Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo.

Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins.

Engineering neprilysin activity and specificity to create a novel therapeutic for Alzheimer's disease.

Neprilysin is a transmembrane zinc metallopeptidase that degrades a wide range of peptide substrates. It has received attention as a potential therapy for Alzheimer's disease due to its ability to degrade the peptide amyloid beta. However, its broad range of peptide substrates has the potential to limit its therapeutic use due to degradation of additional peptides substrates that tightly regulate many physiological processes. We sought to generate a soluble version of the ectodomain of neprilysin with improved activity and specificity towards amyloid beta as a potential therapeutic for Alzheimer's disease. Extensive amino acid substitutions were performed at positions surrounding the active site and inner surface of the enzyme and variants screened for activity on amyloid beta 1-40, 1-42 and a variety of other physiologically relevant peptides. We identified several mutations that modulated and improved both enzyme selectivity and intrinsic activity. Neprilysin variant G399V/G714K displayed an approximately 20-fold improved activity on amyloid beta 1-40 and up to a 3,200-fold reduction in activity on other peptides. Along with the altered peptide substrate specificity, the mutant enzyme produced a markedly altered series of amyloid beta cleavage products compared to the wild-type enzyme. Crystallisation of the mutant enzyme revealed that the amino acid substitutions result in alteration of the shape and size of the pocket containing the active site compared to the wild-type enzyme. The mutant enzyme offers the potential for the more efficient degradation of amyloid beta in vivo as a therapeutic for the treatment of Alzheimer's disease.

Discovery of AZD3839, a potent and selective BACE1 inhibitor clinical candidate for the treatment of Alzheimer disease.

ß-Site amyloid precursor protein cleaving enzyme1 (BACE1) is one of the key enzymes involved in the processing of the amyloid precursor protein (APP) and formation of amyloid ß peptide (Aß) species. Because cerebral deposition of Aß species might be critical for the pathogenesis of Alzheimer disease, BACE1 has emerged as a key target for the treatment of this disease. Here, we report the discovery and comprehensive preclinical characterization of AZD3839, a potent and selective inhibitor of human BACE1. AZD3839 was identified using fragment-based screening and structure-based design. In a concentration-dependent manner, AZD3839 inhibited BACE1 activity in a biochemical fluorescence resonance energy transfer (FRET) assay, Aß and sAPPß release from modified and wild-type human SH-SY5Y cells and mouse N2A cells as well as from mouse and guinea pig primary cortical neurons. Selectivity against BACE2 and cathepsin D was 14 and >1000-fold, respectively. AZD3839 exhibited dose- and time-dependent lowering of plasma, brain, and cerebrospinal fluid Aß levels in mouse, guinea pig, and non-human primate. Pharmacokinetic/pharmacodynamic analyses of mouse and guinea pig data showed a good correlation between the potency of AZD3839 in primary cortical neurons and in vivo brain effects. These results suggest that AZD3839 effectively reduces the levels of Aß in brain, CSF, and plasma in several preclinical species. It might, therefore, have disease-modifying potential in the treatment of Alzheimer disease and related dementias. Based on the overall pharmacological profile and its drug like properties, AZD3839 has been progressed into Phase 1 clinical trials in man.

New aminoimidazoles as ß-secretase (BACE-1) inhibitors showing amyloid-ß (Aß) lowering in brain.

Amino-2H-imidazoles are described as a new class of BACE-1 inhibitors for the treatment of Alzheimer's disease. Synthetic methods, crystal structures, and structure-activity relationships for target activity, permeability, and hERG activity are reported and discussed. Compound (S)-1m was one of the most promising compounds in this report, with high potency in the cellular assay and a good overall profile. When guinea pigs were treated with compound (S)-1m, a concentration and time dependent decrease in Aß40 and Aß42 levels in plasma, brain, and CSF was observed. The maximum reduction of brain Aß was 40-50%, 1.5 h after oral dosing (100 µmol/kg). The results presented highlight the potential of this new class of BACE-1 inhibitors with good target potency and with low effect on hERG, in combination with a fair CNS exposure in vivo.

Design and synthesis of ß-site amyloid precursor protein cleaving enzyme (BACE1) inhibitors with in vivo brain reduction of ß-amyloid peptides.

The evaluation of a series of aminoisoindoles as ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors and the discovery of a clinical candidate drug for Alzheimer's disease, (S)-32 (AZD3839), are described. The improvement in permeability properties by the introduction of fluorine adjacent to the amidine moiety, resulting in in vivo brain reduction of Aß40, is discussed. Due to the basic nature of these compounds, they displayed affinity for the human ether-a-go-go related gene (hERG) ion channel. Different ways to reduce hERG inhibition and increase hERG margins for this series are described, culminating in (S)-16 and (R)-41 showing large in vitro margins with BACE1 cell IC(50) values of 8.6 and 0.16 nM, respectively, and hERG IC(50) values of 16 and 2.8 µM, respectively. Several compounds were advanced into pharmacodynamic studies and demonstrated significant reduction of ß-amyloid peptides in mouse brain following oral dosing.

Synthesis and structure-activity relationship of 4-(1,3-benzothiazol-2-yl)-thiophene-2-sulfonamides as cyclin-dependent kinase 5 (cdk5)/p25 inhibitors.

