Molecular Transformers: Adaptive Multitarget Ligands for Esterase-Induced Transition from Analgesics to Anesthetics.

Multitarget strategies are essential in addressing complex diseases, yet developing multitarget-directed ligands (MTDLs) is particularly challenging when aiming to engage multiple therapeutic targets across different tissues. Here, we present a molecular transformer strategy, enhancing traditional MTDLs. By utilizing esterase-driven hydrolysis, this approach mimics the adaptive nature of transformers for enabling molecules to modify their pharmacological effects in response to the biological milieu. By virtual screening and biological evaluation, we identified KGP-25, a novel compound initially targeting the voltage-gated sodium channel 1.8 (Nav1.8) in the peripheral nervous system (PNS) for analgesia, and later the γ-aminobutyric acid subtype A receptor (GABAA) in the central nervous system (CNS) for general anesthesia. Our findings confirm KGP-25's dual efficacy in cellular and animal models, effectively reducing opioid-related side effects. This study validates the molecular transformer approach in drug design and highlights its potential to overcome the limitations of conventional MTDLs, paving new avenues in innovative therapeutic strategies.

Deep simulated annealing for the discovery of novel dental anesthetics with local anesthesia and anti-inflammatory properties.

Multifunctional therapeutics have emerged as a solution to the constraints imposed by drugs with singular or insufficient therapeutic effects. The primary challenge is to integrate diverse pharmacophores within a single-molecule framework. To address this, we introduced DeepSA, a novel edit-based generative framework that utilizes deep simulated annealing for the modification of articaine, a well-known local anesthetic. DeepSA integrates deep neural networks into metaheuristics, effectively constraining molecular space during compound generation. This framework employs a sophisticated objective function that accounts for scaffold preservation, anti-inflammatory properties, and covalent constraints. Through a sequence of local editing to navigate the molecular space, DeepSA successfully identified AT-17, a derivative exhibiting potent analgesic properties and significant anti-inflammatory activity in various animal models. Mechanistic insights into AT-17 revealed its dual mode of action: selective inhibition of NaV1.7 and 1.8 channels, contributing to its prolonged local anesthetic effects, and suppression of inflammatory mediators via modulation of the NLRP3 inflammasome pathway. These findings not only highlight the efficacy of AT-17 as a multifunctional drug candidate but also highlight the potential of DeepSA in facilitating AI-enhanced drug discovery, particularly within stringent chemical constraints.

Identification of N-(((1S,3R,5S)-adamantan-1-yl)methyl)-3-((4-chlorophenyl)sulfonyl)benzenesulfonamide as novel Nav1.8 inhibitor with analgesic profile.

Chronic pain is a common and challenging clinical problem that significantly impacts patients' quality of life. The sodium channel Nav1.8 plays a crucial role in the occurrence and development of chronic pain, making it one of the key targets for treating chronic pain. In this article, we combined virtual screening with cell membrane chromatography techniques to establish a novel method for rapid high-throughput screening of selective Nav1.8 inhibitors. Using this approach, we identified a small molecule compound 6, which not only demonstrated high affinity and inhibitory activity against Nav1.8 but also exhibited significant inhibitory effects on CFA-induced chronic inflammatory pain. Compared to the positive drug VX-150, compound 6 showed a more prolonged analgesic effect, making it a promising candidate as a Nav1.8 inhibitor with potential clinical applications. This discovery provides a new therapeutic option for the treatment of chronic pain.

Identification of (4-chlorophenyl)(5-hydroxynaphtho[1,2-b]furan-3-yl)methanone as novel COX-2 inhibitor with analgesic profile.

Chronic pain is a serious problem that affects billions of people worldwide, but current analgesic drugs limit their use in chronic pain management due to their respective side effects. As a first-line clinical drug for chronic pain, COX-2 selective inhibitors can relieve mild to moderate pain, but they also have some problems. The most prominent one is that their analgesic intensity is not enough, and they cannot well meet the treatment needs of chronic pain. Therefore, there is an urgent need to develop COX-2 inhibitors with stronger analgesic intensity. In this article, we used virtual screening method to screen out the structurally novel COX-2 inhibitor for chronic pain management, and conducted a preliminary study on its mechanism of action using molecular dynamics simulation.

