Biology subjects

Gray, N.

Publications and source records attributed to Gray, N..

17 recordsLinked to original sources

bioRxiv2026

Computational design of a versatile, zero-radius proximity labeling enzyme

The ability to map protein interactomes and organelle proteomes is foundational for achieving a molecular understanding of living cells. Proximity labeling (PL) provides a powerful strategy for this, but existing enzymes and photocatalysts are limited by their spatial resolution, reliance on biotin, and/or in vivo compatibility. Here we report FlexID, an engineered promiscuous ligase that catalyzes the rapid attachment of diverse small-molecule probes to proximal endogenous proteins. Critically, FlexID operates through a zero-radius, direct-contact mechanism, offering superior spatial precision compared to existing PL tools. We engineered FlexID by combining the strengths of sequence- and structure-trained computational models to enhance its catalytic activity and structural stability. Biophysical analysis revealed that specific conformational changes in FlexID improve its ability to recognize diverse target proteins while simultaneously preventing the premature release of the reactive intermediate. We demonstrate FlexID's versatility through in vivo proximity labeling, comprehensive organelle proteome mapping, and a high-throughput, fluorescence-based screen for molecular glues. Our work shows that computational methods can be harnessed to create mechanistically distinct PL enzymes and establishes FlexID as a flexible, high-resolution tool for mapping protein interactions and proteomes in living cells.

bioengineering↗

bioRxiv2026

A druggable redox switch on SHP1 controls macrophage inflammation

Immunological proteins are major disease targets, yet most remain undrugged. Post-translational redox modification of cysteine residues has emerged as an important mode of immune cell regulation, particularly in macrophage cytokine responses. Here, we develop a strategy for systematic discovery and small-molecule functionalization of redox-regulated cysteines on immunological proteins. Using deep redox proteomics, we annotate 788 in vivo redox-regulated cysteines across diverse immune-relevant protein domains. We demonstrate how these sites enable cysteine-directed pharmacology through discovery of a novel cysteine activation site on the immune regulator SHP1. Targeting Cys102, we develop a highly selective covalent agonist, SCA, which binds the N-SH2 domain to relieve autoinhibition and activate SHP1. In mouse and human macrophages, SCA selectively engages SHP1 Cys102, antagonizing IRAK signaling and LPS-induced pro-inflammatory cytokine production. Together, this work identifies a druggable cysteine redox switch controlling macrophage cytokine responses and provides a compendium of redox-regulated sites for therapeutic development.

biochemistry↗

bioRxiv2026

A small molecule PTER-selective inhibitor reduces food intake and body weight

PTER (phosphotriesterase-related) is an amidohydrolase that mediates catabolism of the anorexigenic taurine metabolite N-acetyltaurine. However, the structural basis of PTER ligand binding and catalysis remain unknown, limiting our ability to harness this pathway therapeutically. Here we solve crystal structures of a eukaryotic PTER in apo and product-bound forms. These structures uncover an unexpected pocket homology between PTER and histone deacetylase (HDAC) enzymes. We exploit this similarity to engineer a first-in-class substrate-competitive PTER inhibitor called PTERi with nanomolar potency and >100-fold selectivity for PTER over HDACs in vitro. Administration of PTERi to diet-induced obese mice reduces feeding, enhances GLP1-RA (glucagon like peptide 1 receptor agonist)-induced weight loss, and prevents weight regain after GLP1-RA discontinuation. The structure of PTER connects histone and metabolite deacetylation into a parallel conceptual framework and enables proof-of-concept data for pharmacological inhibition of PTER in obesity.

