Evaluation of 1,10-phenanthroline-based hydroxamate derivative as dual histone deacetylases/ribonucleotide reductase inhibitor with antitumor activities.

BACKGROUND: Aberrant expression of histone deacetylases (HDACs) and ribonucleotide reductase (RR) enzymes are commonly observed in various cancers. Researchers are focusing on these enzymes in cancer studies with the aim of developing effective chemotherapeutic drugs for cancer treatment. Targeting both HDAC and RR simultaneously with a dual HDAC/RR inhibitor has exhibited enhanced effectiveness compared to monotherapy in cancer treatment, making it a promising strategy. OBJECTIVES: The objective of the study is to synthesize and assess the anti-cancer properties of a 1,10-phenanthroline-based hydroxamate derivative, characterizing it as a novel dual HDAC/RR inhibitor. METHODS: The N1-hydroxy-N8-(1,10-phenanthrolin-5-yl)octanediamide (PA), a 1,10-phenanthroline-based hydroxamate derivative, was synthesized and structurally characterized. The compound was subjected to in vitro assessments of its anti-cancer, HDAC, and RR inhibitory activities. In silico docking and molecular dynamics simulations were further studied to explore its interactions with HDACs and RRM2. RESULTS: The structurally confirmed PA exhibited antiproliferative activity in SiHa cells with an IC50 of 16.43 µM. It displayed potent inhibitory activity against HDAC and RR with IC50 values of 10.80 µM and 9.34 µM, respectively. Co-inhibition of HDAC and RR resulted in apoptosis-induced cell death in SiHa cells, mediated by the accumulation of reactive oxygen species (ROS). In silico docking studies demonstrated that PA can effectively bind to the active sites of HDAC isoforms and RRM2. Furthermore, PA demonstrated a more favorable interaction with HDAC7, displaying a docking score of -9.633 kcal/mol, as compared to the standard HDAC inhibitor suberoylanilide hydroxamic acid (SAHA), which exhibited a docking score of -8.244 kcal/mol against HDAC7. CONCLUSION: The present study emphasizes the prospect of designing a potential 1,10-phenanthroline hydroxamic acid derivative as a novel dual HDAC and RR-inhibiting anti-cancer molecule.

Density functional theory (DFT) studies in HDAC-based chemotherapeutics: Current findings, case studies and future perspectives.

Density Functional Theory (DFT) is a quantum chemical computational method used to predict and analyze the electronic properties of atoms, molecules, and solids based on the density of electrons rather than wavefunctions. It provides insights into the structure, bonding, and behavior of different molecules, including those involved in the development of chemotherapeutic agents, such as histone deacetylase inhibitors (HDACis). HDACs are a wide group of metalloenzymes that facilitate the removal of acetyl groups from acetyl-lysine residues situated in the N-terminal tail of histones. Abnormal HDAC recruitment has been linked to several human diseases, especially cancer. Therefore, it has been recognized as a prospective target for accelerating the development of anticancer therapies. Researchers have studied HDACs and its inhibitors extensively using a combination of experimental methods and diverse in-silico approaches such as machine learning and quantitative structure-activity relationship (QSAR) methods, molecular docking, molecular dynamics, pharmacophore mapping, and more. In this context, DFT studies can make significant contribution by shedding light on the molecular properties, interactions, reaction pathways, transition states, reactivity and mechanisms involved in the development of HDACis. This review attempted to elucidate the scope in which DFT methodologies may be used to enhance our comprehension of the molecular aspects of HDAC inhibitors, aiding in the rational design and optimization of these compounds for therapeutic applications in cancer and other ailments. The insights gained can guide experimental efforts toward developing more potent and selective HDAC inhibitors.

Folding of Class IIa HDAC Derived Peptides into α-helices Upon Binding to Myocyte Enhancer Factor-2 in Complex with DNA.

