NLRP3 Inflammasome Inhibitors for Antiepileptogenic Drug Discovery and Development.

Epilepsy is one of the most prevalent and serious brain disorders and affects over 70 million people globally. Antiseizure medications (ASMs) relieve symptoms and prevent the occurrence of future seizures in epileptic patients but have a limited effect on epileptogenesis. Addressing the multifaceted nature of epileptogenesis and its association with the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated neuroinflammation requires a comprehensive understanding of the underlying mechanisms of these medications for the development of targeted therapeutic strategies beyond conventional antiseizure treatments. Several types of NLRP3 inhibitors have been developed and their effect has been validated both in in vitro and in vivo models of epileptogenesis. In this review, we discuss the advances in understanding the regulatory mechanisms of NLRP3 activation as well as progress made, and challenges faced in the development of NLRP3 inhibitors for the treatment of epilepsy.

Chalcone: A potential scaffold for NLRP3 inflammasome inhibitors.

Overactivated NLRP3 inflammasome has been shown to associate with an increasing number of disease conditions. Activation of the NLRP3 inflammasome results in caspase-1-catalyzed formation of active pro-inflammatory cytokines (IL-1ß and IL-18) resulting in pyroptosis. The multi-protein composition of the NLRP3 inflammasome and its sensitivity to several damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) make this extensively studied inflammasome an attractive target to treat chronic conditions. However, none of the known NLRP3 inhibitors has been approved for clinical use. Sulfonylurea and covalent inhibitors with electrophilic warhead (Michael acceptor) are among the prominent classes of compounds explored for their NLRP3 inhibitory effects. Chalcone, a small molecule with α, ß unsaturated carbonyl group (Michael acceptor), has also been studied as a promising scaffold for the development of NLRP3 inhibitors. Low molecular weight, easy to manipulate lipophilicity and cost-effectiveness have attracted many to use chalcone scaffold for drug development. In this review, we highlight chalcone derivatives with NLRP3 inflammasome inhibitory activities. Recent developments and potential new directions summarized here will, hopefully, serve as valuable perspectives for investigators including medicinal chemists and drug discovery researchers to utilize chalcone as a scaffold for developing novel NLRP3 inflammasome inhibitors.

Influence of ligand geometry on cholinesterase enzyme - A comparison of 1-isoindolinone based structural analog with Donepezil.

Donepezil (DNPZ) is one of the few FDA-approved widely used medication in the clinical care of Alzheimer's disease (AD) patients. To investigate the effect of geometry and to find the significance of an enol form if any in DNPZ on acetylcholinesterase (AChE) inhibition, we changed the tetrahedral geometry of DNPZ to planar trigonal pyramidal geometry by replacing the α-carbon atom next to ketone functionality with a nitrogen atom. To mimic 1-indanone in DNPZ, we selected 1-isoindolinone framework to synthesize 25 new DNPZ derivatives and characterized using 1H NMR, 13C NMR and ESI-MS spectroscopy methods. Drug likeliness profile for each compound was predicted using Molinspiration online software following Lipinski's rule. Commercially available assay kits were used to measure AChE and butyrylcholinesterase (BuChE) inhibitory effects. NIH/3T3 mouse embryonic fibroblast cell line was used to measure cytotoxic and proliferation effects using LDH and MTT assay, respectively. Compound #20 was selected for comparative computational docking, modelling and physicochemical studies. Our results show that DNPZ with tetrahedral geometry has 3-fold higher AChE inhibition as compared to compound #20 with planar trigonal pyramidal geometry. Our approach may be useful as a novel indirect method to study the significance of the enol form in DNPZ (or similar compounds), since constant interconversion between the keto and enol forms does not permit a direct determination of the effect of the enol form of DNPZ in vivo. Overall, we conclude that the tetrahedral is a better fit and any change in geometry significantly drives down the cholinesterase inhibitory effect of DNPZ.

Singlet Oxygen Activatable Prodrugs of Paclitaxel, SN-38, MMC and CA4: Nonmitochondria-Targeted Prodrugs.

