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p97 is an ATPase that works in concert with histone deacetylase 6 (HDAC6), to facilitate the degradation of misfolded proteins by autophagosomes. p97 has also been implicated in DNA repair and maintaining genomic stability. In this study, we determined the effect of combined inhibition of p97 and HDAC6 activities in mantle cell lymphoma (MCL) cells. We report that treatment with p97 inhibitors induces dose-dependent apoptosis in MCL cells. The p97 inhibitor CB-5083 induces ER stress markers GRP78 and CHOP and results in the accumulation of polyubiquitylated proteins. Co-treatment with CB-5083 and the HDAC6 inhibitor ACY-1215 result in marked downregulation of CDK4, Cyclin D1, and BRCA1 levels without inhibiting autophagic flux. Consequently, treatment with CB-5083 accentuates DNA damage in response to treatment with ACY-1215 resulting in enhanced accumulation of H2AX-γ and synergistic apoptosis. Furthermore, ATM loss severely impairs phosphorylation of 53BP1 following co-treatment with CB-5083 and ACY-1215 in response to gamma irradiation. Finally, co-treatment CB-5083 and ACY-1215 results in reduced tumor volumes and improves survival in Z138C and Jeko-1 xenografts in NSG mice. These observations suggest that combined inhibition of p97 and HDAC6 abrogates resolution of proteotoxic stress and impairs DNA repair mechanisms in MCL cells.

Uropathogenic Escherichia coli (UPEC) is the leading cause of community-acquired urinary tract infections (UTIs), with over 100 million UTIs occurring annually throughout the world. Increasing antimicrobial resistance among UPEC limits ambulatory care options, delays effective treatment, and may increase overall morbidity and mortality from complications such as urosepsis. The polysaccharide capsules of UPEC are an attractive target a therapeutic, based on their importance in defense against the host immune responses; however, the large number of antigenic types has limited their incorporation into vaccine development. The objective of this study was to identify small-molecule inhibitors of UPEC capsule biogenesis. A large-scale screening effort entailing 338,740 compounds was conducted in a cell-based, phenotypic screen for inhibition of capsule biogenesis in UPEC. The primary and concentration-response assays yielded 29 putative inhibitors of capsule biogenesis, of which 6 were selected for further studies. Secondary confirmatory assays identified two highly active agents, named DU003 and DU011, with 50% inhibitory concentrations of 1.0 µM and 0.69 µM, respectively. Confirmatory assays for capsular antigen and biochemical measurement of capsular sugars verified the inhibitory action of both compounds and demonstrated minimal toxicity and off-target effects. Serum sensitivity assays demonstrated that both compounds produced significant bacterial death upon exposure to active human serum. DU011 administration in mice provided near complete protection against a lethal systemic infection with the prototypic UPEC K1 isolate UTI89. This work has provided a conceptually new class of molecules to combat UPEC infection, and future studies will establish the molecular basis for their action along with efficacy in UTI and other UPEC infections.

The SR-like splicing factor SFRS10 (Htra2-beta1) is well known to influence various alternatively spliced exons without being an essential splicing factor. We have shown earlier that SFRS10 binds SMN1/SMN2 RNA and restores full-length (FL)-SMN2 mRNA levels in vitro. As SMN1 is absent in patients with spinal muscular atrophy (SMA), the level of FL-SMN2 determines the disease severity. Correct splicing of SMN2 can be facilitated by histone deacetylase inhibitors (HDACis) via upregulation of SFRS10. As HDACis are already used in SMA clinical trials, it is crucial to identify the spectrum of alternatively spliced transcripts modulated by SFRS10, because elevated SFRS10 levels may influence or misregulate also other biological processes. To address this issue, we generated a conditional Sfrs10 allele in mice using the Cre/loxP system. The ubiquitous homozygous deletion of Sfrs10, however, resulted in early embryonic lethality around E7.5, indicating an essential role of Sfrs10 during mouse embryogenesis. Deletion of Sfrs10 with recombinant Cre in murine embryonic fibroblasts (MEFs) derived from Sfrs10(fl/fl) embryos increased the low levels of SmnDelta7 3-4-fold, without affecting FL-Smn levels. The weak influence of Sfrs10 on Smn splicing was further proven by a Hb9-Cre driven motor neuron-specific deletion of Sfrs10 in mice, which developed normally without revealing any SMA phenotype. To assess the role of Sfrs10 on FL-SMN2 splicing, we established MEFs from Smn(-/-);SMN2(tg/tg);Sfrs10(fl/fl) embryos. Surprisingly, deletion of Sfrs10 by recombinant Cre showed no impact on SMN2 splicing but increased SMN levels. Our findings highlight the complexity by which alternatively spliced exons are regulated in vivo.

