VIRTUAL TARGET CONSTRUCTION FOR DISCOVERY OF HUMAN HISTAMINE H 4 RECEPTOR LIGANDS EMPLOYING A STRUCTURE-BASED VIRTUAL SCREENING APPROACH Original Article GERRY NUGRAHA 1,2 , HARNO DWI PRANOWO 3* , MUDASIR MUDASIR 4 , ENADE PERDANA ISTYASTONO 5 1 Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia, 2 Pharmacy, STIKES ‘Aisyiyah, Palembang 30961, Indonesia, 3 Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia, 4 Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia, 5 Faculty of Pharmacy, Universitas Sanata Dharma, Yogyakarta 55281, Indonesia * Email: harnodp@ugm.ac.id Received: 06 Jan 2022, Revised and Accepted: 30 Apr 2022 ABSTRACT Objective: This study aimed to construct a virtual target to be used in structure-based virtual screening (SBVS) campaigns to discover ligands for human histamine receptor H4 (hHRH4). Methods: The virtual targets construction was initiated by hHRH4 homology modeling, followed by molecular docking of seliforant to the homolog model, and the virtual target candidate was constructed. The hHRH4 complexed to seliforant was subjected to molecular dynamics (MD) simulations in 100 ns. Finally, the pose with the least free energy of binding from the MD simulations was selected for further validation through re- docking simulations. All simulations were conducted by using the YASARA-Structure program package. Results: This study resulted in one validated target for SBVS protocols development. All RMSD values in the internal validation in snapshot 519 molecular dynamics simulation results were less than 2 Å, and this hHRH4 homology model is valid as a virtual target in an SBVS protocol. Moreover, using the clusterization module on MD simulations analysis, ten different virtual targets were available for further utilization. Conclusion: Virtual targets resulted from this study offer more possibilities to construct SBVS protocols to identify hHRH4 ligands. The validated virtual target and the ten different virtual targets resulted from clusterization can be accessed in the following GitHub repository: https://github.com/nugrahagerry/hHRH4. Keywords: Histamine H4, Seliforant, SBVS, Homology modeling, Molecular docking, Molecular dynamics © 2022 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open-access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/) DOI: https://dx.doi.org/10.22159/ijap.2022v14i4.44067. Journal homepage: https://innovareacademics.in/journals/index.php/ijap INTRODUCTION Human histamine receptor H4 (hHRH4) is the target for inflammation and allergic therapy [1], such as joints inflammation [2], atopic dermatitis [3], intestines inflammation and digestive allergy [4], also involved with chronic conjunctivitis allergy [5]. Furthermore, the hHRH4 antagonist affects important regulation of lung inflammation allergy [6]. Therefore, the JNJ38518168 antagonist is under Phase II clinical examination as a drug candidate for asthma and rheumatoid arthritis treatment [7]. This receptor antagonist inhibits heart remodeling, maintains contractility, and increases lifetime [8]. Furthermore, the hHRH4 is involved in pruritus pathogenesis that regulates microglia activity [9], giving a new target to inhibit Parkinson's disease (PD) development [10]. In addition, the histamine H4 receptor inhibits cytokine being released in microglia cells [11], immune cells in the central nerves system related to various neurodegenerative diseases such as ischemic stroke and Alzheimer's disorder (AD) [12]. The receptor expresses a potential therapeutic target for inflammation and autoimmune diseases [13]. The hHRH4 is essential in tumor development [14], became the target for cancer and immune cell treatments [15], and increases brain-derived neurotrophic factor (BDNF) in the primary cortical neuron [16]. Several hHRH4 antagonists have reached clinical evaluation, but concerns about the potential for compound-specific toxicity cause premature termination of further studies [17]. Seliforant, a selective hHRH4 antagonist, is well tolerated and binds highly to animal and human receptors. Seliforant has entered phase II clinical trials and is selective for hHRH4 over histamine H1, histamine H2, and histamine H3 receptors [18], providing preclinical support for pharmacokinetics/pharmacodynamics modeling and selection of clinically effective drug concentrations [19]. With its excellent safety profile, seliforant is the first compound accepted for daily oral dosing and the only one so far to enter clinical trials [17]. Considering the importance of hHRH4, especially on inflammation diseases, rheumatoid arthritis, asthma, dermatitis, and psoriasis [20], it is relevant to explore the main aspects of hHRH4. Exploring the receptor as a target for therapy in the future involves the receptor's active site, molecular modeling, and ligand exploration [21]. The main strategy and one of the effective methods in computer-aided drug discovery is structure-based virtual screening (SBVS) [22]. This approach efficiently designs and discovers bioactive compound optimization [23]. Furthermore, SBVS is able to generate direction for advanced drug development [24], accurate structural modeling, activity prediction [25], projecting ligand binding pose, and estimate its affinity towards the target receptor [26]. The development of structure-based drug design and discovery has been hampered by the lack of a crystal structure for the target protein. Meanwhile, information about the 3D structure of proteins is essential for understanding ligand-receptor interactions [27]. Due to the limitations of experimental data, homology modeling is currently the best choice for obtaining structural information. The purpose of homology modeling is to predict the structure of the sequence with the same accuracy as the results obtained experimentally [21]. Furthermore, advances in computational technology, the development of modeling software, and the increasing number of known protein structures have developed homology modeling methods rapidly and reliably to obtain 3D coordinates of proteins [27]. The crystal structure of hHRH4 is not publicly available. Hence, homology modeling is required to generate virtual targets. Therefore, this study is aimed to generate virtual targets in order to develop a screening protocol for hHRH4 ligand identification, started with hHRH4 homology modeling and then followed by molecular docking of seliforant to the homolog model, molecular dynamics (MD) simulations for 100 ns, and validation of the selected virtual target through re-docking simulations. International Journal of Applied Pharmaceutics ISSN- 0975-7058 Vol 14, Issue 4, 2022