Publications by authors named "Neil S Ryder"

25 Publications

  • Page 1 of 1

Involvement of heat shock proteins in Candida albicans biofilm formation.

J Mol Microbiol Biotechnol 2013 7;23(6):396-400. Epub 2013 Aug 7.

Infectious Diseases, Novartis Institutes for Biomedical Research Inc., Cambridge, Mass., USA.

Biofilm growth represents one of the most challenging problems associated with Candida infections, largely due to the natural resistance of biofilm to the common antifungal drugs. As elevated expression of heat shock proteins (HSP) promotes Candida yeast-hyphae switch, which is an essential step in biofilm formation, we investigated the expression of hsp genes during Candida albicans biofilm development. By measuring mRNA levels using qRT-PCR, we found that all three hsp genes that we monitored are overexpressed in the initial stage of C. albicans biofilm formation. To corroborate this finding, we examined the effect of 17-DMAG, a specific Hsp90 inhibitor, on the formation of C. albicans biofilm. Our results indicate the requirement of HSP during the early phase of Candida biofilm development.
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http://dx.doi.org/10.1159/000351619DOI Listing
June 2014

Identification and evaluation of novel acetolactate synthase inhibitors as antifungal agents.

Antimicrob Agents Chemother 2013 May 11;57(5):2272-80. Epub 2013 Mar 11.

Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, USA.

High-throughput phenotypic screening against the yeast Saccharomyces cerevisiae revealed a series of triazolopyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series, we have applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All compounds of the series yielded highly similar profiles that suggested acetolactate synthase (Ilv2p, which catalyzes the first common step in branched-chain amino acid biosynthesis) as a possible target. The high correlation with profiles of known Ilv2p inhibitors like chlorimuron-ethyl provided further evidence for a similar mechanism of action. Genome-wide mutagenesis in S. cerevisiae identified 13 resistant clones with 3 different mutations in the catalytic subunit of acetolactate synthase that also conferred cross-resistance to established Ilv2p inhibitors. Mapping of the mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity, and it was possible to dock the triazolopyrimidine-sulfonamide compound into this pocket in silico. However, fungal growth inhibition could be bypassed through supplementation with exogenous branched-chain amino acids or by the addition of serum to the medium in all of the fungal organisms tested except for Aspergillus fumigatus. Thus, these data support the identification of the triazolopyrimidine-sulfonamide compounds as inhibitors of acetolactate synthase but suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo.
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http://dx.doi.org/10.1128/AAC.01809-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632958PMC
May 2013

An integrated approach for identification and target validation of antifungal compounds active against Erg11p.

Antimicrob Agents Chemother 2012 Aug 21;56(8):4233-40. Epub 2012 May 21.

Novartis Institutes for BioMedical Research, Novartis Campus, Basel, Switzerland.

Systemic life-threatening fungal infections represent a significant unmet medical need. Cell-based, phenotypic screening can be an effective means of discovering potential novel antifungal compounds, but it does not address target identification, normally required for compound optimization by medicinal chemistry. Here, we demonstrate a combination of screening, genetic, and biochemical approaches to identify and characterize novel antifungal compounds. We isolated a set of novel non-azole antifungal compounds for which no target or mechanism of action is known, using a screen for inhibition of Saccharomyces cerevisiae proliferation. Haploinsufficiency profiling of these compounds in S. cerevisiae suggests that they target Erg11p, a cytochrome P450 family member, which is the target of azoles. Consistent with this, metabolic profiling in S. cerevisiae revealed a buildup of the metabolic intermediates prior to Erg11p activity, following compound treatment. Further, human cytochrome P450 is also inhibited in in vitro assays by these compounds. We modeled the Erg11p protein based on the human CYP51 crystal structure, and in silico docking of these compounds suggests that they interact with the heme center in a manner similar to that of azoles. Consistent with these docking observations, Candida strains carrying azole-resistant alleles of ERG11 are also resistant to the compounds in this study. Thus, we have identified non-azole Erg11p inhibitors, using a systematic approach for ligand and target characterization.
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http://dx.doi.org/10.1128/AAC.06332-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3421618PMC
August 2012

Biophysical investigation of the mode of inhibition of tetramic acids, the allosteric inhibitors of undecaprenyl pyrophosphate synthase.

Biochemistry 2010 Jun;49(25):5366-76

Infectious Diseases, Novartis Institutes for BioMedical Research, Inc., Cambridge, Massachusetts 02139, USA.

Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate the C(55) undecaprenyl pyrophosphate (UPP). It has been demonstrated that tetramic acids (TAs) are selective and potent inhibitors of UPPS, but the mode of inhibition was unclear. In this work, we used a fluorescent FPP probe to study possible TA binding at the FPP binding site. A photosensitive TA analogue was designed and synthesized for the study of the site of interaction of TA with UPPS using photo-cross-linking and mass spectrometry. The interaction of substrates with UPPS and with the UPPS.TA complex was investigated by protein fluorescence spectroscopy. Our results suggested that tetramic acid binds to UPPS at an allosteric site adjacent to the FPP binding site. TA binds to free UPPS enzyme but not to substrate-bound UPPS. Unlike Escherichia coli UPPS which follows an ordered substrate binding mechanism, Streptococcus pneumoniae UPPS appears to follow a random-sequential substrate binding mechanism. Only one substrate, FPP or IPP, is able to bind to the UPPS.TA complex, but the quaternary complex, UPPS.TA.FPP.IPP, cannot be formed. We propose that binding of TA to UPPS significantly alters the conformation of UPPS needed for proper substrate binding. As the result, substrate turnover is prevented, leading to the inhibition of UPPS catalytic activity. These probe compounds and biophysical assays also allowed us to quickly study the mode of inhibition of other UPPS inhibitors identified from a high-throughput screening and inhibitors produced from a medicinal chemistry program.
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http://dx.doi.org/10.1021/bi100523cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2889672PMC
June 2010

Discontinued drugs in 2008: anti-infectives.

Authors:
Neil S Ryder

Expert Opin Investig Drugs 2010 Jan;19(1):1-21

Novartis Institutes for BioMedical Research, Inc., Infectious Diseases, Cambridge, MA 02139, USA.

Of the many drugs dropped from development in 2008, 27 were under development for therapy or prophylaxis of infectious diseases. The majority of these were for diseases of viral etiology (15 agents), while nine were antibacterials and the remaining three were directed against fungal and protozoan pathogens. The antiviral agents were primarily against HIV and hepatitis C virus, together with agents against hepatitis B virus and West Nile virus. The antibacterial agents were mostly from known classes (carbapenems, fluoroquinolones, oxazolidinones), but also included several novel compounds and approaches. With regard to drug type, 19 were small-molecule therapeutics, seven were biologics comprising five vaccines and two antibodies, and one was a high-molecular-weight polymer. Each of these agents is briefly reviewed and the reasons for failure are discussed.
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http://dx.doi.org/10.1517/13543780903473150DOI Listing
January 2010

In vivo characterization of the peptide deformylase inhibitor LBM415 in murine infection models.

Antimicrob Agents Chemother 2009 Sep 13;53(9):3777-81. Epub 2009 Jul 13.

Infectious Diseases, Novartis Institutes for BioMedical Research, Inc., Cambridge, MA 02139, USA.

LBM415 is an antibacterial agent belonging to the peptide deformylase inhibitor class of compounds. It has previously been shown to demonstrate good activity in vitro against a range of pathogens. In this study, the in vivo efficacy of LBM415 was evaluated in various mouse infection models. We investigated activity against a systemic infection model caused by intraperitoneal inoculation of Staphylococcus aureus (methicillin [meticillin] susceptible [MSSA] and methicillin resistant [MRSA]) and Streptococcus pneumoniae (penicillin susceptible [PSSP] and multidrug resistant [MDRSP]), a thigh infection model caused by intramuscular injection of MRSA, and a lung infection produced by intranasal inoculation of PSSP. In the systemic MSSA and MRSA infections, LBM415 was equivalent to linezolid and vancomycin. In the systemic PSSP infection, LBM415 was equivalent to linezolid, whereas against systemic MDRSP infection, the LBM415 50% effective dose (ED50) was 4.8 mg/kg (dosed subcutaneously) and 36.6 mg/kg (dosed orally), compared to 13.2 mg/kg for telithromycin and >60 mg/kg for penicillin V and clarithromycin. In the MRSA thigh infection, LBM415 significantly reduced thigh bacterial levels compared to those of untreated mice, with levels similar to those after treatment with linezolid at the same dose levels. In the pneumonia model, the ED50 to reduce the bacterial lung burden by >4 log10 in 50% of treated animals was 23.3 mg/kg for LBM415, whereas moxifloxacin showed an ED50 of 14.3 mg/kg. In summary, LBM415 showed in vivo efficacy in sepsis and specific organ infection models irrespective of resistance to other antibiotics. Results suggest the potential of peptide deformylase inhibitors as a novel class of therapeutic agents against antibiotic-resistant pathogens.
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http://dx.doi.org/10.1128/AAC.00026-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2737836PMC
September 2009

Discontinued drugs in 2007: anti-infectives.

