All reagents, including chemicals and enzymes, were obtained from Sigma-Aldrich (St. Louis, MO, USA) or other reputable suppliers, with detailed information provided where applicable. Compounds 4 (N-Hydroxy-3-[6-(hydroxyamino)-6-oxohexyloxy]benzamide) and 6 (N-hydroxy-4-(7-(hydroxyamino)-7-oxoheptyl)-3-methoxybenzamide) were initially dissolved in dimethyl sulfoxide (DMSO) and subsequently diluted to the required concentrations for enzyme activity assays.

Ligand Preparation: The crystal structure of HDAC1 (PDB ID: 4BKX) served as the receptor template. Avogadro software (version 1.95, Pittsburgh, PA, USA), utilizing the General Amber Force Fields (GAFF), was employed to build and energy-minimize the structures for Compounds 4 and 6 [29]. Docking Setup: Molecular docking of each ligand was conducted with AutoDock Vina (version 1.2.7, La Jolla, CA, USA), allowing full ligand flexibility while keeping the receptor rigid [30]. The docking grid was established around the HDAC1 active site, with center coordinates set (–46.756, 16.290, –7.785). Simplified docking parameters were chosen for clarity. Interaction Analysis: The lowest-energy docking poses for each ligand underwent subsequent molecular dynamics simulations. Protein-ligand interactions derived from both the docking results and the final molecular dynamics structures were analyzed using the Protein-Ligand Interaction Profiler (PLIP) online platform [31].

All molecular dynamics (MD) simulations were executed using GROMACS software (version 2021.4, Stockholm, Sweden). The simulations employed the AMBER99SB-ILDN force field, which was further adjusted with recently reported parameters specific for zinc(II)-binding residues [32, 33]. These adjustments were essential for accurately modeling the zinc ion coordination in the active site, enhancing metalloprotein stability during MD runs. The SPC/E water model was utilized for solvation, and ligand topologies compatible with GROMACS were generated using ACPYPE software (version v2022.7.27, Cambridge, UK) [34]. Each simulation system was initially solvated, followed by charge neutralization through the addition of three chloride ions. Subsequently, energy minimization was performed using the steepest descent algorithm for 500,000 steps. Equilibration occurred in two sequential stages: first, a 100-ps NVT ensemble run stabilized the system temperature at approximately 300 K using the leap-frog integrator. This was followed by another 100-ps NPT equilibration, also employing the leap-frog integrator, stabilizing the system’s pressure at roughly 1 bar. During both equilibration phases, positional restraints were applied to the receptor and ligand atoms. Temperature control was maintained by the Berendsen velocity-rescale thermostat, and pressure control utilized the Parrinello-Rahman barostat. The final MD production run lasted 100 ns without positional restraints. Two independent replicates for each HDAC1-inhibitor complex were carried out using distinct initial velocity conditions. These replicates facilitated the identification of consistent dynamical behaviors across systems, enabling selection of the most representative simulation for subsequent inhibitor comparisons and analyses. The root mean square deviation (RMSD) values for ligand trajectories relative to the protein backbone were determined using the RMSD calculation module of GROMACS. Additionally, RMSD values of the protein backbone relative to the complex was performed to provide insight into the protein stability throughout the simulations.

The purified HDAC1 enzyme was obtained from Abcam (Cambridge, UK) and used for substrate activity assays. The enzyme was added at a final concentration of 10 µg/mL to reaction solutions containing 10 to 500 µM Boc-Lys(Ac)-pNA (N-Boc-Lysine acetyl-p-nitroaniline, Abcam, Cambridge, MA, USA) in 50 mM TBS buffer (Sigma, St. Louis, MI, USA, pH 7.0). Reactions were conducted in a 96-well plate with a final volume of 100 µL and incubated at 37 °C for 1 hour. Following incubation, reactions were terminated by adding 10 µL of color developer (trypsin, Sigma), followed by thorough mixing. The plate was further incubated at 37 °C for 30 minutes, after which absorbance readings were obtained at 405 nm using a 96-well plate reader (Bio-Tek ELx808, Winooski, VT, USA). Enzymatic activity was monitored by measuring the increase in absorbance at 405 nm, corresponding to the release of p-nitroaniline ($\epsilon$ = 8800 M^-1$\cdot$cm^-1). All experiments were performed in triplicate. For IC₅₀ determination, enzymatic assays were conducted under the same conditions, with the reaction mixture pre-incubated for 1 hour with varying concentrations of Compounds 4 and 6 prior to initiation with a fixed substrate concentration. A separate set of assays was performed at fixed inhibitor concentrations (0–100 µM for Compounds 4 and 6), where the substrate concentration was varied from 0.1 to 0.4 mM. All experiments were conducted in triplicate to ensure reproducibility.

