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Irwin Chaiken

Publications and source records attributed to Irwin Chaiken.

16 recordsLinked to original sources
PubMed2007

Recruitment pharmacophore for interleukin 5 receptor alpha antagonism.

Interleukin-5 receptor alpha is a therapeutic target for hypereosinophilic diseases including allergic inflammations and asthma. The cyclic peptide AF17121 (Ac-VDE[CWRIIASHTWFC]AEE-CONH(2)) has been identified as a submicromolar inhibitor of interleukin 5 (IL5)-interleukin 5 receptor alpha (IL5Ralpha) interaction from a random peptide screen. However, this inhibitor has limitations as a drug lead because of its relatively large size. We used chemical synthesis of peptides with natural and non-natural amino acids along with kinetic binding and cell proliferation competition assays to expand definition of structural elements in the peptide that are important for receptor antagonism and to elucidate the underlying pharmacophore. We found that the specific steric array of hydrogen bonding groups in the Arg 6 guanido side chain is critical for receptor inhibition. We also investigated noncharged structural elements in AF17121. Screening a set of five hydrophobic residues showed that peptide function is strongly sensitive to variations in several of these residues, most prominently Ile 7 and Trp 13. We postulate that presentation of charged, hydrogen bonding and hydrophobic structural elements within the disulfide-constrained peptide drives IL5Ralpha recruitment by AF17121. We hypothesize from these results and previous receptor mutagenesis studies that Arg 6 recruitment of IL5Ralpha occurs through hydrogen bonding as well as charge-charge interactions with Asp 55 in site one of domain 1 of IL5Ralpha, and that this interaction is complemented by additional charged and hydrophobic interactions around the Asp 55 locus. Scaffolding a limited set of structural elements in the inhibitor pharmacophore may be useful for small molecule antagonist design inspired by the peptide.

Amino Acid Sequence↗
PubMed2006

Antibody binding is a dominant determinant of the efficiency of human immunodeficiency virus type 1 neutralization.

Primary and laboratory-adapted variants of human immunodeficiency virus type 1 (HIV-1) exhibit a wide range of sensitivities to neutralization by antibodies directed against the viral envelope glycoproteins. An antibody directed against an artificial FLAG epitope inserted into the envelope glycoproteins of three HIV-1 isolates with vastly different neutralization sensitivities inhibited all three viruses equivalently. Thus, naturally occurring HIV-1 isolates that are neutralization resistant are not necessarily more impervious to the inhibitory consequences of bound antibody. Moreover, the binding affinity of the anti-FLAG antibody correlated with neutralizing potency, underscoring the dominant impact on neutralization of antibody binding to the envelope glycoproteins.

Antibody Affinity↗
PubMed2006

Asymmetric usage of antagonist charged residues drives interleukin-5 receptor recruitment but is insufficient for receptor activation.

The cyclic peptide AF17121 (VDECWRIIASHTWFCAEE) is a library-derived antagonist for human Interleukin-5 receptor alpha (IL5Ralpha). We have previously demonstrated that AF17121 mimics Interleukin-5 (IL5) by binding in a region of IL5Ralpha that overlaps the IL5 binding epitope. In the present study, to explore the functional importance of the amino acid residues of AF17121 required for effective binding to, and antagonism of, IL5Ralpha, each charged residue was subjected to site-directed mutagenesis and examined for IL5Ralpha interaction by using a surface plasmon resonance biosensor. One residue, Arg(6), was found to be essential for receptor antagonism; its replacement with either alanine or lysine completely abolished the interaction between AF17121 and IL5Ralpha. Other charged residues play modulatory roles. One class consists of the N-terminal acidic cluster (Asp(2) and Glu(3)) for which alanine replacement decreased the association rate. A second class consists of His(11) and the C-terminal acidic cluster (Glu(17) and Glu(18)) for which alanine replacement increased the dissociation rate. Binding model analysis of the mutants of the latter class of residues indicated the existence of conformational rearrangement during the interaction. On the basis of these results, we propose a model in which Arg(6) and N-terminal acidic residues drive the encounter complex, while Arg(6), His(11), and C-terminal acidic residues are involved in stabilizing the final complex. These data argue that the charged residues of AF17121 are utilized asymmetrically in the pathway of inhibitor-receptor complex formation to deactivate the receptor function. The results also help focus emerging models for the mechanism by which IL5 activates the IL5Ralpha-betac receptor system.

Amino Acid Sequence↗
PubMed2005

Receptor epitope usage by an interleukin-5 mimetic peptide.

