Myotonic dystrophy type 1 (DM1) is a dominantly inherited neuromuscular disorder resulting from expression of RNA containing an expanded CUG repeat (CUG(exp)). The pathogenic RNA is retained in nuclear foci. Poly-(CUG) binding proteins in the Muscleblind-like (MBNL) family are sequestered in foci, causing misregulated alternative splicing of specific pre-mRNAs. Inhibitors of MBNL1-CUG(exp) binding have been shown to restore splicing regulation and correct phenotypes in DM1 models. We therefore conducted a high-throughput screen to identify novel inhibitors of MBNL1-(CUG)12 binding. The most active compound was lomofungin, a natural antimicrobial agent. We found that lomofungin undergoes spontaneous dimerization in DMSO, producing dilomofungin, whose inhibition of MBNL1-(CUG)12 binding was 17-fold more potent than lomofungin itself. However, while dilomofungin displayed the desired binding characteristics in vitro, when applied to cells it produced a large increase of CUG(exp) RNA in nuclear foci, owing to reduced turnover of the CUG(exp) transcript. By comparison, the monomer did not induce CUG(exp) accumulation in cells and was more effective at rescuing a CUG(exp)-induced splicing defect. These results support the feasibility of high-throughput screens to identify compounds targeting toxic RNA, but also demonstrate that ligands for repetitive sequences may have unexpected effects on RNA decay.

Mis-sense mutations in the α-subunit of the G-protein, Gsα, cause fibrous dysplasia of bone/McCune-Albright syndrome. The biochemical outcome of these mutations is constitutively active Gsα and increased levels of cAMP. The aim of this study was to develop an assay system that would allow the identification of small molecule inhibitors specific for the mutant Gsα protein, the so-called gsp oncogene. Commercially available Chinese hamster ovary cells were stably transfected with either wild-type (WT) or mutant Gsα proteins (R201C and R201H). Stable cell lines with equivalent transfected Gsα protein expression that had relatively lower (WT) or higher (R201C and R201H) cAMP levels were generated. These cell lines were used to develop a fluorescence resonance energy transfer (FRET)-based cAMP assay in 1536-well microplate format for high throughput screening of small molecule libraries. A small molecule library of 343,768 compounds was screened to identify modulators of gsp activity. A total of 1,356 compounds with inhibitory activity were initially identified and reconfirmed when tested in concentration dose responses. Six hundred eighty-six molecules were selected for further analysis after removing cytotoxic compounds and those that were active in forskolin-induced WT cells. These molecules were grouped by potency, efficacy, and structural similarities to yield 22 clusters with more than 5 of structurally similar members and 144 singleton molecules. Seven chemotypes of the major clusters were identified for further testing and analyses.

Chronic lymphocytic leukemia (CLL) exhibits high remission rates after initial chemoimmunotherapy, but with relapses with treatment, refractory disease is the most common outcome, especially in CLL with the deletion of chromosome 11q or 17p. In addressing the need of treatments for relapsed disease, we report the identification of an existing U.S. Food and Drug Administration-approved small-molecule drug to repurpose for CLL treatment. Auranofin (Ridaura) is approved for use in treating rheumatoid arthritis, but it exhibited preclinical efficacy in CLL cells. By inhibiting thioredoxin reductase activity and increasing intracellular reactive oxygen species levels, auranofin induced a lethal endoplasmic reticulum stress response in cultured and primary CLL cells. In addition, auranofin displayed synergistic lethality with heme oxygenase-1 and glutamate-cysteine ligase inhibitors against CLL cells. Auranofin overcame apoptosis resistance mediated by protective stromal cells, and it also killed primary CLL cells with deletion of chromosome 11q or 17p. In TCL-1 transgenic mice, an in vivo model of CLL, auranofin treatment markedly reduced tumor cell burden and improved mouse survival. Our results provide a rationale to reposition the approved drug auranofin for clinical evaluation in the therapy of CLL.

The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug-drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell-like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton's tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL.

