular dynamics (MD) simulations of the two known inhibitors with known IC50 values and the resultant inhibitors were performed inside the binding pockets to check their stability within. The free energy barrier of the methyl transfer from S-adenosyl-l-methionine (SAM) to each inhibitor was determined by combining steered molecular dynamics (SMD) and umbrella sampling using the quantum mechanics/molecular mechanics (QM/MM) method.

Photoresponsive transformation from spherical to nanotubular assemblies: anticancer drug delivery using macrocyclic cationic gemini amphiphiles.

We developed NIR-light-responsive macrocyclic cationic gemini amphiphiles, one of which displayed various favorable properties of lipids. The NIR-light-mediated cleavage of the strained dioxacycloundecine ring led to the conversion of the spherical to a nanotubular self-assembly in the aqueous medium. This photo-mediated transformation from the spherical to nanotubular self-assembly resulted in the release of encapsulated hydrophobic anticancer drug molecule doxorubicin (Dox) in a controlled manner. The potent cationic gemini amphiphile also displayed lower cytotoxicity and efficient NIR-light-mediated Dox release efficacy to cancerous cells.

Configuration Flipping in Distal Pocket of Multidrug Transporter MexB Impacts the Efflux Inhibitory Mechanism.

MexAB-OprM efflux pumps, found in Pseudomonas aeruginosa, play a major role in drug resistance by extruding out drugs and antibiotic molecules from cells. Inhibitors are used to cease the potency of the efflux pumps. In this study, in-silico models are used to investigate the nature of the binding pocket of the MexAB-OprM efflux pump. First, we have performed classical molecular dynamics (MD) simulations to shed light on different aspects of protein-inhibitor interaction in the binding pocket of the pump. Using classical MD simulations, quantum mechanics/molecular mechanics (QM/MM), and various types of analyses, it is found that D13-9001 has a higher binding affinity towards the binding pocket compared to D1 and D2; the results are in sync with the experimental dat. Two stable configurations of D13-9001 are discovered inside the distal pocket which could be one of the primary reasons for the greater efficacy of D13-9001. The free energy barrier upon changing one state to another is calculated by employing umbrella sampling method. Finally, F178 is mutated to have the complete picture as it contributes significantly to the binding energy irrespective of the three inhibitors. Our results may help to design a new generation of inhibitors for such an efflux pump.