The Properties of Activated Carbons Functionalized with an Antibacterial Agent and a New SufA Protease Inhibitor.

S. aureus is the cause of many diseases, including numerous infections of the skin. One way to help combat skin infections is to use bandages containing activated carbon. Currently, there are no dressings on the market that use the synergistic effect of activated carbon and antibiotics. Thus, in this study, we point out the adsorption level of an antimicrobial substance on three different active carbons of different origins; by examining the inhibition level of the growth of S. aureus bacteria, we determined the number of live cells adsorbed on activated carbons depending on the presence of gentamicin in the solution. In addition, we designed and synthesized a new antibacterial substance with a new mechanism of action to act as a bacterial protease inhibitor, as well as determining the antibacterial properties conducted through adsorption. Our results demonstrate that activated carbons with adsorbed antibiotics show better bactericidal properties than activated carbon alone or the antibiotic itself. The use of properly modified activated carbons may have a beneficial effect on the development and functioning of new starting materials for bacteria elimination, e.g., in wound-healing treatments in the future.

Antibacterial Agents Adsorbed on Active Carbon: A New Approach for S. aureus and E. coli Pathogen Elimination.

Antibiotic overuse and mass production have led to a global problem with the treatment of antibacterial infections. Thus, any possibility to limit the number of antibacterial drugs used will contribute to a decrease in the development of pathogenic bacterial resistance. In this study, the enhanced bacterial growth reduction of pharmaceutical activated carbon (PAC) material with adsorbed antimicrobial agents compared to the activity of pure antibacterial drugs was investigated. Sulfamethoxazole (SMZ) at a concentration of 1.1 mg/mL retained the growth of S. aureus and E. coli at 20.5% and 26.5%, respectively, whereas SMZ adsorbed on PAC increased the reduction of the tested bacteria in the range of 47-72%. The use of PAC with adsorbed gentamycin (G) over 24 h improved the effectiveness of E. coli growth reduction by 50% compared to the application of pure antibiotic (3.6 µg/mL). The increased reduction of S. aureus growth by 6% using G with PAC for a 24-h incubation time compared to the use of pure antibiotics at a concentration of 3.6 µg/mL was observed. The results provide proof-of-principle that the new approach of activated carbon with adsorbed antimicrobial agents could yield an attractive background with potential as a new starting material for S. aureus and E. coli pathogen elimination, e.g., in wound-healing treatment in the future.

A rapid method for protein staining in polyacrylamide gels using water saturated with chloroform.

Polyacrylamide gel electrophoresis, followed by an appropriate staining, is a popular and useful analytical procedure for protein identification and characterization. The aim of this study was to develop a method for protein visualization in polyacrylamide gels that would be alternative to Coomassie blue or silver staining. The proposed method is simple, fast and inexpensive. The optimized protocol for protein staining and visualization takes as little as 6 minutes and utilizes deionized water and chloroform. Fluorescence of proteins is induced by UV light and can be detected with a standard transilluminator.

Effective Small Molecule Antibacterials from a Novel Anti-Protein Secretion Screen.

