Lisa Wissler

Ligand binding mechanism in steroid receptors; from conserved plasticity to differential evolutionary constraints

Steroid receptor drugs have been available for more than half a century, but details of the ligand binding mechanism have remained elusive. We solved X-ray structures of the glucocorticoid and mineralocorticoid receptors to identify a conserved plasticity at the helix 6-7 region that extends the ligand binding pocket toward the receptor surface. Since none of the endogenous ligands exploit this region, we hypothesized that it constitutes an integral part of the binding event. Extensive all-atom unbiased ligand exit and entrance simulations corroborate a ligand binding pathway that gives the observed structural plasticity a key functional role. Kinetic measurements reveal that the receptor residence time correlates with structural rearrangements observed in both structures and simulations. Ultimately, our findings reveal why nature has conserved the capacity to open up this region, and highlight how differences in the details of the ligand entry process result in differential evolutionary constraints across the steroid receptors.

The Discovery of Potent and Selective Non-Steroidal Glucocorticoid Receptor Modulators, Suitable for Inhalation.

We report the discovery of highly potent and selective non-steroidal glucocorticoid receptor modulators with PK properties suitable for inhalation. A high throughput screen of the AstraZeneca compound collection identified sulfonamide 3 as a potent non-steroidal glucocorticoid receptor ligand. Further optimization of this lead generated indazoles 30 and 48 that were progressed to characterization in in vivo models. X-ray crystallography was used to gain further insight into the binding mode of selected ligands.

Cathepsin C Inhibitors: Property Optimization and Identification of a Clinical Candidate

A lead generation and optimization program delivered the highly selective and potent CatC inhibitor 10 as an in vivo tool compound and potential development candidate. Structural studies were undertaken to generate SAR understanding.

The Discovery of Mmp7 Inhibitors Exploiting a Novel Selectivity Trigger.

