Disruption of XIAP-RIP2 Association Blocks NOD2-Mediated Inflammatory Signaling.

Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.

OTUB1 modulates c-IAP1 stability to regulate signalling pathways.

The cellular inhibitor of apoptosis (c-IAP) proteins are E3 ubiquitin ligases that are critical regulators of tumour necrosis factor (TNF) receptor (TNFR)-mediated signalling. Through their E3 ligase activity c-IAP proteins promote ubiquitination of receptor-interaction protein 1 (RIP1), NF-κB-inducing kinase (NIK) and themselves, and regulate the assembly of TNFR signalling complexes. Consequently, in the absence of c-IAP proteins, TNFR-mediated activation of NF-κB and MAPK pathways and the induction of gene expression are severely reduced. Here, we describe the identification of OTUB1 as a c-IAP-associated deubiquitinating enzyme that regulates c-IAP1 stability. OTUB1 disassembles K48-linked polyubiquitin chains from c-IAP1 in vitro and in vivo within the TWEAK receptor-signalling complex. Downregulation of OTUB1 promotes TWEAK- and IAP antagonist-stimulated caspase activation and cell death, and enhances c-IAP1 degradation. Furthermore, knockdown of OTUB1 reduces TWEAK-induced activation of canonical NF-κB and MAPK signalling pathways and modulates TWEAK-induced gene expression. Finally, suppression of OTUB1 expression in zebrafish destabilizes c-IAP (Birc2) protein levels and disrupts fish vasculature. These results suggest that OTUB1 regulates NF-κB and MAPK signalling pathways and TNF-dependent cell death by modulating c-IAP1 stability.

Structure-guided design of a selective BCL-X(L) inhibitor.

The prosurvival BCL-2 family protein BCL-X(L) is often overexpressed in solid tumors and renders malignant tumor cells resistant to anticancer therapeutics. Enhancing apoptotic responses by inhibiting BCL-X(L) will most likely have widespread utility in cancer treatment and, instead of inhibiting multiple prosurvival BCL-2 family members, a BCL-X(L)-selective inhibitor would be expected to minimize the toxicity to normal tissues. We describe the use of a high-throughput screen to discover a new series of small molecules targeting BCL-X(L) and their structure-guided development by medicinal chemistry. The optimized compound, WEHI-539 (7), has high affinity (subnanomolar) and selectivity for BCL-X(L) and potently kills cells by selectively antagonizing its prosurvival activity. WEHI-539 will be an invaluable tool for distinguishing the roles of BCL-X(L) from those of its prosurvival relatives, both in normal cells and notably in malignant tumor cells, many of which may prove to rely upon BCL-X(L) for their sustained growth.

Development of orthogonally protected hypusine for solid-phase peptide synthesis.

An orthogonally protected hypusine reagent was developed for solid-phase synthesis of hypusinated peptides using the Fmoc/t-Bu protection strategy. The reagent was synthesized in an overall yield of 27% after seven steps from Cbz-Lys-OBzl and (R)-3-hydroxypyrrolidin-2-one. The side-chain protecting groups (Boc and t-Bu) are fully compatible with standard Fmoc chemistry and can be readily removed during the peptide cleavage step. The utility of the reagent was demonstrated by solid-phase synthesis of hypusinated peptides.

c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling.

Ubiquitin ligases are critical components of the ubiquitination process that determine substrate specificity and, in collaboration with E2 ubiquitin-conjugating enzymes, regulate the nature of polyubiquitin chains assembled on their substrates. Cellular inhibitor of apoptosis (c-IAP1 and c-IAP2) proteins are recruited to TNFR1-associated signalling complexes where they regulate receptor-stimulated NF-κB activation through their RING domain ubiquitin ligase activity. Using a directed yeast two-hybrid screen, we found several novel and previously identified E2 partners of IAP RING domains. Among these, the UbcH5 family of E2 enzymes are critical regulators of the stability of c-IAP1 protein following destabilizing stimuli such as TWEAK or CD40 signalling or IAP antagonists. We demonstrate that c-IAP1 and UbcH5 family promote K11-linked polyubiquitination of receptor-interacting protein 1 (RIP1) in vitro and in vivo. We further show that TNFα-stimulated NF-κB activation involves endogenous K11-linked ubiquitination of RIP1 within the TNFR1 signalling complex that is c-IAP1 and UbcH5 dependent. Lastly, NF-κB essential modifier efficiently binds K11-linked ubiquitin chains, suggesting that this ubiquitin linkage may have a signalling role in the activation of proliferative cellular pathways.

