Discovery of a Small-Molecule Modulator of the Autophagy-Lysosome Pathway That Targets Lamin A/C and LAMP1, Induces Autophagic Flux, and Affects Lysosome Positioning in Neurons.

Autophagy is a major catabolic degradation and recycling process that maintains homeostasis in cells and is especially important in postmitotic neurons. We implemented a high-content phenotypic assay to discover small molecules that promote autophagic flux and completed target identification and validation studies to identify protein targets that modulate the autophagy pathway and promote neuronal health and survival. Efficient syntheses of the prioritized compounds were developed to readily access analogues of the initial hits, enabling initial structure-activity relationship studies to improve potency and preparation of a biotin-tagged pulldown probe that retains activity. This probe facilitated target identification and validation studies through pulldown and competition experiments using both an unbiased proteomics approach and western blotting to reveal Lamin A/C and LAMP1 as the protein targets of compound RH1115. Evaluation of RH1115 in neurons revealed that this compound induces changes to LAMP1 vesicle properties and alters lysosome positioning. Dysfunction of the autophagy-lysosome pathway has been implicated in a variety of neurodegenerative diseases, including Alzheimer's disease, highlighting the value of new strategies for therapeutic modulation and the importance of small-molecule probes to facilitate the study of autophagy regulation in cultured neurons and in vivo.

Probing the Cytotoxic Signaling Induced by Eupenifeldin in Ovarian Cancer Models.

High-grade serous ovarian cancer (HGSOC) is the most common and lethal ovarian cancer histotype. Lack of early detection methods, limited therapeutic agents, and low 5-year survival rate reflect the urgent need to develop new therapies. Eupenifeldin, a bistropolone, originally isolated from Eupenicillium brefeldianum, is a cytotoxic fungal metabolite. In three HSGOC cell lines (OVCAR3, OVCAR5, OVCAR8), eupenifeldin was found to have an IC50 value less than 10 nM, while 10 times higher concentrations were required for cytotoxicity in nontumorigenic fallopian tube secretory epithelial cell lines (FTSEC). An in vivo hollow fiber assay showed significant cytotoxicity in OVCAR3. Eupenifeldin significantly increased Annexin V staining in OVCAR3 and -8, but not OVCAR5. Eupenifeldin activated caspases 3/7 in OVCAR3, OVCAR5, and OVCAR8; however, cleaved PARP was only detected in OVCAR3. Quantitative proteomics performed on OVCAR3 implicated ferroptosis as the most enriched cell death pathway. However, validation experiments did not support ferroptosis as part of the cytotoxic mechanism of eupenifeldin. Autophagic flux and LC3B puncta assays found that eupenifeldin displayed weak autophagic induction in OVCAR3. Inhibition of autophagy by cotreatment with bafilomycin reduced the toxicity of eupenifeldin, supporting the idea that induction of autophagy contributes to the cytotoxic mechanism of eupenifeldin.

A case of nocardiosis in a patient with ulcerative colitis on chronic corticosteroids, infliximab, and upadacitinib.

Key Clinical Message: Immunosuppression, malnutrition, and underlying infection can unmask obscure infections which can be challenging to identify. Early diagnosis and treatment of infections in immunosuppressed patients are essential due to high morbidity and mortality. Abstract: The immunosuppressive effects of treatment for ulcerative colitis (UC), including chronic corticosteroids, anti-TNF agents, and JAK inhibitors, can impact the spread of latent or obscure infections. Clinicians should have a low threshold for pursuing aggressive diagnostic and therapeutic intervention in patients who show signs of clinical deterioration while on immunosuppressing medications. Our unique case highlights an immunosuppressed patient with UC who developed Nocardiosis after initiation of upadacitinib while hospitalized for concurrent UC flare and Clostridium difficile infection.

The impact of broadening COVID-19 testing eligibility on communities of color - A lesson for overcoming structural barriers to health care.

