Quantifying KRAS G12C Covalent Drug Inhibitor Activity in Mouse Tumors Using Mass Spectrometry.

The growing opportunities recognized for covalent drug inhibitors, like KRAS G12C inhibitors, are driving the need for mass spectrometry methods that can quickly and robustly measure therapeutic drug activity in vivo for drug discovery research and development. Effective front-end sample preparation is critical for proteins extracted from tumors but is generally labor intensive and impractical for large sample numbers typical in pharmacodynamic (PD) studies. Herein, we describe an automated and integrated sample preparation method for the measurement of activity levels of KRAS G12C drug inhibitor alkylation from complex tumor samples involving high throughput detergent removal and preconcentration followed by quantitation using mass spectrometry. We introduce a robust assay with an average intra-assay coefficient of variation (CV) of 4% and an interassay CV of 6% obtained from seven studies, enabling us to understand the relationship between KRAS G12C target occupancy and the therapeutic PD effect from mouse tumor samples. Further, the data demonstrated that the drug candidate GDC-6036, a KRAS G12C covalent inhibitor, shows dose-dependent target inhibition (KRAS G12C alkylation) and MAPK pathway inhibition, which correlate with high antitumor potency in the MIA PaCa-2 pancreatic xenograft model.

Discovery of a putative blood-based protein signature associated with response to ALK tyrosine kinase inhibition.

BACKGROUND: ALK tyrosine kinase inhibition has become a mainstay in the clinical management of ALK fusion positive NSCLC patients. Although ALK mutations can reliably predict the likelihood of response to ALK tyrosine kinase inhibitors (TKIs) such as crizotinib, they cannot reliably predict response duration or intrinsic/extrinsic therapeutic resistance. To further refine the application of personalized medicine in this indication, this study aimed to identify prognostic proteomic biomarkers in ALK fusion positive NSCLC patients to crizotinib. METHODS: Twenty-four patients with advanced NSCLC harboring ALK fusion were administered crizotinib in a phase IV trial which included blood sampling prior to treatment. Targeted proteomics of 327 proteins using MRM-MS was used to measure plasma levels at baseline (including pre-treatment and early treatment blood samples) and assess potential clinical association. RESULTS: Patients were categorized by duration of response: long-term responders [PFS ≥ 24 months (n = 7)], normal responders [3 < PFS < 24 months (n = 10)] and poor responders [PFS ≤ 3 months (n = 5)]. Several proteins were identified as differentially expressed between long-term responders and poor responders, including DPP4, KIT and LUM. Next, using machine learning algorithms, we evaluated the classification potential of 40 proteins. Finally, by integrating the different analytic methods, we selected 22 proteins as potential candidates for a blood-based prognostic signature of response to crizotinib in NSCLC patients harboring ALK fusion. CONCLUSION: In conjunction with ALK mutation, the expression of this proteomic signature may represent a liquid biopsy-based marker of long-term response to crizotinib in NSCLC. Expanding the utility of prognostic biomarkers of response duration could influence choice of therapy, therapeutic sequencing, and potentially the need for alternative or combination therapy.Trial registration ClinicalTrials.gov, NCT02041468. Registered 22 January 2014, https://clinicaltrials.gov/ct2/show/NCT02041468?term=NCT02041468&rank=1.

Soluble amyloid precursor protein α and ß in CSF in Alzheimer's disease.

OBJECTIVE: Cerebral accumulation of amyloid ß (Aß) is a pathological hallmark of Alzheimer's disease (AD). Proteolytic processing of amyloid precursor protein (APP) by α- or ß-secretase results in two soluble metabolites, sAPPα and sAPPß, respectively. However, previous data have shown that both α- and ß-secretase have multiple cleavage sites. The aim of this study was to characterize the C-termini of sAPPα and sAPPß in cerebrospinal fluid (CSF) by mass spectrometry (MS) and to evaluate whether different combinations of these fragments better separate between AD patients and controls by comparing two different sAPP immunoassays. METHODS: Using immunoprecipitation and high resolution MS, the APP species present in CSF were investigated. CSF levels of sAPPα and sAPPß from patients with AD (n=43) and from non-demented controls (n=44) were measured using AlphaLISA and MSD immunoassays that employ different antibodies for C-terminal recognition of sAPPα. RESULTS: Four different C-terminal forms of sAPP were identified, sAPPß-M671, sAPPß-Y681, sAPPα-Q686, and sAPPα-K687 (APP770 numbering). Neither immunoassay for the sAPP species could separate the two patient groups. The correlation (R(2)) between the two immunoassays was 0.41 for sAPPα and 0.45 for sAPPß. CONCLUSION: Using high resolution MS, we show here for the first time that sAPPα in CSF ends at Q686 and K687. The findings also support the conclusion from several previous studies that sAPPα and sAPPß levels are unaltered in AD.

Aß1-15/16 as a potential diagnostic marker in neurodegenerative diseases.

Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) reflect brain biochemistry. Using combined immunoprecipitation and mass spectrometry, we have shown that amyloid beta 1-15 (Aß1-15) is produced by concerted ß- and α-secretase cleavage of amyloid precursor protein (APP) and that the relative levels of Aß1-16 in AD compared to controls are increased. Furthermore, drug-induced γ-secretase inhibition enhances the relative levels of Aß1-15 and Aß1-16. Here, we investigate a novel immunoassay for Aß1-15/16 in a broad range of neurodegenerative conditions. The CSF level of Aß1-15/16 was measured by the bead-based amplified luminescent proximity homogeneous assay (Alpha technology). Concentrations of Aß1-15/16 were analyzed in subjects with Parkinson disease (PD; n = 90), PD with dementia (PDD) (n = 32), dementia with Lewy bodies (DLB) (n = 68), AD (n = 48), progressive supranuclear palsy (PSP) (n = 45), multiple system atrophy (MSA) (n = 46), and corticobasal degeneration (CBD) (n = 12). The detecting antibody is specific to the C-terminal epitope of Aß15. We found that a carboxypeptidase (CPB) present in fetal bovine serum (FBS), a component of the buffers used, degrades Aß1-16 to Aß1-15, which is then detected by the Aß1-15/16 assay. Significantly, lower levels of Aß1-15/16 were detected in PD, PDD, PSP, and MSA compared to other neurodegenerative diseases and controls. Using the specific Aß1-15/16 assay, a reliable quantification of Aß1-15 or Aß1-15/16 in CSF samples is obtained. We found reduced levels of Aß1-15 in parkinsonian disease groups. The molecular mechanism behind this reduction is at present unknown.

Integrated cell-based platform to study EGFR activation and transactivation.

The epidermal growth factor receptor (EGFR) pathway is one of the most deregulated molecular pathways in human epithelial cancers. Many approved drugs were optimized to directly target EGFR but yielded only modest clinical improvement in cancer patients due to low efficacy and drug resistance. Transactivation of EGFR by other cell surface receptors such as G-protein-coupled receptors (GPCRs) was proposed to explain this lack of efficacy. Even if direct EGFR activation and transactivation by GPCR contribute to the activation of the same signaling pathways, they are often studied as independent events resulting in partial investigation of a drug's mechanism of action. We present a novel high-throughput approach that integrates interrogation of direct activation of EGFR and its transactivation via GPCR activation. Using distinct technology platforms, three readouts were used to measure (1) direct activation of GPCR via cyclic adenosine monophosphate (cAMP) detection, (2) direct activation of EGFR through the release of intracellular Ca(2+), and (3) EGFR transactivation by GPCR using the detection of p-extracellular-signal-regulated kinases 1/2 (p-ERK1/2). In addition to being simple, quick, and homogenous, our methods were shown to be more sensitive than those in current use. These enabling tools should improve the knowledge pertaining to GPCRs and receptor tyrosine kinases trans-regulation and facilitate the design of more potent and better targeted new therapeutic strategies.

AlphaScreen-based characterization of the bifunctional kinase/RNase IRE1alpha: a novel and atypical drug target.

Assay technologies that were originally developed for high-throughput screening (HTS) have recently proven useful in drug discovery for activities located upstream (target identification and validation) and downstream (ADMET) of HTS. Here the authors investigated and characterized the biological properties of a novel target, IRE1alpha, a bifunctional kinase/RNase stress sensor of the endoplasmic reticulum (ER). They have developed a novel assay platform using the HTS technology AlphaScreen to monitor the dimerization/oligomerization and phosphorylation properties of the cytosolic domain of IRE1alpha. They show in vitro that dimerization/oligomerization of the cytosolic domain of IRE1 correlated with the autophosphorylation ability of this domain and its endoribonuclease activity toward XBP1 mRNA. Using orthogonal in vitro and cell-based approaches, the authors show that the results obtained using AlphaScreen were biologically relevant. Preliminary characterization of assay robustness indicates that both AlphaScreen assays should be useful in HTS for the identification of IRE1 activity modulators.

The BRET2/arrestin assay in stable recombinant cells: a platform to screen for compounds that interact with G protein-coupled receptors (GPCRS).

In BRET2 (Bioluminescence Resonance Energy Transfer), a Renilla luciferase (RLuc) is used as the donor protein, while a Green Fluorescent Protein (GFP2) is used as the acceptor protein. In the presence of the cell permeable substrate DeepBlueC, RLuc emits blue light at 395 nm. If the GFP2 is brought into close proximity to RLuc via a specific biomolecular interaction, the GFP2 will absorb the blue light energy and reemit green light at 510nm. BRET2 signals are therefore easily determined by measuring the ratio of green over blue light (510/395nm) using appropriate dual channel luminometry instruments (e.g., Fusion Universal Microplate Analyzer, Packard BioScience). Since no light source is required for BRET2 assays, the technology does not suffer from high fluorescent background or photobleaching, the common problems associated with standard FRET-based assays. Using BRET2, we developed a generic G Protein-Coupled Receptor (GPCR) assay based on the observation that activation of the majority of GPCRs by agonists leads to the interaction of beta-arrestin (a protein that is involved in receptor desensitization and sequestration) with the receptor. We established a cell line stably expressing the GFP2:beta-arrestin 2 fusion protein, and showed that it can be used to monitor the activation of various transiently expressed GPCRs, in BRET2/arrestin assays. In addition, using the HEK 293/GFP2:beta-arrestin 2 cell line as a recipient, we generated a double-stable line co-expressing the vasopressin 2 receptor (V2R) fused to RLuc (V2R:RLuc) and used it for the pharmacological characterization of compounds in BRET2/arrestin assays. This approach yields genuine pharmacology and supports the BRET2/arrestin assay as a tool that can be used with recombinant cell lines to characterize ligand-GPCR interactions which can be applied to ligand identification for orphan receptors.