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Published in final edited form as: Angew Chem Int Ed Engl. 2009;48(26):4718–4724. doi: 10.1002/anie.200805900

Probing the Bioactive Conformation of an Archetypal Natural Product HDAC Inhibitor Using Conformationally Homogeneous Triazole-Modified Cyclic Tetrapeptides**

W Seth Horne 1,, Christian A Olsen 1,, John M Beierle 1, Ana Montero 1, M Reza Ghadiri 1,*
PMCID: PMC3041267  NIHMSID: NIHMS271273  PMID: 19267380

A fundamental strategy in rationally designing synthetic compounds to bind a protein of interest is to use a known ligand as a structural model to specify the precise conformational and pharmacophoric requirements for binding. Despite the remarkable success of this approach, a significant difficulty is that free ligands (in the absence of their cognate receptors) often adopt multiple conformations in solution or in the solid state compared to the receptor-bound structure.[1] These occurrences can render design models based on the free ligand structure difficult to obtain or even misleading.[2] Here we present evidence that the more potent conformation of apicidin, an archetypal member of a family of naturally occurring cyclic tetrapeptide inhibitors of histone deacetylases (HDACs), is not the all-trans (t-t-t-t) structure that predominates in solution,[3,4] but rather a cis-trans-trans-trans (c-t-t-t) conformation. Our studies rely on the design, synthesis, structural characterization, and functional analysis of a series of cyclic pseudo-tetrapeptides bearing 1,4- or 1,5-disubstituted 1,2,3-triazole amino acids that serve as trans- or cis-amide bond surrogates, respectively. We show that by replacing an amide bond with a triazole, the bond in question can be fixed in either a trans-like or cis-like configuration, allowing us to individually probe the binding affinity of distinct conformations. The heterocyclic compounds adopt conformations that overlay closely with the targeted conformations of apicidin and demonstrate potent HDAC inhibitory activities, in some cases equivalent or superior to those of the natural product. This study highlights the utility of triazole-modified cyclic peptides in constructing useful bioactive probe molecules, supports the c-t-t-t conformation as the bioactive conformation of cyclic tetrapeptide HDAC inhibitors, and provides a useful three-dimensional pharmacophoric model for use in advancing design principles for more selective HDAC inhibitors.

HDACs play critical roles in the regulation of gene transcription[5] by cleaving the acetyl groups from specific ε-aminoacetylated lysine residues in nucleosomal histone tails and non-histone proteins.[6] Although the precise roles of HDAC isoforms in cellular function and tumorigenesis are not yet completely understood, inhibition of HDAC activity has emerged as a promising approach in anticancer chemotherapy.[68] An interesting family of non-ribosomal cyclic tetrapeptide natural products, including the apicidins, trapoxins, microcins, and chlamydocin, exert potent cytotoxic activities against cancer cells by inhibiting HDACs.[69] These natural cyclic tetrapeptides are distinguished by the presence of a decanoic acid side chain derivatized with a ketone, terminal α,β-epoxyketone, or hydroxamic acid, which mimics an acetylated lysine residue and interacts with the active site zinc ion.

Despite the considerable number of synthetic analogs that have been reported for the cyclic tetrapeptide HDAC inhibitors,[8,10] the bioactive conformation has remained rather ambiguous. Indeed, several molecular conformations have been reported for apicidin alone (Figure 1). Apicidin was originally reported to adopt a t-t-t-t conformation based on NMR experiments carried out using pyridine-d5 or CD2Cl2.[4] More recently, our laboratory[11] and a group at Merck[3] independently found that apicidin adopts multiple conformations (in a ratio of ~80%:15%:5%) in the more polar solvent DMSO, with the predominant species adopting a t-t-t-t conformation and the second most populated conformation having a cis-configuration at the tertiary amide bond of the Pip residue. Finally, a crystallographic structure obtained from CHCl3/methanol/pentane contained a cis-amide at the Pip residue.[3] The observation of both cis- and trans-tertiary amides in these structures of apicidin (as well as other members of the tetrapeptide natural product family[8,12]), combined with the lack of any co-crystal structure of a cyclic tetrapeptide inhibitor with an HDAC enzyme, led us to question which amide configuration is present in the dominant bioactive conformation. Although the c-t-t-t conformation had previously been tentatively identified as the bioactive structure in related cyclic tetrapeptides based on a correlation of activity with predicted or known solution conformations,[12] we aimed to carry out a more controlled study in which apicidin analogs having either a fixed cis- or trans-amide isostere could be directly compared in an HDAC inhibition assay.

