TAT-RasGAP317-326 kills cells by targeting inner-leaflet-enriched phospholipids.

TAT-RasGAP317-326 is a cell-penetrating peptide-based construct with anticancer and antimicrobial activities. This peptide kills a subset of cancer cells in a manner that does not involve known programmed cell death pathways. Here we have elucidated the mode of action allowing TAT-RasGAP317-326 to kill cells. This peptide binds and disrupts artificial membranes containing lipids typically enriched in the inner leaflet of the plasma membrane, such as phosphatidylinositol-bisphosphate (PIP2) and phosphatidylserine (PS). Decreasing the amounts of PIP2 in cells renders them more resistant to TAT-RasGAP317-326, while reducing the ability of cells to repair their plasma membrane makes them more sensitive to the peptide. The W317A TAT-RasGAP317-326 point mutant, known to have impaired killing activities, has reduced abilities to bind and permeabilize PIP2- and PS-containing membranes and to translocate through biomembranes, presumably because of a higher propensity to adopt an α-helical state. This work shows that TAT-RasGAP317-326 kills cells via a form of necrosis that relies on the physical disruption of the plasma membrane once the peptide targets specific phospholipids found on the cytosolic side of the plasma membrane.

Toward the design and development of peptidomimetic inhibitors of the Ataxin-1 aggregation pathway.

Spinocerebellar ataxia type 1 is a degenerative disorder caused by polyglutamine expansions and aggregation of Ataxin-1. The interaction between Capicua (CIC) and the AXH domain of Ataxin-1 protein has been suggested as a possible driver of aggregation for the expanded Ataxin-1 protein and the subsequent onset of spinocerebellar ataxia 1. Experimental studies have demonstrated that short constructs of CIC may prevent such aggregation and suggested this as a possible candidate to inspire the rational design of peptidomimetics. In this work, molecular modeling techniques, namely the alchemical mutation and force field-based molecular dynamics, have been employed to propose a pipeline for the rational design of a CIC-inspired inhibitor of the ataxin-1 aggregation pathway. In particular, this study has shown that the alchemical mutation can estimate the affinity between AXH and CIC with good correlation with experimental data, while molecular dynamics shed light on molecular mechanisms that occur for stabilization of the interaction between the CIC-inspired construct and the AXH domain of Ataxin-1. This work lays the foundation for a rational methodology for the in silico screening and design of peptidomimetics, which can expedite and streamline experimental studies to identify strategies for inhibiting the ataxin-1 aggregation pathway.

Extended diffusion theory: Recovering dynamics from biased/accelerated molecular simulations.

Dynamical properties are of great importance in determining the behavior of synthetic and natural molecules, but capturing them by computational methods is a nontrivial task. Very often the time scales of the relevant phenomena are far beyond the typical time windows accessible by classical Molecular Dynamics (MD) simulations, currently limited to the order of microseconds on standard laboratory workstations. On the other hand, biased and accelerated simulations allow for fast and thorough exploration of the molecular conformational space, but they lose the dynamic information. The problem of recovering dynamics from biased/accelerated simulations is a very active field of research, but no totally robust/reliable solutions have been given yet. In this paper it is shown how the Smoluchowski equation, in the framework of Diffusion Theory (DT), can be used to bridge this gap, and dynamical properties, in the form of time correlation functions (TCFs), can be extracted also from such kind of simulations. DT is first extended (EDT) to express the mobility tensors entering the Smoluchowski operator in terms of a recently introduced unified and regularized Rotne-Prager-Yamakawa approximation, [P. J. Zuk, E. Wajnryb, K. A. Mizerski, P. Szymczak, J. Fluid. Mech. 2014, 741, R5, 1-13] also involving mixed rotation-translation contributions, and rotation-rotation terms beside the classical translation-translation ones, so far used in DT. Then, the method is applied to recover the dynamics of a nontrivial example of a peptide in explicit water from the first 200 ns of a Replica Exchange Molecular Dynamics simulation, which is a popular computational method that destroys the long time dynamics. EDT dynamics were found to favorably compare against those coming from a standard MD simulation of the same system, requiring a time window of 30 µs to converge. This result shows that EDT is a tool of practical value to recover the long time dynamics of systems in diffusive regimes from biased/accelerated simulations, to be exploited in those cases when direct evaluation by standard MD is unfeasible.