4-(1,3-Benzothiazol-2-yl)thiophene-2-sulfonamide (4a) was found to be a moderately potent inhibitor of cyclin-dependent kinase 5 (cdk5) from a HTS screen. The synthesis and SAR around this hit is described. The X-ray coordinates of ligand 4a with cdk5 are also reported, showing an unusual binding mode to the hinge region via a water molecule.

Aminoimidazoles as BACE-1 inhibitors: the challenge to achieve in vivo brain efficacy.

The evaluation of a series of bicyclic aminoimidazoles as potent BACE-1 inhibitors is described. The crystal structures of compounds 14 and 23 in complex with BACE-1 reveal hydrogen bond interactions with the protein important for achieving potent inhibition. The optimization of permeability and efflux properties of the compounds is discussed as well as the importance of these properties for attaining in vivo brain efficacy. Compound (R)-25 was selected for evaluation in vivo in wild type mice and 1.5h after oral co-administration of 300µmol/kg (R)-25 and efflux inhibitor GF120918 the brain Aß40 level was reduced by 17% and the plasma Aß40 level by 76%.

An integrated approach to fragment-based lead generation: philosophy, strategy and case studies from AstraZeneca's drug discovery programmes.

Fragment-based lead generation (FBLG) has recently emerged as an alternative to traditional high throughput screening (HTS) to identify initial chemistry starting points for drug discovery programs. In comparison to HTS screening libraries, the screening sets for FBLG tend to contain orders of magnitude fewer compounds, and the compounds themselves are less structurally complex and have lower molecular weight. This report summarises the advent of FBLG within the industry and then describes the FBLG experience at AstraZeneca. We discuss (1) optimising the design of screening libraries, (2) hit detection methodologies, (3) evaluation of hit quality and use of ligand efficiency calculations, and (4) approaches to evolve fragment-based, low complexity hits towards drug-like leads. Furthermore, we exemplify our use of FBLG with case studies in the following drug discovery areas: antibacterial enzyme targets, GPCRs (melanocortin 4 receptor modulators), prostaglandin D2 synthase inhibitors, phosphatase inhibitors (protein tyrosine phosphotase 1B), and protease inhibitors (b-secretase).

Discovery of a novel warhead against beta-secretase through fragment-based lead generation.

Fragment-based lead generation was applied to find novel small-molecule inhibitors of beta-secretase (BACE-1), a key target for the treatment of Alzheimer's disease. Fragment hits coming from a 1D NMR screen were characterized by BIAcore, and the most promising compounds were soaked into protein crystals to help the rational design of more potent hit analogues. Problems arising due to our inability to grow BACE-1 crystals at the biologically relevant pH at which the screen was run were overcome by using endothiapepsin as a surrogate aspartyl protease. Among others, we identified 6-substituted isocytosines as a novel warhead against BACE-1, and the accompanying paper in this journal describes how these were optimized to a lead series of nanomolar inhibitors.1.

Application of fragment-based lead generation to the discovery of novel, cyclic amidine beta-secretase inhibitors with nanomolar potency, cellular activity, and high ligand efficiency.

Fragment-based lead generation has led to the discovery of a novel series of cyclic amidine-based inhibitors of beta-secretase (BACE-1). Initial fragment hits with an isocytosine core having millimolar potency were identified via NMR affinity screening. Structure-guided evolution of these fragments using X-ray crystallography together with potency determination using surface plasmon resonance and functional enzyme inhibition assays afforded micromolar inhibitors. Similarity searching around the isocytosine core led to the identification of a related series of inhibitors, the dihydroisocytosines. By leveraging the knowledge of the ligand-BACE-1 recognition features generated from the isocytosines, the dihydroisocytosines were efficiently optimized to submicromolar potency. Compound 29, with an IC50 of 80 nM, a ligand efficiency of 0.37, and cellular activity of 470 nM, emerged as the lead structure for future optimization.

Structure and molecular interactions of a unique antitumor antibody specific for N-glycolyl GM3.

N-glycolyl GM3 ganglioside is an attractive target antigen for cancer immunotherapy, because this epitope is a molecular marker of certain tumor cells and not expressed in normal human tissues. The murine monoclonal antibody 14F7 specifically recognizes N-glycolyl GM3 and shows no cross-reactivity with the abundant N-acetyl GM3 ganglioside, a close structural homologue of N-glycolyl GM3. Here, we report the crystal structure of the 14F7 Fab fragment at 2.5 A resolution and its molecular model with the saccharide moiety of N-glycolyl GM3, NeuGcalpha3Galbeta4Glcbeta. Fab 14F7 contains a very long CDR H3 loop, which divides the antigen-binding site of this antibody into two subsites. In the docking model, the saccharide ligand is bound to one of these subsites, formed solely by heavy chain residues. The discriminative feature of N-glycolyl GM3 versus N-acetyl GM3, its hydroxymethyl group, is positioned in a hydrophilic cavity, forming hydrogen bonds with the carboxyl group of Asp H52, the indole NH of Trp H33 and the hydroxyl group of Tyr H50. For the hydrophobic methyl group of N-acetyl GM3, this environment would not be favorable, explaining why the antibody specifically recognizes N-glycolyl GM3, but not N-acetyl GM3. Mutation of Asp H52 to hydrophobic residues of similar size completely abolished binding. Our model of the antibodycarbohydrate complex is consistent with binding data for several tested glycolipids as well as for a variety of 14F7 mutants with replaced VL domains.