Improved quantitative mass spectrometry methods for characterizing complex ubiquitin signals.

Ubiquitinated substrates can be recruited to macromolecular complexes through interactions between their covalently bound ubiquitin (Ub) signals and Ub receptor proteins. To develop a functional understanding of the Ub system in vivo, methods are needed to determine the composition of Ub signals on individual substrates and in protein mixtures. Mass spectrometry has emerged as an important tool for characterizing the various forms of Ub. In the Ubiquitin-AQUA approach, synthetic isotopically labeled internal standard peptides are used to quantify unbranched peptides and the branched -GG signature peptides generated by trypsin digestion of Ub signals. Here we have built upon existing methods and established a comprehensive platform for the characterization of Ub signals. Digested peptides and isotopically labeled standards are analyzed either by selected reaction monitoring on a QTRAP mass spectrometer or by narrow window extracted ion chromatograms on a high resolution LTQ-Orbitrap. Additional peptides are now monitored to account for the N terminus of ubiquitin, linear polyUb chains, the peptides surrounding K33 and K48, and incomplete digestion products. Using this expanded battery of peptides, the total amount of Ub in a sample can be determined from multiple loci within the protein, minimizing possible confounding effects of complex Ub signals, digestion abnormalities, or use of mutant Ub in experiments. These methods have been useful for the characterization of in vitro, multistage ubiquitination and have now been extended to reactions catalyzed by multiple E2 enzymes. One question arising from in vitro studies is whether individual protein substrates in cells may be modified by multiple forms of polyUb. Here we have taken advantage of recently developed polyubiquitin linkage-specific antibodies recognizing K48- and K63-linked polyUb chains, coupled with these mass spectrometry methods, to further evaluate the abundance of mixed linkage Ub substrates in cultured mammalian cells. By combining these two powerful tools, we show that polyubiquitinated substrates purified from cells can be modified by mixtures of K48, K63, and K11 linkages.

Characterization of ML-IAP protein stability and physiological role in vivo.

ML-IAP [melanoma IAP (inhibitor of apoptosis)] is an anti-apoptotic protein that is expressed highly in melanomas where it contributes to resistance to apoptotic stimuli. The anti-apoptotic activity and elevated expression of IAP family proteins in many human cancers makes IAP proteins attractive targets for inhibition by cancer therapeutics. Small-molecule IAP antagonists that bind with high affinities to select BIR (baculovirus IAP repeat) domains have been shown to stimulate auto-ubiquitination and rapid proteasomal degradation of c-IAP1 (cellular IAP1) and c-IAP2 (cellular IAP2). In the present paper, we report ML-IAP proteasomal degradation in response to bivalent, but not monovalent, IAP antagonists. This degradation required ML-IAP ubiquitin ligase activity and was independent of c-IAP1 or c-IAP2. Although ML-IAP is best characterized in melanoma cells, we show that ML-IAP expression in normal mammalian tissues is restricted largely to the eye, being most abundant in ciliary body epithelium and retinal pigment epithelium. Surprisingly, given this pattern of expression, gene-targeted mice lacking ML-IAP exhibited normal intraocular pressure as well as normal retinal structure and function. The results of the present study indicate that ML-IAP is dispensable for both normal mouse development and ocular homoeostasis.

A Non-immunogenic Bivalent d-Protein Potently Inhibits Retinal Vascularization and Tumor Growth.

We report a general approach to engineering multivalent d-proteins with antibody-like activities in vivo. Mirror-image phage display and structure-guided design were utilized to create a d-protein that uses receptor mimicry to antagonize vascular endothelial growth factor A (VEGF-A). Selections against the d-protein form of VEGF-A using phage-displayed libraries of two different domain scaffolds yielded two proteins that bound distinct receptor interaction sites on VEGF-A. X-ray crystal structures of the d-protein/VEGF-A complexes were used to guide affinity maturation and to construct a heterodimeric d-protein VEGF-A antagonist with picomolar activity. The d-protein VEGF-A antagonist prevented vascular leakage in a rabbit eye model of wet age-related macular degeneration and slowed tumor growth in the MC38 syngeneic mouse tumor model with efficacies comparable to those of approved antibody drugs, and in contrast with antibodies, the d-protein was non-immunogenic during treatment and following subcutaneous immunizations.

