Designer proteins to trigger cell death.

Efforts to generate biologically active proteins by de novo computational design have been limited to creating functional sites within pre-existing scaffolds. Procko et al. use an innovative computational design approach coupled with in-vitro-targeted evolution to produce a potent polypeptide inhibitor of a viral Bcl-2-like protein. This novel inhibitor triggers apoptosis of virus-infected cells.

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.

Targeted degradation via direct 26S proteasome recruitment.

Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.

Primary Amine Tethered Small Molecules Promote the Degradation of X-Linked Inhibitor of Apoptosis Protein.

We hypothesized that the proximity-driven ubiquitylation of E3-interacting small molecules could affect the degradation of E3 ubiquitin ligases. A series of XIAP BIR2 domain-binding small molecules was modified to append a nucleophilic primary amine. This modification transforms XIAP binders into inducers of XIAP degradation. The degradation of XIAP is E1- and proteasome-dependent, dependent on the ligase function of XIAP, and is rescued by subtle modifications of the small molecule that would obviate ubiquitylation. We demonstrate in vitro ubiquitylation of the small molecule that is dependent on its interaction with XIAP. Taken together, these results demonstrate the designed ubiquitylation of an engineered small molecule and a novel approach for the degradation of E3 ubiquitin ligases.

Phosphorylation and linear ubiquitin direct A20 inhibition of inflammation.

Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn's disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death.

A selective peptide inhibitor of Frizzled 7 receptors disrupts intestinal stem cells.

Regeneration of the adult intestinal epithelium is mediated by a pool of cycling stem cells, which are located at the base of the crypt, that express leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5). The Frizzled (FZD) 7 receptor (FZD7) is enriched in LGR5+ intestinal stem cells and plays a critical role in their self-renewal. Yet, drug discovery approaches and structural bases for targeting specific FZD isoforms remain poorly defined. FZD proteins interact with Wnt signaling proteins via, in part, a lipid-binding groove on the extracellular cysteine-rich domain (CRD) of the FZD receptor. Here we report the identification of a potent peptide that selectively binds to the FZD7 CRD at a previously uncharacterized site and alters the conformation of the CRD and the architecture of its lipid-binding groove. Treatment with the FZD7-binding peptide impaired Wnt signaling in cultured cells and stem cell function in intestinal organoids. Together, our data illustrate that targeting the lipid-binding groove holds promise as an approach for achieving isoform-selective FZD receptor inhibition.

Publisher Correction: A selective peptide inhibitor of Frizzled 7 receptors disrupts intestinal stem cells.

The version of this article originally published contained older versions of the Life Sciences Reporting Summary and the Supplementary Text and Figures. The error has been corrected in the HTML and PDF versions of the article.

Picomolar zinc binding modulates formation of Bcl10-nucleating assemblies of the caspase recruitment domain (CARD) of CARD9.

The caspase recruitment domain-containing protein 9 (CARD9)-B-cell lymphoma/leukemia 10 (Bcl10) signaling axis is activated in myeloid cells during the innate immune response to a variety of diverse pathogens. This signaling pathway requires a critical caspase recruitment domain (CARD)-CARD interaction between CARD9 and Bcl10 that promotes downstream activation of factors, including NF-κB and the mitogen-activated protein kinase (MAPK) p38. Despite these insights, CARD9 remains structurally uncharacterized, and little mechanistic understanding of its regulation exists. We unexpectedly found here that the CARD in CARD9 binds to Zn2+ with picomolar affinity-a concentration comparable with the levels of readily accessible Zn2+ in the cytosol. NMR solution structures of the CARD9-CARD in the apo and Zn2+-bound states revealed that Zn2+ has little effect on the ground-state structure of the CARD; yet the stability of the domain increased considerably upon Zn2+ binding, with a concomitant reduction in conformational flexibility. Moreover, Zn2+ binding inhibited polymerization of the CARD9-CARD into helical assemblies. Here, we also present a 20-Å resolution negative-stain EM (NS-EM) structure of these filamentous assemblies and show that they adopt a similar helical symmetry as reported previously for filaments of the Bcl10 CARD. Using both bulk assays and direct NS-EM visualization, we further show that the CARD9-CARD assemblies can directly template and thereby nucleate Bcl10 polymerization, a capacity considered critical to propagation of the CARD9-Bcl10 signaling cascade. Our findings indicate that CARD9 is a potential target of Zn2+-mediated signaling that affects Bcl10 polymerization in innate immune responses.

BCL2 mutations do not confer adverse prognosis in follicular lymphoma patients treated with rituximab.

