DELs enable the development of BRET probes for target engagement studies in cells.

DNA-encoded libraries (DELs) provide unmatched chemical diversity and starting points for novel drug modalities. Here, we describe a workflow that exploits the bifunctional attributes of DEL ligands as a platform to generate BRET probes for live cell target engagement studies. To establish proof of concept, we performed a DEL screen using aurora kinase A and successfully converted aurora DEL ligands as cell-active BRET probes. Aurora BRET probes enabled the validation and stratification of the chemical series identified from primary selection data. Furthermore, we have evaluated the effective repurposing of pre-existing DEL screen data to find suitable leads for BRET probe development. Our findings support the use of DEL workflows as an engine to create cell-active BRET probes independent of structure or compound SAR. The combination of DEL and BRET technology accelerates hit-to-lead studies in a live cell setting.

Psilocybin: Characterization of the Metastable Zone Width (MSZW), Control of Anhydrous Polymorphs, and Particle Size Distribution (PSD).

Psilocybin, a serotonergic agonist, was granted a "breakthrough therapy" status by the Food and Drug Administration for clinical trials involving major depressive disorder and treatment-resistant depression. The direct phosphorylation of psilocin to psilocybin that uses a fast crystallization associated with a kinetically controlled process resulted in a smaller particle size distribution. Herein, the measurement of the metastable zone width (MSZW) and nucleation induction enabled a thermodynamically controlled crystallization process, which leads to the formation of a crystal structure with stronger interactions, controlled particle size distribution (PSD), and improved impurity profile. Employing a high-resolution inline microscopy viewer allowed the real-time monitoring of the crystallization process and the measurement of the particle size. We also present a comprehensive study of the formation of polymorph B (trihydrate), polymorph A (anhydrate), and polymorph H (anhydrate) using water recrystallization, which indicates that the formation of polymorph B (trihydrate) is independent of the crystallization method. However, polymorphs A and H are dependent on the mode of drying: drying at room temperature under vacuum gives rise to mainly polymorph A, and when heated even at relatively low temperatures, a mixture of polymorphs A and H beings to form.

Psilocybin: crystal structure solutions enable phase analysis of prior art and recently patented examples.

Psilocybin {systematic name: 3-[2-(dimethylamino)ethyl]-1H-indol-4-yl dihydrogen phosphate} is a zwitterionic tryptamine natural product found in numerous species of fungi known for their psychoactive properties. Following its structural elucidation and chemical synthesis in 1959, purified synthetic psilocybin has been evaluated in clinical trials and has shown promise in the treatment of various mental health disorders. In a recent process-scale crystallization investigation, three crystalline forms of psilocybin were repeatedly observed: Hydrate A, Polymorph A, and Polymorph B. The crystal structure for Hydrate A was solved previously by single-crystal X-ray diffraction. This article presents new crystal structure solutions for the two anhydrates, Polymorphs A and B, based on Rietveld refinement using laboratory and synchrotron X-ray diffraction data, and density functional theory (DFT) calculations. Utilizing the three solved structures, an investigation was conducted via Rietveld method (RM) based quantitative phase analysis (QPA) to estimate the contribution of the three different forms in powder X-ray diffraction (PXRD) patterns provided by different sources of bulk psilocybin produced between 1963 and 2021. Over the last 57 years, each of these samples quantitatively reflect one or more of the hydrate and anhydrate polymorphs. In addition to quantitatively evaluating the composition of each sample, this article evaluates correlations between the crystal forms present, corresponding process methods, sample age, and storage conditions. Furthermore, revision is recommended on characterizations in recently granted patents that include descriptions of crystalline psilocybin inappropriately reported as a single-phase `isostructural variant.' Rietveld refinement demonstrated that the claimed material was composed of approximately 81% Polymorph A and 19% Polymorph B, both of which have been identified in historical samples. In this article, we show conclusively that all published data can be explained in terms of three well-defined forms of psilocybin and that no additional forms are needed to explain the diffraction patterns.

Direct Phosphorylation of Psilocin Enables Optimized cGMP Kilogram-Scale Manufacture of Psilocybin.

A second-generation kilogram-scale synthesis of the psychedelic tryptamine psilocybin has been developed. The synthesis was designed to address several challenges first encountered with the scale-up of previously described literature procedures, which were not optimized for providing consistent yield and purity of products, atom economy, or being run in pilot plant-scale reactors. These challenges were addressed and circumvented with the design of the second-generation route, which featured an optimized cGMP large-scale Speeter-Anthony tryptamine synthesis to the intermediate psilocin with improved in-process control and impurity removal over the three steps. Psilocin was subsequently phosphorylated directly with phosphorous oxychloride for the first time, avoiding a tedious and poor atom economy benzyl-protecting group strategy common to all previously described methods for producing psilocybin. In this report, the challenges encountered in a 100 g scale first-generation literature-based synthesis are highlighted, followed by a detailed description of the newly developed second-generation synthesis to provide over one kilogram of high-purity psilocybin under cGMP.

