Biology subjects

Galera-Prat, A.

Publications and source records attributed to Galera-Prat, A..

13 recordsLinked to original sources

bioRxiv2025

Automodification of N-terminal serine residues in PARP2 impacts PARP2 release from DNA damage sites

ADP-ribosyltransferases PARP1 and PARP2 are involved in DNA repair mechanisms and play a major role in detecting DNA damage. PARP1/2 enzymes transfer the ADP-ribosyl moiety from NAD+ to DNA damage proteins, histones and to itself, activating the DNA repair cascade. Recent studies have shown that Histone PARylation Factor (HPF1) forms a joint active site with the catalytic domain of PARP1/2 and alters their target modification site from glutamate/aspartate towards serine. In this work, we have identified key serine residues within the N-terminus of full-length PARP2 that are the main targets of PARP1/2 automodification. We demonstrated this using site-directed mutagenesis, gel-based PARylation assays of automodification reactions, and by measuring the release of PARP2 from the DNA damage site using a fluorescence polarization assay. We show that in the presence of HPF1, PARP2 serine 8 and serine 73 are predominantly ADP-ribosylated and serine 8, which is present in both human PARP2 isoforms, is the major site for PARP2 automodification. Our results provide insight into the mechanistic role of the N-terminus of PARP2 in the PARylation-dependent release of PARP2 from DNA damage sites.

biochemistry↗

bioRxiv2025

Discovery of Tankyrase scaffolding inhibitor specifically targeting the ARC4 peptide binding domain

In the past, development of tankyrase inhibitors has focused on the ADP-ribosyltransferase domain. Targeting tankyrases ability to interact with protein substrates through their ARC domains represents an alternative strategy to be explored as a therapeutic approach against specific protein-protein interactions. In this paper, we employed a FRET-based assay to identify ARC4-binding compounds by screening the EU-OPENSCREEN Pilot and Commercials Diversity libraries. We discovered an effective series of compounds with the same scaffold and through chemical synthesis we obtained the compound S8 (ARCher-142), which binds selectively to ARC4 with potency of 8 {micro}M. NMR analysis and X-ray crystallography allowed us to identify the binding site in ARC4 and to rationalize the observed selectivity. Despite binding exclusively to ARC4, the inhibitor can attenuate the WNT/{beta}-catenin signaling pathway in cells. Our work demonstrates that targeting single ARC domains is possible, offering an inhibition approach tailored to tankyrase ARC4 inhibition. SignificanceTankyrases impact a variety of cellular processes by binding proteins through their ARC domains and the inhibition of these scaffolding functions represents an alternative therapeutic approach to catalytic inhibitors. With a FRET-based high-throughput screening of the EU-OPENSCREEN Pilot and Commercials Diversity libraries we discovered a pyrrolone-based scaffold that is interestingly selective towards ARC4, despite the high conservation of the ARC binding site. Our synthesized compound S8 (ARCher-142) displays an 8 {micro}M potency for TNKS2 ARC4. With NMR and X-ray crystallography we demonstrate that S8 (ARCher-142) competes with the peptide optimized for binding and extends to a unique hydrophobic sub-pocket of ARC4. The compound attenuates the WNT/{beta}-catenin signaling pathway in cells and interestingly offers the possibility to target specific protein-protein interactions mediated by ARC4, paving the way for the development of a pyrrolone-based class of tankyrase scaffolding inhibitors.

biochemistry↗

bioRxiv2024

Substitutions at the C-8 position of quinazolin-4-ones improve the potency of nicotinamide site binding tankyrase inhibitors

