Large-scale transcriptomic analysis reveals that pridopidine reverses aberrant gene expression and activates neuroprotective pathways in the YAC128 HD mouse


Huntington Disease (HD) is an incurable autosomal dominant neurodegenerative disorder driven by an expansion repeat giving rise to the mutant huntingtin protein (mHtt), which is known to disrupt a multitude of transcriptional pathways. Pridopidine, a small molecule in development for treatment of HD, has been shown to improve motor symptoms in HD patients. In HD animal models, pridopidine exerts neuroprotective effects and improves behavioral and motor functions. Pridopidine binds primarily to the sigma-1 receptor, (IC50 ~ 100 nM), which mediates its neuroprotective properties, such as rescue of spine density and aberrant calcium signaling in HD neuronal cultures. Pridopidine enhances brain-derived neurotrophic factor (BDNF) secretion, which is blocked by putative sigma-1 receptor antagonist NE-100, and was shown to upregulate transcription of genes in the BDNF, glucocorticoid receptor (GR), and dopamine D1 receptor (D1R) pathways in the rat striatum. The impact of different doses of pridopidine on gene expression and transcript splicing in HD across relevant brain regions was explored, utilizing the YAC128 HD mouse model, which carries the entire human mHtt gene containing 128 CAG repeats.


Structural Changes Due to Antagonist Binding in Ligand Binding Pocket of Androgen Receptor Elucidated through Molecular Dynamics Simulations


When a small molecule binds to the androgen receptor (AR), a conformational change can occur which impacts subsequent binding of co-regulator proteins and DNA. In order to accurately study this mechanism, the scientific community needs a crystal structure of the Wild type AR (WT-AR) ligand binding domain, bound with antagonist. To address this open need, we leveraged molecular docking and molecular dynamics (MD) simulations to construct a structure of the WT-AR ligand binding domain bound with antagonist bicalutamide. The structure of mutant AR (Mut-AR) bound with this same antagonist informed this study. After molecular docking analysis pinpointed the suitable binding orientation of a ligand in AR, the model was further optimized through 1 μs of MD simulations. Using this approach, three molecular systems were studied: (1) WT-AR bound with agonist R1881, (2) WT-AR bound with antagonist bicalutamide, and (3) Mut-AR bound with bicalutamide. Our structures were very similar to the experimentally determined structures of both WT-AR with R1881 and Mut-AR with bicalutamide, demonstrating the trustworthiness of this approach. In our model, when WT-AR is bound with bicalutamide, Val716/Lys720/Gln733, or Met734/Gln738/Glu897 move and thus disturb the positive and negative charge clumps of the AF2 site. This disruption of the AF2 site is key for understanding the impact of antagonist binding on subsequent co-regulator binding. In conclusion, the antagonist induced structural changes in WT-AR detailed in this study will enable further AR research and will facilitate AR targeting drug discovery.


Loss of the Sigma-1 receptor disrupts pridopidine-induced gene expression

Poster at AAN 2018

Objective: Investigate the role of S1R in the mechanism of action of pridopidine.

Background: Sigma-1 receptor (S1R) is an ER chaperone protein involved in neuromodulation and neuroplasticity. Pridopidine is a selective S1R agonist small molecule currently in clinical development for Huntington disease by Teva Pharmaceuticals Ltd.

Design/Methods: Transcriptomic analysis of WT and S1R-deficient mice treated with increasing concentrations of pridopidine (0, 0.3, 3, 30, or 60 mg/kg) for 10 days. Prefrontal cortex, striatum and hippocampus tissues were collected and profiled via RNAseq.


Integrity, standards, and QC-related issues with big-data in pre-clinical drug discovery


The tremendous expansion of data analytics and public and private big datasets presents an important opportunity for pre-clinical drug discovery and development. In the field of life sciences, the growth of genetic, genomic, transcriptomic and proteomic data is partly driven by a rapid decline in experimental costs as biotechnology improves throughput, scalability, and speed. Yet far too many researchers tend to underestimate the challenges and consequences involving data integrity and quality standards. Given the effect of data integrity on scientific interpretation, these issues have significant implications during preclinical drug development. We describe standardized approaches for maximizing the utility of publicly available or privately generated biological data and address some of the common pitfalls. We also discuss the increasing interest to integrate and interpret cross-platform data. Principles outlined here should serve as a useful broad guide for existing analytical practices and pipelines and as a tool for developing additional insights into therapeutics using big data.


