Tuesday, March 30, 2021

The role of reversible and irreversible covalent chemistry in targeted protein degradation [@hannahjanekc, @gbernardes_chem]

Hannah Kiely-Collins, Georg E. Winter, Gonçalo J.L. Bernardes

Cell Chemical Biology, 2021


Proteolysis-targeting chimeras (PROTACs) that degrade disease-causing proteins by hijacking the endogenous ubiquitin-proteasome system have emerged as an exciting and transformative technology in both chemical biology and drug discovery. Currently, the majority of PROTACs use reversible non-covalent ligands for both the target protein of interest (POI) and E3 ligase. In this review, we explore the burgeoning role of reversible and irreversible covalent chemistry in targeted protein degradation. We highlight the key advantages of targeted covalent inhibitors, whether as the target POI or E3 ligase ligand, such as their ability to enhance the selectivity of PROTACs, enable access to more of the “undruggable” proteome and expand the repertoire of recruited E3 ligases.

Thursday, March 25, 2021

Tunable Methacrylamides for Covalent Ligand Directed Release Chemistry

Rambabu N. Reddi, Efrat Resnick, Adi Rogel, Boddu Venkateswara Rao, Ronen Gabizon, Kim Goldenberg, Neta Gurwicz, Daniel Zaidman, Alexander Plotnikov, Haim Barr, Ziv Shulman, and Nir London

Journal of the American Chemical Society 2021

DOI: 10.1021/jacs.0c10644

Targeted covalent inhibitors are an important class of drugs and chemical probes. However, relatively few electrophiles meet the criteria for successful covalent inhibitor design. Here we describe α-substituted methacrylamides as a new class of electrophiles suitable for targeted covalent inhibitors. While typically α-substitutions inactivate acrylamides, we show that hetero α-substituted methacrylamides have higher thiol reactivity and undergo a conjugated addition–elimination reaction ultimately releasing the substituent. Their reactivity toward thiols is tunable and correlates with the pKa/pKb of the leaving group. In the context of the BTK inhibitor ibrutinib, these electrophiles showed lower intrinsic thiol reactivity than the unsubstituted ibrutinib acrylamide. This translated to comparable potency in protein labeling, in vitro kinase assays, and functional cellular assays, with improved selectivity. The conjugate addition–elimination reaction upon covalent binding to their target cysteine allows functionalizing α-substituted methacrylamides as turn-on probes. To demonstrate this, we prepared covalent ligand directed release (CoLDR) turn-on fluorescent probes for BTK, EGFR, and K-RasG12C. We further demonstrate a BTK CoLDR chemiluminescent probe that enabled a high-throughput screen for BTK inhibitors. Altogether we show that α-substituted methacrylamides represent a new and versatile addition to the toolbox of targeted covalent inhibitor design.

Friday, March 19, 2021

Reimagining Druggability Using Chemoproteomic Platforms [@DanNomura]

Jessica N. Spradlin, Erika Zhang, and Daniel K. Nomura

Accounts of Chemical Research 2021

DOI: 10.1021/acs.accounts.1c00065

One of the biggest bottlenecks in modern drug discovery efforts is in tackling the undruggable proteome. Currently, over 85% of the proteome is still considered undruggable because most proteins lack well-defined binding pockets that can be functionally targeted with small molecules. Tackling the undruggable proteome necessitates innovative approaches for ligand discovery against undruggable proteins as well as the development of new therapeutic modalities to functionally manipulate proteins of interest. Chemoproteomic platforms, in particular activity-based protein profiling (ABPP), have arisen to tackle the undruggable proteome by using reactivity-based chemical probes and advanced quantitative mass spectrometry-based proteomic approaches to enable the discovery of “ligandable hotspots” or proteome-wide sites that can be targeted with small-molecule ligands. These sites can subsequently be pharmacologically targeted with covalent ligands to rapidly discover functional or nonfunctional binders against therapeutic proteins of interest. Chemoproteomic approaches have also revealed unique insights into ligandability such as the discovery of unique allosteric sites or intrinsically disordered regions of proteins that can be pharmacologically and selectively targeted for biological modulation and therapeutic benefit. Chemoproteomic platforms have also expanded the scope of emerging therapeutic modalities for targeted protein degradation and proteolysis-targeting chimeras (PROTACs) through the discovery of several new covalent E3 ligase recruiters. Looking into the future, chemoproteomic approaches will unquestionably have a major impact in further expansion of existing efforts toward proteome-wide ligandability mapping, targeted ligand discovery efforts against high-value undruggable therapeutic targets, further expansion of the scope of targeted protein degradation platforms, the discovery of new molecular glue scaffolds that enable unique modulation of protein function, and perhaps most excitingly the development of next-generation small-molecule induced-proximity-based therapeutic modalities that go beyond degradation. Exciting days lie ahead in this field as chemical biology becomes an increasingly major driver in drug discovery, and chemoproteomic approaches are sure to be a mainstay in developing next-generation therapeutics.

