Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications

Wenxu Zhou; Emilio Ramos; Xunlu Zhu; Paxtyn M. Fisher; Medhanie E. Kidane; Boden H. Vanderloop; Crista D. Thomas; Juqiang Yan; Ujjal Singha; Minu Chaudhuri; Michael T. Nagel; W. David Nes

doi:10.1194/jlr.m091587

Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications

The Journal of Lipid Research ◽

10.1194/jlr.m091587 ◽

2019 ◽

Vol 60 (5) ◽

pp. 981-994 ◽

Cited By ~ 4

Author(s):

Wenxu Zhou ◽

Emilio Ramos ◽

Xunlu Zhu ◽

Paxtyn M. Fisher ◽

Medhanie E. Kidane ◽

...

Keyword(s):

Animal Studies ◽

Acanthamoeba Castellanii ◽

Chemical Kinetic ◽

Ergosterol Biosynthesis ◽

Binding Studies ◽

Product Specificity ◽

Phylogenetic Implications ◽

Olefin Production ◽

Novel Scaffold ◽

Suicide Substrates

Pathogenic organisms may be sensitive to inhibitors of sterol biosynthesis, which carry antimetabolite properties, through manipulation of the key enzyme, sterol methyltransferase (SMT). Here, we isolated natural suicide substrates of the ergosterol biosynthesis pathway, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT), and demonstrated their interference in Acanthamoeba castellanii steroidogenesis: CHT and ERGT inhibit trophozoite growth (EC50 of 51 nM) without affecting cultured human cell growth. Washout experiments confirmed that the target for vulnerability was SMT. Chemical, kinetic, and protein-binding studies of inhibitors assayed with 24-AcSMT [catalyzing C28-sterol via Δ24(28)-olefin production] and 28-AcSMT [catalyzing C29-sterol via Δ25(27)-olefin production] revealed interrupted partitioning and irreversible complex formation from the conjugated double bond system in the side chain of either analog, particularly with 28-AcSMT. Replacement of active site Tyr62 with Phe or Leu residues involved in cation-π interactions that model product specificity prevented protein inactivation. The alkylating properties and high selective index of 103 for CHT and ERGT against 28-AcSMT are indicative of a new class of steroidal antibiotic that, as an antimetabolite, can limit sterol expansion across phylogeny and provide a novel scaffold in the design of amoebicidal drugs. Animal studies of these suicide substrates can further explore the potential of their antibiotic properties.

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Inhibition of human neutrophil beta2-integrin-dependent adherence by hyperbaric O2

AJP Cell Physiology ◽

10.1152/ajpcell.1997.272.3.c770 ◽

1997 ◽

Vol 272 (3) ◽

pp. C770-C777 ◽

Cited By ~ 110

Author(s):

S. R. Thom ◽

I. Mendiguren ◽

K. Hardy ◽

T. Bolotin ◽

D. Fisher ◽

...

Keyword(s):

Guanylate Cyclase ◽

Human Neutrophil ◽

Animal Studies ◽

Surface Expression ◽

Cell Surface Expression ◽

Human Neutrophils ◽

Membrane Guanylate Cyclase ◽

Binding Studies ◽

Beta2 Integrins ◽

Cell Fractions

Animal and clinical investigations have reported that exposure to hyperbaric O(2) improved the outcome of some reperfusion injuries. Animal studies have suggested that this may be due to an inhibition of leukocyte adherence to injured endothelium. This investigation tested the hypothesis that exposure to hyperbaric O(2) would inhibit beta2-integrin-dependent adherence of human neutrophils. Subjects were exposed to O(2) at partial pressures of up to 3 atmospheres absolute (ATA; 1 ATA = 0.1 MPa) for 45 min, and neutrophil binding to nylon columns and to fibrinogen-coated surfaces was measured. Exposure to O(2) at 2.8 or 3.0 ATA inhibited beta2-integrin-dependent neutrophil adherence but had no effect on the cell-surface expression of beta2-integrins, respiratory burst in response to phorbol ester, or non-beta2-integrin-dependent adherence to plastic plates coated with a fibronectin-like protein. beta2-Integrin adherence was restored by incubating blood with 8-bromoguanosine 3',5'-cyclic monophosphate (cGMP) and hyperbaric O(2) inhibited synthesis of cGMP by neutrophils stimulated with N-formyl-Met-Leu-Phe (FMLP). In studies of cell fractions, the activity of membrane guanylate cyclase was found to be increased by incubation with FMLP as well as by atrial natriuretic peptide (ANP) plus ATP. Hyperbaric O(2) had no effect on the basal activity of soluble or membrane-bound guanylate cyclase. However, hyperbaric O(2) inhibited the function of both the extracellular binding domain of membrane guanylate cyclase as well as intracellular catalytic activity. There are approximately 7,300 membrane guanylate cyclase molecules per cell, based on binding studies with ANP, with a dissociation constant of approximately 450 pM. Hyperbaric O(2) inhibits the function of human neutrophil beta2-integrins by a process linked to impaired synthesis of cGMP. a

