Prof. Christina M. Woo - Associate Professor in the Department of Chemistry and Chemical Biology at Harvard University
Christina M. Woo is an Associate Professor in the Department of Chemistry and Chemical Biology at Harvard University, and an affiliate member of the Broad Institute. Christina’s research focuses on the design of chemical approaches to alter post-translational modifications and the signaling outcomes they produce in cells. She obtained a BA in Chemistry from Wellesley College (2008). She obtained her PhD in 2013 from Yale University under the guidance of Professor Seth Herzon as an NSF predoctoral fellow in the synthetic and chemical biology studies of diazofluorene antitumor antibiotics. In 2013, Christina joined the laboratory of Professor Carolyn Bertozzi at the University of California Berkeley as a Jane Coffins Child postdoctoral fellow and continued at Stanford University (2015) as a Burroughs Wellcome Fund postdoctoral fellow, where she developed a mass-independent chemical glycoproteomics platform for the identification of non-templated post-translational modifications. Christina joined the faculty at Harvard University in 2016. Her research has been recognized by the David Gin Young Investigator Award, Camille-Dreyfus Teacher-Scholar Award, Sloan Research Foundation, NSF CAREER, Bayer Early Excellence in Science Award, the NIH DP1 Avenir Award, and the Ono Pharma Foundation Breakthrough Science Award.
Dafydd Owen, PhD - Director, Medicinal Chemistry at Pfizer
Dafydd Owen holds a Bachelor’s degree from Imperial College London which was followed by a PhD from Cambridge University under the supervision of Professor Steve Ley. Following a postdoc at Ohio State University he joined Pfizer in the UK at their Sandwich site in 1999. Since 2011 he has worked for Pfizer in Cambridge MA in an outward looking, collaborative group looking at novel protein families and drug targets. Most recently, he led the SARS-CoV-2 oral protease inhibitor discovery team. The resulting clinical candidate PF-7321332 is the subject of today’s talk.
Lei Zhang, PhD - Senior Director of Medicinal Chemistry at Pfizer
Lei Zhang received his B.S. in chemistry from Peking University and pursued graduate work at Stanford University under the supervision of Prof. Paul Wender. After receiving his Ph.D., Lei joined Pfizer laboratories in 2002. During his time at Pfizer, Lei has worked in multiple therapeutic areas and successfully delivered a number of clinical candidates for disorders with high unmet medical needs in the areas of CNS, immuno-oncology and metabolic diseases. He is currently a senior director of medicinal chemistry, leading the molecular design group of internal medicine medicinal chemistry, based at Cambridge, MA.
Student and Postdoctoral Speakers:
Kyan D’Angelo (he/him/his), Mo Movassaghi group, Massachusetts Institute of Technology
Synthesis and Study of Complex Molecule Antibiotics
This presentation will focus on the development and application of a new strategy for radical-based union of complex indole derivatives. Inspired by nature’s mechanistic blueprint for fragment assembly and complemented by our own detailed mechanistic analysis, our strategy enables the concise total synthesis of structurally complex antibiotics.
Mintesinot Kassu, Roman Manetsch group, Northeastern University
Shotgun Kinetic Target-Guided Synthesis Approach Enables the Discovery of Small Molecule Inhibitors against Pathogenic Amoeba Glucokinases
Fragment-based lead discovery approaches have garnered increasing attention in the last two decades. Kinetic Target-Guided Synthesis (KTGS) is a fragment-based lead discovery approach which enables the identification of promising protein inhibitors through template-mediated assembly of reactive small molecule fragments. This strategy involves the biological target participating in the irreversible assembly of its own inhibitory bidentate ligand from a pool of complementary reactive fragments. We previously demonstrated the suitability of in situ amidation reactions between thioacids and sulfonyl azides, known as sulfo-click reactions, for KTGS experiments involved in targeting Bcl-XL – a key regulator of cellular apoptosis. Despite being described in detail in the early 1980s, KTGS is a relatively unexplored technique with a limited number of reported biological targets screened using this approach. Furthermore, all reported KTGS examples commence with the identification of one or more anchor molecules with previously determined binding potentials via X-ray crystallography, or in silico-based methods. In order to investigate the potential of KTGS to identify potent binders against targets with unresolved structures, we attempted a shotgun KTGS approach using an unoptimized, randomly selected fragment library of 38 sulfonyl azides and 45 thioacids (1710 potential N-acylsulfonamide products) against the brain-eating amoeba protein, Naegleria fowleri glucokinase (NfGlck). This screen incorporated improvements made to our previous KTGS report against Bcl-XL including multi-combinatorial mixing of fragments (190 combinations per well), utilization of multiple-reaction monitoring (MRM) LC-MS/MS for unambiguous detection of hits, and in situ generation of thioacids from corresponding thioesters. 157 hits were detected, and subsequent cheminformatic analysis guided the decision to resynthesize 53 compounds, 12 of which displayed potent activity against NfGlck along with two other amoeba targets known to cause devastating CNS diseases. This study demonstrates the utility of KTGS in conjunction with ligand-based drug design methods to identify small molecule binders in biological systems where resolved X-ray crystal structures are not readily accessible.
