(7ak) Engineering Next-Gen Proteases As Therapeutics and As Tools in Biomedicine and Synthetic Biology

Genes encoding proteases comprise more than 2% of the human genome. Due to their substrate specificity, proteases fulfill complex biological functions resulting in immune system modulation through intracellular signaling and gene regulation. In the context of using proteases as therapeutic agents, taking advantage of such specificity to degrade target proteins has been an objective in biomedicine for several decades. There are 13 FDA-approved protease drugs used in the clinic, mainly for the treatment of cardiovascular diseases. However, this number will surely increase, as recent advances in protein engineering has made it increasingly possible to redesign the selectivity of proteases for the creation of new therapeutic proteases.

My current research is involved in the engineering of protease substrate specificity for the treatment of autoimmune diseases (ADs) and chronic inflammations (CIs), which affect 5 to 8% of the world population. Because ADs are idiopathic and protean in manifestations, with periodic flare-ups and remissions, treatment of these disorders is a lifelong burden requiring lifestyle changes, medical management and surgical interventions. A seminal advance has been the introduction of monoclonal antibody therapies, which target disease-associated proteins (i.e. cytokines) inside cells or in serum. However, antibody therapies are costly, lose efficacy over time, and lead to increased risks of opportunistic infections due to prolonged immune suppression. Unlike antibodies and small molecule inhibitors, which bind stoichiometrically to neutralize target proteins, proteases are particularly attractive since they are catalytic and can degrade multiple targets with similar sequences. However, to further expand the therapeutic use of proteases to treat ADs and CIs, many of their properties need to be improved, including: substrate specificity, mitigation of adverse side effects, and resistance to serum inhibitors. Throughout my graduate studies and postdoctoral career, I have amassed expertise in several fields, including protein, pathway and cellular engineering, chemical and biocatalysis. This toolkit provided me with the strong foundation for a successful academic career dedicated to (1) developing novel therapeutic proteases, enzyme replacement therapies and protease-activatable protein drugs to fight ADs, and other chronic inflammatory diseases and (2) creating novel tools for biomedical, analytical, point-of-care diagnostic applications, and synthetic biology.

In my graduate studies, I was part of the multidisciplinary and highly collaborative environment facilitated by the Center for Enabling New Technologies through Catalysis (CENTC), under the tutelage of Profs. John Hartwig, an organic chemist, and Huimin Zhao, a protein engineer and synthetic biologist. My graduate work extended across a variety of disciplines including organic chemistry, heterogeneous catalysis, biocatalysis, reaction and protein engineering. I developed chemo-enzymatic one-pot reactions that bring together the high productivity and substrate scope of man-made catalysts and the high selectivity of enzymes, in a fashion that capitalized on their undiscovered and underutilized synergy and cooperativity. For example, I combined a ruthenium-catalyzed metathesis reaction with a bio-epoxidation catalyzed by engineered P450s. As a result, I obtained stereo- and regioselective epoxides of defined lengths from a pool of alkenes of different lengths and functional groups.

At the center of my graduate work were my ability to effectively communicate and share ideas with researchers in disparate fields of science and look at problems from different points of view. I participated in several other projects, including translational control of gene expression in mammalian cells and transcriptional and translational manipulations of biosynthetic pathways in S. cerevisiae and bacteria. Through these experiences, I developed in-depth analytical, experimental, and problem-solving abilities and firmly enriched my synthetic biology background while developing crucial collaborations which gave me unique insights for my independent career.

For my postdoctoral work, I joined the protease engineering lab of Prof. Brent Iverson where I am combining directed evolution and deep sequencing (Next-Gen) to evolve proteases with enhanced specificity and activity to target ADs. I improved a state-of-the-art multipurpose in vivo platform that enables both high-throughput protease engineering and substrate specificity profiling. I used this platform to fine-tune the substrate specificity and activity of promiscuous human proteases towards the selective cleavage of tumor necrosis factor α (TNF-α) and immunoglobulins, therapeutic targets in inflammatory bowel disease (IBD) and systemic lupus erythematosus (SLE), respectively. In the near future, we will evaluate our engineered variants in ex vivo and in vivo models of IBD and SLE. Using Next-Gen sequencing, I am analyzing the “degradome” of engineered proteases, as well as other disease-associated proteases to investigate their pathology, discover novel subtle patterns of reactivity, which will provide new insights into the design of more specific peptide inhibitors, fluorogenic peptidic substrates and protease-activatable prodrugs. To further advance our engineering capabilities, I developed an in vitro compartmentalization-based directed evolution platform for the engineering and substrate profiling of other amino acid-modifying enzymes, including formylglycine generating enzymes and sortases. These enzymes have important biomedical applications, in particular in biorthogonal chemistry; however, their full potential has not yet been extensively explored. I will build on these discoveries and tools in my independent career, not only to evolve these proteins, but to also study enzyme mechanisms in the context of diseases and enzyme replacement therapies (ERTs).

