Challenges and opportunities of developing small-molecule therapies for age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of vision loss in senior adults. The disease can be categorized into two types: wet AMD and dry AMD. Wet AMD, also known as exudative or neovascular AMD, is less common but more severe than dry AMD and is responsible for 90% of the visual impairment caused by AMD and affects 20 million people worldwide. Current treatment options mainly involve biologics that inhibit the vascular endothelial growth factor or complement pathways. However, these treatments have limitations such as high cost, injection-related risks, and limited efficacy. Therefore, new therapeutic targets and strategies have been explored to improve the outcomes of patients with AMD. A promising approach is the use of small-molecule drugs that modulate different factors involved in AMD pathogenesis, such as tyrosine kinases and integrins. Small-molecule drugs offer advantages, such as oral administration, low cost, good penetration, and increased specificity for the treatment of wet and dry AMD. This review summarizes the current status and prospects of small-molecule drugs for the treatment of wet AMD. These advances are expected to support the development of effective and targeted treatments for patients with AMD.

(S)-3-(3-Fluoro-4-Methoxybenzyl)-5,6,7-Trimethoxychroman-4-One Suppresses the Proliferation of Huh7 Cells by Up-regulating P21 and Inducing G2/M Phase Arrest.

BACKGROUND/AIM: Hepatocellular carcinoma (HCC) is a prevalent type of cancer worldwide. Although sorafenib is the only chemotherapy agent used for HCC, there is a need to discover a more potent anticancer agent with reduced side-effects. The compound, (S)-3-(3-fluoro-4-methoxybenzyl)-5,6,7-trimethoxychroman-4-one (FMTC), was designed to inhibit tubulin assembly but its specific mechanisms of action have not been previously investigated. Herein, we investigated the regulation mechanisms by which FMTC affects the proliferation of the HCC cell line, Huh7. MATERIALS AND METHODS: The effects of FMTC on cell viability and growth were analyzed in the HCC cell line, Huh7. Cell cycle and apoptosis regulated by FMTC were analyzed using flow cytometry. To verify the regulation of mRNA and protein expression of cell proliferation-related factors by FMTC in Huh7 cells, RT-qPCR and western blot analyses were employed. RESULTS: FMTC suppressed cell division dose-dependently by triggering cell cycle arrest at the G2/M phase via p21 up-regulation. The increased phosphorylation of histone H3 on Ser-10 and the condensation of chromatin in FMTC-treated cells indicated mitotic arrest. Prolonged FMTC-induced cell cycle arrest triggered apoptosis. CONCLUSION: FMTC inhibits the proliferation of human liver cancer cells by up-regulating p21, thereby inducing cell cycle arrest at the G2/M phase. These findings highlight FMTC as a novel agent for HCC treatment.

Cremastranone-Derived Homoisoflavanes Suppress the Growth of Breast Cancer Cells via Cell Cycle Arrest and Caspase-Independent Cell Death.

Breast cancer is the most common cancer and a frequent cause of cancer-related deaths among women wordlwide. As therapeutic strategies for breast cancer have limitations, novel chemotherapeutic reagents and treatment strategies are needed. In this study, we investigated the anti-cancer effect of synthetic homoisoflavane derivatives of cremastranone on breast cancer cells. Homoisoflavane derivatives, SH-17059 and SH-19021, reduced cell proliferation through G2/M cell cycle arrest and induced caspase-independent cell death. These compounds increased heme oxygenase-1 (HO-1) and 5-aminolevulinic acid synthase 1 (ALAS1), suggesting downregulation of heme. They also induced reactive oxygen species (ROS) generation and lipid peroxidation. Furthermore, they reduced expression of glutathione peroxidase 4 (GPX4). Therefore, we suggest that the SH-17059 and SH-19021 induced the caspase-independent cell death through the accumulation of iron from heme degradation, and the ferroptosis might be one of the potential candidates for caspase-independent cell death.

Candesartan, an angiotensin-II receptor blocker, ameliorates insulin resistance and hepatosteatosis by reducing intracellular calcium overload and lipid accumulation.