Discovery of a novel series of pyridone amides as NaV1.8 inhibitors.

The NaV1.8 channel, mainly found in the peripheral nervous system, is recognized as one of the key factors in chronic pain. The molecule VX-150 was initially promising in targeting this channel, but the phase II trials of VX-150 did not show expected pain relief results. By analyzing the interaction mode of VX-150 and NaV1.8, we developed two series with a total of 19 molecules and examined their binding affinity to NaV1.8 in vitro and analgesic effect in vivo. One compound, named 2j, stood out with notable activity against the NaV1.8 channel and showed effective pain relief in models of chronic inflammatory pain and neuropathic pain. Our research points to 2j as a strong contender for developing safer pain-relief treatments.

A Novel Bifunctional µOR Agonist and σ1R Antagonist with Potent Analgesic Responses and Reduced Adverse Effects.

Bifunctional ligands possessing both µOR agonism and σ1R antagonism have shown promise in producing strong analgesic effects with reduced opioid-related side effects. However, the µOR agonism activity of most dual ligands diminishes compared with classical opioids, raising concern about their effectiveness in managing nociceptive pain. In this study, a new class of dual µOR agonist/σ1R antagonist was reported. Through structure-activity relationship analyses, we identified the optimal compound, 4x, which displayed picomolar µOR agonism activity (EC50: 0.6 ± 0.2 nM) and good σ1R inhibitory activity (Ki: 363.7 ± 5.6 nM) with excellent selectivity. Compound 4x exhibited robust analgesic effects in various pain models, with significantly reduced side effects. Importantly, compound 4x also possessed good safety profiles and no abnormalities were observed in biological parameters even under a high dosage. Our findings suggest that 4x may be a promising lead compound for developing safer opioids and warrants further in-depth studies.

Engineering Escherichia coli Nissle 1917 as a microbial chassis for therapeutic and industrial applications.

Genetically engineered microbes, especially Escherichia coli, have been widely used in the biosynthesis of proteins and metabolites for medical and industrial applications. As a traditional probiotic with a well-established safety record, E. coli Nissle 1917 (EcN) has recently emerged as a microbial chassis for generating living therapeutics, drug delivery vehicles, and microbial platforms for industrial production. Despite the availability of genetic tools for engineering laboratory E. coli K-12 and B strains, new genetic engineering systems are still greatly needed to expand the application range of EcN. In this review, we have summarized the latest progress in the development of genetic engineering systems in EcN, as well as their applications in the biosynthesis and delivery of valuable small molecules and biomacromolecules of medical and/or industrial interest, followed by a glimpse of how this rapidly growing field will evolve in the future.

Development of probiotic E. coli Nissle 1917 for ß-alanine production by using protein and metabolic engineering.

ß-alanine has been used in food and pharmaceutical industries. Although Escherichia coli Nissle 1917 (EcN) is generally considered safe and engineered as living therapeutics, engineering EcN for producing industrial metabolites has rarely been explored. Here, by protein and metabolic engineering, EcN was engineered for producing ß-alanine from glucose. First, an aspartate-α-decarboxylase variant ADCK43Y with improved activity was identified and over-expressed in EcN, promoting the titer of ß-alanine from an undetectable level to 0.46 g/L. Second, directing the metabolic flux towards L-aspartate increased the titer of ß-alanine to 0.92 g/L. Third, the yield of ß-alanine was elevated to 1.19 g/L by blocking conversion of phosphoenolpyruvate to pyruvate, and further increased to 2.14 g/L through optimizing culture medium. Finally, the engineered EcN produced 11.9 g/L ß-alanine in fed-batch fermentation. Our work not only shows the potential of EcN as a valuable industrial platform, but also facilitates production of ß-alanine via fermentation. KEY POINTS: • Escherichia coli Nissle 1917 (EcN) was engineered as a ß-alanine producing cell factory • Identification of a decarboxylase variant ADCK43Y with improved activity • Directing the metabolic flux to L-ASP and expressing ADCK43Y elevated the titer of ß-alanine to 11.9 g/L.

Robust Impact Effect and Super-Lyophobic Reduced Galinstan on Polymers Applied for Energy Harvester.

