Benjamin L. Miller, Ph.D - US grants

[unreadable] DESCRIPTION (provided by applicant): Carbohydrates play important and varied roles in the cell, and are of interest as targets for therapeutic intervention, clinical diagnosis, and as potential pharmaceuticals. While compounds capable of selectively serving as receptors (or ligands) have been designed for most biological molecules, very few have been described that are capable of high-affinity noncovalent binding to simple carbohydrates in aqueous solution. The P.I.'s laboratory has recently developed a series of compounds, based on highly substituted tercyclopentanes, that bind simple sugars and more complex liposaccharides in water with affinities two to three orders of magnitude tighter than any previously described synthetic receptors. The research described herein seeks to use these receptors, and analogous molecules, to develop a detailed understanding of the structural factors underlying carbohydrate binding. Through systematic variation of tercyclopentane carbohydrate receptors, the relative contributions of hydrogen bonding, conformational variability, stereochemistry, hydrophobic effects, CH-pi, and cation-pi interactions will be examined. NMR analyses of one of these compounds alone and complexed with carbohydrate guests will provide high-resolution structural information about the binding interactions. This research will provide insights vital to our understanding of fundamental glycobiology, to our ability to design carbohydrate-targeted pharmaceutical agents, and to the development of diagnostic devices (biosensors).

CBET-0730469 Fauchet The development of rapid, sensitive and easy-to-use biosensors for the detection of a wide range of pathogens continues to be of national importance. Traditional diagnostic methods for the detection of viruses are complex and require operation by a highly trained technician. This level of complexity and cost is obviously undesirable, as it significantly lengthens the time necessary to make a diagnosis in a clinical setting, and severely limits the deployment of sensors and diagnostics to locations where both fully equipped laboratories and trained personnel are available. The development of optical label-free biosensor platforms, which promise to simplify the system complexity while exhibiting desirable performance, is a high priority.
Intellectual Merit
A novel platform for single bioparticle detection is proposed, which is based on two-dimensional photonic crystal microcavities fabricated on silicon-on-insulator substrates using microelectronic fabrication steps. Capture of bioparticles in these devices locally increases the refractive index, which can be detected as a shift of the wavelength of light propagation. The photonic crystal microcavities will be designed, fabricated, tested, and optimized for maximum light transmission, and for sensitivity and selectivity to the targets of interest. They will be integrated with microfluidics including a novel ultrathin membrane filter made of silicon that will be used to pre-concentrate the targets. The combination of surface-enhanced Raman scattering (SERS) and 2-D PhC microcavities will be explored to make identification unambiguous and eliminate false positive responses.
Broader Impact
The proposed work responds to mounting concern over the rapid rise of new viral pathogens (including Severe Acute Respiratory Syndrome, or SARS, and the H5N1 "bird flu"), as well as continuing concern over the possible use of viral pathogens such as smallpox as biowarfare agents, in response to which the global research community has targeted the development of label-free biosensors capable of detecting single virus particles as a major goal. The proposed research will provide strategies for the detection of not only dangerous viruses but also a wide range of biological targets.

DESCRIPTION (provided by applicant): Direct single- or near-single copy detection of viruses is an important challenge in human health. Applications include the need to monitor emerging viral threats (H5N1 influenza and the SARS virus, for example), and the importance of understanding the long-term implications of nearly undetectable reservoirs of infection following antiretroviral therapy for HIV. While considerable effort has been expended on the development of ultrasensitive detection methods, the need remains for new technologies that are exceptionally sensitive, label-free, physically robust, and inexpensive. We hypothesize that two-dimensional photonic bandgap (2DPBG) sensors can fulfill all of these requirements. We will test that hypothesis by completing the following three Aims. In Aim 1, we will develop and test a new method for nanoscale patterning of single chain antibodies, and will implement that method in single-target 2DPBG sensors for vaccinia. In Aim 2, we will extend this methodology to the production of dual-target sensors. Finally, we will integrate these novel biosensors with microfluidic channels and with features for sample filtration and/or preconcentration in Aim 3. PUBLIC HEALTH RELEVANCE: Current methods for detecting viruses with high sensitivity - down to the single viral particle level - are expensive and complex. We propose to develop and test a new class of optical biosensors based on silicon that will be able to detect viruses simply and rapidly, with single-particle sensitivity. These new sensors will have applications in the detection of HIV, and emerging viral pathogens such as H5N1 influenza and SARS, among others.

