Associations of the Serum KL-6 with Severity and Prognosis in Patients with Acute Exacerbation of Chronic Obstructive Pulmonary Disease.

BACKGROUND: As a biomarker of alveolar-capillary basement membrane injury, Krebs von den Lungen-6 (KL-6) is involved in the occurrence and development of pulmonary diseases. However, the role of the KL-6 in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) has yet to be elucidated. This prospective study was designed to clarify the associations of the serum KL-6 with the severity and prognosis in patients with AECOPD. METHODS: This study enrolled 199 eligible AECOPD patients. Demographic data and clinical characteristics were recorded. Follow-up was tracked to evaluate acute exacerbation and death. The serum KL-6 concentration was measured via an enzyme-linked immunosorbent assay. RESULTS: Serum KL-6 level at admission was higher in AECOPD patients than in control subjects. The serum KL-6 concentration gradually elevated with increasing severity of AECOPD. Pearson and Spearman analyses revealed that the serum KL-6 concentration was positively correlated with the severity score, monocyte count and concentrations of C-reactive protein, interleukin-6, uric acid, and lactate dehydrogenase in AECOPD patients during hospitalization. A statistical analysis of long-term follow-up data showed that elevated KL-6 level at admission was associated with longer hospital stays, an increased risk of future frequent acute exacerbations, and increased severity of exacerbation in COPD patients. CONCLUSION: Serum KL-6 level at admission is positively correlated with increased disease severity, prolonged hospital stay and increased risk of future acute exacerbations in COPD patients. There are positive dose-response associations of elevated serum KL-6 with severity and poor prognosis in COPD patients. The serum KL-6 concentration could be a novel diagnostic and prognostic biomarker in AECOPD patients.

Structural study of the uPA-nafamostat complex reveals a covalent inhibitory mechanism of nafamostat.

Nafamostat mesylate (NM) is a synthetic compound that inhibits various serine proteases produced during the coagulation cascade and inflammation. Previous studies showed that NM was a highly safe drug for the treatment of different cancers, but the precise functions and mechanisms of NM are not clear. In this study, we determined a series of crystal structures of NM and its hydrolysates in complex with a serine protease (urokinase-type plasminogen activator [uPA]). These structures reveal that NM was cleaved by uPA and that a hydrolyzed product (4-guanidinobenzoic acid [GBA]) remained covalently linked to Ser195 of uPA, and the other hydrolyzed product (6-amidino-2-naphthol [6A2N]) released from uPA. Strikingly, in the inactive uPA (uPA-S195A):NM structure, the 6A2N side of intact NM binds to the specific pocket of uPA. Molecular dynamics simulations and end-point binding free-energy calculations show that the conf1 of NM (6A2N as P1 group) in the uPA-S195A:NM complex may be more stable than conf2 of NM (GBA as P1 group). Moreover, in the structure of uPA:NM complex, the imidazole group of His57 flips further away from Ser195 and disrupts the stable canonical catalytic triad conformation. These results not only reveal the inhibitory mechanism of NM as an efficient serine protease inhibitor but also might provide the structural basis for the further development of serine protease inhibitors.

Identification of small-molecule inhibitors of the DNA repair proteins RuvAB from Pseudomonas aeruginosa.

The Holliday junction (HJ) branch migrator RuvAB complex plays a fundamental role during homologous recombination and DNA damage repair, and therefore, is an attractive target for the treatment of bacterial pathogens. Pseudomonas aeruginosa (P. aeruginosa, Pa) is one of the most common clinical opportunistic bacterial pathogens, which can cause a series of life-threatening acute or chronic infections. Here, we performed a high throughput small-molecule screening targeting PaRuvAB using the FRET-based HJ branch migration assay. We identified that corilagin, bardoxolone methyl (BM) and 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SKQ1) could efficiently inhibit the branch migration activity of PaRuvAB, with IC50 values of 0.40 ± 0.04 µM, 0.38 ± 0.05 µM and 4.64 ± 0.27 µM, respectively. Further biochemical and molecular docking analyses demonstrated that corilagin directly bound to PaRuvB at the ATPase domain, and thus prevented ATP hydrolysis. In contrast, BM and SKQ1 acted through blocking the interactions between PaRuvA and HJ DNA. Finally, these compounds were shown to increase the susceptibility of P. aeruginosa to UV-C irradiation. Our work, for the first time, reports the small-molecule inhibitors of RuvA and RuvB from any species, providing valuable chemical tools to dissect the functional role of each individual protein in vivo.

Identification of Antithrombotic Natural Products Targeting the Major Substrate Binding Pocket of Protein Disulfide Isomerase.

