The ability of biologically active molecules to access intracellular targets remains a critical barrier in drug development. While assays for measuring cellular uptake exist, they often fail to distinguish between membrane-associated or endosomal trapped compounds and those that successfully reach the cytosol. Here, we present the Chloroalkane HaloTag Azide-based Membrane Penetration (CHAMP) Assay, a high-throughput method that employs a minimally disruptive azide tag to report the cytosolic accumulation of diverse molecules in mammalian cells. The CHAMP assay utilizes HaloTag-expressing cells and strain-promoted azide-alkyne cycloaddition (SPAAC) chemistry to quantify the presence of azide-tagged test compounds in the cytosol. We demonstrate the versatility of this approach by evaluating the accumulation profiles of small molecules, peptides, and proteins, revealing how structural variations and stereochemical differences influence cytosolic penetration. Our findings with cell-penetrating peptides confirm established structure-activity relationships, with longer polyarginine sequences showing enhanced accumulation. Additionally, we observed that C-terminal amidation and D-amino acid substitutions significantly impact cellular penetration. When applied to supercharged proteins and antibiotics, CHAMP successfully discriminates between compounds with varying accumulation capabilities. This method provides a robust platform for screening cytosolic accumulation while minimizing the confounding effects of large tags on molecular permeability, potentially accelerating the development of therapeutics targeting intracellular pathways.

Gram-negative bacterial pathogens pose a significant challenge in drug development because their outer membranes hinder the permeation of small molecules. The lack of widely adoptable methods for measuring the cytosolic accumulation of compounds in bacterial cells further hinders drug discovery efforts. To address this challenge, we report the development of the Chloroalkane Azide Membrane Permeability (CHAMP) assay, which we designed specifically to assess molecule accumulation in the cytosol of Gram-negative bacteria. The CHAMP analysis utilizes bioorthogonal epitopes anchored within HaloTag-expressing bacteria and measures the cytosolic arrival of azide-bearing test molecules through strain-promoted azide–alkyne cycloaddition. This workflow enables robust and rapid accumulation measurements of thousands of azide-tagged small molecules. Our approach consistently produces comprehensive accumulation profiles that surpass the scale of previous measurements in Escherichia coli (E. coli). We validated the CHAMP assay across various chemical and biological contexts, including hyperporinated cells, membrane-permeabilized cells, and E. coli strains with impaired TolC function, a key component of the efflux pump. The CHAMP platform provides a simple, high-throughput, and accessible method that enables the analysis of over 1000 molecules within hours. This technique addresses a critical gap in antimicrobial research and has the potential to accelerate the development of effective agents against Gram-negative pathogens. In this work, the authors present their Chloroalkane Azide Membrane Permeability (CHAMP) assay that measures cytosolic accumulation of small molecules in Escherichia coli, enabling rapid profiling of compounds, with the goal to simplify and accelerate antimicrobial drug discovery.

Emerging evidence has demonstrated the importance of pattern recognition receptors (PRRs), including the nucleotide-binding and oligomerization domain receptor 2 (NOD2), in human health and disease states. NOD2 activation has shown promise with aiding malnutrition recovery, lessening irritable bowel disease (IBD) symptoms, and increasing the efficacy of cancer immunotherapy. Currently, most NOD2 agonists are derivatives or analogs of the endogenous agonist derived from bacterial peptidoglycan, muramyl dipeptide (MDP). These MDP-based agonists can suffer from low oral bioavailability and cause significant adverse side effects. With the goal of broadly improving NOD2 therapeutic interventions, we sought to discover a small molecule capable of activating NOD2 by screening a library of total 1917 FDA approved drugs in a phenotypic assay. We identified a class of compounds, benzimidazoles, that act as NOD2 agonists, with the most potent member of this class being nocodazole. Nocodazole activates NOD2 with nanomolar potency and causes the release of cytokines canonically associated with MDP-induced NOD2 activation, suggesting its potential to elicit similar therapeutic immune effects as MDP and potentially offer improved pharmacological properties.

