Discovery and Characterization of a Novel Inhibitor of MatrixMetalloprotease-13 That Reduces Cartilage Damage in Vivowithout Joint Fibroplasia Side Effects*

Received for publication, April 19, 2007, and in revised form, June 14, 2007 Published, JBC Papers in Press, July 10, 2007, DOI 10.1074/jbc.M703286200

Adam R. Johnson‡1,2, Alexander G. Pavlovsky§1, Daniel F. Ortwine§, Faith Prior‡, Chiu-Fai Man‡,Dirk A. Bornemeier‡, Craig A. Banotai§, W. Thomas Mueller§, Patrick McConnell§, Chunhong Yan§, Vijay Baragi¶,Charles Lesch¶, W. Howard Roark�, Michael Wilson�, Kaushik Datta**, Roberto Guzman**, Hyo-Kyung Han‡‡,and Richard D. Dyer‡¶

From the Departments of ‡Inflammation Molecular Sciences, §Discovery Technologies, ¶Inflammation Pharmacology,�Inflammation Chemistry, **World Wide Safety Sciences, and ‡‡Pharmacokinetics and Drug Metabolism, Pfizer Global Researchand Development, Ann Arbor, Michigan 48105

Matrix metalloproteinase-13 (MMP13) is a Zn2�-depend-ent protease that catalyzes the cleavage of type II collagen, themain structural protein in articular cartilage. Excess MMP13activity causes cartilage degradation in osteoarthritis, mak-ing this protease an attractive therapeutic target. However,clinically tested MMP inhibitors have been associated with apainful, joint-stiffening musculoskeletal side effect that maybe due to their lack of selectivity. In our efforts to develop adisease-modifying osteoarthritis drug, we have discoveredMMP13 inhibitors that differ greatly from previous MMPinhibitors; they do not bind to the catalytic zinc ion, they arenoncompetitive with respect to substrate binding, and theyshow extreme selectivity for inhibitingMMP13. By structure-based drug design, we generated an orally active MMP13inhibitor that effectively reduces cartilage damage in vivo anddoes not induce joint fibroplasias in a rat model of musculo-skeletal syndrome side effects. Thus, highly selective inhibi-tion of MMP13 in patients may overcome the major safetyand efficacy challenges that have limited previously testednon-selective MMP inhibitors. MMP13 inhibitors such as theones described here will help further define the role of thisprotease in arthritis and other diseases and may soon lead todrugs that safely halt cartilage damage in patients.

The National Institutes of Health has estimated that morethan 20 million adults in the United States suffer from osteo-arthritis (OA),3 a debilitating disease in which the protectivecushion of cartilage is destroyed, resulting in pain andreduced mobility. A critical step in OA pathology is break-

down of the main structural protein of articular cartilage,type II collagen. This triple helical protein is resistant tomost proteases but is efficiently recognized and degraded bythe Zn2�-dependent enzyme, collagenase-3, known asmatrix metalloproteinase-13 (MMP13) (1–3). MMP13 cata-lyzes the hydrolysis of type II collagen at a unique site result-ing in 3⁄4- and 1⁄4-length polypeptide products (2–6). MMP13is not found in normal adult tissues but is expressed in thejoints and articular cartilage of OA patients (4–8). In addi-tion, regulated expression of human MMP13 in hyaline andjoint cartilages induces OA in genetically modified mice (9).Furthermore, a MMP inhibitor that preferentially inhibitsMMP13 has been shown to block the degradation ofexplanted human osteoarthritic cartilage (5). Based on thesefindings, it is likely that MMP13 is the direct cause of irre-versible cartilage damage in OA.The clinical development of drugs that inhibit the actions

of MMPs has been plagued by the association of a painful,joint-stiffening tendonitis-like side effect, termed “musculo-skeletal syndrome” (MSS), with these inhibitors (10, 11).Such joint side effects are not unique to humans. Rats dosedwith non-selective MMP inhibitors (i.e. compounds thatinhibit several or all MMPs) also display MSS-like sideeffects such as soft tissue fibroplasias, inflammation, andpain (12). Although the human joint side effects are reversi-ble upon withdrawal of drug, MSS has halted clinical trials ofmany non-selective MMP inhibitors (10). We began a searchfor MMP13-selective inhibitors with the hypothesis thatthey would effectively prevent cartilage degradation withoutcausing MSS-like side effects.

EXPERIMENTAL PROCEDURES

MMP Activity Assays—MMP13 activity and MMP selectivityassays employed a thioester substrate, acetyl-Pro-Leu-Gly-[2-mercapto-4-methyl-pentanoyl]-Leu-Gly-O-ethyl ester (BachemCorp. #H-7145) (13) and reaction mixtures containing 50 mM

N-2-hydroxyethylpiperazine-N�-2-ethanesulfonic acid (Hepes)buffer (pH 7.0), 10 mM CaCl2, 1 mM 5,5�-dithiobis(2-nitroben-zoic acid), 0.1 mM substrate, 0.005% Brij 35, and test com-pounds dissolved in Me2SO (1% final). A recombinant humanMMP13 catalytic domain (CD) construct encompassing Tyr-

* The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 2OW9 and 2OZR) have beendeposited in the Protein Data Bank, Research Collaboratory for StructuralBioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

1 These authors contributed equally to this work.2 To whom correspondence should be addressed: Pfizer Global Research and

Development 700 Chesterfield Pkwy., St. Louis, MO 63017. Fax: 636-247-6122; E-mail: adam.johnson@pfizer.com.

