Interaction of a Peptidomimetic Aminimide Inhibitor with Elastase

Peisach, Ezra; Casebier, David S.; Gallion, Steven L.; Furth, Paul S.; Petsko, Gregory A.; Hogan, Joseph C.; Ringe, Dagmar

doi:10.1126/science.7604279

Cited by 46 publications

(22 citation statements)

References 18 publications

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“…That technology exists in the form of peptidomimesis, and an analog of the insoluble inhibitor has been synthesized. 37 This analog inhibits the enzyme potently and selectively, as expected, and the crystal structure of its complex with elastase shows that it binds to the enzyme in all of the subsites identified by the probe molecules and the inhibitors studied structurally.…”

Section: E X P E R I M E N T a L P R O B E M O L E C U L E Smentioning

confidence: 88%

Analysis of the binding surfaces of proteins

Ringe

Mattos

1999

Med. Res. Rev.

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We have developed an experimental approach to map the complete binding surface of any crystalline macromolecule that is fast and flexible. Crystals of the target protein are transferred into organic solvents and the crystal structures are determined at high (about 2Å) resolution. The sites where the solvent molecules bind to the protein are thus identified directly. Different solvents serve as probes for different organic functional groups; thus, benzene is a probe for where aromatic groups like to bind, dimethyl formamide is a probe for peptide binding sites, and so forth. A series of about six such experiments suffices to locate the major binding regions on the protein surface unambiguously. These different sites can then be targeted with “Hydra‐headed” inhibitors that interact simultaneously with more that one site, thereby providing specificity for the desired target. We have used this method to map the complete binding surface of elastase, and find that three regions, including the active site cleft, are generally “sticky” and can make interactions with almost any functional group. Analyses of these binding sites on elastase and other proteins suggests that what makes a binding site is amphipathicity and the ease with which water can be displaced. © 1999 John Wiley & Sons, Inc. Med Res Rev, 19, No. 4, 321–331, 1999.

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Section: E X P E R I M E N T a L P R O B E M O L E C U L E Smentioning

confidence: 88%

Analysis of the binding surfaces of proteins

Ringe

Mattos

1999

Med. Res. Rev.

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“…A prime example of this sort of study involves the extensive analysis on the complexes of elastase with trifluoracetyl-dipeptide-analide inhibitors [11]. This has resulted in the design of a new ligand that simultaneously combined functional groups of at least two of the original inhibitors [13]. Inhibitors can also be optimized to make new drugs by using the trial and error method to modify previously known small molecule ligands.…”

Section: Characterization Of Protein-ligand Binding Sitesmentioning

confidence: 99%

“…That subsite was subsequently utilized in an inhibitor (Fig. 4.3; black; 1BMA) designed specifically to place a group into it [13].…”

Section: Organic Solvent Binding Sitesmentioning

confidence: 99%

Location of Binding Sites on Proteins by the Multiple Solvent Crystal Structure Method

Ringe

Mattos

2006

Fragment‐based Approaches in Drug Discovery

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IntroductionOne of the greater challenges of structural biology is to characterize proteins in enough detail so that the structures can be used for computational prediction of function, and for the design of compounds that can be used to modulate or to interfere with that function. In general, the function of a protein is associated with its interaction with other molecules, either small molecules or other large biological molecules, such as nucleic acids or proteins. Invariably, the function can be disrupted when that interaction is prevented. A complete industry exists solely for the purpose of finding such disruptors -they are called drugs. The discovery of drugs had been a serendipitous process, in an age when very little was known about the structures of the small molecules or their targets. The process of discovery has become more sophisticated as such structures became available.The success of this process relies on knowing where an interaction site is located on a protein and characterizing the properties of that site. The first step is therefore the location of such a site. The properties of the site can then be used to design a molecule that fits it perfectly in terms of size, shape and chemical properties. One method to obtain such properties is to map a site with chemical probe molecules. Ideally these probe molecules can be connected to form a larger molecule that takes advantage of as many of the potential interaction partners in the site as possible. Finally, when the process of finding and characterizing becomes robust enough, the process can be accomplished computationally.At this moment in time, locating a binding site on a protein without any extraneous information, such as a fortuitously bound ligand in the crystal structure, is only marginally successful. The characterization of a binding site is still very laborious when done experimentally and is not robust when done computationally. Thus, mapping such a site with chemical probes could help characterize the site, the map then being used to design a specific ligand unique for the site. In addition, such a map can provide the data from which computational approaches can be calibrated. One way in which mapping can be achieved is to use a variety of small probe molecules and observe where they bind to the surface of the protein. 67 Fragment-based Approaches in Drug Discovery. Edited

