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Biomedical subjects

D M Schmatz

Publications and source records attributed to D M Schmatz.

At least 19 recordsLinked to original sources
PubMed2005

Anthrax lethal factor inhibition.

The primary virulence factor of Bacillus anthracis is a secreted zinc-dependent metalloprotease toxin known as lethal factor (LF) that is lethal to the host through disruption of signaling pathways, cell destruction, and circulatory shock. Inhibition of this proteolytic-based LF toxemia could be expected to provide therapeutic value in combination with an antibiotic during and immediately after an active anthrax infection. Herein is shown the crystal structure of an intimate complex between a hydroxamate, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, and LF at the LF-active site. Most importantly, this molecular interaction between the hydroxamate and the LF active site resulted in (i) inhibited LF protease activity in an enzyme assay and protected macrophages against recombinant LF and protective antigen in a cell-based assay, (ii) 100% protection in a lethal mouse toxemia model against recombinant LF and protective antigen, (iii) approximately 50% survival advantage to mice given a lethal challenge of B. anthracis Sterne vegetative cells and to rabbits given a lethal challenge of B. anthracis Ames spores and doubled the mean time to death in those that died in both species, and (iv) 100% protection against B. anthracis spore challenge when used in combination therapy with ciprofloxacin in a rabbit "point of no return" model for which ciprofloxacin alone provided 50% protection. These results indicate that a small molecule, hydroxamate LF inhibitor, as revealed herein, can ameliorate the toxemia characteristic of an active B. anthracis infection and could be a vital adjunct to our ability to combat anthrax.

Animals↗
PubMed2002

The antifungal echinocandin caspofungin acetate kills growing cells of Aspergillus fumigatus in vitro.

Caspofungin acetate is an antifungal antibiotic that inhibits synthesis of 1,3-beta-D-glucan, an essential component of the fungal cell wall. While caspofungin causes cell death in yeasts and dimorphic fungi such as Candida albicans, its effect on Aspergillus fumigatus is less well understood. We used the fluorescent dyes 5,(6)-carboxyfluorescein diacetate (CFDA) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC), which stain live and dead cells, respectively, to further characterize the antifungal activity of caspofungin. For comparison, compounds whose mode of action was either fungistatic (fluconazole, itraconazole) or fungicidal (amphotericin B) were also evaluated. A correlation between caspofungin-induced loss of viability, decreased CFDA staining, and increased DiBAC staining was established first with C. albicans. For A. fumigatus, caspofungin caused similar dye-staining changes, which were quantified by fluorimetric analysis of stained hyphae grown in a medium that promoted dispersed growth. The minimum concentration of caspofungin required to produce these changes also decreased the level of growth-dependent reduction of the indicator dye Alamar Blue. We observed a differential effect of caspofungin as a function of cell position: 88% of apical cells and 61% of subapical branching cells failed to stain with the viable dye CFDA, but only 24% of subapical cells were unstained. Complementary results were seen with germlings from DiBAC-stained, caspofungin-treated cultures. Extended incubation of A. fumigatus with a single dose of caspofungin affected the same proportion of apical and subapical branching cells for up to 72 h. The dye-staining patterns illustrate that the cells at the active centers for new cell wall synthesis within A. fumigatus hyphae are killed when they are exposed to caspofungin.

Anti-Bacterial Agents↗
PubMed2001

Synthesis of nodulisporic acid 2' '-oxazoles and 2' '-thiazoles.

[reaction--see text] The semisynthetic conversion of nodulisporic acid A (1) into a set of three heterocyclic side chain derivatives provided compounds, highlighted by 6, with an improved spectrum of ectoparasiticidal activity and pharmacokinetic profile relative to the natural product.

Animals↗
PubMed2001

Structure, histone deacetylase, and antiprotozoal activities of apicidins B and C, congeners of apicidin with proline and valine substitutions.

