Genetic Incorporation of Olefin Cross-Metathesis Reaction Tags for Protein Modification

Bhaskar Bhushan, Yuya A. Lin, Martin Bak, Anuchit Phanumartwiwath, Nan Yang, Matthew K. Bilyard, Tomonari Tanaka, Kieran L. Hudson, Lukas Lercher, Monika Stegmann, Shabaz Mohammed, and Benjamin G. Davis

J. Am. Chem. Soc., 2018
DOI: 10.1021/jacs.8b09433

Olefin cross-metathesis (CM) is a viable reaction for the modification of alkene-containing proteins. Although allyl sulfide or selenide side-chain motifs in proteins can critically enhance the rate of CM reactions, no efficient method for their site-selective genetic incorporation into proteins has been reported to date. Here, through the systematic evaluation of olefin-bearing unnatural amino acids for their metabolic incorporation, we have discovered S-allylhomocysteine (Ahc) as a genetically encodable Met analogue that is not only processed by translational cellular machinery but also a privileged CM substrate residue in proteins. In this way, Ahc was used for efficient Met codon reassignment in a Met-auxotrophic strain of E. coli (B834 (DE3)) as well as metabolic labeling of protein in human cells and was reactive toward CM in several representative proteins. This expands the use of CM in the toolkit for “tag-and-modify” functionalization of proteins.

Celastrol binds to its target protein via specific noncovalent interactions and reversible covalent bond

Duo Zhang, Ziwen Chen, Caocao Hu, Siwei Yan, Zhuoer Li, Baohuan Lian, Yang Xu, Ding Rong, Zhiping Zeng, Xiao-kun Zhang and Ying Su

Chem. Commun., 2018
DOI: 10.1039/C8CC06140H

Celastrol is one of the most studied natural products. Our studies show for the first time that celastrol can bind to its target protein via specific noncovalent interactions, that position celastrol next to the thiol group of the reactive cysteine for reversible covalent bond formation. Such specific noncovalent interactions confer celastrol’s binding specificity and demonstrate the feasibility of improving the efficacy and selectivity of celastrol for therapeutic application.

Dimethyl fumarate is an allosteric covalent inhibitor of the p90 ribosomal S6 kinases

Jacob Lauwring Andersen, Borbala Gesser, Erik Daa Funder, Christine Juul Fælled Nielsen, Helle Gotfred-Rasmussen, Mads Kirchheiner Rasmussen, Rachel Toth, Kurt Vesterager Gothelf, J. Simon C. Arthur, Lars Iversen & Poul Nissen

Nature Communications 2018, 9, 4344

Dimethyl fumarate (DMF) has been applied for decades in the treatment of psoriasis and now also multiple sclerosis. However, the mechanism of action has remained obscure and involves high dose over long time of this small, reactive compound implicating many potential targets. Based on a 1.9 Å resolution crystal structure of the C-terminal kinase domain of the mouse p90 Ribosomal S6 Kinase 2 (RSK2) inhibited by DMF we describe a central binding site in RSKs and the closely related Mitogen and Stress-activated Kinases (MSKs). DMF reacts covalently as a Michael acceptor to a conserved cysteine residue in the αF-helix of RSK/MSKs. Binding of DMF prevents the activation loop of the kinase from engaging substrate, and stabilizes an auto-inhibitory αL-helix, thus pointing to an effective, allosteric mechanism of kinase inhibition. The biochemical and cell biological characteristics of DMF inhibition of RSK/MSKs are consistent with the clinical protocols of DMF treatment.

A metabolite-derived protein modification integrates glycolysis with KEAP1–NRF2 signalling

Michael J. Bollong, Gihoon Lee, John S. Coukos, Hwayoung Yun, Claudio Zambaldo, Jae Won Chang, Emily N. Chin, Insha Ahmad, Arnab K. Chatterjee, Luke L. Lairson, Peter G. Schultz & Raymond E. Moellering 

Nature (2018) DOI: 10.1038/s41586-018-0622-0

 that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3,4,5 and transcription6. An important ‘sensor’ protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1–NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases.

