Monday, June 28, 2021

Site-selective tyrosine bioconjugation via photoredox catalysis for native-to-bioorthogonal protein transformation

Beryl X. Li, Daniel K. Kim, Steven Bloom, Richard Y.-C. Huang, Jennifer X. Qiao, William R. Ewing, Daniel G. Oblinsky, Gregory D. Scholes & David W. C. MacMillan

Nat. Chem. (2021). 


The growing prevalence of synthetically modified proteins in pharmaceuticals and materials has exposed the need for efficient strategies to enable chemical modifications with high site-selectivity. While genetic engineering can incorporate non-natural amino acids into recombinant proteins, regioselective chemical modification of wild-type proteins remains a challenge. Herein, we use photoredox catalysis to develop a site-selective tyrosine bioconjugation pathway that incorporates bioorthogonal formyl groups, which subsequently allows for the synthesis of structurally defined fluorescent conjugates from native proteins. A water-soluble photocatalyst, lumiflavin, has been shown to induce oxidative coupling between a previously unreported phenoxazine dialdehyde tag and a single tyrosine site, even in the presence of multiple tyrosyl side chains, through the formation of a covalent C–N bond. A variety of native proteins, including those with multiple tyrosines, can successfully undergo both tyrosine-specific and single-site-selective labelling. This technology directly introduces aldehyde moieties onto native proteins, enabling rapid product diversification using an array of well-established bioorthogonal functionalization protocols including the alkyne–azide click reaction.

An automatic pipeline for the design of irreversible derivatives identifies a potent SARS-CoV-2 Mpro inhibitor

Zaidman, Daniel; Gehrtz, Paul; Filep, Mihajlo; Fearon, Daren; Gabizon, Ronen; Douangamath, Alice; Prilusky, Jaime; Duberstein, Shirly; Cohen, Galit; Owen, C. David ; Resnick, Efrat; Strain-Damerell, Claire ; Lukacik, Petra ; Barr, Haim; Walsh, Martin A.; von Delft, Frank; London, Nir

Cell Chemical Biology, 2021


Designing covalent inhibitors is increasingly important, although it remains challenging. Here, we present covalentizer, a computational pipeline for identifying irreversible inhibitors based on structures of targets with non-covalent binders. Through covalent docking of tailored focused libraries, we identify candidates that can bind covalently to a nearby cysteine while preserving the interactions of the original molecule. We found ∼11,000 cysteines proximal to a ligand across 8,386 complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In a prospective evaluation, five out of nine predicted covalent kinase inhibitors showed half-maximal inhibitory concentration (IC50) values between 155 nM and 4.5 μM. Application against an existing SARS-CoV Mpro reversible inhibitor led to an acrylamide inhibitor series with low micromolar IC50 values against SARS-CoV-2 Mpro. The docking was validated by 12 co-crystal structures. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol.

Tuesday, June 22, 2021

Electrophilic Screening Platforms for Identifying Novel Covalent Ligands for E3 Ligases

Sijin Zheng and Craig M. Crews

Biochemistry 2021

Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules comprised of an E3 ligase recruiting molecule and the ligand for a protein of interest (POI) connected by a chemical linker (Figure 1). The first PROTAC utilized a 10-mer amino acid peptide to recruit Skp1-Cullin-F box protein β-TRCP, an E3 ligase complex, to methionine aminopeptidase-2, the POI, for ubiquitination and degradation.(1) In the ensuing years, the field has moved beyond peptidomimetics to small molecules. Currently, only a handful of E3 ligases are commonly recruited by PROTACs, namely Von-Hippel Lindau (VHL), cereblon (CRBN), MDM2, and IAPs. There are, however, >600 E3 ligases in the human proteome. The broad applicability of VHL- and CRBN-based PROTACs, coupled with the lack of tractable ligands for other E3 ligases, has resulted in an E3 ligase space that is underexplored. While current PROTACs have been shown to degrade a wide variety of biologically native and non-native E3 ligase substrates, ranging from traditionally druggable targets, such as kinases, to those previously considered “undruggable”, such as KRASG12C, there are substrates that have remained elusive, notably tubulin and c-Myc.

Thursday, June 17, 2021

Identification and validation of selective deubiquitinase inhibitors [@BuhrlageLab]

Anthony C. Varca, Dominick Casalena, Wai Cheung Chan, Bin Hu, Robert S. Magin, Rebekka M. Roberts, Xiaoxi Liu, He Zhu, Hyuk-Soo Seo, Sirano Dhe-Paganon, Jarrod A. Marto, Douglas Auld, Sara J. Buhrlage,

Cell Chemical Biology, 2021

Deubiquitinating enzymes (DUBs) are a class of isopeptidases that regulate ubiquitin dynamics through catalytic cleavage of ubiquitin from protein substrates and ubiquitin precursors. Despite growing interest in DUB biological function and potential as therapeutic targets, few selective small-molecule inhibitors and no approved drugs currently exist. To identify chemical scaffolds targeting specific DUBs and establish a broader framework for future inhibitor development across the gene family, we performed high-throughput screening of a chemically diverse small-molecule library against eight different DUBs, spanning three well-characterized DUB families. Promising hit compounds were validated in a series of counter-screens and orthogonal assays, as well as further assessed for selectivity across expanded panels of DUBs. Through these efforts, we have identified multiple highly selective DUB inhibitors and developed a roadmap for rapidly identifying and validating selective inhibitors of related enzymes.

