ACS Bio & Med Chem Au

Compounds that bind to the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) main protease (MPro) often produce biphasic concentration-response curves (CRCs) in biochemical assays; low concentrations activate the enzyme and high concentrations inhibit it. This biphasic behavior complicates data analysis. Here, we compare three approaches to data analysis: fitting the Hill equation to the activation phase, fitting it to the inhibition phase, and fitting an enzyme kinetics model that incorporates dimerization and ligand binding to the complete CRC. In the latter case, cellular efficacy is predicted by extrapolating the model to high enzyme concentrations. For compounds in our drug lead series, all three procedures yield inhibitory concentrations that are correlated with live-virus antiviral assays. The latter procedure provides the most accurate forecast of cellular efficacy rank. These data analysis procedures may be valuable for antiviral drug discovery against MERS-CoV MPro and other enzymes with similar kinetics.

Bifunctional diazirine lipids are valuable tools for mapping protein-lipid interactions and cellular localization by photocrosslinking. Yet, the crosslinking efficiency of these probes has not been systematically evaluated. Here, we use the lipid transfer protein STARD10, which binds phospholipids in a 1:1 stoichiometry within a hydrophobic pocket, to measure the upper limit of the photo-crosslinking efficiency of bifunctional lipid probes. We characterize reaction products using native and denaturing mass spectrometry. Our results show that approximately 5% of photoactivated lipids form covalent protein-lipid crosslinks, while the majority follow intramolecular reaction trajectories, resulting in the formation of products featuring alkene, ketone and hydroxyl moieties. These findings provide essential context for the use of bifunctional probes to uncover the cell biology of lipids and highlight the need for continuous improvement to experimental workflows. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/700185v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@15d1641org.highwire.dtl.DTLVardef@6024e0org.highwire.dtl.DTLVardef@1503dcorg.highwire.dtl.DTLVardef@1b067bd_HPS_FORMAT_FIGEXP M_FIG C_FIG

The RNA helicase DHX9 is essential for genomic stability, transcription, translation regulation and other RNA-related processes. DHX9 has emerged as a therapeutic target for cancer treatment as its expression levels are elevated in several different cancer types. Moreover, tumor cells exhibit a strong dependence on DHX9, making its inhibition an effective strategy for tumor regression. As RNA helicases are conserved enzymes, unique features need to be targeted to minimize side effects. Here, we identified an autoregulatory interface between the DHX9 helicase core and double-stranded RNA binding domain 2 (dsRBD2) ideal for a highly specific inhibition of DHX9. By targeting the proposed dsRBD2-core interface in DHX9, we aim to specifically inhibit DHX9 helicase activity by preventing dsRBD2 binding to the helicase core. We developed a protein binder design-based strategy targeting the dsRBD2-core interface of DHX9 by computationally designing novel dsRBDs. By binding this interface without engaging RNA, the dsRBD designs prevent dsRBD2-core interaction and inhibit DHX9 helicase activity. Our strategy of redesigning autoregulatory protein domains as inhibitors offers a computationally efficient alternative to larger-scale library generation and provides a flexible framework applicable to a wide range of therapeutic targets.

Globally, Mycobacterium tuberculosis remains a significant disease burden. Although effective treatment regimens exist, drug resistance continues to emerge. This clinical resistance, combined with side effects and protracted treatment times from the current front-line therapies, means there is a need to identify novel agents to combat this disease. Here we report on a new chemical series, identified by whole-cell phenotypic growth inhibition screening that demonstrates significant activity across multiple media. Mode of action studies indicate that this series targets the same biological pathway as Ethambutol (EMB), a drug used in the current frontline treatment of tuberculosis. Screening selected analogues against clinical isolates, resistant to EMB, demonstrated differential sensitivity both across the molecules and against the different specific resistant mutations. The data obtained suggests that this series has potential to be developed into a viable, alternative to EMB. TOC figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/702510v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@1a80c05org.highwire.dtl.DTLVardef@1ad3ce9org.highwire.dtl.DTLVardef@79fe79org.highwire.dtl.DTLVardef@131ed78_HPS_FORMAT_FIGEXP M_FIG C_FIG