Insulin resistance is a major contributor to the pathogenesis of several human diseases, including type 2 diabetes, hypertension, and hyperlipidemia. Notably, insulin resistance and hypertension share common abnormalities, including increased oxidative stress, inflammation, and organelle dysfunction. Recently, we showed that excess intracellular Ca2+, a known pathogenic factor in hypertension, acts as a critical negative regulator of insulin signaling by forming Ca2+-phosphoinositides that prevent the membrane localization of AKT, a key serine/threonine kinase signaling molecule. Whether preventing intracellular Ca2+ overload improves insulin sensitivity, however, has not yet been investigated. Here, we show that the antihypertensive agent candesartan, compared with other angiotensin-II receptor blockers, has previously unrecognized beneficial effects on attenuating insulin resistance. We found that candesartan markedly reduced palmitic acid (PA)-induced intracellular Ca2+ overload and lipid accumulation by normalizing dysregulated store-operated channel (SOC)-mediated Ca2+ entry into cells, which alleviated PA-induced insulin resistance by promoting insulin-stimulated AKT membrane localization and increased the phosphorylation of AKT and its downstream substrates. As pharmacological approaches to attenuate intracellular Ca2+ overload in vivo, administering candesartan to obese mice successfully decreased insulin resistance, hepatic steatosis, dyslipidemia, and tissue inflammation by inhibiting dysregulated SOC-mediated Ca2+ entry and ectopic lipid accumulation. The resulting alterations in the phosphorylation of key signaling molecules consequently alleviate impaired insulin signaling by increasing the postprandial membrane localization and phosphorylation of AKT. Thus, our findings provide robust evidence for the pleiotropic contribution of intracellular Ca2+ overload in the pathogenesis of insulin resistance and suggest that there are viable approved drugs that can be repurposed for the treatment of insulin resistance and hypertension.

Synthetic Homoisoflavane Derivatives of Cremastranone Suppress Growth of Colorectal Cancer Cells through Cell Cycle Arrest and Induction of Apoptosis.

Colorectal cancer is diagnosed as the third most prevalent cancer; thus, effective therapeutic agents are urgently required. In this study, we synthesized six homoisoflavane derivatives of cremastranone and investigated their cytotoxic effects on the human colorectal cancer cell lines HCT116 and LoVo. We further examined the related mechanisms of action using two of the potent compounds, SH-19027 and SHA-035. They substantially reduced the cell viability and proliferation in a dose-dependent manner. Treatment with SH-19027 and SHA-035 induced cell cycle arrest at the G2/M phase and increased expression of p21 both of which are implicated in cell cycle control. In addition, the apoptotic cell population and apoptosis-associated marker expression were accordingly increased. These results suggest that the synthesized cremastranone derivatives have anticancer effects through the suppression of cell proliferation and induction of apoptosis. Therefore, the synthesized cremastranone derivatives could be applied as novel therapeutic agents against colorectal cancer.

Suppressive effect of α-mangostin for cancer stem cells in colorectal cancer via the Notch pathway.

BACKGROUND: Since colon cancer stem cells (CSCs) play an important role in chemoresistance and in tumor recurrence and metastasis, targeting of CSCs has emerged as a sophisticated strategy for cancer therapy. α-mangostin (αM) has been confirmed to have antiproliferative and apoptotic effects on cancer cells. This study aimed to evaluate the selective inhibition of αM on CSCs in colorectal cancer (CRC) and the suppressive effect on 5-fluorouracil (5-FU)-induced CSCs. METHODS: The cell viability assay was performed to determine the optimal concentration of αM. A sphere forming assay and flow cytometry with CSC markers were carried out to evaluate the αM-mediated inhibition of CSCs. Western blot analysis and quantitative real-time PCR were performed to investigate the effects of αM on the Notch signaling pathway and colon CSCs. The in vivo anticancer efficacy of αM in combination with 5-FU was investigated using a xenograft mouse model. RESULTS: αM inhibited the cell viability and reduced the number of spheres in HT29 and SW620 cells. αM treatment decreased CSCs and suppressed the 5-FU-induced an increase in CSCs on flow cytometry. αM markedly suppressed Notch1, NICD1, and Hes1 in the Notch signaling pathway in a time- and dose-dependent manner. Moreover, αM attenuated CSC markers CD44 and CD133, in a manner similar to that upon DAPT treatment, in HT29 cells. In xenograft mice, the tumor and CSC makers were suppressed in the αM group and in the αM group with 5-FU treatment. CONCLUSION: This study shows that low-dose αM inhibits CSCs in CRC and suppresses 5-FU-induced augmentation of CSCs via the Notch signaling pathway.

