Fragment-based discovery of a chemical probe for the PWWP1 domain of NSD3.

Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.

Morphology, Cell Division, and Phylogeny of Uroleptus longicaudatus (Ciliophora, Hypotricha), a Species of the Uroleptus limnetis Complex.

A Chinese population of the little-known freshwater hypotrich Uroleptus longicaudatus was investigated with emphasis on its living morphology and infraciliature. The characteristic, tripartite body consists of a narrowed (cephalized) anterior portion, a slender trunk, and a long, slender, and strongly contractile tail occupying up to 30% of body length. Contracted specimens with a tail length of about 12% closely resemble Uroleptus limnetis which has, like U. longicaudatus, its type locality on the East Coast of the United States so that it cannot be excluded that these two species are synonymous. Thus, we propose to subsume these and few other little-known species, which are not clearly distinguishable at the present state of knowledge, as U. limnetis complex. The morphogenesis of U. longicaudatus proceeds as in most congeners. The phylogenetic analyses reveal that Uroleptus is a monophyletic group, but due to the lack of detailed morphological data of the populations sequenced so far, the relationships within this taxon remain obscure. For the objective determination of the tail length of hypotrichs, we propose the "1/3-method", which says that the tail commences at that body width which corresponds one-third of the maximum width. Paruroleptus ophryoglena Gelei, 1954 is transferred to Uroleptus: Uroleptus ophryoglena (Gelei, 1954) comb. nov.

Three-gene based phylogeny of the Urostyloidea (Protista, Ciliophora, Hypotricha), with notes on classification of some core taxa.

Classifications of the Urostyloidea were mainly based on morphology and morphogenesis. Since molecular phylogeny largely focused on limited sampling using mostly the one-gene information, the incongruence between morphological data and gene sequences have risen. In this work, the three-gene data (SSU-rDNA, ITS1-5.8S-ITS2 and LSU-rDNA) comprising 12 genera in the "core urostyloids" are sequenced, and the phylogenies based on these different markers are compared using maximum-likelihood and Bayesian algorithms and tested by unconstrained and constrained analyses. The molecular phylogeny supports the following conclusions: (1) the monophyly of the core group of Urostyloidea is well supported while the whole Urostyloidea is not monophyletic; (2) Thigmokeronopsis and Apokeronopsis are clearly separated from the pseudokeronopsids in analyses of all three gene markers, supporting their exclusion from the Pseudokeronopsidae and the inclusion in the Urostylidae; (3) Diaxonella and Apobakuella should be assigned to the Urostylidae; (4) Bergeriella, Monocoronella and Neourostylopsis flavicana share a most recent common ancestor; (5) all molecular trees support the transfer of Metaurostylopsis flavicana to the recently proposed genus Neourostylopsis; (6) all molecular phylogenies fail to separate the morphologically well-defined genera Uroleptopsis and Pseudokeronopsis; and (7) Arcuseries gen. nov. containing three distinctly deviating Anteholosticha species is established.

Molecular phylogeny and ontogeny of a new ciliate genus, Paracladotricha salina n. g., n. sp. (Ciliophora, Hypotrichia).

A hypotrichous ciliate, Paracladotricha salina n. g., n. sp., was discovered in hypersaline waters (salinity about 80‰) from Qingdao, China. Its morphology and some major ontogenetic stages were studied and the phylogenetic position was estimated using standard methods. Paracladotricha salina is characterized by a flexible, more or less slender body (size 50-120 × 20-35 µm), a gonostomatid oral apparatus, one short and two long frontoventral rows, four macronuclear nodules, almost completely reduced dorsal kineties 1-3, and a loss of several parts of the ciliature, namely, the slightly shortened ciliary row of the adoral membranelles, the paroral, and the buccal, the postoral and pretransverse ventral, the transverse, and the caudal cirri. The ontogenesis is rather simple: anlage II of both filial products and anlage III of the opisthe originate de novo, while anlagen IV and V are formed within the parental rows. This combination of features requires the establishment of a new genus, Paracladotricha, which is, according to the morphological data, closely related to Schmidingerothrix and Cladotricha. The small-subunit rRNA gene was sequenced, indicating that P. salina is, as also demonstrated by the oral apparatus, a member of the gonostomatids. We provide a first, vague hypothesis about the phylogenetic relationships of the Gonostomatidae, Cladotrichidae, and Schmidingerotrichidae. However, since molecular data of the type species of these higher taxa are lacking, their validity and relationships remain obscure.