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“…Protein inhibitor⅐PPE complexes include those with elafin (13), chymotrypsin elastase inhibitor (14), and a heptamer from ␤-casomorphin-7 (15). Small molecule inhibitor complexes include: peptidyl fluoromethyl ketones (16 -19), peptidyl chloromethyl ketones (20), peptidyl boronic acids (21), isocoumarin derivatives (22)(23)(24), peptidylketobenzoxazoles (25), and a peptidomimetic aminimide (26). In all cases the elastase active site maintains the same shape, and typical root mean square differences between backbone atoms are less than 0.3 Å.…”

Section: Novel Mechanism Of Inhibition Of Elastase By ␤-Lactamsmentioning

confidence: 99%

Novel Mechanism of Inhibition of Elastase by β-Lactams Is Defined by Two Inhibitor Crystal Complexes

Taylor¹,

Anderson²,

Dowden³

et al. 1999

Journal of Biological Chemistry

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Two structurally related ␤-lactams form different covalent complexes upon reaction with porcine elastase. The high resolution x-ray structures of these two complexes provide a clear insight into the mechanism of the reaction and suggest the design of a new class of serine protease inhibitors that resist enzyme reactivation by hydrolysis of the acyl intermediate. The presence of a hydroxyethyl substituent on the ␤-lactam ring provides a new reaction pathway resulting in the elimination of the hydroxyethyl group and the formation of a stabilizing conjugated double bond system. In contrast, the presence of a diethyl substituent on the ␤-lactam ring leads to addition of water. The two enzyme complexes show very different binding modes in the enzyme active site.␤-Lactam-related compounds are well established irreversible inhibitors of a wide range of serine proteases including elastase (1), ␤-lactamase (2), phospholipase A2 (3), and bacterial signal peptidases (4). Moreover, their efficacy as orally active inhibitors has led to their widespread use in the clinic. In all cases a first step in the inhibition process is acylation of the active site serine by the ␤-lactam ring to generate a covalently bound acyl-enzyme intermediate. Thereafter, the inhibitor may undergo a further fragmentation resulting in a more stable entity that is resistant to deacylation and hence regeneration of enzyme activity. In this study, we report the discovery of a novel mode of action of a class of monocyclic ␤-lactams, which results in a particularly stable acyl-enzyme intermediate. The proposed mechanism, which is based on the x-ray structures of the elastase-ligand complexes presented below, may be generally applicable to other related serine proteases.Human leukocyte elastase is an important therapeutic target in view of its implied role in diseases such as emphysema (5), cystic fibrosis (6), and rheumatoid arthritis (7). Two x-ray structures of ␤-lactams bound to porcine pancreatic elastase (PPE) 1 have been reported, the first involving a cephalosporin sulfone (8) and more recently an N-sulfonylaryl ␤-lactam (9). In the latter case, the monocyclic ␤-lactam simply acylates the active site serine residue without further fragmentation. The class of monocyclic ␤-lactams reported herein contain an aryloxy substituent at the C-4 position (Fig. 1) that departs subsequent to acylation of the serine residue. We have compared two related members of this class of ␤-lactam that differ primarily in the nature of the C-3 substituent. This difference results in distinct modes of action upon reaction with elastase. EXPERIMENTAL PROCEDURESThe crystals were grown by the hanging drop method at room temperature. Porcine pancreatic elastase at 10 mg/ml was incubated with a 3-fold molar excess of inhibitor for 1.5 h at room temperature before drops were laid. The well solution contained 0.1 M sodium acetate buffer at pH 5.1 and 55 mM sodium sulfate. X-ray data for the EI116-elastase crystal complex were collected at 100 K using a MAR300 image plate mount...

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Interaction of a Peptidomimetic Aminimide Inhibitor with Elastase

Cited by 46 publications

References 18 publications

Analysis of the binding surfaces of proteins

Analysis of the binding surfaces of proteins

Location of Binding Sites on Proteins by the Multiple Solvent Crystal Structure Method

Novel Mechanism of Inhibition of Elastase by β-Lactams Is Defined by Two Inhibitor Crystal Complexes

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