[structure: see text]. Isolation and structure elucidation of two novel cyclic tetrapeptides that show a variety of potent antiprotozoal activities by reversibly inhibiting HDAC have been reported. These are the new members of a unique family of cyclic tetrapeptides that do not require the electrophilic alpha-epoxyketone moiety of HC-toxin, trapoxin A, or chlamydocin for their potent activities against HDAC and the malarial parasite.

Amino Acid Substitution↗
PubMed2001

Quantitative PCR assay to measure Aspergillus fumigatus burden in a murine model of disseminated aspergillosis: demonstration of efficacy of caspofungin acetate.

Caspofungin acetate (MK-0991) is an antifungal antibiotic that inhibits the synthesis of 1,3-beta-D-glucan, an essential component of the cell wall of several pathogenic fungi. Caspofungin acetate was recently approved for the treatment of invasive aspergillosis in patients who are refractory to or intolerant of other therapies. The activity of 1,3-beta-D-glucan synthesis inhibitors against Aspergillus fumigatus has been evaluated in animal models of pulmonary or disseminated disease by using prolongation of survival or reduction in tissue CFU as assay endpoints. Because these methods suffer from limited sensitivity or poor correlation with fungal growth, we have developed a quantitative PCR-based (qPCR) (TaqMan) assay to monitor disease progression and measure drug efficacy. A. fumigatus added to naïve, uninfected kidneys as either ungerminated conidia or small germlings yielded a linear qPCR response over at least 4 orders of magnitude. In a murine model of disseminated aspergillosis, a burden of A. fumigatus was detected in each of five different organs at 4 days postinfection by the qPCR assay, and the mean fungal load in these organs was 1.2 to 3.5 log(10) units greater than mean values determined by CFU measurement. When used to monitor disease progression in infected mice, the qPCR assay detected an increase of nearly 4 log(10) conidial equivalents/g of kidney between days 1 and 4 following infection, with a peak fungal burden that coincided with the onset of significant mortality. Traditional CFU methodology detected only a marginal increase in fungal load in the same tissues. In contrast, when mice were infected with Candida albicans, which does not form true mycelia in tissues, quantitation of kidney burden by both qPCR and CFU assays was strongly correlated as the infection progressed. Finally, treatment of mice with induced disseminated aspergillosis with either caspofungin or amphotericin B reduced the A. fumigatus burden in infected kidneys to the limit of detection for the qPCR assay. Because of its much larger dynamic range, the qPCR assay is superior to traditional CFU determination for monitoring the progression of disseminated aspergillosis and evaluating the activity of antifungal antibiotics against A. fumigatus.

Amphotericin B↗
PubMed2001

Systemic efficacy of nodulisporic acid against fleas on dogs.

Nodulisporic acid A (NSA) is a novel natural product from a new structural class that was shown previously to have insecticidal activity against blowfly larvae. To determine if there was useful systemic efficacy against fleas (Ctenocephalides felis). NSA was evaluated in an artificial membrane flea feeding device and in dogs. In the artificial membrane flea feeding device, adult C. felis were allowed to feed on bovine blood containing various concentrations of NSA through a Parafilm membrane. NSA killed the fleas with a 50% lethal concentration of 0.68 microg/ml and was approximately 10-fold more potent than the systemic insecticide ivermectin. In the initial probe dog test, a single beagle was challenged with 100 C. felis before oral dosing with 15 mg/kg of NSA. Flea counts conducted at 72 hr postdosing showed an 88% reduction relative to control. Re-challenge of the same dog at 5 days postdosing showed 50% reduction of fleas at day 7, demonstrating some residual flea activity. In a confirmatory study, 8 dogs were challenged with 100 fleas just before oral dosing with 15 mg/kg of NSA (4 dogs) or vehicle (4 dogs). There was 99% reduction of fleas at 48 hr postdosing in the NSA-treated dogs relative to control. Additional challenges with 100 fleas were performed on these 8 dogs at 48-hr intervals to determine the duration of efficacy, and there was 97, 51, and 0% reduction of fleas relative to control on days 4, 6, and 8, respectively. No adverse effects were observed in the dogs in these studies. These data show that NSA has potent oral activity in the dog for the control of fleas, while lacking overt mammalian toxicity.