Synthesis of Indazoles via N-N Bond-Forming Oxidative Cyclization from 2-Aminomethyl-phenylamines.

Herein we report a method for the synthesis of indazoles from readily available 2-aminomethyl-phenylamines via N-N bond-forming oxidative cyclization. Inspired by indazole formation initially observed as a side product by N. Coskun et al. we developed a robust protocol to access indazoles in all three tautomeric forms. The method selectively gives access to various 2-substituted 2H-indazoles which are frequently used in drug design, and we also demonstrated its applicability to less studied 3H-indazoles.

Chasing Red Herrings: Palladium Metal Salt Impurities Feigning KRAS Activity in Biochemical Assays.

Identifying promising chemical starting points for small molecule inhibitors of active, GTP-loaded KRAS "on" remains of great importance to clinical oncology and represents a significant challenge in medicinal chemistry. Here, we describe broadly applicable learnings from a KRAS hit finding campaign: While we initially identified KRAS inhibitors in a biochemical high-throughput screen, we later discovered that compound potencies were all but assay artifacts linked to metal salts interfering with KRAS AlphaScreen assay technology. The source of the apparent biochemical KRAS inhibition was ultimately traced to unavoidable palladium impurities from chemical synthesis. This discovery led to the development of a Metal Ion Interference Set (MIIS) for up-front assay development and testing. Profiling of the MIIS across 74 assays revealed a reduced interference liability of label-free biophysical assays and, as a result, provided general estimates for luminescence- and fluorescence-based assay susceptibility to metal salt interference.

Two hypotrichs (Ciliophora, Hypotricha) from China: morphology and SSU rDNA sequence of Holosticha aestuarina nov. spec. and H. muuiensis Kim et al., 2017.

The morphology, four ontogenetic stages, and the phylogenetic relationships based on the small subunit RNA-gene of Holosticha aestuarina nov. spec. from the Yangtze River Estuary (Shanghai) were investigated. The new species differs from the congeners by the following combination of features: body size 160-196 × 27-36 µm in vivo, a vesicular structure containing one or two bean-shaped opaque particles, 24-32 adoral membranelles, 16-34 midventral pairs, 17-26 left marginal cirri, 22-32 right marginal cirri, six or seven transverse cirri, four dorsal kineties, and usually two macronuclear nodules. The anlage for the left marginal row of the proter very likely originates de novo (anterior portion) and from parental cirri (posterior portion). Further, we provide a morphological description of a Chinese population of H. muuiensis Kim et al., 2017, originally discovered in South Korea, indicating that it is confined to the Eastern Asia region. Its SSU rDNA sequence is identical with that of the type population and forms a clade with the available Holosticha sequences. By contrast, H. aestuarina nov. spec. is sister to the Holosticha + Uncinata clade, indicating that Uncinata is a subgenus of Holosticha.

Morphology and small subunit (SSU) rRNA gene sequence of the new brackish water ciliate Neobakuella flava n. g., n. sp. (Ciliophora, Spirotricha, Bakuellidae) and SSU rRNA gene sequences of six additional hypotrichs from Korea.

The morphology and the small subunit (SSU) rRNA gene sequence of the hypotrich Neobakuella flava n. g., n. sp. from the estuary of the Taehwagang River (Ulsan, South Korea) were investigated. The three frontal cirri, the composition of the midventral complex of cirral pairs and rows, and the simple dorsal kinety pattern of three bipolar kineties assign it to the urostyloid taxon Bakuellidae. The increased number of buccal and parabuccal cirri, the presence of transverse cirri, and more than one left marginal row, as well as the lack of caudal cirri separate Neobakuella n. g. from the other bakuellids. Neobakuella flava n. sp. has many 0.3 µm sized green and/or yellow usually dark-green cortical granules and some sparsely distributed, 2 × 1 µm sized grass green with yellowish shimmer granules. The gene sequence data indicate a close relationship with Diaxonella and a distinct separation from the bakuellid Metaurostylopsis and parabirojimid Parabirojimia. The SSU rRNA gene sequences of four further urostyloids (i.e. Diaxonella pseudorubra, Anteholosticha monilata, Metaurostylopsis struederkypkeae, Pseudourostyla cristata) and two stylonychines (i.e. Sterkiella cavicola, Sterkiella histriomuscorum) from Korea were analyzed. Anteholosticha monilata, type of the genus, is clearly separated from the Holosticha clade, supporting the morphological separation from Holosticha. Sterkiella cavicola, type of Sterkiella, clusters within the stylonychines and is obviously closely related with S. histriomuscorum.

