Structure-guided identification and characterization of potent inhibitors targeting PhoP and MtrA to combat mycobacteria.

Mycobacteria are causative agents of tuberculosis (TB), which is a global health concern. Drug-resistant TB strains are rapidly emerging, thereby necessitating the urgent development of new drugs. Two-component signal transduction systems (TCSs) are signaling pathways involved in the regulation of various bacterial behaviors and responses to environmental stimuli. Applying specific inhibitors of TCSs can disrupt bacterial signaling, growth, and virulence, and can help combat drug-resistant TB. We conducted a comprehensive pharmacophore-based inhibitor screening and biochemical and biophysical examinations to identify, characterize, and validate potential inhibitors targeting the response regulators PhoP and MtrA of mycobacteria. The constructed pharmacophore model Phar-PR-n4 identified effective inhibitors of formation of the PhoP-DNA complex: ST132 (IC50 = 29 ± 1.6 µM) and ST166 (IC50 = 18 ± 1.3 µM). ST166 (KD = 18.4 ± 4.3 µM) and ST132 (KD = 14.5 ± 0.1 µM) strongly targeted PhoP in a slow-on, slow-off manner. The inhibitory potency and binding affinity of ST166 and ST132 for MtrAC were comparable to those of PhoP. Structural analyses and molecular dynamics simulations revealed that ST166 and ST132 mainly interact with the α8-helix and C-terminal ß-hairpin of PhoP, with functionally essential residue hotspots for structure-based inhibitor optimization. Moreover, ST166 has in vitro antibacterial activity against Macrobacterium marinum. Thus, ST166, with its characteristic 1,2,5,6-tetrathiocane and terminal sulphonic groups, has excellent potential as a candidate for the development of novel antimicrobial agents to combat pathogenic mycobacteria.

Discovery of a potent inhibitor, D-132, targeting AsfvPolX, via protein-DNA complex-guided pharmacophore screening and in vitro molecular characterizations.

The heightened transmissibility and capacity of African swine fever virus (ASFV) induce fatal diseases in domestic pigs and wild boars, posing significant economic repercussions and global threats. Despite extensive research efforts, the development of potent vaccines or treatments for ASFV remains a persistent challenge. Recently, inhibiting the AsfvPolX, a key DNA repair enzyme, emerges as a feasible strategy to disrupt viral replication and control ASFV infections. In this study, a comprehensive approach involving pharmacophore-based inhibitor screening, coupled with biochemical and biophysical analyses, were implemented to identify, characterize, and validate potential inhibitors targeting AsfvPolX. The constructed pharmacophore model, Phar-PolX-S, demonstrated efficacy in identifying a potent inhibitor, D-132 (IC50 = 2.8 ± 0.2 µM), disrupting the formation of the AsfvPolX-DNA complex. Notably, D-132 exhibited strong binding to AsfvPolX (KD = 6.9 ± 2.2 µM) through a slow-on-fast-off binding mechanism. Employing molecular modeling, it was elucidated that D-132 predominantly binds in-between the palm and finger domains of AsfvPolX, with crucial residues (R42, N48, Q98, E100, F102, and F116) identified as hotspots for structure-based inhibitor optimization. Distinctively characterized by a 1,2,5,6-tetrathiocane with modifications at the 3 and 8 positions involving ethanesulfonates, D-132 holds considerable promise as a lead compound for the development of innovative agents to combat ASFV infections.

Structure-Guided Discovery of PD-1/PD-L1 Interaction Inhibitors: Peptide Design, Screening, and Optimization via Computation-Aided Phage Display Engineering.

