The mode of action and the structure of a herbicide in complex with its target: binding of activated hydantocidin to the feedback regulation site of adenylosuccinate synthetase.

(+)-Hydantocidin, a recently discovered natural spironucleoside with potent herbicidal activity, is shown to be a proherbicide that, after phosphorylation at the 5' position, inhibits adenylosuccinate synthetase, an enzyme involved in de novo purine synthesis. The mode of binding of hydantocidin 5'-monophosphate to the target enzyme was analyzed by determining the crystal structure of the enzyme-inhibitor complex at 2.6-A resolution. It was found that adenylosuccinate synthetase binds the phosphorylated compound in the same fashion as it does adenosine 5'-monophosphate, the natural feedback regulator of this enzyme. This work provides the first crystal structure of a herbicide-target complex reported to date.

The mode of action of aspirin-like drugs: effect on inducible nitric oxide synthase.

Nitric oxide synthesized by inducible nitric oxide synthase (iNOS) has been implicated as a mediator of inflammation in rheumatic and autoimmune diseases. We report that exposure of lipopolysaccharide-stimulated murine macrophages to therapeutic concentrations of aspirin (IC50 = 3 mM) and hydrocortisone (IC50 = 5 microM) inhibited the expression of iNOS and production of nitrite. In contrast, sodium salicylate (1-3 mM), indomethacin (5-20 microM), and acetaminophen (60-120 microM) had no significant effect on the production of nitrite at pharmacological concentrations. At suprapharmacological concentrations, sodium salicylate (IC50 = 20 mM) significantly inhibited nitrite production. Immunoblot analysis of iNOS expression in the presence of aspirin showed inhibition of iNOS expression (IC50 = 3 mM). Sodium salicylate variably inhibited iNOS expression (0-35%), whereas indomethacin had no effect. Furthermore, there was no significant effect of these nonsteroidal anti-inflammatory drugs on iNOS mRNA expression at pharmacological concentrations. The effect of aspirin was not due to inhibition of cyclooxygenase 2 because both aspirin and indomethacin inhibited prostaglandin E2 synthesis by > 75%. Aspirin and N-acetylimidazole (an effective acetylating agent), but not sodium salicylate or indomethacin, also directly interfered with the catalytic activity of iNOS in cell-free extracts. These studies indicate that the inhibition of iNOS expression and function represents another mechanism of action for aspirin, if not for all aspirin-like drugs. The effects are exerted at the level of translational/posttranslational modification and directly on the catalytic activity of iNOS.

Chemistry and mode of action of insecticides /

"R-801837."

The mode of action of the plant antimicrobial peptide MiAMP1 differs from that of its structural homologue, the yeast killer toxin WmKT

The plant antimicrobial peptide MiAMP1 from Macadamia integrifolia and the yeast killer toxin peptide WmKT from Williopsis mrakii are structural homologues. Comparative studies of yeast mutants were performed to test their sensitivity to these two antimicrobial peptides. No differences in susceptibility to MiAMP1 were detected between wild-type and several WmKT-resistant mutant yeast strains. A yeast mutant MT1, resistant to MiAMP1 but unaffected in its susceptibility to plant defensins and hydrogen peroxide, also did not show enhanced tolerance towards WmKT. It is therefore probable that the Greek key beta-barrel structure shared by MiAMP1 and WmKT provides a robust structural framework ensuring stability for the two proteins but that the specific action of the peptides depends on other motifs. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Studies on the mode of action and beta-lactamase inhibitory properties of novel oxapenem compounds

