Search Results (169)

Efflux is one of the key mechanisms used by Gram-negative bacteria to reduce internal antibiotic concentrations. These active transport systems recognize and expel a wide range of toxic molecules, including antibiotics, thereby contributing to reduced antibiotic susceptibility and allowing the bacteria to acquire additional resistance mechanisms. To date, unlike other resistance mechanisms such as enzymatic modification or target mutations/masking, efflux is challenging to detect and counteract in clinical settings, and no standardized methods are currently available to diagnose or inhibit this mechanism effectively. This review first outlines the structural and functional features of major efflux pumps in Gram-negative bacteria and their role in antibiotic resistance. It then explores various strategies used to curb their activity, with a particular focus on efflux pump inhibitors under development, detailing their structural classes, modes of action, and pharmacological potential. We discuss the main obstacles to their development, including the structural complexity and substrate promiscuity of efflux mechanisms, the limitations of current screening methods, pharmacokinetic and tissue distribution issues, and the risk of off-target toxicity. Overcoming these multifactorial barriers is essential to the rational development of less efflux-prone antibiotics or of efflux pump inhibitors. Full article

Cholinergic neuron impairment is a significant cause of cognitive decline in Alzheimer’s disease (AD), making acetylcholinesterase (AChE) a key therapeutic target. AChE inhibitors are principal drugs prescribed to alleviate symptoms in AD patients, while up to 50% of these individuals also suffer from depression, frequently treated with selective serotonin reuptake inhibitors (SSRIs). Due to the multisymptomatic nature of AD, there is a growing interest in developing multitargeted ligands that simultaneously enhance cholinergic and serotonergic tone. This study presents the synthesis of novel ligands based on functionalized piperidines, evaluated through radioligand binding assays at the serotonin transporter (SERT) and AChE and butyrylcholinesterase (BuChE) inhibition. The pharmacological results showed that some compounds exhibited moderate inhibitory activity against AChE, with one compound 19 standing out as the most potent, also displaying a moderate BuChE inhibitory activity, while showing low affinity for SERT. On the other hand, compound 21 displayed an interesting polypharmacological profile, with good and selective activity against BuChE and SERT. The results underscore the difficulty of designing promiscuous ligands for these targets and suggest that future structural modifications could optimize their therapeutic potential in AD. Full article

The mevalonate pathway is crucial for synthesizing isopentenyl pyrophosphate (IPP), the universal precursor of terpenoids, with 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) serving as the rate-determining enzyme that catalyzes the reduction of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to mevalonate, requiring NAD(P)H as an electron donor. Improving the cofactor promiscuity of HMGR can facilitate substrate utilization and terpenoid production by overcoming cofactor specificity limitations. In this study, we heterologously expressed rpHMGR from Ruegeria pomeroyi in Escherichia coli BL21(DE3) for the first time and established that it predominantly utilizes NADH. To broaden its cofactor usage, we employed Molecular Operating Environment (MOE)-assisted design to engineer the cofactor binding site, creating a dual-cofactor-utilizing mutant, D154K (the substitution of aspartic acid with lysine at residue 154). This mutant exhibited a significant 53.7-fold increase in activity toward NADPH, without compromising protein stability at physiological temperatures. The D154K mutant displayed an optimal pH of 6, maintaining over 80% of its catalytic activity across the pH range of 6–8, regardless of whether NADH or NADPH was the cofactor. These findings highlight the value of rational design, enhance our understanding of HMGR-cofactor recognition mechanisms, and provide a foundation for future efforts to optimize and engineer HMGR for broader cofactor flexibility. Full article