3 The abbreviations used are: OA, osteoarthritis; MMP, matrix metallopro-tease; MSS, musculoskeletal syndrome; CD, catalytic domain; FL, full-length; AUC0 –24 h, area under the curve from time 0 to 24 h.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 38, pp. 27781–27791, September 21, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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104 to Asp-270 (SWISS-PROT P45452) was generated by syn-thetic genemethods andbacterial expression (14), andwas usedfor both high throughput screening and crystallographic stud-ies. In the MMP IC50 assays the enzyme was used at a level atleast 10-fold lower than the IC50. The initial rate of substratehydrolysis was determined by monitoring the increase inabsorbance at 412 nm on a microplate reader thermostatted at25 °C. The % of control activity was plotted against inhibitorconcentration and fit to the equation % of control activity �100/(1 � ([I]/IC50)slope), where [I] is the inhibitor concentra-tion, IC50 is the inhibitor concentration where the rate is 50%reduced relative to the control, and slope is the slope of thecurve at its inflection point.Steady-state Kinetics of Inhibition of MMP13—Recombinant

full-length (FL) human MMP13 was expressed as the latentproenzyme in a baculovirus expression system, purified tohomogeneity, and activated by incubating it with 1 mMp-aminophenylmercuric acetate at 37 °C for 1 h. To deter-mine the kinetic mechanism of inhibition by Compound 1,MMP13FL (5 nM) was assayed at 25 °C in reaction mixturescontaining 50 mM Hepes buffer (pH 7.0), 10 mM CaCl2, 140mM NaCl, 1 mM 5,5�-dithiobis(2-nitrobenzoic acid), 0–200�M thioester substrate, 0.005% Brij 35, and various concen-trations of Compound 1.Steady-state kinetics data were fit by global non-linear least

squares regression using GraFit Version 5 software (15) to theequation for noncompetitive inhibition (16), 1/v � [(Km/Vmax)(1 � ([I]/Ki))(1/[S])] � [(1/Vmax)(1 � ([I]/Ki))], where [I]and [S] are the concentrations of inhibitor and substrate,respectively, v is the initial velocity, Km is the Michaelis con-stant, Vmax is the maximal rate, and Ki is the inhibition con-stant. To rule out amore elaboratemodel, we also fit the data tothe equation for mixed inhibition (16), 1/v � [(Km/Vmax)(1 �([I]/Ki))(1/[S])]� [(1/Vmax)(1�([I]/�Ki))], where� is the factorby which the Ki is changed when the substrate is bound to theenzyme.Type II Collagen Cleavage Assay—Reactionmixtures to assay

for inhibition of MMP13-catalyzed hydrolysis of type II colla-gen contained 50 mM Tris-HCl buffer (pH 7.4), 10 mM CaCl2,150mMNaCl, 0.5mg/ml soluble bovine collagen (Elastin Prod-ucts Co. # CJ385), 0.005% Brij 35, and inhibitor in Me2SO (1%final). ActivatedMMP13FL (2 nM)was added, and the reactionswere incubated at 23 °C for 18 h. Reactions were stopped byadding an equal volume of 2� SDS sample loading buffer. Pro-teins were separated by denaturing electrophoresis (8% Tris-glycine) and then visualized with Coomassie R-250. The 3⁄4-length product protein bands were scanned and quantifiedusing a Bio-Rad GS-700 imaging densitometer with QuantityOne Version 4.0 software. The IC50 was defined from a plot ofthe % of control 3⁄4-length product density against inhibitorconcentration.Crystallography—MMP13CD protein for crystallography

was expressed in E. coli BL21(DE3) cells harboring pLysS andpGEMEX-1/MMP13CD.Cellswere grown in a 20-liter fermen-tor thermostatted to 30 °C and sparged with �8 liters of air/min. Themedium (15 liters) contained 260 g ofDifco 0127 yeastextract, 260 g of acidicase peptone (BBL 211843), 260 g of Difco0259 casitone, 260 g of gelysate peptone (BBL 211870), 26 g of

KH2PO4, 26 g K2HPO4, 26 g of Na2HPO4.7H2O, and 1.5 g of

ampicillin and was titrated to pH 6.8. The culture was stirredwith an impeller at 600 rpm andwasmaintained at pH 6.8� 0.2by the addition of 85% lactic acid, whereas foam was reducedusing Antifoam 289. At a culture A600 � 10, the temperaturewas raised to 37 °C, and protein expression was induced byadding isopropyl-�-D-thiogalactopyranoside to 3.2 mM. After3 h the cells were collected by centrifugation. Thewet cells (400g) were suspended in 1.3 liters of 50 mM Tris-HCl buffer (pH8.0) plus 1% Triton X-100, 10 mM MgCl2, and 40 �l of Benzo-nase. The cells were twice passed through a Dyno-Mill (GlennMills Type KDL 0.6-liter chamber) with 0.5 liters of0.25–0.50-mm glass beads at an impeller speed of 4200 rpmand a flow rate of 100ml/min. Insolublematerial waswashed bysequentially suspending it in 2 liters each of the following solu-tions and centrifuging the washed mixtures: 1) 50 mM glycinebuffer (pH 10.0), 10mMEDTA, 1%TritonX-100 (3 times); 2) 50mM glycine buffer (pH 10.0), 10 mM EDTA; 3) 50 mM Tris�HClbuffer (pH 8.0), 10 mM dithiothreitol; 4) deionized water (2times). The protein pellet was dissolved by suspending it in 50mMTris�HCl buffer (pH 7.6) plus 6 M guanidine and stirring thesolution at room temperature. After centrifugation, the proteinin the soluble fraction was brought to �0.15 mg/ml and wasrefolded at �15 °C by diafiltration into 50 mM Tris�HCl buffer(pH 7.6) containing 10 mMCaCl2, 0.1 mM ZnCl2, and 10% glyc-erol using an Amicon S3Y10 membrane. The refolded proteinwas passed through a 0.2-�m filter, concentrated to�1mg/ml,and frozen in aliquots at �70 °C.For crystallization, inhibitors in Me2SO were mixed in a 5:1

molar ratio with MMP13CD (1 mg/ml) (Me2SO, �5% final).Acetohydroxamic acid was added to 0.1 M, and the solutionswere incubated 1 h at 4 °C. After centrifugation, the solubleprotein was concentrated to 7–20 mg/ml, and 2–4-�l hangingdrops of 1:1 protein:reservoir solution were equilibrated over0.5 ml of reservoir solution. The reservoir solution for Com-pound 1 contained 18–22% polyethylene glycol monomethylether 5000 and 0.2 M Li2SO4 in 0.1 M Hepes buffer (pH 7.0),whereas that for Compound 2 contained 2.1 M (NH4)2SO4 in0.1 M Hepes buffer (pH 7.5). After several weeks, crystals werecryo-preserved in 15% glycerol, 85% well solution and flash-frozen in a stream of nitrogen.Diffraction datawere collected at 100Kusing theMARCCD-

