Potentiation of neuromuscular transmission by a small molecule calcium channel gating modifier improves motor function in a severe spinal muscular atrophy mouse model.

Spinal muscular atrophy (SMA) is a monogenic disease that clinically manifests as severe muscle weakness owing to neurotransmission defects and motoneuron degeneration. Individuals affected by SMA experience neuromuscular weakness that impacts functional activities of daily living. We have used a mouse model of severe SMA (SMNΔ7) to test whether a calcium channel gating modifier (GV-58), alone or in combination with a potassium channel antagonist (3,4-diaminopyridine; 3,4-DAP), can improve neuromuscular function in this mouse model. Bath application of GV-58 alone or in combination with 3,4-DAP significantly restored neuromuscular transmission to control levels in both a mildly vulnerable forearm muscle and a strongly vulnerable trunk muscle in SMNΔ7 mice at postnatal days 10-12. Similarly, acute subcutaneous administration of GV-58 to postnatal day 10 SMNΔ7 mice, alone or in combination with 3,4-DAP, significantly increased a behavioral measure of muscle strength. These data suggest that GV-58 may be a promising treatment candidate that could address deficits in neuromuscular function and strength and that the addition of 3,4-DAP to GV-58 treatment could aid in restoring function in SMA.

Rhodium(I)-Catalyzed Annulation of Bicyclo[1.1.0]butyl-Substituted Dihydroquinolines and Dihydropyridines.

Bicyclo[1.1.0]butane-containing compounds feature a unique chemical reactivity, trigger "strain-release" reaction cascades, and provide novel scaffolds with considerable utility in the drug discovery field. We report the synthesis of new bicyclo[1.1.0]butane-linked heterocycles by a nucleophilic addition of bicyclo[1.1.0]butyl anions to 8-isocyanatoquinoline, or, alternatively, iminium cations derived from quinolines and pyridines. The resulting bicyclo[1.1.0]butanes are converted with high regioselectivity to unprecedented bridged heterocycles in a rhodium(I)-catalyzed annulative rearrangement. The addition/rearrangement process tolerates a surprisingly large range of functional groups. Subsequent chemo- and stereoselective synthetic transformations of urea, alkene, cyclopropane, and aniline moieties of the 1-methylene-5-azacyclopropa[cd]indene scaffolds provide several additional new heterocyclic building blocks. X-ray structure-validated quantum mechanical DFT calculations of the reaction pathway indicate the intermediacy of rhodium carbenoid and metallocyclobutane species.

JP4-039, a Mitochondria-Targeted Nitroxide, Mitigates the Effect of Apoptosis and Inflammatory Cell Migration in the Irradiated Mouse Retina.

We hypothesize that the injection of JP4-039, a mitochondria-targeted nitroxide, prior to irradiation of the mouse retina may decrease apoptosis and reduce neutrophil and macrophage migration into the retina. In our study, we aimed to examine the effects of JP4-039 in the mouse retina using fluorescent microscopy, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and flow cytometry. Forty-five mice and one eye per mouse were used. In Group 1, fluorescent microscopy was used to determine retinal uptake of 10 µL (0.004 mg/µL) of intravitreally injected BODIPY-labeled JP4-039 at 0, 15, and 60 min after injection. In Group 2, the TUNEL assay was performed to investigate the rate of apoptosis after irradiation in addition to JP4-039 injection, compared to controls. In Group 3, flow cytometry was used to determine the extent of inflammatory cell migration into the retina after irradiation in addition to JP4-039 injection, compared to controls. Maximal retinal uptake of JP4-039 was 15 min after intravitreal injection (p < 0.0001). JP4-039-treated eyes had lower levels of retinal apoptosis (35.8 ± 2.5%) than irradiated controls (49.0 ± 2.7%; p = 0.0066) and demonstrated reduced migration of N1 cells (30.7 ± 11.7% vs. 77.7 ± 5.3% controls; p = 0.004) and M1 cells (76.6 ± 4.2 vs. 88.1 ± 3.7% controls, p = 0.04). Pretreatment with intravitreally injected JP4-039 reduced apoptosis and inflammatory cell migration in the irradiated mouse retina, marking the first confirmed effect of this molecule in retinal tissue. Further studies may allow for safety profiling and potential use for patients with radiation retinopathy.

