Virtual drug repurposing study for the CGRPR identifies pentagastrin and leuprorelin as putative candidates.

Calcitonin gene-related peptide receptor (CGRPR) is a heterodimer consisting of CLR and RAMP1 proteins. Activation of the CGRPR with the endogenous peptide CGRP is known to play a crucial role in migraine pathophysiology. CGRP occupies two regions in the CGRPR upon binding, namely ectodomain and transmembrane sites (sites 1 and 2, respectively). The disruption of the CGRPR heterodimer interface is one of the main strategies to prevent CGRPR activation and its resulting effects. So far, FDA approved monoclonal antibodies and small molecule gepant inhibitors are considered for the treatment of acute or chronic migraine symptoms. However, most of these gepants have severe side effects. Thus, in this study, a virtual drug repurposing approach is applied to CGRPR to find alternative or better molecules that would have a potential to inhibit or block the CLR - RAMP1 interface compared to known gepant molecules. A small molecule library of FDA-approved molecules was screened in these two different binding sites, further simulations were performed and analyzed. The objectives of this study are (i) to repurpose an FDA-approved drug having more potent features for CGRPR inhibition compared to gepants, and (ii) to examine whether the transmembrane binding site (site 2) accepts small molecules or small peptide analogues for binding. As a result of this extensive in silico analysis, two molecules were identified, namely pentagastrin and leuprorelin. It is shown that FDA approved compound rimegepant and the identified pentagastrin molecules form and maintain the interactions through CLR W72 and RAMP1 W74, which are the residues revealed to have an important role in CGRPR antagonism at binding site 1. At binding site 2, the interactions needed to be formed for CGRP binding are not captured by rimegepant nor leuprorelin, yet leuprorelin forms more interactions throughout the simulations, meaning that small molecules are also capable of binding to site 2. Moreover, it is found that the crucial interactions for receptor signaling and heterodimerization occurred between CLR and RAMP1 interface are disrupted more with the ligands bound to ectodomain site, rather than the transmembrane domain. These findings of pentagastrin and leuprorelin molecules are recommended to be considered in further de novo drug development and/or experimental studies related to CGRPR signaling blockade and antagonism.

Screening of Clinically Approved and Investigation Drugs as Potential Inhibitors of SARS-CoV-2: A Combined in silico and in†vitro Study.

In the current study, we used 7922 FDA approved small molecule drugs as well as compounds in clinical investigation from NIH's NPC database in our drug repurposing study. SARS-CoV-2 main protease as well as Spike protein/ACE2 targets were used in virtual screening and top-100 compounds from each docking simulations were considered initially in short molecular dynamics (MD) simulations and their average binding energies were calculated by MM/GBSA method. Promising hit compounds selected based on average MM/GBSA scores were then used in long MD simulations. Based on these numerical calculations following compounds were found as hit inhibitors for the SARS-CoV-2 main protease: Pinokalant, terlakiren, ritonavir, cefotiam, telinavir, rotigaptide, and cefpiramide. In addition, following 3 compounds were identified as inhibitors for Spike/ACE2: Denopamine, bometolol, and rotigaptide. In order to verify the predictions of in silico analyses, 4 compounds (ritonavir, rotigaptide, cefotiam, and cefpiramide) for the main protease and 2 compounds (rotigaptide and denopamine) for the Spike/ACE2 interactions were tested by in vitro experiments. While the concentration-dependent inhibition of the ritonavir, rotigaptide, and cefotiam was observed for the main protease; denopamine was effective at the inhibition of Spike/ACE2 binding.

Molecular simulations reveal the impact of RAMP1 on ligand binding and dynamics of calcitonin gene-related peptide receptor (CGRPR) heterodimer.

Calcitonin gene-related peptide receptor (CGRPR) is a heterodimer protein complex consisting of a class-B G protein-coupled receptor (GPCR) named calcitonin receptor-like receptor (CLR) and an accessory protein, receptor activity modifying protein type 1 (RAMP1). Here in this study, with several molecular modeling approaches and molecular dynamics (MD) simulations, the structural and dynamical effects of RAMP1 on the binding of small molecule CGRPR inhibitors (namely rimegepant and telcagepant) to the CGRPR extracellular ectodomain complex site (site 1) and transmembrane binding site (site 2) are investigated. Results showed that although these molecules stay stable at site 1, they can also bind to site 2, which may be interpreted as non-specificity of the ligands, however, most of these interactions at transmembrane binding site are not sustainable or are weak. Furthermore, to examine the site 2 for gepant binding, different in silico experiments (i.e., alanine scanning mutagenesis, SiteMap, ligand decomposition binding free energy analyses) are also conducted and the results confirmed the putative binding pocket (site 2) of the gepants at the CGRPRs.