Interaction of transcription factor myocyte enhancer factor-2 (MEF2) family members with class IIa histone deacetylases (HDACs) has been implicated in a wide variety of diseases. Though considerable knowledge on this topic has been accumulated over the years, a high resolution and detailed analysis of the binding mode of multiple class IIa HDAC derived peptides with MEF2D is still lacking. To fulfil this gap, we report here the crystal structure of MEF2D in complex with double strand DNA and four different class IIa HDAC derived peptides, namely HDAC4, HDAC5, HDAC7 and HDAC9. All class IIa HDAC derived peptides form extended amphipathic α-helix structures that fit snugly in the hydrophobic groove of MEF2D domain. Binding mode of class IIa HDAC derived peptides to MEF2D is very similar and occur primarily through nonpolar interactions mediated by highly conserved branched hydrophobic amino acids. Further studies revealed that class IIa HDAC derived peptides are unstructured in solution and appear to adopt a folded α-helix structure only upon binding to MEF2D. Comparison of our peptide-protein complexes with previously characterized structures of MEF2 bound to different co-activators and co-repressors, highlighted both differences and similarities, and revealed the adaptability of MEF2 in protein-protein interactions. The elucidation of the three-dimensional structure of MEF2D in complex with multiple class IIa HDAC derived peptides provide not only a better understanding of the molecular basis of their interactions but also have implications for the development of novel antagonist.

The catalytic domain of free or ligand bound histone deacetylase 4 occurs in solution predominantly in closed conformation.

Human histone deacetylase 4 (HDAC4) is a key epigenetic regulator involved in a number of important cellular processes. This makes HDAC4 a promising target for the treatment of several cancers and neurodegenerative diseases, in particular Huntington's disease. HDAC4 is highly regulated by phosphorylation and oxidation, which determine its nuclear or cytosolic localization, and exerts its function through multiple interactions with other proteins, forming multiprotein complexes of varying composition. The catalytic domain of HDAC4 is known to interact with the SMRT/NCOR corepressor complex when the structural zinc-binding domain (sZBD) is intact and forms a closed conformation. Crystal structures of the HDAC4 catalytic domain have been reported showing an open conformation of HDAC4 when bound to certain ligands. Here, we investigated the relevance of this HDAC4 conformation under physiological conditions in solution. We show that proper zinc chelation in the sZBD is essential for enzyme function. Loss of the structural zinc ion not only leads to a massive decrease in enzyme activity, but it also has serious consequences for the overall structural integrity and stability of the protein. However, the Zn2+ free HDAC4 structure in solution is incompatible with the open conformation. In solution, the open conformation of HDAC4 was also not observed in the presence of a variety of structurally divergent ligands. This suggests that the open conformation of HDAC4 cannot be induced in solution, and therefore cannot be exploited for the development of HDAC4-specific inhibitors.

Histone deacetylase 10: A polyamine deacetylase from the crystal structure to the first inhibitors.

Polyamine deacetylase activity was discovered more than 40 years ago, but the responsible histone deacetylase 10 (HDAC10) was described only recently. HDAC10 is a class IIb HDAC, as is its closest relative, the α-tubulin deacetylase HDAC6. HDAC10 has attracted attention over the last 2 years due to its role in diseases, especially cancer. This review summarises chemical and structural biology approaches to the study of HDAC10. Light will be shed on recent advances in understanding the complex structural biology of HDAC10 and the discovery of the first highly selective HDAC10 inhibitors.

Perspectives and new aspects of histone deacetylase inhibitors in the therapy of CNS diseases.

Many populations worldwide are suffering from central nervous system (CNS) diseases such as brain tumors, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease and Huntington's disease) and stroke. There is a shortage of effective drugs for most CNS diseases. As one of the regulatory mechanisms of epigenetics, the particular role and therapeutic benefits of histone deacetylases (HDACs) in the CNS have been extensively studied. In recent years, HDACs have attracted increasing attention as potential drug targets for CNS diseases. In this review, we summarize the recent applications of representative histone deacetylases inhibitors (HDACis) in CNS diseases and discuss the challenges in developing HDACis with different structures and better blood-brain barrier (BBB) permeability, hoping to promote the development of more effective bioactive HDACis for the treatment of CNS diseases.

