Synthetic Derivatives of Natural ent-Kaurane Atractyligenin Disclose Anticancer Properties in Colon Cancer Cells, Triggering Apoptotic Cell Demise.

The antitumor activity of different ent-kaurane diterpenes has been extensively studied. Several investigations have demonstrated the excellent antitumor activity of synthetic derivatives of the diterpene atractyligenin. In this research, a series of new synthetic amides and their 15,19-di-oxo analogues obtained from atractyligenin by modifying the C-2, C-15, and C-19 positions were designed in order to dispose of a set of derivatives with different substitutions at the amidic nitrogen. Using different concentrations of the obtained compounds (10-300 µM) a reduction in cell viability of HCT116 colon cancer cells was observed at 48 h of treatment. All the di-oxidized compounds were more effective than their alcoholic precursors. The di-oxidized compounds had already reduced the viability of two colon cancer cells (HCT116 and Caco-2) at 24 h when used at low doses (2.5-15 µM), while they turned out to be poorly effective in differentiated Caco-2 cells, a model of polarized enterocytes. The data reported here provide evidence that di-oxidized compounds induced apoptotic cell death, as demonstrated by the appearance of condensed and fragmented DNA in treated cells, as well as the activation of caspase-3 and fragmentation of its target PARP-1.

Binding and unbinding of potent melatonin receptor ligands: Mechanistic simulations and experimental evidence.

The labeled ligand commonly employed in competition binding studies for melatonin receptor ligands, 2-[125I]iodomelatonin, showed slow dissociation with different half-lives at the two receptor subtypes. This may affect the operational measures of affinity constants, which at short incubation times could not be obtained in equilibrium conditions, and structure-activity relationships, as the Ki values of tested ligands could depend on either interaction at the binding site or the dissociation path. To address these issues, the kinetic and saturation binding parameters of 2-[125I]iodomelatonin as well as the competition constants for a series of representative ligands were measured at a short (2 h) and a long (20 h) incubation time. Concurrently, we simulated by molecular modeling the dissociation path of 2-iodomelatonin from MT1 and MT2 receptors and investigated the role of interactions at the binding site on the stereoselectivity observed for the enantiomers of the subtype-selective ligand UCM1014. We found that equilibrium conditions for 2-[125I]iodomelatonin binding can be reached only with long incubation times, particularly for the MT2 receptor subtype, for which a time of 20 h approximates this condition. On the other hand, measured Ki values for a set of ligands including agonists, antagonists, nonselective, and subtype-selective compounds were not significantly affected by the length of incubation, suggesting that structure-activity relationships based on data collected at shorter time reflect different interactions at the binding site. Molecular modeling simulations evidenced that the slower dissociation of 2-iodomelatonin from the MT2 receptor can be related to the restricted mobility of a gatekeeper tyrosine along a lipophilic path from the binding site to the membrane bilayer. The enantiomers of the potent, MT2-selective agonist UCM1014 were separately synthesized and tested. Molecular dynamics simulations of the receptor-ligand complexes provided an explanation for their stereoselectivity as due to the preference shown by the eutomer at the binding site for the most abundant axial conformation adopted by the ligand in solution. These results suggest that, despite the slow-binding kinetics occurring for the labeled ligand, affinity measures at shorter incubation times give robust results consistent with known structure-activity relationships and with interactions taken at the receptor binding site.

A Novel Approach for Glioblastoma Treatment by Combining Apoptosis Inducers (TMZ, MTX, and Cytarabine) with E.V.A. (Eltanexor, Venetoclax, and A1210477) Inhibiting XPO1, Bcl-2, and Mcl-1.

