Search

Nuno Martinho,1–3 Liana C Silva,1,4 Helena F Florindo,1 Steve Brocchini,2 Mire Zloh,3 Teresa S Barata2 1Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal; 2Department of Pharmaceutics, UCL School of Pharmacy, London, 3School of Life and Medical Sciences, University of Hertfordshire, Hatfield, UK; 4Centro de Química-Física Molecular and IN – Institute of Nanoscience and Nanotechnology, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal Abstract: Dendrimers are hyperbranched polymers with a multifunctional architecture that can be tailored for the use in various biomedical applications. Peptide dendrimers are particularly relevant for drug delivery applications due to their versatility and safety profile. The overall lack of knowledge of their three-dimensional structure, conformational behavior and structure–activity relationship has slowed down their development. Fluorophores are often conjugated to dendrimers to study their interaction with biomolecules and provide information about their mechanism of action at the molecular level. However, these probes can change dendrimer surface properties and have a direct impact on their interactions with biomolecules and with lipid membranes. In this study, we have used computer-aided molecular design and molecular dynamics simulations to identify optimal topology of a poly(L-glutamic acid) (PG) backbone dendrimer that allows incorporation of fluorophores in the core with minimal availability for undesired interactions. Extensive all-atom molecular dynamic simulations with the CHARMM force field were carried out for different generations of PG dendrimers with the core modified with a fluorophore (nitrobenzoxadiazole and Oregon Green 488) and various surface groups (glutamic acid, lysine and tryptophan). Analysis of structural and topological features of all designed dendrimers provided information about their size, shape, internal distribution and dynamic behavior. We have found that four generations of a PG dendrimer are needed to ensure minimal exposure of a core-conjugated fluorophore to external environment and absence of undesired interactions regardless of the surface terminal groups. Our findings suggest that NBD-PG-G4 can provide a suitable scaffold to be used for biophysical studies of surface-modified dendrimers to provide a deeper understanding of their intermolecular interactions, mechanisms of action and trafficking in a biological system. Keywords: dendrimers, peptide dendrimers, molecular dynamics, fluorescence, CHARMM, structure-activity, surface properties

The design of stable formulations remains a major challenge for protein therapeutics, particularly the need to minimize aggregation. Experimental formulation screens are typically based on thermal transition midpoints ( T m ), and forced degradation studies at elevated temperatures. Both approaches give limited predictions of long-term storage stability, particularly at low temperatures. Better understanding of the mechanisms of action for formulation of excipients and buffers could lead to improved strategies for formulation design. Here, we identified a complex impact of glycine concentration on the experimentally determined stability of an antibody Fab fragment and then used molecular dynamics simulations to reveal mechanisms that underpin these complex behaviors. T m values increased monotonically with glycine concentration, but associated Δ S vh measurements revealed more complex changes in the native ensemble dynamics, which reached a maximum at 30 mg/mL. The aggregation kinetics at 65 °C were similar at 0 and 20 mg/mL glycine, but then significantly slower at 50 mg/mL. These complex behaviors indicated changes in the dominant stabilizing mechanisms as the glycine concentration was increased. MD revealed a complex balance of glycine self-interaction, and differentially preferred interactions of glycine with the Fab as it displaced hydration-shell water, and surface-bound water and citrate buffer molecules. As a result, glycine binding to the Fab surface had different effects at different concentrations, and led from preferential interactions at low concentrations to preferential exclusion at higher concentrations. During preferential interaction, glycine displaced water from the Fab hydration shell, and a small number of water and citrate molecules from the Fab surface, which reduced the protein dynamics as measured by root-mean-square fluctuation (RMSF) on the short time scales of MD. By contrast, the native ensemble dynamics increased according to Δ S vh , suggesting increased conformational changes on longer time scales. The aggregation kinetics did not change at low glycine concentrations, and so the opposing dynamics effects either canceled out or were not directly relevant to aggregation. During preferential exclusion at higher glycine concentrations, glycine could only bind to the Fab surface through the displacement of citrate buffer molecules already favorably bound on the Fab surface. Displacement of citrate increased the flexibility (RMSF) of the Fab, as glycine formed fewer bridging hydrogen bonds to the Fab surface. Overall, the slowing of aggregation kinetics coincided with reduced flexibility in the Fab ensemble at the very highest glycine concentrations, as determined by both RMSF and Δ S vh , and occurred at a point where glycine binding displaced neither water nor citrate. These final interactions with the Fab surface were driven by mass action and were the least favorable, leading to a macromolecular crowding effect under the regime of preferential exclusion that stabilized the dynamics of Fab.