Sirtuins (SIRTs), which remove protein lysine acyl modifications, play crucial roles in diverse cellular processes, including metabolism, gene transcription, DNA damage repair, cell survival, and stress response. Several sirtuins are considered non-oncogene addiction of cancer cells and promising targets for anticancer drug development. High-throughput screening (HTS) methods for sirtuins are critical for the development of potent and isoform-selective sirtuin inhibitors, which are needed to validate the therapeutic potential. Herein, we designed and synthesized a fluorescent polarization (FP) tracer, KP-SC-1. Using this high-affinity tracer, we developed a robust, high-throughput FP competition assay for screening SIRT1-3 inhibitors. The assay was validated by testing known SIRT1-3 inhibitors. The assay can detect NAD+-independent SIRT1-3 inhibitors, as well as NAD+-dependent inhibitors, such as Ex-527 and TM. Finally, our assay showed satisfactory stability and outstanding performance in a pilot library screening. Compared to previous assays, the FP assay uses much less SIRT1-3 enzymes, a feature important for high-throughput library screening. We believe that the FP assay developed here will accelerate the discovery and development of SIRT1-3 inhibitors.

Base excision repair (BER) is the primary pathway that removes oxidatively-induced DNA base damage from the nuclear and mitochondrial genomes, with 8-oxoguanine DNA glycosylase (OGG1) initiating repair at the two most frequently-formed base lesions: 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoGua) and 2,6-diamino-4-oxo-5-formamidopyrimidine (FapyGua). Humans expressing a catalytically-compromised variant of OGG1 (S326C) are at increased risk for type 2 diabetes, Alzheimers disease, and Parkinsons disease. To potentially enhance the overall catalytic efficiency of this variant, a prior medicinal chemistry screen discovered seven chemically distinct agonists of OGG1 that stimulated activity in vitro and attenuated a paraquat (PQ) challenge in cultured cells. Herein, we developed structure-activity relationships around one specific core structure, F01. Using fluorescence-based DNA cleavage assays, we assessed the abilities of these compounds to stimulate the overall rate of OGG1 catalysis. Multiple compounds were identified that increased OGG1 activity on DNAs containing a site-specific 8-oxoGua by 2-fold or greater, with 9 compounds showing EC50 concentrations lower than F01 and were specific for OGG1. Selected agonists were shown to enhance OGG1-catalyzed release of 8-oxoGua and FapyGua from {gamma}-irradiated high-molecular-weight DNA using gas chromatography tandem mass spectrometry analyses. Since these assays did not reveal which step in the overall reaction was stimulated, we used a separation-of-function OGG1 mutant that possessed glycosylase, but not abasic-site (AP) lyase activity to demonstrate that the glycosylase step was not enhanced. In contrast, all agonists stimulated the AP lyase activity to levels equal to or greater than the magnitude of stimulation observed for overall chemistry, implicating agonist-mediated turnover as a potential contributor to the overall rate stimulation. The biological activities of selected agonists were evaluated in OGG1-deficient Kasumi-1 cells under conditions of paraquat (PQ)-induced oxidative stress, with several compounds mitigating PQ challenge.

Kdnases have been reported in a variety of organisms, including marine species such as trout and oysters, the opportunistic Gram-negative bacterium Sphingobacterium multivorum, and several fungal species of the genus Aspergillus, including Aspergillus terreus and Aspergillus fumigatus.. In particular, the Kdnase from the opportunistic airborne pathogen Aspergillus fumigatus (AfKdnase) plays an important role in fungal cell wall integrity and virulence, although the underlying mechanisms remain unclear. To better understand this class of enzymes, selective and sensitive tools are required for discovery, detection and visualization of active Kdnases in complex biological samples. In this work, we report the development of difluoro-Kdn mechanism-based probes functionalized with azide and biotin tags for labeling and detection of Kdnases. We show that the probes exhibit selectivity for Kdnase over the neuraminidases tested and efficiently label recombinantly expressed AfKdnase at micromolar concentrations. In addition, using the azide-bearing probe and click chemistry, we successfully visualized native Kdnases in A. fumigatus mycelia, demonstrating their utility for studying these enzymes in crude biological samples and highlighting their potential for discovering Kdnases in other organisms including fungal and bacterial species.

We developed a series of nitro reduction-reversible acylating reagents. Following optimization of the acylation conditions, these reagents were tested for deacylation with sodium dithionite in vitro. We applied this reversible acylation to modulate RNAzyme-mediated pre-tRNA maturation, demonstrating its ability to regulate RNA-RNA interactions. Furthermore, the in vitro reversible acylation of EGFP mRNA indicated effective control of its translational activity. To explore cellular applications, we validated NQO1-mediated deacylation in vitro and then induced hypoxia in HepG2 cells using cobalt chloride, thereby reactivating the function of acylated EGFP mRNA via endogenous NQO1. Overall, this study highlights the potential for developing nitro reduction-reversible acylation as a new strategy for RNA functional control and RNA-based drug modification.