Administration, Oral↗
PubMed2001

Systemic efficacy of nodulisporamides against fleas on dogs.

Nodulisporic acid A (NSA) has been shown previously to be safe in dogs and to deliver >90% flea control for 4 days following a single oral administration. Three newly prepared nodulisporamide derivatives were subsequently identified from an artificial membrane flea feeding system as exhibiting potency substantially greater than NSA. To determine if they have superior in vivo activity, these 3 nodulisporamides, as well as NSA, were evaluated in dogs at 15 mg/kg/os. Parasite challenges were made by placing 100 live Ctenocephalides felis fleas onto the dorsum of dogs every 48 hr and examining efficacy at each of those intervals over a 22-day period. Results showed that NSA produced >90% efficacy at day 2 and 81% efficacy at day 4, and its residual flea killing fell to approximately 50% by day 6 posttreatment. All dogs treated with the 3 new experimental nodulisporamides were 100% protected from flea challenges to day 8 posttreatment, and 2 of the compounds continued to produce >90% residual activity to 2 wk posttreatment. Pharmacokinetic analysis showed that plasma profiles and half-lives of NSA and these 3 new compounds correlated closely with flea efficacy. These results demonstrate that specific substitutions to the pharmacophore of NSA can substantially increase the duration of activity against fleas.

Administration, Oral↗
PubMed2001

Nitrophenide (Megasul) blocks Eimeria tenella development by inhibiting the mannitol cycle enzyme mannitol-1-phosphate dehydrogenase.

Unsporulated oocysts of the protozoan parasite Eimeria tenella contain high levels of mannitol, which is thought to be the principal energy source for the process of sporulation. Biosynthesis and utilization of this sugar alcohol occurs via a metabolic pathway known as the mannitol cycle. Here, results are presented that suggest that 3-nitrophenyl disulfide (nitrophenide, Megasul), an anticoccidial drug commercially used in the 1950s, inhibits mannitol-1-phosphate dehydrogenase (M1PDH), which catalyzes the committed enzymatic step in the mannitol cycle. Treatment of E. tenella-infected chickens with nitrophenide resulted in a 90% reduction in oocyst shedding. The remaining oocysts displayed significant morphological abnormalities and were largely incapable of further development. Nitrophenide treatment did not affect parasite asexual reproduction, suggesting specificity for the sexual stage of the life cycle. Isolated oocysts from chickens treated with nitrophenide exhibited a dose-dependent reduction in mannitol, suggesting in vivo inhibition of parasite mannitol biosynthesis. Nitrophenide-mediated inhibition of MIPDH was observed in vitro using purified native enzyme. Moreover, MIPDH activity immunoprecipitated from E. tenella-infected cecal tissues was significantly lower in nitrophenide-treated compared with untreated chickens. Western blot analysis and immunohistochemistry showed that parasites from nitrophenide-treated and untreated chickens contained similar enzyme levels. These data suggest that nitrophenide blocks parasite development at the sexual stages by targeting M1PDH. Thus, targeting of the mannitol cycle with drugs could provide an avenue for controlling the spread of E. tenella in commercial production facilities by preventing oocyst shedding.

Animals↗
PubMed2000

Synthesis of apicidin-derived quinolone derivatives: parasite-selective histone deacetylase inhibitors and antiproliferative agents.

Apicidin's indole was efficiently converted into a series of N-substituted quinolone derivatives by indole N-alkylation followed by a two-step, one-pot, ozonolysis/aldol condensation protocol. The new quinolones exhibited good parasite selectivity and potency both at the level of their molecular target, histone deacetylase, and in their whole cell antiproliferative activity in vitro.

Animals↗
PubMed1999

Development of a scintillation proximity assay for histone deacetylase using a biotinylated peptide derived from histone-H4.