Matrilysin or matrix metalloproteinase 7 (MMP7) is a member of a class of zinc-dependent endopeptidases (MMPs) capable of degrading extracellular matrix proteins and thought to play an important role in tissue remodeling associated with various physiological and pathological processes. It has broad specificity and cleaves a number of matrix substances, including proteoglycans and collagen III/IV/V/IX/X/XI, which underlies a potential role for MMP7 inhibitors in the treatment of disease associated with tissue degradation/remodeling. In addition, MMP7 has been reported to have a potential role in tumor metastasis and inflammatory processes. It has also been reported to be expressed in osteoarthritic cartilage, where it is colocalized with the tetraspanin, CD151, which has been implicated in its pericellular activation. As a class, MMPs have been the subject of intense study within the pharmaceutical industry over the last decade, and inhibitors have been described for many of the individual MMPs. However, MMP selectivity remains a significant hurdle for most MMP inhibitors due to a reliance on zinc chelation to provide a significant component of the binding energy. Despite high therapeutic value, there has been a consistent lack of success with such inhibitors in the clinic, and this can largely be attributed to several factors: poor selectivity, poor target validation for the targeted therapy, and poorly defined predictive preclinical animal models for safety and efficacy. The selectivity problem is particularly true with compounds bearing groups that chelate strongly to zinc (e.g. , hydroxamate, reverse hydroxamate) because a large component of the binding energy is derived from a feature common to all MMPs. With this in mind, we initiated a program to identify selective MMP7 inhibitors employing a high-throughput screening approach, with MMP selectivity assessment very early in the screening process. To provide an initial indication of selectivity, actives were screened against MMP1 and MMP14. Compounds showing good selectivity against these two MMPs were then screened against MMP2, MMP12 and MMP13 to give an indication of wider MMP selectivity. As anticipated, potent hydroxamate and reverse hydroxamate inhibitors were identified but none of them showed selectivity over other MMPs. In fact, without exception, hydroxamate, reverse hydroxamate and also hydantoin inhibitors proved more active against many of the other MMPs than against MMP7. One active hit that emerged from these initial selectivity screens was the carboxylic acid compound I. The compound has low potency against MMP7, but there was no indication of activity against any of the other MMPs tested. The low potency limited a true assessment of selectivity but, nonetheless, this initial lead was pursued further, driven largely by two factors: ease of synthesis and crystallization. Firstly, we were able to successfully crystallize this compound within the MMP7 protein and determine the complex structure to enable an understanding of the binding mode. Secondly, the compound was relatively simple and amenable to parallel synthesis from commercial sulfonyl chlorides and amino acids, which would facilitate a rapid initial exploration of structure–activity relationships. Furthermore, the library design for this parallel chemistry could be directed by virtual docking experiments using the X-ray data generated from compound I. We also crystallized one of the nonselective reverse hydroxamate MMP7 inhibitors (compound II) to assess the binding mode of compounds possessing a large S1’ substituent. MMP7 is unusual in the MMP family in the sense that it possesses a very shallow S1’ selectivity pocket, and compound II was expected to provide a poor fit. What was seen from X-ray data is that this compound expands the S1’ pocket to accommodate the large side chain. Using this information, we were able to employ both the open and closed MMP7 binding sites for virtual docking experiments. The structural work carried out around compounds I and II (Figure 1) is described below. The results give some insight into the basis of the selectivity seen with compound I. Also described are the initial results from the library design based on the structural data to increase MMP7 potency whilst retaining selectivity over other MMPs. MMP7 is the smallest member of the MMP family, with the mature enzyme consisting of the catalytic domain alone. The MMP7 structure confirmed the open-faced a-b-sandwich fold (Figure 2), characteristic for most zinc-dependant endopeptidases. The active site is located in a groove adjacent to the central helix B and the antiparallel b-sheet, and it is delineated by the typical HEXGHXXGXXH sequence motif. Upon binding, the scissile peptide bond is positioned with the carbonyl oxygen towards the catalytic zinc ion, which is coordinated by the three histidine residues. The nearby glutamate acts as a general base, promoting nucleophilic attack by a catalytic water molecule and subsequently stabilizes the tetrahedral intermediate. In addition to the catalytic zinc, the structure contains two calcium ions and one additional zinc ion, important for maintaining the structural integrity of the b-sheet. [a] Dr. K. Edman, C. Johansson, Dr. J. Petersen, Dr. A. Tervo, L. Wissler Global Structural Chemistry, AstraZeneca Mçlndal Pepparedsleden 1, 43183 Mçlndal (Sweden) Fax: (+ 46) 31-776-3700 Karl.Edman@astrazeneca.com [b] Dr. M. Furber, P. Hemsley, Dr. G. Pairaudeau, Dr. M. Stocks, Dr. A. Ward, Dr. E. Wells Departments of Medicinal Chemistry and BioSciences AstraZeneca Charnwood Bakewell Road, Loughborough, Leicestershire LE11 5RH (UK) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201000550.

X-Ray Structural Analysis of Tau-Tubulin Kinase 1 and its Interactions with Small Molecular Inhibitors.

Tau‐tubulin kinase 1 (TTBK1) is a serine/threonine/tyrosine kinase that putatively phosphorylates residues including S422 in tau protein. Hyperphosphorylation of tau protein is the primary cause of tau pathology and neuronal death associated with Alzheimer’s disease. A library of 12 truncation variants comprising the TTBK1 kinase domain was screened for expression in Escherichia coli and insect cells. One variant (residues 14–313) could be purified, but mass spectrometric analysis revealed extensive phosphorylation of the protein. Co‐expression with lambda phosphatase in E. coli resulted in production of homogeneous, nonphosphorylated TTBK1. Binding of ATP and several compounds to TTBK1 was characterized by surface plasmon resonance. Crystal structures of TTBK1 in the unliganded form and in complex with ATP, and two high‐affinity ATP‐competitive inhibitors, 3‐[(6,7‐dimethoxyquinazolin‐4‐yl)amino]phenol (1) and methyl 2‐bromo‐5‐(7H‐pyrrolo[2,3‐d]pyrimidin‐4‐ylamino)benzoate (2), were elucidated. The structure revealed two clear basic patches near the ATP pocket providing an explanation of TTBK1 for phosphorylation‐primed substrates. Interestingly, compound 2 displayed slow binding kinetics to TTBK1, the structure of TTBK1 in complex with this compound revealed a reorganization of the L199–D200 peptide backbone conformation together with altered hydrogen bonding with compound 2. These conformational changes necessary for the binding of compound 2 are likely the basis of the slow kinetics. This first TTBK1 structure can assist the discovery of novel inhibitors for the treatment of Alzheimer’s disease.

