Synthesis and biological evaluation of niclosamide PROTACs.

Roughly 268,000 new cases of prostate cancer and 34,000 deaths from prostate cancer are projected by the American Cancer Society to occur in the United States in 2022. Androgen receptor is a key protein in the proliferation and survival of prostate cancer cells and has been revealed to be overexpressed in 30% to 50% of castration-resistant prostate cancer patients. One promising approach to reducing the level of this protein is Proteolysis Targeting Chimeras (PROTACs) that is an emerging drug discovery technology. PROTACs are hetero-bifunctional molecules where one end binds to a protein of interest and the other to an E3 ligase ligand, initiating the Ubiquitin-Proteasome Pathway for protein degradation. Two PROTACs with niclosamide as androgen receptor ligand and VHL-032 as the E3 ligase ligand have been designed and synthesized for suppressing proliferation of androgen receptor-positive prostate cancer cells via degrading androgen receptor. The in vitro antiproliferative assessment suggested that they can selectively suppress PC-3, LNCaP, and 22Rv1 prostate cancer cell proliferation, but cannot inhibit DU145 cell proliferation. However, the mechanism of both compounds in suppressing prostate cancer cell proliferation is not through the AR PROTAC mechanism because they did not degrade AR in our Western Blotting assay up to 1 µM.

GLL398, an oral selective estrogen receptor degrader (SERD), blocks tumor growth in xenograft breast cancer models.

PURPOSE: Selective estrogen receptor degrader (SERD) has proven clinically effective in treating advanced or metastatic breast cancer since the approval of fulvestrant by FDA in 2002. Recent expansion of indications as a first line monotherapy and as combination therapy with CDK4/6 inhibitors further extends its clinical utility as an efficacious breast cancer endocrine regimen. However, the poor pharmacokinetic properties of fulvestrant and its injection-only administration route has driven continued efforts to develop orally bioavailability SERD that could potentially improve clinical response to SERD treatment. GLL398, a boron-modified GW5638 analog, showed superior oral bioavailability, while retaining both antiestrogenic activity and ER degrading efficacy at a potency level comparable to the more active metabolite of GW5638, GW7604. METHODS: Here we used molecular modeling, ER (Y537S) binding assay, MCF-7 Xenograft tumor, and patient-derived xenograft (PDX) tumor model to conduct further studies on the pharmacology and metabolism of GLL398. RESULTS: Consistent with GLL398's robust activities in breast cancer cells that either are tamoxifen resistant or express constitutively active, mutant ESR1 (Y537S), it was found to bind the mutant ERY537S with high affinity. Molecular modeling of the binding mode of GLL398 to ER also found its molecular interactions consistent with the experimentally determined high binding affinity towards WT ER and ERY537S. To test the in vivo efficacy of GLL398, mice bearing MCF-7-derived xenograft breast tumors and patient-derived xenograft tumors harboring ERY537S were treated with GLL398 which potently inhibited tumor growth in mice. CONCLUSIONS: This study demonstrates GLL398 is an oral SERD that has therapeutic efficacy in clinically relevant breast tumor models.

Protective effects of bestatin in the retina of streptozotocin-induced diabetic mice.