Novel Bcl-2 homology-3 domain-like sequences identified from screening randomized peptide libraries for inhibitors of the pro-survival Bcl-2 proteins.

Interactions between Bcl-2 homology-3 (BH3)-only proteins and their pro-survival Bcl-2 family binding partners initiate the intrinsic apoptosis pathway. These interactions are mediated by a short helical motif, the BH3 domain, on the BH3-only protein, which inserts into a hydrophobic groove on the pro-survival molecule. To identify novel peptidic ligands that bind Mcl-1, a pro-survival protein relative of Bcl-2, both human and mouse Mcl-1 were screened against large randomized phage-displayed peptide libraries. We identified a number of 16-mer peptides with sub-micromolar affinity that were highly selective for Mcl-1, as well as being somewhat selective for the species of Mcl-1 (human or mouse) against which the library was panned. Interestingly, these sequences all strongly resembled natural BH3 domain sequences. By switching residues within the best of the human Mcl-1-binding sequences, or extending beyond the core sequence identified, we were able to alter the pro-survival protein interaction profile of this peptide such that it now bound all members tightly and was a potent killer when introduced into cells. Introduction of an amide lock constraint within this sequence also increased its helicity and binding to pro-survival proteins. These data provide new insights into the determinants of BH3 domain:pro-survival protein affinity and selectivity.

Electrostatic docking of a supramolecular host-guest assembly to cytochrome c probed by bidirectional photoinduced electron transfer.

A water-soluble octacarboxyhemicarcerand was used as a shuttle to transport redox-active substrates across the aqueous medium and deliver them to the target protein. The results show that weak multivalent interactions and conformational flexibility can be exploited to reversibly bind complex supramolecular assemblies to biological molecules. Hydrophobic electron donors and acceptors were encapsulated within the hemicarcerand, and photoinduced electron transfer (ET) between the Zn-substituted cytochrome c (MW = 12.3 kD) and the host-guest complexes (MW = 2.2 kD) was used to probe the association between the negatively charged hemicarceplex and the positively charged protein. The behavior of the resulting ternary protein-hemicarcerand-guest assembly was investigated in two binding limits: (1) when K(encaps) ≫ K(assoc), the hemicarcerand transports the ligand to the protein while protecting it from the aqueous medium; and (2) when K(assoc) > K(encaps), the hemicarcerand-protein complex is formed first, and the hemicarcerand acts as an artificial receptor site that intercepts ligands from solution and positions them close to the active site of the metalloenzyme. In both cases, ET mediated by the protein-bound hemicarcerand is much faster than that due to diffusional encounters with the respective free donor or acceptor in solution. The measured ET rates suggest that the dominant binding region of the host-guest complex on the surface of the protein is consistent with the docking area of the native redox partner of cytochrome c. The strong association with the protein is attributed to the flexible conformation and adaptable charge distribution of the hemicarcerand, which allow for surface-matching with the cytochrome.

Ubiquitin binding modulates IAP antagonist-stimulated proteasomal degradation of c-IAP1 and c-IAP2(1).

A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.

Evaluation of assay technologies for the identification of protein-Peptide interaction antagonists.

An increasing number of assay detection technologies are routinely used in small molecule drug discovery and lead optimization. These assays range from solid-phase heterogeneous assays such as enzyme-linked immunosorbent assay and dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA, PerkinElmer Life and Analytical Sciences, Boston, MA) to solution phase, bead-based assays such as electrochemiluminescence assay (BioVeris [Gaithersburg, MD] technology) and amplified luminescent proximity homogeneous assay (AlphaScreen, PerkinElmer Life and Analytical Sciences) to completely solution-based homogeneous assays such as time-resolved fluorescence resonance energy transfer and fluorescence polarization. The aim of this study is to compare these assay technologies and assess the advantages and disadvantages of each in the context of our efforts to develop small molecule antagonists to the melanoma inhibitor of apoptosis protein. In this study, seven peptides have been evaluated for their potencies in each assay format. Our results indicate that these assay technologies produce similar relative potencies; however, some methods may be more susceptible to interference than others. Consequently, the choice of the method used frequently depends on a number of factors in addition to assay reproducibility and performance, such as throughput of the assay, cost, compound interference, and ease of use.

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.