CDC guidelines for COVID-19 testing in March 2020 did not prioritize underserved communities. We present the effect that expanding COVID-19 testing had for residents of the predominantly Hispanic city of Chelsea, MA, which had the highest case rate in the state. Results were compared to another city with similar demographics, Lynn, MA, where testing eligibility remained unchanged. Institutional data were used to identify outpatient visits for influenza-like illness or COVID-19 exposure, COVID-19 tests, and hospitalizations for confirmed COVID-19 between 3/30/2020-4/28/2020. Multivariable logistic regressions were used to compare outcomes before and after the change in testing eligibility occurred on 4/13/2020. A total of 3,060 patients were included, 1,374 Chelsea residents and 1,686 Lynn residents. After guidelines changed, Chelsea residents were more likely to present as outpatients (adjusted odds ratio [AOR] 4.2, p < 0.001) and less likely to be hospitalized (AOR 0.2, p < 0.001). They were more likely to be tested (AOR 8.8, p < 0.001), but less likely to test positive (AOR 0.6, p = 0.05). Lynn residents were also more likely to be tested after 4/13/2020 (AOR 1.9, p < 0.001), but no significant differences in visit acuity or test positivity were observed. This study demonstrates how broadening testing eligibility for one highly affected, predominantly Hispanic community was associated with an increase in outpatient presentations and a concomitant decrease in test positivity and hospitalizations. These results highlight the impact of improved access to care on utilization of services among underserved communities, a lesson that is especially crucial as we continue to grapple with the COVID pandemic.

Discovery of Anticancer Agents of Diverse Natural Origin.

Research progress from mainly over the last five years is described for a multidisciplinary collaborative program project directed toward the discovery of potential anticancer agents from a broad range of taxonomically defined organisms. Selected lead compounds with potential as new antitumor agents that are representative of considerable structural diversity have continued to be obtained from each of tropical plants, terrestrial and aquatic cyanobacteria, and filamentous fungi. Recently, a new focus has been on the investigation of the constituents of U.S. lichens and their fungal mycobionts. A medicinal chemistry and pharmacokinetics component of the project has optimized structurally selected lead natural products, leading to enhanced cytotoxic potencies against selected cancer cell lines. Biological testing has shown several compounds to have in vivo activity, and relevant preliminary structure-activity relationship and mechanism of action studies have been performed. Several promising lead compounds worthy of further investigation have been identified from the most recent collaborative work performed.

PHY34 inhibits autophagy through V-ATPase V0A2 subunit inhibition and CAS/CSE1L nuclear cargo trafficking in high grade serous ovarian cancer.

PHY34 is a synthetic small molecule, inspired by a compound naturally occurring in tropical plants of the Phyllanthus genus. PHY34 was developed to have potent in vitro and in vivo anticancer activity against high grade serous ovarian cancer (HGSOC) cells. Mechanistically, PHY34 induced apoptosis in ovarian cancer cells by late-stage autophagy inhibition. Furthermore, PHY34 significantly reduced tumor burden in a xenograft model of ovarian cancer. In order to identify its molecular target/s, we undertook an unbiased approach utilizing mass spectrometry-based chemoproteomics. Protein targets from the nucleocytoplasmic transport pathway were identified from the pulldown assay with the cellular apoptosis susceptibility (CAS) protein, also known as CSE1L, representing a likely candidate protein. A tumor microarray confirmed data from mRNA expression data in public databases that CAS expression was elevated in HGSOC and correlated with worse clinical outcomes. Overexpression of CAS reduced PHY34 induced apoptosis in ovarian cancer cells based on PARP cleavage and Annexin V staining. Compounds with a diphyllin structure similar to PHY34 have been shown to inhibit the ATP6V0A2 subunit of V(vacuolar)-ATPase. Therefore, ATP6V0A2 wild-type and ATP6V0A2 V823 mutant cell lines were tested with PHY34, and it was able to induce cell death in the wild-type at 246 pM while the mutant cells were resistant up to 55.46 nM. Overall, our data demonstrate that PHY34 is a promising small molecule for cancer therapy that targets the ATP6V0A2 subunit to induce autophagy inhibition while interacting with CAS and altering nuclear localization of proteins.

Development of a High-Throughput, Compound-Multiplexed Fluorescence Polarization Assay to Identify ATG5-ATG16L1 Protein-Protein Interaction Inhibitors.

Macroautophagy is a catabolic process wherein cytosolic cargo is engulfed in an autophagosome that fuses with a lysosome to degrade the cargo for recycling. Autophagy maintains cellular homeostasis and is involved in a myriad of illnesses ranging from cancer to neurodegenerative diseases, but its therapeutic potential remains elusive due to a lack of potent and specific autophagy modulators. To identify specific inhibitors of early autophagy, a target-based, compound-multiplexed, fluorescence polarization, high-throughput screen that targets the ATG5-ATG16L1 protein-protein interaction was developed. This interaction is critical for the formation of LC3-II, which is involved in phagophore maturation, and its disruption should inhibit autophagy. This assay is based on the polarization of light emitted by a fluorescent rhodamine tag conjugated to a peptide corresponding to the N-terminal region of ATG16L1 (ATG16L1-N). It was confirmed that this peptide binds specifically to ATG5, and the assay was validated by rapidly screening 4800 molecules through compound multiplexing. Through these initial screening efforts, a molecule was identified that disrupts the ATG5-ATG16L1 protein-protein interaction with micromolar potency, and this molecule will serve as a starting point for chemical optimization as an autophagy inhibitor.

