Enrichment-Free, Targeted Covalent Drug Discovery in Live Cells.

In the past two decades, drug candidates with a covalent binding mode have gained the interest of medicinal chemists, as several covalent anticancer drugs have successfully reached the clinic. As a covalent binding mode changes the relevant parameters to rank inhibitor potency and investigate structure-activity relationship (SAR), it is important to gather experimental evidence on the existence of a covalent protein–drug adduct. In this work, we review established methods and technologies for the direct detection of a covalent protein–drug adduct, illustrated with examples from (recent) drug development endeavors. These technologies include subjecting covalent drug candidates to mass spectrometric (MS) analysis, protein crystallography, or monitoring intrinsic spectroscopic properties of the ligand upon covalent adduct formation. Alternatively, chemical modification of the covalent ligand is required to detect covalent adducts by NMR analysis or activity-based protein profiling (ABPP). Some techniques are more informative than others and can also elucidate the modified amino acid residue or bond layout. We will discuss the compatibility of these techniques with reversible covalent binding modes and the possibilities to evaluate reversibility or obtain kinetic parameters. Finally, we expand upon current challenges and future applications. Overall, these analytical techniques present an integral part of covalent drug development in this exciting new era of drug discovery.

Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes.

A cell culture microfluidic device has been developed to test the cytotoxicity of anticancer drugs while reproducing multi-organ interactions in vitro. Cells were cultured in separate chambers representing the liver and tumor. The two chambers were connected through a channel to mimick the blood flow. Glioblastoma (GBM) cancer cells (M059K) and hepatoma cells (HepG2) were cultured in the tumor and the liver chambers, respectively. The cytotoxic effect of cancer treatment drug Temolozomide (TMZ) was tested using this two chamber system. The experimental results showed that with the liver cells, the cancer cells showed much higher viability than those without the liver cells. This indicates that the liver metabolism has strong effect on the toxicity of the anticancer drug. The results demonstrated that the perfused two chamber cell culture system has the potential to be used as a platform for drug screening in a more physiologically realistic environment.

We aimed at generating a clinically meaningful in vivo platform for preclinical drug screening in colorectal cancer. Surgical specimens were collected from 143 patients, and 97 carcinomas were engrafted in immunodeficient mouse (tumor take rate 67%). To develop in vivo platform suitable for drug screening, 39 of xenografted carcinomas were selected and expanded satisfactorily in mice with a mean time to reach a size of 1000-1500 mm3 of 90 ± 20 days. In order to validate the similarity of molecular characters between the patient tumor and carcinomas obtained from xenografts, we conducted the mutation status, an expression array, arrayCGH, and other molecular profiling in colon cancer to look at the molecular progression and transition along with primary tumor, metastatic tumor and xenograft model. Histological analysis and the molecular profiles of tumors revealed a high degree of similarity between the patient tumor and xenografted carcinomas. The xenografted tumors maintain their genotypic features of original tumors, suggest that the in vivo platform can be useful for the personalized drug development in colorectal cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1315. doi:1538-7445.AM2012-1315

Activity-based protein profiling (ABPP) is a functional proteomics technique for directly monitoring the expression of active enzymes in cell extracts and living cells. The technique relies on irreversible inhibitors equipped with reactive groups (warheads) that covalently attach to the active site of enzymes and fluorescent or affinity tags for imaging and purification purposes, respectively. Here, a high-throughput and robust protocol for high-resolution quantitative activity-based proteasome profiling is described. We use both panreactive and subunit-specific fluorescent activity-based probes (ABPs) to quantify the proteasome activity in living cells, in the presence or absence of the potent proteasome inhibitor bortezomib. Active proteasome subunits from cell lysates are affinity-purified via a biotinylated ABP. Purification from live cells involves a two-step ABP approach using a reagent with a cell-permeable azide-warhead and postlysis installation of biotin. By means of liquid chromatography-mass spectrometry (LC-MS)-based proteomics, we can accurately identify the enriched proteins and the active site peptides of the enzymes, and relatively quantify all the proteasome activities in one experiment. The fluorescence ABPP protocols takes 2-3 d, and approximately 8-10 d are needed to complete the entire protocol.