Disruption of Ovarian Cancer STAT3 and p38 Signaling with a Small-Molecule Inhibitor of PTP4A3 Phosphatase.

Protein tyrosine phosphatase type IVA member 3 (PTP4A3 or PRL-3) is a nonreceptor, oncogenic, dual-specificity phosphatase that is highly expressed in many human tumors, including ovarian cancer, and is associated with a poor patient prognosis. Recent studies suggest that PTP4A3 directly dephosphorylates SHP-2 phosphatase as part of a STAT3-PTP4A3 feedforward loop and directly dephosphorylates p38 kinase. The goal of the current study was to examine the effect of a PTP4A phosphatase inhibitor, 7-imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione (JMS-053), on ovarian cancer STAT3, SHP-2, and p38 kinase phosphorylation. JMS-053 caused a concentration- and time-dependent decrease in the activated form of STAT3, Y705 phospho-STAT3, in ovarian cancer cells treated in vitro. In contrast, the phosphorylation status of two previously described direct PTP4A3 substrates, SHP-2 phosphatase and p38 kinase, were rapidly increased with JMS-053 treatment. We generated A2780 and OVCAR4 ovarian cancer cells resistant to JMS-053, and the resulting cells were not crossresistant to paclitaxel, cisplatin, or teniposide. JMS-053-resistant A2780 and OVCAR4 cells exhibited a 95% and 50% decrease in basal Y705 phospho-STAT3, respectively. JMS-053-resistant OVCAR4 cells had an attenuated phosphorylation and migratory response to acute exposure to JMS-053. These results support a regulatory role for PTP4A phosphatase in ovarian cancer cell STAT3 and p38 signaling circuits. SIGNIFICANCE STATEMENT: This study demonstrates that chemical inhibition of PTP4A phosphatase activity with JMS-053 decreases STAT3 activation and increases SHP-2 phosphatase and p38 kinase phosphorylation activation in ovarian cancer cells. The newly developed JMS-053-resistant ovarian cancer cells should provide useful tools to further probe the role of PTP4A phosphatase in ovarian cancer cell survival and cell signaling.

C3-Functionalization of indoles with α-heteroaryl-substituted methyl alcohols.

The transition metal-free Cs2CO3/Oxone®-mediated C3-alkylation of indoles proceeds in moderate to high yields with a variety of C4-C7 functionalized indoles and is applicable to 2-, 3- and 4-hydroxymethyl pyridines and related electron-deficient heterocycles, permitting novel late-stage drug functionalizations. Preliminary mechanistic studies support a hydrogen autotransfer-type chain process starting with an initial oxidation of the alcohol to the corresponding aldehyde, followed by a subsequent condensation onto indole and reduction/hydride delivery from another equivalent of the primary alcohol.

Synthesis of 13C-methyl-labeled amino acids and their incorporation into proteins in mammalian cells.

Isotopic labeling of methyl-substituted proteinogenic amino acids with 13C has transformed applications of solution-based NMR spectroscopy and allowed the study of much larger and more complex proteins than previously possible with 15N labeling. Procedures are well-established for producing methyl-labeled proteins expressed in bacteria, with efficient incorporation of 13C-methyl labeled metabolic precursors to enable the isotopic labeling of Ile, Val, and Leu methyl groups. Recently, similar methodology has been applied to enable 13C-methyl labeling of Ile, Val, and Leu in yeast, extending the approach to proteins that do not readily fold when produced in bacteria. Mammalian or insect cells are nonetheless preferable for production of many human proteins, yet 13C-methyl labeling using similar metabolic precursors is not feasible as these cells lack the requisite biosynthetic machinery. Herein, we report versatile and high-yielding synthetic routes to 13C methyl-labeled amino acids based on palladium-catalyzed C(sp3)-H functionalization. We demonstrate the efficient incorporation of two of the synthesized amino acids, 13C-γ2-Ile and 13C-γ1,γ2-Val, into human receptor extracellular domains with multiple disulfides using suspension-cultured HEK293 cells. Production costs are reasonable, even at moderate expression levels of 2-3 mg purified protein per liter of medium, and the method can be extended to label other methyl groups, such as 13C-δ1-Ile and 13C-δ1,δ2-Leu. In summary, we demonstrate the cost-effective production of methyl-labeled proteins in mammalian cells by incorporation of 13C methyl-labeled amino acids generated de novo by a versatile synthetic route.

