Development of a Semi-Mechanistic Modeling Framework for Wet Bead Milling of Pharmaceutical Nanosuspensions.

This study aimed to develop a practical semi-mechanistic modeling framework to predict particle size evolution during wet bead milling of pharmaceutical nanosuspensions over a wide range of process conditions and milling scales. The model incorporates process parameters, formulation parameters, and equipment-specific parameters such as rotor speed, bead type, bead size, bead loading, active pharmaceutical ingredient (API) mass, temperature, API loading, maximum bead volume, blade diameter, distance between blade and wall, and an efficiency parameter. The characteristic particle size quantiles, i.e., x10, x50, and x90, were transformed to obtain a linear relationship with time, while the general functional form of the apparent breakage rate constant of this relationship was derived based on three models with different complexity levels. Model A, the most complex and general model, was derived directly from microhydrodynamics. Model B is a simpler model based on a power-law function of process parameters. Model C is the simplest model, which is the pre-calibrated version of Model B based on data collected from different mills across scales, formulations, and drug products. Being simple and computationally convenient, Model C is expected to reduce the amount of experimentation needed to develop and optimize the wet bead milling process and streamline scale-up and/or scale-out.

Structure of native four-repeat satellite III sequence with non-canonical base interactions.

Tandem-repetitive DNA (where two or more DNA bases are repeated numerous times) can adopt non-canonical secondary structures. Many of these structures are implicated in important biological processes. Human Satellite III (HSat3) is enriched for tandem repeats of the sequence ATGGA and is located in pericentromeric heterochromatin in many human chromosomes. Here, we investigate the secondary structure of the four-repeat HSat3 sequence 5'-ATGGA ATGGA ATGGA ATGGA-3' using X-ray crystallography, NMR, and biophysical methods. Circular dichroism spectroscopy, thermal stability, native PAGE, and analytical ultracentrifugation indicate that this sequence folds into a monomolecular hairpin with non-canonical base pairing and B-DNA characteristics at concentrations below 0.9 mM. NMR studies at 0.05-0.5 mM indicate that the hairpin is likely folded-over into a compact structure with high dynamics. Crystallographic studies at 2.5 mM reveal an antiparallel self-complementary duplex with the same base pairing as in the hairpin, extended into an infinite polymer. The non-canonical base pairing includes a G-G intercalation sandwiched by sheared A-G base pairs, leading to a cross-strand four guanine stack, so called guanine zipper. The guanine zippers are spaced throughout the structure by A-T/T-A base pairs. Our findings lend further insight into recurring structural motifs associated with the HSat3 and their potential biological functions.

Aptamer-Targeted Dendrimersomes Assembled from Azido-Modified Janus Dendrimers "Clicked" to DNA.

Amphiphilic Janus dendrimers (JDs), synthetic alternatives to lipids, have the potential to expand the scope of nanocarrier delivery systems. JDs self-assemble into vesicles called dendrimersomes, encapsulate both hydrophobic cargo and nucleic acids, and demonstrate enhanced stability in comparison to lipid nanoparticles (LNPs). Here, we report the ability to enhance the cellular uptake of Janus dendrimersomes using DNA aptamers. Azido-modified JDs were synthesized and conjugated to alkyne-modified DNAs using copper-catalyzed azide alkyne cycloaddition. DNA-functionalized JDs form nanometer-sized dendrimersomes in aqueous solution via thin film hydration. These vesicles, now displaying short DNAs, are then hybridized to transferrin receptor binding DNA aptamers. Aptamer-targeted dendrimersomes show improved cellular uptake as compared to control vesicles via fluorescence microscopy and flow cytometry. This work demonstrates the versatility of using click chemistry to conjugate functionalized JDs with biologically relevant molecules and the feasibility of targeting DNA-modified dendrimersomes for drug delivery applications.

Interactions of ruthenium(II) polypyridyl complexes with human telomeric DNA.

Eight [Ru(bpy)2L]2+ and three [Ru(phen)2L]2+complexes (where bpy = 2,2'-bipyridine and phen = 1,10-phenanthroline are ancillary ligands, and L = a polypyridyl experimental ligand) were investigated for their G-quadruplex binding abilities. Fluorescence resonance energy transfer melting assays were used to screen these complexes for their ability to selectively stabilize human telomeric DNA variant, Tel22. The best G-quadruplex stabilizers were further characterized for their binding properties (binding constant and stoichiometry) using UV-vis, fluorescence spectroscopy, and mass spectrometry. The ligands' ability to alter the structure of Tel22 was determined via circular dichroism and PAGE studies. We identified me2allox as the experimental ligand capable of conferring excellent stabilizing ability and good selectivity to polypyridyl Ru(II) complexes. Replacing bpy by phen did not significantly impact interactions with Tel22, suggesting that binding involves mostly the experimental ligand. However, using a particular ancillary ligand can help fine-tune G-quadruplex-binding properties of Ru(II) complexes. Finally, the fluorescence "light switch" behavior of all Ru(II) complexes in the presence of Tel22 G-quadruplex was explored. All Ru(II) complexes displayed "light switch" properties, especially [Ru(bpy)2(diamino)]2+, [Ru(bpy)2(dppz)]2+, and [Ru(bpy)2(aap)]2+. Current work sheds light on how Ru(II) polypyridyl complexes interact with human telomeric DNA with possible application in cancer therapy or G-quadruplex sensing.

