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Structural bioinformatics approach to exploring transient binding pockets in proteins

Abstract

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Date Published: 18th Dec 2024

Publication Type: Bachelor's Thesis

Impact of an irreversible β-galactosylceramidase inhibitor on the lipid profile of zebrafish embryos

Abstract

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Authors: Jessica Guerra, Mirella Belleri, Giulia Paiardi, Chiara Tobia, Davide Capoferri, Marzia Corli, Elisa Scalvini, Marco Ghirimoldi, Marcello Manfredi, Rebecca C. Wade, Marco Presta, Luca Mignani

Date Published: 1st Dec 2024

Publication Type: Journal

Computational screening of the effects of mutations on protein-protein off-rates and dissociation mechanisms by τRAMD

Abstract (Expand)

Abstract The dissociation rate, or its reciprocal, the residence time (τ), is a crucial parameter for understanding the duration and biological impact of biomolecular interactions. Accurate prediction …tions. Accurate prediction of τ is essential for understanding protein-protein interactions (PPIs) and identifying potential drug targets or modulators for tackling diseases. Conventional molecular dynamics simulation techniques are inherently constrained by their limited timescales, making it challenging to estimate residence times, which typically range from minutes to hours. Building upon its successful application in protein-small molecule systems, τ-Random Acceleration Molecular Dynamics (τRAMD) is here investigated for estimating dissociation rates of protein-protein complexes. τRAMD enables the observation of unbinding events on the nanosecond timescale, facilitating rapid and efficient computation of relative residence times. We tested this methodology for three protein-protein complexes and their extensive mutant datasets, achieving good agreement between computed and experimental data. By combining τRAMD with MD-IFP (Interaction Fingerprint) analysis, dissociation mechanisms were characterized and their sensitivity to mutations investigated, enabling the identification of molecular hotspots for selective modulation of dissociation kinetics. In conclusion, our findings underscore the versatility of τRAMD as a simple and computationally efficient approach for computing relative protein-protein dissociation rates and investigating dissociation mechanisms, thereby aiding the design of PPI modulators.

Authors: Giulia D’Arrigo, Daria B. Kokh, Ariane Nunes-Alves, Rebecca C. Wade

Date Published: 1st Dec 2024

Publication Type: Journal

Comprehensive characterization of the neurogenic and neuroprotective action of a novel TrkB agonist using mouse and human stem cell models of Alzheimer’s disease

Abstract (Expand)

Abstract Background Neural stem cell (NSC) proliferation and differentiation in the mammalian brain decreases to minimal levels postnatally. Nevertheless, neurogenic niches persist in the adult cortex …rtheless, neurogenic niches persist in the adult cortex and hippocampus in rodents, primates and humans, with adult NSC differentiation sharing key regulatory mechanisms with development. Adult neurogenesis impairments have been linked to Alzheimer’s disease (AD) pathology. Addressing these impairments by using neurotrophic factors is a promising new avenue for therapeutic intervention based on neurogenesis. However, this possibility has been hindered by technical difficulties of using in-vivo models to conduct screens, including working with scarce NSCs in the adult brain and differences between human and mouse models or ethical limitations. Methods Here, we use a combination of mouse and human stem cell models for comprehensive in-vitro characterization of a novel neurogenic compound, focusing on the brain-derived neurotrophic factor (BDNF) pathway. The ability of ENT-A011, a steroidal dehydroepiandrosterone derivative, to activate the tyrosine receptor kinase B (TrkB) receptor was tested through western blotting in NIH-3T3 cells and its neurogenic and neuroprotective action were assessed through proliferation, cell death and Amyloid-β (Aβ) toxicity assays in mouse primary adult hippocampal NSCs, mouse embryonic cortical NSCs and neural progenitor cells (NPCs) differentiated from three human induced pluripotent stem cell lines from healthy and AD donors. RNA-seq profiling was used to assess if the compound acts through the same gene network as BDNF in human NPCs. Results ENT-A011 was able to increase proliferation of mouse primary adult hippocampal NSCs and embryonic cortical NSCs, in the absence of EGF/FGF, while reducing Aβ-induced cell death, acting selectively through TrkB activation. The compound was able to increase astrocytic gene markers involved in NSC maintenance, protect hippocampal neurons from Αβ toxicity and prevent synapse loss after Aβ treatment. ENT-A011 successfully induces proliferation and prevents cell death after Aβ toxicity in human NPCs, acting through a core gene network shared with BDNF as shown through RNA-seq. Conclusions Our work characterizes a novel BDNF mimetic with preferable pharmacological properties and neurogenic and neuroprotective actions in Alzheimer’s disease via stem cell-based screening, demonstrating the promise of stem cell systems for short-listing competitive candidates for further testing.

