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Lehtiö, L.

Publications and source records attributed to Lehtiö, L..

At least 19 recordsLinked to original sources
bioRxiv2026

Discovery of dual thiobarbiturate-indole scaffold as a selective inhibitor targeting chikungunya virus nsP3 macrodomain through a cryptic binding pocket

The chikungunya virus (CHIKV) outbreak imposes a significant burden on healthcare systems and raises an urgent need for effective antiviral therapies. So far there are no specific drugs against CHIKV. A CHIKV macrodomain is critical for virulence and counteracts the host immune response, representing a promising antiviral drug target. Here, we describe small molecule inhibitors targeting the CHIKV macrodomain. Compound 1 (MDOLL-0273) was identified through a high-throughput screening using a fluorescence resonance energy transfer (FRET)-based assay, and its inhibitory activity was validated through multiple orthogonal assays. Compound 1 has a dual thiobarbiturate-indole scaffold and exhibits an IC50 of 8.9 {micro}M. X-ray crystallography revealed that the inhibitor occupies an adenine binding site of the macrodomain and extends into a novel cryptic pocket. Notably, the inhibitor shows high selectivity for the CHIKV macrodomain over a panel of human and viral ADP-ribosyl binding and hydrolyzing proteins. Structure-activity relationship studies and medicinal chemistry efforts provide a promising starting point for further hit optimization.

biochemistry↗
bioRxiv2025

Dimerization of human PARP15 is required for NAD+ binding and automodification

PARP proteins are enzymes catalyzing ADP-ribosylation, a well-conserved post-translational modification. In addition to the catalytic domain present in all PARPs, these proteins have a wide selection of other domains possessing different functions. Additional domains present in human PARP15 are macrodomains, capable of binding ADP-ribose. PARP15 is the least studied of the macrodomain-containing human PARPs, and it is linked to different diseases from infections to cancer. We show that the full-length canonical isoform 1 of PARP15 auto-ADP-ribosylates robustly on glutamate and aspartate residues, which can be hydrolyzed by known ADP-ribosylhydrolases. We have been able to locate several modification sites to the region of tandem macrodomains. In agreement with earlier reports regarding the catalytic domain dimerization being the requirement for enzyme activity, we show this in the full-length context with the recombinant isoform 1 of PARP15. We show that dimerization is required for the efficient substrate NAD+ binding, and we provide structural basis for this requirement with the help of a co-crystal structure of the catalytic domain dimer with an unhydrolyzable substrate analog.

biochemistry↗
bioRxiv2025

A FRET-based high-throughput screening assay for the discovery of Mycobacterium tuberculosis DNA ADP-ribosylglycohydrolase DarG inhibitors

DarTG2 is a conserved toxin-antitoxin ADP-ribosylation system that regulates bacterial survival and the anti-phage response found in many pathogenic bacteria, including Mycobacterium tuberculosis. While DNA ADP-ribosyltransferase (DarT2) toxin mono-ADP-ribosylates a single- stranded DNA sequence motif and potentially induces bacterial dormancy, its cognate DNA ADP- ribosylglycohydrolase (DarG) antitoxin reverses the modification and restores bacterial growth. Therefore, developing DarG selective inhibitors may represent a promising and novel strategy to combat drug-resistant tuberculosis. However, no small molecule inhibitors targeting DarG have been identified to date and establishing a high-throughput screening assay based on its DNA ADP- ribosylhydrolase activity would be challenging. Here, we developed and optimized a simple and robust fluorescence resonance energy transfer (FRET)-based high-throughput screening assay to identify small molecule inhibitors targeting DarG macrodomain in M. tuberculosis. We generated a FRET pair of M. tuberculosis DarG macrodomain and poly-ADP-ribosylated peptide fused with compatible fluorophores. Screening the target-focused phenotypic library using this method led to the identification of pranlukast, which selectively inhibited the DNA ADP-ribosylhydrolase activity of DarG in M. tuberculosis and its orthologues without affecting human mono-ADP- ribosyl binders and erasers. Since pranlukast has previously been reported to reduce M. tuberculosis burden, further investigation into its action mechanism in this context would be valuable.

