Title: Pimelic Diphenylamide 106 Is a Slow, Tight-binding Inhibitor of Class I Histone Deacetylases
Abstract: Histone deacetylase (HDAC) inhibitors, including various benzamides and hydroxamates, are currently in clinical development for a broad range of human diseases, including cancer and neurodegenerative diseases. We recently reported the identification of a family of benzamide-type HDAC inhibitors that are relatively non-toxic compared with the hydroxamates. Members of this class of compounds have shown efficacy in cell-based and mouse models for the neurodegenerative diseases Friedreich ataxia and Huntington disease. Considerable differences in IC50 values for the various HDAC enzymes have been reported for many of the HDAC inhibitors, leading to confusion as to the HDAC isotype specificities of these compounds. Here we show that a benzamide HDAC inhibitor, a pimelic diphenylamide (106), is a class I HDAC inhibitor, demonstrating no activity against class II HDACs. 106 is a slow, tight-binding inhibitor of HDACs 1, 2, and 3, although inhibition for these enzymes occurs through different mechanisms. Inhibitor 106 also has preference toward HDAC3 with Ki of ∼14 nm, 15 times lower than the Ki for HDAC1. In comparison, the hydroxamate suberoylanilide hydroxamic acid does not discriminate between these enzymes and exhibits a fast-on/fast-off inhibitory mechanism. These observations may explain a paradox involving the relative activities of pimelic diphenylamides versus hydroxamates as gene activators. Histone deacetylase (HDAC) inhibitors, including various benzamides and hydroxamates, are currently in clinical development for a broad range of human diseases, including cancer and neurodegenerative diseases. We recently reported the identification of a family of benzamide-type HDAC inhibitors that are relatively non-toxic compared with the hydroxamates. Members of this class of compounds have shown efficacy in cell-based and mouse models for the neurodegenerative diseases Friedreich ataxia and Huntington disease. Considerable differences in IC50 values for the various HDAC enzymes have been reported for many of the HDAC inhibitors, leading to confusion as to the HDAC isotype specificities of these compounds. Here we show that a benzamide HDAC inhibitor, a pimelic diphenylamide (106), is a class I HDAC inhibitor, demonstrating no activity against class II HDACs. 106 is a slow, tight-binding inhibitor of HDACs 1, 2, and 3, although inhibition for these enzymes occurs through different mechanisms. Inhibitor 106 also has preference toward HDAC3 with Ki of ∼14 nm, 15 times lower than the Ki for HDAC1. In comparison, the hydroxamate suberoylanilide hydroxamic acid does not discriminate between these enzymes and exhibits a fast-on/fast-off inhibitory mechanism. These observations may explain a paradox involving the relative activities of pimelic diphenylamides versus hydroxamates as gene activators. The link between post-translational modifications by reversible histone acetylation and deacetylation and mRNA transcription has been shown to be one of the key mechanisms of epigenetic gene regulation (1Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7538) Google Scholar). Acetylation of histone lysine residues, controlled by the histone acetyltransferases and histone deacetylases (HDACs), 2The abbreviations used are:HDAChistone deacetylaseTSAtrichostatin ASAHAsuberoylanilide hydroamic acidMCA4-methylcoumarin-7-amide 2The abbreviations used are:HDAChistone deacetylaseTSAtrichostatin ASAHAsuberoylanilide hydroamic acidMCA4-methylcoumarin-7-amide has been a subject of intense recent research (2Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6507) Google Scholar, 3Marmorstein R. Nat. Rev. Mol. Cell Biol. 2001; 2: 422-432Crossref PubMed Scopus (174) Google Scholar, 4Wade P.A. Hum. Mol. Genet. 