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.

Orientin mediates protection against MRSA-induced pneumonia by inhibiting Sortase A.

Drug-resistant pathogenic Staphylococcus aureus (S. aureus) has severely threatened human health and arouses widespread concern. Sortase A (SrtA) is an essential virulence factor of S. aureus, which is responsible for the covalent anchoring of a variety of virulence-related proteins to the cell wall. SrtA has always been regarded as an ideal pharmacological target against S. aureus infections. In this research, we have determined that orientin, a natural compound isolated from various medicinal plants, can effectively inhibit the activity of SrtA with an IC50 of 50.44 ± 0.51 µM. We further demonstrated that orientin inhibited the binding of S. aureus to fibrinogen and diminished biofilm formation and the attaching of Staphylococcal protein A (SpA) to the cell wall in vitro. Using the fluorescence quenching assay, we demonstrated a direct interaction between orientin and SrtA. Further mechanistic studies revealed that the residues Glu-105, Thr-93, and Cys-184 were the key sites for the binding of SrtA to orientin. Importantly, we demonstrated that treatment with orientin attenuated S. aureus virulence of in vivo and protected mice against S. aureus-induced lethal pneumonia. These findings indicate that orientin is a potential drug to counter S. aureus infections and limit the development of drug resistance.

Mammalian-like type II glutaminyl cyclases in Porphyromonas gingivalis and other oral pathogenic bacteria as targets for treatment of periodontitis.

The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded ß-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.

Sortase A-Inhibitory Coumarins from the Folk Medicinal Plant Poncirus trifoliata.

Thirteen coumarins (1-13), including five new compounds (1-5), were isolated from the folk medicinal plant Poncirus trifoliata. Combined spectroscopic analyses revealed that coumarins 1-4 are bis-isoprenylated coumarins with diverse oxidation patterns, while 5 is an enantiomeric di-isoprenylated coumarin. The absolute configurations of the stereogenic centers in the isoprenyl chains were assigned through MTPA and MPA methods, and those of the known compounds triphasiol (6) and ponciol (7) were also assigned using similar methods. These coumarins inhibited significantly Staphylococcus aureus-derived sortase A (SrtA), a transpeptidase responsible for anchoring surface proteins to the peptidoglycan cell wall in Gram-positive bacteria. The present results obtained indicated that the bioactivity and underlying mechanism of action of these coumarins are associated with the inhibition of SrtA-mediated S. aureus adhesion to eukaryotic cell matrix proteins including fibrinogen and fibronectin, thus potentially serving as SrtA inhibitors.

Identification of potential antivirulence agents by substitution-oriented screening for inhibitors of Streptococcus pyogenes sortase A.

Antimicrobial resistance resulting in ineffective treatment of infectious diseases is an increasing global problem, particularly in infections with pathogenic bacteria. In some bacteria, such as Streptococcus pyogenes, the pathogenicity is strongly linked to the attachment of virulence factors. Their attachment to the cellular membrane is a transpeptidation reaction, catalyzed by sortase enzymes. As such, sortases pose an interesting target for the development of new antivirulence strategies that could yield novel antimicrobial drugs. Using the substitution-oriented fragment screening (SOS) approach, we discovered a potent and specific inhibitor (C10) of sortase A from S. pyogenes. The inhibitor C10 showed high specificity towards S. pyogenes sortase A, with an IC50 value of 10 µM and a Kd of 60 µM. We envision that this inhibitor could be employed as a starting point for further exploration of sortase's potential as therapeutic target for antimicrobial drug development.

Identifying hQC Inhibitors of Alzheimer's Disease by Effective Customized Pharmacophore-Based Virtual Screening, Molecular Dynamic Simulation, and Binding Free Energy Analysis.

Human glutaminyl cyclase (hQC) appeared as a promising new target with its inhibitors attracted much attention for the treatment of Alzheimer's disease (AD) in recent years. But so far, only a few compounds have been reported as hQC inhibitors. To find novel and potent hQC inhibitors, a high-specificity ZBG (zinc-binding groups)-based pharmacophore model comprising customized ZBG feature was first generated using HipHop algorithm in Discovery Studio software for screening out hQC inhibitors from the SPECS database. After purification by docking studies and drug-like ADMET properties filters, four potential hit compounds were retrieved. Subsequently, these hit compounds were subjected to 30-ns molecular dynamic (MD) simulations to explore their binding modes at the active side of hQC. MD simulations demonstrated that these hit compounds formed a chelating interaction with the zinc ion, which was consistent with the finding that the electrostatic interaction was the major driving force for binding to hQC confirmed with MMPBSA energy decomposition. Higher binding affinities of these compounds were also verified by the binding free energy calculations comparing with the references. Thus, these identified compounds might be potential hQC candidates and could be used for further investigation.

