FL, fluorescence; CB, Coomassie blue staining. Up coming, we investigated whether possibly of both chemical substances could inhibit the defatty-acylation activity of SIRT2 simply by evaluating the lysine fatty acylation degree of K-Ras4a, the just reported SIRT2 defatty-acylation focus on. bonding between your hydroxyl SIRT1 and group, SIRT2, and SIRT3. That is backed by research with another little molecule inhibitor additional, NH-TM. These scholarly research offer useful insights for long term SIRT2 inhibitor development. and in cells. Inside our efforts to Sutezolid build up a more powerful SIRT2 inhibitor, we made a decision to carry out framework activity relationship research predicated on the framework of TM. We particularly were thinking about changing the aniline part of the tiny molecule, to review what adjustments would boost or lower SIRT2 selectivity and strength. To this final end, we discovered that adding an individual hydroxyl group for the aniline moiety, resulting in the substance JH-T4 (Shape 1), generates a sirtuin inhibitor with an extremely different account inhibition. We assessed the IC50 ideals (Desk 1) of JH-T4 toward SIRT1, SIRT2, SIRT3, and SIRT6 under pre-incubation circumstances (enzymes, NAD, and inhibitors had been 1st incubated for 15 min before substrates had been added to begin the enzymatic response) and likened these to the IC50 ideals of TM. For SIRT2, we determined the IC50 ideals for both demyritoylation and deacetylation actions. For these assays the H3K9-Myr and H3K9-Ac peptides had been utilized as substrates, as SIRT1,2,3 and 6 possess efficient activity on these peptides, that are employed for Sirtuin studies commonly.[2a, 3b, 7a, 9] Open up in another window Amount 1. Chemical Buildings of different Sirtuin Inhibitors Desk 1. IC50 beliefs (M) of TM, JH-T4 and NH-TM for inhibiting sirtuin deacylation activity (ND = not really determined). Values proven in mounting brackets are from assays without pre-incubation. with IC50 beliefs of 15 M or lower. Oddly enough, beneath the pre-incubation assay condition, TM and JH-T4 inhibited both deacetylation and defatty-acylation activity of SIRT2 comparably (IC50 beliefs in the 30C50 nM range) (Desk 1). To help expand evaluate the defatty-acylation inhibition by TM and JH-T4 we driven the IC50 worth for inhibition of SIRT2 demyristoylation activity without pre-incubating the enzyme with NAD and inhibitor. Without preincubation, the IC50 worth of TM was > 200 M (42% inhibition at 200 M), however the IC50 of JH-T4 was 110 M approximately. This shows that JH-T4 is normally better at inhibiting the defatty-acylation activity of SIRT2 than TM is normally. We also assessed the IC50 beliefs of JH-T4 and TM over the deacetylation activity of SIRT1, SIRT2, and SIRT3 without preincubation. Many IC50 worth for inhibiting the deacetylation activity of SIRT1-3 without pre-incubation didn’t drastically transformation for TM and JH-T4 in comparison to that with pre-incubation (Desk 1).[7a] Nevertheless the IC50 worth of JH-T4 on SIRT1 without pre-incubation increased dramatically JH-T4 (40 M without pre-incubation, in comparison to 0.3 M with pre-incubation). We following wished to review the selectivity and strength of the substances in cells. To judge Sutezolid the inhibition of SIRT1 deacetylation activity, we analyzed p53 acetylation amounts, as Lys382 of p53 is normally a well-established SIRT1 substrate.[10] Needlessly to say, JH-T4, however, not TM, elevated Ac-p53 level in Lys382 in MCF-7 cells (Amount 2A). We further examined if these substances could inhibit the deacetylation activity of SIRT2 in cells predicated on acetyl -tubulin immunofluorescence, as acetyl -tubulin is a used cellular readout of SIRT2 activity widely.[3a] Both TM and JH-T4 treated samples showed a dramatic upsurge in acetyl -tubulin levels set alongside the sample treated with the automobile control, ethanol. Hence, both compounds effectively inhibit SIRT2 deacetylation activity in MCF-7 cells (Amount 2B). Open up in another window Amount 2. In-Cell Sirtuin Inhibition by JH-T4.