Biology subjects

Alaviuhkola, J.

Publications and source records attributed to Alaviuhkola, J..

4 recordsLinked to original sources

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↗

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

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↗