Virtual docking and design of novel Histone deacetylase and Rho- associated protein kinases dual inhibitors (HDAC/ROCKs)

Histone deacetylases (HDACs) belong to a family of epigenetic enzymes that has 18 different isoforms and play an important role in the development and progression of various tumors. To date, five histone deacetylase inhibitors have been approved by the FDA, and are used to treat multiple myeloma, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, and breast cancer (estrogen and/or progesterone positive) [1]. All of them are non-selective. Therefore, their safety profile is poor and their efficacy is low in single therapy. One of our previous research projects demonstrated the synergistic effect of HDAC inhibitors and inhibitors of Rho-associated protein kinases (ROCK) in the treatment of pancreatic ductal adenocarcinoma (PDAC) [2]. This finding led us to design of the dual HDAC/ROCK inhibitors with potential effects on PDAC by using structure-based molecular docking method. Molecular docking study was performed using GOLD software. The crystal structures of ROCK1 (PDB: 6E9W), ROCK2 (PDB: 7JNT), HDAC1 (PDB: 5ICN), and HDAC6 (PDB: 5EDU) enzymes were downloaded from the Protein Data Bank (PDB). The enzymes were prepared for docking study using the online software Play Molecule-ProteinPrepare. The structures of the ROCK1, ROCK2, HDAC1 and HDAC6 inhibitors with their pIC50 values were obtained from the ChEMBL database. The dominant microspecies of all compounds at physiological pH were selected by Marvin Sketch Sketch 6.1.0 program and their further geometrical optimization were performed using the PM3 semi-empirical method and the Hartree-Fock method with 3-21G basis set. The virtual docking procedures for all four enzymes were validated and the calculated RMSD values were below 2Å. The critical parts of the structures that establish the interactions crucial for the inhibition of HDAC1, HDAC6, ROCK1, and ROCK2 were identified. Based on the obtained resultsdual HDAC/ROCK inhibitors were designed and evaluated by validated docking procedures and in silico ADMET profiling. Taking into account all these findings, the most active compounds are selected and will be further synthesized and evaluated using in vitro enzyme and cell tests.10th IAPC Meeting, Book of Abstract

Targeting Histone Deacetylases 6 in Dual-Target Therapy of Cancer

Histone deacetylases (HDACs) are the major regulators of the balance of acetylation of histone and non-histone proteins. In contrast to other HDAC isoforms, HDAC6 is mainly involved in maintaining the acetylation balance of many non-histone proteins. Therefore, the overexpression of HDAC6 is associated with tumorigenesis, invasion, migration, survival, apoptosis and growth of various malignancies. As a result, HDAC6 is considered a promising target for cancer treatment. However, none of selective HDAC6 inhibitors are in clinical use, mainly because of the low efficacy and high concentrations used to show anticancer properties, which may lead to off-target effects. Therefore, HDAC6 inhibitors with dual-target capabilities represent a new trend in cancer treatment, aiming to overcome the above problems. In this review, we summarize the advances in tumor treatment with dual-target HDAC6 inhibitors

Постоји ли граница између цитотоксичног и антиметастатског ефекта селективних инхибитора хистон деацетилазе 6 код солидних тумора?