Cancer immunotherapy harnesses the immune system to combat tumors and has emerged as a major cancer treatment modality. The PD-1/PD-L1 immune checkpoint modulates interactions between tumor cells and T cells and has been extensively targeted in cancer immunotherapy. However, the monoclonal antibodies known to target this immune checkpoint have considerable side effects, and novel PD-1/PD-L1 inhibitors are therefore required. Herein, a peptide inhibitor to disrupt PD-1/PD-L1 interactions was designed through structure-driven phage display engineering coupled to computational modification and optimization. BetaPb, a novel peptide library constructed by using the known structure of PD-1/PD-L, was used to develop inhibitors against the immune checkpoint, and specific peptides with high affinity toward PD-1 were screened through enzyme-linked immunosorbent assays, homogeneous time-resolved fluorescence, and biolayer interferometry. A potential inhibitor, B8, was preliminarily screened through biopanning. The binding affinity of B8 toward PD-1 was confirmed through computation-aided optimization. Assessment of B8 variants (B8.1, B8.2, B8.3, B8.4, and B8.5) demonstrated their attenuation of PD-1/PD-L1 interactions. B8.4 exhibited the strongest attenuation efficiency at a half-maximal effective concentration of 0.1 µM and the strongest binding affinity to PD-1 (equilibrium dissociation constant = 0.1 µM). B8.4 outperformed the known PD-1/PD-L1 interaction inhibitor PL120131 in disrupting PD-1/PD-L1 interactions, revealing that B8.4 has remarkable potential for modification to yield an antitumor agent. This study provides valuable information for the future development of peptide-based drugs, therapeutics, and immunotherapies for cancer.

Development of anti-aflatoxin B1 nanobodies from a novel mutagenesis-derived synthetic library for traditional Chinese medicine and foods safety testing.

BACKGROUND: The main commercially available methods for detecting small molecules of mycotoxins in traditional Chinese medicine (TCM) and functional foods are enzyme-linked immunosorbent assay and mass spectrometry. Regarding the development of diagnostic antibody reagents, effective methods for the rapid preparation of specific monoclonal antibodies are inadequate. METHODS: In this study, a novel synthetic phage-displayed nanobody Golden Glove (SynaGG) library with a glove-like cavity configuration was established using phage display technology in synthetic biology. We applied this unique SynaGG library on the small molecule aflatoxin B1 (AFB1), which has strong hepatotoxicity, to isolate specific nanobodies with high affinity for AFB1. RESULT: These nanobodies exhibit no cross-reactivity with the hapten methotrexate, which is recognized by the original antibody template. By binding to AFB1, two nanobodies can neutralize AFB1-induced hepatocyte growth inhibition. Using molecular docking, we found that the unique non-hypervariable complementarity-determining region 4 (CDR4) loop region of the nanobody was involved in the interaction with AFB1. Specifically, the CDR4's positively charged amino acid arginine directed the binding interaction between the nanobody and AFB1. We then rationally optimized the interaction between AFB1 and the nanobody by mutating serine at position 2 into valine. The binding affinity of the nanobody to AFB1 was effectively improved, and this result supported the use of molecular structure simulation for antibody optimization. CONCLUSION: In summary, this study revealed that the novel SynaGG library, which was constructed through computer-aided design, can be used to isolate nanobodies that specifically bind to small molecules. The results of this study could facilitate the development of nanobody materials to detect small molecules for the rapid screening of TCM materials and foods in the future.

The deficiency of poly-ß-1,6-N-acetyl-glucosamine deacetylase trigger A. baumannii to convert to biofilm-independent colistin-tolerant cells.

Acinetobacter baumannii is a nosocomial pathogen that can be resistant to antibiotics by rapidly modulating its anti-drug mechanisms. The multidrug-resistant A. baumannii has been considered one of the most threatening pathogens to our society. Biofilm formation and persistent cells within the biofilm matrix are recognized as intractable problems, especially in hospital-acquired infections. Poly-ß-1,6-N-acetyl-glucosamine (PNAG) is one of the important building blocks in A. baumannii's biofilm. Here, we discover a protein phosphoryl-regulation on PNAG deacetylase, AbPgaB1, in which residue Ser411 was phosphorylated. The phosphoryl-regulation on AbPgaB1 modulates the product turnover rate in which deacetylated PNAG is produced and reflected in biofilm production. We further uncovered the PgaB deficient A. baumannii strain shows the lowest level of biofilm production but has a high minimal inhibition concentration to antibiotic colistin and tetracycline. Based on bactericidal post-antibiotic effects and time-dependent killing assays with antibacterial drugs, we claim that the PgaB-deficient A. baumannii converts to colistin-tolerant cells. This study utilizes a biofilm-independent colistin-tolerant model of A. baumannii to further investigate its characteristics and mechanisms to better understand clinical outcomes.