Lung cancer remains the leading cause of cancer-related deaths worldwide, with cisplatin as the primary chemotherapy despite its limitations. Organotin(IV) dithiocarbamates have emerged as promising anticancer agents due to their potent cytotoxicity and stability. This study reports the successful synthesis of four novel organotin(IV) dithiocarbamates: dimethyltin(IV) N-methyl-N-benzyldithiocarbamate (DioSn-1), diphenyltin(IV) N-methyl-N-benzyldithiocarbamate (DioSn-2), triphenyltin(IV) N-methyl-N-benzyldithiocarbamate (TriSn-3), and triphenyltin(IV) N-ethyl-N-benzyldithiocarbamate (TriSn-4). Their cytotoxicity against A549 lung carcinoma cells was evaluated via MTT assay, while Annexin V-FITC/PI staining determined the mode of cell death. DioSn-2, TriSn-3, and TriSn-4 exhibited potent cytotoxicity (IC: 0.52-1.86 M), whereas DioSn-1 was inactive (IC > 50 M). Apoptotic features such as cell shrinkage and membrane blebbing were observed, with apoptosis rates ranging from 58% to 91%. DioSn-2 was the most selective (SI = 6.45) and induced early DNA damage within 30 minutes, followed by mitochondrial depolarization and excessive ROS generation. Caspase-9 activation exceeded caspase-8, confirming intrinsic apoptosis. NAC treatment reduced apoptosis by 52%, highlighting oxidative stress as a key cytotoxic mechanism. These findings suggest DioSn-2 as a promising alternative to cisplatin for lung cancer therapy.

The chikungunya virus (CHIKV) outbreak imposes a significant burden on healthcare systems and raises an urgent need for effective antiviral therapies. So far there are no specific drugs against CHIKV. A CHIKV macrodomain is critical for virulence and counteracts the host immune response, representing a promising antiviral drug target. Here, we describe small molecule inhibitors targeting the CHIKV macrodomain. Compound 1 (MDOLL-0273) was identified through a high-throughput screening using a fluorescence resonance energy transfer (FRET)-based assay, and its inhibitory activity was validated through multiple orthogonal assays. Compound 1 has a dual thiobarbiturate-indole scaffold and exhibits an IC50 of 8.9 {micro}M. X-ray crystallography revealed that the inhibitor occupies an adenine binding site of the macrodomain and extends into a novel cryptic pocket. Notably, the inhibitor shows high selectivity for the CHIKV macrodomain over a panel of human and viral ADP-ribosyl binding and hydrolyzing proteins. Structure-activity relationship studies and medicinal chemistry efforts provide a promising starting point for further hit optimization.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are key components of combination antiretroviral therapy (ART) for the treatment of human immunodeficiency virus type 1 (HIV-1) infection, binding an allosteric pocket of reverse transcriptase (RT) and inhibiting viral replication. Although second-generation NNRTIs have improved potency and resistance profiles compared to first-generation NNRTIs, the continued emergence of resistant viral strains and the need for long-acting therapeutic options underscore the importance of developing next-generation compounds. Depulfavirine (VM1500A) is a potent NNRTI being developed as a long-acting formulation. Its prodrug, elsulfavirine (ESV), is approved for HIV-1 treatment in Eurasian countries as a once-daily oral regimen and has demonstrated favorable antiviral efficacy, pharmacokinetics, and tolerability in clinical studies. Here, we report the 2.4 [A] crystal structure of HIV-1 RT in complex with depulfavirine, revealing an extended binding conformation within the NNRTI pocket that reaches from the back of the binding pocket to the entrance. These interactions may shed light on mechanisms of resistance to the F227C mutation, with and without V106 substitution, and Y188L. Notably, depulfavirine maintains potent inhibition of common NNRTI-resistant RT variants, including K103N and Y181C. Combination studies of ESV with antivirals from diverse inhibitor categories demonstrated additive or near-synergistic activity with islatravir (ISL), cabotegravir (CAB), lenacapavir (LEN), and tenofovir (TDF). These findings highlight the broad resistance profile and potential of the depulfavirine combination.

