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Papers by Martin Bryant

Approaches to Disaster Management - Examining the Implications of Hazards, Emergencies and Disasters, 2013

BMJ Supportive & Palliative Care, 2011

Analytical Biochemistry, 1979

1. Anal Biochem. 1979 Jul 1;96(1):84-9. Comparison of two techniques for protein isolation and ra... more 1. Anal Biochem. 1979 Jul 1;96(1):84-9. Comparison of two techniques for protein isolation and radioiodination by tryptic peptide mapping. Bryant ML, Nalewaik RP, Tibbs VL, Todaro GJ. PMID: 495994 [PubMed - indexed for MEDLINE] Publication Types: Comparative Study. ...

Biopolymers, 1997

MyristoylCoA: protein N-myristoyltransferase (NMT) catalyzes the cotranslational covalent attachm... more MyristoylCoA: protein N-myristoyltransferase (NMT) catalyzes the cotranslational covalent attachment of a rare cellular fatty acid, myristate, to the N-terminal Gly residue of a variety of eukaryotic proteins. The myristoyl moiety is often essential for expression of the biological functions for these proteins. Attachment of C14:0 alone provides barely enough hydrophobicity to allow stable association with membranes. The partitioning of N-myrisotylproteins is therefore often modulated by "switches" that function through additional covalent or noncovalent modifications. Candida albicans, the principal cause of systemic fungal infection in immunocompromised humans, contains a single NMT gene that is essential for its viability. The functional properties of the acylCoA binding site of human and C. albicans NMT are very similar. However, there are distinct differences in their peptide binding sites. An ADP ribosylation factor (Arf) is included among the few cellular protein substrates of the fungal enzyme. Alanine scanning mutagenesis of an octapeptide derived from an N-terminal Arf sequence (GLYASKLS-NH2) disclosed that Gly1, Ser5, and Lys6 play predominant roles in binding. ALYASKLS-NH2 is an inhibitor competitive for peptide [Ki(app) = 15.3 +/- 6.4 microM] and noncompetitive for myristoylCoA. Remarkably, replacement of the N-terminal tetrapeptide with an 11-aminoundecanoyl group results in a competitive inhibitor (11-aminoundecanoyl-SKLS-NH2) that is approximately 40-fold more potent [Ki(app) = 0.40 +/- 0.03 microM] than the starting octapeptide. Removal of Leu-Ser from the C-terminus generates a competitive dipeptide inhibitor (11-aminoundecanoyl-SK-NH2) with a Ki(app) of 11.7 +/- 0.4 microM, equivalent to that of the starting octapeptide. A derivative dipeptide inhibitor containing a C-terminal N-cyclohexylethyl lysinamide moiety has the advantage of being more potent (IC50 = 0.11 +/- 0.03 microM) and resistant to digestion by cellular carboxypeptidases. Rigidifying the flexible aminoundecanoyl chain results in very potent general NMT inhibitors (IC50 = 40-50 nM). Substituting a 2-methylimidazole for the N-terminal amine and adding a benzylic alpha-methyl group with R stereochemistry to the rigidifying element produces even more potent inhibitors (IC50 = 20-50 nM) that are up to 500-fold selective for the fungal compared to human enzyme. A related less potent member of this series of compounds is fungistatic. Its growth inhibitory effects are associated with a reduction in cellular protein N-myristoylation, judged using cellular Arf as a reporter. These studies establish that NMT is a new antifungal target.

