CHEMMEDCHEM MINIREVIEWS DOI: 10.1002/cmdc.201402174

Recent Advances in The Discovery of NMyristoyltransferase Inhibitors Can Zhao and Shutao Ma*[a] N-Myristoyltransferase (NMT) is a cytosolic monomeric enzyme present in eukaryotes such as fungi and protozoa, but is not found in prokaryotes. The attachment of a 14-carbon saturated fatty acid, myristate, from myristoyl-CoA (14:0 CoA) to the Nterminal glycine residue in a specific set of cellular proteins is commonly called protein N-myristoylation. The myristoylation reaction catalyzed by the enzyme myristoyl CoA:NMT is both necessary for the growth of various organisms and conclusive for cellular proliferation. Therefore, NMT has been identified as a novel and promising target for antifungal, antiparasitic, and

anticancer agents, and a large number of potent NMT inhibitors with antifungal, antiparasitic, and anticancer activities have been reported. Herein we describe recent advances in the discovery of NMT inhibitors. We introduce not only the functions of NMT, but also some representative natural and synthetic inhibitors, with a focus on their biological activity, selectivity, and structure–activity relationship (SAR) information. In particular, inspiration from NMT inhibitor structures and the future direction of these compounds are highlighted.

Introduction Over the past two decades, fungal infections, parasitic diseases, and cancers have posed a particular threat to hundreds of millions of people globally. For example, the incidence of systemic fungal infections and associated mortality has increased dramatically, due primarily to a significant increase in the number of patients undergoing anticancer chemotherapy, organ transplants, or other procedures that cause potential hosts to become immunocompromised.[1, 2] Parasitic diseases such as malaria, leishmaniasis, and African trypanosomiasis (sleeping sickness) have influenced millions of people worldwide as well.[3] Certain cancers such as lung, stomach, liver, and colorectal cancers are an increasingly prominent cause of death, resulting in a great burden to human healthcare systems.[4] Unfortunately, the chemotherapeutic agents used for treating these diseases also largely suffer drawbacks such as lack sufficient efficacy, undesired drug-related toxicity, serious drug–drug interactions, or severe drug resistance.[5] Therefore, there is an urgent need to develop new chemotherapeutic agent chemotypes with novel mechanisms of action in order to surmount the above shortcomings.[6] A new drug target identified for antifungal, antiparasitic, and anticancer agents has recently attracted a large amount of attention: myristoylCoA:protein N-myristoyltransferase (NMT).[7] NMT is a ubiquitous enzyme in eukaryotes, from protozoa and fungi to larger animals. It catalyzes the covalent transfer of myristic acid (a 14-carbon fatty acid) to the N-terminal glycine [a] C. Zhao, Prof. S. Ma Department of Medicinal Chemistry Key Laboratory of Chemical Biology (Ministry of Education) School of Pharmaceutical Sciences, Shandong University 44, West Culture Road, Jinan 250012 (P.R. China) Fax: (+ 86) 531-88382548 E-mail: [email protected]

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residue of protein substrates.[8] Such substrates include various tyrosine kinases (pp60src, pp60yes, pp56Lck, pp59fyn/syn, and cAbl), the a subunits of many heterotrimeric G-proteins, endothelial nitric oxide synthase, phosphatases, and nonstructural and structural proteins encoded by many viruses including HIV-1.[8a, 9] This is essential for mediating protein–protein interactions and protein–membrane interactions required for the biological activities of many N-myristoyl proteins as well as various newly discovered myristoylated proteins with as yet unidentified roles.[10] NMT-catalyzed protein myristoylation is a lipid modification that occurs through a sequentially ordered bi-bi mechanism. Protein myristoylation consists of a two-step process: the initiator methionine residue on the peptide substrate is removed by methionyl aminopeptidase; the resulting peptide, with a terminal glycine moiety, is now a substrate for NMT activity (Figure 1).[11] The process of protein myristoylation was originally regarded as a co-translational event, which could also occur post-translationally.[12] NMT is constituted by a saddle-shaped b sheet motif flanked by a helices, displaying pseudo-twofold symmetry with regions corresponding to the N- and C-terminal portions of NMT. The myristoyl-CoA binding site is formed by the N-terminal half, and the major portion of the peptide binding site is composed by the C-terminal half.[13] NMT is a member of the GNAT superfamily of proteins, which was first identified in Saccharomyces cerevisiae by Towler, Glaser, and colleagues via the N-terminal sequence (Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg) of the catalytic subunit of cAMP-dependent protein kinase. NMT is also a myristoylated protein in the presence of myristoyl-CoA and ATP.[11b, 14] With advances in the protein myristoylation field, several distinct NMTs have been confirmed in vivo, the molecular weights and subcellular distributions of which clearly vary.[15] Moreover, Raju et al.[16] identified two forms of NMT (NMT-1 and NMT-2) in ChemMedChem 0000, 00, 1 – 14

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Figure 1. NMT catalytic cycle, showing the bi-bi sequential mechanism catalyzed by N-myristoyltransferase.

