Free Energy Profile and Kinetics of Coupled Folding and Binding of the Intrinsically Disordered Protein p53 with MDM2.

Intrinsically disordered proteins (IDPs) exert their functions by binding to partner proteins via a complex process that includes coupled folding and binding. Because inhibiting the binding of the IDP p53 to its partner MDM2 has become a promising strategy for the design of anticancer drugs, we carried out metadynamics simulations to study the coupled folding and binding process linking the IDP p53 to MDM2 in atomic detail. Using bias-exchange metadynamics (BE-MetaD) and infrequent metadynamics (InMetaD), we estimated the binding free energy, the unbinding rate, and the binding rate. By analyzing the stable intermediates, we uncovered the role non-native interactions played in the p53-MDM2 binding/unbinding process. We used a three-state model to describe the whole binding/unbinding process and to obtain the corresponding rate constants. Our work shows that the binding of p53 favors an induced-fit mechanism which proceeds in a stepwise fashion. Our results can be helpful for gaining an in-depth understanding of the coupled folding and binding process needed for the design of MDM2 inhibitors.

Enhanced Sampling Simulations of Ligand Unbinding Kinetics Controlled by Protein Conformational Changes.

Understanding unbinding kinetics of protein-ligand systems is of great importance for the design of ligands with desired specificity and safety. In recent years, enhanced sampling techniques have emerged as effective tools for studying unbinding kinetics of protein-ligand systems at the atomistic level. However, in many protein-ligand systems, the ligand unbinding processes are strongly coupled to protein conformational changes and the disclosure of the hidden degrees of freedom closely related to the protein conformational changes so that sampling is enhanced over these degrees of freedom remains a great challenge. Here, we show how potential-scaled molecular dynamics (sMD) and infrequent metadynamics (InMetaD) simulation techniques can be combined to successfully reveal the unbinding mechanism of 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[18F]fluo-rodibenzo[b,d]thiophene 5,5-dioxide ([18F]ASEM) from a chimera structure of the α7-nicotinic acetylcholine receptor. By using sMD simulations, we disclosed that the "close" to "open" conformational change of loop C plays a key role in the ASEM unbinding process. By carrying out InMetaD simulations with this conformational change taken into account as an additional collective variable, we further captured the key states in the unbinding process and clarified the unbinding mechanism of ASEM from the protein. Our work indicates that combining sMD and InMetaD simulation techniques can be an effective approach for revealing the unbinding mechanism of a protein-ligand system where protein conformational changes control the unbinding process.

Mechanistic insights into peptide and ligand binding of the ATAD2-bromodomain via atomistic simulations disclosing a role of induced fit and conformational selection.

ATAD2 has emerged as a promising bromodomain (BRD)-containing therapeutic drug target in multiple human cancers. However, recent druggability assessment studies predicted ATAD2's BRD as a target 'difficult to drug' because its binding pocket possesses structural features that are unfeasible for ligand binding. Here, by using all-atom molecular dynamics simulations and an advanced metadynamics method, we demonstrate a dynamic view of the binding pocket features which can hardly be obtained from the "static" crystal data. The most important features disclosed from our simulation data, include: (1) a distinct 'open-to-closed' conformational switch of the ZA loop region in the context of peptide or ligand binding, akin to the induced fit mechanism of molecular recognition, (2) a dynamic equilibrium of the BC loop "in" and "out" conformations, highlighting a role in the conformational selection mechanism for ligand binding, and (3) a new binding region identified distal to the histone-binding pocket that might have implications in bromodomain biology and in inhibitor development. Moreover, based on our simulation results, we propose a model for an "auto-regulatory" mechanism of ATAD2's BRD for histone binding. Overall, the results of this study will not only have implications in bromodomain biology but also provide a theoretical basis for the discovery of new ATAD2's BRD inhibitors.

Insight into the adsorption profiles of the Saprolegnia monoica chitin synthase MIT domain on POPA and POPC membranes by molecular dynamics simulation studies.