ABSTRACTImmunotherapy has emerged as a powerful strategy for combating cancer by harnessing the patient immune system to recognize and eliminate malignant cells. The major histocompatibility complex class I (MHC-I) has a pivotal role in the recognition step. These surface proteins present cancer-specific neoantigens to CD8+ T cells, which triggers activation and T cell-mediated killing. However, cancer cells can often evade immune detection by downregulating MHC-I surface expression, which renders the immune response less effective. In turn, this resistance mechanism offers an opportunity to bolster MHC-I surface expressionviatherapeutic interventions. Here, we conducted an initial comprehensive evaluation of previously purported small molecule MHC-I inducers and identified heat shock protein 90 (Hsp90) inhibitors as privileged inducers of MHC-I surface expression. With a core scaffold in hand, we employed anin situclick chemistry-based derivatization strategy to generate 380 novel compounds in the same family. New agents from this library showed high levels of induction, with one of the triazole-based analogs,CliMB-325, also enhancing T cell activation and exhibiting lower toxicity, which could potentiate some immunotherapeutic modalities. Moreover, we demonstrated the potential of a click chemistry-based diversification strategy for the discovery of small molecules to counter immune evasion.

Tuberculosis causes over one million deaths annually and remains the leading cause of death from a single infectious agent. The emergence of multidrug-resistant Mycobacterium tuberculosis strains highlights the urgent need for new antibiotics, a pursuit hindered by the bacterium's complex cell envelope. As most anti-tuberculosis agents act on intracellular targets, assessing cytosolic drug accumulation is critical. Conventional approaches generally quantify whole-cell association without resolving subcellular localization. Moreover, no current method permits real-time monitoring of drug accumulation in live mycobacterial cells. Here, we present a split-luciferin-based assay to quantify molecular accumulation in mycobacteria. Using this approach, we quantified the cytosolic accumulation of diverse small-molecule antibiotics and polyarginine peptides conjugated via a disulfide-linked D-cysteine tag. We also show the localization of a polyarginine peptide inside of mycobacteria in infected macrophage cells, demonstrating that these peptides can cross multiple accumulation barriers. Our findings establish the first assay for real-time quantification of cytosolic molecular accumulation in live mycobacteria, addressing a longstanding methodological gap and enabling mechanistic insights into intracellular drug uptake.

A recent report discovered a chloro-hydantoin lead for COVID-19 antiviral therapy, however, effects of stereochemical changes of the spiro-cycle and halogens were not examined. Herein, we describe how minimal structural variations, such as stereochemistry and atomic size of halogen substituents, affect binding affinity and biological activity. Blind docking derived a correlation between binding affinity and IC 50 of various hydantoin analogs revealed the F compound 8 (2s, 4s) to be more potent than Cl compound 9a (2s, 4s) and Br compound 10a (2s, 4s). However, experimentally, Cl compound 9a and Br compound 10a exhibited greater in vitro potency against M pro than F compound 8. To rationalize the impact of atomic size and stereo configuration on observed biological activity, the volume occupancy and protein-ligand interactions were evaluated. Compounds 9a and 10a occupy 33% and 35% of the cavity space. The Br atom's size optimally fits the cavity with less conformational flexibility, conferring higher stability. MD simulation data of the ligand torsion profile and the solvent accessible surface area also indicated higher conformational stability for compound 10a than compounds 8 and 9a. Compounds 9b and 10b, (2r, 4r) stereoisomers of 9a and 10a respectively, had more conformational flexibility and were less active M pro inhibitors in vitro.

