Discovery of First-in-Class Small Molecule Agonists of the RXFP2 Receptor as Therapeutic Candidates for Osteoporosis

Osteoporosis is a chronic bone disease characterized by decreased bone mass and increased risk of developing fractures, predominantly observed in the elderly. The pathophysiological cause of the disease is a decrease in the activity of the bone-forming cells (osteoblasts) that alters bone remodeling in favor of bone resorption, leading to a decrease in bone mass. Recent studies identified the relaxin family peptide receptor 2 (RXFP2), the G protein-coupled receptor (GPCR) for insulin-like 3 peptide (INSL3), as an attractive target expressed in osteoblast cells to increase bone formation. The goal of this dissertation is to discover and characterize small molecule agonists of RXFP2 that are stable and can be delivered orally to promote bone growth. Several low molecular weight compounds were identified as agonists of the RXFP2 receptor using a cAMP high-throughput screen of the NCATS small molecule library. An extensive structure-activity relationship campaign resulted in highly potent and efficient full RXFP2 agonists. The selectivity and specificity of these compounds for human and mouse RXFP2 was shown in counter-screens against the related relaxin receptor RXFP1 and other GPCRs. Using a series of RXFP2/RXFP1 chimeric receptors, in silico modeling and RXFP2 point mutants, we established that the compounds are allosteric agonists of the RXFP2 receptor and identified the GPCR transmembrane domains as the specific region for compound interaction. We also showed that the candidate compounds promoted mineralization in primary human osteoblasts and had low cytotoxicity in various cell types. The compound with the highest activity in vitro was selected for pharmacokinetics profiling in mice, showing oral bioavailability and bone exposure. Moreover, an efficacy study in wild-type female mice treated orally with the lead compound showed a significant increase of the vertebral trabecular number and thickness compared to vehicle treated controls. Overall, our study has successfully identified and characterized the first-in-class small molecule series of RXFP2 agonists, which may lead to the development of a new class of orally bioavailable drugs for the treatment of diseases associated with bone loss

Microfluidics-Based Single-Cell Functional Proteomics Microchip for Portraying Protein Signal Transduction Networks within the Framework of Physicochemical Principles, with Applications in Fundamental and Translational Cancer Research

Single-cell functional proteomics assays can connect genomic information to biological function through quantitative and multiplex protein measurements. Tools for single-cell proteomics have developed rapidly over the past 5 years and are providing unique opportunities. This thesis describes an emerging microfluidics-based toolkit for single cell functional proteomics, focusing on the development of the single cell barcode chips (SCBCs) with applications in fundamental and translational cancer research. The microchip designed to simultaneously quantify a panel of secreted, cytoplasmic and membrane proteins from single cells will be discussed at the beginning, which is the prototype for subsequent proteomic microchips with more sophisticated design in preclinical cancer research or clinical applications. The SCBCs are a highly versatile and information rich tool for single-cell functional proteomics. They are based upon isolating individual cells, or defined number of cells, within microchambers, each of which is equipped with a large antibody microarray (the barcode), with between a few hundred to ten thousand microchambers included within a single microchip. Functional proteomics assays at single-cell resolution yield unique pieces of information that significantly shape the way of thinking on cancer research. An in-depth discussion about analysis and interpretation of the unique information such as functional protein fluctuations and protein-protein correlative interactions will follow. The SCBC is a powerful tool to resolve the functional heterogeneity of cancer cells. It has the capacity to extract a comprehensive picture of the signal transduction network from single tumor cells and thus provides insight into the effect of targeted therapies on protein signaling networks. We will demonstrate this point through applying the SCBCs to investigate three isogenic cell lines of glioblastoma multiforme (GBM). The cancer cell population is highly heterogeneous with high-amplitude fluctuation at the single cell level, which in turn grants the robustness of the entire population. The concept that a stable population existing in the presence of random fluctuations is reminiscent of many physical systems that are successfully understood using statistical physics. Thus, tools derived from that field can probably be applied to using fluctuations to determine the nature of signaling networks. In the second part of the thesis, we will focus on such a case to use thermodynamics-motivated principles to understand cancer cell hypoxia, where single cell proteomics assays coupled with a quantitative version of Le Chatelier's principle derived from statistical mechanics yield detailed and surprising predictions, which were found to be correct in both cell line and primary tumor model. The third part of the thesis demonstrates the application of this technology in the preclinical cancer research to study the GBM cancer cell resistance to molecular targeted therapy. Physical approaches to anticipate therapy resistance and to identify effective therapy combinations will be discussed in detail. Our approach is based upon elucidating the signaling coordination within the phosphoprotein signaling pathways that are hyperactivated in human GBMs, and interrogating how that coordination responds to the perturbation of targeted inhibitor. Strongly coupled protein-protein interactions constitute most signaling cascades. A physical analogy of such a system is the strongly coupled atom-atom interactions in a crystal lattice. Similar to decomposing the atomic interactions into a series of independent normal vibrational modes, a simplified picture of signaling network coordination can also be achieved by diagonalizing protein-protein correlation or covariance matrices to decompose the pairwise correlative interactions into a set of distinct linear combinations of signaling proteins (i.e. independent signaling modes). By doing so, two independent signaling modes – one associated with mTOR signaling and a second associated with ERK/Src signaling have been resolved, which in turn allow us to anticipate resistance, and to design combination therapies that are effective, as well as identify those therapies and therapy combinations that will be ineffective. We validated our predictions in mouse tumor models and all predictions were borne out. In the last part, some preliminary results about the clinical translation of single-cell proteomics chips will be presented. The successful demonstration of our work on human-derived xenografts provides the rationale to extend our current work into the clinic. It will enable us to interrogate GBM tumor samples in a way that could potentially yield a straightforward, rapid interpretation so that we can give therapeutic guidance to the attending physicians within a clinical relevant time scale. The technical challenges of the clinical translation will be presented and our solutions to address the challenges will be discussed as well. A clinical case study will then follow, where some preliminary data collected from a pediatric GBM patient bearing an EGFR amplified tumor will be presented to demonstrate the general protocol and the workflow of the proposed clinical studies.</p

