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International Journal of Biological Macromolecules 70 (2014) 187–192

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac

n vitro biological evaluation of glyburide as potential inhibitor ofollagenases

ijaya Lakshmi Bodigaa, Sasidhar Reddy Edab, Saishashank Chavalib,agasaisreelekha Nagavalli Revurb, Anita Zhangc, Sandhya Thokalad, Sreedhar Bodigad,∗

Department of Molecular Biology, Institute of Genetics & Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad, IndiaDepartment of Biotechnology, KL University, Green Fields, Vaddeswaram, Andhra Pradesh, IndiaDepartment of Medicine, Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, USADepartment of Biochemistry, Kakatiya University, Vidyaranyapuri, Warangal 506009, Andhra Pradesh, India

r t i c l e i n f o

rticle history:eceived 7 April 2014eceived in revised form 27 June 2014ccepted 28 June 2014vailable online 5 July 2014

eywords:ollagenaseslyburideymography

a b s t r a c t

In tissues with upregulated MMP activity, MMP inhibition remains one of the key strategies. Potentialinhibitors of MMPs have been tested for almost 30 years, but none have reached clinical utility dueto bioavailability issues and adverse effects. This study utilized the approach of drug repurposing forexploring glyburide as a potential inhibitor against collagenases. In silico molecular docking studies werecarried out to probe the interactions of glyburide with the active site Zn. Collagenase enzyme activitymeasurements and zymography analyses using conditioned medium from lung fibroblasts, rheumatoidsynovial fibroblasts, and osteoblasts were carried out to confirm the inhibitory activity. Glyburide bindsand interacts with the catalytic Zn residues of the collagenases, as evidenced by in silico molecular dockingstudies. Fluorescence enzyme activity measurements reveal that glyburide inhibits peptide substrate

cleavage by all three collagenases in a dose-dependent manner. Collagen zymography studies validatedinhibition of these collagenases by glyburide. These results identify glyburide as a potential inhibitor ofcollagenases and provide an insight into the mechanism of action of this small molecule. Thus, glyburidemay offer additional advantages in diabetics, in controlling MMP activation and collagen degradation andcould aid in the treatment of diseases with aberrant MMP activity.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Matrix metalloproteinases (MMPs) are a family of zinc-ontaining endopeptidases that are involved in the degradationf extracellular matrix (ECM). Although the activities of MMPsre regulated precisely during the synthesis as inactive precur-or zymogens, additional regulation at the level of transcription,pecificity of interaction with ECM components, and inhibition byndogenous inhibitors such as Tissue Inhibitor of Matrix Metallo-roteinases (TIMPs) exists in normal physiological conditions [1,2].berrant expression and loss of regulatory control over the MMPsre reported in various diseases such as arthritis, cancer, atheroscle-osis, aneurysms, nephritis, tissue ulcers, and fibrosis [3]. Most

MPs are secreted into extracellular space in a latent proform,

nd require proteolytic cleavage for enzymatic activity. Most cellsn the body express MMPs, even though some enzymes are often

∗ Corresponding author. Tel.: +91 9866646435.E-mail address: sbodiga@gmail.com (S. Bodiga).

ttp://dx.doi.org/10.1016/j.ijbiomac.2014.06.054141-8130/© 2014 Elsevier B.V. All rights reserved.

associated a particular cell type. Collagenases exist as three dis-tinct forms, namely the fibroblast type (MMP-1, collagenase 1) [4],the neutrophil type (MMP-8, collagenase 2) [5], and collagenase-3 (MMP-13) [6]. MMP-1, which is an interstitial collagenaseis expressed ubiquitously in human fibroblasts, keratinocytes,endothelial cells, monocytes, and macrophages and prefers type IIcollagen. Neutrophil collagenase is regarded as being synthesizedexclusively by polymorphonuclear neutrohils. However, MMP-8expression was observed in mononuclear fibroblast-like cells in therheumatoid synovial membrane as well as in cultured rheumatoidsynovial fibroblasts and human endothelial cells [7]. MMP-8 hasbeen found in arthritic lesions, even in the absence of neutrophils,indicating that chondrocytes, and perhaps synovial fiborblasts canproduce this enzyme [8,9]. MMP-8 hydrolyzes the native type Iand II collagens more efficiently than MMP-1. In contrast, MMP-13 has a more restricted pattern of expression within connective

