The initiation and progression of adult-onset periodontitis has been associated with infection of the gingival sulcus byPorphyromonas gingivalis. This organism utilizes a multitude of virulence factors to evade host defenses as it establishes itself as one of the predominant pathogens in periodontal pockets. A feature common to many other oral pathogens is the production of ammonia due to its protective effect during acidic cleansing cycles in the mouth. Additionally, ammonia production byP. gingivalis has been proposed as a virulence factor due to its negative effects on neutrophil function. In this study, we describe the first purification of a peptidylarginine deiminase (PAD) from a prokaryote. PAD exhibits biochemical characteristics and properties that suggest that it may be a virulence agent. PAD deiminates the guanidino group of carboxyl-terminal arginine residues on a variety of peptides, including the vasoregulatory peptide-hormone bradykinin, to yield ammonia and a citrulline residue. The soluble protein has an apparent mass of 46 kDa, while the DNA sequence predicts a full-length protein of 61.7 kDa. PAD is optimally active at 55°C, stable at low pH, and shows the greatest activity above pH 9.0. Interestingly, in the presence of stabilizing factors, PAD is resistant to limited proteolysis and retains significant activity after short-term boiling. We propose that PAD, acting in concert with arginine-specific proteinases from P. gingivalis, promotes the growth of the pathogen in the periodontal pocket, initially by enhancing its survivability and then by assisting the organism in its circumvention of host humoral defenses.

Osteopontin and PPi both suppress hydroxyapatite deposition. Extracellular PPi deficiency causes spontaneous hypercalcification, yet unchallenged osteopontin knockout mice have only subtle mineralization abnormalities. We report that extracellular PPi deficiency promotes osteopontin deficiency and correction of osteopontin deficiency prevents hypercalcification, suggesting synergistic inhibition of hydroxyapatite deposition. Nucleotide pyrophosphatase phosphodiesterase (NPP) isozymes including PC-1 (NPP1) function partly to

SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily A member 2 (SMARCA2), also known as Brahma homologue (BRM), is a Snf2-family DNA-dependent ATPase. BRM and its close homologue Brahma-related gene 1 (BRG1), also known as SMARCA4, are mutually exclusive ATPases of the large ATPdependent SWI/SNF chromatin-remodeling complexes involved in transcriptional regulation of gene expression. No small molecules have been reported that modulate SWI/SNF chromatin-remodeling activity via inhibition of its ATPase activity, an important goal given the well-established dependence of BRG1-deficient cancers on BRM. Here, we describe allosteric dual BRM and BRG1 inhibitors that downregulate BRM-dependent gene expression and show antiproliferative activity in a BRG1mutant-lung-tumor xenograft model upon oral administration. These compounds represent useful tools for understanding the functions of BRM in BRG1-loss-of-function settings and should enable probing the role of SWI/SNF functions more broadly in different cancer contexts and those of other diseases.

Human cathepsin G is a serine proteinase with chymotrypsin-like specificity found in both polymorphonuclear leukocytes (neutrophils) and the U937 leukemic cell line. Utilizing RNA from the latter, we have constructed a cDNA library in lambda gt11 and isolated a clone which apparently codes for the complete amino acid sequence of this enzyme. Analysis of the sequence reveals homology with rat mast cell proteinase II (47%) but a greater degree of identity (56%) with a product of activated mouse cytotoxic T lymphocytes. The close relationship between the three proteins indicates similarities in substrate specificity and in biosynthesis which we predict involves removal of a two amino acid activation peptide during or just before packaging into their respective storage granules.

Cathepsin B is a member of the papain superfamily of cysteine proteases and has been implicated in the pathology of numerous diseases, including arthritis and cancer. As part of an effort to identify potent, reversible inhibitors of this protease, we examined a series of dipeptidyl nitriles, starting with the previously reported Cbz-Phe-NH-CH(2)CN (19, IC(50) = 62 microM). High-resolution X-ray crystallographic data and molecular modeling were used to optimize the P(1), P(2), and P(3) substituents of this template. Cathepsin B is unique in its class in that it contains a carboxylate recognition site in the S(2)' pocket of the active site. Inhibitor potency and selectivity were enhanced by tethering a carboxylate functionality from the carbon alpha to the nitrile to interact with this region of the enzyme. This resulted in the identification of compound 10, a 7 nM inhibitor of cathepsin B, with excellent selectivity over other cysteine cathepsins.

