<i>N</i>‐Myristoyl‐transferase activity in cancer cells

Boutin, Jean A.; Clarenc, Jean-Pierre; Ferry, Gilles; Ernould, Anne-Pascale; Rémond, Georges; Vincent, Michel; Atassi, Ghanem

doi:10.1111/j.1432-1033.1991.tb16282.x

Cited by 13 publications

(20 citation statements)

References 47 publications

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“…The addition of 0.5% DMSO, the vehicle used in the chemical library, caused only a small decrease in NMT activity. A positive control, myristoyl-L-phenylalanine, was tested at the published IC 50 concentration of 100 M (Boutin et al, 1991) and did in fact inhibit the recombinant human NMT by approximately 50%. An example of a hit in the screening assay, 24, is also indicated for comparison.…”

Section: Resultsmentioning

confidence: 99%

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

French¹,

Zhuang²,

Schrecengost³

et al. 2004

J Pharmacol Exp Ther

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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 [3 H]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-octahydropyrrolo[1,2-a]pyrazine (COPP) chemotype as inhibitory toward human NMT-1. Thirty-two compounds containing this substructure inhibited NMT-1, with IC 50 values ranging from 6 M to millimolar concentrations, and a quantitative structure-activity relationship equation (r 2 ϭ 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 myristoylCoA 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 Nmyristoylated 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.The term protein myristoylation refers to the covalent attachment of myristic acid to specific proteins. This process has been shown to target proteins to specific subcellular membranes (Boutin, 1997), stabilize protein conformation (Chow et al., 1987;Zheng et al., 1993;Resh, 1996), and facilitate additional lipidations (Robbins et al., 1995;Yurchak and Sefton, 1995;Song et al., 1997;van't Hof and Resh, 1999). Although the reaction mainly occurs as a cotranslational event, it has been shown to occur after cleavage of some substrates (Zha et al., 2000). N-Myristoyltransferase (NMT; E.C. 2.3.1.97) catalyzes this addition onto the N-terminal glycine residue of specific proteins. The reaction requires only myristoyl-CoA and a protein containing a suitable peptide sequence and occurs through an ordered Bi Bi mechanism Rocque et al., 1993;Bhatnagar et al., 1998). The list of myristoylated proteins is diverse, including viral proteins, G␣ proteins, tyrosine kinases of the Src family, and nitric oxide synthase. Gene replacement studies indicate that NMT is essential for viability of Cryptococcus neoformans (Lodge et al., 1994). In addition, mutation or deletion of NMT results in recess...

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Section: Resultsmentioning

confidence: 99%

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

French¹,

Zhuang²,

Schrecengost³

et al. 2004

J Pharmacol Exp Ther

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“…All biological sources were prepared identically by differential centrifugation as described in Materials and methods. Microsomal fraction activities were measured without addition of detergent [36]. Activity from yeast was partially purified as described in [36].…”

Section: Purification Of Isoform Amentioning

confidence: 99%

“…Microsomal fraction activities were measured without addition of detergent [36]. Activity from yeast was partially purified as described in [36]. All activities are expressed as rate of myristoylation of peptide/mass protein.…”

Section: Purification Of Isoform Amentioning

confidence: 99%

“…Since we found that the NMT activity associated with the microsomal fraction of L1210, a murine leukemia cell line, showed dramatic differences in specificity compared to the yeast enzyme [36], the purification and characterization of the L1210 cytosolic enzyme was completed. These studies on L1210 myristoylation capacity laid the background of our molecular tools for the synthesis and the screening of specific inhibitors of these NMTs (Boutin et al, unpublished).…”

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confidence: 99%

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Myristoyl‐CoA: protein N‐myristoyltransferase activity in cancer cells

Boutin

Ferry

Ernould

et al. 1993

European Journal of Biochemistry

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Myristoylation is a co-translational maturation process of proteins. It is extremely specific for the cosubstrate (myristoyl-CoA) and for the substrate protein that should bear a glycine at the Nterminus of the protein to be myristoylated. This acylation is catalyzed by the myristoyl-CoA :protein N-myristoyltransferase. Most of the molecular biochemistry and biology concerning this enzyme has been done on Saccharomyces cerevisiae. Because of the major importance of this pathway in several types of pathology, it is essential to study intensively the enzyme(s) isolated from mammalian tissue(s) to confirm that the enormous amount of work done on the yeast enzyme can be transposed to mammalian tissues. In earlier studies, we demonstrated the existence of a microsomal N-myristoyltransferase from the murine leukemia cell line L1210 [Boutin, J. A., Clarenc, J.-P., Ferry, G., Ernould, A. P., Remond, G., Vincent, M. & Atassi, G. (1991) E m J. Biochem. 201,257-2631, a feature which is not shared by yeast, and examined the N-myristoyltransferase activities associated with L1210 cytosol. In the present work, we purified to homogeneity one of the isoforms (A) of the transferase from L1210 cytosol. The purified enzyme showed on SDSPAGE an apparent molecular mass of 67.5 kDa, distinct from the 53-kDa yeast cytosolic enzyme. The purified enzyme from L1210 cytosol could be labeled with ['4C]myristoyl-CoA. Rabbit antibodies were raised against the A isoform and used to immunoprecipitate the enzyme and immunoinhibit the activity from the same source. A survey of the specificity of the partially and completely purified isoforms was performed using peptides derived from the NH,-terminus of 42 proteins which are potential substrates for myristoylation, including oncogene products and virus structural proteins. We synthesized a series of compounds capable of inhibiting the cytosol activities of the enzyme. For example, a myristoyltetrahydroquinolein derivative showed an IC,, of about 0.1 pM. Based on both biophysical and biochemical evidence, the N-myristoyltransferases extracted from mammalian cell cytosols seem to be different from the extensively studied yeast enzyme.Myristoyl-CoA :protein N-myristoyltransferase (NMT) is the enzyme responsible for a co-translational modification involving the rare fatty acid, myristic acid, and having as a binding site a glycine residue at the N-terminal of target proteins [l]. Numerous oncogene products from mammalian transforming retroviruses are either myristoylated or expressed as gag-onc fusion proteins which are myristoylated on the N-terminal glycine of the gag protein [2]. This maturation process has been proved to be essential in several types of pathology either by addressing the implicated oncogene product (like src) to a specific site at the plasma membrane level [3-51 or by permitting the regular assembly of the retroviral structural protein gag or the equivalent in other types of viruses [6-91. In both cases, using molecular biology tools, it has been demonstrated that this maturation...

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“…In particular, reducing thiol compounds such as DTT (400 mM) and 2-mercaptoethanol (2 mM) can enhance NMT activity up to 6-fold ( 51 ). Similarly, various types of detergents (Triton 770 and deoxycholate) can increase NMT activity ( 54 ). Conversely, certain oxidizing agents can have an inhibitory effect on NMT ( 51 ).…”

Section: Discussionmentioning

confidence: 99%