Synthetic peptides that specifically bind nuclear hormone receptors offer an alternative approach to small molecules for the modulation of receptor signaling and subsequent gene expression. Here we describe the design of a series of novel stapled peptides that bind the coactivator peptide site of estrogen receptors. Using a number of biophysical techniques, including crystal structure analysis of receptor-stapled peptide complexes, we describe in detail the molecular interactions and demonstrate that all-hydrocarbon staples modulate molecular recognition events. The findings have implications for the design of stapled peptides in general.

Recent literature has claimed that inhibition of the enzyme MTH1 can eradicate cancer. MTH1 is one of the "housekeeping" enzymes that are responsible for hydrolyzing damaged nucleotides in cells and thus prevent them from being incorporated into DNA. We have developed orthogonal and chemically distinct tool compounds to those published in the literature to allow us to test the hypothesis that inhibition of MTH1 has wide applicability in the treatment of cancer. Here we present the work that led to the discovery of three structurally different series of MTH1 inhibitors with excellent potency, selectivity, and proven target engagement in cells. None of these compounds elicited the reported cellular phenotype, and additional siRNA and CRISPR experiments further support these observations. Critically, the difference between the responses of our highly selective inhibitors and published tool compounds suggests that the effect reported for the latter may be due to off-target cytotoxic effects. As a result, we conclude that the role of MTH1 in carcinogenesis and utility of its inhibition is yet to be established.

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) 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 for a range of cysteine targeting warheads, including a novel method based on pK. 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.

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 imidazo[4,5- c]quinolin-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.

No abstract