Monographic treatment of Paraholosticha muscicola (Ciliophora, Keronopsidae), including morphological and molecular biological characterization of a brackish water population from Korea.

Paraholosticha muscicola, type species of Paraholosticha Wenzel, inhabits mainly terrestrial habitats, but also freshwater. A brackish water population from Korea is described, the first record from such a habitat. Principal component analysis shows that this population is more similar to a terrestrial population from Denmark than to a population from Antarctic soil. Keronopsids have two strong morphological/ontogenetic apomorphies (frontal corona formed from anlagen I-III; division in cysts). However, the SSU rRNA sequence of the Korean population does not cluster with that of the Antarctic population in the phylogenetic tree, but both branch off consecutively and immediately before a mixture of other non-dorsomarginalian hypotrichs, including two further keronopsids. Furthermore, the keronopsids cluster in the phylogenetic network, providing phylogenetic conflicts, which cannot be exemplified in the conventional gene tree. To complete the picture of P. muscicola, we provide a detailed overview about nomenclature, history, taxonomy, and its geographic distribution. From the four synonyms proposed so far, we tentatively accept only P. lichenicola and P. ovata. Paraholosticha algivora is likewise very similar. Thus we propose to include these three taxa as members of the P. muscicola complex. Stylonethes sterkii and P. algivora are transferred to Paraholosticha Wenzel. A key to the Paraholosticha species is provided.

Highly Selective PTK2 Proteolysis Targeting Chimeras to Probe Focal Adhesion Kinase Scaffolding Functions.

Focal adhesion tyrosine kinase (PTK2) is often overexpressed in human hepatocellular carcinoma (HCC), and several reports have linked PTK2 depletion and/or pharmacological inhibition to reduced tumorigenicity. However, the clinical relevance of targeting PTK2 still remains to be proven. Here, we present two highly selective and functional PTK2 proteolysis-targeting chimeras utilizing von Hippel-Lindau and cereblon ligands to hijack E3 ligases for PTK2 degradation. BI-3663 (cereblon-based) degrades PTK2 with a median DC50 of 30 nM to >80% across a panel of 11 HCC cell lines. Despite effective PTK2 degradation, these compounds did not phenocopy the reported antiproliferative effects of PTK2 depletion in any of the cell lines tested. By disclosing these compounds, we hope to provide valuable tools for the study of PTK2 degradation across different biological systems.

High mitochondrial haplotype diversity of Coleps sp. (Ciliophora: Prostomatida).

To date the awareness of the temporal population structure in eukaryotic microbes is very limited. This is exemplified in the scarce knowledge about the intraspecific genetic variation in ciliates. To elucidate the genetic variation of Coleps (Ciliophora: Prostomatida), we employed the analysis of the mitochondrial apocytochrome b gene of the Coleps community in a young lake in Germany. The analysis of 111 isolates, sampled from April 2005 to September 2006, revealed a high genetic variation for the two dominant Coleps species (11 mitochondrial haplotypes in Coleps spetai, nine in Coleps hirtus hirtus). The study represents one of the largest datasets of intraspecific diversity in a microbial eukaryote and demonstrates for the first time the suitability of a mitochondrial gene for the detection of genetic variation within populations of eukaryotic microbes. However, the results of our study warrant caution in the application of such an approach, as we amplified some non-orthologous cob-like sequences, whose uncritical acceptance would have led to the erroneous discovery of cryptic species.

Genetic, morphological, and ecological diversity of spatially separated clones of Meseres corlissi Petz & Foissner, 1992 (Ciliophora, Spirotrichea).