tissue, and is usually produced only by cartilage and bone duringdevelopment, and by chondrocytes in osteoarthritis [10]. MMP-13is also expressed in some human malignant breast tumors as well[6]. All the three forms of collagenases are induced in response to

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ytokines and growth factors usually found in arthritic joints. In tis-ues with dysregulation of MMP activity, MMP inhibition remainsne of the key strategies. Drugs that are designed as inhibitors ofMPs have been tested for almost 25 years, yet none have reached

linics. Activation of MMPs, including transformation of zymogennto active enzyme involves the change of the coordination of theinc with the sulfhydryl group of a cysteine residue, for water,hich turns the fourth point of coordination for zinc. Conditions

hat result in the contact between cysteine and zinc can activatehat enzyme, and molecules that can chelate the active zinc canerve as potential inhibitors. Despite the massive investments andailed clinical trials, there is still reason to believe that the MMPnhibitors are likely to emerge as a useful clinical entity, for the

MPs are contributory in a number of disease states. In the cur-ent study, we have tested the ability of glibenclamide (glyburide)ulfonyl urea, namely, 5-chloro-N-[2-(4-{[(cyclohexylcarbamoyl)mino] sulfonyl}-phenyl) ethyl]-2-methoxybenzamide, as a poten-ial inhibitor of MMPs, especially collagenases.

. Experimental procedures

.1. Molecular docking

The two-dimensional structure of glyburide was built using ISIS-raw and exported as a ‘MOL’ file. This was input into the MAESTRO

version 8.5) GUI of the Schrödinger software suite (2008) and sub-ected to ligand preparation using the module LigPrep (MAESTROersion 8.5). The crystal structures of human MMP-1, MMP-8, andMP-13 were obtained from PDB database and their PDB IDs are

TCL, 1ZP5 and 3ZXH, respectively. In order to understand thepecificity of glyburide, we also studied the interactions of gly-uride with MMP 9 (PDB: 1GKC). Crystal structures of MMPs wererepared for docking using protein preparation wizard. Glyburideas docked in the active site of MMPs using Glide extra-precision

XP), version 5.5. For analysing the interactions of docked protein-igand complexes, the Ligplot programme was used [11].

.2. Human fetal lung fibroblast culture for MMP-1 conditionededium

Human fetal lung fibroblasts (HFL-1) obtained from the Amer-can Type Culture Collection (Rockville, MD) were cultured on00-mm culture dishes with Dulbecco’s modified Eagle’s mediumDMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serumFBS; GIbco-BRL/Life Technologies). After fibroblasts were grown toemiconfluence, the medium was changed to fresh DMEM withoutBS. Twenty-four hours later, cells were incubated with 10 ng/ml ofecombinant human MCP-1 (Lyophilized; R&D Systems, Minneapo-is, dissolved in 0.1% BSA in PBS) for 24 h [12]. Conditioned media

ere collected and analyzed by immmunoblotting for MMP-1 using specific antibody, which revealed more than 3-fold induction ofMP-1 in MCP-1 treated cells (data not shown).