Pharmacophore-driven identification of human glutaminyl cyclase inhibitors from foods, plants and herbs unveils the bioactive property and potential of Azaleatin in the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is the leading cause of disabilities in old age and a rapidly growing condition in the elderly population. AD brings significant burden and has a devastating impact on public health, society and the global economy. Thus, developing new therapeutics to combat AD is imperative. Human glutaminyl cyclase (hQC), which catalyzes the formation of neurotoxic pyroglutamate (pE)-modified ß-amyloid (Aß) peptides, is linked to the amyloidogenic process that leads to the initiation of AD. Hence, hQC is an essential target for developing anti-AD therapeutics. Here, we systematically screened and identified hQC inhibitors from natural products by pharmacophore-driven inhibitor screening coupled with biochemical and biophysical examinations. We employed receptor-ligand pharmacophore generation to build pharmacophore models and Phar-MERGE and Phar-SEN for inhibitor screening through ligand-pharmacophore mapping. About 11 and 24 hits identified from the Natural Product and Traditional Chinese Medicine databases, respectively, showed diverse hQC inhibitory abilities. Importantly, the inhibitors TCM1 (Azaleatin; IC50 = 1.1 µM) and TCM2 (Quercetin; IC50 = 4.3 µM) found in foods and plants exhibited strong inhibitory potency against hQC. Furthermore, the binding affinity and molecular interactions were analyzed by surface plasmon resonance (SPR) and molecular modeling/simulations to explore the possible modes of action of Azaleatin and Quercetin. Our study successfully screened and characterized the foundational biochemical and biophysical properties of Azaleatin and Quercetin toward targeting hQC, unveiling their bioactive potential in the treatment of AD.

Deciphering Structure-Function Relationship Unveils Salt-Resistant Mode of Action of a Potent MRSA-Inhibiting Antimicrobial Peptide, RR14.

Multidrug-resistant (MDR) bacteria lead to considerable morbidity and mortality, threatening public health worldwide. In particular, infections of methicillin-resistant Staphylococcus aureus (MRSA) in hospital and community settings are becoming a serious health problem. Antimicrobial peptides (AMPs) are considered novel therapeutic targets against MDR bacteria. However, salt sensitivity reduces the bactericidal potency of AMPs, posing a major obstacle for their development as antibiotics. Thus, the design and development of salt-insensitive peptides with potent antibacterial activity is imperative. Here, we employed biochemical and biophysical examinations coupled with molecular modeling to systematically investigate the structure-function relationship of a novel salt-insensitive AMP, RR14. The secondary structure of RR14 was characterized as an apparent α-helix, a structure that confers strong membrane-permeabilizing ability targeting bacterial-mimetic membranes. Additionally, the bioactive structure of RR14 was determined in complex with dodecylphosphocholine (DPC) micelles, where it possesses a central α-helical segment comprising residues R4 to K13 (R4-K13). RR14 was observed to orient itself into the DPC micelle with its N terminus and the α-helical segment (I5-R10) buried inside the micelles, which is essential for membrane permeabilization and bactericidal activity. Moreover, the specific and featured arrangement of positively charged residues of RR14 on its amphipathic helical conformation has great potential to render its strong salt resistance ability. Our study explored the structure-function relationship of RR14, explaining its possible mode of action against MRSA and other microbes. The insights obtained are of great applicability for the development of new antibacterial agents. IMPORTANCE Many antimicrobial peptides have been observed to become inactive in the presence of high salt concentrations. To further develop new and novel AMPs with potent bactericidal activity and salt insensitivity, understanding the structural basis for salt resistance is important. Here, we employed biochemical and biophysical examinations to systematically investigate the structure-function relationship of a novel salt-insensitive AMP, RR14. RR14 was observed to orient itself into DPC micelles with the N terminus and the α-helical segment (I5-R10) buried inside the micelles, which is essential for membrane permeabilization and bactericidal activity. Moreover, the specific and featured arrangement of cationic residues of RR14 on its amphipathic helical conformation renders its strong salt resistance ability. The insights obtained are of great applicability for developing new antibacterial agents.