We established a light-activatable prodrug strategy that produces the combination effect of photodynamic therapy (PDT) and site-specific chemotherapy. Prodrugs are activated by singlet oxygen (SO), generated from PS and visible or near IR light, in either intra- or inter-molecular manner. The goal of this study is to evaluate cytotoxic effects of nonmitochondria-targeted prodrugs of a number of anticancer drugs with different mechanisms of action. They were tested in both 2D and 3D in vitro conditions via inter-molecular activation of prodrugs by SO generated in mitochondria by protoporphyrin IX-PDT (PpIX-PDT). Prodrugs of anticancer drugs (paclitaxel, SN-38, combretastatin A4 and mitomycin C) were synthesized using facile and high-yielding reactions. Nonmitochondria-targeted prodrugs showed limited dark toxicity while all of them showed greatly enhanced phototoxicity compared to PpIX-PDT in the 2D culture model. Prodrugs generated up to about 95% cell killing at 2.5 µM when administered with hexyl-aminolevulinate (HAL) to produce Protoporphyrin IX in cancer cells in both 2D monolayer and 3D spheroids model. The data demonstrate that mitochondria-targeting of prodrugs is not fully essential for our inter-molecular activation prodrug strategy. The prodrug strategy also worked for anticancer drugs with diverse MOAs.

Chalcone and its analogs: Therapeutic and diagnostic applications in Alzheimer's disease.

Chalcone [(E)-1,3-diphenyl-2-propene-1-one], a small molecule with α, ß unsaturated carbonyl group is a precursor or component of many natural flavonoids and isoflavonoids. It is one of the privileged structures in medicinal chemistry. It possesses a wide range of biological activities encouraging many medicinal chemists to study this scaffold for its usefulness to oncology, infectious diseases, virology and neurodegenerative diseases including Alzheimer's disease (AD). Small molecular size, convenient and cost-effective synthesis, and flexibility for modifications to modulate lipophilicity suitable for blood brain barrier (BBB) permeability make chalcones a preferred candidate for their therapeutic and diagnostic potential in AD. This review summarizes and highlights the importance of chalcone and its analogs as single target small therapeutic agents, multi-target directed ligands (MTDLs) as well as molecular imaging agents for AD. The information summarized here will guide many medicinal chemist and researchers involved in drug discovery to consider chalcone as a potential scaffold for the development of anti-AD agents including theranostics.

1-Isoindolinone scaffold-based natural products with a promising diverse bioactivity.

Isoindolin-1-one or 1-isoindolinone framework is referred to phthalimidines or benzo fused γ-lactams of the corresponding γ-amino carboxylic acids and has been of prime interest for scientists for last several decades. 1-Isoindolinone framework is found in a wide range of naturally occurring compounds with diverse biological activities and therapeutic potential for various chronic diseases. Recent developments in synthetic methods for their procurement have opened a new era of 1-isoindolinone chemistry. This review aims to provide an alphabetical quick reference guide to only 1-isoindolinone based natural products and its variable fused, oxidized and reduced state skeleton with information for advanced chemotaxonomic analyses, cellular targets/pathways and diverse biological activities and future use for medicinal chemistry.

Folate-PEG Conjugates of a Far-Red Light-Activatable Paclitaxel Prodrug to Improve Selectivity toward Folate Receptor-Positive Cancer Cells.