Academia and industry make an extensive effort to discover selective histone deacetylase 6 inhibitors (HDAC6i) which could be delivered to the patients. These attempts are partially obscured by inconsistency in preclinical data regarding the cellular and biochemical changes upon treatment of solid tumours with HDAC6i. In this study, we present computational design, synthesis of three novel HDAC6 inhibitors along with their in vitro pharmacological profile against zinc-dependent HDACs. The in vitro anticancer effects of the synthesized compounds were examined on four types of cancer cell lines, human breast cancer cell lines (MDA-MB-231 and MCF-7) as well as human melanoma cells (A-375 and 518A2). It was found that the synthesized compounds induce apoptosis in high concentrations (IC50 >30 μM) nonetheless, the observed morphological changes of studied cell lines during cell viability assay prompted us to examine their antimetastatic properties. Novel compound MBDR-4 significantly reduces migration and invasiveness of the MDA-MB-231 and A375 cancer cell lines at subapoptotic concentration (5 μM), which open new avenues for redirecting drug development of selective HDAC6 inhibitors as adjuvant chemotherapeutics, with antimetastatic effects.Истраживачи тренутно улажу велике напоре у испитивање селективних инхибитора хистон деацетилазе 6 (HDAC6i) 1,2 који би могли да нађу клиничку примену 3 . Ова истраживања су делимично отежана услед неконзистентних преклиничких резултата о ћелијским и биохемијским променама након третмана солидних тумора HDAC6i(4,5). Наша студија обухвата компјутерски дизајн и синтезу три нова HDAC6 инхибитора, заједно са in vitro ензимским испитивањем активности на сродним изоформама HDAC ензимима. Антинеопластичан ефекат синте- тисаних једињења је испитан in vitro на четири типа ћелија карцинома, хуманим ћелијама карцинома дојке (MDA-MB-231 и MCF-7) и хуманим ће- лијама меланома (A-375 и 518A2). Утврђено је да синтетисана једињења индукују апоптозу при високим концентрацијама (IC 50 >30 μM). Истовреме- но је уочено да ова једињења доводе и до морфолошких промена на тре- тираним ћелијама током извођења теста виабилности што нас је усмери- ло ка испитивању њихових антиметастатских особина. Једињење MBDR-4 снажно инхибира миграцију и инвазивност MDA-MB-231 и A375 ћелија кан- цера на субапоптотској концентрацији (5 μM), што отвара нове могућности за истраживање селективних HDAC6 инхибитора као адјувантних хемоте- рапеутика са антиметастатским ефектом.Канцеролошка секција Српског лекарског друштва Serbian Medical Society Oncology Section Београд, новембар 2019 / Belgrade, November 2019 АНАЛИ КАНЦЕРОЛОШКЕ СЕКЦИЈЕ СЛД ANNALS OF ONCOLOGY SECTION Зборник апстраката / Abstract Boo

Discovery of 1-Benzhydryl-Piperazine-Based HDAC Inhibitors with Anti-Breast Cancer Activity: Synthesis, Molecular Modeling, In Vitro and In Vivo Biological Evaluation

Abstract Isoform-selective histone deacetylase (HDAC) inhibition is promoted as a rational strategy to develop safer anti-cancer drugs compared to non-selective HDAC inhibitors. Despite this presumed benefit, considerably more non-selective HDAC inhibitors have undergone clinical trials. In this report, we detail the design and discovery of potent HDAC inhibitors, with 1-benzhydryl piperazine as a surface recognition group, that differ in hydrocarbon linker. In vitro HDAC screening identified two selective HDAC6 inhibitors with nanomolar IC50 values, as well as two non-selective nanomolar HDAC inhibitors. Structure-based molecular modeling was employed to study the influence of linker chemistry of synthesized inhibitors on HDAC6 potency. The breast cancer cell lines (MDA-MB-231 and MCF-7) were used to evaluate compound-mediated in vitro anti-cancer, anti-migratory, and anti-invasive activities. Experiments on the zebrafish MDA-MB-231 xenograft model revealed that a novel non-selective HDAC inhibitor with a seven-carbon-atom linker exhibits potent anti-tumor, anti-metastatic, and anti-angiogenic effects when tested at low micromolar concentrations

Preclinical Evaluation of an Imidazole-Linked Heterocycle for Alzheimer’s Disease