(A) Ac-p53 levels to judge the inhibition of SIRT1 in cells following 6 hr 25M inhibitor and 200 nM trichostatin A (TSA) treatment in MCF-7 cells. (B) Ac–tubulin amounts to detect inhibition of SIRT2 after 6 hr 25 M inhibitor treatment in MCF-7 cells. (C) Inhibition of SIRT2 by TM and JH-T4 treatment by analyzing K-Ras4a lysine fatty acylation amounts. (D) Recognition of K-Ras4a lysine fatty acylation amounts to.[PubMed] [Google Scholar]. sIRT1 and group, SIRT2, and SIRT3. That is additional backed by research with another little molecule inhibitor, NH-TM. These research offer useful insights for upcoming SIRT2 inhibitor advancement. and in cells. Inside our efforts to build up a more powerful SIRT2 inhibitor, we made a decision to carry out framework activity relationship research predicated on the framework of TM. We particularly were thinking about changing the aniline part of the tiny molecule, to review what adjustments would boost or reduce SIRT2 strength and selectivity. To the end, we discovered that adding an individual hydroxyl group over the aniline moiety, resulting in the substance JH-T4 (Amount 1), creates a sirtuin inhibitor with an extremely different inhibition account. We assessed the IC50 beliefs (Desk 1) of JH-T4 toward SIRT1, SIRT2, SIRT3, and SIRT6 under pre-incubation circumstances (enzymes, NAD, and inhibitors had been initial incubated for 15 min before substrates had been added to begin the enzymatic response) and likened these to the IC50 beliefs of TM. For SIRT2, we driven the IC50 beliefs for both deacetylation and demyritoylation actions. For these assays the H3K9-Ac and H3K9-Myr peptides had been utilized as substrates, as SIRT1,2,3 and 6 possess efficient activity on these peptides, which are generally employed for Sirtuin research.[2a, 3b, 7a, 9] Open up in another window Amount 1. Chemical Buildings of different Sirtuin Inhibitors Desk 1. IC50 beliefs (M) of TM, JH-T4 and NH-TM for inhibiting sirtuin deacylation activity (ND = not really determined). Values proven in mounting brackets are from assays without pre-incubation. with IC50 beliefs of 15 M or lower. Oddly enough, beneath the pre-incubation assay condition, TM and JH-T4 inhibited both deacetylation and defatty-acylation activity of SIRT2 comparably (IC50 beliefs in the 30C50 nM range) (Desk 1). To help expand evaluate the defatty-acylation inhibition by TM and JH-T4 we motivated the IC50 worth for inhibition of SIRT2 demyristoylation activity without pre-incubating the enzyme with NAD and inhibitor. Without preincubation, the IC50 worth of TM was > 200 M (42% inhibition at 200 M), however the IC50 of JH-T4 was around 110 M. This shows that JH-T4 is certainly better at inhibiting the defatty-acylation activity of SIRT2 than TM is certainly. We also assessed the IC50 beliefs of TM and JH-T4 in the deacetylation activity of SIRT1, SIRT2, and SIRT3 without preincubation. Many IC50 worth for inhibiting the deacetylation activity of SIRT1-3 without pre-incubation didn’t drastically transformation for TM and JH-T4 in comparison to that with pre-incubation (Desk 1).[7a] Nevertheless the IC50 worth of JH-T4 on SIRT1 without pre-incubation increased dramatically JH-T4 (40 M without pre-incubation, in comparison to 0.3 M with pre-incubation). We following wanted to evaluate the strength and selectivity of the substances in cells. To judge the inhibition of SIRT1 deacetylation activity, we analyzed p53 acetylation amounts, as Lys382 of p53 is certainly a well-established SIRT1 substrate.[10] Needlessly to say, JH-T4, however, not TM, elevated Ac-p53 level in Lys382 in MCF-7 cells (Body 2A). We further examined if these substances could inhibit the deacetylation activity of SIRT2 in cells predicated on acetyl -tubulin immunofluorescence, as acetyl -tubulin is certainly a trusted mobile readout of SIRT2 activity.