.3. Rheumatoid synovial fibroblast culture for MMP-8onditioned medium

Synovial fibroblasts were isolated from freshly dispersed syn-vium of patients with rheumatoid arthritis as described previously13]. Local ethics committee approval was obtained for this studynd institutional safety and ethical guidelines were followed.thics committee waived the need for written informed consent ofatients as specimens used in the study were intended for discard

n the normal course of surgical procedure. Briefly, the superfi-ial layer of synovial was dissected, minced, and incubated withrequent pipetting for 60–90 min in 4 mg/ml clostridial collagen-se (Worthington Biochemical, St. Louis, MO) and 0.1% DNase I

ical Macromolecules 70 (2014) 187–192

(Sigma Chemicals, St. Louis, MO) in DMEM at 37 ◦C. An equal vol-ume of 0.25% trypsin was then added for an additional 30 min.Liberated cells were washed twice in 50% PBS-50% DMEM +15%FBS at 37 ◦C and seeded at 106–107 cells/100 mm tissue cultureplate in DMEM-FBS. Non-adherent cells were removed by vigorouswashing at 24 h. Conditioned media were obtained by incubatingthe cells for 24 h with medium 199 supplemented with phorbol12-myristate 13-acetate (PMA, 10 nM). A 50-kDa immunoreactiveband was observed by using MMP-8 specific monoclonal antibody(data not shown).

2.4. Normal human osteoblast cell culture for MMP-13conditioned medium

Normal human osteoblasts (3 × 105cells) (NHOst) obtained fromLonza, Walkersville, MD, USA were grown to subconfluence in�-MEM containing 10% FBS before they were subjected to ultra-sound treatment. A UV-sterilized transducer (Exogene 2000; Smith& Nephew, Inc., Memphis, TN) that generated 1.5 MHz ultrasoundin a pulse-wave mode (200 �s pulse burst width with repetitivefrequency of 1 kHz at the intensity of 30 mW/cm2) was immersedvertically into each culture well for 20 min to just touch the sur-face of the medium. The distance between the transducer and thecells was approximately maintained between 5 and 6 mm. Controlsamples were prepared in the same manner without the ultra-sound exposure [14]. MMP-13 secreted into the culture mediumwas collected 4 h after ultrasound exposure.

2.5. Fluorogenic MMP activity measurements

The conditioned media were centrifuged for 4 min at 13,000 × gin a microcentrifuge to remove cells and cellular debris. Culturemedia was concentrated using a 3-kDa cutoff membrane (Cen-triprep and Microcon; Amicon) and used for MMP activity assays.MMP-1, -8 and -13 activities in respective culture media were mea-sured by SensoLyte Plus 520 MMP assay kits (AnaSpec, San Jose,CA). Glyburide at the indicated concentrations (1, 5 and 10 �M)was added to the harvested media aliquots and incubated for 24 h.Prior to assay, conditioned medium (60 �l) containing MMPs wasactivated with 1 mM APMA (4-aminophenylmercuric acetate) andthe assay was performed in proprietary buffers at 37 ◦C in the pres-ence of MMP substrate (1:100 dilution) for 30 min. The reactionwas terminated by adding stop solution.

2.6. Collagen zymography

Aliquots used for fluorogenic enzyme activity measurementswere also used for zymography. Conditioned media appropriatelyincubated with APMA, GM 6001 and glyburide was mixed with2× SDS sample buffer, omitting �-mercaptoethanol and used forzymography. Briefly, aliquots of the conditioned media were elec-trophoresed on a 10% SDS-polyacrylamide gel containing 0.5 mg/mlcollagen (Collagen Type I and Collagen Type IV, Sigma-Aldrich).The zymographic activities were revealed by staining with 1%Coomassie Blue and, subsequently, destaining of the gel.

2.7. Statistical analyses

Data were analyzed by one-way analysis of variance using Sig-maStat 3.5. Values are expressed as mean ± standard error (SE).

3. Results

MMPs generally consist of a prodomain, a catalytic domain, ahinge (linker) region and a hemopexin domain. They are eithersecreted from the cell or anchored to the plasma membrane [15].