Enhanced Suppression of a Protein–Protein Interaction in Cells Using Small-Molecule Covalent Inhibitors Based on an N-Acyl-N-alkyl Sulfonamide Warhead [@AJWilsonGroup]

Tsuyoshi Ueda, Tomonori Tamura, Masaharu Kawano, Keiya Shiono, Fruzsina Hobor, Andrew J. Wilson, and Itaru Hamachi

Journal of the American Chemical Society 2021

DOI: 10.1021/jacs.1c00703

Protein–protein interactions (PPIs) intimately govern various biological processes and disease states and therefore have been identified as attractive therapeutic targets for small-molecule drug discovery. However, the development of highly potent inhibitors for PPIs has proven to be extremely challenging with limited clinical success stories. Herein, we report irreversible inhibitors of the human double minute 2 (HDM2)/p53 PPI, which employ a reactive N-acyl-N-alkyl sulfonamide (NASA) group as a warhead. Mass-based analysis successfully revealed the kinetics of covalent inhibition and the modification sites on HDM2 to be the N-terminal α-amine and Tyr67, both rarely seen in traditional covalent inhibitors. Finally, we demonstrated prolonged p53-pathway activation and more effective induction of the p53-mediated cell death in comparison to a noncovalent inhibitor. This study highlights the potential of the NASA warhead as a versatile electrophile for the covalent inhibition of PPIs and opens new avenues for the rational design of potent covalent PPI inhibitors.

Concise Asymmetric Syntheses of Streptazone A and Abikoviromycin

Gustav J. Wørmer  Dr. Nikolaj L. Villadsen  Dr. Peter Nørby  Prof. Dr. Thomas B. Poulsen

Angew. Chem. Int. Ed. 2021


Streptazone A and abikoviromycin are alkaloids that both feature an unusual arrangement of reactive functionalities within a compact tricyclic ring system. Here, we report a highly concise asymmetric synthesis of both natural products. The route first constructs another family member, streptazone B1, using a rhodium‐catalyzed distal selective allene‐ynamide Pauson–Khand reaction. A regio‐ and enantioselective epoxidation under chiral phase‐transfer catalytic conditions directly afforded streptazone A in 8 steps overall. In one additional step, a chemoselective, iridium‐catalyzed reduction of the enaminone system then gave abikoviromycin. The reactivity of streptazone A towards a cysteine mimic, N‐acetylcysteamine, was studied and revealed unanticipated transformations, including bis‐thiol conjugation which may proceed via formation of a cyclopentadienone intermediate. With flexible access to these compounds, studies aimed to identify their direct biological targets are now possible.

Tuesday, March 16, 2021

Covalent and non-covalent binding free energy calculations for peptidomimetic inhibitors of SARS-CoV-2 main protease

