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Inhibitors targeting the epidermal growth factor receptor (EGFR) are an effective therapy for patients with non-small cell lung cancer harboring drug-sensitive activating mutations in the EGFR kinase domain. Drug resistance due to treatment-acquired mutations has motivated the development of successive generations of inhibitors that bind in the ATP site. The third-generation agent osimertinib is now a first-line treatment for this disease. Recently, allosteric inhibitors have been developed to overcome drug-resistant mutations that confer a resistance to osimertinib. Here, we present the structure-guided design and synthesis of a mutant-selective lead compound, which consists of a pyridinyl imidazole-fused benzylisoindolinedione scaffold that simultaneously occupies the orthosteric and allosteric sites. The compound potently inhibits enzymatic activity in L858R/T790M/C797S mutant EGFR (4.9 nM), with a significantly lower activity for wild-type EGFR (47 nM). Additionally, this compound achieves modest cetuximab-independent and mutant-selective cellular efficacies on the L858R (1.2 μM) and L858R/T790M (4.4 μM) variants.

Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its potential as a therapeutic target. We used chemoproteomic profiling and structure-based design to develop a selective, in vivo-compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA-sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer. A highly selective inhibitor of the DCLK1/2 kinases is used to uncover the consequences of DCLK1 inhibition on viability, phosphosignaling and the transcriptome in patient-derived organoid models of pancreatic ductal adenocarcinoma.

Current small molecule inhibitors of KRAS(G12C) bind irreversibly in the switch-II pocket, exploiting the strong nucleophilicity of the acquired cysteine as well as the preponderance of the GDP-bound form of this mutant. Nevertheless, many oncogenic KRAS mutants lack these two features, and it remains unknown whether targeting the switch-II pocket is a practical therapeutic approach for KRAS mutants beyond G12C. Here we use NMR spectroscopy and a novel cellular KRAS engagement assay to address this question by examining a collection of SII-P ligands from the literature and from our own laboratory. We show that the switch-II pockets of many GTP hydrolysis-deficient KRAS hotspot (G12, G13, Q61) mutants are accessible using non-covalent ligands, and that this accessibility is not necessarily coupled to the GDP state of KRAS. The results we describe here emphasize the switch-II pocket as a privileged drug binding site on KRAS and unveil new therapeutic opportunities in RAS-driven cancer.

Macrocyclic peptides open new opportunities to target intracellular protein-protein interactions (PPIs) that are often considered nondruggable by traditional small molecules. However, engineering sufficient membrane permeability into these molecules is a central challenge for identifying clinical candidates. Currently, there is a lack of high-throughput assays to assess peptide permeability, which limits our capacity to engineer this property into macrocyclic peptides for advancement through drug discovery pipelines. Accordingly, we developed a high throughput and target-agnostic cell permeability assay that measures the relative cumulative cytosolic exposure of a peptide in a concentration-dependent manner. The assay was named NanoClick as it combines in-cell Click chemistry with an intracellular NanoBRET signal. We validated the approach using known cell penetrating peptides and further demonstrated a correlation to cellular activity using a p53/MDM2 model system. With minimal change to the peptide sequence, NanoClick enables the ability to measure uptake of molecules that enter the cell via different mechanisms such as endocytosis, membrane translocation, or passive permeability. Overall, the NanoClick assay can serve as a screening tool to uncover predictive design rules to guide structure-activity-permeability relationships in the optimization of functionally active molecules.

Target engagement and cell permeation are important parameters that may limit the efficacy of proteolysis-targeting chimeras (PROTACs). Here, we present an approach that facilitates both the quantitation of PROTAC binding affinity for an E3 ligase of interest, as well as the assessment of relative intracellular availability. We present a panel of E3 ligase target engagement assays based upon the NanoBRET Target Engagement platform. Querying E3 ligase engagement under live-cell and permeabilized-cell conditions allow calculation of an availability index that can be used to rank order the intracellular availability of PROTACs. Here we present examples where the cellular availability of PROTACs and their monovalent precursors are prioritized using NanoBRET assays for CRBN or VHL E3 ligases.

