identification of novel rab27a/melanophilin blockers by pharmacophore-based virtual screening

Identification of Novel Rab27a/Melanophilin Blockersby Pharmacophore-Based Virtual Screening

Jong Young Joung & Ha Yeon Lee & Jongil Park &

Jee-Young Lee & Byung Ha Chang & Kyoung Tai No &

Ky-Youb Nam & Jae Sung Hwang

Received: 19 March 2013 /Accepted: 30 October 2013 /Published online: 30 November 2013# Springer Science+Business Media New York 2013

Abstract Melanocytes are unique cells that produce specific melanin-containing intracellularorganelles called melanosomes. Melanosomes are transported from the perinuclear area ofmelanocytes toward the plasma membrane as they become more melanized in order to increaseskin pigmentation. In this vesicular trafficking of melanosomes, Rab27a, melanophilin, andmyosin Va play crucial roles in linking melanosomes to actin-based motors. To identify novelcompounds to inhibit binding interface between Rab27a and melanophilin, a pharmacophoremodel was built based on a modeled 3D structure of the protein complex that describes theessential binding residues in the intermolecular interaction. A pharmacophore model wasemployed to screen a chemical library database. Finally, 25 virtual hits were selected for biologicalevaluations. The biological activities of 11 analogues were evaluated in a second assay. Twocompounds were identified as having concentration-dependent inhibitory activity. By analyzingstructure–activity relationships of derivatives of BMD-20, two hydroxyl functional groups werefound to be critical for blocking the intermolecular binding between Rab27a and melanophilin.

Keywords Rab27a/melanophilin . Pharmacophore-based virtual screening . Skin pigmentation .

Mekanosome tansport .Molecular docking

Appl Biochem Biotechnol (2014) 172:1882–1897DOI 10.1007/s12010-013-0615-2

J. Y. Joung and H. Y. Lee contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s12010-013-0615-2)contains supplementary material, which is available to authorized users.

J. Y. Joung : J.<Y. Lee : B. H. Chang :K.<Y. Nam (*)Bioinformatics and Molecular Design Research Center, Seoul 120-749, Republic of Koreae-mail: [email protected]

K.<Y. NamGachon Institute of Pharmaceutical Sciences, Gachon University, Incheon 406-840, Republic of Korea

K. T. NoDepartment of Biotechnology and Translational Research Center for Protein Function Control, YonseiUniversity, Seoul 120-749, Republic of Korea

H. Y. Lee : J. Park : J. S. Hwang (*)Department of Genetic Engineering and Skin Biotechnology Center, Kyung Hee University,Yongin 446-701, Republic of Koreae-mail: [email protected]

http://dx.doi.org/10.1007/s12010-013-0615-2

Introduction

With the increasing demand for functional cosmetics, performance-driven researchefforts have focused on new functional ingredients from both natural and syntheticchemical sources. Skin whitening is the one of the important issues being consideredin the field of functional cosmetics. The discovery of new skin-whitening materialsrequires biological studies of pigmentation in the skin.

