Tracking Prenucleation Molecular Clustering of Salicylamide in Organic Solvents.

Crystal nucleation shapes the structure and product size distribution of solid-state pharmaceuticals and is seeded by early-stage molecular self-assemblies formed in host solution. Here, molecular clustering of salicylamide in ethyl acetate, methanol, and acetonitrile was investigated using photon correlation spectroscopy. Cluster size steadily increased over 3 days and with concentration across the range from undersaturated to supersaturated solutions. Solute concentration normalized by solubility provided more sensitive characterization of molecular-level conditions than concentration alone. In saturated solution, cluster size is independent of solvent, while at equal supersaturation, solvent-dependent cluster size increases as methanol < acetonitrile < ethyl acetate, commensurate with increasing nucleation propensity. In ethyl acetate, with largest prenucleation clusters, the driving force required for nucleation is lowest, compared to methanol with smallest clusters and highest driving force. To understand solvent-solute effects, we performed IR spectroscopy supported by molecular simulations. We observe solute-solvent interaction weakening in the same order: methanol < acetonitrile < ethyl acetate, quantifying the weaker solvent-solute interactions that permit the formation of larger prenucleation clusters. Our results support the hypothesis that nucleation is easier in weaker solvents because weak solute-solvent interactions favor growth of large clusters, as opposed to relying solely on ease of desolvation.

Coupled Electrostatic and Hydrophobic Destabilisation of the Gelsolin-Actin Complex Enables Facile Detection of Ovarian Cancer Biomarker Lysophosphatidic Acid.

Lysophosphatidic acid (LPA) is a promising biomarker candidate to screen for ovarian cancer (OC) and potentially stratify and treat patients according to disease stage. LPA is known to target the actin-binding protein gelsolin which is a key regulator of actin filament assembly. Previous studies have shown that the phosphate headgroup of LPA alone is inadequate to bind to the short chain of amino acids in gelsolin known as the PIP2-binding domain. Thus, the molecular-level detail of the mechanism of LPA binding is poorly understood. Here, we model LPA binding to the PIP2-binding domain of gelsolin in the gelsolin-actin complex through extensive ten-microsecond atomistic molecular dynamics (MD) simulations. We predict that LPA binding causes a local conformational rearrangement due to LPA interactions with both gelsolin and actin residues. These conformational changes are a result of the amphipathic nature of LPA, where the anionic phosphate, polar glycerol and ester groups, and lipophilic aliphatic tail mediate LPA binding via charged electrostatic, hydrogen bonding, and van der Waals interactions. The negatively-charged LPA headgroup binds to the PIP2-binding domain of gelsolin-actin while its hydrophobic tail is inserted into actin, creating a strong LPA-insertion pocket that weakens the gelsolin-actin interface. The computed structure, dynamics, and energetics of the ternary gelsolin-LPA-actin complex confirms that a quantitative OC assay is possible based on LPA-triggered actin release from the gelsolin-actin complex.

Computational Peptide Design Cotargeting Glucagon and Glucagon-like Peptide-1 Receptors.

Peptides are sustainable alternatives to conventional therapeutics for G protein-coupled receptor (GPCR) linked disorders, promising biocompatible and tailorable next-generation therapeutics for metabolic disorders including type-2 diabetes, as agonists of the glucagon receptor (GCGR) and the glucagon-like peptide-1 receptor (GLP-1R). However, single agonist peptides activating GLP-1R to stimulate insulin secretion also suppress obesity-linked glucagon release. Hence, bioactive peptides cotargeting GCGR and GLP-1R may remediate the blood glucose and fatty acid metabolism imbalance, tackling both diabetes and obesity to supersede current monoagonist therapy. Here, we design and model optimized peptide sequences starting from peptide sequences derived from earlier phage-displayed library screening, identifying those with predicted molecular binding profiles for dual agonism of GCGR and GLP-1R. We derive design rules from extensive molecular dynamics simulations based on peptide-receptor binding. Our newly designed coagonist peptide exhibits improved predicted coupled binding affinity for GCGR and GLP-1R relative to endogenous ligands and could in the future be tested experimentally, which may provide superior glycemic and weight loss control.

