Jeanne A. Hardy, Ph.D. - US grants

DESCRIPTION: (provided by applicant) Development of a novel bicombinatorial screen will allow isolation of small molecule compounds (about 180 daltons) that interact specifically with individual intracellular proteins. By screening a combinatorial library of compounds against a combinatorial library of protein targets the chance of discovering specific compound-protein pairs will be maximized. Typically a compound must interact with a protein very strongly in order to be specific. This screen will take advantage of tethering technology, which uses a secondary disulfide tether between the small molecule and a cysteine on the protein to stabilize interacting complexes. This allows specific binding even when the interaction energy is low, so the likelihood of finding interacting molecules is dramatically increased, and the compound library can be relatively small. The compounds can be rapidly converted to fluorescent tags to study protein localization, or to affinity compounds to isolate the protein they recognize alone and in complexes. These compounds will also be useful as novel lead compounds for drug discovery. In contrast to conventional drug screens which focus on a single protein target, the bicombinatorial screen should suggest both which chemical moities are the most effective in binding proteins, and which chemical constituents confer anti-specificity. It may also be able to suggest some unexploited families of proteins that would also be suitable as drug targets.

Sankaran Thayumanavan, Scott Auerbach, Jeanne Hardy and Dhandapani Venkataraman of the University of Massachusetts Amherst and Mark Johnson of Yale University are supported by an award from the Chemical Bonding Centers (CBC) Phase I program to carry out research addressing several fundamental aspects of proton transport, the molecular level process which underlies a number of important biological phenomena as well as the functioning of a central component in fuel cells. Proton transfer is a key aspect of a broad array of chemical problems, including acid-base reactions, energy storage and utilization, and catalysis. The center is focusing its investigation of the chemistry of proton conduction via site-to-site jumps and well-defined scaffolds. The main objectives are to (i) identify optimal functional groups and the underlying dynamics of facile proton transfer; and (ii) fabricate ideal scaffolds that produce stable and rapid proton conduction.

An important application area of this research is the design of better membranes for fuel cells. In keeping with this goal, the CBC will create a web-based entity, the National Chemical Energy Research Network, NCERN, that will (i) act as a dynamic interface among energy researchers and research centers; and (ii) foster better communication between chemists working on energy-related research and the general public. NCERN will facilitate the creation of wiki pages and will moderate interactive chats on these subjects. The center is having a broad impact both through the activities of NCERN as well as through its function as an interdisciplinary training center for graduate students and postdoctorals.

This Integrative Graduate Education and Research Traineeship (IGERT) award establishes a novel interdisciplinary training program at the University of Massachusetts-Amherst to address the emerging field of Cellular Engineering. Engineering cellular form and function is the basis for many ventures in the biomedical and biotechnology industries, including design of bioremediation processes, generation of artificial organs/tissues, production of biologics from cell culture, design of new and improved protein-based pharmaceuticals and targeted drug delivery. Students matriculate in one of 12 degree programs with a research focus in one of three interrelated cellular engineering thrust areas: 1) Applied Systems Biology, 2) Cell Delivery and 3) Protein Engineering. Key features include a novel unifying lecture/laboratory course to train both life scientists and engineers/physical scientists in cellular engineering fundamentals, interdisciplinary research involving "supergroup" projects in which students seek out collaboration with a related training laboratory; interactions with industry through the established UMass-Amherst Institute for Cellular Engineering; weekly research seminars with a mentoring component; and formal professional development activities.

This IGERT has all-female leadership and significant numbers of female faculty participants. Underrepresented students are recruited through the NEAGEP, an NSF-funded project co-led by UMass-Amherst and including ten research-extensive and six minority-serving institutions that collaborate to increase the number of underrepresented students who receive doctoral degrees in science, technology, engineering and mathematics disciplines. This IGERT encourages novel research collaborations in cellular engineering among faculty, creating new bridging programs among departments and providing unique learning opportunities for trainees. Purposeful alignment with the Institute for Cellular Engineering enables substantial interaction with regional cellular engineering companies, significantly broadening student training. IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.