Melanocytes are specialized cells that synthesize and produce melanin. Skin mela-nocytes are localized in the basal layer of the epidermis. In mammalian melanocytes,specific melanin-producing intracellular organelles called melanosomes mature througha series of morphologically defined stages [1]. Typically, membranous organelles aretransported via microtubules (for long-range mobility) and actin filaments (for short-range movement) [2]. Upon maturation of melanosomes in the perinuclear area,kinesins capture melanosomes and transport them to the periphery of the cell throughmicrotubules. At the periphery, melanosomes are released from the microtubules andbind to neighboring actin filaments by molecular motor proteins [3]. From theperiphery, melanosomes are shifted to keratinocytes. In the melanocytes of patientswith Griscelli syndrome, actin-based movement is impaired and clustering of melano-somes in the perinuclear area is observed [3, 4]. Rab7 and Rab27a control two motorprotein activities, and Rab27a, melanophilin, and myosin Va are involved in actin-based transport of melanosomes [5]. In melanocytes, melanophilin can simultaneouslyassociate with activated Rab27a and myosin Va in melanosome transport [6]. Byreconstitution using purified proteins, Wu et al. showed that Rab27a and melanophilinare sufficient to form a transport complex with myosin Va in vitro [7]. Aftermelanosome transport, the second Rab27a effector exophilin4 (Slp2-a) promotes theanchoring of melanosomes to the plasma membrane of melanocytes [8, 9]. Kukimoto-Niino et al. employed biochemical and structural methods to elucidate the molecularmechanism of the activation/inactivation of Rab27a during melanosome transport [9].In three independent mutations [i.e., dilute mice (myosin Va null), ashen mice (Rab27anull), and leaden mice (melanophilin null)], the loss of any of these three componentsresults in defects in melanosome distribution [4, 10]. Rab27a primarily plays an inhibitoryrole in regulating the release of granules at the plasma membrane in Rab27-deficientmutant mice [11].

We investigated molecular interactions between these motor proteins in transportcomplexes to find skin-whitening compounds for use as functional cosmetics ingredients.In Griscelli syndrome, a skin-whitening effect arising from a decrease in skin pigmenta-tion is observed. Missense mutations in the RAB27A gene (Trp73Gly, Leu130Pro, andAla152Pro) are associated with Griscelli syndrome, and these mutations markedly affectboth the guanosine triphosphate (GTP) and guanosine diphosphate (GDP) nucleotide-binding activities of the Rab27a protein [12]. The other residues of Leu84, Phe88, andAsp91 in Rab27a are not conserved in the Rab3 or Rab8 isoforms, but mutations(Tyr6Phe, Leu84Ile, Phe88Tyr, and Asp91Gly) of Rab27a exhibit reduced melanophilin-binding activity [13]. Since protein complexes of Rab27a (which belongs to the mono-meric Ras-like GTPase superfamily [14]) and melanophilin can be modeled based on X-ray structures, the binding interface between Rab27a and melanophilin was selected as themolecular target for virtual screening. Protein–protein inhibition is expected by blockingthe binding interface between Rab27a and melanophilin.

Virtual screening for blockers of the binding interface between Rab27a and melanophilinwas performed according to standard pharmacophore-based screening procedures. The

Appl Biochem Biotechnol (2014) 172:1882–1897 1883

protocol of structure-based pharmacophore modeling involves an analysis of the complemen-tary chemical features of the binding interface between Rab27a and melanophilin and theirspatial relationships. Pharmacophore modeling provides initial hits for further biologicalevaluation and optimization to suggest lead compounds. After biological evaluation, twoblockers of the binding interface between Rab27a and melanophilin were ultimately selected,and structure–activity relationships were analyzed based on the results of a melanosomeaggregation assay. The overall virtual screening workflow is shown in Fig. 1.

Materials and Methods

Molecular Modeling of Rab27a/Melanophilin Complex

The structure of the Rab27a/melanophilin complex has not been determined although thestructures of Rab27a and melanophilin were independently published as X-ray structures ofRab27a/Slp2-a (PDB ID, 3BC1) and Rab27b/melanophilin (Slac2-a) (PDB ID, 2ZET). Be-cause Rab27a and Rab27b are highly similar in sequence homology, Rab27a from the X-raycomplex structure of Rab27a/Slp2-a was superimposed on the Rab27b structure in theRab27b/melanophilin complex to generate the molecular model of Rab27a/melanophilin. In

Fig. 1 Workflow for the identification of blockers of the binding interface between Rab27a and melanophilin

1884 Appl Biochem Biotechnol (2014) 172:1882–1897

order to refine the model, energy minimization for side chains in the binding complex wasperformed with an atomic fixed constraint on backbone structures to prevent dislocation ofbackbone position and orientation from the reference structure. The accuracy of the modelstructure was confirmed based on the location of interacting residues or essential residues(Arg29, Glu32, Gly 116, Tyr121, and Gly133 in melanophilin) in the binding interface, whichwere identified in site-directed mutagenesis experiments [9, 13].