A sequential three-component synthesis of functionalized benzisothiazoline-3-acetic acid 1,1-dioxides utilizing a domino Heck-aza-Michael pathway is reported. This one-pot procedure rapidly assembles functionalized benzylsulfonamides, which undergo a palladium-catalyzed, domino, Heck-aza-Michael transformation in an experimentally straightforward manner. This attractive protocol has been utilized to synthesize three combinatorial sublibraries (I-III) comprising a total of 95 compounds in high purities (> or =95% for 75 compounds), yield and quantities.

Natural IgM antibodies play an important role in the body's defense mechanisms against transformed cells in the human body and are currently being exploited both in prognoses of malignant lesions and in the therapy of cancer patients. However, despite growing interest and clinical promise, thus far the IgM class of antibodies has failed to gain widespread commercial interest as these are considered to be difficult to produce recombinantly. IgMs are polymeric and have a relatively large mass. In addition, IgM molecules are heavily glycosylated and, when produced in non-human cell lines, they may contain non-human glycan structures which may be potentially immunogenic. Clearly, production systems capable of expressing human recombinant IgM antibodies are needed. We have successfully used PER.C6 cells-a human cell line-to generate three separate human recombinant monoclonal IgMs in suspension cultures in protein-free medium. All three of the IgMs were constructed with joining (J) chain and were expressed in the pentameric form. One of the IgMs was also expressed as a hexamer without J chain. Clones with cell specific productivities greater than 20 pg/cell/day were generated, which led to yields of 0.5 g/L to 2g/L in fed-batch production. All the IgMs expressed were biologically active as shown in binding and cytotoxicity assays. These studies demonstrate the potential of PER.C6 cells for the production of high levels of functional recombinant IgM and other polymeric molecules, using a straightforward and rapid stable cell line generation method.

The parallel synthesis of gamma-turn-inspired peptidomimetic libraries has been demonstrated through two approaches toward the preparation of 1,4-diazepin-5-ones. In the first approach, 1,4-diazepin-5-ones scaffolds were prepared on gram scale and subsequently diversified to produce libraries. In a second approach, libraries of 1,4-diazepin-5-ones were produced directly through a three-component strategy that maximizes the diversity obtained in a single step.

Development of an ionic immobilization, diversification, and release method for the generation of methionine aminopeptidase inhibitors is reported. This method involves the immobilization of 5-bromofuran-2-carboxylic acid and 5-bromothiophene-2-carboxylic acid onto PS-BEMP, followed by Suzuki reaction on a resin-bound intermediate and subsequent release to provide products in moderate yields and excellent purities. Compound potencies were evaluated on the Co(II), Mn(II), Ni(II), and Fe(II) forms of Escherichia coli MetAP1. The furoic-acid analogs were found to be Mn(II) selective with IC 50 values in the low micromolar range. Qualitative SAR analysis, supplemented by molecular modeling studies, provides valuable information on structural elements responsible for potency and selectivity.

A synthetic sequence was developed in which a diene containing an attached secondary amine was reacted with maleic anhydride to afford the title structures in one step. The reaction involves a Diels-Alder reaction combined with a transacylation reaction of the imide group. A series of six scaffolds was constructed using this methodology. Each scaffold was subsequently reacted with 12 amines to afford a library containing 72 compounds.

The reactions of 1,2- and 1,3-hydroxyalkyl azides and aldehydes in the presence of Lewis acid result in the one-step construction of oxazolines and dihydrooxazines, respectively. The reaction was adapted to parallel synthesis using a polymer-bound phosphine to scavenge excess hydroxyalkyl azide. Thus, a 60-member library of various disubstituted oxazolines and di- and trisubstituted dihydrooxazines was generated.