Adjuvant treatment for Glioblastoma Grade 4 with Temozolomide (TMZ) inevitably fails due to therapeutic resistance, necessitating new approaches. Apoptosis induction in GB cells is inefficient, due to an excess of anti-apoptotic XPO1/Bcl-2-family proteins. We assessed TMZ, Methotrexate (MTX), and Cytarabine (Ara-C) (apoptosis inducers) combined with XPO1/Bcl-2/Mcl-1-inhibitors (apoptosis rescue) in GB cell lines and primary GB stem-like cells (GSCs). Using CellTiter-Glo® and Caspase-3 activity assays, we generated dose-response curves and analyzed the gene and protein regulation of anti-apoptotic proteins via PCR and Western blots. Optimal drug combinations were examined for their impact on the cell cycle and apoptosis induction via FACS analysis, paralleled by the assessment of potential toxicity in healthy mouse brain slices. Ara-C and MTX proved to be 150- to 10,000-fold more potent in inducing apoptosis than TMZ. In response to inhibitors Eltanexor (XPO1; E), Venetoclax (Bcl-2; V), and A1210477 (Mcl-1; A), genes encoding for the corresponding proteins were upregulated in a compensatory manner. TMZ, MTX, and Ara-C combined with E, V, and A evidenced highly lethal effects when combined. As no significant cell death induction in mouse brain slices was observed, we conclude that this drug combination is effective in vitro and expected to have low side effects in vivo.

Oleoylethanolamide and Palmitoylethanolamide Enhance IFNß-Induced Apoptosis in Human Neuroblastoma SH-SY5Y Cells.

Oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) are endogenous lipids that act as agonists of the peroxisome proliferator-activated receptor α (PPARα). Recently, an interest in the role of these lipids in malignant tumors has emerged. Nevertheless, the effects of OEA and PEA on human neuroblastoma cells are still not documented. Type I interferons (IFNs) are immunomodulatory cytokines endowed with antiviral and anti-proliferative actions and are used in the treatment of various pathologies such as different cancer forms (i.e., non-Hodgkin's lymphoma, melanoma, leukemia), hepatitis B, hepatitis C, multiple sclerosis, and many others. In this study, we investigated the effect of OEA and PEA on human neuroblastoma SH-SY5Y cells treated with IFNß. We focused on evaluating cell viability, cell proliferation, and cell signaling. Co-exposure to either OEA or PEA along with IFNß leads to increased apoptotic cell death marked by the cleavage of caspase 3 and poly-(ADP ribose) polymerase (PARP) alongside a decrease in survivin and IKBα levels. Moreover, we found that OEA and PEA did not affect IFNß signaling through the JAK-STAT pathway and the STAT1-inducible protein kinase R (PKR). OEA and PEA also increased the phosphorylation of p38 MAP kinase and programmed death-ligand 1 (PD-L1) expression both in full cell lysate and surface membranes. Furthermore, GW6471, a PPARα inhibitor, and the genetic silencing of the receptor were shown to lower PD-L1 and cleaved PARP levels. These results reveal the presence of a novel mechanism, independent of the IFNß-prompted pathway, by which OEA and PEA can directly impair cell survival, proliferation, and clonogenicity through modulating and potentiating the intrinsic apoptotic pathway in human SH-SY5Y cells.

Using part of the initial analgesic dose as the epidural test dose did not delay the onset of labor analgesia: a randomized controlled clinical trial.

BACKGROUND: Epidural test dose for labor analgesia is controversial and varies widely in clinical practice. It is currently unclear whether using a portion of the initial dose for analgesia as the test dose delays the onset time of analgesia, compared to the traditional test dose. METHODS: One hundred and twenty-six parturients who chose epidural analgesia during labor were randomly assigned to two groups. The first dose in group L was 3 ml 1.5% lidocaine, and in the RF group was 10 ml 0.1% ropivacaine combined with 2 µg/ml fentanyl. After 3 min of observation, both groups received 8 ml 0.1% ropivacaine combined with 2 µg/ml fentanyl. The onset time of analgesia, motor and sensory blockade level, numerical pain rating scale, patient satisfaction score, and side effects were recorded. RESULTS: The onset time of analgesia in group RF was similar to that in group L (group RF vs group L, 7.0 [5.0-9.0] minutes vs 8.0 [5.0-11.0] minutes, p = 0.197). The incidence of foot numbness (group RF vs group L, 34.9% vs 57.1%, p = 0.020) and foot warming (group RF vs group L, 15.9% vs 47.6%, p < 0.001) in group RF was significantly lower than that in group L. There was no difference between the two groups on other outcomes. CONCLUSIONS: Compared with 1.5% lidocaine 3 ml, 0.1% ropivacaine 10 ml combined with 2 µg/ml fentanyl as an epidural test dose did not delay the onset of labor analgesia, and the side effects were slightly reduced. CLINICAL TRIAL REGISTRATION: http://www.chictr.org.cn (ChiCTR2100043071).

