BE-FLARE: a fluorescent reporter of base editing activity reveals editing characteristics of APOBEC3A and APOBEC3B.

BACKGROUND: Base Editing is a precise genome editing method that uses a deaminase-Cas9 fusion protein to mutate cytidine to thymidine in target DNA in situ without the generation of a double-strand break. However, the efficient enrichment of genetically modified cells using this technique is limited by the ability to detect such events. RESULTS: We have developed a Base Editing FLuorescent Activity REporter (BE-FLARE), which allows for the enrichment of cells that have undergone editing of target loci based on a fluorescence shift from BFP to GFP. We used BE-FLARE to evaluate the editing efficiency of APOBEC3A and APOBEC3B family members as alternatives deaminase domains to the rat APOBEC1 domain used in base editor 3 (BE3). We identified human APOBEC3A and APOBEC3B as highly efficient cytidine deaminases for base editing applications with unique properties. CONCLUSIONS: Using BE-FLARE to report on the efficiency and precision of editing events, we outline workflows for the accelerated generation of genetically engineered cell models and the discovery of alternative base editors.

Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease.

The CRISPR-Cas9 RNA-guided endonuclease system allows precise and efficient modification of complex genomes and is continuously developed to enhance specificity, alter targeting and add new functional moieties. However, one area yet to be explored is the base chemistry of the associated RNA molecules. Here we show the design and optimisation of hybrid DNA-RNA CRISPR and tracr molecules based on structure-guided approaches. Through careful mapping of the ribose requirements of Cas9, we develop hybrid versions possessing minimal RNA residues, which are sufficient to direct specific nuclease activity in vitro and in vivo with reduced off-target activity. We identify critical regions within these molecules that require ribose nucleotides and show a direct correlation between binding affinity/stability and cellular activity. This is the first demonstration of a non-RNA-guided Cas9 endonuclease and first step towards eliminating the ribose dependency of Cas9 to develop a XNA-programmable endonuclease.

Identification and Characterization of Dual Inhibitors of the USP25/28 Deubiquitinating Enzyme Subfamily.

The ubiquitin proteasome system is widely postulated to be a new and important field of drug discovery for the future, with the ubiquitin specific proteases (USPs) representing one of the more attractive target classes within the area. Many USPs have been linked to critical axes for therapeutic intervention, and the finding that USP28 is required for c-Myc stability suggests that USP28 inhibition may represent a novel approach to targeting this so far undruggable oncogene. Here, we describe the discovery of the first reported inhibitors of USP28, which we demonstrate are able to bind to and inhibit USP28, and while displaying a dual activity against the closest homologue USP25, these inhibitors show a high degree of selectivity over other deubiquitinases (DUBs). The utility of these compounds as valuable probes to investigate and further explore cellular DUB biology is highlighted by the demonstration of target engagement against both USP25 and USP28 in cells. Furthermore, we demonstrate that these inhibitors are able to elicit modulation of both the total levels and the half-life of the c-Myc oncoprotein in cells and also induce apoptosis and loss of cell viability in a range of cancer cell lines. We however observed a narrow therapeutic index compared to a panel of tissue-matched normal cell lines. Thus, it is hoped that these probes and data presented herein will further advance our understanding of the biology and tractability of DUBs as potential future therapeutic targets.

Methionine-rich repeat proteins: a family of membrane-associated proteins which contain unusual repeat regions.

We report the protein isolation, cloning and characterization of members of an unusual protein family, which comprise the most abundant proteins present in the squid eye. The proteins in this family have a range of molecular weights from 32 to 36 kDa. Electron microscopy and detergent solubilization demonstrate that these proteins are tightly associated with membrane structures where they may form tetramers. Despite this, these proteins have no stretches of hydrophobic residues that could form typical transmembrane domains. They share an unusual protein sequence rich in methionine, and contain multiple repeating motifs. We have therefore named these proteins Methionine-Rich Repeat Proteins (MRRPs). The use of structure prediction algorithms suggest very little recognized secondary structure elements. At the time of cloning no sequence or structural homologues have been found in any database. We have isolated three closely related cDNA clones from the MRRP family. Coupled in vitro transcription/translation of the MRRP clones shows that they encode proteins with molecular masses similar to components of native MRRPs. Immunoblot analysis of these proteins reveals that they are also present in squid brain, optic lobe, and heart, and also indicate that MRRP-like protein motifs may also exist in mammalian tissues. We propose that MRRPs define a family of important proteins that have an unusual mode of attachment or insertion into cell membranes and are found in evolutionarily diverse organisms.

