Small-Scale Panel Comprising Diverse Gene Family Targets To Evaluate Compound Promiscuity.

Despite the recent advances in the life sciences and the remarkable investment in drug discovery research, the success rate of small-molecule drug development remains low. Safety is the second most influential factor of drug attrition in clinical studies; thus, the selection of compounds with fewer toxicity concerns is crucial to increase the success rate of drug discovery. Compounds that promiscuously bind to multiple targets are likely to cause unexpected pharmacological activity that may lead to adverse effects. Therefore, avoiding such compounds during early research stages would contribute to identifying compounds with a higher chance of success in the clinic. To evaluate the interaction profile against a wide variety of targets, we constructed a small-scale promiscuity panel (PP) consisting of eight targets (ROCK1, PDE4D2, GR, PPARγ, 5-HT2B, adenosine A3, M1, and GABAA) that were selected from diverse gene families. The validity of this panel was confirmed by comparison with the promiscuity index evaluated from larger-scale panels. Analysis of data from the PP revealed that both lipophilicity and basicity are likely to increase promiscuity, while the molecular weight does not significantly contribute. Additionally, the promiscuity assessed using our PP correlated with the occurrence of both in vitro cytotoxicity and in vivo toxicity, suggesting that the PP is useful to identify compounds with fewer toxicity concerns. In summary, this small-scale and cost-effective PP can contribute to the identification of safer compounds that would lead to a reduction in drug attrition due to safety issues.

Structure-based rational design of staurosporine-based fluorescent probe with broad-ranging kinase affinity for kinase panel application.

Selectivity profiling of compounds is important for kinase drug discovery. To this end, we aimed to develop a broad-range protein kinase assay by synthesizing a novel staurosporine-derived fluorescent probe based on staurosporine and kinase-binding related structural information. Upon structural analysis of staurosporine with kinases, a 4'-methylamine moiety of staurosporine was found to be located on the solvent side of the kinases, to which several linker units can be conjugated by either alkylation or acylation. However, such conjugation was suggested to reduce the binding affinities of the modified compound for several kinases, owing to the elimination of hydrogen bond donor moiety of NH-group from 4'-methylamine and/or steric hindrance by acyl moiety. Based on this structural information, we designed and synthesized a novel staurosporine-based probe without methyl group in order to retain the hydrogen bond donor, similar to unmodified staurosporine. The broad range of the kinase binding assay demonstrated that our novel fluorescent probe is an excellent tool for developing broad-ranging kinase binding assay.

Discovery of 7-Oxo-2,4,5,7-tetrahydro-6 H-pyrazolo[3,4- c]pyridine Derivatives as Potent, Orally Available, and Brain-Penetrating Receptor Interacting Protein 1 (RIP1) Kinase Inhibitors: Analysis of Structure-Kinetic Relationships.

We report the discovery of 7-oxo-2,4,5,7-tetrahydro-6 H-pyrazolo[3,4- c]pyridine derivatives as a novel class of receptor interacting protein 1 (RIP1) kinase inhibitors. On the basis of the overlay study between HTS hit 10 and GSK2982772 (6) in RIP1 kinase, we designed and synthesized a novel class of RIP1 kinase inhibitor 11 possessing moderate RIP1 kinase inhibitory activity and P-gp mediated efflux. The optimization of the core structure and the exploration of appropriate substituents utilizing SBDD approach led to the discovery of 22, a highly potent, orally available, and brain-penetrating RIP1 kinase inhibitor with excellent PK profiles. Compound 22 significantly suppressed necroptotic cell death both in mouse and human cells. Oral administration of 22 (10 mg/kg, bid) attenuated disease progression in the mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). Moreover, analysis of structure-kinetic relationship (SKR) for our novel chemical series was also discussed.

CETSA quantitatively verifies in vivo target engagement of novel RIPK1 inhibitors in various biospecimens.

The proof of target engagement (TE) is a key element for evaluating potential investment in drug development. The cellular thermal shift assay (CETSA) is expected to facilitate direct measurement of intracellular TE at all stages of drug development. However, there have been no reports of applying this technology to comprehensive animal and clinical studies. This report demonstrates that CETSA can not only quantitatively evaluate the drug-TE in mouse peripheral blood, but also confirm TE in animal tissues exemplified by using the receptor interacting protein 1 kinase (RIPK1) lead compound we have developed. Our established semi-automated system allows evaluation of the structure-activity relationship using native RIPK1 in culture cell lines, and also enables estimation of drug occupancy ratio in mouse peripheral blood mononuclear cells. Moreover, optimized tissue homogenisation enables monitoring of the in vivo drug-TE in spleen and brain. Our results indicate that CETSA methodology will provide an efficient tool for preclinical and clinical drug development.

Parallel fluorescent probe synthesis based on the large-scale preparation of BODIPY FL propionic acid.

We describe a methodology for quick development of fluorescent probes with the desired potency for the target of interest by using a method of parallel synthesis, termed as Parallel Fluorescent Probe Synthesis (Parallel-FPS). BODIPY FL propionic acid 1 is a widely used fluorophore, but it is difficult to prepare a large amount of 1, which hinders its use in parallel synthesis. Optimization of a synthetic scheme enabled us to obtain 50g of 1 in one batch. With this large quantity of 1 in hand, we performed Parallel-FPS of BODIPY FL-labeled ligands for estrogen related receptor-α (ERRα). An initial trial of the parallel synthesis with various linkers provided a potent ligand for ERRα (Reporter IC50=80nM), demonstrating the usefulness of Parallel-FPS.

