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Chronic obstructive pulmonary disease (COPD) is a leading cause of death and cigarette smoke is the main risk factor. Detecting its earliest stages and preventing a decline in lung function are key goals. The pathogenesis of COPD is complex but has some similarities to cystic fibrosis (CF), a disease caused by mutations in the cftr gene. CF leads to chronic inflammation, abnormal mucus, and cycles of infection. Cigarette smoke exposure also causes CFTR dysfunction, and it is probably not a coincidence that inflammation, mucus obstruction, and infections are also characteristics of COPD, although the exacerbations can be quite different. We review here the acute effects of cigarette smoke on CFTR function and its potential role in COPD. Understanding CFTR regulation by cigarette smoke may identify novel drug targets and facilitate the development of therapeutics that reduce the progression and severity of COPD.

The marine natural product latonduine A (1) shows F508del-cystic fibrosis transmembrane regulator (CFTR) corrector activity in cell-based assays. Pull-down experiments, enzyme inhibition assays, and siRNA knockdown experiments suggest that the F508del-CFTR corrector activities of latonduine A and a synthetic analogue MCG315 (4) result from simultaneous inhibition of PARP3 and PARP16. A library of synthetic latonduine A analogs has been prepared in an attempt to separate the PARP3 and PARP16 inhibitory properties of latonduine A with the goal of discovering selective small-molecule PARP3 and PARP16 inhibitory cell biology tools that could confirm the proposed dual-target F508del-CFTR corrector mechanism of action. The structure activity relationship (SAR) study reported herein has resulted in the discovery of the modestly potent (IC50 3.1 μM) PARP3 selective inhibitor (±)-5-hydroxy-4-phenyl-2,3,4,5-tetrahydro-1H-benzo[c]azepin-1-one (5) that shows 96-fold greater potency for inhibition of PARP3 compared with its inhibition of PARP16 in vitro and the potent (IC50 0.362 μM) PARP16 selective inhibitor (±)-7,8-dichloro-5-hydroxy-4-(pyridin-2-yl)-2,3,4,5-tetrahydro-1H-benzo[c]azepin-1-one (6) that shows 205-fold selectivity for PARP16 compared with PARP3 in vitro. At 1 or 10 μM, neither 5 or 6 alone showed F508del-CFTR corrector activity, but when added together at 1 or 10 μM each, the combination exhibited F508del-CFTR corrector activity identical to 1 or 10 μM latonduine A (1), respectively, supporting its novel dual PARP target mechanism of action. Latonduine A (1) showed additive in vitro corrector activity in combination with the clinically approved corrector VX809, making it a potential new partner for cystic fibrosis combination drug therapies.

Most cases of cystic fibrosis (CF) are caused by class 2 mutations in the cystic fibrosis transmembrane regulator (CFTR). These proteins preserve some channel function but are retained in the endoplasmic reticulum (ER). Partial rescue of the most common CFTR class 2 mutant, F508del-CFTR, has been achieved through the development of pharmacological chaperones (Tezacaftor and Elexacaftor) that bind CFTR directly. However, it is not clear whether these drugs will rescue all class 2 CFTR mutants to a medically relevant level. We have previously shown that the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen can correct F508del-CFTR trafficking. Here, we utilized RNAi and pharmacological inhibitors to determine the mechanism of action of the NSAID glafenine. Using cellular thermal stability assays (CETSAs), we show that it is a proteostasis modulator. Using medicinal chemistry, we identified a derivative with a fourfold increase in CFTR corrector potency. Furthermore, we show that these novel arachidonic acid pathway inhibitors can rescue difficult-to-correct class 2 mutants, such as G85E-CFTR 13%, that of non-CF cells in well-differentiated HBE cells. Thus, the results suggest that targeting the arachidonic acid pathway may be a profitable way of developing correctors of certain previously hard-to-correct class 2 CFTR mutations.

Despite progress in developing pharmacotherapies to rescue F508del-CFTR, the most prevalent Cystic Fibrosis (CF)-causing mutation, individuals homozygous for this mutation still face several disease-related symptoms. Thus, more potent compound combinations are still needed. Here, we investigated the mechanism of action (MoA) of RDR01752, a novel F508del-CFTR trafficking corrector. F508del-CFTR correction by RDR01752 was assessed by biochemical, immunofluorescence microscopy and functional assays in cell lines and in intestinal organoids. To determine the MoA of RDR01752, we assessed its additive effects to those of genetic revertants of F508del-CFTR, the FDA-approved corrector drugs VX-809 and VX-661, and low temperature. Our data demonstrated that RDR01752 rescues F508del-CFTR processing and plasma membrane (PM) expression to similar levels of VX-809 in cell lines, although RDR01752 produced lower functional rescue. However, in functional assays using intestinal organoids (F508del/F508del), RDR01752, VX-809 and VX-661 had similar efficacy. RDR01752 demonstrated additivity to revertants 4RK and G550E, but not to R1070W, as previously shown for VX-809. RDR01752 was also additive to low temperature. Co-treatment of RDR01752 and VX-809 did not increase F508del-CFTR PM expression and function compared to each corrector alone. The lack of additivity of RDR01752 with the genetic revertant R1070W suggests that this compound has the same effect as the insertion of tryptophan at 1070, i.e., filling the pocket at the NBD1:ICL4 interface in F508del-CFTR, similarly to VX-809. Combination of RDR01752 with correctors mimicking the rescue by revertants G550E or 4RK could thus maximize rescue of F508del-CFTR.

These are exciting times in the development of therapeutics for cystic fibrosis (CF). New correctors and potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) are being developed in academic laboratories and pharmaceutical companies, and the field is just beginning to understand their mechanisms of action. Studies of CFTR modulators are also yielding insight into the general principles and strategies that can be used when developing pharmacological chaperones, a new class of drugs. Combining two or even three correctors with a potentiator is an especially promising approach which should lead to further improvements in efficacy and clinical benefit for patients.

