In a previous study (section 2.2), four aurones were found to be active towards HDAC enzymes, nevertheless none of them displayed any significant HDAC6-selective profile 55. In an attempt to enhance the HDAC6 selectivity profile of the aurone series and to broaden the chemical space of potential HDAC6 inhibitors, new series of compounds were processed through two screening campaigns conducted in parallel (Fig. 12C and Fig. 14D, experimental details given in section 2.8).
An in silico – in vitro strategy was applied for the aurone series (ninety-one compounds, Fig. 12A) and for a small series of 1,3,4-oxadiazoles (nine compounds, Fig. 12B). Those compounds were screened into HDAC2 (representative of class I HDACs, PDB code: 4LXZ 52) and HDAC6 (homology model 53) catalytic sites through Gold 5.2 software (CCDC Cambridge) using a previously validated docking protocol 55. The overall aim was to explore the influence of both aurone’s ring A and B modulations on HDAC6 activity. The aurones were divided in eight sub-classes according to the diverse substitutions on the A, B rings. After docking calculations, twenty-eight compounds were selected according to their docking score and through a visual inspection analysis (zinc chelation being the major criteria) for further enzymatic investigations (section 2.10 Table S4-11). A three-step biological screening was then applied. The twenty-eight molecules were first screened on HDAC6. Seven compounds with inhibition of 40 percent and above, when tested at 100 µM, were retained for IC50 determination (Table 4, IC50
plots given in section 2.10, Fig. S10). HDAC screening using a pool of class I HDAC enzymes contained in HeLa nuclear extract was then performed.
With few exceptions, indolyl-aurones were predicted unable to interact with the HDAC2 catalytic zinc ion, whereas zinc chelation was always obtained within HDAC6. Indeed, a substituted indole group is bulkier than the phenyl ring B of classical aurones, and the catalytic pocket in HDAC2 is narrower than in HDAC6 (VHDAC6 = 406.5 Å3 versus VHDAC2 = 190.8 Å3, calculated via CASTp server
56). As an example, 37 was predicted to chelate zinc ion in HDAC6, while making polar interactions with both Glu779 and Tyr782 residues. Although the N-butyl group faced a hydrophobic wall made by Pro501 and Pro748, the indole ring was not favorably oriented as it faced a hydrophilic area characterized by Asp567 and Ser568. On the contrary, 37 interacted with HDAC2 channel rim but not with the zinc binding domain: its ring A was settled into a groove lined by Pro34 and hydrogen-bonded with both Glu103 and Gly32. Its N-butyl group was found in the channel instead (section 2.10 Fig. S9).
A total of ten indolyl-aurones out of thirty-five were selected for further investigations (section 2.10 Table S4). No zinc chelation within HDAC2 but with HDAC6 was also observed when the aurone ring A was 4,6-dihyxdroxysubstituted, and the ring B possessed hydroxyl and/or alkyloxy-substituents on its cycle. Compounds 77, 78, 82, 84, 89 and 94 were chosen from these sub-groups for further biological investigation. Heterogeneous results were obtained in terms of zinc chelation with halogeno- and alkyl-substitutions on the aurone ring B. HDAC6 activity was, nevertheless, checked for seven compounds (96, 98, 99, 104, 109, 110 and 113) from these two sub-classes (section 2.10 Table S6-S9). Finally, one sulfonyl-aurone (72) which obtained good docking results in terms of docking score and zinc-chelation properties towards HDAC6 was also selected (section 2.10 Table S5).
6,7-dihydroxylated aurones, bearing a catechol motif, were always predicted to interact with the HDAC6 catalytic zinc ion. Two compounds from this sub-set were selected for biological tests, as, for both of them, docking results converged towards a unique pose in the catalytic site of HDAC6 (121 and 124, section 2.10 Table S10). Compound 121 was encountered to chelate HDAC6 catalytic zinc in a bidentate manner, while interacting with both Asp649 and Asp742 residues. Similarly to the indolyl-aurone 37 in HDAC2, neither 121 nor 124 chelate the zinc ion, having their ring B first introduced into
52
HDAC2’s channel and being sandwiched between Phe210 and Phe155 (Fig. 13). Finally, one dimeric aurone (127) and one 1,3,4-oxadiazole compound (129) were also checked for HDAC6 inhibitory activity, despite the presence of zinc chelation in both HDAC2 and HDAC6 structures (section 2.10 Table S11 and S12).
