Repurposing of intestinal defensins as multi-target,dual-function amyloid inhibitors via cross-seeding

Amyloid formation and microbial infection are the two common pathological causes of neurogenerative diseases, including Alzheimer''s disease (AD), type II diabetes (T2D), and medullary thyroid carcinoma (MTC). While significant efforts have been made to develop different prevention strategies and preclinical hits for these diseases, conventional design strategies of amyloid inhibitors are mostly limited to either a single prevention mechanism (amyloid cascade vs. microbial infection) or a single amyloid protein (Aβ, hIAPP, or hCT), which has prevented the launch of any successful drug on the market. Here, we propose and demonstrate a new “anti-amyloid and anti-bacteria” strategy to repurpose two intestinal defensins, human α-defensin 6 (HD-6) and human β-defensin 1 (HBD-1), as multiple-target, dual-function, amyloid inhibitors. Both HD-6 and HBD-1 can cross-seed with three amyloid peptides, Aβ (associated with AD), hIAPP (associated with T2D), and hCT (associated with MTC), to prevent their aggregation towards amyloid fibrils from monomers and oligomers, rescue SH-SY5Y and RIN-m5F cells from amyloid-induced cytotoxicity, and retain their original antimicrobial activity against four common bacterial strains at sub-stoichiometric concentrations. Such sequence-independent anti-amyloid and anti-bacterial functions of intestinal defensins mainly stem from their cross-interactions with amyloid proteins through amyloid-like mimicry of β-sheet associations. In a broader view, this work provides a new out-of-the-box thinking to search and repurpose a huge source of antimicrobial peptides as amyloid inhibitors, allowing the blocking of the two interlinked pathological pathways and bidirectional communication between the central nervous system and intestines via the gut–brain axis associated with neurodegenerative diseases.

Amyloid formation and microbial infection are the two common pathological causes of neurogenerative diseases. Here, we proposed a new “anti-amyloid and anti-bacteria” strategy to repurpose two intestinal defensins as multiple-target, dual-function amyloid inhibitors.     

Di-isatin heteronuclear compounds and their antibacterial activity

Twenty propylene and butylene tethered di-isatin heteronuclear compounds 5a-t were synthesized, and their antibacterial activities were evaluated. Most of the synthesized di-isatin heteronuclear derivatives were active against both Gram-positive and Gram-negative strains, and some of them exhibited considerable activities against drug-resistant organisms. In particular, di-isatin 5a (MIC: 32-512 μg/mL) was more active than the reference vancomycin against Gram-negative pathogens, demonstrating the antibacterial potential of di-isatin heteronuclear compounds. The inhibitory activity of di-isatin 5a was higher than mono-isatin against Escherichia coli DNA gyrase, suggesting the dimers could improve the inhibitory activity against DNA gyrase when compared with the isatin. The structure-activity relationship was discussed to provide an insight for rational designs of more efficient antibacterial candidates.     

Expression and Purification of an Antimicrobial Peptide by Fusion with Elastin-like Polypeptides in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Different carrier molecules have been fused to antimicrobial polypeptides (AMPs) to facilitate recombinant protein expression and purification. Some of them have improved the stability of AMPs and reduced the toxicity to host cells, but most current strategies still have some problems to be solved such as poor yield, low purity, high expense, time-consumption, and difficulty in scaling-up. Here, we introduced the elastin-like polypeptides (ELPs) as a fusion partner to express an antimicrobial polypeptide halocidin18 (Hal18). By the reversible soluble–insoluble phase transition, 69 mg of the fusion protein were purified from 1 l of culture medium with the purity of nearly 95%. After cleavage with hydroxylamine, the ELP’s tag was easily separated from Hal18 in the next round of inverse transition cycle and Hal18 (1.7 mg, ∼1.9 kDa) was mainly found in the supernatant with a recovery of about 47% and purity of 60%. Antimicrobial activity showed that Hal18 had strong antimicrobial activity against Escherichia coli and Micrococcus luteus but weak activity against Pichia pastoris.     