Development of an automated screen for Kv7.2 potassium channels and discovery of a new agonist chemotype.

To identify pore domain ligands on Kv7.2 potassium ion channels, we compared wild-type (WT) and W236L mutant Kv7.2 channels in a series of assays with previously validated and novel agonist chemotypes. Positive controls were retigabine, flupirtine, and RL-81; i.e. Kv7.2 channel activators that significantly shift voltage-dependent activation to more negative potentials (ΔV50) at 5 µM. We identified 6 new compounds that exhibited differential enhancing activity between WT and W236L mutant channels. Whole cell patch-clamp electrophysiology studies were conducted to identify Kv7.2. Kv7.2/3, Kv7.4, and Kv7.5 selectivity. Our results validate the SyncroPatch platform and establish new structure activity relationships (SAR). Specifically, in addition to selective Kv7.2, Kv7.2/3, Kv7.4. and Kv7.5 agonists, we identified a novel chemotype, ZK-21, a 4-aminotetrahydroquinoline that is distinct from any of the previously described Kv7 channel modifiers. Using flexible receptor docking, ZK-21 was predicted to be stabilized by W236 and bind perpendicular to retigabine, burying the benzyl carbamate group into a tunnel reaching the core of the pore domain.

Recent syntheses and biological profiling of quassinoids.

Quassinoid natural products have gained considerable recognition for their diverse biological properties and their synthetically challenging, highly oxygenated polycyclic structures. Herein, we discuss strategies and tactics in the total synthesis of quassinoids that have been evolving over the past 15 years. Additionally, recent structure-activity relationships and potential biological mechanisms of actions are briefly summarized.

Synthesis of sp3-rich chiral bicyclo[3.3.1]nonanes for chemical space expansion and study of biological activities.

Chiral sp3-rich bicyclo[3.3.1]nonane scaffolds 10-12 were synthesized as single diastereomers from aldehyde 9, which was prepared from 4,4-dimethoxycyclohexa-2,5-dienone through a copper-catalyzed enantioselective reduction. Three different types of intramolecular addition reactions were studied: SmI2-mediated reductive cyclization, base-promoted aldol reaction, and one-pot Mannich reaction. We succeeded in introducing three side-chains to scaffold 11 and construct an sp3-rich compound library in both enantiomeric variants by simply changing the chirality of the ligands. The biological evaluation revealed that all synthesized compounds exhibited a concentration-dependent inhibition of hypoxia-inducible factor-1 (HIF-1) transcriptional activity, with IC50 values in the range of 17.2-31.7 µM, whereas their effects on cell viability were varied (IC50 = 3.5 to > 100 µM). The most active compound 16f inhibits the accumulation of HIF-1α protein and mRNA in hypoxia, indicating that it has a mechanism of action distinctly different from other known compounds bearing the common bicyclo[3.3.1]nonane skeleton.

Enantioselective synthesis and selective functionalization of 4-aminotetrahydroquinolines as novel GLP-1 secretagogues.

Polysubstituted tetrahydroquinolines were obtained in moderate to high yields (28% to 92%) and enantiomeric ratios (er 89:11 to 99:1) by a three-component Povarov reaction using a chiral phosphoric acid catalyst. Significantly, post-Povarov functional group interconversions allowed a rapid access to a library of 36 enantioenriched 4-aminotetrahydroquinoline derivatives featuring five points of diversity. Selected analogs were assayed for their ability to function as glucagon-like peptide-1 (GLP-1) secretagogues.