The critical role of chitin synthases in oomycete hyphal tip growth has been established. A microtubule interacting and trafficking (MIT) domain was discovered in the chitin synthases of the oomycete model organism, Saprolegnia monoica. MIT domains have been identified in diverse proteins and may play a role in intracellular trafficking. The structure of the Saprolegnia monoica chitin synthase 1 (SmChs1) MIT domain has been recently determined by our group. However, although our in vitro assay identified increased strength in interactions between the MIT domain and phosphatidic acid (PA) relative to other phospholipids including phosphatidylcholine (PC), the mechanism used by the MIT domain remains unknown. In this work, the adsorption behavior of the SmChs1 MIT domain on POPA and POPC membranes was systematically investigated by molecular dynamics simulations. Our results indicate that the MIT domain can adsorb onto the tested membranes in varying orientations. Interestingly, due to the specific interactions between MIT residues and lipid molecules, the binding affinity to the POPA membrane is much higher than that to the POPC membrane. A binding hotspot, which is critical for the adsorption of the MIT domain onto the POPA membrane, was also identified. The lower binding affinity to the POPC membrane can be attributed to the self-saturated membrane surface, which is unfavorable for hydrogen-bond and electrostatic interactions. The present study provides insight into the adsorption profile of SmChs1 and additionally has the potential to improve our understanding of other proteins containing MIT domains.

Theoretical study of the binding profile of an allosteric modulator NS-1738 with a chimera structure of the α7 nicotinic acetylcholine receptor.

Potentiation of the function of the α7 nicotinic acetylcholine receptor (α7-nAChR) is believed to provide a possible way for the treatment of cholinergic system dysfunctions such as Alzheimer's disease and schizophrenia. Positive allosteric modulators (PAMs) are able to augment the peak current response of the endogenous agonist of α7-nAChR by binding to some allosteric sites. In this study, the binding profile of a potent type I PAM, NS-1738, with a chimera structure (termed α7-AChBP) constructed from the extracellular domain of α7-nAChR and an acetylcholine binding protein was investigated with molecular docking, molecular dynamics simulation, and free energy calculation methods. We found that NS-1738 could bind to three allosteric sites of α7-AChBP, namely, the top pocket, the vestibule pocket and the agonist sub-pocket. NS-1738 has moderate binding affinities (-6.76 to -9.15 kcal mol-1) at each allosteric site. The urea group is critical for binding and can form hydrogen-bond interactions with the protein. The bulky trifluoromethyl group also has a great impact on the binding modes and binding affinities. We believe that our study provides valuable insight into the binding profiles of type I PAMs with α7-nAChR and is helpful for the development of novel PAMs.

A theoretical study on the molecular determinants of the affibody protein Z(Aß3) bound to an amyloid ß peptide.

Amyloid beta (Aß) peptides are small cleavage products of the amyloid precursor protein. Aggregates of Aß peptides are thought to be linked with Alzheimer's and other neurodegenerative diseases. Strategies aimed at inhibiting amyloid formation and promoting Aß clearance have been proposed and investigated in in vitro experiments and in vivo therapies. A recent study indicated that a novel affibody protein ZAß3, which binds to an Aß40 monomer with a binding affinity of 17 nM, is able to prevent the aggregation of Aß40. However, little is known about the energetic contribution of each residue in ZAß3 to the formation of the (ZAß3)2:Aß complex. To address this issue, we carried out unbiased molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area calculations. Through the per-residue decomposition scheme, we identified that the van der Waals interactions between the hydrophobic residues of (ZAß3)2 and those at the exterior and interior faces of Aß are the main contributors to the binding of (ZAß3)2 to Aß. Computational alanine scanning identified 5 hot spots, all residing in the binding interface and contributing to the binding of (ZAß3)2 to Aß through the hydrophobic effect. In addition, the amide hydrogen bonds in the 4-strand ß-sheet and the π-π stacking were also analyzed. Overall, our study provides a theoretical basis for future experimental improvement of the ZAß3 peptide binding to Aß.

Selective ligands of estrogen receptor ß discovered using pharmacophore mapping and structure-based virtual screening.