Correction: Discovery of 8-prenylnaringenin from hop (Humulus lupulus L.) as a potent monoacylglycerol lipase inhibitor for treatments of neuroinflammation and Alzheimer's disease.

[This corrects the article DOI: 10.1039/D1RA05311F.].

Structural insights into DNA binding domain of vancomycin-resistance-associated response regulator in complex with its promoter DNA from Staphylococcus aureus.

In Staphylococcus aureus, vancomycin-resistance-associated response regulator (VraR) is a part of the VraSR two-component system, which is responsible for activating a cell wall-stress stimulon in response to an antibiotic that inhibits cell wall formation. Two VraR-binding sites have been identified: R1 and R2 in the vraSR operon control region. However, the binding of VraR to a promoter DNA enhancing downstream gene expression remains unclear. VraR contains a conserved N-terminal receiver domain (VraRN ) connected to a C-terminal DNA binding domain (VraRC ) with a flexible linker. Here, we present the crystal structure of VraRC alone and in complex with R1-DNA in 1.87- and 2.0-Å resolution, respectively. VraRC consisting of four α-helices forms a dimer when interacting with R1-DNA. In the VraRC -DNA complex structure, Mg2+ ion is bound to Asp194. Biolayer interferometry experiments revealed that the addition of Mg2+ to VraRC enhanced its DNA binding affinity by eightfold. In addition, interpretation of NMR titrations between VraRC with R1- and R2-DNA revealed the essential residues that might play a crucial role in interacting with DNA of the vraSR operon. The structural information could help in designing and screening potential therapeutics/inhibitors to deal with antibiotic-resistant S. aureus via targeting VraR.

Antigen-Antibody Complex-Guided Exploration of the Hotspots Conferring the Immune-Escaping Ability of the SARS-CoV-2 RBD.

The COVID-19 pandemic resulting from the spread of SARS-CoV-2 spurred devastating health and economic crises around the world. Neutralizing antibodies and licensed vaccines were developed to combat COVID-19, but progress was slow. In addition, variants of the receptor-binding domain (RBD) of the spike protein confer resistance of SARS-CoV-2 to neutralizing antibodies, nullifying the possibility of human immunity. Therefore, investigations into the RBD mutations that disrupt neutralization through convalescent antibodies are urgently required. In this study, we comprehensively and systematically investigated the binding stability of RBD variants targeting convalescent antibodies and revealed that the RBD residues F456, F490, L452, L455, and K417 are immune-escaping hotspots, and E484, F486, and N501 are destabilizing residues. Our study also explored the possible modes of actions of emerging SARS-CoV-2 variants. All results are consistent with experimental observations of attenuated antibody neutralization and clinically emerging SARS-CoV-2 variants. We identified possible immune-escaping hotspots that could further promote resistance to convalescent antibodies. The results provide valuable information for developing and designing novel monoclonal antibody drugs to combat emerging SARS-CoV-2 variants.

Comprehensive Deep Mutational Scanning Reveals the Immune-Escaping Hotspots of SARS-CoV-2 Receptor-Binding Domain Targeting Neutralizing Antibodies.

