Oral estrogen receptor PROTAC® vepdegestrant (ARV-471) is highly efficacious as monotherapy and in combination with CDK4/6 or PI3K/mTOR pathway inhibitors in preclinical ER+ breast cancer models.

PURPOSE: Estrogen Receptor (ER) alpha signaling is a known driver of ER-positive (ER+)/human epidermal growth factor receptor 2 negative (HER2-) breast cancer. Combining endocrine therapy (ET) such as fulvestrant with CDK4/6, mTOR or PI3K inhibitors is now a central strategy for the treatment of ER+ advanced breast cancer. However, suboptimal ER inhibition and resistance resulting from ESR1 mutation dictates that new therapies are needed. EXPERIMENTAL DESIGN: A medicinal chemistry campaign identified vepdegestrant (ARV-471), a selective, orally bioavailable, potent small molecule PROteolysis-TArgeting Chimera (PROTAC®) degrader of ER. We used biochemical and intracellular target engagement assays to demonstrate the mechanism of action of vepdegestrant, and ESR1 wild-type and mutant ER+ preclinical breast cancer models to demonstrate ER degradation-mediated tumor growth inhibition. RESULTS: Vepdegestrant induced ≥90% degradation of wild-type (WT) and mutant ER, inhibited ER-dependent breast cancer cell line proliferation in-vitro and achieved significant tumor growth inhibition (TGI) (87-123%) in MCF7 orthotopic xenograft models, better than the ET agent fulvestrant (31-80% TGI). In the hormone-independent ER Y537S patient derived xenograft (PDX) breast cancer model ST941/HI, vepdegestrant achieved tumor regressions and was similarly efficacious in the ST941/HI/PBR palbociclib-resistant model (102% TGI). Vepdegestrant induced robust tumor regressions in combination with each of the CDK4/6 inhibitors palbociclib, abemaciclib, and ribociclib, the mTOR inhibitor everolimus, and the PI3K inhibitors alpelisib and inavolisib. CONCLUSIONS: Vepdegestrant achieved greater ER degradation in-vivo compared to fulvestrant, which correlated with improved tumor growth inhibition, suggesting vepdegestrant could be a more effective backbone ET for patients with ER+/HER2- breast cancer.

Baseline Severe Acute Respiratory Syndrome Viral Load Is Associated With Coronavirus Disease 2019 Severity and Clinical Outcomes: Post Hoc Analyses of a Phase 2/3 Trial.

BACKGROUND: Elucidating the relationship between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load and clinical outcomes is critical for understanding coronavirus disease 2019 (COVID-19). METHODS: The SARS-CoV-2 levels were analyzed by quantitative real-time polymerase chain reaction (RT-qPCR) of nasopharyngeal or oropharyngeal swab specimens collected at baseline, and clinical outcomes were recorded over 60 days from 1362 COVID-19 hospitalized patients enrolled in a multicenter, randomized, placebo-controlled phase 2/3 trial of sarilumab for COVID-19 (ClinicalTrials.gov NCT04315298). RESULTS: In post hoc analyses, higher baseline viral load, measured by both RT-qPCR cycle threshold and log10 copies/mL, was associated with greater supplemental oxygenation requirements and disease severity at study entry. Higher baseline viral load was associated with higher mortality, lower likelihood of improvement in clinical status and supplemental oxygenation requirements, and lower rates of hospital discharge. Viral load was not impacted by sarilumab treatment over time versus placebo. CONCLUSIONS: These data support viral load as an important determinant of clinical outcomes in hospitalized patients with COVID-19 requiring supplemental oxygen or assisted ventilation.

Targeting Nuclear Receptors with PROTAC degraders.

