Discovery of Potent Nonpeptide Inhibitors of Stromelysin Using SAR by NMR

Abstract

With the use of an NMR-based method, potent (IC50 < 25 nM) nonpeptide inhibitors of the matrix metalloproteinase stromelysin (MMP-3) were discovered. The method, called SAR by NMR (for structure−activity relationships by nuclear magnetic resonance), involves the identification, optimization, and linking of compounds that bind to proximal sites on a protein. Using this technique, two ligands that bind weakly to stromelysin (acetohydroxamic acid, K D = 17 mM; 3-(cyanomethyl)-4‘-hydroxybiphenyl, K D = 0.02 mM) were identified. On the basis of NMR-derived structural information, the two fragments were connected to produce a 15 nM inhibitor of this enzyme. This compound was rapidly discovered (less than 6 months) and required only a minimal amount of chemical synthesis. These studies indicate that the SAR by NMR method can be effectively applied to enzymes to yield potent lead inhibitorsan important part of the drug discovery process.

Identification of a second aryl phosphate-binding site in protein-tyrosine phosphatase 1B: A paradigm for inhibitor design

“…5. Indeed, the utility of this strategy to obtain high affinity ligands for proteins has been successfully demonstrated by Fesik and colleagues (35,36). By using NMR techniques, a weak inhibitor (acetohydroxamic acid, K d ϭ 17 mM) that is targeted to the catalytic site and a modest inhibitor [3-(cynomethyl)-4Ј-hydroxybiphenyl, K d ϭ 0.02 mM]) that binds to the S 1 Ј site of stromelysin have been identified.…”

Distance mapping of protein‐binding sites using spin‐labeled oligosaccharide ligands

“…Keywords: Nitroxide spin label; galectin-3; drug design; drug screening; distance mapping Knowing the geometric relationship of various small molecules that bind to protein surfaces can be an important point of reference in the design of effective inhibitors of protein function (Shuker et al 1996;Hadjuk et al 1997). Here, we illustrate a method for providing intermolecular distance information based on the use of spin-labeled analogs of known protein ligands to perturb cross-peaks in a 15 N-1 H heteronuclear single quantum coherence (HSQC) spectrum in a distance-dependent fashion.…”

“…This protocol is discussed as part of a drug design strategy in which subsequent perturbation of chemical shifts of distance mapped amide cross-peaks can be used effectively to screen a library of compounds for other ligands that bind to the target protein at distances suitable for chemical linkage to the primary ligand. This approach is novel in that it bypasses the need for structure determination and resonance assignment of the target protein.Keywords: Nitroxide spin label; galectin-3; drug design; drug screening; distance mapping Knowing the geometric relationship of various small molecules that bind to protein surfaces can be an important point of reference in the design of effective inhibitors of protein function (Shuker et al 1996;Hadjuk et al 1997). Here, we illustrate a method for providing intermolecular distance information based on the use of spin-labeled analogs of known protein ligands to perturb cross-peaks in a 15 N-1 H heteronuclear single quantum coherence (HSQC) spectrum in a distance-dependent fashion.…”

Determining the optimal size of small molecule mixtures for high throughput NMR screening

“…Additionally, NMR may be used to evaluate the physical properties of a chemical lead, measure K D 's (Fielding, 2003), identify ligand binding sites (Roberts, 2000), and determine a co-structure (Clore and Gronenborn, 1994;Cooke, 1997;Kay, 1997;Roberts, 2000). A diverse number of NMR screening approaches have been developed, which include SAR by NMR (Shuker et al, 1996;Hajduk et al, 1997a;Hajduk et al, 1997c;Hajduk et al, 1999b;Johnson et al, 2003), SHAPES (Moore et al, 2004;Lepre et al, 2002;Fejzo et al, 1999), and MS/NMR (Moy et al, 2001). NMR spectroscopy is a relatively insensitive technique requiring higher amounts of material and acquisition time compared to standard methods used in traditional HTS assays.…”