ß-Peptides incorporating polyhydroxylated cyclohexane ß-amino acid: synthesis and conformational study.

We describe the synthesis of trihydroxylated cyclohexane ß-amino acids from (-)-shikimic acid, in their cis and trans configuration, and the incorporation of the trans isomer into a trans-2-aminocyclohexanecarboxylic acid peptide chain. Subsequently, the hydroxyl groups were partially or totally deprotected. The structural study of the new peptides by FTIR, CD, solution NMR and DFT calculations revealed that they all fold into a 14-helix secondary structure, similarly to the homooligomer of trans-2-aminocyclohexanecarboxylic acid. This means that the high degree of substitution of the cyclohexane ring of the new residue is compatible with the adoption of a stable helical secondary structure and opens opportunities for the design of more elaborate peptidic foldamers with oriented polar substituents at selected positions of the cycloalkane residues.

Molecular Dynamics Simulations of Transmembrane Cyclic Peptide Nanotubes Using Classical Force Fields, Hydrogen Mass Repartitioning, and Hydrogen Isotope Exchange Methods: A Critical Comparison.

Self-assembled cyclic peptide nanotubes with alternating D- and L-amino acid residues in the sequence of each subunit have attracted a great deal of attention due to their potential for new nanotechnology and biomedical applications, mainly in the field of antimicrobial peptides. Molecular dynamics simulations can be used to characterize these systems with atomic resolution at different time scales, providing information that is difficult to obtain via wet lab experiments. However, the performance of classical force fields typically employed in the simulation of biomolecules has not yet been extensively tested with this kind of highly constrained peptide. Four different classical force fields (AMBER, CHARMM, OPLS, and GROMOS), using a nanotube formed by eight D,L-α-cyclic peptides inserted into a lipid bilayer as a model system, were employed here to fill this gap. Significant differences in the pseudo-cylindrical cavities formed by the nanotubes were observed, the most important being the diameter of the nanopores, the number and location of confined water molecules, and the density distribution of the solvent molecules. Furthermore, several modifications were performed on GROMOS54a7, aiming to explore acceleration strategies of the MD simulations. The hydrogen mass repartitioning (HMR) and hydrogen isotope exchange (HIE) methods were tested to slow down the fastest degrees of freedom. These approaches allowed a significant increase in the time step employed in the equation of the motion integration algorithm, from 2 fs up to 5-7 fs, with no serious changes in the structural and dynamical properties of the nanopores. Subtle differences with respect to the simulations with the unmodified force fields were observed in the concerted movements of the cyclic peptides, as well as in the lifetime of several H-bonds. All together, these results are expected to contribute to better understanding of the behavior of self-assembled cyclic peptide nanotubes, as well as to support the methods tested to speed up general MD simulations; additionally, they do provide a number of quantitative descriptors that are expected to be used as a reference to design new experiments intended to validate and complement computational studies of antimicrobial cyclic peptides.

Inverse Conformational Selection in Lipid-Protein Binding.

Interest in lipid interactions with proteins and other biomolecules is emerging not only in fundamental biochemistry but also in the field of nanobiotechnology where lipids are commonly used, for example, in carriers of mRNA vaccines. The outward-facing components of cellular membranes and lipid nanoparticles, the lipid headgroups, regulate membrane interactions with approaching substances, such as proteins, drugs, RNA, or viruses. Because lipid headgroup conformational ensembles have not been experimentally determined in physiologically relevant conditions, an essential question about their interactions with other biomolecules remains unanswered: Do headgroups exchange between a few rigid structures, or fluctuate freely across a practically continuous spectrum of conformations? Here, we combine solid-state NMR experiments and molecular dynamics simulations from the NMRlipids Project to resolve the conformational ensembles of headgroups of four key lipid types in various biologically relevant conditions. We find that lipid headgroups sample a wide range of overlapping conformations in both neutral and charged cellular membranes, and that differences in the headgroup chemistry manifest only in probability distributions of conformations. Furthermore, the analysis of 894 protein-bound lipid structures from the Protein Data Bank suggests that lipids can bind to proteins in a wide range of conformations, which are not limited by the headgroup chemistry. We propose that lipids can select a suitable headgroup conformation from the wide range available to them to fit the various binding sites in proteins. The proposed inverse conformational selection model will extend also to lipid binding to targets other than proteins, such as drugs, RNA, and viruses.

Aggregation versus inclusion complexes to solubilize drugs with cyclodextrins. A case study using sulphobutylether-ß-cyclodextrins and remdesivir.