Nuclear receptors comprise a class of intracellular transcription factors whose major role is to act as sensors of various stimuli and to convert the external signal into a transcriptional output. Nuclear receptors (NRs) achieve this by possessing a ligand binding domain, which can bind cell permeable agonists, a DNA-binding domain, which binds the upstream sequences of target genes, and a regulatory domain that recruits the transcriptional machinery. The ligand binding alters the activation state of the NR, either by activating or inactivating its transcriptional output. Given the central role of NRs in signal transduction, many currently approved therapeutics modulate the activity of NRs. Here we discuss how PROTAC degraders afford a novel approach to abrogate the downstream signaling activity of NRs. We highlight six broad functional reasons why PROTAC degraders are preferable to the classical ligand binding pocket antagonists, with specific examples provided for each category. Lastly, as Androgen Receptor and Estrogen Receptor PROTAC degraders are being pursued as treatment for prostate cancer and breast cancer, respectively, a rationale is provided for the translational utility for the degradation of these two NRs.

Duvoglustat HCl Increases Systemic and Tissue Exposure of Active Acid α-Glucosidase in Pompe Patients Co-administered with Alglucosidase α.

Duvoglustat HCl (AT2220, 1-deoxynojirimycin) is an investigational pharmacological chaperone for the treatment of acid α-glucosidase (GAA) deficiency, which leads to the lysosomal storage disorder Pompe disease, which is characterized by progressive accumulation of lysosomal glycogen primarily in heart and skeletal muscles. The current standard of care is enzyme replacement therapy with recombinant human GAA (alglucosidase alfa [AA], Genzyme). Based on preclinical data, oral co-administration of duvoglustat HCl with AA increases exposure of active levels in plasma and skeletal muscles, leading to greater substrate reduction in muscle. This phase 2a study consisted of an open-label, fixed-treatment sequence that evaluated the effect of single oral doses of 50 mg, 100 mg, 250 mg, or 600 mg duvoglustat HCl on the pharmacokinetics and tissue levels of intravenously infused AA (20 mg/kg) in Pompe patients. AA alone resulted in increases in total GAA activity and protein in plasma compared to baseline. Following co-administration with duvoglustat HCl, total GAA activity and protein in plasma were further increased 1.2- to 2.8-fold compared to AA alone in all 25 Pompe patients; importantly, muscle GAA activity was increased for all co-administration treatments from day 3 biopsy specimens. No duvoglustat-related adverse events or drug-related tolerability issues were identified.

Development of a Multiantigen Panel for Improved Detection of Borrelia burgdorferi Infection in Early Lyme Disease.

The current standard for laboratory diagnosis of Lyme disease in the United States is serologic detection of antibodies against Borrelia burgdorferi. The Centers for Disease Control and Prevention recommends a two-tiered testing algorithm; however, this scheme has limited sensitivity for detecting early Lyme disease. Thus, there is a need to improve diagnostics for Lyme disease at the early stage, when antibiotic treatment is highly efficacious. We examined novel and established antigen markers to develop a multiplex panel that identifies early infection using the combined sensitivity of multiple markers while simultaneously maintaining high specificity by requiring positive results for two markers to designate a positive test. Ten markers were selected from our initial analysis of 62 B. burgdorferi surface proteins and synthetic peptides by assessing binding of IgG and IgM to each in a training set of Lyme disease patient samples and controls. In a validation set, this 10-antigen panel identified a higher proportion of early-Lyme-disease patients as positive at the baseline or posttreatment visit than two-tiered testing (87.5% and 67.5%, respectively; P < 0.05). Equivalent specificities of 100% were observed in 26 healthy controls. Upon further analysis, positivity on the novel 10-antigen panel was associated with longer illness duration and multiple erythema migrans. The improved sensitivity and comparable specificity of our 10-antigen panel compared to two-tiered testing in detecting early B. burgdorferi infection indicates that multiplex analysis, featuring the next generation of markers, could advance diagnostic technology to better aid clinicians in diagnosing and treating early Lyme disease.

Oral Migalastat HCl Leads to Greater Systemic Exposure and Tissue Levels of Active α-Galactosidase A in Fabry Patients when Co-Administered with Infused Agalsidase.

