Biology subjects

Garaizar, A.

Publications and source records attributed to Garaizar, A..

8 recordsLinked to original sources

bioRxiv2025

Benchmarking Free Energy Computational Methods for Revealing the Interactions Driving PARP1 Selective Inhibition

Accurate prediction of inhibitor selectivity across protein paralogues remains a central challenge in computational drug discovery. Here, we systematically benchmark three computational methods--Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA), free energy perturbation (FEP) and potential of mean force (PMF) calculations--in their ability to recapitulate PARP1 versus PARP2 selectivity for eight clinically relevant PARP enzyme inhibitors used in ovarian, breast and prostate tumors among others. We demonstrate how MM/PBSA calculations offer rapid and qualitative insights, but show pronounced sensitivity to the chosen static conformational pose, being particularly challenging for ligands with subtle energetic differences between distinct protein paralogues. In contrast, both FEP and PMF calculations using atomistic models with explicit solvent result in substantially improved agreement with experimental binding affinities. The FEP method exhibits the strongest quantitative correlation with experimental binding free energy differences, remarkably reproducing selectivity trends even among nearly isoenergetic complexes. Notably, our structural contact analysis reveals how contact connectivity controls ligand selectivity, providing valuable mechanistic and molecular insight into the key residues that stabilize each inhibitor in both protein enzymes. Together, our multi-method computational study contributes to elucidate potential chemical modifications across the ligand chemical space to enhance potency and specificity, informing the future design and evaluation of selective inhibitors for precision oncology, including therapies targeting homologous recombination-deficient cancers.

bioinformatics↗

bioRxiv2025

Decoding PARP1 Selectivity: Atomistic Insights for Next-Generation Cancer Inhibitors

Selective inhibition of PARP1 represents a promising strategy to improve the therapeutic index of PARP inhibitors, a class of anticancer agents that exploit defects in DNA repair pathways. While PARP inhibitors have shown remarkable clinical benefit, particularly in BRCA-mutated tumors, the lack of discrimination between PARP1 and its close homolog PARP2, often leads to hematological toxicity and limits treatment efficacy. Thus, achieving molecular selectivity for PARP1 remains a central challenge in the rational design of safer and more potent inhibitors. To explore the molecular determinants of ligand selectivity, we focus on four clinically relevant PARP inhibitors--two PARP1-selective (saruparib and NMS-P118) and two non-selective (veliparib and olaparib) inhibitors--and perform atomistic potential-of-mean-force calculations of the PARP1 catalytic binding domain in the presence of these molecules. Our simulations near-quantitatively capture the experimental relative binding preferences, demonstrating that our approach reliably reflects selectivity patterns. Based on these findings, we analyze protein-ligand contact frequencies to identify the stabilizing interaction network and contact connectivity inducing protein selectivity. The most frequent protein-inhibitor contacts are primarily mediated by tyrosine triads and electrostatic interactions, showing a cooperative complex network of intermolecular contacts which strongly relies on protein multivalency. To dissect the decisive role of individual residues across the binding site, we also perform targeted mutagenesis of the PARP1 catalytic pocket in complex with saruparib, replacing several active-site amino acids by glycines. Progressively increasing the number of mutations markedly reduces binding stability, with distinct residue combinations exerting two primary effects: destabilization of the final bound state and the emergence of energetic barriers along the ligand association pathway. Together, our results provide a coherent mechanistic framework for understanding PARP1 selectivity and informs the rational design of next-generation inhibitors with improved efficacy and safety.