165 detector installed on the Industrial Macromolecular Crys-tallographer Associations beamline 17-ID at the AdvancedPhoton Source in Argonne, IL. Data were integrated withHKL2000 and reduced using SCALEPACK (17). During struc-ture solution and refinement, initial phases were obtained bymolecular replacement with AMoRe (18) using the crystalstructure coordinates of human MMP3 (PDB 1CIZ) as asearch model (19), modified for sequence differences. Afterseveral rounds of model refinement using REFMAC5 (20)and manual correction, the inhibitor, acetohydroxamate,and water molecules were placed using QUANTA-2000(Accelrys, San Diego, CA).Cytokine-stimulated Cartilage Explant Degradation Assay—

Bovine nasal cartilage explants (1 � 3 mm, 9 per group) werecultured for 2 weeks with interleukin-1� (50 ng/ml) andoncostatin M (50 ng/ml) in the presence of vehicle or up to 10

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�M Compound 2. The culture medium was refreshed twiceweekly, and the conditioned medium was pooled and collectedfor quantification of hydroxyproline (21). At the end of thestudy, the remaining cartilagewas digestedwith papain, and thehydroxyproline in this sample was measured.In Vivo Cartilage Degradation Induced by Exogenous

MMP13—To assess the ability of orally dosed Compound 2 toreach the knee joint target tissue and inhibit MMP13-induceddegradation of cartilage, rats (12 per group) were dosed orallywith vehicle or various doses of Compound 2. After 3 h, theknee joints were injected with 12 �g of activated MMP13FLenzyme. The animals were sacrificed 2 h later, the knee jointswere opened, and the synovial space was lavaged twice with 80�l of saline. The type II collagen neoepitope biomarker in thelavage fluid was quantified by an enzyme-linked immunosor-bent assay (22).Surgical Model of OA—To assess the ability of an MMP13

inhibitor to prevent cartilage damage in vivo in a chronicdisease model, Compound 2 was tested in a surgicallyinduced model of OA in rabbits. The Institutional AnimalCare and Use Committee reviewed and approved the proto-col for this rabbit study. Animals were male specific-patho-gen-free New Zealand White rabbits weighing 2.5–3.5 kg.The rabbits (12 per group) were individually housed in stain-less steel caging in an Association for Assessment andAccreditation of Laboratory Animal Care accredited facility.Rooms were environmentally controlled to provide a tem-perature of 61–71 °F, a relative humidity of 35–65%, 100%fresh air at a rate of 10–12 room exchanges per h, and alight:dark cycle of 12:12 h. The rabbits were fed a designatedamount of rabbit chow (LabDiet 5321, PMI Nutrition, Brent-wood, MO) and provided with a continuous supply of de-ionized drinking water. The anterior cruciate ligament ofone knee joint was transected, and �70% of the medialmeniscus was excised. The joints were closed, and the ani-mals were allowed to recover. Beginning 1 day post-opera-tion, the animals were dosed orally twice daily with 30 mg/kgof Compound 2 or vehicle. Five weeks later, the animals weresacrificed, and the knee joints were removed and stainedwith India ink to evaluate the presence and severity of carti-lage lesions as follows. After joint removal and separation ofthe femoral condyle from the tibial plateau, the joint waswrapped in a paper towel moistened with saline and kept onice. Each bone was unwrapped and dipped into waterproofdrawing India ink (Sanford no. 4418) to immerse the entirearticular surface. The bone was then briefly rinsed in abeaker of normal saline then blotted with a clean paper towelto remove excess ink. The remaining ink reveals damage,fibrillation, and fissuring of the cartilage. Each articular sur-face of the joint was then imaged using three different anglesfor the femoral condyle and one for the tibial plateau. Imagesof the lesions were captured using a Nikon SMZ800 dissect-ing microscope (1� setting) with a SPOT Insight color CCDcamera (Diagnostics Instruments Model 320) and a CadmetMS2000 light source. Images were saved as bitmap files(1600 � 1200, 24 bits per pixel) using SPOT Version 4 soft-ware using auto-exposure setting. Images were burned ontocompact discs, and lesions were demarcated in blinded fash-

ion by two independent, trained investigators. Calibrationwas performed using images of standards taken on the samemicroscope at the same time.Rat Fibroplasia Model of Musculoskeletal Joint Side Effects—

To determine the propensity of an MMP13 inhibitor to inducejoint side effects, Compound 2was tested in a ratmodel ofMSSthat has been used previously to demonstrate joint toxicities ofthe non-selective MMP inhibitor, marimastat (12). We orallydosed Sprague-Dawley rats (6/sex) with vehicle or 2000 mg/kgof Compound 2 per day (i.e. the maximum feasible dose) for aperiod of 2 weeks. A third group of rats given a single daily oraldose of 2000 mg/kg of a non-selective hydroxamic acid MMPinhibitor, Compound 4, (R)-2-(4�-bromobiphenyl-4-sulfo-nylamino)-N-hydroxy-3-methylbutyramide (23), served as thepositive control.The calcaneon tendon and femorotibial, tibiotarsal, and

humeroradial joints were prepared bilaterally for microscopicassessment of potential joint-related lesions. After 14 days oforal dosing to rats of either vehicle, MMP13 inhibitor Com-pound 2 or non-selective MMP inhibitor Compound 4 rep-resentative samples of femorotibial joints were collected atnecropsy, fixed in 10% neutral-buffered formalin, decalci-fied, and processed with hematoxylin and eosin for lightmicrocopy to assess any compound-induced changes in jointhistology.Statistical Analyses—For the MMP13 inhibitor kinetic

mechanism of inhibition study, the initial rate data were fit tothe various models for inhibition using GraFit software (15).This program globally fits the rate data at the various inhibitorconcentrations and provides a reduced X2 value representingthe goodness of fit of the data to the kinetic model. For thestatistical analysis of animal model data, SigmaStat� software(Jandel Scientific, San Rafael, CA) was used. One-way analysisof variance was carried out to see if differences existed amongthe experimental groups, and Dunnett’s test was used to see ifthe mean responses of the experimental groups were statisti-cally significantly different from control group. Statistical testswere two-tailed and carried out at the p � 0.05 level of statisti-cal significance. The rabbit OA study consisted of at least twogroups, and the normality and equal variance tests used for dataanalysis indicated that the samples satisfied the standard statis-tical assumptions required.