Authors:
Neil S Ryder

Expert Opin Investig Drugs 2009 Jan;18(1):1-11

Infectious Diseases, Novartis Institutes for BioMedical Research Inc., 500 Technology Square, Cambridge, MA 02139, USA.

Twelve drugs related to therapy and/or prevention of infectious diseases were discontinued from development during 2007. All of these agents were aimed at viral infections, primarily hepatitis C virus (six agents) and HIV (five agents). Nearly half were vaccines, including three against HIV, a therapeutic vaccine for hepatitis C, and a vaccine for prevention and treatment of herpes simplex virus type 2. Each of these agents is briefly reviewed and the reasons for failure are discussed.
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http://dx.doi.org/10.1517/13543780802614920DOI Listing
January 2009

Discontinued drugs in 2006: anti-infectives.

Authors:
Neil S Ryder

Expert Opin Investig Drugs 2007 Dec;16(12):1867-78

Infectious Diseases, Novartis Institutes for BioMedical Research, Inc., 500 Technology Square, Cambridge, MA 02139, USA.

Of the drugs dropped from development in 2006, 11 were being developed for infectious diseases. Of these, nine were for viral diseases, including four against HIV, two against hepatitis C virus and one each against respiratory syncytial virus, severe acute respiratory syndrome (coronavirus) and a variety of viruses. The nine antiviral agents comprised six synthetic small-molecule compounds, one peptide, one monoclonal antibody and a vaccine. The remaining two agents were a vaccine for Pseudomonas aeruginosa infection, and lipid-based agent for septic shock. Each of these drugs is briefly reviewed and reasons for failure are discussed.
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http://dx.doi.org/10.1517/13543784.16.12.1867DOI Listing
December 2007

Reduced susceptibility of Haemophilus influenzae to the peptide deformylase inhibitor LBM415 can result from target protein overexpression due to amplified chromosomal def gene copy number.

Antimicrob Agents Chemother 2007 Mar 12;51(3):1004-10. Epub 2007 Jan 12.

Infectious Diseases, Novartis Institutes for BioMedical Research, 500 Technology Square, Cambridge, MA 02139, USA.

Previous genetic analysis of Haemophilus influenzae revealed two mechanisms associated with decreased susceptibility to the novel peptide deformylase inhibitor LBM415: AcrAB-TolC-mediated efflux and Fmt bypass, resulting from mutations in the pump repressor gene acrR and in the fmt gene, respectively. We have isolated an additional mutant, CDS23 (LBM415 MIC, 64 microg/ml versus 4 microg/ml against the parent strain NB65044) that lacks mutations in the acrR or fmt structural genes or in the gene encoding Def, the intracellular target of LBM415. Western immunoblot analysis, two-dimensional gel electrophoresis, and tryptic digestion combined with mass spectrometric identification showed that the Def protein was highly overexpressed in the mutant strain. Consistent with this, real-time reverse transcription-PCR revealed a significant increase in def transcript titer. No mutations were found in the region upstream of def that might account for altered expression; however, pulsed-field gel electrophoresis suggested that a genetic rearrangement of the region containing def had occurred. Using a combination of PCR, sequencing, and Southern blot analyses, it was determined that the def gene had undergone copy number amplification, explaining the high level of target protein expression. Inactivation of the AcrAB-TolC efflux pump in this mutant increased susceptibility 16-fold, highlighting the role of efflux in exacerbating the overall reduced susceptibility resulting from target overexpression.
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http://dx.doi.org/10.1128/AAC.01103-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1803149PMC
March 2007

Exploring the antibacterial and hemolytic activity of shorter- and longer-chain beta-, alpha,beta-, and gamma-peptides, and of beta-peptides from beta2-3-aza- and beta3-2-methylidene-amino acids bearing proteinogenic side chains--a survey.

Chem Biodivers 2005 Mar;2(3):401-20

Uppsala University, Institute of Chemistry, Department of Organic Chemistry, Box 599, SE-75124 Uppsala.