In this paper, we present a novel reduced Galinstan-based microfluidic energy harvester, which can converse kinetic energy to electricity from an arbitrary vibration source. Firstly, the wetting behaviors of reduced Galinstan are performed, which shows a robust impact effect on polymer substrates. Moreover, the electric circuit model of the reduced Galinstan-based energy harvester is made and discussed by the use of the EDLCs (electrical double layer capacitors). After modeling, the microfluidic energy harvester with coplanar microfluidic channels is designed and fabricated. Finally, the performance of the microfluidic energy harvester is investigated, which can harvest multi-direction vibration energy. The experiment results demonstrate that the novel reduced Galinstan-based microfluidic energy harvester is suitably and uniquely applied in a complex vibration environment.

Dual drugs decorated bacteria irradiate deep hypoxic tumor and arouse strong immune responses.

Intratumoral environment as a hypoxic, non-inflamed "cold" state is difficult for many agents to accumulate and activate the immune system. Intrinsically, facultative anaerobic Salmonella VNP20009 target the tumor hypoxic areas, invade into tumor cells and exhibit an immune effect. Here we engineer the bacteria by decorating their surface with newly synthesized heptamethine cyanine dyes NHS-N782 and JQ-1 derivatives to obtain the biohybrid agent N-V-J, leading to the deep tumor targeted photothermal therapy and magnified immunotherapy. Due to the mitochondrial targeting capacity of NHS-N782, N-V-J becomes susceptive to the temperature rise when reaching tumors. This synergistic strategy promotes the systemic immunity by creating an inflamed "hot" tumor state from three different dimensions, which include the inherent immunogenicity of bacteria, the near-infrared laser triggered tumor antigens and the downregulation of PD-L1 expression. All these approaches result in effective and long-lasting T cell immune responses to prevent local and distant tumors for extended time. Leveraging the attenuated bacteria to transport dual drugs to the tumor tissues for self-synthetic vaccines provides a novel paradigm to enhance the bacteria-mediated cancer immunotherapy.

A Bioluminescent Probe for Detecting Norepinephrine in Vivo.

As a neurotransmitter, norepinephrine (NE) is critical for psychiatric conditions, neurodegenerative diseases, and pheochromocytoma. A real-time and noninvasive method for the detection of NE as a tracer to investigate the NE-relevant disease treatment process is urgently desirable. Herein, we successfully developed a turn-on NE bioluminescent probe (NBP), which was grounded on p-toluenethiol deprotectrf by nucleophilic substitution. Compared with other analytes, the NBP exhibited high sensitivity and selectivity in vitro. More importantly, the NBP provides a promising strategy for in vivo imaging of NE in living animals with noninvasive visualization and real-time features.

[Yishen Huoxue Decoction for infertility in elderly men with kidney deficiency and blood stasis].

OBJECTIVE: To observe the clinical effect of Yishen Huoxue Decoction on infertility in elderly men with kidney deficiency and blood stasis. METHODS: We randomly assigned 60 infertility male patients with kidney deficiency and blood stasis to two groups of an equal number to receive Yishen Huoxue Decoction, 1 dose a day, twice daily in the morning and evening (the observation group, aged ï¼»42.10 ± 3.82ï¼½ yr) or Shengjing Tablets (4 tablets once, tid) combined with L-carnitine oral solution at 10 ml, bid (the control group, aged ï¼»42.79 ± 3.56ï¼½ yr), both for 3 months. Before and after treatment, we obtained the semen parameters, levels of reproductive hormones and traditional Chinese medicine (TCM) clinical symptoms scores from the patients and compared them between the two groups. RESULTS: A total of 57 patients completed this clinical study, 29 in the observation group, with pregnancy achieved in 2 cases (6.90%), and 28 in the control group, with pregnancy achieved in 1 (3.57%). The patients in the observation group, compared with the controls, showed significantly higher total effectiveness (82.76% vs 71.43%, P < 0.05) and overall therapeutic efficacy on TCM syndromes (93.10% vs 85.71%, P < 0.05), as well as higher semen volume, sperm concentration, total sperm count, total sperm motility, percentages of progressively motile sperm (PMS) and morphologically normal sperm (MNS), and TCM clinical symptoms scores (all P < 0.05), but no statistically significant difference in the levels of reproductive hormones (E2, LH, PRL, T and FSH) (P > 0.05). Remarkable increases were observed in both the observation and control groups after medication in sperm concentration, total sperm count, total sperm motility, PMS, MNS, and TCM clinical symptom scores (all P < 0.05). CONCLUSIONS: Yishen Huoxue Decoction can significantly improve the semen quality and TCM clinical symptoms of elderly infertility males with kidney deficiency and blood stasis.