Propolis is a natural antibacterial medicine with a varied content of phenolic compounds, which determines the activity of the ethanol extract of propolis (EEP). A new attempt was made to standardize ethanol propolis extract via its conversion into a dry concentrate (dEEP) through a two-step process. Four samples of poplar propolis from the same geographical region were used for the study. Obtained dry concentrates reconstituted in 70% ethanol (500 μg/mL) were analyzed for their antioxidant properties, total phenolic and flavonoid contents, as well as HPLC polyphenol profile. It was shown that dEEP solutions in 70% ethanol, regardless of the diversified quality of the raw material, have equalized antioxidant properties and phenolic and flavonoid contents compared to raw EEPs. However, quantitative differences in the nine individual components were still found by HPLC-DAD. The antibacterial activity of the dEEP solutions (0.03–500 µg/mL) was compared with three individual polyphenols’ effect against Klebsiella pneumoniae and Streptococcus agalactiae. Based on the obtained MIC values and anti-biofilm activity of dEEPs compared to pure polyphenols, it was established that the effectiveness of the extract results from the combined action of flavonoids and phenolic acids. The antibacterial effectiveness of p-coumaric acid, galangin, and pinocembrin was additionally modeled using in silico analyses, suggesting promiscuous binding of all tested polyphenolic ligands to target enzymes. Full article

Background/Objectives: Zika fever is a disease caused by the Zika virus (ZIKV). Symptomatic cases may be associated with neurological disorders in adults, as well as congenital Zika syndrome and other birth defects during pregnancy. In 2016, Zika fever was considered a public health problem by the World Health Organization (WHO), highlighting the need to develop new therapies against the disease. Currently, there is no antiviral or vaccine available to treat or prevent severe cases. Due to the lack of available therapeutics and few promising hit molecules, we computationally screened the well-described ZIKV protease (NS3^pro) as a drug target to revisit the small-molecule database Brazilian Compound Library (BraCoLi) and select potential inhibitors. Methods: We employed a consensus docking screening of a library of 1176 compounds using GOLD and DockThor. We selected 28 hits based on predicted binding affinity, and only the remnants of three compounds were available in the library at the time of this study for experimental validation. The hits were evaluated for their cytotoxic (CC₅₀) and effective concentrations (EC₅₀) for their potential antiviral activity in Vero cells. Results: The three hit compounds presented modest CC₅₀ values of 89.15 ± 3.72, >100, and 29.67 ± 1.01 μM, with the latter, a carbohydrate derivative, having an EC₅₀ value of >12.5 μM (~40% inhibition) against ZIKV PE243. Additionally, the essentially non-toxic compound, an arylfuran derivative, also inhibited the ZIKV NS3^pro with an IC₅₀ value of 17 μM but presented evidence of acting through a promiscuous mechanism for enzyme inhibition. Conclusion: This study highlights the relevance of revisiting existing small-molecule assets to identify novel therapeutic starting points against ZIKV, aiming for potential lead candidates in the future. Full article

The family of the β₂-integrin receptors is critically involved in host defense and homeostasis, by mediating immune cell adhesion, migration, and phagocytosis. Due to their key roles in immune surveillance and inflammation, their modulation has been recognized as an attractive drug target. However, the development of therapeutics has been limited, partly due to the high promiscuity of endogenous ligands, their functional responses, and gaps in our understanding of their disease-related molecular mechanisms. The delineation of the molecular role of β₂ integrins and their ligands has been hampered by a shortage of validated assay systems. To facilitate molecular and functional studies on the β₂-integrin family, and to enable screening of modulators, this study provides a uniform and validated assay platform. For this purpose, the major ligand-binding domains (αI) of all four β₂ integrins were recombinantly expressed in both low- and high-affinity states. By optimizing the expression parameters and selecting appropriate purification tags, all αI-domain variants could be produced with high yield and purity. Direct binding studies using surface plasmon resonance (SPR) confirmed the expected activity and selectivity profiles of the recombinant αI domains towards their reported ligands, validating our approach. In addition, the SPR studies provided additional insights into ligand binding, especially for the scarcely described family member CD11d. Alongside characterizing endogenous ligands, the platform can be employed to test pharmacologically active compounds, such as the reported β₂-integrin antagonist simvastatin. In addition, we established a bead-based adhesion assay using the recombinant αI domains, and a cell-based adhesion assay underlining most findings generated with the isolated αI domains. Interestingly, the binding of ligands to the recombinant α_DI is not dependent on divalent cation, in contrast to the full integrin CD11d/CD18, suggesting a binding mode distinct of the metal ion-dependent adhesion site (MIDAS). The setup highlights the applicability of recombinant αI domains for first screenings and direct or competitive interaction studies, while the full integrin is needed to validate those findings. Full article