Autophagy and post-ischemic conditioning in retinal ischemia.

Retinal ischemia is a major cause of vision loss and a common underlying mechanism associated with diseases, such as diabetic retinopathy and central retinal artery occlusion. We have previously demonstrated the robust neuroprotection in retina induced by post-conditioning (post-C), a brief period of ischemia, 24 h, following a prolonged and damaging initial ischemia. The mechanisms underlying post-C-mediated retinal protection are largely uncharacterized. We hypothesized that macroautophagy/autophagy is a mediator of post-C-induced neuroprotection. This study employed an in vitro model of oxygen glucose deprivation (OGD) in the retinal R28 neuronal cell line, and an in vivo rat model of retinal ischemic injury. In vivo, there were significant increases in autophagy proteins, MAP1LC3-II/LC3-II, and decreases in SQSTM1/p62 (sequestosome 1) in ischemia/post-C vs. ischemia/sham post-C. Blockade of Atg5 and Atg7 in vivo decreased LC3-II, increased SQSTM1, attenuated the functional protective effect of post-C, and increased histological damage and TUNEL compared to non-silencing siRNA. TUNEL after ischemia in vivo was found in retinal ganglion, amacrine, and photoreceptor cells. Blockade of Atg5 attenuated the post-C neuroprotection by a brief period of OGD in vitro. Moreover, in vitro, post-C attenuated cell death, loss of cellular proliferation, and defective autophagic flux from prolonged OGD. Stimulating autophagy using Tat-Beclin 1 rescued retinal neurons from cell death after OGD. As a whole, our results suggest that autophagy is required for the neuroprotective effect of retinal ischemic post-conditioning and augmentation of autophagy offers promise in the treatment of retinal ischemic injury.Abbreviations: BECN1: Beclin 1, autophagy related; DAPI: 4',6-diamidino-2-phenylindole; DR: diabetic retinopathy; EdU: 5-ethynyl-2'-deoxyuridine; ERG: Electroretinogram; FITC: Fluorescein isothiocyanate; GCL: Ganglion cell layer; GFAP: Glial fibrillary acidic protein; INL: Inner nuclear layer; IPL: Inner plexiform layer; MAP1LC3/LC3: Microtubule-associated protein 1 light chain 3; OGD: Oxygen-glucose deprivation; ONL: Outer nuclear layer; OP: Oscillatory potential; PFA: Paraformaldehyde; PL: Photoreceptor layer; post-C: post-conditioning; RFP: Red fluorescent protein; RGC: Retinal ganglion cell; RPE: Retinal pigment epithelium; RT-PCR: Real-time polymerase chain reaction; SEM: Standard error of the mean; siRNA: Small interfering RNA; SQSTM1: Sequestosome 1; STR: Scotopic threshold response; Tat: Trans-activator of transcription; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling.

Multiple Chemical Features Impact Biological Performance Diversity of a Highly Active Natural Product-Inspired Library.

A systematic, diversity-oriented synthesis approach was employed to access a natural product-inspired flavonoid library with diverse chemical features, including chemical properties, scaffold, stereochemistry, and appendages. Using Cell Painting, the effects of these diversity elements were evaluated, and multiple chemical features that predict biological performance diversity were identified. Scaffold identity appears to be the dominant predictor of performance diversity, but stereochemistry and appendages also contribute to a lesser degree. In addition, the diversity of chemical properties contributed to performance diversity, and the driving chemical property was dependent on the scaffold. These results highlight the importance of key chemical features that may inform the creation of small-molecule, performance-diverse libraries to improve the efficiency and success of high-throughput screening campaigns.

Beclin 1-ATG14L Protein-Protein Interaction Inhibitor Selectively Inhibits Autophagy through Disruption of VPS34 Complex I.