The increasing problem of bacterial resistance to antibiotics underscores the urgent need for new antibacterials. Protein export pathways are attractive potential targets. The Sec pathway is essential for bacterial viability and includes components that are absent from eukaryotes. Here, we used a new high-throughput in vivo screen based on the secretion and activity of alkaline phosphatase (PhoA), a Sec-dependent secreted enzyme that becomes active in the periplasm. The assay was optimized for a luminescence-based substrate and was used to screen a ~240K small molecule compound library. After hit confirmation and analoging, 14 HTS secretion inhibitors (HSI), belonging to eight structural classes, were identified with IC50 < 60 µM. The inhibitors were evaluated as antibacterials against 19 Gram-negative and Gram-positive bacterial species (including those from the WHO's top pathogens list). Seven of them-HSI#6, 9; HSI#1, 5, 10; and HSI#12, 14-representing three structural families, were bacteriocidal. HSI#6 was the most potent hit against 13 species of both Gram-negative and Gram-positive bacteria with IC50 of 0.4 to 8.7 µM. HSI#1, 5, 9 and 10 inhibited the viability of Gram-positive bacteria with IC50 ~6.9-77.8 µM. HSI#9, 12, and 14 inhibited the viability of E. coli strains with IC50 < 65 µM. Moreover, HSI#1, 5 and 10 inhibited the viability of an E. coli strain missing TolC to improve permeability with IC50 4 to 14 µM, indicating their inability to penetrate the outer membrane. The antimicrobial activity was not related to the inhibition of the SecA component of the translocase in vitro, and hence, HSI molecules may target new unknown components that directly or indirectly affect protein secretion. The results provided proof of the principle that the new broad HTS approach can yield attractive nanomolar inhibitors that have potential as new starting compounds for optimization to derive potential antibiotics.

Determination of mechanisms of action of active carbons as a feed additive.

Activated carbon's porous structure allows it to adsorb a substrates, products and catalysts from the environment thus modificated the biocatalysis processes in digestive tract. Active carbons are currently used to remove solvents from gas streams and for water purification; however, few studies have examined the mechanisms of action of active carbon during the biotransformation processes in the digestive tracks. The potential benefits of using activated carbon in feed are uncertain because both its chemical and physical properties can vary significantly depending on the type of carbonaceous feedstock. However, the use of active carbons as dietary supplements can also bring many benefits during biotransformation processes in the gastrointestinal tract. Active carbons can adsorb toxins from the gastrointestinal tract and reduce excessive intestinal gas accumulation. The study concerning the adsorption of bacteria and vitamins on the porous structure of various species of active carbons is an important factor to determine their mechanism of action in biocatalysis in digestive system. The use of properly modified activated carbons as feed additives may have a beneficial effect on the development and functioning of breeding animals in the future. The results of our research show that the active carbon obtained from beech (KB), which contained, on average, 14% oxygen content by weight adsorbed bacteria, such as E. coli and S. aureus, better than all the other active carbons tested. Moreover, the meso- and macropores of carbon seem to contribute little to bacterial adsorption by active carbons. The electron microscopy studies confirmed that the bacteria adhered mainly to the active carbon surface. Our results also indicate that the examined active carbons from beech (KB), coconut shells (TE50), and hard coal (RB2) do not adsorb (or adsorb with very limited efficiency) the vitamins that are routinely added to feed, such as A, B1, D, and K. Broilers fed with feed mixtures supplemented with activated carbon (KB) resulted in increases in the weight of the chickens (∼2%) after 14 days of application and 2% lower feed consumption (conversion) relative to a control sample. Our data indicate that modifying the surface area and elementary content of active carbon may affect its specificity and selectivity and its capacity to absorb particles used in veterinary, human pharmacy, and cosmetology.

New diaryl ω-(isothiocyanato)alkylphosphonates and their mercapturic acids as potential antibacterial agents.

Thirty-four novel, diaryl ω-(isothiocyanato)alkylphosphonates with chlorine atom and methoxy, dimethoxy, methylsulfanyl, or methoxycarbonyl groups at ortho, meta, or para positions of the phenyl ring, and with an unbranched alkyl chain (n = 2-6) were designed and synthesized in a one-pot reaction in 11-76% yields. All isothiocyanates thus generated were evaluated for the first time for antibacterial activity on Pseudomonas aeruginosa and Staphylococcus aureus bacterial strains, and had satisfactory antibacterial activity in most cases. The highest activity, similar to that of reference gentamicin activity against S. aureus, was seen in compounds 9 and 13 (1.5 ±â€¯0.1 and 2.5 ±â€¯0.2 µM, respectively), whereas for P. aeruginosa more than half of tested compounds proved to be more effective than gentamicin. Additionally, selected isothiocyanates (9, 13, 18, and 23) were transformed in 52-73% yields into mercapturic acids 42-45, which also exhibited satisfactory antibacterial effect against S. aureus strain.