Isovitexin, a Potential Candidate Inhibitor of Sortase A of Staphylococcus aureus USA300.

Staphylococcus aureus (S. aureus) causes a broad variety of diseases. The spread of multidrugresistant S. aureus highlights the need to develop new ways to combat S. aureus infections. Sortase A (SrtA) can anchor proteins containing LPXTG binding motifs to the bacteria surface and plays a key role in S. aureus infections, making it a promising antivirulence target. In the present study, we used aSrtA activity inhibition assay to discover that isovitexin, a Chinese herbal product, can inhibit SrtA activity with an IC50 of 28.98 µg/ml. Using a fibrinogenbinding assay and a biofilm formation assay, we indirectly proved the SrtA inhibitory activity of isovitexin. Additionally, isovitexin treatment decreased the amount of staphylococcal protein A (SpA) on the surface of the cells. These data suggest that isovitexin has the potential to be an anti-infective drug against S. aureus via the inhibition of sortase activity.

Streptococcus suis sortase A is Ca2+ independent and is inhibited by acteoside, isoquercitrin and baicalin.

Sortase A (SrtA) has long been recognized as an ideal drug target for therapeutic agents against Gram-positive pathogens. However, the SrtA of Streptococcus suis (Ss-SrtA), an important zoonotic agent, has not been studied. In this study, the enzymatic properties of Ss-SrtA were investigated, and inhibition of Ss-SrtA by natural products was evaluated. Ss-SrtA was expressed and purified. The purified recombinant Ss-SrtA had maximal activity at pH 6.0-7.5, 45°C, and showed a Km of 6.7 µM for the hydrolysis of substrate abz-LPATG-dnp. Different from Staphylococcus aureus SrtA (Sa-SrtA) which is stimulated by Ca2+, Ss-SrtA was observed to be Ca2+ independent. Structural analysis showed that salt bridges formed between K111 and D180 in Ss-SrtA replaced the function of Ca2+ in Sa-SrtA to stabilize the substrate-binding cleft. Site-directed mutagenesis identified H126, C192 and R200 as the key residues of Ss-SrtA active site. To discover potential inhibitors, the percent inhibition of sortase activity by natural products was measured. Among these selected natural products, acteoside, isoquercitrin and baicalin were discovered as novel SrtA inhibitors, with IC50 values of 36.3 ± 1.3 µM, 100.0 ± 1.3 µM and 85.4 ± 1.5 µM, respectively. The inhibitory effects of these three natural products were further confirmed on endogenous Sa-SrtA. Using a previously established S. aureus model with a fluorescent-labeled Sa-SrtA substrate, acteoside, isoquercitrin, and baicalin showed 86%, 28% and 45% inhibition on endogenous Sa-SrtA activity, respectively. Overall, these findings shed new light on enzymatic properties, Ca2+-independent catalytic mechanism and potential inhibitors of Ss-SrtA.

Spatholobus suberectus Dunn. constituents inhibit sortase A and Staphylococcus aureus cell clumping to fibrinogen.

Sortases are a family of Gram-positive transpeptidases responsible for anchoring surface protein virulence factors to the peptidoglycan cell wall layer. In Staphylococcus aureus (S. aureus), deletion of sortase isoform results in a significant reduction in virulence and infection potential. Twenty flavonoids were isolated from the stem of the folk medicinal plant Spatholobus suberectus Dunn. These compounds were tested against S. aureus-derived sortase A (SrtA), a key transpeptidase for bacterial virulence. Among these active flavonoids, 7-hydroxy-6-methoxy-flavanone (3) and formononetin (10) were identified as compounds with promising SrtA inhibitory activity. These compounds also exhibited inhibitory activity against S. aureus cell clumping to fibrinogen. The suppression of cell-clumping activity indicates the potential of these compounds in the treatment of S. aureus infections via the inhibition of SrtA.

Identification of potential glutaminyl cyclase inhibitors from lead-like libraries by in silico and in vitro fragment-based screening.

A glutaminyl cyclase (QC) fragment library was in silico selected by disconnection of the structure of known QC inhibitors and by lead-like 2D virtual screening of the same set. The resulting fragment library (204 compounds) was acquired from commercial suppliers and pre-screened by differential scanning fluorimetry followed by functional in vitro assays. In this way, 10 fragment hits were identified ([Formula: see text]5 % hit rate, best inhibitory activity: 16 [Formula: see text]). The in vitro hits were then docked to the active site of QC, and the best scoring compounds were analyzed for binding interactions. Two fragments bound to different regions in a complementary manner, and thus, linking those fragments offered a rational strategy to generate novel QC inhibitors. Based on the structure of the virtual linked fragment, a 77-membered QC target focused library was selected from vendor databases and docked to the active site of QC. A PubChem search confirmed that the best scoring analogues are novel, potential QC inhibitors.