Caspases are the cysteine proteases that control apoptotic cell death. If caspases can be activated, cancer cells die; conversely inhibiting caspases could prevent cell death in diseases like heart attack and stroke. Thus there has been significant interest in caspases as drug targets. This interest was heightened when caspase-6 was discovered to play a central role in neurodegenerative diseases. Unfortunately, to date, no caspase-directed therapies are on the market, primarily because work has focused on targeting the active site, which is the most overlapping and conserved region of the family. It is becoming increasingly clear that each caspase is regulated in a unique and complex manner so the most promising avenue for achieving caspase-specific inhibition may be by harnessing allosteric sites. In order to target a specific caspase or group of caspases allosterically, it is essential to understand the differences between individual caspases and the similarities within subgroups in the caspase family. Thus, the goal of this project is understand how phosphorylation and zinc contribute to regulation of caspase activity. Understanding the roles of phosphorylation or zinc binding alone provides critical information about natural regulatory processes for each member of the apoptotic caspases. Together these sites highlight key sensitive regions that allow strategic control of caspase function. Caspases are extensively phosphorylated. Most phosphorylation events lead to inactivation of caspase function. Our first approach uses methods we have developed for structural analysis of phosphomimetic and phosphorylated versions of caspases. These structures uncover the mechanism by which phosphorylation prevents caspase activity and also identify key regions of conformational control, which are functional allosteric sites. Second, various caspases can be inhibited by zinc, which has also been linked to apoptosis and Alzheimer's Disease. We are applying anomalous x-ray diffraction experiments to identify and characterize novel zinc-binding sites in caspases. Both of these approaches: phosphorylation and zinc-binding have helped us previously to identify new allosteric sites in caspases. By systematically applying these approaches, we can comprehensively map allosteric sites that are used across the caspase family as well as unique sites that are found only on one particular caspase. Our approaches are designed to provide the molecular details of allosteric control as well as assess the biological relevance of these mechanisms. The comparative map of caspase allostery by phosphorylation and zinc binding that we are generating will enable us to select the most appropriate regulatory sites for optimal control of caspase function and for effective treatment of diseases that involve caspases.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Our current work focuses on caspase-6 and -7. During this reporting period we worked on five main goals for understanding caspase-6 and -7. First, we have designed two new classes of active site inhibitors. We have crystals of caspase-6 with several of these active site inhibitors. Second, we have designed several mutations to probe the role of the structural changes in the 130's and 90's helix in caspase-6. Third, we have crystallized several versions of caspase-6 in a newly inactivated state. Together these structures will help us to understand and ultimately control caspase-6 in neurodegenerative diseases. Forth, we have grown crystals of caspase-7 with a number of novel active-site ligands. The aim of these studies is to understand the reaction mechanism in caspases. To date the molecular details of catalysis remain hazy because the only active site ligands that have been structurally characterized also distort the geometry around the catalytic diad. Fifth we have crystals of several new mutants of caspase-7 that are aimed at helping us to understand the interplay of caspase-7 active-site and allosteric-site regulation.

DESCRIPTION (provided by applicant): Caspase-6 is a critical factor in the development of several neurodegenerative disorders including Alzheimer's and Huntington's Diseases. Transgenic mice in which the caspase-6 cleavage sites in Amyloid Precursor Protein or Huntingtin Protein were mutated to prevent caspase-6 cleavage are protected from neurodegeneration, making caspase-6 an attractive target for treatment of Alzheimer's and Huntington's Diseases. Caspase-6 is also a member of the family of apoptotic proteins that control apoptotic cell death. To date it has been impossible to enumerate the specific roles caspase-6 plays in the cell that are related to or unique from other family members. This information is of central importance because blocking these additional roles could potentially lead to negative side-effects if caspase-6 were targeted in treatments for Huntington's and Alzheimer's long term. No caspase-specific probes exist because all available probes for use in cell or animal models function at the active sites of caspases and all caspases have very similar active sites. This screen makes use of a recently discovered allosteric site in caspase-6 that is not present in any other caspase. This allosteric site makes it possible to achieve caspase-6 specific inhibition for the very first time. The goal of this project is to develop a probe that specifically targets this unique allosteric site in caspase-6. A chemical probe that is specific an selective for caspase-6 will allow the native biological role of caspase-6 to be distinguished from other apoptotic caspases for the first time. This will allow validation of caspase-6 as a novel target for treatment of neurodegeneration. In addition to validating the allosteric site for drug discovery, the probes will also serve as lead compounds for discovery of novel drugs for neurodegeneration. This screen uses a robust screening protocol for identifying caspase-6 allosteric inhibitors at an allosteric site we have identified by crystallography and mutagenesis. This allosteric site is unique to caspase-6 and is not present in any other caspase. In the pilot screen, a hit rate of 2.9% with a cutoff of 30% inhibition was observed. The hit-rate can be adjusted to the desired level by using more stringent cutoffs. A Z' score of 0.8 was also observed, suggesting that caspase-6 is a tractable target for this type of screening. A panel of 61 hits was retested in the primary assay and subjected to two secondary assays. Two additional secondary assays have also been established and validated. This screen also utilizes a novel tertiary assay based on biophysical and spectroscopic observations. The allosterically inhibited conformation targeted in this screen exhibits a unique signature in the circular dichroism spectra. Thus using the circular dichroism assay provides mechanistic information about both the mode and location of probe binding. This assay is the first and only assay available that allows allosteric site inhibitors to be distinguished from active site inhibitors inany caspase. As this is the first screen enabling identification of caspase-6 selective compounds, our screen will allow discovery of probes to control this important protease.