The Zinc-Binding Group Effect: Lessons from Non-Hydroxamic Acid Vorinostat Analogs.

Histone deacetylases (HDACs) are enzymes pursued as drug targets in various cancers and several non-oncological conditions, such as inflammation and neurodegenerative disorders. In the past decade, HDAC inhibitors (HDACi) have emerged as relevant pharmaceuticals, with many efforts devoted to the development of new representatives. However, the growing safety concerns regarding the established hydroxamic acid-based HDAC inhibitors tend to drive current research more toward the design of inhibitors bearing alternative zinc-binding groups (ZBGs). This Perspective presents an overview of all non-hydroxamic acid ZBGs that have been incorporated into the clinically approved prototypical HDACi, suberoylanilide hydroxamic acid (vorinostat). This provides the unique opportunity to compare the inhibition potential and biological effects of different ZBGs in a direct way, as the compounds selected for this Perspective differ only in their ZBG. To that end, different strategies used to select a ZBG, its properties, activity, and liabilities are discussed.

Exploring structural requirements of HDAC10 inhibitors through comparative machine learning approaches.

Histone deacetylase (HDAC) inhibitors are in the limelight of anticancer drug development and research. HDAC10 is one of the class-IIb HDACs, responsible for cancer progression. The search for potent and effective HDAC10 selective inhibitors is going on. However, the absence of human HDAC10 crystal/NMR structure hampers the structure-based drug design of HDAC10 inhibitors. Different ligand-based modeling techniques are the only hope to speed up the inhibitor design. In this study, we applied different ligand-based modeling techniques on a diverse set of HDAC10 inhibitors (n = 484). Machine learning (ML) models were developed that could be used to screen unknown compounds as HDAC10 inhibitors from a large chemical database. Moreover, Bayesian classification and Recursive partitioning models were used to identify the structural fingerprints regulating the HDAC10 inhibitory activity. Additionally, a molecular docking study was performed to understand the binding pattern of the identified structural fingerprints towards the active site of HDAC10. Overall, the modeling insight might offer helpful information for medicinal chemists to design and develop efficient HDAC10 inhibitors.

Histone deacetylase inhibitors as sanguine epitherapeutics against the deadliest lung cancer.

The back-breaking resistance mechanisms generated by lung cancer cells against epidermal growth factor receptor (EGFR), KRAS and Janus kinase 2 (JAK2) directed therapies strongly prioritizes the requirement of novel therapies which are perfectly tolerated, potentially cytotoxic and can reinstate the drug-sensitivity in lung cancer cells. Enzymatic proteins modifying the post-translational modifications of nucleosome-integrated histone substrates are appearing as current targets for defeating various malignancies. Histone deacetylases (HDACs) are hyperexpressed in diverse lung cancer types. Blocking the active pocket of these acetylation erasers through HDAC inhibitors (HDACi) has come out as an optimistic therapeutic recourse for annihilating lung cancer. This article in the beginning gives an overview about lung cancer statistics and predominant lung cancer types. Succeeding this, compendium about conventional therapies and their serious drawbacks has been provided. Then, connection of uncommon expression of classical HDACs in lung cancer onset and expansion has been detailed. Moreover, keeping the main theme in view this article deeply discusses HDACi in the context of aggressive lung cancer as single agents and spotlights various molecular targets suppressed or induced by these inhibitors for engendering cytotoxic effect. Most particularly, the raised pharmacological effects achieved on using these inhibitors in concerted form with other therapeutic molecules and the cancer-linked pathways altered by this procedure are described. The positive direction towards further heightening of efficacy and the pressing requirement of exhaustive clinical assessment has been proposed as a new focus point.