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Azole Antifungal Agents To Treat the Human Pathogens Acanthamoeba castellanii and Acanthamoeba polyphaga through Inhibition of Sterol 14α-Demethylase (CYP51)

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00476-15 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4707-4713 ◽

Cited By ~ 22

Author(s):

David C. Lamb ◽

Andrew G. S. Warrilow ◽

Nicola J. Rolley ◽

Josie E. Parker ◽

W. David Nes ◽

...

Keyword(s):

Antifungal Agents ◽

Acanthamoeba Castellanii ◽

Therapeutic Agents ◽

Effective Inhibitor ◽

Human Pathogens ◽

Acanthamoeba Polyphaga ◽

Binding Studies ◽

Content Type ◽

Inhibition Concentration

ABSTRACTIn this study, we investigate the amebicidal activities of the pharmaceutical triazole CYP51 inhibitors fluconazole, itraconazole, and voriconazole againstAcanthamoeba castellaniiandAcanthamoeba polyphagaand assess their potential as therapeutic agents againstAcanthamoebainfections in humans. Amebicidal activities of the triazoles were assessed byin vitrominimum inhibition concentration (MIC) determinations using trophozoites ofA. castellaniiandA. polyphaga. In addition, triazole effectiveness was assessed by ligand binding studies and inhibition of CYP51 activity of purifiedA. castellaniiCYP51 (AcCYP51) that was heterologously expressed inEscherichia coli. Itraconazole and voriconazole bound tightly to AcCYP51 (dissociation constant [Kd] of 10 and 13 nM), whereas fluconazole bound weakly (Kdof 2,137 nM). Both itraconazole and voriconazole were confirmed to be strong inhibitors of AcCYP51 activity (50% inhibitory concentrations [IC50] of 0.23 and 0.39 μM), whereas inhibition by fluconazole was weak (IC50, 30 μM). However, itraconazole was 8- to 16-fold less effective (MIC, 16 mg/liter) at inhibitingA. polyphagaandA. castellaniicell proliferation than voriconazole (MIC, 1 to 2 mg/liter), while fluconazole did not inhibitAcanthamoebacell division (MIC, >64 mg/liter)in vitro. Voriconazole was an effective inhibitor of trophozoite proliferation forA. castellaniiandA. polyphaga; therefore, it should be evaluated in trials versus itraconazole for controllingAcanthamoebainfections.

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Structural Basis of Recognition of Pathogen-associated Molecular Patterns and Inhibition of Proinflammatory Cytokines by Camel Peptidoglycan Recognition Protein

Journal of Biological Chemistry ◽

10.1074/jbc.m111.228163 ◽

2011 ◽

Vol 286 (18) ◽

pp. 16208-16217 ◽

Cited By ~ 27

Author(s):

Pradeep Sharma ◽

Divya Dube ◽

Amar Singh ◽

Biswajit Mishra ◽

Nagendra Singh ◽

...

Keyword(s):

