ATM inhibitors, such as 7, have demonstrated the antitumor potential of ATM inhibition when combined with DNA double-strand break-inducing agents in mouse xenograft models. However, the properties of ...7 result in a relatively high predicted clinically efficacious dose. In an attempt to minimize attrition during clinical development, we sought to identify ATM inhibitors with a low predicted clinical dose (<50 mg) and focused on strategies to increase both ATM potency and predicted human pharmacokinetic half-life (predominantly through the increase of volume of distribution). These efforts resulted in the discovery of 64 (AZD0156), an exceptionally potent and selective inhibitor of ATM based on an imidazo4,5-cquinolin-2-one core. 64 has good preclinical phamacokinetics, a low predicted clinical dose, and a high maximum absorbable dose. 64 has been shown to potentiate the efficacy of the approved drugs irinotecan and olaparib in disease relevant mouse models and is currently undergoing clinical evaluation with these agents.
A novel series of small-molecule inhibitors has been developed to target the double mutant form of the epidermal growth factor receptor (EGFR) tyrosine kinase, which is resistant to treatment with ...gefitinib and erlotinib. Our reported compounds also show selectivity over wild-type EGFR. Guided by molecular modeling, this series was evolved to target a cysteine residue in the ATP binding site via covalent bond formation and demonstrates high levels of activity in cellular models of the double mutant form of EGFR. In addition, these compounds show significant activity against the activating mutations, which gefitinib and erlotinib target and inhibition of which gives rise to their observed clinical efficacy. A glutathione (GSH)-based assay was used to measure thiol reactivity toward the electrophilic functionality of the inhibitor series, enabling both the identification of a suitable reactivity window for their potency and the development of a reactivity quantitative structure-property relationship (QSPR) to support design.
•Brain malignancies have a poor prognosis and are a significant unmet need.•The blood brain barrier excludes most xenobiotics from the brain, limiting exposure.•Osimertinib, AZD3759 and AZD1390 cross ...the BBB.•Complementary preclinical assays enable brain penetrant candidates to be optimised.
Due to the blood–brain barrier (BBB) limiting the exposure of therapeutics to the central nervous system (CNS), patients with brain malignancies are challenging to treat, typically have poor prognoses, and represent a significant unmet medical need. Preclinical data report osimertinib to have significant BBB penetration and emerging clinical data demonstrate encouraging activity against CNS malignancies. Here, we discuss the oncology drug candidates AZD3759 and AZD1390 as case examples of discovery projects designing in BBB penetrance. We demonstrate how these innovative kinase inhibitors were recognized as brain penetrant and outline our view of experimental approaches and strategies that can facilitate the discovery of new brain-penetrant therapies for the treatment of primary and secondary CNS malignancies as well as other CNS disorders.
The brain is a major sanctuary site for metastatic cancer cells that evade systemic therapies. Through pre-clinical pharmacological, biological, and molecular studies, we characterize the functional ...link between drug resistance and central nervous system (CNS) relapse in Epidermal Growth Factor Receptor- (EGFR-) mutant non-small cell lung cancer, which can progress in the brain when treated with the CNS-penetrant EGFR inhibitor osimertinib. Despite widespread osimertinib distribution in vivo, the brain microvascular tumor microenvironment (TME) is associated with the persistence of malignant cell sub-populations, which are poised to proliferate in the brain as osimertinib-resistant lesions over time. Cellular and molecular features of this poised state are regulated through a Ras homolog family member A (RhoA) and Serum Responsive Factor (SRF) gene expression program. RhoA potentiates the outgrowth of disseminated tumor cells on osimertinib treatment, preferentially in response to extracellular laminin and in the brain. Thus, we identify pre-existing and adaptive features of metastatic and drug-resistant cancer cells, which are enhanced by RhoA/SRF signaling and the brain TME during the evolution of osimertinib-resistant disease.
By identifying every pair of molecules that differ only by a particular, well-defined, structural transformation in a database of measured properties and computing the corresponding change in ...property, we obtain an overview of the effect that structural change has upon the property and set an expectation for what will happen when that transformation is applied elsewhere. The mean change indicates the expected magnitude of the change in the property and the number of cases in which the property increases give the probability that the structural transformation will cause the property to increase. Outliers indicate potential ways of avoiding the general trend. Comparing to changes in lipophilicity highlights structural transformations that have unusual effects, some of which can be explained by conformational changes. In this paper, we focus upon the effects on aqueous solubility, plasma protein binding and oral exposure of adding substituents to aromatic rings and methylating heteroatoms.
DMPK data and knowledge are critical in maximising the probability of developing successful drugs via the application of in silico, in vitro and in vivo approaches in drug discovery.
The evaluation, ...optimisation and prediction of human pharmacokinetics is now a mainstay within drug discovery. These elements are at the heart of the 'right tissue' component of AstraZeneca's '5Rs framework' which, since its adoption, has resulted in increased success of Phase III clinical trials. With the plethora of DMPK related assays and models available, there is a need to continually refine and improve the effectiveness and efficiency of approaches best to facilitate the progression of quality compounds for human clinical testing.