Protein disulfide isomerase (PDI) is a vital oxidoreductase. Extracellular PDI promotes thrombus formation but does not affect physiological blood hemostasis. Inhibition of extracellular PDI has been demonstrated as a promising strategy for antithrombotic treatment. Herein, we focused on the major substrate binding site, a unique pocket in the PDI b' domain, and identified four natural products binding to PDI by combining virtual screening with tryptophan fluorescence-based assays against a customized natural product library. These hits all directly bound to the PDI-b' domain and inhibited the reductase activity of PDI. Among them, galangin showed the most prominent potency (5.9 µM) against PDI and as a broad-spectrum inhibitor for vascular thiol isomerases. In vivo studies manifested that galangin delayed the time of blood vessel occlusion in an electricity-induced mouse thrombosis model. Molecular docking and dynamics simulation further revealed that the hydroxyl-substituted benzopyrone moiety of galangin deeply inserted into the interface between the PDI-b' substrate-binding pocket and the a' domain. Together, these findings provide a potential antithrombotic drug candidate and demonstrate that the PDI b' domain is a critical domain for inhibitor development. Besides, we also report an innovative high-throughput screening method for the rapid discovery of PDI b' targeted inhibitors.

Film thickness measurement based on nonlinear phase analysis using a Linnik microscopic white-light spectral interferometer.

Based on white-light spectral interferometry and the Linnik microscopic interference configuration, the nonlinear phase components of the spectral interferometric signal were analyzed for film thickness measurement. The spectral interferometric signal was obtained using a Linnik microscopic white-light spectral interferometer, which includes the nonlinear phase components associated with the effective thickness, the nonlinear phase error caused by the double-objective lens, and the nonlinear phase of the thin film itself. To determine the influence of the effective thickness, a wavelength-correction method was proposed that converts the effective thickness into a constant value; the nonlinear phase caused by the effective thickness can then be determined and subtracted from the total nonlinear phase. A method for the extraction of the nonlinear phase error caused by the double-objective lens was also proposed. Accurate thickness measurement of a thin film can be achieved by fitting the nonlinear phase of the thin film after removal of the nonlinear phase caused by the effective thickness and by the nonlinear phase error caused by the double-objective lens. The experimental results demonstrated that both the wavelength-correction method and the extraction method for the nonlinear phase error caused by the double-objective lens improve the accuracy of film thickness measurements.

Effect of a comprehensive therapy plus gushenyutai plaster administered at guanyuan (CV 4) on male infertility associated with semen non-liquefaction.

OBJECTIVE: To observe the clinical effect of Gushenyutai plaster administered at the Guanyuan (CV 4) acupoint on male infertility associated with semen non-liquefaction. METHODS: Sixty-two male patients with infertility caused by semen non-liquefaction were randomized into a treatment and control group. The control group received comprehensive therapy, which included oral administration of clarithromycin sustained release tablets, hip bath with Zhongyaoxiaoyan granules, prostate massage, and transurethral microwave treatment. The treatment group was administered Gushenyutai plaster in addition to the comprehensive therapy. Both groups were treated for 8 weeks. After treatment, the clinical effect and pregnancy rate were assessed and compared between the two groups. RESULTS: The effective rate of the treatment group was significantly higher than that of the control group (96.77% vs 70.97%, P < 0.05) and the pregnancy rates of the treatment group and control groups were 38.71% and 16.13%, respectively (P < 0.05). CONCLUSION: The effect of Gushenyutai plaster plus comprehensive therapy was better than that of the comprehensive therapy alone on male infertility induced by semen non-liquefaction.

Walrycin B, as a novel separase inhibitor, exerts potent anticancer efficacy in a mouse xenograft model.

Proper chromosome segregation during cell division relies on the timely dissolution of chromosome cohesion. Separase (EC3.4.22.49), a cysteine protease, plays a critical role in mitosis by cleaving the kleisin subunit of cohesin, thereby presenting a promising target for cancer therapy. However, challenges in isolating active human separase suitable for high-throughput screening have limited the identification of effective inhibitors. Here, we conducted a high-throughput screening of small-molecule inhibitors using the protease domain of Chaetomium thermophilum separase (ctSPD), which not only shares significant sequence similarity with human separase but is also readily available. After conducting a primary screening of a library containing 9,172 compounds and subsequent validation using human separase, we identified walrycin B and its analogs, toxoflavin, 3-methyltoxoflavin, and 3-phenyltoxoflavin, as potent inhibitors of human separase. Subsequent microscale thermophoresis assays and molecular dynamics simulations revealed that walrycin B binds to the active site of separase and competes with substrates for binding. Additionally, cell-based studies showed that walrycin B and its analogs effectively induce cell cycle arrest at the M phase, activate apoptosis, and ultimately lead to cell death in mitosis. Finally, in a mouse xenograft model, walrycin B exhibited significant antitumor efficacy with minimal side effects. Together, these findings highlight the therapeutic potential of walrycin B for cancer treatment and its utility as a chemical tool in future studies involving separase.