Mitochondrial energetics is impaired in very long-chain acyl-CoA dehydrogenase deficiency and can be rescued by treatment with mitochondria-targeted electron scavengers.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is the most common defect of mitochondrial long-chain fatty acid ß-oxidation. Patients present with heterogeneous clinical phenotypes affecting heart, liver and skeletal muscle predominantly. The full pathophysiology of the disease is unclear and patient response to current therapeutic regimens is incomplete. To identify additional cellular alterations and explore more effective therapies, mitochondrial bioenergetics and redox homeostasis were assessed in VLCAD-deficient fibroblasts, and several protective compounds were evaluated. The results revealed cellular and tissue changes, including decreased respiratory chain (RC) function, increased reactive oxygen species (ROS) production and altered mitochondrial function and signaling pathways in a variety of VLCAD-deficient fibroblasts. The mitochondrially enriched electron and free radical scavengers JP4-039 and XJB-5-131 improved RC function and decreased ROS production significantly, suggesting that they are viable candidate compounds to further develop to treat VLCAD-deficient patients.

The human milk oligosaccharides 2'-fucosyllactose and 6'-sialyllactose protect against the development of necrotizing enterocolitis by inhibiting toll-like receptor 4 signaling.

BACKGROUND: Necrotizing enterocolitis (NEC) develops through exaggerated toll-like receptor 4 (TLR4) signaling in the intestinal epithelium. Breast milk is rich in non-digestible oligosaccharides and prevents NEC through unclear mechanisms. We now hypothesize that the human milk oligosaccharides 2'-fucosyllactose (2'-FL) and 6'-sialyllactose (6'-SL) can reduce NEC through inhibition of TLR4 signaling. METHODS: NEC was induced in newborn mice and premature piglets and infant formula was supplemented with 2'-FL, 6'-SL, or lactose. Intestinal tissue was obtained at surgical resection. HMO inhibition of TLR4 was assessed in IEC-6 enterocytes, mice, and human tissue explants and via in silico modeling. RESULTS: Supplementation of infant formula with either 2'-FL and/or 6'-SL, but not the parent sugar lactose, reduced NEC in mice and piglets via reduced apoptosis, inflammation, weight loss, and histological appearance. Mechanistically, both 2'-FL and 6'-SL, but not lactose, reduced TLR4-mediated nuclear factor kappa light-chain enhancer of activated B cells (NF-kB) inflammatory signaling in the mouse and human intestine. Strikingly, in silico modeling revealed 2'-FL and 6'-SL, but not lactose, to dock into the binding pocket of the TLR4-MD2 complex, explaining their ability to inhibit TLR4 signaling. CONCLUSIONS: 2'-FL and 6'-SL, but not lactose, prevent NEC in mice and piglet models and attenuate NEC inflammation in the human ileum, in part through TLR4 inhibition. IMPACT: Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants that occurs in the setting of bacterial colonization of the gut and administration of formula feeds and activation by the innate immune receptor toll-like receptor 4 (TLR4). Breast milk prevents NEC through unclear mechanisms. We now show that breast milk-enriched human milk oligosaccharides (HMOs) that are derived from lactose prevent NEC through inhibition of TLR4. The human milk oligosaccharides 2'-FL and 6'-SL, but not the backbone sugar lactose, prevent NEC in mice and piglets. 2'-FL and 6'-SL but not lactose inhibited TLR4 signaling in cultured enterocytes, in enteroids derived from mouse intestine, and in human intestinal explants obtained at the time of surgical resection for patients with NEC. In seeking the mechanisms involved, 2'-FL and 6'-SL but not lactose were found to directly bind to TLR4, explaining the inhibition and protection against NEC. These findings may impact clinical practice by suggesting that administration of HMOs could serve as a preventive strategy for premature infants at risk for NEC development.

The mitochondrial-targeted reactive species scavenger JP4-039 prevents sulfite-induced alterations in antioxidant defenses, energy transfer, and cell death signaling in striatum of rats.