bovine brain cortex. NMT-1 was reported to be present in four distinct isoforms, ranging from 49 to 68 kDa, whereas only a single NMT-2 isoform (65 kDa) was found.[17] Perinpanayagam et al.[18] reported NMT-1 to be a substrate of caspases-3 and -8, whereas NMT-2 is a caspase-3 substrate only. Meanwhile, the cleavage sites located at the N termini of both NMTs was identified as well. With respect to the functional importance of NMTs in vivo, NMT-1 from various sources has been extensively studied. A polypeptide of 455 amino acids encoded by nmt1 is necessary for vegetative cell growth in S. cerevisiae and early development of mouse embryos;[19] its knockdown inhibits tumor growth.[20] Although the biochemical characterization of NMT-2 is still in its infancy, the roles played by this enzyme are certain. Using western blot analysis, Selvakumar et al.[21] reported that NMT-2 has generally higher expression levels in cancerous tissues than in normal tissues, leading to the candidacy of NMT-2 as a target for the development of anticancer agents.[22] As a novel target for antifungal, antiparasitic, and anticancer agents, NMT is worthy of further study and exploration. In terms of antifungal agents, genetic experiments have indicated that many species of fungi, including Candida albicans and Cryptococcus neoformans, would not survive in vitro without NMT because it plays a significant role in the eukaryotic cell.[23, 24] Therefore, NMT inhibitors as antifungal agents are extremely promising. The potential of NMT as a target for the development of antiparasitic compounds is highlighted by comparative biochemical studies of NMTs from Plasmodium falciparum (a principal causative agent of malaria) and human NMT, given that nmt is a single-copy gene in protozoan parasites.[25] Gene-targeting and RNA interference (RNAi) studies have shown that Leishmania major and Trypanosoma brucei are not viable without NMT.[26] Moreover, genomic searching and comparisons of NMT protein sequences and other genetic and functional data have also indicated that NMTs from other protozoan parasites including L. major, T. brucei, and T. cruzi (causative vectors of cutaneous leishmaniasis, African trypanosomiasis, and Chagas disease, respectively) are good drug targets.[26a, 27]  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org With regard to anticancer agents, NMT has been observed to possess elevated activity in gallbladder cancer,[28] adenocarcinoma,[29] and various other carcinomas.[30] Additionally, the proliferative capacity of mammary epithelial cells is related to NMT activity,[31] and human NMT-1 expression was discovered by quantitative RT-PCR during the progression of eight different human cancers.[32] The above information indicates that human NMT-1 is up-regulated in ovarian, colon, lung, and breast cancers by 1.8- (p = 0.012), 3.1- (p = 0.001), 2.3- (p = 0.003), and 3.7-fold (p = 0.032) on average, relative to respective healthy tissues. It is clear that human NMT-1 can be used as a biomarker for the detection of breast, colon, lung, and ovarian cancers.[32] Thereby NMT is also considered as a novel anticancer target. There are large differences between human NMTs and homologues in parasites and fungi. In higher non-human eukaryotes, there are two predominant isoforms, NMT-1 and NMT-2, which constitute a distinct family of NMTs due to their sequence similarity of 76–77 %. In contrast, human NMT-1 is present as four distinct isoforms, whereas human NMT-2 only exists as a single protein.[17] Although the myristoyl-CoA binding sites of purified human and fungal NMTs are highly conserved, their peptide binding sites are divergent.[33] Thus, many antifungal agents that target the peptide binding site of NMT can avoid producing toxicity. Furthermore, there are a few differences in residue composition in the peptide binding sites of human and fungal NMTs: Ser339, Ala352, and Glu451 residues in human NMT correspond to Phe339, Ile352, and Leu451 residues in C. albicans NMT.[34] Moreover, two human isozymes (NMT-1 and NMT-2) share 77 % identity and in particular, NMT2 is the closest human homologue to T. brucei NMT, with overall 55 % identity and 69 % similarity.[35] On the basis of the above differences between the substrate specificity of fungal and parasitic versus human NMTs, the design and development of species-selective NMT inhibitors are considered to be very promising.[24] On the whole, NMT has been confirmed as a novel and promising antifungal, antiparasitic, and anticancer target. A large number of NMT inhibitors as potent antifungal, antiparasitic, or anticancer agents have been reported recently, based on a specific NMT target. This review highlights recent advances in the search for new natural and synthetic NMT inhibitors, focusing on their biological activity, selectivity, and structure– activity relationship (SAR) information on their antifungal, antiparasitic, or anticancer activities.

1. Antifungal NMT Inhibitors 1.1. Monocyclic compounds 1.1.1. Imidazoles A series of imodazole derivatives were designed and synthesized by Devadas et al.[22b, 36] as potent and selective NMT inhibitors. In this series, compound 1 was shown to be the most potent and selective NMT inhibitor among the imidazole-substituted dipeptide amide derivatives, with an IC50 value of 0.02  0.001 mm against C. albicans NMT, exhibiting 400-fold seChemMedChem 0000, 00, 1 – 14