Small-molecule inhibitors of ferrochelatase are antiangiogenic agents.

Activity of the heme synthesis enzyme ferrochelatase (FECH) is implicated in multiple diseases. In particular, it is a mediator of neovascularization in the eye and thus an appealing therapeutic target for preventing blindness. However, no drug-like direct FECH inhibitors are known. Here, we set out to identify small-molecule inhibitors of FECH as potential therapeutic leads using a high-throughput screening approach to identify potent inhibitors of FECH activity. A structure-activity relationship study of a class of triazolopyrimidinone hits yielded drug-like FECH inhibitors. These compounds inhibit FECH in cells, bind the active site in cocrystal structures, and are antiangiogenic in multiple in vitro assays. One of these promising compounds was antiangiogenic in vivo in a mouse model of choroidal neovascularization. This foundational work may be the basis for new therapeutic agents to combat not only ocular neovascularization but also other diseases characterized by FECH activity.

Inhibitory Effect of Chlorogenic Acid Analogues Comprising Pyridine and Pyrimidine on α-MSH-Stimulated Melanogenesis and Stability of Acyl Analogues in Methanol.

In continuation of studies for α-MSH stimulated melanogenesis inhibitors, we have evaluated the design, synthesis, and activity of a new series of chlorogenic acid (CGA) analogues comprising pyridine, pyrimidine, and diacyl derivatives. Among nineteen synthesized compounds, most of them (fifteen) exhibited better inhibitions of melanin formation in B16 melanoma cells. The results illustrated that a pyridine analogue 6f and a diacyl derivative 13a of CGA showed superior inhibition profiles (IC50: 2.5 ± 0.7 µM and 1.1 ± 0.1 µM, respectively) of α-MSH activities than positive controls, kojic acid and arbutin (IC50: 54 ± 1.5 µM and 380 ± 9.5 µM, respectively). The SAR studies showed that both -CF3 and -Cl groups exhibited better inhibition at the meta position on benzylamine than their ortho and para positions. In addition, the stability of diacyl analogues of CGA in methanol monitored by HPLC for 28 days indicated the steric bulkiness of acyl substituents as a key factor in their stability.

Allyl Isothiocyanate Protects Acetaminophen-Induced Liver Injury via NRF2 Activation by Decreasing Spontaneous Degradation in Hepatocyte.

Acetaminophen (APAP) is one of the most frequently prescribed analgesic and anti-pyretic drugs. However, APAP-induced hepatotoxicity is a major cause of acute liver failure globally. While the therapeutic dose is safe, an overdose of APAP produces an excess of the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI), subsequently resulting in hepatotoxicity. Allyl isothiocyanate (AITC), a bioactive molecule in cruciferous plants, is reported to exert various biological effects, including anti-inflammatory, anti-cancer, and anti-microbial effects. Notably, AITC is known for activating nuclear factor erythroid 2-related factor 2 (NRF2), but there is limited evidence supporting the beneficial effects on hepatocytes and liver, where AITC is mainly metabolized. We applied a mouse model in the current study to investigate whether AITC protects the liver against APAP-induced injury, wherein we observed the protective effects of AITC. Furthermore, NRF2 nuclear translocation and the increase of target genes by AITC treatment were confirmed by in vitro experiments. APAP-induced cell damage was attenuated by AITC via an NRF2-dependent manner, and rapid NRF2 activation by AITC was attributed to the elevation of NRF2 stability by decreasing its spontaneous degradation. Moreover, liver tissues from our mouse experiment revealed that AITC increases the expression of heme oxygenase-1 (HO-1), an NRF2 target gene, confirming the potential of AITC as a hepatoprotective agent that induces NRF2 activation. Taken together, our results indicate the potential of AITC as a natural-product-derived NRF2 activator targeting the liver.