[3a] Both TM and JH-T4 treated samples showed a dramatic upsurge in acetyl -tubulin amounts set alongside the sample treated with the automobile control, ethanol. Hence, both compounds effectively inhibit SIRT2 deacetylation activity in MCF-7 cells (Body 2B). Open up in another window Body 2. In-Cell Sirtuin Inhibition by JH-T4.(A) Ac-p53 levels to judge the inhibition of SIRT1 in cells following 6 hr 25M inhibitor and 200 nM trichostatin A (TSA) treatment in MCF-7 cells. (B) Ac–tubulin amounts to detect inhibition.[PMC free of charge content] [PubMed] [Google Scholar] [10] Vaziri H, Dessain SK, Ng Eaton E, Imai SI, Frye RA, Pandita TK, Guarente L, Weinberg RA, Cell 2001, 107, 149C159. SIRT2, and SIRT3. That is additional supported by research with another little molecule inhibitor, NH-TM. These research offer useful insights for upcoming SIRT2 inhibitor advancement. and in cells. Inside our efforts to build up a more powerful SIRT2 inhibitor, we made a decision to carry out framework activity relationship research predicated on the framework of TM. We particularly were thinking about changing the aniline part of the tiny molecule, to review what adjustments would boost or reduce SIRT2 strength and selectivity. To the end, we discovered that adding an individual hydroxyl group in the aniline moiety, resulting in the substance JH-T4 (Body 1), creates a sirtuin inhibitor with an extremely different inhibition account. We assessed the IC50 beliefs (Desk 1) of JH-T4 toward SIRT1, SIRT2, SIRT3, and SIRT6 under pre-incubation circumstances (enzymes, NAD, and inhibitors had been initial incubated for 15 min before substrates had been added to begin the enzymatic response) and likened these to the IC50 beliefs of TM. For SIRT2, we motivated the IC50 beliefs for both deacetylation and demyritoylation actions. For these assays the H3K9-Ac and H3K9-Myr peptides had been utilized as substrates, as SIRT1,2,3 and 6 possess efficient activity on these peptides, which are generally employed for Sirtuin research.[2a, 3b, 7a, 9] Open up in another window Body 1. Chemical Buildings of different Sirtuin Inhibitors Desk 1. IC50 beliefs (M) of TM, JH-T4 and NH-TM for inhibiting sirtuin deacylation activity (ND = not really determined). Values proven in mounting brackets are from assays without pre-incubation. with IC50 beliefs of 15 M or lower. Oddly enough, beneath the pre-incubation assay condition, TM and JH-T4 inhibited both deacetylation and defatty-acylation activity of SIRT2 comparably (IC50 beliefs in the 30C50 nM range) (Desk 1). To help expand evaluate the defatty-acylation inhibition by TM and JH-T4 we motivated the IC50 worth for inhibition of SIRT2 demyristoylation activity without pre-incubating the enzyme with NAD and inhibitor. Without preincubation, the IC50 worth of TM was > 200 M (42% inhibition at 200 M), however the IC50 of JH-T4 was around 110 M. This shows that JH-T4 is certainly better at inhibiting the defatty-acylation activity of SIRT2 than TM is certainly. We also assessed the IC50 beliefs of TM and JH-T4 in the deacetylation activity of SIRT1, SIRT2, and SIRT3 without preincubation. Many IC50 worth for inhibiting the deacetylation activity of SIRT1-3 without pre-incubation didn’t drastically transformation for TM and JH-T4 in comparison to that with pre-incubation (Desk 1).[7a] Nevertheless the IC50 worth of JH-T4 on SIRT1 without pre-incubation increased dramatically JH-T4 (40 M without pre-incubation, in comparison to 0.3 M with pre-incubation). We following wanted to evaluate the strength and selectivity of the substances in cells. To judge the inhibition of SIRT1 deacetylation activity, we analyzed p53 acetylation amounts, as Lys382 of p53 is certainly a well-established SIRT1 substrate.[10] Needlessly to say, JH-T4, however, not TM, increased Ac-p53 level on Lys382 in MCF-7 cells (Figure 2A). We further tested if these compounds could inhibit the deacetylation activity of SIRT2 in cells based on acetyl -tubulin immunofluorescence, as acetyl -tubulin Sutezolid is a widely used cellular readout of SIRT2 activity.