Alexander L. Breeze

The high-resolution crystal structure of a 24-kDa gyrase B fragment from E. coli complexed with one of the most potent coumarin inhibitors, clorobiocin

Coumarin antibiotics, such as clorobiocin, novobiocin, and coumermycin A1, inhibit the supercoiling activity of gyrase by binding to the gyrase B (GyrB) subunit. Previous crystallographic studies of a 24‐kDa N‐terminal domain of GyrB from E. coli complexed with novobiocin and a cyclothialidine analogue have shown that both ligands act by binding at the ATP‐binding site. Clorobiocin is a natural antibiotic isolated from several Streptomyces strains and differs from novobiocin in that the methyl group at the 8 position in the coumarin ring of novobiocin is replaced by a chlorine atom, and the carbamoyl at the 3′ position of the noviose sugar is substituted by a 5‐methyl‐2‐pyrrolylcarbonyl group. To understand the difference in affinity, in order that this information might be exploited in rational drug design, the crystal structure of the 24‐kDa GyrB fragment in complex with clorobiocin was determined to high resolution. This structure was determined independently in two laboratories, which allowed the validation of equivalent interpretations. The clorobiocin complex structure is compared with the crystal structures of gyrase complexes with novobiocin and 5′‐adenylyl‐β,γ‐imidodiphosphate, and with information on the bound conformation of novobiocin in the p24‐novobiocin complex obtained by heteronuclear isotope‐filtered NMR experiments in solution. Moreover, to understand the differences in energetics of binding of clorobiocin and novobiocin to the protein, the results from isothermal titration calorimetry are also presented.

Fragment screening to predict druggability (ligandability) and lead discovery success

F A l w i p T s a p i c c 2 e g Target druggability – ligandability It is estimated that only 1% of drug discovery projects make it to market industry-wide. There is increasing regulatory pressure for new products to show significant improvement over existing therapies. Despite genomic initiatives, only three new targets are addressed each year with synthetic drugs [1]. Late-stage failure in clinical trials are costly, therefore significant cost savings will be achieved by improving the selection of protein targets and selecting winning projects early on in the process. Thus, pharmaceutical companies face a major challenge today: we need to reduce attrition throughout the drug discovery process to reduce cost and increase success rates while, at the same time, exploiting novel mechanisms for new drugs – to differentiate from competitors. The term ‘druggability’ usually refers to the likelihood of finding orally bioavailable small molecules that bind to a particular target in a disease-modifying way [2]. Unless there are other known compounds, either on the market or in clinical trials, acting on a particular target, intrinsic druggability is unknown. It is, therefore, useful to distinguish the ability of a target to bind small molecules from the more complex pharmacokinetic and pharmacodynamic mechanisms included in the term druggability. In recent years, the term druggability has increasingly

An Integrated Approach to Fragment-Based Lead Generation:Philosophy, Strategy and Case Studies from AstraZenecas 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).

Design and Synthesis of Novel Lactate Dehydrogenase A Inhibitors by Fragment-Based Lead Generation

Lactate dehydrogenase A (LDHA) catalyzes the conversion of pyruvate to lactate, utilizing NADH as a cofactor. It has been identified as a potential therapeutic target in the area of cancer metabolism. In this manuscript we report our progress using fragment-based lead generation (FBLG), assisted by X-ray crystallography to develop small molecule LDHA inhibitors. Fragment hits were identified through NMR and SPR screening and optimized into lead compounds with nanomolar binding affinities via fragment linking. Also reported is their modification into cellular active compounds suitable for target validation work.

Small Molecule Binding Sites on the Ras:SOS Complex Can be Exploited for Inhibition of Ras Activation.

Constitutively active mutant KRas displays a reduced rate of GTP hydrolysis via both intrinsic and GTPase-activating protein-catalyzed mechanisms, resulting in the perpetual activation of Ras pathways. We describe a fragment screening campaign using X-ray crystallography that led to the discovery of three fragment binding sites on the Ras:SOS complex. The identification of tool compounds binding at each of these sites allowed exploration of two new approaches to Ras pathway inhibition by stabilizing or covalently modifying the Ras:SOS complex to prevent the reloading of Ras with GTP. Initially, we identified ligands that bound reversibly to the Ras:SOS complex in two distinct sites, but these compounds were not sufficiently potent inhibitors to validate our stabilization hypothesis. We conclude by demonstrating that covalent modification of Cys118 on Ras leads to a novel mechanism of inhibition of the SOS-mediated interaction between Ras and Raf and is effective at inhibiting the exchange of labeled GDP in both mutant (G12C and G12V) and wild type Ras.

Small-molecule proteomimetic inhibitors of the HIF-1α-p300 protein-protein interaction.

