Biology subjects

Lee, K.-J.

Publications and source records attributed to Lee, K.-J..

12 recordsLinked to original sources

bioRxiv2026

Pan-Coronavirus Protease Engagement and Differentiated Pharmacology of Antiviral Candidate CMX990

COVID-19 highlighted the need for antivirals that can be rapidly deployed against both current and future coronavirus threats. CMX990 is a clinical stage coronavirus main protease (Mpro) inhibitor bearing a unique trifluoromethoxymethyl ketone warhead. Here, we broadly evaluate its pan-coronavirus target engagement, antiviral breadth, and pharmacologic differentiation. Across a reporter panel of 24 Mpro enzymes spanning alpha-, beta-, delta-, and gamma-coronaviruses, CMX990 retained robust potent activity, including against nirmatrelvir-resistant SARS-CoV-2 variants. In direct comparison, nirmatrelvir and ensitrelvir showed diminished potency against non-SARS-CoV-2 coronaviruses. Live-virus assays corroborated this breadth, with potent, selective activity against SARS-CoV-2, SARS-CoV-1, MERS-CoV, and additional human coronaviruses. Structural studies revealed a conserved covalent binding mode centered on the catalytic Cys of coronavirus Mpro. In serial-passage studies, resistance emerged only after approximately twice as many serial passages under CMX990 selection as under nirmatrelvir selection, without selecting for the canonical nirmatrelvir-resistance substitution E166V. Extracellular washout experiments revealed superior persistence of antiviral activity relative to nirmatrelvir, consistent with durable target engagement. In a murine SARS-CoV-2 infection model, once-daily CMX990 protected against weight loss and reduced lung viral titers to near baseline levels. Collectively, these findings identify CMX990 as a structurally and functionally validated pan-coronavirus protease inhibitor supporting its potential as a pandemic-ready antiviral candidate.

pharmacology and toxicology↗

bioRxiv2026

Orally Bioavailable SARS-CoV-2 Protease Inhibitors Bearing a Hydroxymethyl Ketone Warhead

The use of covalent warheads targeting the catalytic cysteine has been a cornerstone in coronavirus main protease (Mpro) inhibitor development, where various electrophilic motifs have been used including aldehydes, nitriles, ketoamides, and hydroxymethyl ketones (HMKs). Recent efforts have been mostly centered around nitrile warheads, given the success of compounds like Nirmatrelvir and Ensitrelvir in the clinic. However, finding and advancing alternative chemotypes with differentiating chemical and pharmacological profiles is essential for future pandemic preparedness. Among such alternatives, HMKs hold special interest because they balance reduced intrinsic electrophilicity with an excellent selectivity profile. Nevertheless, early HMK-based compounds, such as the clinical-stage Mpro inhibitor PF-00835231, suffered from poor oral bioavailability and therefore required intravenous administration, with or without prodrug derivatization of the hydroxyl group. Here, we describe our efforts in advancing the HMK field via the discovery of mCMX110, a lead that has superior potency, increased unbound exposure in vivo, and favorable oral bioavailability in preclinical studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/725542v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@40222aorg.highwire.dtl.DTLVardef@831b88org.highwire.dtl.DTLVardef@184a910org.highwire.dtl.DTLVardef@779095_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2026

Droplet-compatible single-cell DNA methylation sequencing with PreTIC

Single-cell DNA methylation sequencing has remained technically specialized due to challenges of interfacing conversion chemistry with droplet microfluidics. We introduce pre-tagmentation in situ conversion (PreTIC), which enables whole-methylome profiling on a commercial droplet platform using off-the-shelf reagents. With PreTIC, we produce over 13,000 single-cell methylomes from fixed frozen cells in two days and resolve cell-specific epigenetic variation in human peripheral blood mononuclear cells.