2001; 10: 693-698Crossref PubMed Scopus (277) Google Scholar, 5Thiel G. Lietz M. Hohl M. Eur. J. Biochem. 2004; 271: 2855-2862Crossref PubMed Scopus (94) Google Scholar). Generally, histone hypoacetylation causes transcriptional silencing, whereas histone hyperacetylation results in transcriptional activation of various genes (6Hassig C.A. Schreiber S.L. Curr. Opin. Chem. Biol. 1997; 1: 300-308Crossref PubMed Scopus (336) Google Scholar, 7Kouzarides T. Curr. Opin. Genet. Dev. 1999; 9: 40-48Crossref PubMed Scopus (585) Google Scholar, 8Grozinger C.M. Schreiber S.L. Chem. Biol. 2002; 9: 3-16Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). Eighteen HDACs have been identified in the human genome, including the zinc-dependent HDACs (class I, class II, and class IV), and the NAD+-dependent enzymes (class III or sirtuins) (9de Ruijter A.J. van Gennip A.H. Caron H.N. Kemp S. van Kuilenburg A.B. Biochem. J. 2003; 370: 737-749Crossref PubMed Scopus (2423) Google Scholar, 10Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1290) Google Scholar). HDACs 1, 2, 3, and 8 belong to class I, showing homology to the yeast enzyme RPD3. Class II is further divided into class IIa (HDACs 4, 5, 7, and 9) and IIb (HDAC 6 and 10), according to their sequence homology and domain organization. HDAC11 is the lone member of class IV (9de Ruijter A.J. van Gennip A.H. Caron H.N. Kemp S. van Kuilenburg A.B. Biochem. J. 2003; 370: 737-749Crossref PubMed Scopus (2423) Google Scholar, 11Gao L. Cueto M.A. Asselbergs F. Atadja P. J. Biol. Chem. 2002; 277: 25748-25755Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar). The sirtuins (class III) are related to the yeast Sir2 protein and are involved in regulation of metabolism and aging (10Blander G. Guarente L. Annu. Rev. Biochem. 2004; 73: 417-435Crossref PubMed Scopus (1290) Google Scholar).To date, a number of small molecule inhibitors of the zinc-dependent HDACs have been identified (12Beckers T. Burkhardt C. Wieland H. Gimmnich P. Ciossek T. Maier T. Sanders K. Int. J. Cancer. 2007; 121: 1138-1148Crossref PubMed Scopus (162) Google Scholar). These compounds can be broadly grouped in four chemical classes: the hydroxamates, the benzamides, butyrate analogs, and cyclic peptides, such as depsipeptide and related compounds (12Beckers T. Burkhardt C. Wieland H. Gimmnich P. Ciossek T. Maier T. Sanders K. Int. J. Cancer. 2007; 121: 1138-1148Crossref PubMed Scopus (162) Google Scholar, 13Itoh Y. Suzuki T. Miyata N. Curr. Pharm. Des. 2008; 14: 529-544Crossref PubMed Scopus (106) Google Scholar). Hydroxamate-based inhibitors, such as trichostatin A (TSA) and suberoylanilide hydroamic acid (SAHA; Fig. 1A) are believed to be pan-HDAC inhibitors (14Butler L.M. Agus D.B. Scher H.I. Higgins B. Rose A. Cordon-Cardo C. Thaler H.T. Rifkind R.A. Marks P.A. Richon V.M. Cancer Res. 2000; 60: 5165-5170PubMed Google Scholar, 15Richon V.M. Emiliani S. Verdin E. Webb Y. Breslow R. Rifkind R.A. Marks P.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3003-3007Crossref PubMed Scopus (836) Google Scholar, 16Kelly W.K. O'Connor O.A. Krug L.M. Chiao J.H. 