biochemistry↗

bioRxiv2025

Dried Blood Spot Lipidomics in Diabetes

Comprehensive lipidomic profiling in diabetes has identified disease-associated lipid signatures that may support more personalised monitoring beyond routine glycaemic control. Dried blood spot (DBS) microsampling offers a minimally invasive method for collecting and storing small blood volumes (<50 {micro}L), enabling decentralised and longitudinal sampling. However, its application in lipidomics for cardiometabolic phenotyping remains underexplored. In this study, 34 participants (17 diabetics, 17 non-diabetic controls) self-collected DBS samples using advanced microsampling devices (10 {micro}L, Capitainer(R)B). Lipid extracts were analysed using a validated liquid chromatography-trapped ion mobility-time-of-flight mass spectrometry method optimised for semi-quantitative DBS lipidomics. Multivariate analysis revealed significant differences in lipidomic profiles (n = 432 lipids) between diabetic and non-diabetic individuals (R{superscript 2}Y = 0.82, Q{superscript 2}Y = 0.50, p = 0.05). Key discriminatory lipids (all VIP > 1.44), including HexCer(18:1;O2/24:0), HexCer(18:1;O2/22:0), LPC(16:0), PC(O-34:3), TG(18:0_18:1_20:3), and TG(18:1_18:1_22:6), were consistent with matched venous and capillary plasma samples. Lipid class and structural differences, including elevated long-chain triacylglycerols and reduced lysophosphatidylcholines reflected known dyslipidaemia in diabetes and demonstrate the capacity of DBS to capture biologically meaningful lipid perturbations. Participant-reported outcomes from Problem Areas In Diabetes (PAID-20), Patient Activation Measure (PAM-13), and microsampling perception questionnaires highlighted psychosocial and behavioural insights, and indicated strong support for DBS sampling over venepuncture, citing ease of use and reduced burden. These findings establish DBS lipidomics as a feasible and informative approach for stratifying cardiometabolic disease, supporting broader implementation in remote, personalised monitoring frameworks (Figure 1). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/656742v1_fig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@c53b4org.highwire.dtl.DTLVardef@14b507forg.highwire.dtl.DTLVardef@191e563org.highwire.dtl.DTLVardef@16af172_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Graphical abstract - Chapter 5. C_FIG

biochemistry↗

bioRxiv2024

Unveiling the hidden interactome of CRBN molecular glues with chemoproteomics

Targeted protein degradation and induced proximity refer to strategies that leverage the recruitment of proteins to facilitate their modification, regulation or degradation. As prospective design of glues remains challenging, unbiased discovery methods are needed to unveil hidden chemical targets. Here we establish a high throughput affinity purification mass spectrometry workflow in cell lysates for the unbiased identification of molecular glue targets. By mapping the targets of 20 CRBN-binding molecular glues, we identify 298 protein targets and demonstrate the utility of enrichment methods for identifying novel targets overlooked using established methods. We use a computational workflow to estimate target confidence and perform a biochemical screen to identify a lead compound for the new non-ZF target PPIL4. Our study provides a comprehensive inventory of targets chemically recruited to CRBN and delivers a robust and scalable workflow for identifying new drug-induced protein interactions in cell lysates.

cancer biology↗

bioRxiv2024

Down-regulation of AKT proteins slows the growth of mutant-KRAS pancreatic tumors

Serine/threonine kinase AKT isoforms play a well-established role in cell metabolism and growth. Most pancreatic adenocarcinoma (PDAC) harbors activation mutations of KRAS, which activates the PI3K/AKT signaling pathway. However, AKT inhibitors are not effective in the treatment of pancreatic cancer. To better understand the role of AKT signaling in mutant-KRAS pancreatic tumors, this study utilizes proteolysis-targeting chimeras (PROTACs) and CRISPR-Cas9-genome editing to investigate AKT proteins. PROTAC down-regulation of AKT proteins markedly slowed the growth of three pancreatic tumor cell lines harboring mutant KRAS. In contrast, inhibition of AKT kinase activity alone had very little effect on the growth of these cell lines. Concurrent genetic deletion of all AKT isoforms (AKT1, AKT2, and AKT3) in the KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cell line also dramatically slowed its growth in vitro and when orthotopically implanted in syngeneic mice. Surprisingly, insulin-like growth factor-1 (IGF-1), but not epidermal growth factor (EGF), restored KPC cell growth in serum-deprived conditions and the IGF-1 growth stimulation effect was AKT dependent. RNA-seq analysis of AKT1/2/3-deficient KPC cells suggested that reduced cholesterol synthesis may be responsible for the decreased response to IGF-1 stimulation. These results indicate that the presence of all three AKT isoforms supports pancreatic tumor cell growth and pharmacological degradation of AKT proteins may be more effective than AKT catalytic inhibitors for treating pancreatic cancer.

cancer biology↗

bioRxiv2023

Borrowing Transcriptional Kinases to Activate Apoptosis

Protein kinases are disease drivers whose therapeutic targeting traditionally centers on inhibition of enzymatic activity. Here chemically induced proximity is leveraged to convert kinase inhibitors into context-specific activators of therapeutic genes. Bivalent molecules that link ligands of the transcription factor B-cell lymphoma 6 (BCL6) to ATP-competitive inhibitors of cyclin-dependent kinases (CDKs) were developed to re-localize CDK to BCL6-bound loci on chromatin and direct phosphorylation of RNA Pol II. The resulting BCL6-target proapoptotic gene expression translated into killing of diffuse large B-cell lymphoma (DLBCL) cells at 72 h with EC50s of 0.9 - 10 nM and highly specific ablation of the BCL6-regulated germinal center response in mice. The molecules exhibited 10,000-fold lower cytotoxicity in normal lymphocytes and are well tolerated in mice. Genomic and proteomic evidence corroborated a gain-of-function mechanism where, instead of global enzyme inhibition, a fraction of total kinase activity is borrowed and re-localized to BCL6-bound loci. The strategy demonstrates how kinase inhibitors can be used to context-specifically activate transcription, accessing new therapeutic space.

