Craig A Parish

Merck, Whitehouse Station, New Jersey, United States

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Publications (13)64.83 Total impact

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    ABSTRACT: The Candida albicans fitness test is a whole cell screening platform that utilizes a mixed-pool of C. albicans mutants, each of which carries a heterozygous deletion of a particular gene. In the presence of an antifungal inhibitor, a subset of these mutants exhibits a growth phenotype of hypersensitivity or hyposensitivity. Collectively these mutants reflect aspects of the mechanism of action of the compound in question. In the course of screening natural products a culture of Streptomyces sp. MS-1-4 was discovered to produce a compound, dretamycin, which yielded a fitness profile exhibiting significant hypersensitivity of the DRE2 heterozygote and hyposensitivity of the DIP5 heterozygote. Herein we report the production, isolation, and structure elucidation of dretamycin.
    Journal of Natural Products 06/2014; 77(6). DOI:10.1021/np401019t · 3.95 Impact Factor
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    ABSTRACT: Polymer based carriers that aid in endosomal escape have proven to be efficacious siRNA delivery agents in vitro and in vivo; however, most suffer from cytotoxicity due in part to a lack of selectivity for endosomal versus cell membrane lysis. For polymer based carriers to move beyond the laboratory and into the clinic, it is critical to find carriers that are not only efficacious, but also have margins that are clinically relevant. In this paper we report three distinct categories of polymer conjugates that improve the selectivity of endosomal membrane lysis by relying on the change in pH associated with endosomal trafficking, including incorporation of low pKa heterocycles, acid cleavable amino side chains, or carboxylic acid pH sensitive charge switches. Additionally, we determine the therapeutic index of our polymer conjugates in vivo and demonstrate that the incorporation of pH responsive elements dramatically expands the therapeutic index to 10-15, beyond that of the therapeutic index (less than 3), for polymer conjugates previously reported.
    Bioconjugate Chemistry 01/2014; 25(2). DOI:10.1021/bc400442p · 4.82 Impact Factor
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    ABSTRACT: With the development of new synthesis procedures, an ever increasing number of chemical modifications can now be incorporated into synthetic oligonucleotides, representing new challenges for analytical chemists to efficiently identify and characterize such molecules. While conventional mass spectrometry (MS) has proven to be a powerful tool to study nucleic acids, new and improved methods and software are now needed to address this emerging challenge. In this report, we describe a simple yet powerful program that affords great flexibility in the calculation of theoretical masses for conventional as well as modified oligonucleotide molecules. This easy to use program can accept input oligonucleotide sequences and then calculate the theoretical mass values for full length products, process impurities, potential metabolites, and gas phase fragments. We intentionally designed this software so that modified nucleotide residues can be incorporated into oligonucleotide sequences, and corresponding mass values can be rapidly calculated. To test the utility of this program, two oligonucleotides that contain a large number of chemical modifications were synthesized. We have analyzed these samples using a Q-TOF mass spectrometer and compared the calculated masses to the observed ones. We found that all of the data matched very well with less than 30 ppm mass errors, well within the expectation for our instrument operated in its current mode. These data confirmed the validity of calculations performed with this new software.
    Journal of the American Society for Mass Spectrometry 06/2013; DOI:10.1007/s13361-013-0643-8 · 3.19 Impact Factor
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    ABSTRACT: The resistance of methicillin-resistant Staphylococcus aureus (MRSA) to all β-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of β-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem both in vitro and in vivo with potent efficacy. The in vitro activity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to β-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with β-lactams by preventing the signal peptidase-mediated secretion of proteins required for β-lactam resistance. Combinations of SpsB inhibitors and β-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to β-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections.
    Antimicrobial Agents and Chemotherapy 06/2012; 56(9):4662-70. DOI:10.1128/AAC.00726-12 · 4.45 Impact Factor
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    ABSTRACT: Starting with the discovery of penicillin, the pharmaceutical industry has relied extensively on natural products (NPs) as an unparalleled source of bioactive small molecules suitable for antibiotic development. However, the discovery of structurally novel and chemically tractable NPs with suitable pharmacological properties as antibiotic leads has waned in recent decades. Today, the repetitive "rediscovery" of previously known NP classes with limited antibiotic lead potential dominates most industrial efforts. This limited productivity, exacerbated by the significant financial and resource requirements of such activities, has led to a broad de-emphasis of NP research by most pharmaceutical companies, including most recently Merck. Here we review our strategies--both technological and philosophical--in addressing current antifungal discovery bottlenecks in target identification and validation and how such efforts may improve NP-based antimicrobial discoveries when aligned with NP screening and dereplication.