A bioluminescent probe for in vivo imaging of pyroglutamate aminopeptidase in a mouse model of inflammation.

Pyroglutamate aminopeptidase (PGP) specifically cleaves the peptide bond of pyroglutamic acid linked to the N-terminal end of a polypeptide or protein. Previous studies showed that PGP was associated with several physiological processes and diseases especially those involving inflammation. Utilizing a 'caging' strategy, we designed and synthesized a bioluminescence probe (PBL) with a limit-of-detection of 3.7 * 10-4 mU/mL. In vivo imaging in a mouse model of inflammatory liver disease revealed that the probe has excellent sensitivity and selectivity and provides a powerful tool for studying the physiological and pathological processes involving PGP.

Photoinduced Electron Transfer-Based Fluorescent Agonists for α1-Adrenergic Receptors Imaging.

The novel fluorescent agonists were discovered herein for α1-adrenergic receptors (α1-ARs) based on photoinduced electron transfer (PeT) off-on switch by conjugating the fluorophore 7-(diethylamino)coumarin-3-carboxylic acid with phenylephrine. After careful evaluation, these probes exhibited efficient binding affinity with α1-ARs and could be applied to selectively imaging α1-ARs or successfully tracing the dynamic process of α1-AR internalization in living cells. Meanwhile, a bioluminescence resonance energy transfer binding assay with these new probes has been well-established and applied. Therefore, these PeT-based on-off agonists may serve as powerful tools for the α1-AR-associated study during drug discovery.

Elevated miR-124-3p in the aging colon disrupts mucus barrier and increases susceptibility to colitis by targeting T-synthase.

The risk of colitis and colorectal cancer increases markedly throughout adult life, endangering the health and lives of elderly individuals. Previous studies have proposed that bacterial translocation and infection are the main risk factors for these diseases. Therefore, in the present study, we aimed to identify the underlying mechanism by focusing on the mucus barrier function and mucin-type O-glycosylation. We evaluated alterations in the colon mucus layer in 2-, 16-, and 24-month-old mice and aged humans. Aged colons showed defective intestinal mucosal barrier and changed mucus properties. The miR-124-3p expression level was significantly increased in the aged distal colonic mucosa, which was accompanied by an increase in pathogens and bacterial translocation. Meanwhile, T-synthase, the rate-limiting enzyme in O-glycosylation, displayed an age-related decline in protein expression. Further experiments indicated that miR-124-3p modulated O-glycosylation by directly targeting T-synthase. Moreover, young mice overexpressing miR-124-3p exhibited abnormal glycosylation, early-onset, and more severe colitis. These data suggest that miR-124-3p predisposes to senile colitis by reducing T-synthase, and the miR-124-3p/T-synthase/O-glycans axis plays an essential role in maintaining the physiochemical properties of colonic mucus and intestinal homeostasis.

Biological applications of a turn-on bioluminescent probe for monitoring sulfite oxidase deficiency in vivo.

Sulfites are widely used as preservative and antioxidant additives in food and drug. A non-invasive method for in vivo imaging of sulfite represents a powerful tool for estimating its potential effects in living organisms. Herein, we report the design, development, and application of sulfite bioluminescent probes (SBPs) for the analyte-specific detection of sulfite through sulfite-mediated intramolecular cleavage. Among them, SBP-1 exhibited the excellent responsivity, high selectivity and sensitivity. By taking advantage of this probe, the first in vivo imaging of sulfate was successfully carried out, not only to trace exogenous sulfite level in living animal, but also to investigate endogenous sulfite in a sulfite oxidase deficiency model.

Bioluminescence imaging of exogenous & endogenous cysteine in vivo with a highly selective probe.

Cysteine (Cys) is a semi-essential amino acid that exerts a vital role in numerous biological functions. A noninvasive method for in vivo imaging of cysteine could represent a valuable tool for research cysteine and its complex contributions in living organisms. Thus, we developed a turn-on bioluminescence probe (CBP) not only for detecting exogenous and endogenous cysteine in vitro and in vivo, but also for visualizing these cysteines in whole animal. The current applications may help shed light on the complex mechanisms of cysteine in miscellaneous physiological and pathological processes.