Flavonoid Glycosides Inhibit Sortase A and Sortase A-Mediated Aggregation of Streptococcus mutans, an Oral Bacterium Responsible for Human Dental Caries.

Three flavonoids were isolated from dried flowers of Sophora japonica using repetitive column chromatography and high-performance liquid chromatography. The flavonoids were identified as rutin (1), quercetin-3'-O-methyl-3-O-α-L-rhamnopyranosyl(1 → 6)-ß-D-glucopyranoside (2), and quercetin (3) on the basis of spectroscopic analysis and comparison of values reported in the literature. These compounds inhibited the action of sortase A (SrtA) from Streptococcus mutans, a primary etiologic agent of human dental caries. The onset and magnitude of inhibition of saliva-induced aggregation of S. mutans treated with compound 1 was comparable to that of untreated S. mutans with a deletion of the srtA gene.

Chinese herb medicine against Sortase A catalyzed transformations, a key role in gram-positive bacterial infection progress.

Many Gram-positive bacteria can anchor their surface proteins to the cell wall peptidoglycan covalently by a common mechanism with Sortase A (SrtA), thus escaping from the host's identification of immune cells. SrtA can complete this anchoring process by cleaving LPXTG motif conserved among these surface proteins and thus these proteins anchor on the cell wall. Moreover, those SrtA mutants lose this capability to anchor these relative proteins, with these bacteria no longer infectious. Therefore, SrtA inhibitors can be promising anti-infective agents to cure bacterial infections. Chinese herb medicines (CHMs) (chosen from Science Citation Index) have exhibited inhibition on SrtA of Gram-positive pathogens irreversibly or reversibly. In general, CHMs are likely to have important long-term impact as new antibacterial compounds and sought after by academia and the pharmaceutical industry. This review mainly focuses on SrtA inhibitors from CHMs and the potential inhibiting mechanism related to chemical structures of compounds in CHMs.

Designing of potential inhibitors against Staphylococcus aureus sortase A: Combined analogue and structure based approach with in vitro validation.

Staphylococcus aureus sortase A is an attractive target of Gram-positive bacteria that plays a crucial role in anchoring of surface proteins to peptidoglycan present in bacterial cell wall. Inhibiting sortase A is an elementary and essential effort in preventing the pathogenesis. In this context, in silico virtual screening of in-house database was performed using ligand based pharmacophore model as a filter. The developed pharmacophore model AAHR 11 consists of two acceptors, one hydrophobic and one ring aromatic feature. Top ranked molecule KKR1 was docked into the active site of the target. After profound analysis, it was analyzed and optimized based on the observations from its binding pose orientation. Upgraded version of KKR1 was KKR2 and has improved docking score, binding interactions and best fit in the binding pocket. KKR1 along with KKR2 were further validated using 100 ns molecular dynamic studies. Both KKR1 and KKR2 contain Indole-thiazolidine moiety and were synthesized. The disk diffusion assay has good initial results (ZI of KKR1, KKR2 were 24, 38 mm at 10 µg/mL and ZI of Ampicillin was 22 at 10 µg/mL) and calculated MICs of the molecules (KKR1 5.56±0.28 µg/mL, KKR2 1.32±0.12 µg/mL, Ampicillin 8±1.1 µg/mL) were in good agreement with standard drug Ampicillin. KKR1 has shown IC50 of 1.23±0.14 µM whereas the optimized lead molecule KKR2 show IC50 of 0.008±0.07 µM. Results from in silico were validated by in vitro studies and proved that indole-thiazolidine molecules would be useful for future development as lead molecules against S. aureus sortase A.

siRNA screen identifies QPCT as a druggable target for Huntington's disease.

Huntington's disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified new modifiers of mutant HTT toxicity by performing a large-scale 'druggable genome' siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen and also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone αB-crystallin and reduced the aggregation of diverse proteins. We generated new QPCT inhibitors using in silico methods followed by in vitro screening, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a new HD druggable target affecting mutant HTT aggregation and provide proof of principle for a discovery pipeline from druggable genome screen to drug development.

Streptococcus mutans sortase A inhibitory metabolites from the flowers of Sophora japonica.

A new maltol derivative (2) along with three known maltol derivative (1) and flavonol glycosides (3 and 4) were isolated from the dried flowers of Sophora japonica. Based upon the results of combined spectroscopic methods, the structure of new compound (2) was determined to be maltol-3-O-(4'-O-cis-p-coumaroyl-6'-O-(3-hydroxy-3-methylglutaroyl))-ß-glucopyranoside, an isomer of 1. These compounds strongly inhibited the action of sortase A (SrtA) from Streptococcus mutans, a primary etiologic agent of human dental caries. The onset and magnitude of inhibition of the saliva-induced aggregation in S. mutans treated with compound 2 (4×IC50) were comparable to the behavior of untreated srtA-deletion mutant.