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CHEMMEDCHEM MINIREVIEWS lectivity versus human NMT. It was also found, through detailed enzyme kinetic analyses, to be a competitive inhibitor (Ki = 0.027  0.007 mm) with regard to the peptide substrate GNAASARR-NH2. This compound provided excellent complementarity for the C. albicans NMT peptide site, which demonstrated nearly 1000-fold greater potency and 400-fold higher selectivity than the original template octapeptide ALYASKLSNH2 by optimizing interactions with the four key recognition elements in the parental octapeptide.[37] Compound 2 was confirmed to be a peptidomimetic NMT inhibitor, with IC50 values of 0.056  0.01 mm against C. albicans NMT and 0.035  0.008 mm against S. cerevisiae NMT. It was also considered a competitive inhibitor (Ki = 0.031  0.003 mm) with regard to the peptide substrate GNAASARR-NH2 via kinetic analysis. Further study indicated that the imidazole-substituted phenylacetyl moiety effectively imitates the tetrapeptide motif GLYA and imparts strong affinity to the inhibitors.[22b, 33c, 38] Compounds 3 and 4 were identified by Brown et al.[39] as sub-micromolar inhibitors, which exhibited good selectivity versus the human enzyme in a survey of potential cyclic and acyclic lysine replacements in known l-seryl-l-lysyl dipeptide NMT inhibitors. Compound 3, with an IC50 value of 0.34  0.05 mm against C. albicans NMT, is an acyclic thialysine analogue which is likely to be easily afforded from commercially available thialysine. In contrast, compound 4 is an acyclic glycinamide analogue with an IC50 value of 0.26  0.01 mm against C. albicans NMT. As can be observed from compound 4, the potent and selective peptidomimetic NMT inhibitors could be thought to retain less basic amine replacements at the lysine-6 and N-terminal amino group, which were found to be two key recognition sites. Compound 5, described by Lodge et al.[40] as a tripeptide NMT inhibitor containing l-serine, l-lysine, and cyclohexyl-lalanine at its C terminus, showed modest inhibitory activity, with an IC50 value of 1.45  0.08 mm against C. albicans NMT, and 560-fold higher selectivity for fungal NMT than human NMT. A single 100 mm dose of this peptidomimetic was added to log-phase cultures of a laboratory strain of C. albicans to generate significant suppression of growth for 24 h (EC50 = 51 17 mm) despite its modest inhibitory activity in vitro. Further evidence demonstrated that its fungistatic effect is directly involved with its inhibition of cellular NMT, because compound 5 affects Arf N-myristoylation and growth, whereas the enantiomer of 5 had no effect on NMT activity in vitro, growth of C. albicans in culture, or Arf N-myristoylation in vivo. These findings show that its fungistatic activity is due to a “specific” physical effect on the organism.[37, 40] Devadas et al.[41] synthesized a new class of biologically active non-peptidic NMT inhibitors emanating from the octapeptide ALYASKLS-NH2. Among them, compound 6, with one chiral center in its structure, had the key enzyme recognition elements tethered to novel scaffolds devoid of peptide bonds. The S and R isomers of compound 6 were both found to be competitive NMT inhibitors, with Ki values of 20 and 9 mm, respectively. In addition, the S isomer was only fungistatic against C. albicans, whereas the R isomer was fungicidal against both C. albicans and C. neoformans with minimum fun 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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gicidal concentration (MFC) values of 250 and 200 mm, respectively. Further study indicated that its fungicidal effect is accompanied by a rapid inhibition of protein N-myristoylation in C. neoformans rather than cell lysis.[41]

1.1.2. Others Compound 7 is one of the most potent peptidomimetic NMT inhibitors identified, with an IC50 value of 0.043  0.006 mm against C. albicans NMT. Its potency is likely due in part to the highly basic amine functionality at the N-terminus. However, it was found to be a nonselective inhibitor of the orthologous fungal NMT due to its modest selectivity versus human NMT.[37] ChemMedChem 0000, 00, 1 – 14

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CHEMMEDCHEM MINIREVIEWS In general, as a result of their inability to penetrate fungal cellular membranes, peptide and peptidomimetic NMT inhibitors have generally weak antifungal activity in vitro.[42]

1.2. Bicyclic compounds 1.2.1. Benzofurans Compounds 8 and 9, reported by Miura et al.,[43] are a pair of enantiomers that displayed potent antifungal activity. The S enantiomer 8 showed an IC50 value of 0.96 mm against C. albicans NMT, whereas R enantiomer 9 is less potent against the same enzyme, with an IC50 value of 3.6 mm. It is quite clear that the stereochemical configuration has a significant impact on antifungal activity. In addition, transferred nuclear Overhauser effect (trNOE) experiments were used to research the bioactive configurations of these derivatives for further optimization of the lead structures. A new class of pyridine-substituted dipeptide amides was prepared by Ohtsuka et al.[44] as potent and selective NMT inhibitors. In the series, the (pyridin-3ylmethyl)amino derivative 10 was designed and synthesized by full use of the structural information of C. albicans NMT in complex with compound 3. Compound 10 showed the strongest inhibitory activity against C. albicans NMT, with an IC50 value of 0.1 mm, displaying more than 5000-fold selectivity over human NMT. The differences in primary sequence between C. albicans and human NMTs could be behind the high degree of selectivity. Further studies showed that a methyl group is still the best substituent at the benzofuran C3 position of compound 10. However, it demonstrated little antifungal activity in a mouse systemic candidiasis model, moderate antifungal activity against C. albicans in vitro, and insufficient metabolic stabili 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org ty (elimination half-life t1/2 = 0.42 h in mice). Nevertheless, compound 10 was still selected as a lead compound for further optimization. Compound 11 was one of the most potent antifungal agents among the benzofuran derivatives, with an IC50 value of 0.0052 mm against C. albicans NMT and a minimal inhibitory concentration (MIC) value of 0.021 mm. Its high activity was found to be mainly due to the presence of an electron-withdrawing group on the aromatic ring attached to the C2 position. However, it had only moderate selectivity over human NMT. Accordingly, further modification work is required.[45] As a new class of antifungal agents, compounds 12 and 13 selectively and potently inhibited C. albicans NMT. They were designed and optimized by Kawasaki et al.[46] based on the crystal structure of NMT in complex with a benzofuran inhibitor and SAR analysis through various biological assays involving a quasi-in-vivo assay and cassette dosing pharmacokinetics (PK) studies in rats. Among the benzofuran derivatives, compounds 12 and 13 demonstrated not only the highest quasiin-vivo antifungal activity, with respective IC50 values of 0.0075 and 0.0057 mm against C. albicans NMT, but also > 450- and 430-fold selectivity over human NMT. They also displayed the most potent antifungal activity, with a median effective dose