Figure 1.

Figure 1

Cis-trans isomerization in the cyclic tetrapeptide natural product apicidin (1) gives rise to an equilibrium between the predominant t-t-t-t conformation (80%) and a minor c-t-t-t conformer (15%) in DMSO.[3,11] The molecular structures shown are the t-t-t-t NMR structure determined in pyridine-d5[4] and the c-t-t-t crystal structure.[3]

We and others have shown that the 1,4-disubstituted 1,2,3-triazole regioisomer effectively mimics a trans-amide bond,[13] whereas the 1,5-disubstituted 1,2,3-triazole regioisomer closely models a cis-amide bond.[14,15] Furthermore, the requisite 1,4- and 1,5-regioisomers can both be conveniently synthesized from azide and alkyne substrates,[16,17] making the triazole an attractive choice for an amide bond surrogate. We first synthesized heterocyclic apicidin analogs 2, 3, and 4 (Scheme 1). Bromoketal 5 was converted to the corresponding Grignard reagent and reacted with Boc-Ser β-lactone to yield the protected L-2-amino-8-oxodecanoic acid (L-Aoda) derivative 6.[18] Boc-Ile (7) was converted to propargyl amine derivative 8,[19] which was used along with amino acid 6 in solution phase peptide synthesis to afford linear azido-alkyne tetrapeptides 9 and 10. Copper-(I)-catalyzed intramolecular azide–alkyne cycloaddition[17] of 9 and 10 yielded heterocyclic peptides 2 and 3, respectively, each bearing the trans-amide surrogate. Peptide 4, bearing the 1,5-substituted 1,2,3-triazole, was prepared by a thermal Huisgen [3+2] dipolar cycloaddition reaction of 9 (DMF, microwave, 220 °C), which afforded a mixture of triazole regioisomers 2 and 4 in a 2:1 ratio from which 4 was isolated by preparative HPLC. The preparation of additional 1,5-regioisomers 1113, in which the Ile residue was substituted with Leu, allowed us to investigate the hypothesis that the multiple conformations in 4 (vide infra) might arise from the presence of a β-substituted amino acid (Ile) adjacent to the triazole. Since Ru-mediated cyclization of linear tetrapeptides proved unsuccesful, we developed an alternative strategy employing the Ru-catalyzed formation of 1,5-disubstituted 1,2,3-triazoles on solid phase followed by macrolactamization of the linear pseudotetrapeptides 1416 in solution to give 1113 (Scheme 2, Figure S1).[15,16] Compounds 1113 differ only in the chirality at the Leu and Ala positions of the macrocycle, making possible an examination of how chirality might affect conformational properties of the peptide ring. The Trp(OMe) residue present in apicidin was replaced with Trp in our compounds to simplify the syntheses.

Scheme 1.

Scheme 1

(a) N,O-dimethylhydroxylamine·HCl, EDC·HCl, iPr2EtN; (b) LiAlH4; (c) dimethyl (2-oxopropyl)phosphonate, p-TsN3, K2CO3 (48% overall, 3 steps); (d) TFA; (e) Boc-Trp-OH, HBTU, iPr2EtN (94% over 2 steps); (f) TFA; (g) 6, HBTU, iPr2EtN (74% over two steps); (h) TFA; (i) N3-D-Ala-OH or N3-D-Leu-OH, EDC·HCl, HOBT, iPr2EtN, 0 °C (73% for Ala, 31% for Leu, overall); (j) CuI, 2,6-lutidine, iPr2EtN, tris-(benzyltriazolylmethyl)amine[20] (50% yield by HPLC); (k) microwave, 220 °C (2:1 ratio of 4:2 by HPLC, 8% isolated yield of 4).

Scheme 2.

Scheme 2

a) NMP–piperidine 3:1; b) N3-L-Ala-OH or N3-D-Ala-OH (4 equiv), DIC (4 equiv), HOBt (4 equiv), NMP, 2 h; c) Fmoc-L-Leu-CCH or Fmoc-D-Leu-CCH (2 equiv), Cp*Ru(COD)Cl (20%), toluene, Ar, 16 h, 45 °C; d) NMP-piperidine 3:1; e) Fmoc-Trp-OH (4 equiv), HBTU (4 equiv), iPr2EtN (8 equiv), NMP, 2 h; f) NMP–piperidine 3:1; g) TFA-CH2Cl2 1:1; h) HATU (2 equiv), iPr2EtN (4 equiv), 1.5 h, 0.5 mM in DMF (all cyclization yields were >95% by HPLC [see Figure S1]; isolated yields based on resin loading were 9% for 11, 9% for 12, and 10% for 13).

