Low-dose biliatresone treatment of pregnant mice causes subclinical biliary disease in their offspring: Evidence for a spectrum of neonatal injury.

Biliary atresia is a neonatal disease characterized by damage, inflammation, and fibrosis of the liver and bile ducts and by abnormal bile metabolism. It likely results from a prenatal environmental exposure that spares the mother and affects the fetus. Our aim was to develop a model of fetal injury by exposing pregnant mice to low-dose biliatresone, a plant toxin implicated in biliary atresia in livestock, and then to determine whether there was a hepatobiliary phenotype in their pups. Pregnant mice were treated orally with 15 mg/kg/d biliatresone for 2 days. Histology of the liver and bile ducts, serum bile acids, and liver immune cells of pups from treated mothers were analyzed at P5 and P21. Pups had no evidence of histological liver or bile duct injury or fibrosis at either timepoint. In addition, growth was normal. However, serum levels of glycocholic acid were elevated at P5, suggesting altered bile metabolism, and the serum bile acid profile became increasingly abnormal through P21, with enhanced glycine conjugation of bile acids. There was also immune cell activation observed in the liver at P21. These results suggest that prenatal exposure to low doses of an environmental toxin can cause subclinical disease including liver inflammation and aberrant bile metabolism even in the absence of histological changes. This finding suggests a wide potential spectrum of disease after fetal biliary injury.

Lysosomal lipid peroxidation regulates tumor immunity.

Lysosomal inhibition elicited by palmitoyl-protein thioesterase 1 (PPT1) inhibitors such as DC661 can produce cell death, but the mechanism for this is not completely understood. Programmed cell death pathways (autophagy, apoptosis, necroptosis, ferroptosis, and pyroptosis) were not required to achieve the cytotoxic effect of DC661. Inhibition of cathepsins, or iron or calcium chelation, did not rescue DC661-induced cytotoxicity. PPT1 inhibition induced lysosomal lipid peroxidation (LLP), which led to lysosomal membrane permeabilization and cell death that could be reversed by the antioxidant N-acetylcysteine (NAC) but not by other lipid peroxidation antioxidants. The lysosomal cysteine transporter MFSD12 was required for intralysosomal transport of NAC and rescue of LLP. PPT1 inhibition produced cell-intrinsic immunogenicity with surface expression of calreticulin that could only be reversed with NAC. DC661-treated cells primed naive T cells and enhanced T cell-mediated toxicity. Mice vaccinated with DC661-treated cells engendered adaptive immunity and tumor rejection in "immune hot" tumors but not in "immune cold" tumors. These findings demonstrate that LLP drives lysosomal cell death, a unique immunogenic form of cell death, pointing the way to rational combinations of immunotherapy and lysosomal inhibition that can be tested in clinical trials.

Synthesis of Cyclohexane-Angularly-Fused Triquinanes.

Cyclohexane-angularly-fused triquinanes, 6-5-5-5 tetracycles, have attracted the attention of synthetic chemists due to their highly congested core structures and multiple quaternary carbon centers. This review focuses on the six completed total synthesis of naturally occurring cyclohexane-angularly-fused triquinanes in addition to seven notable methodologies that have been developed for the synthesis of these structures.

Targeted delivery of mPGES-1 inhibitors to macrophages via the folate receptor-ß for inflammatory pain.

Activated macrophages overexpress the folate receptor ß (FR-ß) that can be used for targeted delivery of drugs conjugated to folic acid. FR-expressing macrophages contribute to arthritis progression by secreting prostaglandin E2 (PGE2). Non-steroidal anti-inflammatory drugs (NSAIDs) block PGs and thromboxane by inhibiting the cyclooxygenase (COX) enzymes and are used for chronic pain and inflammation despite their well-known toxicity. New NSAIDs target an enzyme downstream of COXs, microsomal prostaglandin E synthase-1 (mPGES-1). Inhibition of mPGES-1 in inflammatory macrophages promises to retain NSAID efficacy while limiting toxicity. We conjugated a potent mPGES-1 inhibitor, MK-7285, to folate, but the construct released the drug inefficiently. Folate conjugation to the primary alcohol of MK-7285 improved the construct's stability and the release of free drug. Surprisingly, the drug-folate conjugate potentiated PGE2 in FR-positive KB cells, and reduced PGE2 in macrophages independently of the FR. Folate conjugation of NSAIDs is not an optimal strategy for targeting of macrophages.