Phenolic compounds as histone deacetylase inhibitors: binding propensity and interaction insights from molecular docking and dynamics simulations.

Histone deacetylases are well-established target enzymes involved in the pathology of different diseases including cancer and neurodegenerative disorders. The approved HDAC inhibitor drugs are associated with cellular toxicities. Different phenolic compounds have been shown to possess inhibitory activities against HDACs and are, therefore, considered safer alternatives to synthetic compounds. Here, we elucidated the binding mode and calculated the binding propensity of some of the top phenolic compounds against different isoforms representing different classes of Zn2+ ion-containing HDACs using the molecular docking approach. Our data reaffirmed the activity of the studied phenolic compounds against HDACs. Binding interaction analysis suggested that these compounds can block the activity of HDACs with or without binding to the active site zinc metal ion. Furthermore, molecular dynamics (MD) simulations were carried out on the selected crystal and docking complexes of each selected HDAC isoform. Analysis of root-mean-square displacement (RMSD) showed that the phenolic compounds demonstrated a stable binding mode over 50 ns in a way that is comparable to the cocrystal ligands. Together, these findings can aid future efforts in the search for natural inhibitors of HDACs.

Structural exploration of common pharmacophore based berberine derivatives as novel histone deacetylase inhibitor targeting HDACs enzymes.

Histone deacetylase (HDAC) inhibitors, are new class of cancer chemotherapeutics used in clinical development. It plays a pivotal role in restoring the acetylation balance and lysine residual deacetylation in histone and non-histone proteins. Notably, HDAC inhibitors have been approved by FDA to treat different malignancies. Recently, we demonstrated berberine as pan inhibitor for HDAC. However, isoform specific inhibition of HDAC enzyme is highly warranted. Therefore, a pharmacophore based structural exploration of berberine is in need to be developed, berberine is composed of four portions namely: a) zinc binding group (ZBG), b) Linker (scaffold), c) connect unit (CU), and d) surface recognition moiety (SRM). We derived four berberine derivatives based on common HDAC inhibition pharmacophore, compound 4 possesses highest bit score by molecular docking and compound stability by HOMOs-LUMOs analysis. It is concluded that, structurally modified berberine derivatives shown better inhibition of HDAC enzymes offering improved clinical efficacy.

A comparative quantitative structural assessment of benzothiazine-derived HDAC8 inhibitors by predictive ligand-based drug designing approaches.

Histone deacetylase 8 (HDAC8) is a verified biomolecular target associated with diverse diseases including cancer. Though several HDAC inhibitors emerged effective against such diseases, no selective HDAC8 inhibitor is approved to date. Therefore, the development of potent HDAC8-selective inhibitors is inevitable to combat such diseases. Here, some benzothiazine-derived HDAC8 inhibitors were considered for a comparative QSAR analysis which may elucidate the prime structural components responsible for modulating their efficacy. Several outcomes from these diverse modelling techniques justified one another and thus validated each other. The ligand-based pharmacophore modelling study identified ring aromatic, positive ionizable, and hydrophobic features as essential structural attributes for HDAC8 inhibition. Besides, MLR, HQSAR and field-based 3D-QSAR studies signified the utility of the positive ionizable and hydrophobic features for potent HDAC8 inhibition. Again, the field-based 3D-QSAR study provided useful insight regarding the substitution in the fused phenyl ring. Moreover, the current observations also validated the previously reported molecular docking observations. Based on the outcomes, some new molecules were designed and predicted. Therefore, this comparative structural analysis of these HDAC8 inhibitors will surely assist in the development of potent HDAC8 inhibitors as promising anticancer therapeutics in the future.

Phage-assisted, active site-directed ligand evolution of a potent and selective histone deacetylase 8 inhibitor.

