INHIBITORS

Abstract

The present invention relates to compounds having the formula [Co-enzyme A or analogue thereof]-Z1-Z2-Z3-Z4, wherein Z1 is a linker, Z2 and Z3 are peptides or peptide-based moieties, and Z4 is a C-terminal group. The invention also provides pharmaceutical compositions comprising compounds of the invention, and their uses for the treatment of cancer, wound healing and nerve regeneration, inter alia.

Claims

1. A compound of formula Co-enzyme A-acetyl-SS(Nle)P-NH.sub.2 (SEQ ID NO: 4).

2. A compound of Formula I:
[Co-enzyme A or analogue thereof]-Z1-Z2-Z3-Z4 (I) wherein Z1 is a linker moiety or is absent; Z2 consists of a peptide or peptide-based moiety having the Formula II: TABLE-US-00013 (SEQIDNO:1) X1-X2-X3-X4(II) wherein X1 and X2 are independently S or Hse; X3 is M or Nle; a non-natural amino acid; a C.sub.1-5 cyclic or non-cyclic alkyl group; or a C.sub.1-5 ether group; and X4 is P or is absent; or Z2 consists of a peptide wherein the amino acid sequence of the peptide has the Formula III: TABLE-US-00014 (SEQIDNO:2) X5-X6-X7-X8(III) wherein X5 is D, E, M or Q; X6 is D, E or S; X7 is D, E or Q; and X8 is I or L; Z3 is 0-30 amino acids; and Z4 is a C-terminal group or is absent, or a pharmaceutically-acceptable salt thereof.

3. The compound as claimed in claim 2, wherein the Co-enzyme A analogue is ##STR00005## wherein R1 is hydrogen, a phosphate group, acetoacetate, alkyl, aralkyl or a cyclic alkyl.

4. The compound as claimed in claim 2, wherein Z1 is acetyl.

5. The compound as claimed in claim 2, wherein Z2 consists of a peptide or peptide-based moiety having the Formula II: TABLE-US-00015 (SEQIDNO:1) X1-X2-X3-X4(II) wherein X1 and X2 are independently S or Hse; X3 is M or Nle; and X4 is P.

6. The compound as claimed in claim 2, wherein Z2 has the amino acid sequence: TABLE-US-00016 (SEQIDNO:3) SSMPor (SEQIDNO:4) SS(Nle)P.

7. The compound as claimed in claim 2, wherein Z2 consists of a peptide and wherein the amino acid sequence of the peptide is TABLE-US-00017 (SEQIDNO:5) X5-X6-X7-X8 wherein X5 is E or D X6 is D or E X7 is D or E, and X8 is I or L.

8. The compound as claimed in claim 7, wherein Z2 consists of a peptide, wherein the amino acid sequence of the peptide is selected from the group consisting of: TABLE-US-00018 (SEQIDNO:6) DDDI, (SEQIDNO:7) EEEI, (SEQIDNO:8) MDEL, (SEQIDNO:9) DEDI, (SEQIDNO:10) DEEL, (SEQIDNO:11) EDDI, (SEQIDNO:12) EDEI, (SEQIDNO:13) EEDL, (SEQIDNO:14) EEEL, (SEQIDNO:15) DDEI, (SEQIDNO:16) EDQL, (SEQIDNO:17) ESEL, (SEQIDNO:18) DEEI, (SEQIDNO:19) EEDI, (SEQIDNO:20) EDEL,and (SEQIDNO:21) QEEI.

9. The compound as claimed in claim 8, wherein the amino acid sequence of the peptide is selected from the group consisting of: TABLE-US-00019 (SEQIDNO:6) DDDI, (SEQIDNO:7) EEEI, (SEQIDNO:11) EDDI, (SEQIDNO:19) EEDI,and (SEQIDNO:20) EDEL.

10. The compound as claimed in claim 2, wherein Z3 is K or is absent.

11. The compound as claimed in claim 2, wherein Z4 is NH.sub.2 or N-alkyl.

12. The compound as claimed in claim 2, which additionally comprises a targeting moiety.

13. The compound as claimed in claim 12, wherein the targeting moiety is a cancer cell-targeting moiety.

14. The peptidomimetic of a compound as claimed in claim 2.

15. A pharmaceutical composition comprising a compound as claimed in claim 2, optionally together with one or more diluents, carriers or excipients.

16. (canceled)

17. A method of treating or preventing a disease or disorder associated with NatA activity, the method comprising administering an effective amount of a compound as claimed in claim 2, wherein Z2 has the sequence of SEQ ID NO: 1, to a subject in need thereof.

18-19. (canceled)

20. A method of treating or preventing cancer, the method comprising administering an effective amount of a compound as claimed in claim 2, wherein Z2 has the sequence of SEQ ID NO: 1, to a subject in need thereof.

21-22. (canceled)

23. The 4-method as claimed in claim 20, wherein the cancer is selected from the group consisting of lymphomas, leukaemias, neuroblastomas, glioblastomas, carcinomas, adenocarcinomas, melanomas, lung cancer, breast cancer, hepatocellular carcinoma, colorectal cancer, pancreatic cancer, ovarian cancer, gastric cancer, non-small cell lung cancer, papillary thyroid carcinoma, neuroblastoma, prostate cancer and thyroid cancer.

24. (canceled)

25. A method of treating or preventing a disease or disorder associated with Naa80 activity, the method comprising administering an effective amount of a compound as claimed in claim 2, wherein the amino acid sequence of Z2 is SEQ ID NO: 2, to a subject in need thereof.

26-27. (canceled)

28. A method of enhancing cell mobility or wound healing, the method comprising administering an effective amount of a compound as claimed in claim 2, wherein the amino acid sequence of Z2 is SEQ ID NO: 2, to a subject in need thereof.

29-31. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0126] FIG. 1: Immunoprecipitated Naa80-V5 was used in Nt-acetylation assays with [.sup.14C]-Ac-CoA and peptides representing a variety of N-termini.

[0127] FIG. 2: Sequence alignment (first 61-63 amino acids) of the six human mature actin isoforms generated as in (A). Of note, methionines and/or cysteines at the second position are removed prior to Nt-acetylation (arrows) of newly-formed acidic N-termini.