Ernest Awoonor-Williams,  Abd Al-Aziz A. Abu-Saleh

Phys. Chem. Chem. Phys., 2021


COVID-19, the disease caused by the newly discovered coronavirus—SARS-CoV-2, has created a global health, social, and economic crisis. As of mid-January 2021, there are over 90 million confirmed cases and more than 2 million reported deaths due to COVID-19. Currently, there are very limited therapeutics for the treatment or prevention of COVID-19. For this reason, it is important to find drug targets that will lead to the development of safe and effective therapeutics against the disease. The main protease (Mpro) of the virus is an attractive target for the development of effective antiviral therapeutics because it is required for proteolytic cleavage of viral polyproteins. Furthermore, the Mpro has no human homologues, so drugs designed to bind to this target directly have less risk for off-target effects. Recently, several high-resolution crystallographic structures of the Mpro in complex with inhibitors have been determined—to guide drug development and to spur efforts in structure-based drug design. One of the primary objectives of modern structure-based drug design is the accurate prediction of receptor–ligand binding affinities for rational drug design and discovery. Here, we perform rigorous alchemical absolute binding free energy calculations and QM/MM calculations to give insight into the total binding energy of two recently crystallized inhibitors of SARS-CoV-2 Mpro, namely, N3 and α-ketoamide 13b. The total binding energy consists of both covalent and non-covalent binding components since both compounds are covalent inhibitors of the Mpro. Our results indicate that the covalent and non-covalent binding free energy contributions of both inhibitors to the Mpro target differ significantly. The N3 inhibitor has more favourable non-covalent interactions, particularly hydrogen bonding, in the binding site of the Mpro than the α-ketoamide inhibitor. Also, the Gibbs energy of reaction for the Mpro–N3 covalent adduct is greater than the Gibbs reaction energy for the Mpro–α-ketoamide covalent adduct. These differences in the covalent and non-covalent binding free energy contributions for both inhibitors could be a plausible explanation for their in vitro differences in antiviral activity. Our findings are consistent with the reversible and irreversible character of both inhibitors as reported by experiment and highlight the importance of both covalent and non-covalent binding free energy contributions to the absolute binding affinity of a covalent inhibitor towards its target. This information could prove useful in the rational design, discovery, and evaluation of potent SARS-CoV-2 Mpro inhibitors for targeted antiviral therapy.

Monday, March 15, 2021

Chemical proteomic identification of functional cysteines with atypical electrophile reactivities

Kevin Litwin, Vincent M. Crowley, Radu M. Suciu, Dale L. Boger, Benjamin F. Cravatt

Tetrahedron Letters, 2021, 67, 152861


Cysteine-directed covalent ligands have emerged as a versatile category of chemical probes and drugs that leverage thiol nucleophilicity to form permanent adducts with proteins of interest. Understanding the scope of cysteines that can be targeted by covalent ligands, as well as the types of electrophiles that engage these residues, represent important challenges for fully realizing the potential of cysteine-directed chemical probe discovery. Although chemical proteomic strategies have begun to address these important questions, only a limited number of electrophilic chemotypes have been explored to date. Here, we describe a diverse set of candidate electrophiles appended to a common core 6-methoxy-1,2,3,4-tetrahydroquinoline fragment and evaluate their global cysteine reactivity profiles in human cancer cell proteomes. This work uncovered atypical reactivity patterns for a discrete set of cysteines, including residues involved in enzymatic catalysis and located in proximity to protein–protein interactions. These findings thus point to potentially preferred electrophilic groups for site-selectively targeting functional cysteines in the human proteome.


Wednesday, March 10, 2021

Profiling the Proteome-Wide Selectivity of Diverse Electrophiles

Patrick R. A. Zanon, Fengchao Yu, Patricia Musacchio, Lisa Lewald, Michael Zollo, Kristina Krauskopf, Dario Mrdović, Patrick Raunft, Thomas E. Maher, Marko Cigler, Christopher Chang, Kathrin Lang, F. Dean Toste, Alexey I. Nesvizhskii, Stephan M. Hacker

ChemRxiv, 2021


Targeted covalent inhibitors are powerful entities in drug discovery, but their application has so far mainly been limited to addressing cysteine residues. The development of cysteine-directed covalent inhibitors has largely profited from determining their proteome-wide selectivity using competitive residue-specific proteomics. Several probes have recently been described to monitor other amino acids using this technology and many more electrophiles exist to modify proteins. Nevertheless, a direct, proteome‑wide comparison of the selectivity of diverse probes is still entirely missing. Here, we developed a completely unbiased workflow to analyse electrophile selectivity proteome‑wide and applied it to directly compare 54 alkyne probes containing diverse reactive groups. In this way, we verified and newly identified probes to monitor a total of nine different amino acids as well as the N‑terminus proteome‑wide. This selection includes the first probes to globally monitor tryptophans, histidines and arginines as well as novel tailored probes for methionines, aspartates and glutamates.