cancer biology↗

bioRxiv2023

Exploration of the Tunability of BRD4 Degradation by DCAF16 Trans-labelling Covalent Glues

Small molecules that can induce protein degradation by inducing proximity between a desired target and an E3 ligase have the potential to greatly expand the number of proteins that can be manipulated pharmacologically. Current strategies for targeted protein degradation are mostly limited in their target scope to proteins with preexisting ligands. Alternate modalities such as molecular glues, as exemplified by the glutarimide class of ligands for the CUL4CRBN ligase, have been mostly discovered serendipitously. We recently reported a trans-labelling covalent glue mechanism which we named Template-assisted covalent modification, where an electrophile decorated small molecule binder of BRD4 was effectively delivered to a cysteine residue on an E3 ligase DCAF16 as a consequence of a BRD4-DCAF16 protein-protein interaction. Herein, we report our medicinal chemistry efforts to evaluate how various electrophilic modifications to the BRD4 binder, JQ1, affect DCAF16 trans-labeling and subsequent BRD4 degradation efficiency. We discovered a decent correlation between the ability of the electrophilic small molecule to induce ternary complex formation between BRD4 and DCAF16 with its ability to induce BRD4 degradation. Moreover, we show that a more solvent-exposed warhead presentation is optimal for DCAF16 recruitment and subsequent BRD4 degradation. Unlike the sensitivity of CUL4CRBN glue degraders to chemical modifications, the diversity of covalent attachments in this class of BRD4 glue degraders suggests a high tolerance and tunability for the BRD4-DCAF16 interaction. This offers a potential new avenue for a rational design of covalent glue degraders by introducing covalent warheads to known binders.

pharmacology and toxicology↗

bioRxiv2023

Clinical prediction of wound re-epithelisation outcomes in non-severe burn injury using the plasma lipidome

Whilst wound repair in severe burns has received substantial research attention, non-severe burns (<20% total body surface area) remain relatively understudied, despite causing considerable physiological impact and constituting most of the hospital admissions for burns. Early prediction of healing outcomes would decrease financial and patient burden, and aid in preventing long-term complications from poor wound healing. Lipids have been implicated in inflammation and tissue repair and may play essential roles in burn wound healing. In this study, plasma samples were collected from 20 non-severe burn patients over 6 weeks from admission, including surgery, and analysed by liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance spectroscopy to detect 850 lipids and 112 lipoproteins. Orthogonal projections to latent structures-discriminant analysis was performed to identify changes associated with re-epithelialisation and delayed re-epithelisation. We demonstrated that the lipid and lipoprotein profiles at admission could predict re-epithelisation outcomes at 2 weeks post-surgery, and that these discriminatory profiles were maintained up to 6 weeks post-burn. Inflammatory markers GlycB and C-reactive protein indicated divergent systemic responses to the burn injury at admission. Triacylglycerols, diacylglycerols and low-density lipoprotein subfractions were associated with delayed wound closure (p-value <0.02, Cliffs delta >0.7), whilst high-density lipoprotein subfractions, phosphatidylinositols, phosphatidylcholines, and phosphatidylserines were associated with re-epithelisation at 2 weeks post-surgery (p-value <0.01, Cliffs delta <-0.7). Further model validation will potentially lead to personalised intervention strategies to reduce the risk of chronic complications post-burn injury.

systems biology↗

bioRxiv2023

Metabolic consequences of non-severe burn injury are associated with increased plasma markers of inflammation and cardiovascular disease risk.

