Identification and Characterization of 4-[[4-(2- Butynyloxy)phenyl]sulfonyl]-N-hydroxy-2,2-dimethyl-(3S)- thiomorpholinecarboxamide (TMI-1), a Novel Dual Tumor Necrosis Factor- -Converting Enzyme/Matrix Metalloprotease Inhibitor for the Treatment of Rheumatoid Arthritis

Tumor necrosis factor (TNF)is a well validated therapeutic target for the treatment of rheumatoid arthritis. TNFis initially synthesized as a 26-kDa membrane-bound form (pro-TNF) that is cleaved by a Zn-metalloprotease named TNF-converting enzyme (TACE) to generate the 17-kDa, soluble, mature TNF. TACE inhibitors that prevent the secretion of soluble TNFmay be effective in treating rheumatoid arthritis (RA) patients. Using a structure-based design approach, we have identified a novel dual TACE/matrix metalloprotease (MMP) inhibitor 4-[[4(2-butynyloxy)phenyl]sulfonyl]-N-hydroxy-2,2-dimethyl-(3S)thiomorpholinecarboxamide (TMI-1). This molecule inhibits TACE and several MMPs with nanomolar IC50 values in vitro. In cell-based assays such as monocyte cell lines, human primary monocytes, and human whole blood, it inhibits lipopolysaccharide (LPS)-induced TNFsecretion at submicromolar concentrations, whereas there is no effect on the TNFmRNA level as judged by RNase protection assay. The inhibition of LPSinduced TNFsecretion is selective because TMI-1 has no effect on the secretion of other proinflammatory cytokines such as interleukin (IL)-1 , IL-6, and IL-8. Importantly, TMI-1 potently inhibits TNFsecretion by human synovium tissue explants of RA patients. In vivo, TMI-1 is highly effective in reducing clinical severity scores in mouse prophylactic collagen-induced arthritis (CIA) at 5, 10, and 20 mg/kg p.o. b.i.d. and therapeutic CIA model at 100 mg/kg p.o. b.i.d. In summary, TMI-1, a dual TACE/MMP inhibitor, represents a unique class of orally bioavailable small molecule TNF inhibitors that may be effective and beneficial for treating RA. Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease that results in progressive joint destruction and substantial morbidity. Despite a tremendous effort made to understand its etiology over more than two decades, the precise cause of RA remains unresolved. In recent years, the proinflammatory cytokine TNFhas been demonstrated to play a pivotal role in RA (Feldmann et al., 1998). Elevated concentrations of soluble TNFare found in the synovial fluid of RA patients (Feldmann et al., 1994, 2001). TNFalso contributes to cartilage breakdown and bone erosion by 1 These authors contributed equally to this manuscript. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.059675. ABBREVIATIONS: RA, rheumatoid arthritis; TNF, tumor necrosis factor; MMP, matrix metalloproteinase; TACE, tumor necrosis factorconverting enzyme; TMI-1,4-[[4-(2-butynyloxy)phenyl]sulfonyl]-N-hydroxy-2,2-dimethyl-(3S)-thiomorpholinecarboxamide; OA, osteoarthritis; LPS, lipopolysaccharide; CIA, collagen-induced arthritis; FRET, fluorescence-based fluorescence resonance energy transfer; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; CFA, complete Freund’s adjuvant; PD98059, 2-(2 -amino-3 -methoxyphenyl)-oxanaphthalen-4-one; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole; CGS-27023, N-hydroxy-2(R)-(4-methoxysulfonyl)(3-picolyl)-(amino-3 methylbutaneamide hydrochloride monohydrate); BMS-275291, (S)N-[2-mercapto-1-oxo-4-(3,4,4-trimethyl-2,5-dioxo-1-imidazolidinyl)buty]-L-leucyl-N,3-dimethyl-L-valinamide; BAY 12-9566, 4-[4-(chlorophenyl)phenyl]-4-oxo-2S-(phenylthiomethyl)butanoic acid. 0022-3565/04/3091-348–355$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 309, No. 1 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 59675/1135429 JPET 309:348–355, 2004 Printed in U.S.A. 348 at A PE T Jornals on A ril 8, 2017 jpet.asjournals.org D ow nladed from stimulating the production of other proinflammatory cytokines, recruitment of inflammatory cells into the synovium of joints, and induction of degradative enzymes such as various matrix metalloproteases (MMPs) (Choy and Panayi, 2001). The recent clinical success of anti-TNFagents such as the soluble TNFreceptor (Moreland et al., 1997) and antiTNFantibody (Elliott et al., 1994a,b) has further validated TNFas an important therapeutic target for RA. However, despite the success of these biological agents in the treatment of RA, this class of agents has various limitations, including the requirement of parenteral injection, high cost, and the possibility of antibody formation against these agents. Hence, the development of orally bioavailable, small molecule inhibitors of TNFrepresents