AIM: To discover novel ligands of estrogen receptor (ER) ß using pharmacophore mapping and structure-based screening. METHODS: A computer-aided strategy combining pharmacophore mapping and structure-based screening was used to screen the Maybridge and Enamine databases. Yeast two-hybrid (Y2H) assay was used to detect the activity and selectivity of the chosen compounds. The transcriptional activities of the chosen compounds were demonstrated with luciferase reporter assays. The anti-proliferative effects of ER antagonists against MCF-7 and MDA-MB-231 breast cancer cells were examined using MTT assay, and the mechanisms of action were analyzed with flow cytometry analysis and Western blotting. RESULTS: Through in silico screen, 95 compounds were chosen for testing in Y2H assay, which led to 20 potent ligands, including 10 agonists, 8 antagonists and 2 partial agonists with EC50 or IC50 values at µmol/L. Furthermore, 6 agonists exhibited absolute selectivity for ERß, and 3 agonists showed higher selectivity for ERß. The agonists 1g and 1h (10, 25, and 50 µmol/L) dose-dependently increased ER transcriptional activities, whereas the antagonists 2a and 2d (10, 25, and 50 µmol/L) caused dose-dependent inhibition on the activities. The antagonists and partial agonists at 100 µmol/L suppressed the proliferation of ERα positive MCF-7 cells and ERß positive MDA-MB-231 cells, but were more effective against MDA-MB-231 cells. Treatment of MDA-MB-231 cells with antagonists 2a and 2d (25 and 50 µmol/L) dose-dependently increased the population of cells in the S phase. Both 2a and 2d treatment dose-dependently decreased the expression levels of cyclin A and CDK2. Meanwhile, the downregulation of cyclin E was only caused by 2d, while 2a treatment did not cause significant changes in the protein levels of cyclin E. CONCLUSION: The selective ligands discovered in this study are promising drug candidates to be used as molecular probes to explore the differences between ERα and ERß.

Performance evaluation of 2D fingerprint and 3D shape similarity methods in virtual screening.

Virtual screening (VS) can be accomplished in either ligand- or structure-based methods. In recent times, an increasing number of 2D fingerprint and 3D shape similarity methods have been used in ligand-based VS. To evaluate the performance of these ligand-based methods, retrospective VS was performed on a tailored directory of useful decoys (DUD). The VS performances of 14 2D fingerprints and four 3D shape similarity methods were compared. The results revealed that 2D fingerprints ECFP_2 and FCFP_4 yielded better performance than the 3D Phase Shape methods. These ligand-based methods were also compared with structure-based methods, such as Glide docking and Prime molecular mechanics generalized Born surface area rescoring, which demonstrated that both 2D fingerprint and 3D shape similarity methods could yield higher enrichment during early retrieval of active compounds. The results demonstrated the superiority of ligand-based methods over the docking-based screening in terms of both speed and hit enrichment. Therefore, considering ligand-based methods first in any VS workflow would be a wise option.

In silico studies of ASEM analogues targeting α7-nAChR and experimental verification.

The α7 nicotinic acetylcholine receptor (α7-nAChR) is implicated in a variety of neurodegenerative and neuropsychiatric disorders, such as Alzheimer's disease (AD) and schizophrenia. The progress of these disorders can be studied using positron emission tomography (PET) with radiotracers for α7-nAChR. [18F]ASEM and [18F] para-ASEM (also referred to as [18F]DBT-10) are novel and potent α7-nAChR PET radiotracers which have successfully been used in human subjects and nonhuman primates, though further improvement of them is still a pressing task in the community of neurodegeneration research. In this work, we demonstrate the use of modern in silico techniques to predict the binding modes, binding strengths, and residence times for molecular PET tracers binding to proteins, using ASEM and DBT-10 as a showcase of the predictive and interpretational power of such techniques, in particular free energy perturbation theory. The corresponding compounds were synthesized and further tested by in vitro binding experiment for validation. Encouragingly, our in silico modeling can correctly predict the binding affinities of the ASEM analogues. The structure-activity relationships for the ortho- and para-substitutions are well explained at the atomistic level and provide structure-based guiding for the future development of PET tracers for α7-nAChR. A discussion is presented on the complementary use of in silico rational methods based on atomic and electronic principles for in vitro characterization of PET tracers.

Computational Insight into the Binding Profile of the Second-Generation PET Tracer PI2620 with Tau Fibrils.