Protein active states are dynamically regulated by various modifications; thus, endogenous protein modification is an important tool for understanding protein functions and networks in complicated biological systems. Here we developed a new pyridinium-based approach to label lysine residues under physiological conditions that is low-toxicity, efficient, and lysine-selective. Furthermore, we performed a large-scale analysis of the ∼70% lysine-selective proteome in MCF-7 cells using activity-based protein profiling (ABPP). We quantifically assessed 1216 lysine-labeled peptides in cell lysates and identified 386 modified lysine sites including 43 mitochondrial-localized proteins in live MCF-7 cells. Labeled proteins significantly preferred the mitochondria. This pyridinium-based methodology demonstrates the importance of analyzing endogenous proteins under native conditions and provides a robust chemical strategy utilizing either lysine-selective protein labeling or spatiotemporal profiling in a living system.

Rationale Lung cancer is the leading cause of all cancer related deaths and treatment is still suboptimal. Novel biomarkers with a reliable predictive significance which may additionally represent therapeutic targets are therefore of utmost importance. In the post genomic era most biomarker studies aim to measure abundances instead of real enzymatic activities. Therefore crucial changes in enzymatic activities during tumor progression and treatment response might not be detected. In order to circumvent these limitations a new methodology termed Activity Based Protein Profiling (ABPP), developed by Professor B. Cravatt and colleagues, may be a promising option. The methodology employs so-called Activity Based Probes (ABPs) that selectively bind to active sites of members of distinct enzyme superfamilies, thereby making an activity read-out of any given proteome possible. Using ABPs targeting serine hydrolases, a large and diverse class of enzymes that have previously been linked to lung cancer development, we introduced ABPP as a screening platform in a clinical setting. Methods A directed mass spectrometric approach was used for qualitative and quantitative analysis of ABP labeled proteomes. Samples were analyzed on an FTICR mass spectrometer (LTQ-FTMS, Thermo Finnigan, Bremen, Germany). Mass spectrometric data were searched against a recently updated human database (UniProt) using the Mascot search engine (version 2.2). SuperHirn v0.3 was used for label-free quantitation. Inclusion/exclusion lists were generated based on data derived from discovery-driven experiments in data-dependent acquisition mode. Results Approximately 30 serine hydrolases - mostly esterases and proteases - were identified per investigated proteome of the human lung adenocarcinoma cell line CaLu-3. Certain enzymes like Fatty Acid Synthase (FASN) or Kallikrein-6 (KLK6) have previously been associated with lung cancer development or lung cancer diagnosis, respectively. However, other enzymes like Neutral cholesterol ester hydrolase 1 (NCEH1) have not been linked to lung cancer so far or even represent uncharacterized proteins (Abhydrolase domain-containing protein 10, ABHD10). Interestingly, several threonine proteases were identified, indicating that this class of enzymes is also susceptible to the ABP targeting serine hydrolases. Conclusion ABPP allows fast and semi-quantitative analysis of enzymatic activity profiles in cell lines and primary human specimen. We aim to apply the established methodology on >100 pairs of fresh-frozen human lung adenocarcinoma biopsies and corresponding normal lung tissues from our tumor bank and link activity profiles of serine hydrolases to clinical follow-up data. The results of this study will ideally allow the discrimination of low-/ high-risk lung adenocarcinoma patients. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5576.

Methods for the surface patterning of small molecules and biomolecules can yield useful platforms for drug screening, synthetic biology applications, diagnostics, and the immobilization of live cells. However, new techniques are needed to achieve the ease, feature sizes, reliability, and patterning speed necessary for widespread adoption. Herein, we report an easily accessible and operationally simple photoinitiated reaction that can achieve patterned bioconjugation in a highly chemoselective manner. The reaction involves the photolysis of 2-azidophenols to generate iminoquinone intermediates that couple rapidly to aniline groups. We demonstrate the broad functional group compatibility of this reaction for the modification of proteins, polymers, oligonucleotides, peptides, and small molecules. As a specific application, the reaction was adapted for the photolithographic patterning of azidophenol DNA on aniline glass substrates. The presence of the DNA was confirmed by the ability of the surface to capture living cells bearing the sequence complement on their cell walls or cytoplasmic membranes. Compared to other light-based DNA patterning methods, this reaction offers higher speed and does not require the use of a photoresist or other blocking material.