A randomized trial to assess the acceleration of viral clearance by the combination Favipiravir/Ivermectin/Niclosamide in mild-to-moderate COVID-19 adult patients (FINCOV).

BACKGROUND: The efficacy of the viral clearance and clinical outcomes of favipiravir (FPV) in outpatients being treated for coronavirus disease 2019 (COVID-19) is unclear. Ivermectin (IVM), niclosamide (NCL), and FPV demonstrated synergistic effects in vitro for exceed 78% inhibiting severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) replication. METHODS: A phase 2, open-label, 1:1, randomized, controlled trial was conducted on Thai patients with mild-to-moderate COVID-19 who received either combination FPV/IVM/NCL therapy or FPV alone to assess the rate of viral clearance among individuals with mild-to-moderate COVID-19. RESULTS: Sixty non-high-risk comorbid patients with mild-to-moderate COVID-19 were randomized; 30 received FPV/IVM/NCL, and 30 received FPV alone. Mixed-effects multiple linear regression analysis of the cycle threshold value from SARS-CoV-2 PCR demonstrated no statistically significant differences in viral clearance rates between the combined FPV/IVM/NCL therapy group and the FPV-alone group. World Health Organization Clinical Progression scores and symptomatic improvement did not differ between arms on days 3, 6, and 10, and no adverse events were reported. No patients required hospitalization, intensive care unit admission, or supplemental oxygen or died within 28 days. C-reactive protein on day 3 was lower in the FPV/IVM/NCL group. CONCLUSION: Viral clearance rates did not differ significantly between the FPV/IVM/NCL combination therapy and FPV-alone groups of individuals with mild-to-moderate COVID-19, although the combined regimen demonstrated a synergistic effect in vitro. No discernible clinical benefit was observed. Further research is required to explore the potential benefits of FVP beyond its antiviral effects. TRIAL REGISTRATION: TCTR20230403007, Registered 3 April 2023 - Retrospectively registered,https://trialsearch.who.int/Trial2.aspx?TrialID=TCTR20230403007.

HYSCORE and QM/MM Studies of Second Sphere Variants of the Type 1 Copper Site in Azurin: Influence of Mutations on the Hyperfine Couplings of Remote Nitrogens.

Secondary coordination sphere (SCS) interactions have been shown to play important roles in tuning reduction potentials and electron transfer (ET) properties of the Type 1 copper proteins, but the precise roles of these interactions are not fully understood. In this work, we examined the influence of F114P, F114N, and N47S mutations in the SCS on the electronic structure of the T1 copper center in azurin (Az) by studying the hyperfine couplings of (i) histidine remote Nε nitrogens and (ii) the amide Np using the two-dimensional (2D) pulsed electron paramagnetic resonance (EPR) technique HYSCORE (hyperfine sublevel correlation) combined with quantum mechanics/molecular mechanics (QM/MM) and DLPNO-CCSD calculations. Our data show that some components of hyperfine tensor and isotropic coupling in N47SAz and F114PAz (but not F114NAz) deviate by up to ∼±20% from WTAz, indicating that these mutations significantly influence the spin density distribution between the CuII site and coordinating ligands. Furthermore, our calculations support the assignment of Np to the backbone amide of residue 47 (both in Asn and Ser variants). Since the spin density distributions play an important role in tuning the covalency of the Cu-Scys bond of Type 1 copper center that has been shown to be crucial in controlling the reduction potentials, this study provides additional insights into the electron spin factor in tuning the reduction potentials and ET properties.

3D amide proton transfer-weighted imaging may be useful for diagnosing early-stage breast cancer: a prospective monocentric study.

