Discovery of AZD4747, a Potent and Selective Inhibitor of Mutant GTPase KRASG12C with Demonstrable CNS Penetration.

The glycine to cysteine mutation at codon 12 of Kirsten rat sarcoma (KRAS) represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 14, AZD4747, a clinical development candidate for the treatment of KRASG12C-positive tumors, including the treatment of central nervous system (CNS) metastases. Building on our earlier discovery of C5-tethered quinazoline AZD4625, excision of a usually critical pyrimidine ring yielded a weak but brain-penetrant start point which was optimized for potency and DMPK. Key design principles and measured parameters that give high confidence in CNS exposure are discussed. During optimization, divergence between rodent and non-rodent species was observed in CNS exposure, with primate PET studies ultimately giving high confidence in the expected translation to patients. AZD4747 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.

Optimization of hERG and Pharmacokinetic Properties for Basic Dihydro-8H-purin-8-one Inhibitors of DNA-PK.

The DNA-PK complex is activated by double-strand DNA breaks and regulates the non-homologous end-joining repair pathway; thus, targeting DNA-PK by inhibiting the DNA-PK catalytic subunit (DNA-PKcs) is potentially a useful therapeutic approach for oncology. A previously reported series of neutral DNA-PKcs inhibitors were modified to incorporate a basic group, with the rationale that increasing the volume of distribution while maintaining good metabolic stability should increase the half-life. However, adding a basic group introduced hERG activity, and basic compounds with modest hERG activity (IC50 = 10-15 µM) prolonged QTc (time from the start of the Q wave to the end of the T wave, corrected by heart rate) in an anaesthetized guinea pig cardiovascular model. Further optimization was necessary, including modulation of pK a, to identify compound 18, which combines low hERG activity (IC50 = 75 µM) with excellent kinome selectivity and favorable pharmacokinetic properties.

Accelerating the Validation of Endogenous On-Target Engagement and In Cellulo Kinetic Assessment for Covalent Inhibitors of KRASG12C in Early Drug Discovery.

Covalent inhibition is a valuable modality in drug discovery because of its potential ability in decoupling pharmacokinetics from pharmacodynamics by prolonging the residence time of the drug on the target of interest. This increase in target occupancy is limited only by the rate of target turnover. However, a limitation in such studies is to translate the in vitro inhibition assessment to the appropriate in cellulo target engagement parameter by covalent probes. Estimation of such parameters is often impeded by the low-throughput nature of current probe-free approaches. In this study, an ultra-performance liquid chromatography-multiple reaction monitoring mass spectrometry platform was utilized to develop a targeted proteomics workflow that can evaluate cellular on-target engagement of covalent molecules in an increased throughput manner. This workflow enabled a throughput increase of 5-10 fold when compared to traditional nanoLC-based proteomics studies. To demonstrate the applicability of the method, KRASG12C was used as a model system to investigate the interaction of an irreversible covalent small molecule, compound 25, both in vitro and in cellulo. Initial biochemical studies confirmed that the small molecule forms an adduct with the targeted cysteine on the protein, as assessed at the level of both intact protein and on the target peptide. In cellulo studies were carried out to quantify target engagement and allele selectivity assessment for the small molecule in the heterozygous NCI-H358 cell line for KRASG12C with respect to the WT type protein. The workflow enabled evaluation of in vitro and in cellulo target engagement kinetics, providing mechanistic insights into the irreversible mode of inhibition. In summary, the method has the potential for target agnostic application in the assessment of on-target engagement of covalent probes compatible with the high-throughput requirements of early drug discovery.

Discovery of 5-{4-[(7-Ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl]piperazin-1-yl}-N-methylpyridine-2-carboxamide (AZD5305): A PARP1-DNA Trapper with High Selectivity for PARP1 over PARP2 and Other PARPs.