Sulfite oxidase (SO) deficiency is a disorder caused either by isolated deficiency of SO or by defects in the synthesis of its molybdenum cofactor. It is characterized biochemically by tissue sulfite accumulation. Patients present with seizures, progressive neurological damage, and basal ganglia abnormalities, the pathogenesis of which is not fully established. Treatment is supportive and largely ineffective. To address the pathophysiology of sulfite toxicity, we examined the effects of intrastriatal administration of sulfite in rats on antioxidant defenses, energy transfer, and mitogen-activated protein kinases (MAPK) and apoptosis pathways in rat striatum. Sulfite administration decreased glutathione (GSH) concentration and glutathione peroxidase, glucose-6-phosphate dehydrogenase, glutathione S-transferase, and glutathione reductase activities in striatal tissue. Creatine kinase (CK) activity, a crucial enzyme for cell energy transfer, was also decreased by sulfite. Superoxide dismutase-1 (SOD1) and catalase (CAT) proteins were increased, while heme oxygenase-1 (HO-1) was decreased. Additionally, sulfite altered phosphorylation of MAPK by decreasing of p38 and increasing of ERK. Sulfite further augmented the content of GSK-3ß, Bok, and cleaved caspase-3, indicating increased apoptosis. JP4-039 is a mitochondrial-targeted antioxidant that reaches higher intramitochondrial levels than other traditional antioxidants. Intraperitoneal injection of JP4-039 before sulfite administration preserved activity of antioxidant enzymes and CK. It also prevented or attenuated alterations in SOD1, CAT, and HO-1 protein content, as well as changes in p38, ERK, and apoptosis markers. In sum, oxidative stress and apoptosis induced by sulfite injection are prevented by JP4-039, identifying this molecule as a promising candidate for pharmacological treatment of SO-deficient patients.

Synthesis and evaluation of bifunctional PTP4A3 phosphatase inhibitors activating the ER stress pathway.

We developed JMS-053, a potent inhibitor of the dual specificity phosphatase PTP4A3 that is potentially suitable for cancer therapy. Due to the emerging role of the unfolded protein response (UPR) in cancer pathology, we sought to identify derivatives that combine PTP4A3 inhibition with induction of endoplasmatic reticulum (ER) stress, with the goal to generate more potent anticancer agents. We have now generated bifunctional analogs that link the JMS-053 pharmacophore to an adamantyl moiety and act in concert with the phosphatase inhibitor to induce ER stress and cell death. The most potent compound in this series, 7a, demonstrated a ca. 5-fold increase in cytotoxicity in a breast cancer cell line and strong activation of UPR and ER stress response genes in spite of a ca. 13-fold decrease in PTP4A3 inhibition. These results demonstrate that the combination of phosphatase inhibition with UPR/ER-stress upregulation potentiates efficacy.

Improved correction of F508del-CFTR biogenesis with a folding facilitator and an inhibitor of protein ubiquitination.

A growing number of diseases are linked to the misfolding of integral membrane proteins, and many of these proteins are targeted for ubiquitin-proteasome-dependent degradation. One such substrate is a mutant form of the Cystic Fibrosis Transmembrane Conductance Regulator (F508del-CFTR). Protein folding "correctors" that repair the F508del-CFTR folding defect have entered the clinic, but they are unlikely to protect the entire protein from degradation. To increase the pool of F508del-CFTR protein that is available for correction by existing treatments, we determined a structure-activity relationship to improve the efficacy and reduce the toxicity of an inhibitor of the E1 ubiquitin activating enzyme that facilitates F508del-CFTR maturation. A resulting lead compound lacked measurable toxicity and improved the ability of an FDA-approved corrector to augment F508del-CFTR folding, transport the protein to the plasma membrane, and maintain its activity. These data support a proof-of-concept that modest inhibition of substrate ubiquitination improves the activity of small molecule correctors to treat CF and potentially other protein conformational disorders.

Combined injury: irradiation with skin or bone wounds in rodent models.

