A microfluidic system that replicates pharmacokinetic (PK) profiles in vitro improves prediction of in vivo efficacy in preclinical models.

Test compounds used on in vitro model systems are conventionally delivered to cell culture wells as fixed concentration bolus doses; however, this poorly replicates the pharmacokinetic (PK) concentration changes seen in vivo and reduces the predictive value of the data. Herein, proof-of-concept experiments were performed using a novel microfluidic device, the Microformulator, which allows in vivo like PK profiles to be applied to cells cultured in microtiter plates and facilitates the investigation of the impact of PK on biological responses. We demonstrate the utility of the device in its ability to reproduce in vivo PK profiles of different oncology compounds over multiweek experiments, both as monotherapy and drug combinations, comparing the effects on tumour cell efficacy in vitro with efficacy seen in in vivo xenograft models. In the first example, an ERK1/2 inhibitor was tested using fixed bolus dosing and Microformulator-replicated PK profiles, in 2 cell lines with different in vivo sensitivities. The Microformulator-replicated PK profiles were able to discriminate between cell line sensitivities, unlike the conventional fixed bolus dosing. In a second study, murine in vivo PK profiles of multiple Poly(ADP-Ribose) Polymerase 1/2 (PARP) and DNA-dependent protein kinase (DNA-PK) inhibitor combinations were replicated in a FaDu cell line resulting in a reduction in cell growth in vitro with similar rank ordering to the in vivo xenograft model. Additional PK/efficacy insight into theoretical changes to drug exposure profiles was gained by using the Microformulator to expose FaDu cells to the DNA-PK inhibitor for different target coverage levels and periods of time. We demonstrate that the Microformulator enables incorporating PK exposures into cellular assays to improve in vitro-in vivo translation understanding for early therapeutic insight.

Challenges and Opportunities in Cancer Drug Resistance.

There has been huge progress in the discovery of targeted cancer therapies in recent years. However, even for the most successful and impactful cancer drugs which have been approved, both innate and acquired mechanisms of resistance are commonplace. These emerging mechanisms of resistance have been studied intensively, which has enabled drug discovery scientists to learn how it may be possible to overcome such resistance in subsequent generations of treatments. In some cases, novel drug candidates have been able to supersede previously approved agents; in other cases they have been used sequentially or in combinations with existing treatments. This review summarizes the current field in terms of the challenges and opportunities that cancer resistance presents to drug discovery scientists, with a focus on small molecule therapeutics. As part of this review, common themes and approaches have been identified which have been utilized to successfully target emerging mechanisms of resistance. This includes the increase in target potency and selectivity, alternative chemical scaffolds, change of mechanism of action (covalents, PROTACs), increases in blood-brain barrier permeability (BBBP), and the targeting of allosteric pockets. Finally, wider approaches are covered such as monoclonal antibodies (mAbs), bispecific antibodies, antibody drug conjugates (ADCs), and combination therapies.

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.

Development of isotope-enriched phosphatidylinositol-4- and 5-phosphate cellular mass spectrometry probes.

Synthetic phosphatidylinositol phosphate (PtdInsP n ) derivatives play a pivotal role in broadening our understanding of PtdInsP n metabolism. However, the development of such tools is reliant on efficient enantioselective and regioselective synthetic strategies. Here we report the development of a divergent synthetic route applicable to the synthesis of deuterated PtdIns4P and PtdIns5P derivatives. The synthetic strategy developed involves a key enzymatic desymmetrisation step using Lipozyme TL-IM®. In addition, we optimised the large-scale synthesis of deuterated myo-inositol, allowing for the preparation of a series of saturated and unsaturated deuterated PtdIns4P and PtdIns5P derivatives. Experiments in MCF7 cells demonstrated that these deuterated probes enable quantification of the corresponding endogenous phospholipids in a cellular setting. Overall, these deuterated probes will be powerful tools to help improve our understanding of the role played by PtdInsP n in physiology and disease.

AZD0364 Is a Potent and Selective ERK1/2 Inhibitor That Enhances Antitumor Activity in KRAS-Mutant Tumor Models when Combined with the MEK Inhibitor, Selumetinib.

The RAS-regulated RAF-MEK1/2-ERK1/2 (RAS/MAPK) signaling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in BRAF or RAS genes. The clinical benefit of inhibitors of this pathway as single agents has only been realized in BRAF-mutant melanoma, with limited effect of single-agent pathway inhibitors in KRAS-mutant tumors. Combined inhibition of multiple nodes within this pathway, such as MEK1/2 and ERK1/2, may be necessary to effectively suppress pathway signaling in KRAS-mutant tumors and achieve meaningful clinical benefit. Here, we report the discovery and characterization of AZD0364, a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and kinase selectivity. In vitro, AZD0364 treatment resulted in inhibition of proximal and distal biomarkers and reduced proliferation in sensitive BRAF-mutant and KRAS-mutant cell lines. In multiple in vivo xenograft models, AZD0364 showed dose- and time-dependent modulation of ERK1/2-dependent signaling biomarkers resulting in tumor regression in sensitive BRAF- and KRAS-mutant xenografts. We demonstrate that AZD0364 in combination with the MEK1/2 inhibitor, selumetinib (AZD6244 and ARRY142886), enhances efficacy in KRAS-mutant preclinical models that are moderately sensitive or resistant to MEK1/2 inhibition. This combination results in deeper and more durable suppression of the RAS/MAPK signaling pathway that is not achievable with single-agent treatment. The AZD0364 and selumetinib combination also results in significant tumor regressions in multiple KRAS-mutant xenograft models. The combination of ERK1/2 and MEK1/2 inhibition thereby represents a viable clinical approach to target KRAS-mutant tumors.