BACKGROUND: We investigated the value of three-dimensional amide proton transfer-weighted imaging (3D-APTWI) in the diagnosis of early-stage breast cancer (BC) and its correlation with the immunohistochemical characteristics of malignant lesions. METHODS: Seventy-eight women underwent APTWI and dynamic contrast-enhanced (DCE)-MRI. Pathological results were categorized as either benign (n = 43) or malignant (n = 37) lesions. The parameters of APTWI and DCE-MRI were compared between the benign and malignant groups. The diagnostic value of 3D-APTWI was evaluated using the area under the receiver operating characteristic curve (ROC-AUC) to establish a diagnostic threshold. Pearson's correlation was used to analyze the correlation between the magnetization transfer asymmetry (MTRasym) and immunohistochemical characteristics. RESULTS: The MTRasym and time-to-peak of malignancies were significantly lower than those of benign lesions (all p < 0.010). The volume transfer constant, rate constant, and wash-in and wash-out rates of malignancies were all significantly greater than those of benign lesions (all p < 0.010). ROC-AUCs of 3D-APTWI, DCE-MRI, and 3D-APTWI+DCE to differential diagnosis between early-stage BC and benign lesions were 0.816, 0.745, and 0.858, respectively. Only the difference between AUCAPT+DCE and AUCDCE was significant (p < 0.010). When a threshold of MTRasym for malignancy for 2.42%, the sensitivity and specificity of 3D-APTWI for BC diagnosis were 86.5% and 67.6%, respectively; MTRasym was modestly positively correlated with pathological grade (r = 0.476, p = 0.003) and Ki-67 (r = 0.419, p = 0.020). CONCLUSIONS: 3D-APTWI may be used as a supplementary method for patients with contraindications of DCE-MRI. MTRasym can imply the proliferation activities of early-stage BC. RELEVANCE STATEMENT: 3D-APTWI can be an alternative diagnostic method for patients with early-stage BC who are not suitable for contrast injection. KEY POINTS: • 3D-APTWI reflects the changes in the microenvironment of early-stage breast cancer. • Combined 3D-APTWI is superior to DCE-MRI alone for early-stage breast cancer diagnosis. • 3D-APTWI improves the diagnostic accuracy of early-stage breast cancer.

Discovery of new N-Phenylamide Isoxazoline derivatives with high insecticidal activity and reduced honeybee toxicity.

Isoxazoline is a novel structure with strong potential for controlling agricultural insect pests, but its high toxicity to honeybees limits its development in agriculture. Herein, a series of N-phenylamide isoxazoline derivatives with low honeybee toxicity were designed and synthesized using the intermediate derivatization method. Bioassay results showed that these compounds exhibited good insecticidal activity. Compounds 3b and 3f showed significant insecticidal effects against Plutella xylostella (P. xylostella) with median lethal concentrations (LC50) of 0.06 and 0.07 mg/L, respectively, comparable to that of fluralaner (LC50 = 0.02 mg/L) and exceeding that of commercial insecticide fluxametamide (LC50 = 0.52 mg/L). It is noteworthy that the acute honeybee toxicities of compounds 3b and 3f (LD50 = 1.43 and 1.63 µg/adult, respectively) were significantly reduced to 1/10 of that of fluralaner (LD50 = 0.14 µg/adult), and were adequate or lower than that of fluxametamide (LD50 = 1.14 µg/adult). Theoretical simulation using molecular docking indicates that compound 3b has similar binding modes with fluralaner and a similar optimal docking pose with fluxametamide when binding to the GABA receptor, which may contribute to its potent insecticidal activity and relatively low toxicity to honey bees. This study provides compounds 3b and 3f as potential new insecticide candidates and provides insights into the development of new isoxazoline insecticides exhibiting both high efficacy and environmental safety.

Amide proton transfer weighted contrast has diagnostic capacity in detecting diabetic foot: an MRI-based case-control study.