A radiation combined injury is defined as an injury that occurs in the setting of irradiation, such as those expected after a nuclear accident, radiation dispersal device release (a 'dirty bomb'), or a nuclear weapon detonation. There is much research on irradiation-associated burns and their healing, but there is less known about other injuries sustained in the context of irradiation. Animal models are limited in their correlations to clinical situations but can support research on specific questions about injuries and their healing. Mouse models of irradiation with skin or bone wounds are validated as highly reproducible and quantitative. They show dose-dependent impairment of wound healing, with later recovery. Irradiation-induced delay of bone wound healing was mitigated to different extents by single doses of gramicidin S-nitroxide JP4-039, a plasmid expressing manganese superoxide dismutase, amifostine/WR2721, or the bifunctional sulfoxide MMS-350. These models should be useful for research on mechanisms of radiation dermal and osseous damage and for further development of new radioprotectors. They also provide information of potential relevance to the effects of clinical radiation therapies.

Structure of the Complex of an Iminopyridinedione Protein Tyrosine Phosphatase 4A3 Phosphatase Inhibitor with Human Serum Albumin.

Protein tyrosine phosphatase (PTP) 4A3 is frequently overexpressed in human solid tumors and hematologic malignancies and is associated with tumor cell invasion, metastasis, and a poor patient prognosis. Several potent, selective, and allosteric small molecule inhibitors of PTP4A3 were recently identified. A lead compound in the series, JMS-053 (7-imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione), has a long plasma half-life (∼ 24 hours) in mice, suggesting possible binding to serum components. We confirmed by isothermal titration calorimetry that JMS-053 binds to human serum albumin. A single JMS-053 binding site was identified by X-ray crystallography in human serum albumin at drug site 3, which is also known as subdomain IB. The binding of JMS-053 to human serum albumin, however, did not markedly alter the overall albumin structure. In the presence of serum albumin, the potency of JMS-053 as an in vitro inhibitor of PTP4A3 and human A2780 ovarian cancer cell growth was reduced. The reversible binding of JMS-053 to serum albumin may serve to increase JMS-053's plasma half-life and thus extend the delivery of the compound to tumors. SIGNIFICANCE STATEMENT: X-ray crystallography revealed that a potent, reversible, first-in-class small molecule inhibitor of the oncogenic phosphatase protein tyrosine phosphatase 4A3 binds to at least one site on human serum albumin, which is likely to extend the compound's plasma half-life and thus assist in drug delivery into tumors.

A novel androgen receptor antagonist JJ-450 inhibits enzalutamide-resistant mutant ARF876L nuclear import and function.

BACKGROUND: Castration-resistant prostate cancer can develop resistance to enzalutamide because of androgen receptor (AR) point mutations, AR overexpression, constitutively active AR splice variants, and/or elevated intratumoral androgen synthesis. The point mutation ARF876L was reported to be stimulated, instead of inhibited, by enzalutamide, thus contributing to enzalutamide resistance. We have recently developed JJ-450 as a novel AR antagonist with the potential to treat enzalutamide-resistant castration-resistant prostate cancer (CRPC). METHODS: We employed several assays to determine the impact of JJ-450 and enzalutamide on prostate cancer cell lines expressing green fluorescent protein (GFP)-ARF876L . These assays include a prostate-specific antigen enhancer/promoter-based luciferase assay to determine AR transcriptional activity, a quantitative real-time polymerase chain reaction assay, and Western blot analysis to detect expression of AR-target genes at the messenger RNA and protein level, fluorescence microscopy to show AR subcellular localization, and a 5-bromo-2'-deoxyuridine assay to measure prostate cancer cell proliferation. RESULTS: As expected, enzalutamide inhibited wild-type (WT) AR but not ARF876L transcriptional activity in the luciferase assay. In contrast, JJ-450 inhibited both WT-AR and ARF876L transcriptional activity to a similar extent. Also, enzalutamide retarded androgen-induced nuclear import of GFP-AR, but not GFP-ARF876L , whereas JJ-450 retarded nuclear import of both GFP-AR and GFP-ARF876L . To further evaluate JJ-450 inhibition of ARF876L , we stably transfected C4-2 cells separately with GFP-AR or GFP-ARF876L . Enzalutamide inhibited endogenous AR-target gene expression in C4-2-GFP-ARWT , but not in the C4-2-GFP-ARF876L subline, whereas JJ-450 inhibited AR-target gene expression in both C4-2 sublines. More importantly, enzalutamide inhibited proliferation of C4-2-GFP-ARWT , but not of the C4-2-GFP-ARF876L subline, whereas JJ-450 inhibited proliferation of both C4-2 sublines. CONCLUSION: JJ-450 inhibits enzalutamide-resistant ARF876L mutant nuclear translocation and function. Our findings suggest that JJ-450 and its analogs should be further developed to provide a potential new approach for the treatment of enzalutamide-resistant CRPC.