biochemistry↗
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↗
bioRxiv2025

Discovery of inhibitors for bacterial Arr enzymes ADP-ribosylating and inactivating rifamycin antibiotics

ADP-ribosylation is an enzymatic process where an ADP-ribose moiety is transferred from NAD+ to an acceptor molecule. While ADP-ribosylation is well-established as a post-translational modification of proteins, rifamycin antibiotics are its only known small-molecule targets. ADP-ribosylation of rifampicin was first identified in Mycolicibacterium smegmatis, whose Arr enzyme transfers the ADP-ribose moiety to the 23-hydroxy group of rifampicin preventing its interaction with the bacterial RNA polymerase thereby inactivating the antibiotic. Arr homologues are widely spread among bacterial species and present in several pathogenic species often associated with mobile genetic elements. Inhibition of Arr enzymes offers a promising strategy to overcome ADP-ribosylation mediated rifamycin resistance. We developed a high-throughput activity assay, which was applied to screen an in-house library of human ADP-ribosyltransferase-targeted compounds. We identified 15 inhibitors with IC50 values below 5 {micro}M against four Arr enzymes from M. smegmatis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Mycobacteroides abscessus. The observed overall selectivity of the hit compounds over the other homologues indicated structural differences between the proteins. We crystallized M. smegmatis and P. aeruginosa Arr enzymes, the former in complex with its most potent hit compound with an IC50 value of 1.3 {micro}M. We observed structural differences in the NAD+ binding pockets of the two Arr homologues explaining the selectivity. Although the Arr inhibitors did not sensitize M. smegmatis to rifampicin in a growth inhibition assay, the structural information and the collection of inhibitors provide a foundation for rational modifications and further development of the compounds.

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

Oligomerisation mediated by the D2 domain of DTX3L is critical for DTX3L-PARP9 reading function of mono-ADP-ribosylated androgen receptor

Deltex proteins are a family of E3 ubiquitin ligases that encode C-terminal RING and DTC domains that mediate interactions with E2 ubiquitin-conjugating enzymes and recognise ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N-terminal domain architecture. The N-terminal D1 and D2 domains of DTX3L mediate homo-oligomerisation, and the D3 domain interacts with PARP9, a protein that contains tandem macrodomains with ADP-ribose reader function. While DTX3L and PARP9 are known to heterodimerize, they assemble into a high molecular weight oligomeric complex, but the nature of the oligomeric structure, including whether this contributes to the ADP-ribose reader function is unknown. Here, we report a crystal structure of the DTX3L N-terminal D2 domain and show that it forms a tetramer with, conveniently, D2 symmetry. We identified two interfaces in the structure: a major, conserved interface with a surface of 973 [A]2 and a smaller one of 415 [A]2. Using native mass spectrometry, we observed molecular species that correspond to monomers, dimers and tetramers of the D2 domain. Reconstitution of DTX3L knockout cells with a D1-D2 deletion mutant showed the domain is dispensable for DTX3L-PARP9 heterodimer formation, but necessary to assemble an oligomeric complex with efficient reader function for ADP-ribosylated androgen receptor. Our results suggest that homo-oligomerisation of DTX3L is important for mono-ADP-ribosylation reading by the DTX3L-PARP9 complex and to a ligand-regulated transcription factor.

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↗
bioRxiv2023

Discovery of DTX3L inhibitors through a homogeneous FRET-based assay that monitors formation and removal of poly-ubiquitin chains

Ubiquitination is a complex and reversible protein post-translational modification in which the subsequent action of enzymes belonging to three different families, broadly referred to as E1, E2 and E3, results in the covalent linking of ubiquitin to a target protein. While this linkage is canonically an isopeptide bond between the C-terminus of ubiquitin and the lysine residue of the target protein, Ser, Thr, and Tyr can also be susceptible to ubiquitination through an oxyester bond. Once ubiquitinated, multiple units of ubiquitin can be attached to the initial ubiquitin thus extending it to a chain of ubiquitins. Ubiquitination regulates multiple cellular processes, but it is best known as a modification that targets proteins for proteasomal degradation following the formation poly-ubiquitin chains linked through lysine 48 or 63 of ubiquitin. Dysregulation of ubiquitination has been associated with multiple types of cancer and efforts have been carried out to develop technologies that lead to the identification of inhibitors of the enzymes involved in the ubiquitination cascade. Herein, we present the development of a FRET-based assay that allows us to monitor auto-ubiquitination of DTX3L, a RING-type E3 ubiquitin ligase. Our method shows a robust signal window with a robust average Z factor of 0.76. From a validatory screening experiment we have identified the first molecules that inhibit DTX3L with potencies in the low micromolar range. Additionally, we have expanded the system to study deubiquitinases such as USP28 that lead to reduction of FRET due to hydrolysis of fluorescent poly-Ub chains.