Phage-assisted, active site-directed ligand evolution (PADLE) is a recently developed technique that uses an amber codon-encoded noncanonical amino acid (ncAA) as an anchor to direct phage-displayed peptides to a target for an enhanced ligand identification process. 2-Amino-8-oxodecanoic acid (Aoda) is a ketone-containing ncAA residue in the macrocyclic peptide natural product apicidin that is a pan-inhibitor of Zn2+ -dependent histone deacetylases (HDACs). Its ketone serves as an anchoring point to coordinate the catalytic zinc ion in HDACs. Using a previously evolved N𝜀 -acetyl-lysyl-tRNA synthetase in combination with tRNAPyl , we showed that Aoda was efficiently incorporated into proteins in Escherichia coli by amber suppression. By propagating an amber codon-obligate phagemid library in E. coli encoding Aoda, we generated an Aoda-containing phage-displayed peptide library. Using this library to conduct PADLE against HDAC8 revealed a 7-mer peptide GH8P01F1 with Aoda-flanking amino acid residues that matched existing peptide sequences in identified HDAC8 substrates. Switching Aoda in GH8P01F1 to a more Zn2+ -chelating ncAA S-2-amino-8-hydroxyamino-8-oxooctanoic acid (Asuha) led to an extremely potent compound GH8HA01, which has an HDAC8-inhibition Ki value of 0.67 nM. GH8HA01 and its 5-mer truncation analogue Ac-GH8HA01Δ1Δ7 that has an HDAC8-inhibition Ki value of 0.31 nM are two of the most potent HDAC8 inhibitors that have been developed. Furthermore, both are highly selective against HDAC8 compared with other HDACs tested, demonstrating the great potential of using PADLE to identify highly potent and selective ligands for targets with conserved active sites among homologues.

Aza-SAHA Derivatives Are Selective Histone Deacetylase 10 Chemical Probes That Inhibit Polyamine Deacetylation and Phenocopy HDAC10 Knockout.

We report the first well-characterized selective chemical probe for histone deacetylase 10 (HDAC10) with unprecedented selectivity over other HDAC isozymes. HDAC10 deacetylates polyamines and has a distinct substrate specificity, making it unique among the 11 zinc-dependent HDAC hydrolases. Taking inspiration from HDAC10 polyamine substrates, we systematically inserted an amino group ("aza-scan") into the hexyl linker moiety of the approved drug Vorinostat (SAHA). This one-atom replacement (C→N) transformed SAHA from an unselective pan-HDAC inhibitor into a specific HDAC10 inhibitor. Optimization of the aza-SAHA structure yielded the HDAC10 chemical probe DKFZ-748, with potency and selectivity demonstrated by cellular and biochemical target engagement, as well as thermal shift assays. Cocrystal structures of our aza-SAHA derivatives with HDAC10 provide a structural rationale for potency, and chemoproteomic profiling confirmed exquisite cellular HDAC10-selectivity of DKFZ-748 across the target landscape of HDAC drugs. Treatment of cells with DKFZ-748, followed by quantification of selected polyamines, validated for the first time the suspected cellular function of HDAC10 as a polyamine deacetylase. Finally, in a polyamine-limiting in vitro tumor model, DKFZ-748 showed dose-dependent growth inhibition of HeLa cells. We expect DKFZ-748 and related probes to enable further studies on the enigmatic biology of HDAC10 and acetylated polyamines in both physiological and pathological settings.

Molecular architecture of nucleosome remodeling and deacetylase sub-complexes by integrative structure determination.