Humanity is facing a vast prevalence of neurodegenerative diseases, with Alzheimer’s disease (AD) being the most dominant, without efficacious drugs, and with only a few therapeutic targets identified. In this scenario, we aim to find molecular entities that modulate imidazoline I2 receptors (I2-IRs) that have been pointed out as relevant targets in AD. In this work, we explored structural modifications of well-established I2-IR ligands, giving access to derivatives with an imidazole-linked heterocycle as a common key feature. We report the synthesis, the affinity in human I2-IRs, the brain penetration capabilities, the in silico ADMET studies, and the three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of this new bunch of I2-IR ligands. Selected compounds showed neuroprotective properties and beneficial effects in an in vitro model of Parkinson’s disease, rescued the human dopaminergic cell line SH-SY5Y from death after treatment with 6-hydroxydopamine, and showed crucial anti-inflammatory effects in a cellular model of neuroinflammation. After a preliminary pharmacokinetic study, we explored the action of our representative 2-(benzo[b]thiophen-2-yl)-1H-imidazole LSL33 in a mouse model of AD (5xFAD). Oral administration of LSL33 at 2 mg/Kg for 4 weeks ameliorated 5XFAD cognitive impairment and synaptic plasticity, as well as reduced neuroinflammation markers. In summary, this new I2-IR ligand that promoted beneficial effects in a well-established AD mouse model should be considered a promising therapeutic strategy for neurodegeneration

Virtual Docking, design and in silico ADMET profiling of novel Rho- associated protein kinases-1 (ROCK1) inhibitors

Overexpression of Rho-associated protein kinases has been associated with various diseases, including tumors. None of the approved ROCK inhibitors are used for cancer treatment. However, some of them have been shown to have anti-tumor potential. The main objective of this study was to develop novel ROCK1 inhibitors using the structure-based method, molecular docking, and prediction of pharmacokinetic properties using the ADMET predictor. The key interactions that strongly correlate with the activity of ROCK1 inhibitors are hydrogen bonds between amino acid residues Met156, Glu154 and the hinge region of the inhibitors, indicating possible structural changes in the hinge region of studied compounds. On the other hand, the lack of interactions between 1,3-benzoxadiol moiety and the enzyme presents a promising approach for further structural modifications in order to design more effective ROCK1 inhibitors. All the important interactions between the developed ROCK1 inhibitors and the binding site of the enzyme were established. They also showed acceptable pharmacokinetic properties and could be further used for synthesis and evaluation by various biological assays.2nd International Conference on Chemo and Bioinformatics, September 28-29, 2023. Kragujevac, Serbi

3D-Quantitative Structure-Activity Relationship and design of novel Rho- associated protein kinases-1 (ROCK1) inhibitors

Rho-associated coiled-coil kinases (ROCKs) are involved in essential cellular functions such as adhesion, contraction, motility, proliferation, and cell survival/apoptosis. Four ROCK inhibitors have already been approved by the FDA and are used to treat glaucoma (ripasudil and netarsudil), cerebral vasospasm (fasudil), and graft-versus-host disease (belumosudil). Recent studies have focused on exploring the role of ROCK kinase inhibitors in cancer treatment and the development of new ROCK inhibitors. The main objective of this study was to identify critical structural features relevant to the inhibition of ROCK1 using a ligand-based 3D-QSAR (3D quantitative structure-activity relationship) method. The 3D-QSAR model for ROCK1 was created and validated using internal and external validation parameters (R2, Q2, R2pred, rm2, r/2m, ������������������ 2̅̅̅ and ∆r2m). The main structural features that correlate with the inhibition of ROCK1 were identified (e.g., heterocycle with hydrogen donor group like nitrogen atom) and further structural modifications of the ROCK1 inhibitors that contribute to increased activity were proposed (removal of the amino group of the oxadiazole, modification of the substituents of the phenyl ring).2nd International Conference on Chemo and Bioinformatics, September 28-29, 2023. Kragujevac, Serbi

Anticancer evaluation of the selected tetrahydropyrimidines: 3D-QSAR, cytotoxic activities, mechanism of action, DNA, and BSA interactions