Botulinum Neurotoxins (BoNT) are the most potent toxins currently known. However, they also have therapeutic applications for an increasing number of motor related conditions due to their specificity, and low diffusion into the system. Although the start- and end- points for the BoNT mechanism of action are well-studied, a critical step remains poorly understood. It is theorised that BoNTs undergo a pH-triggered conformational shift, activating the neurotoxin by priming it to form a transmembrane (TM) channel. To test this hypothesis, we combined molecular dynamics (MD) simulations and small-angle x-ray scattering (SAXS), revealing a new conformation of serotype E (BoNT/E). This conformation was exclusively observed in simulations below pH 5.5, as determined by principal component analysis (PCA), and its theoretical SAXS profile matched an experimental SAXS profile obtained at pH 4. Additionally, a localised secondary structural change was observed in MD simulations below pH 5.5, in a region previously identified as instrumental for membrane insertion for serotype A (BoNT/A). These changes were found at a critical pH value for BoNTs in vivo, and may be relevant for their therapeutic use., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. K. A. Bunting and D. Higazi are employed by IPSEN., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Dendrimers are well-defined hyperbranched polymers built from a variety of different monomers and with tuneable properties that make them suitable for different biomedical applications. Their three-dimensional (3D) structure cannot be usually determined experimentally due to their inherent nature of repeating patterns in the topology, failure to crystalize, and/or high flexibility. Therefore, their conformations and interactions at the atomistic level can be studied only by using computational chemistry methods, including molecular dynamics, Monte Carlo simulations, and molecular docking., Areas Covered: In this review, the methods that could be utilized in computer-aided dendrimer design are considered, providing a list of approaches to generate initial 3D coordinates and selected examples of applications of relevant molecular modeling methods., Expert Opinion: Computational chemistry provides an invaluable set of tools to study dendrimers and their interactions with drugs and biological targets. There is a gap in the software development that is dedicated to study of these highly variable and complex systems that could be overcome by the integration of already established approaches for topology generation and open source molecular modeling libraries. Furthermore, it would be highly beneficial to collate already built 3D models of various dendrimers with corresponding relevant experimental data.

Computational techniques are useful to predict interaction models and molecular properties for the design of drug delivery systems, such as dendrimers. This work evaluated the impact of surface modifications of mannosamine-conjugated multifunctional poly(glutamic acid) (PG)-dendrimers as nanocarriers of the tumour associated antigens (TAA) MART-1, gp100:44 and gp100:209. Molecular dynamics simulations and docking studies were performed. Nitrobenzoxadiazole (NBD)-PG-G4-dendrimer displayed 64 carboxylic groups, however, the Frontier Molecular Orbital Theory study evidenced that only 32 of those were available to form covalent bonds. When the number of mannosamines conjugated to dendrimer was increased from 16 to 32, the dendrimer interacted with the receptor with higher affinity. However, 16 mannosamines-NBD-PG-G4-dendrimer was chosen to conjugate TAA for added functionality as no carboxylic end groups were available for further conjugation in the 32 mannosamines-dendrimer. Docking results showed that the majority of TAA-conjugated NBD-PG-G4-dendrimer demonstrated a favourable interaction with mannosamine binding site on mannose receptor, thus constituting a promising tool for TAA targeted delivery. Our in silico approach effectively narrows down the selection of the best candidates for the synthesis of functionalised PG-dendrimers with desired functionalities. These results will significantly reduce the time and efforts required to experimentally synthesise modified dendrimers for optimal antigen delivery.

Protein formulation development relies on the selection of excipients that inhibit protein-protein interactions preventing aggregation. Empirical strategies involve screening many excipient and buffer combinations using force degradation studies. Such methods do not readily provide information on intermolecular interactions responsible for the protective effects of excipients. This study describes a molecular docking approach to screen and rank interactions allowing for the identification of protein-excipient hotspots to aid in the selection of excipients to be experimentally screened. Previously published work with Drosophila Su(dx) was used to develop and validate the computational methodology, which was then used to determine the formulation hotspots for Fab A33. Commonly used excipients were examined and compared to the regions in Fab A33 prone to protein-protein interactions that could lead to aggregation. This approach could provide information on a molecular level about the protective interactions of excipients in protein formulations to aid the more rational development of future formulations.

Cancer is one of the most common diseases afflicting people globally. New therapeutic approaches are needed due to the complexity of cancer as a disease. Many current treatments are very toxic and have modest efficacy at best. Increased understanding of tumor biology and immunology has allowed the development of specific immunotherapies with minimal toxicity. It is important to highlight the performance of monoclonal antibodies, immune adjuvants, vaccines and cell-based treatments. Although these approaches have shown varying degrees of clinical efficacy, they illustrate the potential to develop new strategies. Targeted immunotherapy is being explored to overcome the heterogeneity of malignant cells and the immune suppression induced by both the tumor and its microenvironment. Nanodelivery strategies seek to minimize systemic exposure to target therapy to malignant tissue and cells. Intracellular penetration has been examined through the use of functionalized particulates. These nano-particulate associated medicines are being developed for use in imaging, diagnostics and cancer targeting. Although nano-particulates are inherently complex medicines, the ability to confer, at least in principle, different types of functionality allows for the plausible consideration these nanodelivery strategies can be exploited for use as combination medicines. The development of targeted nanodelivery systems in which therapeutic and imaging agents are merged into a single platform is an attractive strategy. Currently, several nanoplatform-based formulations, such as polymeric nanoparticles, micelles, liposomes and dendrimers are in preclinical and clinical stages of development. Herein, nanodelivery strategies presently investigated for cancer immunotherapy, cancer targeting mechanisms and nanocarrier functionalization methods will be described. We also intend to discuss the emerging nano-based approaches suitable to be used as imaging techniques and as cancer treatment options.