Honokiol ameliorates angiotensin II-induced hypertension and endothelial dysfunction by inhibiting HDAC6-mediated cystathionine γ-lyase degradation.

Hypertension and endothelial dysfunction are associated with various cardiovascular diseases. Hydrogen sulphide (H2 S) produced by cystathionine γ-lyase (CSE) promotes vascular relaxation and lowers hypertension. Honokiol (HNK), a natural compound in the Magnolia plant, has been shown to retain multifunctional properties such as anti-oxidative and anti-inflammatory activities. However, a potential role of HNK in regulating CSE and hypertension remains largely unknown. Here, we aimed to demonstrate that HNK co-treatment attenuated the vasoconstriction, hypertension and H2 S reduction caused by angiotensin II (AngII), a well-established inducer of hypertension. We previously found that histone deacetylase 6 (HDAC6) mediates AngII-induced deacetylation of CSE, which facilitates its ubiquitination and proteasomal degradation. Our current results indicated that HNK increased endothelial CSE protein levels by enhancing its stability in a sirtuin-3-independent manner. Notably, HNK could increase CSE acetylation levels by inhibiting HDAC6 catalytic activity, thereby blocking the AngII-induced degradative ubiquitination of CSE. CSE acetylation and ubiquitination occurred mainly on the lysine 73 (K73) residue. Conversely, its mutant (K73R) was resistant to both acetylation and ubiquitination, exhibiting higher protein stability than that of wild-type CSE. Collectively, our findings suggested that HNK treatment protects CSE against HDAC6-mediated degradation and may constitute an alternative for preventing endothelial dysfunction and hypertensive disorders.

SETD1A Promotes Proliferation of Castration-Resistant Prostate Cancer Cells via FOXM1 Transcription.

Androgen deprivation therapy eventually leads to the development of castration-resistant prostate cancer (CRPC). Here, we demonstrate for the first time that the histone H3K4 methyltransferase SETD1A is a major regulator for the proliferation of metastatic CRPC (mCRPC). The expression of SETD1A was significantly correlated with the survival rate of patients with prostate cancer. SETD1A, which is expressed at a higher level in mCRPC than in primary prostate cancer cells, promotes the expression of FOXM1, a gene encoding a cell proliferation-specific transcription factor. SETD1A is recruited to the promoter region of FOXM1 (forkhead box M1) upon binding to E2F1, a protein that regulates the transcription of FOXM1 and contributes to the trimethylation of H3K4 in the FOXM1 promoter region. In addition, SETD1A is essential for the expression of stem cell factor (e.g., OCT4, octamer-binding transcription factor 4) and stem cell formation in mCRPC, suggesting the importance of SETD1A expression in mCRPC tumor formation. Notably, poor prognosis is associated with high expression of the SETD1A-FOXM1 pair in clinical data sets. Therefore, our study suggests that SETD1A plays an important role in the proliferation of mCRPC by regulating FOXM1 transcription.

SG-SP1 Suppresses Mast Cell-Mediated Allergic Inflammation via Inhibition of FcεRI Signaling.

Background: As the number of allergic disease increases, studies to identify new treatments take on new urgency. Epigallocatechin gallate (EGCG), a major component of green tea, has been shown to possess a wide range of pharmacological properties, including anti-inflammation and anti-viral infection. In previous study, gallic acid (GA), a part of EGCG, has shown anti-allergic inflammatory effect. To improve on preliminary evidence that GA has allergy mitigating effect, we designed SG-SP1 based on GA, and aimed to assess the effects of SG-SP1 on mast cell-mediated allergic inflammation using various animal and in vitro models. Methods: For in vitro experiments, various types of IgE-stimulated mast cells (RBL-2H3: mast cell-like basophilic leukemia cells, and primary cultured peritoneal and bone marrow-derived mast cells) were used to determine the role of SG-SP1 (0.1-1 nM). Immunoglobulin (Ig) E-induced passive cutaneous anaphylaxis and ovalbumin-induced systemic anaphylaxis, standard animal models for immediate-type hypersensitivity were also used. Results: For in vitro, SG-SP1 reduced degranulation of mast cells by down-regulating intracellular calcium levels in a concentration-dependent manner. SG-SP1 decreased expression and secretion of inflammatory cytokines in activated mast cells. This suppressive effect was associated with inhibition of the phosphorylation of Lyn, Syk and Akt, and the nuclear translocation of nuclear factor-κB. Due to the strong inhibitory effect of SG-SP1 on Lyn, the known upstream signaling to FcεRI-dependent pathway, we confirmed the direct binding of SG-SP1 to FcεRI, a high affinity IgE receptor by surface plasmon resonance experiment. Oral administration of SG-SP1 hindered allergic symptoms of both anaphylaxis models evidenced by reduction of hypothermia, serum IgE, ear thickness, and tissue pigmentation. This inhibition was mediated by the reductions in serum histamine and interleukin-4. Conclusions: We determined that SG-SP1 directly interacts with FcεRI and propose SG-SP1 as a therapeutic candidate for mast cell-mediated allergic inflammatory disorders via inhibition of FcεRI signaling.