Background/Objectives: Stephania epigaea is a plant from the Menispermaceae family. Its root is an important traditional folk medicine, which is called Diburong in China. Diburong is rich in benzylisoquinoline alkaloids (BIAs), including cepharanthine, which has been demonstrated to exhibit significant anti-inflammatory, antiviral, antineoplastic, and anti-SARS-CoV-2 activities, as well as raising leukocytes. Cepharanthine is composed of (R)- and (S)-1-benzylisoquinoline alkaloid (1-BIA). (S)-norcoclaurine-6-O-methyltransferase (6OMT) is a rate-limiting enzyme in BIA biosynthesis. However, its role in the cepharanthine biosynthetic pathway, particularly with the (R) stereoisomer substrate, remains largely unexplored. This study aimed to identify Se6OMTs involved in the cepharanthine biosynthetic pathway and elucidate the O-methyltransferases (OMTs) responsible for the production of (R)- and (S)-stereoisomer BIAs. Methods: In this study, three OMTs were cloned from S. epigaea and functionally characterized using nine 1-BIAs of (R)- and (S)-configurations as substrates. Results: Two O-methyltransferases, Se6OMT1 and Se6OMT3, showed efficient catalytic activity at the C6 position of both (R)- and (S)-norcoclaurine. Furthermore, Se6OMT3 demonstrated high catalytic activity at the C7 and C4′ positions of other (R)- and (S)-configuration 1-BIAs, which resulted in the generation of multiple products. Conclusions: This study focused on 6OMT enzymes in S. epigaea, identifying Se6OMTs involved in the cepharanthine biosynthetic pathway, determining the OMTs involved in the production of (R)- and (S)-stereoisomer BIAs. This research provides valuable insights into the substrate promiscuity of Se6OMTs on (R)- and (S)-configured 1-BIAs in S. epigaea and highlights the genetic components necessary for the metabolic engineering and synthetic biology approaches to cepharanthine production. Full article

The octapeptide angiotensin II (Ang II) is a circulating hormone as well as a locally formed agonist synthesized by the angiotensin-converting enzyme (ACE) of endothelial cells. It forms a powerful mechanism to control the amount and pressure of body fluids. All main effects are directed to save body salt and water and ensure blood pressure under basic conditions and in emergencies. All blood vessels respond to stimulation by Ang II; the immediate response is smooth muscle contraction, increasing vascular resistance, and elevating blood pressure. Such effects are conveyed by type 1 angiotensin receptors (AT₁Rs) located in the plasma membrane of both endothelial and vascular smooth muscle cells. AT₁Rs are heterotrimeric G protein-coupled receptors (GPCRs), but their signal pathways are much more complicated than other GPCRs. In addition to G_q/11, the G_12/13, JAK/STAT, Jnk, MAPK, and ERK 1/2, and arrestin-dependent and -independent pathways are activated because of the promiscuous attachment of different signal proteins to the intracellular G protein binding site and to the intracellular C terminal loop. Substantial changes in protein expression follow, including the intracellular inflammation signal protein NF-κB, endothelial contact proteins, cytokines, matrix metalloproteinases (MMPs), and type I protocollagen, eliciting the inflammatory transformation of endothelial and vascular smooth muscle cells and fibrosis. Ang II is an important contributor to vascular pathologies in hypertensive, atherosclerotic, and aneurysmal vascular wall remodeling. Such direct vascular effects are reviewed. In addition to reducing blood pressure, AT₁R antagonists and ACE inhibitors have a beneficial effect on the vascular wall by inhibiting pathological wall remodeling. Full article