Intestinal pathogens use the host's excessive inflammatory cytokine response, designed to eliminate dangerous bacteria, to disrupt epithelial gut wall integrity and promote their tissue invasion. We sought to develop a non-antibiotic-based approach to prevent this injury. Molecular docking studies suggested that glycosylated dendrimers block the TLR4-MD-2-LPS complex, and a 13.6 kDa polyamidoamine (PAMAM) dendrimer glucosamine (DG) reduced the induction of human monocyte interleukin (IL)-6 by Gram-negative bacteria. In a rabbit model of shigellosis, PAMAM-DG prevented epithelial gut wall damage and intestinal villous destruction, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. Computational modelling studies identified a 3.3 kDa polypropyletherimine (PETIM)-DG as the smallest likely bioactive molecule. In human monocytes, high purity PETIM-DG potently inhibited Shigella Lipid A-induced IL-6 expression. In rabbits, PETIM-DG prevented Shigella-induced epithelial gut wall damage, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. There was no change in β-defensin, IL-10, interferon-β, transforming growth factor-β, CD3 or FoxP3 expression. Small and orally delivered DG could be useful for preventing gut wall tissue damage in a wide spectrum of infectious diarrhoeal diseases., (Copyright © 2012 EMBO Molecular Medicine.)

The molecular modeling of hyperbranched molecules is currently constrained by difficulties in model building, due partly to lack of parameterization of their building blocks. We have addressed this problem with specific relevance to a class of hyperbranched macromolecules known as dendrimers by describing a new concept and developing a method that translates monomeric linear sequences into a full atomistic model of a hyperbranched molecule. Such molecular-modeling-based advances will enable modeling studies of important biological interactions between naturally occurring macromolecules and synthetic macromolecules. Our results also suggest that it should be possible to apply this sequence-based methodology to generate hyperbranched structures of other dendrimeric structures and of linear polymers.

The partial modification of carboxylic acid terminated polyamidoamine (PAMAM) dendrimers with glucosamine has been reported to give dendrimer glucosamine conjugates novel immuno-modulatory and anti-angiogenic properties. Experimental analysis of these glycosylated dendrimers showed that, on average, eight glucosamine molecules were covalently bound to each dendrimer. In order to better understand the surface loading and distribution of these glucosamine molecules, molecular reactivity was determined by evaluation of electronic properties using frontier molecular orbital theory (FMOT) and molecular dynamics simulations. It was shown that the surface loading and distribution of zero length amide bond-conjugated glucosamine molecules was determined by both electronic effects and by the different dynamic conformations adopted by the modified dendrimer during the incremental addition of glucosamine. Importantly, the structural features and the dynamic behavior of the partially glycosylated generation 3.5 PAMAM dendrimer showed that its flexibility and polarity changed with the incremental addition of glucosamine. These peripheral glucosamine molecules remained available on the dendrimer's surface for interaction with the biological target.

The crystal structure of the TLR4-MD-2-LPS complex responsible for triggering powerful pro-inflammatory cytokine responses has recently become available. Central to cell surface complex formation is binding of lipopolysaccharide (LPS) to soluble MD-2. We have previously shown, in biologically based experiments, that a generation 3.5 PAMAM dendrimer with 64 peripheral carboxylic acid groups acts as an antagonist of pro-inflammatory cytokine production after surface modification with 8 glucosamine molecules. We have also shown using molecular modelling approaches that this partially glycosylated dendrimer has the flexibility, cluster density, surface electrostatic charge, and hydrophilicity to make it a therapeutically useful antagonist of complex formation. These studies enabled the computational study of the interactions of the unmodified dendrimer, glucosamine, and of the partially glycosylated dendrimer with TLR4 and MD-2 using molecular docking and molecular dynamics techniques. They demonstrate that dendrimer glucosamine forms co-operative electrostatic interactions with residues lining the entrance to MD-2's hydrophobic pocket. Crucially, dendrimer glucosamine interferes with the electrostatic binding of: (i) the 4'phosphate on the di-glucosamine of LPS to Ser118 on MD-2; (ii) LPS to Lys91 on MD-2; (iii) the subsequent binding of TLR4 to Tyr102 on MD-2. This is followed by additional co-operative interactions between several of the dendrimer glucosamine's carboxylic acid branches and MD-2. Collectively, these interactions block the entry of the lipid chains of LPS into MD-2's hydrophobic pocket, and also prevent TLR4-MD-2-LPS complex formation. Our studies have therefore defined the first nonlipid-based synthetic MD-2 antagonist using both animal model-based studies of pro-inflammatory cytokine responses and molecular modelling studies of a whole dendrimer with its target protein. Using this approach, it should now be possible to computationally design additional macromolecular dendrimer based antagonists for other Toll Like Receptors. They could be useful for treating a spectrum of infectious, inflammatory and malignant diseases.