CD13/APN (aminopeptidase N) was first identified as a selective angiogenic marker expressed in tumor vasculature and is considered a target for anti-cancer therapy. CD13 was also reported to express in non-diabetic, hypoxia-induced retinal neovascularization. Whether diabetes induces upregulation of CD13 expression in the retina is unknown. We hypothesize that at an early stage of non-proliferative diabetic retinopathy (NPDR) characterized by disruption of blood-retinal barrier (BRB) permeability is related to upregulated expression of CD13 because of its known role in extracellular matrix (ECM) degradation. The purpose of this study is to evaluate the role of CD13/APN and the therapeutic efficacy of a CD13/APN inhibitor in a mouse model of streptozotocin-induced NPDR. Hyperglycemic C57Bl/6 mice 26 weeks after streptozotocin (STZ) injection were intravitreally injected with a sustained release formulation of CD13/APN inhibitor bestatin. At 15th day of post-bestatin treatment, mouse retinas were evaluated for vascular permeability by Evans blue dye extravasation assay, fluorescent angiography of retinal vascular permeability and leukostasis. Retinal protein extracts were analyzed by Western blot to determine the effects of bestatin treatment on the expression of CD13/APN related inflammatory mediators of ECM degradation and angiogenesis. Intravitreal bestatin treatment significantly inhibited retinal vascular permeability and leukostasis. This treatment also significantly inhibited retinal expression of CD13, ECM degrading proteases (heparanase and MMP9 and angiogenic molecules (HIF-1α and VEGF). Intravitreal CD13 inhibition may relate to furthering our knowledge on the protective effect of bestatin against diabetic retinal vasculature abnormalities through inhibition of retinal permeability, leukostasis, inflammatory molecules of ECM degradation and angiogenesis.

A novel rare sugar inhibitor of murine herpes simplex keratitis.

PURPOSE: To determine the therapeutic efficacy of a novel rare sugar, l-psicose, for the treatment of HSV-1 induced herpetic stromal keratitis (HSK) in a mouse eye model. METHODS: One rare sugar l-psicose was assayed for HSV-1 inhibition of in vitro virus adsorption. The IC50 and IC90 values of l-psicose were determined using plaque reduction assay (PRA) in CV-1 cell. Female Balb/c mice were corneally infected with HSV-1, strain KOS-GFP; A topical eye drop treatment of l-psicose was started 24 h after infection and continued four times daily for ten consecutive days. The severity of HSK was monitored by slit lamp examination in a masked fashion and Infectious HSV-1 shedding was determined by PRA. RESULTS: l-psicose was found to have anti-viral activity in vitro at an IC50 dose of 99.5 mM and an IC90 dose of 160 mM. Topical eye drop treatment with 200 mM l-psicose in PBS solution significantly reduced the severity of HSK compared to the mock treatment group. The in vivo mouse ocular model results of l-psicose therapy correlated with accelerated clearance of virus from eye swabs. CONCLUSION: The results suggest that topical treatment with rare sugar l-psicose has efficacy against HSK through inhibition of HSV-1.

A preliminary report on oral glucose tolerance and antinociceptive activity tests conducted with methanol extract of Xanthosoma violaceum aerial parts.

BACKGROUND: Xanthosoma violaceum is commonly observed in fallow areas of Bangladesh but almost no scientific studies exist on this plant. Rural people consume the plant on a frequent basis. The objective of this study was to scientifically analyze the antinociceptive property of methanol extract of aerial parts of the plant along with antihyperglycemic activity. METHODS: Antihyperglycemic activity was measured by oral glucose tolerance test (OGTT). Antinociceptive activity was determined by observed decreases in abdominal constrictions in intraperitoneally administered acetic acid-induced pain model in mice. RESULTS: Administration of methanol extract of aerial parts led to dose-dependent and significant reductions in blood glucose levels in glucose-loaded mice. At doses of 50, 100, 200 and 400 mg per kg body weight, the extract reduced blood sugar levels by 19.3, 23.2, 31.8, and 47.1%, respectively compared to control animals. By comparison, a standard antihyperglycemic drug, glibenclamide, when administered at a dose of 10 mg per kg body weight, reduced blood glucose level by 48.9%. In antinociceptive activity tests, the extract at the above four doses reduced the number of abdominal constrictions by 41.4, 44.8, 48.3, and 55.2%, respectively. A standard pain relieving (antinociceptive) drug, aspirin, reduced the number of writhings by 31.0 and 51.7%, respectively, when administered at doses of 200 and 400 mg per kg body weight. CONCLUSION: To our knowledge, this is the first report on oral glucose tolerance and antinociceptive activity evaluation of aerial parts of the plant. Since the plant is widely available in Bangladesh, the aerial parts can be a readily available source for particularly the rural population for lowering blood sugar in diabetic patients and for alleviating pain.