Authors: Despoina Charou, Thanasis Rogdakis, Alessia Latorrata, Maria Valcarcel, Vasileios Papadogiannis, Christina Athanasiou, Alexandros Tsengenes, Maria Anna Papadopoulou, Dimitrios Lypitkas, Matthieu D. Lavigne, Theodora Katsila, Rebecca C. Wade, M. Zameel Cader, Theodora Calogeropoulou, Achille Gravanis, Ioannis Charalampopoulos

Date Published: 1st Dec 2024

Publication Type: Journal

The accomplices: Heparan sulfates and N-glycans foster SARS-CoV-2 spike:ACE2 receptor binding and virus priming

Abstract (Expand)

Although it is well established that the SARS-CoV-2 spike glycoprotein binds to the host cell ACE2 receptor to initiate infection, far less is known about the tissue tropism and host cell susceptibility …susceptibility to the virus. Differential expression across different cell types of heparan sulfate (HS) proteoglycans, with variably sulfated glycosaminoglycans (GAGs), and their synergistic interactions with host and viral N-glycans may contribute to tissue tropism and host cell susceptibility. Nevertheless, their contribution remains unclear since HS and N-glycans evade experimental characterization. We, therefore, carried out microsecond-long all-atom molecular dynamics simulations, followed by random acceleration molecular dynamics simulations, of the fully glycosylated spike:ACE2 complex with and without highly sulfated GAG chains bound. By considering the model GAGs as surrogates for the highly sulfated HS expressed in lung cells, we identified key cell entry mechanisms of spike SARS-CoV-2. We find that HS promotes structural and energetic stabilization of the active conformation of the spike receptor-binding domain (RBD) and reorientation of ACE2 toward the N-terminal domain in the same spike subunit as the RBD. Spike and ACE2 N-glycans exert synergistic effects, promoting better packing, strengthening the protein:protein interaction, and prolonging the residence time of the complex. ACE2 and HS binding trigger rearrangement of the S2’ functional protease cleavage site through allosteric interdomain communication. These results thus show that HS has a multifaceted role in facilitating SARS-CoV-2 infection, and they provide a mechanistic basis for the development of GAG derivatives with anti-SARS-CoV-2 potential.

Authors: Giulia Paiardi, Matheus Ferraz, Marco Rusnati, Rebecca C. Wade

Date Published: 22nd Oct 2024

Publication Type: Journal

Multiresolution molecular dynamics simulations reveal the interplay between conformational variability and functional interactions in membrane‐bound cytochrome P450 2B4

Abstract (Expand)

Abstract Cytochrome P450 2B4 (CYP 2B4) is one of the best‐characterized CYPs and serves as a key model system for understanding the mechanisms of microsomal class II CYPs, which metabolize most known …hich metabolize most known drugs. The highly flexible nature of CYP 2B4 is apparent from crystal structures that show the active site with either a wide open or a closed heme binding cavity. Here, we investigated the conformational ensemble of the full‐length CYP 2B4 in a phospholipid bilayer, using multiresolution molecular dynamics (MD) simulations. Coarse‐grained MD simulations revealed two predominant orientations of CYP 2B4's globular domain with respect to the bilayer. Their refinement by atomistic resolution MD showed adaptation of the enzyme's interaction with the lipid bilayer, leading to open configurations that facilitate ligand access to the heme binding cavity. CAVER analysis of enzyme tunnels, AquaDuct analysis of water routes, and Random Acceleration Molecular Dynamics simulations of ligand dissociation support the conformation‐dependent passage of molecules between the active site and the protein surroundings. Furthermore, simulation of the re‐entry of the inhibitor bifonazole into the open conformation of CYP 2B4 resulted in binding at a transient hydrophobic pocket within the active site cavity that may play a role in substrate binding or allosteric regulation. Together, these results show how the open conformation of CYP 2B4 facilitates the binding of substrates from and release of products to the membrane, whereas the closed conformation prolongs the residence time of substrates or inhibitors and selectively allows the passage of smaller reactants via the solvent and water channels.

Authors: Sungho Bosco Han, Jonathan Teuffel, Goutam Mukherjee, Rebecca C. Wade

Date Published: 1st Oct 2024

Publication Type: Journal

EDITORIAL: Chemical Compound Space Exploration by Multiscale High-Throughput Screening and Machine Learning

Abstract

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Authors: Ganna Gryn’ova, Tristan Bereau, Carolin Müller, Pascal Friederich, Rebecca C. Wade, Ariane Nunes-Alves, Thereza A. Soares, Kenneth Merz

Date Published: 12th Aug 2024

Publication Type: Journal

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