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 Introduction: The activity of protein kinases plays a critical role in the aberrant activation of oncogenic signaling pathways which can drive tumorigenesis and malignant transformation in cancer. AKT has been shown to be both upregulated and mutated in cancer cells allowing it to serve as a driver of cancer cell growth and progression. Inhibition of AKT activation and activity are both attractive targets for effective cancer drug discovery. Experimental Procedures: We developed continuous, homogeneous assays for unactive AKTs. A peptide substrate, modified with a sulfonamido-oxine fluorophore (Sox), utilizes chelation-enhanced fluorescence to enable a real-time readout of AKT-driven phosphorylation. First, a subset of 30,000 Sox-containing sequences were evaluated for AKT substrate candidates, selecting for assay robustness and specificity. A physiologically relevant peptide substrate was identified and used to develop a kinetic assay to monitor AKT1 activation and activity. Unactive AKT1 was incubated with DOPS/DOPC and PIP3, which mimics the plasma membrane, allowing the PH domain of AKT to bind, leading to a conformational change that enables full activation of AKT by PDK1 and MK2. Upon assay initiation, active AKT phosphorylates the sensor peptide, and the resulting signal is read in kinetic mode using a fluorescence intensity readout. First derivative plots for each progress curve were generated; the slope of the linear regions of each plot, representing acceleration towards a steady-state, were used to determine relative rates of activation for each AKT. Results: We developed a novel assay for AKT activation and substrate phosphorylation utilizing AQT0076, DOPS/DOPC, PIP3, PDK1, and MK2. With a mix of classical AKT inhibitors and allosteric inhibitors that rely on an inactive “PH-in” conformation, we demonstrated inhibition and quantified inhibitor potency of both active “PH-out” AKT activity and inactive “PH-in” AKT activation via dose-response measurements of steady-state rate and rate acceleration, respectively. Conclusions: A robust, homogeneous assay was developed to simultaneously monitor AKT activation and substrate phosphorylation kinetically over time. Through a continuous assay, we captured both steady-state rates and rate acceleration as a function of inhibitor concentration, allowing for accurate quantitation of both classes of AKT inhibitors in a single experimental format. With some inhibitors, we observed potencies that matched reported literature values, while with others we observed marked differences that may reflect a more physiological context that could translate through to higher efficacy in the clinic. This novel assay format provides a new tool that can be used in drug discovery to generate more effective next generation inhibitors of both AKT activation and subsequent substrate phosphorylation to prevent cancer cell growth and progression. Citation Format: Susan Cornell-Kennon, Daniel Urul, Matthew Hakar, Hayley McMahon, Khanh Huynh, Earl May, Erik Schaefer. A novel sox-based continuous and homogeneous assay for the discovery of inhibitors of inactive and active AKT [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 1666.