This article builds on previously published strategies from our laboratories, highlighting recent discoveries and successes, that brings our AstraZeneca Oncology DMPK strategy up to date. We review the core aspects of DMPK in Oncology drug discovery and highlight data recently generated in our laboratories that have influenced our screening cascade and experimental design. We present data and our experiences of employing cassette animal PK, as well as re-evaluating in vitro assay design for metabolic stability assessments and expanding our use of freshly excised animal and human tissue to best inform first time in human dosing and dose escalation studies.
Application of our updated drug-drug interaction and central nervous system drug exposure strategies are exemplified, as is the impact of physiologically based pharmacokinetic and pharmacokinetic-pharmacodynamic modelling for human predictions.
Biophysical methods have become established in many areas of drug discovery. Application of these methods was once restricted to a relatively small number of scientists using specialized, low ...throughput technologies and methods. Now, automated high-throughput instruments are to be found in a growing number of laboratories. Many biophysical methods are capable of measuring the equilibrium binding constants between pairs of molecules crucial for molecular recognition processes, encompassing protein-protein, protein-small molecule, and protein-nucleic acid interactions, and several can be used to measure the kinetic or thermodynamic components controlling these biological processes. For a full characterization of a binding process, determinations of stoichiometry, binding mode, and any conformational changes associated with such interactions are also required. The suite of biophysical methods that are now available represents a powerful toolbox of techniques which can effectively deliver this full characterization.The aim of this chapter is to provide the reader with an overview of the drug discovery process and how biophysical methods, such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), nuclear magnetic resonance, mass spectrometry (MS), and thermal unfolding methods can answer specific questions in order to influence project progression and outcomes. The selection of these examples is based upon the experiences of the authors at AstraZeneca, and relevant approaches are highlighted where they have utility in a particular drug discovery scenario.
The displacement of probes that bind selectively to subdomains IIA or IIIA on human serum albumin (HSA) by competing compounds has been followed using fluorescence spectroscopy, and has therefore ...been used to assign a primary binding site for these compounds in the presence and absence of fatty acids. The crystal structures have also been solved for three compounds: a matched pair of carboxylic acids whose binding strength to HSA unexpectedly decreased as the lipophilicity increased; and a highly bound sulphonamide that appeared not to displace the probes in the displacement assay. The crystallography results support the findings from the fluorescence displacement assay. The results indicate that drug binding to subdomain IB might also be important location for certain compounds.
Targeted covalent inhibition is an established approach for increasing the potency and selectivity of potential drug candidates, as well as identifying potent and selective tool compounds for target ...validation studies. It is evident that identification of reversible recognition elements is essential for selective covalent inhibition, but this must also be achieved with the appropriate level of inherent reactivity of the reactive functionality (or “warhead”). Structural changes that increase or decrease warhead reactivity, guided by methods to predict the effect of those changes, have the potential to tune warhead reactivity and negate issues related to potency and/or toxicity. The half-life to adduct formation with glutathione (GSH t 1/2) is a useful assay for measuring the reactivity of cysteine-targeting covalent warheads but is limited to synthesized molecules. In this manuscript we assess the ability of several experimental and computational approaches to predict GSH t 1/2 for a range of cysteine targeting warheads, including a novel method based on pK a. Furthermore, matched molecular pairs analysis has been performed against our internal compound collection, revealing structure–activity relationships between a selection of different covalent warheads. These observations and methods of prediction will be valuable in the design of new covalent inhibitors with desired levels of reactivity.
Osimertinib is a potent and selective EGFR tyrosine kinase inhibitor (EGFR-TKI) of both sensitizing and T790M resistance mutations. To treat metastatic brain disease, blood-brain barrier (BBB) ...permeability is considered desirable for increasing clinical efficacy.
We examined the level of brain penetration for 16 irreversible and reversible EGFR-TKIs using multiple
and
BBB preclinical models.
osimertinib was the weakest substrate for human BBB efflux transporters (efflux ratio 3.2).
rat free brain to free plasma ratios (Kpuu) show osimertinib has the most BBB penetrance (0.21), compared with the other TKIs (Kpuu ≤ 0.12). PET imaging in Cynomolgus macaques demonstrated osimertinib was the only TKI among those tested to achieve significant brain penetrance (
%ID 1.5, brain/blood Kp 2.6). Desorption electrospray ionization mass spectroscopy images of brains from mouse PC9 macrometastases models showed osimertinib readily distributes across both healthy brain and tumor tissue. Comparison of osimertinib with the poorly BBB penetrant afatinib in a mouse PC9 model of subclinical brain metastases showed only osimertinib has a significant effect on rate of brain tumor growth.
These preclinical studies indicate that osimertinib can achieve significant exposure in the brain compared with the other EGFR-TKIs tested and supports the ongoing clinical evaluation of osimertinib for the treatment of EGFR-mutant brain metastasis. This work also demonstrates the link between low
transporter efflux ratios and increased brain penetrance
supporting the use of
transporter assays as an early screen in drug discovery.
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