[0128] FIG. 3: Naa80-eGFP expressed in HeLa and HAP1 cells shows a cytosolic distribution.

[0129] FIG. 4: NAA80 KO1 cells were generated by deleting 17 bp in exon 2 and NAA80 KO2 by inserting 404 bp into exon 2 of the NAA80 gene. Putative gene product of NAA80 KO1 consists of 193 aa and in NAA80 KO2 of 151 aa protein. Both knockout gene products lack the crucial Ac-CoA binding motif.

[0130] FIG. 5: Phosphorescence imaging shows a distinct 43-kDa band in NAA80 KO1 cell lysates incubated with [.sup.14C]-Ac-CoA and purified recombinant MBP-Naa80, suggesting the incorporation of [.sup.14C]-Ac at the N-terminus of actin.

[0131] FIG. 6A: The isoform- and Nt-acetylation specificity of anti Ac--actin and Ac--actin antibodies is confirmed in dot blot assays with peptides representing the unacetylated and acetylated N-termini of - and -actin.

[0132] FIG. 6B: Naa80 expression is required for actin Nt-acetylation. Immunoblot analysis of HAP1 control and NAA80 KO1 cells transfected with either empty V5 plasmid, wild-type Naa80-V5 or the catalytically inactive mutant Naa80mut-V5. The samples were probed with anti Ac--actin, Ac--actin, pan-actin, V5 and GAPDH antibodies.

[0133] FIG. 7: Actin Nt-acetylation affects cell motility. (A) Representative images of HAP1 control and NAA80 KO1 cells in wound-healing assay showing the degree of gap closure after 18 h. Scalebar, 150 m. (B) Quantification of gap size in wound-healing assays relative to the size at t0. (C) Average cell front velocity (m/h) calculated from (B). (D) Chemotaxis assay showing percentage of cells migrated from a 1% to 10% FBS chamber. (E) Random migration assay showing the percentage of cells migrating from a 10% to 10% FBS chamber. Asterisks represent significance determined by two-sided Student's t-test: *** p0.001.

[0134] FIG. 8: Effects of actin Nt-acetylation on cytoskeletal morphology. (A) and (B) Representative micrographs illustrating a differential morphological phenotype at the level of filopodia between HAP1 ctrl (A) and NAA80 KO1 (B) cells where the absence of Naa80 promotes filopodia formation. (C) and (D) For both cell lines, (C) the number and (D) length of filopodia were determined. (E) G/F-actin ratios from HAP1 control and NAA80 KO1 cells. Asterisks represent significance determined by Student' s t-test: * p0.05; ** p0.01; *** p 0.001. Shown are the meansSD. (F) and (G) Confocal images (top) and STED zoom-in frames of lamellipodia (bottom). The number of cells containing at least one clearly distinguishable lamellipodium supported by filopodia/microspikes was determined. Since HAP1 cells typically grow in clusters, only cells at the borders of clusters (i.e. with a front facing away from the cluster) were considered, as indicated by the numbering. Lamellipodia-positive cells are indicated by green numbers. (H) Quantification of lamellipodia-positive cells.

[0135] FIG. 9: Actin Nt-acetylation affects filament elongation and depolymerization. (A) Coomassie and immunoblot analysis of cytoplasmic actin purified from wild type and NAA80 KO cells. 100% Nt-acetylated a-skeletal actin (15) is shown for reference. (B) The polymerization rate of actin alone (2M actin (6% pyrene-labelled)) is unchangedNt-acetylation. Data shown as the average curve from the number of independent experiments indicated in each panel, with standard deviation error bars in lighter color (Ac-actin, blue; non-Ac-actin, red). (C) Elongation actin alone (1.5 M filament seeds; 0.5 M actin (6% pyrene-labelled)). The barbed end elongation rate of actin filament seeds is 2.2-fold faster for Ac-actin than non-Ac-actin. The polymerization of Ac-actin and non-Ac-actin in the absence of seeds are shown as controls. (D) Depolymerisation actin alone (0.1 M filament seeds; (6% pyrene-labelled). The depolymerization rate of actin filament seeds is 1.7-fold faster for Ac-actin than non-Ac-actin. (E) Nucleation and branching (Arp complex). 2 M actin (6% pyrene-labelled). 100 nM N-WASP WCA [Arp complex]. The concentration dependence of polymerization rates of actin assembly by Arp complex (nucleation and branching) shows no difference for Ac-actin vs. non-Ac-actin. Shown is the means.e.m of three independent experiments. (F) Nucleation and barbed end capping (mDia2). 2 M actin (6% pyrene-labelled). [mDia2]. The concentration dependence of polymerization rates of actin assembly induced by mDia2 (nucleation and barbed end capping) is unchangedNt-acetylation. (G) Nucleation and elongation (mDia1). 2 M actin (6% pyrene-labelled). [mDia1]. The concentration dependence of polymerization rates of actin assembly induced by mDia1 (nucleation and barbed end elongation) is>2-fold faster for Ac-actin than non-Ac-actin. (H) Formin elongation from profilin1-actin. 1.5 M filament seeds. 20 nM mDia1, 0.5 M actin (6% pyrene-labelled), 1.5 M profilin1. The elongation rate of filament seeds by mDia1 from profilin1-actin is 35% faster for Ac-actin than non-Ac-actin. For both actins, the elongation rate of mDia1 from profilin1-actin is faster relative to the elongation rate of actin seeds alone (shown as controls). (I) Formin elongation from profilin2-actin. 1.5 M filament seeds. 20 nM mDia1, 0.5 M actin (6% pyrene-labelled), 1.5 M profilin2. In contrast, barbed end elongation by mDia1 from profilin2-actin is unchanged for non-Ac-actin and reduced 40% for Ac-actin relative to the elongation rate of actin seeds alone.

[0136] FIG. 10: (A) IC50 values for inhibitors measured in an Nt-acetylation inhibitor assay for Naa80. (B) Ki-values for CoA-Ac-DDDI-NH.sub.2 (SEQ ID NO: 6) for different human NATs measured in an Nt-acetylation inhibitor assay. All reactions were performed at least three times and in triplicate; errors bars represent standard deviation of each measurement.