Label-Free Digital Holotomography Reveals Ibuprofen-Induced Morphological Changes to Red Blood Cells.

Understanding the dose-dependent effect of over-the-counter drugs on red blood cells (RBCs) is crucial for hematology and digital pathology. Yet, it is challenging to continuously record the real-time, drug-induced shape changes of RBCs in a label-free manner. Here, we demonstrate digital holotomography (DHTM)-enabled real-time, label-free concentration-dependent and time-dependent monitoring of ibuprofen on RBCs from a healthy donor. The RBCs are segmented based on three-dimensional (3D) and four-dimensional (4D) refractive index tomograms, and their morphological and chemical parameters are retrieved with their shapes classified using machine learning. We directly observed the formation and motion of spicules on the RBC membrane when aqueous solutions of ibuprofen were drop-cast on wet blood, creating rough-membraned echinocyte forms. At low concentrations of 0.25-0.50 mM, the ibuprofen-induced morphological change was transient, but at high concentrations (1-3 mM) the spiculated RBC remained over a period of up to 1.5 h. Molecular simulations confirmed that aggregates of ibuprofen molecules at high concentrations significantly disrupted the RBC membrane structural integrity and lipid order but produced negligible effect at low ibuprofen concentrations. Control experiments on the effect of urea, hydrogen peroxide, and aqueous solutions on RBCs showed zero spicule formation. Our work clarifies the dose-dependent chemical effects on RBCs using label-free microscopes that can be deployed for the rapid detection of overdosage of over-the-counter and prescribed drugs.

Lithiophilic Nanowire Guided Li Deposition in Li Metal Batteries.

Lithium (Li) metal batteries (LMBs) provide superior energy densities far beyond current Li-ion batteries (LIBs) but practical applications are hindered by uncontrolled dendrite formation and the build-up of dead Li in "hostless" Li metal anodes. To circumvent these issues, we created a 3D framework of a carbon paper (CP) substrate decorated with lithiophilic nanowires (silicon (Si), germanium (Ge), and SiGe alloy NWs) that provides a robust host for efficient stripping/plating of Li metal. The lithiophilic Li22 Si5 , Li22 (Si0.5 Ge0.5 )5, and Li22 Ge5 formed during rapid Li melt infiltration prevented the formation of dead Li and dendrites. Li22 Ge5 /Li covered CP hosts delivered the best performance, with the lowest overpotentials of 40 mV (three times lower than pristine Li) when cycled at 1 mA cm-2 /1 mAh cm-2 for 1000 h and at 3 mA cm-2 /3 mAh cm-2 for 500 h. Ex situ analysis confirmed the ability of the lithiophilic Li22 Ge5 decorated samples to facilitate uniform Li deposition. When paired with sulfur, LiFePO4, and NMC811 cathodes, the CP-LiGe/Li anodes delivered 200 cycles with 82%, 93%, and 90% capacity retention, respectively. The discovery of the highly stable, lithiophilic NW decorated CP hosts is a promising route toward stable cycling LMBs and provides a new design motif for hosted Li metal anodes.

Biological effects of the loss of homochirality in a multicellular organism.

Homochirality is a fundamental feature of all known forms of life, maintaining biomolecules (amino-acids, proteins, sugars, nucleic acids) in one specific chiral form. While this condition is central to biology, the mechanisms by which the adverse accumulation of non-L-α-amino-acids in proteins lead to pathophysiological consequences remain poorly understood. To address how heterochirality build-up impacts organism's health, we use chiral-selective in vivo assays to detect protein-bound non-L-α-amino acids (focusing on aspartate) and assess their functional significance in Drosophila. We find that altering the in vivo chiral balance creates a 'heterochirality syndrome' with impaired caspase activity, increased tumour formation, and premature death. Our work shows that preservation of homochirality is a key component of protein function that is essential to maintain homeostasis across the cell, tissue and organ level.

