Stefan Gerhardt

Structure of IL-17A in complex with a potent, fully human neutralizing antibody.

IL-17A is a pro-inflammatory cytokine produced by the newly identified Th17 subset of T-cells. We have isolated a human monoclonal antibody to IL-17A (CAT-2200) that can potently neutralize the effects of recombinant and native human IL-17A. We determined the crystal structure of IL-17A in complex with the CAT-2200 Fab at 2.6 A resolution in order to provide a definitive characterization of the epitope and paratope regions. Approximately a third of the IL-17A dimer is disordered in this crystal structure. The disorder occurs in both independent copies of the complex in the asymmetric unit and does not appear to be influenced by crystal packing. The complex contains one IL-17A dimer sandwiched between two CAT-2200 Fab fragments. The IL-17A is a disulfide-linked homodimer that is similar in structure to IL-17F, adopting a cystine-knot fold. The structure is not inconsistent with the previous prediction of a receptor binding cavity on IL-17 family members. The epitope recognized by CAT-2200 is shown to involve 12 amino acid residues from the quaternary structure of IL-17A, with each Fab contacting both monomers in the dimer. All complementarity-determining regions (CDRs) in the Fab contribute to a total of 16 amino acid residues in the antibody paratope. In vitro affinity optimization was used to generate CAT-2200 from a parental lead antibody using random mutagenesis of CDR3 loops. This resulted in seven amino acid changes (three in VL-CDR3 and four in VH-CDR3) and gave an approximate 30-fold increase in potency in a cell-based neutralization assay. Two of the seven amino acids form part of the CAT-2200 paratope. The observed interaction site between CAT-2200 and IL-17A is consistent with data from hydrogen/deuterium exchange mass spectrometry and mutagenesis approaches.

A combined spectroscopic and crystallographic approach to probing drug-human serum albumin interactions

The displacement of probes that bind selectively to subdomains IIA or IIIA on human serum albumin (HSA) by competing compounds has been followed using fluorescence spectroscopy, and has therefore been used to assign a primary binding site for these compounds in the presence and absence of fatty acids. The crystal structures have also been solved for three compounds: a matched pair of carboxylic acids whose binding strength to HSA unexpectedly decreased as the lipophilicity increased; and a highly bound sulphonamide that appeared not to displace the probes in the displacement assay. The crystallography results support the findings from the fluorescence displacement assay. The results indicate that drug binding to subdomain IB might also be important location for certain compounds.

Discovery of a Potent, Selective, and Orally Bioavailable Acidic 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) Inhibitor: Discovery of 2-[(3S)-1-[5-(Cyclohexylcarbamoyl)-6-propylsulfanylpyridin-2-yl]-3-piperidyl]acetic Acid (AZD4017)

Inhibition of 11β-HSD1 is an attractive mechanism for the treatment of obesity and other elements of the metabolic syndrome. We report here the discovery of a nicotinic amide derived carboxylic acid class of inhibitors that has good potency, selectivity, and pharmacokinetic characteristics. Compound 11i (AZD4017) is an effective inhibitor of 11β-HSD1 in human adipocytes and exhibits good druglike properties and as a consequence was selected for clinical development.

Dipeptidyl nitrile inhibitors of Cathepsin L.

A series of potent Cathepsin L inhibitors with good selectivity with respect to other cysteine Cathepsins is described and SAR is discussed with reference to the crystal structure of a protein-ligand complex.

Novel Acidic 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) Inhibitor with Reduced Acyl Glucuronide Liability: The Discovery of 4-[4-(2-Adamantylcarbamoyl)-5-tert-butyl-pyrazol-1-yl]benzoic Acid (AZD8329)

Inhibition of 11β-HSD1 is viewed as a potential target for the treatment of obesity and other elements of the metabolic syndrome. We report here the optimization of a carboxylic acid class of inhibitors from AZD4017 (1) to the development candidate AZD8329 (27). A structural change from pyridine to pyrazole together with structural optimization led to an improved technical profile in terms of both solubility and pharmacokinetics. The extent of acyl glucuronidation was reduced through structural optimization of both the carboxylic acid and amide substituents, coupled with a reduction in lipophilicity leading to an overall increase in metabolic stability.

Design of selective Cathepsin inhibitors

A number of molecular recognition features have been exploited in structure-based design of selective Cathepsin inhibitors.

Selective non zinc binding inhibitors of MMP13

Directed screening has identified a novel series of MMP13 inhibitors that possess good levels of activity whilst possessing excellent selectivity over related MMPs. The binding mode of the series has been solved by co-crystallisation and demonstrates an interesting mode of inhibition without interaction with the catalytic zinc atom.

Engineering a High-Affinity Anti-IL-15 Antibody: Crystal Structure Reveals an α-Helix in VH CDR3 as Key Component of Paratope

Interleukin (IL) 15 is an inflammatory cytokine that plays an essential role in the activation, proliferation, and maintenance of specific natural killer cell and T-cell populations, and has been implicated as a mediator of inflammatory diseases. An anti-IL-15 antibody that blocked IL-15-dependent cellular responses was isolated by phage display and optimised via mutagenesis of the third complementarity-determining regions (CDRs) of variable heavy (VH) and variable light chains. Entire repertoires of improved variants were recombined with each other to explore the maximum potential sequence space. DISC0280, the most potent antibody isolated using this comprehensive strategy, exhibits a 228-fold increase in affinity and a striking 40,000-fold increase in cellular potency compared to its parent. Such a wholesale recombination strategy therefore represents a useful method for exploiting synergistic potency gains as part of future antibody engineering efforts. The crystal structure of DISC0280 Fab (fragment antigen binding), in complex with human IL-15, was determined in order to map the structural epitope and paratope. The most remarkable feature revealed lies within the paratope and is a novel six-amino-acid α-helix that sits within the VH CDR3 loop at the center of the antigen binding site. This is the first report to describe an α-helix as a principal component of a naturally derived VH CDR3 following affinity maturation.

The design and synthesis of novel N-hydroxyformamide inhibitors of ADAM-TS4 for the treatment of osteoarthritis

Two series of N-hydroxyformamide inhibitors of ADAM-TS4 were identified from screening compounds previously synthesised as inhibitors of matrix metalloproteinase-13 (collagenase-3). Understanding of the binding mode of this class of compound using ADAM-TS1 as a structural surrogate has led to the discovery of potent and very selective inhibitors with favourable DMPK properties. Synthesis, structure-activity relationships, and strategies to improve selectivity and lower in vivo metabolic clearance are described.

Lead Optimisation of Selective Non-Zinc Binding Inhibitors of Mmp13. Part 2.

Directed screening has identified a novel series of non-zinc binding MMP13 inhibitors that possess good levels of activity whilst demonstrating excellent selectivity over related MMPs. A lead optimisation campaign has delivered compounds with enhanced MMP13 potency, good selectivity and acceptable bioavailability profiles leading to a predicted twice-a-day dosing regimen in man.