Pnas, 1991

Covalent linkage of myristate (tetradecanoate; 14:0) to the NH2-terminal glycine residue of the h... more Covalent linkage of myristate (tetradecanoate; 14:0) to the NH2-terminal glycine residue of the human immunodeficiency virus 1 (HIV-1) 55-kDa gag polyprotein precursor (Pr55gag) is necessary for its proteolytic processing and viral assembly. We have shown recently that several analogs of myristate in which a methylene group is replaced by a single oxygen or sulfur atom are substrates for Saccharomyces cerevisiae and mammalian myristoyl-CoA:protein N-myristoyltransferase (EC 2.3.1.97; NMT) despite their reduced hydrophobicity. Some inhibit HIV-1 replication in acutely infected CD4+H9 cells without accompanying cellular toxicity. To examine the mechanism of their antiviral effects, we performed labeling studies with two analogs, 12-methoxydodecanoate (13-oxamyristate; 13-OxaMyr) and 5-octyloxypentanoate (6-oxamyristate; 6-OxaMyr), the former being much more effective than the latter in blocking virus production. [3H]Myristate and [3H]13-OxaMyr were incorporated into Pr55gag with comparable efficiency when it was coexpressed with S. cerevisiae NMT in Escherichia coli. [3H]6-OxaMyr was not incorporated, even though its substrate properties in vitro were similar to those of 13-OxaMyr and myristate. [3H]13-OxaMyr, but not [3H]6-OxaMyr, was also efficiently incorporated into HIV-1 Pr55gag and nef (negative factor) in chronically infected H9 cells. Analog incorporation produced a redistribution of Pr55gag from membrane to cytosolic fractions and markedly decreased its proteolytic processing by viral protease. 13-OxaMyr and 3'-azido-3'-deoxythymidine (AZT) act synergistically to reduce virus production in acutely infected H9 cells. Unlike AZT, the analog is able to inhibit virus production (up to 70%) in chronically infected H9 cells. Moreover, the inhibitory effect lasts 6-8 days. These results suggest that (i) its mechanism of action is distinct from that of AZT and involves a late step in virus assembly; (ii) the analog may allow reduction in the dose of AZT required to affect viral replication; and (iii) combinations of analog and HIV-1 protease inhibitors may have synergistic effects on the processing of Pr55gag.

Proceedings of the National Academy of Sciences of the United States of America, Nov 1, 1989

Myristoyl-CoA:protein N-myristoyltransferase (NMT; EC 2.3.1.97) catalyzes the cotranslational lin... more Myristoyl-CoA:protein N-myristoyltransferase (NMT; EC 2.3.1.97) catalyzes the cotranslational linkage of myristate to the N-terminal glycine residues of several cellular, viral, and oncoproteins. We have recently synthesized a series of sulfur- and oxygen-substituted analogs of myristic acid that are similar in length to the 14:0 fatty acid yet have hydrophobicities equivalent to dodecanoate or decanoate. Previous in vitro enzyme assays and metabolic labeling studies indicate that some of these analogs are excellent substrates for NMT and are incorporated into subsets of cellular N-myristoyl proteins. Their sequence-specific incorporation probably arises from cooperative interactions between the acyl CoA and peptide binding sites of NMT. The human immunodeficiency virus 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) depend on myristoylation of gag polyprotein precursors for assembly. We have tested four analogs--12-methoxydodecanoic acid, 10-propoxydecanoic acid, 5-octyloxypentanoic acid, and 11-ethylthioundecanoic acid--for their ability to block replication of these retroviruses. All reduce HIV-1 replication when incubated with CD4+ H9 cells for 10 days at 10-100 microM. 12-Methoxydodecanoic acid is most effective, producing a concentration-dependent decrease in (i) reverse transcriptase activity (to levels that were 5-10% of control at 20-40 microM), (ii) p24 levels, and (iii) syncytia formation. This degree of inhibition of HIV-1 replication is equivalent to that seen with 5 microM 3'-azido-3'-deoxythymidine and is accomplished without apparent toxicity, as measured by cell viability, protein, and nucleic acid synthesis. 5-Octyloxypentanoic acid inhibits MoMLV assembly in a dose-dependent fashion without accompanying cellular toxicity, while 12-methoxydodecanoic acid has no effect. These data suggest that the use of cellular NMT activity to deliver analogs of myristate with altered physical-chemical properties to proteins that undergo this cotranslational modification may represent an effective anti-viral therapeutic strategy as well as a way to investigate the role of covalently bound fatty acid in viral assembly.