The synthesis of compounds 4 and 6 is essential for developing effective HDAC1 inhibitors, as these compounds contain hydroxamic acid functional groups known to chelate the zinc ion in the active site of HDAC enzymes. The design and synthesis of these molecules aim to optimize their structural features for enhanced binding affinity, stability, and inhibitory potency, as supported by molecular docking and dynamics simulations. Fig. 1 depicts the synthetic pathway for N-Hydroxy-3-[6-(hydroxyamino)-6-oxohexyloxy]benzamide (4). The synthesis begins with 3-hydroxybenzoic acid (1), which undergoes etherification with 6-bromohexanoyl hydroxylamine (2) in the presence of a suitable base, yielding the intermediate 3-(6-bromohexyloxy)benzoic acid (3). Subsequent nucleophilic substitution with hydroxylamine hydrochloride results in the formation of the target compound 4. This reaction sequence involves key transformations, including esterification, halogen displacement, and hydroxylamine incorporation, ensuring the selective introduction of hydroxamic acid functionality. Fig. 2 illustrates the synthetic strategy for N-Hydroxy-4-methoxy-3-[6-(hydroxyamino)-6-oxohexyloxy]benzamide (6). The synthesis commences with 4-methoxy-3-hydroxybenzoic acid (5), which undergoes etherification with 6-bromohexanoyl hydroxylamine under basic conditions to generate 4-methoxy-3-(6-bromohexyloxy)benzoic acid. Subsequent nucleophilic substitution with hydroxylamine hydrochloride affords the final hydroxamic acid derivative 6. By synthesizing and characterizing these compounds, their inhibitory potential against HDAC1 can be experimentally validated, supporting their potential as lead compounds.

Fig. 1.

The scheme for synthesis of compound 4: N-Hydroxy-3-[6-(hydroxyamino)-6-oxohexyloxy]benzamide. Synthesis of compound 4 via etherification of compound 1 and 2, followed by hydroxylamine substitution to yield N-Hydroxy-3-[6-(hydroxyamino)-6-oxohexyloxy]benzamide. MW, microwave.

Fig. 2.

The scheme for synthesis of compound 6: N-Hydroxy-4-methoxy-3-[6-(hydroxyamino)-6-oxohexyloxy]benzamide. Synthesis of compound 6 from 4-methoxy-3-hydroxybenzoic acid via etherification and hydroxylamine substitution.

Understanding the molecular interactions of compound 4 with HDAC1 is critical for elucidating its inhibition mechanism and guiding structure-based drug design. Molecular docking analysis revealed that compound 4 engages in hydrophobic interactions, hydrogen bonding, and $\pi$-stacking within the HDAC1 active site. Two hydrophobic interactions involving Phe150, at distances of 3.62 Å and 3.83 Å, contribute to ligand stabilization. Additionally, five hydrogen bonds were identified: His141 (3.58 Å), His178 (3.81 Å and 2.83 Å), Gly149 (2.81 Å), and Phe205 (3.19 Å), reinforcing binding stability. $\pi$-Stacking interactions further stabilize the complex, with His178 forming a T-shaped $\pi$-stacking interaction at 5.29 Å and Phe205 engaging in a parallel $\pi$-stacking interaction at 3.98 Å. Coordination with the Zn²⁺ ion, at a binding distance of 3.7 and 4.4 Å, plays a crucial role in ligand stabilization. These findings indicate that compound 4’s binding is mediated by a combination of non-covalent interactions and zinc coordination, which enhance its inhibitory potential. The binding conformation of compound 4 within the HDAC1 active site is depicted in Fig. 3A. Similar to compound 4, compound 6 interacts with HDAC1 through hydrophobic contacts, hydrogen bonding, and metal coordination. Molecular docking analysis identified hydrophobic interactions involving Phe205, Pro29, Phe150, and Leu271, with distances ranging from 3.55 Å to 3.73 Å, anchoring the ligand within the active site. Six hydrogen bonds were observed, with His28, His141, Asp99, and Gly149 contributing to ligand stabilization. Asp99 formed two hydrogen bonds with distances of 2.35 Å and 2.94 Å, indicating electrostatic stabilization. The Zn-ligand coordination, at a distance of 3.5 and 5.3 Å, suggests a slightly weaker interaction compared to compound 4. The binding mechanism of compound 6 is primarily governed by hydrophobic interactions and hydrogen bonding, with zinc coordination playing a stabilizing role. The binding conformation of compound 6 within the HDAC1 active site is shown in Fig. 3B. AutoDock Vina binding affinities and molecular dynamics simulations provide quantitative insights into ligand stability. Compound 4 exhibited a binding affinity of –6.2 kcal/mol, while compound 6 showed a slightly weaker binding affinity of –5.7 kcal/mol. The Zn-ligand binding distances, 3.7 and 4.4 Å for compound 4 and 3.5 and 5.3 Å for compound 6, suggest that compound 4 forms a more stable interaction with HDAC1. These values are summarized in Table 1. The binding of both inhibitors to HDAC1 is driven by hydrophobic interactions, hydrogen bonding, and $\pi$-stacking, with Zn coordination playing a key role in stabilizing the complexes. Compound 4 exhibits a stronger binding affinity and a shorter Zn-ligand distance, indicating a more stable coordination within the active site. Although experimental isoform profiling was not performed, we conducted comparative molecular docking simulations with HDAC2 (PDB ID: 3MAX), a closely related Class I isoform. The results show that compound 4 exhibits a reduced binding affinity for HDAC2 (–5.4 kcal/mol) compared to HDAC1 (–6.2 kcal/mol), suggesting a degree of target selectivity. Structural analysis further revealed that compound 4 adopts a more favorable binding conformation within the HDAC1 active site, potentially due to unique interactions with residues such as Phe205 and His178. These findings provide insights into the molecular basis of HDAC1 inhibition and inform future optimization strategies for developing potent inhibitors.