Targeting melanoma's MCL1 bias unleashes the apoptotic potential of BRAF and ERK1/2 pathway inhibitors.

BRAF and MEK1/2 inhibitors are effective in melanoma but resistance inevitably develops. Despite increasing the abundance of pro-apoptotic BIM and BMF, ERK1/2 pathway inhibition is predominantly cytostatic, reflecting residual pro-survival BCL2 family activity. Here, we show that uniquely low BCL-XL expression in melanoma biases the pro-survival pool towards MCL1. Consequently, BRAF or MEK1/2 inhibitors are synthetic lethal with the MCL1 inhibitor AZD5991, driving profound tumour cell death that requires BAK/BAX, BIM and BMF, and inhibiting tumour growth in vivo. Combination of ERK1/2 pathway inhibitors with BCL2/BCL-w/BCL-XL inhibitors is stronger in CRC, correlating with a low MCL1:BCL-XL ratio; indeed the MCL1:BCL-XL ratio is predictive of ERK1/2 pathway inhibitor synergy with MCL1 or BCL2/BCL-w/BCL-XL inhibitors. Finally, AZD5991 delays acquired BRAFi/MEKi resistance and enhances the efficacy of an ERK1/2 inhibitor in a model of acquired BRAFi + MEKi resistance. Thus combining ERK1/2 pathway inhibitors with MCL1 antagonists in melanoma could improve therapeutic index and patient outcomes.

Discovery of a Potent and Selective Oral Inhibitor of ERK1/2 (AZD0364) That Is Efficacious in Both Monotherapy and Combination Therapy in Models of Nonsmall Cell Lung Cancer (NSCLC).

The RAS/MAPK pathway is a major driver of oncogenesis and is dysregulated in approximately 30% of human cancers, primarily by mutations in the BRAF or RAS genes. The extracellular-signal-regulated kinases (ERK1 and ERK2) serve as central nodes within this pathway. The feasibility of targeting the RAS/MAPK pathway has been demonstrated by the clinical responses observed through the use of BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma; however, resistance frequently develops. Importantly, ERK1/2 inhibition may have clinical utility in overcoming acquired resistance to RAF and MEK inhibitors, where RAS/MAPK pathway reactivation has occurred, such as relapsed BRAF V600E/K melanoma. We describe our structure-based design approach leading to the discovery of AZD0364, a potent and selective inhibitor of ERK1 and ERK2. AZD0364 exhibits high cellular potency (IC50 = 6 nM) as well as excellent physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties and has demonstrated encouraging antitumor activity in preclinical models.

Structure-Guided Discovery of Potent and Selective Inhibitors of ERK1/2 from a Modestly Active and Promiscuous Chemical Start Point.

There are a number of small-molecule inhibitors targeting the RAS/RAF/MEK/ERK signaling pathway that have either been approved or are in clinical development for oncology across a range of disease indications. The inhibition of ERK1/2 is of significant current interest, as cell lines with acquired resistance to BRAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition in preclinical models. This article reports on our recent work to identify novel, potent, and selective reversible ERK1/2 inhibitors from a low-molecular-weight, modestly active, and highly promiscuous chemical start point, compound 4. To guide and inform the evolution of this series, inhibitor binding mode information from X-ray crystal structures was critical in the rapid exploration of this template to compound 35, which was active when tested in in vivo antitumor efficacy experiments.

Structure-Guided Design of Highly Selective and Potent Covalent Inhibitors of ERK1/2.

The RAS/RAF/MEK/ERK signaling pathway has been targeted with a number of small molecule inhibitors in oncology clinical development across multiple disease indications. Importantly, cell lines with acquired resistance to B-RAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition by small molecule inhibitors. There are a number of selective, noncovalent ERK1/2 inhibitors reported along with the promiscuous hypothemycin (and related analogues) that act via a covalent mechanism of action. This article reports the identification of multiple series of highly selective covalent ERK1/2 inhibitors informed by structure-based drug design (SBDD). As a starting point for these covalent inhibitors, reported ERK1/2 inhibitors and a chemical series identified via high-throughput screening were exploited. These approaches resulted in the identification of selective covalent tool compounds for potential in vitro and in vivo studies to assess the risks and or benefits of targeting this pathway through such a mechanism of action.

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.