V.L. Bodiga et al. / International Journal of Biological Macromolecules 70 (2014) 187–192 189

Fig. 1. (A) Stereoview of glyburide (shown in green) docked into active site of MMP-1. (B) LIGPLOT scheme showing the interactions between glyburide, catalytic zinc andamino acid residues and backbone peptide chains of MMP-1. (C) Collagen zymography of MMP-1 conditioned medium from human fetal lung fibroblasts, with unstimulatedconditioned medium (Lane 1), MCP-1 treated cells without APMA (Lane 2), and with 1 mM APMA (Lane 3); conditioned medium as in Lane 3, in the presence of 1 mM GM6001 (Lane 4), glyburide (1 �M, Lane 5), glyburide (5 �M, Lane 6) and glyburide (10 �M, Lane 7). Markings depict bands that were intensified by APMA and are consistentwith Pro-MMP-1 (upper arrow) and active MMP-1 (lower arrow) activity. (D) Bar graph showing changes in fluorogenic enzyme activity of MMP-1 from the conditionedmedium using a synthetic substrate in the presence of APMA, GM6001 and glyburide. *P < 0.01 versus untreated control, **P < 0.05 versus MCP-1 treatment, #P < 0.001 versusM † interpw

TatsizgarafcBbtMa1oiBgI

CP-1 + APMA treatment, P < 0.005 versus other concentrations of glyburide (For

eb version of the article.).

he catalytic domain shows the same overall folding in all MMPsnd contains two zinc (II) and two or three calcium (II) ions. One ofhe zinc ions has a catalytic function, while other metal ion plays atructural role. So, three main determinants of the inhibitor-proteinnteractions in the MMP active site are the nature of the catalyticinc-coordination group, the presence of inhibitor-enzyme hydro-en bonds, and the hydrophobic interactions between the inhibitornd the S1′ pocket residues [15]. Stereoviews of the docking studyevealed that glyburide docked into the active sites of MMP-1, -8nd -13 (Panel A, Figs. 1–3). Sulfonyl oxygen and carbonyl oxygenrom the sulfonyl urea interacted in a bidentate manner with theatalytic zinc (Zn170) of MMP-1 and MMP-8, as shown in the Panel, Figs. 1 and 2, respectively. Glyburide also exhibited hydrogenonds with Ala 84, Glu 119 and �–� stacking interactions betweenhe terminal benzene group and imidazole group of His 118 of

MP-1 (Fig. 1, Panel B). In case of MMP-8 (neutrophil collagen-se), second sulfonyl oxygen formed a hydrogen bond with Ala63 of the peptide backbone, while another hydrogen bond wasbserved between Ala 161 and NH of urea, along with �–� stacking

nteractions between Phe 164 and benzamido group (Fig. 2, Panel). For MMP-13, catalytic zinc coordination involved carbonyl oxy-en of urea and �–cation interactions with the phenyl group.n addition, one of the nitrogens from cyclohexyl urea formed

retation of the color information in this figure legend, the reader is referred to the

hydrogen bonding with Ala 186 on the peptide backbone and sidechain of Glu 223. Another hydrogen bond was observed betweenAla 188 and carbamoyl nitrogen. The �–� stacking interactionswere observed between the phenyl group of glyburide and His 226as well as Tyr 176 (Fig. 3, Panel B). Glyburide fulfilled three of thestructural requirements for binding with collagenases and showedbidentate interactions with catalytic zinc. Most interestingly, gly-buride did not show any interactions with the catalytic zinc duringin silico simulations with 1GKC (MMP-9, gelatinase), suggestingthat glyburide is not a universal inhibitor of MMPs (data not shown).Glyburide exhibited glide scores in the range of −6.16 to −7.84, forcollagenases.