The antibacterial activities of 31 different beta-, mixed alpha/beta-, and gamma-peptides, as well as of beta-peptides derived from beta2-3-aza- and beta3-2-methylidene-amino acids were assayed against six pathogens (Enterococcus faecalis, Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa), and the results were compared with literature data. The interaction of these peptides with mammalian cells, as modeled by measuring the hemolysis of human erythrocytes, was also investigated. In addition to those peptides designed to fold into amphiphilic helical conformations with positive charges on one face of the helix, one new peptide with hemolytic activity was detected within the sample set. Moreover, it was demonstrated that neither cationic peptides used for membrane translocation (beta3-oligoarginines), nor mixed alpha/beta- or gamma-peptides with somatostatin-mimicking activities display unwanted hemolytic activity.
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http://dx.doi.org/10.1002/cbdv.200590020DOI Listing
March 2005

Chemical and biological investigations of beta-oligoarginines.

Chem Biodivers 2004 Jan;1(1):65-97

Laboratorium für Organische Chemie, ETH-Zürich, ETH-Hönggerberg, HCI, CH-8093 Zürich.

In view of the important role arginine plays in living organisms as the free amino acid and, especially, as a residue in peptides and proteins, the homologous beta-homoarginines are central in our investigations of beta-peptides (Fig. 1). The preparation of beta2-homoarginine derivatives suitably protected for solution- or solid-phase peptide syntheses is described with full experimental detail (9 and 12 in Scheme 1). The readily available Fmoc-beta3 hArg(Boc)2-OH is used for manual solid-phase synthesis of beta3-oligoarginines (on Rink amide or Rink amide AM resin) either by single amino acid coupling (Scheme 3) or, much better, by dimer-fragment coupling (Scheme 4). In this way, beta3-oligoarginine amides composed of 4, 6, 7, 8, and 10 residues, both with and without fluorescein labelling, were synthesized (Schemes 2-4), purified by preparative HPLC and identified by high-resolution mass spectrometry. The free amino acids (R)- and (S)-H-beta2 hArg-OH and (S)-H-beta3 hArg-OH were tested for their ability to function as substrates for NO synthase (iNOS); the beta3-oligoarginine amides (5, 6, and 7 residues) were tested for antibacterial (against six pathogens) and hemolytic (against rat and human erythrocytes) activities. All test results were negative: none of the free beta-homoarginines induced NO formation (Fig. 3), and there was no lysis of erythrocytes (concentrations up to 100 microM; Table 1), and no significant antibiotic activity (MIC > or = 64 microg/ml; Table 2). Cell-penetration studies with the fluorescence-labelled, peptidase-resistant beta3-oligoarginine amides were carried out with HeLa cells and human foreskin keratinocytes (HFKs). The results obtained with fluorescence microscopy are: i) the longer-chain beta-oligoarginine amides (8 and 10 residues; Figs. 4-6) enter the cells and end up in the nuclei, especially in the nucleoli, irrespective of temperature (37 degrees and 4 degrees with HFKs) or pretreatment with NaN3 (with HFKs), indicating a non-endocytotic and non-energy-dependent uptake mechanism; ii) the beta-tetraarginine derivative occupies the cell surface but does not enter the cells (with HeLa); iii) the cell-growth rate of the HFKs is not affected by a 1-microM concentration of the fluorescence-labelled beta-octaarginine amide (Fig. 7), i.e., there is no antiproliferative effect. In vivo experiments with mouse skin and the beta-octaarginine derivative show migration of the beta-peptide throughout the epidermis (Fig. 8). As a contribution to understanding the mechanism, we have also studied the behavior of fluorescence-labelled beta-octa- and beta-decaarginine amides (TFA salts) towards giant unilamellar vesicles (GUVs) built of neutral (POPC) or anionic (POPC/POPG mixtures) phospholipids: the beta-oligoarginine amides bind tightly to the surface of anionic GUVs but do not penetrate the lipid bilayer (Fig. 9) as they do with living cells. In contrast, a beta-heptapeptide FL-22, which had been used as a negative control sample for the cell-penetration experiments, entered the GUVs of negative surface charge. Thus, the mechanisms of cell and GUV-model penetration appear to be different. Finally, the possible applications and implications of the 'protein transduction' by beta-oligoarginines are discussed.
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http://dx.doi.org/10.1002/cbdv.200490014DOI Listing
January 2004

Peptide deformylase inhibitors as potent antimycobacterial agents.