Introduction: Cystic fibrosis (CF) is the most frequent lethal orphan disease and is caused by mutations in the CFTR gene. The most frequent mutation F508del-CFTR affects multiple organs; infections and subsequent infections and complications in the lung lead to death. Areas covered: This review focuses on new targets and mechanisms that are attracting interest for the development of CF therapies. The F508del-CFTR protein is retained in the endoplasmic reticulum (ER) but has some function if it can traffic to the plasma membrane. Cell-based assays have been used to screen chemical libraries for small molecule correctors that restore its trafficking. Pharmacological chaperones are correctors that bind directly to the F508del-CFTR mutant and promote its folding and trafficking. Other correctors fall into a heterogeneous class of proteostasis modulators that act indirectly by altering cellular homeostasis. Expert opinion: Pharmacological chaperones have so far been the most successful correctors of F508del-CFTR trafficking, but their level of correction means that more than one corrector is required. Proteostasis modulators have low levels of correction but hold promise because some can correct several different CFTR mutations. Identification of their cellular targets and the potential for development may lead to new therapies for CF.

Background and Purpose The most common cystic fibrosis (CF) mutation F508del inhibits the gating and surface expression of CFTR, a plasma membrane anion channel. Optimal pharmacotherapies will probably require both a ‘potentiator’ to increase channel open probability and a ‘corrector’ that improves folding and trafficking of the mutant protein and its stability at the cell surface. Interaction between CF drugs has been reported but remains poorly understood. Experimental Approach CF bronchial epithelial cells were exposed to the corrector VX-809 (lumacaftor) and potentiator VX-770 (ivacaftor) individually or in combination. Functional expression of CFTR was assayed as the forskolin-stimulated short-circuit current (Isc) across airway epithelial monolayers expressing F508del CFTR. Key Results The potentiated Isc response during forskolin stimulation was increased sixfold after pretreatment with VX-809 alone and reached ~11% that measured across non-CF monolayers. VX-770 (100 nM) and genistein (50 μM) caused similar levels of potentiation, which were not additive and were abolished by the CFTR inhibitor CFTRinh-172. The unbound fraction of VX-770 in plasma was 0.13 ± 0.04%, which together with previous measurements in patients given 250 mg p.o. twice daily, suggests a peak free plasma concentration of 1.5–8.5 nM. Chronic exposure to high VX-770 concentrations (>1 μM) inhibited functional correction by VX-809 but not in the presence of physiological protein levels (20–40 mg·mL−1). Chronic exposure to a low concentration of VX-770 (100 nM) together with VX-809 (1 μM) also did not reduce the forskolin-stimulated Isc, relative to cells chronically exposed to VX-809 alone, provided it was assayed acutely using the same, clinically relevant concentration of potentiator. Conclusions and Implications Chronic exposure to clinically relevant concentrations of VX-770 did not reduce F508del CFTR function. Therapeutic benefit of VX-770 + VX-809 (Orkambi) is probably limited by the efficacy of VX-809 rather than by inhibition by VX-770.

Pharmacological chaperones (e.g. VX-809, lumacaftor) that bind directly to F508del-CFTR and correct its mislocalization are promising therapeutics for Cystic Fibrosis (CF). However to date, individual correctors provide only ~4% improvement in lung function measured as FEV1, suggesting that multiple drugs will be needed to achieve substantial clinical benefit. Here we examine if multiple sites for pharmacological chaperones exist and can be targeted to enhance the rescue of F508del-CFTR with the premise that additive or synergistic rescue by multiple pharmacological chaperones compared to single correctors indicates that they have different sites of action. First, we found that a combination of the pharmacological chaperones VX-809 and RDR1 provide additive correction of F508del-CFTR. Then using cellular thermal stability assays (CETSA) we demonstrated the possibility of a third pharmacologically important site using the novel pharmacological chaperone tool compound 4-methyl-N-[3-(morpholin-4-yl) quinoxalin-2-yl] benzenesulfonamide (MCG1516A). All three pharmacological chaperones appear to interact with the first nucleotide-binding domain (NBD1). The triple combination of MCG1516A, RDR1, and VX-809 restored CFTR function to >20% that of non-CF cells in well differentiated HBE cells and to much higher levels in other cell types. Thus the results suggest the presence of at least three distinct sites for pharmacological chaperones on F508del-CFTR NBD1, encouraging the development of triple corrector combinations.

Background Small molecules as shown by VX809 can rescue the mislocalization of F508del-CFTR. The aim of this study was to identify correctors with a clinical history and their targets of action. Methods CFTR correctors were screened using two F508del-CFTR expressing cell based HTS assays. Electrophysiological studies using CFBE41o − and HBE cells and in-vivo mouse assays confirmed CFTR rescue. The target of action was attained using pharmacological inhibitors and siRNA to specific genes. Results Ibuprofen was identified as a CFTR corrector. Ibuprofen treatment of polarized CFBE41o − monolayers increased the short-circuit current (I sc ) response to stimulation. In vivo CF mice treatment with ibuprofen restored the CFTR trafficking. SiRNA knock down of cyclooxygenase expression caused partial F508del-CFTR correction. Conclusion These studies show that ibuprofen is a CFTR corrector and that it causes correction by COX-1 inhibition. Hence ibuprofen may be suitable to be part of a future CF combination therapy.

Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein-protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3-CFTR interface might offer an approach for cystic fibrosis therapeutics.