A comparison of the enzymatic properties of three recombinant isoforms of thrombolytic and antibacterial protein—Destabilase-Lysozyme from medicinal leech

Destabilase-Lysozyme (mlDL) is a multifunctional i-type enzyme that has been found in the secretions from the salivary glands of medicinal leeches. mlDL has been shown to exhibit isopeptidase, muramidase and antibacterial activity. This enzyme attracts interest because it expresses thrombolytic activity through isopeptidolysis of the ε-(γ-Glu)-Lys bonds that cross-link polypeptide chains in stabilised fibrin. To date, three isoforms of mlDL have been identified. The enzymatic properties of pure mlDL isoforms have not yet been described because only destabilase complexes containing other proteins could be isolated from the salivary gland secretion and because low product yield from the generation of recombinant proteins has made comprehensive testing difficult. In the present study, we optimised the procedures related to the expression, isolation and purification of active mlDL isoforms (mlDL-Ds1, mlDL-Ds2, mlDL-Ds3) using an Escherichia coli expression system, and we detected and compared their muramidase, lytic, isopeptidase and antimicrobial activities. After optimisation, the product yield was 30 mg per litre of culture. The data obtained in our study led to the suggestion that the recombinant mlDL isoforms isolated from inclusion bodies form stable oligomeric complexes. Analyses of the tested activities revealed that all isoforms exhibited almost identical patterns of pH and ionic strength effects on the activities. We determined that mlDL-Ds1, 2, 3 possessed non-enzymatic antibacterial activity independent of their muramidase activity. For the first time, we demonstrated the fibrinolytic activity of the recombinant mlDL and showed that only intact proteins possessed this activity, suggesting their enzymatic nature. The recombinant Destabilase-Lysozyme isoforms obtained in our study may be considered potential thrombolytic agents that act through a mechanism different from that of common thrombolytics.     

Antibiotic activities of host defense peptides: more to it than lipid bilayer perturbation

Defensins are small basic amphiphilic peptides (up to 5 kDa) that have been shown to be important effector molecules of the innate immune system of animals, plants and fungi. In addition to immune modulatory functions, they have potent direct antimicrobial activity against a broad spectrum of bacteria, fungi and/or viruses, which makes them promising lead compounds for the development of next-generation antiinfectives. The mode of antibiotic action of defensins was long thought to result from electrostatic interaction between the positively charged defensins and negatively charged microbial membranes, followed by unspecific membrane permeabilization or pore-formation. Microbial membranes are more negatively charged than human membranes, which may explain to some extent the specificity of defensin action against microbes and associated low toxicity for the host. However, research during the past decade has demonstrated that defensin activities can be much more targeted and that microbe-specific lipid receptors are involved in the killing activity of various defensins. In this respect, human, fungal and invertebrate defensins have been shown to bind to and sequester the bacterial cell wall building block lipid II, thereby specifically inhibiting cell wall biosynthesis. Moreover, plant and insect defensins were found to interact with fungal sphingolipid receptors, resulting in fungal cell death. This review summarizes the current knowledge on the mode of action and structure of defensins from different kingdoms, with specific emphasis on their interaction with microbial lipid receptors.     

Isatin‐1,2,3‐triazole‐isatin Scaffolds and Their Antibacterial Activity

Twelve novel isatin‐1,2,3‐triazole‐isatin scaffolds 5a – l were designed, synthesized, and assessed for their in vitro antibacterial activity in this paper. The preliminary results showed that all dimers only endowed with weak antibacterial activity, which was less active than the reference ciprofloxacin. However, the structure–activity relationship was enriched, which may be useful for the further development of more efficient candidates.     