Parthenolide inhibits transforming growth factor ß1-induced epithelial-mesenchymal transition in colorectal cancer cells.

BACKGROUND/AIMS: Transforming growth factor-ß1 (TGF-ß1) induction of epithelial-mesenchymal transition (EMT) is one of the mechanisms by which colorectal cancer (CRC) cells acquire migratory and invasive capacities, and subsequently metastasize. Parthenolide (PT) expresses multiple anti-cancer and anti-inflammatory activities that inhibit nuclear factor κB by targeting the IκB kinase complex. In the present study, we aimed to investigate whether PT can inhibit TGF-ß1-induced EMT in CRC cell lines. METHODS: HT-29 and SW480 cell lines were used in the experiment. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and sub-G1 analysis was measured by flow cytometry. The induction of EMT by TGF-ß1 and inhibition of the process by PT was analyzed by phase contrast microscopy, wounding healing, cellular migration and invasion assays, and Western blotting. RESULTS: TGF-ß1 inhibits HT-29 cell proliferation, but has no effect on SW480 cell proliferation; different concentrations of TGF-ß1 did not induce apoptosis in HT-29 and SW480 cells. PT attenuates TGF-ß1-induced elongated, fibroblast-like shape changing in cells. PT inhibits TGF-ß1-induced cell migration and cell invasion. In addition, other EMT markers such as ß-catenin, Vimentin, Snail, and Slug were suppressed by PT, while E-cadherin was increased by PT. CONCLUSIONS: Our findings show that PT inhibits TGF-ß1-induced EMT by suppressing the expression of the mesenchymal protein and increasing expression of the epithelial protein. These findings suggest a novel approach for CRC treatment by suppression of TGF-ß1-induced EMT.

Enantioselective Synthesis of Homoisoflavanones by Asymmetric Transfer Hydrogenation and Their Biological Evaluation for Antiangiogenic Activity.

Neovascular eye diseases are a major cause of blindness. Excessive angiogenesis is a feature of several conditions, including wet age-related macular degeneration, proliferative diabetic retinopathy, and retinopathy of prematurity. Development of novel antiangiogenic small molecules for the treatment of neovascular eye disease is essential to provide new therapeutic leads for these diseases. We have previously reported the therapeutic potential of anti-angiogenic homoisoflavanone derivatives with efficacy in retinal and choroidal neovascularization models, although these are racemic compounds due to the C3-stereogenic center in the molecules. This work presents asymmetric synthesis and structural determination of anti-angiogenic homoisoflavanones and pharmacological characterization of the stereoisomers. We describe an enantioselective synthesis of homoisoflavanones by virtue of ruthenium-catalyzed asymmetric transfer hydrogenation accompanying dynamic kinetic resolution, providing a basis for the further development of these compounds into novel experimental therapeutics for neovascular eye diseases.

Sulfisoxazole inhibits the secretion of small extracellular vesicles by targeting the endothelin receptor A.

