Validation of ion mobility spectrometry - mass spectrometry as a screening tool to identify type II kinase inhibitors of FGFR1 kinase.

RATIONALE: The protein kinase FGFR1 regulates cellular processes in human development. As over-activity of FGFR1 is implicated with cancer, effective inhibitors are in demand. Type I inhibitors, which bind to the active form of FGFR1, are less effective than type II inhibitors, which bind to the inactive form. Screening to distinguish between type I and type II inhibitors is required. METHODS: X-ray crystallography was used to indicate whether a range of potential inhibitors bind to the active or inactive FGFR1 kinase conformation. The binding affinity of each ligand to FGFR1 was measured using biochemical methods. Electrospray ionisation - ion mobility spectrometry - mass spectrometry (ESI-IMS-MS) in conjunction with collision-induced protein unfolding generated a conformational profile of each FGFR1-ligand complex. The results indicate that the protein's conformational profile depends on whether the inhibitor is type I or type II. RESULTS: X-ray crystallography confirmed which of the kinase inhibitors bind to the active or inactive form of FGFR1 kinase. Collision-induced unfolding combined with ESI-IMS-MS showed distinct differences in the FGFR1 folding landscape for type I and type II inhibitors. Biochemical studies indicated a similar range of FGFR1 affinities for both types of inhibitors, thus providing confidence that the conformational variations detected using ESI-IMS-MS can be interpretated unequivocally and that this is an effective screening method. CONCLUSIONS: A robust ESI-IMS-MS method has been implemented to distinguish between the binding mode of type I and type II inhibitors by monitoring the conformational unfolding profile of FGFR1. This rapid method requires low sample concentrations and could be used as a high-throughput screening technique for the characterisation of novel kinase inhibitors.

AZD9496: An Oral Estrogen Receptor Inhibitor That Blocks the Growth of ER-Positive and ESR1-Mutant Breast Tumors in Preclinical Models.

Fulvestrant is an estrogen receptor (ER) antagonist administered to breast cancer patients by monthly intramuscular injection. Given its present limitations of dosing and route of administration, a more flexible orally available compound has been sought to pursue the potential benefits of this drug in patients with advanced metastatic disease. Here we report the identification and characterization of AZD9496, a nonsteroidal small-molecule inhibitor of ERα, which is a potent and selective antagonist and downregulator of ERα in vitro and in vivo in ER-positive models of breast cancer. Significant tumor growth inhibition was observed as low as 0.5 mg/kg dose in the estrogen-dependent MCF-7 xenograft model, where this effect was accompanied by a dose-dependent decrease in PR protein levels, demonstrating potent antagonist activity. Combining AZD9496 with PI3K pathway and CDK4/6 inhibitors led to further growth-inhibitory effects compared with monotherapy alone. Tumor regressions were also seen in a long-term estrogen-deprived breast model, where significant downregulation of ERα protein was observed. AZD9496 bound and downregulated clinically relevant ESR1 mutants in vitro and inhibited tumor growth in an ESR1-mutant patient-derived xenograft model that included a D538G mutation. Collectively, the pharmacologic evidence showed that AZD9496 is an oral, nonsteroidal, selective estrogen receptor antagonist and downregulator in ER(+) breast cells that could provide meaningful benefit to ER(+) breast cancer patients. AZD9496 is currently being evaluated in a phase I clinical trial. Cancer Res; 76(11); 3307-18. ©2016 AACR.

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.

Tetrahydroisoquinoline Phenols: Selective Estrogen Receptor Downregulator Antagonists with Oral Bioavailability in Rat.

A series of tetrahydroisoquinoline phenols was modified to give an estrogen receptor downregulator-antagonist profile. Optimization around the core, alkyl side chain, and pendant aryl ring resulted in compounds with subnanomolar levels of potency. The phenol functionality was shown to be required to achieve highly potent compounds, but unusually this was compatible with obtaining high oral bioavailabilities in rat.

Optimization of a Novel Binding Motif to (E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic Acid (AZD9496), a Potent and Orally Bioavailable Selective Estrogen Receptor Downregulator and Antagonist.