Near-physiological-temperature serial crystallography reveals conformations of SARS-CoV-2 main protease active site for improved drug repurposing.

The COVID-19 pandemic has resulted in 198 million reported infections and more than 4 million deaths as of July 2021 (covid19.who.int). Research to identify effective therapies for COVID-19 includes: (1) designing a vaccine as future protection; (2) de novo drug discovery; and (3) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determine two apo structures of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease at ambient temperature by serial femtosecond X-ray crystallography. We employ detailed molecular simulations of selected known main protease inhibitors with the structures and compare binding modes and energies. The combined structural and molecular modeling studies not only reveal the dynamics of small molecules targeting the main protease but also provide invaluable opportunities for drug repurposing and structure-based drug design strategies against SARS-CoV-2.

In silico characterization of adipokinetic hormone receptor and screening for pesticide candidates against stick insect, Carausius morosus.

Adipokinetic hormone (AKH) is an insect neuropeptide that plays crucial roles in a variety of physiological functions such as regulation of heartbeat frequency, blood hemolymph trehalose levels, and protein synthesis. It exerts its functions through binding to its cognate G protein-coupled receptor (GPCR), named adipokinetic hormone receptor (AKHR). The aim of this study is to characterize AKHR of stick insect, Carausius morosus, which becomes an agricultural and forest pest during its outbreaks, and to screen pesticide candidates that would act through inhibition of AKHR. To this aim, the sequence of the receptor and its ligand were obtained from previously published transcriptome data and homology modeling, molecular docking, and molecular dynamics (MD) simulations were combined to find the ligand-binding pocket of AKHR. As a result, crucial residues in ligand binding were identified. These residues were located at the 6th and 7th transmembrane (TM) domains and the 2nd extracellular loop (ECL) of AKHR model. In order to propose pesticide candidates, virtual screening was performed, and candidate ligands were obtained. Considering the binding energies and the stability of the interaction between the ligand and the receptor, four hit compounds were selected. In conclusion, this study revealed a possible ligand-binding pocket of AKHR and proposed some high-affinity small-molecules to block its function, which would further facilitate pesticide design studies against the same receptor of various pests.

Integrated Binary QSAR-Driven Virtual Screening and In Vitro Studies for Finding Novel hMAO-B-Selective Inhibitors.

The increased activity of monoamine oxidase (MAO) enzymes may lead to serious consequences since they reduce the level of neurotransmitters and are associated with severe neurodegenerative diseases. The inhibition of this enzyme, especially the B isoform, plays a vital role in the treatment of Parkinson's disease (PD). This study is aimed to find novel human MAO-B (hMAO-B) selective inhibitors. A total of 256.750 compounds from the Otava small molecules database were virtually screened gradually by employing several screening techniques for this purpose. Initially, a high-throughput virtual screening (HTVS) method was employed, and 10% of the molecules having high docking scores were subjected to binary QSAR models for further screening of their therapeutic activities against PD, Alzheimer's disease (AD), and depression as well as for their toxicity and pharmacokinetic properties. Then, enzyme selectivity of the ligands towards the A and B forms that passed through all the filters were studied using the induced-fit docking method and molecular dynamics simulations. At the end of this exhaustive research, we identified two hit molecules ligand 3 (Otava ID: 7131545) and ligand 4 (Otava ID: 7566820). Based on the in vitro results, these two compounds (ligands 3 and 4) together with ligands 1 and 2 found in our previous study showed activity at the nanomolar (nM) level, and the results indicated that these four ligands inhibit hMAO-B better than the FDA-approved drug selegiline.

Current status of multiscale simulations on GPCRs.

Membrane receptors couple signaling pathways using various mechanisms. G Protein-Coupled Receptors (GPCRs) represent the largest class of membrane proteins involved in signal transduction across the biological membranes. They are essential targets for cell signaling and are of great commercial interest to the pharmaceutical industry. Recent advances made in molecular biology and computational chemistry offer a range of simulation and multiscale modeling tools for the definition and analysis of protein-ligand, protein-protein, and protein-membrane interactions. The development of new techniques on statistical methods and free energy simulations help to predict novel optimal ligands, G protein specificity and oligomerization. The identification of the ligand-binding activation mechanisms and atomistic determinants as well as the interactions of intracellular binding partners that bind to GPCR targets in different coupling states will provide greater safety in human life. In this review, recent approaches and applications of multiscale simulations on GPCRs were highlighted.