Staphylococcus aureus is the leading cause of soft tissue infections that can be treated with antibiotics. However, it can also cause significant mortality and morbidity due to systemic infections and infections of surgical implants. Implant infections typically require invasive surgery, and treatment often necessitates removal of the implant because S. aureus biofilms are extremely difficult to eradicate with antibiotic treatment alone. Therefore, there is a significant need for improved diagnostic tools for rapid, non-invasive confirmation of S. aureus infections. We recently developed an activity-based probe containing an oxadiazolone electrophile that selectively labels the S. aureus-specific serine hydrolase, FphE, by covalent binding to its active site serine residue. Here we describe a Cy5-labeled version of the probe, JJ-OX-012, and its characterization as an imaging agent for detecting biofilms both in vitro and in vivo. The probe labeled S. aureus biofilms in vitro, with virtually no background labeling of bacteria that lack FphE expression. Furthermore, we demonstrate that JJ-OX-012 can be used for non-invasive fluorescent imaging as a way to detect S. aureus biofilms in vivo. Overall, these findings support the potential for using covalent probes targeting FphE as imaging agents for rapid detection and diagnosis of staphylococcal infections in vivo.

Oxidative DNA damage caused by endogenous reactive oxygen species (ROS) is a key driver of mutagenesis, cellular dysfunction, and aging, contributing to diseases like cancer, neurodegeneration, rheumatoid arthritis, cardiovascular disorders, and diabetes. Although more than 20 oxidative base lesions have been identified, ROS-induced DNA-protein crosslinks (DPCs) are poorly characterized. ROS-DPCs are unusually bulky and highly toxic lesions that accumulate in metabolically active tissues with age, but their identities, biological consequences, and repair in living cells have remained elusive. In the present work, we characterized ROS-DPCs in human fibrosarcoma (HT1080) cells treated with hydrogen peroxide (H2O2) and elucidated the mechanisms of their removal. Mass spectrometry-based proteomics has identified over 100 cellular proteins that participated in DPC formation, most of which are involved in DNA metabolism. Our data further reveal that DNA replication and transcription facilitate DPC detection and identify a critical role of the ubiquitin-proteasomal system (UPS), replication-coupled activity of SPRTN metalloprotease, and nucleotide excision repair (NER) in removing ROS-induced DPCs. ROS-DPC formation was blocked by pretreatment with metabolically stable and cell-permeable glutathione (GSH) analog ({Psi}-GSH), suggesting a possible therapeutic strategy for preventing diseases associated with increased ROS levels. KEY POINTSMass spectrometry-based proteomics identified over 100 proteins participating in DNA-protein cross-links in human cells treated with ROS Our work reveals the mechanisms through which living cells recognize and remove ROS-DPCs Our study demonstrates the potential of a glutathione analog to prevent ROS-DPC formation GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/704426v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@15d9c33org.highwire.dtl.DTLVardef@ba0307org.highwire.dtl.DTLVardef@1cd46dorg.highwire.dtl.DTLVardef@be80ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

Fluorescent proteins (FPs) are essential tools for biological imaging but are limited by photobleaching, a light-induced loss of fluorescence intensity that reduces spatial and temporal resolution. Despite extensive use, the molecular mechanisms underlying FP photobleaching remain poorly understood due to the diversity of FPs and the complexity of their photochemistry. Existing approaches either monitor fluorescence decay in live cells, reflecting imaging conditions but lacking molecular detail, or rely on in vitro spectroscopy of purified proteins, providing mechanistic insight but often limited to individual FPs. We introduce a quantitative workflow bridging these approaches by combining live-cell measurements with in vitro spectroscopy. In vitro measurements are performed on a dedicated setup that simultaneously monitors absorption, emission, and fluorescence decay during photobleaching. Applied to six FPs spanning different chromophores, emission ranges and sequences, this approach reveals that photobleaching strongly depends on FP. It involves multiple chemical pathways, including oxidation, dimerization, and backbone cleavage. Spectroscopic analysis uncovers a heterogeneous ensemble of photoproducts with distinct photophysical properties that can remain optically active during irradiation, including shortened fluorescence lifetimes or altered absorption spectra. These findings demonstrate that FP photobleaching cannot be described as a simple ON-OFF process but involves complex transformations affecting both fluorescence intensity and lifetime. Such transformations can introduce significant biases in quantitative imaging, particularly in advanced techniques such as FLIM and FRET. Finally, we introduce quantitative indicators enabling robust comparison of FP photostability across experimental conditions. This framework provides a comprehensive approach for understanding and quantifying photobleaching and its implications for fluorescence imaging.

