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Biomedical subjects

James A Wells

Publications and source records attributed to James A Wells.

15 recordsLinked to original sources
PubMed2025

High-affinity CD16A polymorphism associated with reduced risk ofsevere COVID-19.

CD16A is an activating Fc receptor on NK cells that mediates antibody-dependent cellular cytotoxicity (ADCC), a key mechanism in antiviral immunity. However, the role of NK cell-mediated ADCC in SARS-CoV-2 infection remains unclear, particularly whether it limits viral spread and disease severity or contributes to the immunopathogenesis of COVID-19. We hypothesized that the high-affinity CD16AV176 polymorphism influences these outcomes. Using an in vitro reporter system, we demonstrated that CD16AV176 is a more potent and sensitive activator than the common CD16AF176 allele. To assess its clinical relevance, we analyzed 1,027 patients hospitalized with COVID-19 from the Immunophenotyping Assessment in a COVID-19 cohort (IMPACC), a comprehensive longitudinal dataset with extensive transcriptomic, proteomic, and clinical data. The high-affinity CD16AV176 allele was associated with a significantly reduced risk of ICU admission, mechanical ventilation, and severe disease trajectories. Lower anti-SARS-CoV-2 IgG titers were correlated to CD16AV176; however, there was no difference in viral load across CD16A genotypes. Proteomic analysis revealed that participants homozygous for CD16AV176 had lower levels of inflammatory mediators. These findings suggest that CD16AV176 enhances early NK cell-mediated immune responses, limiting severe respiratory complications in COVID-19. This study identifies a protective genetic factor against severe COVID-19, informing future host-directed therapeutic strategies.

Humans↗
PubMed2006

Hot-spot mimicry of a cytokine receptor by a small molecule.

Protein-protein complexes remain enticing, but extremely challenging, targets for small-molecule drug discovery. In a rare example described earlier, a high-affinity small molecule, SP4206 (Kd approximately 70 nM), was found to block binding of the IL-2alpha receptor (IL-2Ralpha) to IL-2 (Kd approximately 10 nM). Recently, the structure of the IL-2/IL-2Ralpha complex was solved [Rickert, M., Wang, X., Boulanger, M. J., Goriatcheva, N., Garcia, K. C. (2005) Science 308:1477-1480]. Using structural and functional analysis, we compare how SP4206 mimics the 83-fold larger IL-2Ralpha in binding IL-2. The binding free energy per contact atom (ligand efficiency) for SP4206 is about twice that of the receptor because of a smaller, but overlapping, contact epitope that insinuates into grooves and cavities not accessed by the receptor. Despite its independent design, the small molecule has a similar, but more localized, charge distribution compared with IL-2Ralpha. Mutational studies show that SP4206 targets virtually the same critical "hot-spot" residues on IL-2 that drive binding of IL-2Ralpha. Moreover, a mutation that enhances binding to the IL-2Ralpha near these hot spots also enhances binding to SP4206. Although the protein and small molecule do bind the same hot spot, they trap very different conformations of IL-2 because of its flexible nature. Our studies suggest that precise structural mimics of receptors are not required for high-affinity binding of small molecules, and they show that there are multiple solutions to tight binding at shared and adaptive hot spots.

Crystallography, X-Ray↗
PubMed2006

A common allosteric site and mechanism in caspases.

We present a common allosteric mechanism for control of inflammatory and apoptotic caspases. Highly specific thiol-containing inhibitors of the human inflammatory caspase-1 were identified by using disulfide trapping, a method for site-directed small-molecule discovery. These compounds became trapped by forming a disulfide bond with a cysteine residue in the cavity at the dimer interface approximately 15 A away from the active site. Mutational and structural analysis uncovered a linear circuit of functional residues that runs from one active site through the allosteric cavity and into the second active site. Kinetic analysis revealed robust positive cooperativity not seen in other endopeptidases. Recently, disulfide trapping identified a similar small-molecule site and allosteric transition in the apoptotic caspase-7 that shares only a 23% sequence identity with caspase-1. Together, these studies show a general small-molecule-binding site for functionally reversing the zymogen activation of caspases and suggest a common regulatory site for the allosteric control of inflammation and apoptosis.

Allosteric Regulation↗
PubMed2005

Disulfide trapping to localize small-molecule agonists and antagonists for a G protein-coupled receptor.