Zymography and fluorescence activity assays were chosen inorder to confirm the results of in silico modeling. We used con-ditioned medium from human fibroblasts in culture stimulatedwith MCP-1 as a source of MMP-1. Similarly, conditioned mediumof synovial fibroblasts from rheumatoid arthroid patients treatedwith 10 nM PMA was used as a source of MMP-8, while mediumfrom ultrasound treated osteoblasts was used as a source of MMP-

13. Unstimulated cultures of fibroblasts secreted pro-MMP-1 atlow levels and therefore conditioned medium was concentratedseveral fold using a 3-kDa cutoff membrane. Secretion of MMP-1into the medium was confirmed by immunoprecipitation, followed

190 V.L. Bodiga et al. / International Journal of Biological Macromolecules 70 (2014) 187–192

Fig. 2. (A) Stereoview of glyburide (shown in green) docked into active site of MMP-8. (B) LIGPLOT scheme showing the interactions between glyburide, catalytic zinc andamino acid residues and backbone peptide chains of MMP-8. (C) Collagen zymography of MMP-8 conditioned medium from human rheumatoid synovial fibroblasts, withunstimulated conditioned medium (Lane 1), PMA-treated cells without APMA (Lane 2), and with 1 mM APMA (Lane 3); conditioned medium as in Lane 3, in the presenceof 1 mM GM 6001 (Lane 4), glyburide (1 �M, Lane 5), glyburide (5 �M, Lane 6) and glyburide (10 �M, Lane 7). Markings depict bands that were intensified by APMA andare consistent with Pro-MMP-8 (upper arrow) and active MMP-8 (lower arrow) activity. (D) Bar graph showing changes in fluorogenic enzyme activity of MMP-8 fromthe conditioned medium using a synthetic substrate in the presence of APMA, GM6001 and glyburide. *P < 0.01 versus untreated control, **P < 0.05 versus PMA treatment,#P < 0.001 versus PMA + APMA treatment, †P < 0.005 versus other concentrations of glyburide (For interpretation of the color information in this figure legend, the reader isr

bnobwgfittftvb(mtpaiAe1gMow(

eferred to the web version of the article.).

y Western blotting using a specific antibody against MM-1 (dataot shown). Collagenolytic activity is indicated by the presencef a clear band on a dark background and the intensity of theand is proportional to the enzyme activity. Low levels of MMP-1ere evidenced as a faint band at 57-kDa on a collagen zymo-

ram (Fig. 1C). Exogenous addition of MCP-1 (10 ng/ml) to thebroblasts markedly enhanced Pro-MMP-1 secretion into the cul-ure medium, seen as enhanced intensity of the 57-kDa protein onhe zymogram. However, there was negligible increase in activeorm of MMP-1, even in MCP-1 treated conditioned medium. Weherefore used 1 mM APMA treatment for 6 h to increase the acti-ation of MMP-1, which resulted in a large increase in 52-kDaand, indicating the conversion of pro-MMP-1 to active-MMP-1Fig. 1C). To one of the aliquots of MMP-1 conditioned medium, Ilo-

astat (GM6001) as a broad-spectrum MMP inhibitor was added,o confirm that the molecule being studied is indeed a metallo-roteinase. Incubation with 1 mM GM6001 abrogated the MMP-1ctivity on the zymogram, indicating that conditioned mediumndeed contained MMP 1 and collagenolytic activity seen withPMA is contributed by MMP-1. To study the possible inhibitoryffect of glyburide on MMP-1, the activity of APMA-activated MMP-

was studied in the presence of increasing concentrations oflyburide. A dose-response inhibition of the pro-MMP-1 to active

MP-1 conversion was revealed with increasing concentrations

f glyburide (1–10 �M). MMP 1 activity was reduced by over 50%ith 5 �M and almost completely abolished with 10 �M glyburide

Fig. 1C). These data were also validated by SensoLyte Plus 520

MMP-1 assay, as shown in Fig. 1D. MCP-1 treatment increased col-lagenenase activity by ∼2.6-fold compared to untreated cells. UsingAPMA, the activity could be increased by more than 6-fold. GM6001effectively reduced the collagenase activity below the basal lev-els. APMA-induced MMP-1 activity decreased to 39.5% with 1 �Mglyburide and further reduced to 21% with 5 �M glyburide. Use of10 �M glyburide almost abrogated the collagenase activity to lev-els seen with GM 6001. These data strongly correlated with thezymographic activity.