Proinflammatory Cytokines ◽

Animal Studies ◽

Short Form ◽

Lipoteichoic Acid ◽

Mortality Rates ◽

Structural Basis ◽

Binding Studies ◽

Peptidoglycan Recognition Protein ◽

Tnf Α ◽

Molecular Patterns

Peptidoglycan recognition proteins (PGRPs) are involved in the recognition of pathogen-associated molecular patterns. The well known pathogen-associated molecular patterns include LPS from Gram-negative bacteria and lipoteichoic acid (LTA) from Gram-positive bacteria. In this work, the crystal structures of two complexes of the short form of camel PGRP (CPGRP-S) with LPS and LTA determined at 1.7- and 2.1-Å resolutions, respectively, are reported. Both compounds were held firmly inside the complex formed with four CPGRP-S molecules designated A, B, C, and D. The binding cleft is located at the interface of molecules C and D, which is extendable to the interface of molecules A and C. The interface of molecules A and B is tightly packed, whereas that of molecules B and D forms a wide channel. The hydrophilic moieties of these compounds occupy a common region, whereas hydrophobic chains interact with distinct regions in the binding site. The binding studies showed that CPGRP-S binds to LPS and LTA with affinities of 1.6 × 10−9 and 2.4 × 10−8m, respectively. The flow cytometric studies showed that both LPS- and LTA-induced expression of the proinflammatory cytokines TNF-α and IL-6 was inhibited by CPGRP-S. The results of animal studies using mouse models indicated that both LPS- and LTA-induced mortality rates decreased drastically when CPGRP-S was administered. The recognition of both LPS and LTA, their high binding affinities for CPGRP-S, the significant decrease in the production of LPS- and LTA-induced TNF-α and IL-6, and the drastic reduction in the mortality rates in mice by CPGRP-S indicate its useful properties as an antibiotic agent.

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The R467K Amino Acid Substitution in Candida albicans Sterol 14α-Demethylase Causes Drug Resistance through Reduced Affinity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.1.63-67.2000 ◽

2000 ◽

Vol 44 (1) ◽

pp. 63-67 ◽

Cited By ~ 82

Author(s):

David C. Lamb ◽

Diane E. Kelly ◽

Theodore C. White ◽

Steven L. Kelly

Keyword(s):

Candida Albicans ◽

Amino Acid ◽

Amino Acid Substitution ◽

Specific Activity ◽

Difference Spectrum ◽

Microsomal Protein ◽

Ergosterol Biosynthesis ◽

Wild Type ◽

Binding Studies ◽

Directed Mutation

ABSTRACT The cytochrome P450 sterol 14α-demethylase (CYP51) ofCandida albicans is involved in an essential step of ergosterol biosynthesis and is the target for azole antifungal compounds. We have undertaken site-directed mutation of C. albicans CYP51 to produce a recombinant mutant protein with the amino acid substitution R467K corresponding to a mutation observed clinically. This alteration perturbed the heme environment causing an altered reduced-carbon monoxide difference spectrum with a maximum at 452 nm and reduced the affinity of the enzyme for fluconazole, as shown by ligand binding studies. The specific activity of CYP51(R467K) for the release of formic acid from 3β-[32-3H]hydroxylanost-7-en-32-ol was 70 pmol/nmol of P450/min for microsomal protein compared to 240 pmol/nmol of P450/min for microsomal fractions expressing wild-type CYP51. Furthermore, inhibition of activity by fluconazole revealed a 7.5-fold-greater azole resistance of the recombinant protein than that of the wild type. This study demonstrates that resistance observed clinically can result from the altered azole affinity of the fungal CYP51 enzyme.

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Reduced accumulation of drug in Candida krusei accounts for itraconazole resistance.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.11.2443 ◽

1996 ◽

Vol 40 (11) ◽

pp. 2443-2446 ◽

Cited By ~ 44

Author(s):

K Venkateswarlu ◽

D W Denning ◽

N J Manning ◽

S L Kelly

Keyword(s):

Acquired Resistance ◽

Sterol Composition ◽

Candida Krusei ◽

Resistant Isolate ◽

Ergosterol Biosynthesis ◽

Intrinsic Resistance ◽

Binding Studies ◽

Intracellular Content ◽

Fluconazole Therapy

Due to intrinsic resistance Candida krusei is emerging as a systemic pathogen in AIDS patients undergoing fluconazole therapy, but acquired resistance to itraconazole has not been studied biochemically. We report here studies on the basis for azole resistance and sterol composition in C. krusei. An itraconazole-resistant isolate showed reduced susceptibility to azole drugs in in vitro growth inhibition studies. Accumulation of 14 alpha-methyl-3,6-diol under azole treatment was associated with growth arrest. In vitro ergosterol biosynthesis and type II binding studies suggested no alteration in the affinity to azole drugs of the target enzyme, the cytochrome P-450 sterol 14 alpha-demethylase, in the resistant isolate. Resistance was associated with a decreased intracellular content of drug in the resistant isolate.