We recently demonstrated the far-red light-activatable prodrug of paclitaxel (PTX), Pc-(L-PTX)2. Upon illumination with a 690 nm laser, Pc-(L-PTX)2 showed combinational cell killing from rapid photodynamic therapy damage by singlet oxygen, followed by sustained chemotherapy effects from locally released PTX. However, its high lipophilicity (log D7.4 > 3.1) caused aggregation in aqueous solutions and has nonselectivity toward cancer cells. To solve these important problems, we prepared folic acid (FA)-conjugated and photoactivatable prodrugs of PTX with a polyethylene glycol (PEG) spacer of various chain lengths: FA-PEG n -Pc-L-PTX [n = 0 (0k, 5), ∼23 (1k, 7a), ∼45 (2k, 7b), ∼80 (3.5k, 7c), or ∼114 (5k, 7d)]. The PEGylated prodrugs 7a-d had a much improved hydrophilicity compared with the non-PEGylated prodrug, Pc-(L-PTX)2. As the PEG length increased, the hydrophilicity of the prodrug increased (log D7.4 values: 1.28, 0.09, -0.24, and -0.59 for 1k, 2k, 3.5k, and 5k PEG prodrugs, respectively). Fluorescence spectral data suggested that the PEGylated prodrugs had good solubility in the culture medium at lower concentrations (<1-2 µM), but showed fluorescence quenching due to limited solubility at higher concentrations (>2 µM). Dynamic light scattering indicated that all of the prodrugs formed nanosized particles in both phosphate-buffered saline and culture medium at a concentration of 5 µM. The PEG length affected both nonspecific and folate receptor (FR)-mediated uptake of the prodrugs. The enhanced cellular uptake was observed for the prodrugs with medium-sized PEGs (1k, 2k, or 3.5k) in FR-positive SKOV-3 cells, but not for the prodrugs with no PEG or with the longest PEG (5k), which suggests the optimal range of PEG length around 1k-3.5k for effective uptake of our prodrug system. Consistent with the cellular uptake pattern, medium-sized PEGylated prodrugs showed more potent phototoxic activity (IC50s, ∼130 nM) than prodrugs with no PEG or the longest PEG (IC50, ∼400 nM). In conclusion, we have developed far-red light-activatable prodrugs with improved water solubility and FR-targeting properties compared with the nontargeted prodrug.

Quantitative modeling of the dynamics and intracellular trafficking of far-red light-activatable prodrugs: implications in stimuli-responsive drug delivery system.

The combination of photodynamic therapy (PDT) with anti-tumor agents is a complimentary strategy to treat local cancers. We developed a unique photosensitizer (PS)-conjugated paclitaxel (PTX) prodrug in which a PS is excited by near-infrared wavelength light to site-specifically release PTX while generating singlet oxygen (SO) to effectively kill cancer cells with both PTX and SO. The aim of the present study was to identify the determinants influencing the combined efficacy of this light-activatable prodrug, especially the bystander killing effects from released PTX. Using PS-conjugated PTX as a model system, we developed a quantitative mathematical model describing the intracellular trafficking. Dynamics of the prodrug and the model predictions were verified with experimental data using human cancer cells in vitro. The sensitivity analysis suggested that parameters related to extracellular concentration of released PTX, prodrug uptake, target engagement, and target abundance are critical in determining the combined killing efficacy of the prodrug. We found that released PTX cytotoxicity was most sensitive to the retention time of the drug in extracellular space. Modulating drug internalization and conjugating the agents targeted to abundant receptors may provide a new strategy for maximizing the killing capacity of the far-red light-activatable prodrug system. These results provide guidance for the design of the PDT combination study in vivo and have implications for other stimuli-responsive drug delivery systems.

Efficient activation of a visible light-activatable CA4 prodrug through intermolecular photo-unclick chemistry in mitochondria.

Photo-unclick chemistry mediates visible and near IR-controlled drug release via a singlet oxygen (SO)-cleavable linker. Due to the limited diffusion distance of SO in biological systems, a photosensitizer and the SO-cleavable linker have been conjugated in one molecule or mixed in nano-drug delivery systems. In this communication, we demonstrate a new strategy to activate prodrugs with photo-unclick chemistry in an intermolecular fashion using an SO-cleavable CA4 prodrug and a mitochondria-specific photosensitizer, protoporphyrin IX, formed from prodrug hexyl-5-aminolevulinate.

Synthesis and biological evaluation of 2-phenol-4-chlorophenyl-6-aryl pyridines as topoisomerase II inhibitors and cytotoxic agents.

A new series of 2-phenol-4-chlorophenyl-6-aryl pyridines were designed, synthesized, and evaluated for topoisomerase (topo) I and II inhibitory activities as well as cytotoxic activity against four different human cancer cell lines such as HCT15, T47D, DU145, and Hela. Most of the tested compounds exhibited stronger topo II inhibitory activity at 100µM as compared to etoposide. All the compounds, except 39, did not show topo I inhibitory activity. Interestingly, compounds that showed better topo II inhibition than etoposide have ortho- or para-chlorophenyl at 4-position of central pyridine, and none of the compounds possess meta-chlorophenyl. SAR study revealed the importance of ortho- or para-chlorophenyl at 4-position of the central pyridine for selective topo II inhibitory activity. Similarly, all compounds possessing meta- or para-hydroxyphenyl moieties showed moderate to significant cytotoxic effects. Particularly, compounds 27-37, and 39 which showed excellent cytotoxicity (IC50=0.68-1.25µM) against T47D breast cancer cells suggest the importance of meta- or para-hydroxyphenyl moiety at 2-position of the central pyridine for the design of anticancer agents with related scaffolds.