Quantifying CDK inhibitor selectivity in live cells.

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.

Synthesis and Biological Evaluation of Tryptamines Found in Hallucinogenic Mushrooms: Norbaeocystin, Baeocystin, Norpsilocin, and Aeruginascin.

A general synthetic method was developed to access known tryptamine natural products present in psilocybin-producing mushrooms. In vitro and in vivo experiments were then conducted to inform speculations on the psychoactive properties, or lack thereof, of the natural products. In animal models, psychedelic activity by baeocystin alone was not evident using the mouse head twitch response assay, despite its putative dephosphorylated metabolite, norpsilocin, possessing potent agonist activity at the 5-HT2A receptor.

Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement.

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.

Novel No-Wash Luminogenic Probes for the Detection of Transporter Uptake Activity.

Luminogenic probes were designed and synthesized for the detection of uptake transporter activity in a lytic cell-based assay. These probes rely on a self-cleavable trimethyl lock quinone-cyanobenzothiazole (TMQ-CNBT) or trimethyl lock quinone-luciferin (TMQ-Luc) linked to the anion transporter substrate fluorescein. Upon cellular transport, the TMQ is reduced by viable cells, resulting in the facile intramolecular lactonization and rapid release of the bioluminescent reporter molecule. The uptake transporter activity can then be detected without removing and washing off the extracellular substrates. Six probes were tested with OATP1B1*1a and OATP1B3 overexpressing HEK293 cells, and all compounds showed up to 10.2-fold enhancement in uptake when compared to control cells. Uptake of TMQ-luciferin compounds 2, 4, and 6 increased linearly over time up to 30 min at a concentration ranging from 40 nM to 20 µM. The apparent Km values obtained at different time intervals up to 30 min were nearly identical for a given compound, which validates the 30 min window as appropriate for uptake transporter assays. The average apparent Km values ranged from 0.3 to 0.8 µM and 0.2 to 1.3 µM for OATP1B1*1a and OATP1B3, respectively, indicating good affinities to these anion transporters. Furthermore, uptake of compound 2 was inhibited by two inhibitors of OATP1B1*1a and OATP1B3: rifampicin and ritonavir. The preliminary results obtained from compound 2 exhibited a time-dependent, saturatable, and inhibitable nature of uptake, indicating the feasibility of using the probe for the detection of a transporter-mediated process. This add-and-read homogeneous assay may provide a convenient, rapid, and facile way to detect changes in transporter activity in a high-throughput format, and this assay design strategy could create a new platform for a general cell uptake assay for biomaterials in the future.

Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening.

Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z' = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples.

Bioluminescent cell-based NAD(P)/NAD(P)H assays for rapid dinucleotide measurement and inhibitor screening.

Abstract The central role of nicotinamide adenine dinucleotides in cellular energy metabolism and signaling makes them important nodes that link the metabolic state of cells with energy homeostasis and gene regulation. In this study, we describe the implementation of cell-based bioluminescence assays for rapid and sensitive measurement of those important redox cofactors. We show that the sensitivity of the assays (limit of detection ∼0.5 nM) enables the selective detection of total amounts of nonphosphorylated or phosphorylated dinucleotides directly in cell lysates. The total amount of NAD+NADH or NADP+NADPH levels can be detected in as low as 300 or 600 cells/well, respectively. The signal remains linear up to 5,000 cells/well with the maximum signal-to-background ratios ranging from 100 to 200 for NAD+NADH and from 50 to 100 for NADP+NADPH detection. The assays are robust (Z' value >0.7) and the inhibitor response curves generated using a known NAD biosynthetic pathway inhibitor FK866 correlate well with the reported data. More importantly, by multiplexing the dinucleotide detection assays with a fluorescent nonmetabolic cell viability assay, we show that dinucleotide levels can be decreased dramatically (>80%) by FK866 treatment before changes in cell viability are detected. The utility of the assays to identify modulators of intracellular nicotinamide adenine dinucleotide levels was further confirmed using an oncology active compound library, where novel dinucleotide regulating compounds were identified. For example, the histone deacetylase inhibitor entinostat was a potent inhibitor of cellular nicotinamide adenine dinucleotides, whereas the selective estrogen receptor modulator raloxifene unexpectedly caused a twofold increase in cellular nicotinamide adenine dinucleotide levels.