RESULTS

Discovery and Biochemical Characterization of MMP13Inhibitors—We screened our compound library for inhibi-tors of the hydrolytic activity of human MMP13CD andfound Compound 1 (6-benzyl-5,7-dioxo-6,7-dihydro-5H-thia-zolo[3,2-c]pyrimidine-2-carboxylic acid benzyl ester) to be apotent inhibitor with an IC50 � 30 � 2 nM (Fig. 1A). Remark-ably, when we tested Compound 1 at up to 100 �M against nineotherMMPs, we found that this novel inhibitor chemotype didnot inhibit these closely related proteases (Table 1). Also high-lighted in Table 1 is the improved potency (IC50 � 0.67 nM) andextremely selective inhibition ofMMP13 by an advanced deriv-ative, Compound 2 (4-[1-methyl-2,4-dioxo-6-(3-phenyl-prop-1-ynyl)-1,4-dihydro-2H-quinazolin-3-ylmethyl]-benzoic acid).The high selectivity of Compounds 1 and 2 for MMP13 is in

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stark contrast to the lack of selectivity of Compound 3, ahydroxamic acid-containing broad-spectrum MMP inhibitorknown as GM-6001, or Galardin. In addition, when testedagainst isolated human ADAMTS4 and -5 in in vitro assays

where cleavage of native aggrecan substratewas quantifiedwiththe BC-3 neoepitope antibody, Compound 2 showed IC50 25�M with both of these aggrecanases.4In some of the MMP selectivity assays, which are carried out

in pH 7 Hepes buffer, Compound 2 showed visible insolubilityat 100�M, as noted in Table 1. In routine solubility tests carriedout in phosphate buffer at pH 7.5 and 6.5, however, Compound2 showed solubility of less than 3 �M, whereas in pH 6 phos-phate buffer it was soluble to 20 �M. Compound 1, on the otherhand, showed no sign of insolubility up to 100 �M, the highestconcentration tested in the MMP selectivity assays. Thus, thesolubility of Compound 2 is sensitive to the buffer system andpH used.In steady-state kinetics experiments, Compound 1 is a linear

noncompetitive inhibitor of MMP13FL (Figs. 1, B and C) withKi � 64� 1 nM,Vmax � 402� 2 pmol min�1,Km � 48 � 1 �M,and a goodness-of-fit reduced X2 value of 0.0262. In noncom-petitive inhibition the inhibitor and the substrate bind inde-pendently and simultaneously to the enzyme at non-overlap-ping sites, and the inhibitor binds with equal affinity to the freeenzyme and to the enzyme�substrate complex. The presence ofbound substrate does not alter the binding affinity of the inhib-itor and vice versa. Attempts to fit the data to the rate equationsfor mixed, competitive, and uncompetitive inhibition modelsresulted in less satisfactory reducedX2 values of 0.0270, 0.2947,and 0.1439, respectively. In addition, we determined that Com-pound 2 is also a linear noncompetitive inhibitor of MMP13.5During MMP-catalyzed peptide bond hydrolysis, it is

thought that the carbonyl oxygen atom or oxyanion of the scis-sile amide group binds to, and is polarized by the catalytic zincion. Nearly all MMP inhibitors described to date possess ahydroxamic acid, carboxylic acid, or other metal-chelatinggroup that binds to this catalytic zinc ion and competes withsubstrate binding (24). In contrast, the noncompetitivemechanism of inhibition of MMP13 displayed by Com-pounds 1 and 2 indicates that these inhibitors do not bind tothe catalytic zinc ion or to the peptide binding active sitecleft. We confirmed that inhibition of MMP13 by Com-pound 2 is rapidly and fully reversible using enzyme-inhibi-tor preincubation-dilution activity studies.5 Furthermore,when tested in an assay using a more physiologically relevantmacromolecular substrate such as type II collagen, Com-pound 1 retains its ability to potently inhibit MMP13-cata-lyzed proteolysis with an IC50 � 51 � 6 nM (Fig. 2).Crystal Structures of Inhibitors Bound toMMP13—The non-

competitive mechanism of inhibition of MMP13 by these newchemotypes indicated that they did not bind to the catalyticzinc ion. Thus, we could not use crystal structure data forMMPs bound to zinc binding inhibitors to construct reliablemodels to predict where these MMP13 inhibitors bind. We,therefore, determined the mode of binding of these uniqueMMP13 inhibitors by crystallography using MMP13CD.6 Dif-

4 G. Munie, A. Wittwer, and M. Tortorella, unpublished data.5 A. R. Johnson, unpublished observations.6 The atomic coordinates of MMP13 in complex with Compounds 1 and 2

have been deposited in the Protein Data Bank as entries 2OW9 and 2OZR,respectively.

FIGURE 1. Inhibition of catalytic activities of MMP13 by Compound 1. A, inhi-bition of MMP13CD-catalyzed cleavage of the thioester substrate by Compound1. Data shown are the means � S.D. of three titrations. B and C, noncompetitivemechanism of inhibition of MMP13FL with Compound 1. Assays were carried outas described under “Experimental Procedures” with 0 (E), 30 (F), 60 (�), and 120(f) nM of Compound 1. The data shown are from one study that has been repli-cated three times. The direct plot of Rate versus substrate concentration, as fit tothe noncompetitive inhibition model, is shown in B, whereas the Lineweaver-Burk linear transformation is plotted in C.