A preliminary evaluation of antihyperglycemic and analgesic activity of Alternanthera sessilis aerial parts.

BACKGROUND: Alternanthera sessilis is used by folk medicinal practitioners of Bangladesh for alleviation of severe pain. The objective of this study was to scientifically analyze the analgesic (non-narcotic) property of aerial parts of the plant along with antihyperglycemic activity. METHODS: Antihyperglycemic activity was measured by oral glucose tolerance tests. Analgesic (non-narcotic) activity was determined by observed decreases in abdominal writhings in intraperitoneally administered acetic acid-induced pain model in mice. RESULTS: Administration of methanol extract of aerial parts led to dose-dependent and significant reductions in blood glucose levels in glucose-loaded mice. At doses of 50, 100, 200 and 400 mg per kg body weight, the extract reduced blood sugar levels by 22.9, 30.7, 45.4 and 46.1%, respectively compared to control animals. By comparison, a standard antihyperglycemic drug, glibenclamide, when administered at a dose of 10 mg per kg body weight, reduced blood glucose level by 48.9%. In analgesic activity tests, the extract at the above four doses reduced the number of abdominal writhings by 27.6, 37.9, 41.4, and 44.8%, respectively. A standard analgesic drug, aspirin, reduced the number of writhings by 31.0 and 51.7%, respectively, when administered at doses of 200 and 400 mg per kg body weight. CONCLUSION: The results validate the folk medicinal use of the plant to alleviate pain. At the same time, the antihyperglycemic activity result suggests that the plant may be a potential source for blood sugar lowering drug(s).

Discovery of Oral Degraders of the ROS1 Fusion Protein with Potent Activity against Secondary Resistance Mutations.

The development of therapeutic resistance in the majority of patients limits the long-term benefit of ROS1 inhibitor treatment. On-target mutations of the ROS1 kinase domain confer resistance to crizotinib and lorlatinib in more than one-third of acquired resistance cases with no current effective treatment option. As an alternative to stoichiometric inhibition, proteolytic degradation of ROS1 could provide an effective tool to combat resistance generated by these mutations. Our study has identified a potent, orally active ROS1 degrader with an excellent pharmacokinetics profile. The degrader can effectively inhibit ROS1-dependent cell proliferation and tumor growth by degrading the ROS1 kinase, thereby eliminating the active phospho-ROS1. More importantly, the degradation-based therapeutic modality can overcome on-target mutation resistance to tyrosine kinase inhibitors by efficient degradation of the mutated kinase to achieve greater potency than inhibition.

Design, Synthesis, and Evaluation of Niclosamide Analogs as Therapeutic Agents for Enzalutamide-Resistant Prostate Cancer.

Niclosamide effectively downregulates androgen receptor variants (AR-Vs) for treating enzalutamide and abiraterone-resistant prostate cancer. However, the poor pharmaceutical properties of niclosamide due to its solubility and metabolic instability have limited its clinical utility as a systemic treatment for cancer. A novel series of niclosamide analogs was prepared to systematically explore the structure-activity relationship and identify active AR-Vs inhibitors with improved pharmaceutical properties based on the backbone chemical structure of niclosamide. Compounds were characterized using 1H NMR, 13C NMR, MS, and elemental analysis. The synthesized compounds were evaluated for antiproliferative activity and downregulation of AR and AR-V7 in two enzalutamide-resistant cell lines, LNCaP95 and 22RV1. Several of the niclosamide analogs exhibited equivalent or improved anti-proliferation effects in LNCaP95 and 22RV1 cell lines (B9, IC50 LNCaP95 and 22RV1 = 0.130 and 0.0997 µM, respectively), potent AR-V7 down-regulating activity, and improved metabolic stability. In addition, both a traditional structure-activity relationship (SAR) and 3D-QSAR analysis were performed to guide further structural optimization. The presence of two -CF3 groups of the most active B9 in the sterically favorable field and the presence of the -CN group of the least active B7 in the sterically unfavorable field seem to make B9 more potent than B7 in the antiproliferative activity.