Inflammasomes are critical to the innate immune system and implicated in numerous chronic and acute inflammatory diseases. Having tools to screen inflammasome modulatory drugs is critical, especially for the NLRP3 inflammasome that is triggered by a wide array of stimuli. We have developed three cell-based methods for quantifying inflammasome function: a NanoBRET™ NLRP3-specific target engagement assay, a caspase-1 activity assay and an IL-1β release assay. For the NLRP3 target engagement assay, cells are transfected with a NLRP3-Nanoluc construct and incubated with a cell-permeable, fluorescent NLRP3 binder (tracer), generating a bioluminescent resonance energy transfer (BRET) signal. The homogeneous caspase-1 and IL-1β assays can be run directly with cells in culture or with transferred culture medium, enabling application of both luminescent assays to the same cell wells via split-sample analysis. Using all three assays, we tested a series of reported NLRP3 inhibitors, including MCC950, oridonin, NBC6, NBC19, CY09, OXSi2 and OLT1177™ (dapansutrile). MCC950 inhibition of caspase-1 activity and IL-1β release was demonstrated in human and mouse cell models. We used LPS-primed THP-1 cells treated with nigericin for testing the series of inhibitors. The NLRP3 target engagement assay used HEK293 cells in suspension in non-binding plates. All three assays demonstrated good inhibition for MCC950, oridonin, NBC6, NBC19 and CY09 with rank order potency consistent with published results. We did not detect inhibition with OxSi2 or OLT1177™. These three assays enable screening for inflammasome antagonists and provide a mechanistic platform for characterizing NLRP3 inflammasome inhibitors.

Concerted multidisciplinary efforts have led to the development of Cyclin-Dependent Kinase inhibitors (CDKi’s) as small molecule drugs and chemical probes of intracellular CDK function. However, conflicting data has been reported on the inhibitory potency of CDKi’s and a systematic characterization of affinity and selectivity against intracellular CDKs is lacking. We have developed a panel of cell-permeable energy transfer probes to quantify target occupancy for all 21 human CDKs in live cells, and present a comprehensive evaluation of intracellular isozyme potency and selectivity for a collection of 46 clinically-advanced CDKi’s and tool molecules. We observed unexpected intracellular activity profiles for a number of CDKi’s, offering avenues for repurposing of highly potent molecules as probes for previously unreported targets. Overall, we provide a broadly applicable method for evaluating the selectivity of CDK inhibitors in living cells, and present a refined set of tool molecules to study CDK function., Cyclin-dependent kinase (CDK) inhibitors are widely used both in the clinic and for basic research aimed at dissecting the specific cellular functions of specific CDKs. Here, the authors report the development of a panel of fluorescent reporter probes and provide a comprehensive profile of the inhibitory activity of several CDK inhibitors towards all 21 CDKs in living cells.

A panel of cell-permeable energy transfer probes has been developed to quantify target occupancy for all 21 CDKs in live, intact cells. Here we present the first comprehensive evaluation of intracellular isozyme potency and selectivity for a collection of 46 clinically-advanced CDKi’s and tool molecules. Here we provide a broadly applicable method for evaluating the selectivity of chemical matter for CDKs in living cells, and present a refined set of tool molecules to study CDK function.

A new generation of heterobifunctional small molecules, termed proteolysis targeting chimeras (PROTACs), targets proteins for degradation through recruitment to E3 ligases and holds significant therapeutic potential. Despite numerous successful examples, PROTAC small molecule development remains laborious and unpredictable, involving testing compounds for end-point degradation activity at fixed times and concentrations without resolving or optimizing for the important biological steps required for the process. Given the complexity of the ubiquitin proteasomal pathway, technologies that enable real-time characterization of PROTAC efficacy and mechanism of action are critical for accelerating compound development, profiling, and improving guidance of chemical structure-activity relationship. Here, we present an innovative, modular live-cell platform utilizing endogenous tagging technologies and apply it to monitoring PROTAC-mediated degradation of the bromodomain and extra-terminal family members. We show comprehensive real-time degradation and recovery profiles for each target, precisely quantifying degradation rates, maximal levels of degradation ( D

Intracellular target affinity and residence time are fundamental aspects of pharmacological mechanism (Lu and Tonge, Curr Opin Chem Biol 14:467-474, 2010). Although various robust biochemical approaches exist to measure these binding characteristics, analysis of compound binding with isolated targets may not accurately reflect engagement in the milieu of living cells. To realize the influence of cellular context, methods are needed that are capable of quantifying affinity and residence time in the presence of the intracellular factors that may impact target engagement. Bioluminescence resonance energy transfer (BRET) offers a solution for intracellular target engagement when quantitative metrics or kinetic analyses are required.