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fraction data collection and refinement statistics arereported in Table 2 for co-crystals of Compounds 1 and 2with a complex of MMP13CD plus acetohydroxamic acid, aligand added to prevent autolysis. The secondary and terti-ary structures for MMP13CD (Fig. 3A) in general resemblethose previously described for MMP13 crystallized with zinc

binding inhibitors (25, 26), except in the N-terminal regionof the protein and in the S1�-specificity loop (S1� nomencla-ture (27)). In various MMP13 crystals we have prepared,including those described here, the N terminus of the pro-tein can adopt different orientations depending on the inhib-itor used, the crystallization conditions, and/or the resultantspace group observed.7 However, it is the unique dispositionof the S1�-specificity loop that is most relevant to our drugdiscovery efforts. We observed that the non-zinc bindingMMP13 inhibitors described here confer an ordered struc-ture to the S1�-specificity loop that is otherwise flexible andpoorly defined.The MMP13-Compound 1-acetohydroxamic acid crystal

structure reveals that the hydroxamate is bound in a biden-tate fashion to the catalytic zinc ion. Compound 1 does notinteract with this zinc ion but instead binds deep within theS1�-specificity loop of the protein and extends past thispocket out toward solvent (Fig. 3B). The benzyl ester ofCompound 1 points toward the substrate binding cleft butoverlaps only slightly with the space that would be occupiedby a P1� leucine amino acid side chain in productively boundsubstrates such as type II collagen or the synthetic thiopep-tolide or in non-selective peptidic MMP inhibitors such asGM-6001 (Fig. 4A). This binding mode for Compound 1 isconsistent with its noncompetitive mechanism of inhibitionand contrasts with the substrate competitive inhibitionexpected for MMP inhibitors that bind to the catalytic zincion (24–26, 28–32).

7 A. G. Pavlovsky, unpublished observations.

FIGURE 2. Inhibition of MMP13-catalyzed cleavage of type II collagen byCompound 1. A, the gel image shown is from one of two replicate experi-ments. Collagen was incubated with MMP13FL (�) or enzyme buffer (�) andvarious concentrations of Compound 1. The density of the 3⁄4-length productwas measured with a densitometer as described under “Experimental Proce-dures.” B, graphical determination of the IC50 for Compound 1 for inhibition ofMMP13-catalyzed cleavage of type II collagen. The % of control data plottedare the averages of two separate experiments.

TABLE 1Contrasting the MMP selectivity of two MMP13 selective inhibitors with that of the non-selective MMP inhibitor, Compound 3 (GM-6001)Inhibition of MMP activity was assayed at least in triplicate inhibitor titrations using the thiopeptolide substrate (13).

*, Compound 2 precipitation was observed in these assays at 100,000 nM.

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In addition to not binding the catalytic zinc ion, Com-pound 1 does not occupy space within the substrate bindingcleft of MMP13. Its inhibitory potency and target specificitycan be explained by complementarities of the inhibitor andthe accommodating S1�-specificity loop of MMP13 in whichit binds (Fig. 3B). Three carbonyl oxygen atoms in Com-pound 1 form hydrogen bonds to the backbone nitrogens ofThr-224 (2.85 Å), Thr-226 (2.98 Å), and Met-232 (2.88 Å)within the S1�-specificity loop. In addition, important inter-actions exist between the phenyl rings of Compound 1 andamino acid residues His-201, Tyr-223, Tyr-225, and Phe-231. The phenyl group of the benzyl ester of Compound 1participates in a nearly co-planar stacking interaction withHis-201 (Fig. 3B), similar to interactions found in co-crystalstructures of MMP3 and MMP8 bound to non-selectiveMMP inhibitors (31, 32). The other hydrophobic interac-tions of Compound 1 with MMP13 are of the aromatic ringedge-to-face variety. An overlay of the orientations of Com-pounds 1 and 2 in the MMP13 crystal structures (Fig. 4B)shows that these structurally different compounds may nev-ertheless share certain crucial interactions with the enzyme.The high selectivity of Compounds 1 and 2 for MMP13

can be rationalized by examination of the residues that com-prise the S1�-specificity loops of the MMPs (Figs. 4C and 5).In most reported MMP structures, including those forMMP13 and MMP3, the S1�-specificity loop is not highlyordered, even in the presence of zinc binding inhibitors (28,29, 32). However, we find that this loop is well defined incomplexes of MMP13 with Compounds 1 and 2. ResiduesLeu-197, Tyr-223, Tyr-225, Gly-227, and Phe-231 play sig-nificant roles in determining the selectivity of inhibitor bind-ing (Fig. 4C). The sequence alignment of related MMPs (Fig.5) reveals that the specificity loops inMMP1, 2, and 9 may betoo short relative to that in MMP13 to accommodate theseinhibitors. A shorter loop makes the S1� pocket of theseenzymes relatively shallow (MMP1) and narrow (MMP2 and-9). On the other hand, the S1�-specificity loops in MMP3

and -17 are longer than in MMP13, which may increase loopflexibility and may in part explain the lack of detectable inhi-bition of these isoforms. Perhaps the most critical featureenabling molecular recognition is the presence of the non-conserved Gly-227 residue in MMP13. Because the �, �angles adopted by Gly-227 residue in MMP13 are in a regionof the Ramachandran plot that is unfavorable for the corre-sponding Glu/Asp residues in MMP8, -12, and -14, the S1�-specificity loop of these enzymes cannot accommodate theseMMP13 inhibitors even though they have large S1� pocketsand the same length specificity loop as MMP13. In additionto different S1�-specificity loop sizes and shapes relative toMMP13, MMPs 1, 3, 7, and 9 lack aromatic residues at eitherposition 225 or 231 (Fig. 5). In MMP13, the side chains ofTyr-225 and Phe-231 make critical hydrophobic stacking oredge-to-face interactions with the terminal phenyl rings ofCompounds 1 and 2. Finally, the S1� pocket in some MMPscontains bulky residues that may prevent these inhibitorsfrom binding (Fig. 5). Leu-197 forms part of the spacious S1�pocket in MMP13, but MMP1 and -7 contain Arg and Tyrresidues, respectively, at this position. Crystal structures ofthese latter MMPs show that these larger residues may blockaccess to the S1� pocket (30).

