Ursula Bodendorf

A Splice Variant of β-Secretase Deficient in the Amyloidogenic Processing of the Amyloid Precursor Protein

β-Secretase (BACE) initiates the amyloidogenic processing of the amyloid precursor protein leading to the generation of the β-amyloid, the main component of Alzheimers disease senile plaques. BACE is a type I transmembrane aspartyl protease of 501 amino acids. Here we describe a novel BACE mRNA lacking 132 base pairs that is expressed in the pancreas but not in the brain. Sequence alignment indicates that the deleted fragment matches the terminal two-thirds of exon 3. The new BACE variant is short of a 44-amino acid region located between the two catalytic aspartyl residues. Accordingly, a 50-kDa form of BACE (BACE457) is detected in the human pancreas. When expressed in cells, BACE457 colocalizes with the marker for the endoplasmic reticulum BiP. Moreover, BACE457 remains in a proenzymatic and endoglycosidase H-sensitive state, suggesting that its transport along the secretory pathway is blocked at the level of the endoplasmic reticulum. Notably, this novel form of BACE does not contribute to the processing of the amyloid precursor protein. Our findings suggest that tissue-specific splicing of the BACE mRNA may explain the observation that in the human pancreas robust transcription of the BACE gene does not translate into recovered enzymatic activity.

The intramembrane protease Sppl2a is required for B cell and DC development and survival via cleavage of the invariant chain

The protease Sppl2a cleaves the N-terminal fragment of invariant chain (CD74) and is required for efficient B cell development and function.

Structural basis for the substrate specificity of bone morphogenetic protein 1/tolloid-like metalloproteases.

Procollagen C-peptidase, also known as bone morphogenetic protein 1 (BMP-1), is a multidomain, zinc endopeptidase of the astacin M12A family. BMP-1 is the prototype of a small group of proteases that have key roles in extracellular matrix formation and morphogenesis. BMP-1, its splice form mTLD, and the related proteases TLL-1 and TLL-2 are considered as promising drug targets for the treatment of excessive fibrosis and muscle wasting. We report here the crystal structures of the protease domains of human BMP-1 and the closely related Tolloid-like protease 1 (TLL-1). The crystal structures reveal an unexpected conformation of a cysteine-rich loop within the active site, and suggest that a flap movement is required in order to allow substrate binding. On the basis of these substantial differences between the BMP-1 and astacin active sites, a structural basis for their differing substrate specificities is proposed.

The disulphide bonds in the catalytic domain of BACE are critical but not essential for amyloid precursor protein processing activity

β‐Site APP‐cleaving enzyme (BACE) initiates the processing of the amyloid precursor protein (APP) leading to the generation of β‐amyloid, the main component of Alzheimers disease senile plaques. BACE (Asp2, memapsin 2) is a type I transmembrane aspartic protease responsible for the β‐secretase cleavage of APP producing a soluble form of the ectodomain (sAPPβ) and the membrane‐bound, carboxy‐terminal intermediates C99 and C89. BACE maturation involves cysteine bridge formation, N‐glycosylation and propeptide removal. We investigated variants of BACE in which the disulphide bonds of the catalytic domain spanning between Cys216/Cys420, Cys278/Cys443 and Cys330/Cys380 were removed by mutagenesis. When transfected in cultured cells, these mutants showed impaired maturation. Nevertheless, a fraction of mutated protein retained both the competence to mature as well as the activity to process APP. For the generation of a functional enzyme the conserved Cys330/Cys380 bond was the most critical, whereas the two bonds between Cys216/Cys420 and Cys278/Cys443, which are typical for the membrane‐bound BACE, appeared to be less important.

Discovery of the First Potent, Selective, and Orally Bioavailable Signal Peptide Peptidase-Like 2a (SPPL2a) Inhibitor Displaying Pronounced Immunomodulatory Effects In Vivo

Signal peptide peptidase-like 2a (SPPL2a) is an aspartic intramembrane protease which has recently been shown to play an important role in the development and function of antigen presenting cells such as B lymphocytes and dendritic cells. In this paper, we describe the discovery of the first selective and orally active SPPL2a inhibitor (S)-2-cyclopropyl-N1-((S)-5,11-dioxo-10,11-dihydro-1H,3H,5H-spiro[benzo[d]pyrazolo[1,2-a][1,2]diazepine-2,1-cyclopropan]-10-yl)-N4-(5-fluoro-2-methylpyridin-3-yl)succinamide 40 (SPL-707). This compound shows adequate selectivity against the closely related enzymes γ-secretase and SPP and a good pharmacokinetic profile in mouse and rat. Compound 40 significantly inhibited processing of the SPPL2a substrate CD74/p8 fragment in rodents at doses ≤10 mg/kg b.i.d. po. Oral dosing of 40 for 11 days at ≥10 mg/kg b.i.d. recapitulated the phenotype seen in Sppl2a knockout (ko) and ENU mutant mice (reduced number of specific B cells and myeloid dendritic cells). Thus, we believe that SPPL2a represents an interesting and druggable pharmacological target, potentially providing a novel approach for the treatment of autoimmune diseases by targeting B cells and dendritic cells.