Genetic and Epigenetic Investigations on Pulmonary Hypertension Syndrome in Meat Type- Chickens

This dissertation presents a collection of studies that investigate the genetic and epigenetic associations to ascites phenotype in broiler chickens. Ascites is a significant metabolic disease associated with fast-growing meat-type chickens (broilers) and is a terminal result of pulmonary hypertension syndrome PHS. It is a multi-factorial syndrome caused by interactions between genetic, physiological, environmental, and managemental factors. It was estimated that ascites accounts for losses of about US$1 billion annually worldwide and for over 25% of broilers mortality. Although traditional and molecular genetic methods in the selection and in performance improvements, has greatly reduced ascites frequency, yet it has not eliminated its occurrence. Therefore, this dissertation aimed to 1) develop SNP assays for the gene region of HTR2B to examine the possible association with ascites phenotype and measure gene and allele specific expression in different tissues at different developmental age stages under hypoxic conditions, 2) investigate the association of mitochondrial prevalence in multiple tissues with ascites susceptibility and resistance in broilers, and genes known to regulate mitochondrial biogenesis were assessed, and 3) mapping genome-wide changes in chromatin accessibility for pulmonary artery tissue in ascites - susceptible and ascites- resistant lines under normal and hypoxic conditions using ATAC-seq technology (Assay for Transposase accessible Chromatin with high-throughput sequencing). Altogether, this collection of studies provides new insights into the genetic and epigenetic basis of the ascites syndrome in chicken

Engineering a feedback-based synthetic gene circuit for targeted continuous evolution of a gene in E. coli

Directed evolution is an invaluable technique for engineering proteins to possess desired physical and chemical properties when very little structural and functional information is known. It is divided into two sequential steps: generating a library of protein variants using mutagenic techniques; and applying a screening or selection strategy to scan the library for variants displaying desired properties. Library generation is performed using either in vitro or in vivo techniques, while screening or selection typically occurs in a suitable host cell. Currently, in vitro methods like error-prone PCR are popular for library generation. However, these techniques can be labour intensive, prone to mutation biases, and generate limited library sizes for screening. In vivo mutagenic techniques overcome these limitations by enabling simultaneous library generation and selection within cells. By generating random mutations in the gene-of-interest within one cell cycle, each cell in a batch culture potentially represents a library variant. Such a continuous evolution system can run for weeks with minimal human intervention, greatly expanding the genetic search space for protein engineering. The challenge lies in developing a mutator system that specifically generates mutations in the target gene, while maintaining the cell’s genomic fidelity. With this goal in mind, a mutator system was engineered in E. coli that introduces targeted cytidine deamination damage and subsequently performs error-prone DNA repair by hijacking the base excision repair pathway. The targeted damage occurs via activation induced cytidine deaminase fused to T7 RNA polymerase, while the error-prone DNA repair is performed by a three-protein fusion comprising a 5’-3’-exonuclease, an AP-endonuclease and an error-prone DNA polymerase. The mutagenic characteristics of this system was tested by knocking out GFP expression and analysing the mutant library using next generation sequencing techniques. The system was also experimentally shown to generate functionally active mutations that reverted inactivated β-lactamase gene variants to confer ampicillin resistance.Open Acces