    Chemistry & biology 02/2011; 18(2):148-64. DOI:10.1016/j.chembiol.2011.01.009 · 6.59 Impact Factor
  • ChemInform 07/2009; 40(28). DOI:10.1002/chin.200928199
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    ABSTRACT: Evaluation of fungal fermentation extracts with whole cell Candida albicans activity resulted in the identification of a novel class of isoxazolidinone-containing metabolites named parnafungins. Chemical-genetic profiling with the C. albicans fitness test identified the biochemical target as inhibition of polyadenosine polymerase, a component of the mRNA cleavage and polyadenylation complex. Parnafungins were discovered from fermentation extracts of fungi resembling F. larvarum isolated from plants, plant litter and lichens. Furthermore authentic strains of F. larvarum var. larvarum and F. larvarum var. rubrum could be induced to produce parnafungins and their degradation products in low titers. Relationships among strains of the F. larvarum complex (FLC), including parnafungin-producing strains, were examined by cladistic analyses of rDNA, mitochondrial rDNA, and two protein-coding genes, comparisons of antifungal activity and antifungal metabolite profiles, and morphological phenotypes. Integrated analyses of these data led to the conclusion that the diversity within the FLC exceeded the one-to-one correspondence between F. larvarum and its teleomorph Cosmospora aurantiicola. Based on multiple gene sequence analyses, strains of the FLC formed a monophyletic clade inclusive of the parnafungin-producing strains. The FLC, including newly discovered parnafungin-producing strains, could be resolved into at least six different lineages, possibly representing cryptic' species, of which one was not fully resolved from F. larvarum var. rubrum. Fusarium larvarum var. rubrum represents a species distinct from var. larvarum. Finally we report that two other species from the Hypocreales, Trichonectria rectipila and Cladobotryum pinarense, are able to produce parnafungins and their open-ring forms.
    Mycologia 06/2009; 101(4):449-72. DOI:10.3852/08-163 · 2.13 Impact Factor
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    ABSTRACT: Parnafungins, natural products containing an isoxazolidinone ring, have been isolated from Fusarium larvarum and have been shown to be potent inhibitors of the fungal polyadenosine polymerase. The extraction and analysis of fermentation broths of taxonomically related organisms identified as closely related Fusarium spp. produce not only parnafungin A and B, but also significant quantities of two related components. These members of the paranfungin family of natural products have been isolated and the structure of each has been elucidated. While structurally analogous to parnafungin A, parnafungin C is further elaborated by methylation of a phenolic hydroxyl group, and parnafungin D has both the methyl phenol ether as well as an epoxide in the xanthone ring system. Parnafungin C and D have potent, broad spectrum antifungal activity and also have been shown to target fungal mRNA cleavage and polyadenylation.
    Bioorganic & medicinal chemistry letters 02/2009; 19(4):1224-7. DOI:10.1016/j.bmcl.2008.12.081 · 2.33 Impact Factor
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    ABSTRACT: Antisense-based screening strategies can be used to sensitize a microorganism and selectively detect inhibitors against a particular cellular target of interest. A strain of Staphylococcus aureus that generates an antisense RNA against SecA,a central member of the protein secretion machinery, has been used to screen for novel antibacterials. Possible inhibitors of the SecA ATP-ase were selected with a high-throughput, two-plate agar-based whole cell differential sensitivity screen. After screening a library of over 115 000 natural products extracts with the SecA antisense strain, an extract of Geomyces pannorum was identified as providing increased activity against the sensitized strain as compared with the wild-type control. Bioassay-guided isolation of the active component from this fungal extract provided a new cis-decalin secondary metabolite, which we have named pannomycin.