Future directions:

My own research laboratory will be develop a highly collaborative and diverse research program whose primary missions will be to:

(1) Evolve human therapeutic proteases for the treatment of ADs, CI, synucleopathies, and other protease-deficiency disorders, such as pancreatic insufficiency, ushering a new wave of mono or combination therapies with existing antibody and small molecule drugs. Specifically, we will engineer proteases to target α-synuclein, whose aggregation correlates with prion-like disorders such as familial Parkinson’s disease and multiple system atrophy. To treat ADs and CI, we will design protease drugs that target not only the pleiotropic TNF-α but importantly, downstream inflammatory cytokines such as IL-6 and IL-13, which are more specific to certain CI diagnostics and less immunosuppressive. Building on my exposure to immunological engineering, we will utilize proteomics, metabolomics and deep sequencing to study the impact and the mechanism of our engineered therapeutics in animal models. With this core mission, we hope to build a vertically integrated research program that take protease drugs from the bench to the clinic.

(2) Evolve proteins for analytical and biomedical applications, disease diagnostics and synthetic biology. My ability to engineer proteases with orthogonal substrate specificity will enable the development of novel tools for highly specific and orthogonal cell and protein labeling, mass spectrometry (MS)- based shotgun proteomics, antibody-drug conjugate design, disease prognostic markers and point-of-care diagnostics. We will design multi-level and multi-input activatable prodrugs which emulate biological reaction cascades for efficient drug targeting of inflammation and cancer, as well as design highly sensitive activatable reporters for point-of-care diagnostics.

Teaching Interests:

I knew I wanted to pursue an academic career on a spring day in 9th grade, when I was asked to teach our physics class to my classmates in the teacher’s absence. I felt very proud after teaching that class both from my own performance and the reception from my peers and friends. In unofficial roles throughout high school, I offered one-on-one tutoring in subjects ranging from Philosophy, English to Natural Sciences. As an undergraduate, I further honed my teaching skills by becoming a certified tutor. In particular, I helped a struggling minority older student earn an A- in General Chemistry. This experience reinforced in me that effective teaching necessitated a hybrid approach, where teaching styles needed to match the student’s learning style.

During my graduate studies, I was a teaching assistant (TA) for three chemical engineering classes. In Biomolecular Engineering, in particular, I designed homework and quiz questions, formulated exam questions and gave guest lectures. I learned how to effectively prepare class material. As a TA, I found that asking students to explain back to me how they understood the material was a valuable teaching tool. As a postdoctoral fellow, I am still sharpening my teaching skills by attending a class on evidence-based teaching.

I am prepared to teach all the core chemical and bimolecular engineering classes and looking forward to applying at least five effective teaching methods to which I myself have been exposed. First, I will engage students in problem-based learning, and will illustrate textbook problems using real world examples. Second, I will encourage peer-teaching through the formation of study groups, in and outside of class. Third, I will guide students to learning how to study, to understand concepts in an organized and layered manner. Fourth, I will ask students to evaluate my teaching, not at the end of the semester, but in the middle of the semester, especially if it’s a class I am new at teaching. That way, I can quickly adjust my teaching methods. Lastly, in the hope of making class material accessible and engaging to all, I will utilize various media to disseminate the class material, including blog posts and short videos on the course website.

Mentorship

I have been fortunate to mentor five undergraduate and exchange students, as well as three graduate students. It has always pleased me to hear from them, the great things they have gone to accomplish and the awards they have won. During graduate studies, I learned various management skills that I will build on. First, encouraging students to read literature and understand their field and the larger implications of their work is critical to their success. My mentees will lead literature review sessions, and will be asked to write a review paper early in their graduate career. Second, to further improve communication skills, graduate students and post-docs will be encouraged to apply for fellowships, participate in grant writing, and attend conferences yearly. Third, while my door will always be open to my mentees, I will hold a formal weekly meeting to discuss their progress, and I will ask students and postdocs to turn in a yearly review of self-assessment, advisor evaluation and goal-setting.