Deubiquitinating enzymes (DUBs) function to remove or cleave ubiquitin from post-translationally modified protein substrates. There are about 100 known DUBs in the proteome, and their dysregulation has been implicated a number of disease states, but the specific function of many subclass members remains poorly understood. Activity-based probes (ABPs) react covalently with an active site residue to report on specific enzyme activity, and thus represent a powerful method to evaluate cellular and physiological enzyme function and dynamics. Ubiquitin-based ABPs, such as HA-Ub-VME, an epitope-tagged ubiquitin carrying a C-terminal reactive warhead, are the leading tool for “DUBome” activity profiling. However, these probes are generally cell membrane impermeable, limiting their use to isolated enzymes or lysates. Development of cell-permeable ABPs would allow engagement of DUB enzymes directly within the context of an intact live cell or organism, refining our understanding of physiological and pathological function, and greatly enhancing opportunities for translational research, including target engagement, imaging and biomarker discovery. This mini-review discusses recent developments in small molecule activity-based probes that target DUBs in live cells, and the unique applications of cell-permeable DUB activity-based probes vs. their traditional ubiquitin-based counterparts.

Induced hepatic (iHep) cells generated by direct reprogramming have been proposed as cell sources for drug screening and regenerative medicine. However, the practical use of a 3D hepatic tissue culture comprised of iHep cells for drug screening and toxicology testing has not been demonstrated. In this study, a 3D vascularized liver organoid composed of iHep cells and a decellularized liver extracellular matrix (LEM) cultured in a microfluidic system is demonstrated. iHep cells are generated by transfection with polymer nanoparticles and plasmids expressing hepatic transcription factors. The iHep cells are cocultured with endothelial cells in the 3D LEM hydrogel in a microfluidic‐based cell culture device with a continuous dynamic flow of media. The resultant 3D vascularized liver organoids maintained under this physiologically relevant culture microenvironment exhibit improved hepatic functionalities, metabolic activity, biosynthetic activity, and drug responses. Finally, the feasibility of using the iHep‐based 3D liver organoid as a high‐throughput drug screening platform, as well as its use in a multiorgan model comprised of multiple internal organoids is confirmed. The study suggests that a combined strategy of direct reprogramming, matrix engineering, and microfluidics can be used to develop a highly functional, standardized, drug screening, and toxicological analysis platform.

Bioorthogonal chemistry combines well with activity-based protein profiling, as it allows for the introduction of detection tags without significantly influencing the physiochemical and biological functions of the probe. In this work, we introduced methyltetrazinylalanine (MeTz-Ala), a close mimic of phenylalanine, into a dipeptide fluoromethylketone cysteine protease inhibitor. Following covalent and irreversible inhibition, the tetrazine allows vizualisation of the captured cathepsin activity by means of inverse electron demand Diels Alder ligation in cell lysates and live cells, demonstrating that tetrazines can be used as live cell compatible, minimal bioorthogonal tags in activity-based protein profiling.