Antimicrob Agents Chemother 2006 Nov 11;50(11):3665-73. Epub 2006 Sep 11.

Novartis Institute for Tropical Diseases, 10 Biopolis Road, 05-01 Chromos, Singapore 138670, Republic of Singapore.

Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from nascent proteins. This is an essential step in bacterial protein synthesis, making PDF an attractive target for antibacterial drug development. Essentiality of the def gene, encoding PDF from Mycobacterium tuberculosis, was demonstrated through genetic knockout experiments with Mycobacterium bovis BCG. PDF from M. tuberculosis strain H37Rv was cloned, expressed, and purified as an N-terminal histidine-tagged recombinant protein in Escherichia coli. A novel class of PDF inhibitors (PDF-I), the N-alkyl urea hydroxamic acids, were synthesized and evaluated for their activities against the M. tuberculosis PDF enzyme as well as their antimycobacterial effects. Several compounds from the new class had 50% inhibitory concentration (IC50) values of <100 nM. Some of the PDF-I displayed antibacterial activity against M. tuberculosis, including MDR strains with MIC90 values of <1 microM. Pharmacokinetic studies of potential leads showed that the compounds were orally bioavailable. Spontaneous resistance towards these inhibitors arose at a frequency of < or =5 x 10(-7) in M. bovis BCG. DNA sequence analysis of several spontaneous PDF-I-resistant mutants revealed that half of the mutants had acquired point mutations in their formyl methyltransferase gene (fmt), which formylated Met-tRNA. The results from this study validate M. tuberculosis PDF as a drug target and suggest that this class of compounds have the potential to be developed as novel antimycobacterial agents.
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http://dx.doi.org/10.1128/AAC.00555-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635232PMC
November 2006

NIM811, a cyclophilin inhibitor, exhibits potent in vitro activity against hepatitis C virus alone or in combination with alpha interferon.

Antimicrob Agents Chemother 2006 Sep;50(9):2976-82

Novartis Institutes for Biomedical Research, Inc., 500 Technology Square, Cambridge, MA 02139, USA.

Host factors involved in viral replication are potentially attractive antiviral targets that are complementary to specific inhibitors of viral enzymes, since resistant mutations against the latter are likely to emerge during long-term treatment. It has been reported recently that cyclosporine, which binds to a family of cellular proteins, cyclophilins, inhibits hepatitis C virus (HCV) replication in vitro. Here, the activities of various cyclosporine derivatives were evaluated in the HCV replicon system. There was a strong correlation between the anti-HCV activity and cyclophilin-binding affinity of these compounds. Of these, NIM811 has been selected as a therapeutic candidate for HCV infection, since it binds to cyclophilins with higher affinity than cyclosporine but is devoid of the significant immunosuppressive activity associated with cyclosporine. NIM811 induced a concentration-dependent reduction of HCV RNA in the replicon cells with a 50% inhibitory concentration of 0.66 microM at 48 h. Furthermore, a greater than three-log(10) viral RNA reduction was achieved after treating the cells with as little as 1 microM of NIM811 for 9 days. In addition, the combination of NIM811 with alpha interferon significantly enhanced anti-HCV activities without causing any increase of cytotoxicity. Taken together, these promising in vitro data warrant clinical investigation of NIM811, an inhibitor of novel mechanism, for the treatment of hepatitis C.
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http://dx.doi.org/10.1128/AAC.00310-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1563518PMC
September 2006

Biological, biochemical, and molecular characterization of a new clinical Trichophyton rubrum isolate resistant to terbinafine.

Antimicrob Agents Chemother 2006 Jun;50(6):2234-6

Infectious Diseases, Novartis Institutes for BioMedical Research, Inc., Cambridge, MA 02139, USA.

We have characterized a new clinical strain of Trichophyton rubrum highly resistant to terbinafine but exhibiting normal susceptibility to drugs with other mechanisms of action. Resistance to terbinafine in this strain is caused by a missense mutation in the squalene epoxidase gene leading to the amino acid substitution F397L.
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http://dx.doi.org/10.1128/AAC.01600-05DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1479141PMC
June 2006

Combination therapy with terbinafine and amphotericin B in a rabbit model of experimental invasive aspergillosis.

Antimicrob Agents Chemother 2005 Nov;49(11):4751-3

Department of Medicine, Division of Infectious Diseases, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, Mail Code 7881, San Antonio, Texas 78229-3900, USA.