Receptor-Based Pharmacophore Design

On the basis of the model structure of the Rab27a/melanophilin complex, pharmacophorequeries were generated to describe intermolecular interactions in the binding interface betweenTyr6 of Rab27a and Glu32 of melanophilin, and Asp91 of Rab27a and Arg29 of melanophilin.Structure-based pharmacophore models [15, 16] were generated using a structure-basedfocusing module in Discovery Studio (Accelrys Inc., San Diego, CA, USA) version 3.0[17]. All pharmacophore features were generated for all atoms within a radius of 10 Å fromthe geometric center of the side chain of Arg29 in melanophilin. The pharmacophore featurescorrespond to inhibitor interactions with binding residues of melanophilin. Exclusion volumespheres were placed on all heavy atoms of the protein by using a 0.9-Å radius. Virtualcompounds library (190,000) was used with commercially available synthetic compoundsfrom ChemBridge, Ltd. (http://www.chembridge.com). Virtual compounds library withmultiple conformers were transferred to the 3D chemical database by applying the catDButility, as implemented in Catalyst™ (Accelrys) [18]. Multiple conformers were generatedusing the FAST search method. All other parameters were applied as default values. Afterassessing the query pharmacophore models, virtual screening was carried out using Catalyst.Among the pharmacophore-based virtual screening hit compounds, those exhibitingunfavorable interactions with the binding site or unrealistic conformations were filtered out byvisual inspection. Finally, we selected and purchased 25 compounds for further melanosomeaggregation measurement.

Lead Optimization by Docking Study

Docking calculations were performed on a selection of derivatives of initial hits [19].Protein coordinates for docking were taken from the melanophilin structure (PDB ID,2ZET). All calculations were performed using default parameters except the ligandconformation. For a given ligand, the method was an iterative procedure in whichrandom ligand conformations were generated up to 5,000 times. The top 10 dockedconformations were saved as results. Docking scores were evaluated using a set ofscoring functions such as Ludi [20], PLP1, PLP2 [21], LigScore1, LigScore2 [22],and PMF [23]. A consensus scoring function was used to evaluate and rank the ligandbinding affinities. These studies were performed using the LigandFit [19] module asimplemented in Discovery Studio (Accelrys).

Melanosome Aggregation Measurement

Melan-a cells were maintained in RPMI 1640 growth medium supplemented with 10 % (v/v)FBS, PMA 200 nmol, and 1 % (v/v) penicillin–streptomycin, in 10 % CO2 at 37 °C. Melan-acells were incubated at a density of 1×105 cells in 24-well plates overnight. After washing inDPBS, melan-a cells were treated with diluted samples of RPMI 1640 with 2 % FBS for3 days. The cells were observed by bright-field microscopy using an Olympus CKX41 culture


http://www.chembridge.com/

microscope (Olympus, Japan), and images were photographed using a DMCe camera (INSIndustry, Korea).

A sample of 20 μM siRNA for melanophilin (MLPH) was used as a positive control. ThesiRNA was mixed with Lipofectamine (Invitrogen) and the cells were treated for 48 h. Thefollowing primer pairs were used for siRNA transfection: MLPH sense (5′-GGGCAAAAUACAAAAGGAGUU-3′) and MLPH antisense (5′-CUCCUUUUGUAUUUUGCCUU-3′).

Evaluation of melanosome aggregation was performed by counting perinuclearmelanosome-aggregated cells in three randommicroscopic fields per well at ×200magnification.Values represent the mean ± SD.