Rheumatoid synovial fibroblast cells in culture showed a veryfaint band corresponding to a 50-kDa pro-MMP-8 on the zymo-gram. Incubation with 10 nM PMA for 24 h significantly increasedthe pro-MMP-8 form in the culture medium, as shown earlier [7].A very faint band corresponding to a 40-kDa protein also wasobserved in the conditioned medium concentrate, indicating it isan active form of MMP-8 (Fig. 2C). To increase the level of activeMMP-8, we incubated the conditioned medium with APMA for24 h that increased the active/inactive MMP-8 ratio. As mentionedearlier, in parallel aliquots, 1 mM GM 6001 significantly attenu-ated APMA-induced formation of active MMP-8. Co-incubation ofconditioned medium with APMA and increasing concentrations ofglyburide showed a clear reduction in the amount of active form ofMMP-8 as shown in the zymogram. MMP 8 activity measurements

using SensoLyte Plus 520 MMP assay kit yielded reproducibleresults. PMA treatment increased collagenenase activity by ∼5.7-fold compared to untreated cells. Using APMA, the activitycould be increased by more than 10-fold. GM6001 effectively

V.L. Bodiga et al. / International Journal of Biological Macromolecules 70 (2014) 187–192 191

Fig. 3. (A) Stereoview of glyburide (shown in green) docked into active site of MMP-13. (B) LIGPLOT scheme showing the interactions between glyburide, catalytic zinc andamino acid residues and backbone peptide chains of MMP-13. (C) Collagen zymography of MMP 13 conditioned medium from osteoblasts, with unstimulated conditionedmedium (Lane 1), ultrasound subjected cells without APMA (Lane 2), and with 1 mM APMA (Lane 3); conditioned medium as in Lane 3, in the presence of 1 mM GM 6001(Lane 4), glyburide (1 �M, Lane 5), glyburide (5 �M, Lane 6) and glyburide (10 �M, Lane 7). Markings depict bands that were intensified by APMA and are consistent withPro-MMP-13 (upper arrow) and active MMP-13 (lower arrow) activity. (D) Bar graph showing changes in fluorogenic enzyme activity of MMP-13 from the conditionedm ide. *Pv ride (Ft

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edium using a synthetic substrate in the presence of APMA, GM6001 and glyburersus ultrasound + APMA treatment, †P < 0.005 versus other concentrations of glybuo the web version of the article.).

educed the collagenase activity to untreated levels. APMA-inducedMP-8 activity decreased to 46.9% with 1 �M glyburide and fur-

her reduced to 16.5% with 5 �M glyburide. Use of 10 �M glyburidelmost abrogated the collagenase activity to levels lower than GM001 (Fig. 2D).

For testing the glyburide inhibition of MMP-13, we used condi-ioned medium of osteoblasts treated with ultrasound for 20 mins a source of MMP-13, as shown earlier [14]. Untreated culturesf osteoblasts secreted very low levels of Pro-MMP-13 seen as aand at 60-kDa. Ultrasound stimulation for 20 min significantly

ncreased Pro-MMP-13 secretion in to the culture medium, seen as large increase in 60-kDa protein on the Western blots developedsing monoclonal MMP-13 antibody as well as enhanced inten-ity of the 60-kDa protein on the zymogram (Fig. 3C). However,here was negligible increase in active form of MMP-13, even afterltrasound treatment. The active form of MMP-13 that is appar-nt in the culture medium concentrate treated with1 mM APMAesulted in a large increase in 50 kDa band, indicating the con-ersion of pro-MMP-13 to active-MMP-13. As shown in Fig. 3C,ncubation of the conditioned medium with GM 6001 completelybrogated the collagenolytic activity. A dose–response inhibitionf the pro-MMP-13 to active MMP-13 conversion was revealedith increasing concentrations of glyburide (1–10 �M). MMP-13

ctivity was reduced by over 60% with 5 �M and almost com-letely abolished with 10 �M glyburide (Fig. 3C). Consistent withhe zymography, ultrasound treatment increased collagenenasectivity by ∼2.0-fold compared to untreated cells. Using APMA, the