A parallel synthesis of a library (80 members) of 2-pyrazolines in solution phase is described. The 2-pyrazoline core was accessed through the [3 + 2] cycloaddition of nitrilimines with enoyl oxazolidinones. The cycloaddition provided two regioisomers, the major product being the C regioisomer. The oxazolidinone moiety was further reduced to the primary alcohol, producing another library of 5-hydroxymethyl-2-pyrazolines. The Lipinski profiles and calculated ADME properties of the compounds are also reported.

The human gene BDP1, localized on chromosome 5q13 in close proximity to the spinal muscular atrophy determining gene SMN, encodes a large protein consisting of 2254 amino acids (aa). In the first third of the gene, the subunit of the RNA polymerase III (Pol III) transcription factor complex (TFIIIB alpha/beta) is encoded. To further characterize the function of BDP1, we carried out a yeast two-hybrid screen using various parts of BDP1. With the clone BDP1-(1-640) we identified a novel interaction partner, ZNF297B. The ZNF297B gene is localized on chromosome 9q24 and encodes a zinc finger protein of 467 aa possessing the typical structure of a transcription factor. The interaction found in yeast was confirmed by co-immunoprecipitation and refined to the N-terminal region of ZNF297B-(1-127) containing the BTB/POZ domain and the N-terminal end of BDP1-(1-299). The ZNF297B transcript is 5.7 kb in length and ubiquitously expressed, with highest levels found in muscles. Immunofluorescence staining revealed a speckled pattern in the nuclei of HEK293 cells. Due to the essential role of BDP1 in Pol III transcription, we propose that ZNF297B may also regulate these transcriptional pathways.

Proximal spinal muscular atrophy (SMA) is a neuromuscular disorder caused by homozygous mutations of the SMN1 gene. SMN1 interacts with multiple proteins with functions in snRNP biogenesis, pre-mRNA splicing and presumably neural transport. SMN2, a nearly identical copy of SMN1, produces predominantly exon 7-skipped transcripts, whereas SMN1 mainly produces full-length transcripts. The SR-like splicing factor Htra2-beta1 facilitates correct splicing of SMN2 exon 7 through direct interaction with an exonic splicing enhancer within exon 7. In rare cases, siblings with identical 5q13-homologues and homozygous absence of SMN1 show variable phenotypes, suggesting that SMA is modified by other factors. By analysing nine SMA discordant families, we demonstrate that in all families unaffected siblings produce significantly higher amounts of SMN, Gemin2, Gemin3, ZPR1 and hnRNP-Q protein in lymphoblastoid cell lines, but not in primary fibroblasts, compared with their affected siblings. Protein p53, an additional SMN-interacting protein, is not subject to an SMN-dependent regulation. Surprisingly, Htra2-beta1 is also regulated by this tissue-specific mechanism. A similar regulation was found in all type I-III SMA patients, although at a different protein level than in discordant families. Thus, our data show that reduced SMN protein levels cause a reduction in the amount of its interacting proteins and of Htra2-beta1 in both discordant and non-discordant SMA families. We provide evidence that an intrinsic SMA modifying factor acts directly on the expression of SMN, thus influencing the SMA phenotype. Further insights into the molecular pathway and the identification of SMA modifying gene(s) may help to find additional targets for a therapy approach.

Almost 20 years of combinatorial chemistry have emphasized the power of numbers, a key issue for drug discovery in the current genomic era, in which it has been estimated that there might be more than 10,000 potential targets for which it would be desirable to have small-molecule modulators. Combinatorial chemistry is best described as the industrialization of chemistry; the chemistry has not changed, just the way in which it is now carried out, which is principally by exploiting instrumentation and robotics coupled to the extensive use of computers to efficiently control the process and analyse the vast amounts of resulting data. Many researchers have contributed to the general concepts as well as to the technologies in present use. However, some interesting challenges still remain to be solved, and these are discussed here in the context of the application of combinatorial chemistry to drug discovery.

Tumour-necrosis factor-alpha (TNF-alpha) is a cytokine that contributes to a variety of inflammatory disease states. The protein exists as a membrane-bound precursor of relative molecular mass 26K which can be processed by a TNF-alpha-converting enzyme (TACE), to generate secreted 17K mature TNF-alpha. We have purified TACE and cloned its complementary DNA. TACE is a membrane-bound disintegrin metalloproteinase. Structural comparisons with other disintegrin-containing enzymes indicate that TACE is unique, with noteable sequence identity to MADM, an enzyme implicated in myelin degradation, and to KUZ, a Drosophila homologue of MADM important for neuronal development. The expression of recombinant TACE (rTACE) results in the production of functional enzyme that correctly processes precursor TNF-alpha to the mature form. The rTACE provides a readily available source of enzyme to help in the search for new anti-inflammatory agents that target the final processing stage of TNF-alpha production.