Results

Molecular Model of Rab27a/Melanophilin Complex

The reliability of the molecular model of the Rab27a/Melanophilin complex was confirmedbased on site-directed mutagenesis data collected from the literature. Kukimoto-Niino et al.determined the X-ray structure of the Rab27b/melanophilin complex and performed mutationexperiments to identify the key residues of the binding interface between Rab27b andmelanophilin [9]. Fukuda performed mutation experiments to investigate recognition betweenSLA1 homology domain 1 (SHD1) of Rab27a and melanophilin by using alanine-based site-directed mutagenesis [13]. In the model structure, Tyr6 of Rab27a forms the hydrogen bondinginteraction with Glu32 of melanophilin in the same way as in the X-ray structure of theRab27b/melanophilin complex. The intermolecular interactions between Asp91 of Rab27a andArg29 and Tyr121 of melanophilin were not experimentally observed [13]. However, Arg29 ofmelanophilin is strongly engaged in an intermolecular electrostatic interaction with the Asp91carbonyl group of Rab27a in the molecular modeling Rab27a/melanophilin complex (Fig. 2).The nonpolar residues (i.e., Val21 and Val18 on melanophilin and Phe88 and Leu84 onRab27a) can form hydrophobic contacts that are essential in recognition and binding in theRab27a/melanophilin complex (Fig. 2). The corresponding residues in the binding interfaceare summarized in Table 1. The single Asp91Gly mutation and the triple mutations(Leu84Ile, Phe88Tyr, and Asp91Gly) of Rab27a markedly reduced the binding activityof melanophilin [13].

Optimizing Pharmacophore Model

The critical residues responsible for melanophilin binding to Rab27a have been identified bysite-directed mutagenesis experiments [9, 13]. For the pharmacophore design of virtualscreening for blockers of the binding interface between Rab27a and melanophilin, we pre-dicted the molecular model of the Rab27a/melanophilin complex (Fig. 2). The correspondingresidues on melanophilin interacting with the key residues of Rab27a were as follows: Glu32for Tyr6 of Rab27a, Arg29 and Tyr121 for Asp91 of Rab27a. The residues Leu84 and Phe88of Rab27a were involved in hydrophobic interactions (Fig. 2 and Table 1). Several bindingfeatures in the pharmacophore model were designed to map onto the key residues in the side ofthe melanophilin interface (Fig. 3). One hydrogen bond donor feature was placed on Glu32and another hydrogen bond donor feature was placed on Gly116, which was positioned on thehelix adjacent to Glu32, to represent the hydrogen bond interaction in the upper side of thebinding interface of melanophilin. One hydrogen bond acceptor feature and one hydrogenbond donor feature were mapped on Arg29 and Tyr121, respectively, in the middle of the


binding interface. One hydrophobic feature was placed on the aromatic ring of Phe88 ofRab27a to describe a hydrophobic interaction.

Virtual Screening of Rab27a/Melanophilin

In order to identify blockers of binding interface between Rab27a and melanophilin, the 3Dchemical database was screened using the optimized pharmacophore model. A total of 323compounds were scored as virtual hits by the pharmacophore model. Drug-likeness filteringwas utilized to yield 225 compounds, which have potential for development as novel activecompounds. Several scoring functions such as Ludi [20], PLP1, PLP2 [21], LigScore1,LigScore2 [22], and PMF [23], and a consensus scoring function were calculated. Afterconsensus scoring (over a score of 5), the remaining compounds were clustered and visuallyinspected. The consensus scoring provided a reduction in the number of false-positives

Fig. 2 The molecular modeling structure of the Rab27a/melanophilin complex and interacting residues in thebinding interface. Rab27b/melanophilin (PDB ID, 2ZET) structure was used as a template

Table 1 Essential residues of Rab27a involved in binding with melanophilin and corresponding residues ofmelanophilin interacting with each residue in the binding interface

Interacting residues (site-directed mutation) [9, 13]

Rab27a Melanophilin

Tyr6 (Y6F) Glu32 (E32A)

Asp91 (D91G)[ Arg29, Tyr121

Leu84 (L84I), Phe88 (F88Y) Val18 (V18A), Val21 (V21A)


compared to individual scoring functions and improved the hit rates from virtual screening[24]. Finally, we selected 25 hit compounds for further biological assays (Fig. 4).