The Impact of Natural Compounds on S-Shaped Aß42 Fibril: From Molecular Docking to Biophysical Characterization.

The pursuit for effective strategies inhibiting the amyloidogenic process in neurodegenerative disorders, such as Alzheimer's disease (AD), remains one of the main unsolved issues, and only a few drugs have demonstrated to delay the degeneration of the cognitive system. Moreover, most therapies induce severe side effects and are not effective at all stages of the illness. The need to find novel and reliable drugs appears therefore of primary importance. In this context, natural compounds have shown interesting beneficial effects on the onset and progression of neurodegenerative diseases, exhibiting a great inhibitory activity on the formation of amyloid aggregates and proving to be effective in many preclinical and clinical studies. However, their inhibitory mechanism is still unclear. In this work, ensemble docking and molecular dynamics simulations on S-shaped Aß42 fibrils have been carried out to evaluate the influence of several natural compounds on amyloid conformational behaviour. A deep understanding of the interaction mechanisms between natural compounds and Aß aggregates may play a key role to pave the way for design, discovery and optimization strategies toward an efficient destabilization of toxic amyloid assemblies.

Fludarabine-Specific Molecular Interactions with Maltose-Modified Poly(propyleneimine) Dendrimer Enable Effective Cell Entry of the Active Drug Form: Comparison with Clofarabine.

Fludarabine is an anticancer antimetabolite essential for modern chemotherapy, but its efficacy is limited due to the complex pharmacokinetics. We demonstrated the potential use of maltose-modified poly(propyleneimine) dendrimer as drug delivery agent to improve the efficiency of therapy with fludarabine. In this study, we elaborated a novel synthesis technique for radioactively labeled fludarabine triphosphate to prove for the first time the direct ability of nucleotide-glycodendrimer complex to enter and kill leukemic cells, without the involvement of membrane nucleoside transporters and intracellular kinases. This will potentially allow to bypass the most common drug resistance mechanisms observed in the clinical setting. Further, we applied surface plasmon resonance and molecular modeling to elucidate the properties of the drug-dendrimer complexes. We showed that clofarabine, a more toxic nucleoside analogue drug, is characterized by significantly different molecular interactions with poly(propyleneimine) dendrimers than fludarabine, leading to different cellular outcomes (decreased rather than increased treatment efficiency). The most probable mechanistic explanation of uniquely dendrimer-enhanced fludarabine toxicity points to a crucial role of both an alternative cellular uptake pathway and the avoidance of intracellular phosphorylation of nucleoside drug form.

The synergistic effect of chlorotoxin-mApoE in boosting drug-loaded liposomes across the BBB.

We designed liposomes dually functionalized with ApoE-derived peptide (mApoE) and chlorotoxin (ClTx) to improve their blood-brain barrier (BBB) crossing. Our results demonstrated the synergistic activity of ClTx-mApoE in boosting doxorubicin-loaded liposomes across the BBB, keeping the anti-tumour activity of the drug loaded: mApoE acts promoting cellular uptake, while ClTx promotes exocytosis of liposomes.

Destabilizing the AXH Tetramer by Mutations: Mechanisms and Potential Antiaggregation Strategies.

The AXH domain of protein Ataxin 1 is thought to play a key role in the misfolding and aggregation pathway responsible for Spinocerebellar ataxia 1. For this reason, a molecular level understanding of AXH oligomerization pathway is crucial to elucidate the aggregation mechanism, which is thought to trigger the disease. This study employs classical and enhanced molecular dynamics to identify the structural and energetic basis of AXH tetramer stability. Results of this work elucidate molecular mechanisms behind the destabilizing effect of protein mutations, which consequently affect the AXH tetramer assembly. Moreover, results of the study draw attention for the first time, to our knowledge, to the R638 protein residue, which is shown to play a key role in AXH tetramer stability. Therefore, R638 might be also implicated in the AXH oligomerization pathway and stands out as a target for future experimental studies focused on self-association mechanisms and fibril formation of full-length ATX1.