The rapid spread of SARS-CoV-2 has caused the COVID-19 pandemic, resulting in the collapse of medical care systems and economic depression worldwide. To combat COVID-19, neutralizing antibodies have been investigated and developed. However, the evolutions (mutations) of the receptor-binding domain (RBD) of SARS-CoV-2 enable escape from neutralization by these antibodies, further impairing recognition by the human immune system. Thus, it is critical to investigate and predict the putative mutations of RBD that escape neutralizing immune responses. Here, we employed computational analyses to comprehensively investigate the mutational effects of RBD on binding to neutralizing antibodies and angiotensin-converting enzyme 2 (ACE2) and demonstrated that the RBD residues K417, L452, L455, F456, E484, G485, F486, F490, Q493, and S494 were consistent with clinically emerging variants or experimental observations of attenuated neutralizations. We also revealed common hotspots, Y449, L455, and Y489, that exerted comparable destabilizing effects on binding to both ACE2 and neutralizing antibodies. Our results provide valuable information on the putative effects of RBD variants on interactions with neutralizing antibodies. These findings provide insights into possible evolutionary hotspots that can escape recognition by these antibodies. In addition, our study results will benefit the development and design of vaccines and antibodies to combat the newly emerging variants of SARS-CoV-2.

Discovery of 8-prenylnaringenin from hop (Humulus lupulus L.) as a potent monoacylglycerol lipase inhibitor for treatments of neuroinflammation and Alzheimer's disease.

Monoacylglycerol lipase (MAGL), a serine hydrolase, converts endocannabinoid 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA) and glycerol in the brain and plays a bidirectional role in controlling nueroinflammation. MAGL, involved in Alzheimer's and Parkinson's diseases, is a promising target for treatment of neurodegenerative disorders. However, the irreversible inhibitors of MAGL lead to the desensitization of CB1 receptors further impairing the benefits associated with the indirect CB1 stimulation. Therefore, development of potent reversible inhibitors from natural products (NPs) and traditional chinese medicines (TCMs) are safer and free from adverse side effects and feasible to avoid drawbacks which irreversible inhibitors cause. Here, we employed pharmacophore-based screening of drug candidates coupled with molecular docking, biochemical assay and Ligplot analyses to identify and characterize inhibitors targeting human MAGL (hMAGL). The built pharmacophore model, Phar-MAGL successfully identified inhibitors NP-2 (IC50 = 9.5 ± 1.2 µM), NP-5 (IC50 = 14.5 ± 1.3 µM), and NP-3 (IC50 = 15.2 ± 1.4 µM), which apparently attenuated the activities of hMAGL in vitro. The evident activities of the identified inhibitors against hMAGL showed that the pharmacophore model, Phar-MAGL is reliable and efficient in screening inhibitors against hMAGL. Our study successfully identified a natrual product inhibitor, NP-2 (8-PN), from the plant Humulus lupulus L. (hops) and its positive effects in neurogenesis and neurodifferentiation along with the evident inhibitory potency against hMAGL revealed the potential for further optimizing and developing into drugs to treat neuroinflammation, Alzheimer's and Parkinson's diseases.

Characterizing the structure-activity relationships of natural products, tanshinones, reveals their mode of action in inhibiting spleen tyrosine kinase.

The cytosolic non-receptor protein kinase, spleen tyrosine kinase (SYK), is an attractive drug target in autoimmune, inflammatory disorder, and cancers indications. Here, we employed pharmacophore-based drug screening combined with biochemical assay and molecular dynamics (MD) simulations to identify and characterize inhibitors targeting SYK. The built pharmacophore model, phar-TanI, successfully identified tanshinone (TanI (IC50 = 1.72 µM)) and its analogs (TanIIA (IC50 = 3.2 µM), ST32da (IC50 = 46 µM), and ST32db (IC50 = 51 µM)) which apparently attenuated the activities of SYK in vitro. Additionally, the MD simulations followed by Ligplot analyses revealed that TanI and TanIIA interfered SYK activity through binding deeply into the active site. Besides, TanI and TanIIA mainly interact with residues L377, A400, V433, M448, M450, A451, E452, L453, G454, P455, and L501, which are functional hotspots for structure-based inhibitor optimization against SYK. The structure-activity relationships (SAR) study of the identified SYK inhibitors demonstrated that the pharmacophore model, phar-TanI is reliable and precise in screening inhibitors against SYK. This study disclosed the structure-function relationships of tanshinones from Traditional Chinese Medicine (Danshen), revealing their binding site and mode of action in inhibiting SYK and provides applicability in developing new therapeutic agents.