EXAMPLES

[0137] The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these

[0138] Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1: Synthesis of Bisubstrate Analogues

[0139] Bisubstrate analogues were prepared by Fmoc-based solid phase peptide synthesis (SPPS) on a Biotage Initiator+ Alstra (Biotage, Sweden) automated microwave peptide synthesizer using a ChemMatrix Rink amide resin (0.47 mmol/g loading) on a 0.2 mmol scale. Each amino acid (4 equiv.) was coupled using DIC (4 equiv.) and oxyma (4 equiv.) or HCTU (4 equiv.) and DIPEA (8 equiv.) in dimethylformamide (DMF) with microwave heating at 75 C. for 5 min. Removal of the Fmoc protecting group was facilitated by treating the resin with piperidine (20% in DMF) at room temperature for 3+10 min. Following Fmoc-deprotection of the N-terminal amino acid, resin-bound peptides were treated with bromoacetic acid (8 equiv.) and DIC (8 equiv.) in DMF for 1 hr as previously described. Final deprotection and cleavage from the solid support was facilitated by treatment of the resin with a mixture of trifluoroacetic acid (TFA), triisopropylsilane (TIS) and water (95:2.5:2.5 v/v, 12.5 mL/g of initial resin used) for 2 h. For the Met-containing inhibitor, a mixture of TFA, TIS, ethane dithiol and water (92.5:2.5:2.5 v/v, 12.5 mL/g of initial resin used) was used. The resin was removed by filtration and washed with an additional portion of cleavage cocktail (12.5 mL/g of initial resin used). The combined TFA fractions were concentrated until approximately 5 mL of the solution remained upon which diethyl ether was added to facilitate precipitation. The diethyl ether was carefully removed using a pipette, and the residues were washed with two portions of fresh diethyl ether. The crude bromoacetyl peptides were dried under vacuum, purified by semipreparative reverse-phase high performance liquid chromatography (RP-HPLC) and lyophilized.

[0140] Purified bromoacetyl peptides (typically 10 mg) and CoA trilithium salt (2 equiv.) were dissolved in triethylammonium bicarbonate buffer (1 M, pH 8.5) and left at room temperature for 16 hr. Finally, the CoA-Ac-peptides were purified by semi-preparative RP-HPLC and lyophilized to give colorless powders. All bisubstrate analogues were purified to a purity of>95% (UV 220 nm) and their structures were confirmed by ESI-MS and NMR analyses.

Example 2: Inhibition of NatA using Bisubstrate Analogues

[0141] A number of bisubstrate analogues were prepared as described in Example 1 with potential inhibitory properties against NatA.

[0142] The IC50 values for these compounds were tested as described in Foyn H, et al. (2013) Design, Synthesis, and Kinetic Characterization of Protein N-Terminal Acetyltransferase Inhibitors. ACS Chem Biol 8:1121-1127; and Foyn H, et al. (2017) DTNB-Based quantification of in vitro enzymatic N-terminal acetyltransferase activity, Methods Mol. Biol. 1574:9-15. Briefly, a colorimetric acetylation assay, 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) assay was used to measure the catalytic activity of NATs after incubation with NAT inhibitors. In the DTNB assay, thiols present in the enzymatic product, CoA, cleaves DTNB and produces 2-nitro-5-thiobenzoate (NTB-), which absorbs light with a wavelength of 412 nm. NTB- forms in a 1:1 molar ratio to the thiol groups present in the sample. Therefore, NTB- was quantified by measuring the absorbance at 412 nm and indirectly measures peptide Nt-acetylation. Recombinant purified NAT enzyme was mixed with different substrate peptides (300 uM, custom-made) and Ac-CoA (300 uM) in acetylation buffer (50 mM Tris/HEPES pH 8.5-7.4, 100 mM NaCl, 10% glycerol and 0.2 mM EDTA) and with inhibitor concentration (0-500 uM). After 30 min-60 min incubation at 37 C., the enzyme activity was quenched with quenching buffer (3.2 M guanidinium-HCl, 100 mM sodium phosphate dibasic pH 6.8). Triplicates were run for all positive samples, while negative controls were duplicated. As a negative control, reactions without enzyme were incubated at 37 C. before the reaction was quenched and added enzyme. In order to measure CoA production, 2 mM DTNB (dissolved in 100 mM sodium phosphate dibasic pH 6.8 and 10 mM EDTA) was added to the quenched reaction and the absorbance at 412 nm was measured with a spectrophotometer (Epoc). Background absorbance (determined in negative controls) was subtracted from the absorbance determined in each individual reaction. Thiophenolate production was quantified assuming =13.7103 M1 cm1. For NatA and NatB enzyme complex inhibition, immunoprecipitation was performed using Naa15 ab (custom made, Arnesen T, Anderson D, Baldersheim C, Lanotte M, Varhaug J E, Lillehaug J R. (2005) Identification and characterization of the human ARD1-NATH protein acetyltransferase complex. Biochemical J 386(Pt 3):433-43.) or Naa20 ab (Nat5, abnova) respectively and protein A/G magnetic agarose beads (Millipore). A similar Nt-acetylation reaction was performed as previously described and the enzymatic activity was quenched with a final concentration of 1% TFA and analyzed by HPLC (Evjenth R, Van Damme P, Gevaert K, Arnesen T (2013) HPLC-based Quantification of in vitro N-terminal Acetylation. Methods Mol Biol 981:95-102.).

[0143] The results are shown in the table below.

TABLE-US-00009 TABLE 1 Overview of IC50 values of all inhibitors tested against NatA Compound IC50 (M) SEQ ID NO: CoA-Ac-SESS 15.4 0.97 26 CoA-Ac-SASE 62.4 35.5 27 CoA-Ac-AASE 247 10.5 28 CoA-Ac-SSSE 9.47 0.21 29 CoA-Ac-SAAE 12.1 1.12 30 CoA-Ac-SSAE 4.7 0.28 31 CoA-Ac-SYAE 36.2 0.85 32 CoA-Ac-SSAA 5.3 0.24 33 CoA-Ac-SSME 1.28 0.18 34 CoA-Ac-SSMP 0.82 0.11 3 CoA-Ac-SSMPV 2.1 0.42 35 CoA-Ac-SS(Nle)P 1.46 0.29 4 CoA-Ac-PS(Nle)P >>1000 36

[0144] All of the above compounds comprised C-terminal amide groups.