Objective: To evaluate the role of foot muscle amide proton transfer weighted (APTw) contrast and tissue rest perfusion in quantifying diabetic foot (DF) infection and its correlation with blood parameters. Materials and methods: With approval from an ethical review board, this study included 40 diabetes mellitus (DM) patients with DF and 31 DM patients without DF or other lower extremity arterial disease. All subjects underwent MRI, which included foot sagittal APTw and coronal arterial spin labeling (ASL) imaging. The normalized MTRasym (3.5 ppm) and the ratio of blood flow (rBF) in rest status of the affected side lesions to the non-affected contralateral side were determined. The inter-group differences of these variables were evaluated. Furthermore, the association between normalized MTRasym (3.5 ppm), rBF, and blood parameters [fasting blood glucose (FBG), glycosylated hemoglobin content, C-reactive protein, neutrophil percentage, and white blood cell count] was explored. Using an ROC curve, the diagnostic capacity of normalized MTRasym (3.5 ppm), BF, and blood biochemical markers in differentiating with or without DF in DM was assessed. Results: In the DF group, MTRasym (3.5 ppm) and BF in lesion and normalized MTRasym (3.5 ppm) were higher than those in the control group (p < 0.05). In addition, correlations were identified between normalized MTRasym (3.5 ppm) and blood parameters, such as C-reactive protein, glycosylated hemoglobin content, FBG, neutrophil ratio, and white blood cell (p < 0.001). Meanwhile, association between BF in lesion and blood parameters, such as C-reactive protein, neutrophil percentage, and FBG (p < 0.01). AUC of normalized MTRasym (3.5 ppm) in identifying with/without DF in patients with DM is 0.986 (95% CI, 0.918-1.00) with the sensitivity of 97.22% and the specificity of 100%. Conclusion: Normalized MTRasym (3.5 ppm) and the BF in lesion may be treated as a safer and more convenient new indicator to evaluate the tissue infection without using a contrast agent, which may be useful in monitoring and preoperatively assessing DF patients with renal insufficiency.

Harnessing potential of avian eggshell membrane derived collagen hydrolysate for bone tissue regeneration.

BACKGROUND: Natural bone grafts are the highly preferred materials for restoring the lost bone, while being constrained of donor availability and risk of disease transmission. As a result, tissue engineering is emerging as an efficacious and competitive technique for bone repair. Bone tissue engineering (TE) scaffolds to support bone regeneration and devoid of aforesaid limitations are being vastly explored and among these the avian eggshell membrane has drawn attention for TE owing to its low immunogenicity, similarity with the extracellular matrix, and easy availability. METHODOLOGY AND RESULTS: In this study, the development of bone ingrowth support system from avian eggshell membrane derived collagen hydrolysates (Col-h) is reported. The hydrolysate, cross-linked with glutaraldehyde, was developed into hydrogels with poly-(vinyl alcohol) (PVA) by freeze-thawing and further characterized with ATR-FTIR, XRD, FESEM. The biodegradability, swelling, mechanical, anti-microbial, and biocompatibility evaluation were performed further for the suitability in bone regeneration. The presence of amide I, amide III, and -OH functional groups at 1639 cm- 1,1264 cm- 1, and 3308 cm- 1 respectively and broad peak between 16°-21° (2θ) in XRD data reinstated the composition and form. CONCLUSIONS: The maximum ratio of Col-h/PVA that produced well defined hydrogels was 50:50. Though all the hydrogel matrices alluded towards their competitive attributes and applicability towards restorative bone repair, the hydrogel with 40:60 ratios showed better mechanical strength and cell proliferation than its counterparts. The prominent E. coli growth inhibition by the hydrogel matrices was also observed, along with excellent biocompatibility with MG-63 osteoblasts. The findings indicate strongly the promising application of avian eggshell-derived Col-h in supporting bone regeneration.

Risk factors and early preventive measures for long COVID in non-hospitalized patients: analysis of a large cohort in the United Arab Emirates.