Eleni Kisty (she/her), Eranthie Weerapana group, Boston College
Unraveling Cysteine Oxidation in the Mitochondria
Throughout eukaryotic evolution, the mitochondrion has been the primary energy generator for cellular function. Equally important, is its vital role as a redox producer, regulator, and reporter allowing the cell to assess its own health through reactive oxygen species. Mitochondrial reactive oxygen species (mtROS) are natural byproducts of cellular respiration and
are crucial for the maintenance of energy resources along with signaling fluctuations in metabolism or the cellular environment. These oxidative species can react with sulfylhydryls on cysteines resulting in oxidative post-translational modifications (PTMs) which are imperative for downstream signaling events. Low, basal levels of mtROS are important for cellular proliferation, differentiation, and metabolic regulation, whereas, higher levels are correlated with a profound array of pathologies such as cancer, type-II diabetes, neurodegenerative diseases, cardiovascular disorders, and senescence. When antioxidant systems become overwhelmed, direct damage to macromolecules can result in cell death. Although it is recognized that mtROS is necessary for cellular signaling and functions, the precise protein targets are predominantly unknown. This talk details methods in determining the delicate rapport between mitochondrial-generated oxidative stress and cysteine reactivity and oxidation by means of quantitative, activity-based protein profiling (isoTOP-ABPP) tandem mass spectrometry and isotope-encoded affinity tags (OxiCAT), respectively. Additionally, we explore novel proximity labeling platforms to analyze mitochondrial sub-compartmental cysteine oxidation and disulfide changes upon oxidative stress. Through treatments with hydrogen peroxide and Antimycin A, endogenous and exogenous sources of ROS, respectively, we disentangle recognized and novel modifications in mitochondrial protein cysteine reactivity and oxidation.
Carly Schissel (She/Her) , Bradley Pentelute group, Massachusetts Institute of Technology
Deep Learning to Design Nuclear-Targeting Abiotic Miniproteins
There are more amino acid permutations within a 40-residue sequence than atoms on Earth. This vast chemical search space hinders the use of human learning to design functional polymers. Here we show how machine learning enables the de novo design of abiotic nuclear-targeting miniproteins to traffic antisense oligomers to the nucleus of cells. We combined high-throughput experimentation with a directed evolution-inspired deep-learning approach in which the molecular structures of natural and unnatural residues are represented as topological fingerprints. The model is able to predict activities beyond the training dataset, and simultaneously deciphers and visualizes sequence–activity predictions. The predicted miniproteins, termed ‘Mach’, reach an average mass of 10 kDa, are more effective than any previously known variant in cells and can also deliver proteins into the cytosol. The Mach miniproteins are non-toxic and efficiently deliver antisense cargo in mice. These results demonstrate that deep learning can decipher design principles to generate highly active biomolecules that are unlikely to be discovered by empirical approaches.
Veronika Shoba, Amit Choudhary group, Broad Institute & Harvard Institutes of Medicine
Rational design of bifunctional molecules that alter the specificity of serine/threonine and tyrosine kinases
Bifunctional molecules that induce posttranslational modifications by forcing proximity between the enzyme and the target protein are providing fundamentally new therapeutic strategies to various maladies. In this presentation, the design principles behind the generation of phosphorylation-inducing chimeric small molecules (PHICS) for serine and threonine phosphorylation of target proteins by Protein Kinase C (PKC) will be discussed. PHICS based on PKC not only induced phosphorylation of non-substrate in cells but also altered the interaction interface between PKC and its natural substrate Bruton’s Tyrosine Kinase (BTK) to induce neo-phosphorylations that inhibit BTK’s activity. The development of PHICS for tyrosine phosphorylation on the target protein and its application for the activation of receptor tyrosine kinase signaling will also be discussed. These studies demonstrate that the chemical and functional diversity of proteins in cells can be further enriched with the help of kinase-engaging bifunctional compounds.