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CHEMMEDCHEM MINIREVIEWS (ED50) value of 7.1 mg kg 1 in the in vivo model. Moreover, they were more active in a rat systemic candidiasis model, had a longer t1/2, a better PK profile, and a broader spectrum against a variety of fungal species than compound 10. However, the two compounds showed little activity against C. neoformans and Aspergillus fumigatus.[44b, 45a, 46] Thus, further modification studies are necessary to enhance in vivo efficacy. A further series of benzofuran derivatives were designed and synthesized by Kawasaki et al.[46] by modifying the C2 substituent of compounds 12 and 13 to overcome their instability in artificial gastric fluid (pH 1.2). Among them, the acid-stable compounds 14 and 15 showed the strongest antifungal activity against C. albicans NMT, with IC50 values of 0.00039 and 0.00058 mm, respectively. They also displayed potent antifungal activity in an animal model, with respective ED50 values of 15 and 8.7 mg kg 1. However, they exhibited weak activity against A. fumigatus NMT. Therefore, further modifications are necessary to obtain new compounds with potent activity against both A. fumigatus and C. albicans NMTs, thereby inhibiting the growth of both A. fumigatus and C. albicans.[46] In another series of benzofuran NMT inhibitors reported by Deokar et al.,[47] compound 16 displayed potent antifungal activity with an IC50 value of 0.001 mm against C. albicans NMT. The mechanistic study showed that the imidazole nitrogen atom is approachable for hydrogen bond interaction with active site residues, and its biological activity is closely linked to the conformational rigidity of the substituent at the C4 position of the benzofuran ring, as determined by three-dimensional quantitative structure–activity relationship (3D-QSAR) analysis. The aryl benzofuran-2-yl ketoxime derivative 17 was found to be another active NMT inhibitor. The oxygen atom of the ketoxime moiety forms a hydrogen bond with Asp412 of C. albicans NMT. Compound 17 also undergoes hydrophobic interactions with Val108, Phe117, Tyr225, and Tyr354 of C. albicans NMT.[48] Therefore, these QSAR models are expected for formimg the basis for the design and development of novel NMT inhibitors. 5-Benzofuran-5-ol derivatives 18 and 19, designed by Ryu et al.[49] based on the benzofuran scaffold, showed potent antifungal activity against all tested fungi with common MIC values of 1.6 mg mL 1 against C. albicans and 3.2 mg mL 1 against C. tropicalis, respectively. The benzofuran-5-ols could be metabolized to benzoquinone derivatives with a quinonoid structure in fungi, which demonstrated potent antifungal activity. This suggests that the benzofuran-5-ol scaffolds are very likely to be lead structures for the development of antifungal agents.[49] In particular, compound 20 has shown the greatest antifungal activity identified to date in the benzofuran derivatives against NMT, with an IC50 value of 0.00039 mm. The mechanism of action indicates that the pyridine nitrogen atom forms a hydrogen bond with Asn392 of NMT, and that the pyridine ring itself fits into the hydrophobic pocket of NMT. Moreover, the electron-donating methyl group on the pyridine ring increases the electron density on the pyridine ring, thereby enhancing its hydrogen bond interaction with Asn392.[42, 50]

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www.chemmedchem.org 1.2.2. Benzothiazoles Ebara et al.[51] synthesized a series of novel benzothiazole derivatives. Among them, compound 21, (1R,3S)-N-{2-[(cyclopeanthylcarbonyl)amino]benzothiazol-6-yl}-3-[(2-naphthylmethyl)amino]cyclohexanecarboxamide, was the most active NMT inhibitor, with an IC50 value of 0.00049 mm, whereas compound 22 had an IC50 value of 0.0024 mm against C. albicans NMT. Compound 21 has much higher inhibitory activity than compound 22 due to the more hydrophobic cyclopentyl group in place of the cyclopropyl group on the C2 side chain. Moreover, compound 21 also displayed strong antifungal activity in vitro, with an MFC for C. albicans at 0.78 mm. Kinetics studies indicated that it is noncompetitive for the myristoyl-CoA binding site, while competitive with the peptide binding site.

Studies into the mechanism of action highlighted four important chemical elements that are indispensable for inhibitory activity in the benzothiazole derivatives against C. albicans NMT. The hydrophobic aromatic core of their structures is located at the center of the C. albicans NMT active site, and their two hydrophobic groups on the aromatic core occupy the two hydrophobic pockets in the active site, forming key hydrophobic interactions. The amino group in their structures also forms a hydrogen bonding interaction with the important functional active site residue Leu451. Moreover, an additional hydrogen bond acceptor or donor introduced into their structure is also required for interaction with some important residues, such as Asp110 and Asp112.[5a, 51] Thus, the above information can be applied to the design of new benzothiazole derivatives with potent in vitro and in vivo activity via combination of the results from molecular docking and 3D-QSAR analysis. 1.2.3. Isoquinolines 4-Arylthiosemicarbazide isoquinoline derivatives were acknowledged by Siwek et al.[52] as NMT inhibitors with potent antifungal activity. In particular, compound 23, with an ortho-methyl group at the terminal phenyl ring, showed antifungal activity at non-cytotoxic concentrations in mammalian cells with an MIC value of 50 mg mL 1 against C. albicans. In contrast, although compound 24 displayed the most potent antifungal activity in the series with an MIC value of 25 mg mL 1 against C. albicans, it possessed the highest cytotoxic activity against mammalian cells. Three factors that affect antifungal activity were validated by molecular modeling and docking studies regarding molecular electron-donating capacity: 1) favorable ChemMedChem 0000, 00, 1 – 14

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binding energy, 2) the presence of an electron-accepting heteroaromatic ring at the N1 position of the thiosemicarbazide scaffold, and 3) high HOMO and dipole moment values.