Approaches to Disaster Management - Examining the Implications of Hazards, Emergencies and Disasters, 2013

BMJ Supportive & Palliative Care, 2011

Analytical Biochemistry, 1979

1. Anal Biochem. 1979 Jul 1;96(1):84-9. Comparison of two techniques for protein isolation and ra... more 1. Anal Biochem. 1979 Jul 1;96(1):84-9. Comparison of two techniques for protein isolation and radioiodination by tryptic peptide mapping. Bryant ML, Nalewaik RP, Tibbs VL, Todaro GJ. PMID: 495994 [PubMed - indexed for MEDLINE] Publication Types: Comparative Study. ...

Biopolymers, 1997

MyristoylCoA: protein N-myristoyltransferase (NMT) catalyzes the cotranslational covalent attachm... more MyristoylCoA: protein N-myristoyltransferase (NMT) catalyzes the cotranslational covalent attachment of a rare cellular fatty acid, myristate, to the N-terminal Gly residue of a variety of eukaryotic proteins. The myristoyl moiety is often essential for expression of the biological functions for these proteins. Attachment of C14:0 alone provides barely enough hydrophobicity to allow stable association with membranes. The partitioning of N-myrisotylproteins is therefore often modulated by "switches" that function through additional covalent or noncovalent modifications. Candida albicans, the principal cause of systemic fungal infection in immunocompromised humans, contains a single NMT gene that is essential for its viability. The functional properties of the acylCoA binding site of human and C. albicans NMT are very similar. However, there are distinct differences in their peptide binding sites. An ADP ribosylation factor (Arf) is included among the few cellular protein substrates of the fungal enzyme. Alanine scanning mutagenesis of an octapeptide derived from an N-terminal Arf sequence (GLYASKLS-NH2) disclosed that Gly1, Ser5, and Lys6 play predominant roles in binding. ALYASKLS-NH2 is an inhibitor competitive for peptide [Ki(app) = 15.3 +/- 6.4 microM] and noncompetitive for myristoylCoA. Remarkably, replacement of the N-terminal tetrapeptide with an 11-aminoundecanoyl group results in a competitive inhibitor (11-aminoundecanoyl-SKLS-NH2) that is approximately 40-fold more potent [Ki(app) = 0.40 +/- 0.03 microM] than the starting octapeptide. Removal of Leu-Ser from the C-terminus generates a competitive dipeptide inhibitor (11-aminoundecanoyl-SK-NH2) with a Ki(app) of 11.7 +/- 0.4 microM, equivalent to that of the starting octapeptide. A derivative dipeptide inhibitor containing a C-terminal N-cyclohexylethyl lysinamide moiety has the advantage of being more potent (IC50 = 0.11 +/- 0.03 microM) and resistant to digestion by cellular carboxypeptidases. Rigidifying the flexible aminoundecanoyl chain results in very potent general NMT inhibitors (IC50 = 40-50 nM). Substituting a 2-methylimidazole for the N-terminal amine and adding a benzylic alpha-methyl group with R stereochemistry to the rigidifying element produces even more potent inhibitors (IC50 = 20-50 nM) that are up to 500-fold selective for the fungal compared to human enzyme. A related less potent member of this series of compounds is fungistatic. Its growth inhibitory effects are associated with a reduction in cellular protein N-myristoylation, judged using cellular Arf as a reporter. These studies establish that NMT is a new antifungal target.