UNLABELLED: Migalastat HCl (AT1001, 1-Deoxygalactonojirimycin) is an investigational pharmacological chaperone for the treatment of α-galactosidase A (α-Gal A) deficiency, which leads to Fabry disease, an X-linked, lysosomal storage disorder. The currently approved, biologics-based therapy for Fabry disease is enzyme replacement therapy (ERT) with either agalsidase alfa (Replagal) or agalsidase beta (Fabrazyme). Based on preclinical data, migalastat HCl in combination with agalsidase is expected to result in the pharmacokinetic (PK) enhancement of agalsidase in plasma by increasing the systemic exposure of active agalsidase, thereby leading to increased cellular levels in disease-relevant tissues. This Phase 2a study design consisted of an open-label, fixed-treatment sequence that evaluated the effects of single oral doses of 150 mg or 450 mg migalastat HCl on the PK and tissue levels of intravenously infused agalsidase (0.2, 0.5, or 1.0 mg/kg) in male Fabry patients. As expected, intravenous administration of agalsidase alone resulted in increased α-Gal A activity in plasma, skin, and peripheral blood mononuclear cells (PBMCs) compared to baseline. Following co-administration of migalastat HCl and agalsidase, α-Gal A activity in plasma was further significantly increased 1.2- to 5.1-fold compared to agalsidase administration alone, in 22 of 23 patients (95.6%). Importantly, similar increases in skin and PBMC α-Gal A activity were seen following co-administration of migalastat HCl and agalsidase. The effects were not related to the administered migalastat HCl dose, as the 150 mg dose of migalastat HCl increased α-Gal A activity to the same extent as the 450 mg dose. Conversely, agalsidase had no effect on the plasma PK of migalastat. No migalastat HCl-related adverse events or drug-related tolerability issues were identified. TRIAL REGISTRATION: ClinicalTrials.gov NCT01196871.

Catalytic in vivo protein knockdown by small-molecule PROTACs.

The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.

Examination of Sec22 Homodimer Formation and Role in SNARE-dependent Membrane Fusion.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complexes play essential roles in catalyzing intracellular membrane fusion events although the assembly pathway and molecular arrangement of SNARE complexes in membrane fusion reactions are not well understood. Here we monitored interactions of the R-SNARE protein Sec22 through a cysteine scanning approach and detected efficient formation of cross-linked Sec22 homodimers in cellular membranes when cysteine residues were positioned in the SNARE motif or C terminus of the transmembrane domain. When specific Sec22 cysteine derivatives are present on both donor COPII vesicles and acceptor Golgi membranes, the formation of disulfide cross-links provide clear readouts on trans- and cis-SNARE arrangements during this fusion event. The Sec22 transmembrane domain was required for efficient homodimer formation and for membrane fusion suggesting a functional role for Sec22 homodimers. We propose that Sec22 homodimers promote assembly of higher-order SNARE complexes to catalyze membrane fusion. Sec22 is also reported to function in macroautophagy and in formation of endoplasmic reticulum-plasma membrane contact sites therefore homodimer assembly may regulate Sec22 activity across a range of cellular processes.

The pharmacological chaperone AT2220 increases recombinant human acid α-glucosidase uptake and glycogen reduction in a mouse model of Pompe disease.

Pompe disease is an inherited lysosomal storage disease that results from a deficiency in the enzyme acid α-glucosidase (GAA), and is characterized by progressive accumulation of lysosomal glycogen primarily in heart and skeletal muscles. Recombinant human GAA (rhGAA) is the only approved enzyme replacement therapy (ERT) available for the treatment of Pompe disease. Although rhGAA has been shown to slow disease progression and improve some of the pathophysiogical manifestations, the infused enzyme tends to be unstable at neutral pH and body temperature, shows low uptake into some key target tissues, and may elicit immune responses that adversely affect tolerability and efficacy. We hypothesized that co-administration of the orally-available, small molecule pharmacological chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride) may improve the pharmacological properties of rhGAA via binding and stabilization. AT2220 co-incubation prevented rhGAA denaturation and loss of activity in vitro at neutral pH and 37°C in both buffer and blood. In addition, oral pre-administration of AT2220 to rats led to a greater than two-fold increase in the circulating half-life of intravenous rhGAA. Importantly, co-administration of AT2220 and rhGAA to GAA knock-out (KO) mice resulted in significantly greater rhGAA levels in plasma, and greater uptake and glycogen reduction in heart and skeletal muscles, compared to administration of rhGAA alone. Collectively, these preclinical data highlight the potentially beneficial effects of AT2220 on rhGAA in vitro and in vivo. As such, a Phase 2 clinical study has been initiated to investigate the effects of co-administered AT2220 on rhGAA in Pompe patients.