genomics↗

bioRxiv2025

Deciphering the oxidative modifications via disulfide mapping

Oxidative stress triggers redox-sensitive post-translational modifications, notably disulfide bond formation involving cysteine residues. However, these bonds are often overlooked in proteomics due to the routine use of reducing agents. Here, we employed LC-MS-based metabolomics and non-reducing tandem mass tag (TMT) proteomics to investigate the effects of H2O2 on MDA-MB-231 cells. Metabolomic analysis revealed pathway-specific inhibition of major metabolic pathways including glycolysis, the TCA cycle, and nucleotide biosynthesis. Proteomic analysis using the DBond algorithm revealed extensive and isoform-specific disulfide crosslinks across more than 1,000 proteins. These linkages were enriched at redox-sensitive cysteines near basic residues and displayed high isoform specificity. Our findings demonstrate that disulfide bond formation serves as a selective mechanism of redox regulation. This study highlights the utility of non-reducing proteomics in elucidating redox-controlled protein networks and structural dynamics under oxidative stress. TeaserNon-reducing proteomics uncovers hidden disulfide-linked networks that rewire protein responses to oxidative stress.

cell biology↗

bioRxiv2025

Innate Immune Remodeling Drives Therapy Resistance via Macrophage-NK Cell Crosstalk.

The dynamic evolution of the immune tumor microenvironment (TME) during targeted therapy is a critical yet poorly understood determinant of treatment response and resistance. While most studies compare immune states before and after treatment, temporal immune changes during therapy remain largely uncharacterized, limiting development of effective combination strategies. Here, we investigated immune dynamics throughout targeted therapy using mouse melanoma models that recapitulate human therapeutic responses. Single-cell RNA sequencing (scRNA-seq) identified a previously unrecognized inflection point where the inflamed TME during tumor regression, characterized by robust NK cell infiltration, transitions to an immune-excluded state upon onset of drug-tolerant residual disease. We uncovered a unique macrophage subset (F4/80hiCCL5MHCIICD63) that orchestrates NK cell recruitment through CCR2/5 signaling during regression. Depletion of these macrophages using LysM-cre;iDTR mice significantly reduced NK cell infiltration. Specifically during residual disease, pharmacological inhibition of Ptpn22, a phosphatase that negatively regulates immune activation, reprogrammed macrophages, restored NK cell recruitment and enhanced therapeutic efficacy. Extending these findings to human cancer, longitudinal scRNA-seq analysis of melanoma and lung cancer patient samples revealed dynamic NK cell infiltration during targeted therapy, establishing a direct link between innate immune remodeling and treatment outcome. Unlike prior prognostic studies assessing immune states at single time points, our results provide mechanistic evidence of a temporal relationship between NK cell infiltration and therapeutic efficacy. Together, these findings position immune evolution as a driver of acquired resistance and identify macrophage-NK cell crosstalk as a therapeutically actionable axis to overcome immune exclusion and improve targeted therapy across multiple cancer types. One Sentence SummaryCancer therapy resistance emerges from a dynamic evolution of the tumor microenvironment, characterized by macrophage-driven NK cell infiltration during initial tumor regression, followed by exclusion of NK cells during residual disease, highlighting macrophage-NK cell interactions as a promising therapeutic target to improve clinical outcomes.

cancer biology↗

bioRxiv2025

Disulfide Crosslinking Induces Rapid Degradation of Arc/Arg3.1 via Hsp70-Mediated Ubiquitin Ligase Pathway

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is an immediate-early gene (IEG) induced by stress and synaptic activity, characterized by transient expression and rapid degradation. However, the mechanisms governing its degradation remain unclear. In this study, we identify a novel degradation pathway for Arc/Arg3.1, driven by its structural features. We demonstrate that the proteasomal degradation of Arc/Arg3.1 is modulated by the Hsp70-CHIP complex, with ubiquitination being impaired in HSF1 knockout cells. The formation of a Cys34-Cys159 disulfide bond crosslinks Arc/Arg3.1 into high-molecular-weight oligomers, altering its ubiquitination pattern and degradation kinetics compared to the C159A mutant. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed that wild-type (WT) Arc/Arg3.1 adopts a more compact structure than the C159A mutant. Notably, the C159A mutant fails to interact with HSF1, resulting in Hsp70 induction upon heat shock. Our findings propose a feedback loop in which disulfide crosslinking of Arc/Arg3.1 induces rapid degradation through Hsp70-mediated ubiquitination, which in turn modulates the heat shock response by inhibiting HSF1 function.

biochemistry↗

bioRxiv2024

Identification of mCMQ069, a novel antimalarial with potential for a single-dose cure and/or 28-day chemoprevention