The cyclic peptide AF17121 is a library-derived antagonist for human interleukin-5 (IL5) receptor alpha (IL5Ralpha) and inhibits IL5 activity. Our previous results have demonstrated that the sixth arginine residue of the peptide is crucial for the inhibitory effect and that several acidic residues in the N- and C-terminal regions also make a contribution, although to a lesser extent (Ruchala, P., Varadi, G., Ishino, T., Scibek, J., Bhattacharya, M., Urbina, C., Van Ryk, D., Uings, I., and Chaiken, I. (2004) Biopolymers 73, 556-568). However, the recognition mechanism of the receptor has remained unresolved. In this study, AF17121 was fused to thioredoxin by recombinant DNA techniques and examined for IL5Ralpha interaction using a surface plasmon resonance biosensor method. Kinetic analysis revealed that the dissociation rate of the peptide.receptor complex is comparable with that of the cytokine.receptor complex. The fusion peptide competed with IL5 for both biological function and interaction with IL5Ralpha, indicating that the binding sites on the receptor are shared by AF17121 and IL5. To define the epitope residues for AF17121, we defined its binding footprint on IL5Ralpha by alanine substitution of Asp(55), Asp(56), Glu(58), Lys(186), Arg(188), and Arg(297) of the receptor. Marked effects on the interaction were observed in all three fibronectin type III domains of IL5Ralpha, in particular Asp(55), Arg(188), and Arg(297) in the D1, D2, and D3 domains, respectively. This footprint represents a significant subset of that for IL5 binding. The fact that AF17121 mimics the receptor binding capability of IL5 but antagonizes biological function evokes several models for how IL5 induces activation of the multisubunit receptor system.

Alanine↗
PubMed2005

Expression, purification, and characterization of recombinant cyanovirin-N for vaginal anti-HIV microbicide development.

Cyanovirin-N (CV-N) is a prokaryotic protein under development as a topical anti-HIV microbicide, an urgent and necessary approach to prevent HIV transmission in at-risk populations worldwide. We have expressed recombinant CV-N as inclusion bodies in the cytoplasm of Escherichia coli. A purification scheme has been developed that exploits the physicochemical properties of this protein, in particular its stability in a harsh inclusion body purification scheme. Under the conditions developed, this system yields 140 mg of highly purified CV-N per liter of high-density cell culture, which represents a 14-fold increase over the best recombinant CV-N yield reported to date. This purification scheme results in monomeric CV-N as analyzed by SDS-PAGE, isoelectric focusing, and reverse phase- and size exclusion-HPLC. This recombinantly expressed and refolded CV-N binds to gp120 with nanomolar affinity and retains its potent anti-HIV activities in cell-based assays. The expression and purification system described herein provides a better means for the mass production of CV-N for further microbicide development.

Animals↗
PubMed2005

Cytokine recognition by human interleukin 5 receptor.

The activation of interleukin 5 (IL-5) receptor is a dynamic process that depends on specific interaction of IL-5 with IL-5 receptor alpha, the formation of oligomeric receptor complexes with receptor beta, and the initiation of cytoplasmic phosphorylation events. These steps culminate in the triggering of a cellular response. Important advances have been made recently in understanding the molecular mechanisms of cytokine recognition, receptor assembly, and signal triggering. Cytokine recognition can be envisioned by relating structure to function in IL-5 and IL-5 receptor alpha. A pair of charge-complementary regions plays an essential role in the specific interaction between IL-5 receptor alpha and IL-5. Moreover, peptide library methodology has led to the discovery of IL-5 receptor alpha antagonists that mimic key elements in IL-5 receptor recognition. Because IL-5 has been implicated in the pathology of eosinophil-related inflammatory diseases, revealing the key recognition elements of IL-5, IL-5 mimetic peptides, and IL-5 receptor alpha could help drive the design of new compounds for therapeutic treatment against allergic inflammatory diseases such as asthma.

Animals↗
PubMed2004

Cyclic peptide interleukin 5 antagonists mimic CD turn recognition epitope for receptor alpha.