A microsomal preparation of human prostatic tissue was used to study the kinetics of interaction of steroid 5 alpha-reductase with finasteride, a known 5 alpha-reductase inhibitor. This molecule has been reported to reversibly bind 5 alpha-reductase in a competitive manner to testosterone with a Ki value in the 10 nM range. The results presented in this paper show that enzyme-inhibitor complex formation does not take place instantaneously as assumed in previous studies. At neutral pH and 37 degrees C, the association of enzyme with inhibitor is governed by a rate constant, kon, of 2.7 x 10(5) M-1 s-1. This low kon value, in combination with the high energy of activation of the association reaction (150 kJ mol-1), indicates that the association process is not diffusion controlled and may proceed through intermediate steps. However, such an intermediate was not detected kinetically under the inhibitor concentrations investigated. We therefore conclude that the equilibrium dissociation constant, Ki*, for the initial binding of the enzyme to the inhibitor is higher than 1.5 x 10(7) M. Even at inhibitor concentrations as low as 1 nM, the reaction was completely displaced to the EI complex and no residual activity detected once the equilibrium was reached. Hence, the interaction between finasteride and 5 alpha-reductase can also be characterized by a very low overall equilibrium dissociation constant (Ki << 10(-9) M), at least 1 order of magnitude lower than previously reported values.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective. Aging and osteoarthritic (OA) cartilage commonly demonstrate enhanced expression of the large, transforming growth factor ␤ (TGF␤)-inducible glycoprotein cartilage intermediate-layer protein (CILP) as well as enhanced extracellular inorganic pyrophosphate (PPi) that promotes the deposition of calcium pyrophosphate dihydrate crystals. In normal chondrocytes, TGF␤ induces elevated chondrocyte extracellular PPi. Insulin-like growth factor 1 (IGF-1) normally blocks this response and reduces extracellular PPi. However, chondrocyte resistance to IGF-1 is observed in OA and aging. Because CILP was reported to chromatographically fractionate with PPi-generating nucleotide pyrophosphatase phosphodiesterase (NPP) activity, it has been broadly assumed that CILP itself has NPP activity. Our objective was to directly define CILP functions and their relationship to IGF-1 in chondrocytes.Methods. Using primary cultures of articular chondrocytes from the knee, we defined the function of the previously described CILP (CILP-1) and of a recently described 50.6% identical protein that we designated the CILP-2 isoform.Results. Both CILP isoforms were constitutively expressed by primary cultured articular chondrocytes, but only CILP-1 expression was detectable in cultured knee meniscal cartilage cells. Neither CILP isoform had intrinsic NPP activity. But CILP-1 blocked the ability of IGF-1 to decrease extracellular PPi, an activity specific for the CILP-1 N-terminal domain. The CILP-1 N-terminal domain also suppressed IGF-1-induced (but not TGF␤-induced) proliferation and sulfated proteoglycan synthesis, and it inhibited ligand-induced IGF-1 receptor autophosphorylation.Conclusion. Two CILP isoforms are differentially expressed by chondrocytes. Neither CILP isoform exhibits PPi-generating NPP activity. But, increased expression of CILP-1, via N-terminal domain-mediated inhibitory effects of CILP-1 on chondrocyte IGF-1 responsiveness, could impair chondrocyte growth and matrix repair and indirectly promote PPi supersaturation in aging and OA cartilage.

The matrix metalloproteinase enzyme MMP-13 plays a key role in the degradation of type II collagen in cartilage and bone in osteoarthritis (OA). An effective MMP-13 inhibitor would therefore be a novel disease modifying therapy for the treatment of arthritis. Our efforts have resulted in the discovery of a series of carboxylic acid inhibitors of MMP-13 that do not significantly inhibit the related MMP-1 (collagenase-1) or tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE). It has previously been suggested (but not proven) that inhibition of the latter two enzymes could lead to side effects. A promising carboxylic acid lead 9 was identified and a convergent synthesis developed. This paper describes the optimization of 9 and the identification of a compound 24f for further development. Compound 24f is a subnanomolar inhibitor of MMP-13 (IC(50) value 0.5 nM and K(i) of 0.19 nM) having no activity against MMP-1 or TACE (IC(50) of >10000 nM). Furthermore, in a rat model of MMP-13-induced cartilage degradation, 24f significantly reduced proteoglycan release following oral dosing at 30 mg/kg (75% inhibition, p < 0.05) and at 10 mg/kg (40% inhibition, p < 0.05).