Cyclohexyl-octahydro-pyrrolo[1,2-a]pyrazine-Based Inhibitors of Human N-Myristoyltransferase-1

Kevin J. French, Yan Zhuang, Randy S. Schrecengost, Jean E. Copper, Zuping Xia, Charles Smith

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

N-Myristoyltransferase (NMT) is an emerging therapeutic target that catalyzes the attachment of myristate to the N terminus of an acceptor protein. We have developed a medium-throughput assay for screening potential small molecule inhibitors of human NMT-1 consisting of recombinant enzyme, biotinylated peptide substrate, and [3H]myristoyl-CoA. Approximately 16,000 diverse compounds have been evaluated, and significant inhibition of NMT was found with 0.8% of the compounds. From these hits, we have identified the cyclohexyl-octahydro-pyrrolo[1,2-a]pyrazine (COPP) chemotype as inhibitory toward human NMT-1. Thirty-two compounds containing this substructure inhibited NMT-1, with IC50 values ranging from 6 μM to millimolar concentrations, and a quantitative structure-activity relationship equation (r2 = 0.72) was derived for the series. The most potent inhibitor (24, containing 9-ethyl-9H-carbazole) demonstrated competitive inhibition for the peptide-binding site of NMT-1 and noncompetitive inhibition for the myristoyl-CoA site. Computational docking studies using the crystal structure of the highly homologous yeast NMT confirmed that 24 binds with excellent complementarity to the peptide-binding site of the enzyme. To evaluate the ability of 24 to inhibit NMT activity in intact cells, monkey CV-1 cells expressing an N-myristoylated green fluorescent protein (GFP) fusion protein were treated with a known NMT inhibitor or with 24. Each compound caused the redistribution of GFP from the plasma membrane to the cytosol. Furthermore, 24 inhibits cancer cell proliferation at doses similar to those that inhibit protein myristoylation. Overall, these studies establish an efficient assay for screening for inhibitors of human NMT and identify a novel family of inhibitors that compete at the peptide-binding site and have activity in intact cells.

Original languageEnglish (US)
Pages (from-to)340-347
Number of pages8
JournalJournal of Pharmacology and Experimental Therapeutics
Volume309
Issue number1
DOIs
StatePublished - Apr 1 2004

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Peptides
Binding Sites
Green Fluorescent Proteins
cyclohexyl-octahydro-pyrrolo(1,2-a)pyrazine
glycylpeptide N-tetradecanoyltransferase
Proteins
Quantitative Structure-Activity Relationship
Myristic Acid
Enzymes
Cytosol
Inhibitory Concentration 50
Haplorhini
Yeasts
Cell Proliferation
Cell Membrane
Neoplasms
S-tetradecanoyl-coenzyme A
Therapeutics
carbazole
1,2-diamino-1,2-N,N'-carbonyl-1,2-dideoxyglucose hydrate

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology

Cite this

French, Kevin J. ; Zhuang, Yan ; Schrecengost, Randy S. ; Copper, Jean E. ; Xia, Zuping ; Smith, Charles. / Cyclohexyl-octahydro-pyrrolo[1,2-a]pyrazine-Based Inhibitors of Human N-Myristoyltransferase-1. In: Journal of Pharmacology and Experimental Therapeutics. 2004 ; Vol. 309, No. 1. pp. 340-347.
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abstract = "N-Myristoyltransferase (NMT) is an emerging therapeutic target that catalyzes the attachment of myristate to the N terminus of an acceptor protein. We have developed a medium-throughput assay for screening potential small molecule inhibitors of human NMT-1 consisting of recombinant enzyme, biotinylated peptide substrate, and [3H]myristoyl-CoA. Approximately 16,000 diverse compounds have been evaluated, and significant inhibition of NMT was found with 0.8{\%} of the compounds. From these hits, we have identified the cyclohexyl-octahydro-pyrrolo[1,2-a]pyrazine (COPP) chemotype as inhibitory toward human NMT-1. Thirty-two compounds containing this substructure inhibited NMT-1, with IC50 values ranging from 6 μM to millimolar concentrations, and a quantitative structure-activity relationship equation (r2 = 0.72) was derived for the series. The most potent inhibitor (24, containing 9-ethyl-9H-carbazole) demonstrated competitive inhibition for the peptide-binding site of NMT-1 and noncompetitive inhibition for the myristoyl-CoA site. Computational docking studies using the crystal structure of the highly homologous yeast NMT confirmed that 24 binds with excellent complementarity to the peptide-binding site of the enzyme. To evaluate the ability of 24 to inhibit NMT activity in intact cells, monkey CV-1 cells expressing an N-myristoylated green fluorescent protein (GFP) fusion protein were treated with a known NMT inhibitor or with 24. Each compound caused the redistribution of GFP from the plasma membrane to the cytosol. Furthermore, 24 inhibits cancer cell proliferation at doses similar to those that inhibit protein myristoylation. Overall, these studies establish an efficient assay for screening for inhibitors of human NMT and identify a novel family of inhibitors that compete at the peptide-binding site and have activity in intact cells.",
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Cyclohexyl-octahydro-pyrrolo[1,2-a]pyrazine-Based Inhibitors of Human N-Myristoyltransferase-1. / French, Kevin J.; Zhuang, Yan; Schrecengost, Randy S.; Copper, Jean E.; Xia, Zuping; Smith, Charles.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 309, No. 1, 01.04.2004, p. 340-347.

Research output: Contribution to journalArticle

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T1 - Cyclohexyl-octahydro-pyrrolo[1,2-a]pyrazine-Based Inhibitors of Human N-Myristoyltransferase-1

AU - French, Kevin J.

AU - Zhuang, Yan

AU - Schrecengost, Randy S.

AU - Copper, Jean E.

AU - Xia, Zuping

AU - Smith, Charles

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AB - N-Myristoyltransferase (NMT) is an emerging therapeutic target that catalyzes the attachment of myristate to the N terminus of an acceptor protein. We have developed a medium-throughput assay for screening potential small molecule inhibitors of human NMT-1 consisting of recombinant enzyme, biotinylated peptide substrate, and [3H]myristoyl-CoA. Approximately 16,000 diverse compounds have been evaluated, and significant inhibition of NMT was found with 0.8% of the compounds. From these hits, we have identified the cyclohexyl-octahydro-pyrrolo[1,2-a]pyrazine (COPP) chemotype as inhibitory toward human NMT-1. Thirty-two compounds containing this substructure inhibited NMT-1, with IC50 values ranging from 6 μM to millimolar concentrations, and a quantitative structure-activity relationship equation (r2 = 0.72) was derived for the series. The most potent inhibitor (24, containing 9-ethyl-9H-carbazole) demonstrated competitive inhibition for the peptide-binding site of NMT-1 and noncompetitive inhibition for the myristoyl-CoA site. Computational docking studies using the crystal structure of the highly homologous yeast NMT confirmed that 24 binds with excellent complementarity to the peptide-binding site of the enzyme. To evaluate the ability of 24 to inhibit NMT activity in intact cells, monkey CV-1 cells expressing an N-myristoylated green fluorescent protein (GFP) fusion protein were treated with a known NMT inhibitor or with 24. Each compound caused the redistribution of GFP from the plasma membrane to the cytosol. Furthermore, 24 inhibits cancer cell proliferation at doses similar to those that inhibit protein myristoylation. Overall, these studies establish an efficient assay for screening for inhibitors of human NMT and identify a novel family of inhibitors that compete at the peptide-binding site and have activity in intact cells.

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