Kathleen Sicinski, Krishna Kumar group, Tufts University
Molecular Design of Secretin Peptides to Modulate Receptor Function
The gut-derived incretin hormone, glucagon-like peptide-1 (GLP-1), plays an important physiological role in attenuating post-prandial blood glucose excursions in part by amplifying pancreatic insulin secretion. Native GLP-1 is rapidly degraded by the serine protease, dipeptidyl peptidase-4 (DPP4); however, enzyme-resistant analogues of this 30-amino-acid peptide provide an effective therapy for type 2 diabetes (T2D) and can curb obesity via complementary functions in the brain. In addition to its medical relevance, the incretin system provides a fertile arena for exploring how to better separate agonist function at cognate receptors versus susceptibility of peptides to DPP4-induced degradation. We have discovered that novel chemical decorations can make GLP-1 and its analogues completely DPP4 resistant while fully preserving GLP-1 receptor activity. This strategy is also applicable to other therapeutic ligands, namely, glucose-dependent insulinotropic polypeptide (GIP), glucagon, and glucagon-like peptide-2 (GLP-2), targeting the secretin family of receptors. The versatility of the approach offers hundreds of active compounds based on any template that target these receptors. These observations should allow for rapid optimization of pharmacological properties and because the appendages are in a position crucial to receptor stimulation, they proffer the possibility of conferring “biased” signaling and in turn minimizing side effects.
Amanda Waterbury, Brian Liau group, Harvard University
Identifying scaffolding and allosteric functions of LSD1 in AML
Using a CRISPR-Cas9 high-density mutagenesis approach called CRISPR-suppressor scanning, we are investigating the role of lysine-specific histone demethylase 1 (LSD1) in AML. We demonstrate that this approach can identify mutations that confer drug resistance to small molecule LSD1 inhibitors. Many of these mutations both impair drug binding and disrupt LSD1 demethylase activity, suggesting that this activity is not required for AML cell survival. We further demonstrate that drug-mediated disruption of a LSD1-GFI1B complex is necessary and sufficient to block AML cell growth. Surprisingly, we also identified mutations enriched in the disordered N-terminus of LSD1, a region often truncated in biochemical and structural studies which therefore lacks functional characterization. Overall, our studies illustrate that CRISPR-suppressor scanning can identify functional hotspots beyond the small molecule binding site that provide critical information on molecular mechanism of action and protein allostery.
Jessie Zhen, Scott Schaus group, Boston University
Novel Reaction Discovery with Rapid HTS via Infrared Spectroscopy and Electrochemical Enzyme Catalysis
A functional high-throughput screening (HTS) system was created to investigate the Petasis Borono-mannich reaction space and to explore possible novel reactivities. Using this method, more than 3000 unique microscale reactions were processed in under 6 months with 74% accuracy. By triaging the IR results, time and resources were spent on analyzing and reproducing medium to high yielding reactions. This helped to identify previously unknown reactivities, allowing for reproduction of novel reactions at bench scales, as well as furthering mechanistic studies of uncommon reaction partners such as thiophenol and pyrone.
This preliminary success suggests that this platform has the potential to generate an enormous volume of empirical standardized data that not only could be used for reaction optimization, but also readily integrated into a near-AI computational system in the future for reaction model prediction and discovery. Since the disappearance of a reagent can be easily observed in the reaction IR, this current HTS process model could also be extended to other reactions such as the Suzuki cross-couplings and Buchwald-Hartwig aminations.
With the broadened knowledge of reagent selection and stereoselectivity of the catalyzed Petasis reactions, we examined the possibility of electrochemical enzymatic catalysis. Galactose oxidase is known to selectively eliminate the pro-S hydrogen during oxidation of the alcohol reagents due to the steric constraints of the active site. This mild, diastereoselective oxidation can provide the aldehyde component in the highly enantioselective, transition-metal-free Petasis reaction.