Anticancer properties of bisaminoquinolines with modified linkers.

We have previously reported the unique features of dimeric bisaminoquinolines as anticancer agents and have identified their cellular target as PPT1, a protein palmitoyl-thioesterase. We now report a systematic study on the role of the linker in these constructs, both with respect to the distance between the heterocycles, the linker hydrophobicity and the methylation status (primary vs. secondary vs. tertiary) of the central nitrogen atom on the observed biological activity.

Synthesis of the Core Ring System of the Antiosteoporotic Citrofulvicin.

Synthesis of the octacyclic ring system of citrofulvicin is described in nine steps from readily available starting materials. Blocking undesired intramolecular cyclization of a reactive ß-diketone intermediate by transient incorporation of a dithiolane ring led to the formation of the requisite 1-hydroxy-2,4,6-trioxaadamantane ring system of citrofulvicin.

Synthesis of and Metal Complexation with a Chiral Cyclam.

Tetraazamacrocycles, like cyclam 1, are well-studied polyamine ligands for metal ions that were first developed to model biological processes. Despite being studied for nearly 60 years, the development of chiral variants of 1 has been limited. We report the synthesis of a chiral variant of 1, the tetraazamacrocycle 2. Outlined herein are the synthesis of 2, a preliminary study of its complexation with metal ions, and the properties of the resulting metal complexes.

SIRT1 is downregulated by autophagy in senescence and ageing.

SIRT1 (Sir2) is an NAD+-dependent deacetylase that plays critical roles in a broad range of biological events, including metabolism, the immune response and ageing1-5. Although there is strong interest in stimulating SIRT1 catalytic activity, the homeostasis of SIRT1 at the protein level is poorly understood. Here we report that macroautophagy (hereafter referred to as autophagy), a catabolic membrane trafficking pathway that degrades cellular components through autophagosomes and lysosomes, mediates the downregulation of mammalian SIRT1 protein during senescence and in vivo ageing. In senescence, nuclear SIRT1 is recognized as an autophagy substrate and is subjected to cytoplasmic autophagosome-lysosome degradation, via the autophagy protein LC3. Importantly, the autophagy-lysosome pathway contributes to the loss of SIRT1 during ageing of several tissues related to the immune and haematopoietic system in mice, including the spleen, thymus, and haematopoietic stem and progenitor cells, as well as in CD8+CD28- T cells from aged human donors. Our study reveals a mechanism in the regulation of the protein homeostasis of SIRT1 and suggests a potential strategy to stabilize SIRT1 to promote productive ageing.

PPT1 inhibition enhances the antitumor activity of anti-PD-1 antibody in melanoma.

New strategies are needed to enhance the efficacy of anti-programmed cell death protein antibody (anti-PD-1 Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of chloroquine derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti-PD-1 Ab in melanoma. The combination resulted in tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen-primed T cells to macrophage-conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma-specific killing. Genetic or chemical Ppt1 inhibition resulted in M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase/stimulator of interferon genes/TANK binding kinase 1 pathway activation and the secretion of interferon-ß in macrophages, the latter being a key component for augmented T cell-mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for this combination in melanoma clinical trials and further investigation in other cancers.

Impaired Redox and Protein Homeostasis as Risk Factors and Therapeutic Targets in Toxin-Induced Biliary Atresia.