1511367
Hardy, Jeanne A.

Various plants make many different chemicals that are of use to human beings in personal healthcare products, neutraceuticals, flavors and fragrances, oils and fats and other high-value foods and pharmaceutical ingredients. In this project it is proposed to increase productivity of plant cell cultures by eliminating the non-producing cells and by keeping the high-producing cells alive with the help of a mammalian protein. This cell-killing protein has a binding site that can be manipulated so that it binds a specific chemical. When that compound binds it inactivates the protein. In cells that make lots of the desired chemical, the cell-killing protein is turned off and cells remain alive. This system will first be developed for plant culture based production of Taxol, a chemotherapy drug. The approach is expected to be applicable to many valuable chemicals produced by plant cells.

Plant natural products represent a promising sector of rare and unique chemical entities impacting healthcare, energy, agriculture, food and nutraceuticals. Production and supply of desirable natural products is often challenging due to their chemical complexity, incompletely characterized biosynthetic pathways and prohibitive numbers of steps in their synthetic preparation. The synthesis of compounds with plant cell hosts often is suboptimal due to low production levels and cellular heterogeneity, with some cells producing high levels and other cells producing no product. In this project a completely novel approach has been designed that enables selection of high-producing cells from heterogeneous plant cultures. This approach relies on the finding that particular mammalian proteases sentence plant cells to death. A fluorescent reporter will be used to engineer an existing protease allosteric site to recognize and be inhibited by paclitaxel (Taxol). This protease that can be inhibited by Taxol, will selectively kill non-paclitaxel producing cells, while leaving productive cells intact. The approach likely will be useful not only for the production of Taxol but for a broad range of plant culture systems.

This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Cellular Dynamics and Function Program of the Division of Molecular and Cellular Biology.

? DESCRIPTION (provided by applicant): Scientists and engineers at the University of Massachusetts, Amherst (UMA) are using state-of-the-art tools and approaches in cellular engineering to address key challenges in biotechnology for human health, including production of anti-cancer drugs from cell cultures, transformation of mammalian cells into useful tissues for organ replacement, and design of novel proteins to serve as advanced therapies. UMA has a history of strength in biotechnology, founding the Institute for Cellular Engineering (ICE) as a platform to sustain and develop novel research and training programs. Given the successful track record of ICE training programs, geographic proximity to a leading biotech hub in Boston, and strong institutional support for development of industry-academic collaborations, UMA is ideally positioned to develop an impactful Biotechnology Training Program (BTP). BTP objectives are to: 1) create a scholarly and social infrastructure to facilitate new and strengthen existing interdisciplinary networks at UMA, particularly those at the interface of engineering and the life sciences; 2) educate students in the fundamentals of quantitative biotechnology through new lecture and laboratory courses; 3) train students to appreciate the impact of biotechnology commercially through a tailored industrial internship and regular interactions with industrial personnel; 4) provide students with opportunities to improve interdisciplinary communication, expand career opportunities, and sharpen professional skills; and 5) increase the number of students, particularly those from URM groups, who pursue careers in biotechnology. This BTP will recruit matriculated predoctoral students from five programs (Chemical Engineering, Chemistry, Polymer Science & Engineering, Veterinary & Animal Sciences, Molecular and Cellular Biology). We request six Trainee slots (matched with two slots from UMA). Traineeships will be awarded to students in their 2nd and 3rd years of study, and we will offer an Associate membership for non-funded students to broaden the impact of the program. Students will complete a prescribed curriculum over two years and obtain a graduate certificate in Cellular Engineering. Innovative features include the Biotechnology Core Course that is informed by and co-taught by industry personnel; specialized Laboratory Modules in biotechnology-relevant approaches and techniques modeled after professional industry workshops; student-run Biotechnology Journal Club to break down discipline barriers in a non-threatening atmosphere; a newly designed course in Biostatistics and Statistical Computing to promote math fluency for all Trainees; collaboration with the STEM Diversity Institute to ensure a diverse student population; an annual Symposium on Biotechnology that offers a unique Speed Dating experience where students pitch their ideas and receive feedback from industry experts; and targeted partnering with the new UMA Graduate School Office of Professional Development to provide career exploration and planning for our students. We have recruited numerous industrial partners to support a hallmark of our program, a formal internship in which all Trainees will participate.