Molecular Engineering of Rigid Hydrogels Co-assembled from Collagenous Helical Peptides Based on a Single Triplet Motif.

The potential of ultra-short peptides to self-assemble into well-ordered functional nanostructures makes them promising minimal components for mimicking the basic ingredient of nature and diverse biomaterials. However, selection and modular design of perfect de novo sequences are extremely tricky due to their vast possible combinatorial space. Moreover, a single amino acid substitution can drastically alter the supramolecular packing structure of short peptide assemblies. Here, we report the design of rigid hybrid hydrogels produced by sequence engineering of a new series of ultra-short collagen-mimicking tripeptides. Connecting glycine with different combinations of proline and its post-translational product 4-hydroxyproline, the single triplet motif, displays the natural collagen-helix-like structure. Improved mechanical rigidity is obtained via co-assembly with the non-collagenous hydrogelator, fluorenylmethoxycarbonyl (Fmoc) diphenylalanine. Characterizations of the supramolecular interactions that promote the self-supporting and self-healing properties of the co-assemblies are performed by physicochemical experiments and atomistic models. Our results clearly demonstrate the significance of sequence engineering to design functional peptide motifs with desired physicochemical and electromechanical properties and reveal co-assembly as a promising strategy for the utilization of small, readily accessible biomimetic building blocks to generate hybrid biomolecular assemblies with structural heterogeneity and functionality of natural materials.

Hydroxychloroquine Does Not Function as a Direct Zinc Ionophore.

Drug-mediated correction of abnormal biological zinc homeostasis could provide new routes to treating neurodegeneration, cancer, and viral infections. Designing therapeutics to facilitate zinc transport intracellularly is hampered by inadequate concentrations of endogenous zinc, which is often protein-bound in vivo. We found strong evidence that hydroxychloroquine, a drug used to treat malaria and employed as a potential treatment for COVID-19, does not bind and transport zinc across biological membranes through ionophoric mechanisms, contrary to recent claims. In vitro complexation studies and liposomal transport assays are correlated with cellular zinc assays in A549 lung epithelial cells to confirm the indirect mechanism of hydroxychloroquine-mediated elevation in intracellular zinc without ionophorism. Molecular simulations show hydroxychloroquine-triggered helix perturbation in zinc-finger protein without zinc chelation, a potential alternative non-ionophoric mechanism.

Single-Particle Resolution of Copper-Associated Annular α-Synuclein Oligomers Reveals Potential Therapeutic Targets of Neurodegeneration.

Metal ions stabilize protein-protein interactions and can modulate protein aggregation. Here, using liquid-based atomic force microscopy and molecular dynamics simulations, we study the concentration-dependent effect of Cu2+ ions on the aggregation pathway of α-synuclein (α-Syn) proteins, which play a key role in the pathology of Parkinson's disease. The full spectrum of α-Syn aggregates in the presence and absence of Cu2+ ions from monomers to mature fibrils was resolved and quantified at the gold-water interface. Raman spectroscopy confirmed the atomic force microscopy (AFM) findings on the heterogeneity in aggregated states of α-Syn. The formation of annular oligomers was exclusively detected upon incubating α-Syn with Cu2+ ions. Our findings emphasize the importance of targeting annular α-Syn protein oligomers for therapeutic intervention and their potential role as biomarkers for early detection and monitoring progression of neurodegeneration.

Predictive Modeling of Neurotoxic α-Synuclein Polymorphs.