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Serafini S. Steinkuhler C. Bioorg. Med. Chem. Lett. 2008; 18: 1814-1819Crossref PubMed Scopus (82) Google Scholar). Studies of benzamide-based HDAC inhibitors have shown that these compounds are class I-specific inhibitors, and have claimed distinct pharmacological properties of the benzamide HDAC inhibitors due to specific inhibition of class I HDACs (mainly HDAC1 and HDAC3) (20Saito A. Yamashita T. Mariko Y. Nosaka Y. Tsuchiya K. Ando T. Suzuki T. Tsuruo T. Nakanishi O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4592-4597Crossref PubMed Scopus (682) Google Scholar, 21Bonfils C. Kalita A. Dubay M. Siu L.L. Carducci M.A. Reid G. Martell R.E. Besterman J.M. Li Z. Clin. Cancer Res. 2008; 14: 3441-3449Crossref PubMed Scopus (78) Google Scholar). Some benzamides (BML-210 and related pimelic diphenylamides 4b and 106; Fig. 1A) have been shown to up-regulate specific genes, including the frataxin gene involved in the neurodegenerative disease Friedriech ataxia, at subtoxic inhibitor concentrations (22Herman D. Jenssen K. Burnett R. Soragni E. Perlman S.L. Gottesfeld J.M. Nat. Chem. Biol. 2006; 2: 551-558Crossref PubMed Scopus (356) Google Scholar, 23Rai M. Soragni E. Jenssen K. Burnett R. Herman D. Coppola G. Geschwind D.H. Gottesfeld J.M. Pandolfo M. PLoS ONE. 2008; 3: e1958Crossref PubMed Scopus (174) Google Scholar, 24Gottesfeld J.M. Pharmacol. Ther. 2007; 116: 236-248Crossref PubMed Scopus (58) Google Scholar). In comparison, the hydroxamates SAHA and TSA were inactive as positive regulators of frataxin gene expression. Thus, the distinct pharmacological properties of the pimelic diphenylamides, as compared with the hydroxamates, cannot be explained by specificity for class I HDACs. Additionally, other benzamide HDAC inhibitors, such as MS-275, have reported IC50 values ranging from ∼740 nm to 8 μm for HDAC3, whereas the reported IC50 values for HDAC1 are more consistent, at around ∼200 nm (12Beckers T. Burkhardt C. Wieland H. Gimmnich P. Ciossek T. Maier T. Sanders K. Int. J. Cancer. 2007; 121: 1138-1148Crossref PubMed Scopus (162) Google Scholar, 25Hu E. Dul E. Sung C.M. Chen Z. Kirkpatrick R. Zhang G.F. Johanson K. Liu R. Lago A. Hofmann G. Macarron R. de los Frailes M. Perez P. Krawiec J. Winkler J. Jaye M. J. Pharmacol. Exp. Ther. 2003; 307: 720-728Crossref PubMed Scopus (324) Google Scholar).In this study, we investigated differences in the kinetic properties between the hydroxamate SAHA and the pimelic diphenylamide inhibitor 106 (Fig. 1A), with recombinant human HDACs. We discovered that the benzamide inhibitor 106, unlike the hydroxamate SAHA, is a slow, tight-binding inhibitor of HDACs 1, 2, and 3, with different inhibitory mechanisms and half-lives of the enzyme-inhibitor complexes. IC50 values for inhibitor 106 decreased significantly during preincubation with HDAC3, but this slow inhibition behavior is less pronounced for HDAC1 or -2. Hydroxamates, on the other hand, are fast-on/fast-off inhibitors of both HDAC1 and HDAC3. The kinetic parameters for these two classes of compounds were determined for HDACs 1 and 3, and compared with the cellular activities of 106 and SAHA.EXPERIMENTAL PROCEDURESMaterials—Recombinant human HDAC1, HDAC2, HDAC3/N-CoR2, and HDAC8 expressed in baculovirus, were purchased from BPS Bioscience (San Diego, CA). Western blots were done to verify that no HDAC1 and HDAC3 cross-contamination was present in these enzyme preparations (supplemental Fig. S1). Acetylated-Lys(acetyl)-4-methylcoumarin-7-amide (acetyl-Lys(Ac)-AMC) was purchased from Biomol International (Plymouth Meeting, PA). Lys-C peptidase was purchased from EMD Chemicals (Gibbstown, NJ). For the class II HDACs, 4, 5, and 7, we obtained cDNAs for these human enzymes from Addgene in pcDNA3.1 with a C-terminal FLAG epitope. Protein overexpression