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.

Drug-induced phospholipidosis is characterized by the accumulation of intracellular phospholipids in cells exposed to cationic amphiphilic drugs. The appearance of unicentric or multicentric multilamellar bodies viewed under an electron microscope (EM) is the morphological hallmark of phospholipidosis. Although the EM method is the gold standard for detecting cellular phospholipidosis, this method has its drawbacks, including low throughput, high cost, and unsuitability for screening a large chemical library. In this study, a cell-based phospholipidosis assay has been developed using the LipidTOX Red reagent in HepG2 cells and miniaturized into a 1536-well plate format. To validate this assay for high-throughput screening (HTS), the LOPAC library of 1280 compounds was screened using a quantitative HTS platform. A group of known phospholipidosis inducers, such as amiodarone, propranolol, chlorpromazine, desipramine, promazine, clomipramine, and amitriptyline, was identified by the screen, consistent with previous reports. Several novel phospholipidosis inducers, including NAN-190, ebastine, GR127935, and cis-(Z)-flupentixol, were identified in this study and confirmed using the EM method. These results demonstrate that this assay can be used to evaluate and profile large numbers of chemicals for drug-induced phospholipidosis.

The microtubule-associated protein (MAP) tau has been implicated in the pathology of numerous neurodegenerative diseases. In the past decade, the hyperphosphorylated and aggregated states of tau protein have been important targets in the drug discovery field for the potential treatment of Alzheimer's disease. Although several compounds have been reported to reduce the hyperphosphorylated state of tau or impact the stabilization of tau, their therapeutic activities are remain to be validated. Recently, reduction of total cellular tau protein has emerged as an alternate intervention point for drug development and a potential treatment of tauopathies. We have developed and optimized homogenous assays, using the AlphaLISA and HTRF assay technologies, for the quantification of total cellular tau protein levels in the SH-SY5Y neuroblastoma cell line. The signal-to-basal ratios were 375 and 5.3, and the Z' factors were 0.67 and 0.60 for the AlphaLISA and HTRF tau assays, respectively. The clear advantages of these homogeneous tau assays over conventional total tau assays, such as ELISA and Western blot, are the elimination of plate wash steps and miniaturization of the assay into 1536-well plate format for the ultra-high-throughput screening of large compound libraries.

A goal of the Tox21 program is to transit toxicity testing from traditional in vivo models to in vitro assays that assess how chemicals affect cellular responses and toxicity pathways. A critical contribution of the NIH Chemical Genomics center (NCGC) to the Tox21 program is the implementation of a quantitative high throughput screening (qHTS) approach, using cell- and biochemical-based assays to generate toxicological profiles for thousands of environmental compounds. Here, we evaluated the effect of chemical compounds on mitochondrial membrane potential in HepG2 cells by screening a library of 1,408 compounds provided by the National Toxicology Program (NTP) in a qHTS platform. Compounds were screened over 14 concentrations, and results showed that 91 and 88 compounds disrupted mitochondrial membrane potential after treatment for 1 or 5 h, respectively. Seventy-six compounds active at both time points were clustered by structural similarity, producing 11 clusters and 23 singletons. Thirty-eight compounds covering most of the active chemical space were more extensively evaluated. Thirty-six of the 38 compounds were confirmed to disrupt mitochondrial membrane potential using a fluorescence plate reader, and 35 were confirmed using a high content imaging approach. Among the 38 compounds, 4 and 6 induced LDH release, a measure of cytotoxicity, at 1 or 5 h, respectively. Compounds were further assessed for mechanism of action (MOA) by measuring changes in oxygen consumption rate, which enabled the identification of 20 compounds as uncouplers. This comprehensive approach allows for the evaluation of thousands of environmental chemicals for mitochondrial toxicity and identification of possible MOAs.

Both pharmacophore models of the human ether-à-go-go-related gene (hERG) channel blockers and phospholipidosis (PLD) inducers contain a hydrophobic moiety and a hydrophilic motif/positively charged center, so it is interesting to investigate the overlap between the ligand chemical spaces of both targets. We have assayed over 4000 non-redundant drug-like compounds for both their hERG inhibitory activity and PLD inducing potential in a quantitative high throughput screening (qHTS) format. Seventy-seven percent of PLD inducing compounds identified from the screening were also found to be hERG channel blockers, and 96.9% of the dually active compounds were positively charged. Among the 48 compounds that induced PLD without inhibiting hERG channel, 24 compounds (50.0%) carried steroidal structures. According to our results, hERG channel blockers and PLD inducers share a large chemical space. In addition, a positively charged hERG channel blocker will most likely induce PLD, while a steroid PLD inducer is less likely a hERG channel blocker.