Enzymes capable of processing a variety of compounds enable plants to adapt to diverse environmental conditions. PRISEs (progesterone-5β-reductase/iridoid synthase-like enzymes), examples of such substrate-promiscuous enzymes, are involved in iridoid and cardenolide pathways and demonstrate notable substrate promiscuity by reducing the activated C=C double bonds of plant-borne and exogenous 1,4-enones. In this study, we identified PRISE genes in Plantago media (PmdP5βR1) and Plantago lanceolata (PlP5βR1), and the corresponding enzymes were determined to share a sequence identity of 95%. Despite the high sequence identity, recombinant expressed PmdP5βR1 was 70 times more efficient than PlP5βR1 for converting progesterone. In order to investigate the underlying reasons for this significant discrepancy, we focused on specific residues located near the substrate-binding pocket and adjacent to the conserved phenylalanine “clamp”. This clamp describes two phenylalanines influencing substrate preferences by facilitating the binding of smaller substrates, such as 2-cyclohexen-1-one, while hindering larger ones, such as progesterone. Using structural analysis based on templates PDB ID: 5MLH and 6GSD from PRISE of Plantago major, along with in silico docking, we identified positions 156 and 346 as hot spots. In PlP5βR1 amino acid residues, A156 and F346 seem to be responsible for the diminished ability to reduce progesterone. Moreover, the double mutant PlP5βR_F156L_A346L, which contains the corresponding amino acids from PmdP5βR1, showed a 15-fold increase in progesterone 5β-reduction. Notably, this modification did not significantly alter the enzyme’s ability to convert other substrates, such as 8-oxogeranial, 2-cyclohexen-1-one, and methyl vinyl ketone. Hence, a rational enzyme design by reducing the number of hotspots selectively, specifically improved the substrate preference of PlP5βR1 for progesterone. Full article

Since its discovery in 1992, the receptor for advanced glycation end products (RAGE) has emerged as a key receptor in many pathological conditions, especially in inflammatory conditions. RAGE is expressed by most, if not all, immune cells and can be activated by many ligands. One characteristic of RAGE is that its ligands are structurally very diverse and belong to different classes of molecules, making RAGE a promiscuous receptor. Many of RAGE ligands are damaged associated molecular patterns (DAMPs) that are released by cells under inflammatory conditions. Although RAGE has been at the center of a lot of research in the past three decades, a clear understanding of the mechanisms of RAGE activation by its ligands is still missing. In this review, we summarize the current knowledge of the role of RAGE and its ligands in inflammation. Full article

The comparative analysis of homologous enzymes is a valuable approach for elucidating enzymes’ structure–function relationships. Glutathione transferases (GSTs, EC. 2.5.1.18) are crucial enzymes in maintaining the homeostatic stability of plant cells by performing various metabolic, regulatory, and detoxifying functions. They are promiscuous enzymes that catalyze a broad range of reactions that involve the nucleophilic attack of the activated thiolate of glutathione (GSH) to electrophilic compounds. In the present work, three highly homologous (96–98%) GSTs from ryegrass Lolium perenne (LpGSTs) were identified by in silico homology searches and their full-length cDNAs were isolated, cloned, and expressed in E. coli cells. The recombinant enzymes were purified by affinity chromatography and their substrate specificity and kinetic parameters were determined. LpGSTs belong to the tau class of the GST superfamily, and despite their high sequence homology, their substrate specificity displays remarkable differences. High catalytic activity was determined towards hydroxyperoxides and alkenals, suggesting a detoxification role towards oxidative stress metabolites. The prediction of the structure of the most active LpGST by molecular modeling allowed the identification of a non-conserved residue (Phe215) with key structural and functional roles. Site-saturation mutagenesis at position 215 and the characterization of eight mutant enzymes revealed that this site plays pleiotropic roles, affecting the affinity of the enzyme for the substrates, catalytic constant, and structural stability. The results of the work have improved our understanding of the GST family in L. perenne, a significant threat to agriculture, sustainable food production, and safety worldwide. Full article

Owing to its promiscuous roles, poly (ADP-ribose) polymerase-1 (PARP-1) is involved in various neurological disorders including several retinal pathologies. Diabetic retinopathy (DR) is the most common microvascular complication of diabetes mellitus affecting the retina. In the present review, we highlight the importance of PARP-1 participation in pathophysiology of DR and discuss promising potential inhibitors for treatment. A high glucose level enhances PARP-1 expression; PARP inhibitors have gained attention due to their potential therapeutic effects in DR. They target different checkpoints (blocking nuclear transcription factor (NF-κB) activation; oxidative stress protection, influence on vascular endothelial growth factor (VEGF) expression, impacting neovascularization). Nowadays, there are several improved clinical PARP-1 inhibitors with different allosteric effects. Combining PARP-1 inhibitors with other compounds is another promising option in DR treatments. Besides pharmacological inhibition, genetic disruption of the PARP-1 gene is another approach in PARP-1-initiated therapies. In terms of future treatments, the limitations of single-target approaches shift the focus onto combined therapies. We emphasize the importance of multi-targeted therapies, which could be effective not only in DR, but also in other ischemic conditions. Full article

