Understanding binding properties at protein-protein interfaces has been limited to structural and mutational analyses of natural binding partners or small peptides identified by phage display. Here, ...we present a high-resolution analysis of a nonpeptidyl small molecule, previously discovered by medicinal chemistry Tilley, J. W., et al. (1997) J. Am. Chem. Soc. 119, 7589-7590, which binds to the cytokine IL-2. The small molecule binds to the same site that binds the IL-2 α receptor and buries into a groove not seen in the free structure of IL-2. Comparison of the bound and several free structures shows this site to be composed of two subsites: one is rigid, and the other is highly adaptive. Thermodynamic data suggest the energy barriers between these conformations are low. The subsites were dissected by using a site-directed screening method called tethering, in which small fragments were captured by disulfide interchange with cysteines introduced into IL-2 around these subsites. X-ray structures with the tethered fragments show that the subsite-binding interactions are similar to those observed with the original small molecule. Moreover, the adaptive subsite tethered many more compounds than did the rigid one. Thus, the adaptive nature of a protein-protein interface provides sites for small molecules to bind and underscores the challenge of applying structure-based design strategies that cannot accurately predict a dynamic protein surface.
Site-Directed Ligand Discovery Erlanson, Daniel A.; Braisted, Andrew C.; Raphael, Darren R. ...
Proceedings of the National Academy of Sciences - PNAS,
08/2000, Letnik:
97, Številka:
17
Journal Article
Recenzirano
Odprti dostop
We report a strategy (called "tethering") to discover low molecular weight ligands (≈ 250 Da) that bind weakly to targeted sites on proteins through an intermediary disulfide tether. A native or ...engineered cysteine in a protein is allowed to react reversibly with a small library of disulfide-containing molecules (≈ 1,200 compounds) at concentrations typically used in drug screening (10 to 200 μ M). The cysteine-captured ligands, which are readily identified by MS, are among the most stable complexes, even though in the absence of the covalent tether the ligands may bind very weakly. This method was applied to generate a potent inhibitor for thymidylate synthase, an essential enzyme in pyrimidine metabolism with therapeutic applications in cancer and infectious diseases. The affinity of the untethered ligand (Ki≈ 1 mM) was improved 3,000-fold by synthesis of a small set of analogs with the aid of crystallographic structures of the tethered complex. Such site-directed ligand discovery allows one to nucleate drug design from a spatially targeted lead fragment.
Small-Molecule Inhibition of TNF-α He, Molly M.; Smith, Annemarie Stroustrup; Oslob, Johan D. ...
Science (American Association for the Advancement of Science),
11/2005, Letnik:
310, Številka:
5750
Journal Article
Recenzirano
We have identified a small-molecule inhibitor of tumor necrosis factor α (TNF-α) that promotes subunit disassembly of this trimeric cytokine family member. The compound inhibits TNF-α activity in ...biochemical and cell-based assays with median inhibitory concentrations of 22 and 4.6 micromolar, respectively. Formation of an intermediate complex between the compound and the intact trimer results in a 600-fold accelerated subunit dissociation rate that leads to trimer dissociation. A structure solved by x-ray crystallography reveals that a single compound molecule displaces a subunit of the trimer to form a complex with a dimer of TNF-α subunits.
Using a site-directed fragment discovery method called tethering, we have identified a 60 nM small molecule antagonist of a cytokine/receptor interaction (IL-2/IL2Rα) with cell-based activity. ...Starting with a low micromolar hit, we employed a combination of tethering, structural biology, and computational analysis to design a focused set of 20 compounds. Eight of these compounds were at least 5-fold more active than the original hit. One of these compounds showed a 50-fold enhancement and represents the highest affinity inhibitor reported against this protein−protein target class. This method of coupling selected fragments with a low micromolar hit shows great potential for generating high-affinity lead compounds.
Minimizing a Binding Domain from Protein A Braisted, Andrew C.; Wells, James A.
Proceedings of the National Academy of Sciences - PNAS,
06/1996, Letnik:
93, Številka:
12
Journal Article
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We present a systematic approach to minimizing the Z-domain of protein A, a three-helix bundle (59 residues total) that binds tightly (Kd = 10 nM) to the Fc portion of an immunoglobin IgG1. Despite ...the fact that all the contacts seen in the x-ray structure of the complex with the IgG are derived from residues in the first two helices, when helix 3 is deleted, binding affinity is reduced > 105-fold (Kd > 1 mM). By using structure-based design and phage display methods, we have iteratively improved the stability and binding affinity for a two-helix derivative, 33 residues in length, such that it binds IgG1 with a Kd of 43 nM. This was accomplished by stepwise selection of random mutations from three regions of the truncated Z-peptide: the 4 hydrophobic residues from helix 1 and helix 2 that contacted helix 3 (the exoface), followed by 5 residues between helix 1 and helix 2 (the interface), and lastly by 19 residues at or near the interface that interacts with Fc (the interface). As selected mutations from each region were compiled (12 in total), they led to progressive increases in affinity for IgG, and concomitant increases in α -helical content reflecting increased stabilization of the two-helix scaffold. Thus, by sequential increases in the stability of the structure and improvements in the quality of the intermolecular contacts, one can reduce larger binding domains to smaller ones. Such mini-protein binding domains are more amenable to synthetic chemistry and thus may be useful starting points for the design of smaller organic mimics. Smaller binding motifs also provide simplified and more tractable models for understanding determinants of protein function and stability.