A meta-metastasis analysis identifies pan-cancer markers and therapeutic targets

Poster - AACR 2017

Metastasis is a leading cause of cancer-associated deaths across several cancer types, yet the molecular details of its development have not been fully elucidated. Transcriptomic analysis can provide insight into the gene expression changes in the tumor that confer metastatic potential even during early stages of tumorigenesis. Here, by leveraging RNA-Seq data collected by The Cancer Genome Atlas (TCGA), we systematically identified mechanisms consistently associated with metastasis in primary tumors from 4844 patients across 13 different cancer types via comparison of primary tumors from patients with primary site, node-negative disease with no recorded distant metastasis (N0 not M1) compared to primary tumors from patients with node-positive disease (N1, N2, or N3). Differentially expressed genes were first determined within each cancer type, to reduce tissue- or disease-specific effects. Subsequently, via meta-analysis, we combine these results to identify commonality across 13 different types of cancer. Next, using a proprietary drug prediction algorithm called Drugfinder, we identified drug candidates that can target metastasis across all cancer types and within specific cancer types.


The international MAQC Society launches to enhance reproducibility of high-throughput technologies

doi:10.1038/nbt.4029 Nature Biotechnology 35, 1127–1128(2017)

Reproducibility is a fundamental hallmark of good science. The US Food and Drug Administration (FDA)-led Microarray and Sequencing Quality Control (MAQC/SEQC) consortia conducted three projects 1,2,3 to assess the reliability and reproducibility of genomics technologies, including microarrays, genome-wide association studies, and next-generation sequencing. Here, we announce that this decade-long effort has led to the formation of a new international society, the Massive Analysis and Quality Control (MAQC) Society (, which is dedicated to quality control and analysis of massive data generated from high-throughput technologies for enhanced reproducibility.


Early pridopidine treatment improves behavioral and transcriptional deficits in YAC128 Huntington disease mice

JCI Insight. 2017;2(23):e95665. doi:10.1172/jci.insight.95665.

Pridopidine is currently under clinical development for Huntington disease (HD), with on-going studies to better characterize its therapeutic benefit and mode of action. Pridopidine was administered either prior to the appearance of disease phenotypes or in advanced stages of disease in the YAC128 mouse model of HD. In the early treatment cohort, animals received 0, 10, or 30 mg/kg pridopidine for a period of 10.5 months. In the late treatment cohort, animals were treated for 8 weeks with 0 mg/kg or an escalating dose of pridopidine (10 to 30 mg/kg over 3 weeks). Early treatment improved motor coordination and reduced anxiety- and depressive-like phenotypes in YAC128 mice, but it did not rescue striatal and corpus callosum atrophy. Late treatment, conversely, only improved depressive-like symptoms. RNA-seq analysis revealed that early pridopidine treatment reversed striatal transcriptional deficits, upregulating disease-specific genes that are known to be downregulated during HD, a finding that is experimentally confirmed herein. This suggests that pridopidine exerts beneficial effects at the transcriptional level. Taken together, our findings support continued clinical development of pridopidine for HD, particularly in the early stages of disease, and provide valuable insight into the potential therapeutic mode of action of pridopidine.


Proteomic Profiling Reveals Targetable Pathways in MGUS (SLAMF6, TNFRSF8, TIMP1, TRL2) That May Contribute to Disease Progression Blood 2017 130:3805;

Poster - ASH 2017

Monoclonal gammopathy of undetermined significance (MGUS), is a pre-malignant plasma cell disorder that affects roughly 4% of the population older than 55 and is associated with a 1% per year risk of progression to multiple myeloma (MM). Successfully intercepting disease progression from MGUS to MM requires a deeper understanding of MGUS biology, identification of prognostic biomarkers and potential targets for interception. With these aims, a cohort of samples were profiled from the Olmstead County Study, which followed >20,000 subjects for over 20 years as they aged and developed a variety of diseases, including MGUS and MM (Kyle, et al. Oncology 2011). Samples were utilized from 2 cohorts: 1) healthy normal individuals with no subsequent diagnosis of plasma cell disorder, and 2) healthy normal individuals who go on to develop MGUS, with a second sample at the time of MGUS diagnosis. Broad proteomic profiling was performed on these samples, to identify pathways involved in the early stages of MGUS disease biology.