Fulvestrant-3-Boronic Acid (ZB716) Demonstrates Oral Bioavailability and Favorable Pharmacokinetic Profile in Preclinical ADME Studies.

Fulvestrant-3-boronic acid (ZB716), an oral selective estrogen receptor degrader (SERD) under clinical development, has been investigated in ADME studies to characterize its absorption, metabolism, and pharmacokinetics. ZB716 was found to have high plasma protein binding in human and animal plasma, and low intestinal mucosal permeability. ZB716 had high clearance in hepatocytes of all species tested. ZB716 was metabolized primarily by CYP2D6 and CYP3A. In human liver microsomes, ZB716 demonstrated relatively low inhibition of CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4 (when testosterone was used as the substrate), and no inhibition of CYP2B6 and 3A4 (when midazolam was used as the substrate). In assays for enzyme activity, ZB716 induced CYP1A2, 2B6, and 3A4 in a concentration-dependent manner. Single-dose and repeated-dose pharmacokinetic studies in rats and dogs showed oral bioavailability, dose-proportional drug exposure, and drug accumulation as measured by maximum concentration and area under the concentration-time curve (AUC).

Intellectual and Developmental Disabilities Research Centers: A Multidisciplinary Approach to Understand the Pathogenesis of Methyl-CpG Binding Protein 2-related Disorders.

Disruptions in the gene encoding methyl-CpG binding protein 2 (MECP2) underlie complex neurodevelopmental disorders including Rett Syndrome (RTT), MECP2 duplication disorder, intellectual disabilities, and autism. Significant progress has been made on the molecular and cellular basis of MECP2-related disorders providing a new framework for understanding how altered epigenetic landscape can derail the formation and refinement of neuronal circuits in early postnatal life and proper neurological function. This review will summarize selected major findings from the past years and particularly highlight the integrated and multidisciplinary work done at eight NIH-funded Intellectual and Developmental Disabilities Research Centers (IDDRC) across the US. Finally, we will outline a path forward with identification of reliable biomarkers and outcome measures, longitudinal preclinical and clinical studies, reproducibility of results across centers as a synergistic effort to decode and treat the pathogenesis of the complex MeCP2 disorders.

Metabolism and Pharmacokinetic Study of the Boron-Containing Prodrug of Belinostat (ZL277), a Pan HDAC Inhibitor with Enhanced Bioavailability.

ZL277 is a prodrug of belinostat with enhanced bioavailability and efficacy as a pan histone deacetylase (HDAC) inhibitor. In this study, we investigated the metabolism and pharmacokinetics of ZL277 in liver S9 fractions, liver microsomes, liver cytosol, and in mice. Metabolic products were identified and quantified by a combination of liquid chromatography and tandem mass spectrometry. The in vitro metabolic profile of ZL277 includes ZL277-B(OH)2-452, the major oxidative metabolite ZL277-OH-424, the active ingredient belinostat, belinostat amide, belinostat acid, and methylated belinostat in liver S9 fractions. Both ZL277-OH-424 and belinostat underwent further glucuronidation in liver microsome, whereas only ZL277-OH-424, but not belinostat, underwent some level of sulfation in rat liver cytosols. These metabolites were examined in plasma and in a breast tumor model in vivo. They were also examined in urine and feces from mice treated with ZL277. The pharmacokinetic study of ZL277 showed the parameters of active drug belinostat with a half-life (t1/2) of 10.7 h, an area under curve value (AUC) of 1506.9 ng/mL*h, and a maximum plasma concentration (Cmax) of 172 ng/mL, reached 3 h after a single dose of 10 mg/kg. The hydrolysis product of the prodrug, ZL277-B(OH)2-452 showed an AUC of 8306 ng/mL*h and Cmax of 931 ng/mL 3 h after drug administration.