BCL2 mutations have been suggested to confer an adverse prognosis to follicular lymphoma (FL) patients, but their prognostic value has not been assessed in patients treated with a rituximab-containing regimen. Here we evaluated the prognostic value of BCL2 mutations in a large prospective cohort of 252 patients with FL treated with immunochemotherapy in the PRIMA randomized trial. Using a DNA-targeted sequencing approach, we detected amino acid altering mutations in 135 patients (54%) and showed that these mutations were probably mediated by the over-activation of AICDA (activation-induced cytidine deaminase) in the context of the t(14;18) translocation. The BCL2 variants identified in PRIMA patients affected the BH1, BH2, and BH3 functional motifs at a lower frequency than the N-terminus and flexible loop domain, with mostly conservative aminoacid changes. With a median follow-up of 6.7 years, we did not observe any impact of BCL2 mutations either on overall survival or progression-free survival.

Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7.

Microtubules have pivotal roles in fundamental cellular processes and are targets of antitubulin chemotherapeutics. Microtubule-targeted agents such as Taxol and vincristine are prescribed widely for various malignancies, including ovarian and breast adenocarcinomas, non-small-cell lung cancer, leukaemias and lymphomas. These agents arrest cells in mitosis and subsequently induce cell death through poorly defined mechanisms. The strategies that resistant tumour cells use to evade death induced by antitubulin agents are also unclear. Here we show that the pro-survival protein MCL1 (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, MCL1 protein levels decline markedly, through a post-translational mechanism, potentiating cell death. Phosphorylation of MCL1 directs its interaction with the tumour-suppressor protein FBW7, which is the substrate-binding component of a ubiquitin ligase complex. The polyubiquitylation of MCL1 then targets it for proteasomal degradation. The degradation of MCL1 was blocked in patient-derived tumour cells that lacked FBW7 or had loss-of-function mutations in FBW7, conferring resistance to antitubulin agents and promoting chemotherapeutic-induced polyploidy. Additionally, primary tumour samples were enriched for FBW7 inactivation and elevated MCL1 levels, underscoring the prominent roles of these proteins in oncogenesis. Our findings suggest that profiling the FBW7 and MCL1 status of tumours, in terms of protein levels, messenger RNA levels and genetic status, could be useful to predict the response of patients to antitubulin chemotherapeutics.

Conformational dynamics control ubiquitin-deubiquitinase interactions and influence in vivo signaling.

Ubiquitin is a highly conserved eukaryotic protein that interacts with a diverse set of partners to act as a cellular signaling hub. Ubiquitin's conformational flexibility has been postulated to underlie its multifaceted recognition. Here we use computational and library-based means to interrogate core mutations that modulate the conformational dynamics of human ubiquitin. These ubiquitin variants exhibit increased affinity for the USP14 deubiquitinase, with concomitantly reduced affinity for other deubiquitinases. Strikingly, the kinetics of conformational motion are dramatically slowed in these variants without a detectable change in either the ground state fold or excited state population. These variants can be ligated into substrate-linked chains in vitro and in vivo but cannot solely support growth in eukaryotic cells. Proteomic analyses reveal nearly identical interaction profiles between WT ubiquitin and the variants but identify a small subset of altered interactions. Taken together, these results show that conformational dynamics are critical for ubiquitin-deubiquitinase interactions and imply that the fine tuning of motion has played a key role in the evolution of ubiquitin as a signaling hub.

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.

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.

Potency-Enhanced Peptidomimetic VHL Ligands with Improved Oral Bioavailability.

The von Hippel-Lindau (VHL) protein plays a pivotal role in regulating the hypoxic stress response and has been extensively studied and utilized in the targeted protein degradation field, particularly in the context of bivalent degraders. In this study, we present a comprehensive peptidomimetic structure-activity relationship (SAR) approach, combined with cellular NanoBRET target engagement assays to enhance the existing VHL ligands. Through systematic modifications of the molecule, we identified the 1,2,3-triazole group as an optimal substitute of the left-hand side amide bond that yields 10-fold higher binding activity. Moreover, incorporating conformationally constrained alterations on the methylthiazole benzylamine moiety led to the development of highly potent VHL ligands with picomolar binding affinity and significantly improved oral bioavailability. We anticipate that our optimized VHL ligand, GNE7599, will serve as a valuable tool compound for investigating the VHL pathway and advancing the field of targeted protein degradation.

The discovery and structural basis of two distinct state-dependent inhibitors of BamA.

BamA is the central component of the essential ß-barrel assembly machine (BAM), a conserved multi-subunit complex that dynamically inserts and folds ß-barrel proteins into the outer membrane of Gram-negative bacteria. Despite recent advances in our mechanistic and structural understanding of BamA, there are few potent and selective tool molecules that can bind to and modulate BamA activity. Here, we explored in vitro selection methods and different BamA/BAM protein formulations to discover peptide macrocycles that kill Escherichia coli by targeting extreme conformational states of BamA. Our studies show that Peptide Targeting BamA-1 (PTB1) targets an extracellular divalent cation-dependent binding site and locks BamA into a closed lateral gate conformation. By contrast, PTB2 targets a luminal binding site and traps BamA into an open lateral gate conformation. Our results will inform future antibiotic discovery efforts targeting BamA and provide a template to prospectively discover modulators of other dynamic integral membrane proteins.