Both 1,4-disubstituted regioisomers (2 and 3) showed a single set of sharp 1H NMR peaks in DMSO-d6, indicating their conformational homogeneity in solution.[21] Of the 1,5-disubstituted regioisomers, peptides 11 and 12 also adopted a single conformation on the NMR timescale, whereas analogs 4 and 13 showed multiple sets of 1H NMR peaks indicative of multiple, slowly interconverting backbone conformations (Figure S2). The observed conformational heterogeneity for 4 and 13 possibly results from cistrans isomerization of one or more amides in the macrocycles.

The bioactivities of the peptides were preliminarily assessed using an in vitro fluorescence assay measuring the inhibition of HDAC activity in HeLa cell nuclear extracts. Along with apicidin, 1,4-disubstituted triazole-modified peptide 2 and 1,5-disubstituted triazole-modified peptides 4, 11, and 12 showed IC50 values ≤100 nM. Compound 3 had an intermediate IC50 of ~200 nM, while peptide 13 exhibited an IC50 value >10 μM. As a control, linear azido-alkyne peptide 9, which contains all the important functional groups present in 3 and 4 but lacks the cyclic structure, had an IC50 more than two orders of magnitude higher than any of the cyclic peptides. The lower inhibitory activity of 3 compared to 2 may be explained by the methyl side chain in 2 acting as a better steric mimic of Pip than the branched isobutyl in 3. Although compound 4 exhibited an IC50 of <100 nM, the multiple conformations observed by 1H NMR for this compound negated its utility as a probe of apicidin’s bioactive conformation. For our more detailed characterizations of HDAC inhibitory activities, we therefore employed only peptides 2, 11, and 12 (all of which adopted a single conformation as indicated by 1H NMR) using a panel of purified recombinant human HDAC enzymes (Table 1). Against HDAC1, 1,5-disubstituted 1,2,3-triazole regioisomer 11 had an IC50 value similar to that of apicidin, whereas the corresponding 1,4-disubstituted 1,2,3-triazole regioisomer 2 showed an eight-fold loss in inhibitory activity. Against HDAC3, the three compounds all had similar activities, although 11 was again somewhat more potent than 2. Interestingly, compound 11 was significantly more active than apicidin against HDAC8, and somewhat more active against HDAC6. Compound 12, which differs in stereochemistry from apicidin at the Leu residue, exhibited reduced activity compared to the other apicidin analogs for all the HDACs tested.

Table 1.

Observed conformations in DMSO and potencies of triazole containing pseudopeptides and control compounds against recombinant human HDAC enzymes.[a]

compound conformation IC50/nM
HDAC1 HDAC3[b] HDAC6 HDAC8
2 t-t-t-t 25 16 >10,000 ND
11 c-t-t-t 7 9 6100 105
12 c-t-c-t 75 119 >10,000 ND
apicidin t-t-t-t (80%)
c-t-t-t (15%)
3 11 >10,000 750
TSA ND ND 6 32
[a]

IC50 values were obtained by testing in triplicate. ND = Values not determined for the compound against this enzyme. TSA = trichostatin A, which was used as a positive control for HDAC6 and HDAC8 because apicidin is a poor inhibitor of these enzymes.

[b]

In complex with NCoR2.