Compensatory increases of select proteostasis networks after Hsp70 inhibition in cancer cells.

Cancer cells thrive when challenged with proteotoxic stress by inducing components of the protein folding, proteasome, autophagy and unfolded protein response (UPR) pathways. Consequently, specific molecular chaperones have been validated as targets for anti-cancer therapies. For example, inhibition of Hsp70 family proteins (hereafter Hsp70) in rhabdomyosarcoma triggers UPR induction and apoptosis. To define how these cancer cells respond to compromised proteostasis, we compared rhabdomyosarcoma cells that were sensitive (RMS13) or resistant (RMS13-R) to the Hsp70 inhibitor MAL3-101. We discovered that endoplasmic reticulum-associated degradation (ERAD) and autophagy were activated in RMS13-R cells, suggesting that resistant cells overcome Hsp70 ablation by increasing misfolded protein degradation. Indeed, RMS13-R cells degraded ERAD substrates more rapidly than RMS cells and induced the autophagy pathway. Surprisingly, inhibition of the proteasome or ERAD had no effect on RMS13-R cell survival, but silencing of select autophagy components or treatment with autophagy inhibitors restored MAL3-101 sensitivity and led to apoptosis. These data indicate a route through which cancer cells overcome a chaperone-based therapy, define how cells can adapt to Hsp70 inhibition, and demonstrate the value of combined chaperone and autophagy-based therapies.This article has an associated First Person interview with the first author of the paper.

Binding Site Interactions of Modulators of Breast Cancer Resistance Protein, Multidrug Resistance-Associated Protein 2, and P-Glycoprotein Activity.

ATP-binding cassette (ABC)-transporters protect tissues by pumping their substrates out of the cells in many physiological barriers, such as the blood-brain barrier, intestine, liver, and kidney. These substrates include various endogenous metabolites, but, in addition, ABC transporters recognize a wide range of compounds, therefore affecting the disposition and elimination of clinically used drugs and their metabolites. Although numerous ABC-transporter inhibitors are known, the underlying mechanism of inhibition is not well characterized. The aim of this study is to deepen our understanding of transporter inhibition by studying the molecular basis of ligand recognition. In the current work, we compared the effect of 44 compounds on the active transport mediated by three ABC transporters: breast cancer resistance protein (BCRP and ABCG2), multidrug-resistance associated protein (MRP2 and ABCC2), and P-glycoprotein (P-gp and ABCB1). Eight compounds were strong inhibitors of all three transporters, while the activity of 36 compounds was transporter-specific. Of the tested compounds, 39, 25, and 11 were considered as strong inhibitors, while 1, 4, and 11 compounds were inactive against BCRP, MRP2, and P-gp, respectively. In addition, six transport-enhancing stimulators were observed for P-gp. In order to understand the observed selectivity, we compared the surface properties of binding cavities in the transporters and performed structure-activity analysis and computational docking of the compounds to known binding sites in the transmembrane domains and nucleotide-binding domains. Based on the results, the studied compounds are more likely to interact with the transmembrane domain than the nucleotide-binding domain. Additionally, the surface properties of the substrate binding site in the transmembrane domains of the three transporters were in line with the observed selectivity. Because of the high activity toward BCRP, we lacked the dynamic range needed to draw conclusions on favorable interactions; however, we identified amino acids in both P-gp and MRP2 that appear to be important for ligand recognition.