Abstract Protein kinases form a network of signaling pathways and are modified by multiple post-translational modifications, including phosphorylation, which regulate the kinase enzymatic activity, protein complexes formation, and its cellular location. Given this rich target complexity, >30% of all drug development is focused on kinases. However, these target only 15% of the human kinome and are primarily just one type (ATP-competitive) that can readily result in off-target inhibition and the development of resistance during cancer treatment. Most kinase inhibitor development is carried out in vitro assays with an epitope-tagged recombinant kinase. As a result, efficacy in patients can be limited due to poor inhibitor performance under physiological conditions (e.g., mM ATP and kinases being complexed with other proteins). In contrast, in vitro assays using unfractionated cell lysates provide a close approximation of the native kinase environment, including the complexity of interactions with other cellular components. Being able to selectively quantify the enzymatic activity of all protein kinases in crude cell or tissue homogenates would enable an improved understanding of kinase signaling biology, drug development, and personalized medicine. However, despite the large amount of literature on kinase assays, only a fraction is compatible with unfractionated lysates since most peptide substrates are not selective for a given kinase. To that end, we utilized a continuous assay format coupled with high-throughput Sox-sensor-peptide library synthesis using physiological sequences to identify selective sensor peptide substrates for multiple high-profile kinases implicated in cancer. As an example, we identified a highly selective sensor peptide for ERK1 and 2 mitogen-activated protein kinases (MAPKs) involved in pro-growth/oncogenic signaling. We demonstrated selectivity initially with the recombinant enzymes for ERK1/2 and a panel of other related MAPKs or CMGC kinases. Next, we extended the selectivity screen to 392 kinases using a large panel kinome profiling service. The screen also demonstrated high selectivity for ERK1/2 isoforms. We then tested our best sensor peptide substrate for ERK1/2 using unfractionated lysates from the HEK293T cell line with ERK2 overexpression and demonstrated high activity that could be increased 50-fold by the addition of recombinant MEK1 or 2, the upstream kinase that activates ERK1 and 2, and completely blocked with the addition of an ERK2-selective inhibitor. These results demonstrate a method to systematically generate a selective sensor peptide for ERK1/2 to continuously monitor this kinase activity in crude cell or tissue lysates. This poster will fully describe the methods and results pertaining to ERK-selective sensor peptide development and validation, and the ability to extend to other kinases of interest. Citation Format: Venkatesh Nemmara, Susan Cornell-Kennon, Zhibing Lu, Gabriela Pikul, Zacchary Belisle, Matthew Hakar, Jefferson Chin, Erik Schaefer, Earl May. A continuous kinetic assay to quantitate specific protein kinase activity in unfractionated cell lysates [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 1676.

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 reengineered 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.

To characterize modulators of enzymatic activity, a continuous assay format will quantify with confidence the initial reaction velocity far better than an end point assay. When the progress curve is linear from the start and remains linear throughout the experiment, a single end point reading can approximate the reaction rate, but a kinetic assay will provide the rate more accurately. However, if there is a delay in the onset of the reaction, or there are other changes in reaction rate over time, then a single end point reading is insufficient. Thus, important information about the reaction can be missed or mischaracterized. While visual inspection is the most common way to determine the range of the progress curve from which to extract rate information, this method is not practical when thousands of progress curves are generated in an experimental day. We have developed an automated protocol to streamline and optimize the process. The heuristic algorithm analyzes the progress curve to identify different regions for characterization (e.g. lag, primary rate, secondary rate, final plateau) and then extract rates of interest for each of these regions. These rates are subsequently used to measure the specific activity of the enzyme under various assay conditions, and to characterize the modulation of that activity: simple dose-response testing, mode of inhibition analysis, time-dependent inhibition determination, reversibility testing, determination of residence time for reversible inhibitors, and kinact/KI analysis for irreversible inhibitors. We describe the algorithm and several applications as they apply to protein kinases using the PhosphoSens assay platform from AssayQuant within the Analyze module of the Scigilian data analysis package. Citation Format: Earl William May, Daniel Urul, Susan Cornell-Kennon, Zhibing Lu, Erik Schaefer, Sam Hoare, France Laliberté, Quay Vong, Paul Payette, Jean Marois. Automation of linear range determination: Enzymatic progress curve applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2766.

Enzyme inhibitors that form covalent bonds with their targets are being increasingly pursued in drug development. Assessing their biochemical activity relies on time-dependent assays, which are distinct and more complex compared with methods commonly employed for reversible-binding inhibitors. To provide general guidance to the covalent inhibitor development community, we explored methods and reported kinetic values and experimental factors in determining the biochemical activity of various covalent epidermal growth factor receptor (EGFR) inhibitors. We showcase how liquid handling and assay reagents impact kinetic parameters and potency interpretations, which are critical for structure-kinetic relationships and covalent drug design. Additionally, we include benchmark kinetic values with reference inhibitors, which are imperative, as covalent EGFR inhibitor kinetic values are infrequently consistent in the literature. This overview seeks to inform best practices for developing new covalent inhibitors and highlight appropriate steps to address gaps in knowledge presently limiting assay reliability and reproducibility.