Selected tetrahydropyrimidines (THPMs) were investigated by means of cytotoxic activities on selected cancer (HeLa, A549, and LS174) and non-cancerous cell lines (MRC-5). Among evaluated compounds, two of them ( B7 and B8 ) showed good cytotoxic activity on the tested cell lines and were selected for fur- ther evaluation that included mechanism of action, DNA and BSA interactions and molecular docking study. Calculated parameters from fluorescence quenching studies indicated that B7 and B8 bind on mi- nor groove of DNA and have great ability to bind on carrier protein. Three-dimensional quantitative struc- ture anti-HeLa activity study was performed with data set of THPMs. Molecular Interaction Fields were used to derive Grid independent descriptors (GRIND), as independent variables in Pentacle software. The quality and predictive capacity of the model was proved by internal statistical parameters: R 2 = 0.992, Q 2 = 0.51, as well as external parameters such as R 2 pred = 0.804 and r m 2 , r / 2 m and r 2 m , that were higher than 0.5. The structural determinants significant for anti-HeLa activity of compounds were identified by using developed 3D-QSAR model. Interpretation of the most impactful GRIND variables on the anti-HeLa activity generated several hypotheses for design of novel and more potent anti-HeLa tetrahydropyrim- idines. Additional molecular targets for the most active synthesized derivatives ( B7 and B8 ) are predicted by use of online web-based tool-SwissTargetPrediction

Discovery of dual-acting HDAC inhibitors for the treatment of pancreatic cancer by combining drug synergy and molecular modeling

Pancreatic ductal adenocarcinoma (PDAC) is one of the most threatening and deadly cancers. The occurrence of chemoresistance in PDAC plays an important role in the unfavorable survival rates, so there is an urgent need to rapidly develop new pharmaceutical strategies to address this problem and improve treatment outcomes for PDAC (1). Considering the numerous epigenetic alterations observed in PDAC, the use of epigenetic drugs, such as histone deacetylase (HDAC) inhibitors, is a promising approach, especially when used in combination therapies (2). In this study, we investigated the potential of using drug sensitivity and basal gene expression data from pancreatic cancer cell lines to develop a bioinformatic screening protocol to predict the available combinatorial options for HDAC inhibitors, including sirtuin (SIRT) inhibitors. Experimental validation of the protocol in two pancreatic cancer cell lines (MIA PaCa-2 cells and PANC-1) confirmed the identified synergies between HDAC inhibitors and the sphingosine-1-phosphate (S1P) receptor agonist – fingolimod or HDAC inhibitors and the Rho-associated protein kinase (ROCK) inhibitor – RKI-1447 (3). The bioinformatic screening protocol developed to predict synergistic drug combinations in PDAC identified several previously unknown interaction partners of HDAC inhibitors. The predicted interaction partners of HDAC inhibitors, including ROCK, aurora kinase A inhibitor (AURKA), glutaminase inhibitor (GLS) and WEE1 kinase inhibitor, were selected using structure-based molecular modeling for the development of novel classes of dual-acting HDAC inhibitors. The novel dual inhibitors (HDAC/ROCK, SIRT/AURKA, HDAC/GLS and HDAC/WEE1) were designed based on the known pharmacophore properties and molecular docking models developed for the respective targets. The molecular docking study was performed using GOLD software. The enzymes were prepared for the docking study using the online software Play Molecule-ProteinPrepare. The docking results of the developed inhibitors showed a remarkable affinity to the specific targets. The critical parts of the structures that produce the interactions crucial for the inhibition of HDAC1, HDAC6, ROCK1, ROCK2, AURKA, GLS and WEE1 were identified. In conjunction with the drug synergy predictions, these designed molecules also show great potential as promising structures for subsequent experimental evaluation. Taking all these findings into account, the most aactive compounds were selected to be further synthesized and evaluated using in vitro enzyme and cell assays. References 1) A. McGuigan et al., World J Gastroenterol. 24, 4846–4861 (2018). 2) X.-S. Xiang, P.-C. Li, W.-Q. Wang, L. Liu, Biochimica et Biophysica Acta (BBA) - Reviews on Cancer. 1877, 188676 (2022). 3) N. Djokovic, A. Djuric, D. Ruzic, T. Srdic-Rajic, K. Nikolic, Pharmaceuticals. 16, 294 (2023)