Host-Guest Interactions between Candesartan and Its Prodrug Candesartan Cilexetil in Complex with 2-Hydroxypropyl-ß-cyclodextrin: On the Biological Potency for Angiotensin II Antagonism.

Renin-angiotensin aldosterone system inhibitors are for a long time extensively used for the treatment of cardiovascular and renal diseases. AT1 receptor blockers (ARBs or sartans) act as antihypertensive drugs by blocking the octapeptide hormone Angiotensin II to stimulate AT1 receptors. The antihypertensive drug candesartan (CAN) is the active metabolite of candesartan cilexetil (Atacand, CC). Complexes of candesartan and candesartan cilexetil with 2-hydroxylpropyl-ß-cyclodextrin (2-HP-ß-CD) were characterized using high-resolution electrospray ionization mass spectrometry and solid state 13C cross-polarization/magic angle spinning nuclear magnetic resonance (CP/MAS NMR) spectroscopy. The 13C CP/MAS results showed broad peaks especially in the aromatic region, thus confirming the strong interactions between cyclodextrin and drugs. This experimental evidence was in accordance with molecular dynamics simulations and quantum mechanical calculations. The synthesized and characterized complexes were evaluated biologically in vitro. It was shown that as a result of CAN's complexation, CAN exerts higher antagonistic activity than CC. Therefore, a formulation of CC with 2-HP-ß-CD is not indicated, while the formulation with CAN is promising and needs further investigation. This intriguing result is justified by the binding free energy calculations, which predicted efficient CC binding to 2-HP-ß-CD, and thus, the molecule's availability for release and action on the target is diminished. In contrast, CAN binding was not favored, and this may allow easy release for the drug to exert its bioactivity.

Oligomerization and cooperativity in GPCRs from the perspective of the angiotensin AT1 and dopamine D2 receptors.

G Protein-Coupled Receptors (GPCRs) can form homo- and heterodimers or constitute higher oligomeric clusters with other heptahelical GPCRs. In this article, multiscale molecular modeling approaches as well as experimental techniques which are used to study oligomerization of GPCRs are reviewed. In particular, the effect of dimerization/oligomerization to the ligand binding affinity of individual protomers and also on the efficacy of the oligomer are discussed by including diverse examples from the literature. In addition, possible allosteric effects that may emerge upon interaction of GPCRs with membrane components, like cholesterol, is also discussed. Investigation of these above-mentioned interactions may greatly contribute to the candidate molecule screening studies and development of novel therapeutics with fewer adverse effects.

Application of Multiscale Simulation Tools on GPCRs. An Example with Angiotensin II Type 1 Receptor.

G protein-coupled receptors (GPCRs) represent the biggest class of membrane proteins included in signal transduction cascade across the biological lipid bilayers. They are essential target structures for cell signaling and are of great commercial interest to the pharmaceutical industry (~50% of marketed drugs and ~25% of top-selling drugs targeting this receptor family). Recent advances made in molecular biology and computational chemistry open new avenues for the design of new therapeutic compounds. Molecular biology has recently provided the crystal structures of a few ligand-bound GPCRs in active and inactive states, which can be used as accurate templates in modeling studies. Computational chemistry offers a range of simulation, multiscale modeling with ligand- and structure-based approaches, and virtual screening tools for definition and analysis of protein-ligand, protein-protein, and protein-DNA interactions. Development of new approaches and algorithms on statistical methods and free energy simulations help to predict novel optimal compounds. Integrated approach to drug discovery that combines quantum mechanics calculations, molecular docking, molecular dynamics (MD) simulations, quantitative structure-activity relationships (QSAR), and de novo design studies under a single umbrella can be used for decreasing the risk of false-positive results. Each method has its own pros and cons and, when used alone, is not likely to yield very useful results. However, when these methods are combined with positive feedback loops, they may enhance each other and successful drug leads may be obtained. Moreover, investigating the activation mechanisms and atomistic determinants of ligand binding to GPCR targets would allow greater safety in the human life.

Proposing Novel MAO-B Hit Inhibitors Using Multidimensional Molecular Modeling Approaches and Application of Binary QSAR Models for Prediction of Their Therapeutic Activity, Pharmacokinetic and Toxicity Properties.