molecular biology↗

bioRxiv2025

Compositional control of ageing kineticsin TDP-43 condensates

Biomolecular compartments orchestrate the spatiotemporal organisation of cells. The spontaneous assembly of proteins and nucleic acids through liquid-liquid phase separation into biomolecular condensates has been shown to ubiquitously contribute to the functional compartmentalisation of the cytoplasm and nucleoplasm. However, some condensates can undergo an additional phase transition from functional liquid states to pathological solid-like assemblies (i.e., ageing). This liquid-to-solid transition, driven by the accumulation of protein cross-{beta}-sheet structures, represents a hallmark of multiple neurodegenerative disorders. In this study, we employ Molecular Dynamics simulations to explore the role of various biomolecules in regulating the ageing kinetics of condensates scaffolded by TDP-43, a key RNA-binding protein linked to amyotrophic lateral sclerosis and frontotemporal dementia. We find that the recruitment of arginine-rich peptides, such as those produced by the C9orf72 gene, accelerates the nucleation of cross-{beta}-sheet structures. In contrast, the inclusion of poly-Uridine RNA and the HSP70 chaperone significantly slows the emergence of these structures. Remarkably, we observe a correlation between the compactness of the low-complexity domain of TDP-43-- which drives the transition to cross-{beta}-sheet structures--and the condensate ageing kinetics as we vary the composition of the condensates. Moreover, we find that near-interfacial regions of TDP-43 condensates exhibit faster {beta}-sheet transitions than the bulk core of the condensate. Together, our findings underscore the critical role of client biomolecules in modulating protein conformational ensembles and intermolecular interactions, thereby controlling the propensity of condensates to transition into harmful solid-like states.

biophysics↗

bioRxiv2022

Ageing critically transforms the network connectivity and viscoelasticity of RNA-binding protein condensates but RNA can prevent it

Biomolecular condensates, some of which are liquid-like during health, can age over time becoming gel-like pathological systems. One potential source of loss of liquid-like properties during ageing of RNA-binding protein condensates is the progressive formation of inter-protein {beta}-sheets. To bridge microscopic understanding between accumulation of inter-protein {beta}-sheets over time and the modulation of FUS and hnRNPA1 condensate viscoelasticity, we develop a multiscale simulation approach. Our method integrates atomistic simulations with sequence-dependent coarse-grained modelling of condensates that exhibit accumulation of inter-protein {beta}-sheets over time. We reveal that inter-protein {beta}-sheets notably increase condensate viscosity but does not transform the phase diagrams. Strikingly, the network of molecular connections within condensates is drastically altered, culminating in gelation when the network of strong {beta}-sheets fully percolates. However, high concentrations of RNA decelerate the emergence of inter-protein {beta}-sheets. Our study uncovers molecular and kinetic factors explaining how the accumulation of inter-protein {beta}-sheets can trigger liquid-to-solid transitions in condensates, and suggests a potential mechanism to slow such transitions down.

biophysics↗

bioRxiv2021

Dual RNA modulation of protein mobility and stability within phase-separated condensates

One of the key mechanisms employed by cells to control their spatiotemporal organization is the formation and dissolution of phase-separated condensates. The balance between condensate assembly and disassembly can be critically regulated by the presence of RNA. In this work, we use a novel sequence-dependent coarse-grained model for proteins and RNA to unravel the impact of RNA in modulating the transport properties and stability of biomolecular condensates. We explore the phase behavior of several RNA-binding proteins such as FUS, hnRNPA1 and TDP-43 proteins along with that of their corresponding prion-like domains and RNA-recognition motifs, from absence to moderately high RNA concentration. By characterising the phase diagram, key molecular interactions, surface tension and transport properties of the condensates, we report a dual RNA-induced behavior: On the one hand, RNA enhances phase separation at low concentration as long as the RNA radius of gyration is comparable to that of the proteins, whilst at high concentration it inhibits the ability of proteins to self-assemble independently of its length. On the other hand, along with the stability modulation, the viscosity of the condensates can be considerably reduced at high RNA concentration as long as the length of the RNA chains is shorter than that of the proteins. Conversely, long RNA strands increase viscosity, even at high concentration, but barely modify protein self-diffusion, which mainly depends on RNA concentration and on its own effect on droplet density. On the whole, our work rationalizes the different routes by which RNA can regulate phase separation and condensate dynamics, as well as the subsequent aberrant rigidification implicated in the emergence of various neuropathologies and age-related diseases.

biophysics↗

bioRxiv2020

Nucleosome plasticity is a critical element of chromatin liquid-liquid phase separation and multivalent nucleosome interactions