Antagonistic effects of combination therapy using amphotericin B (AmB) with agents which block ergosterol synthesis are a concern. Terbinafine was evaluated with AmB to assess antagonism or synergy in a rabbit model of invasive aspergillosis. Terbinafine had relatively little activity but did not demonstrate antagonism against AmB in our model.
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http://dx.doi.org/10.1128/AAC.49.11.4751-4753.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1280177PMC
November 2005

Role of the AcrAB-TolC efflux pump in determining susceptibility of Haemophilus influenzae to the novel peptide deformylase inhibitor LBM415.

Antimicrob Agents Chemother 2005 Aug;49(8):3129-35

Infectious Diseases, Novartis Institute for Biomedical Research, Inc., 100 Technology Square, Cambridge, MA 02139, USA.

Haemophilus influenzae isolates vary widely in their susceptibilities to the peptide deformylase inhibitor LBM415 (MIC range, 0.06 to 32 microg/ml); however, on average, they are less susceptible than gram-positive organisms, such as Staphylococcus aureus and Streptococcus pneumoniae. Insertional inactivation of the H. influenzae acrB or tolC gene in strain NB65044 (Rd strain KW20) increased susceptibility to LBM415, confirming a role for the AcrAB-TolC pump in determining resistance. Consistent with this, sequencing of a PCR fragment generated with primers flanking the acrRA region from an LBM415-hypersusceptible H. influenzae clinical isolate revealed a genetic deletion of acrA. Inactivation of acrB or tolC in several clinical isolates with atypically reduced susceptibility to LBM415 (MIC of 16 microg/ml or greater) significantly increased susceptibility, confirming that the pump is also a determinant of decreased susceptibility in these clinical isolates. Examination of acrR, encoding the putative repressor of pump gene expression, from several of these strains revealed mutations introducing frameshifts, stop codons, and amino acid changes relative to the published sequence, suggesting that loss of pump repression leads to decreased susceptibility. Supporting this, NB65044 acrR mutants selected by exposure to LBM415 at 8 microg/ml had susceptibilities to LBM415 and other pump substrates comparable to the least sensitive clinical isolates and showed increased expression of pump genes.
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http://dx.doi.org/10.1128/AAC.49.8.3129-3135.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1196275PMC
August 2005

Amino acid substitution in Trichophyton rubrum squalene epoxidase associated with resistance to terbinafine.

Antimicrob Agents Chemother 2005 Jul;49(7):2840-4

Infectious Diseases, Novartis Institutes for BioMedical Research, Vienna, Austria.

There has only been one clinically confirmed case of terbinafine resistance in dermatophytes, where six sequential Trichophyton rubrum isolates from the same patient were found to be resistant to terbinafine and cross-resistant to other squalene epoxidase (SE) inhibitors. Microsomal SE activity from these resistant isolates was insensitive to terbinafine, suggesting a target-based mechanism of resistance (B. Favre, M. Ghannoum, and N. S. Ryder, Med. Mycol. 42:525-529, 2004). In this study, we have characterized at the molecular level the cause of the resistant phenotype of these clinical isolates. Cloning and sequencing of the SE gene and cDNA from T. rubrum revealed the presence of an intron in the gene and an open reading frame encoding a protein of 489 residues, with an equivalent similarity (57%) to both yeast and mammalian SEs. The nucleotide sequences of SE from two terbinafine-susceptible strains were identical whereas those of terbinafine-resistant strains, serially isolated from the same patient, each contained the same single missense introducing the amino acid substitution L393F. Introduction of the corresponding substitution in the Candida albicans SE gene (L398F) and expression of this gene in Saccharomyces cerevisiae conferred a resistant phenotype to the transformants when compared to those expressing the wild-type sequence. Terbinafine resistance in these T. rubrum clinical isolates appears to be due to a single amino acid substitution in SE.
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http://dx.doi.org/10.1128/AAC.49.7.2840-2844.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1168638PMC
July 2005

Biochemical characterization of terbinafine-resistant Trichophyton rubrum isolates.

Med Mycol 2004 Dec;42(6):525-9

Novartis Research Institute, Vienna, Austria.