The optimization of linkers that connect fragments within drug binding sites represents an impediment in fragment-based drug discovery (FBDD). To improve our understand of the molecular factors that enable effective fragment linking, we have produced a series of compounds that bind to the ATP and allosteric sites of the EGFR kinase domain connected by two distinct linker structures. We find the linker is responsible for opposing impacts on potency against EGFR mutants, the most potent of which are active in human cancer cells. Comparison of X-ray cocrystal structures of active versus inactive molecules provide unique experimentally derived insights into linker design criteria such as how fragment flexibility and intermolecular interactions can serve compound design broadly in drug optimization.

Introduction: The aberrant activation of oncogenic signaling pathways can drive tumorigenesis and malignant transformation, where deregulation of the activity of protein kinases or phosphatases can play a critical role. Several protein tyrosine phosphatases (PTPs) that are overexpressed in human cancers have recently been shown to activate signaling pathways and promote tumor development and progression (Sivaganesh et al., 2021. Protein Tyrosine Phosphatases: Mechanisms in Cancer. Int J Mol Sci. 22(23):12865). This reinforces the need for protein phosphatase inhibitors for cancer therapy, which requires robust assays to monitor PTP activity. 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 known physiological substrates. PTP enzyme activity was monitored using the PhosphoSens® platform in kinetic mode (a progress curve in every well) using fluorescence intensity (Ex/Em 360/485 nm) or in endpoint mode with Europium and time-resolved fluorescence (Ex/Em 360/620 nm). Results: Using commercially available phosphatases, we demonstrated the ability to rapidly identify novel Sox-based phosphopeptide substrates for each phosphatase. Performance measures included initial reaction rates, Km's, signal/background, linear reaction kinetics, absence of initial lags, assay sensitivity and specificity. Final optimized assay conditions were established for 28 different phosphatases. Each phosphatase was titrated to determine the lowest optimal assay concentration, demonstrating linearity and detection of low nM enzyme. Potency assessments using a set of compounds revealed sub nM inhibitor IC50 values and this included using sodium orthovanadate as a positive control. Finally, we demonstrated the ability to profile a range of novel phosphatase inhibitors to determine selectivity. Conclusions: The generation of robust activity-based Sox-sensor phosphopeptide substrates opens new areas for effective drug discovery with protein phosphatases as an emerging target class. The PhosphoSens-Kinetic assay format is ideal for assessing enzyme regulation and elucidating drug mechanism of action, potency, and profiling. The PhosphoSens-Red endpoint format is ideal for HTS and high-throughput SAR. Together, these formats can be applied across the entire target discovery and drug development workflow, providing a dramatic improvement in performance and productivity needed to address the challenges and opportunities of next generation protein kinase and phosphatase inhibitors. Citation Format: Susan Cornell-Kennon, Erik Schaefer, Earl May, Matthew Hakar. Sox-based sensor phosphopeptides for continuous, homogeneous and quantitative monitoring of protein phosphatase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1426.

Lazertinib (YH25448) is a novel third-generation tyrosine kinase inhibitor (TKI) developed as a treatment for EGFR mutant non-small cell lung cancer. To better understand the nature of lazertinib inhibition, we determined crystal structures of lazertinib in complex with both WT and mutant EGFR and compared its binding mode to that of structurally related EGFR TKIs. We observe that lazertinib binds EGFR with a distinctive pyrazole moiety enabling hydrogen bonds and van der Waals interactions facilitated through hydrophilic amine and hydrophobic phenyl groups, respectively. Biochemical assays and cell studies confirm that lazertinib effectively targets EGFR(L858R/T790M) and to a lesser extent HER2. The molecular basis for lazertinib inhibition of EGFR reported here highlights previously unexplored binding interactions leading to improved medicinal chemistry properties compared to clinically approved osimertinib (AZD9291) and offers novel strategies for structure-guided design of tyrosine kinase inhibitors.