Assembly of monomeric α-synuclein (αS) into aggregation-resistant helically folded tetramers and related multimers is a key target for Parkinson's disease (PD). Protein dynamics hampers experimental characterization of the polymorphism of these structures and so computational modeling and simulation is providing a complementary approach to obtain high-resolution structural information on the assembly of αS and interactions with biological surfaces. These computational techniques are particularly valuable for intrinsically disordered proteins (IDPs) and short-lived peptide and protein assemblies with as yet undetermined 3D structures. Experimental observables such as NMR J-coupling constants and chemical shifts can be predicted directly from simulation data, and compared with available experimental data to generate the most physically realistic atomic-resolution structure. For appropriately validated and benchmarked computational models, macroscopic aggregation properties can be related to the calculated thermodynamic properties at an atomic level. In this chapter, we describe a useful protocol for designing helical αS multimers, especially tetramers, and scanning the peptide-membrane interface for cell-bound αS tetramers. These computationally modeled structures are validated by comparison with the range of available known experimental parameters at time of writing in early 2020, and used to generate predictive design rules to motivate and guide experiments.

Characterization of Amyloidogenic Peptide Aggregability in Helical Subspace.

Prototypical amyloidogenic peptides amyloid-ß (Aß) and α-synuclein (αS) can undergo helix-helix associations via partially folded helical conformers, which may influence pathological progression to Alzheimer's (AD) and Parkinson's disease (PD), respectively. At the other extreme, stable folded helical conformers have been reported to resist self-assembly and amyloid formation. Experimental characterisation of such disparities in aggregation profiles due to subtle differences in peptide stabilities is precluded by the conformational heterogeneity of helical subspace. The diverse physical models used in molecular simulations allow sampling distinct regions of the phase space and are extensive in capturing the ensemble of rich helical subspace. Robust and powerful computational predictive methods utilizing network theory and free energy mapping can model the origin of helical population shifts in amyloidogenic peptides, which highlight their inherent aggregability. In this chapter, we discuss computational models, methods, design rules, and strategies to identify the driving force behind helical self-assembly and the molecular origin of aggregation resistance in helical intermediates of Aß42 and αS. By extensive multiscale mapping of intrapeptide interactions, we show that the computational models can capture features that are otherwise imperceptible to experiments. Our models predict that targeting terminal residues may allow modulation and control of initial pathogenic aggregability of amyloidogenic peptides.

On the origin of chaotrope-modulated electrocatalytic activity of cytochrome c at electrified aqueous|organic interfaces.

Electrochemical, spectroscopic and computational methods are used to demonstrate that electrified aqueous|organic interfaces are a suitable bio-mimetic platform to study and contrast the accelerated electrocatalytic activity of cytochrome c towards the production of reactive oxygen species (ROS) in the presence of denaturing agents such as guanidinium chloride and urea.

Balanced lipase interactions for degradation-controlled paclitaxel release from lipid cubic phase formulations.

Lipid cubic phase (LCP) formulations enhance the intestinal solubility and bioavailability of hydrophobic drugs by reducing precipitation and facilitating their mass transport to the intestinal surface for absorption. LCPs with an ester linkage connecting the acyl chain to the glycerol backbone (monoacylglycerols), are susceptible to chemical digestion by several lipolytic enzymes including lipases, accelerating the release of hydrophobic agents from the lipid bilayers of the matrix. Unlike regular enzymes that transform soluble substrates, lipolytic enzymes act at the interface of water and insoluble lipid. Therefore, compounds that bind to this interface can enhance or inhibit the activity of enzymes to varying extent. Here, we explore how the lipolysis rate can be tuned by the interfacial interaction of porcine pancreatic lipase with monoolein LCPs containing a known lipase inhibitor, tetrahydrolipstatin. Release of the Biopharmaceutical Classification System (BCS) class IV drug, paclitaxel, from the inhibitor-modified LCP was examined in the presence of lipase and its effectors colipase and calcium. By combining experimental dynamic digestion studies, thermodynamic measurements and molecular dynamics simulations of the competitive inhibition of lipase by tetrahydrolipstatin, we reveal the role and mode of action of lipase effectors in creating a precisely-balanced degradation-controlled LCP release system for the poorly soluble paclitaxel drug.

Modulating the pro-apoptotic activity of cytochrome c at a biomimetic electrified interface.