[3a] Both the TM and JH-T4 treated samples showed a dramatic increase in acetyl -tubulin levels compared to the sample treated with the vehicle control, ethanol. Thus, both compounds efficiently inhibit SIRT2 deacetylation activity in MCF-7 cells (Figure 2B). Open in a separate window Figure 2. In-Cell Sirtuin Inhibition by JH-T4.(A) Ac-p53 levels to evaluate the inhibition of SIRT1 in cells after 6 hr 25M inhibitor and 200 nM trichostatin A (TSA) treatment in MCF-7 cells. (B) Ac–tubulin levels to detect inhibition of SIRT2 after 6 hr 25 M inhibitor treatment in MCF-7 cells. (C) Inhibition of SIRT2 by TM and JH-T4 treatment by evaluating K-Ras4a lysine fatty acylation levels. (D) Detection of K-Ras4a lysine fatty acylation levels to evaluate in-cell inhibition of SIRT2 defatty-acylation activity. FL, fluorescence; CB, Coomassie blue staining. Next, we investigated whether either of the two compounds could inhibit the defatty-acylation activity of SIRT2 by evaluating the lysine fatty acylation level.Indeed, NH-TM was potent against SIRT2, exhibiting an IC50 value of 0.088 M (Table 1). molecule inhibitor, NH-TM. These studies provide useful insights for future SIRT2 inhibitor development. and in cells. In our efforts to develop a more potent SIRT2 inhibitor, we decided to carry out structure activity relationship studies based on the structure of TM. We specifically were interested in modifying the aniline portion of the small molecule, to study what modifications would increase or decrease SIRT2 potency and selectivity. To this end, we found that adding a single hydroxyl group on the aniline moiety, leading to the compound JH-T4 (Figure 1), produces a sirtuin inhibitor with a very different inhibition profile. We measured the IC50 values (Table 1) of JH-T4 toward SIRT1, SIRT2, SIRT3, and SIRT6 under pre-incubation conditions (enzymes, NAD, and inhibitors were first incubated for 15 min before substrates were added to start the enzymatic reaction) and compared them to the IC50 values of TM. For SIRT2, we determined the IC50 values for both deacetylation and demyritoylation activities. For these assays the H3K9-Ac and H3K9-Myr peptides were used as substrates, as SIRT1,2,3 and 6 have efficient activity on these peptides, which are commonly used for Sirtuin studies.[2a, 3b, 7a, 9] Open in a separate window Figure 1. Chemical Structures of different Sirtuin Inhibitors Table 1. IC50 values (M) of TM, Rabbit polyclonal to ANGPTL3 JH-T4 and NH-TM for inhibiting sirtuin deacylation activity (ND = not determined). Values shown in brackets are from assays without pre-incubation. with IC50 values of 15 M or lower. Interestingly, under the pre-incubation assay condition, TM and JH-T4 inhibited both the deacetylation and defatty-acylation activity of SIRT2 comparably (IC50 values in the 30C50 nM range) (Table 1). To further compare the defatty-acylation inhibition by TM and JH-T4 we determined the IC50 value for inhibition of SIRT2 demyristoylation activity without pre-incubating the enzyme with NAD and inhibitor. Without preincubation, the IC50 value of TM was > 200 M (42% inhibition at 200 M), but the IC50 of JH-T4 was approximately 110 M. This suggests that JH-T4 is more efficient at inhibiting the defatty-acylation activity of SIRT2 than TM is. We also measured the IC50 values of TM and JH-T4 on the deacetylation activity of SIRT1, SIRT2, and SIRT3 without preincubation. Most IC50 value for inhibiting the deacetylation activity of SIRT1-3 without pre-incubation did not drastically change for TM and JH-T4 compared to that with pre-incubation (Table 1).[7a] However the IC50 value of JH-T4 on SIRT1 without pre-incubation increased dramatically JH-T4 (40 M without pre-incubation, compared to 0.3 M with pre-incubation). We next wanted to compare the potency and selectivity of these compounds in cells. To evaluate the inhibition of SIRT1 deacetylation activity, we examined p53 acetylation levels, as Lys382 of p53 is definitely a well-established SIRT1 substrate.