Design and synthesis of highly potent benzodiazepine gamma-secretase inhibitors: preparation of (2S,3R)-3-(3,4-difluorophenyl)-2-(4-fluorophenyl)-4- hydroxy-N-((3S)-1-methyl-2-oxo-5- phenyl-2,3-dihydro-1H-benzo[e][1,4]-diazepin-3-yl)butyramide by use of an asymmetric Ireland-Claisen rearrangement.

Novel benzodiazepine-containing gamma-secretase inhibitors for potential use in Alzheimer's disease have been designed that incorporate a substituted hydrocinnamide C-3 side chain. A syn combination of alpha-alkyl or aryl and beta-hydroxy or hydroxymethyl substituents was shown to give highly potent compounds. In particular, (2S,3R)-3-(3,4-difluorophenyl)-2-(4-fluorophenyl)-4-hydroxy-N-((3S)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)butyramide (34) demonstrated excellent in vitro potency (IC(50) = 0.06 nM). 34 could also be selectively methylated to give [(3)H]-28, which is of use in radioligand binding assays.

Cell banking for pharmaceutical research.

The provision of high-quality eukaryotic cells through robust cell banking processes is essential for the progression of drug discovery projects throughout the pharmaceutical research process. Numerous models exist to meet this aim, and this review describes many of the underlying principles, challenges and opportunities as well as detailing how these have been addressed within AstraZeneca. Crucial aspects discussed include cell line acquisition, cell bank generation, cryopreservation, storage, tracking and distribution. Because quality assurance underpins much of the process, quality control (QC) testing including mycoplasma screening and cell line authentication are also discussed in detail. Furthermore, because many of the underlying principles of cell banking are applicable in non-pharmaceutical settings, it is hoped that this review will prove a useful resource across the wider scientific community.

Enzymatic characterisation of USP7 deubiquitinating activity and inhibition.

USP7 (HAUSP) is a deubiquitinating enzyme, which plays a crucial role in regulating the levels of the p53 tumour suppressor protein, through its ability to prevent the proteasomal degradation of the Ubiquitin ligase for p53, Hdm2. Supporting evidence suggests that an inhibitor of USP7 would act to abrogate the action of Hdm2, and thereby elevate levels of the p53 protein, with associated therapeutic benefits in cancer and potentially other diseases. In this article, we describe the characterisation of differential enzyme activity of both the full length and putative catalytic domain of human USP7 expressed in both bacterial and insect cell expression systems. We also demonstrate the way in which variations in the reducing environment surrounding the enzyme can dramatically affect both the stability of the enzyme and the range of small molecules able to inhibit the catalytic activity of the enzyme. Furthermore, we describe the validation and use of this assay for a high-throughput screening approach, again highlighting the critical nature of the enzyme's environment. Taken together, these findings not only increase our understanding of the enzymatic activity of deubiquitinating enzymes, but also highlight several key considerations of importance in the development of therapeutic agents against this novel class of therapeutic targets.

Novel orally bioavailable gamma-secretase inhibitors with excellent in vivo activity.

The development of potent gamma-secretase inhibitors having substituted heterocycles attached to a benzobicyclo[4.2.1]nonane core is described. This work led to the identification of [6S,9R,11R]-2',3',4',5,5',6,7,8,9,10-decahydro-2-(5-(4-fluorophenyl)-1-methylpyrazol-3-yl)-5'-(2,2,2-trifluoroethyl)spiro[6,9-methanobenzocyclooctene-11,3'-[1,2,5]thiadiazole] 1',1'-dioxide (16), which has excellent in vitro potency (0.06 nM) and which reduced amyloid-beta in APP-YAC mice with an ED(50) of 1 mg/kg (po). 16 had a good pharmacokinetic profile in three preclinical species.