SummaryMost cases of cystic fibrosis (CF) are attributable to the F508del allele of CFTR, which causes the protein to be retained in the endoplasmic reticulum (ER) and subsequently degraded. One strategy for CF therapy is to identify corrector compounds that help traffic F508del-CFTR to the cell surface. Pharmacological chaperones, or correctors that bind specifically to F508del-CFTR and restore function, would be the most promising drug development candidates, but few pharmacological chaperones exist for F508del-CFTR. Using differential scanning fluorimetry (DSF), we have surveyed corrector compounds and identified one, RDR1, which binds directly to the first nucleotide binding domain (NBD1) of F508del-CFTR. We show that RDR1 treatment partially rescues F508del-CFTR function in both cells and in an F508del-CF mouse model. Thus, RDR1 is a pharmacological chaperone of F508del-CFTR and represents a novel scaffold for drug development.

High-throughput small-molecule screens hold great promise for identifying compounds with potential therapeutic value in the treatment of protein-trafficking diseases such as cystic fibrosis (CF) and nephrogenic diabetes insipidus (NDI). The approach usually involves expressing the mutant form of the gene in cells and assaying function in a multiwell format when cells are exposed to libraries of compounds. Although such functional assays are useful, they do not directly test the ability of a compound to correct defective trafficking of the protein. To address this we have developed a novel corrector-screening assay for CF, in which the appearance of the mutant protein at the cell surface is measured. We used this assay to screen a library of 2000 compounds and have isolated several classes of trafficking correctors that had not previously been identified. This novel screening approach to protein-trafficking diseases is robust and general, and could enable the selection of molecules that could be translated rapidly to a clinical setting.

Cystic fibrosis (CF) is caused by mutations in CF transmembrane conductance regulator (CFTR). The most frequent mutation (F508del-CFTR) results in altered proteostasis, that is, in the misfolding and intracellular degradation of the protein. The F508del-CFTR proteostasis machinery and its homeostatic regulation are well studied, while the question whether ‘classical’ signalling pathways and phosphorylation cascades might control proteostasis remains barely explored. Here, we have unravelled signalling cascades acting selectively on the F508del-CFTR folding-trafficking defects by analysing the mechanisms of action of F508del-CFTR proteostasis regulator drugs through an approach based on transcriptional profiling followed by deconvolution of their gene signatures. Targeting multiple components of these signalling pathways resulted in potent and specific correction of F508del-CFTR proteostasis and in synergy with pharmacochaperones. These results provide new insights into the physiology of cellular proteostasis and a rational basis for developing effective pharmacological correctors of the F508del-CFTR defect. DOI: http://dx.doi.org/10.7554/eLife.10365.001, eLife digest Cystic fibrosis is a genetic disease that commonly affects people of European descent. The condition is caused by mutations in the gene encoding a protein called “cystic fibrosis transmembrane conductance regulator” (or CFTR for short). CFTR forms a channel in the membrane of cells in the lungs that help transport salt across the membrane. Mutated versions of the protein are not as efficient at transporting salts, and eventually this damages the lung tissue. As the damage progresses, individuals become very vulnerable to bacterial infections that further damage the lungs and may eventually lead to death. One of the reasons CFTR mutations are harmful is that they cause the protein to fold up incorrectly and remain trapped inside the cell. Cells have quality control systems that recognize and destroy poorly folded proteins, and so only a few of the mutated CFTR proteins ever make it to the membrane to move salts. New therapies have been developed that improve folding of the protein and/or help the CFTR proteins that make it to the membrane work better. But more and better treatment options are needed. Hegde, Parashuraman et al. have now tested drugs that control how proteins fold and move to the membrane to see how they affect gene expression in cells with the most common cystic fibrosis-causing mutation. These drugs are known to improve the activity of the CFTR mutant, but do so too weakly to be of clinical interest. The experiments revealed that the expression of a few hundred genes was changed in response the drugs. Many of these genes were involved in major signalling pathways that control how CFTR is folded and trafficked within cells. Next, Hegde, Parashuraman et al. tested drugs that inhibit these signalling pathways to see if they improve salt handling in the mutated cells. The experiments demonstrated that these inhibitor drugs efficiently block the breakdown of misfolded CFTR, or boost the likelihood of CFTR making it to the membrane, helping improve salt trafficking in the cells. The inhibitors produced even better results when used in combination with a known CFTR-protecting drug. The results suggest that identifying and targeting signalling pathways involved in the folding, trafficking, and breakdown of CFTR may prove a promising way to treat cystic fibrosis. DOI: http://dx.doi.org/10.7554/eLife.10365.002

Cystic fibrosis (CF) is a major lethal genetic disease caused by mutations in the CF transmembrane conductance regulator gene (CFTR). This encodes a chloride ion channel on the apical surface of epithelial cells. The most common mutation in CFTR (F508del-CFTR) generates a protein that is misfolded and retained in the endoplasmic reticulum. Identifying small molecules that correct this CFTR trafficking defect is a promising approach in CF therapy. However, to date only modest efficacy has been reported for correctors in clinical trials. We identified the marine sponge metabolite latonduine as a corrector. We have now developed a series of latonduine derivatives that are more potent F508del-CFTR correctors with one (MCG315 [2,3-dihydro-1H-2-benzazepin-1-one]) having 10-fold increased corrector activity and an EC50 of 72.25 nM. We show that the latonduine analogs inhibit poly-ADP ribose polymerase (PARP) isozymes 1, 3, and 16. Further our molecular modeling studies point to the latonduine analogs binding to the PARP nicotinamide-binding domain. We established the relationship between the ability of the latonduine analogs to inhibit PARP-16 and their ability to correct F508del-CFTR trafficking. We show that latonduine can inhibit both PARP-3 and -16 and that this is necessary for CFTR correction. We demonstrate that latonduine triggers correction by regulating the activity of the unfolded protein response activator inositol-requiring enzyme (IRE-1) via modulation of the level of its ribosylation by PARP-16. These results establish latonduines novel site of action as well as its proteostatic mechanism of action.