was achieved by transfecting plasmids into HEK293t cells with 293-fectin reagent (Invitrogen). Cells were lysed at 72 h with whole cell lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.5! Triton X-100, 10! glycerol), and passed over FLAG-M2 affinity resin (Sigma) for purification. Following several washes, HDAC proteins were eluted with 5 bead volumes of 100 μg/ml FLAG peptide (Sigma), and assayed without further purification. HDAC inhibitor SAHA was purchased from Biomol International (Plymouth Meeting, PA) through a custom synthesis order, and 106 (N1-(2-aminophenyl)-N7-p-tolylheptanediamide) was synthesized as previous described (23Rai M. Soragni E. Jenssen K. Burnett R. Herman D. Coppola G. Geschwind D.H. Gottesfeld J.M. Pandolfo M. PLoS ONE. 2008; 3: e1958Crossref PubMed Scopus (174) Google Scholar) and provided by Repligen Corporation (Waltham, MA).Assay of HDAC Activities and Inhibition Kinetics—The deacetylase activities of HDACs 1, 2, and 3 were measured by assaying enzyme activity using peptidase (Lys-C peptidase and trypsin) and the synthetic substrate acetyl-Lys(Ac)-AMC, as previously described (26Wegener D. Wirsching F. Riester D. Schwienhorst A. Chem. Biol. 2003; 10: 61-68Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 27Wegener D. Hildmann C. Riester D. Schwienhorst A. Anal. Biochem. 2003; 321: 202-208Crossref PubMed Scopus (97) Google Scholar). Deacetylated lysine-AMC was released by the peptidase and free fluorogenic 4-methylcoumarin-7-amide (MCA) was generated. The fluorogenic MCA could then be read with an excitation wavelength of 370 nm and emission wavelength of 460 nm. Assays for class II HDACs were done using acetyl-Lys(trifluoroacetyl)-AMC under the same conditions (17Lahm A. Paolini C. Pallaoro M. Nardi M.C. Jones P. Neddermann P. Sambucini S. Bottomley M.J. Lo Surdo P. Carfi A. Koch U. De Francesco R. Steinkuhler C. Gallinari P. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 17335-17340Crossref PubMed Scopus (417) Google Scholar) (synthesized at Scripps). All assays for HDAC activity on the acetylated lysine substrates were performed in 96-well, non-binding plates (Greiner Bio-one, NC) in 50 mm Tris-HCl buffer (pH 8.0), containing 137 mm NaCl, 1 mm MgCl2, 2.7 mm KCl, and 0.1 mg/ml bovine serum albumin (standard HDAC buffer) at ambient temperature. The final assay volume was 50 μl, except for the dilution experiment described below, which was at 100 μl. The amount of MCA generated was equal to deacetylated substrate and was normalized with a non-acetylated substrate standard (supplemental Fig. S2A).Determination of the Inhibitor IC50 Values with Preincubation—Deacetylation assays were based on the homogenous fluorescence release assay, described above. Purified recombinant enzymes were incubated with serial-diluted inhibitors at the concentrations indicated in the figures, with preincubation times ranging from 0 to 3 h, in the standard HDAC buffer (as in Fig. 1B). Acetyl-Lys(Ac)-AMC substrate (at 10 μm, corresponding to the Km for both HDAC1 and HDAC3, supplemental Fig. S2B) was added after the preincubation period, and the reaction was allowed to run for 1 h. The trypsin peptidase developer, at a final concentration of 5 mg/ml, was added after 1 h, and the fluorescence emission was then measured using a Tecan M200 96-well plate reader (San Jose, CA). The IC50 was determined by fitting the data using the KaleidaGraph nonlinear regression program (Synergy Software, Reading, PA).Slow, Tight-binding Kinetic Determination Using the Progression Method—Slow tight-binding kinetics of 106 with class I HDACs were evaluated by the progression curve approach described by Morrison and Walsh (28Morrison J.F. Walsh C.T. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 201-301PubMed Google Scholar). In theory, there are three possible slow, tight-binding mechanisms, as previously described (29Duggleby R.G. Attwood P.V. Wallace J.C. Keech D.B. Biochemistry. 1982; 21: 3364-3370Crossref PubMed Scopus (75) Google Scholar). The two most common ones are shown here as Mechanisms 1 and 2. E+S↔kmES↔kcatE+P+|k1↓↑k-1EIMECHANISM 1 E+S↔kmES↔kcatE+P+|k1↓↑k-1EI⇄k-2k2EI*MECHANISM 2 To determine the mechanism and associated kinetic values, a series of inhibition progression curves for HDACs 1, 2, and 3, at different concentrations of inhibitor 106, were generated by adding 100 ng of each enzyme into separate reaction mixtures containing 50 μm acetyl-Lys(Ac)-AMC substrate (5 times the Km) and 2 milliunits of Lys-C peptidase developer. Lys-C was used in the progression curve method to prevent degradation of the HDAC enzymes during the assay, allowing a linear no-inhibitor control. The generation of the fluorogenic MCA, due to the deacetylation of the lysine substrate, was assessed continuously for up to 1 h at ambient temperature. Data from each progression curve, at different inhibitor concentrations, were fit using the nonlinear regression program KaleidaGraph to the integrated rate equation for slow-binding inhibitors: [F]=vst+(v0-vs)(1-exp(-kobst))/kobs(Eq. 1) where [F] is the amount of MCA fluorophore generated, represented in arbitrary fluorescence units (r), which is proportion to the deacetylated substrate at time t. v0 and vs are the initial and the final steady-state velocities, respectively. kobs is the apparent first-order rate constant obtained by the best fit to the data. Because kobs is the only value that is not significantly altered by small systematic errors (29Duggleby R.G. Attwood P.V. Wallace J.C. Keech D.B. Biochemistry. 1982; 21: 3364-3370Crossref PubMed Scopus (75) Google Scholar, 30Cornish-Bowden A. Biochem. J. 1975; 149: 305-312Crossref PubMed Scopus (87) Google Scholar), the kobs values were then plotted against the inhibitor concentrations for which each kobs value was obtained. For Mechanism 1, the relationship between kobs and the inhibitor concentration is linear, kobs=k-1+k1[I]/(1+[S]/Km)(Eq. 2) and Ki=k-1/k1(Eq. 3) For Mechanism 2, the relationship between kobs and the inhibitor concentration is hyperbolic, kobs=k-2+k2[I]/[[I]+Ki*(1+[S]/Km)](Eq. 4) and Ki=Ki*[k-2/(k2+k-2)](Eq. 5) where Ki* is the stable complex forming constant and Ki is the overall final inhibitory constant for the entire process.Determination of Ki for Fast On/Off Inhibitors—For a classical fast on/off competitive inhibitor, such as SAHA, the steady state HDAC enzyme velocities are achieved within seconds. The deacetylation rate in the presence (vi) and absence (v0) of the inhibitor are linear, with no time dependence. Therefore, the Ki of the inhibitor can easily be determined using the ratio of vi over v0 (single step fast on/off) according to the relationship (31Tornheim K. Anal. Biochem. 