Biomedical subjects

Kurt Deshayes

Publications and source records attributed to Kurt Deshayes.

10 recordsLinked to original sources

PubMed2006

Design, synthesis, and biological activity of a potent Smac mimetic that sensitizes cancer cells to apoptosis by antagonizing IAPs.

Designed second mitochondrial activator of caspases (Smac) mimetics based on an accessible [7,5]-bicyclic scaffold bind to and antagonize protein interactions involving the inhibitor of apoptosis (IAP) proteins, X-chromosome-linked IAP (XIAP), melanoma IAP (ML-IAP), and c-IAPs 1 and 2 (cIAP1 and cIAP2). The design rationale is based on a combination of phage-panning data, peptide binding studies, and a survey of potential isosteres. The synthesis of two scaffolds is described. These compounds bind the XIAP-baculoviral IAP repeat 3 (BIR3), cIAP1-BIR3, cIAP2-BIR3, and ML-IAP-BIR domains with submicromolar affinities. The most potent Smac mimetic binds the cIAP1-BIR3 and ML-IAP-BIR domains with a K i of 50 nM. The X-ray crystal structure of this compound bound to an ML-IAP/XIAP chimeric BIR domain protein is compared with that of a complex with a phage-derived tetrapeptide, AVPW. The structures show that these compounds bind to the Smac-binding site on ML-IAP with identical hydrogen-bonding patterns and similar hydrophobic interactions. Consistent with the structural data, coimmunoprecipitation experiments demonstrate that the compounds can effectively block Smac interactions with ML-IAP. The compounds are further demonstrated to activate caspase-3 and -7, to reduce cell viability in assays using MDA-MB-231 breast cancer cells and A2058 melanoma cells, and to enhance doxorubicin-induced apoptosis in MDA-MB-231 cells.

Apoptosis↗

PubMed2005

Engineering ML-IAP to produce an extraordinarily potent caspase 9 inhibitor: implications for Smac-dependent anti-apoptotic activity of ML-IAP.

ML-IAP (melanoma inhibitor of apoptosis) is a potent anti-apoptotic protein that is strongly up-regulated in melanoma and confers protection against a variety of pro-apoptotic stimuli. The mechanism by which ML-IAP regulates apoptosis is unclear, although weak inhibition of caspases 3 and 9 has been reported. Here, the binding to and inhibition of caspase 9 by the single BIR (baculovirus IAP repeat) domain of ML-IAP has been investigated and found to be significantly less potent than the ubiquitously expressed XIAP (X-linked IAP). Engineering of the ML-IAP-BIR domain, based on comparisons with the third BIR domain of XIAP, resulted in a chimeric BIR domain that binds to and inhibits caspase 9 significantly better than either ML-IAP-BIR or XIAP-BIR3. Mutational analysis of the ML-IAP-BIR domain demonstrated that similar enhancements in caspase 9 affinity can be achieved with only three amino acid substitutions. However, none of these modifications affected binding of the ML-IAP-BIR domain to the IAP antagonist Smac (second mitochondrial activator of caspases). ML-IAP-BIR was found to bind mature Smac with low nanomolar affinity, similar to that of XIAP-BIR2-BIR3. Correspondingly, increased expression of ML-IAP results in formation of a ML-IAP-Smac complex and disruption of the endogenous interaction between XIAP and mature Smac. These results suggest that ML-IAP might regulate apoptosis by sequestering Smac and preventing it from antagonizing XIAP-mediated inhibition of caspases, rather than by direct inhibition of caspases.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Hybrid photoactive assemblies: electron injection from host-guest complexes into semiconductor nanoparticles.

Novel ternary assemblies consisting of fully encapsulated host-guest complexes (hemicarceplexes) and wide band gap semiconductor nanoparticles were investigated. The water-soluble amphiphilic host (octacarboxyhemicarcerand) traps the hydrophobic chromophore within its cavity and binds to the surface of metal oxide nanoparticles. Fluorescence quenching and fast charge injection, kforward >/= 7 x 109 s-1, from the S2 state of encapsulated azulene were observed. Charge recombination proceeds at a much lower rate of 2 x 107 s-1. Interestingly, the recombination kinetics is homogeneous, suggesting that electron tunneling through the wall of the "molecular container" is the rate-limiting step of the process.