The most common cystic fibrosis (CF) mutation F508del inhibits the gating and surface expression of CFTR, a plasma membrane anion channel. Optimal pharmacotherapies will probably require both a 'potentiator' to increase channel open probability and a 'corrector' that improves folding and trafficking of the mutant protein and its stability at the cell surface. Interaction between CF drugs has been reported but remains poorly understood.CF bronchial epithelial cells were exposed to the corrector VX-809 (lumacaftor) and potentiator VX-770 (ivacaftor) individually or in combination. Functional expression of CFTR was assayed as the forskolin-stimulated short-circuit current (Isc ) across airway epithelial monolayers expressing F508del CFTR.The potentiated Isc response during forskolin stimulation was increased sixfold after pretreatment with VX-809 alone and reached ~11% that measured across non-CF monolayers. VX-770 (100 nM) and genistein (50 μM) caused similar levels of potentiation, which were not additive and were abolished by the CFTR inhibitor CFTRinh -172. The unbound fraction of VX-770 in plasma was 0.13 ± 0.04%, which together with previous measurements in patients given 250 mg p.o. twice daily, suggests a peak free plasma concentration of 1.5-8.5 nM. Chronic exposure to high VX-770 concentrations (1 μM) inhibited functional correction by VX-809 but not in the presence of physiological protein levels (20-40 mg·mL(-1) ). Chronic exposure to a low concentration of VX-770 (100 nM) together with VX-809 (1 μM) also did not reduce the forskolin-stimulated Isc , relative to cells chronically exposed to VX-809 alone, provided it was assayed acutely using the same, clinically relevant concentration of potentiator.Chronic exposure to clinically relevant concentrations of VX-770 did not reduce F508del CFTR function. Therapeutic benefit of VX-770 + VX-809 (Orkambi) is probably limited by the efficacy of VX-809 rather than by inhibition by VX-770.

The AML-1/ETO fusion protein, created by the (8;21) translocation in M2-type acute myelogenous leukemia (AML), is a dominant repressive form of AML-1. This effect is due to the ability of the ETO portion of the protein to recruit co-repressors to promoters of AML-1 target genes. The t(11;17)(q21;q23)-associated acute promyelocytic leukemia creates the promyelocytic leukemia zinc finger PLZFt/RARα fusion protein and, in a similar manner, inhibits RARα target gene expression and myeloid differentiation. PLZF is expressed in hematopoietic progenitors and functions as a growth suppressor by repressing cyclin A2 and other targets. ETO is a corepressor for PLZF and potentiates transcriptional repression by linking PLZF to a histone deacetylase-containing complex. In transiently transfected cells and in a cell line derived from a patient with t(8;21) leukemia, PLZF and AML-1/ETO formed a tight complex. In transient assays, AML-1/ETO blocked transcriptional repression by PLZF, even at substoichiometric levels relative to PLZF. This effect was dependent on the presence of the ETO zinc finger domain, which recruits corepressors, and could not be rescued by overexpression of co-repressors that normally enhance PLZF repression. AML-1/ETO also excluded PLZF from the nuclear matrix and reduced its ability to bind to its cognate DNA-binding site. Finally, ETO interacted with PLZF/RARα and enhanced its ability to repress through the RARE. These data show a link in the transcriptional pathways of M2 and M3 leukemia.

The AML-1/ETO fusion protein, created by the (8;21) translocation in M2-type acute myelogenous leukemia (AML), is a dominant repressive form of AML-1. This effect is due to the ability of the ETO portion of the protein to recruit co-repressors to promoters of AML-1 target genes. The t(11;17)(q21;q23)-associated acute promyelocytic leukemia creates the promyelocytic leukemia zinc finger PLZFt/RARα fusion protein and, in a similar manner, inhibits RARα target gene expression and myeloid differentiation. PLZF is expressed in hematopoietic progenitors and functions as a growth suppressor by repressing cyclin A2 and other targets. ETO is a corepressor for PLZF and potentiates transcriptional repression by linking PLZF to a histone deacetylase-containing complex. In transiently transfected cells and in a cell line derived from a patient with t(8;21) leukemia, PLZF and AML-1/ETO formed a tight complex. In transient assays, AML-1/ETO blocked transcriptional repression by PLZF, even at substoichiometric levels relative to PLZF. This effect was dependent on the presence of the ETO zinc finger domain, which recruits corepressors, and could not be rescued by overexpression of co-repressors that normally enhance PLZF repression. AML-1/ETO also excluded PLZF from the nuclear matrix and reduced its ability to bind to its cognate DNA-binding site. Finally, ETO interacted with PLZF/RARα and enhanced its ability to repress through the RARE. These data show a link in the transcriptional pathways of M2 and M3 leukemia.

The promyelocytic leukemia zinc finger (PLZF) protein is a transcription factor disrupted in patients with t(11;17)(q23;q21)-associated acute promyelocytic leukemia. PLZF contains an N-terminal BTB/POZ domain which is required for dimerization, transcriptional repression, formation of high-molecular-weight DNA-protein complexes, nuclear sublocalization, and growth suppression. X-ray crystallographic data show that the PLZF BTB/POZ domain forms an obligate homodimer via an extensive interface. In addition, the dimer possesses several highly conserved features, including a charged pocket, a hydrophobic monomer core, an exposed hydrophobic surface on the floor of the dimer, and two negatively charged surface patches. To determine the role of these structures, mutational analysis of the BTB/POZ domain was performed. We found that point mutations in conserved residues that disrupt the dimer interface or the monomer core result in a misfolded nonfunctional protein. Mutation of key residues from the exposed hydrophobic surface suggests that these are also important for the stability of PLZF complexes. The integrity of the charged-pocket region was crucial for proper folding of the BTB/POZ domain. In addition, the pocket was critical for the ability of the BTB/POZ domain to repress transcription. Alteration of charged-pocket residue arginine 49 to a glutamine (mutant R49Q) yields a domain that can still dimerize but activates rather than represses transcription. In the context of full-length PLZF, a properly folded BTB/POZ domain was required for all PLZF functions. However, PLZF with the single pocket mutation R49Q repressed transcription, while the double mutant D35N/R49Q could not, despite its ability to dimerize. These results indicate that PLZF requires the BTB/POZ domain for dimerization and the charged pocket for transcriptional repression.