1994; 221: 53-56Crossref PubMed Scopus (17) Google Scholar, 32Koh C.Y. Kazimirova M. Trimnell A. Takac P. Labuda M. Nuttall P.A. Kini R.M. J. Biol. Chem. 2007; 282: 29101-29113Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) in Equation 6. vi/v0=1/[[I]/Ki(1+[S]/Km)+1](Eq. 6) The velocity of each progression line was calculated, and the ratio of vi/v0 was plotted against the corresponding inhibitor concentration. The Ki was then determined by fitting the data to Equation 6, using the KaleidaGraph nonlinear regression program.Comparative Disassociation through 100-fold Dilution—HDAC1 (10 μg) and HDAC3 (10 μg) were each incubated with 2 μm 106 or 100 nm SAHA, in standard deacetylase assay buffer, containing 0.1 mg/ml bovine serum albumin, for 1 h. After this preincubation time, 1 μl of each mixture was diluted into a final volume of 100 μl, containing 0.1 mg/ml bovine serum albumin, 50 μm acetyl-Lys(Ac)-AMC substrate, and 2 milliunits of Lys-C peptidase developer, without or with each inhibitor at the initial concentration (2 μm 106 or 100 nm SAHA). The amounts of HDAC1 and HDAC3 after dilution were 100 ng in the final assay solution. Progression curves were then measured for an additional hour.Cell Culture, SDS-PAGE, and Western Blot Analysis—A lymphoblastoid cell line derived from a Friedreich ataxia patient (GM15850) was obtained from the NIGMS Genetic Repository (Coriell Medical Institute). Cells were cultured in RPMI 1640 media with 10! fetal bovine serum and 10 mm HEPES, at 37 °C in 5! CO2. After the 5th split, the cells were treated with inhibitor 106 (2 μm) or SAHA (2 μm) for 24 h, in culture medium. The treatment concentrations were determined based on growth inhibition through an MTS cell proliferation assay (Promega) (supplemental Fig. S3). These concentrations correspond approximately to the EC10 for 106, and 2 μm SAHA is near its EC50 for blocking cell proliferation. 24 h after treatment, the cells were washed twice with Hanks' balanced salts buffer (Invitrogen) to remove the inhibitor. A portion of the cell population was then harvested immediately after washing, and referred to as a time 0 point (24 h treatment point), and the rest of the cells were then re-cultured in cell culture media without added inhibitor. The re-cultured cells and controls were then harvested every hour for a total of 7 h. The cells were washed twice before lysis with a low salt lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 10! glycerol, 0.5! Triton X-100, and 1× protease inhibitors, Roche Diagnostics) for 30 min, and followed by a 15-s sonication pulse at 3 watts (Branson Sonifier 150, Branson, CT). The cell lysates were then denatured with LDS loading buffer (Invitrogen) and run on 4–12! SDS gradient polyacrylamide gels (Invitrogen). Total histone H3, acetylated histone H3 (K9+K14), and frataxin were visualized with primary histone H3 antibody from Abcam (Cambridge, MA), acetylated K9+K14 histone H3 antibody from Upstate (Temecula, CA), and anti-frataxin antibody from MitoSciences (Eugene, OR), respectively, followed by rabbit (for histone H3 and acetylated H3) or mouse (for frataxin) IgG-horseradish peroxidase conjugated secondary antibody (Cell Signaling, MA).RESULTSHDAC Inhibition Assays—We assayed each of the recombinant class I (HDACs 1, 2, 3, and 8) and representative class II (HDACs 4, 5, and 7) enzymes with the pimelic diphenylamide HDAC inhibitor 106 (Fig. 1A). For HDAC3, a recombinant fragment of the co-repressor protein N-CoR2 was co-expressed because the deacetylase activating domain of N-CoR is required for HDAC3 activity (reviewed in Ref. 33Karagianni P. Wong J. Oncogene. 2007; 26: 5439-5449Crossref PubMed Scopus (170) Google Scholar). Enzyme and inhibitor were preincubated for 1 to 3 h prior to addition of a fluorogenic substrate (see “Experimental Procedures”), and from compound titrations, IC50 values are calculated. Compound 106 exhibits good inhibitory activity against class I HDACs, with IC50 values of 150 nm for HDAC1 and 370 nm for HDAC3. 