Monoamine oxidase (MAO) enzymes MAO-A and MAO-B play a critical role in the metabolism of monoamine neurotransmitters. Hence, MAO inhibitors are very important for the treatment of several neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS). In this study, 256 750 molecules from Otava Green Chemical Collection were virtually screened for their binding activities as MAO-B inhibitors. Two hit molecules were identified after applying different filters such as high docking scores and selectivity to MAO-B, desired pharmacokinetic profile predictions with binary quantitative structure-activity relationship (QSAR) models. Therapeutic activity prediction as well as pharmacokinetic and toxicity profiles were investigated using MetaCore/MetaDrug platform which is based on a manually curated database of molecular interactions, molecular pathways, gene-disease associations, chemical metabolism, and toxicity information. Particular therapeutic activity and toxic effect predictions are based on the ChemTree ability to correlate structural descriptors to that property using recursive partitioning algorithm. Molecular dynamics (MD) simulations were also performed to make more detailed assessments beyond docking studies. All these calculations were made not only to determine if studied molecules possess the potential to be a MAO-B inhibitor but also to find out whether they carry MAO-B selectivity versus MAO-A. The evaluation of docking results and pharmacokinetic profile predictions together with the MD simulations enabled us to identify one hit molecule (ligand 1, Otava ID: 3463218) which displayed higher selectivity toward MAO-B than a positive control selegiline which is a commercially used drug for PD therapeutic purposes.

Integration of multi-scale molecular modeling approaches with experiments for the in silico guided design and discovery of novel hERG-Neutral antihypertensive oxazalone and imidazolone derivatives and analysis of their potential restrictive effects on cell proliferation.

AT1 antagonists is the most recent drug class of molecules against hypertension and they mediate their actions through blocking detrimental effects of angiotensin II (A-II) when acts on type I (AT1) A-II receptor. The effects of AT1 antagonists are not limited to cardiovascular diseases. AT1 receptor blockers may be used as potential anti-cancer agents - due to the inhibition of cell proliferation stimulated by A-II. Therefore, AT1 receptors and the A-II biosynthesis mechanisms are targets for the development of new synthetic drugs and therapeutic treatment of various cardiovascular and other diseases. In this work, multi-scale molecular modeling approaches were performed and it is found that oxazolone and imidazolone derivatives reveal similar/better interaction energy profiles compared to the FDA approved sartan molecules at the binding site of the AT1 receptor. In silico-guided designed hit molecules were then synthesized and tested for their binding affinities to human AT1 receptor in radioligand binding studies, using [125I-Sar1-Ile8] AngII. Among the compounds tested, 19d and 9j molecules bound to receptor in a dose response manner and with relatively high affinities. Next, cytotoxicity and wound healing assays were performed for these hit molecules. Since hit molecule 19d led to deceleration of cell motility in all three cell lines (NIH3T3, A549, and H358) tested in this study, this molecule is investigated in further tests. In two cell lines (HUVEC and MCF-7) tested, 19d induced G2/M cell cycle arrest in a concentration dependent manner. Adherent cells detached from the plates and underwent cell death possibly due to apoptosis at 19d concentrations that induced cell cycle arrest.

Structure-based design of hERG-neutral antihypertensive oxazalone and imidazolone derivatives.

Angiotensin II receptor type 1 (AT1) antagonists are the most recent drug class against hypertension. Recently first crystal structure of AT1 receptor is deposited to the protein data bank (PDB ID: 4YAY). In this work, several molecular screening methods such as molecular docking and de novo design studies were performed and it is found that oxazolone and imidazolone derivatives reveal similar/better interaction energy profiles compared to the FDA approved sartan molecules at the binding site of the AT1 receptor. A database consisting of 3500-fragments were used to enumerate de novo designed imidazolone and oxazolone derivatives and hereby more than 50000 novel small molecules were generated. These derivatives were then used in high throughput virtual screening simulations (Glide/HTVS) to find potent hit molecules. In addition, virtual screening of around 18 million small drug-like compounds from ZINC database were screened at the binding pocket of the AT1 receptor via Glide/HTVS method. Filtered structures were then used in more sophisticated molecular docking simulations protocols (i.e., Glide/SP; Glide/XP; Glide/IFD; Glide/QPLD, and GOLD). However, the K+ ion channel/drug interactions should also be considered in studies implemented in molecular level against their cardiovascular risks. Thus, selected compounds with high docking scores via all diverse docking algorithms are also screened at the pore domain regions of human ether-a-go-go-related gene (hERG1) K+ channel to remove the high affinity hERG1 blocking compounds. High docking scored compounds at the AT1 with low hERG1 affinity is considered for long molecular dynamics (MD) simulations. Post-processing analysis of MD simulations assisted for better understanding of molecular mechanism of studied compounds at the binding cavity of AT1 receptor. Results of this study can be useful for designing of novel and safe AT1 inhibitors.