MPP+ inhibits mitochondrial complex I and alpha-ketoglutarate dehydrogenase causing necrosis or apoptosis of catecholaminergic neurons. Low glucose levels or glycolytic blockade has been shown to potentiate MPP+ toxicity. We found that MPP+ caused concentration-dependent apoptosis of neuronally differentiated PC12 cells and that glucose, but not pyruvate, supplementation reduced apoptosis. Oligomycin concentrations sufficient to inhibit ATP synthase blocked the decreased apoptosis afforded by glucose supplementation. Laser-scanning confocal microscope imaging of chloromethyl-tetramethylrosamine methyl ester fluorescence to estimate DeltaPsiM showed that MPP+ and atractyloside reduced DeltaPsiM, while cyclosporin A (CSA) and glucose supplementation reversed decreases in DeltaPsiM caused by MPP+. Oligomycin blocked the effect of glucose supplementation on DeltaPsiM. These findings show that (i) MPP+-induced and atractyloside-induced apoptosis are associated with reduced DeltaPsiM; (ii) CSA maintains DeltaPsiM and reduces MPP+-induced apoptosis; and (iii) glucose supplementation maintains DeltaPsiM, likely by glycolytic ATP-dependent proton pumping at ATP synthase and reduces MPP+-induced apoptosis.

The promyelocytic leukaemia (protein) (PML) localizes to multiprotein complexes known as PML nuclear bodies. We found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) co-immunoprecipitates with PML and co-localizes with PML in nuclear bodies. RNase treatment disrupts the ability of PML and GAPDH to both co-localize and co-immunoprecipitate, indicating that the association between PML and GAPDH depends on the presence of RNA. Disruption of PML bodies contributes towards reduced apoptosis in acute promyelocytic leukaemia and GAPDH induces apoptotic neuronal death. The GAPDH–PML interaction may be involved in the regulation of apoptosis.

The promyelocytic leukemia (PML) protein forms nuclear bodies which are relocated to the cytoplasm by the RNA virus lymphocytic choriomeningitis virus (LCMV). The viral Z protein directly binds to PML and can relocate the nuclear bodies. Others have observed that LCMV virions may contain ribosomes; hence, we investigated the effects of infection on the distribution of ribosomal P proteins (P0, P1, and P2) with PML as a reference point. We demonstrate an association of PML bodies with P proteins by indirect immunofluorescence and coimmunoprecipitation experiments, providing the first evidence of nucleic acid-binding proteins associated with PML bodies. We show that unlike PML, the P proteins are not redistributed upon infection. Immunofluorescence and coimmunoprecipitation studies indicate that the viral Z protein binds the nuclear, but not the cytoplasmic, fraction of P0. The nuclear fraction of P0 has been associated with translationally coupled DNA excision repair and with nonspecific endonuclease activity; thus, P0 may be involved in nucleic acid processing activities necessary for LCMV replication. During the infection process, PML, P1, and P2 are downregulated but P0 remains unchanged. Further, P0 is present in virions while PML is not, indicating some selectivity in the assembly of LCMV.

Studies in non-neural cells have suggested that a fall in mitochondrial membrane potential (ΔΨM) is one of the earliest events in apoptosis. It is not known whether neural apoptosis caused by nerve growth factor (NGF) and serum withdrawal involves a decrease in ΔΨM. We used epifluorescence and laser confocal microscopy with the mitochondrial potentiometric dyes chloromethyl-tetramethylrosamine methyl ester and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethybenzimidazol carbocyanine iodide to estimate ΔΨM. PC12 cells were differentiated in media containing serum and NGF for 6 d before withdrawal of trophic support. After washing, the cells were incubated with media containing serum and NGF (M/S+N), media without serum and NGF, or media with the “trophic-like” monoamine oxidase B inhibitor, (−)-deprenyl. Mitochondria in cells without trophic support underwent a progressive shift to lower ΔΨMvalues that was significant by 3 hr after washing. The percentages of cells with nuclear chromatin condensation or nuclear DNA fragmentation were not significantly increased above those for cells in M/S+N until 6 hr after washing. Replacement of cells into M/S+N or treatment with (−)-deprenyl markedly reduced the proportion of mitochondria with decreased ΔΨM. Measurements of cytoplasmic peroxyl radical levels with 2′,7′-dihydrodichlorofluorescein fluorescence and intramitochondrial Ca2+with dihydro-rhodamine-2-acetylmethyl ester indicated that cytoplasmic peroxyl radical levels were not increased until after 6 hr, whereas increases in intramitochondrial Ca2+paralleled the decreases in ΔΨM. (−)-Deprenyl appeared to alter the relationship between intramitochondrial Ca2+levels and ΔΨM, possibly through its reported capacity to increase the synthesis of proteins such as BCL-2.

