Biology subjects

Zhang, S. M.

Publications and source records attributed to Zhang, S. M..

6 recordsLinked to original sources

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↗

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↗

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↗

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

A Multi-pronged Screening Approach Targeting Inhibition of ETV6 PNT Domain Polymerization

ETV6 is an ETS family transcriptional repressor for which head-to-tail polymerization of its PNT domain facilitates cooperative binding to DNA by its ETS domain. Chromosomal translocations frequently fuse the ETV6 PNT domain to one of several protein tyrosine kinases. The resulting chimeric oncoproteins undergo ligand-independent self-association, autophosphorylation, and aberrant stimulation of downstream signaling pathways leading to a variety of cancers. Currently, no small molecules inhibitors of ETV6 PNT domain polymerization are known and no assays targeting PNT domain polymerization have been described. In this study, we developed complementary experimental and computational approaches for identifying such inhibitory compounds. One mammalian cellular approach utilized a mutant PNT domain heterodimer system covalently attached to split Gaussia luciferase fragments. In this protein fragment complementation assay, inhibition of PNT domain heterodimerization reduces luminescence. A yeast assay took advantage of activation of the reporter HIS3 gene upon heterodimerization of mutant PNT domains fused to DNA-binding and transactivation domains. In this two-hybrid screen, inhibition of PNT domain heterodimerization prevents cell growth in medium lacking histidine. The Bristol University Docking Engine (BUDE) was used to identify virtual ligands from the ZINC8 library predicted to bind the PNT domain polymerization interfaces. Over 75 hits from these three assays were tested by NMR spectroscopy for binding to the purified ETV6 PNT domain. Although none were found to bind, lessons learned from this study may facilitate future approaches for developing therapeutics that act against ETV6 oncoproteins by disrupting PNT domain polymerization.

biochemistry↗

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↗