The cyclic peptide AF17121 (Ac-VDECWRIIASHTWFCAEE) that inhibits interleukin 5 (IL-5) function and IL-5 receptor alpha-chain (IL-5Ralpha) binding has been derived from recombinant random peptide library screening and follow-up synthetic variation. To better understand the structural basis of its antagonist activity, AF17121 and a series of analogs of the parent peptide were prepared by solid phase peptide synthesis. Sequence variation was focused on the charged residues Asp(2), Glu(3), Arg(6), Glu(17), and Glu(18). Two of those residues, Glu(3) and Arg(6), form an EXXR motif that was found to be common among library-derived IL-5 antagonists. The E and R in the EXXR motif have a proximity similar to charged residues in a previously identified receptor alpha binding region, the beta-strand between the C- and D-helices of human IL-5. Optical biosensor interaction kinetics and cell proliferation assays were used to evaluate the antagonist activities of the purified synthetic peptides, by measuring competition with the highly active single chain IL-5. Analogs in which acidic residues (Asp(2), Glu(3), Glu(17), and Glu(18)) were replaced individually by Ala retained substantial competition activity, with multiple replacements in these residues leading to fractional loss of potency at most. In contrast, R6A analogs had strongly reduced competition activity. The results reveal that the arginine residue is crucial for the IL-5Ralpha binding of AF17121, while the acidic residues are not essential though likely complex-stabilizing particularly in the Asp(2)-Glu(3) region. By CD, AF17121 exhibited mostly disordered structure with evidence for a small beta-sheet content, and replacement of the arginine had no influence on the observed secondary structure of the peptides. The dominance of Arg(6) in AF17121 activity corresponds to previous findings of dominance of the positive charge balance in the antiparallel beta-sheet of IL-5 composed of (88)EERRR(92) in one strand of the CD turn region of IL-5 and with Arg(32) in the neighboring beta-strand. These results argue that AF17121 and related library-derived peptides function by mimicking the CD turn receptor alpha recognition epitope in IL-5 and open the way to small molecule antagonist design.

Amino Acid Sequence↗
PubMed2004

Small-molecule inhibitors of HIV-1 entry block receptor-induced conformational changes in the viral envelope glycoproteins.

When interacting with the CD4 receptor, the HIV gp120 envelope glycoprotein undergoes conformational changes that allow binding to the chemokine receptor. Receptor binding is proposed to lead to conformational changes in the gp41 transmembrane envelope glycoprotein involving the creation and/or exposure of a coiled coil consisting of three heptad repeat (HR) sequences. The subsequent interaction of the HR2 region of gp41 with this coiled coil results in the assembly of a six-helix bundle that promotes the fusion of the viral and target cell membranes. Here we show that CD4 binding to gp120 induces the formation and/or exposure of the gp41 HR1 coiled coil in a process that does not involve gp120 shedding and that depends on the proteolytic maturation of the gp160 envelope glycoprotein precursor. Importantly, BMS-806 and related HIV-1 entry inhibitors bind gp120 and block the CD4 induction of HR1 exposure without significantly affecting CD4 binding. Moreover, these compounds do not disrupt gp120-chemokine receptor binding or the HR1-HR2 interaction within gp41. These studies thus define a receptor-induced conformational rearrangement of gp120-gp41 that is important for both CD4-dependent and CD4-independent HIV-1 entry and is susceptible to inhibition by low-molecular-weight compounds.

Cell Line↗
PubMed2003

Kinetic interaction analysis of human interleukin 5 receptor alpha mutants reveals a unique binding topology and charge distribution for cytokine recognition.

Human interleukin 5 receptor alpha (IL5Ralpha) comprises three fibronectin type III domains (D1, D2, and D3) in the extracellular region. Previous results have indicated that residues in the D1D2 domains are crucial for high affinity interaction with human interleukin 5 (IL5). Yet, it is the D2D3 domains that have sequence homology with the classic cytokine recognition motif that is generally assumed to be the minimum cytokine-recognizing unit. In the present study, we used kinetic interaction analysis of alanine-scanning mutational variants of IL5Ralpha to define the residues involved in IL5 recognition. Soluble forms of IL5Ralpha variants were expressed in S2 cells, selectively captured via their C-terminal V5 tag by anti-V5 tag antibody immobilized onto the sensor chip and examined for IL5 interaction by using a sandwich surface plasmon resonance biosensor method. Marked effects on the interaction kinetics were observed not only in D1 (Asp(55), Asp(56), and Glu(58)) and D2 (Lys(186) and Arg(188)) domains, but also in the D3 (Arg(297)) domain. Modeling of the tertiary structure of IL5Ralpha indicated that these binding residues fell into two clusters. The first cluster consists of D1 domain residues that form a negatively charged patch, whereas the second cluster consists of residues that form a positively charged patch at the interface of D2 and D3 domains. These results suggest that the IL5 x IL5Ralpha system adopts a unique binding topology, in which the cytokine is recognized by a D2D3 tandem domain combined with a D1 domain, to form an extended cytokine recognition interface.