Structure-based design of hERG-neutral antihypertensive oxazalone and imidazolone derivatives.

Angiotensin II receptor type 1 (AT1) antagonists are the most recent drug class against hypertension. Recently first crystal structure of AT1 receptor is deposited to the protein data bank (PDB ID: 4YAY). In this work, several molecular screening methods such as molecular docking and de novo design studies were performed and it is found that oxazolone and imidazolone derivatives reveal similar/better interaction energy profiles compared to the FDA approved sartan molecules at the binding site of the AT1 receptor. A database consisting of 3500-fragments were used to enumerate de novo designed imidazolone and oxazolone derivatives and hereby more than 50000 novel small molecules were generated. These derivatives were then used in high throughput virtual screening simulations (Glide/HTVS) to find potent hit molecules. In addition, virtual screening of around 18 million small drug-like compounds from ZINC database were screened at the binding pocket of the AT1 receptor via Glide/HTVS method. Filtered structures were then used in more sophisticated molecular docking simulations protocols (i.e., Glide/SP; Glide/XP; Glide/IFD; Glide/QPLD, and GOLD). However, the K+ ion channel/drug interactions should also be considered in studies implemented in molecular level against their cardiovascular risks. Thus, selected compounds with high docking scores via all diverse docking algorithms are also screened at the pore domain regions of human ether-a-go-go-related gene (hERG1) K+ channel to remove the high affinity hERG1 blocking compounds. High docking scored compounds at the AT1 with low hERG1 affinity is considered for long molecular dynamics (MD) simulations. Post-processing analysis of MD simulations assisted for better understanding of molecular mechanism of studied compounds at the binding cavity of AT1 receptor. Results of this study can be useful for designing of novel and safe AT1 inhibitors.

Synthesis, biological activity and multiscale molecular modeling studies for coumaryl-carboxamide derivatives as selective carbonic anhydrase IX inhibitors.

New coumaryl-carboxamide derivatives with the thiourea moiety as a linker between the alkyl chains and/or the heterocycle nucleus were synthesized and their inhibitory activity against the human carbonic anhydrase (hCA) isoforms hCA I, II, VII and IX were evaluated. While the hCA I, II and VII isoforms were not inhibited by the investigated compounds, the tumour-associated isoform hCA IX was inhibited in the high nanomolar range. 2-Oxo-N-((2-(pyrrolidin-1-yl)ethyl)carbamothioyl)-2H-chromene-3-carboxamide (e11) exhibited a selective inhibitory action against hCA IX with the Ki of 107.9 nM. In order to better understand the inhibitory profiles of studied molecules, multiscale molecular modeling approaches were used. Different molecular docking algorithms were used to investigate binding poses and predicted binding energies of studied compounds at the active sites of the CA I, II, VII and IX isoforms.

Identification of novel serotonin reuptake inhibitors targeting central and allosteric binding sites: A virtual screening and molecular dynamics simulations study.

The serotonin (5-hydroxytryptamine, 5HT) transporter (SERT) is a member of neurotransmitter sodium symporter (NSS) family, which maintains neurotransmitter by reuptaking 5HT into synapses. Decrease in serotonin concentrations in synaptic clefts have been reported to cause psychological and neurological disorders. Therefore, inhibition of SERT is a potent strategy for the treatment of related diseases such as depression. In this study, approximately 260,000 small molecules from an available chemical database have been virtually screened both at central and allosteric binding sites of SERT to identify potent novel candidate SERT inhibitors. A set of docking algorithms were used to predict binding modes and energies of compounds. Screening analyses led three top-ranked hit compounds (160234, Otava ID: 7118020138; 159166, Otava ID: 7117171303; and 69419, Otava ID: 118671819) for central binding site (S1) and one compound (93507, Otava ID: 6248262) for allosteric binding site (S2). These promising compounds are then subjected to long multiple molecular dynamics (MD) simulations to elucidate their structural and dynamical profiles at the binding cavities of SERT. Higher predicted binding affinities of identified compounds were also confirmed with binding free energy calculations (MM/GBSA) in comparison with the reference central and allosteric binding site inhibitors, paroxetine (8PR) and escitalopram (68P), respectively. To the best of our knowledge, the present work is the first structure-based high throughput virtual screening study reported using recently revealed crystal structure of SERT for screening inhibitors from chemical databases on S1 and S2 binding sites. Small molecule library screening study yielded candidate compounds both at central and allosteric binding site of SERT, and further experimentation may pave the way for developing novel strong inhibitors.