BACKGROUND & AIMS: Extrahepatic biliary atresia (BA) is a pediatric liver disease with no approved medical therapy. Recent studies using human samples and experimental modeling suggest that glutathione redox metabolism and heterogeneity play a role in disease pathogenesis. We sought to dissect the mechanistic basis of liver redox variation and explore how other stress responses affect cholangiocyte injury in BA. METHODS: We performed quantitative in situ hepatic glutathione redox mapping in zebrafish larvae carrying targeted mutations in glutathione metabolism genes and correlated these findings with sensitivity to the plant-derived BA-linked toxin biliatresone. We also determined whether genetic disruption of HSP90 protein quality control pathway genes implicated in human BA altered biliatresone toxicity in zebrafish and human cholangiocytes. An in vivo screening of a known drug library was performed to identify novel modifiers of cholangiocyte injury in the zebrafish experimental BA model, with subsequent validation. RESULTS: Glutathione metabolism gene mutations caused regionally distinct changes in the redox potential of cholangiocytes that differentially sensitized them to biliatresone. Disruption of human BA-implicated HSP90 pathway genes sensitized zebrafish and human cholangiocytes to biliatresone-induced injury independent of glutathione. Phosphodiesterase-5 inhibitors and other cyclic guanosine monophosphate signaling activators worked synergistically with the glutathione precursor N-acetylcysteine in preventing biliatresone-induced injury in zebrafish and human cholangiocytes. Phosphodiesterase-5 inhibitors enhanced proteasomal degradation and required intact HSP90 chaperone. CONCLUSION: Regional variation in glutathione metabolism underlies sensitivity to the biliary toxin biliatresone and may account for the reported association between BA transplant-free survival and glutathione metabolism gene expression. Human BA can be causatively linked to genetic modulation of protein quality control. Combined treatment with N-acetylcysteine and cyclic guanosine monophosphate signaling enhancers warrants further investigation as therapy for BA.

Synthesis and Applications of the C2-Symmetrical Diamine 2,7-Diazabicyclo[4.4.1]undecane.

Chiral diamines are particularly useful as ligands for asymmetric catalysis. In an effort to expand the library of such diamines, the synthesis and resolution of the C2-symmetric diamine 2,7-diazabicyclo[4.4.1]undecane [(-)-1] are reported. Diamine (-)-1 has been prepared in multigram quantities from the known bicyclic diketone 7 in four steps without the need for chromatographic purification. Derivatives of (-)-1, i.e., the bis-methylated diamine (+)-5 and two diastereomeric tricyclic analogs, were evaluated as potential sparteine surrogates. The solid-state structure of the (+)-5-methyllithium complex was obtained. High levels of asymmetric induction were observed while using (+)-5 as a ligand in palladium-mediated asymmetric allylations.

A Potent Autophagy Inhibitor (Lys05) Enhances the Impact of Ionizing Radiation on Human Lung Cancer Cells H1299.

Autophagy inhibition through small-molecule inhibitors is one of the approaches to increase the efficiency of radiotherapy in oncological patients. A new inhibitor-Lys05-with the potential to accumulate within lysosomes and to block autophagy was discovered a few years ago. Several studies have addressed its chemosensitizing effects but nothing is known about its impact in the context of ionizing radiation (IR). To describe its role in radiosensitization, we employed radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative). Combined treatment of H1299 cells by Lys05 together with IR decreased cell survival in the clonogenic assay and real-time monitoring of cell growth more than either Lys05 or IR alone. Immunodetection of LC3 and p62/SQSTM1 indicated that autophagy was inhibited, which correlated with increased SQSTM1 and decreased BNIP3 gene expression determined by qRT-PCR. Fluorescence microscopy and flow cytometry uncovered an accumulation of lysosomes. Similarly, transmission electron microscopy demonstrated the accumulation of autophagosomes confirming the ability of Lys05 to potentiate autophagy inhibition in H1299 cells. We report here for the first time that Lys05 could be utilized in combination with IR as a promising future strategy in the eradication of lung cancer cells.

A Transannular Rearrangement Reaction of a Pyrroloindoline Diketopiperazine.