Partial p16 staining in oropharyngeal squamous cell carcinoma: extent and pattern correlate with human papillomavirus RNA status.

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma has unique biology and better outcomes. p16 immunostaining is used as a surrogate marker for transcriptionally active HPV. Although diffuse staining is generally accepted as positive, the significance of partial staining has not been established, nor has the cutoff for extent of p16 staining that should be used to identify a tumor as HPV-related. From three other large studies utilizing p16 immunohistochemistry, we identified all cases with partial positive staining. The p16-stained slides were reviewed by three study pathologists for staining (nuclear and cytoplasmic) extent (in quartiles), and also for percentage that was confluent (ie, back-to-back cell staining). Tumors were histologically typed (keratinizing, non-keratinizing, or non-keratinizing with maturation) and tested for high-risk HPV by RNA in-situ hybridization and reverse-transcriptase PCR. For the 16 cases, there were two 4+(13%), five 3+(31%), six 2+(38%), and three 1+(19%) p16 staining tumors. Extent of staining ranged from 5 to 90% of cells positive with 25% or more confluent staining in 4/16 (25%). Of the 16 (31%) cases, 5 were HPV-related on the basis of RNA in-situ hybridization and reverse-transcriptase PCR. All of these cases had >50% p16 staining, 4/5 (80%) had more than 25% confluent staining, and 4/7 (57%) had non-keratinizing histological features. Only one of the p16 1+/2+ tumors was HPV RNA-positive (by reverse-transcriptase PCR only and low level). All 1+/2+ cases were keratinizing type or undifferentiated. By sensitive detection methods, most partial p16-positive squamous cell carcinoma cases with >50% staining harbor transcriptionally active HPV, and most HPV+ tumors have significant amounts of confluent staining. Cases with <50% p16 staining and lacking significant confluent staining rarely harbor HPV. These results support that greater than 75% p16 staining or, alternatively, >50% staining combined with >25% confluent areas, are suitable cutoffs for defining positivity.

Transcriptionally-active high-risk human papillomavirus is rare in oral cavity and laryngeal/hypopharyngeal squamous cell carcinomas--a tissue microarray study utilizing E6/E7 mRNA in situ hybridization.

AIMS: Human papillomavirus is well established in oropharyngeal squamous cell carcinoma as both causative and prognostic, but its significance in non-oropharyngeal tumours is unclear. In particular, the significance of finding viral DNA is not known. We sought to evaluate nonoropharyngeal squamous cell carcinomas for transcriptionally-active human papillomavirus and to compare this with the presence of viral DNA. METHODS: We evaluated an 87 patient tissue microarray cohort of oral cavity and laryngeal/hypopharyngeal squamous cell carcinomas for high risk human papillomavirus DNA and E6 and E7 mRNA transcripts by in situ hybridization, and for p16 expression by immunohistochemistry. RESULTS: We found only two of the 73 (2.7%) evaluable cases to harbour transcriptionally-active human papillomavirus. Both of these tumours were from the larynx, one was positive for human papillomavirus DNA by in situ hybridization, and both were extensively positive for p16. All oral cavity and hypopharyngeal tumours were negative for human papillomavirus. CONCLUSIONS: Transcriptionally-active human papillomavirus appears to be rare in laryngeal, hypopharyngeal, and oral cavity squamous cell carcinomas. As such, it appears unlikely to be a 'driver' or to be clinically significant in most established tumours.

Co-administration with the pharmacological chaperone AT1001 increases recombinant human α-galactosidase A tissue uptake and improves substrate reduction in Fabry mice.