Protonation State-Induced Unfolding of Protein Secondary Structure in the Gas Phase.

The combination of infrared spectroscopy (IR) and ion mobility mass spectrometry (IM-MS) has revealed that protein secondary structures are retained upon transformation from aqueous solution to the gas phase under gentle conditions. Yet the details about where and how these structural elements are embedded in the gas phase remain elusive. In this study, we employ long time scale molecular dynamics (MD) simulations to examine the extent to which proteins retain their solution structures and the impact of protonation state on the stability of secondary structures in the gas phase. Our investigation focuses on two well-studied proteins, myoglobin and ß-lactoglobulin, representing typical helical and ß-sheet proteins, respectively. Our simulations accurately reproduce the experimental collision cross section (CCS) data measured by IM-MS. Based on accurately reproducing previous experimental collision cross section data and dominant secondary structural species obtained from IM-MS and IR, we confirm that both proteins largely retain their native secondary structural components upon passing from aqueous solution to the gas phase. However, we observe significant reductions in secondary structure contents (19.2 ± 1.2% for myoglobin and 7.3 ± 0.6% for ß-lactoglobulin) in specific regions predominantly composed of ionizable residues. Further mechanistic analysis suggests that alterations in protonation states of these residues after phase transition induce changes in their local interaction networks and backbone dihedral angles, which potentially promote the unfolding of secondary structures in the gas phase. We anticipate that similar protonation state induced unfolding may be observed in other proteins possessing distinct secondary structures. Further studies on a broader array of proteins will be essential to refine our understanding of protein structural behavior during the transition to the gas phase.

Multifunctional metal-coordinated Co-assembled carrier-free nanoplatform based on dual-drugs for ferroptosis-mediated cocktail therapy of hepatocellular carcinoma growth and metastasis.

The heterogeneity of hepatocellular carcinoma (HCC) and the complexity of the tumor microenvironment (TME) pose challenges to efficient drug delivery and the antitumor efficacy of combined or synergistic therapies. Herein, a metal-coordinated carrier-free nanodrug (named as USFe3+ LA NPs) was developed for ferroptosis-mediated multimodal synergistic anti-HCC. Natural product ursolic acid (UA) was incorporated to enhance the sensitivity of tumor cells to sorafenib (SRF). Surface decoration of cell penetration peptide and epithelial cell adhesion molecule aptamer facilitated the uptake of USFe3+ LA NPs by HepG2 cells. Meanwhile, Fe3+ ions could react with intracellular hydrogen peroxide, generating toxic hydroxyl radical (·OH) for chemodynamical therapy (CDT) and amplified ferroptosis by cystine/glutamate antiporter system (System Xc-), which promoted the consumption of glutathione (GSH) and inhibited the expression of glutathione peroxidase 4 (GPX4). Notably, these all-in-one nanodrugs could inhibit tumor metastasis and induced immunogenic cell death (ICD). Last but not least, the nanodrugs demonstrated favorable biocompatibility, augmenting the immune response against the programmed death-ligand 1 (PD-L1) by increasing cytotoxic T cell infiltration. In vivo studies revealed significant suppression of tumor growth and distant metastasis. Overall, our work introduced a novel strategy for applications of metal-coordinated co-assembled carrier-free nano-delivery system in HCC combination therapy, especially in the realms of cancer metastasis prevention and immunotherapy.

Exploring the mechanism of photosensitizer conjugation on membrane perturbation of antimicrobial peptide: A multiscale molecular simulation study.

Antimicrobial peptides (AMPs) exert their biological functions by perturbation with cellular membrane. Conjugation of AMPs with photosensitizer (PS) is a promising strategy for enhancing the efficacy and reducing systemic toxicity of AMPs. However, it is still elusive how the conjugated PS impacts the perturbation of AMPs on cell membrane from molecular level. Here, we addressed this issue by a multiscale computational strategy on pyropheophorbide-a (PPA) conjugated K6L9 (PPA-K6L9), a PS-AMP conjugate developed by us previously. Our atomistic molecular dynamics (MD) simulations revealed that the porphyrin moiety of PPA enhanced the stability of the conjugate in a lipid bilayer membrane model. Moreover, such moiety also maintained the amphipathic structure of K6L9, which is crucial for membrane pore formation. Coarse-grained MD simulations further showed that the conjugates aggregated in membrane environment and formed more stable toroidal pores with respect to K6L9 alone, suggesting the conjugation of PPA may enhance the membrane-disruption activity of K6L9. Consistent with this, our cellular experiments confirmed that PPA-K6L9 was more toxic to 4 T1 tumor cells than K6L9. This study provides insights into the mechanism by which PS-AMP conjugates disrupt cellular membranes and could aid in the design of more potent AMP conjugates.