Far-Red Light-Activatable Prodrug of Paclitaxel for the Combined Effects of Photodynamic Therapy and Site-Specific Paclitaxel Chemotherapy.

Paclitaxel (PTX) is one of the most useful chemotherapeutic agents approved for several cancers, including ovarian, breast, pancreatic, and nonsmall cell lung cancer. However, it causes systemic side effects when administered parenterally. Photodynamic therapy (PDT) is a new strategy for treating local cancers using light and photosensitizer. Unfortunately, PDT is often followed by recurrence due to incomplete ablation of tumors. To overcome these problems, we prepared the far-red light-activatable prodrug of PTX by conjugating photosensitizer via singlet oxygen-cleavable aminoacrylate linker. Tubulin polymerization enhancement and cytotoxicity of prodrugs were dramatically reduced. However, once illuminated with far-red light, the prodrug effectively killed SKOV-3 ovarian cancer cells through the combined effects of PDT and locally released PTX. Ours is the first PTX prodrug that can be activated by singlet oxygen using tissue penetrable and clinically useful far-red light, which kills the cancer cells through the combined effects of PDT and site-specific PTX chemotherapy.

Anticancer drug released from near IR-activated prodrug overcomes spatiotemporal limits of singlet oxygen.

Photodynamic therapy (PDT) is a cancer treatment modality where photosensitizer (PS) is activated by visible and near IR light to produce singlet oxygen ((1)O2). However, (1)O2 has a short lifetime (<40 ns) and cannot diffuse (<20 nm) beyond the cell diameter (e.g., ∼ 1800 nm). Thus, (1)O2 damage is both spatially and temporally limited and does not produce bystander effect. In a heterogeneous tumor, cells escaping (1)O2 damage can regrow after PDT treatment. To overcome these limitations, we developed a prodrug concept (PS-L-D) composed of a photosensitizer (PS), an anti-cancer drug (D), and an (1)O2-cleavable linker (L). Upon illumination of the prodrug, (1)O2 is generated, which damages the tumor and also releases anticancer drug. The locally released drug could cause spatially broader and temporally sustained damage, killing the surviving cancer cells after the PDT damage. In our previous report, we presented the superior activity of our prodrug of CA4 (combretastatin A-4), Pc-(L-CA4)2, compared to its non-cleavable analog, Pc-(NCL-CA4)2, that produced only PDT effects. Here, we provide clear evidence demonstrating that the released anticancer drug, CA4, indeed damages the surviving cancer cells over and beyond the spatial and temporal limits of (1)O2. In the limited light illumination experiment, cells in the entire well were killed due to the effect of released anti-cancer drug, whereas only a partial damage was observed in the pseudo-prodrug treated wells. A time-dependent cell survival study showed more cell death in the prodrug-treated cells due to the sustained damage by the released CA4. Cell cycle analysis and microscopic imaging data demonstrated the typical damage patterns by CA4 in the prodrug treated cells. A time-dependent histological study showed that prodrug-treated tumors lacked mitotic bodies, and the prodrug caused broader and sustained tumor size reduction compared to those seen in the tumors treated with the pseudo-prodrug. This data consistently support that the released CA4 overcomes the spatiotemporal limitations of (1)O2, providing far superior antitumor effect.

Design and synthesis of conformationally constrained hydroxylated 4-phenyl-2-aryl chromenopyridines as novel and selective topoisomerase II-targeted antiproliferative agents.

