Global profiling of phosphorylation-dependent changes in cysteine reactivity [@estherkemper1

Kemper, E.K., Zhang, Y., Dix, M.M. & Benjamin F. Cravatt.

Nat Methods, 2022

https://www.nature.com/articles/s41592-022-01398-2

Proteomics has revealed that the ~20,000 human genes engender a far greater number of proteins, or proteoforms, that are diversified in large part by post-translational modifications (PTMs). How such PTMs affect protein structure and function is an  active  area  of  research  but  remains  technically  challenging  to  assess  on  a  proteome-wide  scale.  Here,  we  describe  a  chemical proteomic method to quantitatively relate serine/threonine phosphorylation to changes in the reactivity of cysteine residues, a parameter that can affect the potential for cysteines to be post-translationally modified or engaged by covalent drugs. Leveraging the extensive high-stoichiometry phosphorylation occurring in mitotic cells, we discover numerous cysteines that exhibit phosphorylation-dependent changes in reactivity on diverse proteins enriched in cell cycle regulatory pathways. The discovery  of  bidirectional  changes  in  cysteine  reactivity often  occurring  in  proximity  to  serine/threonine  phosphorylation events points to the broad impact of phosphorylation on the chemical reactivity of proteins and the future potential to create small-molecule probes that differentially target proteoforms with PTMs.

Aryl Fluorosulfate-Based Inhibitors that Covalently Target the SIRT5 Lysine Deacylase [@ChristianAOlsen]

Bolding, J. E.; Martín-Gago, P.; Rajabi, N.; Gamon, L. F.; Hansen, T. N.; Davies, M. J.; Olsen, C. A. ChemRxiv 2022

https://doi.org/10.26434/chemrxiv-2022-zds91

The sirtuin enzymes are a family of lysine deacylases that regulate gene transcription and metabolism. Sirtuin 5 (SIRT5) hydrolyzes malonyl, succinyl, and glutaryl epsilon‐N‐carboxyacyllysine posttranslational modifications and has recently emerged as a vulnerability in certain cancers. However, chemical probes to illuminate its potential as a pharmacological target has been lacking. Here we report the harnessing of aryl fluorosulfate-based electrophiles as an avenue to furnish covalent inhibitors that target SIRT5. Alkyne-tagged affinity-labeling agents recognize and capture SIRT5 in cultured HEK293T cells and can label SIRT5 in the hearts of mice upon intravenous injection of the compound. This work demonstrates the utility of aryl fluorosulfate electrophiles for targeting of SIRT5 and suggests this as a means for the development of potential covalent drug candidates. It is our hope that these results will provide a key reference for future studies investigating SIRT5 and general sirtuin biology in the mitochondria.

Widespread occurrence of covalent lysine–cysteine redox switches in proteins

Rabe von Pappenheim, F., Wensien, M., Ye, J. et al. 

Nat Chem Biol (2022). https://doi.org/10.1038/s41589-021-00966-5

We recently reported the discovery of a lysine–cysteine redox switch in proteins with a covalent nitrogen–oxygen–sulfur (NOS) bridge. Here, a systematic survey of the whole protein structure database discloses that NOS bridges are ubiquitous redox switches in proteins of all domains of life and are found in diverse structural motifs and chemical variants. In several instances, lysines are observed in simultaneous linkage with two cysteines, forming a sulfur–oxygen–nitrogen–oxygen–sulfur (SONOS) bridge with a trivalent nitrogen, which constitutes an unusual native branching cross-link. In many proteins, the NOS switch contains a functionally essential lysine with direct roles in enzyme catalysis or binding of substrates, DNA or effectors, linking lysine chemistry and redox biology as a regulatory principle. NOS/SONOS switches are frequently found in proteins from human and plant pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and also in many human proteins with established roles in gene expression, redox signaling and homeostasis in physiological and pathophysiological conditions.

Electroaffinity Labeling: A New Platform for Chemoproteomic-based Target Identification

Yu  Kawamata†1,  Keun  Ah  Ryu†2,  Gary  N.  Hermann†1,  Alexander  Sandahl1,  Julien  C. Vantourout1,  Aleksandra  K.  Olow3,  La-Tonya  A.  Adams4,  Eva  Rivera-Chao1,  Lee  R.  Roberts2, Rob C. Oslund*2, Olugbeminiyi  O. Fadeyi*2,  Phil  S. Baran

chemrxiv, 2022

10.26434/chemrxiv-2022-s2m8f

Target  identification  is  a  critical  pillar  within  the  drug  discovery  process  that  involves deconvoluting  the  protein  target  of  a  pharmacologically  active  small  molecule  ligand.  While photoaffinity  labeling  strategies  have  become  the  benchmark  for  target  deconvolution  of  small molecules  owing  to  their  reliance  on  external  activation  to  induce  covalent  protein  capture,  the process  of  target  identification  remains  one  of  the  most  technically  challenging  aspects  of  early drug  discovery.  Thus,  there  is  a  strong  demand  for  new  technologies  that  allow  for  controlled activation  of  chemical  probes  to  covalently  label  their  protein  target.  Here,  we  introduce  an electroaffinity  labeling  (ECAL)  platform  which  leverages  the  use  of  a  small,  redox-active diazetidinone  (DZE)  functional  group  to  enable  chemoproteomic-based  target  identification  of pharmacophores  within live  cell  environments. 