With a detailed knowledge of the MMP13 protein struc-ture surrounding Compound 1, we have rationally designedanalogs, such as Compound 2, which are more potent thanCompound 1 and retain extremely high selectivity forMMP13 (Table 1). The ability to modify the central bicyclicring system and the linker between the bicyclic core and thephenyl group that abuts the active site cleft allows the designof highly varied analogs with improved potency and physicalproperties. Compound 2, for example, provides a tool to testwhether inhibition of MMP13 alone can prevent cartilagedamage.Inhibition of Cartilage Degradation in Vitro and in Vivo—In

cartilage explants stimulated with interleukin-1� and oncosta-tin M, Compound 2 significantly inhibited the release of

TABLE 2Data collection and crystallographic refinement statistics

Compound 1 Compound 2Data collectionWavelength (Å) 1.00Source/detector APS 17-ID/MARCCD-165 APS 17-ID/MARCCD-165Space group C2 C2Unit cell: a, b, c (Å) 140.76, 36.68, 71.68 161.84, 71.97, 138.13� (°) 93.5 124.6Resolution range (Å) (highest resolution shell) 70-1.72 (1.78-1.72) 50-2.1 (2.18-2.1)Measured/unique reflections 110,901/37,780 173,489/66,624Completeness (%) 97.5 (67.5) 90.7 (62.8)Rmerge

a 0.0562 (0.226) 0.096 (0.342)Refinement statisticsResolution range (Å) (highest resolution shell) 70-1.74 (1.79-1.74) 50-2.3 (2.36-2.3)Rwork

b 0.167 (0.234) 0.262 (0.328)Rfree

c 0.193 (0.275) 0.334 (0.376)Number of atoms 3,163 11,880Root mean square deviation from idealityBond length (Å) 0.005 0.006Bond angles (°) 1.027 0.975Dihedral angles (°) 5.694 5.721Chiral-center restraints (Å3) 0.06 0.058

aRmerge � hkl�I � �I��/hklI, where I is the intensity of unique reflection hkl, and �I� is the average over symmetry-related observation of unique reflection hkl.bRwork � �Fobs � Fcalc�/Fobs, where Fobs and Fcalc are the observed and the calculated structure factors, respectively.c Rfree is R with 5% of reflections sequestered before refinement.

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hydroxyproline from bovine nasal cartilage (Fig. 6A) with anIC50 � 1.3 � 0.4 �M (Fig. 6B). Given that both MMP1 andMMP13 are induced in this type of explant system (33) andMMPs other than collagenases may contribute to the cleavageof hydroxyproline-containing peptides, this level of inhibitoryactivity for the MMP13 inhibitor Compound 2 is notable.We next assessed the ability of orally dosed Compound 2 to

inhibit the degradation of type II collagen in an acute model ofcartilage damage induced by the injection of active humanMMP13 into rat knee joints. At doses as low as 0.1mg/kg, Com-pound 2 significantly inhibited the formation of the type II col-lagen neoepitope biomarker (Fig. 6C), which appears upon col-lagen degradation and cartilage damage. Thus, by oral dosing

we could deliver to the knee joint an effective amount of anMMP13 inhibitor sufficient to reduce the cartilage damageinduced by exogenously added MMP13.Given the effective inhibition of cartilage damage in these

acute models, we next wanted to determine whether anMMP13 inhibitor could prevent cartilage damage in a chronicin vivo model of osteoarthritis. Several such models have beenreported. In a rat model after medial collateral ligament trans-action and meniscal tear a non-selective MMP inhibitor inhib-ited cartilage degradation and osteophyte formation by 39%(34). In a rabbit anterior cruciate ligament transection model,both MMP1 and -13 are known to be up-regulated (35). In arabbit model in which anterior cruciate ligament transectionwas accompanied by removal of part of the meniscus (partialmeniscectomy), a non-selective MMP inhibitor significantlyreduced cartilage damage (36).Using the rabbit anterior cruciate ligament transection/par-

tial meniscectomy model, we found that Compound 2 dosedorally at 30 mg/kg twice daily provided a dramatic level of car-tilage protection that can be seen qualitatively in photographsshowing the gross morphology of the cartilage surfaces fromrepresentative vehicle and Compound 2-dosed rabbits (Fig.7A). Quantitatively, Compound 2 significantly reduced the car-tilage lesion areas on the tibial plateaus and on the femoralcondyles by 68 and 51%, respectively, relative to vehicle-dosedanimals (Fig. 7B).In this study Compound 2 achieved a total plasma exposure

of 200 �g�h/ml and a free fraction (i.e. not protein bound)AUC0–24 h exposure of 6 �g�h/ml, corresponding to an averageplasma free fraction that is 148-fold higher than the in vitropotency of Compound 2with humanMMP13FL (IC50 � 4 nM).The efficacy demonstrated by oral administration of thisMMP13 inhibitor suggests that most of the cartilage damageobserved in this model is in fact due to MMP13 activity.Because humans and rabbits can express the same collagenases(MMP1, -8, -13, and -14), the response of a compound in arabbit model should be predictive of its response in treatingpatients withOA. Finally, in comparison to the level of cartilageprotection that was afforded in this type of model by a non-selective hydroxamic acid-containing MMP inhibitor (36), ourresults withCompound 2 show that inhibition ofMMP13 aloneis nearly as effective in reducing the cartilage lesion area asbroad-spectrumMMP inhibition.Lack ofMusculoskeletal Joint Side Effects—Achieving efficacy

has been difficult for clinically applied MMP inhibitors, butsafety has also been a challenge due to the occurrence of MSSjoint pathology in subjects dosed with non-selective MMPinhibitors. To assess the potential of an MMP13 inhibitor toinduce MSS-like symptoms, we followed a protocol similar toone recently described for evaluation of MMP inhibitor-in-duced joint toxicity (12). As depicted in the representative pho-tomicrographs of femorotibial joint sections from three treat-ment groups, no MSS-like fibroplasias were observed in any ofthe animals dosed orally for 2 weeks with vehicle (Fig. 8A) orMMP13 inhibitor Compound 2 (Fig. 8B). In contrast, all 12animals treated with the non-selective hydroxamic acid-con-taining experimental MMP inhibitor, Compound 4, displayedjoint fibroplasias characterized by marked expansion of the