Oxaline, glandicoline, and meleagrin contain a unique triazaspirocyclic structure. Attracted by their biological activities, we attempted a novel strategy, mimicking a proposed biosynthetic pathway for glandicoline B in Penicillium chrysogenum and Penicillium oxalicum and using a transannular rearrangement to the desired triazaspirocycle 15.

A 3-(4-nitronaphthen-1-yl) amino-benzoate analog as a bifunctional AKR1C3 inhibitor and AR antagonist: Head to head comparison with other advanced AKR1C3 targeted therapeutics.

Drugs used for the treatment of castration resistant prostate cancer (CRPC) include Abiraterone acetate (Zytiga®) and Enzalutamide (XTANDI®). However, these drugs provide clinical benefit in metastatic disease for only a brief period before drug resistance emerges. One mechanism of drug resistance involves the overexpression of type 5 17-ß-hydroxysteroid dehydrogenase (aldo-keto reductase 1C3 or AKR1C3), a major enzyme responsible for the formation of intratumoral androgens that activate the androgen receptor (AR). 3-((4-Nitronaphthalen-1-yl)amino)benzoic acid 1 is a "first-in-class" AKR1C3 competitive inhibitor and AR antagonist. Compound 1 was compared in a battery of in vitro studies with structurally related N-naphthyl-aminobenzoates, and AKR1C3 targeted therapeutics e.g. GTx-560 and ASP9521, as well as with R-bicalutamide, enzalutamide and abiraterone acetate. Compound 1 was the only naphthyl derivative that was a selective AKR1C3 inhibitor and AR antagonist in direct competitive binding assays and in AR driven reporter gene assays. GTx-560 displayed weak activity as a direct AR antagonist but had high potency in the AR reporter gene assay consistent with its ability to inhibit the co-activator function of AKR1C3. By contrast ASP9521 did not act as either an AR antagonist or block AR reporter gene activity. Compound 1 was the only compound that showed comparable potency to inhibit AKR1C3 and act as a direct AR antagonist. Compound 1 blocked the formation of testosterone in LNCaP-AKR1C3 cells, and the expression of PSA driven by the AKR1C3 substrate (4-androstene-3,17-dione) and by an AR agonist, 5α-dihydrotestosterone consistent with its bifunctional role. Compound 1 blocked the nuclear translocation of the AR at similar concentrations to enzalutamide and caused disappearance of the AR from cell lysates. R-biaclutamide and enzalutamide inhibited AKR1C3 at concentrations 200x greater than compound 1, suggesting that its bifunctionality can be explained by a shared pharmacophore that can be optimized.

PPT1 Promotes Tumor Growth and Is the Molecular Target of Chloroquine Derivatives in Cancer.

Clinical trials repurposing lysosomotropic chloroquine (CQ) derivatives as autophagy inhibitors in cancer demonstrate encouraging results, but the underlying mechanism of action remains unknown. Here, we report a novel dimeric CQ (DC661) capable of deacidifying the lysosome and inhibiting autophagy significantly better than hydroxychloroquine (HCQ). Using an in situ photoaffinity pulldown strategy, we identified palmitoyl-protein thioesterase 1 (PPT1) as a molecular target shared across monomeric and dimeric CQ derivatives. HCQ and Lys05 also bound to and inhibited PPT1 activity, but only DC661 maintained activity in acidic media. Knockout of PPT1 in cancer cells using CRISPR/Cas9 editing abrogates autophagy modulation and cytotoxicity of CQ derivatives, and results in significant impairment of tumor growth similar to that observed with DC661. Elevated expression of PPT1 in tumors correlates with poor survival in patients in a variety of cancers. Thus, PPT1 represents a new target in cancer that can be inhibited with CQ derivatives. SIGNIFICANCE: This study identifies PPT1 as the previously unknown lysosomal molecular target of monomeric and dimeric CQ derivatives. Genetic suppression of PPT1 impairs tumor growth, and PPT1 levels are elevated in cancer and associated with poor survival. These findings provide a strong rationale for targeting PPT1 in cancer. This article is highlighted in the In This Issue feature, p. 151.

