Biology subjects

Kelley, M. R.

Publications and source records attributed to Kelley, M. R..

5 recordsLinked to original sources

bioRxiv2026

A kinetic-aware approach to infer metabolic variations and flux using transcriptomics and metabolomics data

Assessing metabolic variations and flux quantities enable systematic understandings of metabolic shifts, reprogramming, adaptation and interactions in human diseases. However, omics-based estimation of metabolic flux and its variation remains challenging due to several fundamental limitations: the need for disease and tissue context specific metabolic model; nonlinear enzyme kinetic model that links enzyme and substrate changes to reaction flux; partial, unpaired, and snap-shot measurements across omics modalities; and uncertainty in computational prediction. Here, we present Michaelis-Menten model-based Flux Estimation Analysis (mmFEA), a Monte Carlo framework for estimating condition-specific flux changes by integrating paired or unpaired metabolomics and transcriptomics (or proteomics) data. mmFEA separates each reaction-rate change into enzyme- and substrate-associated components and assess reaction rate using Michaelis-Menten kinetics equation. A baseline metabolite saturation rate is introduced by integrating protein language model predicted kinetic parameters and human baseline level metabolic concentration to enable kinetic-aware integration of unpaired substrate and enzyme level measurements. Distribution of metabolic flux and variations between conditions are further computed using MCMC sampling by treating Michaelis-Menten-derived marginal flux distribution as prior and coherency in flux balance as likelihood. To benchmark mmFEA, we generated an in-house multi-omics data set including transcriptomics, metabolomics, metabolic activity functional assay, and CRISPR screening data using pancreatic cancer cell line system treated by APEX1 inhibitors. We demonstrated that mmFEA could accurately capture experimentally observed metabolic changes and achieved a better performance than all baseline methods. Our analysis revealed the necessity in using both substrate and enzyme modality and kinetic aware model in metabolic flux assessment. Further analysis using independent pancreatic cancer cohorts further validated the robustness of mmFEA, supporting integration of condition-linked unpaired data. Pan-cancer and spatial multi-omic applications demonstrated the use of mmFEA for resolving context-dependent metabolic variation when direct flux measurements are unavailable. Together, mmFEA provides a mechanistically grounded framework for estimating relative metabolic flux changes and their uncertainty from heterogeneous omics data.

systems biology↗

bioRxiv2026

Integrated Post-Translational Modification (PTM) Proteomics Reveals Early Redox and Mitochondrial Remodeling Following Ref-1 Inhibition in Pancreatic Cancer

BackgroundApurinic/apyrimidinic endonuclease 1/redox factor-1 (Ref-1/APE1) is a central regulator of redox-dependent transcriptional signaling that promotes pancreatic ductal adenocarcinoma (PDAC) progression, therapeutic resistance, and metabolic adaptation. While pharmacologic inhibition of Ref-1 suppresses tumor growth and alters cellular metabolism, immediate molecular events linking Ref-1 inhibition to downstream cellular adaptation remain poorly understood. We therefore sought to characterize proteome-wide signaling responses induced by second-generation Ref-1 redox inhibitor, APX2014. MethodsWe applied an integrated multiplexed proteomics workflow to simultaneously quantify global protein abundance together with cysteine oxidation, phosphorylation, and lysine acetylation in Pa03C PDAC cells following acute treatment (30-120 min) with selective Ref-1 redox inhibitor APX2014. Differential post-translational modification (PTM) analysis, pathway enrichment, structural mapping of regulated sites, and functional mitochondrial substrate utilization assays were performed to define early signaling responses. ResultsAPX2014 induced rapid and extensive remodeling of PTM landscape while producing minimal changes in global protein abundance. Cysteine oxidation represented the earliest and most sustained response, accompanied by widespread phosphorylation and delayed lysine acetylation. Integrated pathway analyses identified mitochondrial translation, respiratory electron transport, TCA cycle metabolism, and mitochondrial redox homeostasis as the earliest and most consistently regulated processes. Functional mitochondrial assays confirmed impaired utilization of TCA cycle substrates following APX2014 treatment. Coordinated PTM remodeling was observed on Ref-1-associated signaling proteins, including NF-{kappa}B1 and p53, revealing simultaneous regulation of oxidation, phosphorylation, and acetylation within functionally important domains. Early redox-sensitive protein networks were also associated with subsequent disruption of mitotic organization. ConclusionsIntegrated multi-PTM proteomics reveals that pharmacologic Ref-1 redox inhibition rapidly rewires regulatory signaling networks before detectable changes in protein abundance. Our findings identify mitochondrial redox remodeling as an early consequence of Ref-1 inhibition, providing systems-level insight into how Ref-1-targeted therapies disrupt metabolic and stress-adaptive programs in pancreatic cancer. This work establishes a framework for understanding the molecular basis of Ref-1-directed therapeutics and highlights integrated PTM profiling as a powerful strategy for defining early drug response mechanisms. These findings provide a strong translational rationale for advancing next-generation Ref-1 redox inhibitors such as APX2014, developed from the first-in-class inhibitor APX3330 currently in clinical trials, and underscore the broader therapeutic potential of targeting Ref-1 redox signaling in pancreatic cancer.

cancer biology↗

bioRxiv2023

Potentiating NaV1.1 in Dravet syndrome patient iPSC-derived GABAergic neurons increases neuronal firing frequency and decreases network synchrony