In efforts towards eliminating malaria, a discovery program was initiated to identify a novel antimalarial using KAF156 as a starting point. Following the most recent TCP/TPP guidelines, we have identified mCMQ069 with a predicted single oral dose for treatment ([~]40-106 mg) and one-month chemoprevention ([~]96-216 mg). We have improved unbound MPC and predicted human clearance by 18-fold and 10-fold respectively when compared to KAF156.

microbiology↗

bioRxiv2021

Strategic infarct locations for post-stroke depressive symptoms: a lesion- and disconnection-symptom mapping study

BackgroundDepression is the most common neuropsychiatric complication after stroke. Infarct location is associated with post-stroke depressive symptoms (PSDS), but it remains debated which brain structures are critically involved. We performed a large-scale lesion-symptom mapping study to identify infarct locations, and white matter disconnections, associated with PSDS. MethodsWe included 553 patients (age 69{+/-}11 years, 42% female) with acute ischemic stroke. PSDS were measured using the 30-item Geriatric Depression Scale (GDS-30). Multivariable support vector regression (SVR)-based analyses were performed both at the level of individual voxels (SVR-VLSM) and predefined regions of interest (SVR-ROI) to relate infarct location to PSDS. We externally validated our findings in an independent stroke cohort (N=459). Finally, disconnectome-based analyses were performed using SVR-VLSM, in which white matter fibers disconnected by the infarct were analyzed instead of the infarct itself. Results: Infarcts in the right amygdala, right hippocampus and right pallidum were consistently associated with PSDS (permutation-based p<0.05) in SVR-VLSM and SVR-ROI. External validation (N=459) confirmed the association between infarcts in the right amygdala and pallidum, but not the right hippocampus, and PSDS. Disconnectome-based analyses revealed that disconnections in the right parahippocampal white matter, right thalamus and pallidum, and right anterior thalamic radiation were significantly associated (permutation-based p<0.05) with PSDS. ConclusionsInfarcts in the right amygdala and pallidum, and disconnections of right limbic and frontal cortico-basal ganglia-thalamic circuits, are associated with PSDS. Our findings provide a comprehensive and integrative picture of strategic infarct locations for PSDS, and shed new light on pathophysiological mechanisms of depression after stroke.

neuroscience↗

bioRxiv2021

Vaccine Hyporesponse Induced By Individual Antibiotic Treatment In Mice And Non-Human Primates Is Diminished Upon Recovery Of The Gut Microbiome

Emerging evidence demonstrates a connection between microbiome composition and suboptimal response to vaccines (vaccine hyporesponse). Harnessing the interaction between microbes and the immune system could provide novel therapeutic strategies for improving vaccine response. Currently we do not fully understand the mechanisms and dynamics by which the microbiome influences vaccine response. Using both mouse and non-human primate models, we report that short-term oral treatment with a single antibiotic (vancomycin) results in disruption of the gut microbiome and this correlates with a decrease in systemic levels of antigen-specific IgG upon subsequent parenteral vaccination. We further show that recovery of microbial diversity before vaccination prevents antibiotic-induced vaccine hyporesponse, and that the antigen specific IgG response correlates with the recovery of microbiome diversity. RNA-sequencing analysis of small intestine, spleen, whole blood, and secondary lymphoid organs from antibiotic treated mice revealed a dramatic impact on the immune system, and a muted inflammatory signature is correlated with loss of bacteria from Lachnospiraceae, Ruminococcaceae, and Clostridiaceae. These results suggest that microbially modulated immune pathways may be leveraged to promote vaccine response and will inform future vaccine design and development strategies. ImportanceAntibiotic-induced gut microbiome disruption has been linked to reduced vaccine efficacy. Despite these observations, there remains a knowledge gap in the specific mechanisms by which antibiotics and the gut microbiome influence vaccine efficacy. We aim to contribute to the fields growing mechanistic understanding by presenting a detailed analysis of antibiotic treatment and recovery as it relates to vaccine response and the microbiome. Using animal models, we show that short-term antibiotic treatment prior to vaccination results in diminished vaccine-specific immune responses, and that these are correlated with specific microbiome signatures. We also demonstrate the converse, in which gut microbiome recovery can result in improved vaccine response. We further reveal that antibiotics can significantly alter multiple relevant immune pathways and this alteration in immune tone may contribute to the vaccine hyporesponse. We expect our findings will enable the continued prosecution of the role of the microbiome in modulating the host immune system.