Autophagy, a catabolic recycling process, has been implicated as a critical pathway in cancer. Its role in maintaining cellular homeostasis helps to nourish hypoxic, nutrient-starved tumors and protects them from chemotherapy-induced death. Recent efforts to target autophagy in cancer have focused on kinase inhibition, which has led to molecules that lack specificity due to the multiple roles of key kinases in this pathway. For example, the lipid kinase VPS34 is present in two multiprotein complexes responsible for the generation of phosphatidylinositol-3-phosphate. Complex I generates the autophagosome, and Complex II is crucial for endosomal trafficking. Molecules targeting VPS34 inhibit both complexes, which inhibits autophagy but causes undesirable defects in vesicle trafficking. The lack of specific autophagy modulators has limited the utility of autophagy inhibition as a therapeutic strategy. We hypothesize that disruption of the Beclin 1-ATG14L protein-protein interaction, which is required for the formation, proper localization, and function of VPS34 Complex I but not Complex II, will disrupt Complex I formation and selectively inhibit autophagy. To this end, a high-throughput, cellular NanoBRET assay was developed targeting this interaction. An initial screen of 2560 molecules yielded 19 hits that effectively disrupted the interaction, and it was confirmed that one hit disrupted VPS34 Complex I formation and inhibited autophagy. In addition, the molecule did not disrupt the Beclin 1-UVRAG interaction, critical for VPS34 Complex II, and thus had little impact on vesicle trafficking. This molecule is a promising new tool that is critical for understanding how modulation of the Beclin 1-ATG14L interaction affects autophagy. More broadly, its discovery demonstrates that targeting protein-protein interactions found within the autophagy pathway is a viable strategy for the discovery of autophagy-specific probes and therapeutics.

Building a Team-Based Gastroenterology Practice With Advanced Practice Providers.

The use of advanced practice providers (APPs), such as nurse practitioners and physician assistants, has grown substantially in gastroenterology practices in the United States. The first formal training programs appeared in the mid-1960s; however, incorporation of APPs into gastroenterology practices occurred sporadically until the early 1990s, when several large practices began utilizing APPs in both outpatient and inpatient environments. Over the next 20 years, APPs became increasingly more common. In hospital settings, they provide continuity of care, especially for practices that rotate physicians into hospital services on a periodic basis. Efficient use of APPs frees physicians to focus on new patients, procedures, and complex chronic care management. APPs who have independent, appropriately managed schedules can generate revenue that covers salary and benefits. Billing and coding for APPs can be complex, but once regulatory issues are understood, these aspects become straightforward and can be easily applied to gastroenterology practices. There is an ongoing need for more formal training and onboarding resources, which could be met by national gastroenterology and hepatology societies. This article reviews the various ways in which APPs can be incorporated into gastroenterology and hepatology care.

Systematic Diversity-Oriented Synthesis of Reduced Flavones from γ-Pyrones to Probe Biological Performance Diversity.

Diversity-oriented synthesis (DOS) has historically focused on the development of small-molecule collections with considerable chemical diversity with the hypothesis that chemical diversity will lead to diverse biological activities. We took a systematic approach to DOS to develop a focused library of reduced flavones from γ-pyrones with diversity of appendage, stereochemistry, and chemical properties to determine which features of small molecules are most predictive of biological performance diversity. The effects of these systematic modifications on biodiversity were determined using Cell Painting and cytotoxicity assays to compare the results of multiple methods of assessment. We observed that a greater fraction of sp3 hybridized atoms (fsp3) does not always lead to enhanced biodiversity, that stereochemistry and appendage diversity both contribute to biodiversity, and that lipophilicity of the pyrone class of compounds correlates with biodiversity. These results will contribute to the development of a general algorithm to predict which chemical features should be considered during the synthesis of DOS libraries to create biological performance-diverse collections of small molecules for probe and drug discovery.

Next generation diversity-oriented synthesis: a paradigm shift from chemical diversity to biological diversity.

Diversity-oriented synthesis has historically focused on the generation of small-molecule collections with considerable scaffold, stereochemical, and appendage diversity. Recently, this focus has begun to shift to the production of small-molecule libraries with diverse biological activities. It is currently not clear which properties and structural features of molecules are predictive of diverse performance in biological assays, and a better understanding of this relationship is critical for the development of performance-diverse small-molecule libraries for the discovery of novel probes for challenging targets. This review explores recent synthetic strategies for the production of bioactive small molecules and concludes with a presentation of current methods that enable the assessment of the biological performance diversity of small-molecule libraries.

