Covalent binding design strategy: A prospective method for discovery of potent targeted anticancer agents

Luhong Wang, Jingyuan Zhao, Yao Yao, Changyuan Wang, Jianbin Zhang, Xiaohong Shu, Xiuli Sun, Yanxia Li, Kexin Liu, Hong Yuan, Xiaodong Ma

European Journal of Medicinal Chemistry, 2017

DOI: 10.1016/j.ejmech.2017.09.024

Cancer remains the most serious disease that threatens human health. Molecularly targeted cancer therapies, specifically small-molecule protein kinase inhibitors, form an important part of cancer therapy. Targeted covalent modification represents a proven approach to drug discovery with the recent FDA approvals of afatanib, ibrutinib, and osimertinib agents, which were designed to undergo an irreversible hetero-Michael addition reaction with a unique cysteine residue of a specific protein. Covalent inhibitors possess numerous advantages, including increased biochemical efficacy, longer duration of action, the high potential for improved therapeutic index due to lower effective dose, and the potential to inhibit certain drug resistance mechanisms. In this regard, the novel targeted anticancer agents whose activity is presumably dependent upon a hetero-Michael addition reaction with thiols are summarized in this article.

Structure–Activity Relationships of Potent, Targeted Covalent Inhibitors That Abolish Both the Transamidation and GTP Binding Activities of Human Tissue Transglutaminase

Abdullah Akbar, Nicole M. R. McNeil, Marie R. Albert, Viviane Ta, Gautam Adhikary, Karine Bourgeois, Richard L. Eckert, and Jeffrey W. Keillor

J. Med. Chem., Article ASAP

DOI: 10.1021/acs.jmedchem.7b01070

Human tissue transglutaminase (hTG2) is a multifunctional enzyme. It is primarily known for its calcium-dependent transamidation activity that leads to formation of an isopeptide bond between glutamine and lysine residues found on the surface of proteins, but it is also a GTP binding protein. Overexpression and unregulated hTG2 activity have been associated with numerous human diseases, including cancer stem cell survival and metastatic phenotype. Herein, we present a series of targeted covalent inhibitors (TCIs) based on our previously reported Cbz-Lys scaffold. From this structure–activity relationship (SAR) study, novel irreversible inhibitors were identified that block the transamidation activity of hTG2 and allosterically abolish its GTP binding ability with a high degree of selectivity and efficiency (kinact/KI > 105 M–1 min–1). One optimized inhibitor (VA4) was also shown to inhibit epidermal cancer stem cell invasion with an EC50 of 3.9 μM, representing a significant improvement over our previously reported “hit” NC9.

Impact of the structures of macrocyclic Michael acceptors on covalent proteasome inhibition

S. Kitahata,a  F. Yakushijiab  and  S. Ichikawa

Chem. Sci., 2017, 8, 6959-6963

DOI: 10.1039/C7SC02941A

Molecules that have a reactive functional group within a macrocycle represent a class of covalent inhibitor. The relationship between reactivity and affinity for the target is cooperative and complicated. An understanding and characterization of this class of inhibitor are vital for the development of covalent inhibitors as drug candidates. Herein, we describe a systematic analysis of structure–activity relationships using a series of syringolin analogues, which are irreversible covalent inhibitors of proteasomes. We investigate the detailed mechanistic effects of the macrocycles on affinity and reaction rate.

Differential Kinobeads Profiling for Target Identification of Irreversible Kinase Inhibitors

Lars Dittus, Thilo Werner, Marcel Muelbaier and Marcus Bantscheff
GlaxoSmithKline, Meyerhofstrasse 1, D-69117 Heidelberg, Germany

ACS Chem. Biol., Article ASAP

DOI: 10.1021/acschembio.7b00617

Chemoproteomics profiling of kinase inhibitors with kinobeads enables the assessment of inhibitor potency and selectivity for endogenously expressed protein kinases in cell lines and tissues. Using a small panel of targeted covalent inhibitors, we demonstrate the importance of measuring covalent target binding in live cells. We present a differential kinobeads profiling strategy for covalent kinase inhibitors where a compound is added either to live cells or to a cell extract that enables the comprehensive assessment of inhibitor selectivity for covalent and noncovalent targets. We found that Acalabrutinib, CC-292, and Ibrutinib potently and covalently bind TEC family kinases, but only Ibrutinib also potently binds to BLK. ZAK was identified as a submicromolar affinity Ibrutinib off-target due to covalent modification of Cys22. In contrast to Ibrutinib, 5Z-7-Oxozeaenol reacted with Cys150 next to the DFG loop, demonstrating an alternative route to covalent inactivation of this kinase, e.g., to inhibit canonical TGF-β dependent processes.