Biological Evaluation of Virtual Hit Compounds

In order to determine the inhibition activity of melanosome transport, 25 selected virtual hitcompounds were purchased and assayed using melan-a cells. Three compounds—BMD-11,BMD-20, and BMD-25—were initially identified as strong active blockers to reduce melano-some transport. Because BMD-25 was identified as a cytotoxic compound from an MTT assay(data not shown), it was discarded from further biological evaluation. Six compounds—BMD-3, BMD-4, BMD-10, BMD-12, BMD-13, BMD-21, and BMD-25—showed low activity(Fig. 5). The other compounds showed no activity at reducing melanosome transport fromthe nucleus to the dendritic sites of a melanoma cell.

In Fig. 6, the concentration-dependent activities of three highly active compounds in thevirtual hit compounds (BMD-11, BMD-20, and BMD-25) are shown. Compounds BMD-11,BMD-20, and BMD-25 were tested at three different concentrations (7.5 μM, 5 μM, and2.5 μM) and displayed increasing inhibition of melanosome transport with increasing concen-trations. The inhibition activity shown by BMD-25 at 4 μM treatment was similar to thatshown by BMD-20 at 5 μM. Since BMD-20 was shown to be more active without toxicity,additional assays were performed to confirm the inhibitory activity. The assay results areshown in the Supplementary material.

SAR Analysis and Docking Study of Rab27a/Melanophilin Binding Blockers

Alistair et al. investigated the roles of Rab27a and myosin Va as the primary interactionproteins with melanophilin involved in melanosome transport [25]. Treatment withsiRNA of Rab27a and myosin Va disrupted the interactions between melanosometransport machineries, Rab27a, myosin Va, and melanophilin. In this work, we usedsiRNA of melanophilin as the positive control for inhibition of melanosome transportsince the molecular target for designing inhibitors of melanosome transport is the bindinginterface of melanophilin bound to Rab27a.

Fig. 3 Pharmacophore model ofthe binding interface ofmelanophilin


The binding modes of two blockers (BMD-11 and BMD-20) with melanophilin aredescribed in Fig. 7. Two hydroxyl groups on one aromatic ring interact with Glu-32, whichacts as a hydrogen bond acceptor. The binding modes show that the linker region between twoaromatic rings is involved in the hydrogen bond interactions with Arg29 and Tyr121. Theopposite parts of di-hydroxyl aromatic ring are stretched toward Gly133 between two α-helixes. As shown in the binding poses in Fig. 7, hydrogen bond interactions caused by twohydroxyl groups were expected to be the critical features in the binding between blockers andmelanophilin. In order to confirm the need for two hydroxyl groups (as hydrogen-bond

Fig. 4 Hit compounds from pharmacophore-based screening of an in-house commercial library


donors), modified analogue compounds with one or two methoxy groups in place of thehydroxyl groups (to block hydrogen bond donors) were tested in further biological assays(Fig. 8). To evaluate the effect of the pyridine ring in BMD-11 and the acetohydrozide inBMD-20, we selected similar analogues by a docking study. Docking is one of the methods topredict the binding mode between a small molecule (or inhibitor) and a receptor protein. It ishypothesized that if a molecule binds to the active site of a target protein with a physico-chemically stable conformation, this molecule can then control the activity of the protein [26].Although docking can be complicated by finding the optimized conformation of a molecule,this problem can be overcome by flexible docking or protein-induced docking methods. Thedocking results may be estimated by force field-based scoring functions for the interactionenergy between molecule and protein [26]. On the basis of these scoring functions, we canpredict properties of the molecule, such as binding affinity. Therefore, docking plays animportant role in the field of drug design.