Profiling Sulfur(VI) Fluorides as Reactive Functionalities for Chemical Biology Tools and Expansion of the Ligandable Proteome

Katharine Gilbert, Aini Vuorinen, Arron Aatkar Peter Pogány, Jonathan Pettinger, Joanna M. Kirkpatrick, Katrin Rittinger∥, David House, Glenn A. Burley, Jacob T. Bush

chemrxiv, 2022

10.26434/chemrxiv-2022-j8b8d

Chemical probes are valuable tools to explore the function of proteins. Incorporation of electrophiles into small molecules enables covalent capture of protein interactions and provides access to powerful technologies including chemo-proteomic profiling and reactive fragment screening. Current approaches have been largely limited to protein pockets con-taining cysteine, so establishing strategies to target other amino acid residues is essential to expanding the applicability across the proteome. Here, we profiled sulfur(VI) fluorides (SVI-F) as reactive functionalities that can modify multiple residues in-cluding Lys, Tyr, His and Ser, thus offering utility for targeting almost any protein. These studies provided an in-depth under-standing of SVI-F functionalities, including hydrolytic stability, protein reactivity and utility in chemoproteomics. Such insights offer a valuable guide for the prospective design of SVI-F-containing ligands for various chemical biology workflows and illus-trate the wide range of proteins that SVI-Fs can capture, thus highlighting the opportunity for SVI-Fs to expand the liganded proteome.

Umpolung strategies for the functionalization of peptides and proteins

Andrew M. White,   Isabella R. Palombi,  and  Lara R. Malins 

Chemical Science, 2022

https://pubs.rsc.org/en/content/articlelanding/2022/sc/d1sc06133j

Umpolung strategies, defined as synthetic approaches which reverse commonly accepted reactivity patterns, are broadly recognized as enabling tools for small molecule synthesis and catalysis. However, methods which exploit this logic for peptide and protein functionalizations are comparatively rare, with the overwhelming majority of existing bioconjugation approaches relying on the well-established reactivity profiles of a handful of amino acids. This perspective serves to highlight a small but growing body of recent work that masterfully capitalizes on the concept of polarity reversal for the selective modification of proteinogenic functionalities. Current applications of umpolung chemistry in organic synthesis and chemical biology as well as the vast potential for further innovations in peptide and protein modification will be discussed.

Selective inhibitors of JAK1 targeting a subtype-restricted allosteric cysteine

Madeline E Kavanagh, Benjamin D Horning, Roli Khattri, Nilotpal Roy, Justine P Lu, Landon R Whitby, Jaclyn C Brannon, Albert Parker, Joel M Chick, Christie L Eissler, Ashley Wong, Joe L Rodriguez, Socorro Rodiles, Kim Masuda, John R Teijaro, Gabriel M Simon, Matthew P Patricelli, Benjamin F Cravatt

biorxiv https://doi.org/10.1101/2022.01.31.478302

The  JAK  family  of  non-receptor tyrosine  kinases  includes  four subtypes  (JAK1, JAK2, JAK3, and  TYK2)  and  is  responsible  for  signal  transduction  downstream  of  diverse  cytokine  receptors. JAK  inhibitors  have  emerged  as  important  therapies  for  immuno(onc)ological  disorders, but their use  is  limited  by  undesirable  side  effects  presumed  to  arise  from  poor  subtype  selectivity,  a common  challenge  for  inhibitors  targeting  the  ATP-binding  pocket  of  kinases.  Here,  we  describe the  chemical  proteomic  discovery  of a  druggable  allosteric  cysteine  present  in  the  non-catalytic pseudokinase  domain  of  JAK1  (C817)  and  TYK2  (C838),  but  absent  from  JAK2  or  JAK3. Electrophilic  compounds  selectively  engaging  this  site  block  JAK1-dependent transphosphorylation  and  cytokine  signaling, while  appearing  to  act largely  as  “silent”  ligands  for TYK2.  Importantly, the  allosteric  JAK1  inhibitors  do  not  impair  JAK2-dependent  cytokine signaling  and  are  inactive  in  cells  expressing  a  C817A  JAK1  mutant.  Our  findings  thus  reveal  an allosteric  approach  for  inhibiting  JAK1  with  unprecedented  subtype  selectivity.