Tuberculosis persists as a major global health threat, significantly exacerbated by the rise of drug-resistant strains. Cytochrome P450 of 124 family CYP124 from Mycobacterium tuberculosis (CYP124), implicated in host sterol metabolism and bacterial virulence, represents an emerging and promising therapeutic target. While its precise physiological role was previously debated, CYP124s confirmed ability to metabolize immunomodulatory host sterols underscores its pharmacological relevance. Utilizing surface plasmon resonance binding assays and UV-Vis spectral titration screening, we identified nine novel non-azole ligands for CYP124 from a library of 32 plant-derived and marine natural compounds. Among these hits, (25S)-5-cholestane-3{beta},4{beta},6,7,8,15{beta},16{beta},26-octaol (termed 15{beta}-octaol) and henricioside H2 (HD-4) induced characteristic difference spectra and formed long-lived inhibitory complexes with CYP124, exhibiting dissociation half-lives of 181 min and 65 min, respectively. However, their inhibitory potency was moderate, with IC50 values of approximately 86 M for 15{beta}-octaol and exceeding 100 M for HD-4. Complementary in silico molecular docking and analysis identified key conserved hydrophobic residues within the CYP124 active site crucial for ligand binding, suggesting a shared pharmacophore. Furthermore, structural similarity analysis revealed that 37 human endogenous metabolites, including known immunoregulatory sterols, bear resemblance to the identified CYP124 ligands. This finding points towards a potential sterol-mediated interplay at the host-pathogen interface. Collectively, these results provide a foundation for the future development of mechanism-based CYP124 inhibitors as therapeutics against multidrug-resistant tuberculosis.

Inorganic polyphosphate (polyP), a linear polymer of orthophosphate residues, is involved in a range of cellular processes, including phosphate storage, blood clotting, chromatin remodeling, RNA processing, and mitochondrial function. Despite its significance, tools for monitoring polyP with specificity and in real time are lacking, limiting insights into its dynamics. Here, we present FRAPPe (FRET-based Ratiometric Analysis of Polyphosphate), a FRET-based sensor for polyP. FRAPPe consists of an eCFP-eYFP FRET pair flanking CHAD, a polyP-binding domain. The sensor remains unresponsive to nucleic acids and other polyanions, while polyP binding induces a significant decrease in FRET, allowing quantitative estimation of polyP. We further show that FRAPPe can qualitatively estimate polyP levels from crude cell extracts, supporting its utility in high-throughput genetic and pharmacological screens to identify regulators of polyP metabolism and signaling.

De novo melanin design seeks to extend natural melanin colors to new, stable colors (blue, purple, green) with sequence-to-color tunability. Natural melanin, polymerized from tyrosine (Y), is a robust pigment with heterogenous molecular weights. Control of melanin size (length) is challenging; thus, only specific colors (yellow to brown) exist in nature. In this work, we describe the design of blue melanin through the polymerization of Y-containing pentapeptides with two key properties: tight packing during peptide assembly and high solubility in aqueous environments. By motif scaffolding a pentapeptide-repeat protein (PRP) with RFdiffusion, we narrowed 160,000 possible combinations to a library of 905 Y-containing pentapeptides with tight packing features. Two of the most soluble designs successfully formed stable blue melanin with {lambda}max absorbing in 615-620 nm, contributed by homogeneous melanin length achieved around 60 Y units. Other designs also formed new colors (purple, green), along with more known colors (red, yellow, brown). We found that blue melanin exhibited thermal stability at an autoclave temperature of 121{degrees}C and photostability of weeks under 600 lux illumination. We also demonstrated the application of blue melanin as an electrophoretic ink. De novo color design from simple peptides could potentially transform how colorants are sourced and produced. Our approach with computational design should also inspire the development of new deep-learning tools to directly predict colors from amino acid sequences.

Antimicrobial resistance poses major therapeutic challenges, particularly for multidrug-resistant mycobacterial infections caused by Mycobacterium tuberculosis (Mtb) and non-tuberculous mycobacteria (NTM). L,D-Transpeptidases (Ldts) are attractive drug targets due to their essential role in peptidoglycan cell wall crosslinking, yet existing assays suffer from low throughput and limited sensitivity. We report a versatile, bead-based platform for high-throughput analysis of Ldt activity and inhibitor discovery. We incubated peptidoglycan stem peptides, either naturally harvested or synthetically immobilized on abiotic surfaces, with Ldts and a fluorescent acyl acceptor to quantitatively monitor crosslinking. After optimizing assay parameters, we profiled six Mycobacterium smegmatis Ldt paralogs, including the first characterization of a class 6 Ldt with chemically defined substrate sequences. Utilizing a series of acyl acceptors, we demonstrated modifications within the acyl acceptor that are tolerated by mycobacterial Ldts. Screening of {beta}-lactam antibiotics revealed potent inhibition by (carba)penems, while cephalosporins, monobactams and penams showed negligible activity. The assay achieved excellent performance metrics and was successfully adapted to ELISA and 96-well formats, providing a powerful tool for discovering Ldt-targeted therapeutics against tuberculosis and related infections.