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Lectin Binding Studies on RNA Tumor Virus-Infected Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100115250 ◽

1975 ◽

Vol 33 ◽

pp. 326-327

Author(s):

D. C. Hixson

Keyword(s):

Binding Sites ◽

Lectin Binding ◽

Culture Conditions ◽

3T3 Cells ◽

Binding Studies ◽

Con A ◽

Microscopic Studies ◽

Tumor Virus ◽

Infected Cells ◽

Cell Culture Conditions

The abilities of plant lectins to preferentially agglutinate malignant cells and to bind to specific monosaccharide or oligosaccharide sequences of glycoproteins and glycolipids make them a new and important biochemical probe for investigating alterations in plasma membrane structure which may result from malignant transformation. Electron and light microscopic studies have demonstrated clustered binding sites on surfaces of SV40-infected or tryp- sinized 3T3 cells when labeled with concanavalin A (con A). No clustering of con A binding sites was observed in normal 3T3 cells. It has been proposed that topological rearrangement of lectin binding sites into clusters enables con A to agglutinate SV40-infected or trypsinized 3T3 cells (1). However, observations by other investigators have not been consistent with this proposal (2) perhaps due to differences in reagents used, cell culture conditions, or labeling techniques. The present work was undertaken to study the lectin binding properties of normal and RNA tumor virus-infected cells and their associated viruses using lectins and ferritin-conjugated lectins of five different specificities.

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Architecture of Radiolitid Rudist (Mollusca) Shell-Wall Structures and its Phylogenetic Implications

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100117054 ◽

1975 ◽

Vol 33 ◽

pp. 686-687

Author(s):

David H. Sturm ◽

Bob F. Perkins

Keyword(s):

Phylogenetic Relationships ◽

Outer Wall ◽

Shell Microstructure ◽

Shell Wall ◽

Wall Structures ◽

Phylogenetic Implications ◽

Cellular Wall ◽

Superfamily Analysis ◽

Central Texas

Each of the seven families of rudists (Mollusca, Bivalvia, Hippuritacea) is characterized by distinctive shell-wall architectures which reflect phylogenetic relationships within the superfamily. Analysis of the complex, calcareous, cellular wall of the attached valve of the radiolite rudist Eoradiolites davidsoni (Hill) from the Comanche Cretaceous of Central Texas indicates that its wall architecture is an elaboration of the simpler monopleurid rudist wall and supports possible radiolite-monopleurid relationships.Several well-preserved specimens of E. davidsoni were sectioned, polished, etched, and carbon and gold coated for SEM examination. Maximum shell microstructure detail was displayed by etching with a 0.7% HC1 solution from 80 to 100 seconds.The shell of E. davidsoni comprises a large, thick-walled, conical, attached valve (AV) and a small, very thin, operculate, free valve (FV) (Fig. 1a). The AV shell is two-layered with a thin inner wall, in which original structures are usually obliterated by recrystallization, and a thick, cellular, outer wall.

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Decellularized bone extracellular matrix in skeletal tissue engineering

Biochemical Society Transactions ◽

10.1042/bst20190079 ◽

2020 ◽

Vol 48 (3) ◽

pp. 755-764

Author(s):

Benjamin B. Rothrauff ◽

Rocky S. Tuan

Keyword(s):

Tissue Engineering ◽

Bone Regeneration ◽

Tissue Regeneration ◽

Animal Studies ◽

Tumor Resection ◽

Regenerative Capacity ◽

Skeletal Tissue ◽

Large Bone

Bone possesses an intrinsic regenerative capacity, which can be compromised by aging, disease, trauma, and iatrogenesis (e.g. tumor resection, pharmacological). At present, autografts and allografts are the principal biological treatments available to replace large bone segments, but both entail several limitations that reduce wider use and consistent success. The use of decellularized extracellular matrices (ECM), often derived from xenogeneic sources, has been shown to favorably influence the immune response to injury and promote site-appropriate tissue regeneration. Decellularized bone ECM (dbECM), utilized in several forms — whole organ, particles, hydrogels — has shown promise in both in vitro and in vivo animal studies to promote osteogenic differentiation of stem/progenitor cells and enhance bone regeneration. However, dbECM has yet to be investigated in clinical studies, which are needed to determine the relative efficacy of this emerging biomaterial as compared with established treatments. This mini-review highlights the recent exploration of dbECM as a biomaterial for skeletal tissue engineering and considers modifications on its future use to more consistently promote bone regeneration.

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