Targeting quiescent leukemic stem cells using second generation autophagy inhibitors.

In chronic myeloid leukemia (CML), tyrosine kinase inhibitor (TKI) treatment induces autophagy that promotes survival and TKI-resistance in leukemic stem cells (LSCs). In clinical studies hydroxychloroquine (HCQ), the only clinically approved autophagy inhibitor, does not consistently inhibit autophagy in cancer patients, so more potent autophagy inhibitors are needed. We generated a murine model of CML in which autophagic flux can be measured in bone marrow-located LSCs. In parallel, we use cell division tracing, phenotyping of primary CML cells, and a robust xenotransplantation model of human CML, to investigate the effect of Lys05, a highly potent lysosomotropic agent, and PIK-III, a selective inhibitor of VPS34, on the survival and function of LSCs. We demonstrate that long-term haematopoietic stem cells (LT-HSCs: Lin-Sca-1+c-kit+CD48-CD150+) isolated from leukemic mice have higher basal autophagy levels compared with non-leukemic LT-HSCs and more mature leukemic cells. Additionally, we present that while HCQ is ineffective, Lys05-mediated autophagy inhibition reduces LSCs quiescence and drives myeloid cell expansion. Furthermore, Lys05 and PIK-III reduced the number of primary CML LSCs and target xenografted LSCs when used in combination with TKI treatment, providing a strong rationale for clinical use of second generation autophagy inhibitors as a novel treatment for CML patients with LSC persistence.

Design, Synthesis, and Biological Evaluation of Allosteric Effectors That Enhance CO Release from Carboxyhemoglobin.

Carbon monoxide (CO) poisoning causes between 5,000-6,000 deaths per year in the US alone. The development of small molecule allosteric effectors of CO binding to hemoglobin (Hb) represents an important step toward making effective therapies for CO poisoning. To that end, we have found that the synthetic peptide IRL 2500 enhances CO release from COHb in air, but with concomitant hemolytic activity. We describe herein the design, synthesis, and biological evaluation of analogs of IRL 2500 that enhance the release of CO from COHb without hemolysis. These novel structures show improved aqueous solubility and reduced hemolytic activity and could lead the way to the development of small molecule therapeutics for the treatment of CO poisoning.

N-(7-Cyano-6-(4-fluoro-3-(2-(3-(trifluoromethyl)phenyl)acetamido)phenoxy)benzo[d]thiazol-2-yl)cyclopropanecarboxamide (TAK632) Promotes Inhibition of BRAF through the Induction of Inhibited Dimers.

BRAFV600E is the most common activating mutation in melanoma and patients treated with BRAFV600E inhibitors all develop resistance within one year. A significant resistance pathway is paradoxical activation (transactivation) involving BRAF dimers, whereby an inhibitor bound protein subunit allosterically activates the other subunit. We recently reported on dimeric BRAFV600E -selective vemurafenib inhibitors that stabilize an inactive αC-out/αC-out homodimeric conformation with improved inhibitor potency and selectivity in vitro. We set out to extend this strategy to target RAF homo- and heterodimers with the pan-RAF inhibitor TAK632 in dimeric configuration. Surprisingly, we find that monomeric TAK632 induces an active αC-in/αC-in BRAF dimer conformation, while dimeric TAK inhibitors cannot promote BRAF dimers and have significantly compromised potency in vitro. These studies uncover the intimate connection between BRAF dimerization and TAK632 mode of inhibition and highlight the importance of understanding the impact of BRAF inhibitors on kinase dimerization.

Dimeric quinacrines as chemical tools to identify PPT1, a new regulator of autophagy in cancer cells.

DQ661 is a novel dimeric quinacrine that affects multiple lysosomal functions (autophagy and macropinocytosis) and mTORC1 (mechanistic target of rapamycin) activity by specifically targeting protein-palmitoyl thioesterase 1 (PPT1). DQ661 has in vivo activity in immunocompetent mouse models of cancer, and constitutes a new tool compound for the study of lysosomal function in cancer and therapeutic resistance.