Identification and optimization of a novel series of selective PIP5K inhibitors.

Phosphatidyl inositol (4,5)-bisphosphate (PI(4,5)P2) plays several key roles in human biology and the lipid kinase that produces PI(4,5)P2, PIP5K, has been hypothesized to provide a potential therapeutic target of interest in the treatment of cancers. To better understand and explore the role of PIP5K in human cancers there remains an urgent need for potent and specific PIP5K inhibitor molecules. Following a high throughput screen of the AstraZeneca collection, a novel, moderately potent and selective inhibitor of PIP5K, 1, was discovered. Detailed exploration of the SAR for this novel scaffold resulted in the considerable optimization of both potency for PIP5K, and selectivity over the closely related kinase PI3Kα, as well as identifying several opportunities for the continued optimization of drug-like properties. As a result, several high quality in vitro tool compounds were identified (8, 20 and 25) that demonstrate the desired biochemical and cellular profiles required to aid better understanding of this complex area of biology.

From PIM1 to PI3Kδ via GSK3ß: Target Hopping through the Kinome.

Selective inhibitors of phosphoinositide 3-kinase delta are of interest for the treatment of inflammatory diseases. Initial optimization of a 3-substituted indazole hit compound targeting the kinase PIM1 focused on improving selectivity over GSK3ß through consideration of differences in the ATP binding pockets. Continued kinase cross-screening showed PI3Kδ activity in a series of 4,6-disubstituted indazole compounds, and subsequent structure-activity relationship exploration led to the discovery of an indole-containing lead compound as a potent PI3Kδ inhibitor with selectivity over the other PI3K isoforms.

Discovery of 4,6-disubstituted pyrimidines as potent inhibitors of the heat shock factor 1 (HSF1) stress pathway and CDK9.

Heat shock factor 1 (HSF1) is a transcription factor that plays key roles in cancer, including providing a mechanism for cell survival under proteotoxic stress. Therefore, inhibition of the HSF1-stress pathway represents an exciting new opportunity in cancer treatment. We employed an unbiased phenotypic screen to discover inhibitors of the HSF1-stress pathway. Using this approach we identified an initial hit (1) based on a 4,6-pyrimidine scaffold (2.00 µM). Optimisation of cellular SAR led to an inhibitor with improved potency (25, 15 nM) in the HSF1 phenotypic assay. The 4,6-pyrimidine 25 was also shown to have high potency against the CDK9 enzyme (3 nM).

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.

Potent, selective small molecule inhibitors of type III phosphatidylinositol-4-kinase α- but not ß-inhibit the phosphatidylinositol signaling cascade and cancer cell proliferation.

Two series of inhibitors of type III phosphatidylinositol-4-kinase were identified by high throughput screening and optimised to derive probe compounds that independently and selectively inhibit the α- and the ß-isoforms with no significant activity towards related kinases in the pathway. In a cellular environment, inhibition of the α- but not the ß-subtype led to a reduction in phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate concentration, causing inhibition of inositol-1-phosphate formation and inhibition of proliferation in a panel of cancer cell lines.

Inhibiting NF-κB-inducing kinase (NIK): discovery, structure-based design, synthesis, structure-activity relationship, and co-crystal structures.

The discovery, structure-based design, synthesis, and optimization of NIK inhibitors are described. Our work began with an HTS hit, imidazopyridinyl pyrimidinamine 1. We utilized homology modeling and conformational analysis to optimize the indole scaffold leading to the discovery of novel and potent conformationally constrained inhibitors such as compounds 25 and 28. Compounds 25 and 31 were co-crystallized with NIK kinase domain to provide structural insights.

4-Phenyl-7-azaindoles as potent, selective and bioavailable IKK2 inhibitors demonstrating good in vivo efficacy.

The lead optimization of a series of potent azaindole IKK2 inhibitors is described. Optimization of the human whole blood activity and selectivity over IKK1 in parallel led to the discovery of 16, a potent and selective IKK2 inhibitor showing good efficacy in a rat model of neutrophil activation.

Diverse heterocyclic scaffolds as allosteric inhibitors of AKT.