[10] As expected, JH-T4, but not TM, improved Ac-p53 level about Lys382 in MCF-7 cells (Number 2A). We further tested if these compounds could inhibit the deacetylation activity of SIRT2 in cells based on acetyl -tubulin immunofluorescence, as acetyl -tubulin is definitely a widely used cellular readout of SIRT2 activity.[3a] Both the TM and JH-T4 treated samples showed a dramatic increase in acetyl -tubulin levels compared to the sample treated with the vehicle control, ethanol. Therefore, both compounds efficiently inhibit SIRT2 deacetylation activity in MCF-7 cells (Number 2B). Open in a separate window Number 2. In-Cell Sirtuin Inhibition by JH-T4.(A) Ac-p53 levels to evaluate the inhibition of SIRT1 in cells after 6 hr 25M inhibitor and 200 nM trichostatin A (TSA) treatment in MCF-7 cells. (B) Ac–tubulin levels to detect inhibition of SIRT2 after 6 hr 25 M inhibitor treatment in MCF-7 cells. (C) Inhibition of SIRT2 by TM and JH-T4 treatment by evaluating K-Ras4a lysine fatty acylation levels. (D) Detection of K-Ras4a lysine fatty acylation levels to evaluate in-cell inhibition of SIRT2 defatty-acylation activity. FL, fluorescence; CB, Coomassie blue staining. Next, we investigated whether either of the two compounds could inhibit the defatty-acylation activity of SIRT2 by evaluating the lysine fatty acylation level of K-Ras4a, the only reported SIRT2 defatty-acylation target. We made use of the biorthogonal palmitic acid analogue Alk14 following a same methods previously explained.[5] First, we looked at the ability of the compounds to inhibit SIRT2 defatty-acylation on K-Ras4a inhibition of SIRT2 defatty-acylation of K-Ras4a,.Mel B., Kazantsev Aleksey G., Science 2007, 317, 516C519; [PubMed] [Google Scholar]b Rumpf T, Schiedel M, Karaman B, Roessler C, North BJ, Lehotzky A, Olah J, Ladwein KI, Schmidtkunz K, Gajer M, Pannek M, Steegborn C, Sinclair DA, Gerhardt S, Ovadi J, Schutkowski M, Sippl W, Einsle O, Jung M, Nat Commun 2015, 6, 6263; [PMC free article] [PubMed] [Google Scholar]c Lain S, Hollick JJ, Campbell J, Staples OD, Higgins M, Aoubala M, McCarthy A, Appleyard V, Murray KE, Baker L, Thompson A, Mathers J, Holland SJ, Stark MJ, Pass G, Woods J, Lane DP, Westwood NJ, Malignancy Cell 2008, 13, 454C463; [PMC free article] [PubMed] [Google Scholar]d Mellini P, Itoh Y, Tsumoto H, Li Y, Suzuki M, Tokuda N, Kakizawa T, Miura Y, Takeuchi J, Lahtela-Kakkonen M, Suzuki T, Chem Sci 2017, 8, 6400C6408; [PMC free article] [PubMed] [Google Scholar]e Schiedel M, Rumpf T, Karaman B, Lehotzky A, Olah J, Gerhardt S, Ovadi J, Sippl W, Einsle O, Jung M, J Med Chem 2016, 59, 1599C1612; [PubMed] [Google Scholar]f Yang LL, Wang HL, Zhong L, Yuan C, Liu SY, Yu ZJ, Liu S, Yan YH, Wu C, Wang Y, Wang Z, Yu Y, Chen Q, Li GB, Eur J Med Chem 2018, 155, 806C823; [PubMed] [Google Scholar]g Wang C, Wang F, Chen X, Zou Y, Zhu H, Zhao Q, Shen J, Li Y, Li Y, He B, Bioorg Med Chem Lett 2018, 28, 2375C2378; [PubMed] [Google Scholar]h Yang L, Ma X, Yuan C, He Y, Li L, Fang S, Xia W, He T, Qian S, Xu Z, Li G, Wang Z, Eur J Med Chem 2017, 134, 230C241; [PubMed] [Google Scholar]i Tatum PR, Sawada H, Ota Y, Itoh Y, Zhan P, Ieda N, Nakagawa H, Miyata N, Suzuki T, Bioorg Med Chem Lett 2014, 24, 1871C1874; [PubMed] [Google Scholar]j Villalba JM, Alcain FJ, Biofactors 2012, 38, 349C359; [PMC free article] [PubMed] [Google Scholar]k Hu J, Jing H, Lin H, Long term Med Chem 2014, 6, 945C966; [PMC free article] [PubMed] [Google Scholar]l Yoon YK, Oon CE, Anticancer Providers Med Chem 2016, 16, 1003C1016; [PubMed] [Google Scholar]m Rotili D, Tarantino D, Nebbioso A, Paolini C, Huidobro C, Lara E, Mellini P, Lenoci A, Pezzi R, Botta G, Lahtela-Kakkonen M, Poso A, Steinkuhler C, Gallinari P, De Maria R, Fraga M, Esteller M, Altucci L, Mai A, J Med Chem 2012, 55, 10937C10947. and SIRT1, SIRT2, and SIRT3. This is further supported by