Structural Analysis and Optimization of Context-Independent Anti-Hypusine Antibodies.

Context-independent anti-hypusine antibodies that bind to the post-translational modification (PTM), hypusine, with minimal dependence on flanking amino acid sequences, were identified. The antibodies bind to both hypusine and deoxyhypusine or selectively to hypusine but not to deoxyhypusine. Phage display was used to further enhance the affinity of the antibodies. Affinity maturation of these anti-hypusine antibodies improved their performance in affinity capture of the only currently known hypusinated protein, eukaryotic translation initiation factor 5A. These anti-hypusine antibodies may have utility in the identification of novel hypusinated proteins. Crystal structures of the corresponding Fab fragments were determined in complex with hypusine- or deoxyhypusine-containing peptides. The hypusine or deoxyhypusine moiety was found to reside in a deep pocket formed between VH and VL domains of the Fab fragments. Interaction between the antibodies and hypusine includes an extensive hydrogen bond network. These are, to our knowledge, the first reported structures of context-independent anti-PTM antibodies in complex with the corresponding PTM.

Smac mimetic induces cell death in a large proportion of primary acute myeloid leukemia samples, which correlates with defined molecular markers.

Apoptosis is deregulated in most, if not all, cancers, including hematological malignancies. Smac mimetics that antagonize Inhibitor of Apoptosis (IAP) proteins have so far largely been investigated in acute myeloid leukemia (AML) cell lines; however, little is yet known on the therapeutic potential of Smac mimetics in primary AML samples. In this study, we therefore investigated the antileukemic activity of the Smac mimetic BV6 in diagnostic samples of 67 adult AML patients and correlated the response to clinical, cytogenetic and molecular markers and gene expression profiles. Treatment with cytarabine (ara-C) was used as a standard chemotherapeutic agent. Interestingly, about half (51%) of primary AML samples are sensitive to BV6 and 21% intermediate responsive, while 28% are resistant. Notably, 69% of ara-C-resistant samples show a good to fair response to BV6. Furthermore, combination treatment with ara-C and BV6 exerts additive effects in most samples. Whole-genome gene expression profiling identifies cell death, TNFR1 and NF-κB signaling among the top pathways that are activated by BV6 in BV6-sensitive, but not in BV6-resistant cases. Furthermore, sensitivity of primary AML blasts to BV6 correlates with significantly elevated expression levels of TNF and lower levels of XIAP in diagnostic samples, as well as with NPM1 mutation. In a large set of primary AML samples, these data provide novel insights into factors regulating Smac mimetic response in AML and have important implications for the development of Smac mimetic-based therapies and related diagnostics in AML.

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.

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.

Hemicarceplexes That Release Guests upon Irradiation.

Cleavage of one of the four bridging units in the two new types of hemicarceplexes presented here is sufficient to effect immediate guest release into the chloroform solvent (shown schematically on the right). The thermal stability and photoactivity of these hemicarceplexes indicate that incarceration is potentially useful for the controlled delivery of drugs.

Discovery of a Potent and Selective BCL-XL Inhibitor with in Vivo Activity.

A-1155463, a highly potent and selective BCL-XL inhibitor, was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based design. This compound is substantially more potent against BCL-XL-dependent cell lines relative to our recently reported inhibitor, WEHI-539, while possessing none of its inherent pharmaceutical liabilities. A-1155463 caused a mechanism-based and reversible thrombocytopenia in mice and inhibited H146 small cell lung cancer xenograft tumor growth in vivo following multiple doses. A-1155463 thus represents an excellent tool molecule for studying BCL-XL biology as well as a productive lead structure for further optimization.

Stabilizing the pro-apoptotic BimBH3 helix (BimSAHB) does not necessarily enhance affinity or biological activity.

An attractive approach for developing therapeutic peptides is to enhance binding to their targets by stabilizing their α-helical conformation, for example, stabilized BimBH3 peptides (BimSAHB) designed to induce apoptosis. Unexpectedly, we found that such modified peptides have reduced affinity for their targets, the pro-survival Bcl-2 proteins. We attribute this loss in affinity to disruption of a network of stabilizing intramolecular interactions present in the bound state of the native peptide. Altering this network may compromise binding affinity, as in the case of the BimBH3 stapled peptide studied here. Moreover, cells exposed to these peptides do not readily undergo apoptosis, strongly indicating that BimSAHB is not inherently cell permeable.