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Domain-specific BET bromodomain ligands represent an attractive target for drug discovery with the potential to unlock the therapeutic benefits of antagonizing these proteins without eliciting the toxicological aspects seen with pan-BET inhibitors. While we have reported several distinct classes of BD2 selective compounds, namely, GSK620, GSK549, and GSK046, only GSK046 shows high aqueous solubility. Herein, we describe the lead optimization of a further class of highly soluble compounds based upon a picolinamide chemotype. Focusing on achieving >1000-fold selectivity for BD2 over BD1 ,while retaining favorable physical chemical properties, compound 36 was identified as being 2000-fold selective for BD2 over BD1 (Brd4 data) with >1 mg/mL solubility in FaSSIF media. 36 represents a valuable new in vivo ready molecule for the exploration of the BD2 phenotype.

The profound efficacy, yet associated toxicity of pan-BET inhibitors is well documented. The possibility of an ameliorated safety profile driven by significantly selective (>100-fold) inhibition of a subset of the eight bromodomains is enticing, but challenging given the close homology. Herein, we describe the X-ray crystal structure-directed optimization of a novel weak fragment ligand with a pan-second bromodomain (BD2) bias, to potent and highly BD2 selective inhibitors. A template hopping approach, enabled by our parallel research into an orthogonal template (15, GSK046), was the basis for the high selectivity observed. This culminated in two tool molecules, 20 (GSK620) and 56 (GSK549), which showed an anti-inflammatory phenotype in human whole blood, confirming their cellular target engagement. Excellent broad selectivity, developability, and in vivo oral pharmacokinetics characterize these tools, which we hope will be of broad utility to the field of epigenetics research.

Pan-bromodomain and extra terminal domain (BET) inhibitors interact equipotently with the eight bromodomains of the BET family of proteins and have shown profound efficacy in a number of in vitro phenotypic assays and in vivo pre-clinical models in inflammation or oncology. A number of these inhibitors have progressed to the clinic where pharmacology-driven adverse events have been reported. To better understand the contribution of each domain to their efficacy and improve their safety profile, selective inhibitors are required. This article discloses the profile of GSK046, also known as iBET-BD2, a highly selective inhibitor of the second bromodomains of the BET proteins that has undergone extensive pre-clinical in vitro and in vivo characterization.

[Image: see text] Pan-BET inhibitors have shown profound efficacy in a number of in vivo preclinical models and have entered the clinic in oncology trials where adverse events have been reported. These inhibitors interact equipotently with the eight bromodomains of the BET family of proteins. To better understand the contribution of each domain to their efficacy and to improve from their safety profile, selective inhibitors are required. This Letter discloses the profile of GSK973, a highly selective inhibitor of the second bromodomains of the BET proteins that has undergone extensive preclinical in vitro and in vivo characterization.

Domain-specific BET bromodomain ligands represent an attractive target for drug discovery with the potential to unlock the therapeutic benefits of antagonizing these proteins without eliciting the toxicological aspects seen with pan-BET inhibitors. While we have reported several distinct classes of BD2 selective compounds, namely, GSK620, GSK549, and GSK046, only GSK046 shows high aqueous solubility. Herein, we describe the lead optimization of a further class of highly soluble compounds based upon a picolinamide chemotype. Focusing on achieving1000-fold selectivity for BD2 over BD1 ,while retaining favorable physical chemical properties, compound

Pan-bromodomain and extra terminal domain (BET) inhibitors interact equipotently with the eight bromodomains of the BET family of proteins and have shown profound efficacy in a number of

Bromodomain inhibitors revisited Bromodomain and extraterminal domain (BET) proteins contribute to the pathogenesis of cancer and immune diseases through their effects on transcriptional regulation. BET proteins contain two nearly identical bromodomains, BD1 and BD2, structural modules that have attracted great interest as targets for drug development. First-generation drugs that inhibited both BD1 and BD2 showed promising therapeutic activity in preclinical models but proved to be less efficacious in clinical trials. Gilan et al. took a different approach and designed drugs that selectively inhibited BD1 or BD2 (see the Perspective by Filippakopoulos and Knapp). They found that BD1 and BD2 inhibitors altered gene expression in different ways and that BD2 inhibitors had greater therapeutic activity than BD1 inhibitors in preclinical models of inflammation and autoimmune disease. Science , this issue p. 387 ; see also p. 367