Poly-ADP-ribose-polymerase (PARP) inhibitors have achieved regulatory approval in oncology for homologous recombination repair deficient tumors including BRCA mutation. However, some have failed in combination with first-line chemotherapies, usually due to overlapping hematological toxicities. Currently approved PARP inhibitors lack selectivity for PARP1 over PARP2 and some other 16 PARP family members, and we hypothesized that this could contribute to toxicity. Recent literature has demonstrated that PARP1 inhibition and PARP1-DNA trapping are key for driving efficacy in a BRCA mutant background. Herein, we describe the structure- and property-based design of 25 (AZD5305), a potent and selective PARP1 inhibitor and PARP1-DNA trapper with excellent in vivo efficacy in a BRCA mutant HBCx-17 PDX model. Compound 25 is highly selective for PARP1 over other PARP family members, with good secondary pharmacology and physicochemical properties and excellent pharmacokinetics in preclinical species, with reduced effects on human bone marrow progenitor cells in vitro.

A Novel High-Throughput FLIPR Tetra-Based Method for Capturing Highly Confluent Kinetic Data for Structure-Kinetic Relationship Guided Early Drug Discovery.

Target engagement by small molecules is necessary for producing a physiological outcome. In the past, a lot of emphasis was placed on understanding the thermodynamics of such interactions to guide structure-activity relationships. It is becoming clearer, however, that understanding the kinetics of the interaction between a small-molecule inhibitor and the biological target [structure-kinetic relationship (SKR)] is critical for selection of the optimum candidate drug molecule for clinical trial. However, the acquisition of kinetic data in a high-throughput manner using traditional methods can be labor intensive, limiting the number of molecules that can be tested. As a result, in-depth kinetic studies are often carried out on only a small number of compounds, and usually at a later stage in the drug discovery process. Fundamentally, kinetic data should be used to drive key decisions much earlier in the drug discovery process, but the throughput limitations of traditional methods preclude this. A major limitation that hampers acquisition of high-throughput kinetic data is the technical challenge in collecting substantially confluent data points for accurate parameter estimation from time course analysis. Here, we describe the use of the fluorescent imaging plate reader (FLIPR), a charge-coupled device (CCD) camera technology, as a potential high-throughput tool for generating biochemical kinetic data with smaller time intervals. Subsequent to the design and optimization of the assay, we demonstrate the collection of highly confluent time-course data for various kinase protein targets with reasonable throughput to enable SKR-guided medicinal chemistry. We select kinase target 1 as a special case study with covalent inhibition, and demonstrate methods for rapid and detailed analysis of the resultant kinetic data for parameter estimation. In conclusion, this approach has the potential to enable rapid kinetic studies to be carried out on hundreds of compounds per week and drive project decisions with kinetic data at an early stage in drug discovery.

Alkynyl Benzoxazines and Dihydroquinazolines as Cysteine Targeting Covalent Warheads and Their Application in Identification of Selective Irreversible Kinase Inhibitors.

With a resurgence in interest in covalent drugs, there is a need to identify new moieties capable of cysteine bond formation that are differentiated from commonly employed systems such as acrylamide. Herein, we report on the discovery of new alkynyl benzoxazine and dihydroquinazoline moieties capable of covalent reaction with cysteine. Their utility as alternative electrophilic warheads for chemical biological probes and drug molecules is demonstrated through site-selective protein modification and incorporation into kinase drug scaffolds. A potent covalent inhibitor of JAK3 kinase was identified with superior selectivity across the kinome and improvements in in vitro pharmacokinetic profile relative to the related acrylamide-based inhibitor. In addition, the use of a novel heterocycle as a cysteine reactive warhead is employed to target Cys788 in c-KIT, where acrylamide has previously failed to form covalent interactions. These new reactive and selective heterocyclic warheads supplement the current repertoire for cysteine covalent modification while avoiding some of the limitations generally associated with established moieties.

The Discovery of 7-Methyl-2-[(7-methyl[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino]-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one (AZD7648), a Potent and Selective DNA-Dependent Protein Kinase (DNA-PK) Inhibitor.