Human diphtheria toxin-like ADP-ribosyltransferases, PARPs and tankyrases, transfer ADP-ribosyl groups to other macromolecules, thereby controlling various signaling events in cells. They are considered promising drug targets, especially in oncology, and some small molecule inhibitors have already been developed. These inhibitors typically interact with the nicotinamide binding site and extend along the NAD+ binding groove of the catalytic domain. Quinazolin-4-ones have been explored as promising scaffolds for such inhibitors and we have identified a new position within the catalytic domain that has not been extensively studied yet. In this study, we investigate larger substituents at the C-8 position and, using X-ray crystallography, we demonstrate that nitro- and diol-substituents engage in new interactions with TNKS2, improving both affinity and selectivity. Both nitro- and diol-substituents exhibit intriguing inhibition of TNKS2, with compound 49 displaying an IC50 of 65 nM, while compound 40s IC50 value is 14 nM. Both analogues show efficacy in cell assays and attenuate the tankyrase-controlled Wnt/{beta}-catenin signaling with sub-micromolar IC50. When tested against a wider panel of enzymes, compound 40 displayed high selectivity towards tankyrases, whereas 49 also inhibited other PARPs. The results offer new insights for inhibitor development targeting tankyrases and PARPs by focusing on the subsite between a mobile active site loop and the canonical nicotinamide binding site.

biochemistry↗

bioRxiv2023

High-throughput screening assay for PARP-HPF1 interaction inhibitors to affect DNA damage repair

ADP-ribosyltransferases PARP1 and PARP2 play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins. Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations. Recently, a study showed that Histone PARylation Factor (HPF1) forms a joint active site with PARP1/2. The interaction of HPF1 with PARP1/2 alters the automodification site from Aspartate, Glutamate to Serine, which has been shown to be a key ADP-ribosylation event in the context of DNA damage. Therefore disruption of PARP1/2-HPF1 interaction could be an alternative strategy for drug development to block the PARP1/2 activity. In this study, we describe a FRET based high-throughput screening assay to screen inhibitor libraries against PARP-HPF1 interaction. We optimized the conditions for FRET signal and verified the interaction by competing the FRET pair in multiple ways. The assay is robust and easy to automate. Validatory screening showed the robust performance of the assay, and we discovered two compounds, Dimethylacrylshikonin and Alkannin, with {micro}M inhibition potency against PARP1/2-HPF1 interaction. The assay will facilitate the discovery of inhibitors against HPF1-PARP1/2 complex and to develop potentially new effective anticancer agents.

biochemistry↗

bioRxiv2023

Discovery of 2-amide-3-methylester thiophenes inhibiting SARS-CoV-2 ADP-ribosyl hydrolysing macrodomain and coronavirus replication

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has made it clear that further development of antiviral therapies will be needed to combat additional SARS-CoV-2 variants or novel CoVs. Here, we describe small molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation mediated innate immune responses. The compounds inhibiting Mac1 were discovered through high-throughput screening (HTS) using a protein FRET-based competition assay and the best hit compound had an IC50 of 14 {micro}M. Three validated HTS hits have the same 2-amide-3-methylester thiophene scaffold and the scaffold was selected for structure-activity relationship (SAR) studies through commercial and synthesized analogs. We studied the compound binding mode in detail using X-ray crystallography and this allowed us to focus on specific features of the compound and design analogs. Compound 27 (MDOLL-0229) had an IC50 of 2.1 {micro}M and was generally selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human ADP-ribose binding proteins. The improved potency allowed testing of its effect on virus replication and indeed, 27 inhibited replication of both MHVa prototype CoV, and SARS-CoV-2. Furthermore, sequencing of a drug-resistant MHV identified mutations in Mac1, further demonstrating the specificity of 27. Compound 27 is the first Mac1 targeted small molecule demonstrated to inhibit coronavirus replication in a cell model. This, together with its well-defined binding mode, makes 27 a good candidate for further hit/lead-optimization efforts.

biochemistry↗

bioRxiv2022

benzothiazole scaffold as versatile nicotinamide mimic allowing nanomolar inhibition of different PARP enzymes

Here we report [1,2,4]triazolo[3,4-b]benzothiazole (TBT) as a new inhibitor scaffold, which competes with nicotinamide in the binding pocket of human poly- and mono-ADP-ribosylating enzymes. The binding mode was studied through analogs and their crystal structures with TNKS2, PARP2, PARP14 and PARP15. Based on the substitution pattern, we were able to identify The 3-amino derivatives 21 (OUL243) and 27 (OUL232), as inhibitors of mono-ARTs PARP7, PARP10, PARP11, PARP12, PARP14 and PARP15 at nM potencies, with compound 27 being the most potent PARP10 inhibitor described to date with an IC50 of 7.8 nM and the first PARP12 inhibitor ever reported. On the contrary, hydroxy derivative 16 (OUL245) inhibits poly-ARTs with a selectivity towards PARP2. The scaffold does not possess inherent cell toxicity and the inhibitors can enter cells and engage with the target protein. This, together with favorable ADME properties, demonstrates the potential of the TBT scaffold for future drug development efforts towards selective inhibitors against specific enzymes.