A key question regarding the signaling mechanism for G protein-coupled receptors (GPCRs) is what triggers agonism versus antagonism. Peptide analogs derived from the chemokine, complement fragment 5 anaphylatoxin (C5a), can act as agonists or antagonists to the C5a receptor, a member of the GPCR family [Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J. & Bourne, H. R. (2001) J. Biol. Chem. 276, 3394-4000]. Recently, we showed that two Cys residues engineered near a proposed binding site in the C5a receptor on transmembrane helices III and VI can selectively and reversibly trap short Cys-containing 3-mer peptides derived from C5a by disulfide bond formation [Buck, E. A., Bourne, H. & Wells, J. A. (November 18, 2004) J. Biol. Chem., 10.1074/jbc.C400500200]. Here, a library of 10,000 compounds, each containing an exchangeable thiol, was screened to identify specific small-molecule mimics that block binding of C5a. Some of the selected compounds acted as agonists and were as potent as the natural C5a ligand, and some acted as antagonists. A residue near these compounds, Ile-116 in helix III, functions as a "gatekeeper" to modulate these effects. A small substitution, Ile-116-Ala, enhanced affinity for some compounds and allowed antagonists to function as agonists; a larger substitution, Ile-116-Trp, decreased affinity and agonism. Thus, subtle changes in either the structure of the ligand or the receptor at the site between helix III, VI, and VII can switch the receptor on or off. This ligand binding and activation site may be similarly positioned in other members of the chemokine receptor family. Selective ligand trapping by reversible disulfide formation may serve to nucleate the development of small-molecule mimics.

Binding Sites↗
PubMed2005

Malonate-assisted purification of human caspases.

Caspases have in the past decade become some of the most intensely pursued targets for the design of small-molecule inhibitors. Two significant technological roadblocks to developing caspase-binding molecules are the poor solubility of a subset of the bacterially expressed proteins and the instability of the renatured proteins that results from rapid inactivating autolysis at high protein concentrations. In this report, we present a generalized method of renaturing human caspases and inhibiting the self-proteolytic activity of the enzymes without a need for covalent active-site inhibitors. Our method, which involves blocking the S1 region of the active site with malonate, enables one to inhibit fully the inactivating autolysis in human caspases and increases the yields of renatured active enzyme. It furthermore does not necessitate removal of malonate prior to setting up enzymatic assays since as high as 100-mM concentrations of malonate do not compete efficiently with caspase substrates or larger caspase inhibitors for binding to the active site. The method described in this report simplifies greatly caspase purification and makes it possible to stabilize the enzymes against autolysis without a need for costly, and frequently synthetically challenging, small-molecule inhibitors.

Autolysis↗
PubMed2004

Site-specific disulfide capture of agonist and antagonist peptides on the C5a receptor.

The manner by which peptidic ligands bind and activate their corresponding G-protein-coupled receptors is not well understood. One of the better characterized peptidic ligands is the chemotactic cytokine complement factor 5a (C5a), a 74-amino acid helical bundle. Previous studies showed 6-mer peptide analogs derived from the C terminus of the C5a ligand can bind to C5aR (Kd values approximately 0.1-1 microm) and either agonize or antagonize the receptor (Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J., and Bourne, H. R. (2001) J. Biol. Chem. 276, 3394-3400). Here, we provide direct biochemical data using disulfide trapping to support a model that these peptides bind within a transmembrane helical triad formed by alpha-helices III, VI, and VII. We show that the three amino acids on the C terminus of the peptide analogs bind too weakly to exert a functional effect themselves. However, when a cysteine residue is placed on their N terminus they can be trapped by disulfide interchange to specific cysteines in helix III and VI and not to other cysteines, engineered into the C5aR. The trapped peptides function as agonists or partial antagonists, similar to the non-covalent parents from which they were derived. These data help to further refine the binding mode for C5a to the C5aR and suggest an approach and a binding site that may be applicable to studying other peptide binding receptors.

Animals↗
PubMed2004

Discovery of an allosteric site in the caspases.

Allosteric regulation of proteins by conformational change is a primary means of biological control. Traditionally it has been difficult to identify and characterize novel allosteric sites and ligands that freeze these conformational states. We present a site-directed approach using Tethering for trapping inhibitory small molecules at sites away from the active site by reversible disulfide bond formation. We screened a library of 10,000 thiol-containing compounds against accessible cysteines of two members of the caspase family of proteases, caspase-3 and -7. We discovered a previously unreported and conserved allosteric site in a deep cavity at the dimer interface 14 A from the active site. This site contains a natural cysteine that, when disulfide-bonded with either of two specific compounds, inactivates these proteases. The allosteric site is functionally coupled to the active site, such that binding of the compounds at the allosteric site prevents peptide binding at the active site. The x-ray crystal structures of caspase-7 bound by either compound demonstrates that they inhibit caspase-7 by trapping a zymogen-like conformation. This approach may be useful to identify new allosteric sites from natural or engineered cysteines, to study allosteric transitions in proteins, and to nucleate drug discovery efforts.

Allosteric Regulation↗
PubMed2004

Apo cytochrome c inhibits caspases by preventing apoptosome formation.

Caspases are cysteine proteases and potent inducers of apoptosis. Their activation and activity is therefore tightly regulated. There are several mechanisms by which caspases can be activated but one key pathway involves release of holo cytochrome c from mitochondria into the cytoplasm. Cytoplasmic holo cytochrome c binds to apoptotic protease activating factor-1 (Apaf-1), driving the formation of an Apaf-1 oligomer (the apoptosome) which in turn binds and activates caspase-9. Previously we showed that the apo form of cytochrome c (lacking heme) can bind Apaf-1 and block both holo-dependent caspase activation in cell extracts and Bax-induced apoptosis in cells. Here we tested the ability of apo cytochrome c to inhibit caspase-9 activation induced by recombinant Apaf-1. Furthermore, using purified proteins and size exclusion chromatography we show that apo cytochrome c prevents holo cytochrome c-dependent apoptosome formation.