OBJECTIVES: Long COVID is characterized by persistent symptoms lasting for 4 weeks or more following the acute infection with SARS-CoV-2. Risk factors for long COVID and the impact of pre-COVID vaccination and treatment during acute COVID-19 remain uncertain. This study aimed to investigate patient-specific factors associated with long COVID in a large cohort of non-hospitalized adult patients with mild to moderate COVID-19 in Dubai. STUDY DESIGN: Cohort study. METHODS: The study included 28,375 non-hospitalized adult patients diagnosed with mild to moderate COVID-19 between January 1, 2021, and September 31, 2022, in Dubai, who were followed up for 90 days. The presence of long COVID symptoms was documented by physicians during patient visits to the family medicine department. Furthermore, long COVID-related risk factors were collected and analyzed, including patient demographics, comorbidities, pre-COVID vaccination status, and the COVID-related treatments received during the acute phase of the illness. Cox proportional hazard models were applied for the statistical analysis. RESULTS: Among the cohort, 2.8% of patients experienced long COVID symptoms during the 90-day follow-up. Patients with long COVID tended to be younger, female, and of Caucasian race. Common symptoms included fatigue, muscle pain, respiratory symptoms, abdominal and neurological symptoms, allergic reactions, skin rashes, and hair loss. Risk factors for long COVID were identified as diabetes mellitus, asthma, and Vitamin D deficiency. Females and Caucasians had a higher risk of long COVID during the pre-Omicron period compared to the Omicron period. Pre-COVID vaccination was associated with a reduced risk of long COVID in all patient subgroups. Treatment with favipiravir or sotrovimab during the acute phase of COVID-19 was linked to a decreased risk of long COVID, although favipiravir showed limited effectiveness in the high-risk group. CONCLUSION: This study contributes to the existing knowledge by identifying risk factors for long COVID among non-hospitalized patients and emphasizing the potential benefits of pre-COVID vaccination and timely treatment.

Comparative evaluation of neonicotinoids and their metabolites-induced oxidative stress in carp primary leukocytes and CLC cells.

Neonicotinoids (NEOs) have been designed to act selectively on insect nicotinic acetylcholine receptors (nAChRs). However, nAChRs are also expressed in vertebrate immune cells, so NEOs may interfere with the immune system in exposed non-target animals. The present study shows that NEOs: imidacloprid and thiacloprid, and their main metabolites: desnitro-imidacloprid and thiacloprid amide, at sub-micromolar concentrations ranging from 2.25 to 20 µM, affect the immune cells of fish. This was found both in primary cultures of leukocytes isolated from the carp head kidney and in the continuous adherent carp monocyte/macrophage cell line. Moreover, the results revealed that the studied pesticides and metabolites generate oxidative stress in carp immune cells and that this is one of the most important mechanisms of neonicotinoid immunotoxicity. Significant increases were observed in the formation of ROS and malondialdehyde (MDA). The antioxidant status alteration was linked with decrease in antioxidant enzyme activity: superoxide dismutase (SOD), catalase (CAT), and non-enzymatic antioxidant glutathione (GSH). Importantly, the metabolites: desnitro-imidacloprid and thiacloprid amide showed significantly higher cytotoxicity towards fish leukocytes than their parent compounds, imidacloprid and thiacloprid, which emphasizes the importance of including intermediate metabolites in toxicology studies.

[Chinese expert consensus on iruplinalkib for the treatment of locally advanced or metastatic ALK-positive non-small cell lung cancer (2024 edition)].

Anaplastic lymphoma kinase (ALK) fusion gene is one of the most common driver gene in non-small cell lung cancer (NSCLC). Epidemiological data showed that ALK gene fusion is detected in 9.06% of Chinese advanced NSCLC patients. ALK-tyrosine kinase inhibitors (TKIs) have become the standard treatment for advanced NSCLC patients with ALK gene fusion. Seven different ALK-TKIs have been approved by the National Medical Products Administration (NMPA) of China, including crizotinib, ceritinib, alectinib, ensartinib, brigatinib, lorlatinib, and iruplinalkib. Iruplinalkib is a novel new-generation ALK-TKI independently developed in China. On June 27, 2023, the NMPA approved iruplinalkib for the treatment of locally advanced or metastatic ALK-positive NSCLC patients whose disease has progressed after previous treatment with crizotinib or who are intolerant to crizotinib. On January 16, 2024, the NMPA approved iruplinalkib for the first-line treatment of locally advanced or metastatic ALK-positive NSCLC patients. In order to better understand the efficacy and safety of iruplinalkib, and facilitate more rationally clinical application of iruplinalkib, the Medical Oncology Branch of China International Exchange and Promotive Association for Medical and Health Care and the Chinese Association for Clinical Oncologists co-organized experts to compile the "Chinese expert consensus on iruplinalkib for the treatment of locally advanced or metastatic ALK-positive non-small cell lung cancer (2024 edition)".

High anti Diastereoselectivity in a Tandem Oxyhomologation-Coupling Protocol for the Preparation of Amides and Peptides Incorporating α-Hydroxy ß-Amino Acids.

