Covalent-Allosteric Akt Inhibitors: Targeting Akt3 through Molecular Hybridization and Expanding the Isoform-Selective Toolbox

Kosmas Alexandros Pervanidis § ; Giovanni Danilo D’Angelo § ; Ioannis Athanasiadis § ; Hannah Jantzen; Andrea Scrima; Katharina Schäfer; Lena Quambusch # ; Matthias P. Müller; Daniel Rauh *

ACS Chem. Biol. (2026) 21 (10): 2572–2596.

https://doi.org/10.1021/acschembio.6c00415

Akt is a central serine/threonine kinase in the PI3K/Akt/mTOR pathway that regulates cell survival, growth, and metabolic homeostasis. Although Akt1, Akt2, and Akt3 share high sequence homology, they perform distinct physiological and pathological functions, motivating the development of small-molecule isoform-selective chemical probes. Here, we first applied a molecular hybridization strategy to combine structural features that lead to Akt3-selectivity with the covalent pharmacophore of established covalent-allosteric Akt inhibitors, enabling selective and covalent targeting of Akt3. We then expanded the chemical space of covalent-allosteric Akt inhibitors to investigate how subtle structural differences influence selectivity across all three isoforms. Guided by structure–activity relationships and high-resolution co-crystal structures, systematic modifications of substituent patterns, linker geometry, and scaffold architecture revealed distinct isoform-selectivity profiles. Biochemical target inhibition, cellular target engagement, and covalent-binding studies further characterized these compounds and their selectivity. Collectively, these findings expand the covalent-allosteric Akt inhibitor toolbox, provide insight into the structural determinants governing Akt isoform selectivity, and establish molecular hybridization as a strategy for selective covalent targeting of Akt3.

Sub-stoichiometric degradation is dispensable for potent PROTACs as demonstrated by irreversible covalent BTK degraders

Ran Cheng, Hanfeng Lin, Xin Yu, Shrilekha Misra, Andrew D. Mitchell, Jennifer A. Woyach, Xiaoli Qi & Jin Wang

Nat Commun (2026). https://doi.org/10.1038/s41467-026-78239-9

Proteolysis-targeting chimeras (PROTACs) represent a transformative therapeutic modality, yet the viability of covalent PROTACs remains debated, as irreversible binding seemingly contradicts the catalytic mechanism of action. Here, we develop and characterize PSIRC3, a potent covalent PROTAC for Bruton’s tyrosine kinase (BTK). PSIRC3 induces selective BTK degradation with a sub-nanomolar DC50 of 0.75 nM and a Dmax of 85%, while its non-covalent counterpart is inactive. Such activity requires covalent bond formation with Cys481, as efficacy is abolished against the C481S BTK mutant. PSIRC3 achieves maximum BTK degradation within 30 min, a kinetic profile linked to rapid cell permeation and efficient ternary complex formation. In vivo, a single administration of PSIRC3 leads to substantial BTK degradation in both PBMCs ( > 80%) and splenocytes ( > 50%). Computational modeling, parameterized with our experimental data, reveals that degradation efficacy is governed by a balance between E3 ligase and target affinities: excessively high E3 affinity is detrimental by inducing a hook effect, while higher target affinity is generally beneficial. Our findings provide strong evidence that covalent engagement can drive potent and selective protein degradation, challenging the notion that catalytic turnover is indispensable for PROTAC efficacy, and open avenues for targeting intractable proteins.

Sulfonyl-Imidazopyridines Decouple Reactivity from Stability for Ligandability Mapping and Covalent Inhibitor Discovery

Zhihong Li, Madeleine L. Ware, Phillip W. Gingrich, Bissan Al-Lazikani, Ku-Lung Hsu;

 Journal American Chemical Society (2026) 148 (39): 41908–41920.

https://doi.org/10.1021/jacs.6c07487

Covalent probes and therapeutics must balance electrophilicity and stability for function in biological systems, yet gains in stability can sacrifice proteome coverage and limit ligand discovery. Here, we show that single-atom N-to-C substitutions of sulfonyl purines at the nucleofuge position decouple reactivity from stability. Systematic chemical proteomic profiling identifies sulfonyl-imidazopyridines as a class of electrophiles with enhanced cellular and in vivo stability while retaining tunable reactivity toward functional tyrosine and lysine sites. These electrophiles expand access to proteomic sites not engaged by parent sulfonyl-purines or related -triazoles. Importantly, the differential binding of N7- vs N9-sulfonyl-imidazopyridine regioisomer pairs expedites the discovery of proteome-wide-selective inhibitors of metabolic targets, including lanosterol synthase (LSS), phosphoglycerate mutase 1 (PGAM1), and DCTP pyrophosphatase 1 (DCTPP1). Collectively, this work establishes a general strategy for stabilizing electrophiles and introduces a platform for global ligandability mapping guided by regioselective recognition.