Development of bestatin-based activity-based probes for metallo-aminopeptidases

The discovery of novel drug targets enhances the development of novel drugs, and the discovery of novel target proteins depends on highly accurate high-throughput methods of analyzing drug-protein interactions. Protein expression levels, spatial localization, and structural differences directly affect pharmacodynamics. To date, >20000 proteins have been discovered in the human proteome by the genome and proteome projects via gene and protein sequencing. Understanding the biological functions of proteins is critical in identifying and regulating biological processes, with most remaining unidentified. Until recently, >85% of proteins were considered undruggable, mainly because of the lack of binding pockets and active sites targeted by small molecules. Therefore, characterization of the reactive sites of amino acids based on proteomic hierarchy is the key to novel drug design. Recently, with the rapid development of mass spectrometry (MS), the study of drug-target protein interactions based on proteomics technology has been considerably promoted. Activity-based protein profiling (ABPP) is an active chemical probe-based method of detecting functional enzymes and drug targets in complex samples. Compared with classical proteomics strategies, ABPP is based mainly on protein activity. It has been successfully utilized to characterize the activities of numerous protease families with crucial biological functions, such as serine hydrolases, protein kinases, glycosidases, and metalloenzymes. It has also been used to identify key enzymes that are closely related to diseases and develop covalent inhibitors for use in disease treatment. The technology used in proteome analysis ranges from gel electrophoresis to high-throughput MS due to the progress of MS technology. ABPP strategies combined with chemical probe labeling and quantitative MS enable the characterization of amino acid activity, which may enhance the discovery of novel drug targets and the development of lead compounds. Amino acid residues play critical roles in protein structures and functions, and covalent drugs targeting these amino acids are effective in treating numerous diseases. There are 20 main types of natural amino acids, with different reactivities, in the proteins in the human body. In addition, the proteins and amino acids are affected by the spatial microenvironment, leading to significant differences in their spatial reactivities. The key in evaluating the reactivities of amino acids via ABPP is to select those with high reactivities. The core of the ABPP strategy is the use of chemical probes to label amino acid sites that exhibit higher activities in certain environments. The activity-based probe (ABP) at the core of ABPP consists of three components: reactive, reporter groups and a linker. The reactive group is the basis of the ABP and anchors the drug target via strong forces, such as covalent bonds. The reaction exhibits a high specificity and conversion rate and should display a good biocompatibility. Activity probes based on different amino acid residues have been developed, and the screening of amino acid activity combined with isotope labeling is a new focus of research. Currently, different types of ABPs have been developed to target amino acids and characterize amino acid reactivity, such as cysteine labeled with an electrophilic iodoacetamide probe and lysine labeled with activated esters. ABPP facilitates the discovery of potentially therapeutic protein targets, the screening of lead compounds, and the identification of drug targets, thus aiding the design of novel drugs. This review focuses on the development of ABPP methods and the progress in the screening of amino acid reactivity using ABPs, which should be promising methods for use in designing targeted drugs with covalent interactions.

Structural and functional analyses of proteins‐of‐interest (POI) in multimolecular crowding conditions (mMCC) such as live cells and tissues are regarded as inevitable challenges for in depth understanding of the real shapes of POIs in their existing natural environments. Activity‐based protein profiling (ABPP) is a definitely powerful tool capable of analyzing a proteome possessing a particular activity under mMCC. While ABPP usually targets a proteome of interest, study of a particular protein in mMCC is also valuable. Although activity‐based probes (ABPs) are often used for this aim, most of conventional ABPs cause the loss of original activities, and therefore are not perfectly suitable for functional analysis of labeled proteins. Ligand‐directed chemistry (LDchem) developed by our group is an alternative approach of ABPs, that can modify a surface of POI rather than its active site using a cleavable electrophile in a traceless manner. LDchem thus enables the POI labeling with a synthetic fluorophore with no or minimal effects on the original functions of POIs even in mMCC. In this review, we briefly describe a principle of LDchem for native protein labeling and summarize its recent chemical biology applications such as the imaging‐based biological analysis of POI functions and construction of POI‐based biosensors.

Target-identification and understanding of mechanism-of-action (MOA) are challenging for development of small-molecule probes and their application in biology and drug discovery. For example, although aspirin has been widely used for more than 100 years, its molecular targets have not been fully characterized. To cope with this challenge, we developed a novel technique called quantitative acid-cleavable activity-based protein profiling (QA-ABPP) with combination of the following two parts: (i) activity-based protein profiling (ABPP) and iTRAQ™ quantitative proteomics for identification of target proteins and (ii) acid-cleavable linker-based ABPP for identification of peptides with specific binding sites. It is known that reaction of aspirin with its target proteins leads to acetylation. We thus applied the above technique using aspirin-based probes in human cancer HCT116 cells. We identified 1110 target proteins and 2775 peptides with exact acetylation sites. By correlating these two sets of data, 523 proteins were identified as targets of aspirin. We used various biological assays to validate the effects of aspirin on inhibition of protein synthesis and induction of autophagy which were elicited from the pathway analysis of Aspirin target profile. This technique is widely applicable for target identification in the field of drug discovery and biology, especially for the covalent drugs.