Biology subjects

Rudd, S. G.

Publications and source records attributed to Rudd, S. G..

8 recordsLinked to original sources

bioRxiv2025

NUDT5 regulates the global efficacy of nucleoside analog drugs by coordinating purine synthesis and PRPP allocation

Cancer cells require nucleotides to meet proliferation demands, which has been effectively exploited by nucleoside analog (NA) drugs. Despite their success, we still do not completely understand factors dictating their anticancer effect. Here, we report that a NUDT5 scaffolding function indirectly regulates the global efficacy of anti-cancer NA drugs. Mechanistically, PROTAC- or RNAi-mediated NUDT5 depletion, not inhibition, desensitizes cells to 6-thioguanine (6TG) and other NA drugs proportionally with NUDT5 abundance. NUDT5-depleted cells appear locked into de novo purine biosynthesis (DNPB), thus impairing salvage of nucleotide precursors and NA drug activation. Specifically, NUDT5 interacts with phosphoribosyl amidotransferase (PPAT), the rate-limiting DNPB enzyme, to putatively regulate its activity, DNPB flux, and resultant phosphoribosyl pyrophosphate (PRPP) allocation. Collectively, these results suggest that NUDT5 controls NA drug efficacy by coordinating nucleotide synthesis and PRPP utilization, making it a potential biomarker of clinical efficacy and target for finetuning antimetabolite therapies. Statement of significanceNUDT5 non-enzymatically regulates the global efficacy of anti-cancer nucleoside analog drugs by interacting with PPAT to control DNPB and allocation of PRPP, thereby establishing it as a biomarker of clinical significance.

biochemistry↗

bioRxiv2025

Uridine cytidine kinases dictate the therapeutic response of molnupiravir via its bioactivation

Molnupiravir is a nucleoside analogue antiviral drug with activities against a broad spectrum of RNA viruses, including its clinical indication SARS-CoV-2. Whilst its antiviral mechanism-of-action is well defined, host pharmacogenetic factors that regulate its therapeutic responses - efficacy as well as selectivity - have not been mechanistically deciphered and characterized. Here we identified that uridine cytidine kinase (UCK), the first and rate-limiting kinase of the pyrimidine salvage pathway, could effectively phosphorylate N4-hydroxycytidine (NHC), the active compound of molnupiravir, and thereby dictate its anti-SARS-CoV-2 efficacy, as well as its selectivity index. Using target engagement and enzyme kinetic assays, we demonstrate that recombinant UCK isoform 1 (UCK1) and 2 (UCK2) effectively bind and phosphorylate NHC, with UCK2 displaying 9-fold higher catalytic efficiency. Accordingly, in SARS-CoV-2-infected cells, downregulation of UCK2 via siRNA hampered the intracellular accumulation of the triphosphate antiviral metabolite of NHC, resulting in 10-fold reduction of antiviral efficacy. Furthermore, we could recapitulate this using a pan-UCK small molecule inhibitor. Critically, both UCK downregulation and inhibition significantly reduced the selectivity index of molnupiravir/NHC. Altogether, this work underscores the pivotal roles of UCK enzymes in upholding molnupiravir efficacy and therapeutic window, and furthermore, highlights their potential as pharmacologically tractable targets for tailoring the therapeutic response to this antiviral agent.

microbiology↗

bioRxiv2024

Glutamine-addiction in cisplatin resistant cancer cells is mediated by SLC7A11 and can be targeted with asparaginase therapy

Linking disease phenotypes with molecular targets is key to the rational design of treatment interventions. Resistance to the chemotherapeutic cisplatin is one of the major factors limiting the clinical utility of this therapy, which is central to the treatment of a variety of solid malignancies. In this study, we couple the upregulation of a chemoresistant factor, the glutamate-cystine antiporter SLC7A11, with the addiction of cisplatin-resistant cancer cells to extracellular glutamine. In doing so, we thus provide a putative biomarker for this acquired metabolic dependency of chemoresistance. Subsequently, we evaluate various therapeutic strategies to selectively kill SLC7A11high cisplatin-resistant cancer cells, identifying cross-resistance to ferroptosis-inducing compounds and hypersensitivity to glutaminase inhibitor CB-839. We identify enzymatic depletion of extracellular glutamine using the long-standing anti-leukemic therapy asparaginase (ASNase), which possesses glutaminase activity, as a potential approach, and show this can be successfully combined with cisplatin in cell models. In summary, this study mechanistically links an acquired metabolic dependency of chemoresistant cancer cells with a putative biomarker and provides a potentially actionable strategy to target these drug resistant cells warranting further investigation.

cancer biology↗

bioRxiv2023

Identification and evaluation of small-molecule inhibitors against the dNTPase SAMHD1 via a comprehensive screening funnel