Project Summary The past five years have been transformative for the Applied Life Sciences at the University of Massachusetts, Amherst (UMass). During this period a $95M investment in state-of-the art equipment housed in 30 core facilities operated by PhD-level center directors and over $400M in new research buildings has revolutionized the research capacity at our institution. This massive infrastructure growth has been matched with the hiring of over 50 new faculty in the life sciences. During this tremendous expansion we have developed the Biotech Training Program (BTP) in Applied Life Sciences, which leverages campus investments to provide outstanding training to a talented group of graduate students to prepare them for careers in the Biotech workforce and related areas. This training is guided by these objectives: 1) create a scholarly and social environment to facilitate new and strengthen existing interdisciplinary networks at UMass, particularly those at the interface of engineering and the life sciences; 2) educate students in the fundamentals of quantitative biotechnology through lecture and laboratory courses; 3) train students in the commercial impact of biotechnology through a tailored industrial internship and regular interactions with industrial personnel; 4) provide students with opportunities to improve interdisciplinary communication, expand career opportunities, and sharpen professional skills; and 5) increase the number of students, particularly those from underrepresented groups, who pursue careers in biotechnology. BTP faculty are recruited not by departmental affiliation, but by membership in the Institute for Applied Life Sciences (IALS), their research in biotechnology and their commitment to student training. For this reason, the UMass BTP recruits students from ten PhD programs: Biomedical Engineering, Chemical Engineering, Chemistry, Civil Engineering, Mechanical Engineering, Microbiology, Molecular & Cell Biology, Polymer Science & Engineering and Veterinary & Animal Sciences. We request ten Trainee slots (matched with three and a half slots annually from UMass). Traineeships are awarded to students for the 2nd and 3rd years of study during which students complete the BTP curriculum. Innovative features include the Frontiers in Biotechnology course that is co-taught by industry personnel; specialized Laboratory Modules in biotechnology-relevant techniques modeled after professional industry workshops; student-run Journal Club to break down discipline barriers; a new course in Quantitative Biology, Biostatistics & Data Science to promote modern data analysis fluency for all trainees; leadership in campus recruiting efforts for a diverse student population and accessibility to disabled students; an annual Fall Symposium in Biotechnology that offers a unique Biotech Battles experience where students solve real-world problems guided by industry experts; and targeted partnering with the UMass Office of Professional Development to provide career exploration and planning for our students. We have established numerous industrial partnerships to support the hallmark of our BTP, a formal internship in which all Trainees participate. Together this comprehensive training program prepares students well for a variety of careers in biotechnology.