Analysis of the Glutamate Agonist LY404,039 Binding to Nonstatic Dopamine Receptor D2 Dimer Structures and Consensus Docking.

Dopamine receptor D2 (D2R) plays an important role in the human central nervous system and is a focal target of antipsychotic agents. The D2HighR and D2LowR dimeric models previously developed by our group are used to investigate the prediction of binding affinity of the LY404,039 ligand and its binding mechanism within the catalytic domain. The computational data obtained using molecular dynamics simulations fit well with the experimental results. The calculated binding affinities of LY404,039 using MM/PBSA for the D2HighR and D2LowR targets were -12.04 and -9.11 kcal/mol, respectively. The experimental results suggest that LY404,039 binds to D2HighR and D2LowR with binding affinities (Ki) of 8.2 and 1640 nM, respectively. The high binding affinity of LY404,039 in terms of binding to [3H]domperidone was inhibited by the presence of a guanine nucleotide, indicating an agonist action of the drug at D2HighR. The interaction analysis demonstrated that while Asp114 was among the most critical amino acids for D2HighR binding, residues Ser193 and Ser197 were significantly more important within the binding cavity of D2LowR. Molecular modeling analyses are extended to ensemble docking as well as structure-based pharmacophore model (E-pharmacophore) development using the bioactive conformation of LY404,039 at the binding pocket as a template and screening of small-molecule databases with derived pharmacophore models.

Reducing Virulence and Biofilm of Pseudomonas aeruginosa by Potential Quorum Sensing Inhibitor Carotenoid: Zeaxanthin.

Pseudomonas aeruginosa can regulate its virulence gene expressions by using a signal system called quorum sensing. It is known that inhibition of quorum sensing can block biofilm formation and leave the bacteria defenseless. Therefore, it is necessary to determine natural sources to obtain potential quorum sensing inhibitors. This study aims to investigate an alternative treatment approach by utilizing the carotenoid zeaxanthin to reduce the expressions of P. aeruginosa virulence factors through quorum sensing inhibition. The inhibition potential of zeaxanthin was determined by in silico screening from a library of 638 lichen metabolites. Fluorescent monitor strains were utilized for quorum sensing inhibitor screens, and quantitative reverse-transcriptase PCR assay was performed for evaluating gene expression. Results indicate that zeaxanthin is a better inhibitor than the lichen secondary metabolite evernic acid, which was previously shown to be capable of inhibiting P. aeruginosa quorum sensing systems.

Biological Insights of the Dopaminergic Stabilizer ACR16 at the Binding Pocket of Dopamine D2 Receptor.

The dopamine D2 receptor (D2R) plays an important part in the human central nervous system and it is considered to be a focal target of antipsychotic agents. It is structurally modeled in active and inactive states, in which homodimerization reaction of the D2R monomers is also applied. The ASP2314 (also known as ACR16) ligand, a D2R stabilizer, is used in tests to evaluate how dimerization and conformational changes may alter the ligand binding space and to provide information on alterations in inhibitory mechanisms upon activation. The administration of the D2R agonist ligand ACR16 [3H](+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol ((+)PHNO) revealed Ki values of 32 nM for the D2highR and 52 µM for the D2lowR. The calculated binding affinities of ACR16 with post processing molecular dynamics (MD) simulations analyses using MM/PBSA for the monomeric and homodimeric forms of the D2highR were -9.46 and -8.39 kcal/mol, respectively. The data suggests that the dimerization of the D2R leads negative cooperativity for ACR16 binding. The dimerization reaction of the D2highR is energetically favorable by -22.95 kcal/mol. The dimerization reaction structurally and thermodynamically stabilizes the D2highR conformation, which may be due to the intermolecular forces formed between the TM4 of each monomer, and the result strongly demonstrates dimerization essential for activation of the D2R.