The one-pot MAC (Masked Acyl Cyanide) reaction is used to perform the tandem oxyhomologation reaction of N,N-dibenzyl-l-phenylalaninal and coupling with nitrogen nucleophiles to provide a wide selection of amide and peptide derivatives of (2S,3S)-allophenylnorstatin in generally good yields and with high anti selectivity, often with dr >98:2. The procedure works equally well with other selected N,N-dibenzyl α-amino aldehydes, and is used to achieve a very short synthesis of (2S,3S,S)-epibestatin.

Radical SAM Enzyme PylB Generates a Lysyl Radical Intermediate in the Biosynthesis of Pyrrolysine by Using SAM as a Cofactor.

Pyrrolysine, the 22nd amino acid encoded by the natural genetic code, is essential for methanogenic archaea to catabolize methylamines into methane. The structure of pyrrolysine consists of a methylated pyrroline carboxylate that is linked to the ε-amino group of the l-lysine via an amide bond. The biosynthesis of pyrrolysine requires three enzymes: PylB, PylC, and PylD. PylB is a radical S-adenosyl-l-methionine (SAM) enzyme and catalyzes the first biosynthetic step, the isomerization of l-lysine into methylornithine. PylC catalyzes an ATP-dependent ligation of methylornithine and a second l-lysine to form l-lysine-Nε-methylornithine. The last biosynthetic step is catalyzed by PylD via oxidation of the PylC product to form pyrrolysine. While enzymatic reactions of PylC and PylD have been well characterized by X-ray crystallography and in vitro studies, mechanistic understanding of PylB is still relatively limited. Here, we report the first in vitro activity of PylB to form methylornithine via the isomerization of l-lysine. We also identify a lysyl C4 radical intermediate that is trapped, with its electronic structure and geometric structure well characterized by EPR and ENDOR spectroscopy. In addition, we demonstrate that SAM functions as a catalytic cofactor in PylB catalysis rather than canonically as a cosubstrate. This work provides detailed mechanistic evidence for elucidating the carbon backbone rearrangement reaction catalyzed by PylB during the biosynthesis of pyrrolysine.

Characterizing amyloid protein conformation with a multi-resonant metasurface based biosensor in the infrared window.

Misfolding of amyloid protein will cause neurodegeneration and trigger conformational disease. The lack of an effective detection approach is a brake on unveiling the mechanism of protein misfolding. We theoretically proposed a novel metasurface-based biosensor for characterizing the protein's conformation. The coupling complementary split ring resonator (cSRR) was engineered to manipulate incident waves in the near-infrared (NIR) and mid-infrared (MIR) windows at the same sensing surface. The cSRRs had the advantages of intensifying the electric field and sharpening the resonance profile, resulting in a highly qualified biosensing performance. In the NIR window, the biolayer's refractive index and thickness change were detected by the dual-wavelength, which resolved into an optogeometrical parameter of the amyloid biolayer. In the MIR window, the resonant wave specifically triggered the rotation-vibration transition of amyloid protein molecules with different conformations, which was shown as the unique Amide I and II bands in the fingerprint spectrum. Thus, our proposed biosensor presented sensitive detection of biolayer and specific identification of constituent molecules. It is helpful to interpret the protein's misfolding behavior on the molecular level by associating the biolayer's structure and the constituent molecule's conformational change.

Design, synthesis, in silico, and in vitro evaluation of novel benzyloxybenzene substituted (S)-α-amino amide derivatives as cholinesterases and monoaminoxidases inhibitor.

