(4dt) Discovering Enzyme Allosteric Sites to Inform Drug Design

Developmental disorders present a large medical burden in the United States, affecting 1 in 6 children. Genetic mutations are the underlying cause of many developmental disorders and are increasingly detectable due to falling sequencing costs. In my graduate and postdoctoral work, I used synthetic biology, protein engineering, developmental biology, and genomics techniques to classify genetic variants of unknown significance found in developmental disorders and cancer at scale during early embryonic development. Extending this work, my research program will focus on using early embryonic development to find protective mutations and allosteric sites in enzymes that play important roles in biological signaling pathways. By identifying new allosteric sites in enzymes, my research aims to ultimately inform design of new therapeutics for individuals with developmental disorders. My proposed work will inform our understanding of enzyme function, expand our knowledge of how genetic mutations affect development, and guide the diagnosis and treatment of disease.

Research Experience:

I received my Ph.D. from the Princeton University Chemical and Biological Engineering department under the guidance of Prof. Stas Shvartsman and Prof. Becky Burdine, where I used techniques from synthetic biology, protein engineering, and developmental biology to understand how mutations in components of the Ras signaling pathway cause RASopathies, a class of developmental disorders that affect about 1/1000 human births. One challenge in managing RASopathies has been predicting how severe developmental abnormalities will be based on the mutation found. In order to show that different pathogenic mutations within the enzyme MEK1 had different severities, I transiently overexpressed all known MEK1 RASopathy variants in zebrafish and measured the oval shape of the embryo during gastrulation, which provided a readout of excessive Ras signaling. We showed that mutations found in RASopathies are less severe than mutations found in both RASopathies and cancer, which are in turn less strong than mutations found only in cancer, a ranking that we showed also carries over to heart defects and drug dosage for phenotype reversal.

As an American Heart Association postdoctoral fellow and Hartwell foundation postdoctoral fellow with Prof. Emma Farley at UC San Diego, I used techniques from genomics, synthetic biology, and developmental biology to study gain-of-function mutations in enhancers, which are genomic elements that act as switches to turn on gene expression in certain cell types of a developing organism. The binding affinities of transcription factor binding sites (TFBS) in enhancers play a key role in ensuring precise gene expression during development. I investigated what types of genetic variation within TFBSs can alter gene expression and development within the heart. I used the marine chordate Ciona robusta to study the consequence of manipulating TFBS affinities within the enhancer for FoxF, a heart-specific gene critical for migration of heart cells to the ventral midline and for formation of the heart. I found that the FoxF heart enhancer contains five incredibly low-affinity ETS binding sites that are each necessary for specific expression in the heart progenitor cells. I also found single nucleotide variants that significantly increase the affinity of ETS TFBSs greater than 3-fold and cause expression of FoxF within non-heart cells. In embryos harboring these optimizing mutations within the FoxF enhancer, non-heart cells migrate to the ventral midline along with the normal heart cells, leading to severely deformed, multi-chambered, or even extra hearts. In addition, I significantly improved an existing method to make a library of putative enhancers with corresponding transcribable barcodes that can be used to detect enhancer activity, which represents the first whole embryo massively parallel reporter assay using synthesized enhancer elements.

Selected Publications (*co-first, #co-corresponding):

Lim F*, Solvason JJ*, Ryan GE*, Le SH, Jindal GA, Steffen P, Jandu SK, Farley EK. Affinity-optimizing enhancer variants disrupt development. Nature, 2024

Jindal GA, Bantle AT, Solvason JJ, Grudzien JL, D’Antonio-Chronowska A, Lim F, Le SH, Song BP, Ragsac MF, Klie A, Larsen RO, Frazer KA, Farley EK. Single nucleotide variants within heart enhancers increase binding affinity and disrupt heart development. Developmental Cell, 2023

Jindal GA*, Goyal Y*, Humphreys JM, Yeung E, Tian K, Patterson VL, He H, Burdine RD, Goldsmith EJ#, Shvartsman SY#. How activating mutations affect MEK1 regulation and function. Journal of Biological Chemistry, 2017.

Goyal Y*, Jindal GA*, Pelliccia JL, Yamaya K, Yeung E, Futran AS, Burdine RD, Schüpbach T, Shvartsman SY. Divergent effects of intrinsically active MEK variants on developmental Ras signaling. Nature Genetics, 2017

Jindal GA*, Goyal Y*, Yamaya K, Futran AS, Kountouridis I, Balgobin CA, Schüpbach T, Burdine RD, Shvartsman SY. In vivo severity ranking of Ras pathway mutations associated with developmental disorders. PNAS, 2017

Selected Awards:

Schulman Award for Cardiovascular Research (2022)

Hartwell Postdoctoral Fellowship (2020 - 2021)

American Heart Association Postdoctoral Fellowship (2019 - 2020)

UC San Diego Chancellor’s Research Excellence Scholarship (2018 - 2019)

National Science Foundation Graduate Research Fellowship (2013 - 2017)

Amgen Scholars Research Fellowship (2011)

Teaching Interests and Experience:

Based on my undergraduate coursework in bioengineering and graduate coursework in chemical engineering, I am qualified to teach core chemical engineering courses such as chemical reaction engineering, thermodynamics, and transport phenomena, as well as bioengineering-adjacent courses in biochemistry, cell biology, developmental biology, and evolutionary developmental biology. I have served as an Assistant in Instruction for Introduction to Chemical and Biochemical Engineering Principles during my Ph.D. at Princeton University and also served as the Instructor of Record for an upper-level undergraduate class focusing on discussion of recent research papers at UC San Diego. As a faculty member, I am also interested in designing elective courses related to protein engineering, synthetic biology, signal transduction, gene regulation, and great experiments in biological engineering.

Outreach Interests and Experience:

I have been and remain very interested in sharing my interest and love for science with the next generation of scientists. As an undergraduate and graduate student, I tutored local high school students after school in math, which helped me appreciate the importance of broadening access to education resources. During my postdoc, I helped organize and participated in doing science demonstrations for local middle school students. I also volunteer as a mentor to undergraduate students at UC San Diego and meet regularly to build a relationship, foster a sense of belonging, and answer career questions. In my own lab, I want to continue outreach activities that I have been involved in as well as participate in additional new activities.