Learning and confirming with preclinical studies: modeling and simulation in the discovery of GDC-0917, an inhibitor of apoptosis proteins antagonist.

The application of modeling and simulation techniques is increasingly common in the preclinical stages of the drug development process. GDC-0917 [(S)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-N-(2-(oxazol-2-yl)-4-phenylthiazol-5-yl)pyrrolidine-2-carboxamide] is a potent second-generation antagonist of inhibitor of apoptosis (IAP) proteins that is being developed for the treatment of various cancers. GDC-0917 has low to moderate clearance in the mouse (12.0 ml/min/kg), rat (27.0 ml/min/kg), and dog (15.3 ml/min/kg), and high clearance in the monkey (67.6 ml/min/kg). Accordingly, oral bioavailability was lowest in monkeys compared with other species. Based on our experience with a prototype molecule with similar structure, in vitro-in vivo extrapolation was used to predict a moderate clearance (11.5 ml/min/kg) in humans. The predicted human volume of distribution was estimated using simple allometry at 6.69 l/kg. Translational pharmacokinetic-pharmacodynamic (PK-PD) analysis using results from MDA-MB-231-X1.1 breast cancer xenograft studies and predicted human pharmacokinetics suggests that ED50 and ED90 targets can be achieved in humans using acceptable doses (72 mg and 660 mg, respectively) and under an acceptable time frame. The relationship between GDC-0917 concentrations and pharmacodynamic response (cIAP1 degradation) was characterized using an in vitro peripheral blood mononuclear cell immunoassay. Simulations of human GDC-0917 plasma concentration-time profile and cIAP1 degradation at the 5-mg starting dose in the phase 1 clinical trial agreed well with observations. This work shows the importance of leveraging information from prototype molecules and illustrates how modeling and simulation can be used to add value to preclinical studies in the early stages of the drug development process.

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.

Murine insulin growth factor-like (IGFL) and human IGFL1 proteins are induced in inflammatory skin conditions and bind to a novel tumor necrosis factor receptor family member, IGFLR1.

Psoriasis is a human skin condition characterized by epidermal hyperproliferation and infiltration of multiple leukocyte populations. In characterizing a novel insulin growth factor (IGF)-like (IGFL) gene in mice (mIGFL), we found transcripts of this gene to be most highly expressed in skin with enhanced expression in models of skin wounding and psoriatic-like inflammation. A possible functional ortholog in humans, IGFL1, was uniquely and significantly induced in psoriatic skin samples. In vitro IGFL1 expression was up-regulated in cultured primary keratinocytes stimulated with tumor necrosis factor α but not by other psoriasis-associated cytokines. Finally, using a secreted and transmembrane protein library, we discovered high affinity interactions between human IGFL1 and mIGFL and the TMEM149 ectodomain. TMEM149 (renamed here as IGFLR1) is an uncharacterized gene with structural similarity to the tumor necrosis factor receptor family. Our studies demonstrate that IGFLR1 is expressed primarily on the surface of mouse T cells. The connection between mIGFL and IGFLR1 receptor suggests mIGFL may influence T cell biology within inflammatory skin conditions.

Structure of C3b in complex with CRIg gives insights into regulation of complement activation.

The complement system is a key part of the innate immune system, and is required for clearance of pathogens from the bloodstream. After exposure to pathogens, the third component of the complement system, C3, is cleaved to C3b which, after recruitment of factor B, initiates formation of the alternative pathway convertases. CRIg, a complement receptor expressed on macrophages, binds to C3b and iC3b mediating phagocytosis of the particles, but it is unknown how CRIg selectively recognizes proteolytic C3-fragments and whether binding of CRIg to C3b inhibits convertase activation. Here we present the crystal structure of C3b in complex with CRIg and, using CRIg mutants, provide evidence that CRIg acts as an inhibitor of the alternative pathway of complement. The structure shows that activation of C3 induces major structural rearrangements, including a dramatic movement (>80 A) of the thioester-bond-containing domain through which C3b attaches to pathogen surfaces. We show that CRIg is not only a phagocytic receptor, but also a potent inhibitor of the alternative pathway convertases. The structure provides insights into the complex macromolecular structural rearrangements that occur during complement activation and inhibition. Moreover, our structure-function studies relating the structural basis of complement activation and the means by which CRIg inhibits the convertases provide important clues to the development of therapeutics that target complement.