Quantification and Theoretical Analysis of the Electrophilicities of Michael Acceptors

Dominik S. Allgäuer, Harish Jangra, Haruyasu Asahara , Zhen Li, Quan Chen, Hendrik Zipse, Armin R. Ofial, and Herbert Mayr

J. Am. Chem. Soc., Article ASAP
DOI: 10.1021/jacs.7b05106

In order to quantify the electrophilic reactivities of common Michael acceptors, we measured the kinetics of the reactions of monoacceptor-substituted ethylenes (H2C═CH-Acc, 1) and styrenes (PhCH═CH-Acc, 2) with pyridinium ylides 3, sulfonium ylide 4, and sulfonyl-substituted chloromethyl anion 5. Substitution of the 57 measured second-order rate constants (log k) and the previously reported nucleophile-specific parameters N and sN for 3–5 into the correlation log k = sN(E + N) allowed us to calculate 15 new empirical electrophilicity parameters E for Michael acceptors 1 and 2. The use of the same parameters sN, N, and E for these different types of reactions shows that all reactions proceed via a common rate-determining step, the nucleophilic attack of 3–5 at the Michael acceptors with formation of acyclic intermediates, which subsequently cyclize to give tetrahydroindolizines (stepwise 1,3-dipolar cycloadditions with 3) and cyclopropanes (with 4 and 5), respectively. The electrophilicity parameters E thus determined can be used to calculate the rates of the reactions of Michael acceptors 1 and 2 with any nucleophile of known N and sN. DFT calculations were performed to confirm the suggested reaction mechanisms and to elucidate the origin of the electrophilic reactivities. While electrophilicities E correlate poorly with the LUMO energies and with Parr’s electrophilicity index ω, good correlations were found between the experimentally observed electrophilic reactivities of 44 Michael acceptors and their calculated methyl anion affinities, particularly when solvation by dimethyl sulfoxide was taken into account by applying the SMD continuum solvation model. Because of the large structural variety of Michael acceptors considered for these correlations, which cover a reactivity range of 17 orders of magnitude, we consider the calculation of methyl anion affinities to be the method of choice for a rapid estimate of electrophilic reactivities.

Structure-Activity Relationships of potent, targeted covalent inhibitors that abolish both the transamidation and GTP binding activities of human tissue transglutaminase

Abdullah Akbar, Nicole M.R. McNeil, Marie R. Albert, Viviane Ta, Gautam Adhikary, Karine Bourgeois, Richard L. Eckert, and Jeffrey W. Keillor

J. Med. Chem., 2017
DOI: 10.1021/acs.jmedchem.7b01070

Human tissue transglutaminase (hTG2) is a multifunctional enzyme. It is primarily known for its calcium-dependent transamidation activity that leads to formation of an isopeptide bond between glutamine and lysine residues found on the surface of proteins, but it is also a GTP binding protein. Overexpression and unregulated hTG2 activity has been associated with numerous human diseases, including cancer stem cell survival and metastatic phenotype. Herein, we present a series of targeted covalent inhibitors (TCIs) based on our previously reported Cbz-Lys scaffold. From this structure-activity relationship (SAR) study, novel irreversible inhibitors were identified that block the transamidation activity of hTG2 and allosterically abolish its GTP binding ability with a high degree of selectivity and efficiency (kinact/KI > 105 M-1min-1). One optimized inhibitor (VA4) was also shown to inhibit epidermal cancer stem cell invasion with an EC50 of 3.9 µM, representing a significant improvement over our previously reported ‘hit’ NC9.

Lysine-Targeting Covalent Inhibitors

Jonathan Pettinger, Keith Jones, Matthew David Cheeseman

Angewandte Chemie International Edition, 2017

DOI: 10.1002/anie.201707630

Targeted covalent inhibitors have gained widespread attention in drug discovery as a validated method to circumvent acquired resistance in oncology. This strategy exploits small molecule/protein crystal structures to design tight-binding ligands with appropriately positioned electrophilic warheads. Whilst most focus has been on targeting binding site cysteine residues, targeting nucleophilic lysine residues can also represent a viable approach to irreversible inhibition. However, owing to the basicity of the ε-amino group in lysine, this strategy generates a number of specific challenges. Herein, we review the key principles for inhibitor design, give historical examples and present recent developments that demonstrate its potential for future drug discovery.