Dravet syndrome is a developmental and epileptic encephalopathy characterized by seizures, behavioral abnormalities, developmental deficits, and elevated risk of sudden unexpected death in epilepsy (SUDEP). Most patient cases are caused by de novo loss-of-function mutations in the gene SCN1A, causing a haploinsufficiency of the alpha subunit of the voltage-gated sodium channel NaV1.1. Within the brain, NaV1.1 is primarily localized to the axons of inhibitory neurons, and decreased NaV1.1 function is hypothesized to reduce GABAergic inhibitory neurotransmission within the brain, driving neuronal network hyperexcitability and subsequent pathology. We have developed a human in vitro model of Dravet syndrome using differentiated neurons derived from patient iPSC and enriched for GABA expressing neurons. Neurons were plated on high definition multielectrode arrays (HD-MEAs), permitting recordings from the same cultures over the 7-weeks duration of study at the network, single cell, and subcellular resolution. Using this capability, we characterized the features of axonal morphology and physiology. Neurons developed increased spiking activity and synchronous network bursting. Recordings were processed through a spike sorting pipeline for curation of single unit activity and to assess the effects of pharmacological treatments. At 7-weeks, the application of the GABAAR receptor agonist muscimol eliminated network bursting, indicating the presence of GABAergic neurotransmission. To identify the role of NaV1.1 on neuronal and network activity, cultures were treated with a dose-response of the NaV1.1 potentiator {delta}-theraphotoxin-Hm1a. This resulted in a strong increase in firing rates of putative GABAergic neurons, an increase in the intraburst firing rate, and eliminated network bursting. These results validate that potentiation of NaV1.1 in Dravet patient iPSC-derived neurons results in decreased firing synchrony in neuronal networks through increased GABAergic neuron activity and support the use of human neurons and HD-MEAs as viable high-throughput electrophysiological platform to enable therapeutic discovery.

neuroscience↗

bioRxiv2023

Chemically induced partial unfolding of the multifunctional Apurinic/apyrimidinic endonuclease 1

Apurinic/apyrimidinic endonuclease I (APE1) acts as both an endonuclease and a redox factor to ensure cell survival. The two activities require different conformations of APE1. As an endonuclease, APE1 is fully folded. As a redox factor, APE1 must be partially unfolded to expose the buried residue Cys65, which reduces transcription factors including AP-1, NF-{kappa}B, and HIF-1 and thereby enables them to bind DNA. To determine a molecular basis for partial unfolding associated with APE1s redox activity, we characterized specific interactions of a known redox inhibitor APX3330 with APE1 through waterLOGSY and 1H-15N HSQC NMR approaches using ethanol and acetonitrile as co-solvents. We find that APX3330 binds to the endonuclease active site in both co-solvents and to a distant small pocket in acetonitrile. Prolonged exposure of APE1 with APX3330 in acetonitrile resulted in a time-dependent loss of 1H-15N HSQC chemical shifts ([~]35%), consistent with partial unfolding. Regions that are partially unfolded include adjacent N- and C-terminal beta strands within one of the two sheets comprising the core, which converge within the small binding pocket defined by the CSPs. Removal of APX3330 via dialysis resulted in a slow reappearance of the 1H-15N HSQC chemical shifts suggesting that the effect of APX3330 is reversible. APX3330 significantly decreases the melting temperature of APE1 but has no effect on endonuclease activity using a standard assay in either co-solvent. Our results provide insights on reversible partial unfolding of APE1 relevant for its redox function as well as the mechanism of redox inhibition by APX3330. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/547112v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@13f9fe2org.highwire.dtl.DTLVardef@19cbcborg.highwire.dtl.DTLVardef@1927c7aorg.highwire.dtl.DTLVardef@2a3cc0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2018

Ref-1/APE1 inhibition with novel small molecules blocks ocular neovascularization

Ocular neovascular diseases like wet age-related macular degeneration are a major cause of blindness. Novel therapies are greatly needed for these diseases. One appealing antiangiogenic target is reduction-oxidation factor 1-apurinic/apyrimidinic endonuclease 1 (Ref-1/APE1). This protein can act as a redox-sensitive transcriptional activator for NF-{kappa}B and other pro-angiogenic transcription factors. An existing inhibitor of Ref-1s function, APX3330, previously showed antiangiogenic effects. Here, we developed improved APX3330 derivatives and assessed their antiangiogenic activity. We synthesized APX2009 and APX2014 and demonstrated enhanced inhibition of Ref-1 function in a DNA-binding assay compared to APX3330. Both compounds were antiproliferative against human retinal microvascular endothelial cells (HRECs; GI50 APX2009: 1.1 M, APX2014: 110 nM) and macaque choroidal endothelial cells (Rf/6a GI50 APX2009: 26 M, APX2014: 5.0 M). Both compounds significantly reduced the ability of HRECs and Rf/6a cells to form tubes at mid nanomolar concentrations compared to control, and both significantly inhibited HREC and Rf/6a cell migration in a scratch wound assay, reducing NF-{kappa}B activation and downstream targets. Ex vivo, both APX2009 and APX2014 inhibited choroidal sprouting at low micromolar and high nanomolar concentrations respectively. In the laser-induced choroidal neovascularization mouse model, intraperitoneal APX2009 treatment significantly decreased lesion volume by 4-fold compared to vehicle (p < 0.0001, ANOVA with Dunnetts post hoc tests), without obvious intraocular or systemic toxicity. Thus, Ref-1 inhibition with APX2009 and APX2014 blocks ocular angiogenesis in vitro and ex vivo, and APX2009 is an effective systemic therapy for CNV in vivo, establishing Ref-1 inhibition as a promising therapeutic approach for ocular neovascularization.

pharmacology and toxicology↗