In silico-based screening of natural products as potential inhibitors of SARS-CoV-2 macrodomain 1.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide has led to over 600 million cases of coronavirus disease 2019 (COVID-19). Identifying effective molecules that can counteract the virus is imperative. SARS-CoV-2 macrodomain 1 (Mac1) represents a promising antiviral drug target. In this study, we predicted potential inhibitors of SARS-CoV-2 Mac1 from natural products using in silico-based screening. Based on the high-resolution crystal structure of Mac1 bound to its endogenous ligand ADP-ribose (ADPr), we first performed a docking-based virtual screening of Mac1 inhibitors against a natural product library and obtained five representative compounds (MC1-MC5) by clustering analysis. All five compounds were stably bound to Mac1 during 500 ns long molecular dynamics simulations. The binding free energy of these compounds to Mac1 was calculated using molecular mechanics generalized Born surface area and further refined with localized volume-based metadynamics. The results demonstrated that both MC1 (-9.8 ± 0.3 kcal/mol) and MC5 (-9.6 ± 0.3 kcal/mol) displayed more favorable affinities to Mac1 with respect to ADPr (-8.9 ± 0.3 kcal/mol), highlighting their potential as potent SARS-CoV-2 Mac1 inhibitors. Overall, this study provides potential SARS-CoV-2 Mac1 inhibitors, which may pave the way for developing effective therapeutics for COVID-19.Communicated by Ramaswamy H. Sarma.

In silico screening of natural products as uPAR inhibitors via multiple structure-based docking and molecular dynamics simulations.

Cancer remains one of the most pressing challenges to global healthcare, exerting a significant impact on patient life expectancy. Cancer metastasis is a critical determinant of the lethality and treatment resistance of cancer. The urokinase-type plasminogen activator receptor (uPAR) shows great potential as a target for anticancer and antimetastatic therapies. In this work, we aimed to identify potential uPAR inhibitors by structural dynamics-based virtual screenings against a natural product library on four representative apo-uPAR structural models recently derived from long-timescale molecular dynamics (MD) simulations. Fifteen potential inhibitors (NP1-NP15) were initially identified through molecular docking, consensus scoring, and visual inspection. Subsequently, we employed MD-based molecular mechanics-generalized Born surface area (MM-GBSA) calculations to evaluate their binding affinities to uPAR. Structural dynamics analyses further indicated that all of the top 6 compounds exhibited stable binding to uPAR and interacted with the critical residues in the binding interface between uPAR and its endogenous ligand uPA, suggesting their potential as uPAR inhibitors by interrupting the uPAR-uPA interaction. We finally predicted the ADMET properties of these compounds. The natural products NP5, NP12, and NP14 with better binding affinities to uPAR than the uPAR inhibitors previously discovered by us were proven to be potentially orally active in humans. This work offers potential uPAR inhibitors that may contribute to the development of novel effective anticancer and antimetastatic therapeutics.Communicated by Ramaswamy H. Sarma.

Molecular and Supramolecular Approach to Highly Photocytotoxic Phthalocyanines with Dual Cell Uptake Pathways and Albumin-Enhanced Tumor Targeting.

Phototherapy for non-invasive cancer treatment has been extensively studied. An urgent challenge in phototherapy application is to fabricate appropriate targeted agents to achieve efficient therapeutic effect. Herein, a molecular and supramolecular approach for targeting phototherapy was reasonably designed and realized through the axial sulfonate modification of silicon(IV) phthalocyanines (Pcs), followed by supramolecular interaction with albumin. This approach can not only improve the photoactivities (e.g., fluorescence emission and reactive oxygen species production) of the Pcs but also enhance their tumor targeting. Most importantly, one of the deigned Pcs (4) can target HepG2 cells through dual cell pathways, leading to an extremely high phototoxicity with an EC50 (i.e., concentration of Pcs to kill 50% of cells under light irradiation) value of 2.0 nM. This finding presents a feasible strategy to realize efficient targeting phototherapy.