Protein Environment: A Crucial Triggering Factor in Josephin Domain Aggregation: The Role of 2,2,2-Trifluoroethanol.

The protein ataxin-3 contains a polyglutamine stretch that triggers amyloid aggregation when it is expanded beyond a critical threshold. This results in the onset of the spinocerebellar ataxia type 3. The protein consists of the globular N-terminal Josephin domain and a disordered C-terminal tail where the polyglutamine stretch is located. Expanded ataxin-3 aggregates via a two-stage mechanism: first, Josephin domain self-association, then polyQ fibrillation. This highlights the intrinsic amyloidogenic potential of Josephin domain. Therefore, much effort has been put into investigating its aggregation mechanism(s). A key issue regards the conformational requirements for triggering amyloid aggregation, as it is believed that, generally, misfolding should precede aggregation. Here, we have assayed the effect of 2,2,2-trifluoroethanol, a co-solvent capable of stabilizing secondary structures, especially α-helices. By combining biophysical methods and molecular dynamics, we demonstrated that both secondary and tertiary JD structures are virtually unchanged in the presence of up to 5% 2,2,2-trifluoroethanol. Despite the preservation of JD structure, 1% of 2,2,2-trifluoroethanol suffices to exacerbate the intrinsic aggregation propensity of this domain, by slightly decreasing its conformational stability. These results indicate that in the case of JD, conformational fluctuations might suffice to promote a transition towards an aggregated state without the need for extensive unfolding, and highlights the important role played by the environment on the aggregation of this globular domain.

Conformational Dynamics and Stability of U-Shaped and S-Shaped Amyloid ß Assemblies.

Alzheimer's disease is the most fatal neurodegenerative disorder characterized by the aggregation and deposition of Amyloid ß (Aß) oligomers in the brain of patients. Two principal variants of Aß exist in humans: Aß1-40 and Aß1-42. The former is the most abundant in the plaques, while the latter is the most toxic species and forms fibrils more rapidly. Interestingly, fibrils of Aß1-40 peptides can only assume U-shaped conformations while Aß1-42 can also arrange as S-shaped three-stranded chains, as recently discovered. As alterations in protein conformational arrangement correlate with cell toxicity and speed of disease progression, it is important to characterize, at molecular level, the conformational dynamics of amyloid fibrils. In this work, Replica Exchange Molecular Dynamics simulations were carried out to compare the conformational dynamics of U-shaped and S-shaped Aß17-42 small fibrils. Our computational results provide support for the stability of the recently proposed S-shaped model due to the maximized interactions involving the C-terminal residues. On the other hand, the U-shaped motif is characterized by significant distortions resulting in a more disordered assembly. Outcomes of our work suggest that the molecular architecture of the protein aggregates might play a pivotal role in formation and conformational stability of the resulting fibrils.

Self-Assembled Ligands Targeting TLR7: A Molecular Level Investigation.

Toll-like receptors (TLRs) are pattern recognition transmembrane proteins that play an important role in innate immunity. In particular, TLR7 plays a role in detecting nucleic acids derived from viruses and bacteria. The huge number of pathologies in which TLR7 is involved has led to an increasing interest in developing new compounds targeting this protein. Several conjugation strategies were proposed for TLR7 agonists to increase the potency while maintaining a low toxicity. In this work, we focus the attention on two promising classes of TLR7 compounds derived from the same pharmacophore conjugated with phospholipid and polyethylene glycol (PEG). A multidisciplinary investigation has been carried out by molecular dynamics (MD), dynamic light scattering (DLS), electron paramagnetic resonance (EPR), and cytotoxicity assessment. DLS and MD indicated how only the phospholipid conjugation provides the compound abilities to self-assemble in an orderly fashion with a maximal pharmacophore exposition to the solvent. Further EPR and cytotoxicity experiments highlighted that phospholipid compounds organize in stable aggregates and well interact with TLR7, whereas PEG conjugation was characterized by poorly stable aggregates at the cells surface. The methodological framework proposed in this study may be used to investigate, at a molecular level, the interactions generally occurring between aggregated ligands, to be used as drugs, and protein receptors.