Wide-ranging exploration of potential replacements for a quinoline-based inhibitor of activation of AKT kinase led to number of alternative, novel scaffolds with potentially improved potency and physicochemical properties. Examples showed predictable DMPK properties, and one such compound demonstrated pharmacodynamic knockdown of phosphorylation of AKT and downstream biomarkers in vivo and inhibition of tumor growth in a breast cancer xenograft model.

Coumarin-based inhibitors of human NAD(P)H:quinone oxidoreductase-1. Identification, structure-activity, off-target effects and in vitro human pancreatic cancer toxicity.

The enzyme human NAD(P)H quinone oxidoreductase-1 (NQO1), which is overexpressed in several types of tumor cell, is considered a design target for cancer therapeutics. We identify new coumarin-based competitive inhibitors of NQO1, one of which is nanomolar. Using computational docking and molecular dynamics, we obtain insights into the structural basis of inhibition. Selected inhibitors were then assessed for off-target effects associated with dicoumarol and were found to have differing effects on superoxide formation and mitochondrial respiration. A comparison of NQO1 inhibition and off-target effects for dicoumarol and its derivatives suggests that the ability of dicoumarol to kill cancer cells is independent of NQO1 inhibition, that cellular superoxide production by dicoumarol does not seem linked to NQO1 inhibition but may be related to mitochondrial decoupling, and that superoxide does not appear to be a major determinant of cytotoxicity. Implications are discussed for NQO1 inhibition as an anticancer drug design target and superoxide generation as the dicoumarol-mediated mechanism of cytotoxicity.

Time-resolved and static-ensemble structural chemistry of hydroxymethylbilane synthase.

The enzyme hydroxymethylbilane synthase (HMBS, EC 4.3.1.8), 313 amino acid residues and MW 34 kDa, also known as porphobilinogen deaminase (PBGD), catalyses the stepwise polymerization of four molecules of porphobilinogen (PBG) to the linear tetrapyrrole 1-hydroxymethylbilane. Several crystallographic structures of HMBS have been previously determined, most recently including by time-resolved Laue protein crystallography of the Lys59Gln mutant form with reaction initiation undertaken by use of a flow cell carrying the substrate PBG. In this paper we review these structures and add new molecular graphics representations and analyses. Moreover we present a new structure refined at 1.66 A resolution using diffraction data recorded at cryo-temperature (100 K) in an attempt at trapping the polypeptide loop (residues 47 to 58) in the vicinity of the enzyme active site, missing in all previous structure determinations. This loop still has not appeared in the electron density maps, in spite of the advantage of cryo-temperature, but nevertheless the 1.66 A cryo-structure extends the ensemble of known HMBS structures. The cryomodel of protein, cofactor and 320 bound water molecules has been refined to a final R-factor and R-free of 0.198 and 0.247 respectively; the PDB deposition codes, coordinates and structure factors are 1GTK and R1GTKSF respectively. Finally a protein comparison study is presented of the Mycobacterium tuberculosis (MTb) HMBS, with the E. coli HMBS. This has been done as preparation for future structural studies on the MTb HMBS from this important disease bearing organism. The overall amino acid sequence identity is 41%. Most interestingly there is a two-residue reduction in length of the loop referred to above (Asp 50 and Gly 58 being missing in the MTb form). This gives the hope that this loop will be less flexible and thus might become visible to crystallographic analysis.

Direct dynamics calculations of reaction rate and kinetic isotope effects in enzyme catalysed reactions.

Direct dynamics calculations employing hybrid quantum mechanical and molecular mehanical (QM/MM) potentials and molecular dynamics simulation methods have been used to explore the important dynamic role that enzyme structure has on proton transfer in the C-H bond breakage of a methylamine substrate by methylamine dehydrogenase (MADH). Canonical variational transition state theory with optimised multidimensional tunnelling corrections has been used to predict deuterium kinetic isotope effects corresponding to a range of enzyme conformations and to show the importance of donor acceptor separation, and transition state and product stabilisation within the active site. Large kinetic isotope effects can be predicted for proton transfer with both semi-empirical and ab initio electronic structure methods.