Programmed cell death via apoptosis is a natural defence against excessive cell division, crucial for fetal development to maintenance of homeostasis and elimination of precancerous and senescent cells. Here, we demonstrate an electrified liquid biointerface that replicates the molecular machinery of the inner mitochondrial membrane at the onset of apoptosis. By mimicking in vivo cytochrome c (Cyt c) interactions with cell membranes, our platform allows us to modulate the conformational plasticity of the protein by simply varying the electrochemical environment at an aqueous-organic interface. We observe interfacial electron transfer between an organic electron donor decamethylferrocene and O2, electrocatalyzed by Cyt c. This interfacial reaction requires partial Cyt c unfolding, mimicking Cyt c in vivo peroxidase activity. As proof of concept, we use our electrified liquid biointerface to identify drug molecules, such as bifonazole, that can potentially down-regulate Cyt c and protect against uncontrolled neuronal cell death in neurodegenerative disorders.

Long-range Regulation of Partially Folded Amyloidogenic Peptides.

Neurodegeneration involves abnormal aggregation of intrinsically disordered amyloidogenic peptides (IDPs), usually mediated by hydrophobic protein-protein interactions. There is mounting evidence that formation of α-helical intermediates is an early event during self-assembly of amyloid-ß42 (Aß42) and α-synuclein (αS) IDPs in Alzheimer's and Parkinson's disease pathogenesis, respectively. However, the driving force behind on-pathway molecular assembly of partially folded helical monomers into helical oligomers assembly remains unknown. Here, we employ extensive molecular dynamics simulations to sample the helical conformational sub-spaces of monomeric peptides of both Aß42 and αS. Our computed free energies, population shifts, and dynamic cross-correlation network analyses reveal a common feature of long-range intra-peptide modulation of partial helical folds of the amyloidogenic central hydrophobic domains via concerted coupling with their charged terminal tails (N-terminus of Aß42 and C-terminus of αS). The absence of such inter-domain fluctuations in both fully helical and completely unfolded (disordered) states suggests that long-range coupling regulates the dynamicity of partially folded helices, in both Aß42 and αS peptides. The inter-domain coupling suggests a form of intra-molecular allosteric regulation of the aggregation trigger in partially folded helical monomers. This approach could be applied to study the broad range of amyloidogenic peptides, which could provide a new path to curbing pathogenic aggregation of partially folded conformers into oligomers, by inhibition of sites far from the hydrophobic core.

On the ubiquity of helical α-synuclein tetramers.

The experimental finding that α-synuclein (αS) occurs physiologically as a helically folded tetramer begs the question: why are helical tetramers the most populated multimers? While the helical tetramer is known to resist aggregation, the assembly mechanism of αS peptides remains largely unknown. By rationally designing a series of helical multimers from dimer to octamer, we characterized the free energy landscape of wild-type and mutated multimers using molecular dynamics computer simulations. Competition between supramolecular packing and solvation results in well-hydrated dimers and trimers, and more screened pentamers to octamers, with the helical tetramer possessing the most balanced structure with the lowest activation energy. Our data suggest that familial mutants are very sensitive to alterations in monomer packing that would in turn raise the energy barriers for multimerization. Finally, the hypothesis that the αS tetramer forms a soluble, benign "dead end" to circumvent aggregation is supported by its computed very weak association with negatively charged cell membranes.

Molecular Simulations Reveal Terminal Group Mediated Stabilization of Helical Conformers in Both Amyloid-ß42 and α-Synuclein.

The presence of partially structured helices in natively unfolded amyloid-ß42 (Aß42) and α-synuclein (αS) has been shown to accelerate fibrillation in the onset of Alzheimer's and Parkinson's disease, respectively. At the other extreme, folded stable helical conformers have also been reported to resist amyloid formation. Recent studies indicate that amyloidogenic aggregation can be impeded using small molecules that stabilize the α-helical monomers and switch off the neurotoxic pathway. We predict a common intrapeptide route to stabilization based on the plasticity of helical conformations of Aß42 and αS as assessed through extensive atomistic molecular dynamics (MD) computer simulations (∼36 µs) across ten distinct protein force field and water model combinations. Computed free energies and interaction maps (not obtainable from experiments alone) show that flexible terminal groups (N-terminus of Aß42 and C-terminus of αS) show a tendency to stabilize folded helical conformations in both peptides via primary hydrophobic interactions with central hydrophobic domains, and secondary salt bridges with other domains. These interactions confer aggregation resistance by decreasing the population of partially structured helices and are absent in control simulations of complete unfolding. Computed helical stability is also significantly reduced in terminal-deleted variants. The models suggest new strategies to tackle neurodegeneration by rationally re-engineering terminal groups to optimize their predicted ability to deactivate helical monomers.