Antiinfective therapy with a small molecule inhibitor of Staphylococcus aureus sortase.

Methicillin-resistant Staphylococcus aureus (MRSA) is the most frequent cause of hospital-acquired infection, which manifests as surgical site infections, bacteremia, and sepsis. Due to drug-resistance, prophylaxis of MRSA infection with antibiotics frequently fails or incites nosocomial diseases such as Clostridium difficile infection. Sortase A is a transpeptidase that anchors surface proteins in the envelope of S. aureus, and sortase mutants are unable to cause bacteremia or sepsis in mice. Here we used virtual screening and optimization of inhibitor structure to identify 3-(4-pyridinyl)-6-(2-sodiumsulfonatephenyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole and related compounds, which block sortase activity in vitro and in vivo. Sortase inhibitors do not affect in vitro staphylococcal growth yet protect mice against lethal S. aureus bacteremia. Thus, sortase inhibitors may be useful as antiinfective therapy to prevent hospital-acquired S. aureus infection in high-risk patients without the side effects of antibiotics.

Virtual screening of LPXTG competitive SrtA inhibitors targeting signal transduction mechanism in Bacillus anthracis: a combined experimental and theoretical study.

Members of the sortase enzyme super family decorate the surfaces of Bacillus anthracis cell wall with proteins that play key roles in microbial pathogenesis and its biofilm formation. Bacillus anthracis Sortase-A (Ba-SrtA) is a potential target for new therapeutics as it is required for B. anthracis survival and replication within macrophages. An understanding of the binding site pocket and substrate recognition mechanism by SrtA enzymes may serve to be beneficial in the rational development of sortase inhibitors. Here, the LPXTG signal peptide-based competitive inhibitors are screened against the Ba-SrtA and compounds with reasonable inhibition, specificity, and mechanisms of inactivation of SrtA have been covered. The screened compounds are experimentally validated against the phylogenetically similar Gram-positive pathogen B. cereus. In situ microscopic visualizations suggest that these screened compounds showed the microbial and biofilm inhibitory activity against B. cereus. It facilitates the further development of these molecules into useful anti-infective agents to treat infections caused by B. anthracis and other Gram-positive pathogens. These results provide insight into basic design principles for generating new clinically relevant lead molecules. It also provides an alternative strategy where a screened ligand molecule can be used in combination to battle increasingly against the Gram-positive pathogens.

Sortase A inhibitory metabolites from the roots of Pulsatilla koreana.

Three new lignans (3, 4, and 6) along with eight known lignans and phenyl propanoids were isolated from the dried roots of Pulsatilla koreana, an oriental folk medicine. Based upon the results of combined spectroscopic and chemical methods, the structures of new compounds were determined to be lignan glycosides. Included among the known compounds are three compounds (5, 7, and 8) isolated first time from this plant as well as two compounds (2 and 11) previously reported only as synthetic derivatives. These compounds significantly inhibited the action of Sortase A from Streptococcus mutans OMZ65, an isolate from human oral cavity.

In vitro sortase A inhibitory and antimicrobial activity of flavonoids isolated from the roots of Sophora flavescens.

A series of flavonoids (1-14) was isolated from the roots of Sophora flavescens. We evaluated their ability to inhibit both microbial growth and sortase A, an enzyme that plays a key role in cell wall protein anchoring and virulence in Staphylococcus aureus. Most prenylated flavonoids (7-13) displayed potent inhibitory activity against gram-positive and gram-negative bacteria except E. coli, with minimum inhibitory concentrations values ranging from 4.40 to 27.7 µM, and weak or no activity against fungal strains tested. Kurarinol (6) was a potent inhibitor of sortase A, with an IC(50) value of 107.7 ± 6.6 µM. A preliminary structure-activity relationship, including essential structural requirements, is described.

Synthetic analogs of indole-containing natural products as inhibitors of sortase A and isocitrate lyase.

Guided by the inhibitory activities of indole-containing natural products against isocitrate lyase (ICL) from Candida albicans and sortase A (SrtA) from Staphylococcus aureus, a series of compounds structurally analogous to natural products were synthesized. Eight SrtA inhibitors and an ICL inhibitor having higher activities than the natural products were discovered by screening the enzyme inhibitory activities of synthesized compounds. Among the SrtA inhibitors discovered, six exhibited higher activities than p-hydroxymercuribenzoic acid, which suggests that these compounds have great potential as alternative antibacterial agents.