Self-immolative bioluminogenic quinone luciferins for NAD(P)H assays and reducing capacity-based cell viability assays.

Highly sensitive self-cleavable trimethyl lock quinone-luciferin substrates for diaphorase were designed and synthesized to measure NAD(P)H in biological samples and monitor viable cells via NAD(P)H-dependent cellular oxidoreductase enzymes and their NAD(P)H cofactors.

HaloTag as a reporter gene: positron emission tomography imaging with (64)Cu-labeled second generation HaloTag ligands.

THE GOAL OF THIS STUDY IS TO EMPLOY THE HALOTAG TECHNOLOGY FOR POSITRON EMISSION TOMOGRAPHY (PET), WHICH INVOLVES TWO COMPONENTS: the HaloTag protein (a modified hydrolase which covalently binds to synthetic ligands) and HaloTag ligands (HTLs). 4T1 murine breast cancer cells were stably transfected to express HaloTag protein on the surface (termed as 4T1-HaloTag-ECS, ECS denotes extracellular surface). Two new HTLs were synthesized and termed NOTA-HTL2G-S and NOTA-HTL2G-L (2G indicates second generation, S stands for short, L stands for long, NOTA denotes 1,4,7-triazacyclononane-N,N'N''-triacetic acid). Microscopy studies confirmed surface expression of HaloTag in 4T1-HaloTag-ECS cells, which specifically bind NOTA-HTL2G-S/L. Uptake of (64)Cu-NOTA-HTL2G-L in 4T1-HaloTag-ECS tumors (4.3 ± 0.5, 4.1± 0.2, 4.0 ± 0.2, 2.3 ± 0.1, and 2.2 ± 0.1 %ID/g at 0.5, 3, 6, 18, and 24 h post-injection respectively; n = 4) was significantly higher than that in the 4T1 tumors (3.0 ± 0.3, 3.0± 0.1, 3.0 ± 0.2, 2.0 ± 0.4, and 2.4 ± 0.3 %ID/g at 0.5, 3, 6, 18, and 24 h post-injection respectively; n = 4) at early time points. In comparison, (64)Cu-NOTA-HTL2G-S did not demonstrate significant uptake in either 4T1-HaloTag-ECS or 4T1 tumors. Blocking studies and autoradiography of tumor lysates confirmed that (64)Cu-NOTA-HTL2G-L binds specifically to HaloTag protein in the 4T1-HaloTag-ECS tumors, corroborated by histology. HaloTag protein-specific targeting and PET imaging in vivo with (64)Cu-NOTA-HTL2G-L serves as a proof-of-principle for future non-invasive and sensitive tracking of HaloTag-transfected cells with PET, as well as many other studies of gene/protein/cell function in vivo.

A bioluminescence assay for aldehyde dehydrogenase activity.

The aldehyde dehydrogenase (ALDH) family of enzymes is critical for cell survival and adaptation to cellular and environmental stress. These enzymes are of interest as therapeutic targets and as biomarkers of stem cells. This article describes a novel, homogeneous bioluminescence assay to study the activity of the ALDH enzymes. The assay is based on a proluciferin-aldehyde substrate that is recognized and utilized by multiple ALDH enzyme isoforms to generate luciferin. A detection reagent is added to inactivate ALDH and generate light from the luciferin product. The luminescent signal is dependent on the ALDH enzyme concentration and the incubation time in the ALDH reaction; moreover, the luminescent signal generated with the detection reagent is stable for greater than 2 h. This assay provides many advantages over standard NADH fluorescence assays. It is more sensitive and the signal stability provided allows convenient assay setup in batch mode-based high-throughput screens. The assay also shows an accurate pharmacological response for a common ALDH inhibitor and is robust, with a large assay window (S/B=64) and Z'=0.75.

Novel heterocyclic analogues of firefly luciferin.