Fig. 3.

Molecular docking conformations obtained by AutoDock Vina. (A) Binding mode of compound 4 with histone deacetylase 1 (HDAC1). (B) Binding mode of compound 6 with HDAC1. Numbers indicate distances (in Å) for critical interactions between the inhibitors and HDAC1 active site residues.

Molecular docking provides an initial assessment of the binding affinity and interactions between ligands and target proteins. However, docking alone does not account for protein flexibility, solvent effects, and the dynamic nature of ligand binding. To refine these insights, MD simulations were conducted to examine the stability, conformational changes, and binding interactions of compounds 4 and 6 with HDAC1 over time. These simulations help validate docking results and provide a more realistic representation of molecular interactions within a biological system. Fig. 4A shows the complex between compound 4 and HDAC1, where the ligand is stabilized through multiple interactions, including hydrogen bonding and coordination with a metal ion, likely a zinc ion, given HDAC1’s known active site structure. Critical residues, including His178, Tyr303, Tyr204, and Phe205, contribute to the stabilization of the ligand. The hydrogen bonding network, particularly with Gly143, and $\pi$-$\pi$ stacking with aromatic residues suggest a strong binding affinity. The interaction of zinc in the active site of HDAC1 and compound 4 is a divalent covalent coordinate, with distances of 4.3 Å and 4.8 Å, indicating strong coordination that enhances ligand stability. Hydrophobic interactions were observed with key residues such as Phe150, Phe205, and Leu271, with distances ranging from 3.29 to 3.93 Å. MD analysis also revealed significant hydrogen bonds with His141, Gly149, His178, Tyr204, Phe205, and Tyr303, with distances between 2.19 and 3.44 Å. As shown in Table 1, the docking results showed an initial binding affinity of –6.2 kcal/mol, which improved to –7.7 kcal/mol after MD simulations, indicating enhanced stabilization in the binding pocket. In contrast, Fig. 4B illustrates the complex between compound 6 and HDAC1, where fewer stabilizing interactions are observed. The key residues, Tyr287 and Gly143, contribute to hydrogen bonding, but the ligand-metal ion distance is slightly increased to 4.5 Å, which may reduce binding affinity. The interaction of zinc in the active site of HDAC1 and compound 6 is a monovalent covalent coordinate, with a distance of 4.4 Å, suggesting a weaker coordination compared to compound 4. Hydrophobic interactions were observed with Phe144 and Phe199, with distances of 3.82 and 3.73 Å, respectively. The hydrogen bonding network included Gly143 and Tyr297, with distances ranging from 1.76 to 3.04 Å, with Tyr297 forming two hydrogen bonds, reinforcing some level of stability in the binding pocket. As shown in Table 1, the docking results indicated an initial binding affinity of –5.7 kcal/mol, which improved to –7.3 kcal/mol in MD simulations. However, the higher distance from the active site and fewer stabilizing interactions suggest slightly less stable binding compared to compound 4. The MD simulations confirm that compound 4 exhibits a more stable and favorable interaction with HDAC1, characterized by a robust hydrophobic interaction network, a well-defined hydrogen bonding network, and stronger zinc coordination (divalent covalent coordination at 4.3 Å and 4.8 Å). This is further supported by the improved binding free energy from docking (–6.2 kcal/mol) to MD (–7.7 kcal/mol), as reported in Table 1. In contrast, compound 6 shows weaker binding, with an increased metal-ligand distance, fewer stabilizing contacts, and weaker zinc coordination (monovalent covalent coordination at 4.4 Å), leading to a lower overall improvement in free energy (–5.7 kcal/mol to –7.3 kcal/mol). These results suggest that compound 4 is a stronger HDAC1 inhibitor, making it a better candidate for further optimization in HDAC-targeted drug development.