106 exhibits weaker inhibitory activities against HDAC2 and -8, 106 has an IC50 of 760 nm with HDAC2 and an IC50 of 5 μm after a 3-h preincubation with HDAC8 (supplemental Fig. S4). Because recent studies indicate that class II HDACs are not active on standard acetylated lysine peptide substrates (17Lahm A. Paolini C. Pallaoro M. Nardi M.C. Jones P. Neddermann P. Sambucini S. Bottomley M.J. Lo Surdo P. Carfi A. Koch U. De Francesco R. Steinkuhler C. Gallinari P. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 17335-17340Crossref PubMed Scopus (417) Google Scholar), assays with recombinant class II HDACs 4, 5, and 7 used a trifluoracetylated lysine substrate (see Ref. 17Lahm A. Paolini C. Pallaoro M. Nardi M.C. Jones P. Neddermann P. Sambucini S. Bottomley M.J. Lo Surdo P. Carfi A. Koch U. De Francesco R. Steinkuhler C. Gallinari P. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 17335-17340Crossref PubMed Scopus (417) Google Scholar), and we find that 106 has essentially no inhibitory activity against these enzymes (IC50 > 180 μm; supplemental Fig. S5). Previous studies have established that benzamide-type HDAC inhibitors are selective for class I HDAC enzymes and in particular MS-275 and other o-aminobenzamides show a ∼4 to 10-fold preference for HDAC1 over HDAC3 (12Beckers T. Burkhardt C. Wieland H. Gimmnich P. Ciossek T. Maier T. Sanders K. Int. J. Cancer. 2007; 121: 1138-1148Crossref PubMed Scopus (162) Google Scholar, 25Hu E. Dul E. Sung C.M. Chen Z. Kirkpatrick R. Zhang G.F. Johanson K. Liu R. Lago A. Hofmann G. Macarron R. de los Frailes M. Perez P. Krawiec J. Winkler J. Jaye M. J. Pharmacol. Exp. Ther. 2003; 307: 720-728Crossref PubMed Scopus (324) Google Scholar, 34Siliphaivanh P. Harrington P. Witter D.J. Otte K. Tempest P. Kattar S. Kral A.M. Fleming J.C. Deshmukh S.V. Harsch A. Secrist P.J. Miller T.A. Bioorg. Med. Chem. Lett. 2007; 17: 4619-4624Crossref PubMed Scopus (48) Google Scholar). Our results are thus consistent for 106 compared with other benzamides.Effects of Preincubation Time on Observed IC50 Values—SAHA has been shown to be a competitive HDAC inhibitor (12Beckers T. Burkhardt C. Wieland H. Gimmnich P. Ciossek T. Maier T. Sanders K. Int. J. Cancer. 2007; 121: 1138-1148Crossref PubMed Scopus (162) Google Scholar). Dose-response curves of SAHA showed an average IC50 of 17.3 (± 2.1) nm for HDAC1 and 24.1 (± 3.7) nm for HDAC3, independent of preincubation time (Fig. 1B), showing that SAHA rapidly reaches equilibrium with these enzymes. 106 also inhibited the deacetylase activities of HDACs 1,2, 3, and 8; however, unlike SAHA, dose-response curves and calculated IC50 values for 106 vary with enzyme-inhibitor preincubation time (Fig. 1B for HDAC1 and HDAC3; supplemental Fig. S4 for HDAC2 and HDAC8). This was especially true for HDAC3. Even after 2 h of incubation, the IC50 for 106 with HDAC3 was still decreasing. Without preincubation, 106 exhibited an IC50 of 460 nm for HDAC1, and 5.8 μm for HDAC3. After a 15-min preincubation, the IC50 for HDAC1 came to equilibrium at 138 (± 38) nm. For HDAC3, however, the IC50 for 106 did not reach a steady-state value for several hours. After a 3-h preincubation, the IC50 decreased to 380 nm; a 15-fold decrease in the IC50 value compared with the IC50 measured without preincubation. Similar results were also observed for HDAC2 and HDAC8, with higher IC50 values (supplemental Fig. S4). These observations suggest that 106 is a slow, tight-binding inhibitor of class I HDACs. Also, there was a great difference in the on-rate (and presumably the off-rate) for 106 for HDAC1 and HDAC3, respectively. Thus, the IC50 of a particular enzyme/inhibitor pair is not a