[0145] Although CoA-Ac-SSMP (SEQ ID NO: 3) was found to be a very potent inhibitor of NatA, there were problems synthesizing it; this was due in part to a side-reaction of the methionine side chain. A norleucine (Nle) residue was therefore tried in place of methionine in order to try to circumvent the synthesis problem. CoA-Ac-SSNleP (SEQ ID NO: 4) showed only a small increase in IC50 value compared to CoA-Ac-SSMP (SEQ ID NO: 3), but it was much more easily synthesized. The CoA-ac-SSNleP inhibitor (SEQ ID NO: 4) also gave the same phenotype in zebrafish as SSMP (SEQ ID NO: 3).

[0146] Both CoA-Ac-SSNlePK-folic acid (SEQ ID NO: 23) and CoA-Ac-SSNlePK-CPP9 (SEQ ID NOs: 23 and 25) gave comparable phenotypes as CoA-Ac-SSNleP-NH2 (SEQ ID NO: 4) when injected into zebrafish embryos.

Example 3: Selectivity of Bisubstrate Analogues for NatA

[0147] In order to ensure selectivity towards NatA, the CoA-Ac-SSNIeP (SEQ ID NO: 4) was tested against other NATs. Using the Cheng-Prusoff equation for competitive inhibitors, the dissociation constant, K.sub.i, was calculated from IC50 values and [S]/K.sub.m ratios for all the NATs.

[0148] The results (see Table 2 below) clearly indicate that CoA-Ac-SSNleP (SEQ ID NO: 4) selectively inhibits NatA because it is at least 100-fold more potent towards NatA than all the other NATs tested.

TABLE-US-00010 TABLE 2 K.sub.i values for CoA-Ac-SSNleP (SEQ ID NO: 4) against all NATs Compound K.sub.i (M) NatA 0.143 0.03 Naa10 98.9 443 NatB 41.6 51.2 Naa30 132.1 Naa40 41.9 6.4 Naa50 17.7 7.4 Naa60 3.1 0.8

Example 4: Use of NatA Bisubstrate Analogues in Cell-Proliferation Assay

[0149] Cancer cell culture screening of the cell permeable NatA bisubstrate-analogue inhibitors (NatA inhibitor+negative control CoA-Ac-PSNleP (SEQ ID NO: 36)) is carried out. Studies on the NatA inhibitors include, in a step-wise manner, phenotypic determination of cancer cells (cell viability etc.), in depth characterization of activated signalling pathways as well as analysis of N-terminal acetylation status by COFRADIC analyses. Several different cancer cell types are assessed for their sensitivity to NatA-inhibitors, including thyroid cancer cells (Gromyko D, et al. (2010) Depletion of the human N-terminal acetyltransferase A (hNatA) induces p53-dependent apoptosis and p53-independent growth inhibition. Int. J. Cancer 127(12): 2777-89) breast cancer cells, lung cancer cells, and prostate cancer cells. The NCI-60 cancer cell panel in collaboration with SINTEF (Trondheim, Norway) is considered to get a broad cancer profile. Cells are plated in 96 or 384 well plates and grown for 16 hours prior to the addition of test compounds so as to allow cells to adhere to the well and begin growing. A 10-point concentration series, spanning at least 3 log units of concentration, of our NatA inhibitor set is added to cells in assay plates at a constant DMSO concentration empirically determined to be below the maximum tolerated dose. Cells are incubated with inhibitors for at least 2-3 population doublings before measuring the viability response. The widely accepted Z-factor and minimum significant ratio (MSR) metrics are used to quantitatively assess our assays' reproducibility and sensitivity. The activity of our NatA inhibitors in H1299 (lung cancer), CAL-62 and 8305C cells (anaplastic thyroid carcinomas) are initially tested because they have been shown to be sensitive to NatA dosage (Lim J H, Park J W, Chun Y S (2006) Cancer Res 66(22):10677-82.; Gromyko D, Arnesen T, Ryningen A, Varhaug J E, Lillehaug J R (2010) Int J Cancer 127(12):2777-89.). To demonstrate selectivity, each compound's activity to Nthy-ori 3.1 cells (SV40 immortalized thyroid follicular epithelial cells) is compared. The selectivity of each compound is defined as the ratio of the IC50 in CAL-62/8350C to the IC50 in Nthy-ori3.1. The EC50 is defined as the compound concentration that results in 50% inhibition of cell proliferation/viability relative to cells treated with control agents (e.g. negative: DMSO, positive: cytotoxic agent).

[0150] Our hypothesis is that the newly-discovered NatA inhibitors will possess a selective (>10-fold), concentration dependent activity in the CAL-62 and 8305C cells relative to the Nthy-ori3.1 cells. Combination therapies that enhance efficacy or permit reduced dosages to be administered have seen great success in a variety of therapeutic applications. Published data indicates that NatA dosage regulates the sensitivity of thyroid carcinoma cells to cytotoxic agents, including Daunorubicin and KillerTRAIL (Gromyko D, Arnesen T, Ryningen A, Varhaug JE, Lillehaug JR (2010) Int J Cancer 127(12):2777-89.). This observation supports a hypothesis that NatA inhibitors would be useful in combination with clinically used compounds. Synergistic combinations are of particular interest, as they can i) enhance the activity of the mixture relative to that expected from additivity; ii) increase potency; or iii) enhance efficacy. For these synergy experiments, a traditional 8-point matrixed doseresponse analysis is used that tests pairwise combinations of NatA inhibitors with classic cytotoxic agents (e.g. daunorubicin) or molecular targeted therapies (KillerTRAIL, troglitazone, bortezomid). Synergy will be assessed by Bliss and Loewe models as described (Severyn B, Liehr R A, Wolicki A, Nguyen K H, Hudak E M, Ferrer Met al. (2011) ACS Chem Biol 6:1391-1398.). Synergistic combinations that enhance potency and efficacy are prioritized for further characterization.

