Biology subjects

Almlof, I.

Publications and source records attributed to Almlof, I..

3 recordsLinked to original sources

bioRxiv2023

Kinetic and structural characterization of human NUDIX hydrolase NUDT15 and NUDT18 as catalysts of isoprene pyrophosphate hydrolysis

Isoprene pyrophosphates play a crucial role in the synthesis of a diverse array of essential nonsterol and sterol biomolecules, and serve as substrates for post-translational isoprenylation of proteins, enabling specific anchoring to cellular membranes. Hydrolysis of isoprene pyrophosphates would be a means to modulate their levels, downstream products, and protein isoprenylation. While NUDIX hydrolases from plants have been reported to catalyze the hydrolysis of isoprene pyrophosphates, homologous enzymes with this function in animals have not yet been identified. In this study, we screened an extensive panel of human NUDIX hydrolases for activity in hydrolyzing isoprene pyrophosphates. We found that human NUDT15 and NUDT18 efficiently catalyze the hydrolysis of several physiologically relevant isoprene pyrophosphates. Notably, we demonstrate that geranyl pyrophosphate is an excellent substrate for NUDT18, which displays a catalytic efficiency of 2.1{middle dot}105 M-1s-1, thus making it the best substrate identified for NUDT18 to date. Similarly, geranyl pyrophosphate proved to be the best isoprene pyrophosphate substrate for NUDT15, with a catalytic efficiency of 4.0{middle dot}104 M-1s-1. LC-MS analysis of NUDT15 and NUDT18 catalyzed isoprene pyrophosphate hydrolysis revealed the generation of the corresponding monophosphates and inorganic phosphate. Furthermore, we solved the crystal structure of NUDT15 in complex with the hydrolysis product geranyl phosphate at a resolution of 1.70 [A]. This structure revealed that the active site nicely accommodates the hydrophobic isoprenoid moiety and aided in identifying key binding residues. By overexpressing NUDT15 and NUDT18 in cells, we demonstrated a decrease in cellular cholesterol levels. Collectively, our findings strongly imply that isoprene pyrophosphates are endogenous substrates of NUDT15 and NUDT18, and support their involvement in animal isoprene pyrophosphate metabolism.

biochemistry↗

bioRxiv2022

Coupling cellular drug-target engagement to downstream pharmacology with CeTEAM

Cellular target engagement technologies are reforming drug discovery by enabling quantification of intracellular drug binding; however, simultaneous assessment of drug-associated phenotypes has proven challenging. CeTEAM (cellular target engagement by accumulation of mutant) is a platform that can concomitantly evaluate drug-target interactions and phenotypic responses for holistic assessment of drug pharmacology using conditionally-stabilized drug biosensors. We observe that drug-responsive proteotypes are prevalent among reported mutants of known drug targets. CeTEAM-compatible mutants follow structural and biophysical logic that permits intra-protein and paralogous expansion of the biosensor pool, as exemplified by alanine scanning of leucines within the PARP1 helical domain and transfer of PARP1 destabilization to the analogous PARP2 residue. We then apply CeTEAM to uncouple target engagement from divergent cellular activities of MTH1 inhibitors, dissect NUDT15-associated thiopurine metabolism with the R139C pharmacogenetic variant, and profile the live-cell dynamics of PARP1/2 binding and DNA trapping by PARP inhibitors. Further, PARP1-derived biosensors facilitated high-throughput screening of drug-like libraries for PARP1 binders, as well as multimodal ex vivo analysis and non-invasive tracking of PARPi binding in live animals. Our data suggests that CeTEAM can facilitate real-time, comprehensive characterization of target engagement by bridging drug binding events and their biological consequences.

biochemistry↗

bioRxiv2019

MTH1 promotes mitotic progression to avoid oxidative DNA damage in cancer cells

BACKGROUNDWe developed MTH1 inhibitors (MTH1i) TH588 and TH1579 showing broad anti-cancer activity, while structurally distinct MTH1i fail to kill cancer cells. Here, we describe a new role of MTH1 in mitosis and the detailed mechanism of action of TH1579 (karonudib) and other structurally distinct MTH1i.\n\nMATERIALS AND METHODSCancer cell lines or zebrafish embryos were treated with MTH1i or siRNA targeting MTH1 and analysed primarily by live cell and immunofluorescence microscopy, survival assays, DNA fibre or COMET assays. MTH1 and tubulin interactions were analysed in vitro using co-immunoprecipitation and tubulin polymerisation assays.\n\nRESULTSHere, we describe a mitotic role for the MTH1 protein, which binds to tubulin, is required for microtubule polymerisation, correct spindle assembly, mitosis progression and suppression reactive oxygen species (ROS) generation in mitosis. Potent MTH1i display differential abilities to break the MTH1-tubulin interaction and cause mitotic arrest, demonstrating 8-oxodGTPase and mitotic function of MTH1 are mechanistically distinct. TH588 and TH1579 have more profound effect on mitotic arrest than other MTH1i explained by additional direct inhibition of tubulin polymerisation. MTH1i only inhibiting 8-oxodGTPase activity synergize with mitotic poisons.\n\nCONCLUSIONSEfficient MTH1 have a dual mechanism of action: inhibiting mitosis (to generate ROS) and promoting 8-oxodGTP incorporation into DNA during mitotic replication, dependent on ROS generation. Direct inhibition of tubulin polymerisation of TH588 and TH1579 increase their ability to arrest cells and generate ROS in mitosis. Furthermore, non-cytotoxic MTH1 can become effective and increase incorporation of oxidised nucleotides into DNA when combined with sub-therapeutic concentrations of mitotic inhibitors or challenged directly by 8-oxodGTP.

cancer biology↗