To develop novel selective topoisomerase II inhibitors, we designed and synthesized a series of conformationally constrained hydroxylated 4-phenyl-2-aryl chromenopyridines and evaluated their topoisomerase inhibitory activity and cytotoxicity against three human cancer cell lines (DU145, HCT15, and T47D) and a normal cell line (MCF10A). All of the prepared compounds displayed stronger or similar topoisomerase II inhibitory activity as well as cytotoxicity against three human cancer cell lines compared to etoposide. Compounds 10a, 10g, 11a, 11f, 11g, 12a, 12f, and 12g especially showed stronger topoisomerase II inhibitory activity as compared to etoposide at both 100 µM and 20 µM. A structure-activity relationship study revealed that hydroxyphenyl moiety at 4-position of pyridine and ortho-hydroxyphenyl or thienyl moiety at 2-position of pyridine has an important role in displaying selective topoisomerase II inhibition. The compound 12b with para-hydroxyphenyl and meta-hydroxyphenyl at 4- and 2-position of pyridine, respectively, showed the most significant cytotoxicity against all three cancer cell lines, whereas less cytotoxicity to a normal cell line as compared to adriamycin.

Synthesis, topoisomerase I and II inhibitory activity, cytotoxicity, and structure-activity relationship study of 2-phenyl- or hydroxylated 2-phenyl-4-aryl-5H-indeno[1,2-b]pyridines.

A series of novel twenty-eight rigid 2-phenyl- or hydroxylated 2-phenyl-4-aryl-5H-indeno[1,2-b]pyridines were synthesized and evaluated for their topoisomerase inhibitory activity as well as their cytotoxicity against several human cancer cell lines. Generally, hydroxylated compounds (16-18, 22-25, and 29-31) containing furyl or thienyl moiety at 4-position of central pyridine exhibited strong topoisomerase I and II inhibitory activity compared to positive control, camptothecin and etoposide, respectively, in low micromolar range. Structure-activity relationship study revealed that indenopyridine compounds with hydroxyl group at 2-phenyl ring in combination with furyl or thienyl moiety at 4-position are important for topoisomerase inhibition. Compounds (22-25) which contain hydroxyl group at meta position of the 2-phenyl ring at 2-position and furanyl or thienyl substitution at 4-position of indenopyridine, showed concrete correlations between topo I and II inhibitory activity, and cytotoxicity against evaluated human cancer cell lines.

A series of novel terpyridine-skeleton molecule derivants inhibit tumor growth and metastasis by targeting topoisomerases.

A series of novel terpyridine-skeleton molecules containing conformational rigidity, 14 containing benzo[4,5]furo[3,2-b]pyridine core and 15 comprising chromeno[4,3-b]pyridine core, were synthesized, and their biological activities were evaluated. 3-(4-Phenylbenzo[4,5]furo[3,2-b]pyridin-2-yl)phenol (8) was determined to be a nonintercalative topo I and II dual catalytic inhibitor and 3-(4-phenylchromeno[4,3-b]pyridine-2-yl)phenol (22) was determined to be a nonintercalative topo IIα specific catalytic inhibitor by various assays. These two catalytic inhibitors induced apoptosis in addition to G1 arrest in T47D human breast cancer cells with much less DNA toxicity than etoposide. Compounds 8 and 22 significantly inhibited tumor growth in HCT15 subcutaneously implanted xenografted mice. The modification of compounds 8 and 22 with the introduction of a methoxy instead of a hydroxy group enhanced endogenous topo inhibitory activity, metabolic stability in diverse types of liver microsomes and improved pharmacokinetic parameters in rat plasma such as augmentation of bioavailability (41.3% and 33.2% for 2-(3-methoxyphenyl)-4-phenylbenzofuro[3,2-b]pyridine (8-M) and 3-(4-phenylchromeno[4,3-b]pyridine-2-yl)methoxybenzene (22-M), respectively).

Synthesis, antitumor activity, and structure-activity relationship study of trihydroxylated 2,4,6-triphenyl pyridines as potent and selective topoisomerase II inhibitors.

A series of eighteen trihydroxylated 2,4,6-triphenyl pyridines were designed and synthesized which contain hydroxyl groups at ortho, meta or para position of each phenyl rings attached to the central pyridine. They were evaluated for topoisomerase I and II inhibitory activity, and cytotoxicity against several human cancer cell lines for the development of novel anticancer agents. Most of the compounds exhibited strong and selective topoisomerase II inhibitory activity compared to the positive control, etoposide, and also displayed significant cytotoxicity in low micromolar range. Trihydroxylated 2,4,6-triphenyl pyridines were more potent than mono- and di-hydroxylated 2,4,6-triphenyl pyridines, which have been previously studied in our research group. Positive correlation between topoisomerase II inhibitory activity and cytotoxicity was observed for the most compounds. Molecular docking study shows qualitatively consistent with the results of biological assays.