< 0.01 versus untreated control, **P < 0.05 versus ultrasound treatment, #P < 0.001or interpretation of the color information in this figure legend, the reader is referred

activity could be increased by more than 4-fold. GM6001 effectivelyreduced the collagenase activity below the basal levels. APMA-induced MMP-13 activity decreased to 35.3% with 1 �M glyburideand further reduced to 21.2% with 5 �M glyburide. Use of 10 �Mglyburide almost abrogated the collagenase activity to levels seenwith pan-MMP inhibitor, GM 6001 (Fig. 3D).

4. Discussion

Physiological collagenolysis is integral to several biologicalprocesses such as embryogenesis, tissue repair and remodeling,angiogenesis, organ morphogenesis and wound healing [1]. Thecatalytic domain of all MMPs contains a Zn2+ ion coordinatedby a Tris (histidine) motif; the Zn2+ ion is critical for both sub-strate binding and cleavage. As deregulated MMP expression andactivity is involved in multitude of diseased conditions, effortstowards controlling the activity by either increasing the expres-sion of endogenous inhibitory TIMPs or use of synthetic moleculeswith potential for specific and selective inhibition of the MMPs isstrongly recommended. Most MMP inhibitors (MMPi) consist oftwo parts: a zinc-binding group (ZBG) to bind the catalytic metalion and a peptidomimetic backbone to interact non-covalently with

specific subsites neighboring the active site of the protein. Thecatalytic Zn2+ in the active site is surrounded by subsite pocketsdesignated as S1, S2, S3, S1′, S2′, and S3′ [16]. Of the different sub-site pockets, targeting of the S1′ pocket has provided the basis

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[20] M.C. Riddle, J. Clin. Endocrinol. Metab. 88 (2003) 528–530.[21] L. Aguilar-Bryan, C.G. Nichols, S.W. Wechsler, J.P. Clement, A. Boyd 3rd, G. Gon-

92 V.L. Bodiga et al. / International Journal of

f selectivity for many MMPi [17]. Interestingly, glyburide couldnhibit all the collagenases, although the S1′ pockets vary evenmong the three collagenases. MMP 1 possesses a shallow, wheres MMP-8 has a deep and MMP-13 has an intermediate sized S1′

ocket [18]. Although 1GKC (MMP-9) possesses an intermediate1′ pocket similar to MMP-13, glyburide could selectively inter-ct with and inhibit MMP-13, but not MMP-9. The present studyhowed that glyburide, much like the hydroxamates, the strongestnown class of MMP inhibitors binds bidentately to the catalytic ZnII) of the enzyme [19] and showed several strong interactions with-ut any significantly unfavourable contacts. The docking resultslearly suggest that the cyclohexylurea group and sulfonyl groupsre both involved in inhibition of collagenases. Thus, exploitingrug polypharmacology to identify novel modes of actions for drugepurposing has gained significant momentum in the current era ofeak drug pipelines for MMP inhibition. In this regard, glyburide

also known as glibenclamide) is the most widely used sulfonylurearug for the treatment of type 2 diabetes in the United States, for

owering blood glucose levels [20]. It is understood that the sulfonylreas bring about an increase in insulin release through bindingo ATP-dependent K+ channels (KATP) channels on the islets �-cell

embrane and inhibit passive efflux of K+ from the cell [21,22].ecause its pharmacokinetics and bioavailability is established, itan serve as a useful therapeutic in the treatment of complications,here there is increased collagenase activity, as evident from the

trong inhibitory profile of zymograms and fluorescence activityeasurements with glyburide. Therapy with glyburide would be

xpected to reduce pathologically elevated collagenolytic activityuring inflammation and also the collagen turnover required foraintaining the normal tissue integrity, along with controlling the

lasma glucose levels.