Fabry disease is an X-linked lysosomal storage disorder (LSD) caused by mutations in the gene (GLA) that encodes the lysosomal hydrolase α-galactosidase A (α-Gal A), and is characterized by pathological accumulation of the substrate, globotriaosylceramide (GL-3). Regular infusion of recombinant human α-Gal A (rhα-Gal A), termed enzyme replacement therapy (ERT), is the primary treatment for Fabry disease. However, rhα-Gal A has low physical stability, a short circulating half-life, and variable uptake into different disease-relevant tissues. We hypothesized that coadministration of the orally available, small molecule pharmacological chaperone AT1001 (GR181413A, 1-deoxygalactonojirimycin, migalastat hydrochloride) may improve the pharmacological properties of rhα-Gal A via binding and stabilization. AT1001 prevented rhα-Gal A denaturation and activity loss in vitro at neutral pH and 37 °C. Coincubation of Fabry fibroblasts with rhα-Gal A and AT1001 resulted in up to fourfold higher cellular α-Gal A and ~30% greater GL-3 reduction compared to rhα-Gal A alone. Furthermore, coadministration of AT1001 to rats increased the circulating half-life of rhα-Gal A by >2.5-fold, and in GLA knockout mice resulted in up to fivefold higher α-Gal A levels and fourfold greater GL-3 reduction than rhα-Gal A alone. Collectively, these data highlight the potentially beneficial effects of AT1001 on rhα-Gal A, thus warranting clinical investigation.

High-risk human papillomavirus E6/E7 mRNA detection by a novel in situ hybridization assay strongly correlates with p16 expression and patient outcomes in oropharyngeal squamous cell carcinoma.

Human papillomavirus (HPV) is established as causative in oropharyngeal squamous cell carcinomas (OSCCs), being detected in 50% to 80% of tumors by DNA in situ hybridization (ISH) and/or polymerase chain reaction. However, these tests do not assess viral transcription. Many consider E6/E7 messenger ribonucleic acid (mRNA) the best indicator of HPV status, but it has not been detected in situ in OSCC. We constructed tissue microarrays (TMAs) from a cohort of OSCC for which p16 immunohistochemistry and HPV DNA ISH were previously performed on whole sections. We utilized a novel, chromogenic RNA ISH assay called RNAscope to detect E6/E7 mRNA of HPV-16 and other high-risk types on these TMAs. RNA ISH results were obtained for 196 of 211 TMA cases, of which 153 (78.1%) were positive. p16 immunohistochemistry and HPV DNA ISH were positive in 79.0% and 62.4% of cases, respectively. Concordance between RNA and p16, DNA and p16, and RNA and DNA were 96.4%, 78.7%, and 83.5%, respectively. Only 7 cases (3.6%) were discrepant between RNA ISH and p16. In univariate analysis, all 3 tests correlated with better overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS) (all P<0.001). In multivariate analysis, OS correlated significantly with RNA (hazard ratio=0.39, P=0.001), DNA (0.53, P=0.03), and p16 (0.30, P<0.001), but DSS and DFS correlated significantly only with p16 (DSS: 0.36, P=0.006; DFS: 0.42, P=0.016). RNA ISH is more sensitive than DNA ISH in detecting HPV in OSCC, and it correlates strongly with p16. Although both tests were comparable, p16 more strongly stratified patient outcomes.

Identification and characterization of pharmacological chaperones to correct enzyme deficiencies in lysosomal storage disorders.

Many human diseases result from mutations in specific genes. Once translated, the resulting aberrant proteins may be functionally competent and produced at near-normal levels. However, because of the mutations, the proteins are recognized by the quality control system of the endoplasmic reticulum and are not processed or trafficked correctly, ultimately leading to cellular dysfunction and disease. Pharmacological chaperones (PCs) are small molecules designed to mitigate this problem by selectively binding and stabilizing their target protein, thus reducing premature degradation, facilitating intracellular trafficking, and increasing cellular activity. Partial or complete restoration of normal function by PCs has been shown for numerous types of mutant proteins, including secreted proteins, transcription factors, ion channels, G protein-coupled receptors, and, importantly, lysosomal enzymes. Collectively, lysosomal storage disorders (LSDs) result from genetic mutations in the genes that encode specific lysosomal enzymes, leading to a deficiency in essential enzymatic activity and cellular accumulation of the respective substrate. To date, over 50 different LSDs have been identified, several of which are treated clinically with enzyme replacement therapy or substrate reduction therapy, although insufficiently in some cases. Importantly, a wide range of in vitro assays are now available to measure mutant lysosomal enzyme interaction with and stabilization by PCs, as well as subsequent increases in cellular enzyme levels and function. The application of these assays to the identification and characterization of candidate PCs for mutant lysosomal enzymes will be discussed in this review. In addition, considerations for the successful in vivo use and development of PCs to treat LSDs will be discussed.