Discovery of dihydroxylated 2,4-diphenyl-6-thiophen-2-yl-pyridine as a non-intercalative DNA-binding topoisomerase II-specific catalytic inhibitor.

We describe our rationale for designing specific catalytic inhibitors of topoisomerase II (topo II) over topoisomerase I (topo I). Based on 3D-QSAR studies of previously published dihydroxylated 2,4-diphenyl-6-aryl pyridine derivatives, 9 novel dihydroxylated 2,4-diphenyl-6-thiophen-2-yl pyridine compounds were designed, synthesized, and their biological activities were evaluated. These compounds have 2-thienyl ring substituted on the R(3) group on the pyridine ring and they all showed excellent specificity toward topo II compared to topo I. In vitro experiments were performed for compound 13 to determine the mechanism of action for this series of compounds. Compound 13 inhibited topoisomerase II specifically by non-intercalative binding to DNA and did not stabilize enzyme-cleavable DNA complex. Compound 13 efficiently inhibited cell viability, cell migration, and induced G1 arrest. Also from 3D-QSAR studies, the results were compared with other previously published dihydroxylated 2,4-diphenyl-6-aryl pyridine derivatives to explain the structure-activity relationships.

Retinoic acid signaling pathways in development and diseases.

Retinoids comprise a group of compounds each composed of three basic parts: a trimethylated cyclohexene ring that is a bulky hydrophobic group, a conjugated tetraene side chain that functions as a linker unit, and a polar carbon-oxygen functional group. Biochemical conversion of carotenoid or other retinoids to retinoic acid (RA) is essential for normal regulation of a wide range of biological processes including development, differentiation, proliferation, and apoptosis. Retinoids regulate various physiological outputs by binding to nuclear receptors called retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which themselves are DNA-binding transcriptional regulators. The functional response of RA and their receptors are modulated by a host of coactivators and corepressors. Retinoids are essential in the development and function of several organ systems; however, deregulated retinoid signaling can contribute to serious diseases. Several natural and synthetic retinoids are in clinical use or undergoing trials for treating specific diseases including cancer. In this review, we provide a broad overview on the importance of retinoids in development and various diseases, highlighting various retinoids in the drug discovery process, ranging all the way from retinoid chemistry to clinical uses and imaging.

Use of zebrafish in chemical biology and drug discovery.

The zebrafish (Danio rerio) is a small, tropical, freshwater fish that has emerged as a powerful vertebrate model organism for studying genetics and development. Its small size, transparency, cost-effectiveness, close genome homology to humans compared with invertebrates, and capacity for genetic manipulation are all valuable attributes for an excellent animal model. There are additional advantages for using zebrafish specifically in drug discovery, including ease of exposure to chemicals in water. In effect, zebrafish can bridge a gap between in vitro and mammalian work, reducing the use of larger animals and attrition rates. In the drug-discovery process, zebrafish can be used at many stages, including target identification and validation, identification of lead compounds, studying structure-activity relationships and drug safety profiling. In this review, we highlight the potential for the zebrafish model to make the drug-discovery process simpler, more effective and cost-efficient.

Boron chemicals in diagnosis and therapeutics.

Advances in the field of boron chemistry have expanded the application of boron from material use to medicine. Boron-based drugs represent a new class of molecules that possess several biomedical applications including use as imaging agents for both optical and nuclear imaging as well as therapeutic agents with anticancer, antiviral, antibacterial, antifungal and other disease-specific activities. For example, bortezomib (Velcade(®)), the only drug in clinical use with boron as an active element, was approved in 2003 as a proteasome inhibitor for the treatment of multiple myeloma and non-Hodgkin's lymphoma. Several other boron-based compounds are in various phases of clinical trials, which illustrates the promise of this approach for medicinal chemists working in the area of boron chemistry. It is expected that in the near future, several boron-containing drugs should become available in the market with better efficacy and potency than existing drugs. This article discusses the current status of the development of boron-based compounds as diagnostic and therapeutic agents in humans.