Amino Acid Sequence↗
PubMed2003

Salivary agglutinin inhibits HIV type 1 infectivity through interaction with viral glycoprotein 120.

Salivary agglutinin (SAG) is a high molecular mass glycoprotein (340 kDa) that plays important roles in innate immunity. SAG has been found to specifically inhibit HIV-1 infectivity and to bind to virus through the envelope protein gp120. Although SAG binds to gp120 of the virus, the exact nature of this binding has not been characterized. Using surface plasmon resonance technology, we have found that SAG interacts with recombinant envelopes derived from diverse HIV-1 isolates with K(D) values ranging from 10(-7) to 10(-10) M, comparable to gp120-sCD4 binding. Furthermore, SAG binding to gp120 is Ca(2+) dependent. sCD4 prebound to gp120 failed to abrogate SAG binding, suggesting a distinct mechanism for SAG inhibition of HIV-1 infectivity. Inhibition by monoclonal antibodies specific for carbohydrates also implicates the involvement of carbohydrates in the interaction between SAG and gp120. These results argue that the anti-HIV-1 activity of SAG is due to carbohydrate-mediated binding to gp120. A demonstration that SAG is related to lung scavenger receptor, gp-340, further suggests the roles of SAG in preventing pathogen invasion at the entry portal and raises its potential as an anti-HIV-1 drug candidate.

Agglutinins↗
PubMed2002

HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites.

The ability of human immunodeficiency virus (HIV-1) to persist and cause AIDS is dependent on its avoidance of antibody-mediated neutralization. The virus elicits abundant, envelope-directed antibodies that have little neutralization capacity. This lack of neutralization is paradoxical, given the functional conservation and exposure of receptor-binding sites on the gp120 envelope glycoprotein, which are larger than the typical antibody footprint and should therefore be accessible for antibody binding. Because gp120-receptor interactions involve conformational reorganization, we measured the entropies of binding for 20 gp120-reactive antibodies. Here we show that recognition by receptor-binding-site antibodies induces conformational change. Correlation with neutralization potency and analysis of receptor-antibody thermodynamic cycles suggested a receptor-binding-site 'conformational masking' mechanism of neutralization escape. To understand how such an escape mechanism would be compatible with virus-receptor interactions, we tested a soluble dodecameric receptor molecule and found that it neutralized primary HIV-1 isolates with great potency, showing that simultaneous binding of viral envelope glycoproteins by multiple receptors creates sufficient avidity to compensate for such masking. Because this solution is available for cell-surface receptors but not for most antibodies, conformational masking enables HIV-1 to maintain receptor binding and simultaneously to resist neutralization.

Antibody Affinity↗
PubMed2002

Structure-based design of mimetics for granulocyte-macrophage colony stimulating factor (GM-CSF).

Granulocyte-macrophage colony stimulating factor (GM-CSF) activity has been linked to pro-inflammatory effects in autoimmune syndromes, such as rheumatoid arthritis. Thus GM-CSF mimetics with antagonist activity might play a therapeutic role in these diseases. The human GM-CSF core structure consists of a four alpha-helix bundle, and GM-CSF activity is controlled by its binding to a two-subunit receptor. A number of residues located on the B and C helices of GM-CSF are postulated to interact with the alpha chain of the GM-CSF receptor (GM-CSFR). Several approaches have been successfully utilized to develop peptide mimetics of this site, including peptides from the native sequence, a peptide derived from a recombinant antibody (rAb) light chain which mimicked GM-CSF receptor binding activity, and structurally guided de novo design. Analysis of the rAb light chain had suggested mimicry of GM-CSF with residues mostly contributed by the CDR I region. Key residues involved in CDR I peptide/GM-CSFR binding were identified by truncation and alteration of individual residues, while the structural elements required to antagonize the biological action of GM-CSF were separately tested in binding and inhibitory activity assays of multiple cyclic analogues. A peptide designed to retain the loop conformation of the CDR I region of the rAb light chain competed with GM-CSF for both antibody and receptor binding, but the role of specific residues in antibody versus receptor binding differed markedly. These studies suggest that structural analysis of peptide mimetics can reveal differences in receptor and antibody binding, perhaps including key interactions that impact binding kinetics. Peptide mimetics of other four-helix bundle cytokines are reviewed, including helical and reverse turn mimetics of helical structures. Use of peptide mimetics coupled with structural and kinetic analysis provides a powerful approach to identifying important receptor-ligand interactions, which implications for rational design of novel therapeutics.