Five novel firefly luciferin analogues in which the benzothiazole ring system of the natural substrate was replaced with benzimidazole, benzofuran, benzothiophene, benzoxazole, and indole were synthesized. The fluorescence, bioluminescence, and kinetic properties of the compounds were evaluated with recombinant Photinus pyralis wild type luciferase. With the exception of indole, all of the substrates containing heterocycle substitutions produced readily measurable flashes of light with luciferase. Compared to that of luciferin, the intensities ranged from 0.3 to 4.4% in reactions with varying pH optima and times to reach maximal intensity. The heteroatom changes influenced both the fluorescence and bioluminescence emission spectra, which displayed maxima of 479-528 and 518-574 nm, respectively. While there were some interesting trends in the spectroscopic and bioluminescence properties of this group of structurally similar substrate analogues, the most significant findings were associated with the benzothiophene-containing compound. This synthetic substrate produced slow decay glow kinetics that increased the total light-based specific activity of luciferase more than 4-fold versus the luciferin value. Moreover, over the pH range of 6.2-9.4, the emission maximum is 523 nm, an unusual 37 nm blue shift compared to that of the natural substrate. The extraordinary bioluminescence properties of the benzothiophene luciferin should translate into greater sensitivity for analyte detection in a wide variety of luciferase-based applications.

Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ~2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ~150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.

Bioluminescent assays for ADME evaluation: dialing in CYP selectivity with luminogenic substrates.

INTRODUCTION: The cytochrome P450s (CYPs) are central to ADME studies because of their central role in drug metabolism. Proper CYP assay design and a correct understanding of CYP assay selectivity are critical for generating and interpreting biologically relevant data during drug development. Bioluminescent CYP assays use luminogenic probe substrates that have the unique property of producing photons in a second reaction with luciferase. AREAS COVERED: This article presents the general design principles for in vitro CYP assays. Specifically, the article focuses on the bioluminescent approach that couples CYP activity with photon production. EXPERT OPINION: Highly selective luminogenic substrates for CYP1A1, CYP1A2, CYP2C9, CYP3A4, CYP3A7, CYP4A and CYP4F have been developed with utility for interrogating the roles of these enzymes in biochemical and cell-based formats. These selective substrates are part of a larger collection of probes that deliver CYP inhibition and induction data that predict in vivo drug interactions. Furthermore, they support highly sensitive, rapid and scalable assays for cell-based and cell-free biochemical applications, which offer an alternative and often enabling option over conventional assay strategies.

Proluciferin acetals as bioluminogenic substrates for cytochrome P450 activity and probes for CYP3A inhibition.

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.

Improving protein array performance: focus on washing and storage conditions.

For protein microarrays, maintaining protein stability during the slide processing steps of washing, drying, and storage is of major concern. Although several studies have focused on the stability of immobilized antibodies in antibody microarrays, studies on protein-protein interaction arrays and enzyme arrays are lacking. In this paper we used five bait-prey protein interaction pairs and three enzymes to optimize the washing, drying, and storage conditions for protein arrays. The protein arrays for the study were fabricated by combining HaloTag technology and cell-free protein expression. The HaloTag technology, in combination with cell-free expression, allowed rapid expression and immobilization of fusion proteins on hydrogel-coated glass slides directly from cell extracts without any prior purification. Experimental results indicate enzyme captured on glass slides undergoes significant loss of activity when washed and spin-dried using only phosphate buffer, as is typically done with antibody arrays. The impact of washing and spin-drying in phosphate buffer on protein-protein interaction arrays was minimal. However, addition of 5% glycerol to the wash buffer helps retain enzyme activity during washing and drying. We observed significant loss of enzyme activity when slides were stored dry at 4 degrees C, however immobilized enzymes remained active for 30 days when stored at -20 degrees C in 50% glycerol. We also found that cell-free extract containing HaloTag-fused enzymes could undergo multiple freeze/thaw cycles without any adverse impact on enzyme activity. The findings indicate that for large ongoing studies, proteins of interest expressed in cell-free extract can be stored at -70 degrees C and repeatedly used to print small batches of protein array slides to be used over a few weeks.

N-Alkylated 6'-aminoluciferins are bioluminescent substrates for Ultra-Glo and QuantiLum luciferase: new potential scaffolds for bioluminescent assays.

A set of 6'-alkylated aminoluciferins are shown to be bioluminescent substrates for Ultra-Glo and QuantiLum luciferases. These studies demonstrate that both the engineered and wild-type firefly luciferases tolerate much greater steric bulk at the 6' position of luciferin than has been previously reported. The nature of the alkyl substituent strongly affects the strength of the bioluminescent signal, which varies widely based on size, shape, and charge. Several compounds were observed to generate more light than the corresponding unsubstituted 6'-aminoluciferin. Determination of Michaelis-Menten constants for the substrates with Ultra-Glo indicated that the variation arises primarily from differences in V max, ranging from 1.33 x 10 (4) to 332 x 10 (4) relative light units, but in some cases K m (0.73-10.8 microM) also plays a role. Molecular modeling results suggest that interactions of the side chain with a hydrogen-bonding network at the base of the luciferin binding pocket may influence substrate-enzyme binding.