A pharmacogenetic approach to identify mutant forms of α-galactosidase A that respond to a pharmacological chaperone for Fabry disease.

Fabry disease is caused by mutations in the gene (GLA) that encodes α-galactosidase A (α-Gal A). The iminosugar AT1001 (GR181413A, migalastat hydrochloride, 1-deoxygalactonojirimycin) is a pharmacological chaperone that selectively binds and stabilizes α-Gal A, increasing total cellular levels and activity for some mutant forms (defined as "responsive"). In this study, we developed a cell-based assay in cultured HEK-293 cells to identify mutant forms of α-Gal A that are responsive to AT1001. Concentration-dependent increases in α-Gal A activity in response to AT1001 were shown for 49 (60%) of 81 mutant forms. The responses of α-Gal A mutant forms were generally consistent with the responses observed in male Fabry patient-derived lymphoblasts. Importantly, the HEK-293 cell responses of 19 α-Gal A mutant forms to a clinically achievable concentration of AT1001 (10 µM) were generally consistent with observed increases in α-Gal A activity in peripheral blood mononuclear cells from male Fabry patients orally administered AT1001 during Phase 2 clinical studies. This indicates that the cell-based responses can identify mutant forms of α-Gal A that are likely to respond to AT1001 in vivo. Thus, the HEK-293 cell-based assay may be a useful aid in the identification of Fabry patients with AT1001-responsive mutant forms.

Adenosquamous carcinoma of the head and neck: relationship to human papillomavirus and review of the literature.

Adenosquamous carcinoma (ADSC) of the head and neck is an aggressive variant of squamous cell carcinoma (SCC). Certain variants of head and neck SCC are human papillomavirus (HPV)-related and have better prognosis. The relationship of HPV to head and neck ADSC has not been investigated. We searched our files for the term "adenosquamous" and head and neck subsites and found cases from 1998 to 2009. The requisite histologic criteria were the presence of SCC combined with distinct gland formation and/or intracellular mucin. DNA in situ hybridization for high-risk HPV, RNA in situ hybridization for high risk HPV E6 and E7 transcripts, and immunohistochemistry for p16 and p53 were performed. The existing literature on ADSC was also reviewed. Of the 18 cases, eight were from the larynx and hypopharynx, four from the oral cavity, three from the oropharynx, and three from the nasal cavity. Three cases (16%) showed both high risk HPV E6 and E7 and p16 expression, one from the nasal cavity and two from the oropharynx. Both oropharyngeal carcinoma patients were alive and disease free at 34 and 103 months, respectively. ADSCs of the head and neck are a heterogeneous group of tumors. A small minority of cases harbor HPV and most of these, particularly those occurring at sites with known high prevalence of HPV, show active viral transcription with detectable E6 and E7 and overexpression of p16. The HPV-related oropharyngeal cases, though rare, appear to do very well clinically, while the remaining cohort of ADSC patients do quite poorly. Head and neck ADSC appears to be a mixed variant that can be further classified according to its HPV status.

The pharmacological chaperone 1-deoxynojirimycin increases the activity and lysosomal trafficking of multiple mutant forms of acid alpha-glucosidase.