Two molecules involved in signal transduction via the T cell antigen receptor, namely the protein-tyrosine kinase ZAP-70 and the proto-oncoprotein Vav, were found to be constitutively associated with tubulin in Jurkat T cells. Both were able to bind to tubulin independently of one another, as determined by transient transfection into COS-7 cells. The ZAP-70 associated with tubulin was preferentially tyrosine-phosphorylated after T cell antigen receptor stimulation of Jurkat T cells, suggesting that this interaction was functionally significant. Vav was also found to co-immunoprecipitate with ZAP-70 from cell extracts depleted of tubulin. This raised the possibility that Vav might be a substrate for ZAP-70 protein-tyrosine kinase activity. However, tyrosine phosphorylation of Vav preceded that of ZAP-70, indicating that Vav was unlikely to be a downstream target of ZAP-70. The association of ZAP-70 and Vav with tubulin implies that the microtubules may be involved in the signaling function of these two molecules, perhaps by targeting them to their appropriate intracellular location.

SummaryMutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause CF. The most common mutation, F508 deletion, causes CFTR misfolding and endoplasmic reticulum retention, preventing it from trafficking to the cell surface. One approach to CF treatment is to identify compounds that correct the trafficking defect. We screened a marine extract collection and, after extract, deconvolution identified the latonduines as F508del-CFTR trafficking correctors that give functional correction in vivo. Using a biotinylated azido derivative of latonduine, we identified the poly(ADP-ribose) polymerase (PARP) family as latonduine target proteins. We show that latonduine binds to PARPs 1, 2, 3, 4, 5a, and 5b and inhibits PARP activity, especially PARP-3. Thus, latonduine corrects F508del-CFTR trafficking by modulating PARP activity. Latonduines represent pharmacologic agents for F508del-CFTR correction, and PARP-3 is a pathway for the development of CF treatments.

Most cystic fibrosis is caused by mutations in CFTR that prevent its trafficking from the ER to the plasma membrane and is associated with exaggerated inflammation, altered metabolism, and diminished responses to oxidative stress. PARP-1 is activated by oxidative stress and causes energy depletion and cell dysfunction. Inhibition of this enzyme protects against excessive inflammation and recent studies have also implicated it in intracellular protein trafficking. We hypothesized that PARP-1 activity is altered in CF and affects trafficking and function of the most common CF mutant ΔF508 CFTR. Indeed, PARP-1 activity was 2.9-fold higher in CF (ΔF508/ΔF508) human bronchial epithelial primary cells than in non-CF cells, and similar results were obtained by comparing CF vs. non-CF bronchial epithelial cell lines (2.5-fold higher in CFBE41o(-) vs. 16HBE14o(-), P 0.002). A PARP-1 inhibitor (ABT-888, Veliparib) partially restored CFTR channel activity in CFBE41o(-) cells overexpressing ΔF508 CFTR. Similarly, reducing PARP-1 activity by 85% in ileum from transgenic CF mice (Cftr(tm1)Eur) partially rescued ΔF508 CFTR activity to 7% of wild type mouse levels, and similar correction (7.8%) was observed in vivo by measuring salivary secretion. Inhibiting PARP-1 with ABT-888 or siRNA partially restored ΔF508 CFTR trafficking in cell lines, and most ΔF508 CFTR was complex glycosylated when heterologously expressed in PARP-1(-/-) mouse embryonic fibroblasts. Finally, levels of the mature glycoform of CFTR were reduced by peroxynitrite, a strong activator of PARP-1. These results demonstrate that PARP-1 activity is increased in CF, and identify a novel pathway that could be targeted by proteostatic correctors of CFTR trafficking.

Identification of active compounds in high-throughput screening (HTS) contexts can be substantially improved by applying classical experimental design and statistical inference principles to all phases of HTS studies. The authors present both experimental and simulated data to illustrate how true-positive rates can be maximized without increasing false-positive rates by the following analytical process. First, the use of robust data preprocessing methods reduces unwanted variation by removing row, column, and plate biases. Second, replicate measurements allow estimation of the magnitude of the remaining random error and the use of formal statistical models to benchmark putative hits relative to what is expected by chance. Receiver Operating Characteristic (ROC) analyses revealed superior power for data preprocessed by a trimmed-mean polish method combined with the RVM t-test, particularly for small- to moderate-sized biological hits.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated anion channel expressed in epithelial cells. The most common mutation Delta Phe508 leads to protein misfolding, retention by the endoplasmic reticulum, and degradation. One promising therapeutic approach is to identify drugs that have been developed for other indications but that also correct the CFTR trafficking defect, thereby exploiting their known safety and bioavailability in humans and reducing the time required for clinical development. We have screened approved, marketed, and off-patent drugs with known safety and bioavailability using a Delta Phe508-CFTR trafficking assay. Among the confirmed hits was glafenine, an anthranilic acid derivative with analgesic properties. Its ability to correct the misprocessing of CFTR was confirmed by in vitro and in vivo studies using a concentration that is achieved clinically in plasma (10 microM). Glafenine increased the surface expression of Delta Phe508-CFTR in baby hamster kidney (BHK) cells to approximately 40% of that observed for wild-type CFTR, comparable with the known CFTR corrector 4-cyclohexyloxy-2-{1-[4-(4-methoxybenzensulfonyl)-piperazin-1-yl]-ethyl}-quinazoline (VRT-325). Partial correction was confirmed by the appearance of mature CFTR in Western blots and by two assays of halide permeability in unpolarized BHK and human embryonic kidney cells. Incubating polarized CFBE41o(-) monolayers and intestines isolated from Delta Phe508-CFTR mice (treated ex vivo) with glafenine increased the short-circuit current (I(sc)) response to forskolin + genistein, and this effect was abolished by 10 microM CFTR(inh)172. In vivo treatment with glafenine also partially restored total salivary secretion. We conclude that the discovery of glafenine as a CFTR corrector validates the approach of investigating existing drugs for the treatment of CF, although localized delivery or further medicinal chemistry may be needed to reduce side effects.

