Bioactive conformation of stromelysin inhibitors determined by transferred nuclear Overhauser effects.

Abstract

The transferred nuclear Overhauser effect has been used to determine the biologically active conformations of two stromelysin inhibitors. Both inhibitors used in this study were hydroxamic acids generated via chemical synthesis. These structures, representing the conformation of each inhibitor bound to stromelysin, superimposed with excellent agreement.The study also provided information on the shape and orientation of the S2' and Si' pockets of the enzyme relative to thermolysin. Comparisons were made between stromelysin and thermolysin inhibitors to critically examine thermolysin as a template for stromelysin-inhibitor design. The enzyme-bound conformations of these stromelysin inhibitors were determined for use as a template in conformationally restricted drug design.Stromelysin 1 is a zinc metalloendoproteinase that is secreted by synoviocytes and articular chondrocytes in response to inflammatory mediators such as interleukin 1 (1). This enzyme is believed to cause the destruction of cartilage proteoglycans associated with osteo-and rheumatoid arthritis (2). Stromelysin has also been implicated in the acceleration of procollagenase activation, thereby enhancing collagenase-induced cartilage degradation and contributing to the progression of the arthritic disease state (3). Currently there are no therapies available to treat the cartilage degradation that occurs in these arthritic diseases. Because of the link of stromelysin to cartilage degradation, it is anticipated that the design of stromelysin inhibitors could result in the next major class of drugs for the treatment of arthritis.Stromelysin is secreted from cells as a 55-to 57-kDa monomeric proenzyme that, upon activation, loses 80 amino acids to yield a 45-kDa mature enzyme. Recently a Cterminally truncated form of the proenzyme was reported. This 28-kDa protein has been shown to activate identically to the wild type, yielding a 19-kDa protein with binding properties very similar to full-length stromelysin (4, 5). The threedimensional NMR structure of the inhibited catalytic domain of truncated human stromelysin 1 has been solved (6); however, to date the coordinates of these structures have not been released.As part of a rational drug-design strategy, it is important to obtain structural knowledge of the enzyme-inhibitor complex. In particular, it is necessary to know the shape of the binding pocket, as well as the conformation of the interacting inhibitor. Most NMR methods that allow complete structure elucidation of an enzyme-inhibitor complex require significant amounts of enzyme as well as isotopic labeling of the enzyme or inhibitor (7,8). The transferred nuclear Overhauser effect (TR-NOE), however, provides a different approach that focuses on the conformation of the inhibitor in the bound state. This experiment involves the generation of cross-relaxation effects between two protons in the bound state that are subsequently transferred to the free state via chemical exchange (9). The technique has an advantage over other NMR methods in t...

Intricate Functions of Matrix Metalloproteinases in Physiological and Pathological Conditions

“…Firstly, it has an isopropyl substituent which slows down the metabolism and secondly, it has a basic 3‐pryidyl substituent which helps the MMP enter negatively charged tissues such as cartilage (MacPherson et al, ). NMR spectroscopy shows that the inhibitors act by partially covering the S1 specificity pocket while its pyridylmethyl and isobutyl substituents interact with the S2′ and S1′ subsites, respectively (Gonnella et al, , ). Compared to the succinyl hydroxamates, these inhibitors have dual action on MMPs and phosphodiesterase type 4 (PDE4).…”

The Design, Structure, and Therapeutic Application of Matrix Metalloproteinase Inhibitors

“…A para-(biphenyl)ethyl moiety is positioned to allow deep penetration into the S 1ṕ ocket. The mode of binding of CGS 27023A (17) to MMP-3 as well as the full 3-dimensional solution structure of the catalytic domain of MMP-3 complexed with CGS 27023A have been determined by NMR spectroscopy [59,93,140,141]. The para-methoxyphenyl substituent occupies, but does not fill, the S 1´ specificity pocket while the isopropyl and pyridylmethyl substituents occupy the S 1 and S 2ś ubsites, respectively.…”

High-resolution solution structure of the catalytic fragment of human collagenase-3 (MMP-13) complexed with a hydroxamic acid inhibitor

“…There have been a number of X-ray and NMR structures solved for the catalytic domain of MMPs complexed with a variety of inhibitors (Becker et al, 1995;Betz et al, 1997;Bode et al, 1994;Botos et al, 1996;Broutin et al, 1996;Gonnella et al, 1995Gonnella et al, , 1997Gooley et al, 1994Gooley et al, , 1996Lovejoy et al, 1994a,b,c;Moy et al, 1998Moy et al, , 1999Spurlino et al, 1994;Stams et al, 1994;Van Doren et al, 1995) where the X-ray structure of MMP-13 was determined only recently (Lovejoy et al, 1999). There is a close similarity in the overall 3D fold for these proteins consistent with the relatively high level of sequence homology (>40 %).…”