Measurement of histone deacetylase activity is usually accomplished by incubation of the enzyme(s) with acetate-radiolabeled histones or synthetic peptides based on histone sequences, followed by extraction and quantification of released radiolabeled acetic acid. Consequently, this assay is both time consuming and extremely limiting when large numbers of samples are involved. We have now developed a simple, two-step histone deacetylase assay that is based on the scintillation proximity assay (SPA) principle. A biotinylated [3H]acetyl histone H4 peptide substrate was synthesized and shown to generate a radioactive signal upon binding to streptavidin-coated SPA beads. Incubation of biotinylated [3H]acetyl peptide with HeLa nuclear extract (source of histone deacetylase) resulted in a time- and protein-dependent decrease in the SPA signal, providing a measure of enzyme activity. The histone deacetylase-mediated decrease in SPA counts was accompanied by a proportional appearance in free 3H-labeled acetate in the assay mixture. Histone deacetylase activity measured by SPA was concordant with that determined via the traditional ethyl acetate extraction procedure. Furthermore, a broad range of histone deacetylase inhibitors was demonstrated to have comparable effects on the catalytic activity of the HeLa nuclei enzyme using both assays. The histone deacetylase SPA system described here should be readily applicable for automated high-throughput screening and therefore facilitate the discovery of new inhibitors of histone deacetylases.

Biotin↗
PubMed1999

Biosynthesis and catabolism of mannitol is developmentally regulated in the protozoan parasite Eimeria tenella.

The mannitol cycle is a metabolic branch of the glycolytic pathway found in Eimeria tenella. In this paper, we describe the biosynthesis and consumption of mannitol during parasite development. Low micromolar levels of mannitol were detected in all of the asexual stages and mannitol production increased sharply during the sexual phase of the life cycle. Unsporulated oocysts had high mannitol content (300 mM or 25% of the oocyst mass). Mannitol-1-phosphate dehydrogenase (M1PDH), the first committed step of the mannitol cycle, was also elevated in sexual stages and this coincides with mannitol levels. Approximately 90% of the mannitol present in unsporulated oocysts was consumed in the first 15 hr of sporulation, and levels continued to drop until the sporulation process was complete at approximately 35 hr. Thus, mannitol appears to be the "fuel" for sporulation during the vegetative stage of the parasite life cycle. Evaluation of oocyst extracts from 6 additional Eimeria species for mannitol content and the presence of M1PDH indicated that the mannitol cycle was broadly present in this genus. This finding combined with the lack of mannitol metabolism in higher eukaryotes makes this pathway an attractive chemotherapeutic target.

Animals↗
PubMed1998

Novel enzyme-linked immunoassay to determine nanogram levels of pneumocandins in human plasma.

A binding enzyme-linked immunosorbent assay (ELISA) has been developed for measuring nanogram concentrations of semisynthetic pneumocandin antifungal agents in human plasma. Semisynthetic pneumocandin L-733,560 was conjugated to succinylated hemocyanin by water-soluble carbodiimide and was used as an immunogen to produce polyclonal antibodies in rabbits. Pneumocandins were used to directly coat the wells of a microtiter plate, and quantitation was achieved by using rabbit polyclonal antibodies to pneumocandin L-733,560 and goat anti-rabbit immunoglobulin G conjugated to either alkaline phosphatase or horseradish peroxidase. Maximum binding of L-733,560 and most related analogs to the wells of the microtiter plate was found to occur in the first 5 min of incubation at 4 degrees C. Once bound to the plate, these pneumocandins could not be removed from the plate, either by treatment with 4.0 to 6.0 M urea or by treatment with 4.0 to 6.0 M guanidine hydrochloride for 24 h at 4 degrees C. The binding ELISA is linear with drug concentration and can detect levels of L-733,560 as low as 5 ng/ml in human plasma. The assay is also useful for quantitating plasma levels of related semisynthetic pneumocandins including clinical candidate MK-0991.