DESCRIPTION (provided by applicant): Sequestration of MBNL1 by (CUG) repeat RNA is believed to be a key molecular interaction in type 1 myotonic dystrophy (DM1). Thus, new compounds able to inhibit this interaction constitute important potential leads towards the development of effective therapeutic agents. We recently described the discovery of a set of compounds able to bind (CUG) repeat RNA with significant selectivity and inhibit MBNL1 binding. Our proposed research centers on three Aims that will expand on this important initial result, and obtain compounds suitable for clinical development. First, we will determine the minimum binding peptide capable of maintaining selective (CUG) repeat RNA binding, and will explore the effect of peptide N-methylation on affinity. Second, we will employ the highest-affinity lead compound in a displacement-based high throughput screen for new (CUG) repeat-binding chemotypes. Finally, we will carry out a series of cellular assays to determine the ability of new compounds to inhibit (CUG) RNA - MBNL1 binding in a cellular context. PUBLIC HEALTH RELEVANCE: Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults, affecting 1 in 8000 people. The disease is believed to result from the accumulation of a toxic RNA, termed a "CUG repeat", which sequesters a protein critical for proper cellular function. Building on a discovery made in our laboratory of a molecule able to bind CUG repeat RNA, we will carry out a series of experiments designed to yield a new molecule with higher activity and improved drug-like properties. The overall goal of the project will be to obtain at least one compound suitable for development as a therapeutic agent.

DESCRIPTION (provided by applicant): The discovery of RNA sequences of potential biomedical importance has dramatically outpaced chemists' ability to design and synthesize novel selective RNA-binding compounds. This is due largely to a gap in knowledge in the field with regard to fundamental determinants of selectivity. This proposal seeks to test the hypothesis that the sequence selectivity of an RNA-binding compound is directly related to its kinetic off rate or residence time in the desired binding site. While a generally accepted principle in the realm of protein and enzyme recognition, and tested also in the context of DNA recognition, to our knowledge this concept has not been applied to compounds binding RNA. This hypothesis will be tested via three Aims. First, well-validated (but low- throughput) techniques will be used to analyze the binding properties of a series of known RNA-targeted compounds. Second, a new analytical methodology developed in our laboratory termed Arrayed Imaging Reflectometry will be tested in the context of multiplex (high-throughput) assessment of RNA-binding kinetic constants. This will also involve the development of new statistical methods for the analysis of time-dependent array data. Third, we will examine the effect of systematic functional group modification on the binding kinetics and sequence selectivity of a novel compound discovered in our lab that targets a viral RNA critical to the HIV life cycle. Completion of the proposed research will provide a new paradigm for RNA-targeted molecular design based on consideration of binding kinetics, as well as a new analytical tool for high-throughput characterization of RNA binding, and new lead compounds targeting HIV.

Abstract: The human C-C chemokine receptor type 5 (CCR5) is a validated therapeutic target in HIV, and an important emerging target in a host of additional indications including Chagas disease, cancer, and liver and kidney fibrosis. While the FDA-approved drug Marviroc has proven effective as an allosteric inhibitor of CCR5-mediated HIV entry, resistance to Maraviroc is a known complication. An attractive alternative strategy to allosteric inhibition is to ablate production of CCR5. It has recently been discovered that translation of CCR5 is subject to a process known as a -1 programmed ribosomal frameshift, or -1 PRF. In this process, interaction of the ribosome with a structured portion of the CCR5 mRNA causes it to shift backwards 1 nucleotide. This results in production of a non-functional protein, and triggers degradation of CCR5 mRNA via nonsense-mediated decay. We hypothesize that compounds able to specifically bind to and stabilize this structured portion of the CCR5 mRNA, termed a frameshift- stimulatory sequence, or FSS, will act as frameshift enhancers and constitute a new approach to inhibiting CCR5 production. Our hypothesis is grounded on our success with using an innovative library screening strategy, termed Resin-Bound Dynamic Combinatorial Chemistry (RBDCC) to identify compounds able to selectively bind an FSS RNA responsible for frameshifting in HIV. Medicinal chemistry efforts have resulted in the production of compounds with nanomolar affinity, high selectivity, and antiviral activity in human cells. We will apply an analogous approach to the identification of compounds binding the CCR5 FSS RNA, and will develop a new target- competitive method for RBDCC screening. Libraries will incorporate substituted diketopiperazines (a privileged substructure in medicinal chemistry), thus addressing an urgent need for new RNA-binding chemotypes. After prioritizing hit compounds based on biophysical measurements, including assessment of binding to off-target RNA sequences, a panel of cellular assays will be employed to assess the ability of CCR5 FSS-targeting compounds to reduce production of the receptor, and inhibit HIV infectivity. Completion of this research will provide the first compounds able to influence a human frameshifting event, and proof-of-concept for a novel CCR5 RNA-targeted therapeutic strategy.