In Table 2, three compounds derived from the scaffold of the more active compound (BMD-20) are shown along with their activity. Assay results showed no inhibitory activity, confirmingour assumption from SAR analysis that the two hydroxyl groups in themeta- and para- positionsof the phenyl group are key pharmacophore features as hydrogen bond donors. These three

Fig. 5 Quantification of the numbers of aggregated cells treated with 25 compounds. After 72 h of treatment,numbers of aggregated cells were counted. Compounds BMD-10, BMD-11, BMD-20, and BMD-25 displayedsignificant aggregation of melanosomes in the cell

Fig. 6 Quantification of number of aggregated cells with compounds BMD-11, BMD-20, and BMD-25 (n=3)compared to the negative control. After 72 h of treatment with each compound, the number of aggregated cellswas expressed relative to control. A dose-dependent increase in the number of melanosome-aggregated cells wasobserved with the BMD compounds compared to the negative control. Values represent the mean ± SD


analogues that removed sources of hydrogen-bond donation showed that the hydroxyl groups areessential features for hydrogen bonding interactions in the binding of blockers and melanophilin.

After confirming the significance of the two hydroxyl groups, we analyzed the contributionof structural diversification on the opposite aromatic rings. Eleven analogues of BMD-11 werepurchased from ChemBridge Ltd. (San Diego, CA, USA) and assayed using melan-a cells(Fig. 9). Among these analogues, the compounds with bulky substituents in the ortho- andmeta- positions in the benzyl or pyridine rings showed no activity. These results imply that thebulky substituent in ortho- and meta- positions in the benzyl or pyridine rings inhibitedhydrogen bond formation between the blocker and side chains of Arg29 or Tyr121. As shownin Fig. 10, the docking poses of three compounds (from among the 11 analogues) werepredicted to analyze the formation of essential intermolecular interactions. Because of themethyl group in the quinolone ring of the compound assay-2-1, the docking poses forming theessential hydrogen bonding interaction between NH in the amide linker and Tyr121 were notgenerated. The ethoxy substituent in the compound assay-2-3 caused a gap and distorted thehydrogen bonding interaction between Tyr121 and NH in the linker. The compound assay-2-8has a phenanthridine group that binds to the Arg29 surface horizontally, increasing the distanceto the source of the hydrogen bonding interaction, Tyr121. Only the compound assay-2-6, withbromo-benzyl group in the benzyl ring, shows activity to interfere with melanosome transport.The SAR analysis showed that the compounds with substituents that can cause steric hindranceand deplete binding affinity cannot inhibit melanosome transport.

Discussion

In order to find novel hit compounds to inhibit melanosome transportation from the nucleus tothe peripheral dendritic site in melanocytes, virtual screening methods were applied to search acommercially available compound database based on the molecular interaction featuresbetween key transport machineries, Rab27a and melanophilin. A molecular model of theRab27a/melanophilin complex was built because of the lack of an X-ray crystal structure of thecomplete complex. A pharmacophore model defined by experimental evidence of key residues

Fig. 7 Binding modes of BMD-11 and BMD-20, which are highly active in blocking the binding interfacebetween Rab27a and melanophilin


was used in the virtual screening to suggest candidate compounds for melanosome transportinhibition. Through a cell-based assay to observe melanosome aggregation, the inhibitoryactivities of hit compounds were verified. In the cell-based assay, two compounds—BMD-11and BMD-20—showed strong inhibition of melanosome transport. Derivatives of these twocompounds were evaluated in the assay to describe the key binding interactions hypothesizedin the structure–activity relationships. The hydrophobic interaction with the α-helix containingthe essential residues of melanophilin (e.g., Val18 and Val21) in binding with Rab27a was animportant feature in designing inhibitors. From the results of the third cell-based assay, thehydrogen bond interaction by the di-hydroxyl benzyl ring and the hydrophobic interaction

Fig. 8 Cell-based assay of chemical compounds to inhibit melanosome transport. Negative control consists oftreatment with DMSO, while positive control consists of treatment with melanophilin (MLPH) siRNA, whichinhibits melanosome transport by reducing MLPH expression. Melanocytes treated with an active compoundshow melanosome aggregation near the nucleus and cells treated with non-active compounds show no melano-some aggregation


were considered to be the essential features to bind with Glu32 and the adjacent Gly216residue through hydrogen bonding interactions.