biochemistry↗

bioRxiv2022

An evolutionary perspective on the origin, conservation and binding partner acquisition of tankyrases

Tankyrases are poly-ADP-ribosyltransferases that regulate many crucial and diverse cellular processes in humans such as Wnt signaling, telomere homeostasis, mitotic spindle formation and glucose metabolism. While tankyrases are present in most animals, functional differences across species exist. In this work we confirm the widespread distribution of tankyrases throughout the branches of multicellular animal life and identified the single-celled choanoflagellates as earliest origin of tankyrases. We further show that sequence and structural aspects of TNKSs are well conserved even between highly diverged species. We also experimentally characterized an anciently diverged tankyrase homolog from the sponge Amphimedon queenslandica and show that the basic functional aspects, such as poly-ADP-ribosylation activity and interaction with the canonical tankyrase binding peptide motif, are conserved. Conversely, the presence of tankyrase binding motifs in orthologs of confirmed interaction partners vary greatly between species, indicating that tankyrases have different sets of interaction partners depending on the animal lineage. Overall, our analysis suggests a remarkable degree of conservation for tankyrases, although their regulatory functions in cells have likely changed considerably throughout evolution.

bioinformatics↗

bioRxiv2022

Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity

Human mono-ADP-ribosylating PARP enzymes have been linked to several clinically relevant processes and many of these PARPs have been suggested as potential drug targets. Despite recent advances in the field, efforts to discover such compounds have been hindered by the lack of tools to rapidly screen for high potency compounds and profile them against the different PARP enzymes of the ARTD family. We here expanded the methods and engineered mono-ART catalytic fragments to be incorporated into a cellulosome-based octavalent scaffold. Compared to the free enzymes, the scaffold-based system results in an improved activity for the tested PARPs due to improved solubility, stability and the proximity of the catalytic domains, altogether boosting their activity beyond 10-fold in the case of PARP12. This allows us to measure their enhanced activity using a simple and easily accessible homogeneous NAD+ conversion assay, facilitating its automation to reduce the assay volume and lowering the assay costs. The approach will enable the discovery of more potent compounds due to increased assay sensitivity and it can be applied to compound screening campaigns as well as inhibitor profiling.

biochemistry↗

bioRxiv2021

Analogs of TIQ-A as inhibitors of human mono-ADP-ribosylating PARPs

The scaffold of TIQ-A, a previously known inhibitor of human poly-ADP-ribosyltransferase PARP1, was utilized to develop inhibitors against human mono-ADP-ribosyltransferases through structure-guided design and activity profiling. By supplementing the TIQ-A scaffold with small structural changes, based on a PARP10 inhibitor OUL35, selectivity changed from poly-ADP-ribosyltransferases towards mono-ADP-ribosyltransferases. Binding modes of analogs were experimentally verified by determining complex crystal structures with mono-ADP-ribosyltransferase PARP15 and with poly-ADP-ribosyltransferase TNKS2. The best analogs of the study achieved 10 - 20-fold selectivity towards mono-ADP-ribosyltransferases PARP10 and PARP15 while maintaining micromolar potencies. The work demonstrates a route to differentiate compound selectivity between mono- and poly-ribosyltransferases of the human ARTD family.