People with diabetes rely on exogenous insulin to reduce blood glucose levels, compensating for insulin resistance or impaired pancreatic {beta}-cell function. Despite being essential for diabetes management, insulin formulations exhibit inconsistent performance due to their relatively fragile stability. This instability carries significant cost implications: some individuals spend over $1,000 USD per month on insulin, and these high prices influence one in six Americans with diabetes to ration their insulin supplies. Environmental stressors can induce conformational changes that cause insulin to misfold and aggregate into fibrils, which are inactive structures that contribute to long-term diabetic complications. Although insulins instability is well-documented, no test currently exists outside of laboratory settings to determine whether an insulin formulation has degraded. Here, we compare biochemical techniques for assessing bioactivity and structural integrity in three commercial insulin analogs exposed to physiologically relevant stress conditions, showing that fibril formation precedes measurable loss of bioactivity in insulin and that fibrillation depends on both the stressor type and the insulin formulation tested. We then demonstrate proof-of-concept testing for antibody-based degradation detection using commercial monoclonal antibody candidates. Together, these findings underscore the critical need for accessible insulin quality testing and demonstrate the feasibility of antibody-based detection of insulin fibrillation. ImportanceInsulin remains one of the most essential yet fragile biopharmaceuticals used in modern medicine. Globally, 150 million people with diabetes depend on exogenous insulin to regulate blood glucose levels, but the proteins inherent instability can cause degradation during storage or transport. These degradation events can reduce insulins potency and safety, yet patients and healthcare providers currently have no practical means to assess insulin quality before injection. This knowledge gap contributes to inconsistent therapeutic outcomes and increases the risk of complications associated with degraded insulin. Our work directly addresses this unmet clinical and public health need by identifying the molecular changes that occur when insulin analogs are exposed to everyday environmental stressors and by completing proof-of-concept testing for a device to detect insulin fibrillation. We demonstrate that structural transitions to fibrils precede measurable loss of bioactivity and that fibrillation behavior depends on both the insulin analog and the stressor type. By combining biochemical characterization with antibody-based detection, this study establishes a foundation for a low-cost, accessible method to verify insulin integrity outside the laboratory. Such a tool could prevent the use of degraded insulin, improve treatment consistency, and empower patients to ensure the quality of their medication. More broadly, this approach exemplifies how protein stability monitoring can be integrated into biotherapeutic quality assurance, improving safety, efficacy, and trust in life-sustaining biologic medicines.

Accurate replication of mitochondrial genome (mtDNA) integrity, which is essential for cellular metabolism and energy supply, relies primarily on DNA polymerase gamma (Pol {gamma}), Twinkle helicase, and mitochondrial single-stranded DNA binding protein (mtSSB). Twinkle alone exhibits little helicase activity while reports indicate that Pol {gamma} displays from modest to limited unwinding activity. This led us to dissect Pol {gamma} strand displacement activity using structural, biochemical and in silico approaches. Here, we show that human Pol {gamma} carries out robust strand displacement synthesis at physiological concentrations of divalent metal ions which reveals that distinct metal-binding sites can independently regulate DNA synthesis and unwinding activities. We further showed that Pol {gamma} can displace RNA/DNA hybrid with comparable efficiency as DNA/DNA duplex, representing a key implication on RNA primer removal to preserve mtDNA integrity. Our cryo-electron microscopy structures of Pol {gamma} complexed with a template containing downstream dsDNA and an incoming nucleotide revealed the structural mechanism for the strand displacement activity. We identified four conformational states that represent successive stages of DNA unwinding, accompanied by coordinated rearrangement of the downstream DNA and Pol {gamma} elements that mediate strand displacement. This work establishes biochemical and structural mechanisms of Pol {gamma} strand displacement activity, providing fundamental insight into human mitochondrial DNA replication and integrity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/701366v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1be1dborg.highwire.dtl.DTLVardef@8912c5org.highwire.dtl.DTLVardef@12f5e75org.highwire.dtl.DTLVardef@e25d2a_HPS_FORMAT_FIGEXP M_FIG C_FIG