Characterization of the structure-function relationship of a novel salt-resistant antimicrobial peptide, RR12.

Antimicrobial peptides (AMPs) are potential candidates in designing new anti-infective agents. However, many AMPs show poor bactericidal activities in physical salt and serum solutions. Here, we disclosed the structure-function relationships of a novel salt-resistant antimicrobial peptide, RR12, which could further explain its mode of action and show its applicability in developing new antibacterial agents.

Exploring the mode of action of inhibitors targeting the PhoP response regulator of Salmonella enterica through comprehensive pharmacophore approaches.

The PhoQ/PhoP two-component system regulates the physiological and virulence functions of Salmonella enterica. However, the mode of action of known PhoP inhibitors is unclear. We systematically constructed a pharmacophore model of inhibitors to probe the interface pharmacophore model of the PhoP dimer, coupling it with Ligplot analysis. We found that these inhibitors bind on the α5-helix, altering the conformation and interfering with PhoP binding on DNA.

Protein-DNA complex-guided discovery of the antibacterial lead E1 for restoring the susceptibility of Klebsiella Pneumoniae to polymyxin B by targeting the response regulator PmrA.

A new antibacterial drug is urgently needed. We employed a protein-DNA complex-guided pharmacophore modeling approach to screen inhibitors against the response regulator PmrA of polymyxin B-resistant Klebsiella pneumoniae (KP). The identified lead, E1 (IC50 = 10.2 µM), targeted the DNA-binding domain of PmrA (KD = 1.7 µM), whose conserved residues R171, R198, K203, and Y214 have been shown to be hotspots for antimicrobial development. Treatment of E1 restored the susceptibility of KP to polymyxin B.

Characterizing the structure-function relationship reveals the mode of action of a novel antimicrobial peptide, P1, from jumper ant Myrmecia pilosula.

Microbial infections of antibiotic-resistant strains cause serious diseases and have a significant impact on public health worldwide, so novel antimicrobial drugs are urgently needed. Insect venoms, a rich source of bioactive components containing antimicrobial peptides (AMPs), are attractive candidates for new therapeutic agents against microbes. Recently, a novel peptide, P1, identified from the venom of the Australian jumper ant Myrmecia pilosula, showed potent antimicrobial activities against both Gram-negative and Gram-positive bacteria, but its structure-function relationship is unknown. Here, we used biochemical and biophysical techniques coupled with computational simulations to explore the mode of action of P1 interaction with dodecylphosphocholine (DPC) micelles as a model membrane system. Our circular dichroism (CD) and NMR studies revealed an amphipathic α-helical structure for P1 upon interaction with DPC micelles. A paramagnetic relaxation enhancement approach revealed that P1 orients its α-helix segment (F6-G14) into DPC micelles. In addition, the α-helix segment could be essential for membrane permeabilization and antimicrobial activity. Moreover, the arginine residues R8, R11, and R15 significantly contribute to helix formation and membrane-binding affinity. The lysine residue K19 of the C-terminus functionally guides P1 to interact with DPC micelles in the early interaction stage. Our study provides insights into the mode of action of P1, which is valuable in modifying and developing potent AMPs as antibiotic drugs.

Sarkosyl-Induced Helical Structure of an Antimicrobial Peptide GW-Q6 Plays an Essential Role in the Binding of Surface Receptor OprI in Pseudomonas aeruginosa.