1.2.4. Dihydrobenzofurans Laczkowski et al.[53] designed and synthesized 13 novel 6-hydroxybenzofuran-3(2H)-one derivatives based on 2,4-disubstituted 1,3-thiazoles. In this series, compounds 25 and 26 con-

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hibitor, with an MIC value of 0.25 mg mL 1 against C. albicans. Moreover, molecular docking studies confirmed that the benzoxazole ring is surrounded by hydrophobic residues such as Tyr225, Tyr354, and Leu394 at the center of the active site. Accordingly, it is presumed that hydrogen bonding interactions between the benzoheterocyclic core and the active site of C. albicans NMT are likely to be necessary for potent NMT inhibitors.

1.3. Polycyclic compounds 1.3.1. Hexahydrocarbazoles A series of C6- and N9-modified tetrahydrocarbazole derivatives were found to be NMT inhibitors by Sheng et al.[5b] In the series, compound 29, with an MIC value of 0.0625 mg mL 1 against M. gypseum, was more potent than fluconazole. In par-

taining fluorine and bromine atoms at the phenyl ring, respectively, displayed the most active antimicrobial activity, with a common MIC value of 250 mg mL 1 against C. albicans; they were both isomerically pure (100 % Z isomer). Moreover, microbiological studies indicated that the substituent at the para position of the benzene ring plays a considerable role in antifungal activity. In view of this, the compounds obtained are considered to be promising leads for further structural optimization.

ticular, the most active compound 30 possessed MIC values of 0.0156 mg mL 1 against M. gypseum and 1 mg mL 1 against the clinically important pathogenic fungi C. neoformans.

1.2.5. Others Sheng et al.[54] designed and synthesized a series of indole derivatives as isosteric analogues of benzoheterocyclic NMT inhibitors. Among them, compound 27 displayed marginal activity against C. neoformans and moderate activity against M. gypseum and T. rubrum, with MIC80 values of 64, 8, and 32 mg mL 1, respectively. The indole nitrogen atom forms a weak hydrogen bond with Leu451 rather than the C4 secondary amine, which was responsible for its weak antifungal activity. In contrast, another series of benzoxazole derivatives prepared by Sheng et al.[54] showed much more potent antifungal activity and a broader antifungal spectrum than the above indole derivatives. In this series, compound 28 was the most potent NMT in 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

1.3.2. Furan-quinolines Furo[2,3-f]quinolin-5-ol derivatives were designed by Ryu et al.[55] by incorporating a heterocyclic ring into the benzofuran-5-ol skeleton; these compounds demonstrated potent antifungal activity. Among them, compound 31 had an MIC value of 0.8 mg mL 1 against C. albicans and good inhibitory activity against other fungi. Similarly, compound 32, with MIC values of 0.8 mg mL 1 against C. albicans and 0.8 mg mL 1 against C. tropicalis, was the most potent antifungal inhibitor in the series. Overall, the above results are helpful for the development of promising antifungal agents. ChemMedChem 0000, 00, 1 – 14

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www.chemmedchem.org Compound 37, ()-2-methoxy-4-thiatetradecanoic acid, synthesized by Carballeira et al.,[58] was considered to be a novel antifungal agent because of its markedly high activity against C. albicans (MIC = 0.00124 mm), C. neoformans (MIC = 0.00124 mm), and A. niger (ATCC 16404; MIC = 0.00083 mm). Furthermore, a-methoxylation was effective in increasing its antifungal activity as well as its half-life in fungal cells. 1.4.2. Others

1.3.3. Others Compound 33, found by Taha et al.,[42] was found to be a moderately potent NMT inhibitor, with an MIC value of 289 mg mL 1 on the basis of the pharmacophore modeling, QSAR analysis, and screening of NMT in silico. These methods proved to be useful tools in the search for potential antifungal agents based on NMT inhibition.

Myristoyl-CoA S-(2-ketopentadecyl)-CoA 38 was identified to display the most potent inhibitory activity of human NMT, with an IC50 value of 0.06  0.01 mm in the tested series synthesized by Pasha et al.[57] It was also regarded as the substrate of cAMP-dependent protein kinase.

2. Antiparasitic NMT Inhibitors 2.1. Antitrypanocidal NMT inhibitors 1.4. Acyclic compounds 1.4.1. Fatty acids Myristic acid analogues synthesized by Parang et al.,[56] belonging to ()-2-halotetradecanoic acids, displayed potent antifungal activity against such fungi as C. neoformans and S. cerevisiae. In the series, compound 34, ()-2-bromotetradecanoic acid, showed MIC values of 39 mg mL 1 against C. albicans and 20 mg mL 1 against C. neoformans, while compound 35, ()-2-iodotetradecanoic acid, gave MIC values of 100 mg mL 1 against C. albicans and 30 mg mL 1 against C. neoformans. Further studies showed that the pKa and log P values of ()-2-halotetradecanoic acids and the steric volume of their substituents play crucial roles in their antifungal activities. With a clearer understanding of the uptake and metabolism of the myristic acid analogues, their utility as therapeutic agents will be further improved.