Intra- or intercomplex binding to the gamma-secretase enzyme. A model to differentiate inhibitor classes.

Gamma-secretase is one of the critical enzymes required for the generation of amyloid-beta peptides from the beta-amyloid precursor protein. Because amyloid-beta peptides are generally accepted to play a key role in Alzheimer disease, gamma-secretase inhibition holds the promise for a disease-modifying therapy for this neurodegenerative condition. Although recent progress has enhanced the understanding of the biology and composition of the gamma-secretase enzyme complex, less information is available on the actual interaction of various inhibitor classes with the enzyme. Here we show that the two principal classes of inhibitor described in the scientific and patent literature, aspartyl protease transition state analogue and small molecule non-transition state inhibitors, display fundamental differences in the way they interact with the enzyme. Taking advantage of a gamma-secretase enzyme overexpressing cellular system and different radiolabeled gamma-secretase inhibitors, we observed that the maximal binding of non-transition state gamma-secretase inhibitors accounts only for half the number of catalytic sites of the recombinant enzyme complex. This characteristic stoichiometry can be best accommodated with a model whereby the non-transition state inhibitors bind to a unique site at the interface of a dimeric enzyme. Subsequent competition studies confirm that this site appears to be targeted by the main classes of small molecule gamma-secretase inhibitor. In contrast, the non-steroidal anti-inflammatory drug gamma-secretase modulator sulindac sulfide displayed noncompetitive antagonism for all types of inhibitor. This finding suggests that non-steroidal anti-inflammatory drug-type gamma-secretase modulators target an alternative site on the enzyme, thereby changing the conformation of the binding sites for gamma-secretase inhibitors.

3-Substituted gem-cyclohexane sulfone based gamma-secretase inhibitors for Alzheimer's disease: conformational analysis and biological activity.

Previously, chemistry effort on the gem-cyclohexane series of gamma-secretase inhibitors has focused on the 4-position of the cyclohexane ring. Recently chemistry has been directed towards the 3-position and substitution here has also provided compounds with high gamma-secretase activity.

The geminal dimethyl analogue of Flurbiprofen as a novel Abeta42 inhibitor and potential Alzheimer's disease modifying agent.

The subtle modification of a selection of Abeta42 inhibiting non-steroidal anti-inflammatory drugs (NSAIDs), through synthesis of the geminal dimethyl analogues, was anticipated to ablate their cyclooxygenase activity whilst maintaining Abeta42 inhibition. Methylflurbiprofen 6 exhibited similar in vitro Abeta42 inhibition to its parent NSAID Flurbiprofen and was further evaluated in the Tg2576 mouse model of Alzheimer's disease and an animal model of gastro-intestinal (GI) impairment, but proved unviable for further clinical development.

4-substituted cyclohexyl sulfones as potent, orally active gamma-secretase inhibitors.

The protease gamma-secretase plays a pivotal role in the synthesis of pathogenic amyloid-beta in Alzheimer's disease (AD). Here, we report a further extension to a series of cyclohexyl sulfone-based gamma-secretase inhibitors which has allowed the preparation of highly potent compounds which also demonstrate robust Abeta(40) lowering in vivo (e.g., compound 32, MED 1mg/kg p.o. in APP-YAC mice).

Functional overexpression of gamma-secretase reveals protease-independent trafficking functions and a critical role of lipids for protease activity.