Microwave Assisted Synthesis of N-Aryl-N'-[5-(4- Chlorophenyl)-2-Furoyl]-Thioureas And Ureas

Substituted thioureas have attracted much attention due to their herbicidal1, antibacterial2, anti-HIV3 and plant-growth regulating4 activity. Meanwhile substituted ureas are not only used as medicines and agrochemicals because of their antiinflammatory5, analgesic5 and insectcidal6 activity, but also used as intermediates for the synthesis of many important heterocyclic compounds. In addition, 5-aryl-2-furoic acid derivatives have been used as antibacterial agent7, local anesthesia8, analgesic9 and plant-growth regulator10. Therefore, with the objective of obtaining new biologically active compounds, it is necessary to investigate the convenient and efficient method to prepare new compounds bearing 5-aryl-2-furoyl and thiourea or 5-aryl-2-furoyl and urea moieties.     

Synthesis,Characterization, and Antimicrobial Screening of 4″‐methyl‐2,2″‐diaryl‐4,2′:4′,5″‐terthiazole Derivatives

A series of novel 4″‐methyl‐2,2″‐diaryl‐4,2′:4′,5″‐terthiazole ( 8a‐p ) derivatives has been synthesized and screened for antibacterial activity against four pathogenic bacteria, Escherichia coli, Pseudomonas flurescence, Staphylococcus aureus, and Bacillus subtilis. Among them, compounds 8a and 8j exhibited excellent antibacterial activity with minimum inhibitory concentration range of 1.0 to 5.3 μg/mL and compounds 8m and 8p exhibited moderate to good antibacterial activity with minimum inhibitory concentration range of 16.9 to 29.7 μg/mL against all tested strains. All the synthesized compounds were screened for their in vitro antifungal activity against Cocinida candida. Most of the compounds reported moderate antifungal activity. This study provides valuable directions to our ongoing endeavor of rationally designing more potent antimicrobial agent.     

Synthesis of modified peptidoglycan precursor analogues for the inhibition of glycosyltransferase

The peptidoglycan glycosyltransferases (GTs) are essential enzymes that catalyze the polymerization of glycan chains of the bacterial cell wall from lipid II and thus constitute a validated antibacterial target. Their enzymatic cavity is composed of a donor site for the growing glycan chain (where the inhibitor moenomycin binds) and an acceptor site for lipid II substrate. In order to find lead inhibitors able to fill this large active site, we have synthesized a series of substrate analogues of lipid I and lipid II with variations in the lipid, the pyrophosphate, and the peptide moieties and evaluated their biological effect on the GT activity of E. coli PBP1b and their antibacterial potential. We found several compounds able to inhibit the GT activity in vitro and cause growth defect in Bacillus subtilis . The more active was C16-phosphoglycerate-MurNAc-(L-Ala-D-Glu)-GlcNAc, which also showed antibacterial activity. These molecules are promising leads for the design of new antibacterial GT inhibitors.     

Isoflavanones from the heartwood of Swartzia polyphylla and their antibacterial activity against cariogenic bacteria.

The methanolic extract of Swartzia polyphylla DC. heartwood had antibacterial activity against cariogenic bacteria, the mutans Streptococci. The chromatographic purification of the extract afforded seven flavonoids. Among them, three known isoflavanones, dihydrobiochanin A, ferreirin and darbergioidin, and one new isoflavanone, 5,2',4'-trihydroxy-7-methoxyisoflavanone (dihydrocajanin) had potent antibacterial activity against cariogenic bacteria. This effect was not detected on isoflavone derivatives. A comparative antibacterial study of various flavonoids was further performed, and their structure-activity relationship was discussed.     

Bio-Organometallic Derivatives of Antibacterial Drugs

Overuse and misuse of antibacterial drugs has resulted in bacteria resistance and in an increase in mortality rates due to bacterial infections. Therefore, there is an imperative necessity of new antibacterial drugs. Bio-organometallic derivatives of antibacterial agents offer an opportunity to discover new active antibacterial drugs. These compounds are well-characterized products and, in several examples, their antibacterial activities have been studied. Both inhibition of the antibacterial activity and strong increase in the antibiotic activity of the parent drug have been found. The synthesis of the main classes of bio-organometallic derivatives of these drugs, as well as examples of the use of structure–activity relation (SAR) studies to increase the activity and to understand the mode of action of bio-organometallic antimicrobial peptides (BOAMPs) and platensimicyn bio-organometallic mimics is presented in this article.     