To date, no inhibitors targeting the protein–protein interactions of Rab27a and melanophilinhave been discovered to be used for functional materials in medicine or cosmetics. Our

Table 2 Additional chemical compounds tested in third assay

Compounds Structure Active or inactive logP*

BMD-20-1 Inactive 5.379



BMD-20 Active 2.022

* LogP calculated based on Solvation Free Energy Density (SFED) model [36] of compounds, which were abinitio optimized with the HF/6-31G(d) basis set

Fig. 9 Eleven derivative compounds evaluated in second biological assay


research to identify potent hit compounds for blocking the binding interface between Rab27aand melanophilin can be applied to functional cosmetics that are concerned with skinwhitening. Because of limitations in protein–protein binding blockers, the possibility ofsuccess to discover and develop effective blockers of the binding interface between Rab27aand melanophilin remains uncertain. However, recent successes in the discovery of effectiveprotein–protein interaction blockers in tumor-related protein complexes can address theseuncertainties. An important cell cycle regulator, p53, promotes the transcription of genes thatcontrol cell cycle arrest and apoptosis [27]. It is known that in approximately 50 % of all typesof human cancer, p53 is inactivated by mutation and deletion, and wild-type p53 is downreg-ulated by overexpression or amplification of murine double minute 2 (MDM2) [28–31].Several successful cases of discovery of protein–protein inhibitors, which can inhibit theMDM2-p53 protein–protein interaction, have been reported [32, 33], and some clinicalcandidates have advanced into clinical trials [34, 35]. Similar to these achievements in thediscovery of inhibitors for protein–protein interactions, novel inhibitors in blocking thebinding interface between Rab27a and melanophilin can be expected to be a successful casefor discovering lead compounds as functional ingredients for use in cosmetics.

Fig. 10 Binding modes of three compounds evaluated in the second biological assay


In conclusion, we discovered lead compounds that could be developed as protein–proteinblockers for the binding interface between Rab27a and melanophilin with potential pharma-ceutical benefit for the inhibition of melanosomal transport. Pharmacophore models weresuggested based on the study of the intermolecular interaction in the model of the Rab27a/melanophilin complex with experimental evidence from site-directed mutagenesis. Thepharmacophore-based virtual screening proposed 25 virtual hit compounds; these werepurchased and evaluated for their effect on melanosomal transport in melan-a cells. Based onthe scaffold of these active hits, 11 analogues were additionally purchased and evaluated bymelanosome aggregation measurement. From the results of the cell-based assay of these 11analogues, the hypothesis of SAR studies based on the prediction of the binding modes wasconfirmed. The strategy used in this investigation can be applied to the discovery of new leadsfor inhibitors of protein–protein interactions.

Acknowledgments This study was supported by a grant of the Korea Healthcare technology R&D project,Ministry of Health & Welfare, Republic of Korea (grant no. A103017).

Author contribution Jong Young Joung performed virtual screening, analyzed data, and wrote the paper; HaYeon Lee performed biological assays for hit compounds and wrote the paper; Jongil Park performed biologicalassays; Jee-Young Lee analyzed assay results; Byung Ha Chang analyzed SAR results; Kyoung Tai No designedexperiments; Ky-Youb Nam designed experiments and wrote the paper; and Jae Sung Hwang designed thebiological assays.

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identification of novel rab27a/melanophilin blockers by pharmacophore-based virtual screening

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