MEK1 and MEK2 are closely related, dual-specificity tyrosine/threonine protein kinases found in the Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway. Approximately 30% of all human cancers have a constitutively activated MAPK pathway, and constitutive activation of MEK1 results in cellular transformation. Here we present the X-ray structures of human MEK1 and MEK2, each determined as a ternary complex with MgATP and an inhibitor to a resolution of 2.4 A and 3.2 A, respectively. The structures reveal that MEK1 and MEK2 each have a unique inhibitor-binding pocket adjacent to the MgATP-binding site. The presence of the potent inhibitor induces several conformational changes in the unphosphorylated MEK1 and MEK2 enzymes that lock them into a closed but catalytically inactive species. Thus, the structures reported here reveal a novel, noncompetitive mechanism for protein kinase inhibition.

Matrix metalloproteinase-13 (MMP13) is a Zn2؉ -dependent protease that catalyzes the cleavage of type II collagen, the main structural protein in articular cartilage. Excess MMP13 activity causes cartilage degradation in osteoarthritis, making this protease an attractive therapeutic target. However, clinically tested MMP inhibitors have been associated with a painful, joint-stiffening musculoskeletal side effect that may be due to their lack of selectivity. In our efforts to develop a disease-modifying osteoarthritis drug, we have discovered MMP13 inhibitors that differ greatly from previous MMP inhibitors; they do not bind to the catalytic zinc ion, they are noncompetitive with respect to substrate binding, and they show extreme selectivity for inhibiting MMP13. By structurebased drug design, we generated an orally active MMP13 inhibitor that effectively reduces cartilage damage in vivo and does not induce joint fibroplasias in a rat model of musculoskeletal syndrome side effects. Thus, highly selective inhibition of MMP13 in patients may overcome the major safety and efficacy challenges that have limited previously tested non-selective MMP inhibitors. MMP13 inhibitors such as the ones described here will help further define the role of this protease in arthritis and other diseases and may soon lead to drugs that safely halt cartilage damage in patients.The National Institutes of Health has estimated that more than 20 million adults in the United States suffer from osteoarthritis (OA), 3 a debilitating disease in which the protective cushion of cartilage is destroyed, resulting in pain and reduced mobility. A critical step in OA pathology is breakdown of the main structural protein of articular cartilage, type II collagen. This triple helical protein is resistant to most proteases but is efficiently recognized and degraded by the Zn 2ϩ -dependent enzyme, collagenase-3, known as matrix metalloproteinase-13 (MMP13) (1-3). MMP13 catalyzes the hydrolysis of type II collagen at a unique site resulting in 3 ⁄4-and 1 ⁄4-length polypeptide products (2-6). MMP13 is not found in normal adult tissues but is expressed in the joints and articular cartilage of OA patients (4 -8). In addition, regulated expression of human MMP13 in hyaline and joint cartilages induces OA in genetically modified mice (9). Furthermore, a MMP inhibitor that preferentially inhibits MMP13 has been shown to block the degradation of explanted human osteoarthritic cartilage (5). Based on these findings, it is likely that MMP13 is the direct cause of irreversible 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 "musculoskeletal syndrome" (MSS), with these inhibitors (10, 11). Such joint side effects are not unique to humans. Rats dosed with non-selective MMP inhibitors (i.e. compounds that inhibit several or all MMPs) also display MSS-like side effects such as soft tissue fibroplasias, inflammation, and pain (...

Inflammation and thrombosis occur together in many diseases. The leukocyte integrin Mac-1 (also known as integrin αMβ2, or CD11b/CD18) is crucial for leukocyte recruitment to the endothelium, and Mac-1 engagement of platelet GPIbα is required for injury responses in diverse disease models. However, the role of Mac-1 in thrombosis is undefined. Here we report that mice with Mac-1 deficiency (Mac-1−/−) or mutation of the Mac-1-binding site for GPIbα have delayed thrombosis after carotid artery and cremaster microvascular injury without affecting parameters of haemostasis. Adoptive wild-type leukocyte transfer rescues the thrombosis defect in Mac-1−/− mice, and Mac-1-dependent regulation of the transcription factor Foxp1 contributes to thrombosis as evidenced by delayed thrombosis in mice with monocyte-/macrophage-specific overexpression of Foxp1. Antibody and small-molecule targeting of Mac-1:GPIbα inhibits thrombosis. Our data identify a new pathway of thrombosis involving leukocyte Mac-1 and platelet GPIbα, and suggest that targeting this interaction has anti-thrombotic therapeutic potential with reduced bleeding risk.

Structural comparisons of SCD with representative members of the metalloproteinase superfamily clearly highlight the conservation of key secondary structural elements, in spite of major variations in the sequences including insertions and deletions of functional domains. However, the three-dimensional structure of SCD, which is generally closely related to the collagenases, shows significant differences not only in the peripheral regions but also in the specificity pockets; these latter differences should facilitate the rational design of specific inhibitors.

The pH dependence of matrix metalloproteinase (MMP) catalysis is described by a broad bell-shaped curve, indicating the involvement of two unspecified ionizable groups in proteolysis. Stromelysin-1 has a third pK a near 6, resulting in a uniquely sharp acidic catalytic optimum, which has recently been attributed to His 224 . This suggests the presence of a critical, but unidentified, S1 substructure. Integrating biochemical characterizations of inhibitor-enzyme interactions with active site topography from corresponding crystal structures, we isolated contributions to the pH dependence of catalysis and inhibition of active site residues