Globally, burns are a significant cause of injury that can cause substantial acute trauma as well as lead to increased incidence of chronic co-morbidity and disease. To date, research has primarily focused on the systemic response to severe injury, with little in the literature reported on impact of non-severe injuries (<15% total burn surface area; TBSA). To elucidate the metabolic consequences of non-severe burn injury, longitudinal plasma was collected from adults (n=35) who presented at hospital with a non-severe burn injury at admission, and at 6 week follow up. A cross-sectional baseline sample was also collected from non-burn control participants (n=14). Samples underwent multiplatform metabolic phenotyping using 1H nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry to quantify 112 lipoprotein and glycoproteins signatures and 852 lipid species from across 20 subclasses. Multivariate data modelling (Orthogonal projection to latent structures-discriminate analysis) revealed alterations in lipoprotein and lipid metabolism when comparing baseline control to hospital admission samples, with the phenotypic signature found to be sustained at follow up. Univariate (Mann-Whitney U) testing and OPLS-DA indicated specific increases in GlycB (p-value <1.0e-4), low density lipoprotein-2 subfractions (Variable importance in projection score; VIP >6.83e-1) and monoacyglyceride (20:4)(p-value <1.0e-4) and decreases in circulating anti-inflammatory high-density lipoprotein-4 subfractions (VIP >7.75e-1), phosphatidylcholines, phosphatidylglycerols, phosphatidylinositols and phosphatidylserines. The results indicate a persistent systemic metabolic phenotype that occurs even in cases of non-severe burn injury. The phenotype is indicative of an acute inflammatory profile which continues to be sustained post-injury, suggesting an impact on systems health beyond the site of injury. The phenotypes contained metabolic signatures consistent with chronic inflammatory states reported to have elevated incidence post-burn injury. Such phenotypic signatures may provide patient stratification opportunities, to identify individual responses to injury, personalise intervention strategies and improve acute care, reducing risk of chronic co-morbidity.

systems biology↗

bioRxiv2022

Targeted Kinase Degradation via the KLHDC2 Ubiquitin E3 Ligase

Chemically induced protein degradation is a powerful strategy for perturbing cellular biochemistry. The predominant mechanism of action for protein degrader drugs involves induced proximity between the cellular ubiquitin conjugation machinery and the target. Unlike traditional small molecule enzyme inhibition, targeted protein degradation can clear an undesired protein from cells. We demonstrate here the use of peptide ligands for Kelch-Like Homology Domain Containing protein 2 (KLHDC2), a substrate adaptor protein and member of the cullin-2 (CUL2) ubiquitin ligase complex, for targeted protein degradation. Peptide-based bivalent compounds that can induce proximity between KLHDC2 and target proteins cause degradation of the targeted factors. The cellular activity of these compounds depends on KLHDC2 binding. This work demonstrates the utility of KLHDC2 for targeted protein degradation and exemplifies a strategy for the rational design of new peptide-based ligands useful for this purpose.

biochemistry↗

bioRxiv2022

Multi-omic profiling of breast cancer cells uncovers stress MAPK-associated sensitivity to AKT degradation

Over 50% of human tumors display hyperactivation of the serine/threonine kinase AKT. Despite evidence of clinical efficacy, there remains scope to improve upon the therapeutic window of the current generation of AKT inhibitors. Here we report the development of a second-generation AKT degrader, INY-05-040, which outperformed catalytic AKT inhibition with respect to cellular suppression of AKT-driven phenotypes in breast cancer cell lines. A systematic growth inhibition screen across 288 cancer cell lines confirmed a substantially higher potency for INY-05-040 (median GI50adj = 1.1 {micro}M) compared to our first-generation AKT degrader (INY-03-041; median GI50adj = 3.1 {micro}M), with both compounds outperforming catalytic AKT inhibition with GDC-0068 (median GI50adj > 10 {micro}M). Using multi-omic profiling and causal network integration in breast cancer cells, we demonstrate that the enhanced efficacy of INY-05-040 is associated with sustained suppression of AKT signaling, followed by a potent induction of the stress mitogen activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK). Further integration of growth inhibition assays with publicly available transcriptomic, proteomic, and reverse phase protein array (RPPA) measurements established low baseline JNK signaling as a biomarker for breast cancer sensitivity to AKT degradation. Collectively, our study presents a systematic framework for mapping the network-wide signaling effects of therapeutically relevant compounds, and identifies INY-05-040 as a potent pharmacological suppressor of AKT signaling.

cancer biology↗

bioRxiv2022

Covalent Disruptor of YAP-TEAD Association Suppresses Defective Hippo Signaling

The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03-69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03-69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03-69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03-69 led to an in vivo compatible compound MYF-03-176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration.

cancer biology↗

bioRxiv2019

The Salt-Inducible Kinase inhibitor YKL-05-099 suppresses MEF2C function and acute myeloid leukemia progression in vivo