Low power digital baseband core for wireless Micro-Neural-Interface using CMOS sub/near-threshold circuit

This thesis presents the work on designing and implementing a low power digital baseband core with custom-tailored protocol for wirelessly powered Micro-Neural-Interface (MNI) System-on-Chip (SoC) to be implanted within the skull to record cortical neural activities. The core, on the tag end of distributed sensors, is designed to control the operation of individual MNI and communicate and control MNI devices implanted across the brain using received downlink commands from external base station and store/dump targeted neural data uplink in an energy efficient manner. The application specific protocol defines three modes (Time Stamp Mode, Streaming Mode and Snippet Mode) to extract neural signals with on-chip signal conditioning and discrimination. In Time Stamp Mode, Streaming Mode and Snippet Mode, the core executes basic on-chip spike discrimination and compression, real-time monitoring and segment capturing of neural signals so single spike timing as well as inter-spike timing can be retrieved with high temporal and spatial resolution. To implement the core control logic using sub/near-threshold logic, a novel digital design methodology is proposed which considers INWE (Inverse-Narrow-Width-Effect), RSCE (Reverse-Short-Channel-Effect) and variation comprehensively to size the transistor width and length accordingly to achieve close-to-optimum digital circuits. Ultra-low-power cell library containing 67 cells including physical cells and decoupling capacitor cells using the optimum fingers is designed, laid-out, characterized, and abstracted. A robust on-chip sense-amp-less SRAM memory (8X32 size) for storing neural data is implemented using 8T topology and LVT fingers. The design is validated with silicon tapeout and measurement shows the digital baseband core works at 400mV and 1.28 MHz system clock with an average power consumption of 2.2 μW, resulting in highest reported communication power efficiency of 290Kbps/μW to date

Functional impact of inactivating mutations in epigenetic regulators in cancer

Cancer evolution is driven by selection acting on genetic and epigenetic diversity to promote the propagation of the fittest subpopulations. This phenomenon is shaped by the tumor microenvironment which is often characterized by stressful conditions. Epigenetic regulators are frequently mutated during the later stages of tumorigenesis, but the functional impact of their inactivation is poorly understood. In this thesis, I hypothesize that the disruption of the epigenetic regulatory network increases cell fitness in unfavorable environments and thus is selected over time. Through large-scale fitness assays in various cancer models, I demonstrate that epigenetic deregulation leads to a widespread stress-specific survival advantage. This effect is mediated by mutations in all layers of epigenetic regulation, is shared across different stress conditions and is cancer type independent. Then, I explore various cellular mechanisms that can underlie this stress-specific fitness advantage. Genetic diversity, transcriptional heterogeneity or phenotypic plasticity cannot explain the increased survival under stress, as revealed by a combination of reversible epigenetic inhibition, live-cell imaging and single-cell transcriptomics. On the contrary, epigenetically deregulated cells remain phenotypically inert (less responsive) under stress. Transcriptional profiling of cancer populations in hostile conditions, revealed significant alterations in fitness and growth-related signatures. Disruption of the epigenetic machinery results in a defective stress response, thus decreasing the probability of such cells to surpass a stressed threshold and ultimately die. This defective transcriptional rewiring underpins the inert phenotype that emerges upon epigenetic deregulation. Collectively, by investigating the effect of inactivating mutations in epigenetic regulators on cell fitness under environmental stress, I propose that phenotypic inertia is the favorable cellular trait that is selected over time. My findings provide a potential explanation for the widespread subclonal mutations affecting epigenetic regulators and have significant implications for cancer evolution.Open Acces