The nucleosome remodeling and deacetylase (NuRD) complex is a chromatin-modifying assembly that regulates gene expression and DNA damage repair. Despite its importance, limited structural information describing the complete NuRD complex is available and a detailed understanding of its mechanism is therefore lacking. Drawing on information from SEC-MALLS, DIA-MS, XLMS, negative-stain EM, X-ray crystallography, NMR spectroscopy, secondary structure predictions, and homology models, we applied Bayesian integrative structure determination to investigate the molecular architecture of three NuRD sub-complexes: MTA1-HDAC1-RBBP4, MTA1N -HDAC1-MBD3GATAD2CC , and MTA1-HDAC1-RBBP4-MBD3-GATAD2A [nucleosome deacetylase (NuDe)]. The integrative structures were corroborated by examining independent crosslinks, cryo-EM maps, biochemical assays, known cancer-associated mutations, and structure predictions from AlphaFold. The robustness of the models was assessed by jack-knifing. Localization of the full-length MBD3, which connects the deacetylase and chromatin remodeling modules in NuRD, has not previously been possible; our models indicate two different locations for MBD3, suggesting a mechanism by which MBD3 in the presence of GATAD2A asymmetrically bridges the two modules in NuRD. Further, our models uncovered three previously unrecognized subunit interfaces in NuDe: HDAC1C -MTA1BAH , MTA1BAH -MBD3MBD , and HDAC160-100 -MBD3MBD . Our approach also allowed us to localize regions of unknown structure, such as HDAC1C and MBD3IDR , thereby resulting in the most complete and robustly cross-validated structural characterization of these NuRD sub-complexes so far.

Crystal structures of Schistosoma mansoni histone deacetylase 8 reveal a novel binding site for allosteric inhibitors.

Parasitic diseases cause significant global morbidity and mortality particularly in the poorest regions of the world. Schistosomiasis, one of the most widespread neglected tropical diseases, affects more than 200 million people worldwide. Histone deacetylase (HDAC) inhibitors are prominent epigenetic drugs that are being investigated in the treatment of several diseases, including cancers and parasitic diseases. Schistosoma mansoni HDAC8 (SmHDAC8) is highly expressed in all life cycle stages of the parasite, and selective inhibition is required in order to avoid undesirable off-target effects in the host. Herein, by X-ray crystal structures of SmHDAC8-inhibitor complexes, biochemical and phenotypic studies, we found two schistosomicidal spiroindoline derivatives binding a novel site, next to Trp198, on the enzyme surface. We determined that by acting on this site, either by mutation of the Trp198 or by compound binding, a decrease in the activity of the enzyme is achieved. Remarkably, this allosteric site differs from the human counterpart; rather, it is conserved in all Schistosoma species, as well as Rhabidoptera and Trematoda classes, thus paving the way for the design of HDAC8-selective allosteric inhibitors with improved properties.

Design, Synthesis and Biological Characterization of Histone Deacetylase 8 (HDAC8) Proteolysis Targeting Chimeras (PROTACs) with Anti-Neuroblastoma Activity.

In addition to involvement in epigenetic gene regulation, histone deacetylases (HDACs) regulate multiple cellular processes through mediating the activity of non-histone protein substrates. The knockdown of HDAC8 isozyme is associated with the inhibition of cell proliferation and apoptosis enhancement in several cancer cell lines. As shown in several studies, HDAC8 can be considered a potential target in the treatment of cancer forms such as childhood neuroblastoma. The present work describes the development of proteolysis targeting chimeras (PROTACs) of HDAC8 based on substituted benzhydroxamic acids previously reported as potent and selective HDAC8 inhibitors. Within this study, we investigated the HDAC8-degrading profiles of the synthesized PROTACs and their effect on the proliferation of neuroblastoma cells. The combination of in vitro screening and cellular testing demonstrated selective HDAC8 PROTACs that show anti-neuroblastoma activity in cells.

Development of New Inhibitors of HDAC1-3 Enzymes Aided by In Silico Design Strategies.