The emergence of antibiotic-resistant microbial strains has become a public health issue and there is an urgent need to develop new anti-infective molecules. Although natural antimicrobial peptides (AMPs) can exert bactericidal activities, they have not shown clinical efficacy. The limitations of native peptides may be overcome with rational design and synthesis. Here, we provide evidence that the bactericidal activity of a synthetic peptide, GW-Q6, against Pseudomonas aeruginosa is mediated through outer membrane protein OprI. Hyperpolarization/depolarization of membrane potential and increase of membrane permeability were observed after GW-Q6 treatment. Helical structure as well as hydrophobicity was induced by an amphipathic surfactant, sarkosyl, for binding to OprI and possible to membrane. NMR studies demonstrated GW-Q6 is an amphipathic α-helical structure in DPC micelles. The paramagnetic relaxation enhancement (PRE) approach revealed that GW-Q6 orients its α-helix segment (K7-K17) into DPC micelles. Additionally, this α-helix segment is critical for membrane permeabilization and antimicrobial activity. Moreover, residues K3, K7, and K14 could be critical for helical formation and membrane binding while residues Y19 and W20 for directing the C-terminus of the peptide to the surface of micelle. Taken together, our study provides mechanistic insights into the mode of action of the GW-Q6 peptide and suggests its applicability in modifying and developing potent AMPs as therapeutic agents.

Discovery of a potent cyclooxygenase-2 inhibitor, S4, through docking-based pharmacophore screening, in vivo and in vitro estimations.

Cyclooxygenase (COX; EC: 1.14.99.1), the key enzyme in prostaglandin production in the human body, is a major pharmacological target for developing anti-inflammatory agents. Nonsteroidal anti-inflammatory drugs exhibit anti-inflammatory and analgesic activities when inhibiting COX-2 but cause gastrointestinal toxicity and other side effects because of concurrent inhibition of COX-1. Thus, potent and safe inhibitors against COX-2 are urgently required. We constructed a novel docking-based pharmacophore model for screening selective COX-2 inhibitors and discovered compounds S1, S2, S3, and S4, which apparently inhibit COX-2. Particularly, S4 inhibits COX-2 in vitro and shows a potent anti-inflammatory effect in vivo without cytotoxicity. Molecular docking analyses revealed that S4 interacted satisfactorily with the active site of COX-2 but not with that of COX-1. This reveals that S4 more specifically inhibits COX-2 and has potential for application in developing anti-inflammatory and anticancer agents.

Mutations of Cytochrome b559 and PsbJ on and near the QC Site in Photosystem II Influence the Regulation of Short-Term Light Response and Photosynthetic Growth of the Cyanobacterium Synechocystis sp. PCC 6803.

The characteristic features of two types of short-term light adaptations of the photosynthetic apparatus of the cyanobacterium Synechocystis sp. PCC 6803, state transition and blue-green light-induced fluorescence quenching, were compared in wild-type and cytochrome b559 and PsbJ mutant cells with mutations on and near the QC site in photosystem II (PSII). All mutant cells grew photoautotrophically and assembled stable PSII. Thermoluminescence emission experiments showed a decrease in the stability of the S3QB(-)/S2QB(-) charge pairs in the A16FJ, S28Aß, and V32Fß mutant cells. When dark-adapted wild-type and mutant cells were illuminated by medium-intensity blue light, the increase in the PSII fluorescence yield (indicating a transition to state 1) was more prominent in mutant than wild-type cells. Strong blue-light conditions induced a quenching of fluorescence corresponding to nonphotochemical fluorescence quenching (NPQ). The extension of NPQ decreased significantly in the mutants, and the kinetics appeared to be affected. When similar measures were repeated on an orange carotenoid protein (OCP)-deficient background, little or no quenching was observed, which confirms that the decrease in fluorescence under strong blue light corresponded to the OCP-dependent NPQ. Immunoblot results showed that the attenuated effect of blue light-induced NPQ in mutant cells was not due to a lack of OCP. Photosynthetic growth and biomass production were greater for A16FJ, S28Aß, and V32Fß mutant cells than for wild-type cells under normal growth conditions. Our results suggest that mutations of cytochrome b559 and PsbJ on and near the QC site of PSII may modulate the short-term light response in cyanobacteria.