This month we are highlighting two awards from NIH: Dr. Ekaterina Heldwein for her project ‘Biophysical and structural analysis of the herpesviral nuclear budding machinery’ and Dr. James Van Deventer for his project “The yeast surface as a platform for inhibitor discovery.’ Please see the abstracts for these proposals below.
PI: Ekaterina Heldwein
Title: Biophysical and structural analysis of the herpesviral nuclear budding machinery
Abstract: Herpesviruses are double-stranded-DNA enveloped viruses that are among the most complex viruses infecting animals. This proposal focuses on nuclear egress, a critical, conserved step in the assembly and release of progeny virions during which nucleocapsids are translocated from the nucleus into the cytoplasm where they mature into infectious virions. The viral nuclear egress complex (NEC) is the key player in this process. Using in vitro model systems, we previously discovered that the NEC is a complete, virally encoded membrane budding machine that operates at the nuclear envelope. However, a major barrier to understanding nuclear egress is the lack of knowledge of how the NEC generates membrane curvature that results in budding. The long-term goal of this research is to elucidate the detailed mechanism of herpesvirus nuclear egress, both to gain a fundamental knowledge of this unusual process and to identify and characterize novel targets for antiviral therapeutic design. This proposal is driven by the central hypothesis, based on substantial preliminary data, that both NEC/membrane interactions and NEC oligomerization into a coat are the major driving forces that enable negative membrane curvature formation and budding. The objective of this proposal is to systematically dissect the NEC budding mechanism in Herpes Simples virus (HSV) by characterizing essential protein/protein and protein/membrane interactions and budding intermediates by employing a multidisciplinary approach, which includes the cutting-edge approaches of cryoelectron microscopy and electron spin resonance. The scientific premise of the proposed work is that a comprehensive dissection of the NEC-mediated formation of negative membrane curvature is essential for unraveling the unusual mechanism of herpesviral nuclear egress and developing strategies to block it. Beyond viruses, this study will expand our limited mechanistic understanding of the mechanisms of membrane deformation in general. The proposal is innovative because it investigates an unusual mechanism, is guided by an original hypothesis, and employs novel approaches. The proposal is significant because it aims to advance our mechanistic understanding of an essential step in viral replication cycle with the goal of identifying new targets for therapeutic interventions and because it provides an opportunity to develop models of negative curvature formation, currently a black box.
PI: James Van Deventer
Title: The yeast surface as a platform for inhibitor discovery
Abstract: Advances in screening technologies have made ligand discovery against biological targets routine, but converting binding ligands into specific enzyme inhibitors is extremely challenging, even for metalloproteinases and other enzymes with well-defined active sites. The lack of specific inhibitors prevents full elucidation of biological processes as basic as extracellular matrix remodeling. Proteins and small molecules each lack key features of inhibitors. Antibodies and other proteins rarely disrupt enzyme function, but usually exhibit high binding specificity. Small molecules frequently lack single-enzyme specificity, but interfere with enzymatic activity. Neither of these modalities is well-suited for generating potent, specific enzyme inhibitors. My long-term goals are to 1) establish general principles for discovering potent, specific inhibitors against medically relevant enzymes; and 2) utilize the resulting inhibitors to understand the roles of enzymes such as metalloproteinases in normal physiology and pathological processes. I hypothesize that simultaneously leveraging the complementary strengths of proteins and small molecules will give rise to entirely new classes of potent, specific inhibitors. The goal during this proposal period is to convert yeast display, a powerful ligand discovery platform, into a comprehensive inhibitor discovery platform. My lab has already established strategies for expanding the chemical functionality that can be utilized in combination with yeast display. Here, we will enhance our platform further and use it to identify inhibitors against a test set of metalloproteinase targets. In the process, we will gain fundamental insights into how to generate inhibitors that are not accessible using any current inhibitor discovery approaches, setting the stage for 1) a greatly expanded toolkit for studying basic biology; and 2) much broader inhibitor discovery efforts. The initial directions we will pursue are: Direction 1. Expand the range of chemistries that can be encoded in yeast-displayed proteins. Proteins containing canonical amino acids lack key groups found in enzyme inhibitors. We will utilize our quantitative reporter of ncAA incorporation to encode these functionalities in yeast-displayed proteins. Direction 2. Establish assays for quantitatively evaluating enzyme inhibition on the yeast surface. No existing display technologies support quantitative evaluations of enzyme inhibition during high throughput screening. We will utilize dual yeast display technology to establish these capabilities. Direction 3. Use chemically augmented antibody libraries to evolve potent, specific inhibitors. Antibodies rarely inhibit enzymes. We will generate and screen libraries of antibodies containing added chemical groups to establish general principles for inhibitor isolation in this unexplored discovery space. To focus our discovery efforts, we and our collaborators have identified metalloproteinases from multiple families that play important roles in human health and disease. The general discovery principles we establish here will lead directly to new classes of inhibitors for understanding and treating human disease.