DNA-PK is a key component within the DNA damage response, as it is responsible for recognizing and repairing double-strand DNA breaks (DSBs) via non-homologous end joining. Historically it has been challenging to identify inhibitors of the DNA-PK catalytic subunit (DNA-PKcs) with good selectivity versus the structurally related PI3 (lipid) and PI3K-related protein kinases. We screened our corporate collection for DNA-PKcs inhibitors with good PI3 kinase selectivity, identifying compound 1. Optimization focused on further improving selectivity while improving physical and pharmacokinetic properties, notably co-optimization of permeability and metabolic stability, to identify compound 16 (AZD7648). Compound 16 had no significant off-target activity in the protein kinome and only weak activity versus PI3Kα/γ lipid kinases. Monotherapy activity in murine xenograft models was observed, and regressions were observed when combined with inducers of DSBs (doxorubicin or irradiation) or PARP inhibition (olaparib). These data support progression into clinical studies (NCT03907969).

Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor.

Precision medicines exert selective pressure on tumour cells that leads to the preferential growth of resistant subpopulations, necessitating the development of next-generation therapies to treat the evolving cancer. The PIK3CA-AKT-mTOR pathway is one of the most commonly activated pathways in human cancers, which has led to the development of small-molecule inhibitors that target various nodes in the pathway. Among these agents, first-generation mTOR inhibitors (rapalogs) have caused responses in 'N-of-1' cases, and second-generation mTOR kinase inhibitors (TORKi) are currently in clinical trials. Here we sought to delineate the likely resistance mechanisms to existing mTOR inhibitors in human cell lines, as a guide for next-generation therapies. The mechanism of resistance to the TORKi was unusual in that intrinsic kinase activity of mTOR was increased, rather than a direct active-site mutation interfering with drug binding. Indeed, identical drug-resistant mutations have been also identified in drug-naive patients, suggesting that tumours with activating MTOR mutations will be intrinsically resistant to second-generation mTOR inhibitors. We report the development of a new class of mTOR inhibitors that overcomes resistance to existing first- and second-generation inhibitors. The third-generation mTOR inhibitor exploits the unique juxtaposition of two drug-binding pockets to create a bivalent interaction that allows inhibition of these resistant mutants.

Development of a high-throughput method for preparing human urine for two-dimensional electrophoresis.

There is an increasing interest in analysing the human urinary proteome in the search for biomarkers. However, despite the ease of its collection, urine is a difficult fluid to analyse by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) because of its dilute protein content and high salt levels. Here, we describe a method for high-throughput processing of urine for 2-D PAGE. Urine is filtered and applied to solid phase extraction columns. After washing, the urinary proteins are eluted and freeze dried. The lyophilised powder can then be resuspended in an appropriate buffer for downstream proteomic analysis.

Crystal structure of human MMP9 in complex with a reverse hydroxamate inhibitor.

Matrix metalloproteinases (MMPs) and their inhibitors are important in connective tissue re-modelling in diseases of the cardiovascular system, such as atherosclerosis. Various members of the MMP family have been shown to be expressed in atherosclerotic lesions, but MMP9 is consistently seen in inflammatory atherosclerotic lesions. MMP9 over-expression is implicated in the vascular re-modelling events preceding plaque rupture (the most common cause of acute myocardial infarction). Reduced MMP9 activity, either by genetic manipulation or through pharmacological intervention, has an impact on ventricular re-modelling following infarction. MMP9 activity may therefore represent a key mechanism in the pathogenesis of heart failure. We have determined the crystal structure, at 2.3 A resolution, of the catalytic domain of human MMP9 bound to a peptidic reverse hydroxamate inhibitor as well as the complex of the same inhibitor bound to an active-site mutant (E402Q) at 2.1 A resolution. MMP9 adopts the typical MMP fold. The catalytic centre is composed of the active-site zinc ion, co-ordinated by three histidine residues (401, 405 and 411) and the essential glutamic acid residue (402). The main differences between the catalytic domains of various MMPs occur in the S1' subsite or selectivity pocket. The S1' specificity site in MMP9 is perhaps best described as a tunnel leading toward solvent, as in MMP2 and MMP13, as opposed to the smaller pocket found in fibroblast collagenase and matrilysin. The present structure enables us to aid the design of potent and specific inhibitors for this important cardiovascular disease target.