The observed functional data were somewhat surprising, as the greater activity of 1,5-disubstituted 1,2,3-triazole regioisomer 11 compared to 1,4-disubstituted 1,2,3-triazole 2 implicated the c-t-t-t even though the t-t-t-t conformation of apicidin predominates in solution in a variety of solvents. However, a critical requirement for the above analysis to be valid is that peptides 11 and 2 must closely mimic the targeted c-t-t-t and t-t-t-t conformations of apicidin, respectively. To confirm that the apicidin analogs adopted the intended conformations in solution, we carried out a series of structural analyses based on multidimensional NMR spectroscopy (TOCSY, COSY, and ROESY) and distance geometry calculations for compounds 2, 11, and 12 (which are all conformationally homogeneous in DMSO-d6). The three–dimensional NMR structure of 2 indeed revealed that all backbone amide bonds were in the trans configuration (Figures 2a, S3), consistent with the observed large HN–Hα coupling constants (Trp 9.1 Hz, Ile 8.4 Hz, Aoda 8.6 Hz). An overlay of the NMR structure of 2 with the published t-t-t-t NMR structure of apicidin determined in pyridine-d5[4] indicated close similarity of the two structures with high backbone overlap and Cα–Cβ vector alignments (0.55 Å backbone r.m.s.d. for Cα and Cβ atoms) (Figure S4). Likewise, overlaying the NMR structure of 2 with the lowest energy calculated conformation of apicidin, which reportedly closely matches the conformation of the predominant species in DMSO-d6,[3] indicated a significant backbone overlap and good alignment of the Cα–Cβ vectors (0.52 Å r.m.s.d. for Cα and Cβ atoms) (Figures 2a, S3). The reported NMR solution structures for apicidin determined in pyridine-d5 and DMSO-d6 differ only in the rotation of the Aoda–Trp amide bond relative to the plane of the backbone; the corresponding amide bond in compound 2 adopts the conformational rotamer present in the pyridine-d5 structure of apicidin.

Figure 2.

Figure 2

NMR structures for the triazole–modified apicidin analogs. a) Peptide 2 adopted a t-t-t-t conformation (top) that overlays well (bottom, 0.52 Å r.m.s.d. for Cα and Cβ atoms) on the lowest energy calculated conformation of apicidin (yellow) (which reportedly closely matches the predominant conformation of apicidin in DMSO).[3] b) Peptide 11 adopted a c-t-t-t conformation; two families of structures were observed that differ in the rotation of the Trp/Aoda amide relative to the backbone (top). The structures overlay very well on the crystal structure of apicidin[3] (bottom, 0.30 Å and 0.51 Å r.m.s.d. for Cα and Cβ atoms for representative members of the two structural families). c) Peptide 12 adopted a c-t-c-t conformation (top), which overlays very well on the crystal structure of the natural product dihydrotentoxin[24] (yellow) (bottom, 0.24 Å r.m.s.d. for Cα and Cβ atoms). d) Overlay of Cα atoms for compounds 2 (magenta), 11 (two structural families, green and cyan), and 12 (yellow). Due to the c-t-c-t conformation of 12, the Aoda, Trp, and Leu side chains of this peptide project in the same plane as the backbone ring as opposed to projecting upward and out of the plane as for the other peptides. e) Overlay of Cα and Cβ atoms for compounds 2 (magenta) and 11 (two structural families, green and cyan). The Cα atoms of the Ala and Ile/Leu residues are farther apart in 2 and the Cα–Cβ vector of 2 directs the Ile side chain outward away from the ring as opposed to directly above the ring in 11.

Peptides 11 and 12, which differ only in stereochemistry at the Leu residue, adopt considerably different backbone conformations in DMSO-d6. The three amide bonds of peptide 11 (which contains amino acid residues of the same chirality as apicidin) were all in the trans configuration, leading to the intended c-t-t-t conformation when considering the 1,5-triazole as a cis-amide bond isostere (Figure 2b, S3). Two families of structures were observed for 11 that differed only in the rotation of the Aoda–Trp amide bond relative to the backbone plane; however, this amide rotation has little effect on the position of other backbone atoms or on Cα–Cβ vectorial alignments (0.28 Å r.m.s.d. for Cα and Cβ atoms of representative members of the two families). The NMR structures for 11 overlayed very well onthe crystallographic c-t-t-t structure of apicidin[3] (0.30 Å and 0.51 Å r.m.s.d. for Cα and Cβ atoms for representative members of the two families) (Figures 2b, S3), supporting that 11 indeed faithfully represents the c-t-t-t conformation of apicidin that is present as a minor component in DMSO-d6 solution. On the other hand, peptide 12 contained a cis configuration at the Aoda–Trp amide, leading to a cistranscistrans (c-t-c-t) tetrapeptide conformation (Figures 2c, S3). The c-t-c-t structure has been the focus of several calculations and discussions in the literature,[12,22,23] likely because it is among the most common conformations observed in the structures of cyclic tetrapeptides and is reportedly the conformation having the least ring strain. Although our data indicate that the c-t-c-t conformation is poorly suited to exert potent HDAC inhibitory activity, peptide 12 could be useful as a lead compound in constructing conformationally restricted analogs of other cyclic tetrapeptide natural products that are known to adopt the c-t-c-t conformation, such as tentoxin[23] and dihydrotentoxin[24] or the symmetric natural products cyclo(L-Pro-L-Leu)2, cyclo(L-Pro-L-Val)2, or cyclo(L-Pro-L-Phe)2.[25] Indeed, peptide 12 overlayed closely on the crystal structure of dihydrotentoxin[24] (0.24 Å r.m.s.d. for Cα and Cβ atoms) (Figures 2c, S3).