Fig. 4.

Representative snapshots from molecular dynamics simulations illustrating inhibitor interactions within the HDAC1 active site. (A) HDAC1-compound 4 complex. (B) HDAC1-compound 6 complex. Structures represent equilibrated conformations after 100 ns simulations.

Further confirmation of these stability differences is shown in Fig. 5, which presents the RMSD analysis of the protein-ligand complexes and the ligands themselves. Fig. 5A shows the protein backbone RMSD for the HDAC1:compound 4 complex in black and the HDAC1:compound 6 complex in green. The lower fluctuations in the green curve suggest that compound 6 induces less structural stability in the complex. Fig. 5B shows the RMSD of compound 4 in black and compound 6 in red, plotted as running averages for visual clarity. The greater fluctuations in the red curve indicate that compound 6 experiences more conformational instability, further supporting the observation that compound 4 forms a more stable and favorable interaction with HDAC1.

Fig. 5.

Root mean square deviation (RMSD) analyses from molecular dynamics trajectories. (A) Protein backbone RMSD for HDAC1 bound to compound 4 (black) and compound 6 (green). (B) Ligand RMSDs for compound 4 (black) and compound 6 (red) within the HDAC1 active site. Data are presented as running averages for improved visual clarity. The system reached equilibration after 10 ns.

To evaluate the inhibitory effects of compounds 4 and 6 on HDAC1, concentration-response experiments were conducted. These inhibition tests are essential to determine the potency of each compound in suppressing HDAC1 activity, which is crucial for validating computational and structural predictions. The IC₅₀ values obtained provide quantitative insight into the effectiveness of each compound as an HDAC1 inhibitor. Fig. 6A illustrates the inhibition profile of compound 4, showing a sigmoidal curve with a steep decline in remaining HDAC1 activity as the compound concentration increases. The calculated IC₅₀ value for compound 4 is 2.96 $\pm$ 0.4 µM, indicating strong inhibitory potency. In contrast, Fig. 6B represents the inhibition curve for compound 6, which exhibits a similar sigmoidal shape but with a more gradual decline. The IC₅₀ value for compound 6 is 4.76 $\pm$ 0.5 µM, suggesting weaker inhibitory efficiency compared to compound 4. As summarized in Table 2, the lower IC₅₀ value of compound 4 indicates that it is a more potent HDAC1 inhibitor than compound 6. This result aligns with the MD simulations and binding affinity data, which showed that compound 4 forms stronger interactions with HDAC1, including a more stable hydrogen bonding network, stronger hydrophobic interactions, and a more favorable zinc coordination. The higher IC₅₀ value for compound 6 suggests that it requires a higher concentration to achieve the same inhibitory effect as compound 4, reinforcing the conclusion that compound 4 is the superior HDAC1 inhibitor. These findings confirm that compound 4 is a more effective HDAC1 inhibitor than compound 6, making it a better candidate for further drug development targeting HDAC1-mediated pathways. A two-tailed unpaired Student’s t-test was performed to compare the IC₅₀ values of compounds 4 and 6. The analysis yielded a statistically significant difference (p 0.01), confirming that compound 4 exhibits significantly stronger inhibitory potency than compound 6.

Fig. 6.

Concentration-response curves for HDAC1 inhibition by dihydroxamate derivatives. (A) Compound 4. (B) Compound 6. Data points represent mean $\pm$ standard deviation (SD) from three independent experiments. Statistical significance between IC₅₀ values was determined using a two-tailed unpaired Student’s t-test (p 0.01).