LRP-1 Pathway Targeted Inhibition of Vascular Abnormalities in the Retina of Diabetic Mice.

PURPOSE: The cell surface LDL (low-density lipoprotein) receptor-related protein-1 (LRP-1) is important for lipid transport and several cell signaling processes. Human apolipoprotein E (apoE) is a ligand of LRP-1. We previously reported that a short peptide (apoEdp) mimicking the LRP-1 binding region of apoE prevents hyperglycemia-induced retinal endothelial cell dysfunction in vitro. The in-vivo outcome of apoE-based peptidomimetic inhibition of LRP-1 in the treatment of diabetic retinopathy is unknown. METHODS: Six months after streptozotocin induction of diabetes, male C57Bl/6 mice were intravitreally inoculated with apoEdp in a controlled release formulation. On the 15th day post-apoEdp treatment, mouse retinas were harvested to examine (1) blood-retinal-barrier (BRB) permeability by Evans blue dye, inflammatory leukostasis by concanavalin staining of leukocytes and LRP-1 pathway-related protein expression by Western blot analysis and gelatin zymography. RESULTS: Intravitreal apoEdp treatment of diabetic mice significantly reduced Evans blue extravasation and the number of adherent leukocytes in the diabetic mouse retinas. ApoEdp treatment inhibited the expression of extracellular matrix (ECM) degrading proteases heparanase and MMP-2, and restores the BRB tight junction proteins occludin and ZO-1. ApoEdp treatment also inhibited Wnt/ß-catenin-related expression of pro-inflammatory molecules ICAM-1, HIF-1α, and VEGF through negative regulation by LRP-1. CONCLUSION: Intravitreal apoEdp treatment of diabetic mice resulted a significant decrease in retinal vascular abnormalities through downregulation of LRP-1-related ECM protein degradation and Wnt/ß-catenin-related pro-angiogenic molecules.

TLE1 inhibits anoikis and promotes tumorigenicity in human lung cancer cells through ZEB1-mediated E-cadherin repression.

The Transducin-like enhancer of split 1 (TLE1) corepressor protein is overexpressed in human lung tumors and is a putative lung-specific oncogene. However, the molecular mechanism underlying its oncogenic function remains to be delineated. Here, we report an important role of TLE1 in promoting lung tumorigenesis by a mechanism involving induction of anoikis resistance. Using the human lung adenocarcinoma A549 and immortalized bronchial epithelial BEAS-2B cell lines, we observed that TLE1 inhibits anoikis through transcriptional repression of E-cadherin gene. In support of E-cadherin as a downstream target of TLE1 to block anoikis, forced expression of E-cadherin attenuated TLE1-induced anoikis resistance while E-cadherin downregulation decreased the anoikis sensitivity of TLE1 knockdown cells. Furthermore, we determined that E-cadherin expression is transcriptionally induced upon loss of cell attachment and functions as an effector of anoikis. Loss of E-cadherin via the siRNA strategy or exogenous TLE1 expression was sufficient to attenuate anoikis in A549 and BEAS-2B cells. Importantly, we demonstrated that the ZEB1 transcriptional factor is required for TLE1-mediated E-cadherin repression and anoikis resistance. ZEB1 interacted with and recruited the TLE1 to the E-cadherin promoter to impose histone deacetylation and gene silencing. In vivo, TLE1 strongly promoted tumorigenicity of A549 cells in a ZEB1-dependent manner. Underscoring its role in anoikis insensitivity of lung cancer cells, the TLE1-mediated E-cadherin repression was negatively regulated by the tumor suppressor Bcl-2 inhibitor of transcription 1 (Bit1) to effect anoikis. These findings identify the ZEB1/TLE1/E-cadherin transcriptional mechanism as a novel pathway that promotes anoikis resistance and oncogenicity of lung cancer cells.