The bromodomain of ATAD2 has proved to be one of the least-tractable proteins within this target class. Here, we describe the discovery of a new class of inhibitors by high-throughput screening and show how the difficulties encountered in establishing a screening triage capable of finding progressible hits were overcome by data-driven optimization. Despite the prevalence of nonspecific hits and an exceptionally low progressible hit rate (0.001%), our optimized hit qualification strategy employing orthogonal biophysical methods enabled us to identify a single active series. The compounds have a novel ATAD2 binding mode with noncanonical features including the displacement of all conserved water molecules within the active site and a halogen-bonding interaction. In addition to reporting this new series and preliminary structure-activity relationship, we demonstrate the value of diversity screening to complement the knowledge-based approach used in our previous ATAD2 work. We also exemplify tactics that can increase the chance of success when seeking new chemical starting points for novel and less-tractable targets.

Dynamin-1-like protein (DNM1L) gene variants have been linked to childhood refractory epilepsy, developmental delay, encephalopathy, microcephaly, and progressive diffuse cerebral atrophy. However, only a few cases have been reported in the literature and there is still a limited amount of information about the symptomatology and pathophysiology associated with pathogenic variants of DNM1L. We report a 10-year-old girl with a one-year history of mild learning disorder and absence seizures who presented with new-onset focal status epilepticus which progressed to severe encephalopathy and asymmetric hemispheric cerebral atrophy. Differential diagnosis included mitochondrial disease, Rasmussen's encephalitis, and autoimmune encephalitis. Disease progressed from one hemisphere to the other despite anti-seizure medications, hemispherectomy, vagus nerve stimulator, ketogenic diet, and immunomodulators. Continued cerebral atrophy and refractory seizures evolved until death four years after initial presentation. Post-mortem whole-exome sequencing revealed a pathogenic DNM1L variant. This paper presents a novel case of adolescent-onset DNM1L-related intractable epilepsy and encephalopathy.

ATAD2 is a cancer-associated protein whose bromodomain has been described as among the least druggable of that target class. Starting from a potent lead, permeability and selectivity were improved through a dual approach: 1) using CF(2) as a sulfone bio-isostere to exploit the unique properties of fluorine, and 2) using 1,3-interactions to control the conformation of a piperidine ring. This resulted in the first reported low-nanomolar, selective and cell permeable chemical probe for ATAD2.

The BRPF (Bromodomain and PHD Finger-containing) protein family are important scaffolding proteins for assembly of MYST histone acetyltransferase complexes. A selective benzimidazolone BRPF1 inhibitor showing micromolar activity in a cellular target engagement assay was recently described. Herein, we report the optimization of this series leading to the identification of a superior BRPF1 inhibitor suitable for in vivo studies.

Acetylation of histone lysine residues is one of the most well-studied post-translational modifications of chromatin, selectively recognized by bromodomain ‘reader’ modules. Inhibitors of the Bromodomain and Extra Terminal domain (BET) family of bromodomains have shown profound anti-cancer and anti-inflammatory properties, generating much interest in targeting other bromodomain-containing proteins for disease treatment. Herein, we report the discovery of I-BRD9, the first selective cellular chemical probe for Bromodomain-containing protein 9 (BRD9). I-BRD9 was identified through structure-based design, leading to greater than 700-fold selectivity over the BET family and 200-fold over the highly homologous Bromodomain-containing protein 7 (BRD7). I-BRD9 was used to identify genes regulated by BRD9 in Kasumi-1 cells involved in oncology and immune response pathways and to the best of our knowledge, represents the first selective tool compound available to elucidate the cellular phenotype of BRD9 bromodomain inhibition.