Identification of indole inhibitors of human hematopoietic prostaglandin D2 synthase (hH-PGDS).

Human H-PGDS has shown promise as a potential target for anti-allergic and anti-inflammatory drugs. Here we describe the discovery of a novel class of indole inhibitors, identified through focused screening of 42,000 compounds and evaluated using a series of hit validation assays that included fluorescence polarization binding, 1D NMR, ITC and chromogenic enzymatic assays. Compounds with low nanomolar potency, favorable physico-chemical properties and inhibitory activity in human mast cells have been identified. In addition, our studies suggest that the active site of hH-PGDS can accommodate larger structural diversity than previously thought, such as the introduction of polar groups in the inner part of the binding pocket.

Discovery of indazole ethers as novel, potent, non-steroidal glucocorticoid receptor modulators.

A structure-based design approach led to the identification of a novel class of indazole ether based, non-steroidal glucocorticoid receptor (GR) modulators. Several examples were identified that displayed cell potency in the picomolar range, inhibiting LPS-induced TNF-α release by primary peripheral blood mononuclear cells (PBMCs). Additionally, an improved steroid hormone receptor binding selectivity profile, compared to classical steroidal GR agonists, was demonstrated. The indazole ether core tolerated a broad range of substituents allowing for modulation of the physiochemical parameters. A small sub-set of indazole ethers, with pharmacokinetic properties suitable for oral administration, was investigated in a rat antigen-induced joint inflammation model and demonstrated excellent anti-inflammatory efficacy.

Discovery of new selective glucocorticoid receptor agonist leads

We report on the discovery of two new lead series for the development of glucocorticoid receptor agonists. Firstly, the discovery of tetrahydronaphthalenes led to metabolically stable and dissociated compounds. Their binding mode to the glucocorticoid receptor could be elucidated through an X-ray structure. Closer inspection into the reaction path and analyses of side products revealed a new amino alcohol series also addressing the glucocorticoid receptor and demonstrating strong anti-inflammatory activity in vitro.

X-ray Characterization and Structure-Based Optimization of Striatal-Enriched Protein Tyrosine Phosphatase Inhibitors

Excessive activity of striatal-enriched protein tyrosine phosphatase (STEP) in the brain has been detected in numerous neuropsychiatric disorders including Alzheimers disease. Notably, knockdown of STEP in an Alzheimer mouse model effected an increase in the phosphorylation levels of downstream STEP substrates and a significant reversal in the observed cognitive and memory deficits. These data point to the promising potential of STEP as a target for drug discovery in Alzheimers treatment. We previously reported a substrate-based approach to the development of low molecular weight STEP inhibitors with Ki values as low as 7.8 μM. Herein, we disclose the first X-ray crystal structures of inhibitors bound to STEP and the surprising finding that they occupy noncoincident binding sites. Moreover, we utilize this structural information to optimize the inhibitor structure to achieve a Ki of 110 nM, with 15-60-fold selectivity across a series of phosphatases.

Solutions for the storage and handling of SPINE standard pucks.

Currently there is no rack system for the long-term storage of SPINE pucks in spite of their commercial availability and heavy usage at the ESRF. The only way to store pucks is in transport dewar canisters which presents a number of limitations and drawbacks. Here a simple affordable rack for storing SPINE pucks is described, which we believe is accessible to not only synchrotrons but also both academic and industrial research laboratories.