Animals↗
PubMed1997

The mannitol cycle in Eimeria.

A metabolic pathway known as the mannitol cycle has been identified in Eimerian parasites. The pathway is a shunt off of the glycolytic pathway at fructose-6-phosphate (F6P). Two enzymes convert F6P to mannitol and two other enzymes are responsible for converting mannitol back to F6P when it is utilized. Although the pathway is present in various stages of the parasite the most apparent role of this pathway is in the sexual portion of the life cycle, particularly in the formation of oocysts. Extremely high concentrations of mannitol, approaching 0.3 M, are present in unsporulated oocysts. Mannitol functions as the endogenous energy source for oocysts to sporulate in the environment outside of the host. An inhibitory protein which inactivates the first enzyme of the mannitol cycle has been isolated from an oocyst derived inhibited enzyme complex and is believed to prevent the futile cycling of F6P during the maturation of oocysts. Evidence of the vital role of mannitol in the development and maturation of Eimeria tenella oocysts has been facilitated through the use of the drug Nitrophenide, a known anticoccidial which has now been found to be an inhibitor of one of the enzymes responsible for the biosynthesis of mannitol in the parasite. This compound prevents the formation of oocysts and at lower doses reduces mannitol levels in shed oocysts. In addition, oocysts with reduced mannitol levels fail to complete the sporulation process lending further evidence for this polyol's role in the parasite.

Amylopectin↗
PubMed1996

Apicidin: a novel antiprotozoal agent that inhibits parasite histone deacetylase.

A novel fungal metabolite, apicidin [cyclo(N-O-methyl-L-tryptophanyl-L -isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)], that exhibits potent, broad spectrum antiprotozoal activity in vitro against Apicomplexan parasites has been identified. It is also orally and parenterally active in vivo against Plasmodium berghei malaria in mice. Many Apicomplexan parasites cause serious, life-threatening human and animal diseases, such as malaria, cryptosporidiosis, toxoplasmosis, and coccidiosis, and new therapeutic agents are urgently needed. Apicidin's antiparasitic activity appears to be due to low nanomolar inhibition of Apicomplexan histone deacetylase (HDA), which induces hyperacetylation of histones in treated parasites. The acetylation-deacetylation of histones is a thought to play a central role in transcriptional control in eukaryotic cells. Other known HDA inhibitors were also evaluated and found to possess antiparasitic activity, suggesting that HDA is an attractive target for the development of novel antiparasitic agents.

Animals↗
PubMed1995

Selective labeling of intracellular parasite proteins by using ricin.

Studies focused on the synthesis by intracellular parasites of developmentally regulated proteins have been limited due to the lack of a simple method for selectively labeling proteins produced by the parasite. A method has now been developed in which ricin is employed to selectively inhibit host-cell protein synthesis. Ricin is a heterodimer composed of two subunits, a lectin and a glycosidase, and it binds to terminal galactose residues on the cell surface via the lectin. Following endocytosis of the intact molecule, a disulfide bond linking the two subunits is cleaved, and only the glycosidase subunit enters the cytoplasm, where it inhibits cytoplasmic protein synthesis by catalyzing the cleavage of the 28S rRNA. Due to the loss of the receptor-binding lectin subunit, ricin cannot permeate host-cell mitochondria or intracellular parasites, and, therefore, protein synthesis within these compartments continues uninterrupted. This system has been used to selectively label parasite proteins from Eimeria tenella and Toxoplasma gondii by using the avian cell line DU-24. In these cells, mitochondrial protein synthesis was inhibited by using chloramphenicol. The use of the avian rho0 cell line DUS-3 provided an additional advantage, because these cells lack mitochondrial DNA. Therefore, those proteins radiolabeled with [35S]methionine/cysteine in ricin-treated, parasite-infected rho0 cells are exclusively those of the intracellular parasite. This technique should be applicable for studying protein synthesis by other intracellular parasites.

Animals↗