Tuesday, March 9, 2021

Covalent Occlusion of the RORγt Ligand Binding Pocket Allows Unambiguous Targeting of an Allosteric Site

Femke A. Meijer, Maxime C. M. van den Oetelaar, Richard G. Doveston, Ella N. R. Sampers, and Luc Brunsveld

ACS Medicinal Chemistry Letters 2021
DOI: 10.1021/acsmedchemlett.1c00029

The nuclear receptor RORγt is a key positive regulator in the differentiation and proliferation of T helper 17 (Th17) cells and the production of proinflammatory cytokines like IL-17a. Dysregulation of this pathway can result in the development of various autoimmune diseases, and inhibition of RORγt with small molecules thus holds great potential as a therapeutic strategy. RORγt has a unique allosteric ligand binding site in the ligand binding domain, which is distinct from the canonical, orthosteric binding site. Allosteric modulation of RORγt shows high potential, but the targeted discovery of novel allosteric ligands is highly challenging via currently available methods. Here, we introduce covalent, orthosteric chemical probes for RORγt that occlude the binding of canonical, orthosteric ligands but still allow allosteric ligand binding. Ultimately, these probes could be used to underpin screening approaches for the unambiguous and rapid identification of novel allosteric RORγt ligands.

Saturday, March 6, 2021

5-Hydroxy-pyrrolone based building blocks as maleimide alternatives for protein bioconjugation and single-site multi-functionalization

Ewout De Geyter,   Eirini Antonatou,   Dimitris Kalaitzakis,   Sabina Smolen,  Abhishek Iyer,  Laure Tack,   Emiel Ongenae,  Georgios Vassilikogiannakis and  Annemieke Madder

Chem. Sci., 2021


Recent dramatic expansion in potential uses of protein conjugates has fueled the development of a wide range of protein modification methods; however, the desirable single-site multi-functionalization of proteins has remained a particularly intransigent challenge. Herein, we present the application of 5-hydroxy-1,5-dihydro-2H-pyrrol-2-ones (5HP2Os) as advantageous alternatives to widely used maleimides for the chemo- and site-selective labeling of cysteine residues within proteins. A variety of 5HP2O building blocks have been synthesized using a one-pot photooxidation reaction starting from simple and readily accessible furans and using visible light and oxygen. These novel reagents display excellent cysteine selectivity and also yield thiol conjugates with superior stability. 5HP2O building blocks offer a unique opportunity to introduce multiple new functionalities into a protein at a single site and in a single step, thus, significantly enhancing the resultant conjugate's properties.


Thursday, March 4, 2021

Sulfur [18F]Fluoride Exchange Click Chemistry Enabled Ultrafast Late-Stage Radiosynthesis


Qinheng Zheng, Hongtao Xu, Hua Wang, Wen-Ge Han Du, Nan Wang, Huan Xiong, Yuang Gu, Louis Noodleman, K. Barry Sharpless, Guang Yang, and Peng Wu
Journal of the American Chemical Society 2021

DOI: 10.1021/jacs.0c09306

The lack of efficient [18F]fluorination processes and target-specific organofluorine chemotypes remains the major challenge of fluorine-18 positron emission tomography (PET). We report here an ultrafast isotopic exchange method for the radiosynthesis of novel PET agent aryl [18F]fluorosulfate enabled by the emerging sulfur fluoride exchange (SuFEx) click chemistry. The method has been applied to the fully automated 18F-radiolabeling of 25 structurally and functionally diverse aryl fluorosulfates with excellent radiochemical yield (83–100%, median 98%) and high molar activity (280 GBq μmol–1) at room temperature in 30 s. The purification of radiotracers requires no time-consuming HPLC but rather a simple cartridge filtration. We further demonstrate the imaging application of a rationally designed poly(ADP-ribose) polymerase 1 (PARP1)-targeting aryl [18F]fluorosulfate by probing subcutaneous tumors in vivo.

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...