FIGURE 3. Structure of the MMP13-acetohydroxamic acid-compound 1complex. A, ribbon diagram illustrating the unexpected binding mode forCompound 1 within the specificity loop of the enzyme. Zinc and calcium ionsare depicted in magenta and green, respectively. B, the electron density (omitFo � Fc) for Compound 1 in the S1�-specificity pocket of MMP13 (oriented as inA) shows that the inhibitor does not bind to the catalytic zinc ion nor does itprotrude into the substrate binding cleft (upper left corner). Dotted linesdenote the three enzyme-inhibitor hydrogen bonds.

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inner synovial lining of the patellar tendon and adjacent quad-riceps femoris muscle with a population of plump, immaturespindloid fibroblasts contained in a scanty collagenous matrix(Fig. 8C). The AUC0–24 h exposure on day 14 for the combinedsex group dosed with Compound 2 was 1010 �g�h/ml (freefraction exposure of 30�g�h/ml), which translates to an averagefree inhibitor plasma level that is 740-fold higher than the invitro potency of Compound 2 with MMP13. Compound 4

achieved an exposure of 94 �g�h/ml, whereas exposure of thecarboxylic acid active metabolite of Compound 4 reached 684�g�h/ml. Thus, the MMP13 inhibitor Compound 2 did notinduce joint side effects in this 2-week study at exposures 5-foldabove the level needed to effectively protect articular cartilagein the rabbit model and well above the low yet fibroplasia-in-ducing exposures achieved by the non-selective MMP inhibi-tor, Compound 4.

FIGURE 4. Non-selective MMP and MMP13 selective inhibitor binding modes and specificity determinants. A, secondary structure elements of MMP13are colored (loops, green; �-helices, red; �-sheets, blue). The three structural calcium ions (green) as well as the structural and catalytic zinc ions (magenta),respectively, are shown at the top and middle of the figure, the latter were bound by three histidine residues (yellow). The crystal structure orientation of thenon-selective MMP inhibitor GM-6001 (orange) is shown here as bound to MMP13CD. The two hydroxamic acid oxygen atoms of GM-6001 bind to the catalyticzinc ion, whereas the rest of this inhibitor binds in the primed side (right) of the substrate-binding cleft, toward the S1� to S3� subsites. The binding orientationof Compound 1 (colored by atom type: carbon, gray; sulfur, yellow; nitrogen, blue; oxygen, red) in MMP13 shows that it binds deeply and completely within theS1�-specificity loop. B, the orientation of Compound 2 (orange) bound to MMP13 is superimposed on the structure of Compound 1, oriented as in A. C, close upview of the MMP13 S1�-specificity loop that envelops Compound 1, highlighting amino acid residue side chains (orange, labeled) that impart potency andspecificity. Ligands to the catalytic zinc ion (magenta) are three histidine residues (orange) and acetohydroxamic acid, AcNHOH (green).

FIGURE 5. Structure-based sequence alignment of MMPs. Insight gained using an alignment of MMPs (30) with MMP17 added here can be used to rationalizethe selectivity of Compounds 1 and 2 (see “Results” and Fig. 4C). MMP13 residues Phe-196 to Gly-246 are shown.

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DISCUSSION

By mass screening we have discovered an extremely selec-tive inhibitor of MMP13 that is structurally unique in that itdoes not contain an obvious metal binding group. When wecharacterized this initial hit and a rationally designed analog

FIGURE 6. Protection of cartilage in models of accelerated cartilagedegradation. A, Compound 2 inhibits degradation of explants of bovinenasal cartilage that have been stimulated with interleukin-1� (IL-�) andoncostatin M (OSM). The asterisk at 1 and 10 �M Compound 2 denotes p �0.015 and �0.001, respectively. B, graphical determination of the IC50 forinhibition of cartilage degradation for the assay in A. Data plotted are themeans � S.E. C, orally dosed Compound 2 inhibits the degradation of ratknee cartilage induced by injection of activated human MMP13 enzyme. Thelevel of type II collagen neoepitope (TIINE), a biomarker of collagenase-cata-lyzed cleavage of type II collagen, in the synovial fluid lavage is shown. Theasterisk at the 0.1, 1, 10, and 30 mg/kg doses denote p values of 0.004, 0.007,�0.001, and �0.001, respectively.

FIGURE 7. Protection of cartilage by Compound 2 in a surgically inducedmodel of osteoarthritis in rabbits. A, day 35 photographs of the medialfemoral cartilage surface from representative rabbits dosed with vehicle(upper left) or Compound 2 (upper right). The joints shown here are from ani-mals that displayed responses representative of the means that are graphi-cally depicted in B. In the lower images, which are duplicates of the upperimages, the cartilage lesions have been outlined in green for the vehicle (lowerleft) and Compound 2-treated (lower right) rabbit joints. B, the average two-dimensional lesion surface areas of the two treatment groups (vehicle, solidbars; Compound 2, striped bars) are plotted as the means � S.E. from onestudy that has been repeated three independent times with similar quantita-tive results. The erosion areas for both the tibial plateaus and femoral con-dyles were significantly reduced with Compound 2 treatment (asterisk, p �0.002 and �0.001, respectively).