The therapeutically relevant hypoxia inducible factor HIF‐1α–p300 protein–protein interaction can be orthosterically inhibited with α‐helix mimetics based on an oligoamide scaffold that recapitulates essential features of the C‐terminal helix of the HIF‐1α C‐TAD (C‐terminal transactivation domain). Preliminary SAR studies demonstrated the important role of side‐chain size and hydrophobicity/hydrophilicity in determining potency. These small molecules represent the first biophysically characterised HIF‐1α–p300 PPI inhibitors and the first examples of small‐molecule aromatic oligoamide helix mimetics to be shown to have a selective binding profile. Although the compounds were less potent than HIF‐1α, the result is still remarkable in that the mimetic reproduces only three residues from the 42‐residue HIF‐1α C‐TAD from which it is derived.

Real‐Time Monitoring of New Delhi Metallo‐β‐Lactamase Activity in Living Bacterial Cells by 1H NMR Spectroscopy

Disconnections between in vitro responses and those observed in whole cells confound many attempts to design drugs in areas of serious medical need. A method based on 1D 1H NMR spectroscopy is reported that affords the ability to monitor the hydrolytic decomposition of the carbapenem antibiotic meropenem inside Escherichia coli cells expressing New Delhi metallo-β-lactamase subclass 1 (NDM-1), an emerging antibiotic-resistance threat. Cell-based NMR studies demonstrated that two known NDM-1 inhibitors, L-captopril and ethylenediaminetetraacetic acid (EDTA), inhibit the hydrolysis of meropenem in vivo. NDM-1 activity in cells was also shown to be inhibited by spermine, a porin inhibitor, although in an in vitro assay, the influence of spermine on the activity of isolated NDM-1 protein is minimal. This new approach may have generic utility for monitoring reactions involving diffusible metabolites in other complex biological matrices and whole-cell settings, including mammalian cells.

Structural Insights into FGFR Kinase Isoform Selectivity: Diverse Binding Modes of AZD4547 and Ponatinib in Complex with FGFR1 and FGFR4

The fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases has been implicated in a wide variety of cancers. Despite a high level of sequence homology in the ATP-binding site, the majority of reported inhibitors are selective for the FGFR1-3 isoforms and display much reduced potency toward FGFR4, an exception being the Bcr-Abl inhibitor ponatinib. Here we present the crystal structure of the FGFR4 kinase domain and show that both FGFR1 and FGFR4 kinase domains in complex with ponatinib adopt a DFG-out activation loop conformation. Comparison with the structure of FGFR1 in complex with the candidate drug AZD4547, combined with kinetic characterization of the binding of ponatinib and AZD4547 to FGFR1 and FGFR4, sheds light on the observed differences in selectivity profiles and provides a rationale for developing FGFR4-selective inhibitors.

NMR trial models: experiences with the colicin immunity protein Im7 and the p85α C-terminal SH2–peptide complex

Two cases of successful molecular replacement using NMR trial models are presented. One is the crystal structure of the Escherichia coli colicin immunity protein Im7; the other is a heretofore unreported crystal structure of a specific PDGF receptor-derived peptide complex of the carboxy-terminal SH2 domain from the p85alpha subunit of human phosphatidylinositol 3-OH kinase. In both cases, molecular replacement was non-trivial. Success was achieved using trial models that consisted of an ensemble of NMR structures from which the more flexible portions had been excised. Use of maximum-likelihood refinement proved critical to be able to refine the poor starting models. The challenges typical of the use of NMR trial models in molecular replacement are discussed.

Landscape of activating cancer mutations in FGFR kinases and their differential responses to inhibitors in clinical use

Frequent genetic alterations discovered in FGFRs and evidence implicating some as drivers in diverse tumors has been accompanied by rapid progress in targeting FGFRs for anticancer treatments. Wider assessment of the impact of genetic changes on the activation state and drug responses is needed to better link the genomic data and treatment options. We here apply a direct comparative and comprehensive analysis of FGFR3 kinase domain variants representing the diversity of point-mutations reported in this domain. We reinforce the importance of N540K and K650E and establish that not all highly activating mutations (for example R669G) occur at high-frequency and conversely, that some “hotspots” may not be linked to activation. Further structural characterization consolidates a mechanistic view of FGFR kinase activation and extends insights into drug binding. Importantly, using several inhibitors of particular clinical interest (AZD4547, BGJ-398, TKI258, JNJ42756493 and AP24534), we find that some activating mutations (including different replacements of the same residue) result in distinct changes in their efficacy. Considering that there is no approved inhibitor for anticancer treatments based on FGFR-targeting, this information will be immediately translatable to ongoing clinical trials.