Example 5: Plasmid Construction, Recombinant Protein Expression, and Purification of hNAA80

[0151] hNAA80/hNAT6 (Gene ID: 24142) was cloned from human HEK293 cDNA by use of Transcriptor Reverse Transcriptase (Roche) using the following primers: NAA80 sense primer (5-CAACATGCAAGAGCTGACTC-3 SEQ ID NO: 37) and NAA80 antisense primer (5-GATGTCTTTTTCCATCCAGAATATG-3 SEQ ID NO: 38). The PCR product containing the CDS was inserted into the TOPO TA vector pcDNA 3.1/V5-His TOPO (Invitrogen) using the provided kit and resulting in the plasmid pNAA80-V5. Plasmid hNAA80-eGFP was constructed using pNAA80-V5 as template and restriction sites Nhel and Kpnl in peGFP-N1. The MBP-HishNAA80 fusion protein (MBP-Naa80) was constructed by subcloning hNAA80 from the plasmid pNAA80-V5 into the pETM-41 vector using the restriction enzymes Ncol and Acc65I resulting in pETM-41-hNAA80. The plasmid was transformed into E. coli BL21 Star (DE3) cells (Invitrogen) by heat shock. A 300 mL cell culture was cultivated in Luria-Bertani (LB) medium at 37 C. until an OD600 of 0.6 was reached and subsequently transferred to 20 C. Protein expression was induced by addition of 1 mM isopropyl--D-thiogalactopyranoside (IPTG). After 16 h, the cells were harvested by centrifugation and the pellets were stored at 20 C. For purification, the E. coli pellets were thawed at 4 C. and the bacterial cells lysed using mechanical disruption by a French Press (1000 psi pressure) in lysis buffer (50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 20 mM imidazole, 1 mM DTT, 1 x EDTA-free protease inhibitor cocktail (Roche)). After centrifugation (40,000 x g, 25 min, 4 C.), the cell extract was applied on a metal affinity FPLC column (HisTrap HP, GE Healthcare). MBP-Naa80 was eluted with 300 mM imidazole in 50 mM Tris (pH 8.0), 300 mM NaCl, 1 mM DTT. Recombinant protein containing fractions were pooled and further purified via size exclusion chromatography (Superdex 200, GE Healthcare) and purity was determined by analysis of Coomassie stained SDS-PAGE gels. The protein concentrations were determined by absorption at 280 nm using a NanoDrop1000 spectrophotometer (Peqlab, Germany).

Example 6: [.SUP.14.C]-Ac-CoA based N-Terminal Acetyltransferase Assay using Synthetic Oligopeptides

[0152] Whilst Nat6/Naa80 has previously been described as a putative NAT (based on sequence analysis that suggested the presence of a NAT-specific GNAT-fold), no specific substrate has previously been described.

[0153] Here we studied the Nt-acetylation activity of Naa80 towards a representative selection of peptides, using a [.sup.14C]-Ac-CoA based N-terminal acetyltransferase assay using synthetic oligopeptides.

[0154] The assay was performed as described previously (Drazic A & Arnesen T (2017) [14C]-Acetyl-Coenzyme A-Based In Vitro N-Terminal Acetylation Assay. Methods Mol. Biol. 1574:1-8). Briefly, the immuno-precipitates were mixed with 200 M selected synthetic oligopeptides (Biogenes, Germany), 50 M isotope-labelled [.sup.14C]-Ac-CoA (Perkin Elmer) in a total volume of 25 L acetylation buffer (50 mM Tris (pH 7.4), 1 mM EDTA, 1 mM DTT, 10% (v/v) glycerol) The samples were incubated at 37 C. and shaking at 1000 rpm in a Thermomixer block for 60 min. The magnetic beads were removed and the supernatants transferred onto P81 phosphocellulose filter disks (Millipore). The filters disks were washed three times with washing buffer (10 mM HEPES (pH 7.4)) and dried before they were added to 5 mL scintillation cocktail Ultima Gold F (Perkin Elmer). The incorporated [.sup.14C]-Ac was determined by a Perkin Elmer Tri-Carb 2900TR Liquid Scintillation Analyzer.

[0155] The results are shown in FIG. 1. The results revealed a marked preference of Naa80 for peptides with acidic N-termini (e.g. DDDI (SEQ ID NO: 6) and EEEI (SEQ ID NO: 7)). Among all the proteins expressed in animals, these N-termini uniquely correspond to those of cytoplasmic - and -actin, after the intermediate step of post-translational cleavage of the N-terminal acetyl-methionine. This is illustrated in FIG. 2 which shows a sequence alignment (first 61-63 amino acids) of the six human mature actin isoforms.

Example 7: Expression of Naa80-eGFP in Human HAP1 Cells

[0156] The expression pattern of Naa80 in human cells was evaluated as follows:

[0157] HAP1 cells were obtained from Horizon Genomics and cultured as recommended. NAA80 knockout and wildtype HAP1 cells (Horizon C631) were grown in Iscove's Modified Dulbecco's Medium with the addition of 10% fetal bovine serum and 1 penicillin/streptomycin. Prior to use in experiments, all HAP1 cell lines were passaged until diploid status was confirmed by an Accuri BD C6 flow cytometer using propidium iodide staining. HeLa cells (ATTC CCL-2) were cultured in DMEM/10% FBS at 37 C. and 5% CO.sub.2. Cells were seeded on plates or coverslips and transfected with XtremeGene 9 as recommended. For localization and phenotype rescue studies, cells were imaged approx. 24 h and 48 h post- transfection, respectively.

[0158] The results are shown in FIG. 3. Naa80-eGFP expressed in human HAP1 cells displayed a diffuse cytosolic distribution and contrary to most NATs, it did not associate with ribosomes suggesting that it targets its substrate(s) post-translationally.

Example 8: Identification of Naa80-Specific Substrates

[0159] To identify Naa80-specific substrates in cells, we generated two HAP1 NAA80 knockout cell lines (KO1 and KO2) using the CRISPR/Cas9 system (FIG. 4).