β-d-Arabinofuranose-configured Cyclitol Aziridines as Selective, Brain-penetrant Covalent GBA2 Inhibitors

Rob F. Lammers, Qin Su, Laura Mazo, Wendy A. Offen, Roelof Ottenhoff, Mats J. Bulterman, Martijn van der Lienden, Maria J. Ferraz, Florian Küllmer, Zahid Ilhan, Hans van den Elst, Ingeborg van der Made, Cindy P. A. A. van Roomen, Anne de Jongh, Adrianus M. C. H. van den Nieuwendijk, Rolf G. Boot, Noam Zelcer, Gideon J. Davies, Carme Rovira, Herman S. Overkleeft, Johannes M. F. G. Aerts, Marta Artola; 

Journal American Chemical Society, 2026

https://doi.org/10.1021/jacs.6c09754

 Neutral glucosylceramidase GBA2 is a retaining β-d-glucosidase involved in sphingolipid metabolism and implicated in neurological lysosomal storage disorders, yet the lack of highly selective chemical inhibitors has hindered efforts to disentangle its function from that of glucosylceramide synthase (GCS) and lysosomal glucosylceramidase (GBA1). Here, we report the development of the first brain-penetrant, covalent inhibitor that selectively inactivates GBA2 in vivo. Building on β-d-arabinofuranose-configured (β-d-Araf) cyclitol aziridine activity-based probes previously shown to covalently and selectively label GBA2, we designed and synthesized a focused library of electrophile-bearing cyclitols, including epoxides, cyclic sulfates, and a diverse set of N-alkyl aziridines. Biochemical evaluation of their inhibitory activity against GBA1, GBA2, GBA3 and GCS revealed that several N-alkyl aziridines are highly selective, nanomolar GBA2 inhibitors. Lead compounds also demonstrated metabolic stability in murine plasma, robust cellular activity and high selectivity in living cells, with no detectable GCS inhibition. Notably, octyl-alkyne aziridine 5 selectively inhibited GBA2 in mouse brain, representing the first covalent GBA2 inhibitor with demonstrated in vivo activity and brain permeability. Together, these results establish β-d-Araf cyclitol aziridine 5 as a versatile inhibitor for selective, covalent GBA2 inhibition, well suited to dissect GBA2 biology in vivo, particularly in neurological lysosomal storage disorders.

Structure-Guided Design of a Brain-Penetrant Covalent Inhibitor of Human Butyrylcholinesterase with In Vivo Procognitive Efficacy

Peter Mastnak-Sokolov, Urban Košak, Anže Meden, Damijan Knez, Simon Žakelj, Selena Horvat, Anja Pišlar, Jakob Kljun, Alexandre Igert, José Dias, Florian Nachon, Xavier Brazzolotto, Martin Novak, Vendula Rydlova, Jana Zdarova Karasova, Ondřej Soukup, Aleksandra Manik, Jan Detka, Kinga Sałat, Stanislav Gobec

J. Med. Chem. 2026

https://doi.org/10.1021/acs.jmedchem.6c01737

Butyrylcholinesterase (BChE) has emerged as a validated therapeutic target for the management of Alzheimer’s disease symptoms, particularly in advanced stages when acetylcholinesterase activity declines. Despite the numerous BChE inhibitors reported, only the cymserine derivative bisnorcymserine has progressed to clinical evaluation. Here, we report the design and characterization of a novel selective human BChE (hBChE) inhibitor, N-benzylpyrrolidine carbamate (S)-(+)-14. Kinetic analyses and X-ray crystallography of the hBChE−inhibitor complex confirm a covalent mechanism of inhibition. Compound (S)-(+)-14 demonstrates favorable metabolic stability and achieves brain exposure following intraperitoneal administration in mice. In a scopolamine-induced cognitive impairment model, (S)-(+)-14 improves cognitive performance without causing cholinergic adverse effects or motor deficits. Collectively, these findings identify (S)-(+)-14 as a structurally validated, brain-penetrant covalent hBChE inhibitor and highlight its promise as a lead compound for the treatment of Alzheimer’s disease.