Structure-Based Design and Synthesis of 3-Amino-1,5-dihydro-4H-pyrazolopyridin-4-one Derivatives as Tyrosine Kinase 2 Inhibitors.

We report herein the discovery and optimization of 3-amino-1,5-dihydro-4H-pyrazolopyridin-4-one TYK2 inhibitors. High-throughput screening against TYK2 and JAK1-3 provided aminoindazole derivative 1 as a hit compound. Scaffold hopping of the aminoindazole core led to the discovery of 3-amino-1,5-dihydro-4H-pyrazolopyridin-4-one derivative 3 as a novel chemotype of TYK2 inhibitors. Interestingly, initial SAR study suggested that this scaffold could have a vertically flipped binding mode, which prompted us to introduce a substituent at the 7-position as a moiety directed toward the solvent-exposed region. Introduction of a 1-methyl-3-pyrazolyl moiety at the 7-position resulted in a dramatic increase in TYK2 inhibitory activity, and further optimization led to the discovery of 20. Compound 20 inhibited IL-23-induced IL-22 production in a rat PD assay, as well as inhibited IL-23 signaling in human PBMC. Furthermore, 20 showed selectivity for IL-23 signaling inhibition against GM-CSF, demonstrating the unique cytokine selectivity of the novel TYK2 inhibitor.

Development of enzyme-activated photosensitizer based on intramolecular electron transfer.

Photosensitizers produce cytotoxic reactive oxygen species (ROS) upon light illumination, but it is difficult to ablate cells of a specific type (e.g., tumor cells) in the presence of other cell populations, because of the limited precision with which light illumination can be directed to small areas. Here, we report a strategy to achieve cell type-specific ablation by using an enzyme-activated off/on switch for oxidative stress induction. In the unactivated photosensitizer, induction of oxidative stress is quenched by intramolecular electron transfer. However, the target cells express an enzyme that hydrolyzes a substrate moiety of the photosensitizer and the activated photosensitizer induces oxidative stress. As proof of concept, we designed and synthesized a xanthene-based photosensitizer, TGI-betaGal, whose oxidative stress induction ability is switched on following hydrolysis reaction with beta-galactosidase, a widely used gene marker. TGI-betaGal could selectively ablate lacZ-positive cells, whereas it showed no toxicity to lacZ-negative cells, upon light illumination.

Selective photoinactivation of protein function through environment-sensitive switching of singlet oxygen generation by photosensitizer.

Chromophore-assisted light inactivation is a promising technique to inactivate selected proteins with high spatial and temporal resolution in living cells, but its use has been limited because of the lack of a methodology to prevent nonspecific photodamage in the cell owing to reactive oxygen species generated by the photosensitizer. Here we present a design strategy for photosensitizers with an environment-sensitive off/on switch for singlet oxygen ((1)O(2)) generation, which is switched on by binding to the target, to improve the specificity of protein photoinactivation. (1)O(2) generation in the unbound state is quenched by photoinduced electron transfer, whereas (1)O(2) generation can occur in the hydrophobic environment provided by the target protein, after specific binding. Inositol 1,4,5-trisphosphate receptor, which has been suggested to have a hydrophobic pocket around the ligand binding site, was specifically inactivated by an environment-sensitive photosensitizer-conjugated inositol 1,4,5-trisphosphate receptor ligand without (1)O(2) generation in the cytosol of the target cells, despite light illumination, demonstrating the potential of environment-sensitive photosensitizers to allow high-resolution control of generation of reactive oxygen species in the cell.

Highly efficient and photostable photosensitizer based on BODIPY chromophore.

Photosensitizers are reagents that produce reactive oxygen species upon light illumination and are commonly used to study oxidative stress or for photodynamic therapy. There are many available photosensitizers, but most have limitations, such as low photostability, structural instability, or a limited usable range of solvent conditions. Here, we describe a novel photosensitizer scaffold (2I-BDP) based on the unique characteristics of the BODIPY chromophore (i.e., high extinction coefficient, high photostability, and insensitivity to solvent environment). 2I-BDP shows stronger near-infrared singlet oxygen luminescence emission and higher photostability than the well-known photosensitizer, Rose Bengal. Unlike other photosensitizers, this scaffold is widely applicable under various conditions, including lipophilic and aqueous environments. HeLa cells loaded with 2I-BDP could be photosensitized by light illumination, demonstrating that 2I-BDP is potentially useful as a reagent for cell photosensitization, oxidative stress studies, or PDT.

Modification of intracellular Ca2+ dynamics by laser inactivation of inositol 1,4,5-trisphosphate receptor using membrane-permeant probes.

A membrane-permeant malachite green-conjugated IP3 analog (MGIP3/PM) was synthesized as a probe for small molecule-based CALI (smCALI), and its effect on the Ca2+ signaling in intact DT40 chicken B cells was examined. In DT40 B cells treated with the smCALI probe, laser irradiation inhibited IP3-induced Ca2+ oscillations in response to B cell receptor stimulation, demonstrating that IP3R was acutely inactivated. We then applied smCALI to clarify the mechanism of capacitative Ca2+ entry (CCE), in which involvement of IP3R has been suggested. Despite the inactivation of IP3R by smCALI, thapsigargin-induced CCE remained unaffected, providing evidence that functional IP3R is not required for CCE in DT40 cells. These results demonstrate the potency of the smCALI technique for the study of the roles of IP3R in complex intracellular Ca2+ dynamics.