Reversible Covalent Reaction of Levosimendan with Cardiac Troponin C in Vitro and in Situ

Brittney A. Klein, Béla Reiz, Ian M. Robertson, Malcolm Irving, Liang Li, Yin-Biao Sun, and Brian D. Sykes

Biochemistry, 2018, 57 (15), pp 2256–2265

DOI: 10.1021/acs.biochem.8b00109

The development of calcium sensitizers for the treatment of systolic heart failure presents difficulties, including judging the optimal efficacy and the specificity to target cardiac muscle. The thin filament is an attractive target because cardiac troponin C (cTnC) is the site of calcium binding and the trigger for subsequent contraction. One widely studied calcium sensitizer is levosimendan. We have recently shown that when a covalent cTnC–levosimendan analogue is exchanged into cardiac muscle cells, they become constitutively active, demonstrating the potency of a covalent complex. We have also demonstrated that levosimendan reacts in vitro to form a reversible covalent thioimidate bond specifically with cysteine 84, unique to cTnC. In this study, we use mass spectrometry to show that the in vitro mechanism of action of levosimendan is consistent with an allosteric, reversible covalent inhibitor; to determine whether the presence of the cTnI switch peptide or changes in either Ca2+ concentration or pH modify the reaction kinetics; and to determine whether the reaction can occur with cTnC in situ in cardiac myofibrils. Using the derived kinetic rate constants, we predict the degree of covalently modified cTnC in vivo under the conditions studied. We observe that covalent bond formation would be highest under the acidotic conditions resulting from ischemia and discuss whether the predicted level could be sufficient to have therapeutic value. Irrespective of the in vivo mechanism of action for levosimendan, our results provide a rationale and basis for the development of reversible covalent drugs to target the failing heart.

A protein functionalization platform based on selective reactions at methionine residues

Michael T. Taylor, Jennifer E. Nelson, Marcos G. Suero & Matthew J. Gaunt

Nature, 2018, DOI: 10.1038/s41586-018-0608-y

Nature has a remarkable ability to carry out site-selective post-translational modification of proteins, therefore enabling a marked increase in their functional diversity1. Inspired by this, chemical tools have been developed for the synthetic manipulation of protein structure and function, and have become essential to the continued advancement of chemical biology, molecular biology and medicine. However, the number of chemical transformations that are suitable for effective protein functionalization is limited, because the stringent demands inherent to biological systems preclude the applicability of many potential processes2. These chemical transformations often need to be selective at a single site on a protein, proceed with very fast reaction rates, operate under biologically ambient conditions and should provide homogeneous products with near-perfect conversion2,3,4,5,6,7. Although many bioconjugation methods exist at cysteine, lysine and tyrosine, a method targeting a less-explored amino acid would considerably expand the protein functionalization toolbox. Here we report the development of a multifaceted approach to protein functionalization based on chemoselective labelling at methionine residues. By exploiting the electrophilic reactivity of a bespoke hypervalent iodine reagent, the S-Me group in the side chain of methionine can be targeted. The bioconjugation reaction is fast, selective, operates at low-micromolar concentrations and is complementary to existing bioconjugation strategies. Moreover, it produces a protein conjugate that is itself a high-energy intermediate with reactive properties and can serve as a platform for the development of secondary, visible-light-mediated bioorthogonal protein functionalization processes. The merger of these approaches provides a versatile platform for the development of distinct transformations that deliver information-rich protein conjugates directly from the native biomacromolecules.

Type II Kinase Inhibitors Targeting Cys-Gatekeeper Kinases Display Orthogonality with Wild Type and Ala/Gly-Gatekeeper Kinas

Cory A. Ocasio, Alexander A. Warkentin, Patrick J. McIntyre, Krister J. Barkovich, Clare Vesely, John Spencer, Kevan M. Shokat, and Richard Bayliss