[0160] Phospho-imaging of NAA80 KO1 cell lysates treated with purified Naa80 and isotope-labeled [.sup.14C]-acetyl CoA revealed a single band at 43 kDa (FIG. 5), corresponding to the expected molecular mass of actin. Actin's identity was confirmed using N-terminal proteomics, by quantitatively assessing the Nt-acetylation status of 402 unique N-termini from control and NAA80 KO1 cell lysates. Only peptides corresponding to the fully processed N-termini of - and -actin showed altered Nt-acetylation levels in knockout cells. Furthermore, both actin isoforms were 100% and 0% Nt-acetylated in control and NAA80 KO1 cells, respectively. Isoform- and Nt-acetylation-specific actin antibodies recognized a 43 kDa band in control but not NAA80 KO1 cells (FIG. 6A, 6B). The anti Ac--actin and Ac--actin antibody signals reappeared in knockout cell lysates transfected with Naa80 (FIG. 6B), but not in cells transfected with a catalytically inactive mutant, Naa80mut (W105F, R170Q, G173D, Y205F) (FIG. 6B), confirming that a catalytically active Naa80 is absolutely required for actin Nt-acetylation in cells.

[0161] These results were corroborated for both knockout cell lines at the single cell level by immunofluorescence analysis, showing that only control cells and Naa80- eGFP-transfected knockout cells were positive for anti Ac--actin and Ac--actin antibody staining.

Example 9: Actin Nt-Acetylation Controls Cell Motility

[0162] Next, we explored the impact of actin Nt-acetylation in cells. Since the actin cytoskeleton is a major factor in cell motility, we tested HAP1 control and NAA80 knock-out cells in several independent migration assays. We found that the lack of actin Nt-acetylation resulted in increased motility of HAP1 NAA80 knockout cells.

Wound healing/gap closure and chemotaxis assays

[0163] For wound healing assays, cells were seeded in wells of silicone culture inserts (ibidi 80209) placed in slide wells (ibidi 80426). 90 l of 300,000 cells/ml were seeded in each insert well, resulting in 100% confluency after approx. 24 hours at which insets were removed (creating a 400-450 m wide gap), wells washed twice in medium and timelapse started using a Nikon TE2000 microscope with 10x objective. ImagePro plus was used for image processing to measure gap size in m.sup.2. Cell front velocity was calculated according to ibidi Application note 30. The total area of image was multiplied with the centerpiece approximation (the increase of the cell-covered area (in %) per time unit; this number was divided by the length of the image in m and the resulting number divided by 2 for the two cell fronts), resulting in the normalized cell front velocity in m/h. For scratch wounds using IncuCyte ZOOM imaging system, 200 l of 40,000 cells/ml were seeded in 96-well Image lock plates (Essen BioScience) and scratch wounds were prepared with the accompanying scratch wound maker according to manufacturer protocols (creating a 700-m wide gap). Wells were washed twice and filled with 300 l medium. Wounds were imaged in the IncuCyte by 24 hours repeat scanning every 2 h in which 2 positions were imaged per well. Image analysis was performed in the IncuCyte ZOOM software using area masks for wound and cell covered area and calculations of average gap size. Cell front velocity was calculated as above. For chemotaxis assay in the IncuCyte ZOOM, cells were seeded in membrane-containing inserts of ClearView 96-well Cell migration plates with pored membranes. Cells were seeded at a density of 27,000 cells/ml, 60 l/insert well in medium containing 1% FBS. After 3 h, media of different FBS concentrations were added to appropriate top or bottom wells to obtain desired final concentrations of FBS while keeping volumes equal. The membranes were imaged at top and bottom using 10x objective and 24 h repeat scanning every 2 h. Analysis was performed in the IncuCyte ZOOM software measuring cell covered area at top and bottom. Numbers were expressed as amount of cells migrated per tin percent of initial top value. Data were background-subtracted and corrected for growth differences.

[0164] In the wound-healing assays, NAA80 KO1 cells closed the gap approximately 12 h faster than control cells (FIG. 7A, 7B), with cell-front velocity increasing from 6 to 10 m/h (FIG. 7C). We observed similar results for both knockout cell lines in an IncuCyte ZOOM system for live-cell imaging, showing an increase of cell-front velocity from 7 to 9 m/h and 11 m/h for NAA80 KO1 and KO2 cells, respectively. To investigate the migratory properties of single cells, we performed a chemotaxis assay by seeding cells on a transparent pored membrane in medium containing 1% FBS. Cells were attracted to the bottom side by a medium containing 10% FBS, and the amount of migrated cells was normalized to the growth rates of each cell line. NAA80 KO1 cells showed approximately a 50% increase in chemotactic motility compared to control cells (FIG. 7D). In addition to the chemotactic assay for directed migration, we also measured random cell migration in parallel wells in which both chambers contained 10% FBS. In this assay, we also observed increased random motility of NAA80 KO1 cells (FIG. 7E), consistent with an increased capacity of these cells to migrate. Similar results were obtained with NAA80 KO2 cells.

[0165] Taken together, these data demonstrate that cells deficient in actin Nt-acetylation have increased motility.

Example 10: Actin Nt-Acetylation Affects Cytoskeletal Morphology

[0166] The actin cytoskeleton not only regulates cell motility, but also cell shape and morphology. Thus, we compared the morphology of the actin cytoskeleton in HAP1 control and NAA80 KO cells by phalloidin staining.

Phalloidin-based phenotype characterizations

[0167] Lamellipodia phenotype was identified in cells seeded at 70,000 cells/ml in 24-well plates, fixed approx. 24 h post seeding in 3% PFA in cytoskeleton buffer (10 mM MES pH 6.1, 150 mM NaCl, 5 mM EGTA, 5 mM glucose, 5 mM MgCl.sub.2), washed in CB, permeabilized in 0.1% Triton for 10 min, and stained with Rhodamine phalloidin or Phalloidin-Atto-647N, for counting and confocal/STED respectively. Cells at the edge of clusters were counted as either positive or negative for the presence of lamellipodia (mitotic cells were not considered and neither were lamellipodia on top of neighboring cells). Experiment was repeated three times and at least 500 cells were counted per cell line per independent repetition. Filopodia phenotype analysis was performed by seeding cells at 25-50,000 cells/ml in 48-well plates, fixed approx. 24 h post seeding in 3% PFA in 0.1 M phosphate buffer for 30 min and subsequently permeabilized with 0.1% Triton for 10 min, and stained with Rhodamine phalloidin. Filopodia number was counted on isolated cells and length measurements done using the imageJ software (National Institute of Health, Bethesda, MD, USA).