Abnormal deposition of hyperphosphorylated tau as neurofibrillary tangles (NFTs) is an important pathological hallmark of Alzheimer's disease (AD) and of other neurodegenerative disorders. A noninvasive positron emission tomography (PET) tracer that quantifies neurofibrillary tangles in vivo can enhance the clinical diagnosis of AD and can also be used to evaluate the efficacy of therapeutics aimed at reducing the abnormal aggregation of the tau fibril in the brain. In this paper, we study the binding profile of fibrillar tau aggregates with a PET tracer PI2620, which is a new second generation tau PET tracer that is presently experimentally and clinically studied. The target structure for the tau fibril is based on cryo-electron microscopy (cryo-EM) structure. A multiscale simulation workflow including molecular docking, molecular dynamics simulation, metadynamics simulation, and free energy calculations was implemented. We find that PI2620 can bind to eight surface binding sites, three core binding sites, and one entry site. The binding at the core sites and entry site is found to be much more favorable than that on the surface sites due to stronger hydrophobic interactions and less solvent exposure. Furthermore, the entry site which is formed by the terminal ß-sheets of the fibril is found to have the highest binding affinity to PI2620. Importantly, the binding capacity at the entry site can be much higher than that at other core sites, due to its easy accessibility. Therefore, the entry site is believed to be the major binding site for PI2620. A previous computational study on tracers with tau fibrils reports a maximum of four binding sites. Through use of methods that allow us to locate "cryptic binding sites", we report here additional core sites available for binding and we address the limitation of using the cryo-EM structure alone for structure-based tracer design. Our results could be helpful for elucidating the binding mechanism of imaging tracers with the fibrillar form of tau, a knowledge that in turn can be used to guide the development of compounds with higher affinity and selectivity for tau using structure-based design strategies.

Insights into the synergistic mechanism of target resistance: A case study of N. lugens RDL-GABA receptors and fipronil.

It is known that a single mutation exerts moderate resistance to pesticide, while double mutations (DM) cause severe resistance problem through synergistic effect, and even result in failure application of pesticides. However, little is known about how double mutations would synergistically cause much high resistance level. In this work, computational studies were performed on the interaction of fipronil with N. lugens RDL-GABA receptors, to see how single and double mutations changed receptor structure properties and then conferred distinct resistance levels. The A2'S mutation displayed relative weak influence on receptor structure properties. The R0'Q mutation, which has not been detected in the absence of A2'S, however could deeply alter the electrostatic potential around the inner pore region and significantly narrow the bottom region around -2'Pro. For the DM system, the synergistic effect of two mutations lead to similar pore diameters to the WT system, except for the slightly reduced middle part. Docking study and binding free energy calculation revealed that fipronil displayed binding potencies in the order of WT > A2'S > R0'Q > DM systems, coinciding well with the reported fipronil sensitivity trends and resistance levels.

Mechanistic Insight into the Binding Profile of DCVJ and α-Synuclein Fibril Revealed by Multiscale Simulations.

Parkinson's disease (PD) is a serious neurodegenerative disease and is characterized by abnormal α-synuclein (α-syn) accumulation in Lewy bodies (LB) and Lewy neurites (LN), which makes α-syn an important imaging target for PD. An imaging probe that quantifies fibrillar α-syn can enhance the clinical diagnosis of PD and can also be used to evaluate the efficacy of therapeutics aimed at reducing the abnormal aggregation of the α-syn fibril in the brain. In this paper, we study the binding profile of fibrillar α-syn with a fluorescent probe 4-(dicyanovinyl)julolidine (DCVJ), which is being explored for identifying α-syn imaging agents. A multiscale simulation workflow including molecular docking, molecular dynamics, metadynamics, and QM/MM calculations was implemented. We find that DCVJ can bind to multiple sites of α-syn which are located either at the surface or in the core. Free energy calculations using implicit solvent models reveal that the most favorable binding mode for DCVJ is associated with the core binding site and is further confirmed by metadyamics simulation. Besides, a dynamic binding pathway is discovered, which reveals that DCVJ binds gradually into the core of the fibril passing through several intermediate states. The conformational arrest of the dicyano vinyl group in the fibrillar environment could explain the reason behind the fibril-specific fluorescence of DCVJ. Furthermore, based on hybrid QM/MM calculations, the molecular geometry of the dicyano vinyl group is found to be environment specific which explains why DCVJ serves as a staining agent for such fibrillar-like environments. Our results could be helpful for elucidating the binding mechanism of imaging tracers with the fibrillar form of α-syn and explain their fibrillar-specific optical properties, a knowledge that in turn can be used to guide the design and development of compounds with higher affinity and selectivity for α-syn using structure-based strategies.

Free Energy Profile for Penetration of Pittsburgh Compound-B into the Amyloid ß Fibril.