Journal Article↗

PubMed2003

Complex with a phage display-derived peptide provides insight into the function of insulin-like growth factor I.

The dramatic improvement in the NMR spectra of insulin-like growth factor I (IGF-I) in the presence of a peptide identified from a phage display library has allowed for the first time the determination of a high-resolution solution structure for much of IGF-I. The three helices of IGF-I in this complex have an arrangement similar to that seen in high-resolution crystal structures of IGF-I and insulin, although there are differences in the conformation and precise location of helix 3. A cluster of hydrophobic and basic side chains within the turn-helix motif of the peptide contact a hydrophobic patch on helices 1 and 3 of IGF-I. The importance of this patch for tight binding was verified using alanine scanning mutagenesis of the peptide in two different phage display formats. Consistent with its antagonistic activity, the peptide binds to a region implicated by mutagenesis studies to be important for association with IGF binding proteins (IGFBPs). The ability of the peptide to also inhibit signaling has important implications for the manner in which IGF-I interacts with its receptor. Interestingly, the peptide uses the same binding site as detergent and a fragment of IGFBP-5 identified in other IGF-I complexes. The ligand-induced structural variability of helix 3 in these complexes suggests that exchange between such conformations may be the source of the dynamic nature of free IGF-I and likely has functional significance for the ability of IGF-I to recognize two signaling receptors and six binding proteins with high affinity.

Alanine↗

PubMed2003

Structure and function analysis of peptide antagonists of melanoma inhibitor of apoptosis (ML-IAP).

Melanoma inhibitor of apoptosis (ML-IAP) is a potent anti-apoptotic protein that is upregulated in a number of melanoma cell lines but not expressed in most normal adult tissues. Overexpression of IAP proteins, such as ML-IAP or the ubiquitously expressed X-chromosome-linked IAP (XIAP), in human cancers has been shown to suppress apoptosis induced by a variety of stimuli. Peptides based on the processed N-terminus of Smac/DIABLO can negate the ability of overexpressed ML-IAP or XIAP to suppress drug-induced apoptosis. Such peptides have been demonstrated to bind to the single baculovirus IAP repeat (BIR) of ML-IAP and the third BIR of XIAP with similar high affinities (approximately 0.5 microM). Herein, we use phage-display of naïve peptide libraries and synthetic peptides to investigate the peptide-binding properties of ML-IAP-BIR and XIAP-BIR3. X-ray crystal structures of ML-IAP-BIR in complex with Smac- and phage-derived peptides, together with peptide structure-activity-relationship data, indicate that the peptides can be modified to provide increased binding affinity and selectivity for ML-IAP-BIR relative to XIAP-BIR3. For instance, substitution of Pro3' in the Smac-based peptide (AVPIAQKSE) with (2S,3S)-3-methylpyrrolidine-2-carboxylic acid [(3S)-methyl-proline] results in a peptide with 7-fold greater affinity for ML-IAP-BIR and about 100-fold specificity for ML-IAP-BIR relative to XIAP-BIR3.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Exploring protein-protein interactions with phage display.

Protein-protein interactions mediate essentially all biological processes. A detailed understanding of these interactions is thus a major goal of modern biological chemistry. In recent years, genome sequencing efforts have revealed tens of thousands of novel genes, but the benefits of genome sequences will only be realized if these data can be translated to the level of protein function. While genome databases offer tremendous opportunities to expand our knowledge of protein-protein interactions, they also present formidable challenges to traditional protein chemistry methods. Indeed, it has become apparent that efficient analysis of proteins on a proteome-wide scale will require the use of rapid combinatorial approaches. In this regard, phage display is an established combinatorial technology that is likely to play an even greater role in the future of biology. This article reviews recent applications of phage display to the analysis of protein-protein interactions. With combinatorial mutagenesis strategies, it is now possible to rapidly map the binding energetics at protein-protein interfaces through statistical analysis of phage-displayed protein libraries. In addition, naïve phage-displayed peptide libraries can be used to obtain small peptide ligands to essentially any protein of interest, and in many cases, these binding peptides act as antagonists or even agonists of natural protein functions. These methods are accelerating the pace of research by enabling the study of complex protein-protein interactions with simple molecular biology methods. With further optimization and automation, it may soon be possible to study hundreds of different proteins in parallel with efforts comparable to those currently expended on the analysis of individual proteins.