ACS Chemical Biology, 2018,  Article ASAP

DOI: 10.1021/acschembio.8b00592

Analogue-sensitive (AS) kinases contain large to small mutations in the gatekeeper position rendering them susceptible to inhibition with bulky analogues of pyrazolopyrimidine-based Src kinase inhibitors (e.g., PP1). This “bump-hole” method has been utilized for at least 85 of ∼520 kinases, but many kinases are intolerant to this approach. To expand the scope of AS kinase technology, we designed type II kinase inhibitors, ASDO2/6 (analogue-sensitive “DFG-out” kinase inhibitors 2 and 6), that target the “DFG-out” conformation of Cys-gatekeeper kinases with submicromolar potency. We validated this system in vitro against Greatwall kinase (GWL), Aurora-A kinase, and cyclin-dependent kinase-1 and in cells using M110C-GWL-expressing mouse embryonic fibroblasts. These Cys-gatekeeper kinases were sensitive to ASDO2/6 inhibition but not AS kinase inhibitor 3MB-PP1 and vice versa. These compounds, with AS kinase inhibitors, have the potential to inhibit multiple AS kinases independently with applications in systems level and translational kinase research as well as the rational design of type II kinase inhibitors targeting endogenous kinases.

A road map for prioritizing warheads for cysteine targeting covalent inhibitors

Pé. Ábrányi-Balogh, Láó. Petri, Tí. Imre, Pé. Szijj, A. Scarpino, M. Hrast, A. Mitrović, Urš.Peč. Fonovič, K. Németh, Héè. Barreteau, D.I. Roper, K. Horváti, Gyö.G. Ferenczy, J. Kos, J. Ilaš, S. Gobec, Gyö.M. Keserű, A road map for prioritizing warheads for cysteine targeting covalent inhibitors, European Journal of Medicinal Chemistry, 2018, doi: 10.1016/j.ejmech.2018.10.010

Covalent inhibitors have become an integral part of a number of therapeutic protocols and are the subject of intense research. The mechanism of action of these compounds involves the formation of a covalent bond with protein nucleophiles, mostly cysteines. Given the abundance of cysteines in the proteome, the specificity of the covalent inhibitors is of utmost importance and requires careful optimization of the applied warheads. In most of the cysteine targeting covalent inhibitor programs the design strategy involves incorporating Michael acceptors into a ligand that is already known to bind non-covalently. In contrast, we suggest that the reactive warhead itself should be tailored to the reactivity of the specific cysteine being targeted, and we describe a strategy to achieve this goal. Here, we have extended and systematically explored the available organic chemistry toolbox and characterized a large number of warheads representing different chemistries. We demonstrate that in addition to the common Michael addition, there are other nucleophilic addition, addition-elimination, nucleophilic substitution and oxidation reactions suitable for specific covalent protein modification. Importantly, we reveal that warheads for these chemistries impact the reactivity and specificity of covalent fragments at both protein and proteome levels. By integrating surrogate reactivity and selectivity models and subsequent protein assays, we define a road map to help enable new or largely unexplored covalent chemistries for the optimization of cysteine targeting inhibitors.

Elucidating the catalytic power of glutamate racemase by investigating a series of covalent inhibitors

Nicholas Robert Vance  Katie R. Witkin  Patrick W. Rooney  Yalan Li  Marshall Pope Michael Ashley Spies

ChemMedChem 2018, doi: 10.1002/cmdc.201800592

The application of covalent inhibitors has experienced a renaissance within drug discovery programs in the last decade. To leverage the superior potency and drug target residence time of covalent inhibitors, there have been extensive efforts to develop highly specific covalent modifications to reduce off‐target liabilities. Herein, we present a series of covalent inhibitors of an antimicrobial drug target, glutamate racemase, discovered though structure‐based virtual screening. A combination of enzyme kinetics, mass spectrometry, and surface‐plasmon resonance experiments details a highly specific 1,4‐conjugate addition of a small molecule inhibitor with a catalytic cysteine of glutamate racemase. Molecular dynamics simulations and quantum mechanics‐molecular mechanics geometry optimizations reveal the chemistry of the conjugate addition. Two compounds from this series of inhibitors display antimicrobial potency comparable to β‐lactam antibiotics, with significant activity against methicillin‐resistant S. aureus strains. This study elucidates a detailed chemical rationale for covalent inhibition and provides a platform for the development of antimicrobials with a novel mechanism of action against a target in the cell wall biosynthesis pathway.