[0168] Based on the hypermotility phenotype of NAA80 KO cells, we were specifically interested in the formation of cell protrusions, such as lamellipodia and filopodia that are usually linked to cell motility. As anticipated, NAA80 KO cells displayed an increase in both the number and length of filopodia-like structures (FIG. 8A-D). These cells showed an approximate 4-fold increase in the number of filopodia-like structures, defined as finger-like protrusions with a length 0.5 m (FIG. 8C). The average filopodia length increased from 1 pm in control cells to 5 m in NAA80 KO cells (FIG. 8D). Confirming the specificity of this phenotype, the reexpression of Naa80 in NAA80 KO cells reduced the number of filopodia-like structures to near control levels, whereas the expression of the catalytically inactive mutant Naa80mut did not. As with other experiments described above, the NAA80 KO2 clone showed a similar behaviour. Consistent with the apparent elevated number of filamentous actin structures in NAA80 KO cells, we observed a significant reduction in the ratio of globular to filamentous (G/F)-actin (21) from 0.73 in HAP1 control cells to 0.55 in NAA80 KO1 cells (FIG. 8E). This effect was reversed by the expression of wild-type Naa80 but not Naa80mut in both NAA80 KO cell lines, demonstrating that Naa80-dependent actin Nt-acetylation directly impacts the transition between G- and F-actin. Since we observed a hyper-motility phenotype for NAA80 KO cells (FIG. 8), we quantified the number of cells showing at least one distinguishable lamellipodium supported by microspikes or filopodia-like structures. In order to efficiently study these cell protrusions in HAP1 cells, which typically grow in clusters, we included only cells at the borders of the clusters in quantifications. In apparent agreement with the increased cell front velocity in the motility assays, we observed that NAA80 KO cells formed twice as many lamellipodia than control cells (32% vs. 16%) (FIG. 8F-H). These results further emphasize the importance of actin Nt-acetylation in the control of cytoskeletal dynamics.

Example 11: Nt-Acetylation-Dependent Actin Polymerization and Stability

[0169] To address the mechanism behind the altered cytoskeleton organization phenotypes of NAA80 KO cells, we analyzed the recovery rates of cytoskeletal structures in cells treated with the actin depolymerizing drug Latrunculin A (LatA).

[0170] Complete depolymerization of filamentous actin (F-actin) was achieved by incubating cells with 500 nM Latrunculin A (LatA) for 1 h at 37 C. Cells were either fixed with PFA (control) or washed with culture medium to remove LatA and further incubated in drug-free medium at 37 C. Samples were taken every 2 min, fixed and permeabilized with 0.2% Triton X-100 (in PBS) for 10 min and F-actin probed with 500 nM phallodin Atto 647N (STED microscopy) for 30 min.

[0171] Within 60 min of LatA treatment, the actin appeared to be fully depolymerized in control and NAA80 KO cells, and washout of the drug resulted in the recovery of actin filament structures, but the time of recovery was significantly delayed for NAA80 KO cells compared to control cells. Specifically, the formation of actin filament structures with an average length of 1 m was delayed about 4 mins compared to control cells, consistent with a direct role of actin Nt-acetylation in actin polymerization.

[0172] We next explored the in vitro effect of actin Nt-acetylation on the polymerization/depolymerization properties of actin alone or in the presence of some of the most common actin assembly factors in cells. Cytoplasmic actin (a mixture of and isoforms) was purified from control and NAA80 KO cells (FIG. 9A). The time-course of actin polymerization measured using the pyrene-actin polymerization assay revealed no significant difference in the polymerization rate of Nt-acetylated or unacetylated actin (FIG. 9B). However, the elongation of 1.5 M filament seeds in the presence of 0.5 M actin monomers (i.e. above the critical concentration for monomer addition at the barbed end but below that of the pointed end) was more than 2-fold faster for Ac-Actin than non-Ac-Actin (FIG. 9C). In contrast, the depolymerization rate of 0.1 M filament seeds (i.e. below the critical concentration of both the pointed and barbed ends) was 2-fold faster for Ac-actin than non-Ac-actin, suggesting that at least in the absence of other factors, actin filament structures formed by non-Ac-actin might be more stable (FIG. 9D).

[0173] Arp complex strongly enhances nucleation from the side of pre-existing filaments. We found no difference in the relative polymerization rate of Ac-actin and non-Ac-actin induced by Arp complex (FIG. 9E). We next analyzed two formins, mDia1 and mDia2, that while closely related in sequence have very different actin assembly properties. We observed no significant difference in the relative polymerization rates of Ac-actin and non-Ac-actin induced by mDia2 (FIG. 9D), a form in that has strong nucleation but poor elongation activity. In contrast, for mDia1, which has both strong nucleation and elongation activities, the polymerization rate was 2-fold higher for Ac-actin than non-Ac-actin (FIG. 9G). Because Nt-acetylation-dependent differences appear to be emphasized during form in-mediated elongation, we analyzed the role of profilin (isoforms 1 and 2), known to virtually stop pointed end elongation of actin alone while dramatically accelerating barbed end elongation by formins. As expected, mDia1 accelerated the elongation rate of filament seeds from profilin1-actin compared to the elongation of seeds from actin alone, but this effect was greater for Ac-actin than for non-Ac-actin (FIG. 9H). Curiously, no significant difference was observed with profilin2-actin (FIG. 9I), suggesting that mDia1 processes more efficiently profilin1-actin than profilin2-actin. We note, however, that these assays detect the incorporation into filaments of the small fraction of pyrene-labelled actin, which binds profilin with weaker affinity than unlabelled actin, such that the effects of profilin may be far more pronounced than indicated by the bulk assays.