immunology↗

bioRxiv2021

Stepwise Oxidations Play Key Roles in the Structural and Functional Regulations of DJ-1

DJ-1 is known to play neuroprotective roles by eliminating reactive oxygen species (ROS) as an antioxidant protein. However, the molecular mechanism of DJ-1 function has not been well elucidated. This study explored the structural and functional changes of DJ-1 in response to oxidative stress. We found that Cys46 is also reactive cysteine residue in DJ-1, which was identified employing an NPSB-B chemical probe that selectively reacts with redox sensitive cysteine sulfhydryl. Peroxidatic Cys46 readily formed an intra-disulfide bond with resolving Cys53, which was identified with nanoUPLC-ESI-q-TOF tandem mass spectrometry (MS/MS) employing DBond algorithm under the non-reducing condition. We also found that Cys46-Cys53 disulfide crosslinking affects the oxidative state of the third Cys106, which shows the crosstalk among three cysteine residues of DJ-1. Furthermore, we demonstrated that DJ-1 C46A mutant, not forming Cys46-Cys53 intra-disulfide bond, lost structural stability of DJ-1 employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS) analysis. All three Cys mutants lost antioxidant activities in SN4741 cell, a dopaminergic neuronal cell, unlike wild type DJ-1. These findings suggest that DJ-1 regulates its structure and activities by concerted oxidative modifications of three cysteine residues. These studies broaden the understanding of regulatory mechanisms of DJ-1 that operate under oxidative conditions.

biochemistry↗

bioRxiv2020

Cataract-associated New Mutants S175G/H181Q of βB2-Crystallin and P24S/S31G of γD-Crystallin are Involved in Protein aggregation by Structural Changes

{beta}/{gamma}-Crystallins, the main structural protein in human lenses, have highly stable structure for keeping the lens transparent. Their mutations have been linked to congenital cataracts. In this study, we identified 10 new mutations of {beta}/{gamma}-crystallins in lens proteomic dataset of cataract patients using bioinformatic tools. Of these, two double mutants, S175G/H181Q of {beta}B2-crystallin and P24S/S31G of {gamma}D-crystallin, were found mutations occurred in the largest loop linking the distant {beta}-sheets in the Greek key motif. We selected these double mutants for identifying the properties of these mutations, employing biochemical assay, the identification of protein modifications with nanoUPLC-ESI-TOF tandem MS and examining their structural dynamics with hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that both double mutations decrease protein stability and induce the aggregation of {beta}/{gamma}-crystallin, possibly causing cataracts. This finding suggests that both the double mutants can serve as biomarkers of congenital cataracts.

biochemistry↗

bioRxiv2018

Association between activities outside work and presenteeism among Korean wage workers : using nationwide survey

IntroductionPresenteeism is a significant global health issue nowadays and can influence to producitivity loss. So, there were many studies to analyse relationship between workplace factor and presenteeism. But a few studies have considered non-occupational factor. The purpose of this study was to examine the association between presenteeism and activities outside work, such as, volunteering, self-development, leisure/sports, gardening & house repair activity, in Korean wage workers.\n\nMethodsThis study analysed the 4th Korean Working Condition Survey(KWCS) and overall 19,294 wage workers participated. To find the relationship between presenteeism and activities outside work, multivariate logistic regression analysis was used after adjusting for general and occupational characteristics.\n\nResultsAmong self-development, leisure/sports and gardening & house repair activities significantly increased the odds ratio of presenteeism (Odds ratio[OR] =1.33, 95% confidence interval[CI]: 1.23-1.43, OR = 1.40, 95% CI: 1.29-1.51 and OR = 1.09, 95% CI: 1.01-1.18, respectively).\n\nConclusionsSome of activities outside work were related to presenteeism in Korean wage workers. Although many of previous studies addressed positive effect of those activities for health, this study showed negative effect of activities oudside work for health. And we should consider Korean organizational culture for this reason and need more structural studies to find out specific factors.

epidemiology↗