Pompe disease is a lysosomal storage disorder (LSD) caused by mutations in the gene that encodes acid alpha-glucosidase (GAA). Recently, small molecule pharmacological chaperones have been shown to increase protein stability and cellular levels for mutant lysosomal enzymes and have emerged as a new therapeutic strategy for the treatment of LSDs. In this study, we characterized the pharmacological chaperone 1-deoxynojirimycin (DNJ) on 76 different mutant forms of GAA identified in Pompe disease. DNJ significantly increased enzyme activity and protein levels for 16 different GAA mutants in patient-derived fibroblasts and in transiently transfected COS-7 cells. Additionally, DNJ increased the processing of these GAA mutants to their mature lysosomal forms, suggesting facilitated trafficking through the secretory pathway. Immunofluorescence microscopy studies showed increased colocalization of GAA with the lysosomal marker LAMP2 after incubation with DNJ, confirming increased lysosomal trafficking. Lastly, a GAA structural model was constructed based on the related eukaryotic glucosidase maltase-glucoamylase. The mutated residues identified in responsive forms of GAA are located throughout most of the structural domains, with half of these residues located in two short regions within the catalytic domain. Taken together, these data support further evaluation of DNJ as a potential treatment for Pompe disease in patients that express responsive forms of GAA.

Cholesterol exposure at the membrane surface is necessary and sufficient to trigger perfringolysin O binding.

Perfringolysin O (PFO) is the prototype for the cholesterol-dependent cytolysins, a family of bacterial pore-forming toxins that act on eukaryotic membranes. The pore-forming mechanism of PFO exhibits an absolute requirement for membrane cholesterol, but the complex interplay between the structural arrangement of the PFO C-terminal domain and the distribution of cholesterol in the target membrane is poorly understood. Herein we show that PFO binding to the bilayer and the initiation of the sequence of events that culminate in the formation of a transmembrane pore depend on the availability of free cholesterol at the membrane surface, while changes in the acyl chain packing of the phospholipids and cholesterol in the membrane core, or the presence or absence of detergent-resistant domains do not correlate with PFO binding. Moreover, PFO association with the membrane was inhibited by the addition of sphingomyelin, a typical component of membrane rafts in cell membranes. Finally, addition of molecules that do not interact with PFO, but intercalate into the membrane and displace cholesterol from its association with phospholipids (e.g., epicholesterol), reduced the amount of cholesterol required to trigger PFO binding. Taken together, our studies reveal that PFO binding to membranes is triggered when the concentration of cholesterol exceeds the association capacity of the phospholipids, and this cholesterol excess is then free to associate with the toxin.

Dscam2 mediates axonal tiling in the Drosophila visual system.

Sensory processing centres in both the vertebrate and the invertebrate brain are often organized into reiterated columns, thus facilitating an internal topographic representation of the external world. Cells within each column are arranged in a stereotyped fashion and form precise patterns of synaptic connections within discrete layers. These connections are largely confined to a single column, thereby preserving the spatial information from the periphery. Other neurons integrate this information by connecting to multiple columns. Restricting axons to columns is conceptually similar to tiling. Axons and dendrites of neighbouring neurons of the same class use tiling to form complete, yet non-overlapping, receptive fields. It is thought that, at the molecular level, cell-surface proteins mediate tiling through contact-dependent repulsive interactions, but proteins serving this function have not yet been identified. Here we show that the immunoglobulin superfamily member Dscam2 restricts the connections formed by L1 lamina neurons to columns in the Drosophila visual system. Our data support a model in which Dscam2 homophilic interactions mediate repulsion between neurites of L1 cells in neighbouring columns. We propose that Dscam2 is a tiling receptor for L1 neurons.

Dendrite self-avoidance is controlled by Dscam.

Dendrites distinguish between sister branches and those of other cells. Self-recognition can often lead to repulsion, a process termed "self-avoidance." Here we demonstrate that dendrite self-avoidance in Drosophila da sensory neurons requires cell-recognition molecules encoded by the Dscam locus. By alternative splicing, Dscam encodes a vast number of cell-surface proteins of the immunoglobulin superfamily. We demonstrate that interactions between identical Dscam isoforms on the cell surface underlie self-recognition, while the cytoplasmic tail converts this recognition to dendrite repulsion. Sister dendrites expressing the same isoforms engage in homophilic repulsion. By contrast, Dscam diversity ensures that inappropriate repulsive interactions between dendrites sharing the same receptive field do not occur. The selectivity of Dscam-mediated cell interactions is likely to be widely important in the developing fly nervous system, where processes of cells must distinguish between self and nonself during the construction of neural circuits.