a highly desirable strategy for treating RA. TNFis initially expressed on the cell surface as a 26kDa, type II transmembrane pro-form. The membrane-bound pro-TNFcan then be cleaved between Ala-76 and Val-77 by a Zn-metalloprotease, TNFconverting enzyme (TACE), resulting in the formation of the 17-kDa, mature, soluble cytokine. TACE is a member of the ADAM (a disintegrin and metalloprotease-containing enzyme) family of proteases and is the predominant protease responsible for the generation of soluble TNF(Moss et al., 2001). Indeed, T cells derived from TACE Zn/ Zn knockout mice have a 90% reduction in their ability to process pro-TNF(Black et al., 1997). Levels of TACE protein and its enzymatic activity in the synovial tissue of patients with RA are significantly higher than those of patients with osteoarthritis (Ohta et al., 2001). Therefore, synthetic TACE inhibitors, which inhibit the processing of pro-TNFon the plasma membrane, represent an appealing alternative to the neutralization of TNFby biological agents. TACE shares a significant degree of structural homology with the matrix metalloproteases in enzyme structures, particularly around the zinc-containing active site. MMPs have also been implicated to play a critical role in both RA and OA (Shaw et al., 2000). They may contribute to joint destruction directly by degrading the cartilage and bone structure, or indirectly by promoting angiogenesis in the formation of pannus in the joints of RA patients. Several MMP proteins or activities have been demonstrated to be elevated in the synovium of RA patients (Pap et al., 2000; Katrib et al., 2001; Tomita et al., 2002). Therefore, a dual TACE and MMP inhibitor may be clinically beneficial in treating RA patients. Here, we report the discovery of a potent, orally bioavailable, dual TACE/MMP inhibitor, 4-[[4-(2-butynyloxy)phenyl] sulfonyl]-N-hydroxy-2,2-dimethyl-(3S)-thiomorpholinecarboxamide (TMI-1). Structure-based design led to this sulfonamide-hydroxamate inhibitor of TACE, which has a P1 moiety that provides increased potency against TACE, both in enzyme and cellular assays. This compound inhibits TACE with an IC50 value of 8.4 nM. It also inhibits several MMPs, including MMP-1, -7, -9, -13, and -14 with nanomolar activity. In various cell-based assays, including human whole blood, it inhibits lipopolysaccharide (LPS)-induced TNFsecretion at submicromolar concentrations, whereas it has no effect on the TNFmRNA level as judged by RNase protection assays. Furthermore, TMI-1 potently inhibits spontaneous TNFsecretion by human synovium tissue explants of RA patients. In vivo, TMI-1 has been demonstrated to be highly effective in reducing LPS-induced TNFsecretion in mouse serum and clinical severity scores in both prophylactic and therapeutic mouse collagen-induced arthritis (CIA) models. Together, TMI-1, a dual TACE/MMP inhibitor, represents a unique class of orally active small molecules that may be effective and beneficial for treating RA. Materials and Methods TACE and MMP Enzymatic Assays. A proprietary synthetic peptide of pro-TNFcontaining the minimal TACE cleavage sequence, Abz-LAQAVRSSSR-Dpa, developed at Wyeth (Jin et al., 2002) and custom made by AnaSpec, Inc. (San Jose, CA), was used as the substrate for measuring TACE activities. A segment of the extracellular portion of the human TACE that comprises the catalytic domain, the disintegrin domain, the epidermal growth factor-like domain, and the Crambin-like domain was used in the current study. The protein was expressed in Chinese hamster ovary cells and purified by nickel-nitrilotriacetic acid and preparative size exclusion chromatography (Superdex 200 16/60) columns to near homogeneity. Compounds were tested for their ability to inhibit the cleavage of the substrate by the purified enzyme in a fluorescence-based fluorescence resonance energy transfer (FRET) assay. The human TACE protein (1 g/ml) was pretreated with the inhibitors at various concentrations for 10 min at room temperature. The reaction was initiated by the addition of pro-TNFpeptide (50 M final concentration) to the TACE protein, and the increase in fluorescence was monitored at excitation of 320 nm and emission of 420 nm over a period of 10 min as described previously (Jin et al., 2002). Under this assay condition, the IC50 value should be very close to the Ki value because the ratio of the substrate concentration to the Km is 1:10 in the assay. For MMP assays described here, the source of enzymes was the recombinant human catalytic domain either prepared at Wyeth Research (Cambridge, MA) (MMP-1, -13) or purchased from Calbiochem (San Diego, CA) (MMP-2, -7, and -9) or Chemicon International (Temecula, CA) (MMP-14). A continuous assay was used in which the substrate is a synthetic peptide containing a fluorescent group (7methoxycoumarin), which is quenched by energy transfer to a 2,4dinitrophenyl group. When the peptide was cleaved by MMPs, an increase in fluorescence was observed. The substrate used was 7-methoxycoumarin-PQGL-(3-[2,4-dinitrophenyl]-L-2,3-diaminopropionyl)-AR-OH (denoted as Wammp-5, custom synthesized by AnaSpec, Inc.). The assays were carried out at room temperature in a buffer containing 50 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM CaCl2, and 0.005% Brij-35 as reported previously (Knight et al., 1992). Substrates were either colorimetric or fluorescent synthetic peptides purchased from either Bachem (King of Prussia, PA) (MMP-1, -7, -9, -13, and -14) or AnaSpec (MMP-2). The enzymatic reactions were initiated by adding the substrate to a final concentration of 20 M. The initial rate of the cleavage reaction was determined immediately after substrate addition. For most MMP assays reported here, the IC50 value is approximately 2-fold of the Ki value. The ADAM-TS-4 (Aggrecanase-1) assay was performed using a fluorescent peptide Abz-TEGEARGSVI-Dap(Dnp)-KK (denoted as WAAG-3R, custom synthesized by AnaSpec, Inc.). The assay buffer contains 50 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM CaCl2, 0.1% CHAPS, and 5% glycerol. Total reaction volume is 100 l. The recombinant Agg-1 proteins generated at Wyeth Research (final concentration of 5 g/ml in the assay) were pretreated with the various concentrations of the compound for 10 to 15 min at 37°C. The reaction was initiated by addition of the WAAG-3R substrate at a final concentration of 25 M. The reaction is monitored at excitation of 340 nm and emission of 420 nm over a period of 30 min at 37°C in a fluorimeter (GeminiXS; Molecular Devices Corp., Sunnyvale, CA). Cell-Based Assays. The cell-based activity of TMI-1 was evaluated in human and murine monocyte cell lines, including THP-1 and Raw cells, human primary monocytes, and human whole blood for TACE Inhibitor for Treating RA 349 at A PE T Jornals on A ril 8, 2017 jpet.asjournals.org D ow nladed from the inhibition of LPSor zymosan-induced TNF, IL-1 , IL-6, and IL-8 secretion. Cells were treated with LPS (100 ng/ml), zymosan (10 g/ml) for 4 h in growth medium or human whole blood. At the end of the incubation period, the cells or the human whole blood were centrifuged at 1500 rpm for 15 min. The supernatants were collected and frozen at –80°C. The concentrations of soluble TNFand other cytokines were determined by an ELISA assay according to the manufacturer’s instructions (BioSource International, Camarillo, CA). The effect of TMI-1 on TNFRII shedding was determined in human whole blood after 4 h of incubation with phorbol 12-myristate 13-acetate (30 ng/ml). The supernatant was collected at the end of incubation period, and the level of TNFRII was determined by an ELISA assay (BioSource International). Inhibition of TNFSecretion in Human Synovial Tissue Explants. The inflamed synovium tissues from the joints of RA patients were extracted as a by-product of joint replacement therapy with the consent of the patients. To liberate the cells from the connective tissue matrix, the synovium tissues were digested with collagenase IV and DNase. The generated macrophages, T cells, plasma cells, dendritic cells, and fibroblasts were treated with or without TMI-1 for 2 days in an ex vivo cell culture. These synovium explant tissue cultures from RA patients secrete soluble TNFspontaneously with no external stimulation required. The supernatant was collected, and soluble TNFand IL-6 were detected by an ELISA assay according to the manufacturer’s instructions (BioSource International). RNase Protection Assay. Raw cells were pretreated with compounds (TMI-1, PD98059, SB203580) for 1 h at the various concentrations. Both PD98059 and SB203580 were purchased from Calbiochem. Cells were stimulated with 100 ng/ml LPS overnight. The total RNA was extracted using TRIzol reagent from Invitrogen (Carlsbad, CA). Ten micrograms of total RNA from control and treated samples was hybridized with a P-labeled mouse TNFprobe (custom made by BD PharMingen, San Diego, CA) at 56°C overnight. The sample was digested with RNase, separated on a 6% polyacrylamide gel (Sequa Gel; National Diagnostics, Manville, NJ), and autoradiographed as described by the manufacturer’s instructions (BD