The discovery of an orally bioavailable selective estrogen receptor downregulator (SERD) with equivalent potency and preclinical pharmacology to the intramuscular SERD fulvestrant is described. A directed screen identified the 1-aryl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole motif as a novel, druglike ER ligand. Aided by crystal structures of novel ligands bound to an ER construct, medicinal chemistry iterations led to (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (30b, AZD9496), a clinical candidate with high oral bioavailability across preclinical species that is currently being evaluated in phase I clinical trials for the treatment of advanced estrogen receptor (ER) positive breast cancer.

Investigation of (E)-3-[4-(2-Oxo-3-aryl-chromen-4-yl)oxyphenyl]acrylic Acids as Oral Selective Estrogen Receptor Down-Regulators.

A novel estrogen receptor down-regulator, 7-hydroxycoumarin (5, SS5020), has been reported with antitumor effects against chemically induced mammary tumors. Here, we report on our own investigation of 7-hydroxycoumarins as potential selective estrogen receptor down-regulators, which led us to the discovery of potent down-regulating antagonists, such as 33. Subsequent optimization and removal of the 7-hydroxy group led to coumarin 59, which had increased potency and improved rat bioavailability relative to SS5020.

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.

FGFR1 Kinase Inhibitors: Close Regioisomers Adopt Divergent Binding Modes and Display Distinct Biophysical Signatures.

The binding of a ligand to its target protein is often accompanied by conformational changes of both the protein and the ligand. This is of particular interest, since structural rearrangements of the macromolecular target and the ligand influence the free energy change upon complex formation. In this study, we use X-ray crystallography, isothermal titration calorimetry, and surface-plasmon resonance biosensor analysis to investigate the binding of pyrazolylaminopyrimidine inhibitors to FGFR1 tyrosine kinase, an important anticancer target. Our results highlight that structurally close analogs of this inhibitor series interact with FGFR1 with different binding modes, which are a consequence of conformational changes in both the protein and the ligand as well as the bound water network. Together with the collected kinetic and thermodynamic data, we use the protein-ligand crystal structure information to rationalize the observed inhibitory potencies on a molecular level.

Characterization of the complex formed between a potent neutralizing ovine-derived polyclonal anti-TNFα Fab fragment and human TNFα.

TNFα (tumour necrosis factor α) is an early mediator in the systemic inflammatory response to infection and is therefore a therapeutic target in sepsis. AZD9773 is an ovine-derived, polyclonal anti-TNFα Fab fragment derived from a pool of serum and currently being developed as a treatment for severe sepsis and septic shock. In the present study, we show that although AZD9773 has a modest affinity for TNFα in a binding assay, the Ki in a cell-based assay is approximately four orders of magnitude lower. We show using SEC (size exclusion chromatography) that the maximum size of the complex between AZD9773 and TNFα is consistent with approximately 12 Fabs binding to one TNFα trimer. A number of approaches were taken to map the epitopes recognized by AZD9773. These revealed that a number of different regions on TNFα are involved in binding to the polyclonal Fab. The data suggest that there are probably three epitopes per monomer that are responsible for most of the inhibition by AZD9773 and that all three can be occupied at the same time in the complex. We conclude that AZD9773 is clearly demonstrated to bind to multiple epitopes on TNFα and suggest that the polyclonal nature may account, at least in part, for the very high potency observed in cell-based assays.

Structural approaches to obtain kinase selectivity.

One of the grand challenges in kinase drug discovery is the design of small-molecule inhibitors with selectivity profiles that will ultimately be efficacious in the clinic. Current medicinal chemistry strategies make heavy use of structural, biophysical and computational approaches to achieve this multi-faceted goal. Here we review structure-based approaches underlying the development of several molecules that are currently in clinical trials, including the cMet inhibitor ARQ197 and the Bcr-Abl inhibitor ponatinib. We highlight the challenge posed by the emergence of resistance mutants and discuss promising lead generation strategies to obtain selective inhibitors of protein and lipid kinases such as targeting of specific sites, the use of fragment-based approaches and new chemical probes based on metal complexes.