In 2010, the National Institutes of Health (NIH) established the Therapeutics for Rare and Neglected Diseases (TRND) program within the National Center for Advancing Translational Sciences (NCATS), which was created to stimulate drug discovery and development for rare and neglected tropical diseases through a collaborative model between the NIH, academic scientists, nonprofit organizations, and pharmaceutical and biotechnology companies. This paper describes one of the first TRND programs, the development of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) for the treatment of Niemann-Pick disease type C1 (NPC1). NPC is a neurodegenerative, autosomal recessive rare disease caused by a mutation in either the NPC1 (about 95% of cases) or the NPC2 gene (about 5% of cases). These mutations affect the intracellular trafficking of cholesterol and other lipids, which leads to a progressive accumulation of unesterified cholesterol and glycosphingolipids in the CNS and visceral organs. Affected individuals typically exhibit ataxia, swallowing problems, seizures, and progressive impairment of motor and intellectual function in early childhood, and usually die in adolescence. There is no disease modifying therapy currently approved for NPC1 in the US. A collaborative drug development program has been established between TRND, public and private partners that has completed the pre-clinical development of HP-β-CD through IND filing for the current Phase I clinical trial that is underway. Here we discuss how this collaborative effort helped to overcome scientific, clinical and financial challenges facing the development of new drug treatments for rare and neglected diseases, and how it will incentivize the commercialization of HP-β-CD for the benefit of the NPC patient community.

Chordoma is a rare primary bone malignancy that arises in the skull base, spine and sacrum and originates from remnants of the notochord. These tumors are typically resistant to conventional chemotherapy, and to date there are no FDA-approved agents to treat chordoma. The lack of in vivo models of chordoma has impeded the development of new therapies for this tumor. Primary tumor from a sacral chordoma was xenografted into NOD/SCID/IL-2R γ-null mice. The xenograft is serially transplantable and was characterized by both gene expression analysis and whole genome SNP genotyping. The NIH Chemical Genomics Center performed high-throughput screening of 2,816 compounds using two established chordoma cell lines, U-CH1 and U-CH2B. The screen yielded several compounds that showed activity and two, sunitinib and bortezomib, were tested in the xenograft. Both agents slowed the growth of the xenograft tumor. Sensitivity to an inhibitor of IκB, as well as inhibition of an NF-κB gene expression signature demonstrated the importance of NF-κB signaling for chordoma growth. This serially transplantable chordoma xenograft is thus a practical model to study chordomas and perform in vivo preclinical drug testing.

Myotonic dystrophy (DM) is a multi-system neuromuscular disorder for which there is no treatment. We have developed a medium throughput phenotypic assay, based on the identification of nuclear foci in DM patient cell lines using in situ hybridization and high-content imaging to screen for potentially useful therapeutic compounds. A series of further assays based on molecular features of DM have also been employed. Two compounds that reduce and/or remove nuclear foci have been identified, Ro 31-8220 and chromomycin A3. Ro 31-8220 is a PKC inhibitor, previously shown to affect the hyperphosphorylation of CELF1 and ameliorate the cardiac phenotype in a DM1 mouse model. We show that the same compound eliminates nuclear foci, reduces MBNL1 protein in the nucleus, affects ATP2A1 alternative splicing and reduces steady-state levels of CELF1 protein. We demonstrate that this effect is independent of PKC activity and conclude that this compound may be acting on alternative kinase targets within DM pathophysiology. Understanding the activity profile for this compound is key for the development of targeted therapeutics in the treatment of DM.

The discovery and characterization of a novel chemical series of phosphorothioyl-containing imidazopyridines as potent neuropeptide S receptor antagonists is presented. The synthesis of analogues and their structure-activity relationship with respect to the Gq, Gs, and ERK pathways is detailed. The pharmacokinetics and in vivo efficacy of a potent analogue in a food intake rodent model are also included, underscoring its potential therapeutic value for the treatment of sleep, anxiety, and addiction disorders.

Chronic lymphocytic leukemia (CLL) is an adult lymphoid malignancy with a variable clinical course. There is considerable interest in the identification of new treatments, as most current approaches are not curative. While most patients respond to initial chemotherapy, relapsed disease is often resistant to the drugs commonly used in CLL and patients are left with limited therapeutic options. In this study, we used a luminescent cell viability assay based on ATP levels to find compounds that were potent and efficacious in killing CLL cells. We employed an in-house process of quantitative high throughput screening (qHTS) to assess 8 concentrations of each member of a 2,816 compound library (including FDA-approved drugs and those known to be bio-active from commercial suppliers). Using qHTS we generated potency values on each compound in lymphocytes donated from each of six individuals with CLL and five unaffected individuals. We found 102 compounds efficacious against cells from all six individuals with CLL ("consensus" drugs) with five of these showing low or no activity on lymphocytes from a majority of normal donors, suggesting some degree of specificity for the leukemic cells. To our knowledge, this is the first study to screen a drug library against primary CLL cells to identify candidate agents for anti-cancer therapy. The results presented here offer possibilities for the development of novel drug candidates for therapeutic uses to treat CLL and other diseases.