    Journal of Natural Products 01/2009; 72(1):59-62. DOI:10.1021/np800528a · 3.95 Impact Factor
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    ABSTRACT: To discover antifungal treatments that possess the desired characteristics of broad spectrum activity, a strong safety profile, and oral bioavailability, new discovery strategies must be implemented to identify structural classes of molecules capable of combating these microorganisms. One such technique that has been implemented is the Candida albicans Fitness Test, a whole cell screening platform capable of delineating the mechanism of action of compounds that demonstrate activity against the clinically relevant pathogenic fungus, C. albicans. Screening crude natural product extracts with this technology has resulted in the identification of a novel family of antifungal natural products, named the parnafungins, which inhibit the enzyme polyadenosine polymerase (PAP), a key component of the mRNA cleavage and polyadenylation complex. Owing to the rapid interconversion of the structural and stereoisomers of the parnafungins at neutral pH, the determination of the structural isomer with the highest affinity for PAP with standard biochemical assays has not been possible. Herein, we present an application of affinity-selection/mass spectrometry (AS-MS) to determine that the "straight" parnafungin structural isomer (parnafungin A) binds preferentially to PAP compared to the "bent" structural isomer (parnafungin B).
    Journal of the American Chemical Society 01/2009; 130(49):16704-10. DOI:10.1021/ja805531w · 11.44 Impact Factor
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    ABSTRACT: The Candida albicans Fitness Test, a whole-cell screening platform, was used to profile crude fermentation extracts for novel antifungal natural products with interesting mechanisms of action. An extract with intrinsic antifungal activity from the fungus Fusarium larvarum displayed a Fitness Test profile that strongly implicated mRNA processing as the molecular target responsible for inhibition of fungal growth. Isolation of the active components from this sample identified a novel class of isoxazolidinone-containing natural products, which we have named parnafungins. These natural products were isolated as an interconverting mixture of four structural- and stereoisomers. The isomerization of the parnafungins was due to a retro-Michael ring-opening and subsequent reformation of a xanthone ring system. This interconversion was blocked by methylation of an enol moiety. Structure elucidation of purified parnafungin derivatives was accomplished by X-ray crystallography and NMR analysis. The biochemical target of these natural products has been identified as the fungal polyadenosine polymerase. Parnafungins demonstrated broad spectrum antifungal activity with no observed activity against gram-positive or gram-negative bacteria. The intact isoxazolidinone ring was required for antifungal activity. In addition, the natural products were efficacious in a mouse model of disseminated candidiasis.
    Journal of the American Chemical Society 07/2008; 130(22):7060-6. DOI:10.1021/ja711209p · 11.44 Impact Factor
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    ABSTRACT: Natural products provide an unparalleled source of chemical scaffolds with diverse biological activities and have profoundly impacted antimicrobial drug discovery. To further explore the full potential of their chemical diversity, we survey natural products for antifungal, target-specific inhibitors by using a chemical-genetic approach adapted to the human fungal pathogen Candida albicans and demonstrate that natural-product fermentation extracts can be mechanistically annotated according to heterozygote strain responses. Applying this approach, we report the discovery and characterization of a natural product, parnafungin, which we demonstrate, by both biochemical and genetic means, to inhibit poly(A) polymerase. Parnafungin displays potent and broad spectrum activity against diverse, clinically relevant fungal pathogens and reduces fungal burden in a murine model of disseminated candidiasis. Thus, mechanism-of-action determination of crude fermentation extracts by chemical-genetic profiling brings a powerful strategy to natural-product-based drug discovery.
    Chemistry & Biology 05/2008; 15(4):363-74. DOI:10.1016/j.chembiol.2008.02.016 · 6.59 Impact Factor
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    ABSTRACT: The isolation and structure elucidation of 1 from the Basidomycete fungus Baeospora myosura is described. This new ene-triyne antibiotic was most potent against Gram-positive bacteria, while it was less active against Gram-negative bacteria and a yeast. MICs against several strains of Staphylococcus aureus were as low as 0.001 microg/mL. Analogues of 1 that did not contain the ene-triyne moiety were inactive against all microorganisms tested. The isolation of this new natural product was complicated by the highly reactive nature of the conjugated terminal polyacetylene.
    Journal of Natural Products 12/2004; 67(11):1900-2. DOI:10.1021/np0497853 · 3.95 Impact Factor