[0174] Together, these results suggest that the differences in actin assembly between Ac-actin and non-Ac-actin emanate mainly from an Nt-acetylation-dependent increase in the rates of filament elongation and depolymerization, whereas nucleation appears to be mostly unaffected.

Example 12: Naa80 Substrate Screening Assay

[0175] In order to investigate the in vitro activity of Naa80 further, an enzyme activity screening assay on purified Naa80 was undertaken using a broad substrate library (including amino acids, nucleosides, coenzymes, amines, saccharides, vitamins, antioxidants and peptides) modified from Kuhn et al. (Kuhn et al. (2013) Broad-substrate screen as a tool to identify substrates for bacterial Gcn5-related N-acetyltransferases with unknown substrate specificity. Protein Science 22(2):222-230). Among all of the potential substrates tested, only the peptides with N-terminal sequence of MDEL.sub.24 (SEQ ID NO: 45), DDDI.sub.24 (SEQ ID NO: 41) and EEEI.sub.24 (SEQ ID NO: 40) were acetylated, as shown in the following table:

TABLE-US-00011 TABLE Peptidestestedaspotentialsubstratesfor Naa80mediatedacetylation Peptide Productformation(M) SEQIDNO: EEEI.sub.24 42.4(0.95) 40 MLGP.sub.24 2.06(1.49) 49 MDEL.sub.24 119(5.61) 45 MKKS.sub.24 3.37(1.71) 51 DDDI.sub.24 50.5(1.0) 41 SESS.sub.24 1.14(3.35) 43 MTNK.sub.24 4.25(1.75) 48 AVFA.sub.24 0.96(0.84) 44 MAPL.sub.24 2.28(0.61) 50 MELL.sub.24 2.15(0.40) 46

[0176] The 24-mer substrate peptides differed in their 7 N-terminal amino acids while the 17 C-terminal residues (RWGRPVGRRRRPVRVYP, SEQ ID NO: 39) were kept constant: EEEI.sub.24 (EEEIAAL, SEQ ID NO: 40), DDDI.sub.24 (DDDIAAL, SEQ ID NO: 41), MDDD.sub.24 (MDDDIAA, SEQ ID NO: 42), SESS.sub.24 (SESSSKS, SEQ ID NO: 43), AVFA.sub.24 (AVFADLD, SEQ ID NO: 44), MDEL.sub.24 (MDELFPL, SEQ ID NO: 45), MELL.sub.24 (MELLSPP, SEQ ID NO: 46), MLGT.sub.24 (MLGTGPA, SEQ ID NO: 47), MTNK.sub.24 (MTNKSSL, SEQ ID NO: 48), MLGP.sub.24 (MLGPEGG, SEQ ID NO: 49), MAPL.sub.24 (MAPLDLD, SEQ ID NO: 50), MKKS.sub.24 (MKKSYSG, SEQ ID NO: 51).

[0177] Substrate specificity studies in the linear range of enzyme concentration revealed that the best substrate for Naa80 was MDEL.sub.24 (SEQ ID NO: 45), with a 5-fold higher product formation compared to the second best substrate, MDDD.sub.24 (SEQ ID NO: 42), while its cellular substrate, processed -actin, DDDI.sub.24 (SEQ ID NO: 41) ranked third. Both MDEL (p65, SEQ ID NO: 8) and MDDD.sub.24 (unprocessed -actin, SEQ ID NO: 42) represent cellular NatB substrates.

Example 13: Selective and Potent Inhibitors of Naa80

[0178] We then pursued Naa80 bisubstrate analogue inhibitors based on the results of the substrate screening and the sequences of the cellular substrates processed - and -actin. We synthesized bisubstrate conjugates of Coenzyme A coupled to the tetrapeptides MDEL-NH.sub.2 (SEQ ID NO: 8), DDDI-NH.sub.2 (SEQ ID NO: 6), EEEI-NH.sub.2 (SEQ

[0179] ID NO: 7), and MLGT-NH.sub.2 (SEQ ID NO: 52) via an acetamide (Ac) linker. Inhibition studies revealed that CoA-Ac-DDDI-NH.sub.2 was the most potent Naa80 inhibitor with an IC50 value of 0.38 M, 3-fold and 3.3-fold more potent than CoA-Ac-EEEI-NH.sub.2 (SEQ ID NO: 7) and CoA-Ac-MDEL-NH.sub.2 (SEQ ID NO: 8), respectively (FIG. 10A). The negative control, CoA-Ac-MLGT-NH.sub.2 (SEQ ID NO: 52), based on a NatC substrate, did not show detectable inhibition of Naa80 at the highest concentration tested of 1000 M.

[0180] In order to determine the selectivity of the most potent Naa80 inhibitor, CoA-Ac-DDDI-NH.sub.2 (SEQ ID NO: 6) was tested against a panel of human NATs (FIG. 10B). With a K.sub.i value of 43 nM, the inhibitor was established to be both potent and selective.

[0181] Interestingly, the monomeric Naa10, which has a known preference for acetylating acidic N-termini in vitro, was the second most inhibited NAT enzyme. However, CoA-Ac-DDDI-NH.sub.2 (SEQ ID NO: 6),showed a 88-fold reduced potency for Naa10 compared to Naa80.

Example 14: Further Inhibitors of Naa80

[0182] The following additional Naa80 bisubstrate analogue inhibitors were produced and tested as described above:

TABLE-US-00012 SEQIDNO: Peptide IC50(M) StDev(M) 6 DDDI 0.38 0.10 7 EEEI 1.16 0.10 8 MDEL 1.26 0.09 53 PDEL 13.39 2.24 9 DEDI 3.12 0.33 10 DEEL 4.73 1.43 11 EDDI 0.12 0.05 12 EDEI 0.76 0.18 13 EEDL 1.76 0.52 14 EEEL 1.16 0.12 15 DDEI 2.67 0.67 16 EDQL 0.85 0.05 17 ESEL 11.50 3.23 18 DEEI 1.22 0.33 19 EEDI 0.17 0.04 20 EDEL 0.15 0.02 21 QEEI 2.23 0.47