Josephin Domain Structural Conformations Explored by Metadynamics in Essential Coordinates.

The Josephin Domain (JD), i.e. the N-terminal domain of Ataxin 3 (At3) protein, is an interesting example of competition between physiological function and aggregation risk. In fact, the fibrillogenesis of Ataxin 3, responsible for the spinocerebbellar ataxia 3, is strictly related to the JD thermodynamic stability. Whereas recent NMR studies have demonstrated that different JD conformations exist, the likelihood of JD achievable conformational states in solution is still an open issue. Marked differences in the available NMR models are located in the hairpin region, supporting the idea that JD has a flexible hairpin in dynamic equilibrium between open and closed states. In this work we have carried out an investigation on the JD conformational arrangement by means of both classical molecular dynamics (MD) and Metadynamics employing essential coordinates as collective variables. We provide a representation of the free energy landscape characterizing the transition pathway from a JD open-like structure to a closed-like conformation. Findings of our in silico study strongly point to the closed-like conformation as the most likely for a Josephin Domain in water.

Insights into the Effect of the G245S Single Point Mutation on the Structure of p53 and the Binding of the Protein to DNA.

The transcription factor p53 is a potent tumor suppressor dubbed as the "guardian of the genome" because of its ability to orchestrate protective biological outputs in response to a variety of oncogenic stresses. Mutation and thus inactivation of p53 can be found in 50% of human tumors. The majority are missense mutations located in the DNA binding region. Among them, G245S is known to be a structural hotspot mutation. To understand the behaviors and differences between the wild-type and mutant, both a dimer of the wild type p53 (wt-p53) and its G245S mutant (G245S-mp53), complexed with DNA, were simulated using molecular dynamics for more than 1 µs. wt-p53 and G245S-mp53 apo monomers were simulated for 1 µs as well. Conformational analyses and binding energy evaluations performed underline important differences and therefore provide insights to understand the G245S-mp53 loss of function. Our results indicate that the G245S mutation destabilizes several structural regions in the protein that are crucial for DNA binding when found in its apo form and highlight differences in the mutant-DNA complex structure compared to the wt protein. These findings not only provide means that can be applied to other p53 mutants but also serve as structural basis for further studies aimed at the development of cancer therapies based on restoring the function of p53.

Conformational fluctuations of the AXH monomer of Ataxin-1.

In this paper, we report the results of molecular dynamics simulations of AXH monomer of Ataxin-1. The AXH domain plays a crucial role in Ataxin-1 aggregation, which accompanies the initiation and progression of Spinocerebellar ataxia type 1. Our simulations involving both classical and replica exchange molecular dynamics, followed by principal component analysis of the trajectories obtained, reveal substantial conformational fluctuations of the protein structure, especially in the N-terminal region. We show that these fluctuations can be generated by thermal noise since the free energy barriers between conformations are small enough for thermally stimulated transitions. In agreement with the previous experimental findings, our results can be considered as a basis for a future design of ataxin aggregation inhibitors that will require several key conformations identified in the present study as molecular targets for ligand binding.

Characterization of the AXH domain of Ataxin-1 using enhanced sampling and functional mode analysis.

Ataxin-1 is the protein responsible for the Spinocerebellar ataxia type 1, an incurable neurodegenerative disease caused by polyglutamine expansion. The AXH domain plays a pivotal role in physiological functions of Ataxin-1. In Spinocerebellar ataxia 1, the AXH domain is involved in the misfolding and aggregation pathway. Here molecular modeling is applied to investigate the protein-protein interactions contributing to the AXH dimer stability. Particular attention is focused on: (i) the characterization of AXH monomer-monomer interface; (ii) the molecular description of the AXH monomer-monomer interaction dynamics. Technically, an approach based on functional mode analysis, here applied to replica exchange molecular dynamics trajectories, was employed. The findings of this study are consistent with previous experimental results and elucidate the pivotal role of the I580 residue in mediating the AXH monomer-monomer interaction dynamics.

Clickable polyethyleneimine incorporated into triblock copolymeric micelles as an efficient platform in the delivery of siRNA to NSCLC cells.