Four novel oxapenem compounds were evaluated for their ß-lactamase inhibitory and antibacterial properties. Two (AM-112 and AM-113) displayed intrinsic antibacterial activity with MICs of between 2 to 16µg/ml and 0.5-2µg/ml against Escherichia coli and methicillin-sensitive and -resistant Staphylococcus aureus, respectively. The isomers of these compounds, AM-115 and AM-114 did not display significant antibacterial activity. Combination of the oxapenems with ceftazidime afforded protection against ß-lactamase-producing strains, including hyperproducers of class C enzymes and extended-spectrum ß-lactamase enzymes. A fixed 4µg/ml concentration of AM-112 protected a panel of eight cephalosporins against hydrolysis by class A and class C ß-lactamase producers. In vivo studies confirmed the protective effect of AM-112 for ceftazidime against ß-lactamase producing S. aureus, Enterobacter cloacae and E. coli strains in a murine intraperitoneal infection model. Each of the oxapenems inhibited class A, class C and class D ß-lactamases isolated from whole cells and purified by isoelectric focusing. AM-114 and AM-115 were as effective as clavulanic acid against class A enzymes. AM-112 and AM-113 were less potent against these enzymes. Class C and class D enzymes proved very susceptible to inhibition by the oxapenems. Molecular modelling of the oxapenems in the active site of the class A. TEM-1 and class C P99 enzymes identified a number of potential sites of interaction. The modelling suggested that Ser-130 in TEM-1 and Tyr-150 in P99 were likely candidates for cross-linking of the inhibitor, leading to inhibition of the enzyme. Morphology studies indicated that sub-inhibitory concentrations of the oxapenems caused the formation of round-shaped cells in E. coli DC0, indicating inhibition of penicillin-binding protein 2 (PBP2). The PBP affinity profile of AM-112 was examined in isolated cell membranes of E. coli DC0, S. aureus NCTC 6571, Enterococcus faecalis SFZ and E. faecalis ATCC 29213, in competition with a radiolabelled penicillin. PBP2 was identified as the primary target for AM-112 in E. coli DC0. Studies on S. aureus NCTC 6571 failed to identify a binding target. AM-112 bound to all the PBPs of both E. faecalis strains, and a concentration of 10µg/ml inhibited all the PBPs except PBP3.

The mode of action of a lipid mobilising factor in cancer cachexia

Cachexia is characterised by a progressive weight loss due to depletion of both skeletal muscle and adipose tissue. The loss of adipose tissue is due to the production of a tumour-derived lipid mobilising factor (LMF), which has been shown to directly induce lipolysis in isolated epididymal murine white adipocytes. The administration of LMF to a non-tumour bearing mice produced a rapid weight loss, with a specific reduction in carcass lipid with also some redistribution of lipid with the accumulation of lipid in the liver. There was also up-regulation of uncoupling protein-1 and -2 mRNA and protein expression in brown adipose tissue, suggesting that an adaptive process occurs due to increased energy mobilisation. There was also up-regulation of UCP-2 in the livers of LMF treated mice, suggesting a protective mechanism to the build up of lipid in the livers, which would produce free radical by-products. LMF was also shown to stimulate cyclic AMP production in CHO-K1 cells transfected with human -3 adrenergic receptors and inhibited by the -β3 antagonist SR59230A. LMF binding was also inhibited by SR59230A in isolated receptors. This suggests that LMF mediates its effects through a β3 adrenergic receptor. There were also changes in glucose and fatty acid uptake in LMF treated mice, which suggests metabolic changes are occurring. The study suggests that a tumour derived lipolytic factor acts through the 3 adrenoceptor producing effects on lipid mobilisation, energy expenditure and glucose metabolism.