Non-Coding RNAs in Ovarian Cancer

Ovarian cancer (OC) is the most lethal form of gynaecological cancer, with high- grade serous ovarian carcinoma (HGSOC) being the most common and the deadliest subtype. Non-coding RNAs are a recently discovered species of RNAs that do not code for proteins, yet play a crucial role in both normal physiology and disease. The overall goal of this thesis was to apply the power of non-coding RNAs to OC with the following aims: (1) to identify novel small non-coding RNAs present in serum that could separate patients with HGSOC from healthy women as well as predict their surgical outcome, (2) to assess the role of long non-coding RNAs (lncRNAs) in promoting cisplatin resistance in cell line models of OC, and (3) to study the effects of mutant-p53 on mRNAs and lncRNAs using a small compound known as APR-246 as well as investigating the drug’s mechanisms of action. Firstly, the lethality of OC could partially be attributed to the lack of specific symptoms, leading this disease to be termed the ‘silent killer’, as well as low inci- dence rate of 9.4 cases per 100,000 individuals, both requiring a highly accurate test for population screening that remains an ongoing challenge. Measuring the levels of small non-coding RNAs, known as microRNAs, in serum, experiments described in this thesis aimed to identify novel microRNAs that could separate pa- tients with HGSOC from healthy women as well as predict their surgical outcome, one of the most important factors influencing overall patient survival. Because serum microRNAs can be affected by pre-analytical factors such as haemolysis, the sensitivity of four methodologies to detect low levels of haemolysis was first determined. This work is published in Plos One. The work described in this thesis identified a novel serum microRNA, miR-375, that could improve the accuracy of CA-125, a routinely used biomarker in diagnosing OC, in separating patients with HGSOC from healthy women. Next, serum microRNA miR-34a-5p was found to predict the surgical outcome of patients with HGSOC. In fact, miR-34a-5p was found to be superior to CA-125 for this purpose. Although the standard therapy for treating OC consists of surgical removal of the tumour followed by chemotherapy containing platinum/taxane agents, this regimen may be too aggressive for a sub- set of patients who might benefit from neoadjuvant chemotherapy, i.e. chemother- apy followed by the surgery. A pre-operative expectation of the the surgical out- come could help surgeons decide on optimal timing for surgery. Both miR-375 and miR-34a-5p were also unaffected by haemolysis. Secondly, although OC is initially sensitive to chemotherapy, most patients develop resistance within two years, resulting in recurrent disease that is difficult to treat. To identify novel lncRNAs that could promote drug resistance, expression of ninety lncRNAs was profiled in cell line models of cisplatin resistance. Five lncRNAs were found to have the potential to promote cisplatin resistance in vitro, and lncRNA Urothelial Cancer Associated 1 (UCA1) was selected for further investigations. Despite its role in promoting cisplatin resistance in bladder cancer, UCA1 was not found to promote cisplatin resistance in cell line models of OC. Lastly, the tumour suppressor TP53 plays a central role in the biology of cancer and it is almost universally mutated in HGSOC. Recent evidence suggests that p53, the protein encoded by TP53, can significantly influence the expression of both small and long non-coding RNAs. Experiments described in this thesis aimed to investigate the effect of mutant-p53 on protein-coding and non-coding RNAs by using a small compound known as APR-246 which has been reported to restore wild-type p53 activities in multiple cancers by stabilising the structure of mutant- p53. Despite currently undergoing a phase Ib/II clinical trial for potential treatment of recurrent HGSOC, the ability of APR-246 to restore wild-type p53 activities in HGSOC has not been tested. A global transcriptomic analysis conducted in this thesis discovered that p53-responsive mRNAs and lncRNAs were not robustly induced following APR-246 treatment in two cell line models of HGSOC, but indicated that APR-246 could function by inducing high levels of reactive oxidative species (ROS). Overall, data presented in this thesis demonstrated the utility of small non- coding RNAs in identifying patients with HGSOC from healthy women as well as predicting their surgical outcome. This thesis also implicated that lncRNAs, in general, could have a role in promoting cisplatin resistance in OC as well as suggested that APR-246 could, based on evidence obtained from the expression of p53-responsive mRNAs and lncRNAs, act independently of mutant-p53. Together, this research raises novel ways for clinical management of patients with HGSOC and addresses the challenge of drug resistance using non-coding RNAs, as well as questions the assumed mechanisms of action of the ‘p53-activating’ drug APR- 246

Membrane adsorbers and novel affinity peptides for recombinant protein purification