reliable measurement due to the variability with different procedures; i.e. preincubation periods. To better understand the mechanisms for inhibition of these enzymes and to determine the kinetic constants of slow, tight-binding inhibitors, progression curve experiments for class I HDACs were next performed.HDAC Inhibitor 106 Is a Slow, Competitive Tight-binding Inhibitor of HDACs 1 and 2—HDAC1 deacetylation progression curves were measured in the presence of different concentrations of 106 (ranging from 0.5 nm to 20 μm), performed in triplicate (Fig. 2A). The data were fit with Equation 1, using the estimated v0 and vs values to determine the kobs for each run. The kobs was then plotted against the concentration of 106 used for each determination of kobs. A linear trend was observed, indicating a competitive tight-binding mechanism, as described by Mechanism 1 (Fig. 2A). The data were then fit with Equation 2, and Ki, k1, and k–1 were determined using Equations 2 and 3. For HDAC1 and inhibitor 106, k1 was 4.9 × 104 (± 1.2 × 104) m–1 min–1, and k–1 was 0.0072 (± 0.0017) min–1. Based on a presumed first-order decay, k–1 corresponds to a half-life of ∼1.5 h for the 106-HDAC1 complex. The Ki was then determined from triplicate experiments, using Equation 3. The Ki was found to be 148 (± 36) nm for HDAC1. The kinetics for HDAC2 were also investigated at various concentrations of 106, and found to be similar to HDAC1, suggesting that inhibition follows Mechanism 1, as described above (supplemental Fig. S4). From progression curves obtained at different concentrations of 106,a Ki of 102 nm was determined for HDAC2. Due to the high peptidase sensitivity of HDAC8 (data not shown), we were unable to perform a similar kinetic analysis.FIGURE 2Time-dependent inhibition of HDAC1 and HDAC3 as a function of inhibitor 106 concentration. The reaction mixture contained 100 ng of enzyme in the standard HDAC Tris buffer (pH 8.0), 50 μm acetylated-lysine substrate, various concentrations of inhibitor 106 (indicated) and Lys-C peptidase (2 milliunits). A, the progress curves for HDAC1 in the presence of increasing concentrations of inhibitor 106 were measured for 1 h and fit with Equation 1 (left). The rate constant kobs as a function of inhibitor 106 concentration was plotted (right). B, the progress curves for HDAC3 in the presence of increasing concentrations of inhibitor 106 were measured for 1 h and fit with Equation 1 (left). The rate constant kobs as a function of inhibitor 106 concentration was plotted (right).View Large Image Figure ViewerDownload Hi-res image Download (PPT)HDAC Inhibitor 106 Is a Slow, Competitive Tight-binding Inhibitor of HDAC3 with a Conformational Change in the Enzyme—HDAC3 deacetylation progression curves were measured at various concentrations of 106, as described above (Fig. 2B). Again, the data were fit with Equation 1, using the estimated v0 and vs values to determine kobs for each concentration of 106. The kobs values were then plotted against 106 concentration, and a hyperbolic relationship with an increasing inhibitor concentration was observed (Fig. 2B). This upward slope hyperbolic relationship was consistent with Mechanism 2, reflecting slow, tight-binding inhibition (having a slow step for forming a stable complex with a possible conformation change in the protein). The data were fit with Equations 4 and 5 to determine the kinetic parameters. The overall Ki value was determined to be 14 nm (± 3) nm, with Ki* of 224 nm (± 62 nm); k2 was 0.021 (± 0.002) min–1, and k–2 was 0.00143 (± 0.00197) min–1 for HDAC3. Based on a presumed f