We investigated the biochemical basis for resistance in six sequential clinical isolates of Trichophyton rubrum, from the same patient, which exhibited high-level primary resistance to terbinafine. Cellular ergosterol biosynthesis was measured by incorporation of [14C]acetate, and microsomal squalene epoxidase was assayed by conversion of [3H]squalene to squalene epoxide and lanosterol. Direct comparison was made with a terbinafine-susceptible reference strain of T. rubrum in which squalene epoxidase was previously studied. Resistant isolates displayed normal cellular ergosterol biosynthesis, although slight accumulation of radiolabeled squalene suggested reduced squalene epoxidase activity. Ergosterol biosynthesis in the resistant isolates was only inhibited by terbinafine concentrations above 1 microg/ml (IC50 5 microg/ml). In the reference strain, ergosterol biosynthesis was eliminated by terbinafine at 0.03 microg/ml in accordance with historical data. There was no significant difference in sensitivity between the six resistant isolates. Squalene epoxidase from resistant strains was three orders of magnitude less sensitive than normal enzyme to terbinafine (IC50 of 30 micromol/l and 19 n mol/l respectively). The epoxidase in the resistant strains was also unresponsive to tolnaftate. Resistance to terbinafine in these T. rubrum isolates appears to be due to alterations in the squalene epoxidase gene or a factor essential for its activity.
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http://dx.doi.org/10.1080/13693780410001661482DOI Listing
December 2004

Identification of novel potent bicyclic peptide deformylase inhibitors.

Bioorg Med Chem Lett 2004 Mar;14(6):1477-81

Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.

Screening of our compound collection using Staphylococcus aureus Ni-Peptide deformylase (PDF) afforded a very potent PDF inhibitor with an IC(50) in the low nanomolar range but with poor antibacterial activity (MIC). Three-dimensional structural information obtained from Pseudomonas aeruginosa Ni-PDF complexed with the inhibitor suggested the synthesis of a variety of analogues that would maintain high binding affinity while attempting to improve antibacterial activity. Many of the compounds synthesized proved to be excellent PDF-Ni inhibitors and some showed increased antibacterial activity in selected strains.
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http://dx.doi.org/10.1016/j.bmcl.2004.01.014DOI Listing
March 2004

Antibiotic and hemolytic activity of a beta2/beta3 peptide capable of folding into a 12/10-helical secondary structure.

Chembiochem 2003 Dec;4(12):1345-7

Uppsala University, Institute of Chemistry, Department of Organic Chemistry, Box 599, 75124 Uppsala, Sweden.

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http://dx.doi.org/10.1002/cbic.200300698DOI Listing
December 2003

In vitro analysis of the ability of Trichophyton rubrum to become resistant to terbinafine.

Antimicrob Agents Chemother 2003 Nov;47(11):3634-6

Novartis Research Institute, 1235 Vienna, Austria.

In this study, we have investigated in vitro the resistance frequency and development of resistance to terbinafine of Trichophyton rubrum. Results demonstrated that naturally occurring mutants are rare and that T. rubrum appears to have little capacity to develop resistance to terbinafine even after prolonged exposure.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC253782PMC
http://dx.doi.org/10.1128/AAC.47.11.3634-3636.2003DOI Listing
November 2003

Comparison of in vitro activities of 17 antifungal drugs against a panel of 20 dermatophytes by using a microdilution assay.

J Clin Microbiol 2003 Oct;41(10):4817-9

Novartis Research Institute, Vienna, Austria.

The in vitro activities of 17 antifungal drugs against a panel of 20 dermatophytes comprising 6 different species were determined using a microdilution assay according to the NCCLS M38-P method with some modifications. Terbinafine was the most potent systemic drug while tolnaftate and amorolfine were the most active topical agents.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC254304PMC
http://dx.doi.org/10.1128/JCM.41.10.4817-4819.2003DOI Listing
October 2003

Novel high energy intermediate analogues with a triazasterol structure as potential ergosterol biosynthesis inhibitors IV: antimicrobial activity of mono-, bi-, and tricyclic 8, 13, 15-triazasteroid analogues including the synthesis of novel 4-alkylamino- and 4-alkenylamino-9-hydroxypyrimidoisoquinolines.

Arch Pharm (Weinheim) 2003 Aug;336(6-7):336-44

Institute of Pharmaceutical Chemistry and Pharmaceutical Technology, Karl-Franzens-University Graz, Graz, Austria.