Amino Acid Sequence↗
PubMedDate not supplied

Coiled coil miniprotein randomization on phage leads to charge pattern mimicry of the receptor recognition determinant of interleukin 5.

Phage display was used to identify sequences that mimic structural determinants in interleukin5 (IL5) for IL5 receptor recognition. A coiled coil stem loop (CCSL) miniprotein scaffold library was constructed with its turn region randomized and panned for binding variants against human IL5 receptor alpha chain (IL5Ralpha). Competition enzyme-linked immunosorbent assays identified CCSL-phage selectants for which binding to IL5Ralpha was competed by IL5. The most frequently selected and IL5-competed CCSL-phage contain charged residues Arg and Glu in their turn sequences, in this regard resembling a beta strand sequence in the 'CD turn' region, of IL5, that has been proposed to present a key determinant for IL5 receptor alpha chain recognition. The most dominant CCSL-phage selectant sequence, PVEGRV, contains a negative/positive charge pattern similar to that seen in the original CD turn. To test the relatedness of CCSL-phage selectant sequences to the IL5 receptor recognition epitope, PVEGRV was grafted into the sequence 87--92 of a monomeric IL5. The resulting IL5 variant, [(87)PVEGRV(92)]GM1, was able to bind to IL5Ralpha in biosensor assays, to elicit TF-1 cell proliferation and to induce STAT5 phosphorylation in TF-1 cells. The results help discern sequence patterns in the IL5 CD turn region which are key in driving receptor recognition and demonstrate the utility of CCSL miniprotein scaffold phage display to identify local IL5 mimetic sequence arrangements that may ultimately lead to IL5 antagonists.

Amino Acid Sequence↗
PubMedDate not supplied

Proteins, recognition networks and developing interfaces for macromolecular biosensing.

Genomics and proteomics discovery is leading to the identification of all proteins and to the opportunity, and challenge, to reveal the protein recognition networks that drive virtually all biological processes. Over the past decade, biosensors have emerged as a key technology for detection and analysis of biomolecular interactions. An important limitation in developing such biosensors is that the focus has been mainly on sensor platforms, the transducing hardware that converts interaction signals into recorded data, without adequately considering the role of molecular interfaces, the elements of sensors that interact with analytes to produce signals. We have investigated this alternative focus by identifying and, where necessary, designing molecular interfaces that will more effectively drive new biosensor development and utilization in biomedical and biotechnological investigations. Here we describe our recent studies of coiled coil and lipid bilayer interfaces and the potential to use these to expand sensing technologies for multiplexed target detection and analysis in increasingly biologically relevant membrane like environments.

Amino Acid Motifs↗
PubMedDate not supplied

Modular, self-assembling peptide linkers for stable and regenerable carbon nanotube biosensor interfaces.

As part of an effort to develop nanoelectronic sensors for biological targets, we tested the potential to incorporate coiled coils as metallized, self-assembling, site-specific molecular linkers on carbon nanotubes (CNTs). Based on a previously conceived modular anchor-probe approach, a system was designed in which hydrophobic residues (valines and leucines) form the interface between the two helical peptide components. Charged residues (glutamates and arginines) on the borders of the hydrophobic interface increase peptide solubility, and provide stability and specificity for anchor-probe assembly. Two histidine residues oriented on the exposed hydrophilic exterior of each peptide were included as chelating sites for metal ions such as cobalt. Cysteines were incorporated at the peptide termini for oriented, thiol-mediated coupling to surface plasmon resonance (SPR) biosensor surfaces, gold nanoparticles or CNT substrates. The two peptides were produced by solid phase peptide synthesis using Fmoc chemistry: an acidic 42-residue peptide E42C, and its counterpart in the heterodimer, a basic 39-residue peptide R39C. The ability of E42C and R39C to bind cobalt was demonstrated by immobilized metal affinity chromatography and isothermal titration calorimetry. SPR biosensor kinetic analysis of dimer assembly revealed apparent sub-nanomolar affinities in buffers with and without 1 mM CoCl2 using two different reference surfaces. For device-oriented CNT immobilization, R39C was covalently anchored to CNT tips via a C-terminal cysteine residue. Scanning electron microscopy was used to visualize the assembly of probe peptide (E42C) N-terminally labeled with 15 nm gold nanoparticles, when added to the R39C-CNT surface. The results obtained open the way to develop CNT tip-directed recognition surfaces, using recombinant and chemically synthesized chimeras containing binding epitopes fused to the E42C sequence domain.

Biosensing Techniques↗