The efficacy of transfection vectors to cross the endosomal membrane into the cytosol is a central aspect in the development of nucleic acid-based therapeutics. The challenge remains the same: Delivery, Delivery, Delivery. Despite a rational and appropriate construct of triblock polymeric micelles, which could serve as an ideal platform for the co-delivery of siRNAs and hydrophobic anticancer drugs, we show here its inability to properly convey oligonucleotides to their final destination. In order to overcome biological barriers, a linear PEI comprising two orthogonal groups was synthesized, holding an appropriate balance between safety and efficacy. Micellar carriers were then formulated with this polymer to enhance endosomal siRNA release. This chemical technology also addresses the two major challenges to consider when developing novel micellar products for siRNA delivery, namely cytotoxicity of polycations and endosomal escape. Herein, we demonstrate successful release of siRNA using a polymer tailoring strategy combined with a relevant in vitro approach, considering STAT3 as a promising target in the treatment of non-small cell lung cancer (NSCLC).

VirtuousPocketome: a computational tool for screening protein-ligand complexes to identify similar binding sites.

Protein residues within binding pockets play a critical role in determining the range of ligands that can interact with a protein, influencing its structure and function. Identifying structural similarities in proteins offers valuable insights into their function and activation mechanisms, aiding in predicting protein-ligand interactions, anticipating off-target effects, and facilitating the development of therapeutic agents. Numerous computational methods assessing global or local similarity in protein cavities have emerged, but their utilization is impeded by complexity, impractical automation for amino acid pattern searches, and an inability to evaluate the dynamics of scrutinized protein-ligand systems. Here, we present a general, automatic and unbiased computational pipeline, named VirtuousPocketome, aimed at screening huge databases of proteins for similar binding pockets starting from an interested protein-ligand complex. We demonstrate the pipeline's potential by exploring a recently-solved human bitter taste receptor, i.e. the TAS2R46, complexed with strychnine. We pinpointed 145 proteins sharing similar binding sites compared to the analysed bitter taste receptor and the enrichment analysis highlighted the related biological processes, molecular functions and cellular components. This work represents the foundation for future studies aimed at understanding the effective role of tastants outside the gustatory system: this could pave the way towards the rationalization of the diet as a supplement to standard pharmacological treatments and the design of novel tastants-inspired compounds to target other proteins involved in specific diseases or disorders. The proposed pipeline is publicly accessible, can be applied to any protein-ligand complex, and could be expanded to screen any database of protein structures.

In silico investigation of cytochrome bc1 molecular inhibition mechanism against Trypanosoma cruzi.

Chagas' disease is a neglected tropical disease caused by the kinetoplastid protozoan Trypanosoma cruzi. The only therapies are the nitroheterocyclic chemicals nifurtimox and benznidazole that cause various adverse effects. The need to create safe and effective medications to improve medical care remains critical. The lack of verified T. cruzi therapeutic targets hinders medication research for Chagas' disease. In this respect, cytochrome bc1 has been identified as a promising therapeutic target candidate for antibacterial medicines of medical and agricultural interest. Cytochrome bc1 belongs to the mitochondrial electron transport chain and transfers electrons from ubiquinol to cytochrome c1 by the action of two catalytic sites named Qi and Qo. The two binding sites are highly selective, and specific inhibitors exist for each site. Recent studies identified the Qi site of the cytochrome bc1 as a promising drug target against T. cruzi. However, a lack of knowledge of the drug mechanism of action unfortunately hinders the development of new therapies. In this context, knowing the cause of binding site selectivity and the mechanism of action of inhibitors and substrates is crucial for drug discovery and optimization processes. In this paper, we provide a detailed computational investigation of the Qi site of T. cruzi cytochrome b to shed light on the molecular mechanism of action of known inhibitors and substrates. Our study emphasizes the action of inhibitors at the Qi site on a highly unstructured portion of cytochrome b that could be related to the biological function of the electron transport chain complex.

Influence of dexamethasone on the interaction between glucocorticoid receptor and SOX9: A molecular dynamics study.