We investigated the intraspecific variation of the spirotrich freshwater ciliate Meseres corlissi at the level of genes (SSrDNA, ITS), morphology (14 characters), and ecophysiology (response to temperature and pH). Five of the eight clonal M. corlissi cultures isolated from five localities on four continents were studied at all levels. The null hypothesis was that geographic distance plays no role: M. corlissi lacks biogeography. The intraspecific variation was low at the genetic level (0%-4%), moderate at the morphological level (5%-15%), and high at the ecophysiological level (10%-100%). One clone, isolated from subtropical China, differed significantly at all levels from all other clones, suggesting limited dispersal and local adaptation among M. corlissi. However, other clones from distant areas, such as Australia and Austria, were genetically identical and differed only slightly in morphology and temperature response. We speculate that our findings may be typical for rare species; the chances may be equally high for both global dispersal of most and local adaptation of some populations in areas where dispersal has been permanently or temporarily reduced.

Microwave-assisted parallel synthesis of fused heterocycles in a novel parallel multimode reactor.

New rotor types using disposable glass vials for small-scale parallel synthesis in multimode microwave reactors are introduced. One rotor comprises 16 groups of four vials, whereas the second uses four silicon carbide plates with a 6 x 4 matrix to process the vials. Both rotors achieve utmost temperature homogeneity upon microwave irradiation and can be used for microwave-mediated reactions at temperatures of up to 200 degrees C and pressures of 20 bar. The generation of three different heterocycle libraries furnishing thiophenes, oxindoles, and benzimidazoles using the new rotor types is described.

Morphology, cell-division, and phylogeny of Schmidingerothrix elongata spec. nov. (Ciliophora, Hypotricha), and brief guide to hypotrichs with Gonostomum-like oral apparatus.

The hypotrich Schmidingerothrix elongata spec. nov., discovered in saline (20‰) soil of the Longfeng Wetland, Daqing, northern China, was studied using live observation and protargol impregnation. It is characterized, inter alia, by colorless cortical granules arranged in short rows, three frontoventral cirral rows with the rightmost extending far posteriorly, and 4-8, usually six macronuclear nodules. Cell division proceeds as in congeners and confirms the lack of dorsal ciliature. In phylogenetic analyses based on SSU rDNA, S. elongata is sister of S. salinarum+Paracladotricha salina. A re-investigation of the type slides of P. salina, type of Paracladotricha, revealed a misobservation in the original description. Since P. salina lacks, like Schmidingerothrix spp., a dorsal ciliature, Paracladotricha becomes a junior, subjective synonym of Schmidingerothrix with S. salina comb. nov. as fourth species. A review of the phylogenetic analyses dealing with Schmidingerothrix shows that its position is variable. However, together with the gonostomatid oral apparatus it can be hypothesized that Schmidingerothrix is a member of the Gonostomatidae or a close relative. A list of genera (14) and species (58) which have - like Schmidingerothrix - a gonostomatid oral apparatus, as well as a key to these genera are provided.

Morphology, morphogenesis, and molecular phylogeny of Uroleptus (Caudiholosticha) stueberi (Foissner, 1987) comb. nov. (Ciliophora, Hypotricha), and reclassification of the remaining Caudiholosticha species.

The morphology, morphogenesis, and SSU rDNA sequence of a Caudiholosticha stueberi population from Southwest China soil were analyzed. The studies confirm the assumption of previous workers that this species has dorsomarginal kineties and thus does not belong to the urostyloids, but to the non-oxytrichid dorsomarginalian genus Uroleptus whose members have, in contrast to C. stueberi, a distinct tail. On the basis of two morphological features we split Uroleptus into three subgenera: U. (Uroleptus) (tail present; more than five transverse cirri; habitat freshwater), U. (Paruroleptus) (present; five or less; freshwater or soil), and U. (Caudiholosticha) stat. nov. (lacking; five or less; soil). Since Uroleptus (Caudiholosticha) stueberi comb. nov. is the type of Caudiholosticha, the other 16 species so far assigned to Caudiholosticha have to be reclassified because they obviously lack dorsomarginal kineties. Based on published data, six new urostyloid genera are established: Extraholosticha gen. nov. (type: Holosticha sylvatica; monotypic); Adumbratosticha gen. nov. (type: H. tetracirrata; three species); Acuholosticha gen. nov. (type: U. paranotabilis; five species); Limnoholosticha gen. nov. (type: H. (Holosticha) navicularum; four species); Multiholosticha gen. nov. (type: H. multicaudicirrus; two species); and Caudikeronopsis gen. nov. (type: Caudiholosticha marina; monotypic). Urosomoida sejongensis is transferred to Oxytrichella: O. sejongensis comb. nov.

