Abstract Introduction: Protein kinases are a diverse group of 518 enzymes whose dysregulation lies at the center of many diseases. Currently, 30% of all drug development efforts are focused on protein kinases. Although 41 drugs are approved and >120 in clinical trials, these are predominately ATP-competitive inhibitors. More recently, there has been an expanded focus on kinase inhibitors with different modes of action, where new tools are needed to effectively characterize inhibitor mechanism of action, predict drug potency and to drive decisions earlier in the drug development process. We developed a simple yet powerful method for the generation of peptide sensors that can be used for the continuous, quantitative and homogenous detection of kinase and phosphatase activity with recombinant enzymes and crude lysates to enable target discovery and drug development. Experimental Procedures: We harnessed chelation-enhanced fluorescence by combining next generation sulfonamido-oxine (Sox) chromophore technology with high-throughput solid-phase peptide synthesis methods to identify optimized sequences based on physiological substrates. Enzyme activity is monitored kinetically using fluorescence intensity (Ex/Em 360/485 nm) or in endpoint mode using Europium and time-resolved fluorescence (Ex/Em 360/620 nm). Results: We demonstrate the ability to rapidly identify novel optimized substrates, where performance measures included higher reaction rates, lower Km's, higher signal/background, increased sensitivity and specificity. We identified highly generic substrates (for robust detection of 80 Tyrosine kinases) and highly-selective substrates (for quantitative detection of targeted kinases in crude cell or tissue lysates for profiling, potency assessments and SAR). We have developed sensors to monitor activity of high-profile tyrosine kinases, including the EGFR and clinically-relevant mutants, JAK kinases, Tec-kinases, and, serine/threonine kinases, including CDK1-9, MAPK pathway (MAP4Ks, MAP3Ks, MAPKs & MAPKAPKs), PKR/EIF2AKs and PIM1. In addition, CSox-based phosphopeptide substrates are used to monitor protein phosphatases with specificity for tyrosine (PTP1B, SHP1/2) or serine/threonine (PP2A, PP2C, PHLPP). Conclusions: The generation of robust activity-based sensors, even where peptide assays previously weren’t available, opens new areas for effective drug discovery. The Sox-based kinetic assay format is ideal for elucidating drug mechanism of action, potency, and enzyme regulation. The PhosphoSens-Red endpoint format is ideal for HTS, SAR and profiling. Together, these formats can be applied across the entire target discovery and drug development workflow, providing a quantum improvement in performance and productivity needed to address the challenges and opportunities of next generation protein kinase and phosphatase inhibitors. Citation Format: Erik M. Schaefer, Susan Cornell-Kennon, Bill Lu. CSox-based sensors for continuous, homogeneous and quantitative monitoring of protein kinase and phosphatase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1769.
Expression under the control of the mouse transferrin promoter of a transgene encoding a soluble secreted derivative of the ectodomain of the human insulin receptor in transgenic mice results in the accumulation of this high-affinity insulin-binding protein in the plasma. Alterations of glucose homeostasis are observed including postabsorptive hyperglycemia concomitant with increased hepatic glucose production and hyperinsulinemia. Thus, this is the first transgenic animal model of chronic hyperglycemia with alterations in glucose homeostasis that are produced without a targeted alteration of pancreatic function. These mice provide a new experimental model to follow the progression and long-term consequences of chronic hyperglycemia.
Abstract We studied the binding of 125 I‐labeled diphtheria toxin (DTX) to receptors on monolayer cultures of Chinese hamster ovary cells (CHO‐K1) and Vero cells. The number of DTX receptors detected on the cell surface was shown to be dependent on the cell density (number of cells per unit area). Cells at low density (≤23,000 cells per cm 2 for CHO‐K1 cells; ≤80,000 cells per cm 2 for Vero cells) had more receptors for DTX than cells at higher densities. The difference in receptor number between low‐ and high‐density cells was 33‐fold for CHO‐K1 cells and 19‐fold for Vero cells. We estimated the maximum number of DTX receptors on low‐density CHO‐K1 and Vero cells to be 50,000 and 370,000 per cell, respectively. The cell density at which the binding of DTX was reduced to 50% of maximum was considerably lower for CHO‐K1 cells than for Vero cells (33,000 vs. 220,000 cells per cm 2 , respectively). Vero cells grown on a surface that had been conditioned by high‐density cells bound less DTX, suggesting that interaction of these cells with the underlying extracellular matrix might regulate the number of cell surface receptors for DTX. Low‐density cells were more sensitive to DTX than high‐density cells, suggesting that low‐density cells possessed an increased number of functional receptors that actively transported DTX to the cytosol. CHO‐K1 and Vero cells were equally protected by SITS (4‐Acetamido‐4prime;‐Isothiocyano‐Stilbene‐2,2′‐disulfonic Acid), a compound that has been shown to inhibit the binding and entry of DTX in Vero cells, suggesting that intoxication of CHO‐K1 and Vero cells is mediated by a similar mechanism. The data illustrate the importance of taking into account the cell density when measuring the number of DTX receptors on adherent cells.