Lineage-defining transcription factors (TFs) are compelling targets for leukemia therapy, yet they are among the most challenging proteins to modulate directly with small molecules. We previously used CRISPR screening to identify a Salt-Inducible Kinase 3 (SIK3) requirement for the growth of acute myeloid leukemia (AML) cell lines that overexpress the lineage TF MEF2C. In this context, SIK3 maintains MEF2C function by directly phosphorylating histone deacetylase 4 (HDAC4), a repressive cofactor of MEF2C. Here, we evaluated whether inhibition of SIK3 with the tool compound YKL-05-099 can suppress MEF2C function and attenuate disease progression in animal models of AML. Genetic targeting of SIK3 or MEF2C selectively suppressed the growth of transformed hematopoietic cells under in vitro and in vivo conditions. Similar phenotypes were obtained when exposing cells to YKL-05-099, which caused cell cycle arrest and apoptosis in MEF2C-expressing AML cell lines. An epigenomic analysis revealed that YKL-05-099 rapidly suppressed MEF2C function by altering the phosphorylation state and nuclear localization of HDAC4. Using a gatekeeper allele of SIK3, we found that the anti-proliferative effects of YKL-05-099 occurred through on-target inhibition of SIK3 kinase activity. Based on these findings, we treated two different mouse models of MLL-AF9 AML with YKL-05-099, which attenuated disease progression in vivo and extended animal survival at well-tolerated doses. These findings validate SIK3 as a therapeutic target in MEF2C-positive AML and provide a rationale for developing drug-like inhibitors of SIK3 for definitive pre-clinical investigation and for studies in human patients with leukemia.\n\nKey PointsAML cells are uniquely sensitive to genetic or chemical inhibition of Salt-Inducible Kinase 3 in vitro and in vivo.\n\nA SIK inhibitor YKL-05-099 suppresses MEF2C function and AML in vivo.

cancer biology↗

bioRxiv2018

Hypoxia-induced switch in SNAT2/SLC38A2 regulation generates endocrine-resistance in breast cancer

Tumor hypoxia is associated with poor patient outcomes in estrogen receptor- (ER) positive breast cancer. Hypoxia is known to affect tumor growth by reprogramming metabolism and regulating amino acid (AA) uptake. Here we show that the glutamine transporter, SNAT2, is the AA transporter most frequently induced by hypoxia in breast cancer and it is regulated by HIF1 both in-vitro and in-vivo in xenografts. SNAT2 induction in MCF7 cells was also regulated by ER but it became predominantly a HIF-1-dependent gene under hypoxia. Relevant to this, binding sites for both HIF-1 and ER overlap in SNAT2s cis-regulatory elements. In addition, the downregulation of SNAT2 by the ER antagonist fulvestrant was reverted in hypoxia.\n\nOverexpression of SNAT2 in-vitro to recapitulate the levels induced by hypoxia caused enhanced growth, particularly after ER inhibition, in hypoxia, or when glutamine levels were low. SNAT2 upregulation in-vivo caused complete resistance to anti-estrogen and, partially, anti-VEGF therapies. Finally, high SNAT2 expression levels correlate with HIF-1 and worse outcome in patients given anti-estrogen therapy. Our findings show a switch in regulation of SNAT2 between ER and HIF-1, leading to endocrine resistance in hypoxia. Development of drugs targeting SNAT2 may be of value for a subset of hormone-resistant breast cancer.

cancer biology↗

bioRxiv2018

Cheminformatics tools for analyzing and designing optimized small molecule libraries

Libraries of highly annotated small molecules have many uses in chemical genetics, drug discovery and drug repurposing. Many such libraries have become available, but few data-driven approaches exist to compare these libraries and design new ones. In this paper, we describe such an approach that makes use of data on binding selectivity, target coverage and induced cellular phenotypes as well as chemical structure and stage of clinical development. We implement the approach as R software and a Web-accessible tool (http://www.smallmoleculesuite.org) that uses incomplete and often confounded public data in combination with user preferences to score and create libraries. Analysis of six kinase inhibitor libraries using our approach reveals dramatic differences among them, leading us to design a new LSP-OptimalKinase library that outperforms all previous collections in terms of target coverage and compact size. We also assemble a mechanism of action library that optimally covers 1852 targets of the liganded genome. Using our tools, individual research groups and companies can quickly analyze private compound collections and public libraries can be progressively improved using the latest data.

systems biology↗

bioRxiv2017

Progress Towards a Public Chemogenomic Set for Protein Kinases and a Call for Contributions

Protein kinases are highly tractable targets for drug discovery. However, the biological function and therapeutic potential of the majority of the 500+ human protein kinases remains unknown. We have developed physical and virtual collections of small molecule inhibitors, which we call chemogenomic sets, that are designed to inhibit the catalytic function of almost half the human protein kinases. In this manuscript we share our progress towards generation of a comprehensive kinase chemogenomic set (KCGS), release kinome profiling data of a large inhibitor set (Published Kinase Inhibitor Set 2 (PKIS2)), and outline a process through which the community can openly collaborate to create a KCGS that probes the full complement of human protein kinases.

pharmacology and toxicology↗