e15132 Background: Certain heptamethine cyanine dye derivatives like MHI-148 (1a) and IR-783 exhibit strong affinity for cancer cells. These dyes selectively target and accumulate within cancer cells due to overexpression of organic anion-transporting polypeptides (OATPs) and hypoxia-induced factors. In contrast, chlorin e6 (Ce6, 4), a commonly used photosensitizer in cancer treatment, lacks effective cancer targeting, leading to limited therapeutic efficacy and non-specific tissue damage. Methods: To enhance Ce6's tumor specificity, researchers developed novel tumor-targeting photosensitizer-carrier-conjugates (PSCs) by attaching Ce6 to heptamethine cyanine dyes. Results: These PSCs showed improved cancer cell uptake and retention compared to Ce6 alone, as demonstrated in breast cancer cells (MCF7) both in vitro and in mice xenograft models. The synthesis of PSCs involved three steps starting from Ce6 anhydride, yielding moderate results. The conjugates were complexed with either cold Cu for reference or radioactive 64Cu for quantitative studies and PET imaging. Both complexed and non-complexed PSCs were effective for in vitro and in vivo imaging studies. NIRF imaging revealed enhanced cancer cell uptake of PSCs compared to Ce6 alone, while PET imaging provided quantitative tumor visualization. PSCs demonstrated superior tumor imaging efficiency compared to 18F-FDG in mice xenografts. Conclusions: These findings highlight the potential of PSCs as multifunctional theranostic agents for image-guided photodynamic and radiotherapeutic cancer treatment.

Understanding the factors that influence the accumulation of molecules beyond the mycomembrane of Mycobacterium tuberculosis (Mtb)─the main barrier to accumulation─is essential for developing effective antimycobacterial agents. In this study, we investigated two design principles commonly observed in natural products and mammalian cell-permeable peptides: backbone N-alkylation and macrocyclization. To assess how these structural edits impact molecule accumulation beyond the mycomembrane, we utilized our recently developed Peptidoglycan Accessibility Click-Mediated Assessment (PAC-MAN) assay for live-cell analysis. Our findings provide the first empirical evidence that peptide macrocyclization generally enhances accumulation in mycobacteria, while N-alkylation influences accumulation in a context-dependent manner. We examined these design principles in the context of two peptide antibiotics, tridecaptin A1 and griselimycin, which revealed the roles of N-alkylation and macrocyclization in improving both accumulation and antimicrobial activity against mycobacteria in specific contexts. Together, we present a working model for strategic structural modifications aimed at enhancing the accumulation of molecules past the mycomembrane. More broadly, our results also challenge the prevailing belief in the field that large and hydrophilic molecules, such as peptides, cannot readily traverse the mycomembrane.

Immunotherapy has emerged as a powerful strategy for combating cancer by harnessing the patient immune system to recognize and eliminate malignant cells. The major histocompatibility complex class I (MHC-I) has a pivotal role in the recognition step. These surface proteins present cancer-specific neoantigens to CD8+ T cells, which triggers activation and T cell-mediated killing. However, cancer cells can often evade immune detection by downregulating MHC-I surface expression, which renders the immune response less effective. In turn, this resistance mechanism offers an opportunity to bolster MHC-I surface expression via therapeutic interventions. Here, we conducted an initial comprehensive evaluation of previously purported small molecule MHC-I inducers and identified heat shock protein 90 (Hsp90) inhibitors as privileged inducers of MHC-I surface expression. With a core scaffold in hand, we employed an in situ click chemistry-based derivatization strategy to generate 380 novel compounds in the same family. New agents from this library showed high levels of induction, with one of the triazole-based analogs, CliMB-325, also enhancing T cell activation and exhibiting lower toxicity, which could potentiate some immunotherapeutic modalities. Moreover, we demonstrated the potential of a click chemistry-based diversification strategy for the discovery of small molecules to counter immune evasion.

The binding interface between antigens and antibodies is pivotal in humoral immune responses and provides crucial effective defense against pathogens and exogenous threats. Existing predictive computational methodologies, including structure-based and sequence-based approaches, offer valuable insights but face challenges such as unknown antigen structures and reliance on manually curated features. Most current methods primarily predict antigen epitope, often neglecting the specific molecular epitope-paratope interactions essential for immune efficacy. In this study, we introduce a novel approach EPP (Epitope-Paratope Predictor), using the ESM-2 protein language model as a feature encoder and a Bi-LSTM network to predict epitope-paratope interactions. Our method processes antigen and antibody sequences as inputs, leveraging a novel dataset strategy and encoding protein representations to enhance prediction accuracy. The results demonstrate a significant improvement in prediction accuracy compared to existing methods, highlighting the importance of protein feature encoder and temporal dependencies within sequences. The model's performance in different antigen clusters is analyzed, while those predictions are compared with that from AlphaFold3 and Dock method. Our method validation shows superior performance in recognizing distinctive epitopes of the same antigen when bound to different antibodies. This approach offers a new strategy for an in-depth understanding of antigen-antibody interactions, essential for an array of pioneer projects, such as structure-guided design and affinity maturation for precision antibodies targeting a given epitope.