2015 Spring.Includes bibliographical references.The purification of recombinant proteins for use as pharmaceutically active ingredients represents one of the largest costs of drug development and production. Of the different classes of recombinant protein therapeutics monoclonal antibodies represent the largest percentage of protein therapeutics currently on the market with even more in clinical development. The work presented in this thesis describes the evaluation of both commercial and newly designed anion exchange and hydrophobic interaction (HIC) membrane adsorbers as well as identification of novel affinity peptides for the purification of recombinant proteins, specifically monoclonal antibodies. Commercially available anion-exchange membrane adsorbers were evaluated for their potential to remove impurities commonly present at low concentration in recombinant protein solutions expressed in mammalian cell culture. These so-called trace impurities include virus, host cell proteins, and DNA; these impurities are of particular concern because they are highly immunogenic at very low concentrations. Ionic strength and pH were shown to be the dominant factors affecting impurity binding on quaternary amine (Q) membranes indicating these ligands interact with the impurities primarily through electrostatic interactions. It is likely impurity interactions with primary amine ligands involved not only electrostatic but hydrogen bonding interactions which stabilized impurity-ligand interactions enabling greater removal at a broader range of solution pH and ionic strength conditions. Binding of host cell proteins with a broad range of isoelectric points was also demonstrated using the primary amine ligand as compared to the Q ligands. The effect of solution pH, ionic strength, flow rate, and the presence of competing anionic species was investigated. In addition to commercially available anion-exchange membrane adsorbers novel anion-exchange membranes, developed by Dr. Bharat Bhut and Prof. Scott Husson at Clemson University, were evaluated for binding capacity and virus removal. Regenerated cellulose microfiltration membranes were modified with a negatively-charged quaternary amine polymer, systematically varying the polymer chain density and length. IgG and DNA binding capacity, as well as minute virus of mice removal, was evaluated as a function of polymer chain density and length. It was shown that IgG binding capacity increased with polymer chain density indicating IgG access to binding sites was not a limiting factor. Similarly, high polymer chain density and longer polymerization time (translating to longer polymer chain length) resulted in higher DNA binding and virus removal again indicating ligand accessibility was not an issue even with large solutes such as virus. Environmentally-responsive hydrophobic interaction membranes were also developed in the Wickramasinghe lab and evaluated for protein binding capacity and recovery. Three-dimensional polymer brushes were grafted from 0.45 µm pore size regenerated cellulose membrane surfaces. The dynamic binding capacity of human IgG was greater than current commercially available hydrophobic interaction membranes with comparable recoveries. Affinity purification using novel small peptides was also explored as an antibody purification tool. Several heptapeptide affinity ligands were identified that bound specifically to the Fc region of IgG. These peptides have similar function to Staphylococcus Aureus Protein A, which is used extensively as an affinity purification ligand for monoclonal antibodies in the pharmaceutical industry. A large library of seven amino acid-long peptides was screened via M13 Phage Display for specific binding to the Fc, or constant region, of human IgG antibody. After initial identification, specificity of binding only to IgG was demonstrated through subsequent competitive ELISA assays. Though the affinity peptides were initially screened against human IgG₄ Fc, binding to a larger subset of human and non-human antibodies was shown indicating the peptides were binding to highly conserved regions on the antibodies. Because Protein A has some limitations in industrial process applications, these novel heptapeptides may provide an alternative solution for affinity purification of monoclonal antibodies

The microbial ecology and biogeochemistry of cyanobacteria in the arsenic-rich and inorganic carbon-limited geothermal waters of El Tatio Geyser Field, Chile

Geothermal settings are some of the best-known analogs for early earth environments and among the best places to investigate the impact of extreme conditions on microbial life. El Tatio Geyser Field (ETGF) is a geothermal setting located at 4,300m in the Atacama Desert region of Chile. Its high-elevation desert position leads to high UV-flux, rapid evaporation, and mineral precipitation. El Tatio geothermal waters also possess extremely limited concentrations of life-essential nutrients, such as dissolved inorganic carbon (DIC as CO2(aq) + HCO3-), contain among the highest naturally occurring concentrations of the toxic element arsenic (As as H3AsO30 + HXAsO43-X), and are buffered to circumneutral pH by arsenate (H2AsO4-/HAsO42-; pKa ~ 6.9 at 25°C). Cyanobacteria were found to be the most important primary producers supporting microbial communities in El Tatio geothermal waters. The objective of this dissertation work was to characterize the role of cyanobacteria in the ETGF microbial ecosystem, and determine the response of cyanobacteria to the high-As and low-DIC conditions present at ETGF. Field observations, geochemical analyses, and next-generation 16S rRNA gene sequencing approaches were used to determine the geochemical controls on cyanobacterial distribution, the phylogenetic diversity of El Tatio cyanobacteria, and the corresponding microbial community structure at sites with and without cyanobacteria. Four cultured cyanobacterial strains were isolated from ETGF mat material, and experiments were performed to assess the growth and carbon-uptake response of these strains to low DIC, AsIII, and AsV. AsIII and temperature negatively controlled the abundance and distribution of cyanobacteria in geothermal outflows throughout ETGF, whereas AsV positively influenced these factors. In the laboratory, AsIII inhibited the growth of cultured strains, while AsV stimulated growth. Closed-system experiments showed significantly increased carbon uptake and growth in the presence of AsV, due to the ability of arsenate to offset the rapid upward pH shift that often occurs in mats during photosynthesis, thereby maintaining DIC in the preferred forms for cyanobacterial uptake. These results showed that AsV plays a positive role in the ETGF microbial ecosystem by increasing the productivity of cyanobacterial mats under low DIC and arsenate-buffered conditions.Geological Science