Christian Wiesmann

De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling

NF-κB transcription factors mediate the effects of pro-inflammatory cytokines such as tumour necrosis factor-α and interleukin-1β. Failure to downregulate NF-κB transcriptional activity results in chronic inflammation and cell death, as observed in A20-deficient mice. A20 is a potent inhibitor of NF-κB signalling, but its mechanism of action is unknown. Here we show that A20 downregulates NF-κB signalling through the cooperative activity of its two ubiquitin-editing domains. The amino-terminal domain of A20, which is a de-ubiquitinating (DUB) enzyme of the OTU (ovarian tumour) family, removes lysine-63 (K63)-linked ubiquitin chains from receptor interacting protein (RIP), an essential mediator of the proximal TNF receptor 1 (TNFR1) signalling complex. The carboxy-terminal domain of A20, composed of seven C2/C2 zinc fingers, then functions as a ubiquitin ligase by polyubiquitinating RIP with K48-linked ubiquitin chains, thereby targeting RIP for proteasomal degradation. Here we define a novel ubiquitin ligase domain and identify two sequential mechanisms by which A20 downregulates NF-κB signalling. We also provide an example of a protein containing separate ubiquitin ligase and DUB domains, both of which participate in mediating a distinct regulatory effect.

The identification of 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) as a potent, selective, orally bioavailable inhibitor of class I PI3 kinase for the treatment of cancer

Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis, biological activity, and further profiling of these compounds are described. This work resulted in the discovery of 17, GDC-0941, which is a potent, selective, orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer.

Variants of the antibody herceptin that interact with HER2 and VEGF at the antigen binding site

The interface between antibody and antigen is often depicted as a lock and key, suggesting that an antibody surface can accommodate only one antigen. Here, we describe an antibody with an antigen binding site that binds two distinct proteins with high affinity. We isolated a variant of Herceptin, a therapeutic monoclonal antibody that binds the human epidermal growth factor receptor 2 (HER2), on the basis of its ability to simultaneously interact with vascular endothelial growth factor (VEGF). Crystallographic and mutagenesis studies revealed that distinct amino acids of this antibody, called bH1, engage HER2 and VEGF energetically, but there is extensive overlap between the antibody surface areas contacting the two antigens. An affinity-improved version of bH1 inhibits both HER2- and VEGF-mediated cell proliferation in vitro and tumor progression in mouse models. Such “two-in-one” antibodies challenge the monoclonal antibody paradigm of one binding site, one antigen. They could also provide new opportunities for antibody-based therapy.

Macrophage complement receptors and pathogen clearance

Phagocytosis, an important mechanism of the host‐defence system and a primary function of macrophages, is facilitated by opsonization, a process by which serum components tag pathogens for recognition by neutrophils and macrophages. Complement component C3 is central to opsonization. Its first cleavage product, C3b, forms the multisubunit enzyme, C3bBb, which proteolytically cleaves additional C3 molecules on the pathogen surface. C3b is further degraded to iC3b, C3c and C3dg, products that serve as ligands for selective complement receptors on leukocytes. This receptor–ligand interaction subsequently modulates immune responses or directly targets the pathogen for clearance by phagocytosis. Although a central role for C3 in phagocytosis of certain pathogens is well accepted, the receptors orchestrating the phagocytic response have not been well characterized. The recent structures of C3 and its breakdown products have increased our insights into the molecular basis of complement activation and recognition by their receptors. Here we review the biology of macrophage receptors for C3 fragments and discuss their role in the host response to pathogens.

Crystal structure of the HGF β-chain in complex with the sema domain of the met receptor

The Met tyrosine kinase receptor and its ligand, hepatocyte growth factor (HGF), play important roles in normal development and in tumor growth and metastasis. HGF‐dependent signaling requires proteolysis from an inactive single‐chain precursor into an active α/β‐heterodimer. We show that the serine protease‐like HGF β‐chain alone binds Met, and report its crystal structure in complex with the Sema and PSI domain of the Met receptor. The Met Sema domain folds into a seven‐bladed β‐propeller, where the bottom face of blades 2 and 3 binds to the HGF β‐chain ‘active site region’. Mutation of HGF residues in the area that constitutes the active site region in related serine proteases significantly impairs HGF β binding to Met. Key binding loops in this interface undergo conformational rearrangements upon maturation and explain the necessity of proteolytic cleavage for proper HGF signaling. A crystallographic dimer interface between two HGF β‐chains brings two HGF β:Met complexes together, suggesting a possible mechanism of Met receptor dimerization and activation by HGF.

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 Å) 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.

Structure-function studies of two synthetic anti-vascular endothelial growth factor Fabs and comparison with the Avastin Fab.