Issues remain to be elucidated in the developmental regulation of erythropoiesis. In particular the role of Fas, a member of the tumor necrosis factor family of receptors despite much work remains unclear. During erythropoiesis, Fas is expressed at low levels on erythroblasts. For most cell types, Fas to FasL interaction causes apoptotic cell death via caspase activation. Here, we show that in humans, early erythroid progenitors are refractory to apoptosis triggered through Fas. Further during early human erythropoiesis, Fas triggered caspase activation provides a positive stimulus for erythroid maturation, and does not alter cellular proliferation or trigger apoptosis.

The F508del mutation impairs trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) to the plasma membrane and results in a partially functional chloride channel that is retained in the endoplasmic reticulum and degraded. We recently used a novel high-throughput screening (HTS) assay to identify small-molecule correctors of F508del CFTR trafficking and found several classes of hits in a screen of 2000 compounds (Carlile et al., 2007). In the present study, we have extended the screen to 42,000 compounds and confirmed sildenafil as a corrector using this assay. We evaluated structural analogs of sildenafil and found that one such molecule called KM11060 (7-chloro-4-{4-[(4-chlorophenyl) sulfonyl] piperazino}quinoline) was surprisingly potent. It partially restored F508del trafficking and increased maturation significantly when baby hamster kidney (BHK) cells were treated with 10 nM for 24 h or 10 muM for 2 h. Partial correction was confirmed by the appearance of mature CFTR in Western blots and by using halide flux, patch-clamp, and short-circuit current measurements in unpolarized BHK cells, monolayers of human airway epithelial cells (CFBE41o(-)), and intestines isolated from F508del-CFTR mice (Cftr(tm1Eur)) treated ex vivo. Small-molecule correctors such as KM11060 may serve as useful pharmacological tools in studies of the F508del-CFTR processing defect and in the development of cystic fibrosis therapeutics.

Caspase-3 plays a central role in apoptosis. It is also activated in normal erythropoiesis, with its activity peaking early during development (erythroid colony-forming unit [CFU-E] stage). In the present study, we have reduced the expression and subsequent enzymatic activity of caspase-3 by transfection of small interfering RNA (siRNA) directed to caspase-3 in a differentiating human erythroid culture system. We find that siRNA treatment yields a 50% reduction in cells that undergo enucleation with no change in the fraction of cells that undergo apoptosis, measured throughout the culture. Furthermore, a substantial fraction of treated cells are unable to complete the transition from pronormoblasts to basophilic normoblasts. These results demonstrate that caspase-3 is required for efficient erythropoiesis in this model system. (Blood. 2004;103:4310-4316)

Members of the four-and-a-half-LIM domain (FHL) protein family, which are expressed in a tissue- and stage-specific manner, have been reported previously to function as transcriptional coactivators. One of these is the p53-inducible protein DRAL/FHL2 (where DRAL is down-regulated in rhabdomyosarcoma LIM domain protein). In this work, we identified potential binding partners for DRAL/FHL2 using an inducible yeast two-hybrid system. We present evidence of a functional interaction between the promyelocytic leukemia zinc finger protein (PLZF) and DRAL/FHL2. PLZF is a sequence-specific transcriptional repressor whose function relies on recruitment of corepressors that form part of the histone deacetylase complex involved in chromatin remodeling. DRAL/FHL2 interacts specifically with PLZF in vitro and in vivo and augments transcriptional repression mediated by PLZF. This is the first reported incidence of a bona fide FHL protein-mediated corepression and supports the notion of these proteins having a role as coregulators of tissue-specific gene expression.

(-)-Deprenyl and structurally related propargylamines increase neuronal survival independently of monoamine oxidase B (MAO-B) inhibition, in part by decreasing apoptosis. We found that deprenyl and two other propargylamines, one of which does not inhibit monoamine oxidase B, increased survival in trophically withdrawn 6-day nerve growth factor (NGF)- and 9-day NGF-differentiated PC-12 cells but not in NGF naive or 3-day NGF-differentiated PC-12 cells. Four days of prior NGF exposure were required for the propargylamine-mediated antiapoptosis. Studies using actinomycin D, cycloheximide, and camptothecin revealed that the maintenance of both transcription and translation, particularly between 2 and 6 h after trophic withdrawal, was required for propargylamine-mediated antiapoptosis. Metabolic labeling of newly synthesized proteins for two-dimensional protein gel autoradiography and scintillation counting showed that the propargylamines either increased or reduced the levels of new synthesis or induced de novo synthesis of a number of different proteins, most notably proteins in the mitochondrial and nuclear subfractions. Western blotting for whole cell or subcellular fraction lysates showed that the timing of new protein synthesis changes or subcellular redistribution of apoptosis-related proteins induced by the propargylamines were appropriate to antiapoptosis. The apoptosis-related proteins included superoxide dismutases (SOD1 and SOD2), glutathione peroxidase, c-JUN, and glyceraldehyde-3-phosphate dehydrogenase. Most notable were the prevention of apoptotic decreases in BCL-2 levels and increases in mitochondrial BAX levels. In general, (-)-deprenyl-related propargylamines appear to reduce apoptosis by altering the levels or subcellular localization of proteins that affect mitochondrial membrane permeability, scavenge oxidative radicals, or participate in specific apoptosis signaling pathways.