The glucocorticoid receptor (GR) is a nuclear receptor that controls critical biological processes by regulating the transcription of specific genes. GR transcriptional activity is modulated by a series of ligands and coenzymes, where a ligand can act as an agonist or antagonist. GR agonists, such as the glucocorticoids dexamethasone (DEX) and prednisolone, are widely prescribed to patients with inflammatory and autoimmune diseases. DEX is also used to induce osteogenic differentiation in vitro. Recently, it has been highlighted that DEX induces changes in the osteogenic differentiation of human mesenchymal stromal cells by downregulating the transcription factor SRY-box transcription factor 9 (SOX9) and upregulating the peroxisome proliferator-activated receptor γ (PPARG). SOX9 is fundamental in the control of chondrogenesis, but also in osteogenesis by acting as a dominant-negative of RUNX2. Many processes remain to be clarified during cell fate determination, such as the interplay between the key transcription factors. The main objective pursued by this work is to shed light on the interaction between GR and SOX9 in the presence and absence of DEX at an atomic level of resolution using molecular dynamics simulations. The outcome of this research could help the understanding of possible molecular interactions between GR and SOX9 and their role in the determination of cell fate. The results highlight the key residues at the interface between GR and SOX9 involved in the complexation process and shed light on the mechanism through which DEX modulates GR-SOX9 binding and exerts its biological activity.

Reorganization of the outer layer of a model of the plasma membrane induced by a neuroprotective aminosterol.

Trodusquemine is an amphipathic aminosterol that has recently shown therapeutic benefit in neurodegenerative diseases altering the binding of misfolded proteins to the cell membrane. To unravel the underlying mechanism, we studied the interactions between Trodusquemine (TRO) and lipid monolayers simulating the outer layer of the plasma membrane. We selected two different compositions of dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM), cholesterol (Chol) and monosialotetrahexosylganglioside (GM1) lipid mixture mimicking either a lipid-raft containing membrane (Ld+So phases) or a single-phase disordered membrane (Ld phase). Surface pressure-area isotherms and surface compressional modulus-area combined with Brewster Angle Microscopy (BAM) provided the thermodynamic and morphological information on the lipid monolayer in the presence of increasing amounts of TRO in the monolayer. Experiments revealed that TRO forms stable spreading monolayers at the buffer-air interface where it undergoes multiple reversible phase transitions to bi- and tri-layers at the interface. When TRO was spread at the interface with the lipid mixtures, we found that it distributes in the lipid monolayer for both the selected lipid compositions, but a maximum TRO uptake in the rafts-containing monolayer was observed for a Lipid/TRO molar ratio equal to 3:2. Statistical analysis of BAM images revealed that TRO induces a decrease in the size of the condensed domains, an increase in their number and in the thickness mismatch between the Ld and So phase. Experiments and MD simulations converge to indicate that TRO adsorbs preferentially at the border of the So domains. Removal of GM1 from the lipid Ld+So mixture resulted in an even greater TRO-mediated reduction of the size of the So domains suggesting that the presence of GM1 hinders the localization of TRO at the So domains boundaries. Taken together these observations suggest that Trodusquemine influences the organization of lipid rafts within the neuronal membrane in a dose-dependent manner whereas it evenly distributes in disordered expanded phases of the membrane model.

Informed classification of sweeteners/bitterants compounds via explainable machine learning.

Perception of taste is an emergent phenomenon arising from complex molecular interactions between chemical compounds and specific taste receptors. Among all the taste perceptions, the dichotomy of sweet and bitter tastes has been the subject of several machine learning studies for classification purposes. While previous studies have provided accurate sweeteners/bitterants classifiers, there is ample scope to enhance these models by enriching the understanding of the molecular basis of bitter-sweet tastes. Towards these goals, our study focuses on the development and testing of several machine learning strategies coupled with the novel SHapley Additive exPlanations (SHAP) for a rational sweetness/bitterness classification. This allows the identification of the chemical descriptors of interest by allowing a more informed approach toward the rational design and screening of sweeteners/bitterants. To support future research in this field, we make all datasets and machine learning models publicly available and present an easy-to-use code for bitter-sweet taste prediction.