Another myristic acid analogue 36 that was the substrate of cAMP-dependent protein kinase (Gly-Asn-Ala4-Lys2-Arg2) also displayed potent antifungal activity with an IC50 value of 0.66  0.02 mm. The incorporation of a double bond into the alkyl chain at positions 9 and 10 was an important reason for its good antifungal activity.[57]  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

2.1.1. Monocyclic compounds 2.1.1.1. Pyrazole sulfonamides Brand et al.[59] obtained a series of pyrazole sulfonamides, considered as T. brucei NMT inhibitors, by high-throughput screening and chemistry-driven optimization of the hits from the 63362-compound diversity library. In the series, compounds

39, 40, and 41 displayed potent trypanocidal activity. 2,6-Dichloro-substituted pyrazole sulfonamide 39, the design of which was based on the SAR of the aryl group of the pyrazole sulfonamides, showed an IC50 value of 0.34 mm against T. brucei NMT. Similarly, compound 40 designed based on an aminopyridine scaffold and additional protein–ligand interactions, not only demonstrated more potent trypanocidal activity, with an IC50 value of 0.14 mm against T. brucei NMT than compound 39, but also possessed a promising level of selectivity.[59, 60] In particular, compound 41 was the most potent trypanocidal NMT inhibitor in the series. It was able to cure human African trypanosomiasis (HAT) with IC50 values of 0.002 mm against T. brucei NMT and 0.003 mm against human NMT, as well as an ChemMedChem 0000, 00, 1 – 14

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CHEMMEDCHEM MINIREVIEWS EC50 value of 0.002 mm against T. brucei NMT. Furthermore, it showed good oral pharmacokinetics in mice, resulting in sufficient oral levels for the therapy of HAT, and effectively cured mouse models of stage 1 infection with T. brucei and T. brucei rhodesiense.[60] It also has a low volume of distribution (Vd = 0.4 L kg 1), moderate half-life (t1/2 = 1.2 h), and low oral bioavailability (F = 19 %) including low blood clearance (Clb = 6 mL min 1 kg 1). Unfortunately, compound 41 was unable to cross the blood–brain barrier, thereby exhibiting less selectivity against human NMT.[59, 61] Compound 42 was obtained by Frearson et al.[61] by optimizing a screening hit, which involved the design and synthesis of over 200 compounds. It was considered a model NMT inhibitor, with IC50 values of 0.002 mm against T. brucei NMT and 0.004 mm against human NMT. It also inhibited the proliferation of the bloodstream form T. brucei in culture with an EC50 value of 0.002 mm. Thus, compound 42 was assessed for efficacy in an animal model of trypanosomiasis given its impressive potency in inhibiting both T. brucei NMT and proliferation in vitro, together with its promising physicochemical properties. Compound 42 also displayed promising selectivity at the cellular level despite its small window of selectivity between human and T. brucei NMTs.

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1.7 mm against P. falciparum NMT. They were the most distinctive to be used for the treatment of malaria.

2.2.1.2. Others Sheng et al.[26b] identified a series of selective inhibitors against the NMTs of P. falciparum and T. brucei via a “piggyback” approach and high-throughput screening of range of known fungal NMT inhibitors. In this series, compound 46 was found to be the most potent NMT inhibitor, with IC50 values of 0.36 mm against P. falciparum NMT and 0.74 mm against T. brucei NMT. Further binding mode and SAR studies indicated that

2.1.2. Bicyclic compounds Compound 43 was considered to be one of the most potent T. brucei inhibitors among the other bicyclic molecules tested, as it displayed an IC50 value of 0.250  0.04 mm against T. brucei NMT and an ED50 value of 66 mm against mammalian parasite stages. Moreover, it proved to be nontoxic to murine macro-

compound 46 forms five hydrogen bonds with P. falciparum NMT, as the loss of hydrogen bonding could lead to a fivefold decrease in binding affinity for NMT.

2.2.2. Bicyclic compounds phages (ED50 > 1500 mm). Panethymitaki et al.[62] previously demonstrated that NMT was essential for the viability of L. major and T. brucei, using gene targeting and RNAi. However, its inhibitory activity against L. major NMT was demonstrated.

2.2. Antiplasmodia NMT inhibitors 2.2.1. Monocyclic compounds 2.2.1.1. Pyrimidines A novel series of pyrimidine-based antiprotozoal NMT inhibitors were identified by Bell et al.[63] in a high-throughput screen of the Pfizer corporate compound collection. Among them, azetidine pyrimidines 44 and 45 displayed potent antiplasmodial activity with respective IC50 values of 0.482 and  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

2.2.2.1. Benzothiophenes A series of novel benzo[b]thiophene-containing inhibitors against P. falciparum and Plasmodium vivax NMTs were designed and synthesized by Rackham et al.,[64] who used a leadhopping approach. They were found to show antiparasitic activity in vitro, good selectivity against human NMT-1 and excellent ligand efficiency. Among them, compound 47 displayed extraordinarily effective antiparasitic activity in vitro, with Ki values of 1.3 mm against P. falciparum NMT and 1.32 mm against P. vivax NMT. As a result, 47 was considered a lead compound for the development of promising plasmodial NMT inhibitors. In particular, compound 48 showed the most potent antiplasmodial activity in this series, with Ki values of 0.83 mm against P. falciparum NMT and 0.08 mm against P. vivax NMT, as well as an EC50 value of 2.0 mm. It was found to have 10-fold greater affinity for P. vivax NMT over P. falciparum NMT. However, the ChemMedChem 0000, 00, 1 – 14

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presence of labile ester groups may result in unknown metabolic stability; the next step in research will focus on the stability of benzothiophenes with strong potency and high selectivity.