The mode of action of bradykinin

The action of bradykinin on transepithelial transfer of sodium and water in isolated rat jejunum and on smooth muscle contraction of rat terminal ileum has been investigated. (1) Bradykinin was shown to stimulate transfer at low control transfer, inhibit transfer at high control transfer and have no effect at intermediate transfer in rat jejunal sacs. Stimulation of transfer occurred only when bradykinin was in the serosal solutiun while inhibition of transfer occurred whether bradykinin was in the aerosal or mucosal solution. Bradykinin-induced stimulation of transfer was not affected by adrenalectomy, nephrectomy, combined adrenalectomy-nephrectomy,  nor maintenance on 1% saline drinking solution or low sodium diet pretreatment. Meclofenamic acid abolished the bradykinin-induced inhibition of water transfer while prostaglandins A1, E1 aud F2α all potentiated this action. Theophylline inhibited water transfer and potentiated the bradykinin-induced inhibition of water transfer. Cyclic AMP and dibutyryl cyclic AMP both inhibited water transfer and the bradykinin-induced inhibition of water transfer was potentiated by the latter. ( 2 ) Bradykinin-induced contractions of rat terminal ileum were little affected by hyoscine while those of acetylcholine were abolished. Anoxia reduced markedly responses tv bradykinin while those of acetylcholine were little affected . Theophylline reduced the responses of rat terminal ileum to bradykinin significantly more than those to acetylcholine. Aspirin and indomethacin reduced markedly the responses to bradykinin while not affecting those to acetylcholine and PGT2. Meslofenamic acid at a concentration of 3.4 µM blocked bradykinin-induced contractions but had no effect on those to acctylcholine, PGE2 or PGF2 and at a concentration of 17. 0 µM drastically reduced bradykinin responses but also reduced those to acetylcholine, PGE2 and PGF2α• Flufenamic acid drastically reduced responses to bradykinin while not affecting those to acetylcholine and PGE2 and slightly affecting those to PGF2α. Polyphloretin phosphate reduced responses to bradykinin, PGF2α and PGE2 but not acetylcholine . Diphloretin phosphate reduced responses to bradykinin, PGF2 and PGE2 in a dose dependent manner but not those to acetylcholine. SC 19220 , in a dose dependent manner, inhibited responses to bradykinin and PGE2 but not to acetylcholine and PGF2. 7 oxa - 13 -prostynoic acid non specifically reduced responses to acetylcholine, bradykinin and PGE2. Bradykinin, in the presence of SQ 20881 , increased the release of prostaglandin-like activity from rat terminal ileum and this was reduced or abolished in the presence of indomethacin, aspirin, meclofenamic acid or flufenamio acid. The extract of PG-like activity did not appear as PGE, PGA or PGFon TLC, but included a substance with similar mobility as 15-Keto-prosta-glandin E2.

Investigating the mode of action of AN5568, a novel therapeutic against African trypanosomiasis

No abstract available.

Biosynthesis and mode of action of ribosomally synthesized and post-translationally modified antimicrobial peptides

One of the greatest sources of biologically active compounds is natural products. Often these compounds serve as platforms for the design and development of novel drugs and therapeutics. The overwhelming amount of genomic information acquired in recent years has revealed that ribosomally synthesized and post-translationally modified natural products are much more widespread than originally anticipated. Identified in nearly all forms of life, these natural products display incredible structural diversity and possess a wide range of biological functions that include antimicrobial, antiviral, anti-inflammatory, antitumor, and antiallodynic activities. The unique pathways taken to biosynthesize these compounds offer exciting opportunities for the bioengineering of these complex molecules. The studies described herein focus on both the mode of action and biosynthesis of antimicrobial peptides. In Chapter 2, it is demonstrated that haloduracin, a recently discovered two-peptide lantibiotic, possesses nanomolar antimicrobial activity against a panel of bacteria strains. The potency of haloduracin rivals that of nisin, an economically and therapeutically relevant lantibiotic, which can be attributed to a similar dual mode of action. Moreover, it was demonstrated that this lantibiotic of alkaliphile origin has better stability at physiological pH than nisin. The molecular target of haloduracin was identified as the cell wall peptidoglycan precursor lipid II. Through the in vitro biosynthesis of haloduracin, several analogues of Halα were prepared and evaluated for their ability to inhibit peptidoglycan biosynthesis as well as bacterial cell growth. In an effort to overcome the limitations of in vitro biosynthesis strategies, a novel strategy was developed resulting in a constitutively active lantibiotic synthetase enzyme. This methodology, described in Chapter 3, enabled the production of fully-modified lacticin 481 products with proteinogenic and non-proteinogenic amino acid substitutions. A number of lacticin 481 analogues were prepared and their antimicrobial activity and ability to bind lipid II was assessed. Moreover, site-directed mutagenesis of the constitutively active synthetase resulted in a kinase-like enzyme with the ability to phosphorylate a number of peptide substrates. The hunt for a lantibiotic synthetase enzyme responsible for installing the presumed dehydro amino acids and a thioether ring in the natural product sublancin, led to the identification and characterization of a unique post-translational modification. The studies described in Chapter 4, demonstrate that sublancin is not a lantibiotic, but rather an unusual S-linked glycopeptide. Its structure was revised based on extensive chemical, biochemical, and spectroscopic characterization. In addition to structural investigation, bioinformatic analysis of the sublancin gene cluster led to the identification of an S-glycosyltransferase predicted to be responsible for the post-translational modification of the sublancin precursor peptide. The unprecedented glycosyltransferase was reconstituted in vitro and demonstrated remarkable substrate promiscuity for both the NDP-sugar co-substrate as well as the precursor peptide itself. An in vitro method was developed for the production of sublancin and analogues which were subsequently evaluated in bioactivity assays. Finally, a number of putative biosynthetic gene clusters were identified that appear to harbor the necessary genes for production of an S-glycopeptide. An additional S-glycosyltransferase with more favorable intrinsic properties including better expression, stability, and solubility was reconstituted in vitro and demonstrated robust catalytic abilities.