The amyloid ß (Aß) fibril is a hallmark of Alzheimer's disease (AD) and has therefore served as an important target for early diagnosis of AD. The Pittsburgh Compound-B (PiB) is one of the most famous positron emission tomography (PET) tracers commonly used for in vivo detection of Aß fibrils. Many theoretical studies have predicted the existence of various core binding sites with different microenvironments for probes binding to the Aß fibril. However, little attention has been devoted to how the probes actually penetrate into the different core binding sites. In this study, an integrated molecular modeling scheme is used to study the penetration of PiB into the core binding sites of the Aß1-42 fibril structure recently obtained by cryogenic electron microscopy. We find that there are two core binding sites for PiB with dramatic differences in cavity size and microenvironment properties, and furthermore that the penetration of PiB into site-1 is energetically prohibitive, whereas the penetration into site-2 is much more favorable. Therefore, the binding capacity at site-2 may be larger than that at site-1 despite its lower binding affinity. Our results thus suggest that site-2 may be a major binding site for PiB binding to Aß fibril and emphasize the importance to adopt a full dynamical picture when studying tracer-fibril binding problems in general, something that in turn can be used to guide the development of tracers with higher affinity and selectivity for the Aß fibril.

Computational studies of the binding profile of phosphoinositide PtdIns (3,4,5) P3 with the pleckstrin homology domain of an oomycete cellulose synthase.

Saprolegnia monoica is a model organism to investigate Saprolegnia parasitica, an important oomycete which causes considerable loss in aquaculture every year. S. monoica contains cellulose synthases vital for oomycete growth. However, the molecular mechanism of the cellulose biosynthesis process in the oomycete growth is still poorly understood. Some cellulose synthases of S. monoica, such as SmCesA2, are found to contain a plecsktrin homology (PH) domain, which is a protein module widely found in nature and known to bind to phosphoinositides, a class of signaling compounds involved in many biological processes. Understanding the molecular interactions between the PH domain and phosphoinositides would help to unravel the cellulose biosynthesis process of oomycetes. In this work, the binding profile of PtdIns (3,4,5) P3, a typical phosphoinositide, with SmCesA2-PH was studied by molecular docking, molecular dynamics and metadynamics simulations. PtdIns (3,4,5) P3 is found to bind at a specific site located at ß1, ß2 and ß1-ß2 loop of SmCesA2-PH. The high affinity of PtdIns (3,4,5) P3 to SmCesA2-PH is contributed by the free phosphate groups, which have electrostatic and hydrogen-bond interactions with Lys88, Lys100 and Arg102 in the binding site.

A High Affinity Red Fluorescence and Colorimetric Probe for Amyloid ß Aggregates.

A major challenge in the Alzheimer's disease (AD) is its timely diagnosis. Amyloid ß (Aß) aggregates have been proposed as the most viable biomarker for the diagnosis of AD. Here, we demonstrate hemicyanine-based benzothiazole-coumarin (TC) as a potential probe for the detection of highly toxic Aß42 aggregates through switch-on, enhanced (~30 fold) red fluorescence (Emax = 654 nm) and characteristic colorimetric (light red to purple) optical outputs. Interestingly, TC exhibits selectivity towards Aß42 fibrils compared to other abnormal protein aggregates. TC probe show nanomolar binding affinity (Ka = 1.72 × 10(7) M(-1)) towards Aß42 aggregates and also displace ThT bound to Aß42 fibrils due to its high binding affinity. The Aß42 fibril-specific red-shift in the absorption spectra of TC responsible for the observed colorimetric optical output has been attributed to micro-environment change around the probe from hydrophilic-like to hydrophobic-like nature. The binding site, binding energy and changes in optical properties observed for TC upon interaction with Aß42 fibrils have been further validated by molecular docking and time dependent density functional theory studies.

Structural and functional characterization of the microtubule interacting and trafficking domains of two oomycete chitin synthases.

UNLABELLED: Chitin synthases (Chs) are responsible for the synthesis of chitin, a key structural cell wall polysaccharide in many organisms. They are essential for growth in certain oomycete species, some of which are pathogenic to diverse higher organisms. Recently, a microtubule interacting and trafficking (MIT) domain, which is not found in any fungal Chs, has been identified in some oomycete Chs proteins. Based on experimental data relating to the binding specificity of other eukaryotic MIT domains, there was speculation that this domain may be involved in the intracellular trafficking of Chs proteins. However, there is currently no evidence for this or any other function for the MIT domain in these enzymes. To attempt to elucidate their function, MIT domains from two Chs enzymes from the oomycete Saprolegnia monoica were cloned, expressed, and characterized. Both were shown to interact strongly with the plasma membrane component, phosphatidic acid, and to have additional putative interactions with proteins thought to be involved in protein transport and localization. Aiding our understanding of these data, the structure of the first MIT domain from a carbohydrate-active enzyme (MIT1) was solved by NMR, and a model structure of a second MIT domain (MIT2) was built by homology modeling. Our results suggest a potential function for these MIT domains in the intracellular transport and/or regulation of Chs enzymes in the oomycetes. DATABASE: Structural data are available in the Biological Magnetic Resonance Bank (BMRB) database under the accession number 19987 and the PDB database under the accession number 2MPK.