Acute and chronic tendon injuries are among the most common musculoskeletal health problems. Typically, an injured tendon experiences fibrotic scarring that leaves the tissue mechanically compromised and prone to debilitating adhesions that impair joint function. In a fibrotic tendon scar, the cell-cell and paracrine signaling between inflammatory cells, such as macrophages, and tendon fibroblasts activate the latter into fibroproliferative myofibroblasts, ultimately differentiating into a senescent phenotype. Our previous studies using next-generation sequencing and gene set enrichment analysis mechanistically linked fibrosis and senescence in injured mouse tendons with TGF-beta activated mTOR signaling. To further elucidate this pathology, the goal of this proposal is to engineer a microfluidic human tendon-on-chip (hToC) system and use it to more accurately model the biological aspects of the inflammation and fibrosis in injured tendons. In the UG3 phase of this proposal, the chip will be fabricated featuring a multicompartmental design and microfluidic channels to incorporate a fibroblast-seeded collagen hydrogel and simulate vascular blood flow, respectively. Ultrathin, highly permeable, and optically transparent porous silicon membranes (SiM) will separate the hydrogel from circulation and provide a substrate for an endothelial barrier in between. The signaling between the fibroblasts, hydrogel-resident- and circulating-macrophages, and endothelial cells will be enabled through nanoporous SiM (~60 nm), while a microporous SiM (~ 8 µm) will allow extravasation of circulating macrophages and infiltration of the hydrogel under TGF-beta stimulation. To allow for a patient-centric chip, tendon fibroblasts will be used to create the tendon hydrogel and to reprogram donor-matching iPSCs to derive the endothelial cells and macrophages, respectively. An additional innovation will be the integration of label- free photonic sensors into the microfluidic device to allow on-chip sensing, which has been long appreciated as a critical, unmet need for organ-on-chip devices. The UG3 studies will use the chip to validate the role of mTOR in the disease model and identify biologically relevant biomarkers. In the UH3 phase, we will utilize the chip as a pre-clinical trial platform for testing efficacy and safety of FDA-approved mTOR inhibitors as potential disease modifying drugs, and as a drug screening platform to identify and prioritize safer and more potent inhibitors of mTOR and senescence in tendon injury for clinical trials. To successfully complete this innovative project, we have assembled a team of accomplished experts in tendon tissue engineering and surgery, immunology, iPSC technology, GMP cell manufacturing, nano- and micro-fabrication, sensor technology, and high throughput screening. The proposed studies will develop a human microphysiological system to catalyze clinical trials and accelerate drug discovery for acute and chronic tendon injuries.

Abstract The continued advancement of microphysiological systems (MPS) as pre-clinical research tools is vital to overcome the low throughput and inaccuracies inherent in animal models of human disease. The limitations of pre-clinical animal models, most commonly mice, are particularly apparent in inflammatory diseases which are known to have distinct genetic and cytokine responses to inflammation. The establishment of MPS alternatives, however, will require scientific consensus on the protocols and systems best suited to address particular diseases. As the current MPS era is characterized by a proliferation of approaches, the Microphysiological System Data Base (MPS-db) created by the University of Pittsburgh is a valuable tool to hasten the development of MPS standards. Because the success of the MPS-db requires the active participation by MPS developers and users, we seek supplemental funding to contribute the designs, protocols and results for an MPS system that models the interplay between inflammation and fibrosis in tendon healing (UG3TR00287). Importantly, the injury and repair of connective tissue injury is not represented in the current MPS-db but accounts for more than 8.5 million clinical procedures annually, including 2 million major surgeries. Our human tendon-on-a-chip (hToC) model focuses on the early inflammatory stages of tendon repair, where timely interventions may promote scarless healing. The hToC features vascular and collagen compartments which exchange soluble and cellular factors in a simulation of the neovascularized microenvironment established shortly after blood clotting. Monocyte infiltration is hypothesized to play an essential role in the generation of contractile myofibroblasts which progress to senescence and release monocyte activating factors in a positive feedback loop that causes scar tissue. The model uses iPSCs derived from primary human tenocytes to create vascular endothelial cells and monocytes in an isogenic, patient-centric triculture. With supplemental funding we will share: 1) descriptions of the mechanisms of the tendon injury and fibroinflammatory repair process; 2) design details for the hToC including device components and modules for both flow and integrated photonic- based sensing; 3) cell culture and device protocols including phenotypic characteristics and operational parameters such as flow rates for priming of ECs and the introduction of immune cells; 4) The design and rationale for studies under baseline and inflammation/repair conditions; and 5) Results including an analysis of intra-study reproducibility.