Candidacidal potential of licorice phenolic extract: emphasis on its mode of action

Medicinal plants bave gained a special attention in the last years, due to its renowned health benefits, such as antimicrobial effects [I]. In fact, several natural matrices bave been increasingly studied, namely for its antifungal activity against opportunistic fungi [2,3]. Candida species, although commensa! microorganisms, have caused severe organic dysfunctions to the host, once current antifungal agents have lost their recognized efficiency [2]. So, numerous studies have been carried out focusing the mechanisms of acquired drug-resistance by Candida species [of an efficient multi-drug therapy (i.e. chemical drugs and also natural extracts combination), the discovery of the involved mechanisms of actions, morphological changes and related kinetic parameters are of major importance. Glycyrrhiza glabra L. (licorice) hydromethanolic extract have evidenced promissory candidacidal effects, and therefore, the involved mechanisms of action need to be clarified. Thus, in the present study these modes of action were assessed, by using flow cytometry.Overall, the licorice extract induced significant and irreversible primary damages on Candida cells, being membrane disruption and consequent unviability one of the main targets. In fact, after membrane destabilization, cells lost their proper homeostasis, their metabolic functions were blocked and, consequently cells lost functionality. The relevance and interest of the achieved results open new insights towards the upcorning use of the present phenolic matrix, being important to evaluate its in viva efficacy. Therefore, further studies are necessary to deepen knowledge on this field, aiming not only to establish therapeutic and prophylactic doses, but also to improve the clinical intervention in Candida infections.

The use of insect cells to identify potent and selective inhibitors of the replication of the Dengue and Chikungunya viruses and unravel their molecular mechanism of action

Dengue and Chikungunya viruses cause the most important arthropod-borne viral infections for humans. These viruses are predominant in tropical and subtropical regions. In addition, these viruses are predominant in tropical and subtropical regions. Dengue mortality rate is around 1.2 to 3.5% and deaths due to chikungunya fever are around 1 in 1000; however, half of chikungunya-infected patients evolve into a chronic state that can persist for months up to years. There are no antiviral drugs available for DENV and CHIKV treatment and prevention. Moreover, vector control strategies have failed so far. Thus, the development of potent inhibitors for a broad spectrum of RNA viruses is urgently needed. We established and characterized a new embryonic insect cell line from Culex quinquefasciatus mosquito. Also we established the flaviviruses and alphavirus replication, both in C6/36 and Lulo insect cell lines, as well as in Vero cell line. In addition we carried out a reference compound library and reference panel of assays and data for DENV, which provides a benchmark for further studies. During this study, a panel of 9 antiviral molecules, with proven in vitro anti-dengue virus activity and that act at different stages of the DENV life cycle, was selected. Finally, Favipiravir or T-705, was identified as inhibitor in vitro and in vivo of alphaviruses and the mutation K291R in nsP4, which is responsible of the polymerase activity, was found as the mode of action in CHIKV. Interestingly, lysine in motif F1 is also highly conserved in positive-stranded RNA viruses and this might explain the broad spectrum of T-705 antiviral activity.