The Lord of the Bacteria: The Fellowship of the Leader and Other Serine Protease Inhibitors.

Since antibiotics use is currently limited due to undesired side effects and the increasing antibiotic resistance of various bacteria strains, there is a pressing need to develop new strategies and methods preventing epidemic outbreaks. The virulent potency of bacteria relies on a number of different extracellularly secreted factors among which proteases considered as promising, novel drug targets are of special interest. The first evidence that bacterial cysteine, serine and metalloproteinases contributed to the progression of infection was found in the early 70's. This extracellular proteolytic system allows bacteria to penetrate into tissues, escape detection by the host's immune mechanisms and grow despite limited access to nutrition. A molecule able to selectively inhibit the activity of bacterial proteases in the spread of infection may lead to designing novel therapeutics. Moreover, due to their mechanism of action, bacterial protease inhibitors can be used to fight antibiotic-resistant strains. Herein, we undertake a review of various bacterial proteases together with the design and development of their inhibitors (excluding ß-lactams) for the last ten years, and introduce the reader to a brief history of the subject.

The role of serine proteases in the pathogenesis of bacterial infections.

An increasing resistance of pathogenic bacterial species has been considered as one of the major health problems worldwide. The discovery of novel protein targets and development of effective anti-bacterial therapeutics is of high need since for some extremely resistant pathogens we are simply left unarmed. One of new promising therapeutic strategy is the application of specific inhibitors targeting bacterial serine proteases. Pathogenic microorganisms secrete abroad range of hydrolases, including serine proteases which lead to activation of various virulence factors. Herein, we review the specific bacteria serine proteases which have an influence on pathogenicity of bacterial infection as well as we introduce the reader with a brief history of the subject.

Synthesis of novel phosphonic-type activity-based probes for neutrophil serine proteases and their application in spleen lysates of different organisms.

Neutrophils are a type of granulocyte important in the "first line of defense" of the innate immune system. Upon activation, they facilitate the destruction of invading microorganisms by the production of superoxide radicals, as well as the release of the enzymatic contents of their lysozymes. These enzymes include specific serine proteases: cathepsin G, neutrophil elastase, proteinase 3, as well as the recently discovered neutrophil serine protease 4 (NSP4). Under normal conditions, the proteolytic activity of neutrophil proteases is tightly regulated by endogenous serpins; however, this mechanism can be subverted during tissue stress, thereby resulting in the uncontrolled activity of serine proteases, which induce chronic inflammation and subsequent pathology. Herein, we describe the development of low-molecular-weight activity-based probes that specifically target the active sites of neutrophil proteases.

Determination of cathepsin G in endometrial tissue using a surface plasmon resonance imaging biosensor with tailored phosphonic inhibitor.

OBJECTIVE: Cathepsin G is a serine peptidase whose physiological role is mainly associated with an early immune response, anti-microbial activity as well as platelet activation or hydrolysis of coagulation factors. In addition, since the activity of cathepsin G has been associated with the development of various pathological disorders, the measurement of its activity in patient samples is of high interest. Unfortunately, the usefulness of common immunological methods is limited, since they cannot distinguish between catalytically active and inactive protease. STUDY DESIGN: Here we present the application of recently developed Surface Plasmon Resonance-based biosensor for the detection of active cathepsin G in human endometrium samples. The key element of the system is based on the irreversible binding of cathepsin G to its specific phosphonic-type inhibitor immobilized on the surface of the gold chip. The concentration of cathepsin G was measured in tissue samples from the group of patients with endometriosis as well as in the control group. RESULTS: The level of cathepsin G ascertained in endometrium tissue samples was over twice as high for the group of patients suffering from endometriosis as compared to the control group, with the median values of 0.5 pmol/mg and 0.2 pmol/mg, respectively. CONCLUSION: The SPR sensor armed with a specific irreversible phosphonic inhibitor represents a highly useful tool for the determination of catalytically active cathepsin G concentration in endometrial tissue.