Integrins α4β1/ α9β1 are important in the pathogenesis and progression of inflammatory and autoimmune diseases by their roles in leukocyte activation and trafficking. Natalizumab, a monoclonal antibody selectively targeting α4β1 integrin and blocking leukocyte trafficking to the central nervous system, is an immunotherapy for multiple sclerosis (MS). However, due to its adverse effects associated with chronic treatment, alternative strategies using small peptide mimetic inhibitors are being sought. In the present study, we synthesized and characterized visabron c (4-4), a backbone cyclic octapeptide based on the sequence TMLD, a non-RGD unique α4β1 integrin recognition sequence motif derived from visabres, a proteinous disintegrin from the viper venom. Visabron c (4-4) was selected from a minilibrary with conformational diversity based on its potency and selectivity in functional adhesion cellular assays. Visabron c (4-4)'s serum stability, pharmacokinetics, and therapeutic effects following ip injection were assessed in an experimental autoimmune encephalomyelitis (EAE) animal model. Furthermore, visabron c (4-4)'s lack of toxic effects in mice was verified by blood analysis, tissue pathology, immunogenicity, and "off-target" effects, indicating its significant tolerability and lack of immunogenicity. Visabron c (4-4) can be delivered systemically. The in vitro and in vivo data justify visabron c (4-4) as a safe alternative peptidomimetic lead compound/drug to monoclonal anti-α4 integrin antibodies, steroids, and other immunosuppressant drugs. Moreover, visabron c (4-4) design may pave the way for developing new therapies for a variety of other inflammatory and/or autoimmune diseases.

Polyneuropathy is a disease involving multiple peripheral nerves injuries. Axon regrowth remains the major prerequisite for plasticity, regeneration, circuit formation, and eventually functional recovery and therefore, regulation of neurite outgrowth might be a candidate for treating polyneuropathies. In a recent study, we synthesized and established the methylene-cycloalkylacetate (MCAs) pharmacophore as a lead for the development of a neurotropic drug (inducing neurite/axonal outgrowth) using the PC12 neuronal model. In the present study we extended the characterizations of the in vitro neurotropic effect of the derivative 3-(3-allyl-2-methylenecyclohexyl) propanoic acid (MCA-13) on dorsal root ganglia and spinal cord neuronal cultures and analyzed its safety properties using blood biochemistry and cell counting, acute toxicity evaluation in mice and different in vitro “off-target” pharmacological evaluations. This MCA derivative deserves further preclinical mechanistic pharmacological characterizations including therapeutic efficacy in in vivo animal models of polyneuropathies, toward development of a clinically relevant neurotropic drug.

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.

Advanced approaches to stem cell-based therapies is necessary for myocardial regenerative therapy because treatments have yielded modest results in the clinic. Our group previously demonstrated genetic modification of cardiac stem cells with Pim-1 kinase overexpression rejuvenated aged cells and potentiated myocardial repair. Despite these encouraging findings, concerns were raised regarding oncogenic risk associated with Pim-1 kinase overexpression. Testing of these c-kit+ cardiac interstitial cells (cCICs), derived from heart failure patient samples, overexpressing Pim-1 (cCICs-Pim-1) for indices of oncogenic risk was assessed by soft agar colony formation, micronucleation, gamma-Histone 2AX foci, and transcriptome profiling. Collectively, findings demonstrate comparable phenotypic and biological properties of cCICsPim-1 compared to baseline control cCICs with no evidence for oncogenic phenotype. Using a highly-selective and continuous sensor for quantitative assessment of PIM1 kinase activity, a 7-fold increase in cCICs-Pim-1 versus cCICs resulted. Kinase activity was elevated in IKKs, AKT/SGK, CDK1-3, p38, and ERK1/2 in addition to Pim-1, correlating Pim-1 overexpression to contribute to Pim-1-mediated effects. Enhancement of cellular survival, proliferation, and other beneficial properties to augment stem cell-mediated repair without oncogenic risk is a feasible, logical, and safe approach to improve efficacy and overcome current limitations inherent to cellular adoptive transfer therapeutic interventions.