4-alkylamino-and 4-alkenylamino-9-hydroxy-1, 6, 7, 11b-tetrahydro-2H-pyrimido[4, 3-a]isoquinolines were designed as inhibitory tricyclic triaza-analogues of carbocationic high energy intermediates (HEI) of enzymes involved in fungal ergosterol biosynthesis. Various routes for effective synthesis of 9-hydroxypyrimidoisoquinolines from 9-methoxythiones were investigated. The ether cleavage of 9-methoxy-pyrimidoisoquinolines, a key step in the synthesis, was carried out using various protocols. The structures of the obtained 9-hydroxy compounds were confirmed using homo- and hetero-nuclear correlated 1D and 2D NMR experiments. In vitro antifungal susceptibility tests of the alkylaminohydroxypyrimidoisoquinolines revealed weak to good antimycotic effects. The maximum antifungal efficacy was found for 4-[(3R)-6-isopropyl-3-methyl-6-heptenylamino]-9-hydroxypyrimidoisoquinoline. Furthermore, the in vitro activities of the newly synthesized 9-hydroxypyrimidoisoquinolines and of a series of prepared 8, 13, 15-triazasteroid analogues (N-alkyl-N'-(phenethyl- and cyclohexenylethyl)guanidines, N(2) -and N(2), 4-substituted imidazolin-2-amines, and N(4)-alkylaminopyrimidoisoquinolines) against representatives of gram-positive and gram-negative bacteria were investigated. The compounds showed significant antibacterial effects against gram-positive bacteria.
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http://dx.doi.org/10.1002/ardp.200300774DOI Listing
August 2003

Novel high energy intermediate analogues with triazasterol-related structures as inhibitors of ergosterol biosynthesis. Part I: synthesis and antifungal activity of N-alkyl-N'-(phenethyl- and cyclohexenylethyl)guanidines and N2-substituted 2-imidazolinamines.

Arch Pharm (Weinheim) 2002 ;335(11-12):535-46

Institute of Pharmaceutical Chemistry and Pharmaceutical Technology, Karl-Franzens-University Graz, Schubertstrasse 1, A-8010 Graz, Austria.

A series of N-alkyl-N'-(phenethyl- and cyclohexenylethyl) guanidines and N(2)- and N(2), 4-substituted imidazolin-2-amine hydrochlorides with triazasterol-related structures was designed and synthesized as stable analogues to mimic high energy intermediates of ergosterol biosynthesis. The in vitro antifungal susceptibility tests with a standard panel of pathogenic fungi revealed moderate to strong antimycotic effects of the sixteen prepared compounds, in some cases comparable with the activity observed for itraconazole.
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http://dx.doi.org/10.1002/ardp.200290007DOI Listing
April 2003

Clinical Trichophyton rubrum strain exhibiting primary resistance to terbinafine.

Antimicrob Agents Chemother 2003 Jan;47(1):82-6

Center for Medical Mycology, Department of Dermatology, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 44106, USA.

The in vitro antifungal susceptibilities of six clinical Trichophyton rubrum isolates obtained sequentially from a single onychomycosis patient who failed oral terbinafine therapy (250 mg/day for 24 weeks) were determined by broth microdilution and macrodilution methodologies. Strain relatedness was examined by random amplified polymorphic DNA (RAPD) analyses. Data obtained from both broth micro- and macrodilution assays were in agreement and revealed that the six clinical isolates had greatly reduced susceptibilities to terbinafine. The MICs of terbinafine for these strains were >4 microg/ml, whereas they were <0.0002 microg/ml for the susceptible reference strains. Consistent with these findings, the minimum fungicidal concentrations (MFCs) of terbinafine for all six strains were >128 microg/ml, whereas they were 0.0002 microg/ml for the reference strain. The MIC of terbinafine for the baseline strain (cultured at the initial screening visit and before therapy was started) was already 4,000-fold higher than normal, suggesting that this is a case of primary resistance to terbinafine. The results obtained by the broth macrodilution procedure revealed that the terbinafine MICs and MFCs for sequential isolates apparently increased during the course of therapy. RAPD analyses did not reveal any differences between the isolates. The terbinafine-resistant isolates exhibited normal susceptibilities to clinically available antimycotics including itraconazole, fluconazole, and griseofulvin. However, these isolates were fully cross resistant to several other known squalene epoxidase inhibitors, including naftifine, butenafine, tolnaftate, and tolciclate, suggesting a target-specific mechanism of resistance. This is the first confirmed report of terbinafine resistance in dermatophytes.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC148991PMC
http://dx.doi.org/10.1128/AAC.47.1.82-86.2003DOI Listing
January 2003
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