Presenilins appear to form the active center of gamma-secretase but require the presence of the integral membrane proteins nicastrin, anterior pharynx defective 1, and presenilin enhancer 2 for catalytic function. We have simultaneously overexpressed all of these polypeptides, and we demonstrate functional assembly of the enzyme complex, a substantial increase in enzyme activity, and binding of all components to a transition state analogue gamma-secretase inhibitor. Co-localization of all components can be observed in the Golgi compartment, and further trafficking of the individual constituents seems to be dependent on functional assembly. Apart from its catalytic function, gamma-secretase appears to play a role in the trafficking of the beta-amyloid precursor protein, which was changed upon reconstitution of the enzyme but unaffected by pharmacological inhibition. Because the relative molecular mass and stoichiometry of the active enzyme complex remain elusive, we performed size exclusion chromatography of solubilized gamma-secretase, which yielded evidence of a tetrameric form of the complex, yet almost completely abolished enzyme activity. Gamma-secretase activity was reconstituted upon addition of an independent size exclusion chromatography fraction of lower molecular mass and nonproteinaceous nature, which could be replaced by a brain lipid extract. The same treatment was able to restore enzyme activity after immunoaffinity purification of the gamma-secretase complex, demonstrating that lipids play a key role in preserving the catalytic activity of this protease. Furthermore, these data show that it is important to discriminate between intact, inactive gamma-secretase complexes and the active form of the enzyme, indicating the care that must be taken in the study of gamma-secretase.

Generation of C-terminally truncated amyloid-beta peptides is dependent on gamma-secretase activity.

Aberrant production of amyloid-beta peptides by processing of the beta-amyloid precursor protein leads to the formation of characteristic extracellular protein deposits which are thought to be the cause of Alzheimer's disease. Therefore, inhibiting the key enzymes responsible for amyloid-beta peptide generation, beta- and gamma-secretase may offer an opportunity to intervene with the progression of the disease. In human brain and cell culture systems a heterogeneous population of amyloid-beta peptides with various truncations is detected and at present, it is unclear how they are produced. We have used a combination of surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and a specific inhibitor of gamma-secretase to investigate whether the production of all amyloid-beta peptide species requires the action of gamma-secretase. Using this approach, we demonstrate that the production of all truncated amyloid-beta peptides except those released by the action of the nonamyloidogenic alpha-secretase enzyme or potentially beta-site betaAPP cleaving enzyme 2 depends on gamma-secretase activity. This indicates that none of these peptides are generated by a separate enzyme entity and a specific inhibitor of the gamma-secretase enzyme should havethe potential to block the generation of all amyloidogenicpeptides. Furthermore in the presence of gamma-secretase inhibitors, the observation of increased cleavage of the membrane-bound betaAPP C-terminal fragment C99 by alpha-secretase suggests that during its trafficking C99 encounters compartments in which alpha-secretase activity resides.

Topological analysis of peripherin/rds and abnormal glycosylation of the pathogenic Pro216-->Leu mutation.

Peripherin/ rds is an integral membrane glycoprotein found in the rim regions of vertebrate photoreceptor cell discs. The protein is believed to be involved in both formation and maintenance of the characteristic flattened morphology of the outer segment discs and its essential nature is demonstrated by the wide range of retinal degenerative disorders in which the protein has an involvement. Little structural data has been determined for peripherin/ rds, but a topological model of the protein has been proposed. In this paper, we present the first direct evidence for the topology of the protein through the use of scanning glycosylation mutagenesis. Both the topological data and the observation that only the Asn(229) site is efficiently glycosylated in this in vitro transcription/translation system support the common hypotheses. Additionally, expression of the Pro(216)-->Leu mutant demonstrates an abnormal glycosylation pattern, which may explain the mechanism by which this mutation precipitates a retinal degenerative phenotype.

High affinity, bioavailable 3-amino-1,4-benzodiazepine-based gamma-secretase inhibitors.

In this paper, we describe the development of a novel series of high affinity, orally bioavailable 3-amino-1,4 benzodiazepine-based gamma-secretase inhibitors for the potential treatment of Alzheimer's disease. We disclose structure-activity relationships based around the 1, 3 and 5 positions of the benzodiazepine core structure.

Selected non-steroidal anti-inflammatory drugs and their derivatives target gamma-secretase at a novel site. Evidence for an allosteric mechanism.