[

ical Macromolecules 70 (2014) 187–192

References

[1] M.D. Sternlicht, Z. Werb, Annu. Rev. Cell Dev. Biol. 17 (2001) 463–516.[2] H. Nagase, J.F. Woessner Jr., J. Biol. Chem. 274 (1999) 21491–21494.[3] J.F. Woessner, FASEB J. 5 (1991) 2145–2154.[4] G.I. Goldberg, S.M. Wilhelm, A. Kronberger, E.A. Bauer, G.A. Grant, A.Z. Eisen, J.

Biol. Chem. 261 (1986) 6600–6605.[5] K. Hasty, J. Jeffrey, M. Hibbs, H. Welgus, J. Biol. Chem. 262 (1987) 10048–10052.[6] J.M. Freije, I. Diez-Itza, M. Balbín, L.M. Sánchez, R. Blasco, J. Tolivia, C. López-Otín,

J. Biol. Chem. 269 (1994) 16766–16773.[7] R. Hanemaaijer, T. Sorsa, Y.T. Konttinen, Y. Ding, M. Sutinen, H. Visser, V.W.M.

van Hinsbergh, T. Helaakoski, T. Kainulainen, H. Rönkä, J. Biol. Chem. 272 (1997)31504–31509.

[8] L.C. Tetlow, D.J. Adlam, D.E. Woolley, Arthritis Rheum. 44 (2001) 585–594.[9] B.V. Shlopov, W.R. Lie, C.L. Mainardi, A.A. Cole, S. Chubinskaya, K.A. Hasty,

Arthritis Rheum. 40 (2005) 2065–2074.10] M.P. Vincenti, C.I. Coon, J.A. Mengshol, S. Yocum, P. Mitchell, C.E. Brinckerhoff,

Biochem. J. 331 (1998) 341.11] A.C. Wallace, R.A. Laskowski, J.M. Thornton, Protein Eng. 8 (1995) 127–134.12] T. Yamamoto, B. Eckes, C. Mauch, K. Hartmann, T. Krieg, J. Immunol. 164 (2000)

6174–6179.13] E. Unemori, M. Hibbs, E. Amento, J. Clin. Invest. 88 (1991) 1656–1662.14] Y.C. Chiu, T.H. Huang, W.M. Fu, R.S. Yang, C.H. Tang, J. Cell. Physiol. 215 (2007)

356–365.15] H. Nagase, R. Visse, G. Murphy, Cardiovasc. Res. 69 (2006) 562–573.16] P. Cuniasse, L. Devel, A. Makaritis, F. Beau, D. Georgiadis, M. Matziari, A. Yiotakis,

V. Dive, Biochimie 87 (2005) 393–402.17] B.G. Rao, Curr. Pharm. Des. 11 (2005) 295–322.18] H.I. Park, Y. Jin, D.R. Hurst, C.A. Monroe, S. Lee, M.A. Schwartz, Q.X.A. Sang, J.

Biol. Chem. 278 (2003) 51646–51653.19] J.C. Spurlino, A.M. Smallwood, D.D. Carlton, T.M. Banks, K.J. Vavra, J.S. Johnson,

E.R. Cook, J. Falvo, R.C. Wahl, T.A. Pulvino, Proteins: Struct. Funct. Bioinf. (2004)98–109.

zalez, H. Herrera-Sosa, K. Nguy, J. Bryan, D.A. Nelson, Science (New York, NY)268 (1995) 423–426.

22] A.D.B. Harrower, Drug Saf. 22 (2000) 313–320.