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by biochemical, structural, and pharmacological methods,we found that these compounds differ from previouslyreported MMP inhibitors in many other ways. The noncom-petitive mechanism of inhibition by Compounds 1 and 2 isconsistent with crystallographic data that confirm that thesecompounds neither occupy the substrate binding cleft norbind to the catalytic zinc ion. Furthermore, the MMP13selectivity of these inhibitors is dramatic. Compound 2 is atleast 4 orders of magnitude more selective for inhibitingMMP13CD than closely related MMP isoforms, includingthe collagenases MMP1, -8, and -14 and the distantly relatedaggrecanases ADAMTS4 and -5, which are also thought toplay a role in osteoarthritis.One recent report indicated that a weak inhibitor ofMMP13

(IC50 � 10 �M) that did not contain a zinc binding group anddid not inhibit MMP1 or -9 had been identified, but when ametal binding hydroxamic acid functionality was incorporatedinto this structure to improve potency, themetal-chelating ana-log showed lower selectivity and also inhibited MMP9 (37). Incontrast, MMP13 inhibitor Compound 2 does not bind to thecatalytic zinc ion of MMP13, yet it has high inhibitorypotency and selectivity, and it effectively protects cartilagewithout causing joint fibroplasias. Another recent studydescribed some MMP13 inhibitors that are unrelated instructure to Compounds 1 or 2 but did not indicate whetherthose compounds exhibit any desirable pharmacologicalactivity (38).The MMP13 inhibitors Compounds 1 and 2 are unlike tra-

ditional non-selectiveMMP inhibitors that bind to the catalyticzinc ion and occupy the substrate binding cleft and the proxi-mal part of the S1�-specificity pocket. Compounds 1 and 2 binddeeply in the S1� pocket where they interact with residues in the

MMP13 specificity loop to gain remarkable selectivity. Theseinhibitors induce order to what is normally a flexible specificityloop that is poorly resolved in crystal structures. Based on thestrong electron density we observe for the catalytic zinc ion inthe crystal structure data, these MMP13 inhibitors clearly donot inhibit by causing zinc to dissociate from the enzyme. Thenoncompetitive kinetic mechanism of inhibition indicates thatCompounds 1 and 2 do not prevent substrate from binding oralter its affinity. Rather, it is possible that these noncompetitiveinhibitors block catalysis simply by inducing rigidity to the S1�-specificity loop. Dynamics and movement of this loop may beessential for turnover. Alternatively, movement during the cat-alytic cycle of the histidine residues that position the catalyticzinc ion as this metal changes from four- to five- and back tofour-coordinate during the catalytic cycle may be required forcatalysis. By binding in and immobilizing this loop and/or thesecritical histidine residues, the MMP13 inhibitors may interferewith subtle structural or electronic rearrangements of the cat-alytic machinery and/or cause electronic perturbations of crit-ical active site moieties which thereby blocks a step(s) in thecatalytic cycle.MMP inhibitors that have been clinically applied so far have

been relatively non-selective, potently inhibiting all or nearly allof the knownMMPs and even inhibiting more distantly relatedmetalloproteases such as the aggrecanases and the tumornecrosis factor-� converting enzyme. Clinical trials of non-se-lectiveMMP inhibitors have been plagued by the occurrence ofmusculoskeletal syndrome joint side effects in subjects (10).This side effect liability may have prevented clinicians frombeing able to dose suchMMP inhibitors high enough to achieveefficacious exposures. Thus, no MMP inhibitor has yet shownsafety and efficacy in the clinic.

FIGURE 8. Microscopic evaluation of rat knee joints for incidence of MSS-like fibroplasias. The figure shows longitudinal-, hematoxylin-, and eosin-stainedsections of femorotibial joints. A, the patellar tendon (PT) and distal femur (F) from a vehicle-treated rat are shown. The patellar tendon is composed of a dense,highly stained tendon (T) and a less dense inner synovial lining (L). As can be seen in A and B, no lesions were observed in vehicle or MMP13 inhibitor Compound2-treated rat joints, respectively, as denoted by comparable thickness of the patellar tendon in A and B (arrowheads). On the other hand, the non-selectivehydroxamic acid-containing MMP inhibitor Compound 4 produced marked joint fibroplasias characterized by a dramatic expansion of the inner synovial liningof the patellar tendon (arrowheads) and adjacent quadriceps femoris muscle (M) by immature fibroblasts (C). The white scale bar in each photomicrographrepresents 90 �m in length.

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The MMP13 inhibitor Compound 2 appears to have over-come these two major hurdles that have slowed the clinicaldevelopment of MMP inhibitors. Compound 2 effectively pre-vents cartilage degradation in vivo, and it does not induce jointtoxicities in an animal model of MSS. With these promisingdata in hand, we are optimistic that a major hurdle of futureclinical trials, to show that such MMP13 inhibitors are devoidof musculoskeletal syndrome side effects, may now be sur-mountable. Based on the beneficial pharmacology these com-pounds have displayed in animal models of arthritis, we arehopeful that MMP13-selective inhibitors will effectively pro-tect cartilage in patients without causing the MSS side effectsseen previously with non-selective MMP inhibitors. Finally,insofar as aberrant MMP13 activity contributes to otherpathologies such as cancer, heart failure, rheumatoid arthritis,and liver fibrosis (39–43), MMP13 selective inhibitors will beuseful in further characterizing the role of this protease in otherdiseases.

Acknowledgments—We thank GraceMunie, ArtWittwer, andMickyTortorella for ADAMTS data and Jack Kirsch and Shirish Shenolikarfor helpful discussions and critical review of this manuscript.

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Kaushik Datta, Roberto Guzman, Hyo-Kyung Han and Richard D. DyerChunhong Yan, Vijay Baragi, Charles Lesch, W. Howard Roark, Michael Wilson,

Man, Dirk A. Bornemeier, Craig A. Banotai, W. Thomas Mueller, Patrick McConnell, Adam R. Johnson, Alexander G. Pavlovsky, Daniel F. Ortwine, Faith Prior, Chiu-Fai

without Joint Fibroplasia Side Effectsin VivoThat Reduces Cartilage Damage Discovery and Characterization of a Novel Inhibitor of Matrix Metalloprotease-13

doi: 10.1074/jbc.M703286200 originally published online July 10, 20072007, 282:27781-27791.J. Biol. Chem. 

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