Apoenzymes↗
PubMed2004

Mechanistic diversity of cytokine receptor signaling across cell membranes.

Circulating cytokines bind to specific receptors on the cell outer surface to evoke responses inside the cell. Binding of cytokines alters the association between receptor molecules that often cross the membrane only once in a single alpha-helical segment. As a consequence, association of protein domains on the inside of the membrane are also altered. Increasing evidence suggests that an initial "off-state" of associated receptors is perturbed, and brought to an activated state that leads to intracellular signaling and eventually effects a change in DNA transcription. The initial detection event that transduces the change in receptor association is sensitive to both proximity and orientation of the receptors, and probably also to the time that the activated state or receptor association is maintained. Ultimately, a cascade of phosphorylation events is triggered. The initial kinases are sometimes part of the intracellular domains of the receptors. The kinases can also be separate proteins that may be pre-associated with intracellular domains of the receptors, or can be recruited after the intracellular association of the activated receptors. We focus here on each of the cases for which structures of the activated cytokine-receptor complexes are known, in a search for underlying mechanisms. The variations in modes of association, stoichiometries of receptors and cytokines, and orientations before and after activation of these receptors are almost as great as the number of complexes themselves. The principles uncovered nevertheless illustrate the basis for high specificity and fidelity in cytokine-mediated signaling.

Animals↗
PubMed2004

Searching for new allosteric sites in enzymes.

The discovery of new allosteric sites generates opportunities for the identification of novel pharmaceuticals and increases our understanding of basic biological processes. Increasingly, allosteric sites are being discovered in various families of proteins by several methods, paving the way for the development of entirely new classes of drugs with a wide range of chemotypes. New allosteric sites in enzymes have been discovered both incidentally and by directed means, and the mechanisms by which allosteric activation and inhibition occur at these sites have been investigated. By exploring recent structurally well-characterized examples, trends begin to emerge for both the modes of binding and mechanisms of inhibition.

Allosteric Site↗
PubMed2004

Tethering: fragment-based drug discovery.

The genomics revolution has provided a deluge of new targets for drug discovery. To facilitate the drug discovery process, many researchers are turning to fragment-based approaches to find lead molecules more efficiently. One such method, Tethering1, allows for the identification of small-molecule fragments that bind to specific regions of a protein target. These fragments can then be elaborated, combined with other molecules, or combined with one another to provide high-affinity drug leads. In this review we describe the background and theory behind Tethering and discuss its use in identifying novel inhibitors for protein targets including interleukin-2 (IL-2), thymidylate synthase (TS), protein tyrosine phosphatase 1B (PTP-1B), and caspases.

Binding Sites↗
PubMed2003

Potent small-molecule binding to a dynamic hot spot on IL-2.

The complexes between IL-2 and two similar small molecules, one a lead compound and the other a potent, affinity-optimized compound, were determined by X-ray crystallography. The lead compound (IC50 = 6 muM) bound to a hot spot on IL-2 in a groove that is not apparent in either the unliganded protein or a complex between IL-2 and a weakly bound drug fragment. The affinity-optimized compound (IC50 = 0.06 muM), which has an added aromatic acid fragment, bound in the same groove as the lead compound. In addition, a novel binding site was formed for the aromatic acid which is unseen in the complex with the lead compound. Thus, the hot spot on IL-2 is highly dynamic, with the protein changing form at multiple sites to maximize packing for each compound. Binding-site rigidity is often thought to play a role in high-affinity interactions. However, in this case, specific contacts between the small molecule and the protein are made despite the adaptivity of the hot spot. Given the change in morphology that was observed in IL-2, it is unlikely that a potent inhibitor could have been found by rational design. Therefore, fragment assembly methods offer the stochastic advantage of finding fragments in flexible protein regions where structural changes are unpredictable.

Binding Sites↗
PubMed2003

Direct activation of the apoptosis machinery as a mechanism to target cancer cells.

Apoptosis plays a pivotal role in the cytotoxic activity of most chemotherapeutic drugs, and defects in this pathway provide a basis for drug resistance in many cancers. Thus the ability to restore apoptosis by using small molecules could have important therapeutic implications. Using a cell-free assay to simultaneously target multiple components of the apoptosis pathway, we identified a class of compounds that activate caspases in a cytochrome c-dependent manner and induce apoptosis in whole cells. By reconstituting the apoptosis pathway with purified proteins, we determined that these compounds promote the protein-protein association of Apaf-1 into the functional apoptosome. These compounds exert cytostatic and cytotoxic effects on a variety of cancer cell lines while having little or no activity against the normal cell lines tested. These findings suggest that direct activation of the basic apoptosis machinery may be a viable mechanism to selectively target cancer.

Apoptosis↗
PubMed2003

Binding of small molecules to an adaptive protein-protein interface.

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 alpha 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.

Cloning, Molecular↗