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that hijack ubiquitin E3 ligases and induce degradation of intracellular proteins through a tightly regulated proteosomal mechanism. Although several successful PROTACs have been developed against key intracellular target classes including bromodomains, kinases, and nuclear hormone receptors, these bivalent molecules often suffer from poor cell permeability due to high molecular weight. To enable a high-throughput, quantitative readout for PROTAC permeability and E3 ligase occupancy in living cells, we have developed a panel of target engagement (TE) assays for key E3 ligase including CRBN, VHL, XIAP, cIAP, and MDM2. These intracellular assays are the first biophysical method to enable the quantitative determination of compound occupancy, potency, and residence time for specific target proteins inside living cells. The assays provide a direct measure of compound binding or occupancy to a target under physiological conditions using bioluminescent resonance energy transfer (BRET). Citation Format: James Vasta, Cesear Corona, Jennifer Wilkinson, Morgan R. Ingold, Chad Zimprich, Marie Schwinn, Thomas Machleidt, Jim Hartnett, Mei Cong, Frank Fan, Richard L. Somberg, Matthew Robers. A live cell method to assess E3 ligase and target protein occupancy for PROTACs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6407.

Cyclin-dependent kinases (CDK) play key roles in diverse cellular functions including cell cycle control, cell proliferation, and transcriptional regulation. Due to these important roles, CDKs have been targeted for cancer therapeutic development, which has resulted in hundreds of small molecule inhibitors. However, conflicting data of CDK inhibitor potency has been reported and a rigorous comparison of inhibitor affinity and selectivity for intracellular CDKs is lacking. To address this need, we have developed a panel of cell-permeable energy transfer probes or tracers to enable comprehensive quantitation of inhibitor target occupancy and affinity for CDKs in live cells via a bioluminescent energy transfer (BRET) method. Specifically, the quantitative BRET-based capability is achieved via energy transfer from cell-permeable fluorescent tracers reversibly engaged to NanoLuc luciferase-tagged CDK proteins expressed in live cells. An untagged cyclin can be introduced into the cells at the same time as the NanoLuc-CDK, allowing for the interrogation of a specific CDK-cyclin pair. The BRET-based method can be used in equilibrium binding analysis. In addition, time-dependent target-compound occupancy (or residence time) can also be obtained with this method. We will present a comprehensive evaluation of intracellular isozyme potency and selectivity for a collection of clinically-advanced CDK inhibitors and chemical probes across the CDK family. We observed unexpected intracellular activity profiles for some clinically-advanced CDK inhibitors. We further evaluated mechanisms for achieving target selectivity through target residence time under non-equilibrium cell culture conditions. This BRET-based method is broadly applicable for evaluating the occupancy, affinity, and selectivity of chemical matter for CDKs in live cells. Citation Format: James Vasta, Carrow Wells, Cesear Corona, Jennifer Wilkinson, Chad Zimprich, Morgan Ingold, Domenic Ogno, Marie Schwinn, Thomas Machleidt, Mei Cong, Frank Fan, Kristin Huwiler, Amy Landreman, Julie E. Pickett, Kilian Huber, Matthew Robers. Examination of clinically relevant inhibitor selectivity in live cells across the CDK family [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5808.