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.

Rapid generation of a high quality lead for transforming growth factor-beta (TGF-beta) type I receptor (ALK5).

A novel class of 4-pyridinoxy-2-anilinopyridine-based TGF-beta type I receptor (also known as activin-like kinase 5 or ALK5) inhibitors is reported. The binding mode of this scaffold was successfully predicted by analyzing possible docked binding modes of literature inhibitors and novel synthetic ideas. Compounds such as 19 are potent ALK5 inhibitors with good physicochemical and pharmacokinetic properties and thus represent high quality leads for further optimization.

N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5- (tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine, a novel, highly selective, orally available, dual-specific c-Src/Abl kinase inhibitor.

Src family kinases (SFKs) are nonreceptor tyrosine kinases that are reported to be critical for cancer progression. We report here a novel subseries of C-5-substituted anilinoquinazolines that display high affinity and specificity for the tyrosine kinase domain of the c-Src and Abl enzymes. These compounds exhibit high selectivity for SFKs over a panel of recombinant protein kinases, excellent pharmacokinetics, and in vivo activity following oral dosing. N-(5-Chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine (AZD0530) inhibits c-Src and Abl enzymes at low nanomolar concentrations and is highly selective over a range of kinases. AZD0530 displays excellent pharmacokinetic parameters in animal preclinically and in man (t(1/2) = 40 h). AZD0530 is a potent inhibitor of tumor growth in a c-Src-transfected 3T3-fibroblast xenograft model in vivo and led to a significant increase in survival in a highly aggressive, orthotopic model of human pancreatic cancer when dosed orally once daily. AZD0530 is currently undergoing clinical evaluation in man.

The Mycobacterium tuberculosis ino1 gene is essential for growth and virulence.

Inositol is utilized by Mycobacterium tuberculosis in the production of its major thiol and of essential cell wall lipoglycans. We have constructed a mutant lacking the gene encoding inositol-1-phosphate synthase (ino1), which catalyses the first committed step in inositol synthesis. This mutant is only viable in the presence of extremely high levels of inositol. Mutant bacteria cultured in inositol-free medium for four weeks showed a reduction in levels of mycothiol, but phosphatidylinositol mannoside, lipomannan and lipoarabinomannan levels were not altered. The ino1 mutant was attenuated in resting macrophages and in SCID mice. We used site-directed mutagenesis to alter four putative active site residues; all four alterations resulted in a loss of activity, and we demonstrated that a D310N mutation caused loss of the active site Zn2+ ion and a conformational change in the NAD+ cofactor.

Crystal structure of human thymidine phosphorylase in complex with a small molecule inhibitor.

Human thymidine phosphorylase (HTP), also known as platelet-derived endothelial cell growth factor (PD-ECGF), is overexpressed in certain solid tumors where it is linked to poor prognosis. HTP expression is utilized for certain chemotherapeutic strategies and is also thought to play a role in tumor angiogenesis. We determined the structure of HTP bound to the small molecule inhibitor 5-chloro-6-[1-(2-iminopyrrolidinyl) methyl] uracil hydrochloride (TPI). The inhibitor appears to mimic the substrate transition state, which may help explain the potency of this inhibitor and the catalytic mechanism of pyrimidine nucleotide phosphorylases (PYNPs). Further, we have confirmed the validity of the HTP structure as a template for structure-based drug design by predicting binding affinities for TPI and other known HTP inhibitors using in silico docking techniques. This work provides the first structural insight into the binding mode of any inhibitor to this important drug target and forms the basis for designing novel inhibitors for use in anticancer therapy.

Imidazo[1,2-a]pyridines: a potent and selective class of cyclin-dependent kinase inhibitors identified through structure-based hybridisation.

High-throughput screening identified the imidazo[1,2-a]pyridine and bisanilinopyrimidine series as inhibitors of the cyclin-dependent kinase CDK4. Comparison of their experimentally-determined binding modes and emerging structure-activity trends led to the development of potent and selective imidazo[1,2-a]pyridine inhibitors for CDK4 and in particular CDK2.