Abstract Introduction: Protein kinases are a diverse group of 518 enzymes whose dysregulation lies at the center of many diseases across therapeutic areas and especially oncology. Currently, 30% of all drug development efforts are focused on protein kinases. Although many drugs are approved or in clinical trials, these are predominantly ATP-competitive inhibitors. More recently, there has been a surge in the generation of kinase inhibitors with different modes of action, where new tools are needed to effectively characterize inhibitor mechanism of action, predict drug potency, and drive decisions earlier in the drug development process. We developed a simple yet powerful method for the generation of sensors that can be used for the continuous, quantitative, and homogenous detection of kinase activity with recombinant enzymes and crude lysates to enable target discovery and drug development. Experimental Procedures: We harnessed chelation-enhanced fluorescence by integrating the sulfonamido-oxine (Sox) chromophore into high-throughput peptide synthesis methods to identify optimized sequences based on physiologic substrates for Ser, Thr or Tyr kinases. Kinase reactions were monitored using fluorescence intensity in kinetic mode (excitation 360 nm, emission wavelength of 485-505 nm). Results: We demonstrate the ability to rapidly identify novel optimized substrates, where performance measures included higher reaction rates, lower Kms, higher signal/background, increased sensitivity (down to low pM levels), and increased 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 highlight recently developed novel substrates to monitor activity of high-profile tyrosine kinases, including the EGFR and clinically relevant mutants, DDR1/2, JAK kinases (JAK1-3, Tyk2), MET, SYK, Tec-kinases (BTK, ITK, TEC, TXK, BMX), and serine/threonine kinases, including Aurora, CDKs, MAPK pathway (MAP4Ks, Raf, MEK1/2, ERK1/2, RSK), PKR/EIF2AKs and PIM1. Conclusions: The generation of robust activity-based assays opens new areas of the kinome for effective drug discovery. The Sox-based assay technology is ideal for elucidating drug mechanism of action, potency, kinase activation and profiling, and therefore can be applied across the entire target discovery and drug development workflow. These developments provide a quantum improvement in performance and productivity that is needed to address the challenges and opportunities of next-generation protein kinase and phosphatase inhibitors. These assays run on commonly available microplate instruments, providing access across the cancer research and drug development community. Citation Format: Erik M. Schaefer, Susan Cornell-Kennon, Barbara Imperiali. Next-generation CSox-based sensors for continuous, homogeneous, and quantitative monitoring of protein kinase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2393.

Abstract Introduction: Protein kinases are a diverse group of over 500 enzymes whose dysregulation lies at the center of many human diseases, spanning all therapeutic areas. Oncology is the most active area, where 30% of all drug development efforts are focused on protein kinases. Although 30 drugs have been approved by the FDA, and another 120 are in clinical trials, these are predominately ATP-competitive inhibitors. More recently, there has been a surge in the generation of kinase inhibitors with different modes of action, where new tools are needed to effectively and efficiently 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 sensors that can be used for the continuous, quantitative and homogenous detection of kinase activity to enable target discovery and drug development. Experimental Procedures: We have harnessed chelation-enhanced fluorescence by integrating the sulfonamido-oxine (Sox) chromophore into high-throughput peptide synthesis methods to create degenerate peptide libraries containing the consensus sequence for Ser, Thr or Tyr kinases that are implicated in cancer. Kinase reactions included 10 μM substrate, 10 mM MgCl2 and 1 mM ATP and fluorescence intensity was monitored in kinetic mode using a Biotek Synergy Neo2 microplate reader (excitation 360 nm, emission wavelength of 485-505 nm). Results: By exploiting the continuous, quantitative and homogeneous nature of Sox-based detection, we demonstrate the ability to rapidly identify novel optimum substrates simply from observing and analyzing the resulting progress curve (fluorescence intensity over time). Performance measures included higher reaction rates, lower Km's, lower and flatter backgrounds, improved signal/background, increased sensitivity (down to low pM levels), and increased 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 highlight recently developed novel substrates to monitor activity of high-profile tyrosine kinases, including the EGFR and multiple clinically-relevant mutants and Tec-family kinases (BTK, ITK, TEC, TXK, BMX), and serine/threonine kinases, including MAP4K's, MAPKs, CDKs and DRAK1/2. Conclusions: The generation of robust activity-based assays, especially where previously only binding assay formats were available, opens up new areas of the kinome for effective drug discovery. The Sox-based assay technology is ideal for elucidating drug mechanism of action, potency, kinase activation and profiling, and therefore can be applied across the entire target discovery and drug development workflow. These developments provide a quantum improvement in performance and productivity that is needed to address the challenges and opportunities of next generation protein kinase inhibitors. These assays run on commonly available microplate instruments, providing access across the cancer research and drug development community. Citation Format: Erik M. Schaefer, Susan Cornell-Kennon, Eric Lamsa, Erik McCauley, Eric Berg, Jordan Fishman, Barbara Imperiali. Optimized Sox-based sensors for continuous, homogeneous, and quantitative monitoring of protein kinase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-024. doi:10.1158/1538-7445.AM2017-LB-024