ATAD2 is a cancer-associated protein whose bromodomain has been described as among the least druggable of its class. In our recent disclosure of the first chemical probe against this bromodomain, GSK8814 (6), we described the use of a conformationally constrained methoxy piperidine to gain selectivity over the BET bromodomains. Here we describe an orthogonal conformational restriction strategy of the piperidine ring to give potent and selective tropane inhibitors and show structural insights into why this was more challenging than expected. Greater understanding of why different rational approaches succeeded or failed should help in the future design of selectivity in the bromodomain family.

Gene knock outs (KOs) are efficiently engineered through CRISPR-Cas9-induced frameshift mutations. While the efficiency of DNA editing is readily verified by DNA sequencing, a systematic understanding of the efficiency of protein elimination has been lacking. Here we devised an experimental strategy combining RNA sequencing and triple-stage mass spectrometry to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR-Cas9-generated KO lines is necessary for phenotype interpretation.

ATAD2 is a bromodomain-containing protein whose overexpression is linked to poor outcomes in a number of different cancer types. To date, no potent and selective inhibitors of the bromodomain have been reported. This article describes the structure-based optimization of a series of naphthyridones from micromolar leads with no selectivity over the BET bromodomains to inhibitors with sub-100 nM ATAD2 potency and 100-fold BET selectivity.

Tics are a common disorder in children. Some available drugs have unacceptable side effects, and thus alternative treatments are needed. Levetiracetam is an antiepileptic drug reported to be useful for the treatment of tics. Patients age 18 years and younger with tics and Tourette syndrome were enrolled in this prospective, open-label study. Seventy patients were treated with levetiracetam as monotherapy for four years. The initial dose of levetiracetam was 250 mg/day. The dose was titrated over three weeks to 1,000 to 2,000 mg/day. Clinical outcomes were assessed with the Clinical Global Impression Scale, Yale Global Tic Severity Scale, Revised Conners’ Parent Rating Scale, and Revised Conners’ Teacher Rating Scale. After four years of treatment with levetiracetam, all 70 patients showed some improvements based on the scales used. A total of 49 patients improved with regard to behavior and school performance. Levetiracetam was generally well tolerated. Despite the limitations of open-label studies, these results suggest that levetiracetam may be useful in treating tics in children and adolescents. Given its established safety profile, levetiracetam is a candidate for further study.

Some drugs currently used to treat tics have drawbacks, including the risk of side effects such as tardive dyskinesia. Therapeutic options with better safety profiles are needed. Levetiracetam is an antiepileptic drug with atypical GABAergic effects that might be beneficial for this indication. To evaluate the effect of levetiracetam on motor and focal tics, behavior, and school performance in children and adolescents with tics and Tourette syndrome. Twenty-four patients, age 6–18 years, with tics and Tourette syndrome were enrolled in this prospective, double-blinded placebo randomized study for 8 weeks. Each group had 12 patients. The initial starting dose of levetiracetam was 250 mg. The dosage was titrated over 3 weeks to 1,000 to 2,000 mg. Clinical outcomes were assessed with the Clinical Global Impression Scale, Yale Global Tic Severity Scale, and Revised Conners’ Scale. Ten out of 12 patients in the levetiracetam group showed improvements based on all of the scales used and four patients improved with regard to behavior and school performance. Two patients dropped out. Nine patients out of 12 patients in the placebo group showed no improvement, one patient showed a great placebo effect, and two patients dropped out of the study. Levetiracetam was generally well tolerated. Two patients discontinued because of exaggeration of pre-existing behavioral problems. Levetiracetam may be useful in treating tics in children and adolescents. Given its established safety profile, levetiracetam is a candidate for additional evaluation.