Re-designing the α-synuclein tetramer.

A computationally re-designed molecular loop optimizes helical packing of α-synuclein monomers to seal the aggregation-resistant low-weight tetramer, a key target for Parkinson's disease. Helical monomers are pushed into active conformations during supramolecular assembly, and familial missense mutations double the energy barrier to tetramerization, preserving the pool of potentially amyloidogenic monomers.

The fold preference and thermodynamic stability of α-synuclein fibrils is encoded in the non-amyloid-ß component region.

The heterogeneity of the synucleinopathies, neurological disorders that include Parkinson's disease (PD), indicates that toxicity, seeding/cross-seeding ability, and propagation of α-synuclein (αS) assemblies depend on their distinct structural characteristics or "strain". To examine the molecular signature that encodes the aggregation seed, conformational preference, and thermodynamic stability of full-length αS fibrils, we performed molecular dynamics simulations on two non-amyloid-ß component (NAC) fibril structures, containing residues 61-95 of two distinct αS fibrils. We identified several discrete hot spots in the recognized hydrophobic core of NAC (residues 68-82) that could initiate the early assembly of αS. We show that NAC fibrils inherit the preferred fold of their parent αS fibril, but could switch conformational preference in two fibril mutants K80Q and E83Q under different solution conditions. Similar to αS fibrils, NAC fibrils are also sensitive to temperature and salt concentration. The favorable solvation free energy of NAC fibrils at low temperature (280 K) suggests a propensity for cold-denaturation. Our results indicate that the strain-dependent synucleinopathies may be partially imprinted in the fold-dependent thermodynamic properties of NAC fibrils, providing structural insights into the emerging development of anti-PD treatments that target the NAC region of αS.

Ciliary neurotrophic factor receptor regulation of adult forebrain neurogenesis.

Appropriately targeted manipulation of endogenous neural stem progenitor (NSP) cells may contribute to therapies for trauma, stroke, and neurodegenerative disease. A prerequisite to such therapies is a better understanding of the mechanisms regulating adult NSP cells in vivo. Indirect data suggest that endogenous ciliary neurotrophic factor (CNTF) receptor signaling may inhibit neuronal differentiation of NSP cells. We challenged subventricular zone (SVZ) cells in vivo with low concentrations of CNTF to anatomically characterize cells containing functional CNTF receptors. We found that type B "stem" cells are highly responsive, whereas type C "transit-amplifying" cells and type A neuroblasts are remarkably unresponsive, as are GFAP(+) astrocytes found outside the SVZ. CNTF was identified in a subset of type B cells that label with acute BrdU administration. Disruption of in vivo CNTF receptor signaling in SVZ NSP cells, with a "floxed" CNTF receptor α (CNTFRα) mouse line and a gene construct driving Cre recombinase (Cre) expression in NSP cells, led to increases in SVZ-associated neuroblasts and new olfactory bulb neurons, as well as a neuron subtype-specific, adult-onset increase in olfactory bulb neuron populations. Adult-onset receptor disruption in SVZ NSP cells with a recombinant adeno-associated virus (AAV-Cre) also led to increased neurogenesis. However, the maintenance of type B cell populations was apparently unaffected by the receptor disruption. Together, the data suggest that endogenous CNTF receptor signaling in type B stem cells inhibits adult neurogenesis, and further suggest that the regulation may occur in a neuron subtype-specific manner.