Liquid-liquid phase separation (LLPS) of chromatin is an important mechanism that helps explain the membrane-less compartmentalization of the nucleus. Because chromatin compaction and LLPS are collective phenomena, linking their modulation to biophysical features of individual nucleosomes is challenging. Here, we develop a novel multiscale chromatin model that integrates atomistic representations, a chemically-specific coarse-grained model, and a minimal model. In tandem, we devise a transferable Debye-length exchange molecular dynamics approach to achieve enhanced sampling of high-resolution chromatin. We find that nucleosome thermal fluctuations become significant at physiological salt concentrations and destabilize the 30-nm fiber. Nucleosome breathing favors stochastic folding of chromatin and promotes LLPS by simultaneously boosting the transient nature and heterogeneity of nucleosome-nucleosome contacts, and the effective nucleosome valency. Our results put forward the intrinsic plasticity of nucleosomes as a key element in the liquid-like behavior of chromatin, and help reconcile longstanding differences between fiber-based and in vivo chromatin models.

biophysics↗

bioRxiv2020

Targeted modulation of protein liquid-liquid phase separation by evolution of amino-acid sequence

Rationally and efficiently modifying the amino-acid sequence of proteins to control their ability to undergo liquid-liquid phase separation (LLPS) on demand is not only highly desirable, but can also help to elucidate which protein features are important for LLPS. Here, we propose an innovative computational method that couples a genetic algorithm to a sequence-dependent coarse-grained protein model to evolve the amino-acid sequences of phase-separating intrinsically disordered protein regions (IDRs), and purposely enhance or inhibit their capacity to phase-separate. We apply it to the phase-separating IDRs of three naturally occurring proteins, namely FUS, hnRNPA1 and LAF1, as prototypes of regions that exist in cells and undergo homotypic LLPS driven by different types of intermolecular interaction. We find that the evolution of amino-acid sequences towards enhanced LLPS is driven in these three cases, among other factors, by an increase in the average size of the amino acids. However, the direction of change in the molecular driving forces that enhance LLPS (such as hydrophobicity, aromaticity and charge) depends on the initial amino-acid sequence: the critical temperature can be enhanced by increasing the frequency of hydrophobic and aromatic residues, by changing the charge patterning, or by a combination of both. Finally, we show that the evolution of amino-acid sequences to modulate LLPS is strongly coupled to the composition of the medium (e.g. the presence or absence of RNA), which may have significant implications for our understanding of phase separation within the many-component mixtures of biological systems.

biophysics↗

bioRxiv2020

Oligonucleotides can act as superscaffolds that enhance liquid-liquid phase separation of intracellular mixtures

Intracellular liquid-liquid phase separation (LLPS) enables the formation of biomolecular condensates, which play a crucial role in the spatiotemporal organisation of biomolecules (proteins, oligonucleotides). While LLPS of biopolymers has been demonstrated in both experiments and computer simulations, the physical determinants governing phase separation of protein-oligonucleotide systems are not fully understood. Here, we introduce a minimal coarse-grained model to investigate concentration-dependent features of protein-oligonucleotide mixtures. We demonstrate that adding oligonucleotides to biomolecular condensates composed of oligonucleotide-binding scaffold proteins enhances LLPS; since oligonucleotides act as ultra-high-valency molecules (termed superscaffolds) that increase the molecular connectivity among scaffold proteins. Importantly, we find that oligonucleotides promote protein LLPS via a seeding-type mechanism; recruiting numerous protein molecules and reducing the thermodynamic and kinetic barriers for nucleation and phase separation. By probing the conformational properties of oligonucleotides within droplets, we show that these biopolymers can undergo phase separation-driven compaction, which may be entropic in nature. Finally, we provide a quantitative comparison between mixture composition, protein valency, and protein-oligonucleotide interaction strengths. We find that superscaffolds preferentially recruit higher valency proteins to condensates, and that multiphase immiscibility within condensates can be achieved by modulating the relative protein-oligonucleotide binding strengths. These results shed light on the roles of oligonucleotides in ribonu-cleoprotein granule formation, heterochromatin compaction, and internal structuring of the nucleolus and stress granules.

biophysics↗