We propose that the time course of an enzyme reaction following the Michaelis-Menten reaction mechanism can be conveniently described by a newly derived algebraic equation, which includes the Lambert Omega function. Following Northrop's ideas [Anal. Biochem. 321, 457-461,1983], the integrated rate equation contains the Michaelis constant (K-M) and the specificity number (k(s)equivalent to k(cat)/K-M) as adjustable parameters, but not the turnover number k(cat). A modification of the usual global-fit approach involves a combinatorial treatment of nominal substrate concentrations being treated as fixed or alternately optimized model parameters. The newly proposed method is compared with the standard approach based on the "initial linear region" of the reaction progress curves, followed by nonlinear fit of initial rates to the hyperbolic Michaelis-Menten equation. A representative set of three chelation-enhanced fluorescence EGFR kinase substrates is used for experimental illustration. In one case, both data analysis methods (linear and nonlinear) produced identical results. However, in another test case, the standard method incorrectly reported a finite (50-70 mu M) KM value, whereas the more rigorous global nonlinear fit shows that the Km is immeasurably high. (C) 2016 Elsevier Inc. All rights reserved.

Pim-1 kinase exerts potent cardioprotective effects in the myocardium downstream of AKT, but the participation of Pim-1 in cardiac hypertrophy requires investigation. Cardiac-specific expression of Pim-1 (Pim-WT) or the dominant-negative mutant of Pim-1 (Pim-DN) in transgenic mice together with adenoviral-mediated overexpression of these Pim-1 constructs was used to delineate the role of Pim-1 in hypertrophy. Transgenic overexpression of Pim-1 protects mice from pressure-overload-induced hypertrophy relative to wild-type controls as evidenced by improved hemodynamic function, decreased apoptosis, increases in antihypertrophic proteins, smaller myocyte size, and inhibition of hypertrophic signaling after challenge. Similarly, Pim-1 overexpression in neonatal rat cardiomyocyte cultures inhibits hypertrophy induced by endothelin-1. On the cellular level, hearts of Pim-WT mice show enhanced incorporation of BrdU into myocytes and a hypercellular phenotype compared to wild-type controls after hypertrophic challenge. In comparison, transgenic overexpression of Pim-DN leads to dilated cardiomyopathy characterized by increased apoptosis, fibrosis, and severely depressed cardiac function. Furthermore, overexpression of Pim-DN leads to reduced contractility as evidenced by reduced Ca(2+) transient amplitude and decreased percentage of cell shortening in isolated myocytes. These data support a pivotal role for Pim-1 in modulation of hypertrophy by impacting responses on molecular, cellular, and organ levels.