Design,Synthesis, Antibacterial Evaluations and In Silico Studies of Novel Thiosemicarbazides and 1,3,4-Thiadiazoles

The emergence of drug-resistant bacterial strains continues to be one of the major challenges of medicine. For this reason, the importance of searching for novel structures of antibacterial drugs chemically different from the currently known antibiotics is still of great importance. In this study, we synthesized the thiosemicarbazide and 1,3,4-thiadiazole derivatives and tested them for antibacterial activity. In in vitro tests, we examined the activity of the synthesized substances against Gram-positive and Gram-negative bacteria strains. While all 1,3,4-thiadiazoles tested lacked significant activity, the antimicrobial response of the thiosemicarbazides was moderate and it was also dependent on the type and position of the substituent on the phenyl ring. The highest activity towards all Gram-positive bacteria strains was shown by all three linear compounds containing the trifluoromethylphenyl group in the structure. The MIC (minimum inhibitory concentration) values were in the range of 3.9–250 µg/mL. Additionally, we try to explain the mechanism of the antibacterial activity of the tested compounds using the molecular docking to DNA gyrase and topoisomerase IV, following previous reports on the molecular basis of the activity of thiosemicarbazides. Docking simulations allow the purposing dual mechanism of the antibacterial activity of the synthesized compounds through inhibition of topoisomerase IV DNA gyrase with the moderate prevalence of the topoisomerase pathway.     

Synthesis,biological evaluation,and molecular docking study of pyridine clubbed 1,3,4-oxadiazoles as potential antituberculars

A series of pyridine clubbed 1,3,4-oxadiazole derivatives were efficiently synthesized, characterized by standard spectral techniques and evaluated for their in vitro antitubercular activity against Mycobacterium tuberculosis (MTB) H₃₇Ra and Mycobacterium bovis BCG in active and dormant state using an established methods. Compounds 5a, 5m, and 5t were identified as the most active compounds against MTB. Molecular docking was performed against MTB enoyl-ACP (CoA) reductase (FabI/ENR/InhA) enzyme to predict the binding modes and affinity. The theoretical predictions from molecular docking could establish a link between the observed biological activity and the binding affinity shedding light into specific bonded and non-bonded interactions influencing the activity. The active compounds were studied for cytotoxicity against three cell lines and were found to be non-cytotoxic. Specificity of these compounds was checked by screening them for their antibacterial activity against four bacterial strains.     

Synthesis and antibacterial activity of some novel pyrazolopyridine derivatives

Eight pyrazolo[3,4-b]pyridine derivatives have been synthesized by Friedl?nder condensation of 5-aminopyrazole-4-carbaldehyde with active methylene compounds in basic medium. These compounds have been screened for their antibacterial activity against two Gram-negative and two Gram-positive bacterium. Pyrazolopyridines having the carboxamide group at the 5-position showed moderate to good activity against P. aeruginosa, E. coli, S. pneumoniae, and B. cereus.     

Synthesis,Crystal Structures,Lipophilic Properties and Antimicrobial Activity of 5-Pyridylmethylidene-3-rhodanine-carboxyalkyl Acids Derivatives

The constant increase in the resistance of pathogenic bacteria to the commonly used drugs so far makes it necessary to search for new substances with antibacterial activity. Taking up this challenge, we obtained a series of rhodanine-3-carboxyalkyl acid derivatives containing 2- or 3- or 4-pyridinyl moiety at the C-5 position. These compounds were tested for their antibacterial and antifungal activities. They showed activity against Gram-positive bacteria while they were inactive against Gram-negative bacteria and yeast. In order to explain the relationship between the activity of the compounds and their structure, for selected derivatives crystal structures were determined using the X-ray diffraction method. Modeling of the isosurface of electron density was also performed. For all tested compounds their lipophilicity was determined by the RP-TLC method and by calculation methods. On the basis of the carried-out research, it was found that the derivatives with 1.5 N···S electrostatics interactions between the nitrogen atom in the pyridine moiety and the sulfur atom in the rhodanine system showed the highest biological activity.     