Among the twenty-eight biologically assessed compounds, seven were worth for selectivity profile determination, having reached the HDAC6 inhibition threshold of 40% ± SEM (Table 4). Unfortunately, their IC50 values were higher than IC50 values of the best compounds reported in chapter 2.2 (aurones 34 and 35). Compounds 121 and 124, having the lowest HDAC6 IC50 values, did not show any selectivity between HDAC6 and class I HDAC enzymes.
O
O 2 4 3
5 6
7 2'
3' 4' 5'
A
6'B
A
C O
N N
A B
2 3 4 5
6 2'
3' 4' 5' 6' B
Figure 12. Scheme summarizing the screening campaign of one hundred aurone and oxadiazole compounds (ninety-one aurones and nine 1,3,4-oxadiazoles) on HDAC2 and HDAC6.
(A) Aurones general structure: compounds 37-128. (B) 1,3,4-oxadiazoles general structure:
compounds 129-137. (C) Protocol adopted for the in silico – in vitro strategy.
From this study and in line with the results from the chapter 2.2, some hypotheses of structure-activity relationship have emerged. Interestingly, a catechol group was always present as a substituent among the most active aurone compounds. The HDAC6 activity of 4,6-dihydroxylated aurones was abolished if their ring B possessed any halogen or sulfonyl moieties, or if the phenyl ring was replaced by an indole. Moreover, meta substitution of the ring B impaired activity, and so did a para-O-alkyl substitution if the number of carbon exceeded two atoms.
53
A
B
Figure 13. Compound 121 in complex with (A) HDAC2 and (B) HDAC6. Protein structures are shown as grey ribbons. Key residues for molecular interactions are labelled and shown as white sticks. Residues characterizing the zinc chelation triad, are shown as white sticks whereas the zinc ion is represented as a blue sphere. 121 is shown as light orange sticks. Molecular surface is drawn 5 Å around the ligand and is shown as solid surface colored from green (hydrophobic areas) to purple (hydrophilic areas). Figure generated with MOE 2012.
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Table 4. Activity profile of the seven best compounds from the aurone series.
ID Structure enzymes are provided by HeLa nuclear extract. baverage IC50 ± SEM of at least three independent experiments in duplicates.
NA: no activity. IC50 calculated using Prism® 6.0.
An in vitro – in silico strategy was applied for the chalcone series (Fig. 14D). As chalcones are isomers of aurones and are already known as HDAC8 and SIRT1 inhibitors 17,18, it was interesting to directly assess their activity towards HDAC6. Pyrimidinetriones and thiosemicarbazides were also
55
added to this screening campaign. A total of nineteen chalcones, four pyrimidinetriones, and sixteen thiosemicarbazides were screened for HDAC6 inhibition in vitro (Fig. 14A, 14B, 14C). In comparison to aurones and associated series, the test concentration of both chalcones and pyrimidinetriones was divided by two because of solubility issues. Nevertheless, the same 40% inhibition threshold was applied to maximize the chance to identify interesting candidates. Selectivity profile of nine promising compounds (141, 144, 145, 146, 147, 148, 152, 153 and 158) was then defined through HDAC6 IC50
determination and class I HDACs screening. Molecular docking calculations were then run for determining the possible interactions of the compounds with the catalytic site of different HDAC isoforms (section 2.10 Table S13-15).
O
2' 3'
4' 5'
6'
2 3 4 5 6
A
B
A
D B
HN HN
OHO
O
O O
R C
Figure 14. A Scheme summarizing the screening campaign of the three series of compounds on HDAC2 and HDAC6. (A) Chalcones general structure: compounds 138-158. (B) Thiosemicarbazides general structure: compounds: 159-174. (C) Pyrimidinetriones general structure:
compounds 175-179. (D) Protocol adopted for the in vitro – in silico strategy.
The chalcone series displayed an HDAC6 activity with a minimum of 10% of inhibition (section 2.10 Table S13). Nine chalcones reached the threshold for IC50 determination. Unfortunately, because of solubility issues, it was not possible to calculate any IC50, being the plateau never reached (data not shown). Surprisingly, no pyrimidinetriones or thiosemicarbazides were found active, although they were predicted to favorably interact within HDAC6 catalytic site (section 2.10 Table S14 and S15).
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