Redescription of Keronopsis helluo Penard, 1922 from Antarctica and Paraholosticha pannonica Gellért and Tamás, 1959 from Alaska (Ciliophora, Hypotricha).

The morphology of Keronopsis helluo Penard, 1922, type species of Keronopsis, and Paraholosticha pannonica Gellért and Tamás, 1959, two little-known members of the Keronopsidae Jankowski, 1979, was described using standard methods. In addition, we sequenced the SSU rRNA of both species. Keronopsis helluo was isolated from a mossy soil from Robert Island (Antarctica) while P. pannonica was found in terrestrial moss from Alaska. Our data correspond very well with the original descriptions. The frontal ciliature of K. helluo is identical with that of Paraholosticha spp., indicating that some Keronopsis species (K. tasmaniensis, K. dieckmanni) are misclassified in the keronopsids. The type species has distinctly more transverse cirri (8-13) than K. wetzeli (1-3), type species of Parakeronopsis, which is thus perhaps a valid genus or subgenus. The phylogenetic analyses confirm the position of the keronopsids outside the Dorsomarginalia. The species sequenced so far (K. helluo, Paraholosticha muscicola, P. pannonica) emerge from a soft polytomy, which also comprises Bistichella-like species and a large cluster composed of amphisiellids, trachelostylids, and gonostomatids, that is, the method failed to resolve the relationships within the keronopsids. The Keronopsidae and the two species studied are characterized based on previous studies and our data.

Chemically Induced Degradation of the Oncogenic Transcription Factor BCL6.

The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B cell lymphoma (DLBCL). Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We used a structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also causes rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and anti-proliferative effects than compounds that merely inhibit co-repressor interactions. This work establishes the BTB domain as a highly druggable structure, paving the way for the use of other members of this protein family as drug targets. The magnitude of effects elicited by this class of BCL6-degrading compounds exceeds that of our equipotent non-degrading inhibitors, suggesting opportunities for the development of BCL6-based lymphoma therapeutics.

Typification of the genus Dileptus Dujardin, 1841 (Ciliophora, Rhynchostomatia).

In their monograph of the dileptids, Vdacný and Foissner (2012) could not clarify the type species of the genus DileptusDujardin, 1841. Thus, they suggested that the problem be referred to the International Commission on Zoological Nomenclature. However, recently we discovered that Dujardin (1841) has originally typified Dileptus with Amphileptus anser sensu Ehrenberg (1838) which is in fact a misidentified Amphileptus margaritiferEhrenberg, 1833, a common species also originally classified in Dileptus. Under Article 70.3.2 of the Code, Dileptus margaritifer (Ehrenberg, 1833) Dujardin, 1841, thoroughly redescribed by Foissner et al. (1995), is now the type of Dileptus. This has the great advantages of historical continuity and that new combinations (names) are not required.

Systematics and species-specific response to pH of Oxytricha acidotolerans sp. nov. and Urosomoida sp. (Ciliophora, Hypotricha) from acid mining lakes.

We investigated the morphology, phylogeny of the 18S rDNA, and pH response of Oxytricha acidotolerans sp. nov. and Urosomoida sp. (Ciliophora, Hypotricha) isolated from two chemically similar acid mining lakes (pH~2.6) located at Langau, Austria, and in Lusatia, Germany. Oxytricha acidotolerans sp. nov. from Langau has 18 frontal-ventral-transverse cirri but a very indistinct kinety 3 fragmentation so that the assignment to Oxytricha is uncertain. The somewhat smaller species from Lusatia has a highly variable cirral pattern and the dorsal kineties arranged in the Urosomoida pattern and is, therefore, preliminary designated as Urosomoida sp. The pH response was measured as ciliate growth rates in laboratory experiments at pH ranging from 2.5 to 7.0. Our hypothesis was that the shape of the pH reaction norm would not differ between these closely related (3% difference in their SSU rDNA) species. Results revealed a broad pH niche for O. acidotolerans, with growth rates peaking at moderately acidic conditions (pH 5.2). Cyst formation was positively and linearly related to pH. Urosomoida sp. was more sensitive to pH and did not survive at circumneutral pH. Accordingly, we reject our hypothesis that similar habitats would harbour ciliate species with virtually identical pH reaction norm.