Tuesday, June 8, 2021

Rapid and robust cysteine bioconjugation with vinylheteroarenes [@SpringGroupChem]

Hikaru Seki,   Stephen J. Walsh, Jonathan D. Bargh, Jeremy S. Parker,  Jason Carroll  and  David R. Spring 

Chem. Sci., 2021 

DOI: 10.1039/D1SC02722K 

Methods for residue-selective and stable modification of canonical amino acids enable the installation of distinct functionality which can aid in the interrogation of biological processes or the generation of new therapeutic modalities. Herein, we report an extensive investigation of reactivity and stability profiles for a series of vinylheteroarene motifs. Studies on small molecule and protein substrates identified an optimum vinylheteroarene scaffold for selective cysteine modification. Utilisation of this lead linker to modify a number of protein substrates with various functionalities, including the synthesis of a homogeneous, stable and biologically active antibody–drug conjugate (ADC) was then achieved. The reagent was also efficient in labelling proteome-wide cysteines in cell lysates. The efficiency and selectivity of these reagents as well as the stability of the products makes them suitable for the generation of biotherapeutics or studies in chemical biology.

Saturday, June 5, 2021

Viral ADP-ribosyltransferases attach RNA chains to host protein

Katharina H&oumlfer, Maik Schauerte, Julia Grawenhoff, Alexander Wulf, Luisa M. Welp, Franziska A. Billau, Henning Urlaub, Andres J&aumlschke

bioRxiv 2021.06.04.446905; 


he mechanisms by which viruses hijack their host's genetic machinery are of enormous current interest. One mechanism is adenosine diphosphate (ADP) ribosylation, where ADP-ribosyltransferases (ARTs) transfer an ADP-ribose fragment from the ubiquitous coenzyme nicotinamide adenine dinucleotide (NAD) to acceptor proteins. When bacteriophage T4 infects Escherichia coli, three different ARTs reprogram the host's transcriptional and translational apparatus. Recently, NAD was identified as a 5'-modification of cellular RNA molecules in bacteria and higher organisms. Here, we report that bacteriophage T4 ARTs accept not only NAD, but also NAD-RNA as substrate, thereby covalently linking entire RNA chains to acceptor proteins in an "RNAylation" reaction. One of these ARTs, ModB, efficiently RNAylates its host protein target, ribosomal protein S1, at arginine residues and strongly prefers NAD-RNA over NAD. Mutation of a single arginine at position 139 abolishes ADP-ribosylation and RNAylation. Overexpression of mammalian ADP-ribosylarginine hydrolase 1 (ARH1), which cleaves arginine-phosphoribose bonds, shows a decelerated lysis of E. coli when infected with T4. Our findings not only challenge the established views of the phage replication cycle, but also reveal a distinct biological role of NAD-RNA, namely activation of the RNA for enzymatic transfer. Our work exemplifies the first direct connection between RNA modification and post-translational protein modification. As ARTs play important roles in different viral infections, as well as in antiviral defence by the host, RNAylation may have far-reaching implications.

Friday, June 4, 2021

Controlling the Covalent Reactivity of a Kinase Inhibitor with Light

Reynders, M., Chaikuad, A., Berger, B., Bauer, K., Koch, P., Laufer, S., Knapp, S. and Trauner, D.

Angew. Chem. Int. Ed. 2021 

Covalent kinase inhibitors account for some of the most successful drugs that have recently entered the clinic and many others are in preclinical development. A common strategy is to target cysteines in the vicinity of the ATP binding site using an acrylamide electrophile. To increase the tissue selectivity of kinase inhibitors, it could be advantageous to control the reactivity of these electrophiles with light. Here, we introduce covalent inhibitors of the kinase JNK3 that function as photoswitchable affinity labels (PALs). Our lead compounds contain a diazocine photoswitch, are poor non-covalent inhibitors in the dark, and becomes effective covalent inhibitors after irradiation with visible light. Our proposed mode of action is supported by X-ray structures that explain why these compounds are unreactive in the dark and undergo proximity-based covalent attachment following exposure to light.

Tuesday, June 1, 2021

Functionalized Scout Fragments for Site-Specific Covalent Ligand Discovery and Optimization

Vincent M. Crowley, Marvin Thielert, and Benjamin F. Cravatt

ACS Central Science 2021 7 (4), 613-623

Covalent ligands are a versatile class of chemical probes and drugs that can target noncanonical sites on proteins and display differentiated pharmacodynamic properties. Chemical proteomic methods have been introduced that leverage electrophilic fragments to globally profile the covalent ligandability of nucleophilic residues, such as cysteine and lysine, in native biological systems. Further optimization of these initial ligandability events without resorting to the time-consuming process of individualized protein purification and functional assay development, however, presents a persistent technical challenge. Here, we show that broadly reactive electrophilic fragments, or “scouts”, can be converted into site-specific target engagement probes for screening small molecules against a wide array of proteins in convenient gel- and ELISA-based assay formats. We use these assays to expediently optimize a weak potency fragment hit into a sub-μM inhibitor that selectively engages an active-site cysteine in the retinaldehyde reductase AKR1B10. Our findings provide a road map to optimize covalent fragments into more advanced chemical probes without requiring protein purification or structural analysis.

Thiol Reactivity of N-Aryl α-Methylene-γ-lactams: Influence of the Guaianolide Structure [@KayBrummond]

 Daniel P. Dempe, Chong-Lei Ji, Peng Liu, and Kay M. Brummond The Journal of Organic Chemistry, 2020 DOI: 10.1021/acs.joc.2c01530 The α-meth...