Animals↗

PubMed2003

Bacteriophage that display small molecules.

Technology has been developed to display small molecules on phage particles. This innovation enables the generation of libraries of phage-tagged compounds with novel properties that are well suited for in vivo assays.

Bacteriophages↗

PubMed2002

The Erbin PDZ domain binds with high affinity and specificity to the carboxyl termini of delta-catenin and ARVCF.

Erbin is a recently described member of the LAP (leucine-rich repeat and PDZ domain) protein family. We used a C-terminally displayed phage peptide library to identify optimal ligands for the Erbin PDZ domain. Phage-selected peptides were type 1 PDZ ligands that bound with high affinity and specificity to the Erbin PDZ domain in vitro. These peptides most closely resembled the C-terminal PDZ domain-binding motifs of three p120-related catenins: delta-catenin, ARVCF, and p0071 (DSWV-COOH). Analysis of the interactions of the Erbin PDZ domain with synthetic peptides matching the C termini of ARVCF or delta-catenin also demonstrated specific high affinity binding. We characterized the interactions between the Erbin PDZ domain and both ARVCF and delta-catenin in vitro and in vivo. The Erbin PDZ domain co-localized and coprecipitated with ARVCF or delta-catenin complexed with beta-catenin and E/N-cadherin. Mutagenesis and peptide competition experiments showed that the association of Erbin with the cadherin-catenin complex was mediated by the interaction of its PDZ domain with the C-terminal PDZ domain-binding motifs (DSWV-COOH) of ARVCF and delta-catenin. Finally, we showed that endogenous delta-catenin and Erbin co-localized in and co-immunoprecipitated from neurons. These results suggest that delta-catenin and ARVCF may function to mediate the association of Erbin with the junctional cadherin-catenin complex. They also demonstrate that C-terminal phage-display technology can be used to predict physiologically relevant ligands for PDZ domains.

Adaptor Proteins, Signal Transducing↗

PubMed2002

SMAC negatively regulates the anti-apoptotic activity of melanoma inhibitor of apoptosis (ML-IAP).

Inhibitors of apoptosis (IAPs) physically interact with a variety of pro-apoptotic proteins and inhibit apoptosis induced by diverse stimuli. X-linked IAP (X-IAP) is a prototype IAP family member that inhibits several caspases, the effector proteases of apoptosis. The inhibitory activity of X-IAP is regulated by SMAC, a protein that is processed to its active form upon receipt of a death stimulus. Cleaved SMAC binds X-IAP and antagonizes its anti-apoptotic activity. Here we show that melanoma IAP (ML-IAP), a potent anti-cell death protein and caspase inhibitor, physically interacts with SMAC through its BIR (baculovirus IAP repeat) domain. In addition to binding full-length SMAC, ML-IAP BIR associates with SMAC peptides that are derived from the amino terminus of active, processed SMAC. This high affinity interaction is very specific and can be completely abolished by single amino acid mutations either in the amino terminus of active SMAC or in the BIR domain of ML-IAP. In cells expressing ML-IAP and X-IAP, SMAC coexpression or addition of SMAC peptides abrogates the ability of the IAPs to inhibit cell death. These results demonstrate the feasibility of using SMAC peptides as a way to sensitize IAP-expressing cells to pro-apoptotic stimuli such as chemotherapeutic agents.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Rapid identification of small binding motifs with high-throughput phage display: discovery of peptidic antagonists of IGF-1 function.

A panel of 22 naïve peptide libraries was constructed in a polyvalent phage display format and sorted against insulin-like growth factor-1 (IGF-1). The libraries were pooled to achieve a total diversity of 4.4 x 10(11). After three rounds of selection, the majority of the phage clones bound specifically to IGF-1, with a disulfide-constrained CX(9)C scaffold dominating the selection. Four monovalently displayed sub-libraries were designed on the basis of these conserved motifs. Sub-library maturation in a monovalent format yielded an antagonistic peptide that inhibited the interactions between IGF-1 and two cell-surface receptors and those between IGF-1 and two soluble IGF binding proteins with micromolar potency. NMR analysis revealed that the peptide is highly structured in the absence of IGF-1, and peptides that preorganize the binding elements were selected during the sorting.

Amino Acid Motifs↗