Homopurine guanine-rich sequences in complex with N-methyl mesoporphyrin IX form parallel G-quadruplex dimers and display a unique symmetry tetrad.

DNA can fold into G-quadruplexes (GQs), non-canonical secondary structures formed by π-π stacking of G-tetrads. GQs are important in many biological processes, which makes them promising therapeutic targets. We identified a 42-nucleotide long, purine-only G-rich sequence from human genome, which contains eight G-stretches connected by A and AAAA loops. We divided this sequence into five unique segments, four guanine stretches each, named GA1-5. In order to investigate the role of adenines in GQ structure formation, we performed biophysical and X-ray crystallographic studies of GA1-5 and their complexes with a highly selective GQ ligand, N-methyl mesoporphyrin IX (NMM). Our data indicate that all variants form parallel GQs whose stability depends on the number of flexible AAAA loops. GA1-3 bind NMM with 1:1 stoichiometry. The Ka for GA1 and GA3 is modest, ∼0.3 µM -1, and that for GA2 is significantly higher, ∼1.2 µM -1. NMM stabilizes GA1-3 by 14.6, 13.1, and 7.0 °C, respectively, at 2 equivalents. We determined X-ray crystal structures of GA1-NMM (1.98 Å resolution) and GA3-NMM (2.01 Å). The structures confirm the parallel topology of GQs with all adenines forming loops and display NMM binding at the 3' G-tetrad. Both complexes dimerize through the 5' interface. We observe two novel structural features: 1) a 'symmetry tetrad' at the dimer interface, which is formed by two guanines from each GQ monomer and 2) a NMM dimer in GA1-NMM. Our structural work confirms great flexibility of adenines as structural elements in GQ formation and contributes greatly to our understanding of the structural diversity of GQs and their modes of interaction with small molecule ligands.

The first crystal structures of hybrid and parallel four-tetrad intramolecular G-quadruplexes.

G-quadruplexes (GQs) are non-canonical DNA structures composed of stacks of stabilized G-tetrads. GQs play an important role in a variety of biological processes and may form at telomeres and oncogene promoters among other genomic locations. Here, we investigate nine variants of telomeric DNA from Tetrahymena thermophila with the repeat (TTGGGG)n. Biophysical data indicate that the sequences fold into stable four-tetrad GQs which adopt multiple conformations according to native PAGE. Excitingly, we solved the crystal structure of two variants, TET25 and TET26. The two variants differ by the presence of a 3'-T yet adopt different GQ conformations. TET25 forms a hybrid [3 + 1] GQ and exhibits a rare 5'-top snapback feature. Consequently, TET25 contains four loops: three lateral (TT, TT, and GTT) and one propeller (TT). TET26 folds into a parallel GQ with three TT propeller loops. To the best of our knowledge, TET25 and TET26 are the first reported hybrid and parallel four-tetrad unimolecular GQ structures. The results presented here expand the repertoire of available GQ structures and provide insight into the intricacy and plasticity of the 3D architecture adopted by telomeric repeats from T. thermophila and GQs in general.

Evolution of the SARS-CoV-2 proteome in three dimensions (3D) during the first 6 months of the COVID-19 pandemic.

Understanding the molecular evolution of the SARS-CoV-2 virus as it continues to spread in communities around the globe is important for mitigation and future pandemic preparedness. Three-dimensional structures of SARS-CoV-2 proteins and those of other coronavirusess archived in the Protein Data Bank were used to analyze viral proteome evolution during the first 6 months of the COVID-19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in >48 000 viral isolates revealed how each one of 29 viral proteins have undergone amino acid changes. Catalytic residues in active sites and binding residues in protein-protein interfaces showed modest, but significant, numbers of substitutions, highlighting the mutational robustness of the viral proteome. Energetics calculations showed that the impact of substitutions on the thermodynamic stability of the proteome follows a universal bi-Gaussian distribution. Detailed results are presented for potential drug discovery targets and the four structural proteins that comprise the virion, highlighting substitutions with the potential to impact protein structure, enzyme activity, and protein-protein and protein-nucleic acid interfaces. Characterizing the evolution of the virus in three dimensions provides testable insights into viral protein function and should aid in structure-based drug discovery efforts as well as the prospective identification of amino acid substitutions with potential for drug resistance.

Evolution of the SARS-CoV-2 proteome in three dimensions (3D) during the first six months of the COVID-19 pandemic.

Three-dimensional structures of SARS-CoV-2 and other coronaviral proteins archived in the Protein Data Bank were used to analyze viral proteome evolution during the first six months of the COVID-19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in >48,000 viral proteome sequences showed how each one of the 29 viral study proteins have undergone amino acid changes. Structural models computed for every unique sequence variant revealed that most substitutions map to protein surfaces and boundary layers with a minority affecting hydrophobic cores. Conservative changes were observed more frequently in cores versus boundary layers/surfaces. Active sites and protein-protein interfaces showed modest numbers of substitutions. Energetics calculations showed that the impact of substitutions on the thermodynamic stability of the proteome follows a universal bi-Gaussian distribution. Detailed results are presented for six drug discovery targets and four structural proteins comprising the virion, highlighting substitutions with the potential to impact protein structure, enzyme activity, and functional interfaces. Characterizing the evolution of the virus in three dimensions provides testable insights into viral protein function and should aid in structure-based drug discovery efforts as well as the prospective identification of amino acid substitutions with potential for drug resistance.