Substrate profiling of Finegoldia magna SufA protease, inhibitor screening and application to prevent human fibrinogen degradation and bacteria growth in vitro.

SufA, which belongs to the subtilisin-like serine protease family, contains a non-canonical Asp-His-Ser catalytic triad. Under in vitro conditions, SufA is capable of human fibrinogen hydrolysis leading to inhibition of fibrin network formation, thus suggesting its important role in the development and progression of Finegoldia magna infections. In addition, it has been demonstrated that SufA can hydrolyze antibacterial peptides such as LL-37 and the chemokine MIG/CXCL 9, hence evading host defence mechanisms. Although the SufA protease from F. magna was discovered several years ago, its optimal substrate preference has not yet been identified. Considering the role of SufA, we have focused on the profiling of its substrate sequence preference spanning S1-S3 binding pockets using the FRET (fluorescence resonance energy transfer) approach. Next, based on the structure of the P1 residue of the developed substrate, we narrowed the inhibitor screening to the phosphonic analogues of amino acids containing an arginine-like side chain. Among all the compounds tested, only Cbz-6-AmNphth(P)(OPh)2 showed any inhibitory activity against SufA displaying k2/Ki value of 10,800 M(-1) s(-1). In addition, it prevented SufA-mediated human fibrinogen hydrolysis in vitro and exhibited potent antibacterial activity against F. magna, Staphylococcus aureus and Escherichia coli. Herein, we report on the substrate specificity, synthesis and kinetic evaluation of phosphonic inhibitors of SufA protease from F. magna which could help to establish its function in pathogenesis development and may lead to the elaboration of new antibacterial drugs.

Development and binding characteristics of phosphonate inhibitors of SplA protease from Staphylococcus aureus.

Staphylococcus aureus is responsible for a variety of human infections, including life-threatening, systemic conditions. Secreted proteome, including a range of proteases, constitutes the major virulence factor of the bacterium. However, the functions of individual enzymes, in particular SplA protease, remain poorly characterized. Here, we report development of specific inhibitors of SplA protease. The design, synthesis, and activity of a series of α-aminoalkylphosphonate diaryl esters and their peptidyl derivatives are described. Potent inhibitors of SplA are reported, which may facilitate future investigation of physiological function of the protease. The binding modes of the high-affinity compounds Cbz-Phe(P) -(OC6 H4 -4-SO2 CH3 )2 and Suc-Val-Pro-Phe(P) -(OC6 H5 )2 are revealed by high-resolution crystal structures of complexes with the protease. Surprisingly, the binding mode of both compounds deviates from previously characterized canonical interaction of α-aminoalkylphosphonate peptidyl derivatives and family S1 serine proteases.

Phosphonic analogues of glutamic acid as irreversible inhibitors of Staphylococcus aureus endoproteinase GluC: an efficient synthesis and inhibition of the human IgG degradation.

Endoproteinase GluC (V8 protease) is one of many virulence factors released by the Staphylococcus aureus species in vivo. The V8 protease is able to hydrolyze some serpins and all classes of mammalian immunoglobulins. The application of specific and potent inhibitors of V8 protease may lead to the development of new antibacterial agents. Herein, we present the synthesis and the inhibitory properties of novel peptidyl derivatives of a phosphonic glutamic acid analogue. One of the compounds Boc-Phe-Leu-Glu(P)(OC(6)H(4))(2) displayed an apparent second-order inhibition rate value of 8540 M(-1)s(-1). The Boc-Phe-Leu-Glu(P)(OC(6)H(4))(2) compound with the highest inhibitory potency showed the ability to prevent V8-mediated human IgG proteolysis in vitro.