Inhibitors of the secretion of cancer exosomes, which promote cancer progression and metastasis, may not only accelerate exosome biology research but also offer therapeutic benefits for cancer patients. Here we identify sulfisoxazole (SFX) as an inhibitor of small extracellular vesicles (sEV) secretion from breast cancer cells through interference with endothelin receptor A (ETA). SFX, an FDA-approved oral antibiotic, showed significant anti-tumor and anti-metastatic effects in mouse models of breast cancer xenografts, the reduced expression of proteins involved in biogenesis and secretion of sEV, and triggered co-localization of multivesicular endosomes with lysosomes for degradation. We demonstrate the important role of ETA, as target of SFX, by gain- and loss-of-function studies of the ETA protein, through a direct binding assay, and pharmacological and genetic approaches. These findings may provide a foundation for sEV-targeted cancer therapies and the mechanistic studies on sEV biology.

Novel Hypoxia-Inducible Factor 1α (HIF-1α) Inhibitors for Angiogenesis-Related Ocular Diseases: Discovery of a Novel Scaffold via Ring-Truncation Strategy.

Ocular diseases featuring pathologic neovascularization are the leading cause of blindness, and anti-VEGF agents have been conventionally used to treat these diseases. Recently, regulating factors upstream of VEGF, such as HIF-1α, have emerged as a desirable therapeutic approach because the use of anti-VEGF agents is currently being reconsidered due to the VEGF action as a trophic factor. Here, we report a novel scaffold discovered through the complete structure-activity relationship of ring-truncated deguelin analogs in HIF-1α inhibition. Interestingly, analog 6i possessing a 2-fluorobenzene moiety instead of a dimethoxybenzene moiety exhibited excellent HIF-1α inhibitory activity, with an IC50 value of 100 nM. In particular, the further ring-truncated analog 34f, which showed enhanced HIF-1α inhibitory activity compared to analog 2 previously reported by us, inhibited in vitro angiogenesis and effectively suppressed hypoxia-mediated retinal neovascularization. Importantly, the heteroatom-substituted benzene ring as a key structural feature of analog 34f was identified as a novel scaffold for HIF-1α inhibitors that can be used in lieu of a chromene ring.

Chemical Proteomics Reveals Soluble Epoxide Hydrolase as a Therapeutic Target for Ocular Neovascularization.

The standard-of-care therapeutics for the treatment of ocular neovascular diseases like wet age-related macular degeneration (AMD) are biologics targeting vascular endothelial growth factor signaling. There are currently no FDA approved small molecules for treating these blinding eye diseases. Therefore, therapeutic agents with novel mechanisms are critical to complement or combine with existing approaches. Here, we identified soluble epoxide hydrolase (sEH), a key enzyme for epoxy fatty acid metabolism, as a target of an antiangiogenic homoisoflavonoid, SH-11037. SH-11037 inhibits sEH in vitro and in vivo and docks to the substrate binding cleft in the sEH hydrolase domain. sEH levels and activity are up-regulated in the eyes of a choroidal neovascularization (CNV) mouse model. sEH is overexpressed in human wet AMD eyes, suggesting that sEH is relevant to neovascularization. Known sEH inhibitors delivered intraocularly suppressed CNV. Thus, by dissecting a bioactive compound's mechanism, we identified a new chemotype for sEH inhibition and characterized sEH as a target for blocking the CNV that underlies wet AMD.

Synthesis, biological evaluation, and metabolic stability of chlorogenic acid derivatives possessing thiazole as potent inhibitors of α-MSH-stimulated melanogenesis.

A series of catechol and dioxolane analogs containing thiazole CGA derivatives have been synthesized and evaluated for their inhibitory activity against α-MSH. The inhibitory activity was improved by replacing an α,ß-unsaturated carbonyl of previously reported caffeamides with thiazole motif. Surprisingly, compound 7d, one of the derivatives of dioxolane analogs, displayed the most potent inhibitory activity with an IC50 of 0.90µM. Further studies on metabolic stability and bioactivation potential were also accomplished.

Synthesis and biological evaluation of caffeic acid derivatives as potent inhibitors of α-MSH-stimulated melanogenesis.

We have disclosed our effort to develop caffeic acid derivatives as potent and non-toxic inhibitors of α-MSH-stimulated melanogenesis to treat pigmentation disorders and skin medication including a cosmetic skin-whitening agent. The SAR studies revealed that cyclohexyl ester and secondary amide derivatives of caffeic acid showed significant inhibitory activities.