The Safety and Antiviral Activity of BZF961 with or without Ritonavir in Patients Infected with Hepatitis C Virus: A Randomized, Multicenter Trial

PURPOSEnInfection with hepatitis C virus is the leading cause of infectious disease mortality in the United States. BZF961 is a novel small molecule inhibitor of the hepatitis C virus NS3-4A protease. Here we present the results of a randomized, double-blinded, placebo-controlled, multicentered study in treatment-naïve patients with chronic hepatitis C virus genotype-1 infection.nnnMETHODSnPatients were enrolled sequentially in 2 parts and treated for 3days. BZF961 was administered as monotherapy (500mg BID for 3 days) or in combination with the cytochrome P450 3A4 inhibitor ritonavir to boost its exposure (BZF961 10, 20, or 50mg QD or BID).nnnFINDINGSnBZF961 was safe and well tolerated in the patients studied with no serious adverse events. There were no appreciable differences in adverse events among patients who received BZF961, BZF961 with ritonavir, or placebo. There was a significant, clinically meaningful reduction in viral load from baseline in patients treated either with BZF961 500mg every 12hours alone or BZF961 50mg every 12hours in combination with ritonavir. Activity against the hepatitis C virus of the lower-dose regimens was apparent but more modest. There were no relevant changes from baseline viral loads in placebo-treated patients.nnnIMPLICATIONSnCoadministration of ritonavir with BZF961 boosted BZF961 exposure (including Cmin, which is the clinically relevant parameter associated with antiviral activity) in a therapeutic range with less variability compared with BZF961 alone. For strategic reasons, BZF961 is no longer under development.

Degradation of recombinant proteins by Chinese hamster ovary host cell proteases is prevented by matriptase-1 knockout: LAUX et al.

An increasing number of nonantibody format proteins are entering clinical development. However, one of the major hurdles for the production of nonantibody glycoproteins is host cell–related proteolytic degradation, which can drastically impact developability and timelines of pipeline projects. Chinese hamster ovary (CHO) cells are the preferred production host for recombinant therapeutic proteins. Using protease inhibitors, transcriptomics, and genetic knockdowns, we have identified, out of the >700 known proteases in rodents, matriptase‐1 as the major protease involved in the degradation of recombinant proteins expressed in CHO‐K1 cells. Subsequently, matriptase‐1 was deleted in CHO‐K1 cells using “transcription activator‐like effector nucleases” (TALENs) as well as zinc‐finger nucleases (ZFNs). This resulted in a superior CHO‐K1 matriptase (KO) cell line with strongly reduced or no proteolytic degradation activity toward a panel of recombinantly expressed proteins. The matriptase KO cell line was evaluated in spike‐in experiments and showed little or no degradation of proteins incubated in culture supernatant derived from the KO cells. This effect was confirmed when the same proteins were recombinantly expressed in the KO cell line. In summary, the combination of novel cell line engineering tools, next‐generation sequencing screening methods, and the recently published Chinese hamster genome has enabled the development of this novel matriptase KO CHO cell line capable of improving expression yields of intact therapeutic proteins.

Identification of SPPL2a Inhibitors by Multiparametric Analysis of a High-Content Ultra-High-Throughput Screen

The intramembrane protease signal peptide peptidase-like 2a (SPPL2a) is a potential drug target for the treatment of autoimmune diseases due to an essential role in B cells and dendritic cells. To screen a library of 1.4 million compounds for inhibitors of SPPL2a, we developed an imaging assay detecting nuclear translocation of the proteolytically released cytosolic substrate fragment. The state-of-the-art hit calling approach based on nuclear translocation resulted in numerous false-positive hits, mainly interrupting intracellular protein trafficking. To filter the false positives, we extracted 340 image-based readouts and developed a novel multiparametric analysis method that successfully triaged the primary hit list. The identified scaffolds were validated by demonstrating activity on endogenous SPPL2a and substrate CD74/p8 in B cells. The multiparametric analysis discovered diverse cellular phenotypes and provided profiles for the whole library. The principle of the presented imaging assay, the screening strategy, and multiparametric analysis are potentially applicable in future screening campaigns.

Technology toolbox for cell line development - next generation cell line development technologies

Background Chinese hamster ovary (CHO) cells are the most widely used host for large scale production of recombinant therapeutic proteins. A combination of several gene editing approaches applied to Novartis proprietary CHO cell line resulted in a superior cell line with a significant increase of titer and improved product quality. Inter alia we have surprisingly identified a key protease responsible for proteolytic degradation of mainly non-antibody format therapeutic proteins. The recently published CHO genome in combination with screening methods and cell line engineering tools has enabled the development of this novel CHO cell line.