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Download Tool by Submitted in partial satisfaction of the requirements for degree of in in the GRADUATE DIVISION of the UNIVERSITY OF CALIFORNIA, SAN FRANCISCO Approved: ______________________________________________________________________________ Chair ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ Committee Members Copyright 2019 by Adolfo Cuesta ii Acknowledgements For me, completing a doctoral dissertation was a huge undertaking that was only possible with the support of many people along the way. First, I would like to thank my PhD advisor, Jack Taunton. He always gave me the space to pursue my own ideas and interests, while providing thoughtful guidance. Nearly every aspect of this project required a technique that was completely new to me. He trusted that I was up to the challenge, supported me throughout, helped me find outside resources when necessary. I remain impressed with his voracious appetite for the literature, and ability to recall some of the most subtle, yet most important details in a paper. Most of all, I am thankful that Jack has always been so generous with his time, both in person, and remotely. I’ve enjoyed our many conversations and hope that they will continue. I’d also like to thank my thesis committee, Kevan Shokat and David Agard for their valuable support, insight, and encouragement throughout this project. My lab mates in the Taunton lab made this such a pleasant experience, even on the days when things weren’t working well. I worked very closely with Tangpo Yang on the mass spectrometry aspects of this project. Xiaobo Wan taught me almost everything I know about protein crystallography. Thank you as well to Geoff Smith, Jordan Carelli, Pat Sharp, Yazmin Carassco, Keely Oltion, Nicole Wenzell, Haoyuan Wang, Steve Sethofer, and Shyam Krishnan, Shawn Ouyang and Qian Zhao. Sharing an office with this wonderful group of people has been a highlight of my graduate career. About half-way through this PhD project, it became clear that mass spectrometry was going to be a central component. As a result, I joined the Burlingame lab to learn everything I could about it. Thank you to Al Burlingame for letting me join the lab and giving me the opportunity to learn from the many talented people there. In particular, I would like to thank Giselle Knudsen, Robert Chalkley and Mark Burlingame who all volunteered to sit down with me and a few lab new lab members to teach us all the very basics about mass spectrometry. Thank iii you as well to Juan Oses-Prieto, Jason Maynard, Jim Wilkins, Mike Trnka, Anatoly Urisman who have been especially generous with their time and expertise. Many thanks to Nancy Phillips, Krista Kaasik and Shenheng Guan. From long before coming to UCSF, my family was instrumental in their unconditional support on this journey. Thank you to Adolfo Sr. and Edilma for their loving encouragement at every stage, and the many sacrifices they have made to ensure that I (and my siblings) had everything we needed. Thank you to Julian and Melina for their support, and the very often needed comic relief. Thanks as well to Jan, David and Simone, who have been endlessly generous and supportive over the last 5 years. I value our time together. A special thanks to Mollie. Her positive energy, devoted, caring, and loving demeanor have made this process so much easier. Her optimism and excitement on the good days, and cheerleading and support on the bad days helped keep me going. Thank you. iv Contributions Chapter 1 of this thesis is reproduced in its entirety from previously published work cited below: Cuesta, A. & Taunton, J. Lysine-Targeted Inhibitors and Chemoproteomic Probes. Annu. Rev. Biochem. 88, 1–17 (2019) v Development of lysine-reactive covalent inhibitors and chemoproteomic probes Adolfo Cuesta Abstract Covalent inhibitors have numerous applications as drugs, as tools for drug discovery, and as probes for chemical biology. This class of compounds depends on the availability of a suitably reactive nucleophile in the target protein of interest. Among the non-catalytic nucleophiles, cysteine is the most reactive at physiological pH, but also the least prevalent. Based on the analysis presented herein, ~80% of known binding sites in the human proteome lack a cysteine residue. In contrast, ~80% of known binding sites contain either a lysine or a tyrosine—nucleophilic residues that are much less reactive than cysteine at physiological pH. Novel methods of covalent inhibition are therefore necessary to target a larger proportion of the proteome. We mined the Protein Data Bank (PDB) for small molecules within 5 Å of a nucleophilic atom (the epsilon amine in lysine, the phenol hydroxyl in tyrosine or the side chain thiol in cysteine). From the resulting compendium of proteins and ligands, we started with PU-H71 as an affinity scaffold to develop arylsulfonyl fluoride-based inhibitors that target a non-catalytic, non-reactive, surface-exposed lysine residue adjacent to the ATP binding site of Hsp90. We used high-resolution X-ray crystallography to decipher the molecular basis by which constrained, chiral linkers enantioselectively increase the reactivity of sulfonyl fluoride probes toward Hsp90 Lys58, without affecting the reversible binding affinity or intrinsic chemical reactivity. The in vivo use of covalent inhibitors requires electrophiles with the proper balance of metabolic stability and reactivity for their targets. We used the salicylaldehyde group as a reversible covalent electrophile to develop probes that react with the catalytic lysine in kinases. Using these probes, in combination with mass spectrometry, we found that ~50% of the kinome vi can react with the probes under equilibrium labeling conditions, including 80 kinases from living mice treated with the probes. Despite this promiscuity, we discovered that these probes exhibit remarkable kinetic selectivity for a small subset of kinases. The salicylaldehyde probes exhibit an apparent dissociation half-life of greater than 6 hours from a handful of kinases. These are among the first lysine-reactive kinase probes to exhibit the requisite stability and reactivity for in vivo applications. These novel probes demonstrate how to achieve kinase selectivity, despite targeting the conserved, catalytically essential lysine residue. vii Table of Contents Chapter 1: Lysine-targeted inhibitors and chemoproteomic probes ......................................... 1 Chapter 2: Structural bioinformatic mapping of ligandable nucleophiles ............................... 21 Chapter 3: Ligand pre-organization drives enantioselective covalent targeting of a surface- exposed lysine ..................................................................................................................... 35 Chapter 4: Development and characterization of lysine-reactive aryl aldehyde kinase probes by mass spectrometry .......................................................................................................... 71 References ........................................................................................................................... 89 Appendix A: NMR spectra ................................................................................................. 104 Appendix B: Python scripts for data processing .................................................................. 131 1. AA_per_pocket.py ............................................................................................................. 131 2. has_good_K_v3.3_CSV_Output.py ................................................................................. 132 3. PP_LFQ_v5.py .................................................................................................................... 142 4. format_and_combine_protein_value-v2.py .................................................................... 146 5. batch_exp_fit_triplicates.py ............................................................................................. 150 Appendix C: Human proteins with cofactors ≤ 5Å from a Lys e-amine .................................. 152 viii List of figures Figure 1-1: Mining the PDB for lysines proximal to metabolite/cofactor binding sites. ...... 7 Figure 1-2: Proteome-wide determination of lysine reactivity and ligandability using the clickable probe sulfotetrafluorophenyl pentynoate (STP-alkyne) and quantitative mass spectrometry. .............................................................................................................................. 11 Figure 1-3: Lysine-targeted chemoproteomic probes for kinases and other ATP binding proteins. ....................................................................................................................................... 13 Figure 1-4: XO44, a broad-spectrum probe for quantifying kinase occupancy in living cells. ............................................................................................................................................. 15 Figure 1-5: Targeting lysine and N-terminal amines with ortho-substituted aldehydes. ... 17 Figure 2-1: Analysis of amino acids in Swiss-prot and the representative “pocketome” . 24 Figure 2-2: Drug-like molecules within 5 Å of nucleophilic residues in human proteins .. 27 Figure 2-3: Histograms of
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