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↗
bioRxiv2022

Discovery of compounds that inhibit SARS-CoV-2 Mac1-ADP-ribose binding by high-throughput screening

The emergence of several zoonotic viruses in the last twenty years, especially the pandemic outbreak of SARS-CoV-2, has exposed a dearth of antiviral drug therapies for viruses with pandemic potential. Developing a diverse drug portfolio will be critical for our ability to rapidly respond to novel coronaviruses (CoVs) and other viruses with pandemic potential. Here we focus on the SARS-CoV-2 conserved macrodomain (Mac1), a small domain of non-structural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that cleaves mono-ADP-ribose (MAR) from target proteins, protects the virus from the anti-viral effects of host ADP-ribosyltransferases, and is critical for the replication and pathogenesis of CoVs. In this study, a luminescent-based high-throughput assay was used to screen [~]38,000 small molecules for those that could inhibit Mac1-ADP-ribose binding. We identified 5 compounds amongst 3 chemotypes that inhibit SARS-CoV-2 Mac1-ADP-ribose binding in multiple assays with IC50 values less than 100{micro}M, inhibit ADP-ribosylhydrolase activity, and have evidence of direct Mac1 binding. These chemotypes are strong candidates for further derivatization into highly effective Mac1 inhibitors.

biochemistry↗
bioRxiv2021

The zinc-binding motif in tankyrases is required for the structural integrity and proper function of the catalytic domain

Tankyrases are ADP-ribosylating enzymes that regulate many physiological processes in the cell and they are therefore possible drug targets for cancer and fibrotic diseases. The catalytic ADP-ribosyl-transferase domain of tankyrases contains a unique zinc-binding motif of unknown function. Recently, this motif was suggested to be involved in the catalytic activity of tankyrases. In this work, we set out to study the effect of the zinc-binding motif on activity, stability and structure of human tankyrases. We generated mutants of human TNKS1 and TNKS2 abolishing the zinc-binding capabilities and characterized the proteins biochemically and biophysically in vitro. We further generated a crystal structure of TNKS2, in which the zinc ion was oxidatively removed. Our work shows that the zinc-binding motif in tankyrases is a crucial structural element which is particularly important for the structural integrity of the acceptor site. While mutation of the motif rendered TNKS1 inactive likely due to introduction of major structural defects, the TNKS2 mutant remained active and displayed a different activity profile compared to the wild type.

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

Reconstitution of the DTX3L-PARP9 complex reveals determinants for high affinity heterodimer formation and enzymatic function

Ubiquitination and ADP-ribosylation are post-translational modifications that play major roles in pathways like DNA damage response and infection, making them attractive targets for therapeutic intervention. DTX3L, an E3 ubiquitin ligase, forms a heterodimer with PARP9. The complex has ubiquitin ligase activity and also ADP-ribosylates the C-terminus of ubiquitin on Gly76. NAD+-dependent ADP-ribosylation of ubiquitin by DTX3L-PARP9 prevents ubiquitin from conjugating to protein substrates. By using individually produced proteins, we have studied the interaction between DTX3L and PARP9. We identify that the D3 domain (230 - 510) of DTX3L mediates interaction with PARP9 with nanomolar affinity and an apparent 1:1 stoichiometry. Our results also suggest the formation of a higher molecular weight oligomer mediated by the N-terminus of DTX3L (1-200). Furthermore, we show that ADP-ribosylation of ubiquitin at Gly76 is a reversible modification that can be removed by several macrodomain-type hydrolases. Our study provides a framework to understand how DTX3L-PARP9 mediates ADP-ribosylation and ubiquitination in an inter-regulatory manner.

biochemistry↗