Pnas, 1991

Covalent linkage of myristate (tetradecanoate; 14:0) to the NH2-terminal glycine residue of the h... more Covalent linkage of myristate (tetradecanoate; 14:0) to the NH2-terminal glycine residue of the human immunodeficiency virus 1 (HIV-1) 55-kDa gag polyprotein precursor (Pr55gag) is necessary for its proteolytic processing and viral assembly. We have shown recently that several analogs of myristate in which a methylene group is replaced by a single oxygen or sulfur atom are substrates for Saccharomyces cerevisiae and mammalian myristoyl-CoA:protein N-myristoyltransferase (EC 2.3.1.97; NMT) despite their reduced hydrophobicity. Some inhibit HIV-1 replication in acutely infected CD4+H9 cells without accompanying cellular toxicity. To examine the mechanism of their antiviral effects, we performed labeling studies with two analogs, 12-methoxydodecanoate (13-oxamyristate; 13-OxaMyr) and 5-octyloxypentanoate (6-oxamyristate; 6-OxaMyr), the former being much more effective than the latter in blocking virus production. [3H]Myristate and [3H]13-OxaMyr were incorporated into Pr55gag with comparable efficiency when it was coexpressed with S. cerevisiae NMT in Escherichia coli. [3H]6-OxaMyr was not incorporated, even though its substrate properties in vitro were similar to those of 13-OxaMyr and myristate. [3H]13-OxaMyr, but not [3H]6-OxaMyr, was also efficiently incorporated into HIV-1 Pr55gag and nef (negative factor) in chronically infected H9 cells. Analog incorporation produced a redistribution of Pr55gag from membrane to cytosolic fractions and markedly decreased its proteolytic processing by viral protease. 13-OxaMyr and 3'-azido-3'-deoxythymidine (AZT) act synergistically to reduce virus production in acutely infected H9 cells. Unlike AZT, the analog is able to inhibit virus production (up to 70%) in chronically infected H9 cells. Moreover, the inhibitory effect lasts 6-8 days. These results suggest that (i) its mechanism of action is distinct from that of AZT and involves a late step in virus assembly; (ii) the analog may allow reduction in the dose of AZT required to affect viral replication; and (iii) combinations of analog and HIV-1 protease inhibitors may have synergistic effects on the processing of Pr55gag.

Proceedings of the National Academy of Sciences of the United States of America, Nov 1, 1989

Myristoyl-CoA:protein N-myristoyltransferase (NMT; EC 2.3.1.97) catalyzes the cotranslational lin... more Myristoyl-CoA:protein N-myristoyltransferase (NMT; EC 2.3.1.97) catalyzes the cotranslational linkage of myristate to the N-terminal glycine residues of several cellular, viral, and oncoproteins. We have recently synthesized a series of sulfur- and oxygen-substituted analogs of myristic acid that are similar in length to the 14:0 fatty acid yet have hydrophobicities equivalent to dodecanoate or decanoate. Previous in vitro enzyme assays and metabolic labeling studies indicate that some of these analogs are excellent substrates for NMT and are incorporated into subsets of cellular N-myristoyl proteins. Their sequence-specific incorporation probably arises from cooperative interactions between the acyl CoA and peptide binding sites of NMT. The human immunodeficiency virus 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) depend on myristoylation of gag polyprotein precursors for assembly. We have tested four analogs--12-methoxydodecanoic acid, 10-propoxydecanoic acid, 5-octyloxypentanoic acid, and 11-ethylthioundecanoic acid--for their ability to block replication of these retroviruses. All reduce HIV-1 replication when incubated with CD4+ H9 cells for 10 days at 10-100 microM. 12-Methoxydodecanoic acid is most effective, producing a concentration-dependent decrease in (i) reverse transcriptase activity (to levels that were 5-10% of control at 20-40 microM), (ii) p24 levels, and (iii) syncytia formation. This degree of inhibition of HIV-1 replication is equivalent to that seen with 5 microM 3'-azido-3'-deoxythymidine and is accomplished without apparent toxicity, as measured by cell viability, protein, and nucleic acid synthesis. 5-Octyloxypentanoic acid inhibits MoMLV assembly in a dose-dependent fashion without accompanying cellular toxicity, while 12-methoxydodecanoic acid has no effect. These data suggest that the use of cellular NMT activity to deliver analogs of myristate with altered physical-chemical properties to proteins that undergo this cotranslational modification may represent an effective anti-viral therapeutic strategy as well as a way to investigate the role of covalently bound fatty acid in viral assembly.