In the quest to discover new research tools and to develop better agents in the fight against cancer, two antibodies, G6 and B20-4, were isolated from synthetic antibody phage libraries. Unlike the AVASTIN™ antibody, a recently approved agent for the treatment of patients with colorectal cancer, B20-4 and G6 bind and block both human and murine vascular endothelial growth factor (VEGF). Here we have analyzed and compared the binding epitopes on VEGF for these three antibodies using alanine-scanning mutagenesis and structural analyses. The epitopes recognized by both synthetic antibodies are conserved between human and mouse VEGF, and they match closely to the receptor epitopes both structurally and functionally. In contrast, the Avastin epitope overlaps minimally with the receptor binding surface and centers around a residue that is not conserved in mouse. Our structural and functional analyses elucidate the cross-species reactivity of all three antibodies and emphasize the potential advantages of antibody generation using phage display as the resulting antibodies do not depend on sequence differences across species and preferentially target natural protein-protein interaction surfaces.

Structural studies of neuropilin/antibody complexes provide insights into semaphorin and VEGF binding

Neuropilins (Nrps) are co‐receptors for class 3 semaphorins and vascular endothelial growth factors and important for the development of the nervous system and the vasculature. The extracellular portion of Nrp is composed of two domains that are essential for semaphorin binding (a1a2), two domains necessary for VEGF binding (b1b2), and one domain critical for receptor dimerization (c). We report several crystal structures of Nrp1 and Nrp2 fragments alone and in complex with antibodies that selectively block either semaphorin or vascular endothelial growth factor (VEGF) binding. In these structures, Nrps adopt an unexpected domain arrangement in which the a2, b1, and b2 domains form a tightly packed core that is only loosely connected to the a1 domain. The locations of the antibody epitopes together with in vitro experiments indicate that VEGF and semaphorin do not directly compete for Nrp binding. Based upon our structural and functional data, we propose possible models for ligand binding to neuropilins.

Mechanism of MEK inhibition determines efficacy in mutant KRAS- versus BRAF-driven cancers

KRAS and BRAF activating mutations drive tumorigenesis through constitutive activation of the MAPK pathway. As these tumours represent an area of high unmet medical need, multiple allosteric MEK inhibitors, which inhibit MAPK signalling in both genotypes, are being tested in clinical trials. Impressive single-agent activity in BRAF-mutant melanoma has been observed; however, efficacy has been far less robust in KRAS-mutant disease. Here we show that, owing to distinct mechanisms regulating MEK activation in KRAS- versus BRAF-driven tumours, different mechanisms of inhibition are required for optimal antitumour activity in each genotype. Structural and functional analysis illustrates that MEK inhibitors with superior efficacy in KRAS-driven tumours (GDC-0623 and G-573, the former currently in phase I clinical trials) form a strong hydrogen-bond interaction with S212 in MEK that is critical for blocking MEK feedback phosphorylation by wild-type RAF. Conversely, potent inhibition of active, phosphorylated MEK is required for strong inhibition of the MAPK pathway in BRAF-mutant tumours, resulting in superior efficacy in this genotype with GDC-0973 (also known as cobimetinib), a MEK inhibitor currently in phase III clinical trials. Our study highlights that differences in the activation state of MEK in KRAS-mutant tumours versus BRAF-mutant tumours can be exploited through the design of inhibitors that uniquely target these distinct activation states of MEK. These inhibitors are currently being evaluated in clinical trials to determine whether improvements in therapeutic index within KRAS versus BRAF preclinical models translate to improved clinical responses in patients.

Discovery of a Potent, Selective, and Orally Available Class I Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Kinase Inhibitor (GDC-0980) for the Treatment of Cancer.

The discovery of 2 (GDC-0980), a class I PI3K and mTOR kinase inhibitor for oncology indications, is described. mTOR inhibition was added to the class I PI3K inhibitor 1 (GDC-0941) scaffold primarily through the substitution of the indazole in 1 for a 2-aminopyrimidine. This substitution also increased the microsomal stability and the free fraction of compounds as evidenced through a pairwise comparison of molecules that were otherwise identical. Highlighted in detail are analogues of an advanced compound 4 that were designed to improve solubility, resulting in 2. This compound, is potent across PI3K class I isoforms with IC(50)s of 5, 27, 7, and 14 nM for PI3Kα, β, δ, and γ, respectively, inhibits mTOR with a K(i) of 17 nM yet is highly selective versus a large panel of kinases including others in the PIKK family. On the basis of the cell potency, low clearance in mouse, and high free fraction, 2 demonstrated significant efficacy in mouse xenografts when dosed as low as 1 mg/kg orally and is currently in phase I clinical trials for cancer.