The formation of small hybrid aggregates between excipient and drug molecules is one of the mechanisms that contributes to the solubilization of active principles in pharmaceutical formulations. The characterization of the formation, governing interactions and structure of such entities is not trivial since they are highly flexible and dynamic, quickly exchanging molecules from one to another. In the case of cyclodextrins, this mechanism and the formation of inclusion complexes synergistically cooperate to favour the bioavailability of drugs. In a previous study we reported a detailed characterization of the possible formation of inclusion complexes with 1:1 stoichiometry between remdesivir, the only antiviral medication currently approved by the United States Food and Drug Administration for treating COVID-19, and sulphobutylether-ß-cyclodextrins. Here we extend our study to assess the role of the spontaneous aggregation in the solubilization of the same drug, by molecular dynamics simulations at different relative concentrations of both compounds. The number of sulphobutylether substitutions in the cyclodextrin structure and two different protonation states of the remdesivir molecule are considered. We aim to shed light in the solubilization mechanism of sulphobutylether-ß-cyclodextrins, broadly used as an excipient in many pharmaceutical formulations, in particular in the case of remdesivir as an active compound.

Parallel Versus Antiparallel ß-Sheet Structure in Cyclic Peptide Hybrids Containing γ- or δ-Cyclic Amino Acids.

Cyclic peptides with disc-shaped structures have emerged as potent building blocks for the preparation of new biomaterials in fields ranging from biological to material science. In this work, we analyze in depth the self-assembling properties of a new type of cyclic peptides based on the alternation of α-residues and cyclic δ-amino acids (α,δ-CPs). To examine the preferred stacking properties adopted by cyclic peptides bearing this type of amino acids, we carried out a synergistic in vitro/in silico approximation by using simple dimeric models and then extended to nanotubes. Although these new cyclic peptides (α,δ-CPs) can interact either in a parallel or antiparallel fashion, our results confirm that although the parallel ß-sheet is more stable, it can be switched to the antiparallel stacking by choosing residues that can establish favorable cross-strand interactions. Moreover, the subsequent comparison by using the same methodology but applied to α,γ-CPs models, up to the moment assumed as antiparallel-like d,l-α-CPs, led to unforeseen conclusions that put into question preliminary conjectures about these systems. Surprisingly, they tend to adopt a parallel ß-sheet directed by the skeleton interactions. These results imply a change of paradigm with respect to cyclic peptide designs that should be considered for dimers and nanotubes.

Competitive double-switched self-assembled cyclic peptide nanotubes: a dual internal and external control.

"Intelligent" materials based on synthetic small molecules that become functional only under specific conditions provide new opportunities for developing regulated systems aimed at a large number of applications. For instance, biologically active supramolecular entities that are sensitive to environmental conditions, such as the presence of bacterial membranes, are extremely interesting in biomedicine. In this work, we have designed and investigated, using molecular dynamics simulations, a doubly modulable nanotube formed by the self-assembly of cyclic peptides sensitive to both the presence of a lipid membrane and the pH of the aqueous media. The cyclic peptides were designed to self-assemble into peptide nanotubes in the presence of a lipid bilayer and at low pH values. Under these conditions, the residual side chains point outside the cyclic peptides, being exposed to the lipid bilayer, and the inner groups (carboxylic acids) are protonated, thus allowing the permeation of water and preventing that of ions. Higher pH values are expected to create carboxylate groups at the lumen of the peptides, leading to the disassembly of the nanotube, the attraction and translocation of ions towards the hydrophobic core of the bilayer, and eventually killing the target malignant cells. Our results suggest that by introducing a second switch in a membrane sensitive system, it is possible to modulate its interaction with the lipid bilayer. This opens the door to new strategies for the preparation of antimicrobial peptides that interact at the membrane level.

Rhodium-Catalyzed Annulation of ortho-Alkenyl Anilides with Alkynes: Formation of Unexpected Naphthalene Adducts.

o-Alkenyl N-triflylanilides underwent rhodium(III)-catalyzed oxidative annulations with alkynes to produce different types of naphthylamides in a process which involves the cleavage of two C-H bonds. Remarkably, besides formal dehydrogenative (4C+2C) cycloadducts, the reaction also produces variable amounts of isomeric naphthylamides, whose formation requires a formal migration of the alkenyl moiety from the ortho to the meta position of the anilide. The annulation reaction can be efficiently carried out in the absence of external oxidants, such as Cu(OAc)2 .

Tight Xenon Confinement in a Crystalline Sandwich-like Hydrogen-Bonded Dimeric Capsule of a Cyclic Peptide.