Discovery of Environment-Sensitive Fluorescent Agonists for α1-Adrenergic Receptors.

A series of novel fluorescent agonists were well developed herein with turn-on switch for α1-adrenergic receptors (α1-ARs) by conjugating the environment-sensitive fluorophore 4-chloro-7-nitrobenzoxadiazole with phenylephrine. Overall, these probes exhibited efficient binding and apparent fluorescence intensity changes (up to 10-fold) upon binding with α1-ARs. Moreover, these probes have been successfully applied for selectively imaging α1-ARs in the living cells. The dynamic process of α1-ARs internalization was traced successfully with these newly designed fluorescent agonists. Fluorescence polarization assay demonstrated specific interactions between these probes and α1-ARs. With these new probes, a bioluminescence resonance energy transfer binding assay has been well established and applied to the high-throughput screening of unlabeled α1-ARs agonist and antagonist. It is expected that these environment-sensitive fluorescent turn-on agonists may provide useful new tools in studying pharmacology and physiology of α1-ARs during drug discovery.

Lipopolysaccharide induces the early enhancement of mice colonic mucosal paracellular permeability mainly mediated by mast cells.

The alteration of intestinal mucosal barrier is considered to be the central pathophysiological process in response to gastrointestinal infections, and mucosal microstructural damage is a major factor for enhancing epithelial permeability in persistent bacterial infections. However, the mechanism involved in hyperpermeability in the early stage of acute bacterial infections is not fully understood. In the present study, fluorescein isothiocyanate-dextran across and transepithelial resistance measured in Ussing chambers were used to assess the intestinal paracellular permeability. Mast cell activation was evaluated by western blotting for the presence of tryptase released from mast cells. Serum levels of interleukin-6 were evaluated using enzyme-linked immunosorbent assay. Our results indicated that mast cells played a pivotal role in colonic mucosal hyperpermeability in wild type mice treated with lipopolysaccharide (LPS) for 2 h. And the effect of LPS was mainly dependent on mast cell degranulation, while no change in permeability was observed in the mast cell-deficient mice (Wads⁻/⁻) after LPS administration. No obvious changes of the mucosal structure including histomorphological architecture and expression of intercellular junction proteins were obtained in either wild type or Wads⁻/⁻ mice after LPS stimulation by hematoxylin and eosin staining, immunofluorescence staining and western blot analysis. Furthermore, the self-renewal of intestinal epithelia, detected by using proliferation marker 5'-bromo-2'-deoxyuridine, was not involved in increased permeability. Collectively, activation of mast cells induced by LPS mediated intestinal hyperpermeability in the initial stage, and played a crucial role in barrier dysfunction rather than mucosal microstructural damage in acute enterogenous bacterial infection.

Aging-dependent decrease in the numbers of enteric neurons, interstitial cells of Cajal and expression of connexin43 in various regions of gastrointestinal tract.

Aging is a significant risk factor for gastrointestinal dysmotility, but aging-associated differences between different organs and the exact time to start degenerating have remained obscure. Here we evaluated alterations of interstitial cells of Cajal, enteric neurons and connexin43 expression in the stomach, jejunum and colon in 2-, 12-, 16-, 20- and 24-month-old mice, as well as in aged human colon. Interstitial cells of Cajal, cholinergic and nitrergic neurons within the whole digestive tract were reduced over time, but their loss first appeared in stomach, then in intestine, helping to understand that gastric function was first impaired during aging. The decrease of connexin43 expression occurred before interstitial cells of Cajal and neurons loss, suggesting that connexin43 might be the major target influenced during senescence. Furthermore, changes in expressions of pro-inflammatory cytokines (tumour necrosis factor-α, interleukin-1ß, interleukin-6) and apoptosis-related proteins (B-cell lymphoma-2, caspase-3) which indicated "inflammaging", might contribute to the loss of enteric neurons and interstitial cells of Cajal in aged gastrointestinal tract. Our results provide possible therapeutic time window for beneficial intervention for geriatric patients with gastrointestinal motility disorders.