Current treatment guidelines for infective endocarditis focus on left-sided infective endocarditis. Because right-sided infective endocarditis has different presentations and outcomes, it is crucial ...to further delineate surgical outcomes for isolated tricuspid valve endocarditis (TVE).
This retrospective study reviewed 374 surgically treated patients with isolated TVE from January 2012 through April 2022 who underwent isolated tricuspid valve surgical procedures. Primary outcomes were in-hospital mortality, permanent pacemaker need, and postsurgical inotropic support.
The in-hospital mortality was 4% (n = 15). Patients with liver disease had 3.81-times higher odds of death vs no liver disease (odds ratio OR, 3.81; 95% CI, 1.22-12.17). A pacemaker was required in 17% of patients without a prior pacemaker, which was 4.07 times the odds with tricuspid valve replacement (OR, 4.07; 95% CI, 1.72-11.60) vs tricuspid valve repair. Each yearly increase in patient age demonstrated lower odds of permanent pacemaker requirement by 7% (OR, 0.93; 95% CI, 0.89-0.97). The odds for postoperative inotropic support increased 2.55-times higher in patients receiving preoperative inotropic agents (OR, 2.55; 95% CI, 1.29-5.05), 2.27-times higher with renal failure (OR, 2.27; 95% CI, 1.38-3.74), and 86% higher in patients administered preoperative heparin (OR, 1.86; 95% CI, 1.14-3.02).
Mortality of surgical treatment for TVE was 4%, with higher risks with liver disease. Tricuspid valve replacement was associated with higher odds for permanent pacemaker vs repair. Renal failure, preoperative inotropic support, and preoperative heparin were associated with higher odds for postoperative inotropic support. These findings further illustrate surgical outcomes with TVE.
The affinity between molecules depends both on the nature and presentation of the contacts. Here, we observe coupling of functional and structural elements when a protein binding domain is evolved to ...a smaller functional mimic. Previously, a 38-residue form of the 59-residue B-domain of protein A, termed Z38, was selected by phage display. Z38 contains 13 mutations and binds IgG only 10-fold weaker than the native B-domain. We present the solution structure of Z38 and show that it adopts a tertiary structure remarkably similar to that observed for the first two helices of B-domain in the B-domain/Fc complex Deisenhofer, J. (1981) Biochemistry 20, 2361-2370, although it is significantly less stable. Based on this structure, we have improved on Z38 by designing a 34-residue disulfide-bonded variant (Z34C) that has dramatically enhanced stability and binds IgG with 9-fold higher affinity. The improved stability of Z34C led to NMR spectra with much greater chemical shift dispersion, resulting in a more precisely determined structure. Z34C, like Z38, has a structure virtually identical to the equivalent region from native protein A domains. The well-defined hydrophobic core of Z34C reveals key structural features that have evolved in this small, functional domain. Thus, the stabilized two-helix peptide, about half the size and having one-third of the remaining residues altered, accurately mimics both the structure and function of the native domain.
Fragment assembly has shown promise for discovering small-molecule antagonists for difficult targets, including protein−protein interactions. Here, we describe a process for identifying a 60 nM ...inhibitor of the interleukin-2 (IL-2)/IL-2 receptor (IL-2Rα) interaction. By use of fragment-based approaches, a compound with millimolar affinity was evolved to a hit series with low micromolar activity, and these compounds were optimized into a lead series with nanomolar affinity. Fragment assembly was useful not only for hit identification, but also for lead optimization. Throughout the discovery process, biophysical methods and structural biology demonstrated that compounds bound reversibly to IL-2 at the IL-2 receptor binding site.
The cytokine hormone interleukin-2 (IL-2) contains a highly adaptive region that binds small, druglike molecules. The binding properties of this adaptive region have been explored using a “tethering” ...method that relies on the formation of a disulfide bond between the protein and small-molecule ligands. Using tethering, surface plasmon resonance (SPR), and X-ray crystallography, we have discovered that the IL-2 adaptive region contains at least two cooperative binding sites where the binding of a first ligand to one site promotes or antagonizes the binding of a second ligand to the second site. Cooperative energies of interaction of −2 kcal/mol are observed. The observation that the adaptive region contains two adjacent sites may lead to the development of tight-binding antagonists of a protein−protein interaction. Cooperative ligand binding in the adaptive region of IL-2 underscores the importance of protein dynamics in molecular recognition. The tethering approach provides a novel and general strategy for discovering such cooperative binding interactions in specific, flexible regions of protein structure.
A method for constraining short peptides (<20 residues) of arbitrary sequence to an α-helical conformation (∼100% helical in H2O at 25 °C) is presented. Glutamine residues at positions i and i + 7 of ...the peptides were tethered with an alkanediyl chain between the side chain nitrogen atoms. Peptides containing this tether were readily synthesized on the solid phase by amide formation between an α,ω-diaminoalkane and the side chain carboxylates of glutamate residues. The resulting cyclic peptides were studied by NMR and CD and were found to adopt an α-helical conformation in aqueous solution. The α-helix was thermally stable to ≥40 °C. Corresponding untethered control peptides with N-methylglutamine at the i and i + 7 positions lacked helicity under the same conditions. Analogous peptides were also prepared for comparison using the thiolysine cross-linking method described previously Jackson, D. Y.; King, D. S.; Chmielewski, J.; Singh, S.; Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 9391−9392.
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