Summary For kinase inhibitors, intracellular target selectivity is fundamental to pharmacological mechanism. Although a number of acellular techniques have been developed to measure kinase binding or enzymatic inhibition, such approaches can fail to accurately predict engagement in cells. Here we report the application of an energy transfer technique that enabled the first broad-spectrum, equilibrium-based approach to quantitatively profile target occupancy and compound affinity in live cells. Using this method, we performed a selectivity profiling for clinically relevant kinase inhibitors against 178 full-length kinases, and a mechanistic interrogation of the potency offsets observed between cellular and biochemical analysis. For the multikinase inhibitor crizotinib, our approach accurately predicted cellular potency and revealed improved target selectivity compared with biochemical measurements. Due to cellular ATP, a number of putative crizotinib targets are unexpectedly disengaged in live cells at a clinically relevant drug dose., Graphical Abstract, Highlights • The approach enables quantitative profiling of 178 full-length kinases • Compared with biochemical approaches, this is a better predictor of cellular potency • An unexpected intracellular selectivity is observed for certain kinase inhibitors • A mechanistic analysis of ATP interference on target engagement is performed, Vasta et al. describe a broad-spectrum approach (BRET) to quantitatively measure target engagement for kinases inside live cells. Compared with biochemical measurements, the analysis revealed an improved intracellular selectivity profile for clinically relevant kinase inhibitors. This serves as a mechanistic tool to determine the effect of cellular ATP on engagement potency.

In vivo fluorescence cancer imaging is an important tool in understanding tumor growth and therapeutic monitoring and can be performed either with endogenously produced fluorescent proteins or with exogenously introduced fluorescent probes bound to targeting molecules. However, endogenous fluorescence proteins cannot be altered after transfection, thus requiring rederivation of cell lines for each desired color, while exogenously targeted fluorescence probes are limited by the heterogeneous expression of naturally occurring cellular targets. In this study, we adapted the dehalogenase-based protein-Tag (HaloTag) system to in vivo cancer imaging, by introducing highly expressed HaloTag receptors (HaloTagR) in cancer cells coupled with a range of externally injected fluorophore-conjugated dehalogenase-reactive reactive linkers. Tumor nodules arising from a single transfected cell line were stably labeled with fluorescence varying in emission spectra from green to near-infrared. After establishing and validating a SHIN3 cell line stably transfected with HaloTagR (HaloTagR-SHIN3), in vivo spectral fluorescence imaging studies were performed in live animals using a peritoneal dissemination model. The tumor nodules arising from HaloTagR-SHIN3 could be successfully labeled by four different fluorophore-conjugated HaloTag-ligands each emitting light at different wavelengths. These fluorophores could be alternated on serial imaging sessions permitting assessment of interval growth. Fluorescence was retained in histological specimens after fixation. Thus, this tagging system proves versatile both for in vivo and in vitro imaging without requiring modification of the underlying cell line. Thus, this strategy can overcome some of the limitations associated with the use of endogenous fluorescent proteins and exogenous targeted optical agents in current use.

Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies – Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR) – we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be beneficial for screening a large number of antibody samples during early monoclonal development phase.

Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs.

[reaction: see text] An efficient synthesis of a terminal alkyne derived from d-biotin was developed to provide an alternative for carboxyl-based biotinylation. This approach was illustrated by the preparation of alkyne- and alkene-linked phenylalanine derivatives using palladium-catalyzed Sonogashira and Oh methodology. (Strept)avidin binding was observed using soluble and immobilized receptors. These results demonstrate the applicability of carbon-linked biotin derivatives for use in affinity-based purification and bioanalytical applications.

Cytochrome P450 (P450) assays use probe substrates to interrogate the influence of new chemical entities toward P450 enzymes. We report the synthesis and study of a family of bioluminogenic luciferin acetal substrates that are oxidized by P450 enzymes to form luciferase substrates. The luciferin acetals were screened against a panel of purified P450 enzymes. In particular, one proluciferin acetal has demonstrated sensitive and selective CYP3A4-catalyzed oxidation to a luciferin ester-K(m) and k(cat) are 2.88 μM and 5.87 pmol metabolite · min(-1) · pmol enzyme(-1), respectively. The proluciferin acetal was used as a probe substrate to measure IC(50) values of known inhibitors against recombinant CYP3A4 or human liver microsomes. IC(50) values for the known inhibitors correlate strongly with IC(50) values calculated from the traditional high-performance liquid chromatography-based probe substrate testosterone. Luciferin acetals are rapidly oxidized to unstable hemi-orthoesters by CYP3A resulting in luciferin esters and, therefore, are conducive to simple rapid CYP3A bioluminescent assays.