Abstract Bivalent molecules consisting of groups connected through bridging linkers often exhibit strong target binding and unique biological effects. However, developing bivalent inhibitors with the desired activity is challenging due to the dual motif architecture of these molecules and the variability that can be introduced through differing linker structures and geometries. We report a set of alternatively linked bivalent EGFR inhibitors that simultaneously occupy the ATP substrate and allosteric pockets. Crystal structures show that initial and redesigned linkers bridging a trisubstituted imidazole ATP-site inhibitor and dibenzodiazepinone allosteric-site inhibitor proved successful in spanning these sites. The re-engineered linker yielded a compound that exhibited significantly higher potency (~60 pM) against the drug-resistant EGFR L858R/T790M and L858R/T790M/C797S, which was superadditive as compared with the parent molecules. The enhanced potency is attributed to factors stemming from the linker connection to the allosteric-site group and informs strategies to engineer linkers in bivalent agent design.
Abstract Time-dependent inhibitors (TDIs) of enzyme targets offer distinct advantages for the development of potent and selective compounds with favorable pharmacokinetic and pharmacodynamic properties. Such inhibitors are characterized by non-linear progress curves: after an initial inhibited velocity, a rate constant governs the transition to a final steady-state reaction rate of the inhibited enzyme. A final rate of zero indicates irreversible inhibition, whereas a non-zero final rate indicates slow-binding inhibition. Characterizing these inhibitory modes of action is enabled with a continuous assay format that avoids the common pitfalls and misleading results seen with end-point assays. A continuous assay format enables efficient and robust determination of the kinetic parameters required to drive structure-activity relationship optimization to streamline the development of more effective drugs. It is important to note that simple IC50s for TDIs will not suffice, and can, indeed, also be misleading. We have developed a robust three-step workflow based on kinetic catalytic activity measurements to quickly identify and characterize TDIs. First, dose-response experiments are conducted with and without an enzyme-inhibitor preincubation step. The curvature of the reaction progress curve in the non-preincubated experiment and a shift in IC50 from the preincubated experiment are indicative of TDI. In the absence of TDI, simple IC50s are reported with, if possible, Ki values. If TDI is present, a second experiment is conducted to assess compound reversibility using either a jump-dilution protocol or a novel free-compound clearance method that uses gel filtration spin columns or spin plates. In either protocol, forward progress curve analysis is used to monitor the recovery of enzymatic activity after dilution of inhibitor in solution. Lastly, the potency of the inhibitor is evaluated using kinetic experiments tailored to the nature of the inhibition – either reversible or irreversible. If reversible, then the rate constant from the reversibility experiment is used to determine the residence time of the molecule. If irreversible, then a 24-point dose-response experiment with serial 1.5-fold dilutions is performed, and all the progress curves are globally fit to determine kinact/KI, and, if possible, kinact and KI separately. The method will be fully described through the characterization of known EGFR inhibitors of three inhibition types: fast-off (Gefitinib), slow binding (Lapatinib), and irreversible (Osimertinib). Citation Format: Earl May, Daniel Urul, Khanh Huynh, Susan Cornell-Kennon, Venkatesh Nemmara, Zhibing Lu, Samuel Hoare, Michelle Lyles, Erik Schaefer. A proven activity-based workflow for the identification and characterization of time-dependent kinase inhibitors using a continuous assay format [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2061.
Abstract Purpose: c-MET is believed to be an attractive receptor target for molecular therapeutic inhibition. TPR-MET, a constitutively active oncogenic variant of MET, serves as excellent model for testing c-MET inhibitors. Here, we characterized a small molecule c-MET inhibitor, PHA665752, and tested its cooperation with the mammalian target of rapamycin inhibitor as potential targeted therapy. Experimental Design: The effect of PHA665752 treatment was determined on cell growth, motility and migration, apoptosis, and cell-cycle arrest of TPR-MET-transformed cells. Moreover, the effect of PHA665752 on the phosphorylation on MET, as well as its downstream effectors, p-AKT and p-S6K, was also determined. Finally, growth of TPR-MET-transformed cells was tested in the presence of PHA665752 and rapamycin. H441 non–small cell lung cancer (NSCLC) cells (with activated c-Met) were also tested against both PHA665752 and rapamycin. Results: PHA665752 specifically inhibited cell growth in BaF3. TPR-MET cells (IC50 < 0.06 μmol/L), induced apoptosis and cell cycle arrest. Constitutive cell motility and migration of the BaF3. TPR-MET cells was also inhibited. PHA665752 inhibited specific phosphorylation of TPR-MET as well as phosphorylation of downstream targets of the mammalian target of rapamycin pathway. When combined with PHA665752, rapamycin showed cooperative inhibition to reduce growth of BaF3. TPR-MET- and c-MET-expressing H441 NSCLC cells. Conclusions: PHA665752 is a potent small molecule–selective c-MET inhibitor and is highly active against TPR-MET-transformed cells both biologically and biochemically. PHA665752 is also active against H441 NSCLC cells. The c-MET inhibitor can cooperate with rapamycin in therapeutic inhibition of NSCLC, and in vivo studies of this combination against c-MET expressing cancers would be merited.