Histone deacetylases (HDACs) are overexpressed in cancer, and their inhibition shows promising results in cancer therapy. In particular, selective class I HDAC inhibitors such as entinostat are proposed to be more beneficial in breast cancer treatment. Computational drug design is an inevitable part of today's drug discovery projects because of its unequivocal role in saving time and cost. Using three HDAC inhibitors trichostatin, vorinostat, and entinostat as template structures and a diverse fragment library, all synthetically accessible compounds thereof (∼3200) were generated virtually and filtered based on similarity against the templates and PAINS removal. The 298 selected structures were docked into the active site of HDAC I and ranked using a calculated binding affinity. Top-ranking structures were inspected manually, and, considering the ease of synthesis and drug-likeness, two new structures (3a and 3b) were proposed for synthesis and biological evaluation. The synthesized compounds were purified to a degree of more than 95% and structurally verified using various methods. The designed compounds 3a and 3b showed 65-80 and 5% inhibition on HDAC 1, 2, and 3 isoforms at a concentration of 10 µM, respectively. The novel compound 3a may be used as a lead structure for designing new HDAC inhibitors.

Identification of potent HDAC 2 inhibitors using E-pharmacophore modelling, structure-based virtual screening and molecular dynamic simulation.

Histone deacetylase 2 (HDAC 2) of class I HDACs plays a major role in embryonic and neural developments. However, HDAC 2 overexpression triggers cell proliferation by diverse mechanisms in cancer. Over the decades, many pan and class-specific inhibitors of HDAC were discovered. Limitations such as toxicity and differential cell localization of each isoform led researchers to hypothesize that isoform selective inhibitors may be relevant to bring about desired effects. In this study, we have employed the PHASE module to develop an e-pharmacophore model and virtually screened four focused libraries of around 300,000 compounds to identify isoform selective HDAC 2 inhibitors. The compounds with phase fitness score greater than or equal to 2.4 were subjected to structure-based virtual screening with HDAC 2. Ten molecules with docking score greater than -12 kcal/mol were chosen for selectivity study, QikProp module (ADME prediction) and dG/bind energy identification. Compound 1A with the best dock score of -13.3 kcal/mol and compound 1I with highest free binding energy, -70.93 kcal/mol, were selected for molecular dynamic simulation studies (40 ns simulation). The results indicated that compound 1I may be a potent and selective HDAC 2 inhibitor. Further, in vitro and in vivo studies are necessary to validate the potency of selected lead molecule and its derivatives.

Identification of novel leads as potent inhibitors of HDAC3 using ligand-based pharmacophore modeling and MD simulation.

In the landscape of epigenetic regulation, histone deacetylase 3 (HDAC3) has emerged as a prominent therapeutic target for the design and development of candidate drugs against various types of cancers and other human disorders. Herein, we have performed ligand-based pharmacophore modeling, virtual screening, molecular docking, and MD simulations to design potent and selective inhibitors against HDAC3. The predicted best pharmacophore model 'Hypo 1' showed excellent correlation (R2 = 0.994), lowest RMSD (0.373), lowest total cost value (102.519), and highest cost difference (124.08). Hypo 1 consists of four salient pharmacophore features viz. one hydrogen bond acceptor (HBA), one ring aromatic (RA), and two hydrophobic (HYP). Hypo 1 was validated by Fischer's randomization with a 95% of confidence level and the external test set of 60 compounds with a good correlation coefficient (R2 = 0.970). The virtual screening of chemical databases, drug-like properties calculations followed by molecular docking resulted in identifying 22 representative hit compounds. Performed 50 ns of MD simulations on top three hits were retained the salient π-stacking, Zn2+ coordination, hydrogen bonding, and hydrophobic interactions with catalytic residues from the active site pocket of HDAC3. Total binding energy calculated by MM-PBSA showed that the Hit 1 and Hit 2 formed stable complexes with HDAC3 as compared to reference TSA. Further, the PLIP analysis showed a close resemblance between the salient pharmacophore features of Hypo 1 and the presence of molecular interactions in co-crystallized FDA-approved drugs. We conclude that the screened hit compounds may act as potent inhibitors of HDAC3 and further preclinical and clinical studies may pave the way for developing them as effective therapeutic agents for the treatment of different cancers and neurodegenerative disorders.