Estrogen mediates its effects through multiple cellular receptors.In addition to the classical nuclear estrogen receptors (ER and ER ), estrogenalsosignalsthroughtheseven-transmembraneG-protein-coupledreceptor(GPCR)GPR30. Although estrogen is a cell-permeable ligand, it is often assumed that allGPCRs function solely as cell surface receptors. Our previous results showed thatGPR30 appeared to be expressed predominantly in the endoplasmic reticulum. Acriticalquestionthatarisesiswhetherthislocalizationrepresentsthesiteoffunc-tional receptor. To address this question, we synthesized a collection of cell-permeable and cell-impermeable estrogen derivatives. We hypothesized that iffunctional GPR30 were expressed at the cell surface, both permeable and imper-meable derivatives would show activity. However, if functional GPR30 were pre-dominantly intracellular, like ER , only the permeable ligands should show activ-ity. Cell permeability was assessed using cells expressing ER as a modelintracellular estrogen-binding receptor. Our results reveal that despite exhibitingsimilarbindingaffinitiesforGPR30,onlythecell-permeableligandsarecapableofstimulating rapid calcium mobilization and phosphoinositide 3-kinase (PI3K) acti-vation. We conclude that GPR30 expressed intracellularly is capable of initiatingcellular signaling and that there is insufficient GPR30 expressed on the cell sur-face to initiate signaling in response to impermeable ligands in the cell lines ex-amined.Toourknowledge,thisisthefirstdefinitivedemonstrationofafunctionalintracellular transmembrane estrogen receptor.Estrogen is a critical hormone in the development andhomeostasisofmanyorgans,particularlythoseinvolvedinreproduction.Italsoplaysanimportantroleinthede-velopmentandtreatmentofbreastcancerduetotheex-pressionoftheclassicalestrogenreceptor,ER ,amem-berofthenuclearfamilyoftranscriptionfactorreceptors,in a high percentage of breast cancers, making it a tar-get of therapeutic treatment with selective ER modula-tors(SERMs),suchastamoxifen,andmorerecentlyaro-matase inhibitors. Estrogen is also involved in thedevelopment of osteoporosis, coronary heart disease,and other conditions (

The development of (99m)Tc-estradiol radiopharmaceuticals would be advantageous for the detection of estrogen receptor-positive breast tumors. Estradiol derivatives conjugated to organometallic tricarbonyl-Tc(I) and related Re(I) complexes are capable of achieving high receptor binding affinity, but effective methods for synthesizing radiolabeled complexes in water are not available. Our interest in the synthesis of 2-hydrazinopyridines as ligands for Tc and Re led us to investigate Pd-catalyzed amination reactions of halo-pyridine substrates with di-tert-butyl hydrazodiformate. Both 2- and 4-substituted halo-pyridine substrates undergo C-N coupling with di-tert-butyl hydrazodiformate to produce Boc-protected pyridine hydrazine derivatives. Only highly electrophilic 3-pyridine halides were converted to the hydrazine. The Boc-protected 5-bromopyridin-2-yl hydrazine substrate 3 was prepared by regioselective substitution at the 2-position of 2,5-dibromopyridine. This bifunctional chelate was attached to ethynyl or vinyl groups at the 17alpha position of estradiol, using Sonogashira and Suzuki/Miyaura coupling reactions to synthesize 1 and 2 in high yields, respectively. Deprotection of 1 under acidic conditions provided the hydrazine hydrochloride salt 25. The 17alpha-estradiol-tricarbonylrhenium(I) complex 4 was synthesized by labeling 25 with fac-[Re(OH(2))(3)(CO)(3)](+) in aqueous ethanol. This complex exhibited excellent stability and high receptor binding affinity for the estrogen receptor, and it is a promising model for evaluation of the analogous Tc-99m complexes as diagnostic imaging agents for breast tumors.

Background: Membrane-permeable macromolecular (MPM) probes are designed to deliver functional proteins to disease sites and into cells by using protein transduction domain (PTD). To visualize intracellular