Metallo-β-lactamases (MBLs) are members of the structurally conserved but functionally diverse MBL-fold superfamily of metallohydrolases. MBLs are a major concern for global health care as they efficiently inactivate β-lactam antibiotics, including the “last-resort” carbapenems, and no clinically suitable inhibitors are currently available. Increasingly, promiscuous β-lactamase activity is also observed in other members of the superfamily, including from viruses, which represents an underexplored reservoir for future pathways to antibiotic resistance. Here, two such MBL-fold enzymes from Bacillus phages, the cyclic mononucleotide-degrading proteins Apyc_Goe3 and Apyc_Grass, are shown to degrade β-lactam substrates efficiently in vitro. In particular, Apyc_Grass displays a distinct preference for carbapenem substrates with a catalytic efficiency that is within one order of magnitude of the clinically relevant MBL NDM-1. Mutagenesis experiments also demonstrate that the loss of a metal-bridging aspartate residue reduces nuclease activity up to 35-fold but improves carbapenemase activity. In addition, we hypothesise that the oligomeric state significantly influences β-lactamase activity by modifying access to the active site pocket. Together, these observations hint at a possible new avenue of resistance via the spread of phage-borne MBL-fold enzymes with β-lactamase activity. Full article

University women are vulnerable to engaging in risky sexual behaviors (RSBs), which are related to academic stress, alcohol, and tobacco consumption. The aim of this study was to identify profiles of university women who associate RSBs with alcohol consumption, tobacco consumption level, and frequency of academic stress. A total of 534 female university students from Mexican universities answered an online questionnaire with five instruments related to their sexual behavior, alcohol consumption, tobacco consumption, and academic stress. They were studying to become professionals in health sciences, administration and social sciences, engineering, and “others” with grade point averages ranging from 5.7 to 10/10. They had to sign an informed consent and acknowledge having had at least one sexual relationship with anal or vaginal penetration before participating. A multiple correspondence analysis yielded a parsimonious solution with the following three dimensions that explained 38.60% of the variance: 1 “pregnancy”; 2 “number of sexual partners”; 3 “substance use”. Using a point cloud clustering strategy based on the Euclidean distance between categories in a two-dimensional space, five profiles were identified: Conservative and Prudent, Active Promiscuous, Vulnerable Reproductive Health, STI Risk, and Moderate Risk Behaviors. The estimation of ellipses with 95% confidence from the calculation of centroids allowed for the integration of two profiles: Lower probability of RSB and Higher probability of RSB. The contribution of academic stress was null with respect to the model solution, so this variable was discarded. The results identified particular risk profiles in female university students. These findings are useful for the development of differentiated intervention strategies to reduce RSB in this vulnerable group. Full article

The intrinsically disordered polyglutamine-binding protein 1 (PQBP1) has been linked to various cellular processes including transcription, alternative splicing, translation and innate immunity. Mutations in PQBP1 are causative for neurodevelopmental conditions collectively termed as the Renpenning syndrome spectrum. Intriguingly, cells of Renpenning syndrome patients exhibit a reduced innate immune response against human immunodeficiency virus 1 (HIV-1). PQBP1 is responsible for the initiation of a two-step recognition process of HIV-1 reverse-transcribed DNA products, ensuring a type 1 interferon response. Recent investigations revealed that PQBP1 also binds to the p17 protein of avian reovirus (ARV) and is affected by the ORF52 of Kaposi’s sarcoma-associated herpesvirus (KSHV), possibly also playing a role in the innate immune response towards these RNA- and DNA-viruses. Moreover, PQBP1-mediated microglia activation in the context of tauopathies has been reported, highlighting the role of PQBP1 in sensing exogenous pathogenic species and innate immune response in the central nervous system. Its unstructured nature, the promiscuous binding of various proteins and its presence in various tissues indicate the versatile roles of PQBP1 in cellular regulation. Here, we systematically review the available data on the structure of PQBP1 and its cellular functions and interactome, as well as possible implications for innate immune responses and neurodegenerative disorders. Full article