PROJECT SUMMARY Caspases are cysteine proteases that control apoptotic cell death. If caspases are activated, cancer cells die; conversely, inhibiting caspases can prevent cell death in diseases such as heart attack and stroke. Thus, there has been significant interest in caspases as drug targets. This interest heightened further when caspase-6 was discovered to play a central role in neurodegeneration. Unfortunately, to date, no caspase- directed therapies are on the market, primarily because work has focused on the active site, which is the most overlapping and conserved region of the family. It is becoming increasingly clear that each caspase is regulated in a unique and nuanced manner, so the only hope for achieving caspase-specific inhibition is by harnessing their regulation, which is usually mediated at allosteric sites and exosites. In order to target a specific caspase, group of caspases, or subset of caspase substrates, it is essential to understand the dif- ferences between individual caspases and the similarities within caspase subgroups. Thus, our long-term project goal has been to define and exploit unique regulatory features for each of the apoptotic caspases. By identifying allosteric sites and exosites, we have observed and described four major mechanistic classes of exosite and allosteric regulation. The first, shared by many disparate regulators, is the indirect disruption of the loops that cooperatively form the substrate-binging groove and impact catalysis. Based on our studies of caspase regulation via loop disruption, we developed an allosteric inhibitor that is more potent than any reported and is also by far the most selective, preferring caspase-6 by 500-fold over all other caspases. This selectivity is achievable because this new allosteric site is present exclusively in caspase-6. Given this success, we aim to investigate the remaining three classes of allosteric regulation. In Aim 1, we focus on class II, identifying exosites on caspase-6 and its substrates. This concerted analysis is possible for the first time due to our development of a hybrid caspase with the active site specificity of caspase-6 but the exosites of caspase-7. We aim to block particular exosites and explore the impact on a proteome-wide basis. We anticipate that this approach will enable the development of new inhibitors that block cleavage of disease-causing caspase-6 substrates like DJ-1,Tau or huntingtin in Alzheimer and Huntington but not other substrates. In Aim 2, we focus on class III, native small molecule binding. Our recent discovery that ATP binds to an orphan allosteric cavity and new methods will allow us to identify both covalent and non- covalent native ligands that regulate caspase-6 from this site. This goal is significant as it will provide need- ed insights into the intersection between caspases and metabolism. In Aim 3, we focus on class IV, which impact the folded state. We interrogate a caspase-9 site that when phosphorylated leads to disassembly of the core. This is the only site of phosphorylation that is conserved among all human caspases. Together this work has significant therapeutic implications in both proliferative and neurodegenerative diseases.

The severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, is the cause of the novel coronavirus infectious disease 2019, COVID-19. During a key step in the viral lifecycle, infected human cells release not only virus particles but also enzymes that cut viral and host proteins, called proteases. Detection of the proteases linked to SARS-CoV-2 would provide an alternative path for testing for COVID-19. With this award, the Chemical Measurement and Imaging Program in the Division of Chemistry is supporting the research of Drs. Sankaran Thayumanavan, Jeanne Hardy, and Trisha L. Andrew at the University of Massachusetts, Amherst to develop "smart" sampling swabs whose color changes when the main protease of SARS-CoV-2 is present. The color change of the swab results when the main protease of SARS-CoV-2 breaks down a colorless, protein-derived molecule into individual pieces, with one of those pieces having its chemical properties changed during the break-down process, so that it absorbs or produces light that can be seen by the human eye. The investigators design and make the protein-derived probe molecules and then incorporate them into the fibers of currently-used clinical swabs. Their chemical design allows the probe molecules to be selectively broken down by the main protease of SARS-CoV-2 and to produce strong color in the smart swabs even when extremely low concentrations of the main protease are present. Creation of the necessary chemical reactions and their application to develop the protease-responsive smart swabs may lead to inexpensive testing methods that are practical for use by individuals at home. Due to the generality of the release of proteases in the lifecycle of viral infections, the new chemical reactions and resulting technologies are anticipated to be useful for visual detection of other viruses, which may be especially meaningful during future potential outbreaks. Educational, training, and outreach activities focus on graduate students working with Drs. Thayumanavan, Hardy, and Andrew in a cross-cutting, team-oriented research environment to address the development of front-line measurement science approaches, with anticipated highlights shared with the public and K-12 students about their experiences and lessons learned about how research funding can be used to directly impact a major societal crisis.

The goal of this project is to develop rapid, accurate, straightforward probe-based sensors that detect the inherent catalytic properties of viral protease enzymes, which produce a clear optical readout. Efforts are focused on creating new sensing chemistries that utilize specifically designed dye-based reporters and using them to develop inexpensive, disposable smart swabs to detect the presence of catalytically-active severe acute respiratory syndrome coronavirus 2 main protease (CoV2-MP). The general research approach Drs. Thayumanavan, Hardy, and Andrew take is to first generate protease recognition elements by designing, producing, and validating peptides that provide the optimal specificity to the targeted viral protease. The resulting protease recognition elements are then linked to donor-acceptor chromophores to create "pro-chromophoric" probes that remain in a dark (uncolored, discolored, or quenched) state. Upon exposure to the main viral protease, the free reporter is liberated and produces a highly intense colorimetric or fluorescence signal, with the estimated limits of detection offering sensing of sub-picomolar concentrations of CoV2-MP. Sophisticated reporter formulations with integrated chemical amplification routes are predicted to provide access to these limits of detection. Importantly, the protease-based detection strategy and swab test platform are easily and generally transferable for detecting potential future viral pathogens based on their specific protease structures.

This grant is being awarded using funds made available by the Coronavirus Aid, Relief, and Economic Security (CARES) Act supplemental funds allocated to MPS.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.