Investigation of the Binding Profiles of AZD2184 and Thioflavin T with Amyloid-ß(1-42) Fibril by Molecular Docking and Molecular Dynamics Methods.

Detecting deposits of amyloid ß fibrils in the brain is of paramount importance for an early diagnosis of Alzheimer's disease. A number of PET tracers have been developed for amyloid imaging, but many suffer from poor specificity and large signal to background ratio. Design of tracers with specificity and improved binding affinity requires knowledge about various potential binding sites in the amyloid ß fibril available for the tracers and the nature of the local microenvironment of these sites. In this study we investigate the local structure of fibrils using two important probes, namely, thioflavin T (a fluorescent probe) and AZD2184 (a PET tracer). The target structures for amyloid-ß(1-42) fibril are based on reported NMR solution models. By explicitly considering the effect of fibril flexibility on the available binding sites for all these models, the binding affinity of these probes has been investigated. The binding profiles of AZD2184 and thioflavin T were studied by molecular docking and molecular dynamics simulation methods. The two compounds were found to bind at the same sites of the fibril: three of which are within the fibril, and one is on the two sides of the Met35 residue on the surface. The binding affinity of AZD2184 and thioflavin T is found to be higher at the core sites than on the surface due to more contact residues. The binding affinity of AZD2184 is much higher than that of thioflavin T at every site due to electrostatic interaction and spatial restriction, which is in good agreement with experimental observation. However, the structural change of thioflavin T is much more significant than that of AZD2184, which is the chemical basis for its usage as a fluorescent probe. The ramifications of these results for the design and optimization of PET radioligands and fluorescent probes are briefly discussed.

In silico investigation of interactions between human cannabinoid receptor-1 and its antagonists.

Cannabinoid receptor-1 (CB(1)) is widely expressed in the central nervous system and plays a vital role in regulating food intake and energy expenditure. CB(1) antagonists such as Rimonabant have been used in clinic to inhibit food intake, and therefore reduce body weight in obese animals and humans. To investigate the binding modes of CB(1) antagonists to the receptor, both receptor- and ligand-based methods were implemented in this study. At first, a pharmacophore model was generated based on 31 diverse CB(1) antagonists collected from literature. A test set validation and a simulated virtual screening evaluation were then performed to verify the reliability and discriminating ability of the pharmacophore. Meanwhile, the homology model of CB(1) receptor was constructed based on the crystal structure of human ß (2) adrenergic receptor (ß (2)-AR). Several classical antagonists were then docked into the optimized homology model with induced fit docking method. A hydrogen bond between the antagonists and Lys192 on the third transmembrane helix of the receptor was formed in the docking study, which has proven to be critical for receptor-ligand interaction by biological experiments. The structure obtained from induced fit docking was then confirmed to be a reliable model for molecular docking from the result of the simulated virtual screening. The consistency between the pharmacophore and the homology structure further proved the previous observation. The built receptor structure and antagonists' pharmacophore should be useful for the understanding of inhibitory mechanism and development of novel CB(1) antagonists.

Discovery and structure-activity analysis of selective estrogen receptor modulators via similarity-based virtual screening.

A number of selective estrogen receptor modulators (SERMs) were discovered from the SPECS database via a simple protocol. Based on two reference SERMs we identified via structure-based virtual screening previously, ligand-based similarity search and molecular docking filtering were conducted to identify novel SERMs from SPECS library. Among the 36 purchased compounds, 21 were confirmed to be active by in vitro assays, and 10 showed dual profile properties, namely as antagonists of ERα and agonists of ERß. The anti-proliferative potency of these ligands was also evaluated against MCF-7 cell lines. Further investigation of the anti-proliferative mechanism of compound 3a suggested that it induced a G1 cell cycle arrest in ERα positive MCF-7 cell through ERα mediated cyclin D1 down-regulation.