A cyclic hexapeptide with three pyridyl moieties connected to its backbone forms a hydrogen-bonded dimer, which tightly encapsulates a single xenon atom, like a pearl in its shell. The dimer imprints its shape and symmetry to the captured xenon atom, as demonstrated by 129 Xe NMR spectroscopy, single-crystal X-ray diffraction, and computational studies. The dimers self-assemble hierarchically into tubular structures to form a porous supramolecular architecture, whose cavities are filled by small molecules and gases.

A Single Mutation is Sufficient to Modify the Metal Selectivity and Specificity of a Eukaryotic Manganese Superoxide Dismutase to Encompass Iron.

We have generated a site-directed mutant of the manganese superoxide dismutase SOD-3 of C.elegans (MnSOD-3) which modifies the metal specificity of the enzyme. While wild-type MnSOD-3 functions with manganese in the active site (3600 U mg-1 of protein) it has little or no activity when iron is incorporated. However, when histidine replaces glutamine 142 in the active site, the enzyme retains 50 % of its activity and becomes cambialistic for its metal cofactor exhibiting very similar specific activity with either manganese or iron.

Cyclo-lib: a database of computational molecular dynamics simulations of cyclodextrins.

MOTIVATION: Cyclodextrins (CDs) are amongst the most versatile/multi-functional molecules used in molecular research and chemical applications. They are natural cyclic oligosaccharides typically employed to encapsulate hydrophobic groups in their central cavity. This allows solubilizing, protecting or reducing the toxicity of a large variety of different molecules including drugs, dyes and surfactant agents. In spite of their great potential, atomic level information of these molecules, which is key for their function, is really scarce. Computational Molecular Dynamics (MD) simulations have the potential to efficiently fill this gap, providing structural-dynamic information at atomic level in time scales ranging from ps to µs. RESULTS: Cyclo-lib is a database with a publicly accessible web-interface containing structural and dynamic analysis obtained from computational MD simulation trajectories (250 ns long) of native and modified CDs in explicit water molecules. Cyclo-lib currently includes 70 CDs typically employed for fundamental and industrial research. Tools for comparative analysis between different CDs, as well as to restrict the analysis to specific time-segments within the trajectories are also available. Cyclo-lib provides atomic resolution information aimed to complement experimental results performed with the same molecules. AVAILABILITY AND IMPLEMENTATION: The database is freely available under http://cyclo-lib.mduse.com/ CONTACT: Angel.Pineiro@usc.es.

Molecular dynamics simulations for designing biomimetic pores based on internally functionalized self-assembling α,γ-peptide nanotubes.

A molecular dynamics study on internally functionalized peptide nanotubes composed of α- and γ-amino acids self-assembled in lipid bilayers is presented. One of the main advantages of peptide nanotubes composed of γ-amino acids is that the properties of their inner cavities can be tuned by introducing different functions on ß-carbon of the γ-amino acid. In the work described here we studied the effect of the presence of different numbers of hydroxyl groups in different positions in the lumen of these channels when they are inserted into a lipid bilayer and assessed how they affect the structural and dynamic behavior of the modified peptide nanotubes as well as the transmembrane transport of different ions. The results provided atomic information about the effect of polar groups on the dynamic, structural and transport properties of this type of peptidic channel upon insertion into lipid bilayers, projecting a promising future for their use as biomimetic channels when properly inner-derivatized. Furthermore, the chemical versatility of the hydroxyl groups in the lumen of the peptide nanotubes would enable appealing applications for these channels, such as a controlled method for the activation/inactivation of the transmembrane transport along the nanopore.

Designing biomimetic pores based on carbon nanotubes.

Biomimetic nanopores based on membrane-spanning single-walled carbon nanotubes have been designed to include selectivity filters based on combinations of anionic and cationic groups mimicking those present in bacterial porins and in voltage-gated sodium and calcium channels. The ion permeation and selectivity properties of these nanopores when embedded in a phospholipid bilayer have been explored by molecular dynamics simulations and free energy profile calculations. The interactions of the nanopores with sodium, potassium, calcium, and chloride ions have been explored as a function of the number of anionic and cationic groups within the selectivity filter. Unbiased molecular dynamics simulations show that the overall selectivity is largely determined by the net charge of the filter. Analysis of distribution functions reveals considerable structuring of the distribution of ions and water within the nanopores. The distributions of ions along the pore axis reveal local selectivity for cations around filter, even in those nanopores (C0) where the net filter charge is zero. Single ion free energy profiles also reveal clear evidence for cation selectivity, even in the C0 nanopores. Detailed analysis of the interactions of the C0 nanopore with Ca(2+) ions reveals that local interactions with the anionic (carboxylate) groups of the selectivity filter lead to (partial) replacement of solvating water as the ion passes through the pore. These studies suggest that a computational biomimetic approach can be used to evaluate our understanding of the design principles of nanopores and channels.