Immunotherapy has emerged as a powerful strategy for combating cancer by harnessing the patient's immune system to recognize and eliminate malignant cells. Major histocompatibility complex class I (MHC-I) plays a pivotal role by presenting neoantigens to CD8+ T cells, triggering T cell-mediated killing. However, cancer cells often evade detection by downregulating the MHC-I surface expression, hindering the immune response. This resistance mechanism offers an opportunity to bolster MHC-I surface expression via therapeutic interventions. We conducted a comprehensive evaluation of previously purported small molecule MHC-I inducers and identified heat shock protein 90 inhibitors as privileged enhancers. Using a core scaffold, we employed an in situ click chemistry-based derivatization strategy to generate 380 novel compounds. New agents showed high induction levels, with one triazole-based analogue, CliMB-325, also enhancing T cell activation and exhibiting lower toxicity. Altogether, we demonstrated the potential of click chemistry-based diversification for discovering small molecules to counter immune evasion.

The recalcitrance of Mycobacterium tuberculosis to antibiotic treatment has been broadly attributed to the impermeability of the organism's outer mycomembrane. However, the studies that support this inference have been indirect or reliant on bulk population measurements. We previously developed the Peptidoglycan Accessibility Click-Mediated AssessmeNt (PAC-MAN) method to covalently trap azide-modified small molecules in the peptidoglycan cell wall of live mycobacteria after they have traversed the mycomembrane. Using PAC-MAN, we now show that the mycomembrane differentially restricts access to fluorophores and antibiotic derivatives. Mycomembranes of both M. tuberculosis and the model organism Mycobacterium smegmatis discriminate between divergent classes of antibiotics as well as between antibiotics within a single family, the fluoroquinolones. By analyzing subpopulations of M. tuberculosis and M. smegmatis, we also found that some fluorophores and vancomycin are heterogeneously restricted by the mycomembrane. Our data indicate that the mycomembrane is a molecule- and cell-specific barrier to antibiotic permeation.

Staphylococcus aureus ( S. aureus ) has evolved the ability to persist after uptake into host immune cells. This intracellular niche enables S. aureus to potentially escape host immune responses and survive the lethal actions of antibiotics. While the elevated tolerance of S. aureus to small-molecule antibiotics is likely to be multifactorial, we pose that there may be contributions related to permeation of antibiotics into phagocytic vacuoles, which would require translocation across two mammalian bilayers. To empirically test this, we adapted our recently developed permeability assay to determine the accumulation of FDA-approved antibiotics into phagocytic vacuoles of live macrophages. Bioorthogonal reactive handles were metabolically anchored within the surface of S. aureus, and complementary tags were chemically added to antibiotics. Following phagocytosis of tagged S. aureus cells, we were able to specifically analyze the arrival of antibiotics within the phagosomes of infected macrophages. Our findings enabled the determination of permeability differences between extra- and intracellular S. aureus , thus providing a roadmap to dissect the contribution of antibiotic permeability to intracellular pathogens.

Antibiotic resistance is an alarming public health concern that affects millions of individuals across the globe each year. A major challenge in the development of effective antibiotics lies in their limited ability to permeate cells, noting that numerous susceptible antibiotic targets reside within the bacterial cytosol. Consequently, improving the cellular permeability is often a key consideration during antibiotic development, underscoring the need for reliable methods to assess the permeability of molecules across cellular membranes. Currently, methods used to measure permeability often fail to discriminate between the arrival within the cytoplasm and the overall association of molecules with the cell. Additionally, these techniques typically possess throughput limitations. In this work, we describe a luciferase-based assay designed for assessing the permeability of molecules in the cytosolic compartment of Gram-negative bacteria. Our findings demonstrate a robust system that can elucidate the kinetics of intracellular antibiotic accumulation in live bacterial cells in real time.