2.2.2.2. Quinolines A series of novel quinoline inhibitors of P. vivax NMT, 3-butyl-4[(2-cyanoethyl)thio]-6-methoxy-2-methylquinolines, were discovered by Goncalves et al.[65] in a high-throughput screen of 59 500 compounds from Medical Research Council Technology. Among them, compound 49 was found to be the most promising NMT inhibitor, with a Ki value of 1.73  0.16 mm against P. vivax NMT. In addition, another series of quinoline NMT in-

hibitors were designed and synthesized on the basis of the SARs established and the binding modes as confirmed by high-resolution X-ray crystallographic data. Among them, compound 50 showed micromolar activity against P. vivax NMT, with a Ki value of 4.74  0.24 mm, improved lead-like properties (Clog P = 3.0, LipE > 2, Mr = 316 Da) and high selectivity over human NMT isoforms relative to compound 49. The SAR study indicated that the aliphatic chain in the quinoline structure replaced by an ethyl ester both strongly enhances selectivity, particularly over human NMT-2, and retains potent activity against P. vivax NMT. In addition, compound 51, obtained by Crowther et al.[66] in a high-throughput screen of 5655 low-molecular-weight compounds, displayed potent antimalarial activity, with an IC50 value of 1.4 mm against P. vivax NMT.

ly influenced by potential biological instability of this ester moiety. Compound 53, bearing a 1,2,4-oxadiazole moiety, also exhibited high selectivity and promising activity with an IC50 value of 2.0 mm against P. falciparum NMT, but was still less active than 51.

2.2.3. Polycyclic compounds Compound 54 had potent inhibitory activity with an IC50 value of 0.460  0.04 mm against T. brucei NMT and an ED50 value of 16 mm against mammalian parasite stages. It was more significant that some weak inhibitory activity remained against L. major NMT; 54 was also remarkably nontoxic to murine macrophages.[62] Compound 55 was found to be another potent NMT inhibitor similar to compound 46; it was identified by Sheng et al.[26b] via a “piggyback” approach and high-throughput screening. It had IC50 values of 0.28 mm against P. falciparum NMT and 0.77 mm against T. brucei NMT, and sixfold selectivity for P. falciparum NMT over human NMT. Moreover, it docked into the active site of T. brucei NMT, forming five hydrogen bonds with active site residues.

2.2.2.3. Benzofurans Yu et al.[67] designed and synthesized a series of novel benzofuran derivatives with potent and selective activity against P. falciparum NMT based on the hit compound obtained during chemistry-driven optimization. In the series, compound 52 was found to be the most potent NMT inhibitor, demonstrating the highest antiparasitic activity in vitro with an IC50 value of 0.27 mm against P. falciparum NMT. Further studies showed that the ester group in the C2 side chain plays a key role in enhanced potency. Unfortunately, its scope was serious 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

3. Anticancer NMT Inhibitors 3.1. Monocyclic compounds 3.1.1. Metal complexes A series of metal complexes were designed and synthesized by Shrivastav et al.[68] for the first time as novel therapeutic agents targeting NMT for cancer. In this series, compounds 56 and 57, ChemMedChem 0000, 00, 1 – 14

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www.chemmedchem.org action indicated that the low IC50 values toward four malignant cell lines could be attributed, in part, to their interference with NMT activity.[69] Another series of 6-arylidene-2-dimethylaminomethylcyclohexanone hydrochlorides, synthesized by Dimmock et al.,[70]

copper(II) and manganese(III) complexes of N-[(2-hydroxyphenyl)carbonothioyl]pyridine-2-carbohydrazide, were found to be the most promising agents, with IC50 values of 12.2 and 16.1 mm, respectively. Their cytotoxicity against the colon cancer cell line HT29 was due to induction of apoptosis and inhibition of endogenous NMT activity. Interestingly, they had different formation processes: the loss of one proton from the ligand via enolization/thioenolization gave compound 56, whereas the loss of one and two protons from each ligand by aerobic oxidation generated compound 57. In addition, the endogenous NMT activity was inhibited by 48 and 30 %, respectively, when treated with the two compounds.

3.1.2. Benzenesulfonamides A library of novel N-heterocyclic sulfonamide derivatives were identified as effective anticancer inhibitors of human NMT-1. Compounds 58 and 59 were the representatives in this series.

showed potent cytotoxicity toward a wide range of tumor cell lines. In particular, compound 62, with weak inhibitory activity toward human NMT (IC50 : 500 mm) gave IC50 values of 2.29  0.11, 2.40  0.18, and 2.18  0.04 mm toward L1210, Molt 4/C8, and CEM cell lines, respectively. Furthermore, it possessed some selectivity toward colon and breast tumors. It was also indicated that the small electronegative aryl substituents had a strong effect on anticancer potency. Considering this, more potent anticancer NMT inhibitors could be generated by further molecular modifications.

3.2. Bicyclic compounds 3.2.1. Naphthalene ketones

It was confirmed that compound 58 showed powerful cytotoxic activity, with an IC50 value of 4 mm against human NMT and retained good CNS penetration and stability. Compound 59, obtained by replacing the 4-bromo group of compound 58 with a 4-(2-(piperazin-1-yl)pyridin-4-yl) group, showed strong cytotoxic activity, with IC50 values < 1 mm against various colon and breast cancer cell lines and 0.005 mm against human NMT.[7b]

Das et al.[71] designed a series of compounds containing a 5aryl-1-(4-nitrophenyl)-3-oxo-1,4-pentadienyl pharmacophore as novel cytotoxic and anticancer agents. In this series, the most active compounds, 63 and 64, which comprised a cluster of 3arylidene-1-(4-nitrophenylmethylene)-2-oxo-3,4-dihydro-1Hnaphthalenes, demonstrated marked cytotoxicity with IC50 values of 3.20  0.48, 0.968  0.186, and 0.953  0.377 (63), 4.82  2.43, 1.13  0.64 and 1.62  0.11 mm (64) toward L1210, Molt 4/C8, and CEM cell lines, respectively. They were also identified to have activity with an IC50 value of 54  2 mm against human tumor cell lines from nine different neoplastic diseases and showed weak inhibitory activity toward human NMT. Clearly, their particular potency toward malignant cells in

3.1.3. Others A series of Mannich bases of a,b-unsaturated ketones were confirmed to have potent cytotoxic activity. Among them, compounds 60 and 61 as thiol alkylators were the representative compounds with IC50 values of 4 and 1.5 mm against human NMT, respectively. Studies into the mechanism of  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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CHEMMEDCHEM MINIREVIEWS general and leukemic and colon cancers make them suitable lead molecules. In short, naphthalene ketones should be pursued further in the development of potential anticancer agents given the lack of rapid onset of toxicity in rodents and their ability to inhibit of the growth of H. pylori.

www.chemmedchem.org the optimal length of the alkyl chain and the effect of various alkyl chain substituents with different hydrophobicities, steric and electronic properties are questions that have yet to be answered for future modifications.