Self-Sorting of cyclic peptide homodimers into a heterodimeric assembly featuring an efficient photoinduced intramolecular electron-transfer process.

We describe the thermodynamic characterisation of the self-sorting process experienced by two homodimers assembled by hydrogen-bonding interactions through their cyclopeptide scaffolds and decorated with Zn-porphyrin and fullerene units into a heterodimeric assembly that contains one electron-donor (Zn­porphyrin) and one electron-acceptor group (fullerene). The fluorescence of the Zn-porphyrin unit is strongly quenched upon heterodimer formation. This phenomenon is demonstrated to be the result of an efficient photoinduced electron-transfer (PET) process occurring between the Zn-porphyrin and the fullerene units of the heterodimeric system. The recombination lifetime of the charge-separated state of the heterodimer complex is in the order of 180 ns. In solution, both homo- and heterodimers are present as a mixture of three regioisomers: two staggered and one eclipsed. At the concentration used for this study, the high stability constant determined for the heterodimer suggests that the eclipsed conformer is the main component in solution. The application of the bound-state scenario allowed us to calculate that the heterodimer exists mainly as the eclipsed regioisomer (75-90 %). The attractive interaction that exists between the donor and acceptor chromophores in the heterodimeric assembly favours their arrangement in close contact. This is confirmed by the presence of charge-transfer bands centred at 720 nm in the absorption spectrum of the heterodimer. PET occurs in approximately 75% of the chromophores after excitation of both Zn-porphyrin and fullerene chromophores. Conversely, analogous systems, reported previously, decorated with extended tetrathiafulvalene and fullerene units showed a PET process in a significantly reduced extent (33%). We conclude that the strength (stability constant (K) x effective molarity (EM)) of the intramolecular interaction established between the two chromophores in the Zn-porphyrin/fullerene cyclopeptide-based heterodimers controls the regioisomeric distribution and regulates the high extent to which the PET process takes place in this system.

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.

Cyclodextrins: Establishing building blocks for AI-driven drug design by determining affinity constants in silico.

Cyclodextrins (CDs) are cyclic carbohydrate polymers that hold significant promise for drug delivery and industrial applications. Their effectiveness depends on their ability to encapsulate target molecules with strong affinity and specificity, but quantifying affinities in these systems accurately is challenging for a variety of reasons. Computational methods represent an exceptional complement to in vitro assays because they can be employed for existing and hypothetical molecules, providing high resolution structures in addition to a mechanistic, dynamic, kinetic, and thermodynamic characterization. Here, we employ potential of mean force (PMF) calculations obtained from guided metadynamics simulations to characterize the 1:1 inclusion complexes between four different modified ßCDs, with different type, number, and location of substitutions, and two sterol molecules (cholesterol and 7-ketocholesterol). Our methods, validated for reproducibility through four independent repeated simulations per system and different post processing techniques, offer new insights into the formation and stability of CD-sterol inclusion complexes. A systematic distinct orientation preference where the sterol tail projects from the CD's larger face and significant impacts of CD substitutions on binding are observed. Notably, sampling only the CD cavity's wide face during simulations yielded comparable binding energies to full-cavity sampling, but in less time and with reduced statistical uncertainty, suggesting a more efficient approach. Bridging computational methods with complex molecular interactions, our research enables predictive CD designs for diverse applications. Moreover, the high reproducibility, sensitivity, and cost-effectiveness of the studied methods pave the way for extensive studies of massive CD-ligand combinations, enabling AI algorithm training and automated molecular design.

Unraveling the molecular dynamics of sugammadex-rocuronium complexation: A blueprint for cyclodextrin drug design.

Sugammadex, marketed as Bridion™, is an approved cyclodextrin (CD) based drug for the reversal of neuromuscular blockade in adults undergoing surgery. Sugammadex forms an inclusion complex with the neuromuscular blocking agent (NMBA) rocuronium, allowing rapid reversal of muscle paralysis. In silico methods have been developed for studying CD inclusion complexes, aimed at accurately predicting their structural, energetic, dynamic, and kinetic properties, as well as binding constants. Here, a computational study aimed at characterizing the sugammadex-rocuronium system from the perspective of docking calculations, free molecular dynamics (MD) simulations, and biased metadynamics simulations with potential of mean force (PMF) calculations is presented. The aim is to provide detailed information about this system, as well as to use it as a model system for validation of the methods. This method predicts results in line with experimental evidence for both the optimal structure and the quantitative value for the binding constant. Interestingly, there is a less profound preference for the orientation than might be assumed based on electrostatic interactions, suggesting that both orientations may exist in solution. These results show that this technology can efficiently analyze CD inclusion complexes and could be used to facilitate the development and optimization of novel applications for CDs.