In this study, a series of novel benzyloxybenzene substituted (S)-α-amino acid methyl esters and their amide derivatives were synthesized and evaluated for their inhibitory actions against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), monoamine oxidase A (MAO-A), and monoamine oxidase B (MAO-B). The synthetic strategy was based on starting from benzyl bromide (5) and 4-hydroxybenzaldehyde (6). The reaction of 5 and 6 in the presence of K2 CO3 gave benzyloxybenzaldehyde 7. Benzyloxybenzene substituted (S)-α-amino acid methyl esters 11, 12, 13, (±)-19, and (±)-20 were obtained from the reaction of  L-amino acid methyl esters with benzyloxybenzaldehyde (7) followed by in situ reduction with NaBH4 . The reaction of (S)-11, (S)-12, 13, (±)-19, and (±)-20 with excess ammonia gave amides (S)-14, (S)-15, 16, (±)-21, and (±)-22. The in vitro inhibitory activities of compounds against MAO-A, MAO-B, AChE, and BChE were investigated. Within the α-amino acid methyl ester series, 13 (21.32 ± 0.338 µM) showed selectivity by inhibiting the MAO-B better than MAO-A. 13 emerged as the most active member of this series, exhibiting a 12-fold selectivity for MAO-B. 14 (4.501 ± 0.295 µM) demonstrated a pronounced selectivity for MAO-A over MAO-B, with a selectivity ratio of 110-fold. In addition, it was determined that compound 15 (95.65 ± 3.09 µM) had high selectivity for BChE inhibition. 21 was demonstrated the most potent inhibition (18.36 ± 1.36 µM) against AChE.

Evaluation of the antimalarial activity of SAM13-2HCl with morpholine amide (SKM13 derivative) against antimalarial drug-resistant Plasmodium falciparum and Plasmodium berghei infected ICR mice.

Antimalarial drugs are an urgently need and crucial tool in the campaign against malaria, which can threaten public health. In this study, we examined the cytotoxicity of the 9 antimalarial compounds chemically synthesized using SKM13-2HCl. Except for SKM13-2HCl, the 5 newly synthesized compounds had a 50% cytotoxic concentration (CC50) > 100 µM, indicating that they would be less cytotoxic than SKM13-2HCl. Among the 5 compounds, only SAM13-2HCl outperformed SKM13-2HCl for antimalarial activity, showing a 3- and 1.3-fold greater selective index (SI) (CC50/IC50) than SKM13-2HCl in vitro against both chloroquine-sensitive (3D7) and chloroquine -resistant (K1) Plasmodium falciparum strains, respectively. Thus, the presence of morpholine amide may help to effectively suppress human-infectious P. falciparum parasites. However, the antimalarial activity of SAM13-2HCl was inferior to that of the SKM13-2HCl template compound in the P. berghei NK65-infected mouse model, possibly because SAM13-2HCl had a lower polarity and less efficient pharmacokinetics than SKM13-2HCl. SAM13-2HCl was more toxic in the rodent model. Consequently, SAM13-2HCl containing morpholine was selected from screening a combination of pharmacologically significant structures as being the most effective in vitro against human-infectious P. falciparum but was less efficient in vivo in a P. berghei-infected animal model when compared with SKM13-2HCl. Therefore, SAM13-2HCl containing morpholine could be considered a promising compound to treat chloroquine-resistant P. falciparum infections, although further optimization is crucial to maintain antimalarial activity while reducing toxicity in animals.

Tuning Molecular Motion Enhances Intrinsic Fluorescence in Peptide Amphiphile Nanofibers.

Peptide amphiphiles (PAs) are highly tunable molecules that were recently found to exhibit aggregation-induced emission (AIE) when they self-assemble into nanofibers. Here, we leverage decades of molecular design and self-assembly study of PAs to strategically tune their molecular motion within nanofibers to enhance AIE, making them a highly useful platform for applications such as sensing, bioimaging, or materials property characterization. Since AIE increases when aggregated molecules are rigidly and closely packed, we altered the four most closely packed amino acids nearest to the hydrophobic core by varying the order and composition of glycine, alanine, and valine pairs. Of the six PA designs studied, C16VVAAK2 had the highest quantum yield at 0.17, which is a more than 10-fold increase from other PA designs including the very similar C16AAVVK2, highlighting the importance of precise amino acid placement to anchor rigidity closest to the core. We also altered temperature to increase AIE. C16VVAAK2 exhibited an additional 4-fold increase in maximum fluorescence intensity when the temperature was raised from 5 to 65 °C. As the temperature increased, the secondary structure transitioned from ß-sheet to random coil, indicating that further packing an already aligned molecular system makes it even more readily able to transfer energy between the electron-rich amides. This work both unveils a highly fluorescent AIE PA system design and sheds insights into the molecular orientation and packing design traits that can significantly enhance AIE in self-assembling systems.