Thiazolidin-4-Ones as Potential Antimicrobial Agents: Experimental and In Silico Evaluation

Herein, we report computational and experimental evaluations of the antimicrobial activity of twenty one 2,3-diaryl-thiazolidin-4-ones. All synthesized compounds exhibited an antibacterial activity against six Gram-positive and Gram-negative bacteria to different extents. Thus, the MIC was in the range of 0.008–0.24 mg/mL, while the MBC was 0.0016–0.48 mg/mL. The most sensitive bacterium was S. Typhimurium, whereas S. aureus was the most resistant. The best antibacterial activity was observed for compound 5 (MIC at 0.008–0.06 mg/mL). The three most active compounds 5, 8, and 15, as well as compound 6, which were evaluated against three resistant strains, MRSA, P. aeruginosa, and E. coli, were more potent against all bacterial strains used than ampicillin. The antifungal activity of some compounds exceeded or were equipotent with those of the reference antifungal agents bifonazole and ketoconazole. The best activity was expressed by compound 5. All compounds exhibited moderate to good drug-likeness scores ranging from −0.39 to 0.39. The docking studies indicated a probable involvement of E. coli Mur B inhibition in the antibacterial action, while CYP51 inhibition is likely responsible for the antifungal activity of the tested compounds. Finally, the assessment of cellular cytotoxicity of the compounds in normal human MRC-5 cells revealed that the compounds were not toxic.     

Pentacyclic Cytochalasins and Their Derivatives from the Endophytic Fungus Phomopsis sp. xz-18

Eight new cytochalasins 1–8 and ten known analogs 9–18 were isolated from the endophytic fungus Phomopsis sp. xz-18. The planar structures of the cytochalasins were determined by HR-ESI-MS and NMR analysis. Compounds 1, 2, 9 and 10 were 5/6/6/7/5-fused pentacyclic cytochalasins; compounds 3 and 4 had conjugated diene structures in the macrocycle; and compound 6 had a β,γ-unsaturated ketone. The absolute configuration of 6 was confirmed for the first time by the octant rule. The acid-free purification process proved that the pentacyclic system was a natural biosynthetic product and not an acid-mediated intramolecular cyclized artifact. The new compounds did not exhibit activities against human cancer cell lines in cytotoxicity bioassays or antipathogenic fungal activity, but compounds 1, 3 and 4 showed moderate antibacterial activity in disk diffusion assays.     

Antibacterial Activities of Prenylated Isoflavones from Maclura tricuspidata against Fish Pathogenic Streptococcus: Their Structure-Activity Relationships and Extraction Optimization

Streptococcus zoonotic bacteria cause serious problems in aquaculture with clinical effects on humans. A structure-antibacterial activity relationships analysis of 22 isoflavones isolated from M. tricuspidata (leaves, ripe fruits, and unripe fruits) against S. iniae revealed that prenylation of the isoflavone skeleton was an important key for their antibacterial activities (minimum inhibitory concentrations: 1.95–500 μg/mL). Through principal component analysis, characteristic prenylated isoflavones such as 6,8-diprenlygenistein (4) were identified as pivotal compounds that largely determine each part’s antibacterial activities. M. tiricuspidata ripe fruits (MTF), which showed the highest antibacterial activity among the parts tested, were optimized for high antibacterial activity and low cytotoxicity on fathead minnow cells using Box–Behnken design. Optimized extraction conditions were deduced to be 50%/80 °C/7.5 h for ethanol concentration/extraction temperature/time, and OE-MTF showed contents of 6,8-diprenlygenistein (4), 2.09% with a MIC of 40 µg/mL. These results suggest that OE-MTF and its active isoflavones have promising potential as eco-friendly antibacterial agents against streptococcosis in aquaculture.