Cerebral palsy (CP) is a severely debilitating condition. The underlying insult results in irreversible damage to the central nervous system, treatment is essentially palliative. Botulinum toxin type-A (BTX/A) Botox® Allergan, Inc. injections have been found to be effective palliation for dynamic spasticity in CP. There is a paucity of data regarding the maximum dose tolerated in the pediatric population. The present study examines the safety and efficacy of BTX/A therapy at up to 40 U/kg. Twenty-two patients were treated from 1995 to 1999. The population included four adults and 18 pediatric patients. Patients were in three groups: diplegia (n=12), hemiplegia (n=5), and quadriplegia (n=5). They were evaluated by the Modified Ashworth Scale. The incidence and severity of complications is also reported. The significance of these data is also commented upon. This study suggests that BTX/A therapy is safe, efficacious, and cost effective at higher doses, and should be considered as part of a comprehensive therapeutic regimen in appropriate candidates. (J Pediatr Neurol 2004; 2(2): 91-96).

This non-randomized, uncontrolled, single-center study was conducted to evaluate the effects of zonisamide on intractable epilepsy in a primarily pediatric population. Twelve patients ranging in age from 18 months to 23 years were included in the study. All patients were diagnosed with intractable seizures. Adjunctive therapy with zonisamide was initiated at 100 mg once a day and increased, if needed, by 50-100 mg after one week to reach the maximal or therapeutic dosage depending on patient age and weight. Follow-up occurred in person or by phone at two weeks, one month, and every three months. All 12 patients responded favorably to adjunctive zonisamide. Five patients had subclinical status interrupted and stopped as evidenced by clinical examinations and electroencephalographic (EEG) findings. These patients also became awake, alert, more attentive, and more interactive. An additional six patients experienced clinical and EEG improvement in their seizure frequency, duration, and severity. One patient with autonomic seizures showed EEG improvement in seizure frequency, duration, and severity; however, seizure prodrome control was not obtained. Adjunctive therapy with zonisamide benefited all patients in this study, despite the variation in symptoms and etiology. Zonisamide is thought to have multiple mechanisms of action, which may benefit more patient types than antiepileptic drugs with a single mechanism of action. Zonisamide was well tolerated by all patients.

The BRPF (bromodomain and PHD finger-containing) protein family are important scaffolding proteins for assembly of MYST histone acetyltransferase complexes. Here, we report the discovery, binding mode, and structure-activity relationship (SAR) of the first potent, selective series of inhibitors of the BRPF1 bromodomain.

p300/CREB binding protein associated factor (PCAF/KAT2B) and general control nonderepressible 5 (GCN5/KAT2A) are multidomain proteins that have been implicated in retroviral infection, inflammation pathways, and cancer development. However, outside of viral replication, little is known about the dependence of these effects on the C-terminal bromodomain. Herein, we report GSK4027 as a chemical probe for the PCAF/GCN5 bromodomain, together with GSK4028 as an enantiomeric negative control. The probe was optimized from a weakly potent, nonselective pyridazinone hit to deliver high potency for the PCAF/GCN5 bromodomain, high solubility, cellular target engagement, and ≥18000-fold selectivity over the BET family, together with ≥70-fold selectivity over the wider bromodomain families.

Overexpression of ATAD2 (ATPase family, AAA domain containing 2) has been linked to disease severity and progression in a wide range of cancers, and is implicated in the regulation of several drivers of cancer growth. Little is known of the dependence of these effects upon the ATAD2 bromodomain, which has been categorized as among the least tractable of its class. The absence of any potent, selective inhibitors limits clear understanding of the therapeutic potential of the bromodomain. Here, we describe the discovery of a hit from a fragment-based targeted array. Optimization of this produced the first known micromolar inhibitors of the ATAD2 bromodomain.

The purpose of this article is to describe outcomes of intrathecal baclofen therapy for 29 patients with cerebral palsy, focusing on impairments, functional limitations, and disability. Patients received individualized rehabilitation and were followed up to 24 months. The primary outcome measures were the Ashworth Scale and the functional skills and caregiver assistance scales of the Pediatric Evaluation of Disability Inventory (PEDI). Ashworth Scale scores were significantly reduced ( P .0005). All areas of functional skills and caregiver assistance improved. Comparing groups of adults and patients less than 18 years, there were no significant differences, but there was a relationship between age and dose. Comparing groups of patients in high and low levels of independent functional mobility, no significant differences were found. These results provide suggestive evidence that the combination of intrathecal baclofen therapy and rehabilitation has positive effects across the dimensions of disablement. This study serves as a basis for high-level scientific studies of these effects. ( J Child Neurol 2003; 18: 26—34).