biochemistry↗

bioRxiv2021

A molecular toolbox for ADP-ribosyl binding proteins

Proteins interacting with ADP-ribosyl groups are often involved in disease-related pathways or in viral infections, which makes them attractive targets for the development of inhibitors. Our goal was to develop a robust and accessible assay technology that is suitable for high-throughput screening and applicable to a wide range of proteins acting as either hydrolysing or non-hydrolysing binders of mono- and poly-ADP-ribosyl groups. As a foundation of our work, we developed a C-terminal protein fusion tag based on a Gi protein alpha subunit peptide (GAP), which allows for site-specific introduction of cysteine-linked mono- and poly-ADP-ribosyl groups as well as chemical ADP-ribosyl analogs. By fusion of the GAP-tag and ADP-ribosyl binders to fluorescent proteins, we were able to generate robust FRET signals and the interaction with 22 previously described ADP-ribosyl-binders was confirmed. To demonstrate the applicability of this binding assay for high-throughput screening, we utilized it to screen for inhibitors of the SARS-CoV-2 nsp3 macrodomain and identified the drug suramin as a moderate yet unspecific inhibitor of this protein. To complement the binding technology, we prepared high-affinity ADP-ribosyl binders fused to a nanoluciferase, which enabled simple blot-based detection of mono- and poly-ADP-ribosylated proteins. These tools can be expressed recombinantly in E. coli using commonly available agents and will help to investigate ADP-ribosylation systems and aid in drug discovery.

biochemistry↗

bioRxiv2020

Activation of ARTD2/PARP2 by DNA damage induces conformational changes relieving enzyme autoinhibition

Human ARTD2/PARP2 is an ADP-ribosyltransferase which, when activated by 5- phosphorylated DNA ends, catalyzes poly-ADP-ribosylation of itself, other proteins and DNA. A crystal structure of ARTD2 in complex with an activating 5-phosphorylated DNA shows that the WGR domain bridges the dsDNA gap and joins the DNA ends. This DNA binding results in major conformational changes, reorganization of helical fragments, in the ARTD2 regulatory domain. Comparison of ARTD1-3 crystal structures reveal how binding to a DNA damage site leads to formation of a catalytically competent conformation capable of binding substrate NAD+ and histone PARylation factor 1 changing the ARTD2 residue specificity from glutamate to serine when initiating DNA repair processes. The structure also reveals how the conformational changes in the autoinhibitory regulatory domain would promote the flexibility needed by the enzyme to reach the target macromolecule for ADP-ribosylation.

biochemistry↗

bioRxiv2020

Divergent CPEB prion-like domains reveal different assembly mechanisms for a generic amyloid-like fold

Functional amyloids are present in a wide variety of organisms ranging from bacteria to humans. Experience-dependent aggregation of the cytoplasmic polyadenylation element-binding (CPEB) prion-like protein to a translationally active state has emerged as a plausible biochemical substrate of long-lasting memories. CPEB aggregation is driven by prion-like domains (PLD) that are highly divergent in sequence across species. Here, we describe the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD in vitro using single-molecule and bulk biophysical methods and compare them with those previously reported for neuronal Drosophila CPEB, Orb2 PLD. The existence of transient oligomers and mature filaments suggests similarities in the late stages of the assembly pathway for both PLDs. However, while prior to aggregation the Orb2 PLD monomer remains as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising -helical structures that evolve to coiled-coil species, suggesting structural differences at the beginning of their amyloid assembly pathways. Our results show how divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms.

neuroscience↗

bioRxiv2019

smFRET Detects Dual Binding Modes Modulated by Proline Isomerization in a Mega-Dalton Multi-Enzyme Complex

Cellulose is the most abundant organic molecule on Earth and represents a renewable and practically everlasting feedstock for the production of biofuels and chemicals. Self-assembled owing to the high-affinity cohesin-dockerin interaction, cellulosomes are huge multi-enzyme complexes with unmatched efficiency in the degradation of recalcitrant lignocellulosic substrates. The recruitment of diverse dockerin-borne enzymes into a multicohesin protein scaffold dictates the three-dimensional layout of the complex, and interestingly two alternative binding modes have been proposed. Using single-molecule Fluorescence Resonance Energy Transfer, molecular dynamics simulations and NMR measurements on a range of cohesin-dockerin pairs, we directly detect varying distributions between these binding modes that follow a built-in cohesin-dockerin code. Surprisingly, we uncover a prolyl isomerase-modulated allosteric control mechanism, mediated by the isomerization state of a single proline residue, which regulates the distribution and kinetics of binding modes. Overall, our data provide a novel mechanistic understanding of the structural plasticity and dynamics of cellulosomes.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