Tumor necrosis factor-alpha is a potent cytokine, secreted primarily by activated monocytes and macrophages, that possesses a broad range of immunomodulating properties. Involvement of this cytokine has been validated in disease states such as arthritis and Crohn's disease and implicated in diverse neuroimmunological pathologies such as multiple sclerosis, Alzheimers and stroke. TNF-alpha is initially synthesized as a 26 kDa precursor molecule that is subsequently processed to the mature form by cleavage of the Ala76 Val77 bond. The 17 kDa carboxy-terminal protein is then secreted to function in a paracrine manner. The enzyme that processes precursor TNF-alpha has previously been identified as a microsomal metalloprotease called TNF-alpha converting enzyme (TACE). We have now purified and partially cloned the enzyme. TACE represents a novel target for therapeutic intervention in a variety of inflammatory and neuroimmunological diseases.

Background: Combinatorial chemistry using solid-phase synthesis is a rapidly developing technology that can result in a significant reduction in the time required to find and optimize lead compounds. The application of this approach to traditional medicinal chemistry has led to the construction of libraries of small organic molecules on resin beads. A major difficulty in developing large combinatorial libraries is the lack of a facile encoding and decoding methodology to identify active compounds., Results: Several encoding schemes are described which use the ability of mass spectrometry to ascertain isotopic distributions. Molecular tags are attached to resin beads in parallel or on the linker used for chemical library synthesis. The tags are encoded via a controlled ratio of a number of stable isotopes on the tagging molecules, and range from a single to a complex isotopic distribution., Conclusions: A novel coding scheme is described that is useful for the generation of large encoded combinatorial libraries. The code can be cleaved after assay and analyzed by mass spectrometry in an automated fashion. An important element of the combinatorial discovery process is the ability to extract the structure-activity relationship (SAR) information made available by library screening. The speed and sensitivity of the mass-encoding scheme has the potential to determine the full SAR for a given library.

The synthesis and biological evaluation of cAMP-specific phosphodiesterase (PDE IV) inhibitors is described. The PDE IV inhibitor 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro 20-1724, 2) was used as a template from which to design a set of rigid oxazolidinones, imidazolidinones, and pyrrolizidinones that mimic Ro 20-1724 but differ in the orientation of the carbonyl group. The endo isomer of each of these heterocycles was more potent than the exo isomer in an enzyme inhibition assay and a cellular assay, which measured TNF alpha secretion from activated human peripheral blood monocytes (HPBM). Imidazolidinone 4a inhibited human PDE IV with a Ki of 27 nM and TNF alpha secretion from HPBM with an IC50 of 290 nM. By comparison, Ro 20-1724 is significantly less active in these assays with activities of 1930 and 1800nM, respectively.

The synthesis of 1,3-disubstituted pyrrolidines 2 and their activities as type IV phosphodiesterase (PDE) inhibitors are described. Various groups were appended to the nitrogen of the pyrrolidine nucleus to enable structure-activity relationships to be assessed. Groups which render the pyrrolidine nitrogen of 2 nonbasic yielded potent PDE-IV inhibitors. Analogs of amides, carbamates, and ureas of 2 were synthesized to determine the effects that substitution on these functional groups had on PDE-IV inhibitor potency. The structural requirements for PDE-IV inhibitor potency differed among the three classes. A representative amide, carbamate, and urea (2c,d,h) were shown to be > 50-fold selective for inhibiting PDE-IV versus representative PDEs from families I-III and V. Furthermore, these same three inhibitors demonstrated potent functional activity (IC50 < 1 microM) by inhibiting tumor necrosis factor-alpha (TNF-alpha) release from lipopolysaccharide (LPS)-activated purified human peripheral blood monocytes and mouse peritoneal macrophages. These compounds were also tested orally in LPS-injected mice and demonstrated dose-dependent inhibition of serum TNF-alpha levels.