The role of the intestinal microbiota in host health is increasingly revealed in its contributions to disease states. The host-microbiome interaction is multifactorial and dynamic. One of the factors that has recently been strongly associated with host physiological responses is peptidoglycan from bacterial cell walls. Peptidoglycan from gut commensal bacteria activates peptidoglycan sensors in human cells, including the nucleotide-binding oligomerization domain-containing protein 2. When present in the gastrointestinal tract, both the polymeric form (sacculi) and depolymerized fragments can modulate host physiology, including checkpoint anticancer therapy efficacy, body temperature and appetite, and postnatal growth. To utilize this growing area of biology toward therapeutic prescriptions, it will be critical to directly analyze a key feature of the host-microbiome interaction from living hosts in a reproducible and noninvasive way. Here we show that metabolically labeled peptidoglycan/sacculi can be readily isolated from fecal samples collected from both mice and humans. Analysis of fecal samples provided a noninvasive route to probe the gut commensal community including the metabolic synchronicity with the host circadian clock. Together, these results pave the way for noninvasive diagnostic tools to interrogate the causal nature of peptidoglycan in host health and disease.

Introduction Acute hyperglycemia (HG) enhances inflammatory and oxidative stress and exacerbates myocardial infarct size during ischemia-reperfusion injury by activating splenic leukocytes. Formyl peptide receptor 1 (FPR1) on leukocytes is activated by and mediates myocardial ischemia-reperfusion injury. We hypothesize that selective FPR1 antagonist cinnamoyl-F-(D)L-F-(D)L-F (CF) or potent reducing agent tris (2-carboxyethyl) phosphine hydrochloride (TCEP) could abrogate hyperglycemic infarct exacerbation, both alone and synergistically via a novel CF-TCEP compound that would target leukocytes for antioxidative effect. Methods Acute HG was induced in wild type mice with an intraperitoneal dextrose injection followed by left coronary artery occlusion (30 min) and reperfusion (60 min). In treatment groups, CF (0.1 mg/kg or 1 mg/kg), TCEP (1 mg/kg or 20 mg/kg), or the CF-TCEP conjugate (0.1 mg/kg) was administered intravenously before reperfusion. The hearts were harvested to measure infarct size (IF). Results HG resulted in >50% increase in IF compared to euglycemic mice (52.1 ± 3.0 versus 34.0 ± 3.2%, P < 0.05). Neither CF nor TCEP independently exerted an infarct-sparing effect at lower doses (46.2 ± 2.1% or 50.9 ± 4.1%, P > 0.05 versus HG control) but at high doses, significantly attenuated IF exacerbation (23.2 ± 5.2% or 33.9 ± 3.6%, P < 0.05 versus HG control). However, the low-dose CF-TCEP conjugate significantly reduced IF (39.1 ± 1.7%, P < 0.05 versus HG control). IF was decreased to near euglycemic control levels (P > 0.05). Conclusions The CF-TECP conjugate synergistically attenuated HG infarct exacerbation at significantly lower respective doses of CF and TCEP. In addition to the intrinsic anti-inflammatory effect of blocking FPR1, CF is also a feasible tool for leukocyte-targeted therapy to treat IRI.