Gamma-secretase is a multi-component enzyme complex that performs an intramembranous cleavage, releasing amyloid-beta (Abeta) peptides from processing intermediates of the beta-amyloid precursor protein. Because Abeta peptides are thought to be causative for Alzheimer's disease, inhibiting gamma-secretase represents a potential treatment for this neurodegenerative condition. Whereas inhibitors directed at the active center of gamma-secretase inhibit the cleavage of all its substrates, certain non-steroidal antiinflammatory drugs (NSAIDs) have been shown to selectively reduce the production of the more amyloidogenic Abeta(1-42) peptide without inhibiting alternative cleavages. In contrast to the majority of previous studies, however, we demonstrate that in cell-free systems the mode of action of selected NSAIDs and their derivatives, depending on the concentrations used, can either be classified as modulatory or inhibitory. At modulatory concentrations, a selective and, with respect to the substrate, noncompetitive inhibition of Abeta(1-42) production was observed. At inhibitory concentrations, on the other hand, biochemical readouts reminiscent of a nonselective gamma-secretase inhibition were obtained. When these compounds were analyzed for their ability to displace a radiolabeled, transition-state analog inhibitor from solubilized enzyme, noncompetitive antagonism was observed. The allosteric nature of radioligand displacement suggests that NSAID-like inhibitors change the conformation of the gamma-secretase enzyme complex by binding to a novel site, which is discrete from the binding site for transition-state analogs and therefore distinct from the catalytic center. Consequently, drug discovery efforts aimed at this site may identify novel allosteric inhibitors that could benefit from a wider window for inhibition of gamma (42)-cleavage over alternative cleavages in the beta-amyloid precursor protein and, more importantly, alternative substrates.

Conserved residues within the putative active site of gamma-secretase differentially influence enzyme activity and inhibitor binding.

Gamma-secretase performs the final processing step in the generation of amyloid-beta (Abeta) peptides, which are believed to be causative for Alzheimer's disease. Presenilins (PS) are required for gamma-secretase activity and the presence of two essential intramembranous aspartates (D257 and D385) has implicated this region as the putative catalytic centre of an aspartyl protease. The presence of several key hydrogen-bonding residues around the active site of classical aspartyl proteases led us to investigate the role of both the critical aspartates and two nearby conserved hydrogen bond donors in PS1. Generation of cell lines stably overexpressing the D257E, D385E, Y256F and Y389F engineered mutations has enabled us to determine their role in enzyme catalysis and binding of a transition state analogue gamma-secretase inhibitor. Here we report that replacement of either tyrosine residue alters gamma-secretase cleavage specificity, resulting in an increase in the production of the more pathogenic Abeta42 peptide in both cells and membranous enzyme preparations, without affecting inhibitor binding. In contrast, replacement of either of the aspartate residues precludes inhibitor binding in addition to inactivation of the enzyme. Together, these data further incriminate the region around the intramembranous aspartates as the active site of the enzyme, targeted by transition state analogue inhibitors, and highlight the roles of individual residues.

A new series of potent benzodiazepine gamma-secretase inhibitors.

A new series of benzodiazepine-containing gamma-secretase inhibitors with potential use in the treatment of Alzheimer's disease is disclosed. Structure-activity relationships of the pendant hydrocinnamate side-chain which led to the preparation of highly potent inhibitors are described.

In vitro characterization of the presenilin-dependent gamma-secretase complex using a novel affinity ligand.

Gamma-secretase is the enzyme activity releasing the amyloid-beta peptide from membrane-bound processing intermediates derived from the beta-amyloid precursor protein. Cellular release and subsequent aggregation of the amyloid-beta peptide is thought to be causative for the pathogenesis of Alzheimer's disease. Gamma-secretase performs an unusual intramembranous cleavage and has been closely linked to a macromolecular complex containing presenilins. To generate a molecular probe for gamma-secretase, we have developed a novel biotinylated affinity ligand which is based on a specific inhibitor containing a hydroxyethylene dipeptide isostere, known to serve as a transition state analogue for aspartic proteinases. Using this probe we confirmed the presence of the presenilin heterodimer and mature nicastrin in the active enzyme complex and, furthermore, that substrate binding site(s) and active center(s) are spatially separated. Affinity precipitations suggest that only a discrete fraction of cellular presenilin is present in the active gamma-secretase complex and that both gamma(40)- and gamma(42)-activities are mediated by the same molecular entity. This was also reflected by a co-distribution of both enzyme activities in subcellular fractions enriched for trans-Golgi network membranes.