CYCLOPEp Builder: Facilitating cyclic peptide and nanotube research through a user-friendly web platform.

The study of cyclic peptides (CPs) and self-assembling cyclic peptide nanotubes (SCPNs) is pivotal in advancing applications in diverse fields such as biomedicine, nanoelectronics, and catalysis. Recognizing the limitations in the experimental study of these molecules, this article introduces CYCLOPEp Builder, a comprehensive web-based application designed to facilitate the design, simulation, and visualization of CPs and SCPNs. The tool is engineered to generate molecular topologies, essential for conducting Molecular Dynamics simulations that span All-Atom to Coarse-Grain resolutions. CYCLOPEp Builder's user-friendly interface simplifies the complex process of molecular modeling, providing researchers with the ability to readily construct CPs and SCPNs. The platform is versatile, equipped with various force fields, and capable of producing structures ranging from individual CPs to complex SCPNs with different sequences, offering parallel and antiparallel orientations among them. By enhancing the capacity for detailed visualization of molecular assemblies, CYCLOPEp Builder improves the understanding of CP and SCPN molecular interactions. This tool is a step forward in democratizing access to sophisticated simulations, offering an invaluable resource to the scientific community engaged in the exploration of supramolecular structures. CYCLOPEp is accessible at http://cyclopep.com/.

Unraveling lipid and inflammation interplay in cancer, aging and infection for novel theranostic approaches.

The synergistic relationships between Cancer, Aging, and Infection, here referred to as the CAIn Triangle, are significant determinants in numerous health maladies and mortality rates. The CAIn-related pathologies exhibit close correlations with each other and share two common underlying factors: persistent inflammation and anomalous lipid concentration profiles in the membranes of affected cells. This study provides a comprehensive evaluation of the most pertinent interconnections within the CAIn Triangle, in addition to examining the relationship between chronic inflammation and specific lipidic compositions in cellular membranes. To tackle the CAIn-associated diseases, a suite of complementary strategies aimed at diagnosis, prevention, and treatment is proffered. Our holistic approach is expected to augment the understanding of the fundamental mechanisms underlying these diseases and highlight the potential of shared features to facilitate the development of novel theranostic strategies.

Overlay databank unlocks data-driven analyses of biomolecules for all.

Tools based on artificial intelligence (AI) are currently revolutionising many fields, yet their applications are often limited by the lack of suitable training data in programmatically accessible format. Here we propose an effective solution to make data scattered in various locations and formats accessible for data-driven and machine learning applications using the overlay databank format. To demonstrate the practical relevance of such approach, we present the NMRlipids Databank-a community-driven, open-for-all database featuring programmatic access to quality-evaluated atom-resolution molecular dynamics simulations of cellular membranes. Cellular membrane lipid composition is implicated in diseases and controls major biological functions, but membranes are difficult to study experimentally due to their intrinsic disorder and complex phase behaviour. While MD simulations have been useful in understanding membrane systems, they require significant computational resources and often suffer from inaccuracies in model parameters. Here, we demonstrate how programmable interface for flexible implementation of data-driven and machine learning applications, and rapid access to simulation data through a graphical user interface, unlock possibilities beyond current MD simulation and experimental studies to understand cellular membranes. The proposed overlay databank concept can be further applied to other biomolecules, as well as in other fields where similar barriers hinder the AI revolution.

Defining the nature of thermal intermediate in 3 state folding proteins: apoflavodoxin, a study case.

The early stages of the thermal unfolding of apoflavodoxin have been determined by using atomistic multi microsecond-scale molecular dynamics (MD) simulations complemented with a variety of experimental techniques. Results strongly suggest that the intermediate is reached very early in the thermal unfolding process and that it has the properties of an "activated" form of the native state, where thermal fluctuations in the loops break loop-loop contacts. The unrestrained loops gain then kinetic energy corrupting short secondary structure elements without corrupting the core of the protein. The MD-derived ensembles agree with experimental observables and draw a picture of the intermediate state inconsistent with a well-defined structure and characteristic of a typical partially disordered protein. Our results allow us to speculate that proteins with a well packed core connected by long loops might behave as partially disordered proteins under native conditions, or alternatively behave as three state folders. Small details in the sequence, easily tunable by evolution, can yield to one or the other type of proteins.