Conclusions and Outlook 3.3. Polycyclic compounds In a high-throughput screen of ~ 16 000 “drug-like” small molecules, French et al.[72] discovered members of a novel chemotype, cyclohexyl-octahydro-pyrrolo[1,2-a]pyrazine (COPP), as anticancer NMT-1 inhibitors with micromolar potency. In this series, 9-ethyl-9H-carbazole compound 65 was observed to be the most active inhibitor, with an IC50 value of 6  1 mm against human NMT. Docking studies indicated that the cyclohexyl

group plays a key role in the inhibition of NMT, and the carbazole side chain closely interacts with further NMT residues. Compounds with substituted benzyl, phenyl, or thiophenyl groups were less potent than those with multiple conjugated ring systems. Further studies showed that compound 65 is competitive with the peptide substrate and is noncompetitive with regard to the lipid substrate. In general, these polycyclic molecules are likely have great potential as anticancer agents, and the following stages of research will focus on their in vivo therapeutic effects.

3.4. Acyclic compounds 3.4.1. Myristoyl coenzyme A A series of lipidic myristoyl coenzyme A derivatives were synthesized by Bajaj et al.[73] as anticancer agents. In this series, S(2-ketopentadecyl)-CoA 66 and 2-dodecylglycidoyl-S-CoA 67 were the most potent NMT inhibitors, with respective IC50 values of 0.06 and 0.45 mm against human NMT. The affinity of

In general, NMT as a novel target for antifungal, antiparasitic, and anticancer agents is a well-characterized enzyme. A large number of NMT inhibitors have been reported so far. Moreover, NMT, in its capacity as a target for antifungal agents, is the earliest and the most in-depth studied. However, NMT’s role as a target for antiparasitic agents is still emerging, and investigations into NMT as a target for anticancer therapies is still in its infancy. This review describes the key representative inhibitors reported recently from these three therapeutic fields. Firstly, although antifungal NMT inhibitors are represented by various chemotypes such as peptidomimetics, benzofurans, and benzothiazoles,[38] only benzofuran and benzothiazole inhibitors have displayed high selectivity and good antifungal activity. In contrast, peptidomimetic NMT inhibitors are generally devoid of in vivo antifungal activity owing to their inability to penetrate fungal cellular membranes.[41] For instance, benzofuran derivatives 14 and 20 showed the most potent activity against C. albicans NMT with identical IC50 values of 0.00039 mm. Secondly, selective and potent antiparasitic NMT inhibitors derived from the “piggyback” approach demonstrate excellent ligand efficiency, which is a promising starting point for the development of highly selective inhibitors. For example, the pyrazole sulfonamide 41 is the most potent trypanocidal NMT inhibitor, which is able to cure human African trypanosomiasis with IC50 values of 0.002 mm against T. brucei NMT and 0.003 mm against human NMT. Nevertheless, a good translation of enzyme to cellular activity and efficacy in vivo have yet to overcome some significant barriers.[74] Finally, anticancer NMT inhibitors possess cytotoxicity against cancer cells. In particular, compound 58 shows strong cytotoxic activity, with IC50 values < 1 mm against various colon and breast cancer cell lines, and 0.005 mm against human NMT, making it a promising anticancer agent. Although the development of NMT-based chemotherapeutic agents is still a very new field of research, the development of potent anticancer NMT inhibitors is desirable. This review also focuses on the functions of NMT, known representative natural and synthetic inhibitors, as well as their biological activity, selectivity, and SAR information. On the whole, it is believed that on the basis of NMT as a novel drug target, more novel potent NMT inhibitors as antifungal, antiparasitic, and anticancer agents will be discovered in the near future.

Acknowledgements the myristoyl group [CH3(CH2)12CO] in their structures for human NMT-1 could hinder the catalytic transfer of myristoylCoA to the substrate. Further SAR studies showed that the carbonyl group plays an important role in the activity of these lipidic NMT inhibitors. Replacement of the methylene group at position 3 would strongly decrease their potency. In addition,  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

This research was supported financially by the National Natural Science Foundation of China (20872081 and 21072114), the Natural Science Foundation of Shandong (ZR2010M092), and the China–Australia Centre for Health Sciences Research (CACHSR, 2014J06). ChemMedChem 0000, 00, 1 – 14

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CHEMMEDCHEM MINIREVIEWS Keywords: anticancer · antifungal · antiparasite myristoyltransferase · structure–activity relationships

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Received: May 2, 2014 Revised: July 17, 2014 Published online on && &&, 0000

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MINIREVIEWS C. Zhao, S. Ma* && – && Recent Advances in The Discovery of N-Myristoyltransferase Inhibitors

 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

A key target: Fungal infections, parasitic diseases, and cancers remain three major global health problems. N-Myristoyltransferase (NMT) has been identified as a novel and promising target for antifungal, antiparasitic, and anticancer agents. This review highlights recent advances in the discovery of NMT inhibitors, with particular focus on their activities, structure–activity relationships, and mechanisms.

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