Although there are subtle variations in the chemical structures of the natural product 1 and analogues 2, 11, and 12, it is our hypothesis that the major determinant of the observed HDAC inhibition potencies is the respective cis/trans configurations and resulting backbone conformations in each cyclic peptide.[26] For instance, peptide 12 exhibited reduced HDAC inhibitory activity compared to 2 and 11 across our panel of HDAC enzymes. An overlay of Cα atoms for the three compounds clearly shows that compound 12 differs structurally from the other two peptides in having a significantly shorter distance between the Trp and Aoda Cα atoms (2.8 Å for 12 compared to 3.8 Å for both 2 and 11) and, perhaps more importantly, the chair-like c-t-c-t conformation of 12 causes the Cα–Cβ vectors for the Trp, Aoda, and Leu side chains to all project in the same plane as the backbone ring, whereas these side chains in 2 and 11 project above the plane of the ring (Figure 2d). Peptides 2 and 11 both exhibited similar IC50 values against HDAC3, whereas 11 was four-fold more potent than 2 against HDAC1. An overlay of Cα and Cβ atoms for the two compounds indicated that the most obvious structural differences for the two compounds are the greater distance between Cα atoms of the Ile/Leu and Ala residues (5.1 Å for 2 compared to 3.2 Å for both structure families of 11) and the different directions at which the Leu or Ile residue is projected relative to the backbone ring (directly above the ring in 11 compared to outward from the ring in 2) (Figure 2e). Together with the biological data, this finding suggests that the position and orientation of the Leu/Ile residue influences potency for HDAC1, but does not have a pronounced effect for HDAC3. It is also possible that the difference in the aliphatic residue for 2 and 11 (Ile in 2 and Leu in 11) contributes to the observed differences in activity for these compounds.

In summary, the structural and functional data presented here support that a cis-configuration at the Pip residue in the cyclic tetrapeptide HDAC inhibitor apicidin affords improved HDAC-inhibitory activities compared to the t-t-t-t conformation that predominates in solution. We have presented the rational design of conformationally constrained, triazole modified cyclic peptide scaffolds with potent biological activity and have established the ability to probe the biologically relevant conformation of a natural peptide ligand by introducing different triazole regioisomers in place of amide bonds in its backbone. It is our hope that this study will help guide future efforts aimed at improving on the cyclic tetrapeptide HDAC inhibitors and lead to more selective HDAC ligands.

Supplementary Material

Supplementary data

Footnotes

**

We thank Drs. Dee Huang and Laura Pasternack for assistance with NMR, Dr. Sheo Singh for providing coordinates of the published NMR structure of apicidin in pyridine-d5, Dr. Michael Kranz for providing coordinates of the calculated lowest energy conformations of apicidin, Dr. Michael Pique for assistance in the preparation of graphics, and Dr. L. J. Leman for assistance in manuscript preparation. We acknowledge the National Science Foundation (W.S.H.) and NASA Earth and Space Science Fellowship Program (Grant NNX07AR35H) (J.M.B.) for predoctoral fellowships. A.M. thanks the Spanish Ministry for Science and Education for a Fulbright/MEC postdoctoral fellowship. C.A.O. thanks the Lundbeck Foundation and the Danish Research Council for Technology and Production Sciences (274-06-0317) for postdoctoral fellowships, and the Danish Independent Research Council for a Young Researcher’s Award. This work was supported in part by a grant from National Institute of General Medical Sciences (GM52190) and the Skaggs Institute for Chemical Biology.

Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author. Coordinates for the NMR structures reported in this manuscript have been deposited at the BMRB databank (www.bmrb.wisc.edu, accession numbers 20071–20073).

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