Monocyte differentiation induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is interrupted during the course of acute promyelocytic leukemia (APL). One form of APL is associated with the translocation t(11;17), which joins the promyelocytic leukemia zinc finger (PLZF) and retinoic acid receptor α (RARα) genes. Because PLZF is coexpressed in the myeloid lineage with the vitamin D3 receptor (VDR), the interplay between PLZF and VDR was examined. It was found that PLZF interacts directly with VDR. This occurred at least partly through contacts in the DNA-binding domain of VDR and the broad complex, tram-trak, bric-a-brac/pox virus zinc finger (BTB/POZ) domain of PLZF. Moreover, PLZF altered the mobility of VDR derived from nuclear extracts when bound to its cognate binding site, forming a slowly migrating DNA-protein complex. Overexpression of PLZF in a monocytic cell line abrogated 1,25(OH)2D3 activation from both a minimal VDR responsive reporter and the promoter of p21WAF1/CIP1, a target gene of VDR. Deletion of the BTB/POZ domain significantly relieved PLZF-mediated repression of 1,25(OH)2D3-dependent activation. In addition, stable, inducible expression of PLZF in U937 cells inhibited the ability of 1,25(OH)2D3 to induce surface expression of the monocytic marker CD14 and morphologic changes associated with differentiation. These results suggest that PLZF may play an important role in regulating the process by which 1,25(OH)2D3 induces monocytic differentiation in hematopoietic cells.

Myeloid and hematopoietic cell development is a complex process regulated by an extensive network of transcription factors (reviewed in references 58 and 61). These proteins coordinate the sequential expression of gene products which results in progressive stages of progenitor cell commitment and differentiation (14, 57, 59). In hematological malignancies, transcription factors are often disrupted by chromosomal translocations and fused to genes encoding other transcriptional regulators (42, 51, 52). The resulting aberrant factors are oncoproteins that yield altered transcriptional patterns leading to the development of leukemia (54, 61). One such event disrupts ETO (for eight-Twenty One), a protein identified as part of a fusion product resulting from the translocation (8;21) found in 50% of patients with the M2 variant of acute myelogenous leukemia (AML) (see reference 48 and references within). Translocation (8;21) fuses ETO to AML-1, a critical regulator of hematopoiesis (36) that activates a number of myeloid genes, including those coding for granulocyte/macrophage–colony-stimulating factor (CSF), macrophage-CSF, and myeloperoxidase (61) through recruitment of the CREB binding protein (CBP) or p300 and other histone acetyl transferases to the promoters of these genes (31). In contrast, the AML-1–ETO oncoprotein is a dominant-negative form of AML-1 which represses the promoters of genes normally activated by AML-1 (16, 17, 44, 46). This model is highly supported by the similar phenotypes of AML-1 knockout mice and heterozygous AML-1/ETO knockin mice (49, 66), which include a severe block in hematopoiesis at the fetal liver stage and fatal hemorrhages within the central nervous system. At the molecular level, the dominant-negative effect of AML-1–ETO is due to the ability of the ETO moiety of the fusion protein to associate with the corepressors N-CoR, SMRT, and Sin3A, as well as histone deacetylases 1 and 2 (HDAC1 and -2) (17, 44, 62). Despite its ability to interact with other corepressors and HDAC, ETO itself was not previously identified as a corepressor for any sequence-specific transcription factor. The promyelocytic leukemia zinc finger (PLZF) protein is fused to the retinoic acid receptor α (RARα) in the retinoic acid-resistant t(11;17)(q23;q21) variant of acute promyelocytic leukemia (APL) (6, 19, 38). As in the case of t(8;21), this translocation yields an aberrant transcription factor. While RARα activates key genes required for normal myelopoiesis, PLZF-RARα represses expression of such genes in a dominant-negative manner (7, 9, 40, 45). We showed that PLZF was a sequence-specific DNA binding transcriptional repressor (2, 37, 67). This is due to the ability of the PLZF moiety to attract corepressor molecules, such as N-CoR, Sin3A, and SMRT, as well as HDAC1 (8, 20, 22, 25, 41). This interaction is, at least in part, mediated through the N-terminal POZ/BTB (poxvirus and zinc finger/Broad Complex, tramtrack, Bric a Brac) domain of PLZF (25) and indicates that PLZF may repress transcription by altering chromatin conformation. In its basal state, RARα also recruits N-CoR, SMRT, and HDACs to its target promoters, thus keeping them repressed in the absence of ligand. In the presence of the ligand all-trans retinoic acid (ATRA), corepressors are released and coactivators are recruited, resulting in transactivation of RARα target genes (5, 23, 26). However, in APL, the association of the PLZF portion of PLZF/RARα with corepressors and HDACs prohibits activation of RARα targets, even in the presence of high doses of ATRA (18, 20). PLZF is expressed in CD34+ myeloid progenitor cells and is down-regulated during differentiation of myeloid cell lines (53). In addition, PLZF causes growth suppression, differentiation blocking and cell cycle delay and/or arrest in myeloid cell lines (56, 67). These findings suggest that the transcriptional repression mediated by PLZF needs to be switched off for cells to differentiate and proliferate. The identity of potential PLZF targets, including cyclin A and the interleukin 3 receptor alpha (IL-3R α) chain, supports this hypothesis (45). Like PLZF, ETO is expressed in CD34+ cells and several leukemic cell lines (10, 12) and is down-regulated as hematopoietic progenitors mature (12). However, in contrast to PLZF, ETO was not found to bind to a specific DNA sequence (11, 35, 44). The facts that ETO can function as a powerful transcriptional repressor when fused to AML-1 and that it associates with corepressors suggest that it may normally act as a cofactor for certain sequence-specific DNA binding repressors. In light of this notion, we demonstrated that ETO interacts with PLZF in vivo and in vitro through specific domains of the two proteins. Coexpression of ETO with PLZF augments the ability of PLZF to repress transcription through its cognate binding site. This effect was abrogated by sodium butyrate and trichostatin A (TSA), inhibitors of HDACs. Our results suggest that, like the N-CoR and SMRT corepressors, ETO amplifies the transcriptional effects of PLZF by enhancing recruitment of HDACs to target promoters.