studies with another small molecule inhibitor, NH-TM. These studies provide useful insights for long term SIRT2 inhibitor development. and in cells. In our efforts to develop a more potent SIRT2 inhibitor, we decided to carry out structure activity relationship studies based on the structure of TM. We specifically were interested in modifying the aniline portion of the small molecule, to study what modifications would increase or decrease SIRT2 potency and selectivity. To this end, we found that adding a single hydroxyl group within the aniline moiety, leading to the compound JH-T4 (Number 1), generates a sirtuin inhibitor with a very different inhibition profile. We measured the IC50 ideals (Table 1) of JH-T4 toward SIRT1, SIRT2, SIRT3, and SIRT6 under pre-incubation conditions (enzymes, NAD, and inhibitors were 1st incubated for 15 min before substrates were added to start the enzymatic reaction) and compared them to the IC50 ideals of TM. For SIRT2, we identified the IC50 ideals for both deacetylation and demyritoylation activities. For these assays the H3K9-Ac and H3K9-Myr peptides were used as substrates, as SIRT1,2,3 and 6 have efficient activity on these peptides, which are commonly utilized for Sirtuin studies.[2a, 3b, 7a, 9] Open in a separate window Number 1. Chemical Constructions of different Sirtuin Inhibitors Table 1. IC50 ideals (M) of TM, JH-T4 and NH-TM for inhibiting sirtuin deacylation activity (ND = not determined). Values demonstrated in brackets are from assays without pre-incubation. with IC50 ideals of 15 M or lower. Interestingly, under the pre-incubation assay condition, TM and JH-T4 inhibited both the deacetylation and defatty-acylation activity of SIRT2 comparably (IC50 ideals in the 30C50 nM range) (Table 1). To further compare the defatty-acylation inhibition by TM and JH-T4 we identified the IC50 value for inhibition of SIRT2 demyristoylation activity without pre-incubating the enzyme with NAD and inhibitor. Without preincubation, the IC50 value of TM was > 200 M (42% inhibition at 200 M), but the IC50 of JH-T4 was approximately 110 M. This suggests that JH-T4 is usually more efficient at inhibiting the defatty-acylation activity of SIRT2 than TM is usually. We also measured the IC50 values of TM and JH-T4 around the deacetylation activity of SIRT1, SIRT2, and SIRT3 without preincubation. Most IC50 value for inhibiting the deacetylation activity of SIRT1-3 without pre-incubation did not drastically switch for TM and JH-T4 compared to that with pre-incubation (Table 1).[7a] However the IC50 value of JH-T4 on SIRT1 without pre-incubation increased dramatically JH-T4 (40 M without pre-incubation, compared to 0.3 M with pre-incubation). We next wanted to compare the potency and selectivity of these compounds in cells. To evaluate the inhibition of SIRT1 deacetylation activity, we examined p53 acetylation levels, as Lys382 of p53 is usually a well-established SIRT1 substrate.[10] As expected, JH-T4, but not TM, increased Ac-p53 level on Lys382 in MCF-7 cells (Determine 2A). We further tested if these compounds could inhibit the deacetylation activity of SIRT2 in cells based on acetyl -tubulin immunofluorescence, as acetyl -tubulin is usually a widely used cellular readout of SIRT2 activity.[3a] Both the TM and JH-T4 treated samples showed a dramatic increase in acetyl -tubulin levels compared to the sample treated with the vehicle control, ethanol. Thus, both compounds efficiently inhibit SIRT2 deacetylation activity in MCF-7 cells (Physique 2B). Open in a separate window Physique 2. In-Cell Sirtuin Inhibition by JH-T4.(A) Ac-p53 levels to evaluate the inhibition of SIRT1 in cells after 6 hr 25M inhibitor and 200 nM trichostatin A (TSA) treatment in MCF-7 cells. (B) Ac–tubulin levels to detect inhibition of SIRT2 after 6 hr 25 M inhibitor treatment in MCF-7 cells. (C) Inhibition of SIRT2 by TM and JH-T4 treatment by evaluating K-Ras4a lysine fatty acylation levels. (D) Detection of K-Ras4a.