The development of selective histone deacetylase (HDAC) inhibitors with anti-cancer and anti-inflammatory properties remains challenging in large part owing to the difficulty of probing the interaction of small molecules with megadalton protein complexes. A combination of affinity capture and quantitative mass spectrometry revealed the selectivity with which 16 HDAC inhibitors target multiple HDAC complexes scaffolded by ELM-SANT domain subunits, including a novel mitotic deacetylase complex (MiDAC). Inhibitors clustered according to their target profiles with stronger binding of aminobenzamides to the HDAC NCoR complex than to the HDAC Sin3 complex. We identified several non-HDAC targets for hydroxamate inhibitors. HDAC inhibitors with distinct profiles have correspondingly different effects on downstream targets. We also identified the anti-inflammatory drug bufexamac as a class IIb (HDAC6, HDAC10) HDAC inhibitor. Our approach enables the discovery of novel targets and inhibitors and suggests that the selectivity of HDAC inhibitors should be evaluated in the context of HDAC complexes and not purified catalytic subunits.

Keratin filament polypeptides were purified from calf hoof stratum corneum with the aim of studying the in vitro assembly process and determining structural parameters of reconstituted filaments. Anion exchange chromatography was used to obtain the most complete fractionation and identification of the acidic and basic components in the purified polypeptide mixture to date. The reassembly products of the fractionated components were investigated by electron microscopy. Fully reconstituted filaments yield homogeneous solutions, and values of 9.8 nm for the filament diameter and 25 kDa/nm for the mass per unit length (M/L) were obtained by X-ray solution scattering. The structures formed in solution at various stages of filament assembly were not sufficiently homogeneous to be studied by this technique. X-ray diffraction patterns from native stratum corneum display strong maxima at 3.6 and 5.4 nm. Contrary to previous reports, these maxima do not appear to be due to lipids since they are also observed with delipidated rehydrated specimens. A series of weak maxima is also detected in the patterns of dry tissue. The absence of these features in the patterns of reconstituted filaments suggests that, in contrast to some electron microscopic observations, there are no prominent regularities in the structure of calf hoof keratin filaments.

Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. Here we report the first genome-wide screen for complexes in an organism, budding yeast, using affinity purification and mass spectrometry. Through systematic tagging of open reading frames (ORFs), the majority of complexes were purified several times, suggesting screen saturation. The richness of the data set enabled a de novo characterization of the composition and organization of the cellular machinery. The ensemble of cellular proteins partitions into 491 complexes, of which 257 are novel, that differentially combine with additional attachment proteins or protein modules to enable a diversification of potential functions. Support for this modular organization of the proteome comes from integration with available data on expression, localization, function, evolutionary conservation, protein structure and binary interactions. This study provides the largest collection of physically determined eukaryotic cellular machines so far and a platform for biological data integration and modelling.

Some drugs currently used to treat tics in pediatric patients have drawbacks, including the risk of side effects. New therapeutic options with better safety profiles are needed. Levetiracetam is an antiepileptic drug with atypical mechanisms of action that might be beneficial for this indication. We evaluated the effects of levetiracetam on motor and vocal tics, behavior, and school performance in children and adolescents with tics and Tourette syndrome (TS). Sixty patients, < or =18 years of age, with tics and TS were enrolled in this prospective, open-label study. The initial starting dose of levetiracetam was 250 mg/day. The dosage was titrated over 3 weeks to 1,000 to 2,000 mg/day. Clinical outcomes were assessed with the Clinical Global Impression Scale, Yale Global Tic Severity Scale, and Revised Conners' Parent Rating Scale. Behavior and school performance were also recorded. All 60 patients showed improvements based on all of the scales used, and 43 patients improved with regard to behavior and school performance. Levetiracetam was generally well tolerated. Three patients discontinued treatment because of exaggeration of preexisting behavioral problems. Levetiracetam may be useful in treating tics in children and adolescents. Given its established safety profile, levetiracetam is a candidate for evaluation in a well-controlled trial.

Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha triggers a signalling cascade, converging on the activation of the transcription factor NF-kappa B, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-alpha/NF-kappa B pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-alpha/NF-kappa B pathway and is generally applicable to other pathways relevant to human disease.

Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.

Autoimmune neurologic disease management has been significantly modified by the use of high-dose intravenous immunoglobulin (HDIVIG) during the past 15 years. Venous access, readily available IgG (until recently), and the relative lack of serious identifiable complications have prompted its use in myasthenia gravis. In adults, its effectiveness has been inconsistent, with variable acetylcholine receptor (AChR) antibody responses. Ten children were evaluated for clinical responses to, and complications of, HDIVIG. Weekly anti-AChR antibody titers in three patients were obtained. The HDIVIG dosage was 2 gm/kg body weight, infused at variable rates of 2 gm/kg for 1 day, 0.66 gm/kg daily for 3 days, and 0.5 g/kg daily for 4 days; in one patient the total dose was 0.8 gm/kg to correct to the ideal body weight. All children but one tolerated HDIVIG without complications. Eight patients exhibited definite improvement in functional strength after HDIVIG, but a decreasing response to HDIVIG was evident after multiple monthly treatments, warranting the additional use of corticosteroids in two patients. A decrease in anti-AChR antibody levels was observed in the three patients tested, but this decrease was constant in one patient. No correlation was observed between clinical response and antibody titers. HDIVIG is safe and effective in most patients for short-term management of juvenile myasthenia gravis, in myasthenic crises, and in preparing patients for surgery but appears to be of limited long-term benefit.

At the posterior pole of the Drosophila oocyte, oskar induces a tightly localized assembly of pole plasm. This spatial restriction of oskar activity has been thought to be achieved by the localization of oskar mRNA, since mislocalization of the RNA to the anterior induces anterior pole plasm. However, ectopic pole plasm does not form in mutant ovaries where oskar mRNA is not localized, suggesting that the unlocalized mRNA is inactive. As a first step towards understanding how oskar activity is restricted to the posterior pole, we analyzed oskar translation in wild type and mutants. We show that the targeting of oskar activity to the posterior pole involves two steps of spatial restriction, cytoskeleton-dependent localization of the mRNA and localization-dependent translation. Furthermore, our experiments demonstrate that two isoforms of Oskar protein are produced by alternative start codon usage. The short isoform, which is translated from the second in-frame AUG of the mRNA, has full oskar activity. Finally, we show that when oskar RNA is localized, accumulation of Oskar protein requires the functions of vasa and tudor, as well as oskar itself, suggesting a positive feedback mechanism in the induction of pole plasm by oskar.

THE localization of oskar (osk) RNA to the posterior pole of the developing fruit fly (Drosophila) oocyte induces the assembly of pole plasm, causing development of the abdomen and germ line1,2. Failure to localize oskar RNA results in embryos that lack abdomen and germ cells. Conversely, mis-targeting of oskar RNA to the anterior of the oocyte causes formation of ectopic abdomen and germ cells at the anterior pole3. Maternal mutants that have reduced pole plasm activity produce sterile adults with normal abdominal development, suggesting that germ cells are more sensitive than abdomen to defects in pole plasm assembly4. Thus mutations in genes that reduce oskar RNA localization or activity can be recovered as viable sterile adults. In a screen for mutants defective in germ cell formation, we isolated nine alleles of the tropomyosin II gene5. Here we show that mutations in tropomyosin II (TmII) virtually abolish oskar RNA localization to the posterior pole, suggesting an involvement of the actin network in oskar RNA localization.