Compositional differences between Copaxone and Glatopa are reflected in altered immunomodulation ex vivo in a mouse model.

doi: 10.1111/nyas.13547 Annals of the New York Academy of Science

Copaxone (glatiramer acetate, GA), a structurally and compositionally complex polypeptide nonbiological drug, is an effective treatment for multiple sclerosis, with a well-established favorable safety profile. The short antigenic polypeptide sequences comprising therapeutically active epitopes in GA cannot be deciphered with state-of-the-art methods; and GA has no measurable pharmacokinetic profile and no validated pharmacodynamic markers. The study reported herein describes the use of orthogonal standard and high-resolution physicochemical and biological tests to characterize GA and a U.S. Food and Drug Administration-approved generic version of GA, Glatopa (USA-FoGA). While similarities were observed with low-resolution or destructive tests, differences between GA and USA-FoGA were measured with high-resolution methods applied to an intact mixture, including variations in surface charge and a unique, high-molecular-weight, hydrophobic polypeptide population observed only in some USA-FoGA lots. Consistent with published reports that modifications in physicochemical attributes alter immune-related processes, genome-wide expression profiles of ex vivo activated splenocytes from mice immunized with either GA or USA-FoGA showed that 7-11% of modulated genes were differentially expressed and enriched for immune-related pathways. Thus, differences between USA-FoGA and GA may include variations in antigenic epitopes that differentially activate immune responses. We propose that the assays reported herein should be considered during the regulatory assessment process for nonbiological complex drugs such as GA.


Transcriptomic analysis of the YAC128 HD mouse model shows disease mechanisms are ameliorated by pridopidine.

Poster - Society for Neuroscience

Huntington Disease (HD) is a neurodegenerative disorder hallmarked by the expression of a mutant form of the huntingtin gene (mHtt). A therapeutic goal for HD treatment involves the restoration of neurobiological pathways disrupted by mHtt. Pridopidine, an investigational HD drug candidate, has been shown to improve motor symptoms in both preclinical models as well as in HD patients. While originally described as a dopaminergic stabilizer, in vitro binding studies show highest affinity of pridopidine to the sigma-1 receptor. We previously reported that pridopidine upregulates BDNF, glucocorticoid receptor (GR), and dopamine 1 receptor (D1R) signaling pathways in WT rat striatum. To expand on these findings and explore molecular changes specific to HD, we repeated this study in a YAC128 HD mouse model whereby mice were treated with pridopidine or vehicle starting at postnatal week 6 and sacrificed after 11.5 months of age. We then performed RNAseq data analysis on the striatum. We identified 1346 differentially expressed genes (DEGs) in vehicle treated YAC128 mice vs. healthy controls, 221 DEGs in YAC128 mice treated with 30 mg/kg of pridopidine vs. vehicle, and 73 DEGs in YAC128 mice treated with 10 mg/kg of pridopidine vs. vehicle (all adj.p<0.05). In addition, the previously reported pridopidine-induced upregulation of BDNF, GR, and D1R pathways was confirmed after treatment with either dose (adj.p<0.05), and alternatively spliced genes after 30 mg/kg of pridopidine compared to YAC128 vehicle are enriched for the BDNF pathway (adj.p<0.05). Further, pathway analysis of DEGs after 30 mg/kg dosage revealed enrichment for biological processes related to synaptic transmission, including upregulation in the key HD-impaired processes – cAMP and calcium signaling (adj.p<0.05). Lastly, since mHtt was shown to induce neurotoxicity by promoting M1 microglia that secrete proinflammatory cytokines, enrichment analysis tested for microglial markers. Treatment with 30 mg/kg of pridopidine led to the downregulation of genes associated with M1 activation (adj.p<0.05). To summarize, pridopidine induces transcriptional modifications reversing genes in HD-impaired pathways involved in neuronal transmission and protection in the YAC128 striatum.