Efficacy of Induction Therapy With High-Intensity Tofacitinib in 4 Patients With Acute Severe Ulcerative Colitis.

As many as 25% of patients diagnosed with ulcerative colitis are hospitalized with an episode of acute severe ulcerative colitis (ASUC).1 The standard of care for patients hospitalized with ASUC relies on rapid induction with intravenous (IV) corticosteroids. Up to 30% of patients do not respond to corticosteroids alone.2 Rescue therapy with infliximab or cyclosporine has been shown to reduce rates of colectomy to 20% by 90 days.3,4 This still represents a significant rate of treatment failure, which leads to an unplanned and irreversible surgery. In recent years, increasing numbers of patients admitted with ASUC have already failed infliximab therapy, highlighting the need for additional treatment options for these patients. Tofacitinib is a rapidly acting, oral, small-molecule Janus kinase inhibitor that was recently approved by the Food and Drug Administration for treatment of ulcerative colitis.5 We present the first reported use of off-label, high-intensity tofacitinib in 4 patients admitted to our institution with ASUC predicted to fail medical management.

Phyllanthusmin Derivatives Induce Apoptosis and Reduce Tumor Burden in High-Grade Serous Ovarian Cancer by Late-Stage Autophagy Inhibition.

High-grade serous ovarian cancer (HGSOC) is a lethal gynecological malignancy with a need for new therapeutics. Many of the most widely used chemotherapeutic drugs are derived from natural products or their semi-synthetic derivatives. We have developed potent synthetic analogues of a class of compounds known as phyllanthusmins, inspired by natural products isolated from Phyllanthus poilanei Beille. The most potent analogue, PHY34, had the highest potency in HGSOC cell lines in vitro and displayed cytotoxic activity through activation of apoptosis. PHY34 exerts its cytotoxic effects by inhibiting autophagy at a late stage in the pathway, involving the disruption of lysosomal function. The autophagy activator, rapamycin, combined with PHY34 eliminated apoptosis, suggesting that autophagy inhibition may be required for apoptosis. PHY34 was readily bioavailable through intraperitoneal administration in vivo where it significantly inhibited the growth of cancer cell lines in hollow fibers, as well as reduced tumor burden in a xenograft model. We demonstrate that PHY34 acts as a late-stage autophagy inhibitor with nanomolar potency and significant antitumor efficacy as a single agent against HGSOC in vivo This class of compounds holds promise as a potential, novel chemotherapeutic and demonstrates the effectiveness of targeting the autophagic pathway as a viable strategy for combating ovarian cancer. Mol Cancer Ther; 17(10); 2123-35. ©2018 AACR.

Microwave-Assisted, Asymmetric Synthesis of 3-Amino-2,3-Dihydrobenzofuran Flavonoid Derivatives from Chalcones.

A route to access 3-amino-2,3-dihydrobenzofurans that utilizes microwave-assisted organic synthesis to rapidly generate analogues has been developed. The route begins with an acid-catalyzed, microwave-assisted aldol condensation to generate chalcone intermediates, followed by a Corey-Bakshi-Shibata reduction and subsequent Sharpless asymmetric epoxidation to access stereoisomeric epoxyalcohols. The final step is a one-pot, microwave-assisted, regioselective, acid-catalyzed epoxide opening with various amines followed by an intramolecular nucleophilic aromatic substitution reaction to generate the 3-amino-2,3-dihydrobenzofurans. This route provides ready access to stereochemically and structurally diverse analogues of these flavonoid scaffolds. Additionally, a pilot library was synthesized, and the biological activity diversity of the chalcones and dihydrobenzofurans was explored in human carcinoma cell lines.

Small-molecule enhancers of autophagy modulate cellular disease phenotypes suggested by human genetics.

Studies of human genetics and pathophysiology have implicated the regulation of autophagy in inflammation, neurodegeneration, infection, and autoimmunity. These findings have motivated the use of small-molecule probes to study how modulation of autophagy affects disease-associated phenotypes. Here, we describe the discovery of the small-molecule probe BRD5631 that is derived from diversity-oriented synthesis and enhances autophagy through an mTOR-independent pathway. We demonstrate that BRD5631 affects several cellular disease phenotypes previously linked to autophagy, including protein aggregation, cell survival, bacterial replication, and inflammatory cytokine production. BRD5631 can serve as a valuable tool for studying the role of autophagy in the context of cellular homeostasis and disease.