Background Cardiac metabolic abnormalities are present in heart failure. Few studies have followed metabolic changes accompanying diastolic and systolic heart failure in the same model. We examined metabolic changes during the development of diastolic and severe systolic dysfunction in spontaneously hypertensive rats (SHR). Methods and Results We serially measured myocardial glucose uptake rates with dynamic 2‐[ 18 F] fluoro‐2‐deoxy‐ d ‐glucose positron emission tomography in vivo in 9‐, 12‐, and 18‐month‐old SHR and Wistar Kyoto rats. Cardiac magnetic resonance imaging determined systolic function (ejection fraction) and diastolic function (isovolumetric relaxation time) and left ventricular mass in the same rats. Cardiac metabolomics was performed at 12 and 18 months in separate rats. At 12 months, SHR hearts, compared with Wistar Kyoto hearts, demonstrated increased isovolumetric relaxation time and slightly reduced ejection fraction indicating diastolic and mild systolic dysfunction, respectively, and higher (versus 9‐month‐old SHR decreasing) 2‐[ 18 F] fluoro‐2‐deoxy‐ d ‐glucose uptake rates (Ki). At 18 months, only few SHR hearts maintained similar abnormalities as 12‐month‐old SHR, while most exhibited severe systolic dysfunction, worsening diastolic function, and markedly reduced 2‐[ 18 F] fluoro‐2‐deoxy‐ d ‐glucose uptake rates. Left ventricular mass normalized to body weight was elevated in SHR, more pronounced with severe systolic dysfunction. Cardiac metabolite changes differed between SHR hearts at 12 and 18 months, indicating progressive defects in fatty acid, glucose, branched chain amino acid, and ketone body metabolism. Conclusions Diastolic and severe systolic dysfunction in SHR are associated with decreasing cardiac glucose uptake, and progressive abnormalities in metabolite profiles. Whether and which metabolic changes trigger progressive heart failure needs to be established.

The general lack of permeability of small molecules observed for Mycobacterium tuberculosis (Mtb) is most ascribed to its unique cell envelope. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. We describe a novel assay that combines metabolic tagging of the peptidoglycan, which sits directly beneath the mycomembrane, click chemistry of test molecules, and a fluorescent labeling chase step, to measure the permeation of small molecules. We showed that the assay workflow was robust and compatible with high-throughput analysis in mycobacteria by testing a small panel of azide-tagged molecules. The general trend is similar across the two types of mycobacteria with some notable exceptions. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly-discovered compounds into mycobacteria.

AbstractStaphylococcus aureus(S. aureus) has evolved the ability to persist after uptake into host immune cells. This intracellular niche enablesS. aureusto potentially escape host immune responses and survive the action of antibiotics. The elevated tolerance ofS. aureusto small molecule antibiotics is likely to be multifactorial. We pose that there may be contributions related to permeation into phagosome, which would require translocation across two mammalian bilayers. To empirically test this, we adapted our recently developed permeability assay to determine the accumulation of FDA-approved antibiotics in phagocytic vacuoles. Bioorthogonal reactive handles were metabolically anchored within the surface ofS. aureus, and complementary tags were chemically added to antibiotics. Following phagocytosis of labeledS. aureuscells, we were able to specifically analyze the accumulation levels of antibiotics within the phagosomes of infected macrophages. Our findings enabled the determination of differences between the permeability of antibiotics to extra- and intracellularS. aureus, thus providing a roadmap to dissect the contribution of antibiotic permeability to intracellular pathogens.

Background: Splenocyte contribution to ischemic brain injury has been suggested. It is not known whether this effect is due to systemic action or direct influence in ischemic brain tissues. It is also not known how splenocytes migrate into the brain and worsen neurological outcome after brain ischemia. We determined the role of formyl peptide receptor 1 (FPR1), a receptor expressed in monocytes, in the migration of splenocytes into ischemic brain tissues and the contribution of these splenocytes to ischemic brain injury. Methods: Mice with or without fpr1 knockout were subjected to transient focal brain ischemia. The migration of splenocytes was assessed under in vivo and in vitro conditions . Results: cFLFLF, a FPR1 antagonist, inhibited splenocyte migration into the brain and neuroinflammation after ischemic stroke. cFLFLF improved neurological outcome assessed 24 hours or 28 days after stroke. cFLFLF did not alter blood-brain barrier permeability in the ischemic brain. fpr1-/- mice had an attenuated peripheral monocyte and neutrophil infiltration into the brain, a reduced proinflammatory cytokine level and an improved neurological outcome compared with wild-type mice after brain ischemia. cFLFLF did not affect the proinflammatory cytokine levels in the spleen and brain of fpr1-/- mice after ischemic stroke. Conclusions: These results suggest that FPR1 facilitates splenocyte migration into the brain and proinflammatory cytokine production to worsen neurological outcome after brain ischemia, indicating a direct effect of splenocytes on ischemic brain tissues. Our results support the notion that cFLFLF via blocking FPR1 signaling inhibits those pathological processes and is a potential agent for neuroprotection.