Sterile alpha motif and histidine-aspartic acid domain containing protein-1 (SAMHD1) is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase central to cellular nucleotide pool homeostasis. Recent literature has also demonstrated how SAMHD1 can detoxify chemotherapy metabolites thereby controlling their clinical responses. To further understand SAMHD1 biology and to investigate the potential of targeting this enzyme as a neoadjuvant to existing chemotherapies we set out to discover selective small molecule-based inhibitors of SAMHD1. Here we report a discovery pipeline encompassing a biochemical screening campaign and a set of complementary biochemical, biophysical, and cell-based readouts for further characterisation of the screen output. The identified hit compound TH6342 and its analogues, accompanied by their inactive negative control analogue TH7126, demonstrated specific, low M potency in inhibiting the hydrolysis of both natural substrates and nucleotide analogue therapeutics, shown using complementary enzyme-coupled and direct enzymatic activity assays. Their mode of inhibition was subsequently detailed by coupling kinetic studies with thermal shift assays, where TH6342 and analogues were shown to engage with pre-tetrameric SAMHD1 and deter the oligomerisation and allosteric activation of SAMHD1 without occupying nucleotide binding pockets. We further outline the development and application of multiple cellular assays for assessing cellular target engagement and associated functional effects, including CETSA and an in-cell dNTP hydrolase activity assay, which highlighted future optimisation strategies of this chemotype. In summary, with a novel mode of inhibition, TH6342 and analogues broaden the set of tool compounds available in deciphering SAMHD1 enzymology and functions, and furthermore, the discovery pipeline reported herein represents a thorough framework for future SAMHD1 inhibitor development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/524275v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@dc706forg.highwire.dtl.DTLVardef@59a9b4org.highwire.dtl.DTLVardef@9473e2org.highwire.dtl.DTLVardef@4427dc_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Drug synergy scoring using minimal dose response matrices

ObjectiveCombinations of pharmacological agents are essential for disease control and prevention, offering many advantages over monotherapies, with one of these being drug synergy. The state-of-the-art method to profile drug synergy in preclinical research is by using dose-response matrices in disease-appropriate models, however this approach is frequently labour intensive and cost-ineffective, particularly when performed in a medium- to high-throughput fashion. Thus, in this study, we set out to optimise a parameter of this methodology, determining the minimal matrix size that can be used to robustly detect and quantify synergy between two drugs. ResultsWe used a drug matrix reduction workflow that allowed the identification of a minimal drug matrix capable of robustly detecting and quantifying drug synergy. These minimal matrices utilise substantially less reagents and data processing power than their typically used larger counterparts. Focusing on the antileukemic efficacy of the chemotherapy combination of cytarabine and inhibitors of ribonucleotide reductase, we could show that detection and quantification of drug synergy by three common synergy models was well-tolerated despite reducing matrix size from 8x8 to 4x4. Overall, the optimisation of drug synergy scoring as presented here could inform future medium- to high-throughput drug synergy screening strategies in pre-clinical research. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/362103v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@fd5e27org.highwire.dtl.DTLVardef@30570dorg.highwire.dtl.DTLVardef@1a33039org.highwire.dtl.DTLVardef@b2f00c_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2020

SAMHD1 promotes oncogene-induced replication stress

Oncogenes induce DNA replication stress in cancer cells. Although this was established more than a decade ago, we are still unravelling the molecular underpinnings of this phenomenon, which will be critical if we are to exploit this knowledge to improve cancer treatment. A key mediator of oncogene-induced replication stress is the availability of DNA precursors, which will limit ongoing DNA synthesis by cellular replicases. In this study, we identify a potential role for nucleotide catabolism in promoting replication stress induced by oncogenes. Specifically, we establish that the dNTPase SAMHD1 slows DNA replication fork speeds in human fibroblasts harbouring an oncogenic RAS allele, elevating levels of endogenous DNA damage, and ultimately limiting cell proliferation. We then show that oncogenic RAS-driven tumours express reduced SAMHD1 levels, suggesting they have overcome this tumour suppressor barrier, and that this correlates with worse overall survival for these patients. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/226282v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@bb425forg.highwire.dtl.DTLVardef@2a1b7org.highwire.dtl.DTLVardef@c70bf3org.highwire.dtl.DTLVardef@1e1b028_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2019

MTH1 inhibitor TH588 induces mitosis-dependent accumulation of genomic 8-oxodG and disturbs mitotic progression

Reactive oxygen species (ROS) oxidise nucleotide triphosphate pools (e.g., 8-oxodGTP), which may kill cells if incorporated into DNA. Whether cancers avoid poisoning from oxidised nucleotides by preventing incorporation via the oxidised purine diphosphatase MTH1 remains under debate. Also, little is known about DNA polymerases incorporating oxidised nucleotides in cells or how oxidised nucleotides in DNA become toxic. We show replacement of one of the main DNA replicases in human cells, DNA polymerase delta (Pol {delta}), to an error-prone variant allows increased 8-oxodG accumulation into DNA following treatment with the MTH1 inhibitor (MTH1i) TH588. The resulting elevated genomic 8-oxodG correlates with increased cytotoxicity of TH588. Interestingly, no substantial perturbation of replication fork progression is observed, but rather mitotic progression is impaired and mitotic DNA synthesis triggered. Reducing mitotic arrest by reversin treatment prevents accumulation of genomic 8-oxodG and reduces cytotoxicity of TH588, in line with the notion that mitotic arrest is required for ROS build-up and oxidation of the nucleotide pool. Furthermore, we demonstrate delayed mitosis and increased mitotic cell death following TH588 treatment in cells expressing the error-prone Pol {delta} variant, which is not observed following treatments with anti-mitotic agents, thus linking incorporation of oxidised nucleotides and disturbed mitotic progression.

cancer biology↗

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↗